Advances in Experimental Medicine and Biology 1122

Alexander Birbrair Editor

Pericyte
Biology in
Different
Organs
Advances in Experimental Medicine and
Biology

Volume 1122

Editorial Board:

IRUN R. COHEN, The Weizmann Institute of Science, Rehovot, Israel

ABEL LAJTHA, N.S. Kline Institute for Psychiatric Research,
Orangeburg, NY, USA
JOHN D. LAMBRIS, University of Pennsylvania, Philadelphia, PA, USA
RODOLFO PAOLETTI, University of Milan, Milan, Italy
NIMA REZAEI, Tehran University of Medical Sciences, Children’s Medical
Center Hospital, Tehran, Iran

More information about this series at http://www.springer.com/series/5584

Alexander Birbrair
Editor

Pericyte Biology in Different

Organs
Editor
Alexander Birbrair
Department of Radiology
Columbia University Medical Center
New York, NY, USA

Department of Pathology
Federal University of Minas Gerais
Belo Horizonte, MG, Brazil

ISSN 0065-2598     ISSN 2214-8019 (electronic)

Advances in Experimental Medicine and Biology
ISBN 978-3-030-11092-5    ISBN 978-3-030-11093-2 (eBook)
https://doi.org/10.1007/978-3-030-11093-2

Library of Congress Control Number: 2019934955

© Springer Nature Switzerland AG 2019

This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of
the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation,
broadcasting, reproduction on microfilms or in any other physical way, and transmission or information
storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology
now known or hereafter developed.
The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication
does not imply, even in the absence of a specific statement, that such names are exempt from the relevant
protective laws and regulations and therefore free for general use.
The publisher, the authors and the editors are safe to assume that the advice and information in this book
are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the
editors give a warranty, express or implied, with respect to the material contained herein or for any errors
or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims
in published maps and institutional affiliations.

This Springer imprint is published by the registered company Springer Nature Switzerland AG
The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland
Preface

This book’s initial title was “Pericyte Biology: Development, Homeostasis and
Disease.” However, due to the current great interest in this topic, we were able to
assemble more chapters than would fit in one book, covering pericyte biology under
distinct circumstances. Therefore, the book was subdivided into three volumes enti-
tled Pericyte Biology - Novel Concepts, Pericyte Biology in Different Organs, and
Pericyte Biology in Disease.
This book Pericyte Biology in Different Organs presents contributions by expert
researchers and clinicians in the multidisciplinary areas of medical and biological
research. The chapters provide timely detailed overviews of recent advances in the
field. This book describes the major contributions of pericytes to different organs’
biology in physiological and pathological conditions. Further insights into the biol-
ogy of pericytes will have important implications for our understanding of organ
development, homeostasis, and disease. The authors focus on the modern method-
ologies and the leading-edge concepts in the field of cell biology. In recent years,
remarkable progress has been made in the identification and characterization of
pericytes in several tissues using state-of-the-art techniques. These advantages facil-
itated the identification of pericyte subpopulations and definition of the molecular
basis of pericytes’ role within different organs. Thus, the present book is an attempt
to describe the most recent developments in the area of pericyte behavior which is
one of the emergent hot topics in the field of molecular and cellular biology today.
Here, we present a selected collection of detailed chapters on what we know so far
about the pericytes in various tissues and under distinct pathophysiological condi-
tions. Thirteen chapters written by experts in the field summarize the present knowl-
edge about the roles of pericytes in different organs.
Herbert A. Reitsamer and colleagues from Paracelsus Medical University/SALK
discuss the role of pericytes in the retina. Limor Landsman from Tel Aviv University
describes pericytes in the pancreas. Lynn M. Schnapp and colleagues from the
Medical University of South Carolina compile our understanding of pericyte biol-
ogy in the lung. Jyoti Gautam and Yao Yao from the University of Georgia update us
with what we know about skeletal muscle pericytes. Mercedes Fernandez and col-
leagues from the University of Barcelona summarize current knowledge on gut

v
vi Preface

pericytes. Yuya Kunisaki from Kyushu University Hospital addresses the impor-
tance of pericytes in the bone marrow. Martin Canis and Mattis Bertlich from the
University Hospital Munich focus on cochlear pericytes. Maria Angelica Miglino
and colleagues from the University of São Paulo introduce our current knowledge
about placental pericytes. Enis Kostallari and Vijay H. Shah from the Mayo Clinic
discuss the roles of pericytes in the liver. Motohiro Komaki from Kanagawa Dental
University introduces what we know about pericytes in the periodontal ligament.
Linda L. Lee and Vishnu Chintalgattu from Amgen Inc. talk about pericytes in the
heart. Clifford L. Librach and colleagues from the University of Toronto focus on
umbilical cord pericytes. Finally, Michail S. Davidoff from the University Medical
Center Hamburg-Eppendorf gives an overview of pericytes in the testis.
It is hoped that the articles published in this book will become a source of refer-
ence and inspiration for future research ideas. I would like to express my deep grati-
tude to my wife Veranika Ushakova and Mr. Murugesan Tamilsevan from Springer,
who helped at every step of the execution of this project.
This book is dedicated to the memory of my grandfather Pavel Sobolevsky, PhD,
a renowned mathematician, who passed away during the creation of this piece.

My grandfather Pavel Sobolevsky z”l, PhD (March 26, 1930–August 16, 2018)

New York, NY, USA Alexander Birbrair

Belo Horizonte, MG, Brazil
Contents

1 Pericytes in the Retina ������������������������������������������������������������������������������   1

Andrea Trost, Daniela Bruckner, Francisco J. Rivera,
and Herbert A. Reitsamer
2 Pancreatic Pericytes in Glucose Homeostasis and Diabetes ������������������ 27
Limor Landsman
3 Pericytes in the Lung �������������������������������������������������������������������������������� 41
Chi F. Hung, Carole L. Wilson, and Lynn M. Schnapp
4 Pericytes in Skeletal Muscle �������������������������������������������������������������������� 59
Jyoti Gautam and Yao Yao
5 Pericytes in the Gut ���������������������������������������������������������������������������������� 73
Marta Ramirez, Nuria Pell, Marc Mejias, and Mercedes Fernandez
6 Pericytes in Bone Marrow ������������������������������������������������������������������������ 101
Yuya Kunisaki
7 Cochlear Capillary Pericytes ������������������������������������������������������������������ 115
Martin Canis and Mattis Bertlich
8 Pericytes in the Placenta: Role in Placental Development and
Homeostasis ���������������������������������������������������������������������������������������������� 125
Rodrigo S. N. Barreto, Patricia Romagnolli, Andressa Daronco Cereta,
Leda M. C. Coimbra-Campos, Alexander Birbrair,
and Maria Angelica Miglino
9 Pericytes in the Liver �������������������������������������������������������������������������������� 153
Enis Kostallari and Vijay H. Shah
10 Pericytes in the Periodontal Ligament ���������������������������������������������������� 169
Motohiro Komaki

vii
viii Contents

11 Pericytes in the Heart ������������������������������������������������������������������������������ 187

Linda L. Lee and Vishnu Chintalgattu
12 Pericytes in the Umbilical Cord �������������������������������������������������������������� 211
Andrée Gauthier-Fisher, Peter Szaraz, and Clifford L. Librach
13 The Pluripotent Microvascular Pericytes Are the Adult Stem
Cells Even in the Testis ���������������������������������������������������������������������������� 235
Michail S. Davidoff
Index ������������������������������������������������������������������������������������������������������������������ 269
Contributors

Rodrigo S. N. Barreto School of Veterinary Medicine and Animal Sciences,

University of São Paulo, Butantã, Sao Paulo, Brazil
Mattis Bertlich The Department of Otorhinolaryngology, Head and Neck Surgery,
University Hospital, Munich, Federal Republic of Germany
Alexander Birbrair Department of Radiology, Columbia University Medical
Center, New York, NY, USA
Department of Pathology, Federal University of Minas Gerais, Pampulha, Belo
Horizonte, Brazil
Daniela Bruckner Department of Ophthalmology, University Clinic of
Ophthalmology and Optometry, Research Program for Experimental Ophthalmology
and Glaucoma Research, Paracelsus Medical University/SALK, Salzburg, Austria
Martin Canis The Department of Otorhinolaryngology, Head and Neck Surgery,
University Hospital, Munich, Federal Republic of Germany
Andressa Daronco Cereta School of Veterinary Medicine and Animal Sciences,
University of São Paulo, Butantã, Sao Paulo, Brazil
Vishnu Chintalgattu Department of CardioMetabolic Disorders, Amgen Research
and Discovery, Amgen Inc., South San Francisco, CA, USA
Leda M. C. Coimbra-Campos Department of Pathology, Federal University of
Minas Gerais, Pampulha, Belo Horizonte, Brazil
Michail S. Davidoff University Medical Center Hamburg-Eppendorf, Hamburg
Museum of Medical History, Hamburg, Germany
Mercedes Fernandez Angiogenesis in Liver Disease Research Group, IDIBAPS
Biomedical Research Institute, Hospital Clinic, University of Barcelona, Barcelona,
Spain
Biomedical Research Networking Center on Hepatic and Digestive Disease
(CIBEREHD), Spanish National Institute of Health, Barcelona, Spain

ix
x Contributors

Jyoti Gautam Department of Pharmaceutical and Biomedical Sciences, University

of Georgia, Athens, GA, USA
Andrée Gauthier-Fisher CReATe Fertility Centre, University of Toronto, Toronto,
ON, Canada
Chi F. Hung Division of Pulmonary, Critical Care and Sleep Medicine, University
of Washington, Seattle, WA, USA
Motohiro Komaki Department of Highly Advanced Stomatology, Graduate
School of Dentistry, Kanagawa Dental University, Yokohama City, Kanagawa,
Japan
Enis Kostallari Division of Gastroenterology and Hepatology, Mayo Clinic,
Rochester, MN, USA
Yuya Kunisaki Kyushu University Hospital, Center for Cellular and Molecular
Medicine, Fukuoka, Japan
Limor Landsman Department of Cell and Developmental Biology, Sackler
Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel
Linda L. Lee Department of CardioMetabolic Disorders, Amgen Research and
Discovery, Amgen Inc., South San Francisco, CA, USA
Clifford L. Librach CReATe Fertility Centre, University of Toronto, Toronto, ON,
Canada
Department of Obstetrics and Gynecology, University of Toronto, Toronto, ON,
Canada
Department of Physiology, University of Toronto, Toronto, ON, Canada
Institute of Medical Sciences, University of Toronto, Toronto, ON, Canada
Department of Obstetrics and Gynecology, Women’s College Hospital, Toronto,
ON, Canada
Marc Mejias Angiogenesis in Liver Disease Research Group, IDIBAPS
Biomedical Research Institute, Hospital Clinic, University of Barcelona, Barcelona,
Spain
Biomedical Research Networking Center on Hepatic and Digestive Disease
(CIBEREHD), Spanish National Institute of Health, Barcelona, Spain
Maria Angelica Miglino School of Veterinary Medicine and Animal Sciences,
University of São Paulo, Butantã, Sao Paulo, Brazil
Nuria Pell Angiogenesis in Liver Disease Research Group, IDIBAPS Biomedical
Research Institute, Hospital Clinic, University of Barcelona, Barcelona, Spain
Marta Ramirez Angiogenesis in Liver Disease Research Group, IDIBAPS
Biomedical Research Institute, Hospital Clinic, University of Barcelona, Barcelona,
Spain
Contributors xi

Herbert A. Reitsamer Department of Ophthalmology, University Clinic of

Ophthalmology and Optometry, Research Program for Experimental Ophthalmology
and Glaucoma Research, Paracelsus Medical University/SALK, Salzburg, Austria
Francisco J. Rivera Institute of Mol. Regenerative Medicine, Spinal Cord Injury
and Tissue Regeneration Center Salzburg (SCI-TReCS), Paracelsus Medical
University Salzburg, Salzburg, Austria
Laboratory of Stem Cells and Neuroregeneration, Institute of Anatomy, Histology
and Pathology, Faculty of Medicine and Center for Interdisciplinary Studies on the
Nervous System (CISNe), Universidad Austral de Chile, Valdivia, Chile
Patricia Romagnolli School of Veterinary Medicine and Animal Sciences,
University of São Paulo, Butantã, Sao Paulo, Brazil
Lynn M. Schnapp Division of Pulmonary, Critical Care, Allergy and Sleep
Medicine, Medical University of South Carolina, Charleston, SC, USA
Vijay H. Shah Division of Gastroenterology and Hepatology, Mayo Clinic,
Rochester, MN, USA
Peter Szaraz CReATe Fertility Centre, University of Toronto, Toronto, ON,
Canada
Andrea Trost Department of Ophthalmology, University Clinic of Ophthalmology
and Optometry, Research Program for Experimental Ophthalmology and Glaucoma
Research, Paracelsus Medical University/SALK, Salzburg, Austria
Carole L. Wilson Division of Pulmonary, Critical Care, Allergy and Sleep
Medicine, Medical University of South Carolina, Charleston, SC, USA
Yao Yao Department of Pharmaceutical and Biomedical Sciences, University of
Georgia, Athens, GA, USA
Chapter 1
Pericytes in the Retina

Andrea Trost, Daniela Bruckner, Francisco J. Rivera,

and Herbert A. Reitsamer

Abstract Pericytes (PCs) are specialized cells located abluminal of endothelial

cells (ECs) on capillaries, embedded within the same basement membrane. They are
essential regulators of vascular development, remodeling, and blood-retina-barrier
(BRB) tightness and are therefore important components to maintain tissue homeo-
stasis. The perivascular localization and expression of contractile proteins suggest
that PCs participate in capillary blood flow regulation and neurovascular coupling.
Due to their ability to differentiate into various cell types in vitro, they are regarded
as potential cells for tissue repair and therapeutic approaches in regenerative medi-
cine. Altered function or loss of PCs is associated with a multitude of CNS diseases,
including diabetic retinopathy (DR). In this chapter, we will provide a short over-
view of retinal vascular development, the origin of PCs, and focus on PCs in reti-
nopathy of prematurity (ROP) and in the diabetic retina. Further, animal models to
study the fate of PCs and the potential role of (retinal) PCs in regeneration and
wound healing will be discussed.

Keywords Pericyte · Retina · Origin · Pericyte marker · PDGFRb · NG2 · tbx18 ·

Diabetic retinopathy (DR) · Retinopathy of prematurity (ROP) · Wound healing ·
Regeneration

A. Trost (*) · D. Bruckner · H. A. Reitsamer

Department of Ophthalmology, University Clinic of Ophthalmology and Optometry,
Research Program for Experimental Ophthalmology and Glaucoma Research, Paracelsus
Medical University/SALK, Salzburg, Austria
e-mail: [email protected]
F. J. Rivera
Institute of Mol. Regenerative Medicine, Spinal Cord Injury and Tissue Regeneration Center
Salzburg (SCI-TReCS), Paracelsus Medical University Salzburg, Salzburg, Austria
Laboratory of Stem Cells and Neuroregeneration, Institute of Anatomy,
Histology and Pathology, Faculty of Medicine and Center for Interdisciplinary Studies
on the Nervous System (CISNe), Universidad Austral de Chile, Valdivia, Chile

© Springer Nature Switzerland AG 2019 1

A. Birbrair (ed.), Pericyte Biology in Different Organs,
Advances in Experimental Medicine and Biology 1122,
https://doi.org/10.1007/978-3-030-11093-2_1
2 A. Trost et al.

Abbreviations

Angs Angiopoietins
BBB Blood-brain barrier
BM Bone marrow
BRB Blood-retina barrier
CNS Central nervous system
DME Diabetic macular edema
DR Diabetic retinopathy
ECs Endothelial cells
INL Inner nuclear layer
IPL and OPL Inner and outer plexiform layer
MSCs Mesenchymal stem cells
NG2 Neuron-glial antigen 2
NVU Neurovascular unit
ON Optic nerve
ONL Outer nuclear layer
P0 Postnatal day 0
PCs Pericytes
PDGFRb PDGF-receptor beta
RPE Retinal pigment epithelial cells
tbx 18 T-box family transcription factor 18
TGF-b Transforming growth factor beta
VEGF Vascular endothelial growth factor
vSMCs Vascular smooth muscle cells

1.1 Retinal Structure and Function

The human eye is composed of three different layers. The outermost layer is formed
by the cornea and sclera. The middle layer is divided into an anterior part (iris and
ciliary body) and a posterior part (choroid). The light-sensitive organ, the retina,
forms the innermost layer and lines the inner surface of the eye, extending from the
papilla to the ora serrata. In the center of the retina, axons of the ganglion cells are
bundled within the optic nerve (ON) running to the visual cortex in the brain. In
addition to cells of the oligodendroglial lineage, the ON contains incoming blood
vessels, that vascularize the inner retina. Temporal of the ON, the blood vessel free
fovea, the sharpest point of vision and most essential part of the retina for human
vision, is located (Fig. 1.1a). The retina can be divided into the neurosensory retina
and the retinal pigment epithelium. The neurosensory retina is composed of three
layers of nerve cell bodies and two layers of synapses. The outer nuclear layer
(ONL) contains cell bodies of the rods and cones. Bipolar, horizontal, and amacrine
1 Pericytes in the Retina 3

Fig. 1.1 (a) Schematic drawing of an eye cross section. (b) Schematic drawing of a retinal cross
section: the neurosensory retina is composed of three layers of nerve cell bodies and two layers of
synapses. The outer nuclear layer (ONL) contains cell bodies of the rods and cones. Bipolar, hori-
zontal, and amacrine cells are located in the inner nuclear layer (INL). The innermost layer, the
ganglion cell layer, contains cell bodies of ganglion cells and displaced amacrine cells. Within the
inner and outer plexiform layer (IPL and OPL), located between the nerve cell layers, synaptic
contacts occur. Nutrition to retina is provided by the choroid and the inner retinal vasculature: the
three retinal vascular plexi are located in the GCL (superficial), in the IPL (intermediate), and in
the OPL (deep)

cells are located in the inner nuclear layer (INL). The innermost layer, the ganglion
cell layer contains cell bodies of ganglion cells and displaced amacrine cells. Within
the inner and outer plexiform layer (IPL and OPL), located between the nerve cell
layers, synaptic contacts occur (Fig. 1.1b) (Kolb 1995). One of the main functions
of the retina is the conversion of light into an electric impulse, the first stage of
image processing. The light passes through the entire retina, to reach the pigment
molecules in the photoreceptors. The light signal is converted into an electrical
impulse and transmitted to the bipolar cells and the ganglion cells where the signal
is finally sent through the ON to the visual cortex. Photoreceptor cell metabolism
and functioning of the visual cycle is maintained by a monolayer of retinal pigment
epithelial cells (RPE), located in direct contact to the ONL.

1.2 Retinal Vascular Development

Nutrition of the metabolically highly active neural retina is provided by two vas-
cular beds: the outer retina, including photoreceptors and RPE cells, is supplied by
diffusion from the choriocapillaries. The inner retina is nourished by retinal blood
vessels (Fig. 1.1b). During embryonal development, the inner retina is metabolically
4 A. Trost et al.

supported by the hyaloid vasculature, an arterial network in the vitreous. In humans,

the hyaloid vasculature is replaced by retinal vasculature around 15 weeks of gesta-
tion and by the formation of the primary plexus. This remodels into three parallel
connected vascular networks located in the nerve fiber layer and the inner and outer
plexiform layer, until retinal vascularization is completed by 38–40 weeks of gesta-
tion (Lutty and McLeod 2017). In mouse, however, retinal vascular development
starts by sprouting of vessels out of the optic nerve head at birth (P0). At this time
point, a cellular network of astrocytes, already developed, provides a template for
blood vessel sprouting and for the establishment of the primary vascular network.
Within the first 3 postnatal weeks, the three vascular plexi are developed (Dorrell
et al. 2002; Fruttiger 2002, 2007; Selvam et al. 2017): the vessels spread along the
inner retinal surface to the ora serrata until the inner vascular layer is completed at
postnatal day 7 to 10 (P7–P10). They subsequently spread into the retina and form
the deep capillary layer. Finally, the intermediate capillary is formed from P14–P21.
The mammalian retina is dedicated to the central nervous system (CNS) since it
derives from the neural tube and is formed through evagination from the diencepha-
lon. Like in other CNS tissues, paracellular and transendothelial transport from the
vasculature to the surrounding retinal tissue is highly regulated by the BRB, ensur-
ing an optimal chemical composition of the neuronal microenvironment. The BRB
is composed of the inner BRB (retinal capillary endothelial cells) and the outer BRB
(retinal pigment epithelial cells). Although BRB tightness is mainly mediated by
tight and adherent junctions between ECs, PCs have been proven to be an essential
constituent of the BRB and blood-brain barrier (BBB). The contribution of PCs to
the BRB/BBB is discussed in the subheading entitled: “Pericytes and their impact
on the blood-retina barrier (BRB).”

1.3 Identification of (Retinal) Pericytes (PCs)

Currently, there is an urgent need to study and determine the role of retinal PCs in
health and disease. To achieve this goal, the proper identification of this barely
explored cell type is essential. However, following the gene and protein expression
pattern, an identification of a specific PC molecular signature has been quite chal-
lenging. Up to now, no unique PC marker has been identified. Currently, the estab-
lished marker panel for PC characterization comprises PDGF-receptor beta
(PDGFRβ), neuron-glial antigen 2 (NG2, Cspg4, Fig. 1.2a), CD13 (brain PCs),
Desmin, Vimentin, and RGS5 (Armulik et al. 2011). More recently, additional
markers like Gli1 (Kramann et al. 2015) and Tbx18 (Guimaraes-Camboa et al.
2017) were proposed to identify PCs. A recent comparative single-cell transcripto-
mal study identified genes specifically expressed in (brain) PCs: in addition to the
known markers Pdgfrb, Cspg4, Rgs5, and Anpep, Kcnj8, Cd248, Abcc9, Vtn, and
S1pr3 were identified (Vanlandewijck et al. 2018). Of note, many markers used to
identify PCs (e.g., NG2/Cspg4, PDGFRb) are also positive in vascular smooth
1 Pericytes in the Retina 5

Fig. 1.2 (a) NG2-specific labeling of pericytes (PCs, green, arrowheads) on capillaries and
vSMCs (green, open arrowheads) on arterioles and arteries in the ganglion cell layer (GCL) of a
retinal whole mount preparation. (b) Schematic drawing of PCs (green) on capillaries, showing
also a capillary cross section comprising PCs and endothelial cells. (c) Retinal whole mount, show-
ing retinal ganglion cells (RGCs, labeled with Brn3a in red), axons of RGCs (labeled with
NF200 in white), and PCs/vSMCs (green) in the GCL

muscle cells (vSMCs); therefore the localization of PCs on microvessels is an

important identification criteria. Further, alpha-SMA allows for discrimination of
PCs and vSMCs, being negative in PCs on capillaries, but positive in vSMCs on
arterioles/arteries in vivo. Therefore, a combination of NG2 and PDGFRb with
a-SMA and their capillary localization can be recommended for the identification of
retinal PCs (Trost et al. 2013). Depending on their differentiation stage and tissue
localization, PCs may exhibit a heterogeneous morphology. However, in general,
PCs located on microvessels show a spherical-shaped soma with a prominent
nucleus and processes that extend longitudinal on and around the vessel wall, cover-
ing several ECs (Armulik et al. 2011; Shepro and Morel 1993). As a part of the
CNS, the retina exhibits a high PC density with a PC to EC ratio of about 1:1 to 1:3
(Armulik et al. 2011; Frank et al. 1990), which is constant in healthy conditions.

1.4 Origin of (Retinal) PCs

PCs are generated during embryonic and postnatal life (Winkler et al. 2011). During
developmental stages two different primary sources have been described, a neuro-
ectodermal and a mesodermal origin. Quail chick transplantations and a multitude
of reporter mouse models have been used to investigate and discriminate neuroecto-
dermal and mesodermal origin of PCs in different organs. Neuroectodermal or neu-
ral crest origin of PCs and vSMCs has been demonstrated by quail chick
transplantations of brain anlagen and mesoderm in cerebral blood vessels (Korn
et al. 2002) or in brain surface vessels by using Foxs1+/ß-Gal mice (Heglind et al.
2005). Using a Wnt-1-Cre mouse model, neural crest origin was proven for PCs of
embryonic hyaloid blood vessels (Gage et al. 2005), however without providing PC
characterization by additional markers. Along this line, a neural crest origin was
6 A. Trost et al.

demonstrated for the majority of retinal and choroidal PCs and vSMCs using a
Sox10-Cre mouse model (Trost et al. 2013) and of cortical grey matter PCs using an
inducible Sox10-CreERT2 mouse (E7.5 induction) (Simon et al. 2012). These find-
ings were recently confirmed in the brain, eye, and thymus PCs and vSMCs using
Cre-driven Sox10 and Wnt1 mouse models (Wang et al. 2017). To keep in mind,
Wnt1 is a strict marker for neural crest cells and is not detected after migration of
neural crest cells; however, Sox10 is expressed in postmigratory neural crest cells,
as well as in glial cells of the developing nervous system and during adulthood. To
distinguish in the adult tissue, cells with an embryonic neural crest origin vs. cells
that derived from a postnatal Sox10-expressing tissue source, tamoxifen induction
around E8.5–E12.5 in an inducible Sox10-CreERT2 mouse model (Hong and Saint-­
Jeannet 2005), would be desirable.
In addition to a neural crest origin of PCs and vSMCs (forebrain) (Etchevers et al.
2001), also a mesodermal origin has been described for PCs and vSMCs in the mid-
brain, brainstem, spinal cord, and peripheral organs (Korn et al. 2002). These experi-
ments highlight that PCs and vSMC of both, neural crest and mesodermal origin,
coexist within the same organ. Using an XlacZ4 reporter under the control of an
adipose tissue-specific promoter, mesodermal origin was demonstrated in PCs and
vSMCs throughout the vascular bed (Tidhar et al. 2001). Recent studies suggest a
heterogeneous mesodermal origin of PCs: Yamamoto et al. described CD31+F4/80+
macrophages as potential source for a subset of cerebrovascular NG2+ PCs
(Yamamoto et al. 2017), however lacking verification with a second PC marker. In
line with this, tissue myeloid progenitor cells are suggested to differentiate into a
subset of dermal PCs in embryonic skin vasculature through transforming growth
factor-ß signaling (Yamazaki et al. 2017): using a Vav-iCre mouse model to label
hematopoietic cells, one third of NG2+ and PDGFRb+ PCs revealed reporter expres-
sion in the embryonic skin (E15.5). Further investigations on the subtype of hema-
topoietic cells revealed that tissue localized myeloid cells contributes to PC
development in the skin vasculature (CD11b-Cre mouse model). The depletion of
the respective cells [myeloid lineage (PU.1−/−, Ncx1−/−) and macrophages (Csf1op/
op
)] resulted in a defective PC development in skin and brain. In addition, the authors
concluded a limited contribution of neural crest cells to PC development in the skin
(E15.5) using a Wnt-1-Cre reporter mouse and further suggested a minimal contri-
bution of ECs (Tie2-Cre mouse model) (Yamazaki et al. 2017). In contrast, endocar-
dial ECs have been demonstrated to give rise to a subset of cardiac PCs in the murine
embryonic heart through endothelial-mesenchymal transition (Chen et al. 2016):
using EC-specific reporter mice (cdh5-CreERT2, Tie2-Cre, Nfatc1-CreERT2),
reporter-specific expression was detected in PDGFRb+ NG2+ cardiac PCs.
Postnatally, PCs can be recruited by proliferative expansion of preexisting PCs
or from the bone marrow (BM) during vasculature remodeling, e.g., under ischemic
conditions (Kokovay et al. 2006) or in tumors (Song et al. 2005). In line with this,
BM-derived neovascular PCs were also reported following bFGF-induced corneal
neovascularization (Ozerdem et al. 2005).
A multitude of studies investigated the origin of PCs and vSMCs, which is of
great interest in developmental biology and further for the diagnosis and treatment
1 Pericytes in the Retina 7

of diseases associated with PC dysfunction. Up to now, these cells revealed hetero-

geneous origins, even within the same tissue. As recent data suggest that the dif-
ferentiation potential of PCs is limited depending on their origin and tissue
localization (Herrmann et al. 2016), knowledge about the origin may be crucial for
the use of distinct PCs in regenerative approaches. In line with this, several PC
subtypes have been identified, participating in tissue repair and regeneration. Since
both PCs and vSMCs express the respective reporter, a common ancestor as well as
a morphological and biochemical continuum from vSMC to PCs can be assumed.
Indeed, an evolutionary conserved gradual phenotype change along the arteriove-
nous axis was proposed between PCs and (venous) SMCs as well as SMCs form
arteries and arterioles applying single-cell transcriptomal analysis (Vanlandewijck
et al. 2018). However, the authors detected no evidence for the existence of brain
PC subtypes, but they clearly demonstrated an organotypicity, between PCs isolated
from the brain and the lung (Vanlandewijck et al. 2018).

1.5 Animal Models to Study PCs

The contribution of PCs to angiogenesis, vessel stabilization, and BBB has been
studied in mouse models targeting signaling pathways essential for PC-EC interac-
tion and communication. Several molecules, like platelet-derived growth factor
beta (PDGF-B), transforming growth factor beta (TGF-b), vascular endothelial
growth factor (VEGF), and angiopoietins (Angs) (Armulik et al. 2011; Ribatti et al.
2011), are involved in the modulation and control of PC-EC interaction. ECs and
PCs are interdependent; defects or impaired signaling in one cell type affect the
other cell type.

1.5.1 PDGFRb/PDGFB

PDGFRb is expressed on PCs and vascular SMCs, and PDGFRb positive PCs/
vSMC are recruited via endothelial bound PDGF-B to angiogenic sprouts (Betsholtz
2004; Gerhardt and Betsholtz 2003). The PDGF-B/PDGFRb pathway is crucial for
PC proliferation, migration, and recruitment to angiogenic active sites. Disruption
of PDGF-B/PDGFRb signaling components lead to impaired PC recruitment,
reduced PC coverage, dilated capillaries, microaneurysm, and BBB breakdown
(Bell et al. 2010; Lindahl et al. 1997; Tallquist et al. 2003; Winkler et al. 2010).
Finally, impaired PDGFRb signaling and subsequent PC loss results in pathologi-
cally altered blood vessels and impaired tissue homeostasis in these mice. PDGFRb
or PDGF-B knockout mice are not viable and die as embryos, displaying a loss of
PC coverage on brain capillaries and showing evidence of lethal hemorrhages
(Leveen et al. 1994; Lindahl et al. 1997; Soriano 1994).
8 A. Trost et al.

Cre recombinase under the control of the PDGFRb promoter fragment

(−4.7/+0.1 kb) crossed with a reporter strain was used to label PCs and vSMCs
in vivo (Cuttler et al. 2011), showing a close correlation between the endogenous
PDGFRb staining and the transgenic reporter (Cuttler et al. 2011). However, during
embryonic development, PDGFRb is expressed broadly throughout the embryo and
therefore not suitable to trace selectively the fate of PCs (Guimaraes-Camboa et al.
2017). However, in adult animals, PDGFRb expression is confined to PCs and
vSMCs. To trace the fate of PCs, an inducible PDGFRb-CreERT2 mouse model has
been generated (Sheikh et al. 2015), revealing distinct PC labeling in the retina after
tamoxifen induction at P5, P6, and P7 (Park et al. 2017). A second inducible
PDGFRb-P2A-CreERT2 mouse model was established, labeling 84.17 ± 3.48% of
NG2+/PDGFRb+ PCs after P1/P2/P3 tamoxifen induction via the lactating mother if
combined with the reporter line Rosa-tdtomato (Cuervo et al. 2017). Interestingly, a
significantly reduced labeling of NG2+ PCs (41.94 ± 18.67%) was detected by
crossing PDGFRb-P2A-CreERT2 with the reporter line Rosa-mT/mG (Cuervo et al.
2017). These discrepancies between the recombination efficiency of different mouse
reporter lines were reported previously (Liu et al. 2013) and should be taken into
consideration if using reporter lines.

1.5.2 NG2 Proteoglycan

In 2001, NG2 proteoglycan (neural glial antigen 2, CSPG4) was first described as a
PC-specific marker (Ozerdem et al. 2001), showing its impact on angiogenesis by
reduced proliferation of both PCs and ECs in the retina of NG2 null mice (Ozerdem
and Stallcup 2004). Until now, NG2-based mouse models have been used predomi-
nantly in the oligodendroglial lineage and myelin research fields; nevertheless sev-
eral reports exist using these particular animal models to study PCs. For instance,
Schallek et al. imaged retinal PCs noninvasively in the living eye using the NG2-­
dsRed mouse model (Schallek et al. 2013). This model was also used to investigate
cerebral blood flow (Hall et al. 2014). Furthermore, inducible and non-inducible
NG2-Cre reporter mouse models were used to study regional blood flow in brain
(Hill et al. 2015). Similarly, the EYFP-NG2 mouse model was used to perform live
cell imaging of PCs (Zehendner et al. 2013). Tamoxifen induction at different reti-
nal vascular developmental stages in a NG2-CreERT2 reporter mouse revealed spe-
cific PC and vSMC labeling in all retinal vascular layers, although the recombination
efficiency (15%) was rather low (Bruckner et al. 2018).
1 Pericytes in the Retina 9

1.5.3 Tbx18

The T-box family transcription factor tbx 18 has been shown to be essential in kid-
ney vascular development, being expressed in vSMCs and PCs (Xu et al. 2014).
Recently, the specific expression of tbx18 in PCs and vSMCs was identified in the
adult mouse and an inducible reporter mouse model established (tbx18-CreERT2) to
trace the fate of PCs and vSMCs. The authors verified a specific reporter expression
in 90–95% of PDGFRb+/NG2+/CD146+ PCs and vSMCs in different tissues, includ-
ing the retina using the Rosa26tdtomato reporter mouse line (Guimaraes-Camboa et al.
2017), following 3x i.p. tamoxifen injections to adult animals (8-week-old).
Currently, three powerful fate mapping models, the PDGFRb-CreERT2, PDGFRb-­
P2A-­CreERT2, and the tbx18-CreERT2, labeling specifically the majority of PCs and
vSMC following tamoxifen induction in the adulthood, are available. Combining
the NG2-CreERT2 mouse with a more efficient reporter mouse may also increase the
applicability of this fourth model. In general, these models allow to investigate the
in vivo behavior of PCs under healthy conditions as well as their response and cell
fate upon injury or under other pathological conditions. Of note, all these models
label both, PCs and vSMCs, therefore being not able to distinguish between these
two cell types based on the reporter expression. Nevertheless, taking the vascular
diameter into consideration, PCs can be only identified on microvessels, the
capillaries.

1.6  Cs and Their Impact on the Blood-Retina Barrier

P
(BRB)

CNS homeostasis is maintained by a highly coordinated neurovascular unit (NVU),

composed of ECs, PCs (capillaries), vSMC (arteries), glial cells, and neurons. The
interrelation of these cells provides the formation and maintenance of the BRB/
BBB, ensuring a tightly regulated barrier function and tissue/CNS homeostasis. In
the last decades, PCs have been proven to be a substantial component of the BRB
and BBB. Loss or dysfunction of PCs and disrupted BRB/BBB is associated with a
variety of neuropathological diseases, like DR (Eshaq et al. 2017), Alzheimer’s dis-
ease (Sweeney et al. 2018), multiple sclerosis, or CADASIL (Ghosh et al. 2015).
The impact of PC on BBB function has been studied in mouse models with modi-
fied PC-EC cell signaling, drawing emphasis on the PDGFB/PDGFRb signaling
pathway. The use of PDGFRb signaling-deficient mice demonstrated that reduced
PC coverage in brain results in BBB breakdown and an accumulation of plasma-­
derived proteins, resulting in secondary neuronal degenerative alterations (Bell
et al. 2010). In line with this, PC deficiency was associated with increased BBB
permeability in further studies in adult (Armulik et al. 2010) and embryonic tissue
(Daneman et al. 2010). PCs’ contribution to the formation of the BRB in the devel-
oping retinal vasculature was demonstrated by the increased expression of the tight
10 A. Trost et al.

junction protein ZO-1 after direct contact of ECs and PCs and further astrocytes
(Kim et al. 2009), however with the limitation that the authors used aSMA to iden-
tify PCs. A recent study emphasized that PC loss during embryonic development
differs in its impact on BBB stability from adult PC loss: a reduction of PC coverage
during vascular maturation, by PDGF-B depletion at P5, resulted in severe hemor-
rhage, disrupted vascular plexus formation, and macrophage infiltration (Park et al.
2017). PC dropout in capillaries of adult PDGFRb-Cre-ERT2 mice via diphtheria
toxin resulted in a pronounced ablation of PDGFRb+ and NG2+ PCs and SMA+
vSMCs in retinal vessels, however without apparent changes in vascular remodeling
or leakage in the retina and brain (Park et al. 2017). These results indicate that PC
loss from stabilized (retinal) vessels is not sufficient to induce alterations in BRB
and retinal vessel integrity, although adequate PC coverage is essential for BRB
formation and maturation during vascular development. Nevertheless, in many age-­
related diseases, the breakdown of BBB and/or BRB is discussed as one of the main
disease contributing factors.

1.7 PCs in Retinal Diseases

Pathological retinal neovascularization is a main factor of sight-threatening dis-

eases, including diabetic retinopathy (DR), retinopathy of prematurity (ROP), and
age-related macular degeneration (AMD). Therefore, understanding angiogenic
mechanisms in the physiological as well as pathological retina is essential to iden-
tify involved cells, being potential targets for therapeutic interventions. The poten-
tial role of PC in the course of ROP and DR will be discussed.

1.7.1 PCs in Retinopathy of Prematurity (ROP)

The impact of PCs in neovascularization in ROP as well as the interaction of EC and

PC in pathologic neovascularization is poorly understood. Contradictory reports,
regarding the presence of PC on neovascular vessels/tufts in ROP, are published and
will be discussed. ROP affects prematurely born babies, receiving intensive neona-
tal care, including oxygen therapy. Excess oxygen and subsequent hypoxia mainly
contributed to ROP development, however, in case of controlled oxygen supple-
mentation other risk factors get more prominent (gestational age, lack of growth
factor, low birth weight, pre−/postnatal growth). The normal growth of blood ves-
sels is directed by low oxygen in non-vascularized retinal areas and is completed by
38–40 weeks of gestation (see retinal vascular development). In utero, the partial
pressure of oxygen is rather low (50 mmHg), which is increased at ambient room air
(160 mmHg) and even further in case of oxygen supply during neonatal care.
Subsequently, vascular development is reduced or even ceased resulting in avascu-
lar, thus hypoxic retina. This in turn leads to excess production of oxygen-sensitive
1 Pericytes in the Retina 11

growth factors, e.g., VEGF, resulting in neovascularization, including pathological

vessels, also growing into the a-vascular vitreous [reviewed in Liegl et al. (2016)].
Although these vessels may regress during retinal development, fibrous scar tissue
may remain and cause traction to the retina, potentially resulting in retinal detach-
ment and blindness. Furthermore, due to impaired BRB, these pathological vessels
are often leaky. In case of retinal neovascularization, laser photocoagulation and
anti-VEGF therapies are used to treat infants, whereas the latter is discussed contro-
versially regarding dosage and long-term safety and efficacy (Sankar et al. 2018).
The aim to find a balance between low oxygen tension to minimize ROP and high
oxygen tension to prevent brain hypoxia and death remains challenging as shown by
a meta-analysis study (Askie et al. 2011). Another important factor will be the sup-
plementation of IGF-1 in preterm infants to increase very low serum levels, enabling
normal postnatal growth. First animal studies supported the role of supplemental
IGF-1 in preventing ROP (Vanhaesebrouck et al. 2009). Pharmacokinetic and dos-
ing studies of IGF-1/IGFBP-3 were performed in preterm infants (Lofqvist et al.
2009), and its impact on ROP investigated in a phase II study (NTC01096784),
showing no effect on ROP, but reduction in bronchopulmonary dysplasia and intra-
ventricular hemorrhage (Hansen-Pupp et al. 2017).
As retinal vasculature in rodents develops after birth, animal models are used to
study pathologies in retinal vascular development postnatal. To gain insight into
mechanisms of ROP, neovascularization following oxygen-induced retinopathy
(OIR), mimicking the neovascular response in ROP, is analyzed in a multitude of
studies. An established OIR model involves hyperoxia (75% oxygen) from postna-
tal day (P)7 to P12, inducing capillary regression in central retina. Following return
to room air at P12, hypoxia triggers neovascularization which peaks around P17
(Smith et al. 1994). Lee et al. analyzed the impact of CCN1 expression on angio-
genic processes under OIR. CCN1, an extracellular matrix protein active during
angiogenesis, is expressed in ECs during vascular development. However, under
ischemic conditions, CCN1 expression is detected on PCs. Following OIR-induced
retinal neovascularization, retinal tufts, growing toward the vitreous, are formed and
covered by PCs. PC-specific deletion of CCN1 under ischemic conditions results in
reduced angiogenic signals and reduced neovascular outgrowth (Lee et al. 2017),
that was associated with reduced expression of Wnt5a. The authors emphasized a
change in EC-PC communication in angiogenesis under ischemic conditions. In
line with the study of Lee et al., a recent study reported regular PC coverage in the
three-layered vascular plexus as well as in neovascular tufts at P17 following OIR
(Choi et al. 2018). Further, N-cadherin, essential for the maintenance of the BRB
and EC-PC interaction, was detected in neovascular tufts, indicating an intact
EC-PC interaction under ischemic conditions (Choi et al. 2018). However, whether
this EC-PC interaction is intact and sufficient to mediate BRB tightness remains to
be elucidated. In contrast, previous studies suggested a loss of NG2+ PCs following
OIR in rats; however, PC specificity is hard to assess with the presented pictures
(Saito et al. 2007). In line with them, Hughes et al. reported impaired mural cell
differentiation and loss in the neovasculature of an obliterative as well as the surviv-
ing vasculature of a nonobliterative OIR model, both resulting in reduced mural cell
12 A. Trost et al.

coverage and enlarged vessels (Hughes et al. 2007). The authors suggested impaired
perfusion and EC cell death as possible causes for mural cell changes. Reduction of
mural cells in the hypoxic phase, through the application of a PDGFR inhibitor
(STI571), increased neovascularization in ROP rats and was further correlated with
an increase in VEGF and VEGFR-2 mRNA (Wilkinson-Berka et al. 2004).
Concordantly, PDGF-B+/− mice, showing a 30% reduction in PC coverage at P7,
developed twice as many new blood vessels as wild-type littermates under ROP
(Hammes et al. 2002). This indicates that PC deficiency results in reduced inhibition
of EC proliferation, especially under high VEGF levels in hypoxic conditions,
resulting in the formation of new, potential pathologic blood vessels. In contrast, a
more recent study suggested that the development of the deep capillary network
rather than the recruitment of PCs to the vessel sprouts is important to mediate resis-
tance to vascular regression processes induced by hyperoxia (Hoffmann et al. 2005).
Regarding the tightness of neovascular vessels, increased BRB permeability was
demonstrated in newly formed vessels by HRP leakage in the hypoxic phase of a cat
ROP model; however, these newly developed vessels matured and became tight
(Chan-Ling et al. 1992).
Although contradictory results are published regarding the impact of PC cover-
age on newly formed vessels in hypoxic retina, the presence of PCs seems to be vital
for the maintenance of normal angiogenesis by controlling EC proliferation. While
more recent studies report an intact PC-EC interaction and PC coverage (Choi et al.
2018; Lee et al. 2017), the physical contact may not be sufficient to prevent neovas-
cularization in ROP/OIR. Further, the tightness of these neovascular tufts has not
been tested in these studies and remains to be elucidated and compared to previous
studies reporting BRB leakage in neovasculature after OIR. All together, these find-
ings suggest an important role of PCs in neovascularization in OIR, rendering PCs
as potential targets in therapeutic approaches to treat ROP.

1.7.1.1 Cell-Based Therapy Approaches in ROP

Cell-based therapies to ameliorate and repair retinopathic insults were studied in

models of OIR. First attempts to ameliorate ischemia-induced neovascularization
were performed using adult bone marrow-derived myeloid progenitor cells. Upon
intravitreal injection during the hyperoxia or normoxia phase (P9–P12), these
HIF-1a positive cells migrated to avascular regions of the OIR damaged retina and
accelerated vascular repair and normalization. An even better effect was obtained
injecting BM cells prior to exposure to hyperoxia (P2–P7) (Ritter et al. 2006).
Detailed characterization of transplanted cells did not reveal NG2 or CD31 expres-
sion, and the authors excluded a differentiation into PCs or ECs. However, trans-
planted cells were positive for microglial/macrophagic marker and their
differentiation into microglial cells concluded, improving and stabilizing the
response of retinal vessels to hypoxia (Ritter et al. 2006). In contrast, EC-specific
IGFBP-3 expression increased the differentiation of hematopoietic stem cells into
1 Pericytes in the Retina 13

PCs and astrocytes and resulted in increased PC coverage and reduced PC apoptosis
in OIR mice, followed by normalization in vessel morphology (Kielczewski et al.
2011). Further, the authors suggested a beneficial effect of reducing the number of
activated microglial cells by IGFBP-3 expression, which is opposed to findings of
Ritter et al. suggesting a benefit of differentiated microglial cells. The IGFBP-3
mediated reduction in preretinal neovascularization is already under investigation in
clinical ROP studies as discussed above. Besides endothelial progenitor cells,
adipose-­derived stem cells, or bone marrow-derived cells, the transplantation of
mesenchymal stem cells (MSCs) is considered beneficial for tissue repair and thera-
peutic angiogenesis (Sieveking and Ng 2009). In line with this, a TGF-ß1-mediated
suppression of excessive neovascularization in an OIR mouse model was demon-
strated after i.p. injection of human placental amniotic membrane-derived MSCs
(AMSCs). The cells migrated to the retina; however, the lack of integration into the
vascular network suggested absence of differentiation of AMSCs into ECs or PCs
and therefore a regulation of angiogenesis through paracrine mechanisms (Kim
et al. 2016b). Adipose-derived stem cells (ASCs) are able to differentiate into PCs
and were tested for their ability to stabilize retinal vessels in models of retinal vas-
culopathy. Following OIR, intravitreally injected, differentiated ASCs were able to
integrate into retinal vasculature and accelerated recovery from OIR through
enhanced vessel regrowth. Further, injection of these cells before OIR vessel desta-
bilization resulted in reduced retinal capillary dropout. Additionally, injection of
ASCs prevented capillary dropout in a DR model (Akimba mouse) (Mendel et al.
2013). Additional approaches using cell-based therapies for therapeutic revascular-
ization of ischemic injury is reviewed in Trinh et al. (2016). Although many studies
report a beneficial effect following transplantation of diverse cells on OIR pathol-
ogy, the precise mechanisms of neovascularization need additional investigations.
Further, administration routes, mobilization/homing, time points, and appropriate
cell numbers or survival and differentiation of transplanted cells need to be evalu-
ated preclinical to achieve optimized therapeutic use in neovascularization.

1.7.2 PCs in Diabetic Retinopathy (DR)

The second pathology including retinal neovascularization and involvement of PCs

is diabetic retinopathy (DR). DR is a major complication of diabetes leading to
visual impairment and ultimately blindness (Yau et al. 2012). DR is characterized
by increased vascular permeability and microaneurysms, which is most likely
caused by PC loss (Frank 2004; Hammes et al. 2002). This PC loss and formation
of microaneurysms are hallmarks of an early disease state, leading to decreased reti-
nal microcirculation and subsequent hypoxia. Progressive retinal hypoxia, loss of
retinal capillaries, and oxidative stress activate the release of inflammatory cyto-
kines and hypoxia-induced expression of angiogenic growth factors (e.g., vascular
endothelial growth factor, VEGF), resulting in retinal neovascularization: new
14 A. Trost et al.

blood vessels develop, which now extend into the normally a-vascular vitreous and
anterior chamber angle, causing loss of vision and neovascular glaucoma, respec-
tively. Neovascularization can be considered as a central element in the progression
of DR. In a recent review, the effects of reactive metabolites, transient and chronic
hyperglycemia in correlation to neurodegeneration, edema, and neovascularization
are summarized (Hammes 2018).
DR has long been considered a vascular disease based on vascular pathologies
detected in diabetic patients during ophthalmologic examinations. Increasing
reports demonstrating dysfunction and degeneration of nonvascular cells [reviewed
in Kern and Barber (2008)] changed the classification of DR to a neurodegenerative
disease. The exact pathomechanisms for the development and progression of DR
are still unclear, and the mechanism of PC loss in DR remains controversial. PC loss
through apoptosis and destructive pathways under hyperglycemic conditions has
been suggested (Behl et al. 2008). Altered glutamate excitation, reduced trophic
factor signaling, oxidative stress, deposition of hyperglycemia-induced advanced
glycation end products, alterations in intracellular pathways, dysregulation of
endogenous neuroprotective factors, and neuroinflammation are among many
potential causes for an increased apoptosis [reviewed in Barber et al. (2011), Eshaq
et al. (2017) and Hernandez et al. (2016)]. Others suggest active PC depletion by
non-apoptotic mechanisms via growth factor systems like angiopoietin-2/Tie-2
(Pfister et al. 2008). Further, the impact, interdependence, and chronology of neuro-
nal and vascular degeneration in DR are still unclear and discussed controversially.

1.7.2.1 BRB Breakdown in DR

The breakdown of BRB in DR has been reviewed in an excellent article (Klaassen

et al. 2013). Although the molecular mechanisms of PC loss and vascular leakage
have been studied in different DR mouse models [e.g., Enge et al. (2002) and Huang
et al. (2011)], PC loss and subsequent BRB breakdown are not fully understood.
Disruption of endothelial-specific PDGF-B and subsequent reduced PC coverage
resulted in a loss of neuronal layers and folding of the photoreceptor layer in the
retina, resembling signs of DR (Enge et al. 2002; Lindblom et al. 2003). In line with
this, a mutation in PDGFRb resulted in a CNS restricted reduced PC coverage,
impaired vascular permeability, and aberrant BRB development, associated with
retinal ganglion cell (RGC) apoptosis, mimicking features of non-proliferative DR
(Jadeja et al. 2013). Consistently, transient inhibition of PC recruitment by an i.p.
injection of anti-PDGFRb antibody (APB5) to developing retinal vessels (P1)
resulted in BRB breakdown in adult mouse retina (Ogura et al. 2017), resembling
DR pathology with sustained vascular abnormalities and autonomous disease pro-
gression. Furthermore, a clear correlation of APB5 concentration and retinal col-
lapse could be demonstrated. However, APB5 injection in adult mice failed to
induce PC loss from mature vessels, indicating that PDGFRb signaling is not essen-
tial for the maintenance of EC-PC associations. On the other hand, retinal
1 Pericytes in the Retina 15

overexpression of Ang-2 induces PC migration and vascular pathology, suggesting

that Ang-2 plays an important role in diabetic vasoregression via PC destabilization
(Pfister et al. 2010). The mouse model Ins2AktiaVEGF+/−, mimicking signs of
advanced clinical DR (diabetic macular edema, proliferative DR), was established
to study molecular mechanisms at later stages of DR (Wisniewska-Kruk et al.
2014). As the mechanisms of PC loss and BRB breakdown are not fully resolved
until now and (PC-) specific treatment strategies are missing, current therapeutic
interventions aim to reduce neovascularization in proliferative DR.

1.7.2.2 Current Treatments in DR and Potential Therapeutic Strategies

As poor glycemic control is the most relevant risk factor for DR development, tight
blood glucose and blood pressure control are inevitable. In case of proliferative DR,
surgical interventions like laser photocoagulation (Hammes 2005), vitrectomy
(Newman 2010), and intravitreal anti-VEGF injections (Krebs et al. 2013) are cur-
rent approaches to slow down the progression of DR. Although anti-VEGF thera-
pies mediate the desired anti-angiogenic effect and significantly lower the incidence
of vision loss, they also block the neuroprotective and neurotrophic VEGF effects,
which may result in a decreased RGCs survival as demonstrated in rat ischemia-­
reperfusion experiments (Nishijima et al. 2007). Nevertheless, the preventive impact
of an intravitreal ranibizumab (anti-VEGF(−A)) injection was tested in early dia-
betic rats, showing an amelioration in PC loss and increased RGC survival (Xiao
et al. 2017). However, as anti-VEGF treatments in many patients do not inhibit
disease progression (Ip et al. 2015), blocking of different angiogenic factors
(PDGFRb, bFGF, HGF, CTGF) is necessary to target VEGF-independent pathways
when aiming for a more effective therapy.
To date, a multitude of substances has been tested in animal models for their
neuro- and vascular protective properties in DR progression, e.g., growth factors
like insulin-like growth factor, pigment epithelium-derived factor, brain-derived
neurotrophic factor, and nerve growth factor (Hernandez et al. 2016). Focusing on
anti-inflammatory therapy approaches, in addition to corticosteroids, several agents
are in preclinical testing and clinical trials: intravitreal injection of infliximab, a
TNF-a inhibitor, showing positive effects on visual acuity in human patients with a
diabetic macular edema (DME) (Sfikakis et al. 2010), or topical application of
nepafenac, a potent cyclooxygenase inhibitor, revealing a reduction of DME devel-
opment after cataract surgery in patients with DR (Pollack et al. 2016).

1.7.2.3 Cell-Based Therapy Approaches in DR

Endothelial progenitor cells (EPCs) are discussed for their use in therapeutic revas-
cularization and potential vascular repair in diabetic patients [reviewed in Shaw
et al. (2011)]. The beneficial effect of the intravitreal injection of outgrowth
16 A. Trost et al.

endothelial cells (OECs) in ischemic retina (OIR model) was demonstrated by

decreased avascular areas and reduced pathologic neovascularization, due to the
incorporation of OECs in the resident vasculature (Medina et al. 2010). In line with
this, CD34+ cells are regarded as important therapeutic option for revascularization
of ischemic vascular areas and have been tested successfully in clinical trials
(Mackie and Losordo 2011). The use of CD34+ cells isolated from diabetic patients
revealed impaired ability to attach and assimilate into the retinal vasculature after
transplantation, highlighting that the diabetic environment alter EPC phenotype and
function (Caballero et al. 2007). The transplantation of diabetic, transient TGF-ß-
inhibited peripheral CD34+ cells resulted in retinal vascular repair in a retinal isch-
emia reperfusion injury mouse model, suggesting the potential use of (dysfunctional)
cells of diabetic patients for autologous transplantation following ex vivo treatment
(Bhatwadekar et al. 2010). A different cell-based therapeutic approach in DR was
performed by intravitreal injection of TGF-ß1-preconditioned mouse adipose-­
derived stem cells (mASCs) into a model of DR (Akimba mouse). Injection of DiI-­
labeled mASCs at 5 weeks of age revealed retinal microvasculature integration of
these cells 4 weeks later, suggesting their differentiation into PCs. Additionally, reti-
nal stabilization was further suggested by DiI-labeled mASCs without direct incor-
poration on retinal vessels, by conditioning the retinal microenvironment (Mendel
et al. 2013). The ability of mASCs to prevent diabetic retinal microvascular dropout
was tested by their injection at P9 and resulted in reduced capillary loss 2 months
later (Mendel et al. 2013). A similar approach was performed by intravitreal injec-
tion of adipose-derived mesenchymal stem cells (MSCs) into diabetic mice.
Although an increase in intraocular neurotrophic factors and reduced retinal oxida-
tive damage was detected, the cells remained in the vitreous and did not differentiate
into neural- or perivascular-like cells (Ezquer et al. 2016). Therefore, the benefit of
MSC administration in this model was mediated by the generation of a neuroprotec-
tive, anti-apoptotic microenvironment, thereby reducing RGC loss. Recently, human
embryonic stem cells were differentiated into cells revealing similar phenotypic and
functional characteristics of PCs. These cells (hESC-PVPCs) were intravitreally
injected into one eye of diabetic rats and an amelioration in vascular leakage
detected in comparison to the contralateral vehicle-injected eyes (Kim et al. 2016a).
Whether these cells compensate for early PC loss or additionally stabilize the vas-
culature remains to be elucidated. Further, whether an equal amelioration can be
achieved by injecting (retinal) PCs instead of hESC-PVPCs will be of high interest.
An important parameter for the survival and integration of transplanted cells is the
microenvironment in the recipient tissue, being rather inhospitable under diabetic
conditions. In line with this, Ingram et al. demonstrated diminished vasculogenesis
of diabetic versus nondiabetic cord blood-derived progenitor cells upon implanta-
tion (Ingram et al. 2008). In general, the combination of two or more cell popula-
tions for transplantation may result in superior neovascularization and regeneration,
replacing different cell types of a vessel. However, absence of standard cell isolation
protocols and the use of heterogeneous cell populations without detailed character-
ization impair the possibility to compare beneficial effects in animal and clinical
studies, even within the same cell population.
1 Pericytes in the Retina 17

Although some of these substances and cells revealed promising therapeutic

potential, counteracting neuronal and vascular degeneration, ameliorating DR
­progression in experimental and clinical studies, their safe and efficient future thera-
peutic application in the clinic is uncertain. Even though great efforts and improve-
ments in understanding DR pathogenesis and mechanisms have been achieved, DR
development and progression seem to be a multifactorial, complicated process, and
thus, the development of new therapeutic approaches remains challenging.

1.8  otential Role of PCs in Tissue Regeneration and Wound

P
Healing

The capacity of PCs to differentiate into mesenchymal cell types (e.g., osteoblasts,
chondrocytes, adipocytes) has been confirmed in a multitude of studies, and the
contribution of in vitro differentiated PCs to tissue repair and regeneration after
transplantation has been demonstrated in several injury models. The osteogenic
potential of retinal PCs has been proven in vitro and in a diffusion chamber assay
in vivo, also demonstrating formation of cartilage, adipose, and fibrous tissue
(Doherty et al. 1998; Farrington-Rock et al. 2004). Apart from retinal PCs, the dif-
ferentiation potential of PCs isolated from the microvasculature of human muscle
into skeletal muscle was demonstrated in vitro and also in vivo after transplantation
in dystrophic mice (Dellavalle et al. 2007). The contribution of a PC subpopulation
to fiber development was further confirmed in an alkaline phosphatase-based
lineage-­tracing mouse model, during postnatal growth and during acute and chronic
tissue regeneration (Dellavalle et al. 2011). In line with this, PCs purified from dif-
ferent tissues exhibited myogenic potential and regenerated myotubes in cardiotoxin-­
injured skeletal muscle after transplantation (Crisan et al. 2008). The cells further
exhibited osteogenic, chondrogenic, and adipogenic potentials and expressed MSC
markers. Comparing CD34−/CD146+ PCs isolated from human adipose tissue and
bone marrow, differences in the trilineage differentiation potential were detected,
suggesting a tissue-dependent regenerative potential of PCs in vitro (Herrmann
et al. 2016). Tissue dependency was also demonstrated for myocardial PCs, which
were not able to differentiate into the myogenic lineage, as proven for skeletal mus-
cle PCs (Chen et al. 2015). Furthermore, the ability of CNS PCs to differentiate into
non-mesenchymal cells was proven by their differentiation into cells of the neural
and glial lineage (Dore-Duffy et al. 2006; Paul et al. 2012). In addition, the multi-
lineage potential of PCs isolated from ischemic brain was demonstrated, suggesting
their potential repair capacity at the site of brain injuries (Nakagomi et al. 2015).
However, beside tissue-dependent differences, the lack of a commonly used isola-
tion and cultivation protocol (culture conditions like media, serum, cultivation/dif-
ferentiation time period) impedes comparison of experimental findings. Nevertheless,
this in vitro multilineage potential raised expectations that endogenous PCs may
contribute to wound healing and regeneration by replacing specific cells types lost
18 A. Trost et al.

due to injury or disease. As the unequivocal identification of PCs is hampered by its

differing morphological phenotype and protein expression according to the tissue
located in and the differentiation state, a multitude of PC subtypes has been
described and characterized until now. A subpopulation of PCs was shown to give
rise to scar-forming stromal cells in an injured spinal cord (SCI) model using a
Glast-CreER transgenic mouse (Goritz et al. 2011). In line with this, the participa-
tion of PCs in scar formation was described for Nestin−/NG2+ PCs (Birbrair et al.
2014) and PDGFRb+ PCs (Matsushita et al. 2015) after SCI. Further, PCs have been
described to differentiate into myofibroblasts in two kidney fibrosis models, using
an inducible FoxD1-CreERT2 mouse model (Humphreys et al. 2010). Identifying
PCs as potential precursors of myofibroblast and collagen producing cells high-
lighted them as target for anti-fibrotic therapies. Recently, PDGFRb+ brain PCs have
been proposed as potential source of perivascular neural stem cells following tran-
sient brain ischemia/reperfusion injury (Nakata et al. 2017). To confirm these find-
ings, the use of a fate-mapping mouse models, like the PDGFRb-P2A-CreERT2
(Cuervo et al. 2017), will be desirable.
Whether PC heterogeneity is present within the same tissue is under debate, the
distinct responsiveness of subpopulations of PCs to injury/stimuli, however, argues
in favor of this hypothesis (Santos et al. 2017). In contrast, a recent work suggested
the lack of brain PC subtypes but clearly demonstrated a difference between PCs
isolated from the brain versus the lung (Vanlandewijck et al. 2018). The existence
of retinal PC subtypes, however, remains unknown. Recent studies using fate-­
mapping mouse models (PDGFRb, tbx18) reported a > 85% efficiency in targeting
retinal PC, verified by co-labeling with additional PC markers (Cuervo et al. 2017;
Guimaraes-Camboa et al. 2017; Park et al. 2017), suggesting a quite homogeneous
PC population in the retina regarding marker expression. Whether labeling of just a
subpopulation of PCs in other reporter models [e.g., NG2 Hill et al. (2015)] is due
to a specific lower recombination efficiency or due to the presence of distinct sub-
types remains to be elucidated. The expression of different markers as well as the
presence of additional cell types with perivascular location may hamper a confident
identification of proper PCs, emerging the concept of PC subtypes.
Although a multitude of publications describe a potential contribution of PC
(subtypes) to tissue repair and regeneration, in vivo (trans-) differentiation using
fate mapping mouse models has not been proven until now. The recently established
tbx18-CreERT2 mouse model, labeling ~90% of PDGFRb+/NG2+/CD146+ PCs and
vSMCs, was used to investigate the in vivo transdifferentiation potential of PCs
(Guimaraes-Camboa et al. 2017). The authors tested five conditions, revealing no
significant contribution of tbx18+ cells to other cell lineages but retaining PDGFRb
expression. Comparing the brain, heart, skeletal muscle, and brown and white adi-
pose depots of 8- and 87-week-old reporter animals, absence of transdifferentiation
was detected. In line with this, tbx18+-PCs did not transdifferentiate following
trans-aortic constriction (fibrosis model), high-fat diet (adipogenic progenitors),
myofiber degeneration (myogenic potential), or cortical stab wound (neuronal
transdifferentiation or scar tissue formation) (Guimaraes-Camboa et al. 2017).
Although FACS sorted tbx18+ PCs displayed lineage plasticity in vitro, they did not
1 Pericytes in the Retina 19

reveal adipogenic, fibrogenic, myogenic, or neuronal progenitor potential in vivo in

the injury models tested. Hence, plasticity observed in vitro or following transplan-
tation in vivo might be a result of the artificial cell culture environment. Nevertheless,
whether the lack of in vivo transdifferentiation potential of PCs holds true in differ-
ent injury models has to be tested. Importantly, this study highlights the use of
inducible reporter mouse models to perform lineage-tracing studies. And, although
PCs have been excluded as endogenous progenitors for regeneration in these tis-
sues, the use and beneficial effect of in vitro cultivated PCs in regeneration
persists.

1.9 A Commentary on Likely Future Trends or Directions

In addition to their central role in vascular development and maintenance, the mul-
tipotency of PCs emphasizes their role in tissue repair and regeneration. The poten-
tial of PC-associated applications seems to be continuously increasing, ranging
from anti-fibrotic therapies, anti-angiogenic therapies to transplanted PCs for regen-
erative approaches. The identification of PCs remains challenging; however, the use
of inducible fate-mapping models promises facilitation to gain insight into PC func-
tion and mechanism under physiological and pathological conditions. Whether PCs
can be stimulated endogenously to participate in tissue regeneration and replace-
ment of damaged cells remains elusive. In case of effective transdifferentiation of
PCs into damaged cells in vitro or in vivo, their integration into the existing (neuro-
nal) cell network will be essential for successful tissue regeneration, repair, and
function. In fact, we need a deeper understanding of (retinal) PC function, interac-
tion, and differentiation capacity in order to translate findings obtained from animal
models to humans.

References

Armulik A, Genove G, Betsholtz C (2011) Pericytes: developmental, physiological, and pathologi-

cal perspectives, problems, and promises. Dev Cell 21:193–215
Armulik A, Genove G, Mae M, Nisancioglu MH, Wallgard E, Niaudet C, He L, Norlin J, Lindblom
P, Strittmatter K, Johansson BR, Betsholtz C (2010) Pericytes regulate the blood-brain barrier.
Nature 468:557–561
Askie LM, Brocklehurst P, Darlow BA, Finer N, Schmidt B, Tarnow-Mordi W, Ne OCG (2011)
NeOProM: neonatal oxygenation prospective Meta-analysis collaboration study protocol.
BMC Pediatr 11:6
Barber AJ, Gardner TW, Abcouwer SF (2011) The significance of vascular and neural apoptosis to
the pathology of diabetic retinopathy. Invest Ophthalmol Vis Sci 52:1156–1163
Behl Y, Krothapalli P, Desta T, DiPiazza A, Roy S, Graves DT (2008) Diabetes-enhanced tumor
necrosis factor-alpha production promotes apoptosis and the loss of retinal microvascular cells
in type 1 and type 2 models of diabetic retinopathy. Am J Pathol 172:1411–1418
20 A. Trost et al.

Bell RD, Winkler EA, Sagare AP, Singh I, LaRue B, Deane R, Zlokovic BV (2010) Pericytes
control key neurovascular functions and neuronal phenotype in the adult brain and during brain
aging. Neuron 68:409–427
Betsholtz C (2004) Insight into the physiological functions of PDGF through genetic studies in
mice. Cytokine Growth Factor Rev 15:215–228
Bhatwadekar AD, Guerin EP, Jarajapu YP, Caballero S, Sheridan C, Kent D, Kennedy L, Lansang
MC, Ruscetti FW, Pepine CJ, Higgins PJ, Bartelmez SH, Grant MB (2010) Transient inhibition
of transforming growth factor-beta1 in human diabetic CD34+ cells enhances vascular repara-
tive functions. Diabetes 59:2010–2019
Birbrair A, Zhang T, Files DC, Mannava S, Smith T, Wang ZM, Messi ML, Mintz A, Delbono
O (2014) Type-1 pericytes accumulate after tissue injury and produce collagen in an organ-­
dependent manner. Stem Cell Res Ther 5:122
Bruckner D, Kaser-Eichberger A, Bogner B, Runge C, Schrödl F, Strohmaier C, Silva ME,
Zaunmair P, Couillard-Despres S, Aigner L, Rivera FJ, Reitsamer HA, Trost A (2018) Retinal
pericytes: characterization of vascular development-dependent induction time points in an
inducible NG2 reporter mouse model. Curr Eye Res 43(10):1274–1285. https://doi.org/10.108
0/02713683.2018.1493130. Epub 2018 Jul 18. PMID:29939774.
Caballero S, Sengupta N, Afzal A, Chang KH, Li Calzi S, Guberski DL, Kern TS, Grant MB
(2007) Ischemic vascular damage can be repaired by healthy, but not diabetic, endothelial
progenitor cells. Diabetes 56:960–967
Chan-Ling T, Tout S, Hollander H, Stone J (1992) Vascular changes and their mechanisms in the
feline model of retinopathy of prematurity. Invest Ophthalmol Vis Sci 33:2128–2147
Chen Q, Zhang H, Liu Y, Adams S, Eilken H, Stehling M, Corada M, Dejana E, Zhou B, Adams
RH (2016) Endothelial cells are progenitors of cardiac pericytes and vascular smooth muscle
cells. Nat Commun 7:12422
Chen WC, Baily JE, Corselli M, Diaz ME, Sun B, Xiang G, Gray GA, Huard J, Peault B (2015)
Human myocardial pericytes: multipotent mesodermal precursors exhibiting cardiac specific-
ity. Stem Cells 33:557–573
Choi SH, Chung M, Park SW, Jeon NL, Kim JH, Yu YS (2018) Relationship between Pericytes and
endothelial cells in retinal neovascularization: a histological and immunofluorescent Study of
retinal angiogenesis. Korean J Ophthalmol 32:70–76
Crisan M, Yap S, Casteilla L, Chen CW, Corselli M, Park TS, Andriolo G, Sun B, Zheng B, Zhang
L, Norotte C, Teng PN, Traas J, Schugar R, Deasy BM, Badylak S, Buhring HJ, Giacobino
JP, Lazzari L, Huard J, Peault B (2008) A perivascular origin for mesenchymal stem cells in
multiple human organs. Cell Stem Cell 3:301–313
Cuervo H, Pereira B, Nadeem T, Lin M, Lee F, Kitajewski J, Lin CS (2017) PDGFRbeta-P2A-­
CreER(T2) mice: a genetic tool to target pericytes in angiogenesis. Angiogenesis 20:655–662
Cuttler AS, LeClair RJ, Stohn JP, Wang Q, Sorenson CM, Liaw L, Lindner V (2011) Character-­
ization of Pdgfrb-Cre transgenic mice reveals reduction of ROSA26 reporter activity in remod-
eling arteries. Genesis 49:673–680
Daneman R, Zhou L, Kebede AA, Barres BA (2010) Pericytes are required for blood-brain barrier
integrity during embryogenesis. Nature 468:562–566
Dellavalle A, Maroli G, Covarello D, Azzoni E, Innocenzi A, Perani L, Antonini S, Sambasivan
R, Brunelli S, Tajbakhsh S, Cossu G (2011) Pericytes resident in postnatal skeletal muscle dif-
ferentiate into muscle fibres and generate satellite cells. Nat Commun 2:499
Dellavalle A, Sampaolesi M, Tonlorenzi R, Tagliafico E, Sacchetti B, Perani L, Innocenzi A,
Galvez BG, Messina G, Morosetti R, Li S, Belicchi M, Peretti G, Chamberlain JS, Wright
WE, Torrente Y, Ferrari S, Bianco P, Cossu G (2007) Pericytes of human skeletal muscle are
myogenic precursors distinct from satellite cells. Nat Cell Biol 9:255–267
Doherty MJ, Ashton BA, Walsh S, Beresford JN, Grant ME, Canfield AE (1998) Vascular pericytes
express osteogenic potential in vitro and in vivo. J Bone Miner Res 13:828–838
Dore-Duffy P, Katychev A, Wang X, Van Buren E (2006) CNS microvascular pericytes exhibit
multipotential stem cell activity. J Cereb Blood Flow Metab 26:613–624
1 Pericytes in the Retina 21

Dorrell MI, Aguilar E, Friedlander M (2002) Retinal vascular development is mediated by endo-
thelial filopodia, a preexisting astrocytic template and specific R-cadherin adhesion. Invest
Ophthalmol Vis Sci 43:3500–3510
Enge M, Bjarnegard M, Gerhardt H, Gustafsson E, Kalen M, Asker N, Hammes HP, Shani M,
Fassler R, Betsholtz C (2002) Endothelium-specific platelet-derived growth factor-B ablation
mimics diabetic retinopathy. EMBO J 21:4307–4316
Eshaq RS, Aldalati AMZ, Alexander JS, Harris NR (2017) Diabetic retinopathy: breaking the bar-
rier. Pathophysiology 24:229–241
Etchevers HC, Vincent C, Le Douarin NM, Couly GF (2001) The cephalic neural crest provides
pericytes and smooth muscle cells to all blood vessels of the face and forebrain. Development
128:1059–1068
Ezquer M, Urzua CA, Montecino S, Leal K, Conget P, Ezquer F (2016) Intravitreal administration
of multipotent mesenchymal stromal cells triggers a cytoprotective microenvironment in the
retina of diabetic mice. Stem Cell Research and Therapy 7:42
Farrington-Rock C, Crofts NJ, Doherty MJ, Ashton BA, Griffin-Jones C, Canfield AE (2004)
Chondrogenic and adipogenic potential of microvascular pericytes. Circulation 110:2226–2232
Frank RN (2004) Diabetic retinopathy. N Engl J Med 350:48–58
Frank RN, Turczyn TJ, Das A (1990) Pericyte coverage of retinal and cerebral capillaries. Invest
Ophthalmol Vis Sci 31:999–1007
Fruttiger M (2002) Development of the mouse retinal vasculature: angiogenesis versus vasculo-
genesis. Invest Ophthalmol Vis Sci 43:522–527
Fruttiger M (2007) Development of the retinal vasculature. Angiogenesis 10:77–88
Gage PJ, Rhoades W, Prucka SK, Hjalt T (2005) Fate maps of neural crest and mesoderm in the
mammalian eye. Invest Ophthalmol Vis Sci 46:4200–4208
Gerhardt H, Betsholtz C (2003) Endothelial-pericyte interactions in angiogenesis. Cell Tissue Res
314:15–23
Ghosh M, Balbi M, Hellal F, Dichgans M, Lindauer U, Plesnila N (2015) Pericytes are involved
in the pathogenesis of cerebral autosomal dominant arteriopathy with subcortical infarcts and
leukoencephalopathy. Ann Neurol 78:887–900
Goritz C, Dias DO, Tomilin N, Barbacid M, Shupliakov O, Frisen J (2011) A pericyte origin of
spinal cord scar tissue. Science 333:238–242
Guimaraes-Camboa N, Cattaneo P, Sun Y, Moore-Morris T, Gu Y, Dalton ND, Rockenstein E,
Masliah E, Peterson KL, Stallcup WB, Chen J, Evans SM (2017) Pericytes of multiple organs
do not behave as mesenchymal stem cells in vivo. Cell Stem Cell 20:345–359.e5
Hall CN, Reynell C, Gesslein B, Hamilton NB, Mishra A, Sutherland BA, O'Farrell FM, Buchan
AM, Lauritzen M, Attwell D (2014) Capillary pericytes regulate cerebral blood flow in health
and disease. Nature 508:55–60
Hammes HP (2005) Pericytes and the pathogenesis of diabetic retinopathy. Horm Metab Res
37(Suppl 1):39–43
Hammes HP (2018) Diabetic retinopathy: hyperglycaemia, oxidative stress and beyond.
Diabetologia 61:29–38
Hammes HP, Lin J, Renner O, Shani M, Lundqvist A, Betsholtz C, Brownlee M, Deutsch U (2002)
Pericytes and the pathogenesis of diabetic retinopathy. Diabetes 51:3107–3112
Hansen-Pupp I, Hellstrom A, Hamdani M, Tocoian A, Kreher NC, Ley D, Hallberg B (2017)
Continuous longitudinal infusion of rhIGF-1/rhIGFBP-3 in extremely preterm infants: evalua-
tion of feasibility in a phase II study. Growth Hormon IGF Res 36:44–51
Heglind M, Cederberg A, Aquino J, Lucas G, Ernfors P, Enerback S (2005) Lack of the central
nervous system- and neural crest-expressed forkhead gene Foxs1 affects motor function and
body weight. Mol Cell Biol 25:5616–5625
Hernandez C, Dal Monte M, Simo R, Casini G (2016) Neuroprotection as a therapeutic target for
diabetic retinopathy. J Diabetes Res 2016:9508541
Herrmann M, Bara JJ, Sprecher CM, Menzel U, Jalowiec JM, Osinga R, Scherberich A, Alini M,
Verrier S (2016) Pericyte plasticity—comparative investigation of the angiogenic and multilin-
eage potential of pericytes from different human tissues. Eur Cell Mater 31:236–249
22 A. Trost et al.

Hill RA, Tong L, Yuan P, Murikinati S, Gupta S, Grutzendler J (2015) Regional blood flow in the
Normal and ischemic brain is controlled by arteriolar smooth muscle cell contractility and not
by capillary Pericytes. Neuron 87:95–110
Hoffmann J, Feng Y, vom Hagen F, Hillenbrand A, Lin J, Erber R, Vajkoczy P, Gourzoulidou E,
Waldmann H, Giannis A, Wolburg H, Shani M, Jaeger V, Weich HA, Preissner KT, Hoffmann
S, Deutsch U, Hammes HP (2005) Endothelial survival factors and spatial completion, but not
pericyte coverage of retinal capillaries determine vessel plasticity. FASEB J 19:2035–2036
Hong CS, Saint-Jeannet JP (2005) Sox proteins and neural crest development. Semin Cell Dev
Biol 16:694–703
Huang H, Gandhi JK, Zhong X, Wei Y, Gong J, Duh EJ, Vinores SA (2011) TNFalpha is required
for late BRB breakdown in diabetic retinopathy, and its inhibition prevents leukostasis and
protects vessels and neurons from apoptosis. Invest Ophthalmol Vis Sci 52:1336–1344
Hughes S, Gardiner T, Baxter L, Chan-Ling T (2007) Changes in pericytes and smooth muscle
cells in the kitten model of retinopathy of prematurity: implications for plus disease. Invest
Ophthalmol Vis Sci 48:1368–1379
Humphreys BD, Lin SL, Kobayashi A, Hudson TE, Nowlin BT, Bonventre JV, Valerius MT,
McMahon AP, Duffield JS (2010) Fate tracing reveals the pericyte and not epithelial origin of
myofibroblasts in kidney fibrosis. Am J Pathol 176:85–97
Ingram DA, Lien IZ, Mead LE, Estes M, Prater DN, Derr-Yellin E, DiMeglio LA, Haneline LS
(2008) In vitro hyperglycemia or a diabetic intrauterine environment reduces neonatal endothe-
lial colony-forming cell numbers and function. Diabetes 57:724–731
Ip MS, Domalpally A, Sun JK, Ehrlich JS (2015) Long-term effects of therapy with ranibi-
zumab on diabetic retinopathy severity and baseline risk factors for worsening retinopathy.
Ophthalmology 122:367–374
Jadeja S, Mort RL, Keighren M, Hart AW, Joynson R, Wells S, Potter PK, Jackson IJ (2013) A CNS-­
specific hypomorphic Pdgfr-beta mutant model of diabetic retinopathy. Invest Ophthalmol Vis
Sci 54:3569–3578
Kern TS, Barber AJ (2008) Retinal ganglion cells in diabetes. J Physiol 586:4401–4408
Kielczewski JL, Hu P, Shaw LC, Li Calzi S, Mames RN, Gardiner TA, McFarland E, Chan-Ling T,
Grant MB (2011) Novel protective properties of IGFBP-3 result in enhanced pericyte ensheath-
ment, reduced microglial activation, increased microglial apoptosis, and neuronal protection
after ischemic retinal injury. Am J Pathol 178:1517–1528
Kim JH, Kim JH, Yu YS, Kim DH, Kim KW (2009) Recruitment of pericytes and astrocytes is
closely related to the formation of tight junction in developing retinal vessels. J Neurosci Res
87:653–659
Kim JM, Hong KS, Song WK, Bae D, Hwang IK, Kim JS, Chung HM (2016a) Perivascular pro-
genitor cells derived from human embryonic stem cells exhibit functional characteristics of
Pericytes and improve the retinal vasculature in a rodent model of diabetic retinopathy. Stem
Cells Transl Med 5:1268–1276
Kim KS, Park JM, Kong T, Kim C, Bae SH, Kim HW, Moon J (2016b) Retinal angiogenesis effects
of TGF-beta1 and paracrine factors secreted from human placental stem cells in response to a
pathological environment. Cell Transplant 25:1145–1157
Klaassen I, Van Noorden CJ, Schlingemann RO (2013) Molecular basis of the inner blood-retinal
barrier and its breakdown in diabetic macular edema and other pathological conditions. Prog
Retin Eye Res 34:19–48
Kokovay E, Li L, Cunningham LA (2006) Angiogenic recruitment of pericytes from bone marrow
after stroke. J Cereb Blood Flow Metab 26:545–555
Kolb H (1995) Simple anatomy of the retina. In: Kolb H, Fernandez E, Nelson R (eds) Webvision:
the organization of the retina and visual system. University of Utah Health Sciences Center,
Salt Lake City, UT
Korn J, Christ B, Kurz H (2002) Neuroectodermal origin of brain pericytes and vascular smooth
muscle cells. J Comp Neurol 442:78–88
1 Pericytes in the Retina 23

Kramann R, Schneider RK, DiRocco DP, Machado F, Fleig S, Bondzie PA, Henderson JM, Ebert
BL, Humphreys BD (2015) Perivascular Gli1+ progenitors are key contributors to injury-­
induced organ fibrosis. Cell Stem Cell 16:51–66
Krebs I, Schmetterer L, Boltz A, Told R, Vecsei-Marlovits V, Egger S, Schonherr U, Haas A,
Ansari-Shahrezaei S, Binder S (2013) A randomised double-masked trial comparing the visual
outcome after treatment with ranibizumab or bevacizumab in patients with neovascular age-­
related macular degeneration. Br J Ophthalmol 97:266–271
Lee S, Elaskandrany M, Lau LF, Lazzaro D, Grant MB, Chaqour B (2017) Interplay between
CCN1 and Wnt5a in endothelial cells and pericytes determines the angiogenic outcome in a
model of ischemic retinopathy. Sci Rep 7:1405
Leveen P, Pekny M, Gebre-Medhin S, Swolin B, Larsson E, Betsholtz C (1994) Mice deficient for
PDGF B show renal, cardiovascular, and hematological abnormalities. Genes Dev 8:1875–1887
Liegl R, Hellstrom A, Smith LE (2016) Retinopathy of prematurity: the need for prevention. Eye
Brain 8:91–102
Lindahl P, Johansson BR, Leveen P, Betsholtz C (1997) Pericyte loss and microaneurysm forma-
tion in PDGF-B-deficient mice. Science 277:242–245
Lindblom P, Gerhardt H, Liebner S, Abramsson A, Enge M, Hellstrom M, Backstrom G,
Fredriksson S, Landegren U, Nystrom HC, Bergstrom G, Dejana E, Ostman A, Lindahl P,
Betsholtz C (2003) Endothelial PDGF-B retention is required for proper investment of peri-
cytes in the microvessel wall. Genes Dev 17:1835–1840
Liu J, Willet SG, Bankaitis ED, Xu Y, Wright CV, Gu G (2013) Non-parallel recombination limits
Cre-LoxP-based reporters as precise indicators of conditional genetic manipulation. Genesis
51:436–442
Lofqvist C, Niklasson A, Engstrom E, Friberg LE, Camacho-Hubner C, Ley D, Borg J, Smith LE,
Hellstrom A (2009) A pharmacokinetic and dosing study of intravenous insulin-like growth
factor-I and IGF-binding protein-3 complex to preterm infants. Pediatr Res 65:574–579
Lutty GA, McLeod DS (2017) Development of the hyaloid, choroidal and retinal vasculatures in
the fetal human eye. Prog Retin Eye Res 62:58–76
Mackie AR, Losordo DW (2011) CD34-positive stem cells: in the treatment of heart and vascular
disease in human beings. Tex Heart Inst J 38:474–485
Matsushita T, Lankford KL, Arroyo EJ, Sasaki M, Neyazi M, Radtke C, Kocsis JD (2015) Diffuse
and persistent blood-spinal cord barrier disruption after contusive spinal cord injury rapidly
recovers following intravenous infusion of bone marrow mesenchymal stem cells. Exp Neurol
267:152–164
Medina RJ, O'Neill CL, Humphreys MW, Gardiner TA, Stitt AW (2010) Outgrowth endothelial
cells: characterization and their potential for reversing ischemic retinopathy. Invest Ophthalmol
Vis Sci 51:5906–5913
Mendel TA, Clabough EB, Kao DS, Demidova-Rice TN, Durham JT, Zotter BC, Seaman SA,
Cronk SM, Rakoczy EP, Katz AJ, Herman IM, Peirce SM, Yates PA (2013) Pericytes derived
from adipose-derived stem cells protect against retinal vasculopathy. PLoS One 8:e65691
Nakagomi T, Kubo S, Nakano-Doi A, Sakuma R, Lu S, Narita A, Kawahara M, Taguchi A,
Matsuyama T (2015) Brain vascular pericytes following ischemia have multipotential stem
cell activity to differentiate into neural and vascular lineage cells. Stem Cells 33:1962–1974
Nakata M, Nakagomi T, Maeda M, Nakano-Doi A, Momota Y, Matsuyama T (2017) Induction of
perivascular neural stem cells and possible contribution to neurogenesis following transient
brain ischemia/reperfusion injury. Transl Stroke Res 8:131–143
Newman DK (2010) Surgical management of the late complications of proliferative diabetic reti-
nopathy. Eye (Lond) 24:441–449
Nishijima K, Ng YS, Zhong L, Bradley J, Schubert W, Jo N, Akita J, Samuelsson SJ, Robinson
GS, Adamis AP, Shima DT (2007) Vascular endothelial growth factor-a is a survival factor for
retinal neurons and a critical neuroprotectant during the adaptive response to ischemic injury.
Am J Pathol 171:53–67
24 A. Trost et al.

Ogura S, Kurata K, Hattori Y, Takase H, Ishiguro-Oonuma T, Hwang Y, Ahn S, Park I, Ikeda W,

Kusuhara S, Fukushima Y, Nara H, Sakai H, Fujiwara T, Matsushita J, Ema M, Hirashima M,
Minami T, Shibuya M, Takakura N, Kim P, Miyata T, Ogura Y, Uemura A (2017) Sustained
inflammation after pericyte depletion induces irreversible blood-retina barrier breakdown. JCI
Insight 2:e90905
Ozerdem U, Alitalo K, Salven P, Li A (2005) Contribution of bone marrow-derived pericyte pre-
cursor cells to corneal vasculogenesis. Invest Ophthalmol Vis Sci 46:3502–3506
Ozerdem U, Grako KA, Dahlin-Huppe K, Monosov E, Stallcup WB (2001) NG2 proteoglycan is
expressed exclusively by mural cells during vascular morphogenesis. Dev Dyn 222:218–227
Ozerdem U, Stallcup WB (2004) Pathological angiogenesis is reduced by targeting pericytes via
the NG2 proteoglycan. Angiogenesis 7:269–276
Park DY, Lee J, Kim J, Kim K, Hong S, Han S, Kubota Y, Augustin HG, Ding L, Kim JW, Kim
H, He Y, Adams RH, Koh GY (2017) Plastic roles of pericytes in the blood-retinal barrier. Nat
Commun 8:15296
Paul G, Ozen I, Christophersen NS, Reinbothe T, Bengzon J, Visse E, Jansson K, Dannaeus K,
Henriques-Oliveira C, Roybon L, Anisimov SV, Renstrom E, Svensson M, Haegerstrand A,
Brundin P (2012) The adult human brain harbors multipotent perivascular mesenchymal stem
cells. PLoS One 7:e35577
Pfister F, Feng Y, vom Hagen F, Hoffmann S, Molema G, Hillebrands JL, Shani M, Deutsch U,
Hammes HP (2008) Pericyte migration: a novel mechanism of pericyte loss in experimental
diabetic retinopathy. Diabetes 57:2495–2502
Pfister F, Wang Y, Schreiter K, vom Hagen F, Altvater K, Hoffmann S, Deutsch U, Hammes HP,
Feng Y (2010) Retinal overexpression of angiopoietin-2 mimics diabetic retinopathy and
enhances vascular damages in hyperglycemia. Acta Diabetol 47:59–64
Pollack A, Staurenghi G, Sager D, Mukesh B, Reiser H, Singh RP (2016) Prospective randomised
clinical trial to evaluate the safety and efficacy of nepafenac 0.1% treatment for the prevention
of macular oedema associated with cataract surgery in patients with diabetic retinopathy. Br
J Ophthalmol 101:423–427
Ribatti D, Nico B, Crivellato E (2011) The role of pericytes in angiogenesis. Int J Dev Biol
55:261–268
Ritter MR, Banin E, Moreno SK, Aguilar E, Dorrell MI, Friedlander M (2006) Myeloid progeni-
tors differentiate into microglia and promote vascular repair in a model of ischemic retinopa-
thy. J Clin Invest 116:3266–3276
Saito Y, Geisen P, Uppal A, Hartnett ME (2007) Inhibition of NAD(P)H oxidase reduces apoptosis
and avascular retina in an animal model of retinopathy of prematurity. Mol Vis 13:840–853
Sankar MJ, Sankar J, Chandra P (2018) Anti-vascular endothelial growth factor (VEGF) drugs for
treatment of retinopathy of prematurity. Cochrane Database Syst Rev 1:CD009734
Santos GSP, Prazeres P, Mintz A, Birbrair A (2017) Role of pericytes in the retina. Eye (Lond)
32:483–486
Schallek J, Geng Y, Nguyen H, Williams DR (2013) Morphology and topography of retinal peri-
cytes in the living mouse retina using in vivo adaptive optics imaging and ex vivo characteriza-
tion. Invest Ophthalmol Vis Sci 54:8237–8250
Selvam S, Kumar T, Fruttiger M (2017) Retinal vasculature development in health and disease.
Prog Retin Eye Res 63:1–19
Sfikakis PP, Grigoropoulos V, Emfietzoglou I, Theodossiadis G, Tentolouris N, Delicha E, Katsiari
C, Alexiadou K, Hatziagelaki E, Theodossiadis PG (2010) Infliximab for diabetic macular
edema refractory to laser photocoagulation: a randomized, double-blind, placebo-controlled,
crossover, 32-week study. Diabetes Care 33:1523–1528
Shaw LC, Neu MB, Grant MB (2011) Cell-based therapies for diabetic retinopathy. Curr Diab
Rep 11:265–274
Sheikh AQ, Misra A, Rosas IO, Adams RH, Greif DM (2015) Smooth muscle cell progenitors are
primed to muscularize in pulmonary hypertension. Sci Transl Med 7:308ra159
Shepro D, Morel NM (1993) Pericyte physiology. FASEB J 7:1031–1038
1 Pericytes in the Retina 25

Sieveking DP, Ng MK (2009) Cell therapies for therapeutic angiogenesis: back to the bench. Vasc
Med 14:153–166
Simon C, Lickert H, Gotz M, Dimou L (2012) Sox10-iCreERT2 : a mouse line to inducibly trace
the neural crest and oligodendrocyte lineage. Genesis 50:506–515
Smith LE, Wesolowski E, McLellan A, Kostyk SK, D’Amato R, Sullivan R, D'Amore PA (1994)
Oxygen-induced retinopathy in the mouse. Invest Ophthalmol Vis Sci 35:101–111
Song S, Ewald AJ, Stallcup W, Werb Z, Bergers G (2005) PDGFRbeta+ perivascular progenitor
cells in tumours regulate pericyte differentiation and vascular survival. Nat Cell Biol 7:870–879
Soriano P (1994) Abnormal kidney development and hematological disorders in PDGF beta-­
receptor mutant mice. Genes Dev 8:1888–1896
Sweeney MD, Sagare AP, Zlokovic BV (2018) Blood-brain barrier breakdown in Alzheimer dis-
ease and other neurodegenerative disorders. Nat Rev Neurol 14:133–150
Tallquist MD, French WJ, Soriano P (2003) Additive effects of PDGF receptor beta signaling
pathways in vascular smooth muscle cell development. PLoS Biol 1:E52
Tidhar A, Reichenstein M, Cohen D, Faerman A, Copeland NG, Gilbert DJ, Jenkins NA, Shani
M (2001) A novel transgenic marker for migrating limb muscle precursors and for vascular
smooth muscle cells. Dev Dyn 220:60–73
Trinh TLP, Li Calzi S, Shaw LC, Yoder MC, Grant MB (2016) Promoting vascular repair in the
retina: can stem/progenitor cells help? Eye Brain 8:113–122
Trost A, Schroedl F, Lange S, Rivera FJ, Tempfer H, Korntner S, Stolt CC, Wegner M, Bogner B,
Kaser-Eichberger A, Krefft K, Runge C, Aigner L, Reitsamer HA (2013) Neural crest origin of
retinal and choroidal pericytes. Invest Ophthalmol Vis Sci 54:7910–7921
Vanhaesebrouck S, Daniels H, Moons L, Vanhole C, Carmeliet P, De Zegher F (2009) Oxygen-­
induced retinopathy in mice: amplification by neonatal IGF-I deficit and attenuation by IGF-I
administration. Pediatr Res 65:307–310
Vanlandewijck M, He L, Mae MA, Andrae J, Ando K, Del Gaudio F, Nahar K, Lebouvier T, Lavina
B, Gouveia L, Sun Y, Raschperger E, Rasanen M, Zarb Y, Mochizuki N, Keller A, Lendahl U,
Betsholtz C (2018) A molecular atlas of cell types and zonation in the brain vasculature. Nature
554:475–480
Wang D, Wu F, Yuan H, Wang A, Kang GJ, Truong T, Chen L, McCallion AS, Gong X, Li S (2017)
Sox10(+) cells contribute to vascular development in multiple organs-brief report. Arterioscler
Thromb Vasc Biol 37:1727–1731
Wilkinson-Berka JL, Babic S, De Gooyer T, Stitt AW, Jaworski K, Ong LG, Kelly DJ, Gilbert RE
(2004) Inhibition of platelet-derived growth factor promotes pericyte loss and angiogenesis in
ischemic retinopathy. Am J Pathol 164:1263–1273
Winkler EA, Bell RD, Zlokovic BV (2010) Pericyte-specific expression of PDGF beta receptor
in mouse models with normal and deficient PDGF beta receptor signaling. Mol Neurodegener
5:32
Winkler EA, Bell RD, Zlokovic BV (2011) Central nervous system pericytes in health and disease.
Nat Neurosci 14:1398–1405
Wisniewska-Kruk J, Klaassen I, Vogels IM, Magno AL, Lai CM, Van Noorden CJ, Schlingemann
RO, Rakoczy EP (2014) Molecular analysis of blood-retinal barrier loss in the Akimba mouse,
a model of advanced diabetic retinopathy. Exp Eye Res 122:123–131
Xiao A, Zhou Q, Shao Y, Zhong HF (2017) Effect of intravitreal injection of ranibizumab on
retinal ganglion cells and microvessels in the early stage of diabetic retinopathy in rats with
streptozotocin-induced diabetes. Exp Ther Med 13:3360–3368
Xu J, Nie X, Cai X, Cai CL, Xu PX (2014) Tbx18 is essential for normal development of vascula-
ture network and glomerular mesangium in the mammalian kidney. Dev Biol 391:17–31
Yamamoto S, Muramatsu M, Azuma E, Ikutani M, Nagai Y, Sagara H, Koo BN, Kita S, O'Donnell E,
Osawa T, Takahashi H, Takano KI, Dohmoto M, Sugimori M, Usui I, Watanabe Y, Hatakeyama
N, Iwamoto T, Komuro I, Takatsu K, Tobe K, Niida S, Matsuda N, Shibuya M, Sasahara M
(2017) A subset of cerebrovascular pericytes originates from mature macrophages in the very
early phase of vascular development in CNS. Sci Rep 7:3855
26 A. Trost et al.

Yamazaki T, Nalbandian A, Uchida Y, Li W, Arnold TD, Kubota Y, Yamamoto S, Ema M,

Mukouyama YS (2017) Tissue myeloid progenitors differentiate into Pericytes through TGF-­
beta Signaling in developing skin vasculature. Cell Rep 18:2991–3004
Yau JW, Rogers SL, Kawasaki R, Lamoureux EL, Kowalski JW, Bek T, Chen SJ, Dekker JM,
Fletcher A, Grauslund J, Haffner S, Hamman RF, Ikram MK, Kayama T, Klein BE, Klein R,
Krishnaiah S, Mayurasakorn K, O’Hare JP, Orchard TJ, Porta M, Rema M, Roy MS, Sharma T,
Shaw J, Taylor H, Tielsch JM, Varma R, Wang JJ, Wang N, West S, Xu L, Yasuda M, Zhang X,
Mitchell P, Wong TY, Meta-Analysis for Eye Disease Study Group (2012) Global prevalence
and major risk factors of diabetic retinopathy. Diabetes Care 35:556–564
Zehendner CM, Wedler HE, Luhmann HJ (2013) A novel in vitro model to study pericytes in the
neurovascular unit of the developing cortex. PLoS One 8:e81637
Chapter 2
Pancreatic Pericytes in Glucose
Homeostasis and Diabetes

Limor Landsman

Abstract Glucose homeostasis relies on tightly regulated insulin secretion from

pancreatic beta-cells, and its loss in diabetes is associated with the dysfunction of
these cells. Beta-cells reside in the islets of Langerhans, which are highly vascular-
ized by a dense capillary network comprised of endothelial cells and pericytes.
While the requirement of the endothelium for the proper pancreatic function is well
established, the role of pancreatic pericytes has only recently begun to unveil.
Recent studies described multiple roles for pancreatic pericytes in glucose homeo-
stasis, highlighting their function as both regulators of islet blood flow and as a
source of critical signals that support proper beta-cell function and mass.
Furthermore, recent findings point to the contribution of pericytic abnormalities to
beta-cell dysfunction in type 2 diabetes, implicating the involvement of pancreatic
pericytes in both the initiation and the progression of this disease. This newly gained
data implicate pancreatic pericytes as critical components of the cellular network
required for glucose regulation.

Keywords Pancreatic pericytes · Islets of Langerhans · Islet vasculature · Islet

pericytes · Islet capillaries · Beta-cells · Beta-cell function · Beta-cell dysfunction ·
Beta-cell mass · Glucose homeostasis · Glucose-stimulated insulin secretion · Type
2 diabetes

L. Landsman (*)
Department of Cell and Developmental Biology, Sackler Faculty of Medicine,
Tel Aviv University, Tel Aviv, Israel
e-mail: [email protected]

© Springer Nature Switzerland AG 2019 27

A. Birbrair (ed.), Pericyte Biology in Different Organs,
Advances in Experimental Medicine and Biology 1122,
https://doi.org/10.1007/978-3-030-11093-2_2
28 L. Landsman

2.1 Introduction

2.1.1  he Pancreas and Its Role in Glucose Regulation

T
and Diabetes

The pancreas is required for both food digestion and blood glucose regulation. The
pancreas is frequently described as “two organs in one,” as it comprises of two dif-
ferent cellular compartments, the exocrine and the endocrine, each is responsible
for a distinct pancreatic function. Pancreatic exocrine cells, which produce and
transport digestive enzymes to the gut, consist of the majority of the adult pancreatic
tissue (Longnecker et al. 2018). Pancreatic endocrine cells, which produce and
secrete hormones to regulate blood glucose levels, are organized in islets of
Langerhans, structures that are embedded in the exocrine pancreas (Mastracci and
Sussel 2012). Cells of both the exocrine and the endocrine compartment are epithe-
lial cells of endoderm origin (Gittes 2009).
The islets of Langerhans comprise multiple endocrine cell populations, primarily
insulin-producing β-cells, glucagon-producing α-cells, and somatostatin-producing
δ-cells (Mastracci and Sussel 2012). Both α- and β-cells are equipped with a sophis-
ticated mechanism allowing them to sense minute fluctuation in blood glucose levels
and respond by accurately secreting hormones to the bloodstream. An increase in
blood glucose levels prompts insulin secretion from β-cells, in a process termed “glu-
cose-stimulated insulin secretion.” In response to insulin, glucose is being uptaken by
the liver, muscles, adipose tissues, and other tissues, lowering its levels in the blood.
In contrast, glucagon acts to break glucose storages, to ensure sufficient glucose lev-
els in the blood for proper brain function. Thus, pancreatic α- and β-cells cooperate
to maintain tightly regulated blood glucose levels (Mastracci and Sussel 2012).
Diabetes is a chronic disease, now reaching pandemic proportions (DeFronzo et al.
2015). It is characterized by hyperglycemia due to insufficient β-cell function and
mass, either alone or accompanied by insulin resistance. Type 2 diabetes, the most
predominant form of this disease, was regarded for many years as a disease of insulin
resistance. However, there is now a widespread recognition that β-cell failure is a criti-
cal factor in this disease (Ashcroft and Rorsman 2012). β-Cell failure could result
from their insufficient mass, impaired function, or combination of the two (Costes
et al. 2013; Dor and Glaser 2013). Loss of the β-cell mature phenotype, in a process
named “β-cell dedifferentiation,” was recently shown to contribute to β-cell dysfunc-
tion during type 2 diabetes (Talchai et al. 2012; Cinti et al. 2016). However, the under-
lying cause(s) of β-cell failure in diabetes is yet to be uncovered (Cerasi 2011).

2.1.2 The Islet Vasculature

The pancreas vasculature integrates this organ’s endocrine and exocrine compart-
ments, allowing both intra-islets and the islet-acinar interactions (Bonner-Weir
1993). While the islets comprise only 1–2% of the pancreatic volume, they receive
2 Pancreatic Pericytes in Glucose Homeostasis and Diabetes 29

approximately 10% of its blood flow. Islet capillaries are highly permeable, and
their endothelial cells are highly fenestrated to facilitate a quick pancreatic response
to fluctuations in blood glucose levels (Bonner-Weir 1993; Richards et al. 2010).
Each β-cell is in contact with a capillary to allow proper sensing of blood glucose
and the subsequent hormone secretion to the bloodstream (Bonner-Weir 1993;
Granot et al. 2009). In addition to transporting hormones and nutrient to and from
the peripheral circulation, the islet vasculature is required for proper pancreas devel-
opment and function (Lammert et al. 2001; Nikolova et al. 2006; Reinert et al. 2013;
Brissova et al. 2014; Kragl et al. 2016; Hogan and Hull 2017). Similar to capillaries
of other organs, the islet vasculature constitutes of endothelial cells and pericytes.
While the role of endothelial cells in pancreas function has been extensively studied
(Richards et al. 2010; Hogan and Hull 2017), the role of pericytes has only recently
begun to unveiled.

2.1.3 Pancreatic Pericytes

Together with endothelial cells, pericytes make the islets dense capillary network
(Armulik et al. 2011; Tang et al. 2013; Sasson et al. 2016) (Fig. 2.1). Similar to
pericytes of other organs, pancreatic pericytes are contractile cells that extend pri-
mary cytoplasmic processes along the endothelial tube (Armulik et al. 2011; Tang
et al. 2013; Sasson et al. 2016) (Fig. 2.1). Pericytes constitute less than 3% of the
islet cell population and cover around 40% of their capillary area (Almaça et al.
2018). These cells express the pericytic markers PDGFRβ (platelet-derived growth
factor receptor β), NG2 (neural/glial antigen 2), αSMA (α-smooth muscle actin),
and desmin (Armulik et al. 2011; Tang et al. 2013; Sasson et al. 2016). Pericytes of
the adult pancreas originate from the embryonic pancreatic mesenchyme, a multi-
cellular layer surrounding the developing pancreatic epithelium (Harari et al., 2019).
In addition to the islets, pericytes are found around capillaries of the exocrine
pancreas (Morikawa et al. 2002; Sakhneny et al. 2018). Although the vasculature of
the endocrine and the exocrine display distinct characteristics (Bonner-Weir 1993),
whether islet pericytes differ from these reside in the exocrine pancreas awaits
investigation. Although pancreatic stellate cells, which reside in the exocrine pan-
creas and contribute to the pathology of pancreatitis and pancreatic cancer, have
pericytic characteristics (Apte et al. 2015), the relation between these two cell popu-
lations is unclear. Together with vascular smooth muscle cells (vSMCs) that encir-
cle large blood vessels, pericytes make up the pancreatic mural cell population
(Armulik et al. 2011).
While the role of endothelial cells in supporting β-cell function and mass has been
vastly investigated (Richards et al. 2010; Hogan and Hull 2017), the understanding of
the role of pericytes in these process lags behind. In recent years, work by us and oth-
ers pointed to pericytes as critical players in pancreatic function. Here, I review cur-
rent knowledge on the contribution of these cells to glucose regulation and its loss in
diabetes, highlighting the role of pancreatic pericytes as both regulators of islet blood
flow, and as a source of critical signals that support proper β-cell function and mass.
30 L. Landsman

Fig. 2.1 Pancreatic

pericytes are associated
with islet of Langerhans.
Mouse pancreatic section
is stained with NG2 to
label pericytes (green),
insulin to label β-cells
(red), and DAPI to label
nuclei (blue)

2.2 Pancreatic Pericytes and Glucose Homeostasis

Proper β-cell function and mass have been shown to depend on cells of the islet
microenvironment, including endothelial, neuronal, and immune cells (Nikolova
et al. 2006; Eberhard and Lammert 2009; Tarussio et al. 2014; Brissova et al. 2014;
Riley et al. 2015; Hogan and Hull 2017). Recent studies pointed to a role of pericytes
in glucose homeostasis, implicating the direct and indirect roles of these cells in
glucose-stimulated insulin secretion (Sasson et al. 2016; Houtz et al. 2016; Epshtein
et al. 2017; Sakhneny et al. 2018; Almaça et al. 2018) (Illustrated in Fig. 2.2).

2.2.1 Pancreatic Pericytes and β-Cell Function

2.2.1.1 Pancreatic Pericytes and β-Cell Maturity

The ability of β-cells to properly secrete insulin depends on their mature phenotype.
While mature β-cells are capable of precise glucose-stimulated insulin secretion,
secretion of insulin from immature β-cells is not tightly linked to blood glucose
levels (Bonner-Weir and Aguayo-Mazzucato 2016). The β-cell mature phenotype is
maintained through the activity of an array of transcription factors (i.e., MafA,
Pdx1, Unc3, and NeuroD1) (Bramswig and Kaestner 2014). These factors regulate
the expression of functional genes required for glucose-stimulated insulin secretion,
including insulin, the glucose transporter Glut2, the ATP-sensitive potassium
2 Pancreatic Pericytes in Glucose Homeostasis and Diabetes 31

Fig. 2.2 Pancreatic pericytes regulate multiple aspects of glucose-stimulated insulin secretion.
Right, pericytes regulate β-cell function through promoting both their mature phenotype and
glucose-­stimulated insulin exocytosis. Upper left, pericytes further regulate β-cell mass by stimu-
lating their proliferation in the neonatal period. Lastly, pericytes modulate local blood flow in the
islet through adjusting capillaries dilation and constriction (lower left)

channel Kir6.2, and the sulfonylurea receptor Sur1. In addition to cell-intrinsic fac-
tors, proper β-cell maturity and function rely on their appropriate interactions with
cells in their microenvironment, including endocrine and non-endocrine cell popu-
lations (Eberhard and Lammert 2009). Loss of the β-cell mature phenotype, due to
their dedifferentiation, contributes to the loss of glycemic control during type 2
diabetes (Dor and Glaser 2013).
We recently showed that pancreatic pericytes are required for proper glucose
regulation (Sasson et al. 2016). To determine their role in vivo, we combined the use
of transgenic mouse lines to selectively ablate pericytes in the pancreas (Sasson
et al. 2016). Of note, other cell populations, such as pancreatic endothelial cells and
pericytes outside of the pancreas, were not targeted by this manipulation. Shortly
after ablation of pancreatic pericytes, mice became glucose intolerant due to
diminished glucose-stimulated insulin secretion (Sasson et al. 2016). This data point
to the requirement of pancreatic pericytes in supporting glucose homeostasis.
Depletion of pancreatic pericytes resulted in the loss of the mature β-cell pheno-
type, as evident by both their impaired function and the reduced levels of genes and
proteins associated with β-cell maturity and function (Sasson et al. 2016). Islets
lacking their pericytes displayed decreased levels of components of the glucose
sensing and insulin secretion machinery, including insulin, Glut2, Kir6.2, and Sur1.
Pericyte-depleted islets also exhibited diminished expression of transcription fac-
tors required for β-cell maturity, including MafA and Pdx1, pointing to β-cell dedif-
ferentiation (Sasson et al. 2016). Loss of β-cell maturity was further observed when
pericytes were depleted ex vivo in isolated islets, indicating that pericytes act within
the islets to support β-cell function, independently of their role as regulators of
blood flow (Sasson et al. 2016). These observations indicate that the mature β-cell
phenotype, required for glucose-stimulated insulin secretion, depends on proper
interactions of these cells with islet pericytes (Sasson et al. 2016).
Thus, pancreatic pericytes directly regulate β-cell maturity, in a blood flow inde-
pendent manner, to control proper glucose regulation (Fig. 2.2).
32 L. Landsman

2.2.1.2 Pancreatic Pericytes and Insulin Secretion

β-Cells store insulin in granules and release it in a tightly regulated manner in

response to glucose. To ensure tight control of glucose levels, the regulation of insu-
lin exocytosis is multilayered and involves complex signaling events (Rorsman and
Braun 2013; Prentki et al. 2013).
Recently, the neurotrophin signaling pathway was implicated in pericyte-­
dependent β-cell function, through regulating glucose-stimulated insulin exocyto-
sis. Houtz et al. showed that nerve growth factor (NGF) acutely regulates
glucose-stimulated insulin secretion from β-cells (Houtz et al. 2016). The NGF
receptor TrkA is expressed by β-cells, and its NGF-mediated activation promotes
the release of insulin from its granules. Accordingly, selective loss of TrkA expres-
sion in β-cells leads to loss of glucose regulation (Houtz et al. 2016). Houtz et al.
further showed that NGF is expressed by pancreatic pericytes and vSMCs when
high glucose levels stimulate its secretion from these cells (Houtz et al. 2016).
Transgenic mice lacking expression of NGF in their pericytes and vSMCs were
glucose intolerant and displayed impaired glucose-stimulated insulin secretion
(Houtz et al. 2016). Thus, this study showed that through glucose-dependent NGF
secretion, pericytes directly promote insulin secretion from β-cells (Fig. 2.2).

2.2.2 Pancreatic Pericytes and the Expansion of β-Cells

After birth, the primary route of β-cell generation is their replication (Dor et al.
2004; Georgia and Bhushan 2004; Meier et al. 2008). β-Cell replication rates decline
with age, and it is significantly higher during the neonatal period than during adult-
hood (Finegood et al. 1995; Gregg et al. 2012; Wang et al. 2015). While β-cell rep-
lication is primarily restricted to the neonatal period, the evidence is accumulating
that adult β-cells maintain a proliferative capacity even as they age (Chen et al. 2009;
Salpeter et al. 2010; Stolovich-Rain et al. 2012). For example, β-cells display com-
pensatory proliferation during increased metabolic demand (Ouaamari et al. 2016).
β-Cell replication rate is regulated by both intrinsic and extrinsic cues, when
cells of the islet microenvironment, including endothelial, neuronal, and immune
cells, have been shown to promote this process (Nikolova et al. 2006; Eberhard and
Lammert 2009; Tarussio et al. 2014; Brissova et al. 2014; Riley et al. 2015).
Recently, we showed that pancreatic pericytes promote β-cell proliferation during
the neonatal period (Epshtein et al. 2017). Depletion of pancreatic pericytes in vivo
was sufficient to reduce the rate of β-cell replication in neonatal mice, pointing to
the requirement of these cells for physiological expansion of β-cells (Epshtein et al.
2017) (Fig. 2.2).
Vascular basement membrane, a specialized extracellular matrix produced by
endothelial cells and pericytes, is located within and around islets (Nikolova et al.
2006; Otonkoski et al. 2008; Kragl and Lammert 2010; Armulik et al. 2011;
Stratman and Davis 2012). Components of the islet vascular basement membrane
2 Pancreatic Pericytes in Glucose Homeostasis and Diabetes 33

promote β-cell proliferation, through their binding to integrins expressed on these

cells and the initiation of downstream signaling (Nikolova et al. 2006; Kragl and
Lammert 2010; Diaferia et al. 2013). We recently showed that conditioned media of
cultured neonatal pericytes stimulate β-cell proliferation (Epshtein et al. 2017).
Furthermore, pericyte-stimulated β-cell replication was dependent on integrin activ-
ity (Epshtein et al. 2017). These findings suggest that pericytes produce basement
membrane components to promote β-cell proliferation.
To conclude, pancreatic pericytes promote β-cell proliferation during the neona-
tal period to establish proper β-cell mass, likely through the production of vascular
basement membrane components.

2.2.3 Pancreatic Pericytes in the Regulation of Blood Flow

In addition to their direct role in regulating β-cell function and mass, pancreatic
pericytes may contribute to glucose homeostasis through regulating islet blood
flow. The islet vascularization adapts to hormone secretion from endocrine cells,
likely to facilitate the distribution of these hormones into the bloodstream
(Schaeffer et al. 2011). In addition to this short-term response, islet vascularization
adapts to prolonged metabolic demand, such as during insulin resistance, through
capillaries dilation (Dai et al. 2013). However, whether the increase in islet blood
flow promotes the lowering of blood glucose levels remains unclear (Hogan and
Hull 2017).
The intra-islet density of pericytes is increased in response to insulin resistance
and obesity, suggesting a role for these cells in the adaptation of the islet vasculature
to high metabolic demand (Tang et al. 2013; Dai et al. 2013). Recently, pericyte-­
dependent dilation of islet capillaries was linked to a short-term increase in glucose
levels, providing a potential mechanism through which increased demand for insu-
lin facilitates its circulation (Almaça et al. 2018) (Fig. 2.2). As suggested by Almaça
et al., glucose metabolism increases the levels of ATP and its metabolite adenosine,
when the latter promotes the relaxation of islet pericytes, resulting in capillaries
dilation (Almaça et al. 2018). Whereas this study implicated β-cells as the source of
adenosine in the pancreas (Almaça et al. 2018), other cell types, including pericytes
(Mandarino et al. 1994), are capable of metabolizing glucose and thus represent a
potential source for this metabolite. Thus, glucose-­dependent pericytes relaxation
may represent a pericyte-autonomous or a nonautonomous mechanism through
which these cells respond to the demand for insulin by increasing blood flow. In
contrast to glucose, noradrenaline, produced by sympathetic innervation to the
islets, promotes pericytes contraction and capillaries constriction to reduce blood
flow (Almaça et al. 2018). These observations suggest that pericyte-dependent cap-
illary dilation is a highly regulated process, which depends on proper cell-cell inter-
actions within the islets of Langerhans. How pericyte-regulated local changes in
islet blood flow influent glucose homeostasis remain to be elucidated.
34 L. Landsman

To conclude, dilation of islet capillaries characterizes both prolonged and acute

increase in insulin demands and is associated with changes in pericytes density and
contractility.

2.3  ericytes in the Loss of Pancreatic Function during Type

P
2 Diabetes

The clinical onset of type 2 diabetes occurs when β-cells fail to secrete sufficient
amounts of insulin to maintain normoglycemia during insulin resistance (Costes
et al. 2013; Dor and Glaser 2013; DeFronzo et al. 2015). The evidence is accumulat-
ing that abnormal pericyte function contributes to β-cell failure and disease develop-
ment, whereas chronic hyperglycemia leads to loss of pancreatic pericytes (Hayden
et al. 2007; Hayden et al. 2010; Sakhneny et al. 2018; Almaça et al. 2018) (Fig. 2.3).
Thus, pancreatic pericytes potentially play a role in both the initiation and progres-
sion of type 2 diabetes.

2.3.1  ancreatic Pericytes as Contributors to β-Cell

P
Dysfunction and Type 2 Diabetes

While type 2 diabetes is influenced by several lifestyle factors, including age, preg-
nancy, and obesity, this disease has a strong genetic component (DeFronzo et al.
2015; Fuchsberger et al. 2016). Linkage and genome-wide association studies have
identified more than 80 genes associated with an increased risk of type 2 diabetes,
many of which are thought to be essential for β-cell function, development, or mass
(Ashcroft and Rorsman 2012; Fuchsberger et al. 2016). In particular, polymorphism
in TCF7L2 (TCF4) is associated with an increased risk of diabetes (Grant et al.
2006). This gene encodes a member of T-cell factor/lymphoid enhancer factor
(TCF/LEF) transcription factors family, which functions downstream of the canoni-
cal Wnt signaling pathway by recruiting β-catenin to target genes (Clevers and
Nusse 2012). Polymorphism in TCF7L2 has a strong correlation to type 2 diabetes
in humans, when carriers of diabetes-associated single nucleotide polymorphism
(SNP) display reduced basal and glucose-stimulated insulin secretion but maintain
normal hepatic function (Grant et al. 2006; Helgason et al. 2007; Lyssenko et al.
2007).
Recently, we showed that Tcf7l2 is expressed at high levels by pancreatic peri-
cytes (Sakhneny et al. 2018). Inactivation of this transcription factor specifically in
these cells leads to glucose intolerance of transgenic mice, which was further aug-
mented upon obesity. The impaired glucose regulation in Tcf7l2-deficient mice was
due to compromised β-cell function and diminished glucose-stimulated insulin
secretion (Sakhneny et al. 2018). Inactivation of pericytic Tcf7l2 was associated
with impaired expression of genes required for β-cell function and maturity, includ-
2 Pancreatic Pericytes in Glucose Homeostasis and Diabetes 35

Fig. 2.3 Pancreatic pericytes can both contribute to and be affected by type 2 diabetes. Left,
Impaired pericyte function leads to β-cell dysfunction and a blunted glucose-stimulated insulin
secretion, thus contributing to diabetes progression. Right, type 2 diabetes leads to loss of pericyte
coverage of islet capillaries, potentially due to the associated chronic hyperglycemia

ing transcription factors (MafA, Pdx1, and NeuroD1) and functional genes (Glut2,
Kir6.2, and Sur1) (Sakhneny et al. 2018). Thus, loss of pericytic Tcf7l2 activity is
sufficient to cause β-cell dysfunction and impaired glucose regulation.
Tcf7l2 is a transcription factor, and its inactivation in pancreatic pericytes alters
their transcriptome (Clevers and Nusse 2012; Sakhneny et al. 2018). In particular,
our analysis revealed the reduced expression of secreted factors in pericytes lacking
Tcf7l2 (Sakhneny et al. 2018). Among these factors is the known Tcf7l2 target gene
BMP4 (bone morphogenetic protein 4) (van de Wetering et al. 2002). β-Cells were
shown to depend on the activity of the BMP4 receptor, BMPR1A, for their proper
function and gene expression in vivo (Goulley et al. 2007). Treating mice lacking
pericytic Tcf7l2 activity with exogenous BMP4 was sufficient to rescue their
impaired glucose-stimulated insulin secretion. Thus, impaired activation of the
BMP4/BMPR1A pathway represents a potential mechanism through which
­abnormal pericytic Tcf7l2 activity leads to β-cell dysfunction and type 2 diabetes
progression.
To conclude, diabetes-associated genetic changes in pancreatic pericytes are
sufficient to cause β-cell dysfunction and loss of glycemic control (Fig. 2.3), sug-
gesting the abnormalities in pancreatic pericytes contributes to the development of
type 2 diabetes.

2.3.2 Hyperglycemia and Loss of Pancreatic Pericytes

One of the significant complications of diabetes is diabetic retinopathy, which is a

leading cause of vision loss and visual disability (DeFronzo et al. 2015; Hammes
2017). Pericytes are considered the primary trigger of vascular damage in the dia-
betic retina, and diabetic retinopathy occurs when these cells are lost (Hammes
2017). Similar to pericytes in the retina, pancreatic pericytes were shown to be
sensitive to chronic hyperglycemia that characterizes diabetes (Almaça et al. 2018).
36 L. Landsman

The morphology of islet vasculature is abnormal in individuals with type 2 dia-

betes (Hogan and Hull 2017). This disease is further associated with morphological
and phenotypical changes in islet-associated pericytes, resulting in widening of the
islet exocrine interface (Hayden and Sowers 2007; Hayden et al. 2010). Islet capil-
laries of individuals with type 2 diabetes display significantly reduced coverage of
pericytes (Almaça et al. 2018). More so, the decline in the coverage of islet capillar-
ies by pericytes correlates with the duration of diabetes, pointing to a progressive
loss of pancreatic pericytes in this disease (Almaça et al. 2018).
Considering their requirement for proper β-cell function and insulin secretion,
the progressive loss of pancreatic pericytes may contribute to further deterioration
in glucose regulation (Fig. 2.3).

2.4 Conclusions

The role of pericytes in proper pancreatic function has only recently begun to unveil.
Recent studies by us and others showed that pancreatic pericytes play multiple roles
in glucose regulation, including support of proper β-cell mass and function and
adjustment of local blood flow in the islets. Evidence began to accumulate that type
2 diabetes is associated with functional changes in pancreatic pericytes. Furthermore,
the progressive loss of these cells during diabetes may contribute to further deterio-
ration in glycemic control. Additional study is required to elucidate the molecular
basis of pericytes function, their interactions with other pancreatic cell types, and
the contribution of pericyte dysfunction to diabetes progression. Nevertheless, the
newly gained data implicate pancreatic pericytes as key components of the cellular
network required for glucose regulation and potential targets for therapeutic
intervention.

References

Almaça J, Weitz J, Rodriguez-Diaz R, Pereira E, Caicedo A (2018) The Pericyte of the pancreatic
islet regulates capillary diameter and local blood flow. Cell Metab 27:630–644.e4. https://doi.
org/10.1016/j.cmet.2018.02.016
Apte M, Pirola RC, Wilson JS (2015) Pancreatic stellate cell: physiologic role, role in fibrosis and can-
cer. Curr Opin Gastroenterol 31:416–423. https://doi.org/10.1097/MOG.0000000000000196
Armulik A, Genové G, Betsholtz C (2011) Pericytes: developmental, physiological, and patho-
logical perspectives, problems, and promises. Dev Cell 21:193–215. https://doi.org/10.1016/j.
devcel.2011.07.001
Ashcroft FM, Rorsman P (2012) Diabetes mellitus and the β cell: the last ten years. Cell 148:1160–
1171. https://doi.org/10.1016/j.cell.2012.02.010
Bonner-Weir S (1993) The microvasculature of the pancreas, with emphasis on that of the islets of
Langerhans. In: Go VLW et al (eds) The pancreas: biology, pathobiology, and disease. Raven
Press, New York, pp 759–768
2 Pancreatic Pericytes in Glucose Homeostasis and Diabetes 37

Bonner-Weir S, Aguayo-Mazzucato C (2016) Physiology: pancreatic [beta]-cell heterogeneity

revisited. Nature 535:365–366. https://doi.org/10.1038/nature18907
Bramswig NC, Kaestner KH (2014) Transcriptional and epigenetic regulation in human islets.
Diabetologia 57:451–454. https://doi.org/10.1007/s00125-013-3150-3
Brissova M, Aamodt K, Brahmachary P, Prasad N, Hong J-Y, Dai C, Mellati M, Shostak A,
Poffenberger G, Aramandla R, Levy SE, Powers AC (2014) Islet microenvironment, modulated
by vascular endothelial growth factor-a signaling, promotes β cell regeneration. Cell Metab
19:498–511. https://doi.org/10.1016/j.cmet.2014.02.001
Cerasi E (2011) β-Cell dysfunction vs insulin resistance in type 2 diabetes: the eternal “chicken
and egg” question. Medicographia 33:35–41
Chen X, Zhang X, Chen F, Larson CS, Wang L-J, Kaufman DB (2009) Comparative study of
regenerative potential of beta cells from young and aged donor mice using a novel islet trans-
plantation model. Transplantation 88:496–503. https://doi.org/10.1097/TP.0b013e3181b0d2ee
Cinti F, Bouchi R, Kim-Muller JY, Ohmura Y, Sandoval PR, Masini M, Marselli L, Suleiman M,
Ratner LE, Marchetti P, Accili D (2016) Evidence of β-cell dedifferentiation in human type 2
diabetes. J Clin Endocrinol Metab 101:1044–1054. https://doi.org/10.1210/jc.2015-2860
Clevers H, Nusse R (2012) Wnt/β-catenin signaling and disease. Cell 149:1192–1205. https://doi.
org/10.1016/j.cell.2012.05.012
Costes S, Langen R, Gurlo T, Butler PC (2013) β-Cell failure in type 2 diabetes: a case of asking
too much of too few? Diabetes 62:327–335. https://doi.org/10.2337/db12-1326
Dai C, Brissova M, Reinert RB, Nyman L, Liu EH, Thompson C, Shostak A, Shiota M, Takahashi
T, Powers AC (2013) Pancreatic islet vasculature adapts to insulin resistance through dilation
and not angiogenesis. Diabetes 62:4144–4153. https://doi.org/10.2337/db12-1657
DeFronzo RA, Ferrannini E, Groop L, Henry RR, Herman WH, Holst JJ, Hu FB, Kahn CR, Raz I,
Shulman GI, Simonson DC, Testa MA, Weiss R (2015) Type 2 diabetes mellitus. Nat Rev Dis
Primers 1:15019. https://doi.org/10.1038/nrdp.2015.19
Diaferia GR, Jimenez-Caliani AJ, Ranjitkar P, Yang W, Hardiman G, Rhodes CJ, Crisa L, Cirulli
V (2013) β1 integrin is a crucial regulator of pancreatic β-cell expansion. Development
140:3360–3372. https://doi.org/10.1242/dev.098533
Dor Y, Glaser B (2013) β-Cell dedifferentiation and type 2 diabetes. N Engl J Med 368:572–573.
https://doi.org/10.1056/NEJMcibr1214034
Dor Y, Brown J, Martinez O, Melton D (2004) Adult pancreatic beta-cells are formed by self-­
duplication rather than stem-cell differentiation. Nature 429:41–46. https://doi.org/10.1038/
nature02520
Eberhard D, Lammert E (2009) The pancreatic beta-cell in the islet and organ community. Curr
Opin Genet Dev 19:469–475. https://doi.org/10.1016/j.gde.2009.07.003
Epshtein A, Rachi E, Sakhneny L, Mizrachi S, Baer D, Landsman L (2017) Neonatal pancre-
atic pericytes support β-cell proliferation. Molecular Metabolism 6:1330–1338. https://doi.
org/10.1016/j.molmet.2017.07.010
Finegood DT, Scaglia L, Bonner-Weir S (1995) Dynamics of beta-cell mass in the growing rat
pancreas. Estimation with a simple mathematical model. Diabetes 44:249–256
Fuchsberger C, Flannick J, Teslovich TM et al (2016) The genetic architecture of type 2 diabetes.
Nature 536:41–47. https://doi.org/10.1038/nature18642
Georgia S, Bhushan A (2004) Beta cell replication is the primary mechanism for maintaining
postnatal beta cell mass. J Clin Investig 114:963–968. https://doi.org/10.1172/JCI200422098
Gittes GK (2009) Developmental biology of the pancreas: a comprehensive review. Dev Biol
326:4–35. https://doi.org/10.1016/j.ydbio.2008.10.024
Goulley J, Dahl U, Baeza N, Mishina Y, Edlund H (2007) BMP4-BMPR1A signaling in beta
cells is required for and augments glucose-stimulated insulin secretion. Cell Metab 5:207–219.
https://doi.org/10.1016/j.cmet.2007.01.009
Granot Z, Swisa A, Magenheim J, Stolovich-Rain M, Fujimoto W, Manduchi E, Miki T, Lennerz
JK, Stoeckert CJ Jr, Meyuhas O, Seino S, Permutt MA, Piwnica-Worms H, Bardeesy N, Dor Y
(2009) LKB1 regulates pancreatic β cell size, polarity, and function. Cell Metab 10:296–308.
https://doi.org/10.1016/j.cmet.2009.08.010
38 L. Landsman

Grant SFA, Thorleifsson G, Reynisdottir I, Benediktsson R, Manolescu A, Sainz J, Helgason

A, Stefansson H, Emilsson V, Helgadottir A, Styrkarsdottir U, Magnusson KP, Walters GB,
Palsdottir E, Jonsdottir T, Gudmundsdottir T, Gylfason A, Saemundsdottir J, Wilensky RL,
Reilly MP, Rader DJ, Bagger Y, Christiansen C, Gudnason V, Sigurdsson G, Thorsteinsdottir
U, Gulcher JR, Kong A, Stefansson K (2006) Variant of transcription factor 7-like 2 (TCF7L2)
gene confers risk of type 2 diabetes. Nat Genet 38:320–323. https://doi.org/10.1038/ng1732
Gregg BE, Moore PC, Demozay D, Hall BA, Li M, Husain A, Wright AJ, Atkinson MA, Rhodes
CJ (2012) Formation of a human β-cell population within pancreatic islets is set early in life.
J Clin Endocrinol Metab 97:3197–3206. https://doi.org/10.1210/jc.2012-1206
Hammes H-P (2017) Diabetic retinopathy: hyperglycaemia, oxidative stress and beyond.
Diabetologia 61:1–10. https://doi.org/10.1007/s00125-017-4435-8
Harari N, Sakhneny L, Khalifa-Malka L, Busch A, Hertel KJ, Hebrok M, Landsman L (2019)
Pancreatic pericytes originate from the embryonic pancreatic mesenchyme. Developmental
Biology In press
Hayden MR, Sowers JR (2007) Isletopathy in type 2 diabetes mellitus: implications of islet RAS,
islet fibrosis, islet amyloid, remodeling, and oxidative stress. Antioxid Redox Signal 9:891–
910. https://doi.org/10.1089/ars.2007.1610
Hayden MR, Karuparthi PR, Habibi J, Wasekar C, Lastra G, Manrique C, Stas S, Sowers JR (2007)
Ultrastructural islet study of early fibrosis in the Ren2 rat model of hypertension. Emerging
role of the islet pancreatic pericyte-stellate cell. JOP 8:725–738
Hayden MR, Yang Y, Habibi J, Bagree SV, Sowers JR (2010) Pericytopathy oxidative stress and
impaired cellular longevity in the pancreas and skeletal muscle in metabolic syndrome and type
2 diabetes. Oxidative Med Cell Longev 3:290–303. https://doi.org/10.4161/oxim.3.5.13653
Helgason A, Pálsson S, Thorleifsson G, Grant SFA, Emilsson V, Gunnarsdottir S, Adeyemo A,
Chen Y, Chen G, Reynisdottir I, Benediktsson R, Hinney A, Hansen T, Andersen G, Borch-­
Johnsen K, Jørgensen T, Schäfer H, Faruque M, Doumatey A, Zhou J, Wilensky RL, Reilly MP,
Rader DJ, Bagger Y, Christiansen C, Sigurdsson G, Hebebrand J, Pedersen O, Thorsteinsdottir
U, Gulcher JR, Kong A, Rotimi C, Stefansson K (2007) Refining the impact of TCF7L2
gene variants on type 2 diabetes and adaptive evolution. Nat Genet 39:218–225. https://doi.
org/10.1038/ng1960
Hogan MF, Hull RL (2017) The islet endothelial cell: a novel contributor to beta cell secretory
dysfunction in diabetes. Diabetologia 60:952–959
Houtz J, Borden P, Ceasrine A, Minichiello L, Kuruvilla R (2016) Neurotrophin signaling is
required for glucose-induced insulin secretion. Dev Cell 39:329–345. https://doi.org/10.1016/j.
devcel.2016.10.003
Kragl M, Lammert E (2010) Basement membrane in pancreatic islet function. Adv Exp Med Biol
654:217–234. https://doi.org/10.1007/978-90-481-3271-3_10
Kragl M, Schubert R, Karsjens H, Otter S, Bartosinska B, Jeruschke K, Weiss J, Chen C, Alsteens
D, Kuss O, Speier S, Eberhard D, Müller DJ, Lammert E (2016) The biomechanical properties
of an epithelial tissue determine the location of its vasculature. Nat Commun 7:13560. https://
doi.org/10.1038/ncomms13560
Lammert E, Cleaver O, Melton D (2001) Induction of pancreatic differentiation by signals from
blood vessels. Science 294:564–567. https://doi.org/10.1126/science.1064344
Longnecker DS, Gorelick F, Thompson ED (2018) Anatomy, histology, and fine structure of the
pancreas, 2nd ed. Wiley-Blackwell, Chichester
Lyssenko V, Lupi R, Marchetti P, Del Guerra S, Orho-Melander M, Almgren P, Sjögren M, Ling
C, Eriksson K-F, Lethagen A-L, Mancarella R, Berglund G, Tuomi T, Nilsson P, Del Prato S,
Groop L (2007) Mechanisms by which common variants in the TCF7L2 gene increase risk of
type 2 diabetes. J Clin Investig 117:2155–2163. https://doi.org/10.1172/JCI30706
Mastracci TL, Sussel L (2012) The endocrine pancreas: insights into development, differen-
tiation, and diabetes. Wiley Interdiscip Rev Developmental Biology 1:609–628. https://doi.
org/10.1002/wdev.44
Meier JJ, Butler AE, Saisho Y, Monchamp T, Galasso R, Bhushan A, Rizza RA, Butler PC (2008)
Beta-cell replication is the primary mechanism subserving the postnatal expansion of beta-cell
mass in humans. Diabetes 57:1584–1594. https://doi.org/10.2337/db07-1369
2 Pancreatic Pericytes in Glucose Homeostasis and Diabetes 39

Mandarino LJ, Finlayson J, Hassell JR (1994) High glucose downregulates glucose transport activity
in retinal capillary pericytes but not endothelial cells. Invest Ophthalmol Vis Sci 35:964–972.
Morikawa S, Baluk P, Kaidoh T, Haskell A, Jain RK, McDonald DM (2002) Abnormalities in peri-
cytes on blood vessels and endothelial sprouts in tumors. Am J Pathol 160:985–1000. https://
doi.org/10.1016/S0002-9440(10)64920-6
Nikolova G, Jabs N, Konstantinova I, Domogatskaya A, Tryggvason K, Sorokin L, Fässler R,
Gu G, Gerber H-P, Ferrara N, Melton DA, Lammert E (2006) The vascular basement mem-
brane: a niche for insulin gene expression and Beta cell proliferation. Dev Cell 10:397–405.
https://doi.org/10.1016/j.devcel.2006.01.015
Otonkoski T, Banerjee M, Korsgren O, Thornell L-E, Virtanen I (2008) Unique basement mem-
brane structure of human pancreatic islets: implications for beta-cell growth and differentiation.
Diabetes Obes Metab 10(Suppl 4):119–127. https://doi.org/10.1111/j.1463-1326.2008.00955.x
Ouaamari El A, Dirice E, Gedeon N, Hu J, Zhou J-Y, Shirakawa J, Hou L, Goodman J, Karampelias
C, Qiang G, Boucher J, Martinez R, Gritsenko MA, De Jesus DF, Kahraman S, Bhatt S, Smith
RD, Beer H-D, Jungtrakoon P, Gong Y, Goldfine AB, Liew CW, Doria A, Andersson O, Qian
W-J, Remold-O’Donnell E, Kulkarni RN (2016) SerpinB1 promotes pancreatic β cell prolif-
eration. Cell Metab 23:194–205. https://doi.org/10.1016/j.cmet.2015.12.001
Prentki M, Matschinsky FM, Madiraju SRM (2013) Metabolic signaling in fuel-induced insulin
secretion. Cell Metab 18:162–185. https://doi.org/10.1016/j.cmet.2013.05.018
Reinert RB, Brissova M, Shostak A, Pan FC, Poffenberger G, Cai Q, Hundemer GL, Kantz J,
Thompson CS, Dai C, McGuinness OP, Powers AC (2013) Vascular endothelial growth factor-
­a and islet vascularization are necessary in developing, but not adult, pancreatic islets. Diabetes
62:4154–4164. https://doi.org/10.2337/db13-0071
Richards OC, Raines SM, Attie AD (2010) The role of blood vessels, endothelial cells, and vascu-
lar pericytes in insulin secretion and peripheral insulin action. Endocr Rev 31:343–363. https://
doi.org/10.1210/er.2009-0035
Riley KG, Pasek RC, Maulis MF, Dunn JC, Bolus WR, Kendall PL, Hasty AH, Gannon M (2015)
Macrophages are essential for CTGF-mediated adult β-cell proliferation after injury. Mol
Metab 4:584–591. https://doi.org/10.1016/j.molmet.2015.05.002
Rorsman P, Braun M (2013) Regulation of insulin secretion in human pancreatic islets. Annu Rev
Physiol 75:155–179. https://doi.org/10.1146/annurev-physiol-030212-183754
Sakhneny L, Rachi E, Epshtein A, Guez HC, Wald-Altman S, Lisnyansky M, Khalifa-Malka
L, Hazan A, Baer D, Priel A, Weil M, Landsman L (2018) Pancreatic Pericytes support
β-cell function in a Tcf7l2-dependent manner. Diabetes 67:437–447. https://doi.org/10.2337/
db17-0697
Salpeter SJ, Klein AM, Huangfu D, Grimsby J, Dor Y (2010) Glucose and aging control the qui-
escence period that follows pancreatic beta cell replication. Development 137:3205–3213.
https://doi.org/10.1242/dev.054304
Sasson A, Rachi E, Sakhneny L, Baer D, Lisnyansky M, Epshtein A, Landsman L (2016) Islet
Pericytes are required for β-cell maturity. Diabetes 65:3008–3014. https://doi.org/10.2337/
db16-0365
Schaeffer M, Hodson DJ, Lafont C, Mollard P (2011) Endocrine cells and blood vessels work in
tandem to generate hormone pulses. J Mol Endocrinol 47:R59–R66. https://doi.org/10.1530/
JME-11-0035
Stolovich-Rain M, Hija A, Grimsby J, Glaser B, Dor Y (2012) Pancreatic beta cells in very old
mice retain capacity for compensatory proliferation. J Biol Chem 287:27407–27414. https://
doi.org/10.1074/jbc.M112.350736
Stratman AN, Davis GE (2012) Endothelial cell-pericyte interactions stimulate basement mem-
brane matrix assembly: influence on vascular tube remodeling, maturation, and stabilization.
Microsc Microanal 18:68–80. https://doi.org/10.1017/S1431927611012402
Talchai C, Xuan S, Lin HV, Sussel L, Accili D (2012) Pancreatic β cell dedifferentiation as a mech-
anism of diabetic β cell failure. Cell 150:1223–1234. https://doi.org/10.1016/j.cell.2012.07.029
Tang S-C, Chiu Y-C, Hsu C-T, Peng S-J, Fu Y-Y (2013) Plasticity of Schwann cells and peri-
cytes in response to islet injury in mice. Diabetologia 56:2424–2434. https://doi.org/10.1007/
s00125-013-2977-y
40 L. Landsman

Tarussio D, Metref S, Seyer P, Mounien L, Vallois D, Magnan C, Foretz M, Thorens B (2014)

Nervous glucose sensing regulates postnatal β cell proliferation and glucose homeostasis.
J Clin Investig 124:413–424. https://doi.org/10.1172/JCI69154
van de Wetering M, Sancho E, Verweij C, de Lau W, Oving I, Hurlstone A, van der Horn K, Batlle
E, Coudreuse D, Haramis AP, Tjon-Pon-Fong M, Moerer P, Van Den Born M, Soete G, Pals
S, Eilers M, Medema R, Clevers H (2002) The beta-catenin/TCF-4 complex imposes a crypt
progenitor phenotype on colorectal cancer cells. Cell 111:241–250
Wang P, Fiaschi-Taesch NM, Vasavada RC, Scott DK, García-Ocaña A, Stewart AF (2015)
Diabetes mellitus--advances and challenges in human β-cell proliferation. Nat Rev Endocrinol
11:201–212. https://doi.org/10.1038/nrendo.2015.9
Chapter 3
Pericytes in the Lung

Chi F. Hung, Carole L. Wilson, and Lynn M. Schnapp

Abstract The lung has numerous roles, including gas exchange, immune surveil-
lance, and barrier function. Being a highly vascularized organ, the lung receives
dual blood supply from both the pulmonary and bronchial circulation. Therefore,
pericytes likely play a prominent role in lung physiology given their localization in
the perivascular niche. New genetic approaches have increased our understanding
of the origin and the diverse functions of lung pericytes. Lung pericytes are myofi-
broblast progenitors, contributing to development of fibrosis in mouse models.
Lung pericytes are also capable of responding to danger signals and amplify the
inflammatory response through elaboration of cytokines and adhesion molecules. In
this chapter, we describe the molecular, anatomical, and phenotypical characteriza-
tion of lung pericytes. We further highlight their potential roles in the pathogenesis
of lung diseases including pulmonary fibrosis, asthma, and pulmonary hyperten-
sion. Finally, current gaps in knowledge and areas of ongoing investigation in lung
pericyte biology are also discussed.

Keywords Lung pericytes · PDGFRβ · Lung development · Lung myofibroblasts ·

Pulmonary fibrosis · Lung injury · Lung inflammation

C. F. Hung
Division of Pulmonary, Critical Care and Sleep Medicine, University of Washington,
Seattle, WA, USA
C. L. Wilson · L. M. Schnapp (*)
Division of Pulmonary, Critical Care, Allergy and Sleep Medicine,
Medical University of South Carolina, Charleston, SC, USA
e-mail: [email protected]

© Springer Nature Switzerland AG 2019 41

A. Birbrair (ed.), Pericyte Biology in Different Organs,
Advances in Experimental Medicine and Biology 1122,
https://doi.org/10.1007/978-3-030-11093-2_3
42 C. F. Hung et al.

3.1 Introduction

A critical but vastly understudied cell in the lung is the pericyte. Pericytes are mes-
enchymal cells defined by their anatomical association with endothelial cells
(Armulik et al. 2011; von Tell et al. 2006). Pericytes directly interact with microvas-
cular endothelium, including capillaries, precapillary arterioles, postcapillary, and
collecting venules, and are critical to blood vessel maturation and stability. However,
it is increasingly clear that lung pericytes play important roles not only in vascular
homeostasis but in lung injury and repair. In this chapter, we will review what is
known about lung pericytes in normal lung homeostasis, including their develop-
mental origins, discuss characterization of these cells in vitro and in vivo, and high-
light current knowledge of lung pericytes in lung diseases, such as pulmonary
fibrosis and pulmonary hypertension. Finally, we will speculate on the potential role
of pericytes as a regenerative niche in the lung.

3.2 Definition of Lung Pericytes

Anatomic Characterization Pericytes are classically defined by ultrastructural

localization demonstrating direct contact with microvascular endothelial cells
through gaps in the shared basement membrane. Using transmission electron
microscopy, Epling was the first to observe pericytes as “filament-containing cells”
in bovine and porcine lungs (Epling 1966), and Weibel confirmed the presence of
pericytes in capillaries of the lungs of humans and other mammals (Weibel 1974).
Compared to organs such as the heart or pancreas, pericytes are more rare in the
lung, and their frequency correlates with body size. For example, pericytes were
readily detected in human and dog lungs but were sparser in rodent lungs and could
not be found in lungs of the Etruscan shrew, the smallest mammal known (Weibel
1974). Pericytes are sensitive to oxygen levels and thus are less abundant in areas of
gas exchange (Shepro and Morel 1993). The pericyte-to-endothelial ratio in the
lung has been estimated to be about 1:10 (Shepro and Morel 1993), less than that in
the retina and central nervous system (~1:1 to 1:3), where barrier function is para-
mount and pericytes play a key role in regulating permeability. By genetically trac-
ing pericytes in mice, Kato et al. found a pericyte-to-endothelial ratio from 1:7 to
1:9 (Kato et al. 2018). Coverage of the abluminal surface of capillaries also varies
by organ and ranges between 18 and 26% in bovine lungs, depending on develop-
mental stage of the animal (Sims and Westfall 1983).
Perivascular stromal cells are found along all levels of the bronchovascular bun-
dle. Classically, lung pericytes are defined at the level of the alveolar capillary bed
(Armulik et al. 2011). Electron microscopy lungs show pericytes embedded within
the capillary basement membrane of alveoli in humans, mice, and other mammals
(Hung et al. 2013) (Epling 1966; Weibel 1974). In human lung samples, Weibel
observed that while most of the pericyte cell body and its large cellular processes
3 Pericytes in the Lung 43

are separated from endothelial cells by the intervening basement membrane, finer
processes penetrate the basement membrane and make contacts with the endothelial
cells in capillaries (Weibel 1974). Some pericytes were even seen to bridge separate
capillary segments (Weibel 1974). The nature of pericyte interactions with endothe-
lial cells in larger microvessels (i.e., the arterioles and venules) of the lung has not
yet been described at the ultrastructural level.
Developmental Origins Current understanding of lung pericytes indicates that
they primarily originate from the mesoderm and mesothelium (Que et al. 2008). In
the mouse, a multipotent cardiopulmonary progenitor positive for the transcription
factors Gli1 and Isl1 and the signaling molecule Wnt2 gives rise to mesodermal
lineages in the lung, including pericytes (Peng et al. 2013). We showed that the
forkhead transcription factor FoxD1 is transiently expressed in pulmonary mesen-
chymal progenitors as they infiltrate the lung buds during mouse embryogenesis
between days 11.5 and 12.5 (Hung et al. 2013). Expression of FoxD1 is then silenced
by day 15.5 once the progenitors differentiate into mature mesenchymal cells, mak-
ing this factor a useful marker for fate mapping these progenitors in the mouse lung
(see section on Molecular Identification below). For most organs, including the
lung, the PDGF-B-PDGFRβ signaling axis is critical in recruiting pericytes to their
proper anatomical niche within the microvascular environment during embryogen-
esis (Armulik et al. 2011). Disruption of this axis by knockout of PDGFRβ or
PDGF-B results in virtually total absence of pericytes in the mouse embryonic lung
(Hellstrom et al. 1999). The developmental origin of human lung pericytes is not
known, but we presume that they derive from a mesodermal precursor as well. As
discussed below, PDGFRβ is a key, robust marker of lung pericytes.
Molecular Identification and Pericyte Subtypes Whereas identification of peri-
cytes in early studies of lung tissue specimens used electron microscopy to define
the cells at the ultrastructural level, this method is cumbersome and difficult to carry
out in most laboratories. Advances in molecular characterization of pericytes and in
fate-mapping transgenic models have enabled investigators to study lung pericytes
with greater ease and flexibility than in the past. Nevertheless, lack of a truly
pericyte-­specific marker means that researchers need to use multiple criteria includ-
ing location relative to endothelial cells in situ, morphology, and genetic markers to
identify pericytes.
Only in the last decade or so have a limited number of studies been undertaken
to isolate and characterize pericytes from “normal” human lung (typically from
failed donor organs or tissue adjacent to carcinoma). The commonalities in these
studies were that pericytes were selected from dissociated lung cells using magnetic
bead-linked antibodies against one or two markers and then expanded in commer-
cial medium especially designed for growth of pericytes. In culture, isolated lung
pericytes have a stellate or elongated morphology with long extensive cellular pro-
cesses (Yuan et al. 2015; Wilson et al. 2018) (Fig. 3.1). In addition, lung pericytes
form primitive tubelike structures on the basement membrane preparation Matrigel
(Wilson et al. 2018; Bagley et al. 2006) (Fig. 3.1).
44 C. F. Hung et al.

Fig. 3.1 Cell morphology and characteristics of isolated human lung pericytes. Representative
phase-contrast light microscopic images of pericytes from normal lungs after plating on plastic (a)
or Matrigel™ (b). Note the extensive number of elongated cellular processes emanating from the
cells on plastic. On Matrigel™, pericytes assemble into primitive networks composed of nodes of
cells from which cellular processes project and connect with other nodes (b). Human lung peri-
cytes are positive for PDGFRβ (c) and NG2 (d) by immunofluorescence. Scale bar = 500 μm in (a),
(c), and (d) and 100 μm in (b)

Table 3.1 summarizes the markers that were used for isolation of human lung
pericytes and the other markers that were detected in these cells in different studies.
Expression of the proteoglycan neural glial antigen-2 (NG2 or chondroitin sulfate
proteoglycan 4) (Yuan et al. 2015; Wilson et al. 2018; Bagley et al. 2005; Bichsel
et al. 2015; Ricard et al. 2014) was reported in all the studies (Fig. 3.2). NG2+ cells
localized to perivascular regions in human lung tissue by immunofluorescence
(Sava et al. 2017; Rock et al. 2011). Most studies showed that lung pericytes are
PDGFRβ+, as expected (Fig. 3.2), and we used this marker in our own work for cell
selection from both human and mouse lungs (Wilson et al. 2018; Hung et al. 2017a).
Other prominent mesenchymal markers found in isolated human lung pericytes
include CD73 (ecto-5′-nucleotidase), CD90 (Thy1, also a fibroblast marker), and
the hyaluronan receptor CD44, although not all studies examined the cells for
these markers. Expression of endoglin (CD105) and PDGFRα is also characteristic
of human lung pericytes. The monoclonal antibody 3G5-defined ganglioside anti-
gen has emerged as a useful pericyte marker for the lung as well as the skin
Table 3.1 Gene markers of isolated human lung pericytes
Yuan et al. Bichsel et al. Wilson et al.
(2015) Bagley et al. Ricard et al. (2015) (CD73/ (2018)
Markers (3G5) (2006) (NG2) (2014) (3G5) CD90) (CD140b)
CD44 ND ND ND + +
CD73 ND ND ND + +
CD90 + + ND + +
CD105 (endoglin) ND + ND + ND
CD140a (PDGFRα) ND ND ND + +
CD140b (PDGFRβ) + + ND ND +
CD146 + ND ND − –
NG2 (CSPG4) + + + + +
3G5 + + + ND ND
αSMA + + − − –
Desmin ND + ND ND ND
Marker(s) used for cell selection shown in parentheses. ND not done

Fig. 3.2 Fate-mapping of Foxd1 progenitor-derived pericytes identifies them as a major source of
myofibroblast precursors in lung injury. (a, b) Confocal images showing fibrotic foci on d7 and
d14 after bleomycin lung injury in Foxd1-Cre; Rs26-TdT-R mice stained for the myofibroblast
marker αSMA (green). Co-expression of myofibroblast marker αSMA and the tdTomato fate
marker of Foxd1-derived pericytes indicated by ( ). Graph indicating proportion of tdTomato+
cells co-expressing αSMA+ in fibroblastic foci at indicated time points after bleomycin injury
(mean ± SEM, n = 3). (c, d) Confocal images showing fibrotic foci on d7 after bleomycin lung
injury in triple transgenic mouse Foxd1-Cre; Rs26-TdT-R; Coll-GFPTg and graph summarizing
proportion of tdTomato cells co-expressing Coll-GFP in fibrotic foci (mean ± SEM, n = 3)
(Bar = 50 μm). Modified and reprinted with permission of the American Thoracic Society.
Copyright © 2018 American Thoracic Society (Hung et al. 2013). The American Journal of
Respiratory and Critical Care Medicine is an official journal of the American Thoracic Society
46 C. F. Hung et al.

(Helmbold et al. 2001a, b) and was used by two investigator teams to isolate these
cells (Yuan et al. 2015; Ricard et al. 2014). There are discrepant reports on CD146
(also known as MCAM or melanoma cell adhesion molecule) expression: one study
found that the cells are positive for CD146 (Yuan et al. 2015), whereas two others
did not (Wilson et al. 2018; Bichsel et al. 2015). The lack of significant CD146
expression is surprising as it is a typical marker of pericytes from other organs, such
as the placenta and brain (Crisan et al. 2008), and was detected in mouse lung peri-
cytes (Hung et al. 2013). Potential explanations for these conflicting data on CD146
positivity include the presence of subpopulations of lung pericytes, differences in
underlying pathology of donor lung tissue, and/or differences in culture conditions.
Also, while α-SMA has been described as a pericyte marker, in general, only mural
cells associated with precapillary arterioles and postcapillary venules express this
contractile protein, whereas quiescent capillary pericytes do not (Nehls and
Drenckhahn 1991). Further complicating interpretations, while pericytes on arteri-
oles and capillaries are NG2+, venular pericytes are NG2−, at least in rat mesenteric
and subcutaneous tissue and skeletal muscle (Murfee et al. 2005). NG2 is induced
in venular pericytes during angiogenesis and vascular remodeling (Murfee et al.
2006). Although this differential expression of NG2 has not yet been established in
the microvasculature of the human lung, it raises the possibility that a cell selection
strategy based on NG2 may not include the entire spectrum of pericytes. Because
the markers are not pericyte specific [e.g., CD146 is expressed by endothelial cells
(Bardin et al. 2001) and 3G5 by mast cells (Gushi et al. 2008)], it is equally ­important
to exclude expression of other cell-specific markers such as CD31 and CD144
(endothelial cell markers PECAM and VE-cadherin, respectively), CD45 (pan leu-
kocyte marker), and CD326 (EpCAM, an epithelial cell marker).
Animal Models Much of our knowledge on the origin and fate of lung pericytes
comes from genetic approaches in mouse models, mostly using Cre-LoxP technol-
ogy (Table 3.2). In these models, Cre recombinase expression is driven by a pro-
moter selective for pericytes or pericyte progenitors, and the recombinase activates
expression of a reporter allele by targeting LoxP sequences that flank a transcrip-
tional stop site upstream of the reporter. This technique irreversibly labels cells with
active promoter expression and their progeny. We showed that expression of the
transcriptional factor FoxD1 marks progenitors destined to become pericytes in the
lung (Hung et al. 2013). Using triple transgenic mice expressing Cre recombinase
from the FoxD1 promoter and GFP from the collagen I promoter (Yata et al. 2003),
in conjunction with a tdTomato reporter allele at the ROSA locus, we found that
FoxD1-derived progenitors give rise to vascular smooth muscle cells and two popu-
lations of pericytes. The major population does not express collagen I or α-SMA
and is positive for PDGFRβ; a second, minor population expresses collagen I and
PDGFRα rather than PDGFRβ (Hung et al. 2013). In this study, all FoxD1-derived
cells were negative for CD31, an important distinction given that a group has
reported that FoxD1 positivity also marks endothelial progenitors in the mouse lung
(Sims-Lucas et al. 2013).
3 Pericytes in the Lung 47

Table 3.2 Transgenic mice for identifying and tracking lung pericytes
Major findings in uninjured
Promoter Insert Type lung References
FoxD1 GFP-Cre Knock-in Cells are PDGFRβ+, Hung et al. (2013)
CD146+, 60% NG2+,
αSMA-; variable expression
of collagen I and PDGFRα
FoxD1 GFP-­ Knock-in, Promoter active between Hung et al. (2013)
CreERT2 TAM days 11.5 and 15.5 in
inducible embryogenesis
FoxJ1 CreER Transgenic, Expressed in NG2+ cells Rock et al. (2011)
TAM
inducible
NG2 DsRedBAC Transgenic Cells are PDGFRβ+, Ricard et al. (2014),
CD146+, αSMA− Chow et al. (2013)
and Akamatsu et al.
(2013)
NG2 CreER™BAC Transgenic, Only 15% recombination Rock et al. (2011)
TAM efficiency; cells are
inducible PDGFRβ+, αSMA−
PDGFRβ Cre Transgenic PDGFRβ expressed in other Guimaraes-Camboa
lineages in addition to et al. (2017)
pericytes during
embryogenesis
PDGFRβ CreER Transgenic, Cells are NG2+ Kato et al. (2018)
(BAC) TAM
inducible
TAM tamoxifen

One important limitation in lung pericyte research using lineage tracing animal
models is the lack of a definitive marker for pericytes. Commonly recognized mark-
ers such as PDGFRβ, NG2, and CD146 are not uniformly expressed in all pericytes.
Moreover, their expression may be spatially and temporally dynamic throughout
development and after injury. For example, PDGFRβ promoter is active in multiple
cell lineages during embryogenesis and is not specific to pericytes (Guimaraes-­
Camboa et al. 2017). Furthermore, activated myofibroblasts derived from non-­
pericyte populations following lung injury may upregulate expression of PDGFRβ
(Henderson et al. 2013; Hung et al. 2018).
Pulse labeling of pericyte populations circumvents the dynamic expression of
pericyte markers that are observed in other cell lineages during development and
with physiologic stress. Modification of Cre recombinase to make its activity induc-
ible allows temporal control in labeling pericytes. For example, a tamoxifen-­
inducible PDGFRβ-CreER transgenic animal enables postnatal marking of
PDGFRβ+ cells; tamoxifen administration at days 1 through 3 after birth labeled
cells tightly juxtaposed to endothelial cells in the lung (Kato et al. 2018). Isolated
labeled cells were positive for expression of PDGFRβ and NG2 and were negative
for markers of endothelial and epithelial cells (Kato et al. 2018).
48 C. F. Hung et al.

There are important limitations to Cre-loxP models. As discussed previously,

promoter activity driving Cre recombinase expression may not be restricted to peri-
cytes, depending on the developmental stage and the presence of physiologic stress.
Secondly, Cre recombinase activity is rarely 100% efficient. Promoter activity,
accessibility of the target loxP sequences in the genome, and experimental condi-
tions used to induce Cre activity in inducible models can all influence labeling effi-
ciency. In one report, postnatal tamoxifen administration labeled only ~15% of the
NG2+ lung cells in NG2-CreER mice (Rock et al. 2011), highlighting a potential
drawback of these types of models. Furthermore, bioavailability of tamoxifen can
influence labeling efficiency in tamoxifen-inducible Cre models. Some investiga-
tors have leveraged this property to study lineage-labeled single cells distributed
throughout the lung by administering low-dose tamoxifen (Kato et al. 2018;
Barkauskas et al. 2013). Finally, there is currently no single marker that identifies
the entire lung pericyte population. The commonly used markers do not completely
overlap, suggesting heterogeneity within the lung pericyte population. We showed
that only a subset of PDGFRβ+ cells (~60%) express NG2 (Hung et al. 2013), sug-
gesting that NG2 may not be an all-inclusive marker of mouse lung pericytes.
Indeed, NG2 has been described as a marker of pericytes in angiogenesis and tissue
remodeling rather than homeostasis (Murfee et al. 2006). Interestingly, Rock et al.
found that a FoxJ1-CreER transgene also marks NG2+ pericytes in the lung (Rock
et al. 2011), although there have been no further reports in the literature using this
transgenic mouse for pericyte fate mapping. Our group investigated NG2 and
CD146 expression in human pericytes. We found that PDGFRβ+ human lung peri-
cytes have low CD146 expression unlike mouse lung pericytes (Hung et al. 2017a,
b). Taken together, the studies suggest that the lung pericyte population is heteroge-
neous. Understanding functional differences in lung pericyte subpopulations is an
area of ongoing investigation.
Cre-loxP technology can also induce specific gene deletions in pericyte-lineage
lung cells. A cross of mice with loxP-flanked Yap1 and Wwtr1 (encoding TAZ pro-
tein) alleles to PDGFRβ-CreER mice generated pericyte-specific inducible dele-
tions of these genes in postnatal lung (Kato et al. 2018). The disruptions in YAP1
and TAZ signaling in pericytes altered postnatal lung morphogenesis. Another novel
application of Cre-loxP technology involves targeted ablation of pericytes within
the lung. Mice are inherently insensitive to diphtheria toxin (DT) as they lack its
receptor (DTR). We crossed ROSA26iDTR mice, in which the expression of the
simian diphtheria toxin receptor (iDTR) is Cre inducible, to FoxD1-Cre mice to
generate the double transgenic FoxD1-Cre;ROSA26iDTR model. In this mouse
model, iDTR is expressed in FoxD1-derived cells, rendering them sensitive to
­ablation upon exposure to DT. Conventional delivery of DT by intraperitoneal injec-
tion resulted in neurologic side effects such as seizures within 72 hours of DT
administration (unpublished data). This observation reflected the critical role of
brain pericytes in the regulation of blood-brain barrier (Hung et al. 2017b). To cir-
cumvent this limitation, we administered low-dose DT by oropharyngeal aspiration.
Using this method, we achieved approximately 40% ablation of PDGFRβ+ stromal
3 Pericytes in the Lung 49

cells in the lung at 7 days after DT delivery (Hung et al. 2017b). Long-term ablation,
however, remains elusive as progenitor populations replenish ablated cells.

3.3 Pericytes During Lung Homeostasis and Repair

The exact roles of pericytes in lung homeostasis remain largely uncharacterized.

Functional studies that disrupt the pericyte-endothelial signaling axis (e.g., PDGF-­
PDGFRß) result in early embryonic lethality due to abnormal vasculogenesis. Most
models that induce pericyte loss focus on developing vessels rather than fully
mature pericyte-endothelial units. In one study, pericytes were depleted by admin-
istering a PDGFRβ-blocking antibody in postnatal pups, which caused abnormal
retinal capillary formation (Ogura et al. 2017) and demonstrated a critical role of
pericytes in retinal angiogenesis. However, administration of the same antibody in
adult mice did not result in pericyte coverage loss, suggesting the PDGF-PDGFRβ
signaling axis is dispensable in the developed microvascular unit. Akt/Jagged1 sig-
naling maintains perivascular stromal cell coverage in homeostasis in the heart and
retina as disruption of endothelial Akt production led to pericyte apoptosis and
breakdown of perivascular matrix (Kerr et al. 2016). Whether blockade of the Akt/
Jagged1/Notch signaling axis results in similar findings in the lung remains
unknown. When retinal pericytes were ablated with DT in adult mice, microangi-
opathy developed, suggesting that maintenance of vascular integrity is a critical
function of retinal pericytes (Valdez et al. 2014). In our model of lung pericyte abla-
tion with DT, we did not observe changes in lung permeability (Hung et al. 2017b),
suggesting that lung pericytes may not be as critical to normal barrier function as in
other organs. Thus, our understanding of pericyte biology in mature lungs under
homeostatic conditions is limited.
The lung has limited ability to regenerate compared to skin and gut epithelium.
Acute lung injury from a variety of insults leads to denudement of damaged alveolar
epithelium and a concurrent local inflammatory response. This is normally followed
by resolution of inflammation, transient scarring, and repopulation of the lost epi-
thelial barrier. In some instances, a pathological response results in persistent scar-
ring or fibrosis, leading to distortion of normal architecture and impairment of lung
function. The precise cellular mechanisms that control regenerative versus patho-
logical responses in the lung remain an area of intense interest.
Accumulating evidence suggests pericytes are important in regulating lung repair
as progenitors of myofibroblasts and mesenchymal progenitor cells. The multidrug
resistance transporter ATP-binding cassette G (ABCG2) was identified as a marker
in lineage-tracing experiments that labeled mesenchymal progenitor cells (MPCs)
that give rise to pericytes (Jun et al. 2011; Marriott et al. 2014; Chow et al. 2013;
Gaskill et al. 2017). Differentiation of ABCG2-lineage cells to mature pericytes was
important in attenuation of bleomycin-induced lung fibrosis and restoration of nor-
mal vascular function following injury (Gaskill et al. 2017). Overexpression of the
Wnt/ß-catenin pathway in ABCG2-lineage cells led to increased pericyte specifica-
50 C. F. Hung et al.

tion without maturation. These mice developed worse lung fibrosis and increased
microvascular dysfunction, suggesting MPC differentiation into mature, functional
pericytes is integral to normal repair in the lung. There may be other pools of pro-
genitors that give rise to renewal of pericytes in different physiologic contexts such
as aging and apoptosis. Identification of the progenitor pool and understanding the
molecular signals that direct regeneration will greatly advance the field’s under-
standing of normal and abnormal repair in the lung interstitium, with relevance to
many chronic pulmonary diseases such as COPD and interstitial lung diseases
where effective medical therapy continues to be lacking.
One aspect of the regenerative response that has garnered significant attention is
the identity of adult progenitor cells that are capable of self-renewal or even renewal
of other cell lineages (multipotency). These elusive adult progenitor cells are
thought to be of mesenchymal origin and are often referred as mesenchymal stem
cells (MSCs). Pericytes have been postulated to be a source of MSCs in multiple
organs (Crisan et al. 2008). MSCs are marked by expression of the transcription
factor Gli1 and ABCG2 and comprise a small subset of the PDGFRβ+ cells in the
lung (Marriott et al. 2014; Chow et al. 2013; Kramann et al. 2015). Although these
MSCs have been called “pericytes,” this term primarily reflects their anatomical
location, as they do not express other markers associated with mature pericytes in
the mouse, such as NG2 and CD146. Using a genetic tracing approach to isolate
ABCG2+ cells, Chow et al. showed that lung MSCs can differentiate into NG2+
pericytes and endothelial cells in vitro (Chow et al. 2013). In contrast, a study using
the transcription factor Tbx18 as a marker of pericytes found that Tbx18-lineage
cells did not exhibit multipotency in vivo (Guimaraes-Camboa et al. 2017).
Likewise, we did not detect expression of MSC markers on human lung pericytes
(defined by NG2+ PDGFRß+) in vitro (Wilson et al. 2018). One possibility to
account for the conflicting reports on pericyte multipotency is that cell culture envi-
ronments often introduce non-physiologic cues that may induce phenotypic changes
in pericytes, cues that are not representative of the native tissue environment where
pericytes reside. To date, there is no evidence to suggest lung pericytes possess
multipotent cell fate plasticity in vivo.

3.4 Pericytes in Lung Disease

Lung Inflammation and Injury Historically, focus on pericytes has been limited
to their functions as structural mural cells that support vascular maturation and
endothelial homeostasis. Accumulating evidence, however, suggests these
­specialized cells may have broader biological functions. One area that has captured
the interest of investigators is the potential role of pericytes in immunity. Much of
the evidence for pericytes in inflammation comes from studies in the central ner-
vous system where pericytes elaborate chemoattractants, adhesion molecules, and
paracrine signaling to amplify local inflammation (Jansson et al. 2014; Rustenhoven
et al. 2017).
3 Pericytes in the Lung 51

Similar to findings in other organs, our group has shown that mouse lung peri-
cytes respond to pro-inflammatory signaling. Murine lung pericytes possess multi-
ple functional TLRs and elaborate cytokines in response to specific TLR agonists
in vitro (Table 3.3) (Hung et al. 2017a). Similarly, cultured human lung pericytes
respond to inflammatory cues and upregulate cytokine expression. Compared to
mouse lung pericytes, human lung pericytes exhibited a narrower range of TLR
responses, with TLR 2/6 and TLR 4 signaling leading to the most robust pro-­
inflammatory responses (Table 3.3). We showed that depletion of pericytes by DT
exposure attenuated the acute inflammatory response in a sterile lung injury model
(Hung et al. 2017a, b). How lung pericyte ablation affects the inflammatory profile
in other models of sterile lung injury and infectious models is yet to be defined.
Based on our experience, we speculate that pericytes play an important supportive
role in mediating local lung inflammation. Their perivascular position in the alveoli
suggests they may play important roles in the activation of the endothelium through
paracrine signaling and direct contact during inflammation. Furthermore, they
reside in a prime location to sense pro-inflammatory alveolar components and may
be one of the very first responders to alveolar damage. Beyond the production of
inflammatory cytokines, pericytes may also direct trafficking of inflammatory cells
that exit systemic circulation through upregulation of adhesion molecules as well as
modification of the extracellular matrix in the perivascular space and the lung
interstitium.
Pulmonary Fibrosis During pulmonary fibrosis, the main effector cell for matrix
production is the lung myofibroblast. However, the origin of lung myofibroblasts
remains controversial. In our transgenic mouse model work, we found that FoxD1-­

Table 3.3 Inflammatory response of lung pericytesa

Inflammatory cytokines and adhesion
Species Stimulus molecules
Mouse IL-1r CXCL1, CXCL2, CXCL10, CCL2,
ICAM1
TLR2/1 CXCL1, CXCL2, CXCL10, CCL2,
TNFα, ICAM1
TLR2/6 CXCL1, CXCL2, CXCL10, CCL2,
TNFα, ICAM1
TLR4 CXCL1, CXCL2, CXCL10, CCL2,
TNFα, ICAM1
TLR5 ICAM1
TLR7 CXCL1, CXCL2, CXCL10, CCL2,
TNFα, ICAM1
TLR9 CXCL2, TNFα
Human TLR2/6 CXCL1, CXCL2, CXCL8, CCL2,
ICAM1
TLR4 CXCL1, CXCL2, CXCL8, CCL2,
ICAM1
a
Pericytes were selected based on PDGFRβ positivity
52 C. F. Hung et al.

derived cells express common markers used to identify pericytes such as PDGFRß,
NG2, and CD146, while they lack expression of endothelial-, epithelial-, or
hematopoietic-­specific markers. In addition, FoxD1-derived cells are located adja-
cent to endothelial cells, suggesting they are indeed pericytes (Fig. 3.2) (Hung et al.
2013). Importantly, they contributed to >50% of α-smooth muscle actin (αSMA)-
positive myofibroblasts in the bleomycin model of lung fibrosis (Hung et al. 2013)
(Fig. 3.2). Our study and Rock et al. (Rock et al. 2011) found other cell types con-
tribute significantly to the myofibroblast pool in the lung. In contrast, in both kidney
and liver models of fibrosis, pericytes were the predominant source of myofibro-
blasts (Lin et al. 2008; Humphreys et al. 2010; Mederacke et al. 2013). These find-
ings establish the novel concept that pericytes are key effectors and potential
therapeutic targets in pulmonary fibrosis.

Pulmonary Arterial Hypertension Pulmonary Arterial Hypertension (PAH),

defined by elevated mean arterial pressure ≥ 25 mmHg at rest, is characterized by
progressive remodeling of the distal pulmonary arterials with resultant increase in
pulmonary vascular resistance, leading to right heart failure and ultimately death.
Pulmonary endothelial dysfunction is a key feature of PAH pathogenesis. The pul-
monary artery wall includes a secondary layer of vascular smooth muscle cells
(SMCs) or smooth muscle-like pericytes (Hemnes and Humbert 2017). Pericytes
isolated from PAH lungs had impaired association with endothelial cells (Yuan et al.
2015). Another study showed increased number of pericytes (defined by 3G5 stain-
ing) surrounding pulmonary vessels of all sizes in explanted human PAH lungs and
in rodent models of pulmonary hypertension (Ricard et al. 2014). Furthermore, the
increased number of pericytes was detected before changes in arterial pressures
were detected, suggesting the changes in pericyte were not secondary to hemody-
namic derangements. Their data suggested that dysregulated FGF2 and IL-6 signal-
ing from endothelial cells drive pericyte proliferation during PAH and contribute to
pathogenesis.
Interestingly, there were two case reports of infants with Adams-Oliver syn-
drome, a rare genetic disorder characterized by congenital scalp defects and limb
defects of unknown pathogenesis, who developed severe infantile pulmonary hyper-
tension, intracranial bleeding, and cutis marmorata telangiectatica congenita (Patel
et al. 2004). At autopsy, lack of pericyte coverage was found areas of vascular dila-
tation, while increased pericyte coverage and proliferation were found in associa-
tion with vessel stenosis. They hypothesized that aberrant pericyte recruitment
caused pulmonary hypertension in this syndrome (Patel et al. 2004). These studies
support the role of pericyte recruitment and proliferation in the pathogenesis of
pulmonary arterial hypertension.
Hereditary Hemorrhagic Telangiectasia Hereditary Hemorrhagic Telangiectasia
(HHT) also known as Osler-­Weber-­Rendu syndrome is an autosomal dominant dis-
order characterized by arteriovenous malformations (AVM) affecting major organs,
particularly the lung, liver, and brain. Lung AVMs are more commonly seen in
HHT1 patients. Most HHT1 patients have mutations in ENG encoding endoglin
3 Pericytes in the Lung 53

(McAllister et al. 1994), a receptor for transforming growth factor-β (TGFβ)/bone

morphogenetic protein (BMP) expressed primarily in endothelial cells and peri-
cytes. Impaired communication between endothelial cell and pericyte, including
loss of normal TGFβ activation and signaling, is postulated to lead to impaired
pericyte differentiation and vessel maturation in HHT (Thalgott and Lebrin 2015).
One group reported that thalidomide increased pericyte number, α-SMA expres-
sion, and their recruitment to blood vessels in a mouse model of HHT, leading to
vasculature stabilization (Thalgott and Lebrin 2015). A speculated mechanism was
a thalidomide-induced increase of PDGF-B expression by endothelial cells.
Pericytes and Asthma Asthma is a common disease characterized by recurrent
inflammatory episodes with airway and vascular remodeling. The airway remodel-
ing includes myofibroblast accumulation, collagen deposition, and increased
smooth muscle mass leading to subepithelial fibrosis and airway narrowing. Our
understanding of the contributions of pericytes in asthma is limited. In a murine
model of chronic house dust mite (HDM) exposure, investigators noted decreased
capillary-associated pericyte coverage (defined by NG2 positivity) and increased
subepithelial (peribronchial) pericytes. These findings were exaggerated when mice
were treated with a PDFGRβ inhibitor and resulted in worsened airway hyperre-
sponsiveness and airway smooth muscle thickening (Johnson et al. 2015). Given
evidence supporting pericyte transition to myofibroblast in other conditions, it is
interesting to speculate that pericytes likewise contribute to subepithelial fibrosis in
asthma. However, more studies are needed to define a role of pericytes in asthma
pathogenesis.

3.5 Conclusion

We are only beginning to appreciate the multiple roles pericytes play in lung disease
pathogenesis. Studies in animal models suggest lung pericytes exhibit a degree of
biological plasticity that was previously underappreciated. They participate in
inflammation and fibrosis, and they also regulate microvascular permeability and
stability (Fig. 3.3). Clinically, some disorders such as pulmonary hypertension and
HHT are associated with changes in pericyte coverage. So are pericytes beneficial
or harmful in disease? To date, there is not enough information on lung pericytes to
answer this question. The timing of pericyte activation during disease, the interac-
tion of pericytes with their cellular neighbors and their microenvironmental niche
when homeostasis is disturbed, and the environmental cues that activate pericytes
are some of the important knowledge gaps in lung pericyte biology that require
further study. Refining methods to identify, isolate, and manipulate lung pericytes in
the laboratory will be essential to advancing knowledge on these cells in pulmonary
health and disease.
54 C. F. Hung et al.

Fig. 3.3 Schematic of potential roles of pericytes in the lung

Acknowledgments The authors thank all the members of the Schnapp and Hung laboratories
who have contributed to studies described in this chapter. We also thank Seth Bollenbecker for
providing the artwork in Fig. 3.3.

References

Akamatsu T, Arai Y, Kosugi I, Kawasaki H, Meguro S, Sakao M, Shibata K, Suda T, Chida K,

Iwashita T (2013) Direct isolation of myofibroblasts and fibroblasts from bleomycin-injured
lungs reveals their functional similarities and differences. Fibrogenesis Tissue Repair 6(1):15.
https://doi.org/10.1186/1755-1536-6-15. PubMed PMID: 23927729; PMCID: PMC3751789
Armulik A, Genove G, Betsholtz C (2011) Pericytes: developmental, physiological, and pathologi-
cal perspectives, problems, and promises. Dev Cell 21(2):193–215. https://doi.org/10.1016/j.
devcel.2011.07.001. PubMed PMID: 21839917
Bagley RG, Weber W, Rouleau C, Teicher BA (2005) Pericytes and endothelial precursor cells:
cellular interactions and contributions to malignancy. Cancer Res 65(21):9741–9750. https://
doi.org/10.1158/0008-5472.CAN-04-4337. PubMed PMID: 16266995
Bagley RG, Rouleau C, Morgenbesser SD, Weber W, Cook BP, Shankara S, Madden SL,
Teicher BA (2006) Pericytes from human non-small cell lung carcinomas: an attractive tar-
get for ­ anti-­
angiogenic therapy. Microvasc Res 71(3):163–174. https://doi.org/10.1016/j.
mvr.2006.03.002. Epub 2006/04/19. PubMed PMID: 16624341
Bardin N, Anfosso F, Masse JM, Cramer E, Sabatier F, Le Bivic A, Sampol J, Dignat-George F
(2001) Identification of CD146 as a component of the endothelial junction involved in the con-
trol of cell-cell cohesion. Blood 98(13):3677–3684. PubMed PMID: 11739172
Barkauskas CE, Cronce MJ, Rackley CR, Bowie EJ, Keene DR, Stripp BR, Randell SH, Noble
PW, Hogan BL (2013) Type 2 alveolar cells are stem cells in adult lung. J Clin Invest
123(7):3025–3036. https://doi.org/10.1172/JCI68782. Epub 2013/08/08. PubMed PMID:
23921127; PMCID: PMC3696553
3 Pericytes in the Lung 55

Bichsel CA, Hall SR, Schmid RA, Guenat OT, Geiser T (2015) Primary human lung pericytes sup-
port and stabilize in vitro perfusable microvessels. Tissue Eng Part A 21(15–16):2166–2176.
https://doi.org/10.1089/ten.TEA.2014.0545. PubMed PMID: 25891384
Chow K, Fessel JP, Kaoriihida S, Schmidt EP, Gaskill C, Alvarez D, Graham B, Harrison DG,
Wagner DH Jr, Nozik-Grayck E, West JD, Klemm DJ, Majka SM (2013) Dysfunctional resi-
dent lung mesenchymal stem cells contribute to pulmonary microvascular remodeling. Pulm
Circ 3(1):31–49. https://doi.org/10.4103/2045-8932.109912. PubMed PMID: 23662173;
PMCID: PMC3641738
Crisan M, Yap S, Casteilla L, Chen CW, Corselli M, Park TS, Andriolo G, Sun B, Zheng B, Zhang
L, Norotte C, Teng PN, Traas J, Schugar R, Deasy BM, Badylak S, Buhring HJ, Giacobino JP,
Lazzari L, Huard J, Peault B (2008) A perivascular origin for mesenchymal stem cells in mul-
tiple human organs. Cell Stem Cell 3(3):301–313. https://doi.org/10.1016/j.stem.2008.07.003.
PubMed PMID: 18786417
Epling GP (1966) Electron microscopic observations of pericytes of small blood vessels in the
lungs and hearts of normal cattle and swine. Anat Rec 155:513–530
Gaskill CF, Carrier EJ, Kropski JA, Bloodworth NC, Menon S, Foronjy RF, Taketo MM, Hong
CC, Austin ED, West JD, Means AL, Loyd JE, Merryman WD, Hemnes AR, De Langhe S,
Blackwell TS, Klemm DJ, Majka SM (2017) Disruption of lineage specification in adult pul-
monary mesenchymal progenitor cells promotes microvascular dysfunction. J Clin Invest
127(6):2262–2276. https://doi.org/10.1172/JCI88629. Epub 2017/05/04. PubMed PMID:
28463231; PMCID: PMC5451236
Guimaraes-Camboa N, Cattaneo P, Sun Y, Moore-Morris T, Gu Y, Dalton ND, Rockenstein E,
Masliah E, Peterson KL, Stallcup WB, Chen J, Evans SM (2017) Pericytes of multiple organs
do not behave as mesenchymal stem cells in vivo. Cell Stem Cell 20(3):345–359.e5. https://doi.
org/10.1016/j.stem.2016.12.006. PubMed PMID: 28111199; PMCID: PMC5337131
Gushi A, Tanaka M, Tsuyama S, Nagai T, Kanzaki T, Kanekura T, Matsuyama T (2008) The 3G5
antigen is expressed in dermal mast cells but not pericytes. J Cutan Pathol 35(3):278–284.
https://doi.org/10.1111/j.1600-0560.2007.00809.x. PubMed PMID: 18251741
Hellstrom M, Kalen M, Lindahl P, Abramsson A, Betsholtz C (1999) Role of PDGF-B and PDGFR-­
beta in recruitment of vascular smooth muscle cells and pericytes during embryonic blood
vessel formation in the mouse. Development 126(14):3047–3055. PubMed PMID: 10375497
Helmbold P, Nayak RC, Marsch WC, Herman IM (2001a) Isolation and in vitro characteriza-
tion of human dermal microvascular pericytes. Microvasc Res 61(2):160–165. https://doi.
org/10.1006/mvre.2000.2292. PubMed PMID: 11254395
Helmbold P, Wohlrab J, Marsch WC, Nayak RC (2001b) Human dermal pericytes express 3G5
ganglioside—a new approach for microvessel histology in the skin. J Cutan Pathol 28(4):206–
210. PubMed PMID: 11426828
Hemnes AR, Humbert M (2017) Pathobiology of pulmonary arterial hypertension: understanding
the roads less travelled. Eur Respir Rev 26(146). https://doi.org/10.1183/16000617.0093-2017
Henderson NC, Arnold TD, Katamura Y, Giacomini MM, Rodriguez JD, McCarty JH, Pellicoro
A, Raschperger E, Betsholtz C, Ruminski PG, Griggs DW, Prinsen MJ, Maher JJ, Iredale
JP, Lacy-Hulbert A, Adams RH, Sheppard D (2013) Targeting of alphav integrin identifies a
core molecular pathway that regulates fibrosis in several organs. Nat Med 19(12):1617–1624.
https://doi.org/10.1038/nm.3282. PubMed PMID: 24216753; PMCID: PMC3855865
Humphreys BD, Lin SL, Kobayashi A, Hudson TE, Nowlin BT, Bonventre JV, Valerius MT,
McMahon AP, Duffield JS (2010) Fate tracing reveals the pericyte and not epithelial origin
of myofibroblasts in kidney fibrosis. Am J Pathol 176(1):85–97. https://doi.org/10.2353/
ajpath.2010.090517. PubMed PMID: 20008127; PMCID: PMC2797872
Hung C, Linn G, Chow YH, Kobayashi A, Mittelsteadt K, Altemeier WA, Gharib SA, Schnapp
LM, Duffield JS (2013) Role of lung pericytes and resident fibroblasts in the pathogenesis
of pulmonary fibrosis. Am J Respir Crit Care Med 188(7):820–830. https://doi.org/10.1164/
rccm.201212-2297OC. PubMed PMID: 23924232; PMCID: 3826269
Hung CF, Mittelsteadt KL, Brauer R, McKinney BL, Hallstrand TS, Parks WC, Chen P, Schnapp
LM, Liles WC, Duffield JS, Altemeier WA (2017a) Lung pericyte-like cells are functional inter-
56 C. F. Hung et al.

stitial immune sentinel cells. Am J Physiol Lung Cell Mol Physiol 312(4):L556–L567. https://
doi.org/10.1152/ajplung.00349.2016. PubMed PMID: 28188224; PMCID: PMC5407093
Hung CF, Chow YH, Liles WC, Altemeier WA, Schnapp LM (2017b) Ablation of Pericyte-like
cells in lungs by oropharyngeal aspiration of diphtheria toxin. Am J Respir Cell Mol Biol
56(2):160–167. https://doi.org/10.1165/rcmb.2016-0083MA. PubMed PMID: 27779900
Hung CF, Wilson CL, Chow YH, Schnapp LM (2018) Role of integrin alpha8 in murine model of
lung fibrosis. PLoS One 13(5):e0197937. https://doi.org/10.1371/journal.pone.0197937. Epub
2018/05/29. PubMed PMID: 29813125
Jansson D, Rustenhoven J, Feng S, Hurley D, Oldfield RL, Bergin PS, Mee EW, Faull RL, Dragunow
M (2014) A role for human brain pericytes in neuroinflammation. J Neuroinflammation 11:104.
https://doi.org/10.1186/1742-2094-11-104. PubMed PMID: 24920309; PMCID: PMC4105169
Johnson JR, Folestad E, Rowley JE, Noll EM, Walker SA, Lloyd CM, Rankin SM, Pietras K,
Eriksson U, Fuxe J (2015) Pericytes contribute to airway remodeling in a mouse model of
chronic allergic asthma. Am J Physiol Lung Cell Mol Physiol 308(7):L658–L671. https://
doi.org/10.1152/ajplung.00286.2014. Epub 2015/02/01. PubMed PMID: 25637607; PMCID:
PMC4385988
Jun D, Garat C, West J, Thorn N, Chow K, Cleaver T, Sullivan T, Torchia EC, Childs C, Shade T,
Tadjali M, Lara A, Nozik-Grayck E, Malkoski S, Sorrentino B, Meyrick B, Klemm D, Rojas
M, Wagner DH, Jr., Majka SM. The pathology of bleomycin-induced fibrosis is associated with
loss of resident lung mesenchymal stem cells that regulate effector T-cell proliferation. Stem
Cells 2011;29(4):725–735. doi: https://doi.org/10.1002/stem.604. Epub 2011/02/12. PubMed
PMID: 21312316; PMCID: PMC3322548
Kato K, Dieguez-Hurtado R, Park DY, Hong SP, Kato-Azuma S, Adams S, Stehling M, Trappmann
B, Wrana JL, Koh GY, Adams RH (2018) Pulmonary pericytes regulate lung morphogene-
sis. Nat Commun 9(1):2448. https://doi.org/10.1038/s41467-018-04913-2. PubMed PMID:
29934496; PMCID: PMC6015030
Kerr BA, West XZ, Kim YW, Zhao Y, Tischenko M, Cull RM, Phares TW, Peng XD, Bernier-­
Latmani J, Petrova TV, Adams RH, Hay N, Naga Prasad SV, Byzova TV (2016) Stability and
function of adult vasculature is sustained by Akt/Jagged1 signalling axis in endothelium. Nat
Commun 7:10960. https://doi.org/10.1038/ncomms10960. Epub 2016/03/15. PubMed PMID:
26971877; PMCID: PMC4793084
Kramann R, Schneider RK, DiRocco DP, Machado F, Fleig S, Bondzie PA, Henderson JM, Ebert
BL, Humphreys BD (2015) Perivascular Gli1+ progenitors are key contributors to injury-­
induced organ fibrosis. Cell Stem Cell 16(1):51–66. https://doi.org/10.1016/j.stem.2014.11.004.
PubMed PMID: 25465115; PMCID: PMC4289444
Lin SL, Kisseleva T, Brenner DA, Duffield JS (2008) Pericytes and perivascular fibroblasts are the
primary source of collagen-producing cells in obstructive fibrosis of the kidney. Am J Pathol
173(6):1617–1627. https://doi.org/10.2353/ajpath.2008.080433. ajpath.2008.080433 [pii].
Epub 2008/11/15. PubMed PMID: 19008372; PMCID: 2626374
Marriott S, Baskir RS, Gaskill C, Menon S, Carrier EJ, Williams J, Talati M, Helm K, Alford CE,
Kropski JA, Loyd J, Wheeler L, Johnson J, Austin E, Nozik-Grayck E, Meyrick B, West JD,
Klemm DJ, Majka SM (2014) ABCG2pos lung mesenchymal stem cells are a novel pericyte
subpopulation that contributes to fibrotic remodeling. Am J Physiol Cell Physiol 307(8):C684–
C698. https://doi.org/10.1152/ajpcell.00114.2014. PubMed PMID: 25122876; PMCID:
PMC4200000
McAllister KA, Grogg KM, Johnson DW, Gallione CJ, Baldwin MA, Jackson CE, Helmbold
EA, Markel DS, McKinnon WC, Murrell J et al (1994) Endoglin, a TGF-beta binding protein
of endothelial cells, is the gene for hereditary haemorrhagic telangiectasia type 1. Nat Genet
8(4):345–351. https://doi.org/10.1038/ng1294-345. PubMed PMID: 7894484
Mederacke I, Hsu CC, Troeger JS, Huebener P, Mu X, Dapito DH, Pradere JP, Schwabe RF (2013)
Fate tracing reveals hepatic stellate cells as dominant contributors to liver fibrosis independent
of its aetiology. Nat Commun 4:2823. https://doi.org/10.1038/ncomms3823. Epub 2013/11/23.
PubMed PMID: 24264436; PMCID: PMC4059406
3 Pericytes in the Lung 57

Murfee WL, Skalak TC, Peirce SM (2005) Differential arterial/venous expression of NG2 pro-
teoglycan in perivascular cells along microvessels: identifying a venule-specific phenotype.
Microcirculation 12(2):151–160. https://doi.org/10.1080/10739680590904955. PubMed
PMID: 15824037
Murfee WL, Rehorn MR, Peirce SM, Skalak TC (2006) Perivascular cells along venules upreg-
ulate NG2 expression during microvascular remodeling. Microcirculation 13(3):261–273.
https://doi.org/10.1080/10739680600559153. PubMed PMID: 16627368
Nehls V, Drenckhahn D (1991) Heterogeneity of microvascular pericytes for smooth muscle type
alpha-actin. J Cell Biol 113(1):147–154. PubMed PMID: 2007619; PMCID: PMC2288926
Ogura S, Kurata K, Hattori Y, Takase H, Ishiguro-Oonuma T, Hwang Y, Ahn S, Park I, Ikeda W,
Kusuhara S, Fukushima Y, Nara H, Sakai H, Fujiwara T, Matsushita J, Ema M, Hirashima M,
Minami T, Shibuya M, Takakura N, Kim P, Miyata T, Ogura Y, Uemura A (2017) Sustained
inflammation after pericyte depletion induces irreversible blood-retina barrier breakdown. JCI
Insight 2(3):e90905. https://doi.org/10.1172/jci.insight.90905. Epub 2017/02/15. PubMed
PMID: 28194443; PMCID: PMC5291729 employees of Astellas Pharma Inc.
Patel MS, Taylor GP, Bharya S, Al-Sanna'a N, Adatia I, Chitayat D, Lewis MES, Human DG
(2004) Abnormal pericyte recruitment as a cause for pulmonary hypertension in Adams–Oliver
syndrome. Am J Med Genet A 129A(3):294–299. https://doi.org/10.1002/ajmg.a.30221
Peng T, Tian Y, Boogerd CJ, Lu MM, Kadzik RS, Stewart KM, Evans SM, Morrisey EE (2013)
Coordination of heart and lung co-development by a multipotent cardiopulmonary progenitor.
Nature 500(7464):589–592. https://doi.org/10.1038/nature12358. PubMed PMID: 23873040;
PMCID: PMC3758448
Que J, Wilm B, Hasegawa H, Wang F, Bader D, Hogan BL (2008) Mesothelium contributes to
vascular smooth muscle and mesenchyme during lung development. Proc Natl Acad Sci U S A
105(43):16626–16630. https://doi.org/10.1073/pnas.0808649105. PubMed PMID: 18922767;
PMCID: PMC2567908
Ricard N, Tu L, Le Hiress M, Huertas A, Phan C, Thuillet R, Sattler C, Fadel E, Seferian A,
Montani D, Dorfmuller P, Humbert M, Guignabert C (2014) Increased pericyte coverage medi-
ated by endothelial-derived fibroblast growth factor-2 and interleukin-6 is a source of smooth
muscle-like cells in pulmonary hypertension. Circulation 129(15):1586–1597. https://doi.
org/10.1161/CIRCULATIONAHA.113.007469. PubMed PMID: 24481949
Rock JR, Barkauskas CE, Cronce MJ, Xue Y, Harris JR, Liang J, Noble PW, Hogan BL (2011)
Multiple stromal populations contribute to pulmonary fibrosis without evidence for epithe-
lial to mesenchymal transition. Proc Natl Acad Sci U S A 108(52):E1475–E1483. https://doi.
org/10.1073/pnas.1117988108. PubMed PMID: 22123957; PMCID: PMC3248478
Rustenhoven J, Jansson D, Smyth LC, Dragunow M (2017) Brain pericytes as mediators of neuroin-
flammation. Trends Pharmacol Sci 38(3):291–304. https://doi.org/10.1016/j.tips.2016.12.001.
Epub 2016/12/27. PubMed PMID: 28017362
Sava P, Ramanathan A, Dobronyi A, Peng X, Sun H, Ledesma-Mendoza A, Herzog EL, Gonzalez
AL (2017) Human pericytes adopt myofibroblast properties in the microenvironment of the IPF
lung. JCI Insight. 2(24). https://doi.org/10.1172/jci.insight.96352. PubMed PMID: 29263297;
PMCID: PMC5752282
Shepro D, Morel NM (1993) Pericyte physiology. FASEB J 7(11):1031–1038. PubMed PMID:
8370472
Sims DE, Westfall JA (1983) Analysis of relationships between pericytes and gas exchange capil-
laries in neonatal and mature bovine lungs. Microvasc Res 25(3):333–342. PubMed PMID:
6855632
Sims-Lucas S, Schaefer C, Bushnell D, Ho J, Logar A, Prochownik E, Gittes G, Bates CM
(2013) Endothelial progenitors exist within the kidney and lung mesenchyme. PLoS One
8(6):e65993. https://doi.org/10.1371/journal.pone.0065993. PubMed PMID: 23823180;
PMCID: PMC3688860
Thalgott J, Lebrin F (2015) Pericytes as targets in hereditary hemorrhagic telangiectasia. Front
Genet 6:37
58 C. F. Hung et al.

Valdez CN, Arboleda-Velasquez JF, Amarnani DS, Kim LA, D'Amore PA (2014) Retinal micro-
angiopathy in a mouse model of inducible mural cell loss. Am J Pathol 184(10):2618–2626.
https://doi.org/10.1016/j.ajpath.2014.06.011. S0002-9440(14)00366-6 [pii]. Epub 2014/08/06.
PubMed PMID: 25092275
von Tell D, Armulik A, Betsholtz C (2006) Pericytes and vascular stability. Exp Cell Res
312(5):623–629. https://doi.org/10.1016/j.yexcr.2005.10.019. PubMed PMID: 16303125
Weibel ER (1974) On pericytes, particularly their existence on lung capillaries. Microvasc Res
8(2):218–235. PubMed PMID: 4140459
Wilson CL, Stephenson SE, Higuero JP, Feghali-Bostwick C, Hung CF, Schnapp LM (2018)
Characterization of human PDGFRβ-positive pericytes from IPF and non-IPF lungs. Am
J Physiol Lung Cell Mol Physiol. https://doi.org/10.1152/ajplung.00289.2018
Yata Y, Scanga A, Gillan A, Yang L, Reif S, Breindl M, Brenner DA, Rippe RA (2003) DNase
I-hypersensitive sites enhance alpha1(I) collagen gene expression in hepatic stellate cells.
Hepatology 37(2):267–276. https://doi.org/10.1053/jhep.2003.50067. PubMed PMID:
12540776
Yuan K, Orcholski ME, Panaroni C, Shuffle EM, Huang NF, Jiang X, Tian W, Vladar EK, Wang
L, Nicolls MR, Wu JY, de Jesus Perez VA (2015) Activation of the Wnt/planar cell polar-
ity pathway is required for pericyte recruitment during pulmonary angiogenesis. Am J Pathol
185(1):69–84. https://doi.org/10.1016/j.ajpath.2014.09.013. PubMed PMID: 25447046;
PMCID: PMC4278244
Chapter 4
Pericytes in Skeletal Muscle

Jyoti Gautam and Yao Yao

Abstract Skeletal muscle regeneration is a highly orchestrated process and

involves the activation of many cellular and molecular pathways. Although satellite
cells (SCs) are the major cell type responsible for muscle regeneration, pericytes
show remarkable myogenic potential and various advantages as cell therapy in mus-
cular disorders. This chapter first introduces the structure, marker expression, origin,
and category of pericytes. Next, we discuss their functions in muscular dystrophy
and/or muscle injuries, focusing on their myogenic, adipogenic, fibrogenic,
chondrogenic, and osteogenic activities. Understanding this knowledge will pro-
mote the development of innovative cell therapies for muscle disorders, including
muscular dystrophy.

Keywords Pericytes · Satellite cells · Mesoangioblasts · Myogenesis ·

Adipogenesis · Fibrosis · Chondrogenesis · Osteogenesis · Ossification · Muscle
injury · Muscular dystrophy · Muscle regeneration · Differentiation

4.1 Introduction

Skeletal muscle, which constitutes around 40% of body weight in humans (Yin et al.
2013), is composed of a heterogeneous collection of muscle fibers. During develop-
ment, myofibers are formed via fusion of mesoderm progenitors called myoblasts
(Yablonka-Reuveni 2011). In neonatal/juvenile stages, the number of myofibers
remains constant, but each myofiber grows in size through fusion with SCs and
postnatal muscle stem/progenitor cells (Yin et al. 2013; Yablonka-Reuveni 2011).

J. Gautam • Y. Yao (*)

Department of Pharmaceutical and Biomedical Sciences, University of Georgia,
Athens, GA, USA
e-mail: [email protected]

© Springer Nature Switzerland AG 2019 59

A. Birbrair (ed.), Pericyte Biology in Different Organs,
Advances in Experimental Medicine and Biology 1122,
https://doi.org/10.1007/978-3-030-11093-2_4
60 J. Gautam and Y. Yao

Fig. 4.1 Anatomical locations of pericytes and satellite cells in skeletal muscle. Pericytes are
associated with capillaries in skeletal muscle, while satellite cells are located between the plasma
membrane of myofiber (sarcolemma) and the basal lamina

SCs are located between the plasma membrane of myofiber (sarcolemma) and the
basal lamina (Mauro 1961; Muir et al. 1965) (Fig. 4.1). These cells normally remain
in the quiescent state but become activated in response to injury. Activated SCs
­proliferate and differentiate along the myogenic pathway to repair and/or replace
damaged myofibers (Yin et al. 2013; Relaix and Zammit 2012; Dumont et al. 2015;
Verdijk et al. 2014). As the primary myogenic cells in skeletal muscle, SCs have
been tested for their therapeutic potential in a variety of muscular disorders, includ-
ing muscular dystrophy. Although SCs are able to undergo myogenic differentiation
and promote muscle regeneration (Collins et al. 2005; Montarras et al. 2005; Sacco
et al. 2008; Boldrin et al. 2009), a few disadvantages limit their potential clinical
use. First, cultured SC-derived myoblasts gradually lose their myogenic activity/
regenerative property. Compared to freshly isolated SCs, cultured SCs induce
­regeneration with much lower efficiency after transplantation (Montarras et al.
2005; Gilbert et al. 2010; Hashimoto et al. 2004). Second, SCs have a low survival
rate in vivo. It has been shown that less than 1% of SC-derived myoblasts survive
the first day after transplantation (Beauchamp et al. 1999; Fan et al. 1996).
Third, SCs are unable to cross the endothelial lining of blood vessels if given
­intravenously, which makes them only suitable for intramuscular delivery. Due to
these drawbacks, alternative myogenic cells in skeletal muscle have been
investigated.
In addition to SCs, many other cell populations in skeletal muscle have myo-
genic potential. These cells include pericytes, pericyte-like mesoangioblasts,
muscle-­derived stem cells, side population cells, and CD133+ stem cells (Ten Broek
et al. 2010; Asakura et al. 2002; Torrente et al. 2004; Galvez et al. 2006; Peault et al.
2007; Negroni et al. 2009; Dellavalle et al. 2007). Among them, we mainly focus on
pericytes and pericyte-like mesoangioblasts owing to their unique features and
4 Pericytes in Skeletal Muscle 61

promising therapeutic potential. Similar to SCs, pericytes demonstrate strong myo-

genic capability in vitro (Birbrair et al. 2013a, 2013b) and in vivo after muscle
injury and/or during muscular dystrophy (Dellavalle et al. 2007, 2011; Birbrair et al.
2013b; Kostallari et al. 2015; Yao et al. 2016). Ablation of muscle pericytes has been
shown to result in both myofiber hypotrophy and impaired establishment of stem
cell quiescence (Kostallari et al. 2015). Unlike SCs, pericytes maintain high myo-
genic activity after in vitro amplification (Dellavalle et al. 2007; Costamagna et al.
2016; Quattrocelli et al. 2014; Bonfanti et al. 2015). In addition, pericytes can be
delivered systemically. It has been shown that pericytes colonize host skeletal mus-
cle and generate dystrophin-positive muscle fibers after intra-arterial injection into
dystrophin-null mdx mice, a mouse model of Duchenne muscular dystrophy (DMD)
(Dellavalle et al. 2007). Additionally, injected pericytes can also replenish the SC
pool in dystrophic mice (Dellavalle et al. 2011). Furthermore, transplanted pericytes
are incorporated into the vasculature and muscle and promote their regeneration in
a limb ischemia model (Dar et al. 2012). These results suggest a great therapeutic
potential of pericytes in muscular dystrophy and other muscle disorders. Here in
this chapter, we first introduce pericyte physiology (structure, marker, origin,
and category), followed by their functions (myogenesis, adipogenesis, fibrosis,
chondrogenesis, osteogenesis, and others) in skeletal muscle.

4.2 Pericyte Physiology

4.2.1 Pericyte Structure and Density

Unlike SCs, pericytes are associated with capillaries in skeletal muscle (Cappellari
and Cossu 2013; Attwell et al. 2016) (Fig. 4.1). At ultrastructural level, pericytes
make numerous direct contacts with endothelial cells (Armulik et al. 2005, 2011;
Sims 1986). It should be noted that the basement membrane is often absent at places
where pericytes and endothelial cells are in close juxtaposition (Tilton et al. 1979).
Pericyte density varies greatly in different tissues/organs. The ratios of pericyte
to endothelium have been estimated to be as high as 1:1 in the retina and CNS and
as low as 1:100 in the capillary bed of skeletal muscle in the upper extremities
(Armulik et al. 2005; Bodnar et al. 2016; Shepro and Morel 1993; Daneman and
Prat 2015; von Tell et al. 2006). Quantitative analysis in rat and human skeletal
muscle shows around 21–24% pericyte coverage/distribution around the capillaries
(Sims 1986; Sims et al. 1994). The difference in pericyte coverage and density
implies that pericytes may exert distinct functions in different tissues/organs. In
skeletal muscle, pericytes have been shown to contribute to muscle development/
homeostasis and muscle regeneration after injury.
62 J. Gautam and Y. Yao

4.2.2 Pericyte Markers

Many cellular markers have been used to identify pericytes. The most commonly
used ones include neural-glial antigen 2 (NG2), platelet-derived growth factor
receptor β (PDGFRβ), smooth muscle α-actin (α-SMA), desmin, CD13, regulator
of G protein signaling 5 (RGS5), CD146, and nestin (Armulik et al. 2011; Birbrair
et al. 2011; Kumar et al. 2017). In addition, alkaline phosphatase (AP) has also been
reported as a pericyte marker in skeletal muscle (Dellavalle et al. 2007; Crisan et al.
2008). It should be noted, however, that none of these markers are pericyte-specific.
They either also label other cells or only mark a subpopulation of pericytes. For
example, NG2 is also expressed in macrophages and oligodendrocyte progenitor
cells (Polito and Reynolds 2005; Moransard et al. 2011). PDGFRβ, α-SMA, and
CD13 also label smooth muscle cells (Kumar et al. 2017; Hellstrom et al. 1999; Jin
et al. 2008; Owens et al. 2004; Wang et al. 2015; Chen et al. 2016). Similarly, RGS5
expression is also found in smooth muscle cells and tumor-derived endothelial cells
(Crisan et al. 2008; Silini et al. 2012), and CD146 is detected in endothelial cells
(Chen et al. 2017). Nestin marks neural stem/progenitor cells and cancer stem cells,
in addition to a subpopulation of pericytes (Birbrair et al. 2013a, 2011, 2014a;
Neradil and Veselska 2015; Suzuki et al. 2010). Pericyte-specific markers are
urgently needed and will significantly advance this field.

4.2.3 Pericyte Origins

Numerous lineage-tracing experiments show that pericytes have different embry-

onic origins in different organs (Armulik et al. 2011; Majesky 2007; Majesky et al.
2011). For example, pericytes in the CNS and thymus are derived from neural crest
(Foster et al. 2008; Muller et al. 2008; Bergwerff et al. 1998; Etchevers et al. 2001;
Korn et al. 2002), whereas pericytes in the gut, lung, and liver are derived from
mesothelium (Wilm et al. 2005; Que et al. 2008; Asahina et al. 2011). Pericytes in
the aorta have multiple developmental origins, including neural crest, somite, and
secondary heart field (Armulik et al. 2011; Majesky 2007; Majesky et al. 2011). A
recent study demonstrates that mesodermal mural cells originate from a clonal pre-
cursor mesenchymoangioblast (MB) (Kumar et al. 2017). These MBs differentiate
into primitive PDGFRβ+CD271+CD73− mesenchymal progenitors, which give rise
to proliferative pericytes, smooth muscle cells, and mesenchymal stem/stromal cells
(Kumar et al. 2017). MB-derived PCs can be further specified to CD274+ capillary
and DLK1+ arteriolar PCs with a proinflammatory and contractile phenotype,
respectively (Kumar et al. 2017). Furthermore, constitutive activation of Notch-1
has been shown to be critical for the conversion of mesoderm-derived somite cells
and neural crest-derived frontonasal mesenchyme to a perivascular cell fate (Miller
et al. 2017). The developmental origin of pericytes in skeletal muscle, however,
remains not fully clear.
4 Pericytes in Skeletal Muscle 63

4.2.4 Pericyte Subtypes

As a heterogeneous population, various subtypes of pericytes have been identified.

These subpopulations have been speculated to exert different functions. In earlier
studies, pericytes have been categorized into three subtypes: precapillary, midcapil-
lary, and postcapillary pericytes, based on their location and morphology (Krueger
and Bechmann 2010; Nehls and Drenckhahn 1991). Precapillary pericytes have sev-
eral circular branches, which tend to wrap around blood vessels. Midcapillary peri-
cytes are spindle-shaped highly elongated cells, which extend mainly in the long
axis of the vessels and have many short secondary processes. Postcapillary pericytes
are shorter stellate-shaped cells that cover the abluminal surface of postcapillaries
and postcapillary venules (Nehls and Drenckhahn 1991). These pericytes differ in
their expression of α-SMA. Midcapillary pericytes show lack of α-SMA, while pre-
and postcapillary pericytes are α-SMA+ (Nehls and Drenckhahn 1991).
In addition, pericytes are also categorized into different subtypes according to
their expression of cellular markers. For example, two subtypes of pericytes, named
type 1 and type 2, have been described in skeletal muscle (Birbrair et al. 2013a;
Majesky 2007). In the Nestin-GFP/NG2-DsRed double transgenic line, type-1 and
type-2 pericytes are defined as Nestin-GFP−/NG2-DsRed+ and Nestin-GFP+/NG2-­
DsRed+ cells, respectively (Birbrair et al. 2013a, 2013b, 2014a, 2013c). It has been
shown that type-1 pericytes generate α-SMA+ classical pericytes, contributing to fat
accumulation and fibrosis in skeletal muscle and other tissues (Birbrair et al. 2013a,
2013b, 2014a, 2013c). Type-2 pericytes, on the other hand, can give rise to either
Tuj1+ cells or α-SMA+ classical pericytes and participate in muscle regeneration and
normal angiogenesis (Birbrair et al. 2013a, 2013b, 2014b). Both type-1 and type-2
pericytes also express PDGFRβ and CD146 and are associated with capillaries
(Birbrair et al. 2013a, 2013b).

4.3  ericytes in Skeletal Muscle Injury and Muscular

P
Dystrophy

4.3.1 Myogenesis

Skeletal muscle has the capability to regenerate itself in response to injury and/or
degenerative diseases, such as muscular dystrophy. SCs, postnatal muscle progeni-
tor cells, play an important role in this process. In addition, there is evidence show-
ing that pericytes and pericyte-like cells also contribute to muscle regeneration/
repair (Fig. 4.2). For example, like SCs, pericytes can express myogenic markers
upon in vitro differentiation, although with different expression patterns and dynam-
ics (Dellavalle et al. 2007). When transplanted into immunodeficient mdx mice,
pericytes undergo myogenesis in vivo, increase dystrophin-positive fibers, and ame-
liorate muscle injury (Dellavalle et al. 2007). Next, it has been demonstrated that
64 J. Gautam and Y. Yao

Fig. 4.2 Differentiation plasticity of skeletal muscle pericytes. In vitro and/or in vivo studies show
that pericytes are able to differentiate into myoblasts, adipocytes, myofibroblasts, chondrocytes,
osteoblasts, and possibly Schwann cells

AP+ pericytes can generate SCs in early postnatal period and contribute to myogen-
esis in both cardiotoxin-induced acute muscle injury and chronic muscular dystro-
phy (Dellavalle et al. 2011). Consistent with these reports, we have demonstrated
that skeletal muscle PDGFRβ+ pericytes can differentiate along either myogenic or
adipogenic pathway depending on the availability of laminin signaling (Fig. 4.2),
and that their myogenic differentiation contributes to muscle development and
regeneration (Yao et al. 2016; Gautam et al. 2017). Further studies show that it is
type-2 but not type-1 pericytes that have myogenic potential (Birbrair et al. 2013a,
2013b; Gautam et al. 2017). It has been reported that transplanted type-2 rather than
type-1 pericytes undergo myogenesis, contributing to muscle regeneration in BaCl2-­
induced muscle injury model (Birbrair et al. 2013b). In addition, intraperitoneal
injection of human adipose tissue-derived pericytes significantly increased the life
span of dystrophin/utrophin double knockout (mdx/utrn−/−) mice (Valadares et al.
2014), another DMD model (Grady et al. 1997; Deconinck et al. 1997), suggesting
a beneficial role of pericytes in DMD. It should be noted, however, that this benefi-
cial effect is due to immune modulation rather than muscle regeneration.
Mesoangioblasts, multipotent cells that reside in the dorsal aorta of the develop-
ing embryo, are pericyte-like cells (Pierantozzi et al. 2016; Vezzani et al. 2016).
Accumulating evidence shows that mesoangioblasts can improve muscle regenera-
tion in various types of muscular dystrophy in mice, dogs, and humans (Galvez
et al. 2006; Berry et al. 2007; Sampaolesi et al. 2003, 2006; Cossu et al. 2015). First,
intra-arterial delivery of mesoangioblasts is able to reverse the dystrophic p­ henotype
4 Pericytes in Skeletal Muscle 65

both morphologically and functionally in α-sarcoglycan-null mice, a limb-­girdle

dystrophy model (Galvez et al. 2006; Sampaolesi et al. 2003). Next, intra-arterial
delivery of wild-type canine mesoangioblasts substantially enhances dystrophin
expression and improves muscle morphology and function in dogs with golden
retriever muscular dystrophy, which mimics the DMD in humans (Sampaolesi et al.
2006). Similar beneficial effects are observed in mdx/utrn−/− mice after administration
of aorta-derived mesoangioblasts (Berry et al. 2007). Furthermore, the therapeutic
effect of intra-arterial transplantation of mesoangioblasts in DMD has been tested in
humans and found to be relatively safe and effective (Cossu et al. 2015).

4.3.2 Adipogenesis

Intramuscular adipose tissue (IMAT) deposition is the accumulation of adipocytes

between muscle cells and beneath the muscle fascia (Pagano et al. 2015; Vettor et al.
2009). It is often seen after skeletal muscle injury or in muscular dystrophy
(Lukjanenko et al. 2013) and has been shown to negatively affect skeletal muscle
regeneration. Fibro/adipogenic progenitors (FAPs), muscle-resident mesenchymal
stem cells that express platelet-derived growth factor receptor alpha (PDGFRα), are
a major source of adipocytes in skeletal muscle (Uezumi et al. 2010; Motohashi
et al. 2008). It has been reported that FAPs proliferate and differentiate into adipo-
cytes in a DMD model, contributing to the accumulation of IMAT (Vettor et al.
2009; Takegahara et al. 2014), which correlates with a decrease in SC number
(Heslop et al. 2000; Blau et al. 1983).
In addition to FAPs, pericytes also contribute to adipogenesis and fat accumula-
tion in skeletal muscle (Fig. 4.2). For example, pericytes isolated from various tis-
sues, including skeletal muscle, are able to undergo adipogenic differentiation
in vitro (Crisan et al. 2008). Using lineage-tracing technique, we have demonstrated
that PDGFRβ+ pericytes are able to differentiate into perilipin+ adipocytes in skele-
tal muscle in vivo in a congenital muscular dystrophy model (Yao et al. 2016).
Further studies show that type-1 but not type-2 pericytes undergo adipogenesis,
contributing to IMAT deposition and skeletal muscle fatty degeneration (Birbrair
et al. 2013a, 2013b; Gautam et al. 2017).

4.3.3 Fibrosis

Fibrosis is a pathological condition characterized by excessive deposition of extra-

cellular matrix and formation of fibrous connective tissue in an organ (Wynn 2008).
It is observed in both pathological conditions and normal aging and generally
referred to as “scarring” when induced in response to injury. Under chronic disease
conditions, such as DMD, progressive fibrosis leads to skeletal muscle dysfunction
66 J. Gautam and Y. Yao

(Morales et al. 2013; Mann et al. 2011; Acuna et al. 2014). During normal aging,
increased infiltration of fibrous tissue results in skeletal muscle stiffness, weakness,
and atrophy (Ryall et al. 2008; Thompson 2009; Kragstrup et al. 2011; Walston
2012). Moreover, formation of fibrous tissue also affects the regeneration potential
of skeletal muscle due to diminished innervation and contractile properties (Juhas
and Bursac 2013; Birbrair et al. 2014c). The major cell type responsible for fibrosis
is myofibroblasts, which are derived from a variety of cell types (Wynn 2008; Willis
et al. 2006; Quan et al. 2006; Zeisberg et al. 2007; Duffield et al. 2013; Lin et al.
2008; Humphreys et al. 2010).
Recent studies suggest that pericytes may serve as a source of myofibroblasts
during skeletal muscle fibrosis (Fig. 4.2). For example, ADAM12 (disintegrin and
metalloproteinase domain-containing protein 12), which labels PDGFRβ+/NG2+
pericytes, has been shown to participate in the formation of fibrotic scar in skeletal
muscle after acute injury (Dulauroy et al. 2012), suggesting that ADAM12 identifies
a myofibroblast progenitor lineage with pericyte features. Additionally, type-1 but
not type-2 pericytes are fibrogenic in vitro and in vivo and contribute to fibrous tis-
sue deposition in skeletal muscle and many other organs (Birbrair et al. 2014a,
2013c). Furthermore, GLAST+ pericytes have been found to participate in the for-
mation of scar tissue in a spinal cord injury model, although GLAST does not label
pericytes specifically (Goritz et al. 2011).

4.3.4 Chondrogenesis and Osteogenesis/Ossification

Heterotopic ossification is the formation of lamellar bone in non-osseous tissues,

such as skeletal muscle, after traumatic injury or in pathological conditions. The
general process of ossification involves an initial formation of ectopic cartilage and
a subsequent formation of endochondral bone (Shimono et al. 2013). Skeletal mus-
cle ossification has been observed following muscle injury due to aberrant activa-
tion of the chondrogenic and osteogenic pathways (Shimono et al. 2013; Bosse
et al. 1994). There is evidence showing that pericytes have chondrogenic and osteo-
genic potential and contribute to skeletal muscle ossification (Fig. 4.2). First, osteo-
progenitor cells isolated from human skeletal muscles express high levels of pericyte
markers, including AP (Levy et al. 2001). Next, pericytes (CD146+CD34−CD45−C
D56−AP+NG2+PDGFRβ+ cells) isolated from human adult skeletal muscle show
chondrogenic and osteogenic potential in vitro (Crisan et al. 2008). Like skeletal
muscle pericytes, pericytes from retina also have chondrogenic potential in vitro
(Farrington-Rock et al. 2004). Consistent with these reports, various studies have
demonstrated that pericytes can undergo chondrogenic and osteogenic differentia-
tion in vitro (Birbrair et al. 2013b, 2014c; Zhang et al. 2011; James et al. 2012).
Whether pericytes contribute to skeletal muscle ossification in vivo, however,
remains unknown.
Ossification of fibrous tissue, including skeletal muscle, is observed in fibrodys-
plasia ossificans progressiva (FOP) (Ramirez et al. 2014; Shore and Kaplan 2010),
4 Pericytes in Skeletal Muscle 67

a genetic disorder caused by an autosomal dominant mutation of BMP receptor—

activin-like kinase 2 (ALK2) (Shore et al. 2006). These findings suggest that ALK2
ligands (e.g., BMPs) may be primary inducers of heterotopic ossification. Consistent
with this speculation, BMP-2, BMP-4, and BMP-9 are highly expressed in human
lesions with heterotopic ossification (Gannon et al. 1997; Grenier et al. 2013) and
promote osteogenesis when mixed with Matrigel and injected into skeletal muscle
(Shimono et al. 2013; Chen et al. 2012; Nishimura et al. 2012). In addition, we have
demonstrated that type-1 pericytes express both BMP-2 and BMP-4 in vitro
(Gautam et al. 2017), again suggesting a possible role of BMP signaling in pericyte
osteogenic differentiation.

4.3.5 Nerve Injury

Motor nerves innervate skeletal muscle (Carlson 1981). It has been shown that
nerve innervation is essential for the mass and function of skeletal muscle (Delbono
2003, 2011). Upon denervation, which usually results from aging, trauma, or immo-
bility, skeletal muscle undergoes reinnervation. Although the molecular mechanism
underlying reinnervation remains largely unclear, Schwann cells, which cover nerve
terminal branches with their processes, have been shown to play a critical role in
this process. It has been shown that Schwann cells from denervated synaptic sites
form a “bridge” to guide the growth of nerve terminal sprouts (Love and Thompson
1999; Sugiura and Lin 2011). There is evidence supporting that pericytes may con-
tribute to skeletal muscle reinnervation via differentiating into myelinating Schwann
cells (Fig. 4.2). For example, under optimized skeletal muscle culture conditions,
type-2 pericytes are able to differentiate into oligodendrocyte progenitor cells
(Birbrair et al. 2013a), which generate mature oligodendrocytes and Schwann cells
(Zawadzka et al. 2010).

4.4 Conclusions

Pericytes are an alternative stem/progenitor population with myogenic potential in

skeletal muscle. Recent studies show that pericytes actively affect muscle regenera-
tion after injury or in muscular dystrophy through their myogenic and non-­myogenic
activities. It is hypothesized that enhancing pericyte myogenesis and inhibiting their
non-myogenic differentiation have a therapeutic potential in muscle disorders.
Future studies should focus on addressing the following key questions on pericytes.
First, what determines the fate of pericytes in their differentiation (myogenesis,
adipogenesis, fibrosis, osteogenesis, and others)? Second, what are the key signal-
ing pathways that regulate pericyte differentiation and fate determination? Third,
what is the difference between pericytes and other stem/progenitor cells in term of
myogenic differentiation? Fourth, what are the molecular markers specific for
68 J. Gautam and Y. Yao

pericytes and/or subtypes of pericytes? Fifth, do different subtypes of pericytes have

distinct differentiation capability? Sixth, how are different pericyte subpopulations
distributed in the vasculature in skeletal muscle? Answers to these questions will
significantly enrich our knowledge in pericyte biology and promote the develop-
ment of effective therapies for various muscle disorders, including muscular
dystrophy.

References

Acuna MJ et al (2014) Restoration of muscle strength in dystrophic muscle by angiotensin-1-7

through inhibition of TGF-beta signalling. Hum Mol Genet 23(5):1237–1249
Armulik A, Abramsson A, Betsholtz C (2005) Endothelial/pericyte interactions. Circ Res
97(6):512–523
Armulik A, Genove G, Betsholtz C (2011) Pericytes: developmental, physiological, and pathologi-
cal perspectives, problems, and promises. Dev Cell 21(2):193–215
Asahina K et al (2011) Septum transversum-derived mesothelium gives rise to hepatic stellate cells
and perivascular mesenchymal cells in developing mouse liver. Hepatology 53(3):983–995
Asakura A et al (2002) Myogenic specification of side population cells in skeletal muscle. J Cell
Biol 159(1):123–134
Attwell D et al (2016) What is a pericyte? J Cereb Blood Flow Metab 36(2):451–455
Beauchamp JR et al (1999) Dynamics of myoblast transplantation reveal a discrete minority of
precursors with stem cell-like properties as the myogenic source. J Cell Biol 144(6):1113–1122
Bergwerff M et al (1998) Neural crest cell contribution to the developing circulatory system:
implications for vascular morphology? Circ Res 82(2):221–231
Berry SE et al (2007) Multipotential mesoangioblast stem cell therapy in the mdx/utrn−/− mouse
model for Duchenne muscular dystrophy. Regen Med 2(3):275–288
Birbrair A et al (2011) Nestin-GFP transgene reveals neural precursor cells in adult skeletal
muscle. PLoS One 6(2):e16816
Birbrair A et al (2013a) Skeletal muscle pericyte subtypes differ in their differentiation potential.
Stem Cell Res 10(1):67–84
Birbrair A et al (2013b) Role of pericytes in skeletal muscle regeneration and fat accumulation.
Stem Cells Dev 22(16):2298–2314
Birbrair A et al (2013c) Type-1 pericytes participate in fibrous tissue deposition in aged skeletal
muscle. Am J Physiol Cell Physiol 305(11):C1098–C1113
Birbrair A et al (2014a) Type-1 pericytes accumulate after tissue injury and produce collagen in an
organ-dependent manner. Stem Cell Res Ther 5(6):122
Birbrair A et al (2014b) Type-2 pericytes participate in normal and tumoral angiogenesis. Am
J Physiol Cell Physiol 307(1):C25–C38
Birbrair A et al (2014c) Pericytes: multitasking cells in the regeneration of injured, diseased, and
aged skeletal muscle. Front Aging Neurosci 6:245
Blau HM, Webster C, Pavlath GK (1983) Defective myoblasts identified in Duchenne muscular
dystrophy. Proc Natl Acad Sci U S A 80(15):4856–4860
Bodnar RJ et al (2016) Pericytes: a newly recognized player in wound healing. Wound Repair
Regen 24(2):204–214
Boldrin L et al (2009) Mature adult dystrophic mouse muscle environment does not impede effi-
cient engrafted satellite cell regeneration and self-renewal. Stem Cells 27(10):2478–2487
Bonfanti C et al (2015) PW1/Peg3 expression regulates key properties that determine mesoangio-
blast stem cell competence. Nat Commun 6:6364
Bosse A et al (1994) Collagens and growth factors in heterotopic ossification. Pathologe
15(4):216–225
4 Pericytes in Skeletal Muscle 69

Cappellari O, Cossu G (2013) Pericytes in development and pathology of skeletal muscle. Circ
Res 113(3):341–347
Carlson BM (1981) Denervation, reinnervation, and regeneration of skeletal muscle. Otolaryngol
Head Neck Surg 89(2):192–196
Chen G, Deng C, Li YP (2012) TGF-beta and BMP signaling in osteoblast differentiation and bone
formation. Int J Biol Sci 8(2):272–288
Chen L et al (2016) Smooth muscle-alpha actin inhibits vascular smooth muscle cell proliferation
and migration by inhibiting Rac1 activity. PLoS One 11(5):e0155726
Chen J et al (2017) CD146 coordinates brain endothelial cell-pericyte communication for blood-­
brain barrier development. Proc Natl Acad Sci U S A 114(36):E7622–E7631
Collins CA et al (2005) Stem cell function, self-renewal, and behavioral heterogeneity of cells
from the adult muscle satellite cell niche. Cell 122(2):289–301
Cossu G et al (2015) Intra-arterial transplantation of HLA-matched donor mesoangioblasts in
Duchenne muscular dystrophy. EMBO Mol Med 7(12):1513–1528
Costamagna D et al (2016) Smad1/5/8 are myogenic regulators of murine and human mesoangio-
blasts. J Mol Cell Biol 8(1):73–87
Crisan M et al (2008) A perivascular origin for mesenchymal stem cells in multiple human organs.
Cell Stem Cell 3(3):301–313
Daneman R, Prat A (2015) The blood-brain barrier. Cold Spring Harb Perspect Biol 7(1):a020412
Dar A et al (2012) Multipotent vasculogenic pericytes from human pluripotent stem cells promote
recovery of murine ischemic limb. Circulation 125(1):87–99
Deconinck AE et al (1997) Utrophin-dystrophin-deficient mice as a model for Duchenne muscular
dystrophy. Cell 90(4):717–727
Delbono O (2003) Neural control of aging skeletal muscle. Aging Cell 2(1):21–29
Delbono O (2011) Expression and regulation of excitation-contraction coupling proteins in aging
skeletal muscle. Curr Aging Sci 4(3):248–259
Dellavalle A et al (2007) Pericytes of human skeletal muscle are myogenic precursors distinct from
satellite cells. Nat Cell Biol 9(3):255–267
Dellavalle A et al (2011) Pericytes resident in postnatal skeletal muscle differentiate into muscle
fibres and generate satellite cells. Nat Commun 2:499
Duffield JS et al (2013) Host responses in tissue repair and fibrosis. Annu Rev Pathol 8:241–276
Dulauroy S et al (2012) Lineage tracing and genetic ablation of ADAM12(+) perivascular cells
identify a major source of profibrotic cells during acute tissue injury. Nat Med 18(8):1262–1270
Dumont NA et al (2015) Satellite cells and skeletal muscle regeneration. Compr Physiol
5(3):1027–1059
Etchevers HC et al (2001) The cephalic neural crest provides pericytes and smooth muscle cells to
all blood vessels of the face and forebrain. Development 128(7):1059–1068
Fan Y et al (1996) Rapid death of injected myoblasts in myoblast transfer therapy. Muscle Nerve
19(7):853–860
Farrington-Rock C et al (2004) Chondrogenic and adipogenic potential of microvascular pericytes.
Circulation 110(15):2226–2232
Foster K et al (2008) Contribution of neural crest-derived cells in the embryonic and adult thymus.
J Immunol 180(5):3183–3189
Galvez BG et al (2006) Complete repair of dystrophic skeletal muscle by mesoangioblasts with
enhanced migration ability. J Cell Biol 174(2):231–243
Gannon FH et al (1997) Bone morphogenetic protein 2/4 in early fibromatous lesions of fibrodys-
plasia ossificans progressiva. Hum Pathol 28(3):339–343
Gautam J, Nirwane A, Yao Y (2017) Laminin differentially regulates the stemness of type I and
type II pericytes. Stem Cell Res Ther 8(1):28
Gilbert PM et al (2010) Substrate elasticity regulates skeletal muscle stem cell self-renewal in
culture. Science 329(5995):1078–1081
Goritz C et al (2011) A pericyte origin of spinal cord scar tissue. Science 333(6039):238–242
Grady RM et al (1997) Skeletal and cardiac myopathies in mice lacking utrophin and dystrophin:
a model for Duchenne muscular dystrophy. Cell 90(4):729–738
70 J. Gautam and Y. Yao

Grenier G et al (2013) BMP-9 expression in human traumatic heterotopic ossification: a case

report. Skelet Muscle 3(1):29
Hashimoto N et al (2004) Muscle reconstitution by muscle satellite cell descendants with stem
cell-like properties. Development 131(21):5481–5490
Hellstrom M et al (1999) Role of PDGF-B and PDGFR-beta in recruitment of vascular smooth
muscle cells and pericytes during embryonic blood vessel formation in the mouse. Development
126(14):3047–3055
Heslop L, Morgan JE, Partridge TA (2000) Evidence for a myogenic stem cell that is exhausted in
dystrophic muscle. J Cell Sci 113(Pt 12):2299–2308
Humphreys BD et al (2010) Fate tracing reveals the pericyte and not epithelial origin of myofibro-
blasts in kidney fibrosis. Am J Pathol 176(1):85–97
James AW et al (2012) Perivascular stem cells: a prospectively purified mesenchymal stem cell
population for bone tissue engineering. Stem Cells Transl Med 1(6):510–519
Jin S et al (2008) Notch signaling regulates platelet-derived growth factor receptor-beta expression
in vascular smooth muscle cells. Circ Res 102(12):1483–1491
Juhas M, Bursac N (2013) Engineering skeletal muscle repair. Curr Opin Biotechnol 24(5):880–886
Korn J, Christ B, Kurz H (2002) Neuroectodermal origin of brain pericytes and vascular smooth
muscle cells. J Comp Neurol 442(1):78–88
Kostallari E et al (2015) Pericytes in the myovascular niche promote post-natal myofiber growth
and satellite cell quiescence. Development 142(7):1242–1253
Kragstrup TW, Kjaer M, Mackey AL (2011) Structural, biochemical, cellular, and func-
tional changes in skeletal muscle extracellular matrix with aging. Scand J Med Sci Sports
21(6):749–757
Krueger M, Bechmann I (2010) CNS pericytes: concepts, misconceptions, and a way out. Glia
58(1):1–10
Kumar A et al (2017) Specification and diversification of Pericytes and smooth muscle cells from
Mesenchymoangioblasts. Cell Rep 19(9):1902–1916
Levy MM et al (2001) Osteoprogenitor cells of mature human skeletal muscle tissue: an in vitro
study. Bone 29(4):317–322
Lin SL et al (2008) Pericytes and perivascular fibroblasts are the primary source of collagen-­
producing cells in obstructive fibrosis of the kidney. Am J Pathol 173(6):1617–1627
Love FM, Thompson WJ (1999) Glial cells promote muscle reinnervation by responding to
activity-­dependent postsynaptic signals. J Neurosci 19(23):10390–10396
Lukjanenko L et al (2013) Genomic profiling reveals that transient adipogenic activation is a hall-
mark of mouse models of skeletal muscle regeneration. PLoS One 8(8):e71084
Majesky MW (2007) Developmental basis of vascular smooth muscle diversity. Arterioscler
Thromb Vasc Biol 27(6):1248–1258
Majesky MW et al (2011) Vascular smooth muscle progenitor cells: building and repairing blood
vessels. Circ Res 108(3):365–377
Mann CJ et al (2011) Aberrant repair and fibrosis development in skeletal muscle. Skelet Muscle
1(1):21
Mauro A (1961) Satellite cell of skeletal muscle fibers. J Biophys Biochem Cytol 9:493–495
Miller SR, Perera SN, Baker CV (2017) Constitutively active Notch1 converts cranial neural crest-­
derived frontonasal mesenchyme to perivascular cells in vivo. Biol Open 6(3):317–325
Montarras D et al (2005) Direct isolation of satellite cells for skeletal muscle regeneration. Science
309(5743):2064–2067
Morales MG et al (2013) Reducing CTGF/CCN2 slows down mdx muscle dystrophy and improves
cell therapy. Hum Mol Genet 22(24):4938–4951
Moransard M et al (2011) NG2 expressed by macrophages and oligodendrocyte precursor cells is
dispensable in experimental autoimmune encephalomyelitis. Brain 134(Pt 5):1315–1330
Motohashi N et al (2008) Muscle CD31(−) CD45(−) side population cells promote muscle regen-
eration by stimulating proliferation and migration of myoblasts. Am J Pathol 173(3):781–791
Muir AR, Kanji AH, Allbrook D (1965) The structure of the satellite cells in skeletal muscle.
J Anat 99(Pt 3):435–444
4 Pericytes in Skeletal Muscle 71

Muller SM et al (2008) Neural crest origin of perivascular mesenchyme in the adult thymus.
J Immunol 180(8):5344–5351
Negroni E et al (2009) In vivo myogenic potential of human CD133+ muscle-derived stem cells: a
quantitative study. Mol Ther 17(10):1771–1778
Nehls V, Drenckhahn D (1991) Heterogeneity of microvascular pericytes for smooth muscle type
alpha-actin. J Cell Biol 113(1):147–154
Neradil J, Veselska R (2015) Nestin as a marker of cancer stem cells. Cancer Sci 106(7):803–811
Nishimura R et al (2012) Regulation of bone and cartilage development by network between BMP
signalling and transcription factors. J Biochem 151(3):247–254
Owens GK, Kumar MS, Wamhoff BR (2004) Molecular regulation of vascular smooth muscle cell
differentiation in development and disease. Physiol Rev 84(3):767–801
Pagano AF et al (2015) Muscle regeneration with intermuscular adipose tissue (IMAT) accumula-
tion is modulated by mechanical constraints. PLoS One 10(12):e0144230
Peault B et al (2007) Stem and progenitor cells in skeletal muscle development, maintenance, and
therapy. Mol Ther 15(5):867–877
Pierantozzi E et al (2016) Tissue-specific cultured human Pericytes: perivascular cells from smooth
muscle tissue have restricted mesodermal differentiation ability. Stem Cells Dev 25(9):674–686
Polito A, Reynolds R (2005) NG2-expressing cells as oligodendrocyte progenitors in the normal
and demyelinated adult central nervous system. J Anat 207(6):707–716
Quan TE, Cowper SE, Bucala R (2006) The role of circulating fibrocytes in fibrosis. Curr
Rheumatol Rep 8(2):145–150
Quattrocelli M et al (2014) Notch signaling regulates myogenic regenerative capacity of murine
and human mesoangioblasts. Cell Death Dis 5:e1448
Que J et al (2008) Mesothelium contributes to vascular smooth muscle and mesenchyme during
lung development. Proc Natl Acad Sci U S A 105(43):16626–16630
Ramirez DM et al (2014) Molecular and cellular mechanisms of heterotopic ossification. Histol
Histopathol 29(10):1281–1285
Relaix F, Zammit PS (2012) Satellite cells are essential for skeletal muscle regeneration: the cell
on the edge returns centre stage. Development 139(16):2845–2856
Ryall JG, Schertzer JD, Lynch GS (2008) Cellular and molecular mechanisms underlying age-­
related skeletal muscle wasting and weakness. Biogerontology 9(4):213–228
Sacco A et al (2008) Self-renewal and expansion of single transplanted muscle stem cells. Nature
456(7221):502–506
Sampaolesi M et al (2003) Cell therapy of alpha-sarcoglycan null dystrophic mice through intra-­
arterial delivery of mesoangioblasts. Science 301(5632):487–492
Sampaolesi M et al (2006) Mesoangioblast stem cells ameliorate muscle function in dystrophic
dogs. Nature 444(7119):574–579
Shepro D, Morel NM (1993) Pericyte physiology. FASEB J 7(11):1031–1038
Shimono K et al (2013) The pathophysiology of heterotopic ossification: current treatment consid-
erations in dentistry. Jpn Dent Sci Rev 50:1–8
Shore EM, Kaplan FS (2010) Inherited human diseases of heterotopic bone formation. Nat Rev
Rheumatol 6(9):518–527
Shore EM et al (2006) A recurrent mutation in the BMP type I receptor ACVR1 causes inherited
and sporadic fibrodysplasia ossificans progressiva. Nat Genet 38(5):525–527
Silini A et al (2012) Regulator of G-protein signaling 5 (RGS5) protein: a novel marker of cancer
vasculature elicited and sustained by the tumor’s proangiogenic microenvironment. Cell Mol
Life Sci 69(7):1167–1178
Sims DE (1986) The pericyte—a review. Tissue Cell 18(2):153–174
Sims D et al (1994) Heterogeneity of pericyte populations in equine skeletal muscle and dermal
microvessels: a quantitative study. Anat Histol Embryol 23(3):232–238
Sugiura Y, Lin W (2011) Neuron-glia interactions: the roles of Schwann cells in neuromuscular
synapse formation and function. Biosci Rep 31(5):295–302
Suzuki S et al (2010) The neural stem/progenitor cell marker nestin is expressed in proliferative
endothelial cells, but not in mature vasculature. J Histochem Cytochem 58(8):721–730
72 J. Gautam and Y. Yao

Takegahara Y et al (2014) Myotube formation is affected by adipogenic lineage cells in a cell-to-­

cell contact-independent manner. Exp Cell Res 324(1):105–114
Ten Broek RW, Grefte S, Von den Hoff JW (2010) Regulatory factors and cell populations involved
in skeletal muscle regeneration. J Cell Physiol 224(1):7–16
Thompson LV (2009) Age-related muscle dysfunction. Exp Gerontol 44(1–2):106–111
Tilton RG, Kilo C, Williamson JR (1979) Pericyte-endothelial relationships in cardiac and skeletal
muscle capillaries. Microvasc Res 18(3):325–335
Torrente Y et al (2004) Human circulating AC133(+) stem cells restore dystrophin expression and
ameliorate function in dystrophic skeletal muscle. J Clin Invest 114(2):182–195
Uezumi A et al (2010) Mesenchymal progenitors distinct from satellite cells contribute to ectopic
fat cell formation in skeletal muscle. Nat Cell Biol 12(2):143–152
Valadares MC et al (2014) Human adipose tissue derived pericytes increase life span in Utrn
(tm1Ked) Dmd (mdx)/J mice. Stem Cell Rev 10(6):830–840
Verdijk LB et al (2014) Satellite cells in human skeletal muscle; from birth to old age. Age (Dordr)
36(2):545–547
Vettor R et al (2009) The origin of intermuscular adipose tissue and its pathophysiological implica-
tions. Am J Physiol Endocrinol Metab 297(5):E987–E998
Vezzani B, Pierantozzi E, Sorrentino V (2016) Not all pericytes are born equal: pericytes from
human adult tissues present different differentiation properties. Stem Cells Dev 25(20)
von Tell D, Armulik A, Betsholtz C (2006) Pericytes and vascular stability. Exp Cell Res
312(5):623–629
Walston JD (2012) Sarcopenia in older adults. Curr Opin Rheumatol 24(6):623–627
Wang G et al (2015) Origin and differentiation of vascular smooth muscle cells. J Physiol
593(14):3013–3030
Willis BC, du Bois RM, Borok Z (2006) Epithelial origin of myofibroblasts during fibrosis in the
lung. Proc Am Thorac Soc 3(4):377–382
Wilm B et al (2005) The serosal mesothelium is a major source of smooth muscle cells of the gut
vasculature. Development 132(23):5317–5328
Wynn TA (2008) Cellular and molecular mechanisms of fibrosis. J Pathol 214(2):199–210
Yablonka-Reuveni Z (2011) The skeletal muscle satellite cell: still young and fascinating at 50.
J Histochem Cytochem 59(12):1041–1059
Yao Y et al (2016) Laminin regulates PDGFRbeta(+) cell stemness and muscle development. Nat
Commun 7:11415
Yin H, Price F, Rudnicki MA (2013) Satellite cells and the muscle stem cell niche. Physiol Rev
93(1):23–67
Zawadzka M et al (2010) CNS-resident glial progenitor/stem cells produce Schwann cells as well
as oligodendrocytes during repair of CNS demyelination. Cell Stem Cell 6(6):578–590
Zeisberg EM et al (2007) Endothelial-to-mesenchymal transition contributes to cardiac fibrosis.
Nat Med 13(8):952–961
Zhang X et al (2011) The Nell-1 growth factor stimulates bone formation by purified human peri-
vascular cells. Tissue Eng Part A 17(19–20):2497–2509
Chapter 5
Pericytes in the Gut

Marta Ramirez, Nuria Pell, Marc Mejias, and Mercedes Fernandez

Abstract This review chapter describes the current knowledge about the nature of
pericytes in the gut, their interaction with endothelial cells in blood vessels, and
their pathophysiological functions in the setting of chronic liver disease. In particu-
lar, it focuses on the role of these vascular cell types and related molecular signaling
pathways in pathological angiogenesis associated with liver disease and in the
establishment of the gut-vascular barrier and the potential implications in liver
disease through the gut-liver axis.

Keywords Chronic liver disease · Pathological angiogenesis · Gut-vascular

barrier · Portosystemic collateral vessels · Mesenteric vascular bed · Intestinal
circulation

5.1 Introduction

This chapter reviews our current understanding of the role of pericytes and endothelial
cells in the gut vasculature during homeostasis and in disease. Specifically, we high-
light the function and importance of these cell types as key components of the major
vascular beds involved in the pathophysiology of chronic liver disease, which are
leading causes of death and liver transplantation worldwide. These vascular beds

Marta Ramirez and Nuria Pell are Co-first authors.

M. Ramirez · N. Pell
Angiogenesis in Liver Disease Research Group, IDIBAPS Biomedical Research Institute,
Hospital Clinic, University of Barcelona, Barcelona, Spain
M. Mejias · M. Fernandez (*)
Angiogenesis in Liver Disease Research Group, IDIBAPS Biomedical Research Institute,
Hospital Clinic, University of Barcelona, Barcelona, Spain
Biomedical Research Networking Center on Hepatic and Digestive Disease (CIBEREHD),
Spanish National Institute of Health, Barcelona, Spain
e-mail: [email protected]; https://www.mercedesfernandezlab.com

© Springer Nature Switzerland AG 2019 73

A. Birbrair (ed.), Pericyte Biology in Different Organs,
Advances in Experimental Medicine and Biology 1122,
https://doi.org/10.1007/978-3-030-11093-2_5
74 M. Ramirez et al.

include not only the intrahepatic circulation but also the intestinal and mesenteric
microvasculature, as well as the portosystemic collaterals and gastroesophageal
varices. All these vessels are active structures affected by the dynamic interplay of
distinct pathophysiological events and complex signals capable of increasing vaso-
dilation, vascular remodeling, angiogenesis, vasculogenesis, and hemodynamic
pressure stress in this pathological setting. We also describe here our current knowl-
edge on the gut-vascular barrier and the potential implications in liver disease
through the gut-liver axis. Another important focus of this chapter relates with the
use of pericytes and endothelial cells as novel therapeutic targets to improve the
outcome in patients suffering from chronic liver disease. This is of particular signifi-
cance due to the lack of effective treatment options beyond liver transplantation,
which is also not the cure since many patients die on the waiting list due to a short-
age of organ donors.

5.2 Pericytes in Blood Vessels

Blood vessels are composed of two interacting cell types. Endothelial cells form the
inner lining of the vessel wall, while pericytes envelop the surface of the vascular
tube. For many years, pericytes have been thought to exclusively provide structural
support to the vessel tube. However, we now know that these cells are functionally
significant and play important roles in microvascular physiology, capillary blood flow
regulation, and angiogenesis (Hirschi and D’Amore 1996; Bergers and Song 2005).
Pericytes are enveloped in a vascular basement membrane that is continuous
with the endothelial basement membrane. At distinct points in the basement mem-
brane, pericytes and endothelial cells form specialized junctions with each other.
These sites are thought to play an anchoring role (Armulik et al. 2011). The number,
distribution, and structure of pericytes vary among different organs and vascular
beds, suggesting that they may have vascular bed- or tissue-specific roles (Hirschi
and D’Amore 1996). Common pericyte molecular markers include platelet-derived
growth factor receptor beta (PDGFRβ), neuron-glial antigen 2 (NG2), alpha smooth
muscle actin (αSMA), or desmin, but no single entirely pericyte-specific marker is
known given that expression of all of them can change depending on developmental
states or pathological reactions (Armulik et al. 2011; Bergers and Song 2005).

5.2.1 Interaction with Endothelial Cells

Pericytes maintain active communication with the underlying endothelial cells, via
direct contact or paracrine signaling. In addition, each pericyte can contact several
endothelial cells at a time, even from different capillaries, and thus integrate coordi-
nate signals between them (Armulik et al. 2011). Pericytes play an important role in
regulation of endothelial cell proliferation and differentiation, contractility and
tone, and stabilization and permeability.
5 Pericytes in the Gut 75

Several molecular signaling pathways have been described to be involved in the

cross talk between pericytes and endothelial cells. Platelet-derived growth factor-B
(PDGFB), angiopoietins, and transforming growth factor beta (TGFβ) are the best
described factors that paracrinely regulate the interactions between both cell types
and will be further discussed below, but also other signaling pathways are impli-
cated, like heparin-binding epidermal growth factor (HB-EGF), which is expressed
by endothelial cells to recruit mural cells, or Notch and Ephrin signaling, which
have been described to be implicated in mural cell association to the endothelium
(Armulik et al. 2011; Gaengel et al. 2009).

5.2.2 Functions

The location of pericytes in capillaries and their muscle cell characteristics point at
a role of these cells in the regulation of capillary blood flow through the contraction
of the endothelium, but their functions are more diverse, including regulation of
sprouting tubes for new capillary growth or maturation and stability of blood vessels
(Bergers and Song 2005).
Direct in vivo evidence of pericyte contraction is limited since very subtle nar-
rowing of capillaries would be sufficient to reduce blood flow and thus this contrac-
tion may be below the level of resolution of any morphological method. However,
the presence of a contractile machinery in pericytes, which express αSMA, suggests
that they could function in a way similar to vascular smooth muscle cells, being able
to produce vasodilation and vasoconstriction. It has also been found that pericytes
express tropomyosin and muscle myosin, besides cyclic-GMP-dependent protein
kinase, which is involved in the contraction of smooth muscle cells. Receptors for
both cholinergic and adrenergic responses are expressed in pericytes, and also mol-
ecules like angiotensin II and endothelin-1 have been reported to bind to pericytes
and paracrinely regulate capillary vascular tone (Hirschi and D’Amore 1996;
Armulik et al. 2011).
Pericytes also have an important role in angiogenesis and in the maturation of the
newly formed vessels (Bergers and Song 2005; Stapor et al. 2014). In vessel sprout-
ing, endothelial cells are stimulated by vascular endothelial growth factor (VEGF)
and other angiogenic factors to degrade basal membrane, migrate, and proliferate
(Gerhardt et al. 2003; Jakobsson et al. 2010). In this stage, pericytes contribute to
the production of VEGF and to the establishment of VEGF gradients which guide
the sprouting process (Bergers and Song 2005). After the microvessel forms a
lumen, endothelial cells secrete growth factors, such as PDGFB, to attract pericytes,
which express the PDGFRβ receptor (Potente et al. 2011). Pericyte attachment to
the endothelial wall stabilizes the nascent vessel and induces endothelial differen-
tiation and growth arrest once the angiogenic process is completed (Bergers and
Song 2005; Armulik et al. 2011).
The angiopoietin-Tie2 axis has also been described to be important in the peri-
cyte coating of blood vessels. The receptor Tie2 is expressed in the endothelial cells,
76 M. Ramirez et al.

and it induces pericyte recruitment and vessel stabilization, while its ligands,
angiopoietin-­1 and angiopoietin-2, are predominantly expressed in perivascular
cells and endothelial cells, respectively. The former, Ang-1, is mainly an agonistic
ligand of Tie2, so it is believed to promote vessel maturation, while the latter, Ang-2,
is considered a destabilizing factor in blood vessels, and its expression is mainly
associated with sites of vascular remodeling (Bergers and Song 2005; Armulik et al.
2011).
TGFβ has been described as an important factor in stabilization of newly formed
blood vessels. Once pericyte precursors begin the coating of a forming endothelial
tube, TGFβ is activated, leading to inhibition of endothelial cell proliferation and
migration and induction of differentiation of perivascular precursors into pericytes
(Bergers and Song 2005; Armulik et al. 2011).
Summarizing, pericytes are essential components of microvessels, not only giv-
ing structural support to endothelial cells but also contributing to capillary blood
flow and angiogenesis through mechanical and signaling roles. Thus, many studies
have pointed at the benefits of targeting both pericytes and endothelial cells as an
angioregulatory therapy in different pathologies, including chronic liver disease, as
described later on in this chapter (Kelly-Goss et al. 2014; Bergers et al. 2003;
Fernandez et al. 2007).

5.3 Blood Supply of the Intestines

The intestines are the main components of the lower part of the gastrointestinal
tract, and they have the essential function of digestion and absorption of all the
ingested food and water. For that purpose, a sufficient amount of tissue oxygenation
is mandatory. Therefore, the gastrointestinal circulation delivers the required oxy-
gen for the secretory, absorptive, and motor activity of the intestines allowing the
posterior delivery of nutrients and water to all the organs of the body using both the
blood and the lymphatic vasculature. The anatomy and physiology of the intestinal
tissue are complex and so is the blood supply that allows its homeostasis and vital
functions, describing that complexity is the aim of this section.

5.3.1 Macroscopic Anatomical Considerations

The intestines are the last part of the continuous passageway that conforms the
entire gastrointestinal tract, and they are divided into two main sections which are
the small and the large intestine. The small intestine is subdivided into duodenum,
jejunum, and ileum macroscopically but has also a microscopically division as
named previously. The large intestine begins after the ileum and starts with the
cecum and the vermiform appendix and continues with the ascending, transverse,
descending, and sigmoid colon to end with the rectum and the anus (Gray and Lewis
2000) (Fig. 5.1).
5 Pericytes in the Gut 77

Fig. 5.1 Blood supply to the gastrointestinal tract. Intestinal tissues are written in gray, while
vascular components are written in black. Gastrointestinal tract blood supply comes from three
aortic branches called celiac trunk and superior and inferior mesenteric arteries. Each one of those
branches has additional branches that allow blood irrigation of the whole tissue from the duode-
num to the transverse colon. Modified from Gray’s Anatomy: superior mesenteric artery (2015)

Blood supply for the intestines comes from different vessels, but all of them
derive from the aorta which is divided into celiac trunk, superior mesenteric artery,
and inferior mesenteric artery (Kachlik et al. 2010) (Fig. 5.1). The first branch is the
celiac trunk, which subdivides into three branches and provide blood irrigation for
the pylorus of the stomach and the duodenum by the branch named gastroduodenal
artery. Below that ramification and also coming from the aorta, there is the superior
mesenteric artery (Fig. 5.2), which branches into five major ramifications that pro-
vide blood to the small intestine and the proximal large intestine. The distal end of
the duodenum is irrigated by the inferior pancreaticoduodenal artery, the smallest
branch, and then there are the intestinal arteries divided into those providing blood
to the ileum (ileal arteries) and the jejunum (jejunal arteries) (Matheson et al. 2000).
All those interconnected branches are supported by the mesentery, and there are
arches helping the continuous blood flow and preventing interruptions that would
lead toward tissue death. Following those branches, there are the ileocolic artery
feeding the terminal ileum, the cecum, and the appendix and the right colic artery
providing for the ascending colon, and finally, the transverse colon receives blood
from the middle colic artery (Fig. 5.1). The last aortic branch irrigating intestines is
the inferior mesenteric artery which provides blood to the large intestine by the fol-
lowing branches: the left colic artery for the descending colon, the sigmoid artery
for the sigmoid colon, and the superior rectal artery for the rectum. However, the
blood flow oxygenating the anus comes from lower rectal arteries branched from
the ileal arteries (Harper and Chandler 2016). Arterial vessels go into the villus giv-
78 M. Ramirez et al.

Fig. 5.2 Superior and inferior mesenteric arteries. Intestinal tissues are written in gray, while vas-
cular components are written in black. The hand-fan-like structure of the mesentery and the vascu-
lature going through it allows the formation of a very stable and organized mesh. Notice that most
of the intestinal structures are supplied by the superior mesenteric artery ant its branches. The
inferior mesenteric artery irrigates the last segment of the intestines beginning at the descendant
colon. Modified from Anatomy and Physiology web site: http://cnx.org/content/col11496/1.6/
(2013)

ing rise to capillary networks that will drain intro the venous channel. The venous
system draining the whole gastrointestinal tract is almost parallel to the blood sup-
ply system even at the arcade points. The superior and inferior mesenteric veins will
drain into the portal vein sending it to the liver and subsequently back to the heart
(Kvietys 2010).

5.3.2 Microscopic Anatomical Considerations

The intestinal layers are interconnected by its microvasculature. It conforms a

branched network made of arterioles, venules, and capillaries that go all over the
three main tissue layers: mucosa, muscularis, and submucosa (Granger et al. 2015).
The blood supply enters the intestine by the surface of the serosa which is in contact
with the mesentery (structure that gives support to the vasculature in the perito-
neum). The first-order arterioles are found in the submucosa coming from the mus-
cular layer and from them derive the second-order arterioles that will end up with
5 Pericytes in the Gut 79

the third order that will get into the mucosa and go down to the tip of the villi where
they become capillaries and form a mesh-like structure (Thorburn et al. 2018)
(Fig. 5.3). The morphology of the villi microcirculation at the small intestine is
generally in an eccentrical manner having a single arteriole that goes up to the tip
from which capillaries derive making up a network with numerous anastomoses. In
contrast, the microvasculature at the colon is more similar to the stomach in which
branches of arterioles and capillaries pass along the luminal surface of the mucosa
and form a network around the glands being much closer to the epithelium than in
the small intestine (Kvietys 2010). For the draining of the products of the chime and
the collection for the posterior distribution of all the nutrients, there are the venules.
They start at the villi and pierce the submucosa where they will become submucosal
collecting venules and bring all the absorbed products to the portal circulation.
Further details on this microvasculature and its physiological and pathological role
as intestinal vascular barrier will be described later on in this chapter.

5.3.3 Functional Considerations

The blood passing through the intestines account for approximately 20–25% of the
whole cardiac output in a non-fed state, but once the digestion is done and absorp-
tion takes place, the blood flow of each artery is increased up to 200% creating a

Vessel carrying
blood
Villi

Lumen

Muscle Villi
layers

Fig. 5.3 The villi vasculature. (a) Cut of an intestine fragment with the lumen and the tissue layers
visible: muscle, mucosa, and submucosa (inside de villi). Veins and arteries pierce the tissue in a
parallel manner. (b) Villi amplification. The submucosa contains the first-order arterioles that will
become second-order arterioles and subsequently capillaries that will carry on the absorption func-
tion. It also contains glands for mucus and enzyme secretions. Modified from Anatomy and
Physiology web site: http://cnx.org/content/col11496/1.6/ (2016)
80 M. Ramirez et al.

state of hyperemia as all the nutrients pass over the mucosal surface and enter inside
the villi (Matheson et al. 2000). That increase will depend uniquely on the chime
composition and will later decrease when interluminal pressure is raised (Granger
et al. 2015). Depending on the functional importance of the tissue layer, it will
receive more or less blood flow (Sparks 2011). The intestinal mucosa, which is full
of capillaries and is the first layer of interplay between nutrients and vasculature as
previously described, is essential for proper nutrient absorption and thus is the one
receiving more blood input. The submucosa is the one preceding the submucosa and
has the first-order arterioles that will allow the blood supply required at the villi.
Besides, it contains glandular cells that produce serous and mucous secretions and
immature enterocytes which will play a valuable role in nutrient absorption
(Fig. 5.3). Finally, the muscular layers provide the ability to contract and thus allow
the mixing and the movement of the chime, they are pierced by the vasculature com-
ing from the mesentery and the serosa. Those two layers receive the rest of the blood
addressed to the intestine.
There are several metabolic factors that maintain homeostasis of intestinal tissues
such as decreased or increased PO2, pH, osmolarity, or adenosine (Matheson et al.
2000). The gastrointestinal circulation contributes also to the defense against ingested
toxins or noxious agents protecting intestine tissue from excessive tissue damage by
a profound and complex vasoregulation (Granger et al. 2015). The nature of the
blood flow in the gastrointestinal tract makes it essential for the proper perfusion and
function of all the vital organs therefore becoming a compelling subject of study.

5.4 Blood Supply of the Mesentery

The mesentery is a double layer of peritoneum composed of mesothelium, connec-

tive tissue, and adipocytes that surrounds, holds, and gives support to the intestines,
allowing blood and lymphatic vessels and nerves to supply them (Fig. 5.2). It was
previously thought to be a discontinuous collection of discrete structures separately
attached into the posterior wall, but recent research has found the mesentery to be one
contiguous structure, which has led to proposals for its reclassification as an organ.
The mesentery of the small intestine emerges from the back of the abdominal
cavity, the area known as “root region,” which corresponds to the attachment of the
superior mesenteric artery to the aorta (Coffey and O’Leary 2016). The mesentery
spans the entire gastrointestinal tract, being classified into different areas depending
on the intestinal region that it covers. There are six flexures in the gastrointestinal
tract: duodenojejunal, ileocaecal, hepatic, splenic, and those between the descend-
ing and sigmoid colon and the sigmoid and rectum. These flexures are used to
delimitate the different portions of the mesentery: small intestine mesentery; right,
transverse, and left mesocolon; mesosigmoid area; and mesorectum, respectively
(Coffey and O’Leary 2016). The mesentery is compactly folded within the perito-
neal cavity, combining mobile regions, like the small intestinal mesentery, and other
regions flattened against the abdominal wall. This structure prevents intestines col-
5 Pericytes in the Gut 81

lapsing into the pelvis by its attachment points to the posterior abdominal wall,
specifically at the right and left mesocolon, and by its connection with the pelvic
wall at the mesosigmoid regions (Coffey and O’Leary 2016).
As mentioned before, the mesentery is essential for blood supply to the intes-
tines. It holds the superior and inferior mesenteric arteries (SMA and IMA) and
veins (SMV and IMV) that will irrigate and drain the gastrointestinal tract, respec-
tively (Drake et al. 2015) (Fig. 5.2). The SMA and IMA arise from the abdominal
aorta and travel in the mesentery to irrigate the splanchnic organs, including the
mesentery itself. The SMV and IMV, which both run alongside their associated
arteries, drain the blood to the portal vein.
Focusing in the mesenteric microcirculation, the capillaries between arterioles
and venules are responsible for the exchange of O2 and nutrients with the paren-
chyma. Besides, the arterioles are responsible for most of the resistance to blood
flow, so the regulation of blood flow in the mesentery takes place mainly on the
walls of the arterioles through contraction or dilation (Jacobson 1982).
The mesentery is a key piece in chronic liver disease as it is affected by excessive
vasodilatation and pathological angiogenesis (Fig. 5.4), which work together to pro-
mote and perpetuate the increased splanchnic blood flow and portal hypertension, as
described below. However, it is also important in other abdominal and non-­
abdominal pathologies such as colorectal cancer, diverticular disease, cardiovascu-
lar disease, inflammatory bowel disease, obesity, and the metabolic syndrome
(Coffey and O’Leary 2016).

5.5 Vascular Alterations in Chronic Liver Disease

Chronic liver diseases, including cirrhosis of the liver, are leading causes of death
and liver transplantation worldwide. They are accompanied by profound circulatory
disturbances that are not limited to the intrahepatic circulation but involve also the
splanchnic and systemic vascular beds. These hemodynamic disturbances are
responsible for the development of a hyperdynamic circulatory state and portal
hypertension, which are major complications of chronic liver diseases.

5.5.1 Portal Hypertension

The portal hypertensive syndrome is characterized by a pathological increase in

blood pressure in the portal vein. It is initiated by an increase in vascular resistance
to portal blood flow at a presinusoidal (portal vein thrombosis), sinusoidal (cirrhosis
of the liver, chronic hepatitis, alcoholic liver disease, and hepatic schistosomiasis),
or postsinusoidal level (Budd-Chiari syndrome). The dominant cause of portal
hypertension relates to liver cirrhosis, which increases resistance through the hepatic
sinusoids due to distortion of the liver vascular architecture caused by fibrosis,
82 M. Ramirez et al.

Fig. 5.4 The mesenteric vascular bed. Angiogenesis and vasodilation are markedly increased in
the mesenteric vascular bed during portal hypertension and cirrhosis, as demonstrated using immu-
nofluorescence for the endothelial cell marker CD31 and hematoxylin and eosin histological
staining

scarring, and nodule formation, as well as by hepatic sinusoidal cellular alterations,

with imbalance between vasodilators and vasoconstrictors, promoting sinusoidal
constriction (Gupta et al. 1998; Rockey and Chung 1998; Schuppan and Afdhal
2008). Another major driver of portal hypertension locates extrahepatically and is
an increase in blood flow in splanchnic organs, as described below.

5.5.2 Increased Splanchnic Blood Flow

The increase in blood flow in splanchnic organs draining into the portal vein and the
subsequent increase in portal venous inflow represents a significant factor maintain-
ing and worsening portal hypertension (Fig. 5.5). It perpetuates and exacerbates
portal pressure elevation, promotes ascites and spontaneous bacterial peritonitis,
and is associated with the formation of an extensive network of portosystemic col-
lateral vessels (i.e., vascular channels linking portal and systemic venous
circulations).
5 Pericytes in the Gut 83

Cirrhosis

Obstruction to portal flow ( Hepatic resistance)

Variceal bleeding
Hepatic encephalopathy Portosystemic collaterals Portal hypertension
Sepsis
Portal venous inflow
RxF)
Vasodilation
Shunting of
Hypocontractility
vasodilators
Angiogenesis

Splanchnic blood flow

Endotoxin
TNF Low effective blood volume
Endocannabinoid

Cardiomyopathy Cardiac output Plasma volume

Bacterial translocation Systemic hyperdynamic

Hepatopulmonary syndrome circulation

Spontaneous bacterial peritonitis Activation of Na2++H2O-retaining

Ascites and vasoconstrictor systems
Hepatorenal syndrome

Fig. 5.5 Pathophysiology of portal hypertension in chronic liver disease. The portal hypertensive
syndrome is characterized by a pathological increase in blood pressure in the portal vein. In cir-
rhosis, portal hypertension is initiated by an increased hepatic resistance to portal blood flow
caused by the distortion of liver vascular architecture associated with fibrogenesis and angiogen-
esis and by an increased hepatic vascular tone due to intrahepatic vasoconstriction secondary to an
imbalance between decreased endogenous dilators and increased vasoconstrictor stimuli. Portal
hypertension is aggravated by an increased blood flow in splanchnic organs draining into the portal
vein due to enhanced vasodilation and angiogenesis and hypocontractility to endogenous vasocon-
strictors, with subsequent elevation in portal venous inflow. Such increased portal venous inflow is
a significant factor maintaining and worsening portal pressure elevation. Increased splanchnic
blood flow also leads to systemic hyperdynamic circulation. Another characteristic feature of por-
tal hypertension is the formation of portosystemic collateral vessels responsible for life-­threatening
consequences like gastroesophageal variceal bleeding, portosystemic encephalopathy, and sepsis

The development of splanchnic hyperdynamic circulation with increased

splanchnic blood flow and portal venous inflow in chronic liver disease is mainly
due to mesenteric arteriolar vasodilation, decreased vascular responsiveness to
endogenous vasoconstrictors, and neovascularization, including angiogenesis and
vasculogenesis (Fernandez 2015). This feature also highlights the potential clinical
relevance of applying combination therapies acting both on prevention/regression
of new splanchnic vessels by antiangiogenic agents and on modulation of vasomo-
tor dynamics by vasoactive substances.
84 M. Ramirez et al.

5.5.3 Portosystemic Collaterals

The most common and clinically threatening collateral vessels are the gastroesopha-
geal varices, which are fragile and particularly prone to leak blood and even rupture,
causing upper gastrointestinal tract bleeding (Fig. 5.6). This hemorrhage is often
torrential and difficult to staunch, and, despite many advances made in this field, it
continues to be the most dramatic and lethal complication of portal hypertension
(Garcia-Tsao et al. 2007; Garcia-Tsao and Bosch 2010; Sharara and Rockey 2001).
Furthermore, because portosystemic venous shunts bypass the liver, several sub-
stances normally metabolized by the liver, such as drugs, toxins, hormones, and
bacteria, can escape from collaterals to the systemic circulation, leading to other
potentially lethal consequences, such as portosystemic encephalopathy, spontaneous
bacterial peritonitis, or systemic infections (Fernandez 2015; Iwakiri et al. 2014).
The current understanding is that portosystemic collateralization is a highly
complex multifactorial process that involves two different but complementary
mechanisms: the opening, dilation, and remodeling of preexisting collaterals and

Fig. 5.6 Gastroesophageal varices. VEGF-dependent angiogenesis plays a crucial part in the for-
mation of portosystemic collateral vessels and gastroesophageal varices. These varices are fragile
and particularly prone to leak blood and even rupture, causing upper gastrointestinal tract bleeding.
This hemorrhage is often torrential and difficult to staunch, and, despite many advances made in
this field, it continues to be the cause of significant morbidity and mortality in patients. Furthermore,
because portosystemic venous shunts bypass the liver, noxious substances that are normally
metabolized by the liver can escape from collaterals to the central venous system, leading to other
potentially lethal consequences, such as portosystemic encephalopathy, spontaneous bacterial
peritonitis, or systemic infections
5 Pericytes in the Gut 85

also the de novo formation and maturation of new collateral vessels, through active
neoangiogenesis (Reiberger et al. 2009, 2013; Van Steenkiste et al. 2009; Fernandez
et al. 2007). Both mechanisms may be driven by similar environmental variables
and work in a coordinated manner with the final common goal of developing more
abundant and more functional collateral vessels capable of conducting blood effi-
ciently, to accommodate the sustained portal pressure elevation and the greatly
increased portal venous flow. Worthy of note is that advanced portal hypertension is
typically associated with an extensive network of high-flow portosystemic collater-
als that may carry over even 90% of the blood entering the portal system.

5.5.4 Pathological Angiogenesis

As described above, angiogenesis represents a critical and clinically important path-

ological hallmark and driving force in the processes of hyperdynamic splanchnic
circulation and portosystemic collateralization during chronic liver disease
(Fernandez et al. 2007, 2009; Fernandez 2015) (Fig. 5.7). It also promotes the estab-
lishment and maintenance of the abnormal angioarchitecture distinctive of the cir-
rhotic liver, being functionally linked with fibrogenesis and inflammation (Tugues
et al. 2007; Fernandez et al. 2009; Mejias et al. 2009; Fernandez 2015). Accordingly,
growing evidence suggests that therapeutic inhibition of angiogenesis could be a
valuable treatment strategy with multiple beneficial effects, reducing the develop-
ment of new collaterals and ameliorating portal hypertension and liver cirrhosis
(Fernandez et al. 2009; Fernandez 2015).

5.5.4.1 Vascular Endothelial Growth Factor

VEGF is the main proangiogenic growth factor implicated in angiogenesis during

cirrhosis. VEGF and its signaling pathway are switched on very early during the
natural history of chronic liver disease, preceding the increase in splanchnic blood
flow and, therefore, occurring prior to shear stress augmentation (Fernandez et al.
2004, 2007; Abraldes et al. 2006; Calderone et al. 2016). Interestingly, we have
recently demonstrated that this early VEGF overexpression in portal hypertension
and cirrhosis is posttranscriptionally regulated by cytoplasmic polyadenylation ele-
ment binding proteins (CPEB), which are in turn activated by Aurora kinase-A
(Fig. 5.8), as described later on (Calderone et al. 2016).
VEGF signaling pathway promotes an extensive neovascularization in the cir-
rhotic liver and the mesenteric vascular bed during cirrhosis, increasing the splanch-
nic blood flow and contributing to the development of portosystemic collateral
vessels, intrahepatic fibrosis, and inflammation (Fernandez et al. 2009; Fernandez
2015). An important consideration is that VEGF not only is involved in neovessel
formation but also has an active role in hemodynamic processes, inducing sys-
temic, splanchnic, and portocollateral hyperdynamic circulation by stimulation of
86 M. Ramirez et al.

Portal hypertension

VEGF

Angiogenesis in the Formation of Angiogenesis in

mesenteric vascular bed portosystemic collaterals the liver

Oxygen and nutrient Recruitment of Activation of hepatic

supply to hepatocytes inflammatory cells stellate cells

Fibrogenesis

Intrahepatic
Splanchnic blood flow
vascular resistance

Portal hypertension

Fig. 5.7 Pathological angiogenesis. Pathological neovascularization stimulated mainly by VEGF

represents a critical and clinically important hallmark in portal hypertension. Neovessel formation
in the mesentery contributes to increase splanchnic blood flow draining into the portal vein, which,
in turn, augments portal venous inflow. Such increased portal venous inflow perpetuates and exac-
erbates portal pressure elevation during chronic liver disease. Angiogenesis also plays a pivotal
role in the development of an extensive network of portosystemic collateral vessels, which include
gastroesophageal varices. These varices are particularly prone to rupture, causing massive, life-­
threatening gastroesophageal bleeding. Collateral vessels are also responsible for other major con-
sequences of chronic liver disease, including portosystemic encephalopathy and sepsis.
Neovascularization has also been implicated as a crucial player in the establishment and mainte-
nance of the abnormal angioarchitecture distinctive of the cirrhotic liver, being intimately linked to
the progression of fibrogenesis and inflammation and playing a major contribution to the aggrava-
tion of portal hypertension

vasodilating agents such as endothelial nitric oxide synthase (eNOS) (Abraldes

et al. 2006). This hyperdynamic circulation, in turn, enhances vascular shear stress,
further stimulating the production of VEGF and NO and perpetuating and aggravat-
ing the portal hypertensive syndrome.
Several studies have shown that the blockade of VEGF or its receptor (VEGFR2),
using several angiogenesis inhibitors with different modes of action and prophylac-
tic and therapeutic strategies, effectively decreases portal pressure and mesenteric
neoangiogenesis, reduces splanchnic blood flow and portosystemic collateralization,
and attenuates liver fibrosis (Fernandez et al. 2004, 2005, 2007; Mejias et al. 2009;
Gallego et al. 2017) (Figs. 5.9 and 5.10).
5 Pericytes in the Gut 87

P
P
Aurora Aurora
kinase kinase
inactive active

VEGF

P
P
Portal hypertension
CPEB1 CPEB1 Alternative processing

P=FxR
VEGF

Shear stress
CPEB4 Cytoplasmic polyadenylation
Splanchnic blood flow Intrahepatic
Collateral formation resistance
VEGF AAAAAAAAA

Vasodilation Neovascularization Fibrogenesis Translation

Hypocontractility

NO eNOS VEGF

Fig. 5.8 Molecular pathophysiology. Portal hypertension activates Aurora kinase-A, which is a
well-known activator of CPEB1. Then, CPEB1 and CPEB4 sequentially lead to increased VEGF
protein, which induces angiogenesis and also stimulates nitric oxide (NO) production. In the cir-
rhotic liver, angiogenesis is intimately linked to liver fibrogenesis, contributing to increase intrahe-
patic vascular resistance. Extrahepatically, angiogenesis works together with vasodilation and
hypocontractility to increase splanchnic blood flow and portosystemic collateral formation. And
the combination of all these factors perpetuates portal hypertension

5.5.4.2 Platelet-Derived Growth Factor

Besides VEGF, the expression of platelet-derived growth factor (PDGF) and its
cognate receptor PDGFRβ is also markedly upregulated during chronic liver dis-
ease and portal hypertension in mesentery and cirrhotic liver and plays a signifi-
cant role in the excessive neovascularization of these vascular beds. Thus, during
angiogenesis, endothelial cells secrete PDGF and, thereby, paracrinically stimu-
late the recruitment of PDGFRβ-positive pericytes, which, in turn, act as support-
ive vascular smooth muscle cells, intervening in neovessel stabilization and
maturation, as described above. Binding of PDGF to its PDGFRβ is also an essen-
tial step in the conversion of quiescent hepatic stellate cell, a liver-specific peri-
cyte, into myofibroblasts and the ensuing recruitment of these cells to sites of liver
fibrosis.
88 M. Ramirez et al.

Fig. 5.9 Multiple beneficial effects of angiogenesis inhibition. Splanchnic neovasculature gener-
ated by angiogenesis during portal hypertension is functional, has vascular integrity, is perfused,
and contributes to increase splanchnic blood flow. Accordingly, inhibition of angiogenesis using
different strategies translates into marked decrease in splanchnic blood flow, portal pressure, and
portosystemic collateral vessels. These beneficial effects are more efficient when using a combined
antiangiogenic treatment directed against pericytes and endothelial cells. This is due to the fact that
in the formation of a new blood vessel, there is first the production of an angiogenic factor, which
activates endothelial cells; these cells proliferate, migrate, and form an endothelial tube. This tube
is then covered by pericytes that provide stabilization and maturation to the newly formed vessel.
The first steps of this angiogenic process are mainly modulated by VEGF, whereas the stabilization
and maturation of the neovessel are mainly modulated by platelet-derived growth factor (PDGF).
Therefore, simultaneous targeting of the VEGF signaling pathway, that is the endothelial cells, and
the PDGF signaling pathway, that is the pericytes, gives a greater vascular destabilization and a
better vascular regression than the targeting of either alone. This is what we found using a combi-
nation of rapamycin (to inhibit the VEGF pathway) and Gleevec (to inhibit the PDGF pathway) or
a multikinase inhibitor such as sorafenib

5.5.4.3  ombined Antiangiogenic Treatment Directed against Pericytes

C
and Endothelial Cells

As mentioned before, in the process of neovascularization, VEGF plays a predomi-

nant role in the initial stages of formation of new blood vessels, activating the pro-
liferation of endothelial cells and the subsequent formation of an endothelial tubule,
while maturation of the newly formed vessels is mainly modulated by the proangio-
genic growth factor PDGF, which regulates the investiture of the endothelial tubule
5 Pericytes in the Gut 89

Fig. 5.10 Gene therapy with cell-targeted molecule-targeted liposomal siRNAs. We have recently
used siRNAKDR lipoplexes to efficiently and specifically target the kinase insert domain receptor
KDR (also known as VEGF receptor-2) in vascular endothelial cells in vivo after systemic intrave-
nous administration. This therapy markedly ameliorates the development of portosystemic collat-
eral vessels and impairs the pathological angiogenic potential of endothelial cells in a murine
model of portal hypertension

with mural cell and pericyte populations, thereby stabilizing the vascular architec-
ture of the nascent vessel. Based on these considerations, it was hypothesized that
the simultaneous targeting of the VEGF and PDGF signaling pathways, which is the
simultaneous targeting of endothelial cells and pericytes, could provide a greater
vascular destabilization and a better vascular regression than targeting either alone.
Indeed, combined antiangiogenic treatment directed against endothelial cells and
pericytes, using VEGF and PDGF inhibitors simultaneously, provides a synergistic
benefit in reducing circulatory abnormalities in portal hypertension (Fernandez
et al. 2007). This is due to the fact that removal of pericyte coverage by anti-PDGF
molecules leads to exposed endothelial tubes, making endothelial cells much more
susceptible to anti-VEGF treatment (Fig. 5.9). In this regard, concurrent targeting of
VEGFR-2 and PDGFR-β signaling pathways using low doses of the multikinase
inhibitor sorafenib, used in clinical practice for the treatment of hepatocellular car-
cinoma (Llovet and Bruix 2009), significantly reduces portosystemic collateraliza-
tion, hyperdynamic splanchnic circulation, intrahepatic fibrosis, and portal pressure
in experiments in rats with portal hypertension and cirrhosis (Reiberger et al. 2009;
Mejias et al. 2009; Tugues et al. 2007), with potential beneficial effects also in
humans (Pinter et al. 2012). In addition, the continued administration of the VEGF
signaling inhibitor rapamycin plus the PDGF signaling inhibitor Gleevec markedly
decreased the splanchnic neovascularization and the pericyte coverage of neoves-
sels in portal hypertensive rats (Fernandez et al. 2007). This combined treatment
also resulted in a virtually complete reversal of the increased portal pressure (40%
90 M. Ramirez et al.

reduction) and the increased portal venous blood inflow of these animals. This is
important since clinical studies have shown a dramatic reduction of the risk of portal
hypertensive complications and improved survival in patients achieving a decrease
in portal pressure of at least 20% under drug therapy. Notably, the magnitude of the
effects of the combination treatment was superior than the addition of the effects of
either drug alone, suggesting a synergistic regulatory interaction between the VEGF
and PDGF signaling pathways in mediating the maintenance of the vascular and
hemodynamic abnormalities observed in portal hypertensive rats. These findings
further suggest that in the absence of proliferating pericytes (i.e., after PDGF signal-
ing inhibition), the endothelium is more vulnerable to antiangiogenic therapies tar-
geting endothelial cells, such as VEGF signaling blockade.

5.5.4.4 Endogenous Angiogenesis Inhibitors

Several lines of evidence support the critical role of endogenous angioinhibitors in

the pathophysiology of chronic liver disease and the therapeutic benefit of interven-
tions aimed at augmenting the expression of these naturally occurring molecules.
Thus, we have found that the powerful endogenous angiogenesis inhibitor pigment
epithelium-derived factor (PEDF) is unidirectionally upregulated together with
VEGF, in an attempt to counteract the proangiogenic activity of VEGF (Fig. 5.11).
Exogenous PEDF overexpression by adenovirus-mediated gene transfer moves the

Portal hypertension

Proangiogenic Antiangiogenic
VEGF PEDF
VEGF PEDF
Adenoviral PEDF
gene transfer

Mesenteric angiogenesis
Portal pressure
Fibrosis

Fig. 5.11 Antiangiogenic and antifibrogenic activity of pigment epithelium-derived factor.

Pigment epithelium-derived factor (PEDF) is an endogenous angiogenesis inhibitor that is unidi-
rectionally upregulated together with VEGF in portal hypertension in an attempt to counteract the
proangiogenic activity of VEGF. Exogenous overexpression of PEDF moves the balance in favor
to inhibition of angiogenesis, translating into beneficial effects
5 Pericytes in the Gut 91

balance between VEGF and PEDF in favor of inhibition and angiogenesis, translat-
ing into portal pressure decrease and partial correction of excessive angiogenesis in
experimental portal hypertension (Mejias et al. 2015; Vespasiani-Gentilucci and
Rombouts 2015).
Another novel approach for halting chronic liver disease progression could be
disruption of the negative feedback loop between VEGF and vasohibin-1, which is
a protein that is normally present in the organism and has antiangiogenic activity.
This therapeutic strategy is a good way to bring VEGF levels back to normal, but
not below normal, preserving the baseline VEGF levels necessary for homeostasis
of healthy vessels and other angiogenic physiologic events and translating into ame-
lioration of hemodynamic disturbances and reduction of liver fibrosis and portal
pressure in portal hypertension (Coch et al. 2014; Chatterjee 2014) (Fig. 5.12).

5.5.4.5 Cytoplasmic Polyadenylation Element Binding Proteins

We have recently identified a new mechanism of regulation of pathologic VEGF

expression and angiogenesis through sequential and nonredundant functions of two
members of the family of cytoplasmic polyadenylation element binding proteins,

Normal Angiogenesis Negative feedback loop

Portal VEGF VASH

hypertension

Normal VEGF High VEGF

levels levels Ectopic VASH
overexpression

Normalized Regressed
Mesenteric angiogenesis
Portal pressure
Splanchnic blood flow
Portosystemic collateralization
Fibrosis VEGF levels back But not below
to normal normal

Fig. 5.12 Disruption of negative feedback loop between vasohibin-1 and VEGF decreases portal
pressure, angiogenesis, and fibrosis in cirrhotic rats. Vasohibin-1 is an endogenous protein with
antiangiogenic activity implicated in a negative feedback loop with VEGF. Disruption of this nega-
tive feedback loop in portal hypertension brings VEGF levels back to normal, but not below nor-
mal, preserving the physiological VEGF required for vascular homeostasis of healthy vessels and
for physiologic angiogenic processes. And that translates into amelioration of several portal
hypertension-­associated abnormalities
92 M. Ramirez et al.

coding region

pre-mRNA transcript

pre-mRNA with

nucleus

cytoplasm
translation activation

CPEB4: cytoplasmic polyadenylation

Fig. 5.13 Sequential functions of CPEB1 and CPEB4 regulate pathologic expression of VEGF
and angiogenesis in chronic liver disease. CPEB proteins are RNA-binding proteins that bind to
and regulate the expression of a specific group of mRNAs, which have, on their 3’untranslated
regions, some sequences, call CPEs, cytoplasmic polyadenylation elements. One of these mRNAs
is VEGF. The binding of CPEB1 to VEGF mRNA promotes alternative 3’UTR processing of
VEGF mRNA in the nuclei. And subsequently, CPEB4 promotes the cytoplasmic polyadenylation
of VEGF mRNA in the cytoplasm, which activates translation and generation of high levels of
VEGF proteins. Importantly, this regulatory mechanism operates only in pathologic conditions,
when CPEB1 and CPEB4 are overexpressed, as we have seen in portal hypertension

CPEB1 and CPEB4 (Figs. 5.8 and 5.13). Importantly, this regulatory mechanism
operates only in pathologic conditions, when CPEB1 and CPEB4 are overexpressed,
as we have seen in portal hypertension and cirrhosis (Calderone et al. 2016). CPEB
proteins are RNA-binding proteins that bind to and regulate the expression of a
specific group of mRNAs, which have, on their non-coding 3’-untranslated regions
(3’UTR), some sequences named cytoplasmic polyadenylation elements (CPEs)
(Bava et al. 2013; Fernandez-Miranda and Mendez 2012; Pique et al. 2008). We
have found that one of these CPEB-regulated mRNAs is VEGF mRNA. Upon portal
hypertension and cirrhosis induction, there is a rapid upregulation and activation by
autophosphorylation of the serine/threonine kinase Aurora kinase-A in the mesen-
tery and the liver. Activated Aurora kinase-A, in turn, phosphorylates and activates
CPEB1 (Mendez et al. 2000a, b; Sarkissian et al. 2004). Activation of CPEB1 then
promotes alternative nuclear processing within 3’UTRs of VEGF and CPEB4
mRNAs, resulting in deletion of translation repressor elements. The subsequent
overexpression of CPEB4 promotes cytoplasmic polyadenylation of VEGF mRNA,
5 Pericytes in the Gut 93

increasing its translation and generating high levels of VEGF (Calderone et al.
2016). In addition to promoting pathologic angiogenesis and remodeling in splanch-
nic, portocollateral, and intrahepatic circulations (Fernandez et al. 2009; Fernandez
2015), excessive VEGF production also contributes to the splanchnic vasodilation
and hypocontractility that characterizes portal hypertension through augmentation
of NO production (Abraldes et al. 2006). These pathogenic processes synergisti-
cally contribute to increase splanchnic blood flow and portosystemic collateraliza-
tion, sustaining and aggravating portal hypertension.
Importantly, this CPEB-mediated regulatory mechanism is essential for patho-
logic angiogenesis but dispensable for physiologic neovascularization. Thus, tar-
geting CPEBs could lead to safer treatment outcomes by specifically reducing
excessive pathologic VEGF production instead of indiscriminately perturbing both
pathologic and physiologic VEGF synthesis, minimizing potential adverse side
effects. Consistent with this notion, CPEB depletion reduced portosystemic col-
lateralization and mesenteric neovascularization and attenuated the progression of
the portal hypertensive syndrome in portal vein-ligated mice, without affecting the
normal vasculature or physiological angiogenesis (Calderone et al. 2016). It is
worth mentioning that CPEBs could also play a role in pathological angiogenesis
during cancer (Ortiz-Zapater et al. 2011), and we have recently demonstrated an
important role of these RNA-binding proteins in hepatic steatosis as well (Maillo
et al. 2017).

5.5.5 Pathological Postnatal Vasculogenesis

Most research in the neovascularization field has focused on angiogenesis, the for-
mation of neovessels from activation and proliferation of mature endothelial cells in
existing vasculature. However, it is now evident that alternative vascularization
mechanisms may occur postnatally, including vasculogenesis, the de novo forma-
tion of vessels out of vascular stem/progenitor cells, which historically was thought
to occur exclusively during embryological development. Indeed, recent studies
from our group have identified vascular stem/progenitor cells residing dormant in
the vascular wall of postnatal mesenteric vessels under normal conditions but pos-
sessing sphere-forming ability and proliferative potential, producing large numbers
of daughter cells (i.e., proliferative progenitors or transit-amplifying cells) that dif-
ferentiate toward either endothelial cells or smooth muscle cells when activated by
injury stimuli, such as upon portal hypertension and cirrhosis induction (Garcia-­
Pras et al. 2017). Importantly, these cells structurally and functionally contribute to
abnormal neovessel formation (Fig. 5.14), indicating that abnormal neovasculariza-
tion in this pathological setting might conceivably be a heterogeneous process, aris-
ing through a combination of both angiogenesis and vasculogenesis. Mechanistically,
we also found that the RNA-binding protein CPEB4 is an important factor respon-
sible for the proliferative activity of stem/progenitor cell progeny, adding therefore
94 M. Ramirez et al.

Fig. 5.14 Vascular stem cells: new culprits uncovered. Recent studies from our lab highlight the
functional significance of pathologic neovascularization derived from vascular stem/progenitor
cells as an important mechanism of formation of new blood vessels in adults, in the setting of
chronic liver disease, and identify these stem cells as potential new therapeutic targets. Thus, we
have demonstrated the existence in the vascular wall of adult mesenteric blood vessels of a distinc-
tive population of vascular stem/progenitor cells. These cells display some of the most widely
accepted criteria for stem cell recognition, including quiescence and slow-cycling properties, high
proliferative potentiality, capability of growing as cellular spheres in suspension culture, expres-
sion of specific biomarkers of stem cells, and the ability to self-renew and generate daughter cells
in response to liver disease induction. This vascular stem cell progeny is able to differentiate into
endothelial and smooth muscle cell lineages and readily contribute physically and functionally to
neovascularization in vivo during chronic liver disease. Hence, chronic liver disease-associated
abnormal neovascularization might conceivably be a heterogeneous process, arising through a
combination of both neoangiogenesis and neovasculogenesis. Accordingly, therapeutic targeting
of both vascular stem cell-derived neovascularization (vasculogenesis) and new vessel growth
mechanisms that utilize non-stem cell constituents (angiogenesis) may effectively block abnormal
neovessel formation and improve antiangiogenic therapeutics

another facet to the “proangiogenic” activity of CPEB4, namely, regulation of cell

proliferation in vascular stem/progenitor cell descendants, which could coordinately
act with the recently demonstrated VEGF-dependent function (Calderone et al.
2016). These findings may also have direct translational implications. Thus,
therapeutic targeting of both vascular stem cell-derived neovascularization (vascu-
logenesis) and new vessel growth mechanisms that utilize non-stem cell constitu-
ents (angiogenesis) may effectively block abnormal neovessel formation and
improve antiangiogenic therapeutics.
5 Pericytes in the Gut 95

5.6 Gut-Vascular Barrier in Chronic Liver Disease

In the intestine, the epithelial lining separates internal organs from the enteric envi-
ronment loaded with various foreign substances, including microbiota and its meta-
bolic products as well as nutrients and wastes (Garrett et al. 2010) (Fig. 5.3). The
lamina propria lying beneath the enterocytes in the intestinal villi especially that in
the lower part houses a largest pool of macrophages for maintaining mucosal
homeostasis against the gut microbiota and for the constant need of epithelial
renewal (Bain and Mowat 2014). The lamina propria contains microvessels as well
as a central lacteal (lymph vessel) and lymphoid tissue, in addition to immune cells.
The portal venous system ensures that substances absorbed in the intestine transit
first through the liver, where they can be further metabolized and detoxified.

5.6.1 Gut-Liver Axis

It was in the early 1950s when a relationship between liver disease and intestine was
first recognized, after hepatic coma was associated with the absorption of nitroge-
nous metabolites from the intestine (Phillips et al. 1952). Following it, Volta et al.
(1987) described for the first time the so-called gut-liver axis being critical in the
progression of chronic liver disease. From there on, many advances have been done
in clarifying the role of the gut in chronic liver disease.
Both quantitative and qualitative changes in the intestinal microbiota have been
associated with liver disease, even though the causes and/or consequence is still
unknown (Schnabl and Brenner 2014; Usami et al. 2015; Bhat et al. 2016). For
instance, in cirrhotic patients, gut dysbiosis is characterized by an overgrowth of
pathogenic bacteria that would favor an increase of bacterial translocation, systemic
inflammation, and systemic infection characteristic of liver cirrhosis. Furthermore,
it is widely known that the portal venous system is another key component in ensur-
ing that substances absorbed in the intestine transit through the liver. In this regard,
many studies have also supported a role of the endotoxemia from gut-derived bacte-
rial translocation in the hyperdynamic circulation characteristic of cirrhotic patients.
In a study conducted by Guarner et al. in the early 1990s, endotoxemia was corre-
lated with nitrite serum levels of cirrhotic patients (Guarner et al. 1993). After treat-
ment with colistin, cirrhotic patients had both nitrite and endotoxin levels reduced
suggesting a lineal relationship between the intestine and liver. A series of recent
studies have further demonstrated that translocation of bacteria and their products
across the intestinal barrier is a common place in patients with liver disease
(Tsiaoussis et al. 2015).
The mainly growing attention in the last years on the gut-liver axis has been in
the microbiota recirculation through the lymphatics during liver pathologies, focus-
ing on the epithelium barrier and leaving rather unexplored the possible role of
blood circulation in this scenario. The normal intestinal protection system against
96 M. Ramirez et al.

pathogens depends on both non-specific mechanisms and specific immunological

responses (Jankowski et al. 1994; Alverdy 1990). As it has been extensively reported,
gut epithelium acts as the first barrier against the bacterial translocation of gut
microbiota into the lymphatics (Artis 2008; Backhed et al. 2005), together with
luminal and submucosal factors. When normal microbiota from the gut, present in
the mucus layer, suffers alterations both in number and composition, viable bacteria
from the lumen go through the intestinal wall and translocate to extraintestinal sites
promoting immunological responses. Upon liver disease, tight and adherent junc-
tions present in attaching epithelial cells get deregulated, allowing selective migra-
tion of microorganisms and bacterial products. Thus, how bacteria participate or are
excluded from the blood circulation was completely unexplored for many years
even though the location of the intestinal capillaries under the epithelial layer
suggested an important role.

5.6.2 Gut-Vascular Barrier

In the year 2015, Spadoni et al. described for the first time a new anatomical struc-
ture in the murine and human intestines, called the gut-vascular barrier (GVB)
(Spadoni et al. 2015). The GVB plays a critical role in health and disease by limiting
systemic dissemination of microbes and toxins while allowing nutrients to access
the circulation. Therefore, in addition to the epithelial barrier, the GVB presents a
second, independent barrier that regulates the translocation of luminal bacteria and
their ligands, as well as innocuous food antigens.
The GVB shares many key morphological and functional characteristics with the
well-known blood-brain barrier, which separates circulating blood from the brain’s
extracellular fluid. It is composed of closely interacting intestinal vascular endothe-
lial cells, pericytes, and enteroglial cells. Endothelial cells in the GVB develop
elaborate junctional complexes that include tight junction and adherens junction,
which strictly control paracellular trafficking of solutes and fluids. Pericytes associ-
ated with GVB microvasculature seem to be necessary for the stabilization and
maintenance of the vascular barrier (Armulik et al. 2010; Daneman et al. 2010).
Spadoni et al. proposed how bacteria can disrupt the GVB, allowing their pas-
sage into the bloodstream (portal vein) and spread to the liver, spleen, or other
peripheral tissues (Spadoni et al. 2015) (Fig. 5.15). They found that the expression
of the plasmalemma vesicle-associated protein-1 (PV1) was upregulated in blood
capillaries upon infection with bacterial pathogens, such as Salmonella typhimurium.
This correlated with increased barrier permeability, with dissemination of the bac-
terium to the liver and the spleen, and with liver damage. In addition to PV1, the
Wnt/β-catenin signaling pathway seems to play a role in regulating GVB and pre-
venting the translocation of bacteria under homeostatic and pathological conditions
(Liebner et al. 2008; Spadoni et al. 2016). Other pathways found upregulated in a
transcriptome analysis (Spadoni et al. 2016), such as SHH signaling pathways,
could also be altered after pathogen infection.
5 Pericytes in the Gut 97

Fig. 5.15 Gut-vascular barrier. In a steady state, pericytes and glial cell surround the vascular
endothelial cells forming the barrier that regulates the diffusion of molecules of 4 kD from the
intestine to the bloodstream and liver. Under bacterial infection, both epithelium and vascular
barriers become leaky, allowing and promoting bacterial spreading through the portal vein

Importantly, disruption of the GVB in the human gut may lead to liver damage,
as observed in patients with celiac disease (Spadoni et al. 2016). The microvascular
endothelium of the gut barrier also plays a crucial role during inflammation in
inflammatory bowel disease. Understanding mechanistically how alterations of the
GVB may disrupt systemic immune homeostasis and promote liver damage warrants
investigation in different pathological settings. This knowledge may also open a new
window of therapeutic treatments to block the entrance of undesirable pathogens.

Grant Support This is supported by grants from the Spanish Ministry of Economy and
Competitiveness (MINECO; SAF2014–55473-R and BES2015–071399); the Spanish Ministry of
Science, Innovation and Universities (SAF2017–87988-R); the European Union FEDER funds;
the Spanish Association Against Cancer (AECC); the Worldwide Cancer Research Foundation;
and the CERCA Programme (Generalitat de Catalunya, Spain). CIBERehd is an initiative from the
Instituto de Salud Carlos III.

Disclosures All authors declare no competing financial interests.

98 M. Ramirez et al.

References

Abraldes JG, Iwakiri Y, Loureiro-Silva M et al (2006) Mild increases in portal pressure upregulate
vascular endothelial growth factor and endothelial nitric oxide synthase in the intestinal micro-
circulatory bed, leading to a hyperdynamic state. Am J Phys 290:G980–G987
Alverdy JC (1990) Effects of glutamine-supplemented diets on immunology of the gut. JPEN
14:109S–113S
Armulik A, Genove G, Mäe M et al (2010) Pericytes regulate the blood–brain barrier. Nature
468:557–561
Armulik A, Genove G, Betsholtz C (2011) Pericytes: developmental, physiological, and pathologi-
cal perspectives, problems, and promises. Dev Cell 21:193–215
Artis D (2008) Epithelial-cell recognition of commensal bacteria and maintenance of immune
homeostasis in the gut. Nat Rev Immunol 8:411–420
Backhed F, Ley RE, Sonnenburg JL et al (2005) Host-bacterial mutualism in the human intestine.
Science 307:1915–1920
Bain CC, Mowat AM (2014) Macrophages in intestinal homeostasis and inflammation. Immunol
Rev 260:102–117
Bava FA, Eliscovich C, Ferreira PG et al (2013) CPEB1 coordinates alternative 3’-UTR formation
with translational regulation. Nature 495:121–125
Bergers G, Song S (2005) The role of pericytes in blood-vessel formation and maintenance. Neuro-­
Oncology 7:452–464
Bergers G, Song S, Meyer-Morse M et al (2003) Benefits of targeting both pericytes and endothe-
lial cells in the tumor vasculature with kinase inhibitors. J Clin Invest 111:1287–1295
Bhat M, Arendt BA, Bhat V et al (2016) Implication of the intestinal microbiome in complications
of cirrhosis. World J Hepatol 8:1128–1136
Calderone V, Gallego J, Fernandez-Miranda G et al (2016) Sequential functions of CPEB1 and
CPEB4 regulate pathologic expression of VEGF and angiogenesis in chronic liver disease.
Gastroenterology 150:982–997
Chatterjee S (2014) Reversal of vasohibin-driven negative feedback loop of vascular endothelial
growth factor/angiogenesis axis promises a novel antifibrotic therapeutic strategy for liver
diseases. Hepatology 60:458–460
Coch L, Mejias M, Berzigotti A et al (2014) Disruption of negative feedback loop between
vasohibin-1 and VEGF decreases portal pressure, angiogenesis and fibrosis in cirrhotic rats.
Hepatology 60:633–647
Coffey JC, O’Leary DP (2016) The mesentery: structure, function, and role in disease. Lancet
Gastroenterol Hepatol 1:238–247
Daneman R, Zhou L, Kebede AA et al (2010) Pericytes are required for blood–brain barrier integ-
rity during embryogenesis. Nature 468:562–566
Drake RL, Vogl AW, Mitchell A (2015) Gray’s anatomy for students, 3rd edn. Churchill
Livingstone/Elsevier, Philadelphia, PA, pp 271–275
Fernandez M (2015) Molecular pathophysiology of portal hypertension. Hepatology 61:1406–1415
Fernandez M, Vizzutti F, Garcia-Pagan JC et al (2004) Anti-VEGF receptor-2 monoclonal
antibody prevents portal-systemic collateral vessel formation in portal hypertensive mice.
Gastroenterology 126:886–894
Fernandez M, Mejias M, Angermayr B et al (2005) Inhibition of VEGF receptor-2 decreases the
development of hyperdynamic splanchnic circulation and portal-systemic collateral vessels in
portal hypertensive rats. J Hepatol 43:98–103
Fernandez M, Mejias M, Garcia-Pras E et al (2007) Reversal of portal hypertension and hyper-
dynamic splanchnic circulation by combined vascular endothelial growth factor and platelet-­
derived growth factor blockade in rats. Hepatology 46:1208–1217
Fernandez M, Semela D, Bruix J et al (2009) Angiogenesis in liver disease. J Hepatol 50:604–620
Fernandez-Miranda G, Mendez R (2012) The CPEB-family of proteins, translational control in
senescence and cancer. Ageing Res Rev 11:460–472
5 Pericytes in the Gut 99

Gaengel K, Genove G, Armulik A et al (2009) Endothelial-mural cell signaling in vascular devel-

opment and angiogenesis. Arterioscler Thromb Vasc Biol 29:630–638
Gallego J, Garcia-Pras E, Mejias M et al (2017) Therapeutic siRNA targeting endothelial KDR
decreases portosystemic collateralization in portal hypertension. Sci Rep 7:14791
Garcia-Pras E, Gallego J, Coch L et al (2017) Role and therapeutic potential of vascular stem/
progenitor cells in pathological neovascularisation during chronic portal hypertension. Gut
66:1306–1320
Garcia-Tsao G, Bosch J (2010) Management of varices and variceal hemorrhage in cirrhosis. N
Engl J Med 362:823–832
Garcia-Tsao G, Sanyal AJ, Grace N et al (2007) Prevention and management of gastroesophageal
varices and variceal hemorrhage in cirrhosis. Hepatology 46:922–938
Garrett WS, Gordon JI, Glimcher LH (2010) Homeostasis and inflammation in the intestine. Cell
140:859–870
Gerhardt H, Golding M, Fruttiger M et al (2003) VEGF guides angiogenic sprouting utilizing
endothelial tip cell filopodia. J Cell Biol 161:1163–1177
Granger DN, Holm L, Kvietys PR (2015) The gastrointestinal circulation: physiology and patho-
physiology. In: Terjung R (ed) Comprehensive physiology. Wiley, Hoboken, NJ. https://doi.
org/10.1002/cphy.c150007
Gray H, Lewis WH (2000) Gray’s anatomy of the human body, 20th edn. Bartleby, New York, NY
Guarner C, Soriano G, Tomas A et al (1993) Increased serum nitrite and nitrate levels in patients
with cirrhosis: relationship to endotoxemia. Hepathology 18:1139–1143
Gupta TK, Toruner M, Chung MK et al (1998) Endothelial dysfunction and decreased production
of nitric oxide in the intrahepatic microcirculation of cirrhotic rats. Hepatology 28:926–931
Harper D, Chandler B (2016) Splanchnic circulation. BJA Education 16:66–71
Hirschi K, D’Amore PA (1996) Pericytes in the microvasculature. Cardiovasc Res 32:687–698
Iwakiri Y, Shah V, Rockey DC (2014) Vascular pathobiology in chronic liver disease and cirrhosis -
current status and future directions. J Hepatol 61:912–924
Jacobson ED (1982) Physiology of the mesenteric circulation. Physiologist 25:439–443
Jakobsson L, Franco CA, Bentley K et al (2010) Endothelial cells dynamically compete for the tip
cell position during angiogenic sprouting. Nat Cell Biol 12:943–953
Jankowski JA, Goodlad RA, Wright NA (1994) Maintenance of normal intestinal mucosa: func-
tion, structure, and adaptation. Gut 35(Suppl 1):S1–S4
Kachlik D, Baca V, Stingl J (2010) The spatial arrangement of the human large intestinal wall
blood circulation. J Anat 216:335–343
Kelly-Goss MR, Sweat RS, Stapor PC et al (2014) Targeting pericytes for angiogenic therapies.
Microcirculation 21:345–357
Kvietys PR (2010) The gastrointestinal circulation (chap. 2: anatomy). Morgan & Claypool Life
Sciences, San Rafael, CA
Liebner S, Corada M, Bangsow T et al (2008) Wnt/−catenin signaling controls development of the
blood—brain barrier. J Cell Biol 183:409–417
Llovet JM, Bruix J (2009) Testing molecular therapies in hepatocellular carcinoma: the need for
randomized phase II trials. J Clin Oncol 27:833–835
Maillo C, Martin J, Sebastian D et al (2017) Circadian- and UPR-dependent control of CPEB4
mediates a translational response to counteract hepatic steatosis under ER stress. Nat Cell Biol
19:94–105
Matheson PJ, Wilson MA, Garrison RN (2000) Regulation of intestinal blood flow. J Surg Res
93:182–196
Mejias M, Garcia-Pras E, Tiani C et al (2009) Beneficial effects of sorafenib on splanchnic, intra-
hepatic, and portocollateral circulations in portal hypertensive and cirrhotic rats. Hepatology
49:1245–1256
Mejias M, Coch L, Berzigotti A et al (2015) Antiangiogenic and antifibrogenic activity of pigment
epithelium-derived factor (PEDF) in bile duct-ligated portal hypertensive rats. Gut 64:657–666
Mendez R, Hake LE, Andresson T et al (2000a) Phosphorylation of CPE binding factor by Eg2
regulates translation of c-Mos mRNA. Nature 404:302–307
100 M. Ramirez et al.

Mendez R, Murthy KG, Ryan K et al (2000b) Phosphorylation of CPEB by Eg2 mediates the recruit-
ment of CPSF into an active cytoplasmic polyadenylation complex. Mol Cell 6:1253–1259
Ortiz-Zapater E, Pineda D, Martínez-Bosch N et al (2011) Key contribution of CPEB4-mediated
translational control to cancer progression. Nat Med 18:83–90
Phillips GB, Schwartz R, Gabuzda GJ Jr et al (1952) The syndrome of impending hepatic coma
in patients with cirrhosis of the liver given certain nitrogenous substances. N Engl J Med
247:239–246
Pinter M, Sieghart W, Reiberger T et al (2012) The effects of sorafenib on the portal hypertensive
syndrome in patients with liver cirrhosis and hepatocellular carcinoma-a pilot study. Aliment
Pharmacol Ther 35:83–91
Pique M, Lopez JM, Foissac S et al (2008) A combinatorial code for CPE-mediated translational
control. Cell 132:434–448
Potente M, Gerhardt H, Carmeliet P (2011) Basic and therapeutic aspects of angiogenesis. Cell
146:873–887
Reiberger T, Angermayr B, Schwabl P et al (2009) Sorafenib attenuates the portal hypertensive
syndrome in partial portal vein ligated rats. J Hepatol 51:865–873
Reiberger T, Payer BA, Schwabl P et al (2013) Nebivolol treatment increases splanchnic blood
flow and portal pressure in cirrhotic rats via modulation of nitric oxide signalling. Liver Int
33:561–568
Rockey DC, Chung JJ (1998) Reduced nitric oxide production by endothelial cells in cirrhotic rat
liver: endothelial dysfunction in portal hypertension. Gastroenterology 114:344–351
Sarkissian M, Mendez R, Richter JD (2004) Progesterone and insulin stimulation of CPEB-­
dependent polyadenylation is regulated by Aurora a and glycogen synthase kinase-3. Genes
Dev 18:48–61
Schnabl B, Brenner DA (2014) Interactions between the intestinal microbiome and liver diseases.
Gastroenterology 146:1513–1524
Schuppan D, Afdhal NH (2008) Liver cirrhosis. Lancet 371:838–851
Sharara AI, Rockey DC (2001) Gastroesophageal variceal hemorrhage. N Engl J Med 345:669–681
Spadoni I, Zagato E, Bertocchi A et al (2015) A gut-vascular barrier controls the systemic dissemi-
nation of bacteria. Science 350:830–834
Spadoni I, Pietrelli A, Pesole G et al (2016) Gene expression profile of endothelial cells during
perturbation of the gut vascular barrier. Gut Microbes 7:540–548
Sparks HV (2011) Effect of local metabolic factors on vascular smooth muscle. Supplement 7:
handbook of physiology, the cardiovascular system, vascular smooth muscle, pp 475–513
Stapor PC, Sweat RS, Dashti DC et al (2014) Pericyte dynamics during angiogenesis: new insights
from new identities. J Vasc Res 51:163–174
Thorburn T, Aali M, Lehmann C (2018) Immune response to systemic inflammation in the intesti-
nal microcirculation. Front Biosci (Landmark Ed) 23:782–795
Tsiaoussis GI, Assimakopoulos SF, Tsamandas AC et al (2015) Intestinal barrier dysfunction
in cirrhosis: current concepts in pathophysiology and clinical implications. World J Hepatol
7:2058–2068
Tugues S, Fernandez-Varo G, Muñoz-Luque J et al (2007) Antiangiogenic treatment with sunitinib
ameliorates inflammatory infiltrate, fibrosis, and portal pressure in cirrhotic rats. Hepatology
46:1919–1926
Usami M, Miyoshi M, Yamashita H (2015) Gut microbiota and host metabolism in liver cirrhosis.
World J Gastroenterol 21:11597–11608
Van Steenkiste C, Geerts A, Vanheule E et al (2009) Role of placental growth factor in mesenteric
neoangiogenesis in a mouse model of portal hypertension. Gastroenterology 137:2112–2124
Vespasiani-Gentilucci U, Rombouts K (2015) Boosting pigment epithelial-derived factor: a prom-
ising approach for the treatment of early portal hypertension. Gut 64:523–524
Volta U, Bonazzi C, Bianchi FB et al (1987) IgA antibodies to dietary antigens in liver cirrhosis.
Ric Clin Lab 17:235–242
Chapter 6
Pericytes in Bone Marrow

Yuya Kunisaki

Abstract Bone marrow environments are composed of multiple cell types, most of
which are thought to be derived from mesenchymal stem cells. In mouse bone mar-
row, stromal cells with CD45− Tie2− CD90− CD51+ CD105+ phenotype, Nestin-­
GFP+, CXCL12-abundant reticular (CAR) cells, PDGFRα+ Sca-1+ or CD51+
PDGFRα+, and Prx-1-derived CD45− Ter119− PDGFRα+ Sca-1+ populations select
for MSC activity. There is evidence that these stromal cell populations display some
significant overlap with each other and comprise important cellular constituents of
the hematopoietic stem cell niche. Moreover, these mesenchymal cell populations
share characteristics in their location as they all are found around bone marrow ves-
sels (can be called “pericytes”). In this chapter, with reviewing the recent literatures,
how the pericytes relate to physiological and pathological hematopoiesis is argued.

Keywords Pericytes · Perivascular cells · Hematopoietic stem cells · Niche ·

Mesenchymal stem cells · Skeletal progenitor · Microenvironments · Bone marrow
vessels · Leukemia · Cancer · Cancer microenvironments · Cytokine niche

6.1 Introduction

Somatic stem cells self-renew to maintain tissue homeostasis for the lifetime of
organisms through tightly controlled proliferation and differentiation (Orkin and
Zon 2008; Li and Clevers 2010; Hsu et al. 2011). Hematopoietic stem cells (HSCs)
are essentially required for the hematopoietic homeostasis. Therefore, they do not
only ensure lifelong replenishment of all blood lineages but also keep their pool
constant. Recent studies have highlighted the importance of bone marrow microen-
vironments that regulate HSC functions (HSC niches) (Kunisaki and Frenette 2012;
Morrison and Scadden 2014). In the HSC biology, there has been a considerable
interest and debate regarding whether or not quiescence and proliferation of HSCs

Y. Kunisaki (*)
Kyushu University Hospital, Center for Cellular and Molecular Medicine, Fukuoka, Japan
e-mail: [email protected]

© Springer Nature Switzerland AG 2019 101

A. Birbrair (ed.), Pericyte Biology in Different Organs,
Advances in Experimental Medicine and Biology 1122,
https://doi.org/10.1007/978-3-030-11093-2_6
102 Y. Kunisaki

are regulated by distinct niches (Orford and Scadden 2008). Recent studies have
revealed that bone marrow perivascular cells, “pericytes,” have mesenchymal stem
cell (MSC) activity and that two types of vessels, arterioles and sinusoids, are
accompanied with distinct pericytes that are associated with quiescent and prolif-
erative HSCs, respectively (Kunisaki et al. 2013; Asada et al. 2017).

6.2 What Does Bone Marrow Do?

Bone marrow is the main organ that maintains the number of circulating leukocytes
and HSCs by controlling their release and recruitment. HSCs self-renew and differen-
tiate into all blood types in response to various demands during life (Orkin and Zon
2008). Functions of HSCs are finely regulated by specialized microenvironments
commonly referred to as “niches” in bone marrow (Kunisaki and Frenette 2012;
Morrison and Scadden 2014; Mendelson and Frenette 2014). In addition, HSCs are
constitutively present at low level in the circulation and constantly released into
peripheral blood and home to bone marrow during homeostasis (Laird et al. 2008).
HSCs are released into peripheral blood in a circadian manner, which is con-
trolled by the sympathetic nervous system through the regulation of CXCL12 levels
in bone marrow (Mendez-Ferrer et al. 2008; Scheiermann et al. 2013). Vessels bor-
der blood circulation in all organs and are important to recruit leukocytes to the
specific organs by expressing adhesion molecules and secreting chemokines accord-
ing to the situations (Druzd et al. 2017; Scheiermann et al. 2012). Endothelial selec-
tins (P-selectin and E-selectin) and vascular cell adhesion molecule-1 (VCAM-1)
are constitutively expressed in bone marrow and have been shown to be important
for the rolling and homing process of the HSCs (Scheiermann et al. 2012). E-selectin,
an adhesion molecule constitutively expressed in certain BM sinusoid microdo-
mains, promotes HSC proliferation, and blockade of E-selectin protects HSC fol-
lowing chemotherapy or irradiation, suggesting that sinusoids represent a
proliferative niche for HSCs (Winkler et al. 2012). Therefore, vessels are structur-
ally and functionally an important constituent of bone marrow.

6.3 Bone Marrow Vascular Structure

In bone marrow, there is an even distribution of the sinusoidal network that occupies
~30% of bone marrow volume, and individual sinusoidal vessels are regularly
spaced and draining into the central vein. In addition to sinusoids, there are small-­
caliber (~20 μm) arterioles, which comprise a much smaller volumetric fraction
~1% of bone marrow. The vessels are distinguished from sinusoids in respect to
their pronounced Tie-2-GFP and CD31 expression (Li et al. 2009), absence of
in vivo staining with the sinusoid-specific Dil-Ac-LDL (Li et al. 2009), and strong
staining with the artery-specific dye Alexa Fluor 633 (Shen et al. 2012). Bone mar-
row vasculature, hence, is composed of distinct vascular components, arterioles and
sinusoids (Fig. 6.1a, b) (Kunisaki et al. 2013).
6 Pericytes in Bone Marrow 103

Fig. 6.1 Bone marrow vessels and pericytes. (a) Immunofluorescence images of mouse sternal
bone marrow stained with anti-VE cadherin and PECAM1 antibodies (left). Two vessel types,
arterioles and sinusoids, are ensheathed by distinct pericytes, Nestin-GFPhigh and Nestin-GFPdim
cells, respectively (right). Scale bar: 100 μm. (b) Immunofluorescence images of cross-sectioned
mouse femoral bone marrow stained with anti-VE cadherin and PECAM1 antibodies (left, right).
Nestin-GFPdim cells are not appreciated due to the contrast to Nestin-GFPhigh cells. Nestin-GFPhigh
cells exist an outer layer wrapping endothelial cells forming a vascular lumen (right)

6.4  esenchymal Precursor and Osteoprecursor Cells

M
as Bone Marrow Components

Recent studies have highlighted the critical importance of bone marrow stromal
cells as essential constituents for the HSC niche through the production of factors
such as CXCL12 and SCF. Bone marrow stromal cells are derived from mesenchy-
mal stem cells (MSCs) (Caplan 1991; Rodda and McMahon 2006; Frenette et al.
2013). Stromal progenitor activity in bone marrow was initially isolated from clonal
populations of fibroblastic colony-forming units (CFU-F) that exhibit self-renewal
and capacity to differentiate into the major mesenchymal lineages (Zhou et al.
104 Y. Kunisaki

2010). In mouse bone marrow, the cell populations exhibiting the mesenchymal
stem/progenitor activity are isolated as Nestin-GFP+ (Mendez-Ferrer et al. 2010),
CD45− Tie2− CD90− CD51+ CD105+ phenotype (Chan et al. 2009), CXCL12-­
abundant reticular (CAR) cells (Omatsu et al. 2010; Sugiyama et al. 2006), leptin
receptor-positive cells (Ding et al. 2012), PDGFRα+ Sca-1+ (Morikawa et al. 2009)
or CD51+ PDGFRα+ (Pinho et al. 2013), and Prx-1-derived CD45− Ter119−
PDGFRα+ Sca-1+ populations (Greenbaum et al. 2013). Recent lineage tracing anal-
yses using tamoxifen-inducible Osx-cre (Osx-creERT2) have revealed that expression
of Osx is not restricted in osteoblasts and that Osx-expressing cells at the neonatal
stage are bone marrow stromal precursors (Mizoguchi et al. 2014). Owen and
Friedenstein predicted 25 years ago that the differentiation tree of stromal cells of
the bone marrow is as complex as their hematopoietic counterparts (Owen and
Friedenstein 1988). However, the hierarchical organization of stromal cells remains
unknown. In addition, stromal cells marked by various genetic reporter mouse
strains, e.g., Prx-1-cre, Osterix-cre, NG2-creER™, leptin receptor-cre, and NG2-cre,
have been reported as important niche constituents (Kunisaki et al. 2013; Omatsu
et al. 2010; Greenbaum et al. 2013; Ding and Morrison 2013). There is evidence
that these stromal cell populations display some significant overlap with each other
and comprise important cellular constituents of the HSC niche, suggesting that
MSCs organize the bone marrow environment. Moreover, these mesenchymal cell
populations share characteristics in their location as they all are found around bone
marrow vessels (can be called “pericytes”).

6.5 Developmental Origins of the Pericytes

Developmental origins of pericytes in bone marrow have still been under investiga-
tions. There are several studies reporting that mesenchymal cells arise from the
neural crest or mesoderm. The neural crest stem cells (NCSCs), which exhibit the
potentials to self-renew and differentiate into neurons, glial cells, and myofibro-
blasts (Morrison et al. 1999), have been described in various adult tissues. Okano’s
group report the presence of NCSCs in bone marrow (Nagoshi et al. 2008) and sug-
gest the possibility that NCSCs might have overlap MSCs in bone marrow. Another
study from Yamazaki’s group examined the origins of bone marrow stromal cells by
using the genetically labeled mouse models as Mesp1-cre for mesoderm-derived
cells and P0-cre for neural crest-derived cells and showed their presence in bone
marrow (Komada et al. 2012). These studies demonstrate that bone marrow NCSCs
exhibit mesenchymal activity as they differentiate into osteo-, chondro-, and adipo-­
lineages in vitro as do bone marrow MSCs, suggesting that there are two stromal
cell types exhibiting MSC activity that are derived from distinct origins in bone
marrow. However, whether NCSCs overlap the pericytes, such as Nestin-GFP+,
LepR+, and CAR cells that comprise the HSC niches, is yet known.
6 Pericytes in Bone Marrow 105

6.6  rterioles and Sinusoids Are Ensheathed by Distinct

A
Mesenchymal Pericytes

One study using the genetic mouse model, where GFP is expressed under nestin
promoter (Nestin-GFP), shows anatomical relationships of pericytes and distinct
vascular structures, arterioles and sinusoids (Kunisaki et al. 2013). Nestin-GFP+
pericytes contain all bone marrow mesenchymal stem cell activity (Mendez-Ferrer
et al. 2010) and two distinct subsets that are associated with distinct vessel types:
rare Nestin-GFP cells expressing the NG2 antigen (~10% of Nestin-GFP+ cells) but
not leptin receptor are exclusively associated with arterioles, whereas NG2− and
LEPR+ Nestin-GFP cells (~90% of Nestin-GFP+ cells) are associated with sinusoids
(Fig. 6.1a, b) (Kunisaki et al. 2013).

6.7  rteriolar Pericytes Are Quiescent and Protected

A
from Myeloablation

The Nestin-GFP+ pericytes associated with arterioles are more quiescent than the
sinusoidal pericytes, as expression of the nuclear proliferation markers, Ki-67 and
proliferation cell nuclear antigen (PCNA), in arteriolar pericytes was significantly
lower than in sinusoidal pericytes and other stromal cells. As the arteriolar pericytes
are largely preserved structurally and numerically as compared to the sinusoidal
pericytes after 5-fluorouracil (5FU), a drug that kills cycling cells, the quiescent
status of the arteriolar pericytes is functionally demonstrated (Kunisaki et al. 2013).

6.8 HSC Niches

The “niche” concept has been validated by numerous studies since Schofield postu-
lated its presence in 1978 (Schofield 1978). Most of the HSCs divide infrequently and
are quiescent in the niche (Kiel et al. 2007). They, however, are reversibly activated in
response to hematopoietic stresses (Wilson et al. 2008). Cell cycle quiescence is a key
behavior of stem cells, which protects them from being exhausted by exogenous
insults and is also assumed to prevent them from acquiring genetic mutations that
potentially result in consequent malignant transformations (Lobo et al. 2007).
The identification of cellular constituents of the HSC niche has recently been the
subject of intense investigations. Osteoblasts have been proposed to promote HSC
quiescence via direct contact (Sugimura et al. 2012) and the secretion of angiopoi-
etin-­1 (Arai et al. 2004) or osteopontin (Nilsson et al. 2005; Stier et al. 2005). On
the other hand, an emerging role of bone marrow vasculature has recently gained
support and interest (Kiel et al. 2007, 2005; Takakura 2012) as other studies have
106 Y. Kunisaki

found that most HSCs are localized adjacent to blood vessels (sinusoids), near peri-
vascular cell populations characterized as CXCL12-abundant reticular (CAR) cells
(Omatsu et al. 2010; Sugiyama et al. 2006), Nestin-GFP+ mesenchymal progenitors
(Mendez-Ferrer et al. 2010), and leptin receptor (LEPR)+ cells (Ding et al. 2012).
The proximity of HSCs to sinusoidal vessels has been reported in many previous
studies. The relationship between HSCs and arteries also has been well documented
in the emergence of HSCs during development as definitive hematopoiesis is begin-
ning in the aorta-gonad-mesonephros (AGM) region (Mikkola and Orkin 2006).

6.9  ssessment of Spatial Relationships by Computational

A
Simulation

Unbiased assessment by computational simulation is useful to evaluate the signifi-

cance of the association between HSCs and bone marrow structures observed in situ
(Kunisaki et al. 2013; Chen et al. 2016; Acar et al. 2015). In a null model, HSCs are
randomly placed on the unoccupied regions of binary spatial maps of bone marrow
structures, sinusoids, arterioles, and bone lining osteoblasts, defined from the
images of whole-mount prepared bone, and Euclidean distance of the random HSCs
to the structures is measured. The means of 1000 simulations define a distribution
of mean distances one would observe for non-preferentially localized HSCs in rela-
tion to the respective structures. If the in situ distance measurements are not statisti-
cally different from those obtained by a random placement of HSCs on the same
structures in silica, this will indicate a non-preferential spatial HSC distribution.
In one study using this modeling (Kunisaki et al. 2013), the mean distance
observed in situ to sinusoids could not be statistically differentiated from that of
randomly placed HSCs. By contrast, the observed mean distance to arterioles was
highly statistically different from that of randomly placed HSCs. The analyses using
the combination of high-quality imaging and the computational simulation have
revealed that quiescent HSCs are specifically associated with small-caliber arteri-
oles (arteriolar niches) (Fig. 6.2).

6.10  ontributions by Distinct Pericytes to HSC

C
Maintenance

There are two types of Nestin-GFP+ pericytes expressing different surface markers,
nerve/glial antigen 2 (NG2) and leptin receptor (Lepr) that are associated with arte-
rioles and sinusoid, respectively, in bone marrow (Kunisaki et al. 2013). Both arte-
riolar and sinusoidal pericytes show high gene expression of cytokines essential for
HSC maintenance such as CXCL12 and stem cell factor (SCF) (Asada et al. 2017).
To investigate contributions of arteriolar and sinusoidal pericytes, we can utilize
genetic mouse models with a cre/flox targeted gene deleting system in which
6 Pericytes in Bone Marrow 107

Fig. 6.2 A computational modeling of the HSC localization in relation to bone marrow arterioles
or sinusoids. (a) Computational simulation of randomly distributed HSCs on images of whole-­
mount prepared sterna. To establish the null-model, binary spatial maps of the sinusoids, arterioles
were defined from the images of whole-mount prepared sterna. To simulate a null model in which
HSCs are not preferentially localized in the marrow, we randomly placed 20 HSCs (to reflect the
mean HSCs/sternum observed in situ) on the unoccupied regions of the spatial maps and measured
the Euclidean distance of HSCs to the nearest vascular structure. Scale bar: 100 μm. (b) The means
of 1000 simulations defined a distribution of mean distances one would observe for non-­
preferentially (random) localized HSCs in relation to the respective structures. If the in situ dis-
tance measurements are not statistically significantly different from those obtained by a random
placement of HSCs on the same structures in silica, this would indicate a non-preferential spatial
HSC distribution. The cumulative probability of observing the in situ mean was calculated based
on the normal distribution obtained from our simulation on a map of each bone marrow structure

CXCL12 or SCF (e.g., CXCL12-flox or SCF-flox animals) can be deleted in spe-

cific cell types (e.g., NG-2 creER™ or leptin receptor cre) (Asada et al. 2017; Ding
et al. 2012; Greenbaum et al. 2013; Ding and Morrison 2013). CXCL12 deletions in
sinusoidal pericytes by using Lepr-cre/Cxcl12fl/− mice mobilize HSCs but have no
effect on bone marrow HSC numbers (Asada et al. 2017; Ding and Morrison 2013).
To examine further a role of CXCL12 produced by NG2+ arteriolar pericytes on
HSC maintenance, tamoxifen-inducible NG2-creER™/Cxcl12fl/− mice are used.
Deletion of CXCL12 postnatally in NG2+ arteriolar niche cells significantly reduces
108 Y. Kunisaki

Fig. 6.3 Hematopoietic stem cell niches in bone marrow. Hematopoietic stem cells, HSCs, reside
in a specialized microenvironment called the niche. A variety of cells, osteoblasts, endothelial
cells, pericytes, non-myelinating Schwann cells, and megakaryocytes, have been reported as niche
components. Arterioles and sinusoids that are ensheathed by distinct perivascular NG2+ Nestin-­
GFPbright or Lepr+ Nestin-GFPdim cells, respectively, comprise distinct cytokine niches for HSCs as
the arteriolar niche promotes quiescence of HSCs, whereas sinusoids form another niche, which
play major roles in HSC proliferation

the number of HSCs in bone marrow. Deletion of SCF in Lepr-cre targeted cells
shows a significant reduction of HSC numbers in bone marrow (Ding et al. 2012),
whereas no significant change is observed in NG2-creER™/SCFfl/− mice, suggesting
that Lepr+ vascular niches rather than NG2+ arteriolar niches are the most important
source of SCF in bone marrow. These results highlight distinct contributions of
pericytes primarily located in separate vascular niches, arteriolar and sinusoidal, in
HSC maintenance (Fig. 6.3).

6.11 Bone Marrow Pericytes in Pathological Conditions

6.11.1  one Marrow Microenvironments Remodeled

B
by Leukemia Cells

In recent years, there have been several studies reporting that leukemia cells remodel
bone marrow microenvironments to favor leukemia progression. These changes
suppress normal hematopoiesis that could lead to myelosuppression in leukemia.
6 Pericytes in Bone Marrow 109

Passegue’s group reported that BCR-/ABL-positive leukemia cells in the mouse

chronic myelogenous leukemia model promote bone marrow pericytes (=mesen-
chymal progenitor cells) to differentiate into bone lineages that suppress normal
hematopoiesis (Schepers et al. 2013). Hong’s group, likewise, demonstrated that
leukemia precursor cells reconstruct bone marrow that helps the leukemia cells
escape from chemotherapy (Duan et al. 2014). In the study from Frenette’s group,
Nestin-GFP-positive mesenchymal pericytes are induced to proliferate and differ-
entiate into osteo-lineages in mice transplanted with MLL-AF9 AML cells, result-
ing in altered distribution of normal HSCs apart from the arteriolar niche with
reduction in their number (Hanoun et al. 2014).
Furthermore, the group of Mendez-Ferrer reported that in the myeloproliferative
disorder model mice induced by the human JAK2 (V617F) mutant gene, tumor cells
impair nerves by producing IL-1β and transform bone marrow microenvironments
that promote tumor development. These changes induce apoptosis of pericytes,
resulting in suppressed HSC functions (Arranz et al. 2014). Nowak et al. also
reported that human myelodysplastic syndrome cells facilitate patient-derived
xenografts in mice by transplanting simultaneously with mesenchymal stromal cells
derived from the same patient (Medyouf et al. 2014). These results collectively sup-
port the notion that cancer cells hijack bone marrow microenvironments to support
cancer growth by involving the pericytes.

6.12 Leukemia Development and Pericytes

Studies using genetic modification mouse models show that hematopoietic tumors
can occur by dysfunction of bone marrow microenvironments. Two studies from the
groups of Orkin et al. and Purton et al. demonstrated that deficiency of the retinoic
acid receptor γ gene or Rb oncogene causes myeloproliferative disorders (Walkley
et al. 2007a, b). More recently, using the tissue cell-specific mouse genetic modifi-
cation model, Scadden’s group shows that spontaneous development of myelodys-
plastic syndrome by deletion of a microRNA processing enzyme, Dicer 1,
specifically in bone precursor cells (Raaijmakers et al. 2010). In addition, the group
of Kousteni reported that acute myeloid leukemia develops by constitutively activat-
ing β-catenin specifically in osteoblasts (Kode et al. 2014).

6.13 Metastatic Bone Cancer and Pericytes

Recent studies have revealed that environmental cues regulate not only primary
tumor growth but also the establishment and progression of metastasis (Yoneda and
Hiraga 2005). Bones are preferred for metastases in various malignancies including
110 Y. Kunisaki

malignant melanoma and breast and prostate cancer. The metastatic processes
highly depend on the vasculature that provides a route for cancer cell to disseminate
into the sites and access oxygen and nutrients. Tumor vessels are also embedded
with pericytes, which are reported to play important roles in capturing circulating
tumor cells at the metastasized tissues (Caplan 2017). One study from Caplan’s
group shows that bone marrow pericytes attract the circulating melanoma cells by
releasing their “fingers” through the fenestrations in the endothelial layers. These
processes are enhanced by platelet-derived growth factor (PDGF)-BB released from
broken platelets(Correa et al. 2016). Collectively, the vasculature wrapped with
“pericytes” from metastatic niches for cancer not only for leukemia in bone
marrow.

6.14 Concluding Remarks

“Pericytes,” synonymous to perivascular mesenchymal progenitors in bone marrow,

border vascular endothelial cells and bone marrow cavities and play important roles
in supporting hematopoiesis as niches as well as in organ regeneration and
angiogenesis.
HSCs are essentially required for hematopoietic homeostasis, for that signals
from the HSC niche are critical. Studies herein report that such selected microenvi-
ronment exists and highlight the possibility of heterogeneity among niche factor-­
producing pericytes. As oligopotent HSCs are identified (Notta et al. 2016;
Sanjuan-Pla et al. 2013; Yamamoto et al. 2013; Pinho et al. 2018), further studies
will determine the extent by which HSC heterogeneity is matched by niche
heterogeneity.
Recently, cellular heterogeneity within a tumor has been highlighted, and among
aggressively proliferating tumor cells, a small fraction is found in quiescent status,
termed “cancer stem cells,” causing refractoriness to anticancer therapy and relapsed
diseases. Even in cancer cells that appear to proliferate in an unlimited manner,
environmental cues are believed to control their cell cycle status, quiescence, or
proliferation. Therefore, “decision” whether symmetric or asymmetric division is
essential for hematologic tumors such as leukemia to maintain “leukemic stem cell
(LSC)” clones. The anatomical and functional interactions between hematopoietic
cells or leukemia and their microenvironments are essential to efficiently control
normal and pathological hematopoiesis (Fig. 6.4). The outcome that further studies
will achieve with the niche is expected to add new concept as “anticancer niche” to
cancer therapy.
6 Pericytes in Bone Marrow 111

Fig. 6.4 Leukemia microenvironments as a therapeutic target. In steady state, the fate of HSCs,
quiescence, proliferation, or differentiation, is determined by their special microenvironments
(HSC niche). Arteriolar niches keep HSCs to be quiescent, whereas sinusoidal niches rather
enforce them to be actively proliferated or mobilized. With gaining our knowledge about environ-
mental cues for pathological hematopoiesis, a new concept “anticancer niche therapy” will be
emerging

References

Acar M, Kocherlakota KS, Murphy MM, Peyer JG, Oguro H, Inra CN et al (2015) Deep imag-
ing of bone marrow shows non-dividing stem cells are mainly perisinusoidal. Nature
526(7571):126–130
Arai F, Hirao A, Ohmura M, Sato H, Matsuoka S, Takubo K et al (2004) Tie2/angiopoietin-1
signaling regulates hematopoietic stem cell quiescence in the bone marrow niche. Cell
118(2):149–161
Arranz L, Sanchez-Aguilera A, Martin-Perez D, Isern J, Langa X, Tzankov A et al (2014)
Neuropathy of haematopoietic stem cell niche is essential for myeloproliferative neoplasms.
Nature 512(7512):78–81
Asada N, Kunisaki Y, Pierce H, Wang Z, Fernandez NF, Birbrair A et al (2017) Differential cytokine
contributions of perivascular haematopoietic stem cell niches. Nat Cell Biol 19(3):214–223
Caplan AI (1991) Mesenchymal stem cells. J Orthop Res 9(5):641–650
Caplan AI (2017) New MSC: MSCs as pericytes are sentinels and gatekeepers. J Orthop Res
35(6):1151–1159
Chan CK, Chen CC, Luppen CA, Kim JB, DeBoer AT, Wei K et al (2009) Endochondral ossifica-
tion is required for haematopoietic stem-cell niche formation. Nature 457(7228):490–494
Chen JY, Miyanishi M, Wang SK, Yamazaki S, Sinha R, Kao KS et al (2016) Hoxb5 marks
long-term haematopoietic stem cells and reveals a homogenous perivascular niche. Nature
530(7589):223–227
Correa D, Somoza RA, Lin P, Schiemann WP, Caplan AI (2016) Mesenchymal stem cells regulate
melanoma cancer cells extravasation to bone and liver at their perivascular niche. Int J Cancer
138(2):417–427
112 Y. Kunisaki

Ding L, Morrison SJ (2013) Haematopoietic stem cells and early lymphoid progenitors occupy
distinct bone marrow niches. Nature 495(7440):231–235
Ding L, Saunders TL, Enikolopov G, Morrison SJ (2012) Endothelial and perivascular cells main-
tain haematopoietic stem cells. Nature 481(7382):457–462
Druzd D, Matveeva O, Ince L, Harrison U, He W, Schmal C et al (2017) Lymphocyte circa-
dian clocks control lymph node trafficking and adaptive immune responses. Immunity
46(1):120–132
Duan CW, Shi J, Chen J, Wang B, Yu YH, Qin X et al (2014) Leukemia propagating cells rebuild
an evolving niche in response to therapy. Cancer Cell 25(6):778–793
Frenette PS, Pinho S, Lucas D, Scheiermann C (2013) Mesenchymal stem cell: keystone of the
hematopoietic stem cell niche and a stepping-stone for regenerative medicine. Annu Rev
Immunol 31:285–316
Greenbaum A, Hsu YM, Day RB, Schuettpelz LG, Christopher MJ, Borgerding JN et al (2013)
CXCL12 in early mesenchymal progenitors is required for haematopoietic stem-cell mainte-
nance. Nature 495(7440):227–230
Hanoun M, Zhang D, Mizoguchi T, Pinho S, Pierce H, Kunisaki Y et al (2014) Acute myelogenous
leukemia-induced sympathetic neuropathy promotes malignancy in an altered hematopoietic
stem cell niche. Cell Stem Cell 15(3):365–375
Hsu YC, Pasolli HA, Fuchs E (2011) Dynamics between stem cells, niche, and progeny in the hair
follicle. Cell 144(1):92–105
Kiel MJ, Yilmaz OH, Iwashita T, Terhorst C, Morrison SJ (2005) SLAM family receptors distin-
guish hematopoietic stem and progenitor cells and reveal endothelial niches for stem cells. Cell
121(7):1109–1121
Kiel MJ, He S, Ashkenazi R, Gentry SN, Teta M, Kushner JA et al (2007) Haematopoietic stem
cells do not asymmetrically segregate chromosomes or retain BrdU. Nature 449(7159):238–242
Kode A, Manavalan JS, Mosialou I, Bhagat G, Rathinam CV, Luo N et al (2014) Leukaemogenesis
induced by an activating beta-catenin mutation in osteoblasts. Nature 506(7487):240–244
Komada Y, Yamane T, Kadota D, Isono K, Takakura N, Hayashi S et al (2012) Origins and proper-
ties of dental, thymic, and bone marrow mesenchymal cells and their stem cells. PLoS One
7(11):e46436
Kunisaki Y, Frenette PS (2012) The secrets of the bone marrow niche: enigmatic niche brings chal-
lenge for HSC expansion. Nat Med 18(6):864–865
Kunisaki Y, Bruns I, Scheiermann C, Ahmed J, Pinho S, Zhang D et al (2013) Arteriolar niches
maintain haematopoietic stem cell quiescence. Nature 502(7473):637–643
Laird DJ, von Andrian UH, Wagers AJ (2008) Stem cell trafficking in tissue development, growth,
and disease. Cell 132(4):612–630
Li L, Clevers H (2010) Coexistence of quiescent and active adult stem cells in mammals. Science
327(5965):542–545
Li XM, Hu Z, Jorgenson ML, Slayton WB (2009) High levels of acetylated low-density lipoprotein
uptake and low tyrosine kinase with immunoglobulin and epidermal growth factor homology
domains-2 (Tie2) promoter activity distinguish sinusoids from other vessel types in murine
bone marrow. Circulation 120(19):1910–1918
Lobo NA, Shimono Y, Qian D, Clarke MF (2007) The biology of cancer stem cells. Annu Rev Cell
Dev Biol 23:675–699
Medyouf H, Mossner M, Jann JC, Nolte F, Raffel S, Herrmann C et al (2014) Myelodysplastic
cells in patients reprogram mesenchymal stromal cells to establish a transplantable stem cell
niche disease unit. Cell Stem Cell 14(6):824–837
Mendelson A, Frenette PS (2014) Hematopoietic stem cell niche maintenance during homeostasis
and regeneration. Nat Med 20(8):833–846
Mendez-Ferrer S, Lucas D, Battista M, Frenette PS (2008) Haematopoietic stem cell release is
regulated by circadian oscillations. Nature 452(7186):442–447
Mendez-Ferrer S, Michurina TV, Ferraro F, Mazloom AR, Macarthur BD, Lira SA et al (2010)
Mesenchymal and haematopoietic stem cells form a unique bone marrow niche. Nature
466(7308):829–834
6 Pericytes in Bone Marrow 113

Mikkola HK, Orkin SH (2006) The journey of developing hematopoietic stem cells. Development
133(19):3733–3744
Mizoguchi T, Pinho S, Ahmed J, Kunisaki Y, Hanoun M, Mendelson A et al (2014) Osterix marks
distinct waves of primitive and definitive stromal progenitors during bone marrow develop-
ment. Dev Cell 29(3):340–349
Morikawa S, Mabuchi Y, Kubota Y, Nagai Y, Niibe K, Hiratsu E et al (2009) Prospective identifica-
tion, isolation, and systemic transplantation of multipotent mesenchymal stem cells in murine
bone marrow. J Exp Med 206(11):2483–2496
Morrison SJ, Scadden DT (2014) The bone marrow niche for haematopoietic stem cells. Nature
505(7483):327–334
Morrison SJ, White PM, Zock C, Anderson DJ (1999) Prospective identification, isolation by flow
cytometry, and in vivo self-renewal of multipotent mammalian neural crest stem cells. Cell
96(5):737–749
Nagoshi N, Shibata S, Kubota Y, Nakamura M, Nagai Y, Satoh E et al (2008) Ontogeny and mul-
tipotency of neural crest-derived stem cells in mouse bone marrow, dorsal root ganglia, and
whisker pad. Cell Stem Cell 2(4):392–403
Nilsson SK, Johnston HM, Whitty GA, Williams B, Webb RJ, Denhardt DT et al (2005)
Osteopontin, a key component of the hematopoietic stem cell niche and regulator of primitive
hematopoietic progenitor cells. Blood 106(4):1232–1239
Notta F, Zandi S, Takayama N, Dobson S, Gan OI, Wilson G et al (2016) Distinct routes of lineage
development reshape the human blood hierarchy across ontogeny. Science 351(6269):aab2116
Omatsu Y, Sugiyama T, Kohara H, Kondoh G, Fujii N, Kohno K et al (2010) The essential func-
tions of adipo-osteogenic progenitors as the hematopoietic stem and progenitor cell niche.
Immunity 33(3):387–399
Orford KW, Scadden DT (2008) Deconstructing stem cell self-renewal: genetic insights into cell-­
cycle regulation. Nat Rev Genet 9(2):115–128
Orkin SH, Zon LI (2008) Hematopoiesis: an evolving paradigm for stem cell biology. Cell
132(4):631–644
Owen M, Friedenstein AJ (1988) Stromal stem cells: marrow-derived osteogenic precursors. Ciba
Found Symp 136:42–60
Pinho S, Lacombe J, Hanoun M, Mizoguchi T, Bruns I, Kunisaki Y et al (2013) PDGFRalpha and
CD51 mark human nestin+ sphere-forming mesenchymal stem cells capable of hematopoietic
progenitor cell expansion. J Exp Med 210(7):1351–1367
Pinho S, Marchand T, Yang E, Wei Q, Nerlov C, Frenette PS (2018) Lineage-biased hematopoietic
stem cells are regulated by distinct niches. Dev Cell 44(5):634–641.e4
Raaijmakers MH, Mukherjee S, Guo S, Zhang S, Kobayashi T, Schoonmaker JA et al (2010)
Bone progenitor dysfunction induces myelodysplasia and secondary leukaemia. Nature
464(7290):852–857
Rodda SJ, McMahon AP (2006) Distinct roles for hedgehog and canonical Wnt signaling
in specification, differentiation and maintenance of osteoblast progenitors. Development
133(16):3231–3244
Sanjuan-Pla A, Macaulay IC, Jensen CT, Woll PS, Luis TC, Mead A et al (2013) Platelet-
biased stem cells reside at the apex of the haematopoietic stem-cell hierarchy. Nature
502(7470):232–236
Scheiermann C, Kunisaki Y, Lucas D, Chow A, Jang JE, Zhang D et al (2012) Adrenergic nerves
govern circadian leukocyte recruitment to tissues. Immunity 37(2):290–301
Scheiermann C, Kunisaki Y, Frenette PS (2013) Circadian control of the immune system. Nat Rev
Immunol 13(3):190–198
Schepers K, Pietras EM, Reynaud D, Flach J, Binnewies M, Garg T et al (2013) Myeloproliferative
neoplasia remodels the endosteal bone marrow niche into a self-reinforcing leukemic niche.
Cell Stem Cell 13(3):285–299
Schofield R (1978) The relationship between the spleen colony-forming cell and the haemopoietic
stem cell. Blood Cells 4(1–2):7–25
114 Y. Kunisaki

Shen Z, Lu Z, Chhatbar PY, O'Herron P, Kara P (2012) An artery-specific fluorescent dye for
studying neurovascular coupling. Nat Methods 9(3):273–276
Stier S, Ko Y, Forkert R, Lutz C, Neuhaus T, Grunewald E et al (2005) Osteopontin is a hema-
topoietic stem cell niche component that negatively regulates stem cell pool size. J Exp Med
201(11):1781–1791
Sugimura R, He XC, Venkatraman A, Arai F, Box A, Semerad C et al (2012) Noncanonical Wnt
signaling maintains hematopoietic stem cells in the niche. Cell 150(2):351–365
Sugiyama T, Kohara H, Noda M, Nagasawa T (2006) Maintenance of the hematopoietic stem cell
pool by CXCL12-CXCR4 chemokine signaling in bone marrow stromal cell niches. Immunity
25(6):977–988
Takakura N (2012) Guest editorial: mutual relationship between vascular biology and hematology.
Int J Hematol 95(2):117–118
Walkley CR, Olsen GH, Dworkin S, Fabb SA, Swann J, McArthur GA et al (2007a) A
microenvironment-­ induced myeloproliferative syndrome caused by retinoic acid receptor
gamma deficiency. Cell 129(6):1097–1110
Walkley CR, Shea JM, Sims NA, Purton LE, Orkin SH (2007b) Rb regulates interactions between
hematopoietic stem cells and their bone marrow microenvironment. Cell 129(6):1081–1095
Wilson A, Laurenti E, Oser G, van der Wath RC, Blanco-Bose W, Jaworski M et al (2008)
Hematopoietic stem cells reversibly switch from dormancy to self-renewal during homeostasis
and repair. Cell 135(6):1118–1129
Winkler IG, Barbier V, Nowlan B, Jacobsen RN, Forristal CE, Patton JT et al (2012) Vascular niche
E-selectin regulates hematopoietic stem cell dormancy, self renewal and chemoresistance. Nat
Med 18(11):1651–1657
Yamamoto R, Morita Y, Ooehara J, Hamanaka S, Onodera M, Rudolph KL et al (2013) Clonal
analysis unveils self-renewing lineage-restricted progenitors generated directly from hemato-
poietic stem cells. Cell 154(5):1112–1126
Yoneda T, Hiraga T (2005) Crosstalk between cancer cells and bone microenvironment in bone
metastasis. Biochem Biophys Res Commun 328(3):679–687
Zhou X, Zhang Z, Feng JQ, Dusevich VM, Sinha K, Zhang H et al (2010) Multiple functions of
Osterix are required for bone growth and homeostasis in postnatal mice. Proc Natl Acad Sci U
S A 107(29):12919–12924
Chapter 7
Cochlear Capillary Pericytes

Martin Canis and Mattis Bertlich

Abstract Capillary pericytes in the cochlea of mammals are—compared to peri-

cytes in other tissues, like the CNS—relatively poorly researched. To begin with,
there is still a considerable debate as to whether the very last precapillary arterioles
should—due to their contractile properties—may be considered to be pericytes.
However, cochlear capillary pericytes have shifted into the center of attention in
the past decade. Most mammals show a considerable number of pericytes in the
stria vascularis of the cochlea—up to 1300 in a mouse alone. This high number may
be explained by the observation that cochlear capillary pericytes may be differenti-
ated into different subgroups, depending on the immune markers that are expressed
by them. Corresponding with these subpopulations, cochlear pericytes fulfill three
core functions in the physiology of the cochlea:
• Formation of the intrastrial blood-fluid barrier—Pericytes monitor the ion, fluid,
and nutrient household and aid in the homeostasis thereof.
• Regulation of cochlear blood flow—By contraction on relaxation, pericytes con-
tribute to the regulation of cochlear blood flow, a paramount function parameter
of the cochlea.
• Immune response—Pericytes actually contribute to the immune response in
inflammation of the cochlea.
Due to these central roles in the physiology of the cochlea, pericytes actually
play a major role in numerous cochlear pathologies, including, but not limited to,
sudden sensorineural hearing loss, acoustic trauma, and inflammation of the cochlea.

Keywords Capillary pericytes · Cochlea · Cochlear blood flow · Cochlear blood

flow regulation · Strial blood-fluid barrier · Immune response · Fluid homeostasis ·
Cochlear pathology

M. Canis · M. Bertlich (*)

The Department of Otorhinolaryngology, Head and Neck Surgery, University Hospital,
Munich, Federal Republic of Germany
e-mail: [email protected]

© Springer Nature Switzerland AG 2019 115

A. Birbrair (ed.), Pericyte Biology in Different Organs,
Advances in Experimental Medicine and Biology 1122,
https://doi.org/10.1007/978-3-030-11093-2_7
116 M. Canis and M. Bertlich

7.1 Introduction

Pericytes in the cochlea are generally considered to be those cells that adhere to the
outer wall of capillaries of the stria vascularis or the spiral ligament. While pericytes
in many other tissues have been characterized relatively well, the function of
cochlear capillary pericytes remains widely unclear.
In the cochlea, there are two distinct subpopulations of pericytes—those that
adhere to the capillaries of the stria vascularis and those that adhere to the capillaries
of the spiral ligament. Both show remarkable differences in terms of cellular mark-
ers these express—while the pericytes of the stria vascularis are very rich in the
structural protein desmin, those of the spiral ligament show a rich expression of
contractile proteins such as smooth muscle actin and tropomyosin. It is probable
that while those pericytes that are rich in desmin fulfill a structural purpose, those
rich in contractile proteins may take an active part in regulating cochlear
microcirculation.
In the following chapter, we therefore discuss the role of cochlear capillary peri-
cytes in various pathologies of the cochlea, like physical or acoustic trauma, bacte-
rial inflammation, or sudden sensorineural hearing loss. Moreover, we discuss the
signaling pathways involved and the role each pericyte subpopulation plays in these
pathologies.
Finally, we will discuss the pharmacological properties of pericytes in the
cochlea and give an outlook as to where research on cochlear capillary pericytes
may head in the following years.

7.2 Main Text

7.2.1 What Is a Cochlear Pericyte?

Generally speaking, pericytes are cells that adhere to the external walls of capillar-
ies and postcapillary venules; they appear in most tissues when energy demand is
high and a dense net of capillaries is present, e.g., the central nervous system (CNS)
or the kidneys. Knowing this, it is not surprising that pericytes also appear in the
stria vascularis as well as the spiral ligament of the cochlea.
However, as the recent debate about the pericytes of the CNS has pointed out
(Hill et al. 2015; Attwell et al. 2016), there is no black-and-white line as to what is
already or still a pericyte and what is a functionally or morphologically similar cell,
but not yet a pericyte. For example, while some authors have decided to already
consider the precapillary arteriolar cells to be pericytes (Attwell et al. 2016), many
authors do not agree with this division (Hill et al. 2015; Fernandez-Klett et al. 2010).
However, this is not so much the case in the cochlea, where a relatively common
7 Cochlear Capillary Pericytes 117

Fig. 7.1 Capillary pericytes in the stria vascularis of a guinea pig, visualized by in vivo fluores-
cence microscopy; pericytes were marked with topical application diaminofluorescein-
2-diacetate; vessels of the stria vascularis were contrasted by i.v. injection of
fluorescein-isothiocyanate-dextran

definition has been agreed upon: pericytes are commonly considered to be those
cells that explicitly adhere to the outer walls of the capillaries of the stria vascularis
or the spiral ligament and express a defined set of cellular markers (Fig. 7.1) (Dai
et al. 2009; Shi et al. 2008).

7.2.2 Blood Supply of the Cochlea

Blood supply to the cochlea is of paramount importance for the function of the
organ; in particular the endocohlear potential (Lamm and Arnold 2000) and the
subsequent production of endolymph directly correlate to steady blood flow in the
stria vascularis (Shi 2011). The entire blood supply to the cochlea makes up 10−7 of
the entire cardiac output (Nakashima et al. 2003) and derives from the anterior infe-
rior cerebellar artery, from which the spiral modiolar artery branches off, radiating
over the scala vestibuli and across the spiral lamina. During its course, the spiral
modiolar artery has numerous radial branches that eventually form the capillaries of
the stria vascularis and the spiral ligament (Shi 2011).
118 M. Canis and M. Bertlich

7.2.3 Morphology and Cellular Markers

Cochlear pericytes are found numerously (up to 1300 in the cochlea of a C57/BL6
mouse) (Neng et al. 2015) and exhibit a very heterogeneous range in terms of mor-
phology and function in the cochlea. Generally speaking, pericytes of the cochlea
commonly express platelet-derived growth factor receptor ß, desmin, neural proteo-
glycan 2, and CD90. However, those pericytes that are commonly found in the spi-
ral ligament typically express connexin 40, a gap junction protein, α-smooth muscle
actin (α-SMA), and tropomyosin, while those present at capillaries of the stria vas-
cularis regularly show a rich expression of desmin without any expression of α-SMA
or tropomyosin (Shi 2011). This is a defining difference between these different
subpopulations of cochlear capillary pericyte and may be indicative of different
functions of the cochlear pericytes.

7.2.4 Physiological Functions of Cochlear Capillary Pericytes

Cochlear pericytes form part of the intrastrial blood-fluid barrier that controls the
exchange between the intrastitial space and blood. The main objective of this highly
specialized network is to shield intrastitial space from any toxins and other poten-
tially harmful substances that might be dissolved in the blood, just like the blood-­
brain barrier. Precise regulation of the transport into and out of the intrastrial space
is also paramount for maintaining exact composition of the inner ear fluids (Juhn
et al. 2001) and therefore, ultimately, the ability to hear. Impairment of the integrity
of the blood labyrinth barrier has been associated with numerous inner ear patholo-
gies, including suppurative labyrinthitis (Zhang et al. 2015), acoustic trauma (Shi
2009), and age-related hearing loss (Neng et al. 2015).
The intrastrial blood-fluid barrier is mainly formed by endothelial cells, which
are connected by tight junctions, and the underlying basement membrane. However,
pericytes do contribute in a large number to the formation of the barrier (Shi et al.
2008; Shi 2009). By means of close biochemical and anatomical linkage, pericytes
monitor the ion, fluid, and nutrient household of the stria vascularis and may respond
accordingly (Peppiatt et al. 2006; Hall et al. 2014; Shi 2016).
Additionally, cochlear pericytes seem to be directly involved in the regulation of
cochlear microcirculation in the stria vascularis. Since these pericytes evidently
show the ability to contract (Dai et al. 2009) and may even do so upon presentation
of a physiological stimulus (Dai et al. 2009; Bertlich et al. 2017a) as well as express-
ing contractile proteins(Dai et al. 2009), a role in the local regulation of blood flow
in the stria vascularis seems very likely. Fittingly, a very similar phenomenon has
been reported with the capillary pericytes of the central nervous system, where peri-
cytes may locally increase or decrease cochlear blood flow according to local
demand (Peppiatt et al. 2006).
7 Cochlear Capillary Pericytes 119

Additionally, pericytes may actually contribute to the immune response in the

stria vascularis. Pericytes have been reported to show very close interlinking with
perivascular-resident macrophage-like melanocytes (Zhang et al. 2013), cells that
contribute to the immune reaction of the stria vascularis. Since pericytes, like the
perivascular-resident macrophage-like melanocytes, show a considerable reaction
after stimulation with bacterial lipopolysaccharide (Zhang et al. 2013, 2015), a con-
tribution of pericytes to the immune response seems likely.

7.2.5 Pathophysiology of Cochlear Pericytes

It has been established that cochlear capillary pericytes contribute to the regulation
of cochlear blood flow; however, (permanent) impairment of cochlear blood flow
has repeatedly been discussed as the final common pathological pathway in numer-
ous entities. Among those are acoustic trauma, where longtime exposure to noise
may lead to increased oxygen demand of the cochlea, eventually causing a relative
lack of oxygen (Arpornchayanon et al. 2013). A longer-lasting lack of oxygen even-
tually activates the TNF pathway, which is known to cause pericytes to contract and
eventually reduce cochlear blood flow (Bertlich et al. 2017a; Arpornchayanon et al.
2013).
In addition to this, the pericytes alter their physical configuration after longer-­
lasting exposure to loud noise—they migrate from the endothelial wall and express
significantly elevated levels of the structural protein desmin (Shi 2009). This migra-
tion of pericytes from the endothelial cells is known to decrease the integrity of the
blood-fluid barrier of the cochlea (Shi 2016).
In addition to this, impairment of cochlear blood flow has been discussed to be
the leading cause for sudden sensorineural hearing loss (SSNHL). It has been
observed that fibrinogen, a serum protein that is part of the plasmatic coagulation
system, is both a risk factor for and a potential treatment target for SSNHL (Suckfull
2002). Since increased fibrinogen levels have been shown to impair hearing thresh-
olds as well as microcirculation (Ihler et al. 2012a) [and thus partial oxygen pres-
sure (Lamm and Arnold 2000)], a contribution of cochlear capillary pericytes is
very probable.
Fittingly, it has been shown that capillary pericytes of the CNS typically exhibit
a rigor mortis-like state after continuous insufficient partial oxygen pressures and
thus continuously impair cochlear blood flow (Hall et al. 2014). Assuming that the
cochlear capillary pericytes exhibit similar properties, this could also explain why
when causal treatment is administered, hearing thresholds may return to basal val-
ues without any permanent alterations (Weiss et al. 2017).
Cochlear capillary pericytes also play a major role in bacterial suppurative laby-
rinthitis, a severe complication of inflammation of the middle ear. Inflammation of
the middle ear is commonly caused by gram-positive bacteria such as Streptococcus
pneumoniae or Streptococcus pyogenes—bacteria which are known to secrete
120 M. Canis and M. Bertlich

exotoxins called streptolysin or pneumolysin. In addition to this, the gram-negative

bacteria that regularly cause otitis media, like Escherichia coli, Haemophilus influ-
enzae, and Pseudomonas aeruginosa, are known to contain an endotoxin, lipopoly-
saccharide, which is known to be set free during an immune response. In suppurative
labyrinthitis, a transition of these toxins into the cochlea has repeatedly been
hypothesized (Ishihara et al. 2016; Buckiova et al. 2012). Since both the exotoxin
and the endotoxin are well known to activate the tumor necrosis factor alpha path-
way, impairment of cochlear blood flow (Ihler et al. 2013) by pericyte contraction is
most likely. Moreover, there is also a second role which pericytes play in suppura-
tive labyrinthitis. Capillary pericytes significantly contribute to the upkeep of the
blood-fluid barrier of the inner ear. In otitis media that has been caused by injection
of lipopolysaccharide into the tympanic cavity, pericytes show significant migration
from the vessel wall, their original site, and thus destabilize the blood-fluid barrier
(Zhang et al. 2015). This observation is similar to those made in acoustic trauma
(Shi 2009) as well as other tissues like the retina (Pfister et al. 2008).
Finally, cochlear capillary pericytes probably also contribute in the immune
response to physical trauma to the cochlea. This is of particular importance since the
insertion of an electrode during the surgical implantation of a cochlear implant is a
procedure that is becoming more and more common in otorhinolaryngology (Wang
et al. 2014). Surgical placement of the electrode of the cochlear implant inside the
cochlea commonly causes direct damage to the basilar membrane (Roland and
Wright 2006) as well as causing a local, abacterial inflammation. Together, both
pose a risk for residual hearing in patients undergoing cochlear implants. However,
it has been shown that application of etanercept, a fusion protein inhibiting the TNF
pathway, is effective in preserving residual hearing under cochlear electrode implan-
tation in an animal model (Ihler et al. 2014). Thus, an involvement of cochlear peri-
cytes seems very probable. Fittingly, similar changes in pericyte morphology have
been described in the central nervous system reacting to physical trauma (Dore-­
Duffy et al. 2000).
Overall, it seems evident that both the impairment of cochlear blood flow and the
breakdown of the cochlear blood-fluid barrier play a major role in the pathophysiol-
ogy of numerous pathologies of the inner hear. However, it is unlikely that both
mechanisms are separate reactions to different stimuli. It is much more likely that
both reactions—impairment of cochlear blood flow and breakdown of the blood-­
fluid barrier of the cochlea—are distinct reactions to inflammatory stimuli. Due to
the considerable heterogeneity of the pericytes (Dai et al. 2009; Shi et al. 2008), it
seems probably that different subpopulations that are present in the stria vascularis
or the spiral ligament react to the same stimulus in a different manner, according to
each subpopulation specification. This would be in line with the different immuno-
histochemical markers that have been described for these populations and would
make both reactions that have been described different sides of the same medal.
Bearing this in mind, it is more than likely that pericytes are involved in numer-
ous more pathologies of the inner ear, like radiation-induced hearing loss (Mujica-­
Mota et al. 2014), chemotoxicity (Sheth et al. 2017; Jeong et al. 2007), and also
possibly hereditary hearing impairments.
7 Cochlear Capillary Pericytes 121

7.2.6  harmacological Features of Cochlear Capillary

P
Pericytes

The aforementioned properties make pericytes valid targets for pharmacotherapy of

numerous inner ear pathologies. For example, the neutralization of tumor necrosis
factor alpha by the use of etanercept seems to be a promising treatment for many of
the aforementioned diseases; in addition to this, testing of various other remedies is
still undergoing both in vitro (Jeong et al. 2007) and the animal model (Bertlich
et al. 2017b; Sharaf et al. 2016). In addition to this, it has been hypothesized that the
blood flow-promoting effect of betahistine on cochlear microcirculation (Ihler et al.
2012b; Bertlich et al. 2014) is mediated by pericytes. However, it has been shown
that this is not the case, as this effect seems to be mediated by precapillary arterioles
(Bertlich et al. 2017c). However, several authors do already consider these cell pop-
ulations to be pericytes in other tissues (Attwell et al. 2016).

7.3 Outlook and Future Trends

When it comes to where research in cochlear capillary pericytes is heading, the most
important findings are the differences but more importantly the similarities to the
capillary pericytes of the central nervous system. Many functions of the pericytes of
the central nervous have been described, and it will be crucial to see of the pericytes
of the cochlea behave the same or in a different manner.
The most important aspect in this respect will be the role of pericytes in the dam-
age to the stria vascularis and the spiral ligament. This includes proliferation and
migration in response to injury—in particular in the long term—as well as regenera-
tive and in particular stem cell properties of pericytes, since all these are consider-
ably better described in pericytes of the central nervous system. A better
understanding of the role of pericytes in the (patho)physiology of the cochlea will
lead to a better understanding of cochlear homeostasis and function overall.
In addition to this, the selective pharmacological targeting of relevant structures
within the cochlea—and this explicitly includes pericytes—will also see a steady
increase.

References

Arpornchayanon W, Canis M, Ihler F, Settevendemie C, Strieth S (2013) TNF-alpha inhibition

using etanercept prevents noise-induced hearing loss by improvement of cochlear blood flow
in vivo. Int J Audiol 52:545–552. https://doi.org/10.3109/14992027.2013.790564
Attwell D, Mishra A, Hall CN, O’Farrell FM, Dalkara T (2016) What is a pericyte? J Cereb Blood
Flow Metab 36:451–455
122 M. Canis and M. Bertlich

Bertlich M, Ihler F, Sharaf K, Weiss BG, Strupp M, Canis M (2014) Betahistine metabolites,
Aminoethylpyridine, and Hydroxyethylpyridine increase cochlear blood flow in Guinea pigs
in vivo. Int J Audiol 53:753–759. http://www.ncbi.nlm.nih.gov/pubmed/25014609
Bertlich M, Ihler F, Weiss BG, Freytag S, Strupp M, Canis M (2017a) Cochlear Pericytes are
capable of reversibly decreasing capillary diameter in vivo after tumor necrosis factor expo-
sure. Otol Neurotol 38:e545–e550
Bertlich M, Ihler F, Weiss BG, Freytag S, Jakob M, Strupp M et al (2017b) Fingolimod (FTY-720)
is capable of reversing tumor necrosis factor induced decreases in Cochlear blood flow. Otol
Neurotol 38:1213–1216
Bertlich M, Ihler F, Weiss BG, Freytag S, Strupp M, Jakob M et al (2017c) Role of capillary peri-
cytes and precapillary arterioles in the vascular mechanism of betahistine in a Guinea pig inner
ear model. Life Sci 187:17–21
Buckiova D, Ranjan S, Newman TA, Johnston AH, Sood R, Kinnunen PKJ et al (2012) Minimally
invasive drug delivery to the cochlea through application of nanoparticles to the round window
membrane. Nanomedicine (Lond) 7:1339–1354
Dai M, Nuttall A, Yang Y, Shi X (2009) Visualization and contractile activity of cochlear pericytes
in the capillaries of the spiral ligament. Hear Res 254:100–107
Dore-Duffy P, Owen C, Balabanov R, Murphy S, Beaumont T, Rafols JA (2000) Pericyte migra-
tion from the vascular wall in response to traumatic brain injury. Microvasc Res 60:55–69
Fernandez-Klett F, Offenhauser N, Dirnagl U, Priller J, Lindauer U (2010) Pericytes in capillaries
are contractile in vivo, but arterioles mediate functional hyperemia in the mouse brain. Proc
Natl Acad Sci U S A 107:22290–22295
Hall CN, Reynell C, Gesslein B, Hamilton NB, Mishra A, Sutherland BA et al (2014) Capillary
pericytes regulate cerebral blood flow in health and disease. Nature 508:55–60. https://doi.
org/10.1038/nature13165
Hill RA, Tong L, Yuan P, Murikinati S, Gupta S, Grutzendler J (2015) Regional blood flow in the
Normal and ischemic brain is controlled by arteriolar smooth muscle cell contractility and not
by capillary Pericytes. Neuron 87:95–110
Ihler F, Strieth S, Pieri N, Gohring P, Canis M (2012a) Acute hyperfibrinogenemia impairs cochlear
blood flow and hearing function in Guinea pigs in vivo. Int J Audiol 51:210–215. https://doi.
org/10.3109/14992027.2011.622302
Ihler F, Bertlich M, Sharaf K, Strieth S, Strupp M, Canis M (2012b) Betahistine exerts a dose-­
dependent effect on cochlear stria vascularis blood flow in Guinea pigs in vivo. PLoS One
7:e39086
Ihler F, Sharaf K, Bertlich M, Strieth S, Reichel CA, Berghaus A et al (2013) Etanercept prevents
decrease of cochlear blood flow dose-dependently caused by tumor necrosis factor alpha. Ann
Otol Rhinol Laryngol 122:468–473
Ihler F, Pelz S, Coors M, Matthias C, Canis M (2014) Application of a TNF-alpha-inhibitor into the
scala tympany after cochlear electrode insertion trauma in Guinea pigs: preliminary audiologic
results. Int J Audiol 53:810–816. https://doi.org/10.3109/14992027.2014.938369
Ishihara H, Kariya S, Okano M, Zhao P, Maeda Y, Nishizaki K (2016) Expression of macrophage
migration inhibitory factor and CD74 in the inner ear and middle ear in lipopolysaccharide-­
induced otitis media. Acta Otolaryngol 136:1011–1016
Jeong H-J, Kim S-J, Moon P-D, Kim N-H, Kim J-S, Park R-K et al (2007) Antiapoptotic mecha-
nism of cannabinoid receptor 2 agonist on cisplatin-induced apoptosis in the HEI-OC1 audi-
tory cell line. J Neurosci Res 85:896–905
Juhn SK, Hunter BA, Odland RM (2001) Blood-labyrinth barrier and fluid dynamics of the inner
ear. Int Tinnitus J 7:72–83
Lamm K, Arnold W (2000) The effect of blood flow promoting drugs on cochlear blood flow, peri-
lymphatic pO(2) and auditory function in the normal and noise-damaged hypoxic and ischemic
Guinea pig inner ear. Hear Res 141:199–219
Mujica-Mota MA, Lehnert S, Devic S, Gasbarrino K, Daniel SJ (2014) Mechanisms of radiation-­
induced sensorineural hearing loss and radioprotection. Hear Res 312:60–68
7 Cochlear Capillary Pericytes 123

Nakashima T, Naganawa S, Sone M, Tominaga M, Hayashi H, Yamamoto H et al (2003) Disorders

of cochlear blood flow. Brain Res Brain Res Rev 43:17–28
Neng L, Zhang J, Yang J, Zhang F, Lopez IA, Dong M et al (2015) Structural changes in thestrial
blood-labyrinth barrier of aged C57BL/6 mice. Cell Tissue Res 361:685–696
Peppiatt CM, Howarth C, Mobbs P, Attwell D (2006) Bidirectional control of CNS capillary diam-
eter by pericytes. Nature 443:700–704
Pfister F, Feng Y, vom Hagen F, Hoffmann S, Molema G, Hillebrands J-L et al (2008) Pericyte
migration: a novel mechanism of pericyte loss in experimental diabetic retinopathy. Diabetes
57:2495–2502
Roland PS, Wright CG (2006) Surgical aspects of cochlear implantation: mechanisms of inser-
tional trauma. Adv Otorhinolaryngol 64:11–30
Sharaf K, Ihler F, Bertlich M, Reichel CA, Berghaus A, Canis M (2016) Tumor necrosis factor-­
induced decrease of Cochlear blood flow can be reversed by Etanercept or JTE-013. Otol
Neurotol 37:e203–e208
Sheth S, Mukherjea D, Rybak LP, Ramkumar V (2017) Mechanisms of Cisplatin-induced ototox-
icity and Otoprotection. Front Cell Neurosci 11:338
Shi X (2009) Cochlear pericyte responses to acoustic trauma and the involvement of hypoxia-­
inducible factor-1alpha and vascular endothelial growth factor. Am J Pathol 174:1692–1704
Shi X (2011) Physiopathology of the cochlear microcirculation. Hear Res 282:10–24
Shi X (2016) Pathophysiology of the cochlear intrastrial fluid-blood barrier (review). Hear Res
338:52–63
Shi X, Han W, Yamamoto H, Tang W, Lin X, Xiu R et al (2008) The cochlear pericytes.
Microcirculation 15:515–529
Suckfull M (2002) Fibrinogen and LDL apheresis in treatment of sudden hearing loss: a ran-
domised multicentre trial. Lancet (London, England) 360:1811–1817
Wang JT, Wang AY, Psarros C, Da Cruz M (2014) Rates of revision and device failure in cochlear
implant surgery: a 30-year experience. Laryngoscope 124:2393–2399
Weiss BG, Bertlich M, Bettag SA, Desinger H, Ihler F, Canis M (2017) Drug-induced
Defibrinogenation as new treatment approach of acute hearing loss in an animal model for
inner ear vascular impairment. Otol Neurotol 38:648–654
Zhang F, Zhang J, Neng L, Shi X (2013) Characterization and inflammatory response of
perivascular-­resident macrophage-like melanocytes in the vestibular system. J Assoc Res
Otolaryngol 14:635–643
Zhang J, Chen S, Hou Z, Cai J, Dong M, Shi X (2015) Lipopolysaccharide-induced middle ear
inflammation disrupts the cochlear intra-strial fluid-blood barrier through down-regulation of
tight junction proteins. PLoS One 10:e0122572
Chapter 8
Pericytes in the Placenta: Role in Placental
Development and Homeostasis

Rodrigo S. N. Barreto, Patricia Romagnolli, Andressa Daronco Cereta,

Leda M. C. Coimbra-Campos, Alexander Birbrair,
and Maria Angelica Miglino

Abstract The placenta is the most variable organ, in terms of structure, among the
species. Besides it, all placental types have the same function: production of viable
offspring, independent of pregnancy length, litter number, or invasion level. The
angiogenesis is a central mechanism for placental functionality, due to proper
maternal-fetal communication and exchanges. Much is known about the vasculature
structure, but little is known about vasculature development and cellular interac-
tions. Pericytes are perivascular cells that were described to control vasculature sta-
bility and permeability. Nowadays there are several new functions discovered, such
as lymphocyte modulation and activation, macrophage-like phagocytic properties,
tissue regenerative and repair processes, and also the ability to modulate stem cells,
majorly the hematopoietic. In parallel, placental tissues are known to be a particu-
larly immune microenvironment and a rich stem cell niche. The pericyte function
plethora could be similar in the placental microenvironment and could have a cen-
tral role in placental development and homeostasis.

Keywords Capillary system · Maternal-fetal communication · Placental vascular-

ization · Placentation · Perivascular cell

R. S. N. Barreto · P. Romagnolli · A. D. Cereta · M. A. Miglino (*)

School of Veterinary Medicine and Animal Sciences, University of São Paulo,
Butantã, Sao Paulo, Brazil
e-mail: [email protected]
L. M. C. Coimbra-Campos
Department of Pathology, Federal University of Minas Gerais,
Pampulha, Belo Horizonte, Brazil
A. Birbrair
Department of Radiology, Columbia University Medical Center, New York, NY, USA
Department of Pathology, Federal University of Minas Gerais, Pampulha,
Belo Horizonte, Brazil

© Springer Nature Switzerland AG 2019 125

A. Birbrair (ed.), Pericyte Biology in Different Organs,
Advances in Experimental Medicine and Biology 1122,
https://doi.org/10.1007/978-3-030-11093-2_8
126 R. S. N. Barreto et al.

8.1 Introduction

The eutherian mammals had a common ancestor that had the placenta with hemot-
rophic and histiotrophic bipotential (Vogel 2005). From this ancestor, all phyloge-
netical clades evolved. In the lower clades (Afrotheria and Xenarthra), there are
only mammals with hemotrophic placentation or the ones whose mothers have low
or no control of blood support to the fetus (Murphy et al. 2001; Vogel 2005; Carter
and Mess 2007). However, in the upper clades (Euarchontoglires and Laurasiatheria),
there are also mammals with histiotrophic placentation, where maternal blood ves-
sels remain intact and the mother controls the blood supply (Murphy et al. 2001;
Vogel 2005; Carter and Mess 2007). The appearance of less invasive placentas in
upper clades, an evolutionary response, maybe to produce a more efficient placenta,
by means of maternal blood control (Mess and Carter 2007), maternal-fetal transfer
(Leiser and Kaufmann 1994), and increasing immune system barrier (Moffett and
Loke 2006). The mechanisms that lead to the eutherian diversification are still not
completely known; however, the genes that control the early embryo and placental
development are largely phylogenetically conserved (Knox and Baker 2008).
Regarding placenta-specific gene expression, human and cattle are more genetically
similar than human and mouse, even with several morphological differences in
between (Barreto et al. 2011). The plethora of the placenta’s gross and fine morpho-
logical characteristics is distributed in mammals, although in all of them the same
general function is achieved: production of healthy offspring. Then, in this chapter,
we will discuss the placental diversity and speculate some possible roles of peri-
cytes in placental development.

8.2 Placental Variability

In several mammals, placental morphology was thoroughly revised by Mossman

(1987) and more recently by Wooding and Burton (2008a); also an actualization of
nomenclature was made by Leiser and Kaufmann (1994). Herein, we will focus on
some general morphological differences and afterward, in more detail, on the pla-
cental vascularization.
The placenta could be classified by the (1) arrangement of extraembryonic mem-
branes, (2) gross morphological shape of maternal-fetal communication, (3) fine
arrangement of maternal-fetal interdigitation, (4) number of tissue layers between
maternal and fetal bloodstreams, and (5) arrangement of maternal and fetal vessels
(Leiser and Kaufmann 1994).
The four extraembryonic membranes could be in contact with the uterus and
arranged in several ways. The amnion, the non-vascularized and in close contact
with the fetus. The yolk sac is transitory in some species and well developed in oth-
ers, arising from the midgut as an endothelial and hematopoietic stem cell niche.
The allantois arises from the hindgut and is the analogous extraembryonic urinary
8 Pericytes in the Placenta: Role in Placental Development and Homeostasis 127

bladder, being also highly vascularized. And the chorion, which is derived from the
trophectoderm, initially has its own capillary system, later fusing with allantois ves-
sels, in several species, and it is the outer membrane being the first barrier (Leiser
and Kaufmann 1994).
The arrangement of those four membranes could produce four models:
1. Chorionic placenta, present in early phases of all eutherians, with the chorion in
major contact with the uterus.
2. Choriovitelline placenta, showing the chorion connected to vitelline vasculariza-
tion, i.E., parts of Perissodactyla and carnivore placenta.
3. Chorioallantoic placenta, where the chorion is connected to allantoic vascular-
ization, i.E., the predominant area in several eutherians.
4. Vitelline placenta, with the fetal circulation connected to the vitelline vessels,
i.E., the area of rodent placenta, fish, and amphibia (Mossman 1987; Leiser and
Kaufmann 1994; Wooding and Flint 1994; Wooding and Burton 2008a).
The maternal-fetal communication units could be spread across all the uterine
wall extension or restricted to some area(s), forming an intense and deep contact or
only a tenuous apposition of tissues. In the diffuse placenta, the maternal-fetal com-
munication is spread (or diffused) by the entire uterine wall and just with apposition
of maternal-fetal tissues, i.e., in pigs, dolphins, Perissodactyla, and some lower pri-
mates. In the cotyledonary placenta, the communication is organized in specialized
areas intercalated by smooth areas and distributed along almost all the uterine wall,
i.e., ruminants. In the zonary placenta, the communication is organized in a belt sur-
rounding all the uterine wall inner circumference, i.e., most carnivores. Being more
restricted, in the bidiscoidal placenta, the communication is organized in only two
discs in close contact, i.e., some lower and higher primates. The highest concentra-
tion of communication is reached with the discoidal placenta, where only one disc
is apparent, i.e., rodents, lagomorphs, great apes, and humans (Mossman 1987;
Leiser and Kaufmann 1994; Wooding and Flint 1994; Wooding and Burton 2008a).
Microscopically, the maternal-fetal communication units could be arranged as:
1. Folded, when the uterine wall and chorion are folded, the simplest one, i.e., mar-
supials and some lower primates.
2. Lamellar, with the uterine wall and chorion forming some branched folds, i.e.,
carnivores.
3. Villous, in which the chorion forms inverted tree shape inside the uterine wall,
i.e., ruminants, horses, higher primates, and humans.
4. Trabecular, a mixture of lamellar and villous types, i.e., some new-world
monkeys.
5. Labyrinthine, most complex, where the chorion is bathed in channels or lacunas
of the maternal bloodstream, i.e., rodents, lagomorphs, and some lower primates
(Mossman 1987; Leiser and Kaufmann 1994; Wooding and Flint 1994; Wooding
and Burton 2008a).
In the maternal-fetal communication, six tissue layers could be present, three on
the fetal side (trophoblast, mesenchyme, and fetal endothelium) and three others on
128 R. S. N. Barreto et al.

the maternal side (endometrial epithelium, endometrial stroma, and maternal endo-
thelium). Moreover, different invasive deepness also happens between placental
types, resulted by losing maternal tissue layers. In the noninvasive epitheliochorial
placenta, all six tissue layers are preserved forming a complete barrier, i.e., lower
primates, horses, and pigs. A specialization of this type, synepitheliochorial, pres-
ents migratory cells that detach from trophoblast and fuse with endometrial epithe-
lium, conferring tenuous invasive behavior (Pereira et al. 2013), i.e., ruminants.
Going deeper, in the endotheliochorial placenta, the chorion stays in contact with
maternal endothelium, i.e., carnivores. The most intimate contact is made by hemo-
chorial placenta, in which the chorion is directly batched by maternal blood,
although one to three trophoblast layers could be present in hemomonochorial, i.e.,
caviomorph rodents, great apes, and humans at term; hemodichorial, i.e., lago-
morphs and human; and hemotrichorial, i.e., some rodents, respectively (Mossman
1987; Leiser and Kaufmann 1994; Wooding and Flint 1994; Wooding and Burton
2008a).
The arrangement of maternal and fetal blood at arterial and venous levels (mac-
roflow) is countercurrent, with some rare exceptions, where maternal and fetal ves-
sels are in parallel and flow in opposite directions. However, at capillary level
(microflow), the arrangement of maternal and fetal capillaries varies. It could be
also countercurrent, efficient for passive diffusion, and present in small placentas,
i.e., rodents and lagomorphs. Also, two other arrangements are possible, the cross-
current flow, in which the maternal and fetal capillaries are arranged in transversal
directions, i.e., carnivores and lower primates, and the multivillous flow which is a
mixture of countercurrent and crosscurrent flows, i.e., ruminants, higher primates,
and humans (Mossman 1987; Leiser and Kaufmann 1994; Wooding and Flint 1994;
Wooding and Burton 2008a).

8.3 Placental Blood Flow

As discussed above, the placental morphology has a plethora of differences among

species; consequently, the structure of maternal and fetal blood capillaries in the
communication units varies in accordance with other morphological variations to
maintain the efficiency of gas, nutrient, and waste exchanges (Mossman 1987;
Leiser and Kaufmann 1994; Wooding and Flint 1994; Wooding and Burton 2008a).
On the fetal side, the capillary bed is formed by non-fenestrated capillaries supplied
by a fetal arteriole (Wooding and Burton 2008a). However, the phylogenetical evo-
lution of eutherians modified majorly the maternal tissue arrangement, generating
four different contact levels between trophoblast and maternal blood, resulting in
significant differences in maternal blood flow organization and control. From the
most distant to the closest contact, the epitheliochorial placenta is formed by two
complex capillary beds (Enders et al. 1998). In the synepitheliochorial and
8 Pericytes in the Placenta: Role in Placental Development and Homeostasis 129

endotheliochorial, there is modification or elimination of the endometrial epithe-

lium, whereas in the hemochorial, observed in humans and rodents, the endometrial
epithelium, the connective tissue, and endothelium are no longer present, and mater-
nal blood is delivered in maternal blood spaces (Heinrich et al. 1988; Kaufmann
et al. 1988).
In addition, in hemochorial, maternal arteries are reminiscent; however, their
endothelium is replaced by migratory and invasive trophoblast cells. In humans,
those arteries end abruptly at maternal blood spaces without channels to carry to the
fetal surface (Wooding and Flint 1994). In hemochorial placentas, no exact control
of maternal blood flow is possible, due to the absence of maternal arterial precapil-
lary sphincters regulated by vasoactive compounds (i.e., estrogen, NO, prostacyclin,
relaxin, and calcitonin) that are present in other placental types (Wooding and Flint
1994; Thornburg et al. 2006). Also, as other placental types, no neural innervation
is present to control vasoconstriction or dilatation (Lachenmayer 1971a; Nikolov
and Schiebler 1973; Myatt 1992), maybe by myometrial axon degeneration medi-
ated by estrogen (Mónica Brauer and Smith 2015). Then, metabolic factors proba-
bly control the vascular walls (Lachenmayer 1971b; Heinrich et al. 1988), and
myometrial contractility could reduce the blood flow in intervillous spaces in hemo-
chorial placentas (Ramsey et al. 1967).
Throughout the pregnancy, both the placenta and fetus increase in size, and, as a
result, more nutrients are needed for development and maintenance of those tissues
(Baur 1977). Firstly, the mother increases her blood volume, blood flow, and cardiac
output to be able to perfuse the developing placenta (Wooding and Burton 2008a).
In humans, the blood volume increases around 1.5 to 2.0 L, and the cardiac output
is around 50% (Thornburg et al. 2006); however, hemochorial placenta presents
lower maternal blood rates than epithelio- or synepitheliochorial placentas (Wooding
and Burton 2008a). As another mechanism, in some primates at late pregnancy,
anastomoses are present between ovarian and uterine arteries to increase the mater-
nal placental blood influx (Wehrenberg et al. 1977), such as in viscacha, a South
American rodent (unpublished data). On the other side, umbilical blood flow needs
to increase to supply the blood channels that occupy around 50% of fetal placental
volume (Wooding and Flint 1994), but no relation of umbilical flow rate with pla-
cental type is observed (Wooding and Burton 2008a). The villous surface-placental
volume ratio also varies (i.e., from 300 cm2 kg−1 in the mare to 10,400 cm2 kg−1 in
the cat) and is lower in large animals than in small ones and higher in species with
compact placentas (cattle, rats, cats, humans) than in diffuse ones (horses, pigs)
(Baur 1977).
Altogether, placental blood flow, transport capacity, villous surface area, and
several other parameters vary greatly between eutherian species, and none of them
have direct influence in fetal growth rate (Wooding and Flint 1994), indicating that
each type of placenta has its own control mechanisms.
130 R. S. N. Barreto et al.

8.4 Placental Vascular Architecture

In comparative aspects, the vascular architecture is not well explored in the litera-
ture. Also, in the capillary system, the formation during placental development and
establishment is performed by different mechanisms and involves different placen-
tal cell population and regions (Mossman 1987; Leiser and Kaufmann 1994;
Wooding and Flint 1994; Wooding and Burton 2008a). Herein, we will discuss the
vascular architecture and formation of the capillary systems, or microvasculature, in
four different placental models: human chorioallantoic, discoidal, villous, hemo-
monochorial, and multivillous flow; mouse choriovitellinic, discoidal, labyrinthine,
hemotrichorial, and countercurrent flow; bovine chorioallantoic, cotyledonary, vil-
lous, synepitheliochorial, and multivillous flow; and feline chorioallantoic, zonary,
labyrinthine, endotheliochorial, and crosscurrent flow.

8.4.1 Human Model: Hemochorial Villous Placenta

A detailed description of the fetal vasculature in the human placenta was made by
Kaufmann et al. (1988) and could be observed in Fig. 8.1. The two umbilical arter-
ies ramify in several chorionic arteries and enter the chorionic plate. From the cho-
rionic plate, the fetal villous tree shape of communication is formed. The stem villi
(supplied by chorionic arterioles) are ramified in intermediated villi (supplied by
arterioles) and finally in terminal villi; the last have around 30 to 80 μm and are sup-
plied only by fetal capillaries that occupy half of their stroma (Kaufmann et al.
1988). In the intermediated villi, the arterioles are surrounded by only one or two
smooth muscle cell layers; however, some venules are surrounded only by pericytes
(Rhodin 1968; Kaufmann et al. 1988). Also, precapillary sphincters, or structures
with similar function, are absent, and then the arteries continue to capillaries by
reducing their luminal diameters and losing the smooth muscle cell layers. The
capillaries in the terminal villi form loops and sinusoids (only at mature placenta)
near to villi tip; those sinusoids possess continuous endothelial wall and basement
membrane, without fenestrations or pores. Functionally, those sinusoids decrease
the blood flow, favoring the fetal-maternal exchange (Arts 1961; Kaufmann et al.
1988). As terminal villi development is sensitive to oxygen concentrations, in some
hypoxia conditions (i.e., preeclampsia, intrauterine growth restriction, diabetes,
etc.), there is an increase of development of those villi and, consequently, sinusoids
(Firth and Leach 1996; Macara et al. 1996; Zygmunt et al. 2003; Kaufmann et al.
2004; van der Heijden et al. 2005; Resta et al. 2006; Burton et al. 2009; Dubova
et al. 2013). In summary, the fetal microvasculature is formed by branching of
umbilical arteries until the sinusoid in the tip of terminal villi.
However, the maternal microvasculature formation is more complex and
involves different cell populations, such as from the maternal immune system and
8 Pericytes in the Placenta: Role in Placental Development and Homeostasis 131

Fig. 8.1 Schema of human placental vascularization. In (a), human fetus and placenta. In (b),
squared area of (a), showing layers and maternal and fetal vascularization. In (c), squared area of
(b), showing maternal microvascularization. In (d), squared area of (b), showing fetal
microvascularization
132 R. S. N. Barreto et al.

even fetal trophoblast cells. Firstly, branches of uterine artery enter the uterine wall
to form the arcuate artery in the myometrium, branching again the radial artery,
and basal artery enters the endometrium to form the spiral arterioles (1955). The
spiral arterioles have no muscular and elastic tissue throughout the wall, and the
lumen is dilated and tortuous. The blood flow from spiral arterioles goes directly
to the placental bed, forming the blood lakes that are drained by openings in the
basal plate and flow by the venous system until the uterine vein (Kaufmann et al.
1988). During the progress of the pregnancy, the spiral arteriole wall is remodeled
by fetal cells with the interference of maternal immune system cells. Some migra-
tory trophoblast cells, the extravillous trophoblast (EVT), secrete human leukocyte
antigen (HLA)-G directly in the maternal blood lakes (Jiang et al. 2015) that
attracts and activates uterine natural killer (uNK) cells. Then, uNK attract EVT
cells to replace the spiral arteriole endothelium; once there, this mechanism of
chemoattraction of EVT and uNK is increased (Arck and Hecher 2013; Tilburgs
et al. 2015), having an important role in maternal immune system regulation.
Therefore, defects in EVT cell migration to spiral arterioles cause deficiency both
in vascular and immune placental systems at the maternal side (Cecati et al. 2011).
In addition, during early pregnancy, EVT is known to invade the maternal
venules that drain the blood lakes (Moser et al. 2017); however, this migration is
more related to fetal-maternal recognition than to vascular system remodeling
(He et al. 2017).

8.4.2 Mouse Model: Hemochorial Labyrinthine Placenta

The general structure of the mouse placenta was reviewed by Croy et al. (2015).
Differently from human, no villous tree is formed, and then the fetal umbilical
artery enters the chorionic plate, branched in innumerous chorionic arterioles, newly
branching in the labyrinth into a dense capillary bed (Adamson et al. 2002; Murrant
2014). The uterine artery branches in the arcuate artery and then in several radial
arteries that enter the uterine wall and branch now in spiral arterioles in the decidua
(Murrant 2014). In mice, the intravascular invasion by trophoblast giant cells (TGC)
is more discrete than what was observed in humans (Murrant 2014). Then, in the
central junctional zone, the spiral arterioles are connected to one to four trophoblast
tubes, named as arteriolar channels, that branch again forming the sinusoid mesh-
work layered by labyrinthine trophoblast cells (Adamson et al. 2002). Finally, the
maternal blood in the labyrinthine sinusoids bath the trophoblast in the maternal
spaces of the labyrinth and are drained through several venulous channels located in
the junctional zone until the decidual venous sinusoids, a conditioned chronic
hypoxia region (Leno-Durán et al. 2010). This condition may explain the glycogen-­
rich cells in the junctional zone, probably to deliver the substrate to permit
8 Pericytes in the Placenta: Role in Placental Development and Homeostasis 133

glycogenolysis in nearby cells (Abrahamsohn and Zorn 1993). Almost similarly to

humans, uNK cells also have a role in trophoblast invasion into maternal vessels
(Yamada et al. 2014).

8.4.3 Cattle Model: Synepitheliochorial Villous Placenta

The umbilical artery is branched in several arteries to form the allantochorionic

arterial system. Each branch enters the cotyledon and branches again into stem
artery to irrigate each stem villus (Leiser et al. 1997b). Then, from the stem artery
branches terminal arteriole to follow the villi ramification until the capillaries in the
terminal villi of the arcade zone. The capillaries are initially tightly arranged, and
then convolutions are made by capillaries coiling and anastomosing to create the
sinusoids (Leiser et al. 1997a; b). After capillarization, terminal venules rise to form
a tubelike venous system in the stem villi. This tubelike venous system converges to
the allantochorionic venous system intended to the umbilical vein (Leiser et al.
1998).
Branches of the uterine artery penetrate the uterine wall, and in the caruncular
stalk, they become vigorously spiraled, just after branching to form the less spiraled
septal arterioles, which finally ramify to form the capillary complexes in the arcade
zone (Leiser et al. 1997a). After capillarization, venules are formed, ­converging
successively to create the stem veins and ultimately the uterine vein (Fig. 8.2).

8.4.4 Feline Model: Endotheliochorial Lamellar Placenta

The umbilical artery enters the chorioallantoic membrane in the placental girdle
region and branches in stem arteries, which in turn branch in arterioles that overlay
the funnel-like maternal system. Those arterioles branch to form the lamellar capil-
lary system, from which are formed venules, which converge to the umbilical vein
(Leiser and Kohler 1984).
The uterine artery penetrates the myometrium stablishing a network-like pattern.
From this network, only one branch of the stem artery enters the endometrium in the
girdle region that is larger than other stem arteries that irrigate the para- or interpla-
cental regions. In direction of endometrial lamellae, the stem artery branches around
five more times, forming a funnel-like system of arterioles. From those arterioles,
one or two branches form the septal capillary network. The vascular connections in
the funnel-like systems are performed by anastomosis with peripheral arterioles of
each system. The capillary network continues forming venules and then converges
to the stem vein in the junctional zone until the umbilical vein (Leiser and Kohler
1983; Dantzer 2002; Wooding and Burton 2008a) (Fig. 8.3).
134 R. S. N. Barreto et al.

Fig. 8.2 Schema of bovine placental vascularization. In (a), bovine fetus and placenta. In (b),
squared area of (a), showing layers and maternal and fetal vascularization inside the placentome.
In (c), squared area of (b), showing maternal (or caruncular) microvascularization. In (d), squared
area of (b), showing fetal (or cotyledonary) microvascularization
8 Pericytes in the Placenta: Role in Placental Development and Homeostasis 135

Fig. 8.3 Schema of cat placental vascularization. In (a), cat fetus and placenta. In (b), squared
area of (a), showing layers and maternal and fetal macro- and microvascularization inside the
placental girdle

8.5 Pericytes in the Placenta

As discussed in other chapters of this book, pericytes were firstly defined by their
anatomical localization, i.e., adhered in the endothelium around the blood vessel
wall with a general function related to vascular stability (Rouget 1873). However,
other pericyte functions have been described, such as vascular permeability and
blood flow control (Pallone et al. 1998, 2003; Pallone and Silldorff 2001); vascular
development, maturation, and remodeling (Levéen et al. 1994; Soriano 1994;
Lindahl et al. 1997; Hellström et al. 2001; Enge et al. 2002); blood-brain barrier
integrity (Cuevas et al. 1984; Nakagawa et al. 2007; Nakamura et al. 2008; Shimizu
et al. 2008; Krueger and Bechmann 2010; Bell et al. 2010; Armulik et al. 2010;
Daneman et al. 2010; Al Ahmad et al. 2011; Kamouchi et al. 2011; Thanabalasundaram
et al. 2011); and regulation of blood coagulation (Bouchard et al. 1997; Kim et al.
2006; Fisher 2009). In addition, in the last decade, new functions related to the
immune system (Armulik et al. 2011; Hellerbrand 2013; Pfister et al. 2013; Nees
et al. 2013; Hurtado-Alvarado et al. 2014; Pan et al. 2014) have been also described,
i.e., lymphocyte modulation and activation (Fabry et al. 1993; Verbeek et al. 1995;
Balabanov et al. 1999; Tu et al. 2011), macrophage-like phagocytic properties
(Jeynes 1985; Hasan and Glees 1990; Balabanov et al. 1996; Thomas 1999; Castejón
2011), and dendritic cell modulation (Krautler et al. 2012). Besides those functions,
the pericytes could also participate in several regenerative tissue and repair
136 R. S. N. Barreto et al.

processes, i.e., formation of skeletal muscle cells (Crisan et al. 2008; Dellavalle
et al. 2011), dental tissues (Feng et al. 2011; Zhao et al. 2014), adipocytes (Crisan
et al. 2008), cartilage (Farrington-Rock et al. 2004), and bone (James et al. 2012),
increasing heart function after infarct (Katare et al. 2011; Chen et al. 2013), healing
(Zebardast et al. 2010), and fibrous tissue formation (Dulauroy et al. 2012; Duffield
et al. 2013).
Pericytes can also modulate stem cells (Crisan et al. 2008; Bandeira et al. 2017),
majorly the hematopoietic (Méndez-Ferrer et al. 2010; Ding et al. 2012; Corselli
et al. 2013; Kunisaki et al. 2013). In parallel, placental tissues are known to be a rich
stem cell niche, and those stromal cells have several applications similar as described
for pericytes [reviewed by Antoniadou and David (2016) and Oliveira and Barreto-­
Filho (2015)]. So, this diverseness of pericyte function could be similar in the pla-
cental microenvironment. Therefore, pericytes could be important for placental
development and homeostasis. Then, due to the absence of pericyte function knowl-
edge in placenta-specific tissue, we will discuss what is known and speculate some
possible functions including hypothesis from deficient or diseased models.

8.5.1 Major Genes Involved in Pericyte Angiogenesis Signaling

During the vasculogenesis in early human pregnancy (around the third week), it is
possible to find two different cell groups directly derived from mesenchymal cells.
Most of those cells are flattened, they stay in contact with the luminal surface of the
forming vessel wall, and during this period, the basement membrane is absent. The
second group of cells is in contact with the outer surface that is more polygonal and
arranged in a weblike shape. Those cells are in contact with other mesenchymal
cells, and they are surrounding the forming vessel wall and had been considered
pericytes. Instead of intimate contact with flattened luminal cells (endothelium), the
processes do not reach the lumen (Demir et al. 1989). This second group of cells,
pericytes, could be differentiated directly from mesenchymal stem cells, or neural
crest, by tumor growth factor (TGF)-β1 signaling (Chen and Lechleider 2004; Ding
et al. 2004). In more mature placenta, it is possible to find decidual (Maier and
Pober 2011) and placental (Robin et al. 2009) pericytes that apparently are hetero-
geneous [as in others (Prazeres et al. 2017)], and both participate in the angiogene-
sis processes (Bergers and Song 2005).
The vascular endothelial growth factor (VEGF) is expressed in the placental peri-
cytes and induces angiogenesis (Aronoff et al. 2017). A hypoxia-induced prolifera-
tion could be influenced by autocrine VEGF signaling (Nomura et al. 1995; Yamagishi
et al. 1999a) to promote angiogenesis (Yonekura et al. 1999). It can also influence
pericyte proliferation and migration (Yamagishi et al. 1999b). In the placenta, pla-
cental growth factor (PGF) also participates in this process (Yonekura et al. 1999).
Together with VEGF, the platelet-derived growth factor B (PDGF-B), a potent
mitogenic agent for connective tissue cells, is involved in the angiogenesis (Moreau
et al. 2014). PDGF-B can be produced by several cells, like megakaryocytes/­
8 Pericytes in the Placenta: Role in Placental Development and Homeostasis 137

platelets, monocytes/macrophages, and villous cytotrophoblast cells, and can

­control pericyte recruitment (Holmgren et al. 1991; Bjarnegard 2004). In addition,
non-­endothelial source of PDGF-B may have long-range effects on pericyte growth
and recruitment (Bjarnegard 2004). The PDGF-B expression could be controlled by
several factors, such as hypoxia (Herrmann et al. 2016), similar to VEGF.
The PDGF signaling is important to attract pericytes by PDGF receptor β
(PDGFRβ) and control pericyte recruitment, proliferation, and differentiation
(Moreau et al. 2014). Apparently, PDGFRβ is also expressed in mice sinusoidal
trophoblast giant cells (Moreau et al. 2014). The PDGF-B/PDGFRβ signaling
between endothelium and pericytes controls the pericyte recruitment. The lack of
this signaling, by knockout of those genes or in some placental diseases (that will be
discussed later in this chapter), could decrease pericyte number and coverage and
increase capillary diameter and permeability, and it also interferes in trophoblast
development. Likewise, Cited2 is involved in pericyte recruitment, proliferation,
and differentiation, acting directly on capillary formation (Moreau et al. 2014);
however, the mechanism is still unknown.
In swine placenta, an epitheliochorial model, the PDGF receptors in the endothe-
lial/perivascular areas of the subepithelial layer from day 24 to 40 of swine preg-
nancy show stronger expression compared to earlier stages (Rodriguez-Martinez
et al. 1992; Persson and Rodriguez-Martinez 1997) suggesting that PDGF plays
also a role in stroma reorganization, particularly during maternal angiogenesis.
After maturation of the vessels, the maternal-fetal interface is composed by the
capillary system at the fetal side and another capillary system of blood space at the
maternal side. Then, the arterioles before maternal-fetal interface are surrounded by
only one or two smooth muscle cell layers; however, some venules after maternal-­
fetal interface are surrounded only by pericytes (Rhodin 1968; Kaufmann et al.
1988). Moreover, maternal precapillary sphincters, before blood spaces in hemo-
chorial placentas, due to the absence neural innervation to control vasoconstriction
or vasodilatation (Lachenmayer 1971a; Nikolov and Schiebler 1973; Myatt 1992)
could be controlled by pericytes, as in neural capillary beds, by alpha smooth mus-
cle actin (α-SMA) and some cholinergic and adrenergic receptors (Rucker et al.
2000).

8.5.2  ericytes in Knockout-/Null-Induced Placental

P
Abnormalities or Diseases

It is a consensus that pericytes participate in capillary wall formation also in the

placenta (Challier et al. 1999). For decades, in some hemochorial placenta types,
pericytes were described to form an incomplete layer adjacent to the endothelial
cells (Heinrich et al. 1988). However, in late human pregnancy, during angiogene-
sis, the capillaries sprouted by the proliferation of endothelial cells and pericytes are
evolved by thick layers of basement membrane (Demir et al. 1989). Nevertheless,
138 R. S. N. Barreto et al.

knowledge about the distribution and function of placental pericytes still needs
more investigation. Something is known about the pericyte ultrastructure (Challier
et al. 1997; Ohlsson et al. 1999; Bjarnegard 2004; Kučera et al. 2010; Jones and
Desoye 2011; Maier and Pober 2011; Deveci et al. 2013; Kim et al. 2015), their
perivascular localization in the capillaries, and the presence of vesicles nearby
(Jones and Desoye 2011). Those vesicles are probably delivery by pericytes in the
direction of the trophoblast basement membrane and some macrophages (Jones and
Desoye 2011).
Even though the placental pericytes are poorly characterized, it is known that
they express α-SMA and NG2 (Ohlsson et al. 1999; Sims 2000; Looman et al.
2007), and pericyte dysfunction, coverage, and distribution may be modified in rela-
tion to pregnancy abnormalities (Maier et al. 2010). In high-altitude pregnancies
that have higher blood pressure, the placental capillaries have lower pericyte cover-
age and increased diameter than in lowlands (Zhang et al. 2002). In pregnancies
complicated by type I diabetes mellitus, no differences in pericyte coverage in pla-
cental vessels were observed (Kučera et al. 2010). In complete hydatidiform mole
pregnancies, a lack in pericyte recruitment is evident, with no association of peri-
cytes and the villi stroma vessels, spaces in between endothelial cells, and no basal
lamina that results in an immature vasculature (Kim et al. 2015). Similarly, lesions
of villous capillary (such as chorangioma, chorangiomatosis, and chorangiosis) also
decrease pericyte number in the capillaries; however, they form a more continuous
and preserved layer (Ogino and Redline 2000).
Pericytes are the most permissive cell for congenital human cytomegalovirus
(HCMV) infection in the villi, such as in the blood-brain barrier and blood-retinal
barrier. This results in the decrease of pericytes due to infection and could interfere
in the placental vascular permeability and increase placental inflammation, angio-
genesis, and microvascular abnormalities (Aronoff et al. 2017).
On the other side, in the mice model of pregnancy-associated hypertension, the
small vessels have abnormal diameter and number and also were not lined by a
basement membrane and poorly covered by pericytes, majorly in the labyrinth,
showing immature vascular network (Furuya et al. 2008). A similar phenotype is
observed in Cited2 null mice, where pericytes around embryonic vessels were dis-
organized (Moreau et al. 2014).
In turn, the PDGF-B, a mitogenic agent that can be produced by several cells
(Holmgren et al. 1991), controls the proliferative potential of extravillous tropho-
blast (Holmgren et al. 1991, 1992). A deficiency in PDGF-B, or its receptor, could
lead to a perinatal death in mice with several malformations due to lack of micro-
vascular pericytes and rupture of microvasculature (Ohlsson et al. 1999). On the
other hand, it induces dilated blood vessels; and it reduces the number of pericytes,
the trophoblast layer, and the surface area of maternal blood spaces in mice laby-
rinth; and it increases hemorrhage due to microvasculature rupture (Ohlsson et al.
1999).
8 Pericytes in the Placenta: Role in Placental Development and Homeostasis 139

8.5.3 Pericytes in Placental Immune Cell Regulation

It is known that the placental mesenchymal stem cells from the maternal origin are
more responsible to immunomodulatory stimulus than fetal ones. Those maternal
cells regulate the induction of tolerogenic dendritic cells and regulatory T cells (Zhu
et al. 2014), having a better immunomodulatory potential than cells from the umbil-
ical funiculus (Talwadekar et al. 2015). Such stem cells too can increase the survival
of skin grafts (Zhu et al. 2014). Mesenchymal stem cells are stromal cells, and they
can be localized perivascularly in placental tissues [reviewed by Lobo et al. (2016)
and Favaron and Miglino (2017)], and they share some markers with pericytes
(Crisan et al. 2008; Muñoz-Fernández et al. 2018). In parallel, human decidual stro-
mal cells are also perivascular cells distributed in the endometrium and decidua
(Wynn 1974) and recently are described as decidual pericytes, due to the expression
of pericyte-associated antigens (such as α-SMA, nestin, PDGF-B, and PDGFRβ),
expression of angiogenic factors, cytokine-induced contraction, and attraction of
peripheral blood NK cells (Muñoz-Fernández et al. 2018). Then those cells may be
pericytes specialized and involved in immune responses during the pregnancy.
Placental pericytes, like other stromal or mesenchymal cells, decrease T cell pro-
liferation by cytokine signaling. Besides that, placental pericytes have low stimula-
tion of T cell; however, MHC-II molecules from pericytes are recognized by and
desensitize T cell through induction of T cell anergy by TCR-dependent mechanism
after IFN-γ stimulation (Maier and Pober 2011). Placenta-derived pericytes, such as
brain pericytes, can induce the formation of putative Treg cells (CD4+CD25highFOXP3+)
with suppressive activity and reduce effector T cells, and this pericyte phenotype
was repressed using TGF-β inhibitor (Domev et al. 2014).
Abnormal interaction between extravillous trophoblast and decidual NK cells
could be observed in impaired pseudovasculogenesis in early human preeclamptic
placenta (Furuya et al. 2008). And pericytes from microvasculature are important in
guiding leukocytes to inflammation site (Stark et al. 2013).
The extravillous trophoblast (EVT) downregulates the expression of MHC class
Ia (MHC1a) (Helige et al. 2008; Jiang et al. 2015) to inhibit maternal immune rejec-
tion by inhibition of uNK, macrophages, and other immune cell receptors (Caumartin
et al. 2007; Li et al. 2009). However, EVT overexpresses MHC1b that active uNK
cells against fetal tissues, altogether works to activate Treg cells (CD4+CD25+FOXP3+)
that inhibits effector T cells (Djurisic et al. 2014; Tilburgs et al. 2015). The Treg cells
have a bidirectional signaling with dendritic cells (DC) (Guerin et al. 2009). When
the CTLA4 in the Treg surface stimulates the CD80/CD86 in DC surface (Aluvihare
et al. 2004), the DC produces indoleamine 2,3-dioxygenase (IDO) (Sato et al. 2003)
that is metabolized and induces FOXP3 production in CD4 + CD25+ T cell, induc-
ing the generation of more Treg cells (Mezrich et al. 2010). As pericytes could induce
dendritic and Treg cells (Zhu et al. 2014), probably by a unknown signaling to DC
and in consequence increase Treg cells. Also, decidual pericytes could be related to
the attraction of peripheral blood NK cells to the decidua (Muñoz-Fernández et al.
2018). As the decrease of uNK (CD56brightCD16−) could reduce EVT migration
140 R. S. N. Barreto et al.

(Lash et al. 2010) and cause defects in maternal spiral arteries (Leonard et al. 2006)
resulting in miscarriage increase (Rizzo et al. 2015). Then, indirectly, pericytes
could be related to EVT recognition by the maternal immune system and spiral
arteries formation, as hypothesized in Fig. 8.4.

Fig. 8.4 Schema of human

spiral arteriole. During
spiral arteriole formation,
there is interaction between
trophoblast cells and some
immune cells. In the
terminal villi (TV), some
cells with migratory
profile, the extravillous
trophoblast (EVT), migrate
to spiral artery replacing
endothelial cells. EVT
have downregulation of
MCH1a that could activate
uterine natural killer (uNK)
cells. However, EVT
upregulates MHC1b that
activates regulatory T (Treg)
cells. Treg and dendritic
cells (DC) activate each
other, and this inhibits the
attack of EVT by
uNK. Also, the pericytes
(PC) from spiral arteriole
wall can interact with some
DC (by unknown
mechanism) and help in
immune cell activation and
trophoblast cell migration.
Dark blue arrow
(activation), red arrow
(inactivation), and light
blue arrow (activation by
unknown mechanism)
8 Pericytes in the Placenta: Role in Placental Development and Homeostasis 141

8.5.4 Pericytes in Placental Cell Differentiation

The pericyte function and plasticity potential are tissue dependent, i.e., skeletal
muscle pericytes have better ability to differentiate to myogenic lineage than
myocardial-­derived ones (Chen et al. 2015). Besides that, pericyte heterogeneity
could be present even inside the same tissue (Prazeres et al. 2017). However, little
is known about specific pericyte differentiation in the placenta.
In general, mesenchymal stem cells can be localized perivascularly [reviewed by
Lobo et al. (2016) and Favaron and Miglino (2017)] such as in placental tissues.
Besides mesenchymal stem cells, placental tissues are a niche of hematopoietic
stem cells (Lee et al. 2010; Dzierzak and Robin 2010), since their early differentia-
tion from hemangioblast during yolk sac formation (Ueno and Weissman 2010;
Golub and Cumano 2013), in the labyrinth development in mice placenta [reviewed
by Ottersbach and Dzierzak (2010)], or in the major vessels of the chorionic plate
(Gekas et al. 2010). Therefore, the placenta is a niche of hematopoietic stem cells
acting in the generation, renewal, and maintenance of undifferentiated status (Gekas
et al. 2010), with stem cell factor (a pericyte-related marker) as a central molecule
to maintain this function (Khodadi et al. 2016).
Endometrial stromal cells have pericyte phenotype with immunogenic profile
(Muñoz-Fernández et al. 2018), as cited before. Also, pericytes that support hema-
topoiesis are present in small number in maternal tissue as early as week 3, but it
increases considerably at the fetal side in week 6 (Robin et al. 2009), suggesting that
maternal pericytes support initial hematopoiesis, and just after fetal pericytes
assume this function in the growing and development placenta. In addition, one
should consider the existence of different populations of pericytes, evidenced by the
distinct functions performed by these cells within the same tissue (Prazeres et al.
2017). A similar event happens in mice, where at E11 low pericyte amount is recov-
ered; however, between E12 and E13, they reach the highest number (Gekas et al.
2010; Ottersbach and Dzierzak 2010), on the same period of higher influx of uNK
and trophoblast hyperplasia (Wooding and Burton 2008b).
The ERK/MAPK pathway in pericytes must be required for self-function main-
tenance and allow differentiation and formation of syncytiotrophoblast layers. Then
a deficiency in this pathway affects syncytiotrophoblast layer formation that impli-
cates in reduced maternal labyrinthic vascularization due to a decrease in interac-
tion with neighbor trophoblast, but without interfering in pericyte migration (Nadeau
and Charron 2014).
The endothelial cell hyperplasia, hypervariable diameter, abundant microaneu-
rysm, abnormal endothelial ultrastructure, and altered permeability could be
observed in the microvasculature of PDGF-B or PDGFRβ null mice. Probably by
reduction of pericytes recruitment in the placenta. Withal, reduction of trophoblast
development and proliferation was observed in these mice null model and in endo-
thelial PDGF-B-deficient mice (Bjarnegard 2004).
142 R. S. N. Barreto et al.

8.6 Final Remarks

Despite the variability of placental morphology among species and even more in the
maternal-fetal interface, the gestational goal is acquired—the production of viable
offspring. Also, the placental microenvironment is unique, due to the interaction
between maternal and fetal tissues, constant cell differentiation, peripheral maternal
immune cell influx, and maintenance of stem cell niche. In parallel, pericytes have
a plethora of functions that meet placental homeostasis. Pericytes have been associ-
ated with some placental function, such as vascular control, immune cell activation,
and trophoblast differentiation; however, the detailed mechanisms are still unknown.

Acknowledgments Alexander Birbrair is supported by a grant from Instituto Serrapilheira/

Serra-­1708-15285, a grant from Pró-reitoria de Pesquisa/Universidade Federal de Minas Gerais
(PRPq/UFMG) (Edital 05/2016), a grant from the National Institute of Science and Technology in
Theranostics and Nanobiotechnology (CNPq/CAPES/FAPEMIG, Process No. 465669/2014-0), a
grant from FAPEMIG [Rede Mineira de Engenharia de Tecidos e Terapia Celular (REMETTEC,
RED-00570-16)], and a grant from FAPEMIG [Rede De Pesquisa Em Doenças Infecciosas
Humanas E Animais Do Estado De Minas Gerais (RED-00313-16)].

References

(1955) The normal anatomy of the uterine artery. Acta Radiol. 43:21–36. doi: 10.3109/
00016925509170755, http://dx.doi.org/10.3109/00016925509170755
Abrahamsohn PA, Zorn TMT (1993) Implantation and decidualization in rodents. J Exp Zool
266:603–628. https://doi.org/10.1002/jez.1402660610
Adamson SL, Lu Y, Whiteley KJ, Holmyard D, Hemberger M, Pfarrer C, Cross JC (2002)
Interactions between trophoblast cells and the maternal and fetal circulation in the mouse pla-
centa. Dev Biol 250:358–373
Al Ahmad A, Taboada CB, Gassmann M, Ogunshola OO (2011) Astrocytes and Pericytes differ-
entially modulate blood—brain barrier characteristics during development and hypoxic insult.
J Cereb Blood Flow Metab 31:693–705. https://doi.org/10.1038/jcbfm.2010.148
Aluvihare VR, Kallikourdis M, Betz AG (2004) Regulatory T cells mediate maternal tolerance to
the fetus. Nat Immunol 5:266–271. https://doi.org/10.1038/ni1037
Antoniadou E, David AL (2016) Placental stem cells. Best Pract Res Clin Obstet Gynaecol 31:13–
29. https://doi.org/10.1016/J.BPOBGYN.2015.08.014
Arck PC, Hecher K (2013) Fetomaternal immune cross-talk and its consequences for maternal and
offspring’s health. Nat Med 19:548–556. https://doi.org/10.1038/nm.3160
Armulik A, Genové G, Mäe M, Nisancioglu MH, Wallgard E, Niaudet C, He L, Norlin J, Lindblom
P, Strittmatter K, Johansson BR, Betsholtz C (2010) Pericytes regulate the blood–brain barrier.
Nature 468:557–561. https://doi.org/10.1038/nature09522
Armulik A, Genové G, Betsholtz C (2011) Pericytes: developmental, physiological, and patho-
logical perspectives, problems, and promises. Dev Cell 21:193–215. https://doi.org/10.1016/j.
devcel.2011.07.001
Aronoff DM, Correa H, Rogers LM, Arav-Boger R, Alcendor DJ (2017) Placental pericytes and
cytomegalovirus infectivity: implications for HCMV placental pathology and congenital dis-
ease. Am J Reprod Immunol 78. https://doi.org/10.1111/aji.12728
Arts NF (1961) Investigations on the vascular system of the placenta. I. General introduction and
the fetal vascular system. Am J Obstet Gynecol 82:147–158
8 Pericytes in the Placenta: Role in Placental Development and Homeostasis 143

Balabanov R, Washington R, Wagnerova J, Dore-Duffy P (1996) CNS microvascular pericytes

express macrophage-like function, cell surface integrin alpha M, and macrophage marker
ED-2. Microvasc Res 52:127–142. https://doi.org/10.1006/mvre.1996.0049
Balabanov R, Beaumont T, Dore-Duffy P (1999) Role of central nervous system microvascular
pericytes in activation of antigen-primed splenic T-lymphocytes. J Neurosci Res 55:578–587.
https://doi.org/10.1002/(SICI)1097-4547(19990301)55:5<578::AID-JNR5>3.0.CO;2-E
Bandeira DS, Casamitjana J, Crisan M (2017) Pharmacology & Therapeutics Pericytes , integral
components of adult hematopoietic stem cell niches. Pharmacol Ther 171:104–113. https://doi.
org/10.1016/j.pharmthera.2016.11.006
Barreto RSN, Bressan FF, Oliveira LJ, Pereira FTV, Perecin F, Ambrósio CE, Meirelles FV,
Miglino MA (2011) Gene expression in placentation of farm animals: an overview of gene
function during development. Theriogenology 76:589–597. https://doi.org/10.1016/j.
theriogenology.2011.03.001
Baur R (1977) Morphometry of the placental exchange area. Adv Anat Embryol Cell Biol 53:
3–65
Bell RD, Winkler EA, Sagare AP, Singh I, LaRue B, Deane R, Zlokovic BV (2010) Pericytes
control key neurovascular functions and neuronal phenotype in the adult brain and during brain
aging. Neuron 68:409–427. https://doi.org/10.1016/j.neuron.2010.09.043
Bergers G, Song S (2005) The role of pericytes in blood-vessel formation and maintenance. Neuro-­
Oncology 7:452–464. https://doi.org/10.1215/S1152851705000232
Bjarnegard M (2004) Endothelium-specific ablation of PDGFB leads to pericyte loss and glomeru-
lar, cardiac and placental abnormalities. Development 131:1847–1857. https://doi.org/10.1242/
dev.01080
Bouchard BA, Shatos MA, Tracy PB (1997) Human brain pericytes differentially regulate expres-
sion of procoagulant enzyme complexes comprising the extrinsic pathway of blood coagula-
tion. Arterioscler Thromb Vasc Biol 17:1–9
Burton GJ, Charnock-Jones DS, Jauniaux E (2009) Regulation of vascular growth and function in
the human placenta. Reproduction 138:895–902. https://doi.org/10.1530/REP-09-0092
Carter AM, Mess A (2007) Evolution of the placenta in eutherian mammals. Placenta 28:259–262.
https://doi.org/10.1016/j.placenta.2006.04.010
Castejón OJ (2011) Ultrastructural pathology of cortical capillary pericytes in human traumatic
brain oedema. Folia Neuropathol 49:162–173
Caumartin J, Favier B, Daouya M, Guillard C, Moreau P, Carosella ED, LeMaoult J (2007)
Trogocytosis-based generation of suppressive NK cells. EMBO J 26:1423–1433. https://doi.
org/10.1038/sj.emboj.7601570
Cecati M, Giannubilo SR, Emanuelli M, Tranquilli AL, Saccucci F (2011) HLA-G and pregnancy
adverse outcomes. Med Hypotheses 76:782–784. https://doi.org/10.1016/j.mehy.2011.02.017
Challier JC, Kacemi A, Galtier M, Boucher M, Tangapregassom MJ, Vervelle C, Bintein T, Espié
MJ, Olive G (1997) Phenotype of cultured fetal perivascular cells from human placenta stud-
ied by scanning electron microscopy. Anat Embryol (Berl) 195:79–86. https://doi.org/10.1007/
s004290050027
Challier JC, Galtier M, Kacemi A, Guillaumin D (1999) Pericytes of term human foeto-placental
microvessels: ultrastructure and visualization. Cell Mol Biol (Noisy-le-Grand) 45:89–100
Chen S, Lechleider RJ (2004) Transforming growth factor—induced differentiation of smooth
muscle from a neural crest stem cell line. Circ Res 94:1195–1202. https://doi.org/10.1161/01.
RES.0000126897.41658.81
Chen C-W, Okada M, Proto JD, Gao X, Sekiya N, Beckman SA, Corselli M, Crisan M, Saparov
A, Tobita K, Péault B, Huard J (2013) Human pericytes for ischemic heart repair. Stem Cells
31:305–316. https://doi.org/10.1002/stem.1285
Chen WCW, Baily JE, Corselli M, Díaz ME, Sun B, Xiang G, Gray GA, Huard J, Péault B (2015)
Human myocardial pericytes: multipotent mesodermal precursors exhibiting cardiac specific-
ity. Stem Cells 33:557–573. https://doi.org/10.1002/stem.1868
144 R. S. N. Barreto et al.

Corselli M, Chin CJ, Parekh C, Sahaghian A, Wang W, Ge S, Evseenko D, Wang X, Montelatici

E, Lazzari L, Crooks GM, Péault B (2013) Perivascular support of human hematopoietic stem/
progenitor cells. Blood 121:2891–2901. https://doi.org/10.1182/blood-2012-08-451864
Crisan M, Yap S, Casteilla L, Chen C, Corselli M, Park TS, Andriolo G, Sun B, Zheng B, Zhang L,
Norotte C, Teng P, Traas J, Schugar R, Deasy BM, Badylak S, Lazzari L, Huard J, Pe B (2008)
A perivascular origin for mesenchymal stem cells in multiple human organs. Cell Stem Cell
3:301–313. https://doi.org/10.1016/j.stem.2008.07.003
Croy BA, Yamada AT, DeMayo FJ, Adamson SL (2015) The guide to investigation of mouse preg-
nancy. Elsevier, San Diego, CA
Cuevas P, Gutierrez-Diaz JA, Reimers D, Dujovny M, Diaz FG, Ausman JI (1984) Pericyte endo-
thelial gap junctions in human cerebral capillaries. Anat Embryol (Berl) 170:155–159
Daneman R, Zhou L, Kebede AA, Barres BA (2010) Pericytes are required for blood-brain bar-
rier integrity during embryogenesis. Nature 468:562–566. https://doi.org/10.1038/nature09513
Dantzer V (2002) Endometrium of epitheliochorial and endotheliochorial placentae. In: Glasser
SR, Aplin JD, Giudice LC, Tabibzadeh S (eds) The endometrium. Taylor & Francis, New York,
pp 352–368
Dellavalle A, Maroli G, Covarello D, Azzoni E, Innocenzi A, Perani L, Antonini S, Sambasivan
R, Brunelli S, Tajbakhsh S, Cossu G (2011) Pericytes resident in postnatal skeletal muscle
differentiate into muscle fibres and generate satellite cells. Nat Commun 2:499. https://doi.
org/10.1038/ncomms1508
Demir R, Kaufmann P, Castelluce M, Erbeng T, Kotowsk A (1989) Fetal vasculogenesis and
angiogenesis in human placental villi. Cells Tissues Organs 136:190–203. https://doi.
org/10.1159/000146886
Deveci E, Soker S, Turgut A, Aktas A, Ayaz E, Sak S, Seker U (2013) The immunohistochemical
and ultrastructural evaluation of pericytes in human full term placentas of gestasyonal diabetes
mellitus. Acta Medica Mediterr 29:697–700
Ding R, Darland DC, Parmacek MS, D’amore PA (2004) Endothelial–mesenchymal interactions
in vitro reveal molecular mechanisms of smooth muscle/Pericyte differentiation. Stem Cells
Dev 13:509–520. https://doi.org/10.1089/scd.2004.13.509
Ding L, Saunders TL, Enikolopov G, Morrison SJ (2012) Endothelial and perivascular cells main-
tain haematopoietic stem cells. Nature 481:457–462. https://doi.org/10.1038/nature10783
Djurisic S, Teiblum S, Tolstrup CK, Christiansen OB, Hviid TVF (2014) Allelic imbalance modu-
lates surface expression of the tolerance-inducing HLA-G molecule on primary trophoblast
cells. Mol Hum Reprod 21:281–295. https://doi.org/10.1093/molehr/gau108
Domev H, Milkov I, Itskovitz-Eldor J, Dar A (2014) Immunoevasive Pericytes from human plu-
ripotent stem cells preferentially modulate induction of allogeneic regulatory T cells. Stem
Cells Transl Med 3:1169–1181. https://doi.org/10.5966/sctm.2014-0097
Dubova EA, Buranova FB, Fyodorova TA, Shchyogolev AI, Sukhikh GT (2013) Morphological
characteristics of the terminal villi in placental failure. Bull Exp Biol Med 155:507–511
Duffield JS, Lupher M, Thannickal VJ, Wynn TA (2013) Host responses in tissue repair and fibro-
sis. Annu Rev Pathol 8:241–276. https://doi.org/10.1146/annurev-pathol-020712-163930
Dulauroy S, Di Carlo SE, Langa F, Eberl G, Peduto L (2012) Lineage tracing and genetic ablation
of ADAM12+ perivascular cells identify a major source of profibrotic cells during acute tissue
injury. Nat Med 18:1262–1270. https://doi.org/10.1038/nm.2848
Dzierzak E, Robin C (2010) Placenta as a source of hematopoietic stem cells. Trends Mol Med
16:361–367. https://doi.org/10.1016/j.molmed.2010.05.005
Enders AC, Blankenship TN, Lantz KC, Enders SS (1998) Morphological variation in the inter-
hemal areas of chorioallantoic placentae: a review. Placenta 19:1–19. https://doi.org/10.1016/
S0143-4004(98)80030-1
Enge M, Bjarnegård M, Gerhardt H, Gustafsson E, Kalén M, Asker N, Hammes H-P, Shani M,
Fässler R, Betsholtz C (2002) Endothelium-specific platelet-derived growth factor-B ablation
mimics diabetic retinopathy. EMBO J 21:4307–4316
Fabry Z, Fitzsimmons KM, Herlein JA, Moninger TO, Dobbs MB, Hart MN (1993) Production of
the cytokines interleukin 1 and 6 by murine brain microvessel endothelium and smooth muscle
pericytes. J Neuroimmunol 47:23–34
8 Pericytes in the Placenta: Role in Placental Development and Homeostasis 145

Farrington-Rock C, Crofts NJ, Doherty MJ, Ashton BA, Griffin-Jones C, Canfield AE (2004)
Chondrogenic and adipogenic potential of microvascular pericytes. Circulation 110:2226–
2232. https://doi.org/10.1161/01.CIR.0000144457.55518.E5
Favaron PO, Miglino MA (2017) Fetal membranes-derived stem cells microenvironment. Adv Exp
Med Biol 1041:235–244
Feng J, Mantesso A, De Bari C, Nishiyama A, Sharpe PT (2011) Dual origin of mesenchymal
stem cells contributing to organ growth and repair. Proc Natl Acad Sci U S A 108:6503–6508.
https://doi.org/10.1073/pnas.1015449108
Firth JA, Leach L (1996) Not trophoblast alone: a review of the contribution of the fetal micro-
vasculature to transplacental exchange. Placenta 17:89–96. https://www.ncbi.nlm.nih.gov/
pubmed/8730878
Fisher M (2009) Pericyte Signaling in the neurovascular unit. Stroke 40:S13–S15. https://doi.
org/10.1161/STROKEAHA.108.533117
Furuya M, Ishida J, Inaba S, Kasuya Y, Kimura S, Nemori R, Fukamizu A (2008) Impaired placen-
tal neovascularization in mice with pregnancy-associated hypertension. Lab Investig 88:416–
429. https://doi.org/10.1038/labinvest.2008.7
Gekas C, Rhodes KE, Vanhandel B, Chhabra A, Ueno M, Mikkola HKA (2010) Hematopoietic
stem cell development in the placenta. Int J Dev Biol 54:1089–1098. https://doi.org/10.1387/
ijdb.103070cg
Golub R, Cumano A (2013) Embryonic hematopoiesis. Blood Cells Mol Dis 51:226–231. https://
doi.org/10.1016/j.bcmd.2013.08.004
Guerin LR, Prins JR, Robertson SA (2009) Regulatory T-cells and immune tolerance in preg-
nancy: a new target for infertility treatment? Hum Reprod Update 15:517–535. https://doi.
org/10.1093/humupd/dmp004
Hasan M, Glees P (1990) The fine structure of human cerebral perivascular pericytes and jux-
tavascular phagocytes: their possible role in hydrocephalic edema resolution. J Hirnforsch
31:237–249
He N, van Iperen L, de Jong D, Szuhai K, Helmerhorst FM, van der Westerlaken LAJ, Chuva de
Sousa Lopes SM (2017) Human extravillous trophoblasts penetrate decidual veins and lym-
phatics before remodeling spiral arteries during early pregnancy. PLoS One 12:e0169849.
https://doi.org/10.1371/journal.pone.0169849
Heinrich D, Aoki A, Metz J (1988) Fetal capillary organization in different types of placenta.
Trophobl Res 3:149–162. https://doi.org/10.1007/978-1-4615-8109-3_11
Helige C, Ahammer H, Hammer A, Huppertz B, Frank H-G, Dohr G (2008) Trophoblastic inva-
sion in vitro and in vivo: similarities and differences. Hum Reprod 23:2282–2291. https://doi.
org/10.1093/humrep/den198
Hellerbrand C (2013) Hepatic stellate cells—the pericytes in the liver. Pflügers Arch Eur J Physiol
465:775–778. https://doi.org/10.1007/s00424-012-1209-5
Hellström M, Gerhardt H, Kalén M, Li X, Eriksson U, Wolburg H, Betsholtz C (2001) Lack of
Pericytes leads to endothelial hyperplasia and abnormal vascular morphogenesis. J Cell Biol
153:543–553
Herrmann M, Bara JJ, Sprecher CM, Menzel U, Jalowiec JM, Osinga R, Scherberich A, Alini M,
Verrier S (2016) Pericyte plasticity—comparative investigation of the angiogenic and multilin-
eage potential of pericytes from different human tissues. Eur Cells Mater 31:236–249. https://
doi.org/10.22203/eCM.v031a16
Holmgren L, Glaser A, Pfeifer-Ohlsson S, Ohlsson R (1991) Angiogenesis during human extra-
embryonic development involves the spatiotemporal control of PDGF ligand and receptor gene
expression. Development 113:749–754
Holmgren L, Claesson-welsh L, Heldin C-H, Ohlsson R (1992) The expression of PDGF α- and
β-receptors in subpopulations of PDGF-producing cells implicates autocrine stimulatory loops
in the control of proliferation in Cytotrophoblasts that have invaded the maternal endometrium.
Growth Factors 6:219–231. https://doi.org/10.3109/08977199209026929
Hurtado-Alvarado G, Cabañas-Morales AM, Gómez-Gónzalez B (2014) Pericytes: brain-immune
interface modulators. Front Integr Neurosci 7:80. https://doi.org/10.3389/fnint.2013.00080
146 R. S. N. Barreto et al.

James AW, Zara JN, Zhang X, Askarinam A, Goyal R, Chiang M, Yuan W, Chang L, Corselli M,
Shen J, Pang S, Stoker D, Wu B, Ting K, Péault B, Soo C (2012) Perivascular stem cells: a
prospectively purified mesenchymal stem cell population for bone tissue engineering. Stem
Cells Transl Med 1:510–519. https://doi.org/10.5966/sctm.2012-0002
Jeynes B (1985) Reactions of granular pericytes in a rabbit cerebrovascular ischemia model.
Stroke 16:121–125
Jiang F, Zhao H, Wang L, Guo X, Wang X, Yin G, Hu Y, Li Y, Yao Y (2015) Role of HLA-G1 in tro-
phoblast cell proliferation, adhesion and invasion. Biochem Biophys Res Commun 458:154–
160. https://doi.org/10.1016/j.bbrc.2015.01.085
Jones CJP, Desoye G (2011) A new possible function for placental pericytes. Cells Tissues Organs
194:76–84. https://doi.org/10.1159/000322394
Kamouchi M, Ago T, Kitazono T (2011) Brain Pericytes: emerging concepts and functional
roles in brain homeostasis. Cell Mol Neurobiol 31(2):175–193. https://doi.org/10.1007/
s10571-010-9605-x
Katare R, Riu F, Mitchell K, Gubernator M, Campagnolo P, Cui Y, Fortunato O, Avolio E, Cesselli
D, Beltrami AP, Angelini G, Emanueli C, Madeddu P (2011) Transplantation of human peri-
cyte progenitor cells improves the repair of infarcted heart through activation of an angio-
genic program involving micro-RNA-132. Circ Res 109:894–906. https://doi.org/10.1161/
CIRCRESAHA.111.251546
Kaufmann P, Luckhardt M, Leiser R (1988) Three-dimensional representation of the
Fetal vessel system in the human placenta. Trophobl Res 3:113–137. https://doi.
org/10.1007/978-1-4615-8109-3_9
Kaufmann P, Mayhew TM, Charnock-Jones DS (2004) Aspects of human Fetoplacental
Vasculogenesis and angiogenesis. II. Changes during Normal pregnancy. Placenta 25:114–126.
https://doi.org/10.1016/j.placenta.2003.10.009
Khodadi E, Shahrabi S, Shahjahani M, Azandeh S, Saki N (2016) Role of stem cell factor in the
placental niche. Cell Tissue Res 366:523–531. https://doi.org/10.1007/s00441-016-2429-3
Kim JA, Tran ND, Li Z, Yang F, Zhou W, Fisher MJ (2006) Brain endothelial Hemostasis regulation
by Pericytes. J Cereb Blood Flow Metab 26:209–217. https://doi.org/10.1038/sj.jcbfm.9600181
Kim KR, Sung CO, Kwon TJ, Lee JB, Robboy SJ (2015) Defective pericyte recruitment of villous
stromal vessels as the possible etiologic cause of hydropic change in complete hydatidiform
mole. PLoS One 10:1–13. https://doi.org/10.1371/journal.pone.0122266
Knox K, Baker JC (2008) Genomic evolution of the placenta using co-option and duplication and
divergence. Genome Res 18:695–705. https://doi.org/10.1101/gr.071407.107
Krautler NJ, Kana V, Kranich J, Tian Y, Perera D, Lemm D, Schwarz P, Armulik A, Browning
JL, Tallquist M, Buch T, Oliveira-Martins JB, Zhu C, Hermann M, Wagner U, Brink R,
Heikenwalder M, Aguzzi A (2012) Follicular dendritic cells emerge from ubiquitous perivas-
cular precursors. Cell 150:194–206. https://doi.org/10.1016/j.cell.2012.05.032
Krueger M, Bechmann I (2010) CNS pericytes: concepts, misconceptions, and a way out. Glia
58:1–10. https://doi.org/10.1002/glia.20898
Kučera T, Vyletěl I, Moravcová M, Krejčí V, Žižka Z, Jirkovská M (2010) Pericyte coverage
of fetoplacental vessels in pregnancies complicated by type 1 diabetes mellitus. Placenta
31:1120–1122. https://doi.org/10.1016/j.placenta.2010.09.014
Kunisaki Y, Bruns I, Scheiermann C, Ahmed J, Pinho S, Zhang D, Mizoguchi T, Wei Q, Lucas D,
Ito K, Mar JC, Bergman A, Frenette PS (2013) Arteriolar niches maintain haematopoietic stem
cell quiescence. Nature 502:637–643. https://doi.org/10.1038/nature12612
Lachenmayer L (1971a) Adrenergic innervation of the umbilical vessels. Light- and fluorescence
microscopic studies. Z Zellforsch Mikrosk Anat 120:120–136
Lachenmayer L (1971b) Adrenergic innervation of the umbilical vessels. Zeitschrift für Zellforsch
und Mikroskopische Anat 120:120–136. https://doi.org/10.1007/BF00331246
Lash GE, Otun HA, Innes BA, Percival K, Searle RF, Robson SC, Bulmer JN (2010) Regulation of
extravillous trophoblast invasion by uterine natural killer cells is dependent on gestational age.
Hum Reprod 25:1137–1145. https://doi.org/10.1093/humrep/deq050
8 Pericytes in the Placenta: Role in Placental Development and Homeostasis 147

Lee LK, Ueno M, Van Handel B, Mikkola HK (2010) Placenta as a newly identified source
of hematopoietic stem cells. Curr Opin Hematol 17:313–318. https://doi.org/10.1097/
MOH.0b013e328339f295
Leiser R, Kaufmann P (1994) Placental structure: in a comparative aspect. Exp Clin Endocrinol
102:122–134
Leiser R, Kohler T (1983) The blood vessels of the cat girdle placenta. Observations on corrosion
casts, scanning electron microscopical and histological studies—I. Maternal vasculature. Anat
Embryol (Berl) 167:85–93. https://doi.org/10.1007/BF00304602
Leiser R, Kohler T (1984) The blood vessels of the cat girdle placenta. Observations on corrosion
casts, scanning electron microscopical and histological studies—II. Fetal vasculature. Anat
Embryol (Berl) 170:209–216. https://doi.org/10.1007/BF00319006
Leiser R, Krebs C, Ebert B, Dantzer V (1997a) Placental vascular corrosion cast studies: a com-
parison between ruminants and humans. Microsc Res Tech 38:76–87. https://doi.org/10.1002/
(SICI)1097-0029(19970701/15)38:1/2<76::AID-JEMT9>3.0.CO;2-S
Leiser R, Krebs C, Klisch K, Ebert B, Dantzer V, Schuler G, Hoffmann B (1997b) Fetal villosity
and microvasculature of the bovine placentome in the second half of gestation. J Anat 191:517–
527. https://doi.org/10.1017/S0021878297002690
Leiser R, Pfarrer C, Abd-Elnaeim M, Dantzer V (1998) Feto-maternal anchorage in epitheliocho-
rial and endotheliochorial placental types studied by histology and microvascular corrosion
casts. Placenta 19:21–39. https://doi.org/10.1016/S0143-4004(98)80031-3
Leno-Durán E, Hatta K, Bianco J, Yamada ÁT, Ruiz-Ruiz C, Olivares EG, Croy BA (2010) Fetal-­
placental hypoxia does not result from failure of spiral arterial modification in mice. Placenta
31:731–737. https://doi.org/10.1016/j.placenta.2010.06.002
Leonard S, Murrant C, Tayade C, Vandenheuvel M, Watering R, Croy B (2006) Mechanisms
regulating immune cell contributions to spiral artery modification—facts and hypotheses—a
review. Placenta 27:40–46. https://doi.org/10.1016/j.placenta.2005.11.007
Levéen P, Pekny M, Gebre-Medhin S, Swolin B, Larsson E, Betsholtz C (1994) Mice deficient for
PDGF B show renal, cardiovascular, and hematological abnormalities. Genes Dev 8:1875–1887
Li C, Houser BL, Nicotra ML, Strominger JL (2009) HLA-G homodimer-induced cytokine secre-
tion through HLA-G receptors on human decidual macrophages and natural killer cells. Proc
Natl Acad Sci U S A 106:5767–5772. https://doi.org/10.1073/pnas.0901173106
Lindahl P, Johansson BR, Levéen P, Betsholtz C (1997) Pericyte loss and microaneurysm forma-
tion in PDGF-B-deficient mice. Science 277:242–245
Lobo SE, Leonel LCPC, Miranda CMFC, Coelho TM, Ferreira GAS, Mess A, Abrão MS, Miglino
MA (2016) The placenta as an organ and a source of stem cells and extracellular matrix: a
review. Cells Tissues Organs 201:239–252. https://doi.org/10.1159/000443636
Looman C, Sun T, Yu Y, Zieba A, Ahgren A, Feinstein R, Forsberg H, Hellberg C, Heldin CH,
Zhang XQ, Forsberg-Nilsson K, Khoo N, Fundele R, Heuchel R (2007) An activating mutation
in the PDGF receptor-beta causes abnormal morphology in the mouse placenta. Int J Dev Biol
51:361–370. https://doi.org/10.1387/ijdb.072301cl
Macara L, Kingdom JC, Kaufmann P, Kohnen G, Hair J, More IA, Lyall F, Greer IA (1996)
Structural analysis of placental terminal villi from growth-restricted pregnancies with abnor-
mal umbilical artery Doppler waveforms. Placenta 17:37–48
Maier CL, Pober JS (2011) Human placental pericytes poorly stimulate and actively regulate
allogeneic CD4 T cell responses. Arterioscler Thromb Vasc Biol 31:183–189. https://doi.
org/10.1161/ATVBAHA.110.217117
Maier CL, Shepherd BR, Yi T, Pober JS (2010) Explant outgrowth, propagation and
characterization of human pericytes. Microcirculation 17:367–380. https://doi.
org/10.1111/j.1549-8719.2010.00038.x
Méndez-Ferrer S, Michurina TV, Ferraro F, Mazloom AR, MacArthur BD, Lira SA, Scadden DT,
Ma’ayan A, Enikolopov GN, Frenette PS (2010) Mesenchymal and haematopoietic stem cells
form a unique bone marrow niche. Nature 466:829–834. https://doi.org/10.1038/nature09262
Mess A, Carter AM (2007) Evolution of the placenta during the early radiation of placental mam-
mals. Comp Biochem Physiol A Mol Integr Physiol 148(4):769–779
148 R. S. N. Barreto et al.

Mezrich JD, Fechner JH, Zhang X, Johnson BP, Burlingham WJ, Bradfield CA (2010) An interac-
tion between kynurenine and the aryl hydrocarbon receptor can generate regulatory T cells.
J Immunol 185:3190–3198. https://doi.org/10.4049/jimmunol.0903670
Moffett A, Loke C (2006) Immunology of placentation in eutherian mammals. Nat Rev Immunol
6:584–594. https://doi.org/10.1038/nri1897
Mónica Brauer M, Smith PG (2015) Estrogen and female reproductive tract innervation: cellular
and molecular mechanisms of autonomic neuroplasticity. Auton Neurosci 187:1–17
Moreau JLM, Artap ST, Shi H, Chapman G, Leone G, Sparrow DB, Dunwoodie SL (2014) Cited2
is required in trophoblasts for correct placental capillary patterning. Dev Biol 392:62–79.
https://doi.org/10.1016/j.ydbio.2014.04.023
Moser G, Weiss G, Sundl M, Gauster M, Siwetz M, Lang-Olip I, Huppertz B (2017) Extravillous
trophoblasts invade more than uterine arteries: evidence for the invasion of uterine veins.
Histochem Cell Biol 147:353–366. https://doi.org/10.1007/s00418-016-1509-5
Mossman HW (1987) Vertebrate fetal membranes. Rutgers University Press, New Brunswisk
Muñoz-Fernández R, de la Mata C, Prados A, Perea A, Ruiz-Magaña MJ, Llorca T, Fernández-­
Rubio P, Blanco O, Abadía-Molina AC, Olivares EG (2018) Human predecidual stromal cells
have distinctive characteristics of pericytes: cell contractility, chemotactic activity, and expres-
sion of pericyte markers and angiogenic factors. Placenta 61:39–47. https://doi.org/10.1016/j.
placenta.2017.11.010
Murphy WJ, Eizirik E, Johnson WE, Zhang YP, Ryder OA, O’Brien SJ (2001) Molecular
phylogenetics and the origins of placental mammals. Nature 409:614–618. https://doi.
org/10.1038/35054550
Murrant CL (2014) Intravital imaging of Vasoactivity in the uterine arterial vasculature tree during
pregnancy. In: Croy BA, Yamada A, DeMayo F, Lee Adamson S (eds) The guide to investiga-
tion of mouse pregnancy. Elseiver, San Diego, CA
Myatt L (1992) Control of vascular resistance in the human placenta. Placenta 13:329–341. https://
doi.org/10.1016/0143-4004(92)90057-Z
Nadeau V, Charron J (2014) Essential role of the ERK/MAPK pathway in blood-placental barrier
formation. Development 141:2825–2837. https://doi.org/10.1242/dev.107409
Nakagawa S, Deli MA, Nakao S, Honda M, Hayashi K, Nakaoke R, Kataoka Y, Niwa M (2007)
Pericytes from brain microvessels strengthen the barrier integrity in primary cultures of rat brain
endothelial cells. Cell Mol Neurobiol 27:687–694. https://doi.org/10.1007/s10571-007-9195-4
Nakamura K, Kamouchi M, Kitazono T, Kuroda J, Matsuo R, Hagiwara N, Ishikawa E, Ooboshi
H, Ibayashi S, Iida M (2008) Role of NHE1 in calcium signaling and cell proliferation in
human CNS pericytes. AJP Hear Circ Physiol 294:H1700–H1707. https://doi.org/10.1152/
ajpheart.01203.2007
Nees S, Weiss DR, Juchem G (2013) Focus on cardiac pericytes. Pflügers Arch - Eur J Physiol
465:779–787. https://doi.org/10.1007/s00424-013-1240-1
Nikolov SD, Schiebler TH (1973) Über das fetale Gefäßsystem der reifen menschlichen Placenta.
Zeitschrift für Zellforsch und Mikroskopische Anat 139:333–350. https://doi.org/10.1007/
BF00306590
Nomura M, Yamagishi SI, Harada SI, Hayashi Y, Yamashima T, Yamashita J, Yamamoto H (1995)
Possible participation of autocrine and paracrine vascular endothelial growth factors in hypoxia-­
induced proliferation of endothelial cells and pericytes. J Biol Chem 270:28316–28324. https://
doi.org/10.1074/jbc.270.47.28316
Ogino S, Redline RW (2000) Villous capillary lesions of the placenta: distinctions between choran-
gioma, chorangiomatosis, and chorangiosis. Hum Pathol 31:945–954. https://doi.org/10.1053/
hupa.2000.9036
Ohlsson R, Falck P, Hellstrom M, Lindahl P, Bostrom H, Franklin G, Ährlund-Richter L, Pollard
J, Soriano P, Betsholtz C (1999) PDGFB regulates the development of the labyrinthine layer
of the mouse fetal placenta. Dev Biol 212:124–136. https://doi.org/10.1006/dbio.1999.9306
Oliveira MS, Barreto-Filho JB (2015) Placental-derived stem cells: culture, differentiation and
challenges. World J Stem Cells 7:769–775. https://doi.org/10.4252/wjsc.v7.i4.769
8 Pericytes in the Placenta: Role in Placental Development and Homeostasis 149

Ottersbach K, Dzierzak E (2010) The placenta as a haematopoietic organ. Int J Dev Biol 54:1099–
1106. https://doi.org/10.1387/ijdb.093057ko
Pallone TL, Silldorff EP (2001) Pericyte regulation of renal medullary blood flow. Exp Nephrol
9:165–170. doi: 52608
Pallone TL, Silldorff EP, Turner MR (1998) Intrarenal blood flow: microvascular anatomy and the
regulation of medullary perfusion. Clin Exp Pharmacol Physiol 25:383–392
Pallone TL, Zhang Z, Rhinehart K (2003) Physiology of the renal medullary microcirculation. Am
J Physiol Ren Physiol 284:F253–F266. https://doi.org/10.1152/ajprenal.00304.2002
Pan S-Y, Chang Y-T, Lin S-L (2014) Microvascular pericytes in healthy and diseased kidneys. Int
J Nephrol Renovasc Dis 7:39–48. https://doi.org/10.2147/IJNRD.S37892
Pereira FTV, Oliveira LJ, Barreto RSN, Mess A, Perecin F, Bressan FF, Mesquita LG, Miglino
MA, Pimentel JR, Neto PF, Meirelles FV (2013) Fetal-maternal interactions in the
Synepitheliochorial placenta using the eGFP cloned cattle model. PLoS One 8:e64399. https://
doi.org/10.1371/journal.pone.0064399
Persson E, Rodriguez-Martinez H (1997) Immunocytochemical localization of growth fac-
tors and intermediate filaments during the establishment of the porcine placenta. Microsc
Res Tech 38:165–175. https://doi.org/10.1002/(SICI)1097-0029(19970701/15)38:1/2<165::
AID-JEMT17>3.0.CO;2-N
Pfister F, Przybyt E, Harmsen MC, Hammes H-P (2013) Pericytes in the eye. Pflügers Arch Eur
J Physiol 465:789–796. https://doi.org/10.1007/s00424-013-1272-6
Prazeres PHDM, Sena IFG, IDT B, de Azevedo PO, Andreotti JP, de Paiva AE, de Almeida VM, de
Paula Guerra DA, Pinheiro Dos Santos GS, Mintz A, Delbono O, Birbrair A (2017) Pericytes
are heterogeneous in their origin within the same tissue. Dev Biol 427:6–11. https://doi.
org/10.1016/j.ydbio.2017.05.001
Ramsey EM, Martin CB, Donner MW (1967) Fetal and maternal placental circulations.
Simultaneous visualization in monkeys by radiography. Am J Obstet Gynecol 98:419–423
Resta L, Capobianco C, Marzullo A, Piscitelli D, Sanguedolce F, Schena FP, Gesualdo L (2006)
Confocal laser scanning microscope study of terminal villi vessels in Normal term and pre-­
eclamptic placentas. Placenta 27:735–739. https://doi.org/10.1016/j.placenta.2005.07.006
Rhodin J (1968) Ultrastructure of mammalian venous capillaries, venules, and small collecting
veins. J Ultrastruct Res 25:452–500. https://doi.org/10.1016/S0022-5320(68)80098-X
Rizzo R, Lo Monte G, Bortolotti D, Graziano A, Gentili V, Di Luca D, Marci R (2015) Impact of
soluble HLA-G levels and endometrial NK cells in uterine flushing samples from primary and
secondary unexplained infertile women. Int J Mol Sci 16:5510–5516. https://doi.org/10.3390/
ijms16035510
Robin C, Bollerot K, Mendes S, Haak E, Crisan M, Cerisoli F, Lauw I, Kaimakis P, Jorna R,
Vermeulen M, Kayser M, van der Linden R, Imanirad P, Verstegen M, Nawaz-Yousaf H,
Papazian N, Steegers E, Cupedo T, Dzierzak E (2009) Human placenta is a potent hematopoi-
etic niche containing hematopoietic stem and progenitor cells throughout development. Cell
Stem Cell 5(4):385–395. https://doi.org/10.1016/j.stem.2009.08.020
Rodriguez-Martinez H, Persson E, Hurst M, Stanchev P (1992) Immunohistochemical localiza-
tion of platelet-derived growth factor receptors in the porcine uterus during the oestrous cycle
and pregnancy. J Vet Med Ser A 39:1–10. https://doi.org/10.1111/j.1439-0442.1992.tb00151.x
Rouget C-MB (1873) Mémoire sur le développement, la structure et les proprietés physiologiques
des capillaires sanguins et lymphatiques. Arch Psys 5:603–610
Rucker HK, Wynder HJ, Thomas WE (2000) Cellular mechanisms of CNS pericytes. Brain Res
Bull 51:363–369
Sato K, Yamashita N, Baba M, Matsuyama T (2003) Modified myeloid dendritic cells act as regu-
latory dendritic cells to induce anergic and regulatory T cells. Blood 101:3581–3589. https://
doi.org/10.1182/blood-2002-09-2712
Shimizu F, Sano Y, Maeda T, Abe M, Nakayama H, Takahashi R, Ueda M, Ohtsuki S, Terasaki T,
Obinata M, Kanda T (2008) Peripheral nerve pericytes originating from the blood-nerve barrier
expresses tight junctional molecules and transporters as barrier-forming cells. J Cell Physiol
217:367–380. https://doi.org/10.1002/jcp.21508
150 R. S. N. Barreto et al.

Sims DE (2000) Diversity within Pericytes. Clin Exp Pharmacol Physiol 27:842–846. https://doi.
org/10.1046/j.1440-1681.2000.03343.x
Soriano P (1994) Abnormal kidney development and hematological disorders in PDGF beta-­
receptor mutant mice. Genes Dev 8:1888–1896
Stark K, Eckart A, Haidari S, Tirniceriu A, Lorenz M, Von Brühl ML, Gärtner F, Khandoga AG,
Legate KR, Pless R, Hepper I, Lauber K, Walzog B, Massberg S (2013) Capillary and arteriolar
pericytes attract innate leukocytes exiting through venules and “instruct” them with pattern-­
recognition and motility programs. Nat Immunol 14(1):41–51. https://doi.org/10.1038/ni.2477
Talwadekar MD, Kale VP, Limaye LS (2015) Placenta-derived mesenchymal stem cells possess
better immunoregulatory properties compared to their cord-derived counterparts–a paired sam-
ple study. Sci Rep 5:15784. https://doi.org/10.1038/srep15784
Thanabalasundaram G, Schneidewind J, Pieper C, Galla H-J (2011) The impact of pericytes on
the blood-brain barrier integrity depends critically on the pericyte differentiation stage. Int
J Biochem Cell Biol 43(9):1284–1293. https://doi.org/10.1016/j.biocel.2011.05.002
Thomas WE (1999) Brain macrophages: on the role of pericytes and perivascular cells. Brain Res
Brain Res Rev 31:42–57
Thornburg KL, Bagby SP, Giraud GD (2006) Maternal adaptation to pregnancy. In: Neill JD (ed)
Knobil and Neill’s physiology of reproduction. Elseiver, Amsterdam
Tilburgs T, Crespo ÂC, van der Zwan A, Rybalov B, Raj T, Stranger B, Gardner L, Moffett A,
Strominger JL (2015) Human HLA-G+ extravillous trophoblasts: immune-activating cells that
interact with decidual leukocytes. Proc Natl Acad Sci 112:7219–7224. https://doi.org/10.1073/
pnas.1507977112
Tu Z, Li Y, Smith DS, Sheibani N, Huang S, Kern T, Lin F (2011) Retinal pericytes inhibit acti-
vated T cell proliferation. Invest Ophthalmol Vis Sci 52:9005–9010. https://doi.org/10.1167/
iovs.11-8008
Ueno H, Weissman IL (2010) The origin and fate of yolk sac hematopoiesis: application of chi-
mera analyses to developmental studies. Int J Dev Biol 54:1019–1031. https://doi.org/10.1387/
ijdb.093039hu
van der Heijden GW, Dieker JW, Derijck AAHA, Muller S, Berden JHM, Braat DDM, van der
Vlag J, de Boer P (2005) Asymmetry in histone H3 variants and lysine methylation between
paternal and maternal chromatin of the early mouse zygote. Mech Dev 122:1008–1022. https://
doi.org/10.1016/j.mod.2005.04.009
Verbeek MM, Westphal JR, Ruiter DJ, de Waal RM (1995) T lymphocyte adhesion to human brain
pericytes is mediated via very late antigen-4/vascular cell adhesion molecule-1 interactions.
J Immunol 154:5876–5884
Vogel P (2005) The current molecular phylogeny of eutherian mammals challenges previ-
ous interpretations of placental evolution. Placenta 26:591–596. https://doi.org/10.1016/j.
placenta.2004.11.005
Wehrenberg WB, Chaichareon DP, Dierschke DJ, Rankin JH, Ginther OJ (1977) Vascular dynam-
ics of the reproductive tract in the female rhesus monkey: relative contributions of ovarian and
uterine arteries. Biol Reprod 17:148–153
Wooding FBP, Burton GJ (2008a) Comparative placentation: structures, functions and evolution.
Springer, New York
Wooding P, Burton GJ (2008b) Haemochorial placentation: mouse, rabbit, man, apes, monkeys. In:
Comparative placentation. Springer, Berlin, pp 185–230
Wooding FBP, Flint APF (1994) Placentation. In: Lamming GE (ed) Marshall’s physiology of
reproduction, 4th edn. Springer-Science, London, pp 233–429
Wynn RM (1974) Ultrastructural development of the human decidua. Am J Obstet Gynecol
118(5):652–670. https://doi.org/10.1016/S0002-9378(16)33740-1
Yamada AT, Bianco JR, Lippe EMO, Degaki KY, Dalmorin AF, Edwards AK, Lima PDA, Paffaro
VA (2014) Unique features of endometrial dynamics during pregnancy. In: Anne Croy B,
Yamada A, DeMayo F, Lee Adamson S (eds) The guide to investigation of mouse pregnancy.
Elseiver, San Diego, CA
8 Pericytes in the Placenta: Role in Placental Development and Homeostasis 151

Yamagishi S, Kawakamia T, Fujimoria H, Yonekura H, Tanaka N, Yamamoto Y, Urayama H,

Watanabe Y, Yamamoto H (1999a) Insulin stimulates the growth and tube formation of
human microvascular endothelial cells through autocrine vascular endothelial growth factor.
Microvasc Res 57:329–339
Yamagishi S, Yonekura H, Yamamoto Y, Fujimori H, Sakurai S, Tanaka N, Yamamoto H (1999b)
Vascular endothelial growth factor acts as a pericyte mitogen under hypoxic conditions. Lab
Investig 79(4):501–509
Yonekura H, Sakurai S, Liu X, Migita H, Wang H, Yamagishi S, Nomura M, Abedin MJ, Unoki
H, Yamamoto Y, Yamamoto H (1999) Placenta growth factor and vascular endothelial growth
factor B and C expression in microvascular endothelial cells and Pericytes. J Biol Chem
274:35172–35178
Zebardast N, Lickorish D, Davies JE (2010) Human umbilical cord perivascular cells (HUCPVC):
a mesenchymal cell source for dermal wound healing. Organogenesis 6:197–203. https://doi.
org/10.4161/org.6.4.12393
Zhang EG, Burton GJ, Smith SK, Charnock-Jones DS (2002) Placental vessel adaptation during
gestation and to high altitude: changes in diameter and perivascular cell coverage. Placenta
23(10):751–762. https://doi.org/10.1016/S0143-4004(02)90856-8
Zhao H, Feng J, Seidel K, Shi S, Klein O, Sharpe P, Chai Y (2014) Secretion of shh by a neuro-
vascular bundle niche supports mesenchymal stem cell homeostasis in the adult mouse incisor.
Cell Stem Cell 14:160–173. https://doi.org/10.1016/j.stem.2013.12.013
Zhu Y, Yang Y, Zhang Y, Hao G, Liu T, Wang L, Yang T, Wang Q, Zhang G, Wei J, Li Y (2014)
Placental mesenchymal stem cells of fetal and maternal origins demonstrate different therapeu-
tic potentials. Stem Cell Res Ther 5:48. https://doi.org/10.1186/scrt436
Zygmunt M, Herr F, Münstedt K, Lang U, Liang OD (2003) Angiogenesis and vasculogenesis in
pregnancy. Eur J Obstet Gynecol Reprod Biol 110(Suppl 1):S10–S18
Chapter 9
Pericytes in the Liver

Enis Kostallari and Vijay H. Shah

Abstract Liver pericytes, commonly named hepatic stellate cells (HSCs), reside in
the space between liver sinusoidal endothelial cells (LSECs) and hepatocytes. They
display important roles in health and disease. HSCs ensure the storage of the major-
ity of vitamin A in a healthy body, and they represent the major source of fibrotic
tissue in liver disease. Surrounding cells, such as LSECs, hepatocytes, and Kupffer
cells, present a significant role in modulating HSC behavior. Therapeutic strategies
against liver disease are being currently developed, where HSCs represent an ideal
target. In this chapter, we will discuss HSC quiescence and activation in the context
of healthy liver and diseases, such as fibrosis, steatohepatitis, and hepatocellular
carcinoma.

Keywords Liver · Hepatic stellate cells · Pericytes · Healthy liver · Regeneration ·

Fibrosis · NASH · NAFLD · Hepatocellular carcinoma · Hepatocytes · Sinusoidal
endothelial cells · Kupffer cells

9.1 Introduction

The liver-specific pericytes are called hepatic stellate cells (HSCs). They reside in
the subendothelial space of Disse, defined as the space between the liver sinusoidal
endothelial cells and the parenchymal cells. HSCs were first described in 1876 by
the German anatomist Carl von Kupffer who visualized them using gold chloride
preparations of human liver. He called these cells “sternzellen” meaning stellate
cells due to their shape (Kupffer 1876). In 1952, Toshito Ito described perisinusoi-
dal cells as fat-storing cells based on their capacity to accumulate lipid droplets (Ito
and Nemoto 1952), also called later lipocytes or Ito cells. The term HCS was reused
in 1971 by the Japanese Kenjiro Wake (Wake 1971), who also described the storage
of vitamin A in rat quiescent HSCs (Wake 1974) and who opened the era of their

E. Kostallari · V. H. Shah (*)

Division of Gastroenterology and Hepatology, Mayo Clinic, Rochester, MN, USA
e-mail: [email protected]

© Springer Nature Switzerland AG 2019 153

A. Birbrair (ed.), Pericyte Biology in Different Organs,
Advances in Experimental Medicine and Biology 1122,
https://doi.org/10.1007/978-3-030-11093-2_9
154 E. Kostallari and V. H. Shah

characterization. The existence of many names describing the same cell, such as Ito
cell, fat-storing cell, lipocyte, and perisinusoidal or parasinusoidal cell, prompted
the investigators of the field in 1996 to standardize the term HSC when referring to
these cells (Hepatology 1996;23:193). In this chapter, we will discuss the origin of
HSCs, their role in the adult healthy liver, and finally their implication in liver dis-
eases where HSCs are designated as the main mediators of fibrosis.

9.2 The Origin of HSCs During Development

The developmental origin of HSCs has been debated since they express both neuro-
nal and mesenchymal cell markers, such as nestin, glial fibrillary acidic protein
(GFAP), p75 neurotrophin receptor (p75NTR), desmin, type 1 collagen, and vimen-
tin. For this reason, it has been thought that HSCs derive from neural crest and from
the mesenchyme (Asahina 2012). To investigate the neural crest origin of HSCs,
yellow fluorescent protein (YFP)+ neural crest descendants were obtained by cross-
ing wingless-type MMTV integration site family member 1 (Wnt1)Cre mice with
Rosa26YFP reporter mice (Cassiman et al. 2006). In this model, HSCs failed to
express the YFP leading to the conclusion that they do not derive from the neural
crest (Cassiman et al. 2006). Nevertheless, a more recent study shows that in zebraf-
ish, HSCs express heart and neural crest derivatives expressed 2 (hand2), a meso-
derm and neural crest marker suggesting a link between HSCs and neural crest (Yin
et al. 2012). This controversy may be explained by interspecies differences and
needs further investigation.
The most accepted developmental origin for HSCs is a sheet of mesoderm that
develops along the foregut endoderm near the cardiac mesoderm, called the septum
transversum (Asahina et al. 2009, 2011; Loo and Wu 2008; Pérez-Pomares et al.
2004; Toi et al. 2018). The septum transversum is also important for the prenatal
development of the liver by secreting bone morphogenetic proteins (BMPs) and
fibroblast growth factors (FGFs) (Zaret 2002). To test whether septum transversum
contributes to HSC population in mice, Wilm’s tumor 1 (WT1) was used as a marker.
Before liver development, WT1 is only expressed in the septum transversum, while
in later stages of liver morphogenesis, WT1 is expressed in mesothelial and subme-
sothelial cells. These cells give rise to WT1−/β-galactosidase+ HSCs (Asahina et al.
2011). The septum transversum origin of HSCs is also demonstrated in rats and in
developing human liver (Toi et al. 2018; Loo and Wu 2008). Moreover, in human
fetuses a cell population expressing hematopoietic stem cell markers (CD34 and
cytokeratin 7/8) and HSC markers [desmin and α-smooth muscle actin (αSMA)]
was identified suggesting a link between hematopoietic and hepatic systems
(Suskind and Muench 2004). In addition to these studies, further investigation is
necessary to understand the developmental origin of HSCs in different species and
whether there exist several subpopulations of HSCs deriving from different
precursors.
9 Pericytes in the Liver 155

9.3 Role of HSCs in Healthy Liver

In a healthy adult liver, quiescent HSCs represent ~10% of total resident cells
(Tsuchida and Friedman 2017). They are mainly recognized for their role in storage
and controlled release of vitamin A. Storage of retinols or vitamin A in cytoplasmic
lipid droplets is the most distinctive feature of quiescent HSCs. These lipid droplets
are routinely used for HSC isolation and are autofluorescent when excited with the
light of ~328 nm (Popper 1944) (Fig. 9.1). In a healthy liver, 50–80% of total body
retinol is stocked in the liver (Blomhoff et al. 1990), of which 80–90% is stored in
HSCs (Hendriks et al. 1985). While it remains unknown how retinol is transferred
to HSCs, more studies exist regarding retinol metabolism in HSCs. Upon entering
in HSCs, retinol is esterified and mostly stored as retinyl esters by lecithin retinol
acyltransferase (LRAT) (Friedman 2008). Retinyl esters constitute 30–50% of the

Fig. 9.1 (Left) Primary quiescent HSCs isolated from mouse liver presenting autofluorescent lipid
droplets (day 1 after isolation). (Right) Primary activated HSC isolated from mouse liver (day 10
after isolation). It presents several processes, and lipid droplets are absent. (Upper panels)
Autofluorescent lipid droplets. (Lower panels) Bright-field capture of the cells merged with the
autofluorescent lipid droplets. Scale bar: 10 μm
156 E. Kostallari and V. H. Shah

lipids of HSC lipid droplets (Senoo 2004). Vitamin A is important for maintaining
HSCs in quiescence since treatment of cultured HSCs with vitamin A induced its
accumulation in cytoplasmic lipid droplets and decreased the expression of HSC
activation markers (Yoneda et al. 2016). Moreover, all-trans-retinoic acid, a derivate
of vitamin A, was demonstrated to reduce HSC activation (Shimizu et al. 2018).
However, the absence of lipid droplets in LRAT-deficient mice was not associated
with liver fibrosis (Kluwe et al. 2011). This controversy raises the question whether
vitamin A loss is the cause or the consequence of HSC activation, leading to the
need of further studies to elucidate this question.
HSCs also secrete molecules that act in an autocrine or paracrine manner, part of
which are lipoproteins, growth factors, and cytokines (Friedman 2008). As other
quiescent perivascular cells, HSCs secrete apolipoprotein E (ApoE) (Ramadori
et al. 1989). At basal level, ApoE-deficient mice showed increased transforming
growth factor beta (TGFβ), monocyte chemoattractant protein-1 (MCP-1), and tis-
sue inhibitor of metalloproteinase-1 (TIMP-1) expression in the liver when com-
pared to wild-type (WT) mice (Ferré et al. 2009), suggesting a role for ApoE in
maintaining liver homeostasis. It would have been interesting to investigate whether
HSC-specific ApoE deletion would have an incidence on liver injury. Follistatin and
interleukin 10 (IL-10) are other molecules secreted by HSCs that have shown anti-­
fibrotic effects (Friedman 2008). Indeed, treatment of carbon tetrachloride (CCl4)-
exposed rats with follistatin reduced liver fibrosis and constrained HSC proliferation
(Patella et al. 2006). Nevertheless, another study showed that follistatin is expressed
only by activated HSCs and could be a marker of liver fibrosis (Boers et al. 2006).
HSCs can also secrete IL-10, a hepatoprotective cytokine recognized for its role in
reducing liver injury (Thompson et al. 1998a, b; Byun et al. 2013). Indeed, IL-10-­
deficient mice develop more severe liver fibrosis following CCl4 administration
(Thompson et al. 1998a, b). The anti-fibrotic molecules produced by HSCs deserve
more elucidation as they have a therapeutic potential.
HSCs have also been shown to have an important role in liver regeneration
following a partial hepatectomy (Preziosi and Monga 2017). In the earlier phase of
liver regeneration, they regulate matrix degradation by secreting matrix metallopro-
teinase and several proteoglycans and induce hepatocyte proliferation by secreting
pleiotrophin and the potent mitogen hepatocyte growth factor (HGF) (Asahina et al.
2002; Taub 2004; Preziosi and Monga 2017). When HSC death was caused by glio-
toxin 24 hours before partial hepatectomy, hepatocyte proliferation was signifi-
cantly impaired due to a lack of HGF (Nejak-Bowen et al. 2013). At later phases of
liver regeneration, HSCs secrete the mitoinhibitory TGFβ to inhibit hepatocyte pro-
liferation (Preziosi and Monga 2017). When gliotoxin was administered 5 days after
partial hepatectomy, hepatocyte proliferation was prolonged due to decreased HSC-­
derived TGFβ and collagen deposition (Nejak-Bowen et al. 2013). An increasing
interest has been shown on the aspect of HSCs as progenitor cells. Several groups
have demonstrated that HSCs can differentiate in hepatocytes during liver regenera-
tion (Yang et al. 2008b; Kordes et al. 2014), suggesting a mesenchymal stem cell
role. All these studies show that HSC presence is essential for normal liver function
and regeneration. Nevertheless, further investigation is needed to elucidate the
existing controversy on the role of HSC-derived signaling in healthy liver.
9 Pericytes in the Liver 157

9.4 Role of HSCs in Liver Disease

HSCs have mainly been studied in liver diseases, due to their capacity to transdif-
ferentiate in myofibroblasts, the major source of collagen deposition during fibro-
genesis. In this sub-chapter, the HSC behavior will be discussed in the context of
liver fibrosis, steatohepatitis, and hepatocellular carcinoma.

9.4.1 HSCs and Liver Fibrosis

Liver fibrosis is the deposition of excessive scar tissue in response to a chronic

injury. The identification of HSC activation in hepatic fibrosis has been a significant
discovery in the understanding of liver’s response to injury. HSC activation refers to
the passage from a quiescent vitamin-rich cell to a proliferative, contractile, migra-
tory, and fibrogenic cell (Friedman 2008) (Fig. 9.1). Some of the most studied sig-
nals that activate HSCs are platelet growth factor (PDGF), TGFβ, Fas cell surface
death receptor (Fas), apoptotic bodies, extracellular vesicles, and stiffness (Friedman
2008; Kostallari and Shah 2016; Dou et al. 2018). These signals derive from several
cell types, such as sinusoidal endothelial cells (SECs) (Kostallari and Shah 2016),
Kupffer cells (Bilzer et al. 2006), injured hepatocytes (Canbay et al. 2002, 2003),
and HSCs (Kostallari et al. 2018) (Fig. 9.2).
In liver injury, SECs become activated or capillarized by losing their fenestrae
(Brunt et al. 2014). Defenestrated SECs increase endothelin-1 (ET-1) secretion and
decrease nitric oxide (NO) release, which contribute to HSC contraction (Kostallari
and Shah 2016; Tsuchida and Friedman 2017). ET-1 is found to also be secreted by
HSCs and to act in an autocrine manner (Pinzani et al. 1996). SECs stimulate HSC
migration by producing stromal-derived factor-1 (SDF-1)/C-X-C motif chemokine
ligand 12 (CXCL12) (Kordes and Häussinger 2013) and releasing sphingosine
kinase (SK)-1-containing EVs (Wang et al. 2015). Moreover, in liver fibrogenesis,
SECs upregulate fibroblast growth factor receptor 1 (FGFR1) and C-X-C motif che-
mokine receptor 4 (CXCR4) expressions, enforcing the fibrogenic phenotype of
HSCs (Ding et al. 2014). Another cell type influencing HSC behavior is the Kupffer
cell. Kupffer cells are the liver resident macrophages lying in the sinusoids and
penetrating between the hepatocytes during liver injury. They have an important
role in HSC migration through PDGF secretion and HSC differentiation into myo-
fibroblasts through TGFβ and reactive oxygen species (ROS) release (Tsuchida and
Friedman 2017; Kiagiadaki et al. 2018). Moreover, Kupffer cells secrete MCP-1 in
a SK-1-dependent manner (Lan et al. 2018). MCP-1 binds to C-C motif chemokine
receptor 2 (CCR2), which is expressed by both HSCs and Kupffer cells, leading to
their respective migration (Marra et al. 1999; Lan et al. 2018) and ultimately to liver
fibrosis (Seki et al. 2009). Treatment of mice with a specific inhibitor of SK-1 pre-
vented liver fibrosis in CCl4- and bile duct ligation (BDL)-induced liver injuries
(Lan et al. 2018). Hepatocytes, which constitute 70–85% of the liver mass, also
158 E. Kostallari and V. H. Shah

Fig. 9.2 Cross-talking between HSCs and the surrounding cells in the context of liver fibrosis.
Defenestrated LSEC upregulates the expression of CXCR4 and FGFR1 and secretion of ET-1,
SDF-1, and SK-1-containing EVs (small red circles), which induce HSC activation. Kupffer cells
participate in this process by secreting PDGF, TGF, ROS, and MCP-1. ROS are also released by
injured hepatocytes. HSCs secrete PDGF, TGF collagen 1, and PDGFRα-enriched EVs (small
purple circles) which act in an autocrine manner. In addition, activated HSCs release IFNβ that
induces hepatocyte apoptosis and hepatocyte-derived apoptotic bodies (pink circles) implicated in
HSC further activation

have a role in HSC activation. In a healthy liver, hepatocytes are polarized cells
presenting microvilli, which contribute to the absorption of proteins and other
molecules coming from the blood. Injured hepatocytes lose their microvilli and can
participate to HSC activation (Canbay et al. 2003; Tsuchida and Friedman 2017).
Indeed, injured hepatocytes underwent apoptosis through a Fas-dependent mecha-
nism and released apoptotic bodies, which were engulfed by HSCs (Canbay et al.
2002, 2003). This induced HSC fibrogenic activity by increasing collagen α1,
TGFβ1, and αSMA expression (Canbay et al. 2003). Moreover, damaged hepato-
cytes release ROS, which activate HSCs and lead to liver fibrogenesis (Jiang et al.
2010; Tsuchida and Friedman 2017).
Besides SECs, Kupffer cells, and hepatocytes, activated HSCs can also release
signals in a paracrine manner, which contribute to fibrosis. Activated HSCs are well
recognized to secrete the canonical pro-fibrogenic molecules such as collagen 1α1,
TGFβ, and PDGF-BB (Drinane et al. 2017; Friedman 2008). Moreover, a decrease
of retinoic acid signaling in HSCs is associated with induction of TGFβ1 expression
and activation of HSCs, suggesting a role for retinoic acid in HSCs quiescence
(Ohata et al. 1997). The presence of bacterial lipopolysaccharide (LPS), due to
9 Pericytes in the Liver 159

increased gut permeability in liver disease, has been shown to have a significant role
in retinoic acid signaling decrease by inducing the autophagy of HSC lipid droplets
and leading to HSC activation (Chen et al. 2017). LPS-dependent activation of
HSCs induces interferon beta (IFNβ) release, which promotes hepatocyte apoptosis
(Dangi et al. 2016). In turn, apoptotic hepatocytes participate to further activation of
HSCs (Canbay et al. 2002, 2003). Moreover, activated HSCs and myofibroblasts
secrete vascular endothelial growth factor (VEGF), a potent angiogenic molecule
that binds to VEGF receptor (VEGFR) expressed by SECs, and induce fibrosis-­
associated angiogenesis (Das et al. 2010). In a recent study, activated HSCs also
secrete PDGFRα-enriched EVs, which have a paracrine effect in promoting in vitro
cell migration and in vivo fibrogenesis (Kostallari et al. 2018). The PDGFRα enrich-
ment in HSC-derived EVs is SHP2 dependent, and treatment of mice with SHP2
inhibitor, SHP099, reduced significantly liver fibrosis (Kostallari et al. 2018).
Interestingly, G protein-coupled receptors (GPCRs) are emerging as new targets in
liver fibrosis. Indeed, protease-activated receptor-2 (PAR2) is expressed in activated
HSCs. Inhibiting PAR2 by PZ-235 decreased HSC proliferation and collagen depo-
sition (Shearer et al. 2016).
Another interesting stimulator of HSC activation is liver stiffness. Indeed, when
plated on a soft substrate, freshly isolated HSCs maintain their lipid droplets and
high levels of PPARγ. However, when plated on a stiff substrate, HSCs lose their
lipid droplets and upregulate αSMA and collagen 1 expression (Guvendiren et al.
2014). Moreover, in a recent study, it has been demonstrated that substrate stiffness
induces HSC activation through translocation into the nucleus of histone acetyl-
transferase p300, which upregulated transcription of HSC activation genes
(Dou et al. 2018). In line with these studies and in a therapeutic perspective, it could
be of interest to further investigate the cause of the stiffness in order to modulate it
in vivo and to explore the role of other GPCRs in liver fibrosis.

9.4.2 HSCs and Steatohepatitis

Steatohepatitis is one of the leading causes of liver diseases in the United States. It
can occur due to a high consumption of alcohol leading to alcoholic steatohepatitis
(ASH) or a high-fat diet leading to nonalcoholic steatohepatitis (NASH). Despite
the different etiologies, these two entities have similar pathogenic mechanisms.
They usually start with steatosis, which is the fat accumulation within the hepato-
cytes (ballooned hepatocytes), followed by hepatocyte cell death response, inflam-
mation, and ultimately fibrogenesis (Greuter et al. 2017; Ibrahim et al. 2018). HSCs
have a significant role in driving inflammation and fibrogenesis in ASH and NASH
by receiving and emitting signals. Lipotoxicity-induced hepatocyte-derived extra-
cellular vesicles promote HSC activation, proliferation, and migration by inhibiting
the quiescence marker peroxisome proliferator-activated receptor gamma (PPARγ)
through miR-128-3p (Povero et al. 2015). Lipotoxicity-induced hepatocyte can also
secrete sonic hedgehog (Shh), which is an activator of HSCs (Yang et al. 2008a;
160 E. Kostallari and V. H. Shah

Rangwala et al. 2011). In turn, activated HSCs can increase the inflammatory reac-
tion by secreting pro-inflammatory cytokines such as MCP1, IL6, TGFβ, and neural
cell adhesion molecules (Friedman 2008; Lee and Jeong 2012).
In alcoholic and nonalcoholic liver disease, intestinal epithelial permeability is
increased, which can be detected by a high level of bacterial LPS in the liver. LPS
can stimulate HSCs through Toll-like receptors (TLRs) (Tsuchida and Friedman
2017). HSCs express several TLRs, such as TLR2, TLR3, TLR4, TLR7, and TLR9
(Seki et al. 2007; Gäbele et al. 2008; Chou et al. 2012; Miura et al. 2013; Byun et al.
2013). In some mouse models of steatohepatitis, activation of TLR4 induces Kupffer
cell chemotaxis and inflammation (Seki et al. 2007; Guo et al. 2009). Moreover
TLR activation in HSCs induces liver fibrosis (Huang et al. 2007; Seki et al. 2007).
HSC nuclear receptors, such as farnesoid X receptor (FXR), peroxisome proliferator-­
activated receptor (PPAR), or vitamin D3 receptor (VDR), are other factors involved
in liver disease. Indeed, deficiency of FXR in HSCs promotes hepatic inflammation
and fibrosis (Kong et al. 2009). Furthermore, a dual PPARα-PPARδ agonist,
GFT505, which may target both hepatocytes and HSCs, protects liver from steato-
sis, inflammation, and fibrosis in several mouse models (Staels et al. 2013). In line
with this, inhibition of VDR signaling by sequestosome 1 (SQSTM1/p62) knockout
attenuates liver inflammation and fibrosis in experimental steatohepatitis (Beilfuss
et al. 2015; Duran et al. 2016).
The interaction between HSCs and neutrophils also seems to play an important
role in the establishment of steatohepatitis through HSCs. Indeed, neutrophils stim-
ulate the secretion of HSC-derived GM-CSF and IL-15, which in turn prolong neu-
trophil survival and may serve as a positive forward loop to promote liver damage
and fibrosis (Zhou et al. 2018). Cholesterol, a component of high-fat diet, is another
factor that can accumulate in HSCs and induce their activation (Tomita et al. 2014).
Cholesterol-lowering drugs, such as ezetimibe or statins, attenuate steatohepatitis
and fibrosis in a mouse model of NASH (Van Rooyen et al. 2013). Nevertheless,
the effect of activated HSCs on recruiting pro-inflammatory cells is not fully
understood and deserves further investigation.

9.4.3 HSCs and Hepatic Metastases

The combination of the unique microenvironment and its hemodynamics character-

istics makes the liver one of the most targeted organs of cancer metastasis.
Furthermore, liver metastases are dependent on the interactions between the tumor
cells and the hepatic stromal cells, such as HSCs. HSCs have several important roles
in liver tumors, such as promoting tumor growth, releasing growth factors and cyto-
kines, regulating extracellular matrix (ECM) turnover and tumor-associated angio-
genesis, and suppressing antitumor immune response (Kang et al. 2011). In 130
cases of hepatocellular carcinoma, peritumoral activated HSCs independently con-
tributed to high recurrence or death rates (Ju et al. 2009). Similar to liver fibrosis,
tumor-stimulated HSCs differentiate into myofibroblasts, upregulate αSMA
9 Pericytes in the Liver 161

expression, proliferate, migrate, and contribute significantly to collagen deposition

(Vidal-Vanaclocha 2008). This behavior is stimulated by tumor cells through para-
crine signaling, including secretion of TGFβ (Kang et al. 2011). In turn, activated
HSCs induce tumor cell proliferation, migration, and invasion through secretion of
several secreted growth factors and cytokines, such as TGFβ, HGF, PDGF, SDF-1,
VEGF, and CXCL12 (Okabe et al. 2009; Amann et al. 2009; Zhao et al. 2011; Kang
et al. 2011; Yaqoob et al. 2012; Dou et al. 2018), and by downregulating other mol-
ecules such as endosialin (Mogler et al. 2017). Indeed, conditioned media of acti-
vated HSCs promoted tumor cell invasion and proliferation in vitro (Okabe et al.
2009; Amann et al. 2009) and in vivo (Dou et al. 2018). Furthermore, HSCs cultured
on a stiff environment induced a higher tumor growth than HSCs cultured on soft
matrix (Dou et al. 2018), suggesting a role for stiffness in tumor progression.
Activated HSCs at the invasive front of liver metastasis can regulate the ECM by
producing matrix metalloproteinase 2 (MMP2), TIMP2, and a disintegrin and
metalloproteinase 9 (ADAM9) allowing cancer cells to increase their invasive
behavior (Musso et al. 1997; Mazzocca et al. 2005). Furthermore, HSC-derived
ECM components, such as collagen, fibronectin, laminin, and CCN family member
1 (CCN1), regulate adhesion, migration, and survival of tumor cells by activating
the integrins on tumor cell surface (Kang et al. 2011; Azzariti et al. 2016; Li et al.
2018). For example, inhibiting α3 integrin on HCC cells abrogated the anti-­apoptotic
effect of laminin-332 (Azzariti et al. 2016). Moreover, the expression of neuropilin-
­1 by HSC-derived myofibroblasts promoted the secretion of fibronectin and there-
fore the increase of tumor microenvironment stiffness leading to a greater tumor
growth (Yaqoob et al. 2012).
Activated HSCs can also promote tumor-associated angiogenesis since they
release VEGF, angiopoietins, and IL-8 (Semela et al. 2008; Taura et al. 2008; Das
et al. 2010; Zhu et al. 2015). Indeed, IL-8 derives mainly from activated HSCs, and
IL-8 inhibition with a blocking antibody significantly reduced angiogenesis (Zhu
et al. 2015). Moreover, activated HSCs protect the hepatic tumor by inhibiting anti-
tumor cytotoxic T cells (Xia et al. 2017). Indeed, tumor-associated HSCs induce
dendritic cell-derived immunoglobulin receptor 2 (DIgR2) in dendritic cells, which
in turn inhibit the inflammatory response of T cells (Xia et al. 2017). It could be of
interest to better understand the role of activated HSCs on the inflammatory cell
behavior in the context of HCC.

9.5 Conclusion

In this chapter, we have identified HSCs as a crucial cell for liver development,
homeostasis, and disease through paracrine and autocrine signaling, which includes
growth factors, cytokines, and extracellular vesicles. While most of the studies
describe HSC-dependent ECM regulation in response to microenvironment stimuli,
less attention is brought to the understanding of other HSC-derived signals and their
effect on the surrounding cell types. The HSC-derived signaling study remains
162 E. Kostallari and V. H. Shah

incomplete, especially in the context of HSC heterogeneity. The isolation of differ-

ent HSC subpopulations and specifically targeting the most fibrogenic ones are
essential for developing novel therapies. Several encouraging anti-fibrotic strategies
aiming at the prevention or reversal of HSC-induced liver fibrosis have been or are
being developed (Schuppan et al. 2018). Recently, a dual C-C chemokine receptor
type 2 and 5 (CCR2/CCR5) antagonist, cenicriviroc, is in phase 2 clinical trial and
is being evaluated in liver inflammation and fibrosis in the context of NASH
(Friedman et al. 2016). This is the first prospective study of an oral agent in patients
with exclusively NASH and liver fibrosis, which aims to assess correlations between
inflammation and fibrosis (Friedman et al. 2016). In parallel, other clinical trials are
ongoing (clinicaltrials.gov) and throw a glimmer of hope in finding treatments for
liver diseases.

References

(1996) Hepatic stellate cell nomenclature. Hepatology 23:193. No authors listed

Amann T, Bataille F, Spruss T, Mühlbauer M, Gäbele E, Schölmerich J, Kiefer P, Bosserhoff AK,
Hellerbrand C (2009) Activated hepatic stellate cells promote tumorigenicity of hepatocellular
carcinoma. Cancer Sci 100(4):646–653
Asahina K (2012) Hepatic stellate cell progenitor cells. J Gastroenterol Hepatol 27(Suppl 2):80–84
Asahina K, Sato H, Yamasaki C, Kataoka M, Shiokawa M, Katayama S, Tateno C, Yoshizato K
(2002) Pleiotrophin/heparin-binding growth-associated molecule as a mitogen of rat hepato-
cytes and its role in regeneration and development of liver. Am J Pathol 160(6):2191–2205
Asahina K, Tsai SY, Li P, Ishii M, Maxson RE Jr, Sucov HM, Tsukamoto H (2009) Mesenchymal
origin of hepatic stellate cells, submesothelial cells, and perivascular mesenchymal cells during
mouse liver development. Hepatology 49(3):998–1011
Asahina K, Zhou B, Pu WT, Tsukamoto H (2011) Septum transversum-derived mesothelium gives
rise to hepatic stellate cells and perivascular mesenchymal cells in developing mouse liver.
Hepatology 53(3):983–995
Azzariti A, Mancarella S, Porcelli L, Quatrale AE, Caligiuri A, Lupo L, Dituri F, Giannelli G
(2016) Hepatic stellate cells induce hepatocellular carcinoma cell resistance to sorafenib
through the laminin-332/α3 integrin axis recovery of focal adhesion kinase ubiquitination.
Hepatology 64(6):2103–2117
Beilfuss A, Sowa JP, Sydor S, Beste M, Bechmann LP, Schlattjan M, Syn WK, Wedemeyer I,
Mathé Z, Jochum C, Gerken G, Gieseler RK, Canbay A (2015) Vitamin D counteracts fibro-
genic TGF-β signalling in human hepatic stellate cells both receptor-dependently and indepen-
dently. Gut 64(5):791–799
Bilzer M, Roggel F, Gerbes AL (2006) Role of Kupffer cells in host defense and liver disease.
Liver Int 26(10):1175–1186
Blomhoff R, Green MH, Berg T, Norum KR (1990) Transport and storage of vitamin A. Science
250(4979):399–404
Boers W, Aarrass S, Linthorst C, Pinzani M, Elferink RO, Bosma P (2006) Transcriptional profil-
ing reveals novel markers of liver fibrogenesis: gremlin and insulin-like growth factor-binding
proteins. J Biol Chem 281(24):16289–16295
Brunt EM, Gouw AS, Hubscher SG, Tiniakos DG, Bedossa P, Burt AD, Callea F, Clouston AD,
Dienes HP, Goodman ZD, Roberts EA, Roskams T, Terracciano L, Torbenson MS, Wanless IR
(2014) Pathology of the liver sinusoids. Histopathology 64(7):907–920
Byun JS, Suh YG, Yi HS, Lee YS, Jeong WI (2013) Activation of toll-like receptor 3 attenuates
alcoholic liver injury by stimulating Kupffer cells and stellate cells to produce interleukin-10 in
mice. J Hepatol 58(2):342–349
9 Pericytes in the Liver 163

Canbay A, Higuchi H, Bronk SF, Taniai M, Sebo TJ, Gores GJ (2002) Fas enhances fibrogen-
esis in the bile duct ligated mouse: a link between apoptosis and fibrosis. Gastroenterology
123(4):1323–1330
Canbay A, Taimr P, Torok N, Higuchi H, Friedman S, Gores GJ (2003) Apoptotic body engulfment
by a human stellate cell line is profibrogenic. Lab Investig 83(5):655–663
Cassiman D, Barlow A, Vander Borght S, Libbrecht L, Pachnis V (2006) Hepatic stellate cells do
not derive from the neural crest. J Hepatol 44(6):1098–1104
Chen M, Liu J, Yang W, Ling W (2017) Lipopolysaccharide mediates hepatic stellate cell activa-
tion by regulating autophagy and retinoic acid signaling. Autophagy 13(11):1813–1827
Chou MH, Huang YH, Lin TM, Du YY, Tsai PC, Hsieh CS, Chuang JH (2012) Selective activa-
tion of toll-like receptor 7 in activated hepatic stellate cells may modulate their profibrogenic
phenotype. Biochem J 447(1):25–34
Dangi A, Huang C, Tandon A, Stolz D, Wu T, Gandhi CR (2016) Endotoxin-stimulated rat
hepatic stellate cells induce autophagy in hepatocytes as a survival mechanism. J Cell Physiol
231(1):94–105
Das A, Shergill U, Thakur L, Sinha S, Urrutia R, Mukhopadhyay D, Shah VH (2010) Ephrin B2/
EphB4 pathway in hepatic stellate cells stimulates Erk-dependent VEGF production and sinu-
soidal endothelial cell recruitment. Am J Physiol Gastrointest Liver Physiol 298(6):G908–G915
Ding BS, Cao Z, Lis R, Nolan DJ, Guo P, Simons M, Penfold ME, Shido K, Rabbany SY, Rafii S
(2014) Divergent angiocrine signals from vascular niche balance liver regeneration and fibro-
sis. Nature 505(7481):97–102
Dou C, Liu Z, Tu K, Zhang H, Chen C, Yaqoob U, Wang Y, Wen J, van Deursen J, Sicard D,
Tschumperlin D, Zou H, Huang WC, Urrutia R, Shah VH, Kang N (2018) P300 acetyltransfer-
ase mediates stiffness-induced activation of hepatic stellate cells into tumor-promoting myofi-
broblasts. Gastroenterology 154(8):2209–2221.e14
Drinane MC, Yaqoob U, Yu H, Luo F, Greuter T, Arab JP, Kostallari E, Verma VK, Maiers J, De
Assuncao TM, Simons M, Mukhopadhyay D, Kisseleva T, Brenner DA, Urrutia R Lomberk
G, Gao Y, Ligresti G, Tschumperlin DJ, Revzin A, Cao S, Shah VH. Synectin promotes fibro-
genesis by regulating PDGFR isoforms through distinct mechanisms. JCI Insight. 2017;2(24).
pii: 92821
Duran A, Hernandez ED, Reina-Campos M, Castilla EA, Subramaniam S, Raghunandan S,
Roberts LR, Kisseleva T, Karin M, Diaz-Meco MT, Moscat J (2016) p62/SQSTM1 by binding
to vitamin D receptor inhibits hepatic stellate cell activity, fibrosis, and liver Cancer. Cancer
Cell 30(4):595–609
Ferré N, Martínez-Clemente M, López-Parra M, González-Périz A, Horrillo R, Planagumà A,
Camps J, Joven J, Tres A, Guardiola F, Bataller R, Arroyo V, Clària J (2009) Increased sus-
ceptibility to exacerbated liver injury in hypercholesterolemic ApoE-deficient mice: potential
involvement of oxysterols. Am J Physiol Gastrointest Liver Physiol 296(3):G553–G562
Friedman SL (2008) Hepatic stellate cells: protean, multifunctional, and enigmatic cells of the
liver. Physiol Rev 88(1):125–172. https://doi.org/10.1152/physrev.00013.2007
Friedman S, Sanyal A, Goodman Z, Lefebvre E, Gottwald M, Fischer L, Ratziu V (2016) Efficacy
and safety study of cenicriviroc for the treatment of non-alcoholic steatohepatitis in adult sub-
jects with liver fibrosis: CENTAUR phase 2b study design. Contemp Clin Trials 47:356–365
Gäbele E, Mühlbauer M, Dorn C, Weiss TS, Froh M, Schnabl B, Wiest R, Schölmerich J, Obermeier
F, Hellerbrand C (2008) Role of TLR9 in hepatic stellate cells and experimental liver fibrosis.
Biochem Biophys Res Commun 376(2):271–276
Greuter T, Malhi H, Gores GJ, Shah VH. Therapeutic opportunities for alcoholic steatohepatitis
and nonalcoholic steatohepatitis: exploiting similarities and differences in pathogenesis. JCI
Insight. 2017;2(17). pii: 95354
Guo J, Loke J, Zheng F, Hong F, Yea S, Fukata M, Tarocchi M, Abar OT, Huang H, Sninsky
JJ, Friedman SL (2009) Functional linkage of cirrhosis-predictive single nucleotide polymor-
phisms of toll-like receptor 4 to hepatic stellate cell responses. Hepatology 49(3):960–968
Guvendiren M, Perepelyuk M, Wells RG, Burdick JA (2014) Hydrogels with differential and pat-
terned mechanics to study stiffness-mediated myofibroblastic differentiation of hepatic stellate
cells. J Mech Behav Biomed Mater 38:198–208
164 E. Kostallari and V. H. Shah

Hendriks HF, Verhoofstad WA, Brouwer A, de Leeuw AM, Knook DL (1985) Perisinusoidal fat-­
storing cells are the main vitamin A storage sites in rat liver. Exp Cell Res 160(1):138–149
Huang H, Shiffman ML, Friedman S, Venkatesh R, Bzowej N, Abar OT, Rowland CM, Catanese
JJ, Leong DU, Sninsky JJ, Layden TJ, Wright TL, White T, Cheung RC (2007) A 7 gene signa-
ture identifies the risk of developing cirrhosis in patients with chronic hepatitis C. Hepatology
46(2):297–306
Ibrahim SH, Hirsova P, Gores GJ (2018) Non-alcoholic steatohepatitis pathogenesis: sublethal
hepatocyte injury as a driver of liver inflammation. Gut 67(5):963–972
Ito T, Nemoto M (1952) Kupfer's cells and fat storing cells in the capillary wall of human liver.
Okajimas Folia Anat Jpn 24(4):243–258
Jiang JX, Venugopal S, Serizawa N, Chen X, Scott F, Li Y, Adamson R, Devaraj S, Shah V,
Gershwin ME, Friedman SL, Török NJ (2010) Reduced nicotinamide adenine dinucleotide
phosphate oxidase 2 plays a key role in stellate cell activation and liver fibrogenesis in vivo.
Gastroenterology 139(4):1375–1384
Ju MJ, Qiu SJ, Fan J, Xiao YS, Gao Q, Zhou J, Li YW, Tang ZY (2009) Peritumoral activated
hepatic stellate cells predict poor clinical outcome in hepatocellular carcinoma after curative
resection. Am J Clin Pathol 131(4):498–510
Kang N, Gores GJ, Shah VH (2011) Hepatic stellate cells: partners in crime for liver metastases.
Hepatology 54(2):707–713
Kiagiadaki F, Kampa M, Voumvouraki A, Castanas E, Kouroumalis E, Notas G (2018) Activin-a
causes hepatic stellate cell activation via the induction of TNFα and TGFβ in Kupffer cells.
Biochim Biophys Acta 1864(3):891–899
Kluwe J, Wongsiriroj N, Troeger JS, Gwak GY, Dapito DH, Pradere JP, Jiang H, Siddiqi M,
Piantedosi R, O'Byrne SM, Blaner WS, Schwabe RF (2011) Absence of hepatic stellate cell
retinoid lipid droplets does not enhance hepatic fibrosis but decreases hepatic carcinogenesis.
Gut 60(9):1260–1268
Kong B, Luyendyk JP, Tawfik O, Guo GL (2009) Farnesoid X receptor deficiency induces nonal-
coholic steatohepatitis in low-density lipoprotein receptor-knockout mice fed a high-fat diet.
J Pharmacol Exp Ther 328(1):116–122
Kordes C, Häussinger D (2013) Hepatic stem cell niches. J Clin Invest 123(5):1874–1880
Kordes C, Sawitza I, Götze S, Herebian D, Häussinger D (2014) Hepatic stellate cells contribute to
progenitor cells and liver regeneration. J Clin Invest 124(12):5503–5515
Kostallari E, Shah VH (2016) Angiocrine signaling in the hepatic sinusoids in health and disease.
Am J Physiol Gastrointest Liver Physiol 311(2):G246–G251
Kostallari E, Hirsova P, Prasnicka A, Verma VK, Yaqoob U, Wongjarupong N, Roberts LR, Shah
VH (2018) Hepatic stellate cell-derived PDGFRα-enriched extracellular vesicles promote liver
fibrosis in mice through SHP2. Hepatology 68:333–348
Kupffer CV (1876) Ueber Sternzellen der Leber. Briefliche Mitteilung an Prof. Waldeyer. Arch
Mikr Anat 12:353–358
Lan T, Li C, Yang G, Sun Y, Zhuang L, Ou Y, Li H, Wang G, Kisseleva T, Brenner D, Guo J (2018)
Sphingosine kinase 1 promotes liver fibrosis by preventing miR-19b-3p-mediated inhibition of
CCR2. Hepatology 68:1070–1086
Lee YS, Jeong WI (2012) Retinoic acids and hepatic stellate cells in liver disease. J Gastroenterol
Hepatol 27(Suppl 2):75–79
Li ZQ, Wu WR, Zhao C, Zhao C, Zhang XL, Yang Z, Pan J, Si WK (2018) CCN1/Cyr61 enhances
the function of hepatic stellate cells in promoting the progression of hepatocellular carcinoma.
Int J Mol Med 41(3):1518–1528
Loo CK, Wu XJ (2008) Origin of stellate cells from submesothelial cells in a developing human
liver. Liver Int 28(10):1437–1445
Marra F, Romanelli RG, Giannini C, Failli P, Pastacaldi S, Arrighi MC, Pinzani M, Laffi G,
Montalto P, Gentilini P (1999) Monocyte chemotactic protein-1 as a chemoattractant for
human hepatic stellate cells. Hepatology 29(1):140–148
9 Pericytes in the Liver 165

Mazzocca A, Coppari R, De Franco R, Cho JY, Libermann TA, Pinzani M, Toker A (2005) A
secreted form of ADAM9 promotes carcinoma invasion through tumor-stromal interactions.
Cancer Res 65(11):4728–4738
Miura K, Yang L, van Rooijen N, Brenner DA, Ohnishi H, Seki E (2013) Toll-like receptor 2
and palmitic acid cooperatively contribute to the development of nonalcoholic steatohepatitis
through inflammasome activation in mice. Hepatology 57(2):577–589
Mogler C, König C, Wieland M, Runge A, Besemfelder E, Komljenovic D, Longerich T,
Schirmacher P, Augustin HG (2017) Hepatic stellate cells limit hepatocellular carcinoma pro-
gression through the orphan receptor endosialin. EMBO Mol Med 9(6):741–749
Musso O, Théret N, Campion JP, Turlin B, Milani S, Grappone C, Clément B (1997) In situ
detection of matrix metalloproteinase-2 (MMP2) and the metalloproteinase inhibitor TIMP2
transcripts in human primary hepatocellular carcinoma and in liver metastasis. J Hepatol
26(3):593–605
Nejak-Bowen KN, Orr AV, Bowen WC Jr, Michalopoulos GK (2013) Gliotoxin-induced changes
in rat liver regeneration after partial hepatectomy. Liver Int 33(7):1044–1055
Ohata M, Lin M, Satre M, Tsukamoto H (1997) Diminished retinoic acid signaling in hepatic stel-
late cells in cholestatic liver fibrosis. Am J Phys 272(3 Pt 1):G589–G596
Okabe H, Beppu T, Hayashi H, Horino K, Masuda T, Komori H, Ishikawa S, Watanabe M,
Takamori H, Iyama K, Baba H (2009) Hepatic stellate cells may relate to progression of intra-
hepatic cholangiocarcinoma. Ann Surg Oncol 16(9):2555–2564
Patella S, Phillips DJ, Tchongue J, de Kretser DM, Sievert W (2006) Follistatin attenuates early
liver fibrosis: effects on hepatic stellate cell activation and hepatocyte apoptosis. Am J Physiol
Gastrointest Liver Physiol 290(1):G137–G144
Pérez-Pomares JM, Carmona R, González-Iriarte M, Macías D, Guadix JA, Muñoz-Chápuli
R. (2004) Contribution of mesothelium-derived cells to liver sinusoids in avian embryos. Dev
Dyn. Mar;229(3):465–74
Pinzani M, Milani S, De Franco R, Grappone C, Caligiuri A, Gentilini A, Tosti-Guerra C, Maggi
M, Failli P, Ruocco C, Gentilini P (1996) Endothelin 1 is overexpressed in human cirrhotic liver
and exerts multiple effects on activated hepatic stellate cells. Gastroenterology 110(2):534–548
Popper H (1944) Distribution of vitamin A in tissue as visualized by flourescence microscopy.
Physiol Rev 24:205–224
Povero D, Panera N, Eguchi A, Johnson CD, Papouchado BG, de Araujo Horcel L, Pinatel EM,
Alisi A, Nobili V, Feldstein AE (2015) Lipid-induced hepatocyte-derived extracellular vesicles
regulate hepatic stellate cell via microRNAs targeting PPAR-γ. Cell Mol Gastroenterol Hepatol
1(6):646–663.e4
Preziosi ME, Monga SP (2017) Update on the mechanisms of liver regeneration. Semin Liver Dis
37(2):141–151
Ramadori G, Rieder H, Theiss F (1989) Meyer zum Büschenfelde KH. Fat-storing (Ito) cells of rat
liver synthesize and secrete apolipoproteins: comparison with hepatocytes. Gastroenterology
97(1):163–172
Rangwala F, Guy CD, Lu J, Suzuki A, Burchette JL, Abdelmalek MF, Chen W, Diehl AM (2011)
Increased production of sonic hedgehog by ballooned hepatocytes. J Pathol 224(3):401–410
Schuppan D, Ashfaq-Khan M, Yang AT, Kim YO (2018) Liver fibrosis: direct antifibrotic agents
and targeted therapies. Matrix Biol 68:435–451. pii: S0945-053X(18)30160-4
Seki E, De Minicis S, Osterreicher CH, Kluwe J, Osawa Y, Brenner DA, Schwabe RF (2007) TLR4
enhances TGF-beta signaling and hepatic fibrosis. Nat Med 13(11):1324–1332
Seki E, de Minicis S, Inokuchi S, Taura K, Miyai K, van Rooijen N, Schwabe RF, Brenner DA
(2009) CCR2 promotes hepatic fibrosis in mice. Hepatology 50(1):185–197
Semela D, Das A, Langer D, Kang N, Leof E, Shah V. (2008) Platelet-derived growth factor sig-
naling through ephrin-b2 regulates hepatic vascular structure and function. Gastroenterology.
Aug;135(2):671–9
Senoo H (2004) Structure and function of hepatic stellate cells. Med Electron Microsc 37(1):3–15
166 E. Kostallari and V. H. Shah

Shearer AM, Rana R, Austin K, Baleja JD, Nguyen N, Bohm A, Covic L, Kuliopulos A (2016)
Targeting liver fibrosis with a cell-penetrating protease-activated Receptor-2 (PAR2) Pepducin.
J Biol Chem 291(44):23188–23198
Shimizu H, Tsubota T, Kanki K, Shiota G (2018) All-trans retinoic acid ameliorates hepatic stel-
late cell activation via suppression of thioredoxin interacting protein expression. J Cell Physiol
233(1):607–616
Staels B, Rubenstrunk A, Noel B, Rigou G, Delataille P, Millatt LJ, Baron M, Lucas A, Tailleux A,
Hum DW, Ratziu V, Cariou B, Hanf R (2013) Hepatoprotective effects of the dual peroxisome
proliferator-activated receptor alpha/delta agonist, GFT505, in rodent models of nonalcoholic
fatty liver disease/nonalcoholic steatohepatitis. Hepatology 58(6):1941–1952
Suskind DL, Muench MO (2004) Searching for common stem cells of the hepatic and hematopoi-
etic systems in the human fetal liver: CD34+ cytokeratin 7/8+ cells express markers for stellate
cells. J Hepatol 40(2):261–268
Taub R (2004) Liver regeneration: from myth to mechanism. Nat Rev Mol Cell Biol 5(10):836–
847. Review
Taura K, De Minicis S, Seki E, Hatano E, Iwaisako K, Osterreicher CH, Kodama Y, Miura K,
Ikai I, Uemoto S, Brenner DA (2008) Hepatic stellate cells secrete angiopoietin 1 that induces
angiogenesis in liver fibrosis. Gastroenterology 135(5):1729–1738
Thompson KC, Trowern A, Fowell A, Marathe M, Haycock C, Arthur MJ, Sheron N (1998a)
Primary rat and mouse hepatic stellate cells express the macrophage inhibitor cytokine inter-
leukin-­10 during the course of activation in vitro. Hepatology 28(6):1518–1524
Thompson K, Maltby J, Fallowfield J, McAulay M, Millward-Sadler H, Sheron N (1998b)
Interleukin-10 expression and function in experimental murine liver inflammation and fibrosis.
Hepatology 28(6):1597–1606
Toi M, Hayashi Y, Murakami I (2018) Hepatic stellate cells derived from the nestin-positive cells
in septum transversum during rat liver development. Med Mol Morphol 51:199–207
Tomita K, Teratani T, Suzuki T, Shimizu M, Sato H, Narimatsu K, Usui S, Furuhashi H, Kimura
A, Nishiyama K, Maejima T, Okada Y, Kurihara C, Shimamura K, Ebinuma H, Saito H,
Yokoyama H, Watanabe C, Komoto S, Nagao S, Sugiyama K, Aosasa S, Hatsuse K, Yamamoto
J, Hibi T, Miura S, Hokari R, Kanai T (2014) Acyl-CoA:cholesterol acyltransferase 1 mediates
liver fibrosis by regulating free cholesterol accumulation in hepatic stellate cells. J Hepatol
61(1):98–106
Tsuchida T, Friedman SL (2017) Mechanisms of hepatic stellate cell activation. Nat Rev
Gastroenterol Hepatol 14(7):397–411
Van Rooyen DM, Gan LT, Yeh MM, Haigh WG, Larter CZ, Ioannou G, Teoh NC, Farrell GC
(2013) Pharmacological cholesterol lowering reverses fibrotic NASH in obese, diabetic mice
with metabolic syndrome. J Hepatol 59(1):144–152
Vidal-Vanaclocha F (2008) The prometastatic microenvironment of the liver. Cancer Microenviron
1(1):113–129
Wake K (1971) “Sternzellen” in the liver: perisinusoidal cells with special reference to storage of
vitamin A. Am J Anat 132(4):429–462
Wake K (1974) Development of vitamin A-rich lipid droplets in multivesicular bodies of rat liver
stellate cells. J Cell Biol 63(2 Pt 1):683–691
Wang R, Ding Q, Yaqoob U, de Assuncao TM, Verma VK, Hirsova P, Cao S, Mukhopadhyay D,
Huebert RC, Shah VH (2015) Exosome adherence and internalization by hepatic stellate cells
triggers sphingosine 1-phosphate-dependent migration. J Biol Chem 290(52):30684–30696
Xia YH, Lu Z, Zhao M, Dai WT, Ding L, Hu LX, Jiang GL (2017) Tumor-specific hepatic stel-
late cells (tHSCs) induces DIgR2 expression in dendritic cells to inhibit T cells. Oncotarget
8(33):55084–55093
Yang L, Wang Y, Mao H, Fleig S, Omenetti A, Brown KD, Sicklick JK, Li YX, Diehl AM (2008a)
Sonic hedgehog is an autocrine viability factor for myofibroblastic hepatic stellate cells.
J Hepatol 48(1):98–106
9 Pericytes in the Liver 167

Yang L, Jung Y, Omenetti A, Witek RP, Choi S, Vandongen HM, Huang J, Alpini GD, Diehl AM
(2008b) Fate-mapping evidence that hepatic stellate cells are epithelial progenitors in adult
mouse livers. Stem Cells 26(8):2104–2113
Yaqoob U, Cao S, Shergill U, Jagavelu K, Geng Z, Yin M, de Assuncao TM, Cao Y, Szabolcs A,
Thorgeirsson S, Schwartz M, Yang JD, Ehman R, Roberts L, Mukhopadhyay D, Shah VH
(2012) Neuropilin-1 stimulates tumor growth by increasing fibronectin fibril assembly in the
tumor microenvironment. Cancer Res 72(16):4047–4059
Yin C, Evason KJ, Maher JJ, Stainier DY (2012) The basic helix-loop-helix transcription factor,
heart and neural crest derivatives expressed transcript 2, marks hepatic stellate cells in zebraf-
ish: analysis of stellate cell entry into the developing liver. Hepatology 56(5):1958–1970
Yoneda A, Sakai-Sawada K, Niitsu Y, Tamura Y (2016) Vitamin A and insulin are required for the
maintenance of hepatic stellate cell quiescence. Exp Cell Res 341(1):8–17
Zaret KS (2002) Regulatory phases of early liver development: paradigms of organogenesis. Nat
Rev Genet 3(7):499–512
Zhao W, Zhang L, Yin Z, Su W, Ren G, Zhou C, You J, Fan J, Wang X (2011) Activated hepatic
stellate cells promote hepatocellular carcinoma development in immunocompetent mice. Int
J Cancer 129(11):2651–2661
Zhou Z, Xu MJ, Cai Y, Wang W, Jiang JX, Varga ZV, Feng D, Pacher P, Kunos G, Torok NJ, Gao
B (2018) Neutrophil-hepatic stellate cell interactions promote fibrosis in experimental steato-
hepatitis. Cell Mol Gastroenterol Hepatol 5(3):399–413
Zhu B, Lin N, Zhang M, Zhu Y, Cheng H, Chen S, Ling Y, Pan W, Xu R (2015) Activated hepatic
stellate cells promote angiogenesis via interleukin-8 in hepatocellular carcinoma. J Transl Med
13:365
Chapter 10
Pericytes in the Periodontal Ligament

Motohiro Komaki

Abstract Teeth are exposed to hundreds of oral bacteria and also challenged by the
mastication forces; because teeth are situated in oral cavity, the entrance of the
digestive tract, and penetrates through the oral epithelium. The periodontal ligament
is a noncalcified tissue that possesses abundant blood vessels, which exist between
tooth root and alveolar bone. The ligament is thought to play an important role in
absorbing the impact of mastication, in the maintenance of periodontal homeostasis,
and in periodontal wound healing. We succeeded in isolating mesenchymal stem
cells (MSCs), so-called periodontal stem cells (PDLSCs), with self-renewability
and multipotency from the periodontal ligament. We also demonstrated that PDLSCs
share some cell surface markers with pericytes and that PDLSCs distribute them-
selves to stay with the endothelial cell networks and that PDLSCs maintain the
endothelial cell networks when added to endothelial cell network formation sys-
tems. Pericytes are located in the proximity of microvascular endothelial cells and
thought to stabilize and supply nutrients to blood vessels. Recently, it was also
reported that pericytes possess multipotency and can be the source of tissue stem
cells and/or progenitor cells. This review explores the distinctive features of the
periodontal ligament tissue and PDLSCs as well as the puzzling similarities between
PDLSCs and pericytes.

Keywords Periodontal ligament · Mesenchymal stem cells · Pericytes ·

Periodontal ligament stem cells · Vascular network · Blood vessels · Regenerative
therapy · Twist · Periostin · SDF-1 · Hypoxia

M. Komaki (*)
Department of Highly Advanced Stomatology, Graduate School of Dentistry,
Kanagawa Dental University, Yokohama City, Kanagawa, Japan
e-mail: [email protected]

© Springer Nature Switzerland AG 2019 169

A. Birbrair (ed.), Pericyte Biology in Different Organs,
Advances in Experimental Medicine and Biology 1122,
https://doi.org/10.1007/978-3-030-11093-2_10
170 M. Komaki

10.1  natomy, Function, and Distinctiveness

A
of Periodontal Tissue

Teeth not only pulverize food brought to the mouth through mastication but also
enable a person to perceive the texture of food through the periodontal ligament. It is
also believed that mastication plays important role in the maintenance of periodontal
ligament homeostasis. Teeth penetrate through the oral epithelium into the oral cav-
ity. The root does not directly bind to the alveolar bone, rather it is drawn into the
alveolar bone like a hammock by predominately collagen fibers of periodontal liga-
ment. Periodontal ligament is situated in a space of approximately 0.25 mm between
two calcified tissues, tooth root and alveolar bone. Thickening of the cementum due
to aging gradually decreases this size; however, this space sustains the uncalcified
zones throughout one’s lifetime. Thus far, the inhibitory mechanism of periodontal
ligament’s calcification through homeobox protein Msx2 and extracellular matrix
protein PLAP-1/asporin has been reported (Yamada et al. 2007; Yoshizawa et al.
2004). We also have reported the expression of Twist, a basic helix-­loop-­helix
(bHLH) transcription factor and also known as a causal factor of Saethre-­Chotzen
syndrome, which causes the premature closure of cranial sutures, within the peri-
odontal ligament (Komaki et al. 2007). Twist is also known as a regulatory factor of
periostin that is involved in the maintenance of integrity of periodontal tissue in
response to occlusal load. We demonstrated that Twist suppressed osteoblastic dif-
ferentiation of periodontal ligament cells as a possible inhibitory mechanism in peri-
odontal ligament (Komaki et al. 2007). Another feature of the periodontal ligament is
abundant blood vessels that cover the root’s surface akin to a bird’s nest (Fig. 10.1).
Periodontal ligament is a noncalcified tissue predominately composed of collagen
fiber types I and III as well as small amounts of oxytalan fibers. A different type of
gene expression in the periodontal ligament as compared to that of dermal connective
tissue has been reported (Lallier et al. 2005). Compared to general connective tissue,
which has a vascular volume of 5%, periodontal ligament has an abundant supply of
blood vessels of ~20% (Lew 1987). The blood vessels of the periodontal ligament
consist of routes that branch from the inferior alveolar artery inside the alveolar bone
and reach the periodontal ligament through Volkmann’s canal in alveolar bone, routes
where anastomosis occurs with blood vessels of the gingiva near the tooth cervix, and
routes where blood vessels, which diverge near the apex into the tooth pulp and peri-
odontal ligament, diverge into the periodontal ligament (Berglundh et al. 1994;
Matsuo and Takahashi 2002). The network of blood vessels formed a double-layered
mesh on the root surface is made of terminal arterioles, capillaries, and postcapillary
venules. Few valves that staunch regurgitation of blood are seen among the veins in
the alveolar bone. Blood vessel system in periodontal ligament dissipates intermit-
tently occurring occlusal loads because blood flows freely in and out between the
periodontal ligament and alveolar bone. Fenestrated capillaries observed around the
root apex enable efficient gas and substance exchange between blood vessels and
surrounding tissues in response to high metabolic turnover of periodontal ligament
(Lew 1987). It has been reported that vascular network in periodontal ligament can
10 Pericytes in the Periodontal Ligament 171

Fig. 10.1 Vascular

network of the molar. The
vascular network of
periodontal ligament is
supplied from three
directions: (1) bifurcated in
the alveolar bone (AB), (2)
anastomosis with the
gingival blood vessels, and
(3) bifurcated into the
apical area. (G) vascular
network of gingiva [figures
from Matsuo and
Takahashi (2002)]

be influenced by external force including orthodontic tooth movement or local

inflammation such as periodontitis (Rygh et al. 1986; Murrell et al. 1996; Zoellner
et al. 2002). The compression of blood vessels due to orthodontic tooth movement
causes local ischemia, which leads to angiogenesis via hypoxia inducible factor-1α
(HIF1α) induction and alveolar bone resorption. The compression of the periodontal
ligament due to orthodontic tooth movement and, in contrast, hypofunction of tooth
due to the lack of adequate occlusal load may indirectly affect blood vessels via the
temporarily altered expression of periostin (Wilde et al. 2003; Afanador et al. 2005;
Lindner et al. 2005; Rios et al. 2008; Li et al. 2010). Besides, infections caused by
periodontal pathogen such as Porphyromonas gingivalis (P.g.) cause disruption of
blood vessels leading to local hypoxia via the production of protease, and chronic
periodontitis further increases in the proportion of anaerobic bacteria. However, this
mechanism is not fully understood.

10.2  he Importance of Periodontal Ligament in Periodontal

T
Tissue Regeneration

Previous in vivo studies have indicated that the periodontal ligament has an
important function in the restoration of periodontal tissue and the maintenance of
homeostasis. In 1976, Melcher proposed that it is important to restore all periodontal
172 M. Komaki

tissue, not only the bone, when treating periodontal tissue destroyed by periodonti-
tis, and that the periodontal ligament is important for that very purpose (Melcher
1976). In 1980, Nyman and Karring et al. conducted fervent in vivo research based
on this hypothesis by Melcher and reported that the periodontal ligament plays an
important role in the regeneration of periodontal tissue (Karring et al. 1980; Nyman
et al. 1980). In other words, connective tissue attachment occurs continuing from
the remaining periodontal ligament if there is any remaining healthy periodontal
ligament on the surface of the tooth root, similar to that observed in normal tissues.
In addition, it has been demonstrated that when periodontal ligament-derived cells
are cultured in vitro and transplanted with decalcified tooth root, they restore the
periodontal ligament (Boyko et al. 1981). In addition, even in tooth replantation
studies on monkeys, it has been shown that root resorption does not occur when
there is healthy periodontal ligament remaining on replanted teeth (Andreasen
1981). By contrast, when bone or gingival connective tissue contacts a root surface
where the periodontal ligament is completely gone, tooth root resorption or ankylo-
sis can take place due to the difference in adjacent tissues. In this regard, it is
believed that the periodontal ligament can form collagen fibers that connect cemen-
tum to the alveolar bone called Sharpey’s fibers, which are a supporting tissue of the
teeth; in short, it may have the ability to regenerate periodontal tissue. In the 1990s,
much research was published on the characteristics of fibroblastic cells derived
from the periodontal ligament. According to Nojima and Basdra et al., fibroblasts
derived from the periodontal ligament show osteoblast-related marker expression
in vitro, and periodontal ligament fibroblasts possess mineralization capabilities
similar to those of osteoblasts and can differentiate into cementoblasts or osteo-
blasts, the cells that form two hard tissues that flank the periodontal ligament,
cementum, and alveolar bone (Nojima et al. 1990; Basdra and Komposch 1997).
However, at this point, osteogenic activity of cells is derived from the periodontal
ligament, but they are not described as being stem cells.

10.3 Periodontal Ligament Stem Cells

MSCs were first identified from bone marrow cells as plastic culture dish adhesive,
colony-forming unit-fibroblastic (CFU-f). In the 1970s, Friedenstein et al. reported
that CFU-f exist in bone marrow from low-density cultures of a bone marrow extract
in vitro and demonstrated osteogenicity of the cells and that when the cells were
transplanted into sub-renal capsule, fibrous tissue, bone, and bone marrow were
formed where the recipient hematopoietic stem cells (HSCs) were homing, suggesting
multipotency of CFU-f (Friedenstein et al. 1970). In the 1980s and 1990s, Pittenger
et al. indicated that this cell type has the multipotency to differentiate into adipo-
cytes, chondroblasts, and osteoblasts, suggesting that stem cells widely known as
MSCs exist in postnatal adult tissue and bone marrow (Pittenger et al. 1999).
In the 1990s, it was shown that fibroblasts derived from the periodontal ligament
possess osteogenic capacity (Nojima et al. 1990; Basdra and Komposch 1997).
10 Pericytes in the Periodontal Ligament 173

Subsequently, it was confirmed from the results of gene expression analyses in

fibroblasts by Lallier et al. that the gene expression of fibroblasts derived from the
periodontal ligament is closer to that of osteoblasts than dermal fibroblasts (Lallier
et al. 2005); however, the function or origin was not fully comprehended long after-
ward. McCulloch, using experiments involving mice with pulse labeled with H3-­
thymidine, published results indicate that stem cells that have a slow rate of
proliferation exist near the blood vessels in the periodontal ligament of mouse
molars (McCulloch 1985). Chen et al., using antibodies against STRO-1, CD146,
and CD44, investigated the localization of possible MSCs in healthy human teeth
and periodontitis-affected teeth and reported the localization of putative MSCs near
the blood vessels (Chen et al. 2006). In 2004, the first report came out regarding the
existence of stem cells being implicated in the importance of periodontal ligament
in the repair and maintenance of periodontal tissue homeostasis (Seo et al. 2004).
This event happened 30 years after Friedenstein et al.’s quest for the discovery of
bone marrow stem cells in the 1970s. MSCs were first identified as CFU-f from
bone marrow. Currently, the minimum criteria of MSCs are (1) colony formation on
plastic culture plates; (2) positive for mesenchymal cell markers such as CD73,
CD90, and CD105 and negative for hematopoietic cell markers, such as CD11b,
CD14, and CD45; and (3) differentiation capability into osteoblasts, chondrocytes,
and adipocytes in vitro (Dominici et al. 2006). Seo et al. reported in vitro differen-
tiation (into osteoblasts and adipocytes) of cells harvested from human periodontal
ligaments using anti-STRO-1 antibodies that bind to an epitope on the cellular
membrane of bone marrow MSCs; they reported the periodontal restorative ability
in transplantations of these cells mixed with hydroxyapatite/tricalcium phosphate
ceramic particles into periodontal tissue defects of immunocompromised rats (Seo
et al. 2004). The anti-STRO-1 antibodies bind to CFU-f on plastic culture plates,
one of minimum criteria for MSCs, but not to hematopoietic progenitor cells from
human bone marrow. Therefore, compared to using whole bone marrow cells with-
out fractionation, it is possible to increase the emergence rate of CFU-f 100-fold.
Besides, it has been demonstrated that 50–70% of these cells bind to antibodies
specific to smooth muscle actin (SMA) (Simmons and Torok-Storb 1991). Recently
it has been reported that anti-STRO-1 antibodies cross-reacts with both heat shock
cognate (HSC) 70 and heat shock protein (HSP) 70 and that STRO-1bright/ HSP70−
cell population demonstrates clonogenicity (Fitter et al. 2017). However, the func-
tion of HSC70 in the mesenchymal stem cell is still not fully understood. Nagatomo
et al. demonstrated the presence of MSCS in the human periodontal ligament that
are positive for markers of bone marrow stem cells, including STRO-1, and have
colony-forming ability and differentiation capability into osteoblasts and adipocytes
(Nagatomo et al. 2006). However, with this approach, the harvested MSCs represent
a group of heterogeneous cells; and cells with clonogenicity and multipotency con-
stitute a very small percentage of the cell group’s population. Whether or not they
are identical to stem cells that exist in tissues, their distribution in the adult organism
and their origin are still not fully understood. With great changes in the notion that
MSCs originate from fibroblasts due to their resemblance to clonogenic fibroblasts,
along with reports that pericytes (which are localized near blood vessels and are
174 M. Komaki

instrumental for blood vessel nutrition and stabilization) possess mineralization

activity (Doherty et al. 1998), multipotency (Pierantozzi et al. 2015), and tissue-­
reparative activity (Morgan and Muntoni 2007), it is now thought that MSCs also
originate from pericytes (da Silva Meirelles et al. 2008; Lv et al. 2014; Sorrentino
et al. 2008). It is believed that, because of their clonogenic properties and multipo-
tency, MSCs are engaged in maintaining tissue homeostasis and restoration, but
their true role in tissues remains unclear. It is unclear whether MSCs are involved in
the repair process of periodontal tissue destruction associated with periodontitis. It
is difficult to imagine, however, that MSCs dominantly contribute to the restoration
of severe tissue loss because of the small number of cells isolated from the peri-
odontal ligament. It is assumed that there is possibly another more biologically
effective mechanism available to repair local tissue destruction in vivo. Sahara et al.
demonstrated, using murine mechanically injured blood vessel models, that bone
marrow-derived cells repair blood vessels via the blood circulation (Sahara et al.
2005). In murine spleen-injured model, it was reported that bone marrow cells enter
peripheral blood and are mobilized to the wound site (Chen et al. 2010). We con-
ducted an observational study using chimeric mice with green florescence protein
(GFP)-labeled bone marrow cells (Fig. 10.2). The results showed that bone marrow-­
derived cells are present around the blood vessels of intact periodontal ligament.
Furthermore, we demonstrated that the number of bone marrow-derived cells
increases when the periodontal tissue is mechanically lesioned and that bone

Fig. 10.2 Localization of GFP-positive cells in intact periodontal tissue. Visualization of bone
marrow cells in vivo on periodontal tissue by using chimeric mice with GFP-labeled bone marrow
cells. Arrows indicate osteoclast-like multinucleated giant cells (a) and macrophage-like cells in
gingival epithelium (b) were GFP-positive, implying that GFP-positive images were confirmed to
match bone marrow cells. GFP-positive cells were also observed at the periodontal ligament (c),
blood vessels (d), and dental pulp (e). (a) Multinucleated giant cells in resorption lacunae of alveo-
lar bone; (b) gingival epithelium; (c) periodontal ligament; (d) blood vessels in periodontal liga-
ment; (e) dental pulp. (g), gingiva; AB alveolar bone, P periodontal ligament, DP dental pulp, D
dentin
10 Pericytes in the Periodontal Ligament 175

marrow-­derived cells mobilize upon damage to the periodontal ligament tissue. In

addition, we extract cells from bone marrow by enzymatic digestion and determine
stem cell quantity using PDGFRα+Sca-1+CD45−TER119− cell numbers according
to Houlihan et al. method and realized that the number of MSCs in bone marrow
decreases when the periodontal tissue is lesioned (Kimura et al. 2014). Although
these results do not prove that they are pericytes, this evidence demonstrates that
bone marrow cells are localized near the periodontal ligament’s blood vessels and
bone marrow cells, reacting to tissue destruction, are mobilized to the periodontal
ligament as well (Fig. 10.3). Using FACS to compare the antigens on the surface of
periodontal ligament cells with those of bone marrow stem cells and dermal fibro-
blasts, in addition to the mesenchymal marker expression, it was shown that PDLSCs
have an expression pattern of marker CD34 similar to that of dermal fibroblasts and
an expression pattern of marker CD166 similar to that of bone marrow stem cells
(Iwasaki et al. 2013). This finding may suggest that stem cells harvested from the
periodontal ligament may partly originate from bone marrow stem cells. Zhou et al.
intravenously injected mice with GFP-labeled bone marrow cells and investigated
the involvement of bone marrow cells in periodontal tissue regeneration. They
reported that bone marrow stem cells are observed in periodontal reparative tissue
and that stromal-derived factor 1 (SDF-1, also known as CXCL12) is involved in the

Fig. 10.3 Mobilization of bone marrow cells to periodontal ligament. Using chimeric mice with
GFP-labeled bone marrow cells, we found that bone marrow cells are located around blood vessels
in intact periodontal ligament. When periodontal lesion was mechanically made, hypoxia and/or
inflammation was observed in periodontium, and the number of bone marrow cells increased in
periodontal ligament. We found that bone marrow cells express SDF-1 receptor, CXCR4, and that
bone marrow cells mobilize toward SDF in chemotaxis chamber. Taken together, the results imply
that bone marrow cells were recruited to periodontal ligament via SDF-1 signal
176 M. Komaki

mobilization of these cells (Zhou et al. 2011). This information is the essential proof
needed for demonstrating that bone marrow cells are involved in the restoration of
periodontal tissue. We also confirmed that bone marrow stem cells express SDF-1
receptor, CXCR4, and in experiments with a chemotaxis chamber to test cell migra-
tion, bone marrow stem cells migrate toward SDF-1 (Kimura et al. 2014).

10.4  pplication of Mesenchymal Cell in Regenerative

A
Periodontal Therapy and Related Findings

Based on in vitro functional assessments, MSCs have been harvested from various
tissues including somatic tissues [bone marrow, fat, synovium (Hatakeyama et al.
2017), and cartilage (Xue et al. 2016)] and tooth and its surrounding tissues (dental
pulp, periodontal ligament, gingiva), as well as fetal appendage [umbilical cord
(Pirjali et al. 2013), cord blood (Zhang et al. 2011), and placenta (Komaki et al.
2017; Tooi et al. 2016)]. Stem cells are expected to find applications in tissue regen-
erative therapy because of their self-proliferation and differentiation potential.
Various experimental evaluations of their ability to restore periodontal tissue have
been conducted, and some clinical tests are being carried out (Monsarrat et al.
2014). However, there is some disparity seen in the restorative effect of stem cells,
and it is still not safe to say at present whether they yield an adequate quantity of
restored tissue or an adequate tissue regeneration efficiency. Currently, there is not
a single marker to identify MSCs, and the cells are extracted according to in vitro
functional evaluations. It is known that the phenotype of MSCs change easily
depending on the tissue extraction method and culture condition such as cellular
density, serum batch, and culture time. This situation may cause considerable varia-
tion in the regenerative effect of stem cells. Tanaka et al. used the enzymatic diges-
tion and cell outgrowth methods to extract cells from the periodontal ligament to
conduct a comparison. The periodontal ligament cells extracted via the enzymatic
digestion method have higher colony-forming and cell differentiation potentials
(Tanaka et al. 2011). Using the cell outgrowth and enzymatic digestion methods, we
extracted cells from the periodontal ligament and obtained MSCs in accordance
with conventional methods. Using commercial bone marrow MSCs as the control,
we checked their cell surface markers again by FACS analysis after incubating these
cells in a differentiation culture medium. We confirmed that there is no change in
mesenchymal cell marker expression, even after differentiation of the stem cells due
to incubation conditions. This experiment at least demonstrates that cell surface
markers CD73, CD90, and CD105, used during in vitro identification of MSCs, are
not affected by the cell differentiation (unpublished data). The heretofore seen peri-
odontal tissue restoration via mesenchymal cells extracted from various sources
[bone marrow (Kawaguchi et al. 2004; Li et al. 2009; Simsek et al. 2012), fat (Tobita
and Mizuno 2013), or periodontal ligament (Dogan et al. 2002, 2003; Akizuki et al.
2005; Iwasaki et al. 2014)] has been demonstrated. We would like to cite an
10 Pericytes in the Periodontal Ligament 177

outstanding systematic review that presents a detailed comparison of periodontal

ligament stem cells and bone marrow stem cells for periodontal tissue regeneration
(Monsarrat et al. 2014; Yan et al. 2015). Tsumanuma et al. have conducted histo-
logical evaluations of MSCs extracted from bone marrow, synovium, and the peri-
odontal ligament and transplanted them into experimentally created canine
periodontal tissue defects. Cell sheets were manufactured before transplantation by
taking cells incubated on temperature-responsive culture plates in a mineralization-­
inducing medium supplemented with ascorbic acid, β-glycerophosphate, and dexa-
methasone. Although no significant results were obtained with stem cells when used
for alveolar bone regeneration, Tsumanuma et al. have reported superior results
from the periodontal ligament stem cell transplantation, when using them to restore
cementum, which makes up supporting dental tissue, and the periodontal ligament
compared to stem cells derived from another tissue (Tsumanuma et al. 2011). This
result may suggest tissue specificity of MSCs when they are used for regenerative
therapy and/or the necessity to consider the histological idiosyncrasies of the lesion
that is to be repaired.
MSCs taken from adults tissues are thought to include cells at varying stages of
division and stemness depending on the local environmental factors such as inflam-
mation or tissue metabolism. The establishment of a harvesting method that allows
for the isolation of mesenchymal stem cell clones that retain their stemness, such as
a single specific marker is expected.
It has been reported that stem cells in the periodontal ligament and bone marrow
exist near blood vessels (McCulloch and Melcher 1983; Shi and Gronthos 2003).
Furthermore, bone marrow cells have been observed near the blood vessels of the
periodontal ligament (Kimura et al. 2014). Dohdrty et al. have found that bovine
pericytes express osteoblastic markers and STRO-1 and can induce mineralization
(Doherty et al. 1998). Although mesenchymal cells derived from avascular tissue
such as cartilage have also been discovered (Xue et al. 2016), based on the previ-
ously discussed localization of periodontal ligament stem cells near blood vessels
and stem cells being reported in the various vascular tissues (da Silva Meirelles
et al. 2006), it is surmised that PDLSCs originate from pericytes and/or bone mar-
row stem cells located near capillaries. There are extremely small amounts of infor-
mation regarding pericytes in the periodontal ligament tissues, and it remains
unclear whether periodontal ligament stem cells and such cells near the blood ves-
sels (e.g., pericytes) are identical.

10.5  imilarities Between Pericytes and Periodontal

S
Ligament Stem Cells

It is thought that pericytes, along with endothelial cells, anatomically constitute the
blood vessels of microvasculature including the terminal arterioles, capillaries, and
postcapillary venules and are providing nutrient and stabilizing blood vessels. As
178 M. Komaki

with MSCs, there is no single marker that identifies pericytes; several markers such
as alpha smooth muscle actin (αSMA), neuron-glial 2 (NG2), CD140a and CD140b
also known as platelet-derived growth factor receptor (PDGFR) α and β, and CD146
are used in conjunction (Dore-Duffy and Cleary 2011). There are recent reports that
pericytes possess multipotency, drawing focus to creating a new role for them
(Yamazaki and Mukouyama 2018; Markovic et al. 2018). Shi et al. reported that
STRO-1+ bone marrow MSCs and dental pulp stem cells (DPSCs) express the mark-
ers widely used as pericyte markers: α smooth muscle action (αSMA) and CD146
(Shi and Gronthos 2003). As mentioned, thus far, MSCs and pericytes are localized
near blood vessels and possess differentiation potentials. There is still debate about
these cells’ identities, their origins, their biological distribution, and their frequency.
Meirelles et al. have conducted long-term incubation of MSC-like cells from vari-
ous organs and tissues including the brain, pancreas, liver, bone marrow, and mus-
cles to investigate their distribution in the adult organism. MSCs were observed in
large and small blood vessels; thus, they concluded that MSCs exist in vascular
niche (da Silva Meirelles et al. 2006). Xu et al. have conducted a review of studies
on whether stem cells can differentiate into functional pericytes. Their criteria were
(1) the study has an appropriate control; (2) it involves pericyte markers such as
PDGFRβ, αSMA, NG2, and desmin; (3) the study functionally evaluates pericytes;
and (4) it involves a differentiation efficiency protocol. They concluded that stem
cells are the origins of pericytes (Xu et al. 2017). In contrast, based on the concept
that the blood vessels in internal organs are reservoir for stem cells, Corselli et al.
have isolated endothelial and perivascular cells by FACS and reported that micro-
vascular pericytes and adventitial cells of large vessels express MSC markers
(Corselli et al. 2010).
The periodontal ligament, supportive tissue for the teeth, is a connective tissue rich
with blood vessels, but there is little information in relation to pericytes. Gould et al.
conducted a cell kinetic analysis and cytology of murine injured molar model and
reported that dividing cells can be observed around the blood vessels and on the out-
side of the blood vessel’s basal lamina after wounding. Besides, they allude to the fact
that accompanying wounding, the origins and functions of the dividing cells in the
periodontal ligament are possibly different from those of classical pericytes (Gould
et al. 1977, 1980). In 1985, McCulloch demonstrated, in autoradiographic investiga-
tion of mitotic cells with H3-thymidine, that cells showing characteristics of stem
cells exist perivascularly in the periodontal ligament in uninjured mice (McCulloch
1985). In 1986, Lew conducted a detailed study on the blood vessels of periodontal
tissues in rats using scanning electron microscopy (SEM). There are three types of
blood vessels in the periodontal ligament: terminal arterioles, ­capillaries, and the
postcapillary venules. Using morphometry, Lew showed that the total vascular vol-
ume of the periodontal ligament root apex was approximately 20%: abundance of
blood vessels compared to the 5% of general connective tissue. We found that MSCs
in the periodontal ligament that form colonies are STRO-1-, CD105-, and CD166-
positive and differentiate into osteoblasts, adipocytes, and chondrocytes (Nagatomo
et al. 2006). We conducted additional experiments to test our hypothesis that bone
marrow-derived cells were situated in periodontal ligament by using GFP transgenic
10 Pericytes in the Periodontal Ligament 179

mice to create bone marrow-substituted chimeric mice, during examination of the

periodontal ligament tissue in both intact and injured sites, and found that GFP-
positive cells were localized perivascularly in the intact periodontal ligament tissue,
while damage leading to increase in the quantity of GFP-positive cells in the peri-
odontal ligament. On the contrary, it was discovered that the number of MSCs
(PDGFR-α+Sca-1+ and CD45−TER119−) extracted from bone marrow decreased
(Kimura et al. 2014). These results possibly suggest that bone marrow stem cells are
one source of the MSCs that exist in the periodontal ligament at least when the tissue
was injured. Zhou et al. have shown that bone marrow MSCs are involved in peri-
odontal ligament repair tissue via the chemotaxis of bone marrow stem cells toward
SDF-1. Recently, it was reported that pericytes express CXCR4 much like bone mar-
row cells (Armulik et al. 2011); therefore, precaution is necessary when investigating
the involvement of pericytes and bone marrow MSCs. We tested whether PDLSCs
express stem cell and pericyte markers NG2, CD146, and CD140b (PDGFRβ),
whether NG2- and CD146-positive cells can be observed near the vascular endothe-
lium in rat periodontal ligament tissue, and whether periodontal ligament stem cells
maintain and/or stabilize blood vessels formed by endothelial cells as do pericytes in
the endothelial cell network formation assay or on cell pattern substrates we have
reported previously (Akahori et al. 2010; Mukai et al. 2008). The results showed that
PDLSCs express both stem cell and pericyte markers, including NG2, CD146, and
CD140b and participate in the maintenance of blood vessel-like structures (Fig. 10.4).
Furthermore, NG2- and CD146-­positive cells are situated near the vascular endothe-
lium in rat periodontal ligament (Iwasaki et al. 2013). There is a study stating that the
blood vessels of the dental root apex are predominately postcapillary venules and that
there is a full coverage of pericytes (Lew 1987). In contrast, there is a paper stating
that pericytes exist in the periodontal ligament’s blood vessels (Rhodin 1968). The
content is not presented here because the paper is unobtainable. Whether or not
PDLSCs and pericytes are the same must be investigated in more detail. Koike et al.
have three-­dimensionally cultured vascular endothelial cell with or without murine
fetus-derived fibroblasts, 10 T1/2 cells in vitro, and then transplanted them into mice
to investigate whether 10 T1/2 cells are involved in the maintenance of vascular net-
work. They demonstrated that 10 T1/2 cells contribute to a long-term maintenance
(56 days) of blood vessel density (Koike et al. 2004). The 10 T1/2 cells are a hetero-
geneous cell population whose phenotype can only be maintained under strict culture
conditions. Some reports show their multipotency, and Koike et al. used 10 T1/2 cells
as mesenchymal progenitor cells. The ontogenetic relation between fibroblasts and
mural cells, including pericytes, is known (Armulik et al. 2011). The relation of bone
marrow stem cells and pericytes needs to be clarified.
Cells that constitute the periodontal tissue are embryologically derived from the
neural crest. We have shown that cells derived from bone marrow exist around the
blood vessels of the periodontal ligament in 8- to 12-week-old mice. Using the lin-
eage tracing method, Takashima et al. have revealed that MSCs in fetal mouse trunk
are partially generated from the neuroepithelium through the intermediate stages of
the neural crest but not from the mesoderm (Takashima et al. 2007). Subsequently,
it was demonstrated that some of the MSCs within adult murine bone marrow come
180 M. Komaki

a HUVEC Fibroblast PDLSC BM-MSC Brain pericyte

3h

6h

9h

18h

24h

b c
250 180
Fibroblast Fibroblast
PDLSC 160 PDLSC
200 * ** BM-MSC
140
BM-MSC
Branching points (per field)

Brain Pericyte Brain Pericyte

120
Meshes (per field)

150 *
100
* ** 80
100 ** *
** * 60
* **
50 40
20
0 0
3 6 9 18 24 3 6 9 18 24
Time(h) Time(h)

Fig. 10.4 Chronologic changes in capillary formation in co-culture of Human Umbilical Vein
Endothelial Cells (HUVECs) with fibroblasts, PDLSCs, BM-MSCs, and brain pericytes. (a)
PKH26-labeled fibroblasts, PDLSCs, BM-MSCs, and brain pericytes were co-cultured with GFP-­
HUVECs (cell-HUVEC ratio, 20:1) on a gel matrix. Photographs of red and green merged images
from each experimental group are shown. Pictures in the leftmost column are from HUVECs
alone. The capillary-like structures of HUVECs began to lose their shape at 9 hours after co-­
culture, and most of the structures had disappeared at 24 hours. However, the capillary structures
were stably observed at 24 hours in the PDLSC, BM-MSC, and brain pericyte groups.
Representative pictures from more than three independent experiments are shown. Scale bar = 300
µm (original magnification × 2) The time course changes in branching point (b) and mesh (c)
counts are also indicated. (Asterisk) Statistically significant differences from the fibroblast group
(P < 0.05)
10 Pericytes in the Periodontal Ligament 181

from the neural crest, in which they were transiently observed during the develop-
mental stage (Morikawa et al. 2009). From this information, it follows that the ori-
gin of MSCs in the adult organism is varied, much like that of pericytes.
Unfortunately, there is no description regarding pericytes in any of the relevant
reports. We must wait for more research to reveal the whole picture of the origins of
MSCs and pericytes as well as their function in the adult organism.

10.6 Possibility of Treatments Targeting Pericytes

The blood vessels constitute a closed system that circulates throughout the body,
and its length is two and a half that of the outer circumference of the Earth. It is
known that blood vessels play both a physiologically and pathologically important
role in tissues. Blood vessels are lined with a single layer of vascular endothelial
cells along their lumen and are specialized according to the needs of the organs to
which they deliver nutrients and oxygen (organ-specific endothelial cell differentia-
tion). It is known that smooth muscle cells and pericytes join around the lumen
formed by endothelial cells and work together with endothelial cells to stabilize and
contract the blood vessels through receptors, including ephrin-B2, platelet-drived
growth factor receptors, and epidermal growth factor receptor 2, and their ligands.
Their diversity in abnormalities such as inflammation and their organ specificity are
acknowledged (Armulik et al. 2011; Tomlinson and Topper 2003; Yoshida and
Owens 2005; Sims 2000; Potente and Makinen 2017). In the adult, rapid metabolic
turnover is known in tissues that face constant challenges, for example, the peri-
odontal tissues, which are exposed to oral bacteria and mastication. The blood ves-
sels in which pericytes and MSCs are believed to be localized are diversified as well.
For this reason, it is thought that there are PDLSCs with various degrees of differen-
tiation in the periodontal ligament. Currently, trials have been conducted using all
sorts of cell surface antigens, including STRO-1, SSEA-4, CD271, and CD146, as
indicators for enrichment of PDLSC subpopulations with high self-­proliferation and
multipotency (Lv et al. 2014). If pericytes and MSCs are tissue-­specific cells that
maintain and repair tissues, and if these cells ceaselessly self-proliferate and differ-
entiate in response to a constantly changing environment, current MSC extraction
protocols make highly efficient and stable extraction for clinical application diffi-
cult. Additionally, the quantity and quality of MSCs taken from the adult organism
decrease with inflammation (Yang et al. 2013) and age (Zhang et al. 2012; Zheng
et al. 2009). It is extremely difficult to maintain the phenotype of the obtained cells,
in addition to securing the cell numbers needed for treatment, all the while avoiding
senescent deterioration. Although still vague, expert use of lineage tracing is eluci-
dating the true origin of pericytes and MSCs. On the basis of the plethora of infor-
mation about pericytes and MSCs, if one could create pericytes or MSCs from
induced pluripotent stem (iPS) cells (Fukuta et al. 2014; Chijimatsu et al. 2017), this
advance would bring us closer to using these cells, which boosts such effective treat-
ment results in various diseases and in periodontal regeneration.
182 M. Komaki

10.7 Conclusion

MSCs and pericytes are cells thought to be harvestable from the adult organism and
have multipotency. They have been used in research on disease models and have seen
partial clinical application. On the other hand, the origin, the distribution in the adult
organism, and the function of these perplexing cells are still not fully understood.
There are still mixed results regarding the therapeutic effects, but the feasibility of the
application of MSCs and pericytes to cell therapy has been demonstrated. Safe, stable,
high-efficiency, and cheap cells will possibly be developed because of the abundance
of accumulated information about them, leading to their application to therapy.

References

Afanador E et al (2005) Messenger RNA expression of periostin and twist transiently decrease by
occlusal hypofunction in mouse periodontal ligament. Arch Oral Biol 50:1023–1031. https://
doi.org/10.1016/j.archoralbio.2005.04.002
Akahori T et al (2010) Implantation of capillary structure engineered by optical lithography
improves hind limb ischemia in mice. Tissue Eng Part A 16:953–959. https://doi.org/10.1089/
ten.tea.2009.0097
Akizuki T et al (2005) Application of periodontal ligament cell sheet for periodontal
regeneration: a pilot study in beagle dogs. J Periodontal Res 40:245–251. https://doi.
org/10.1111/j.1600-0765.2005.00799.x
Andreasen JO (1981) Periodontal healing after replantation and autotransplantation of incisors in
monkeys. Int J Oral Surg 10:54–61
Armulik A, Genove G, Betsholtz C (2011) Pericytes: developmental, physiological, and
pathological perspectives, problems, and promises. Dev Cell 21:193–215. https://doi.
org/10.1016/j.devcel.2011.07.001
Basdra EK, Komposch G (1997) Osteoblast-like properties of human periodontal ligament cells:
an in vitro analysis. Eur J Orthod 19:615–621
Berglundh T, Lindhe J, Jonsson K, Ericsson I (1994) The topography of the vascular systems in the
periodontal and peri-implant tissues in the dog. J Clin Periodontol 21:189–193
Boyko GA, Melcher AH, Brunette DM (1981) Formation of new periodontal ligament by
periodontal ligament cells implanted in vivo after culture in vitro. A preliminary study of
transplanted roots in the dog. J Periodontal Res 16:73–88
Chen SC, Marino V, Gronthos S, Bartold PM (2006) Location of putative stem cells in
human periodontal ligament. J Periodontal Res 41:547–553. https://doi.org/10.1111/j.
1600-0765.2006.00904.x
Chen Y et al (2010) Recruitment of endogenous bone marrow mesenchymal stem cells towards
injured liver. J Cell Mol Med 14:1494–1508. https://doi.org/10.1111/j.1582-4934.2009.00912.x
Chijimatsu R et al (2017) Characterization of mesenchymal stem cell-like cells derived from
human iPSCs via neural crest development and their application for osteochondral repair. Stem
Cells Int 2017:1960965. https://doi.org/10.1155/2017/1960965
Corselli M, Chen CW, Crisan M, Lazzari L, Peault B (2010) Perivascular ancestors of adult
multipotent stem cells. Arterioscler Thromb Vasc Biol 30:1104–1109. https://doi.org/10.1161/
ATVBAHA.109.191643
da Silva Meirelles L, Chagastelles PC, Nardi NB (2006) Mesenchymal stem cells reside in virtually
all post-natal organs and tissues. J Cell Sci 119:2204–2213. https://doi.org/10.1242/jcs.02932
10 Pericytes in the Periodontal Ligament 183

da Silva Meirelles L, Caplan AI, Nardi NB (2008) In search of the in vivo identity of mesenchymal
stem cells. Stem Cells 26:2287–2299. https://doi.org/10.1634/stemcells.2007-1122
Dogan A, Ozdemir A, Kubar A, Oygur T (2002) Assessment of periodontal healing by seeding of
fibroblast-like cells derived from regenerated periodontal ligament in artificial furcation defects
in a dog: a pilot study. Tissue Eng 8:273–282. https://doi.org/10.1089/107632702753725030
Dogan A, Ozdemir A, Kubar A, Oygur T (2003) Healing of artificial fenestration defects by seeding
of fibroblast-like cells derived from regenerated periodontal ligament in a dog: a preliminary
study. Tissue Eng 9:1189–1196. https://doi.org/10.1089/10763270360728099
Doherty MJ et al (1998) Vascular pericytes express osteogenic potential in vitro and in vivo. J Bone
Miner Res 13:828–838. https://doi.org/10.1359/jbmr.1998.13.5.828
Dominici M et al (2006) Minimal criteria for defining multipotent mesenchymal stromal cells. The
International Society for Cellular Therapy position statement. Cytotherapy 8:315–317. https://
doi.org/10.1080/14653240600855905
Dore-Duffy P, Cleary K (2011) Morphology and properties of pericytes. Methods Mol Biol
686:49–68. https://doi.org/10.1007/978-1-60761-938-3_2
Fitter S, Gronthos S, Ooi SS, Zannettino AC (2017) The mesenchymal precursor cell marker
antibody STRO-1 binds to cell surface heat shock cognate 70. Stem Cells 35:940–951. https://
doi.org/10.1002/stem.2560
Friedenstein AJ, Chailakhjan RK, Lalykina KS (1970) The development of fibroblast colonies in
monolayer cultures of Guinea-pig bone marrow and spleen cells. Cell Tissue Kinet 3:393–403
Fukuta M et al (2014) Derivation of mesenchymal stromal cells from pluripotent stem cells
through a neural crest lineage using small molecule compounds with defined media. PLoS One
9:e112291. https://doi.org/10.1371/journal.pone.0112291
Gould TR, Melcher AH, Brunette DM (1977) Location of progenitor cells in periodontal ligament
of mouse molar stimulated by wounding. Anat Rec 188:133–141. https://doi.org/10.1002/
ar.1091880202
Gould TR, Melcher AH, Brunette DM (1980) Migration and division of progenitor cell populations
in periodontal ligament after wounding. J Periodontal Res 15:20–42
Hatakeyama A et al (2017) Isolation and characterization of synovial mesenchymal stem cell
derived from hip joints: a comparative analysis with a matched control knee group. Stem Cells
Int 2017:9312329. https://doi.org/10.1155/2017/9312329
Iwasaki K et al (2013) Periodontal ligament stem cells possess the characteristics of pericytes.
J Periodontol 84:1425–1433. https://doi.org/10.1902/jop.2012.120547
Iwasaki K et al (2014) Periodontal regeneration using periodontal ligament stem cell-transferred
amnion. Tissue Eng Part A 20:693–704. https://doi.org/10.1089/ten.TEA.2013.0017
Karring T, Nyman S, Lindhe J (1980) Healing following implantation of periodontitis affected
roots into bone tissue. J Clin Periodontol 7:96–105
Kawaguchi H et al (2004) Enhancement of periodontal tissue regeneration by transplantation of
bone marrow mesenchymal stem cells. J Periodontol 75:1281–1287. https://doi.org/10.1902/
jop.2004.75.9.1281
Kimura Y et al (2014) Recruitment of bone marrow-derived cells to periodontal tissue defects.
Front Cell Dev Biol 2:19. https://doi.org/10.3389/fcell.2014.00019
Koike N et al (2004) Tissue engineering: creation of long-lasting blood vessels. Nature 428:138–
139. https://doi.org/10.1038/428138a
Komaki M et al (2007) Twist negatively regulates osteoblastic differentiation in human periodontal
ligament cells. J Cell Biochem 100:303–314. https://doi.org/10.1002/jcb.21038
Komaki M et al (2017) Exosomes of human placenta-derived mesenchymal stem cells stimulate
angiogenesis. Stem Cell Res Ther 8:219. https://doi.org/10.1186/s13287-017-0660-9
Lallier TE, Spencer A, Fowler MM (2005) Transcript profiling of periodontal fibroblasts and
osteoblasts. J Periodontol 76:1044–1055. https://doi.org/10.1902/jop.2005.76.7.1044
Lew KK (1987) The periodontal microvasculature—a morphological and morphometric study.
J Nihon Univ Sch Dent 29:262–269
184 M. Komaki

Li H, Yan F, Lei L, Li Y, Xiao Y (2009) Application of autologous cryopreserved bone marrow

mesenchymal stem cells for periodontal regeneration in dogs. Cells Tissues Organs 190:94–
101. https://doi.org/10.1159/000166547
Li G et al (2010) Periostin mediates vascular smooth muscle cell migration through the integrins
alphavbeta3 and alphavbeta5 and focal adhesion kinase (FAK) pathway. Atherosclerosis
208:358–365. https://doi.org/10.1016/j.atherosclerosis.2009.07.046
Lindner V, Wang Q, Conley BA, Friesel RE, Vary C (2005) Vascular injury induces expression
of periostin: implications for vascular cell differentiation and migration. Arterioscler Thromb
Vasc Biol 25:77–83. https://doi.org/10.1161/01.ATV.0000149141.81230.c6
Lv FJ, Tuan RS, Cheung KM, Leung VY (2014) Concise review: the surface markers and identity of
human mesenchymal stem cells. Stem Cells 32:1408–1419. https://doi.org/10.1002/stem.1681
Markovic BS et al (2018) Molecular and cellular mechanisms involved in mesenchymal stem
cell-based therapy of inflammatory bowel diseases. Stem Cell Rev 14:153–165. https://doi.
org/10.1007/s12015-017-9789-2
Matsuo M, Takahashi K (2002) Scanning electron microscopic observation of microvasculature in
periodontium. Microsc Res Tech 56:3–14. https://doi.org/10.1002/jemt.10008
McCulloch CA (1985) Progenitor cell populations in the periodontal ligament of mice. Anat Rec
211:258–262. https://doi.org/10.1002/ar.1092110305
McCulloch CA, Melcher AH (1983) Continuous labelling of the periodontal ligament of mice.
J Periodontal Res 18:231–241
Melcher AH (1976) On the repair potential of periodontal tissues. J Periodontol 47:256–260.
https://doi.org/10.1902/jop.1976.47.5.256
Monsarrat P et al (2014) Concise review: mesenchymal stromal cells used for periodontal
regeneration: a systematic review. Stem Cells Transl Med 3:768–774. https://doi.org/10.5966/
sctm.2013-0183
Morgan J, Muntoni F (2007) Mural cells paint a new picture of muscle stem cells. Nat Cell Biol
9:249–251. https://doi.org/10.1038/ncb0307-249
Morikawa S et al (2009) Development of mesenchymal stem cells partially originate from the
neural crest. Biochem Biophys Res Commun 379:1114–1119. https://doi.org/10.1016/j.
bbrc.2009.01.031
Mukai N et al (2008) A comparison of the tube forming potentials of early and late endothelial
progenitor cells. Exp Cell Res 314:430–440. https://doi.org/10.1016/j.yexcr.2007.11.016
Murrell EF, Yen EH, Johnson RB (1996) Vascular changes in the periodontal ligament after
removal of orthodontic forces. Am J Orthod Dentofac Orthop 110:280–286
Nagatomo K et al (2006) Stem cell properties of human periodontal ligament cells. J Periodontal
Res 41:303–310. https://doi.org/10.1111/j.1600-0765.2006.00870.x
Nojima N et al (1990) Fibroblastic cells derived from bovine periodontal ligaments have the
phenotypes of osteoblasts. J Periodontal Res 25:179–185
Nyman S, Karring T, Lindhe J, Planten S (1980) Healing following implantation of periodontitis-­
affected roots into gingival connective tissue. J Clin Periodontol 7:394–401
Pierantozzi E et al (2015) Human pericytes isolated from adipose tissue have better differentiation
abilities than their mesenchymal stem cell counterparts. Cell Tissue Res 361:769–778. https://
doi.org/10.1007/s00441-015-2166-z
Pirjali T et al (2013) Isolation and characterization of human mesenchymal stem cells derived
from human umbilical cord Wharton's jelly and amniotic membrane. Int J Organ Transplant
Med 4:111–116
Pittenger MF et al (1999) Multilineage potential of adult human mesenchymal stem cells. Science
284:143–147
Potente M, Makinen T (2017) Vascular heterogeneity and specialization in development and
disease. Nat Rev Mol Cell Biol 18:477–494. https://doi.org/10.1038/nrm.2017.36
Rhodin JA (1968) Ultrastructure of mammalian venous capillaries, venules, and small collecting
veins. J Ultrastruct Res 25:452–500
10 Pericytes in the Periodontal Ligament 185

Rios HF et al (2008) Periostin is essential for the integrity and function of the periodontal ligament
during occlusal loading in mice. J Periodontol 79:1480–1490. https://doi.org/10.1902/
jop.2008.070624
Rygh P, Bowling K, Hovlandsdal L, Williams S (1986) Activation of the vascular system: a
main mediator of periodontal fiber remodeling in orthodontic tooth movement. Am J Orthod
89:453–468
Sahara M et al (2005) Comparison of various bone marrow fractions in the ability to participate in
vascular remodeling after mechanical injury. Stem Cells 23:874–878. https://doi.org/10.1634/
stemcells.2005-0012
Seo BM et al (2004) Investigation of multipotent postnatal stem cells from human periodontal
ligament. Lancet 364:149–155. https://doi.org/10.1016/S0140-6736(04)16627-0
Shi S, Gronthos S (2003) Perivascular niche of postnatal mesenchymal stem cells in human
bone marrow and dental pulp. J Bone Miner Res 18:696–704. https://doi.org/10.1359/
jbmr.2003.18.4.696
Simmons PJ, Torok-Storb B (1991) Identification of stromal cell precursors in human bone marrow
by a novel monoclonal antibody, STRO-1. Blood 78:55–62
Sims DE (2000) Diversity within pericytes. Clin Exp Pharmacol Physiol 27:842–846
Simsek SB, Keles GC, Baris S, Cetinkaya BO (2012) Comparison of mesenchymal stem cells and
autogenous cortical bone graft in the treatment of class II furcation defects in dogs. Clin Oral
Investig 16:251–258. https://doi.org/10.1007/s00784-010-0486-7
Sorrentino A et al (2008) Isolation and characterization of CD146+ multipotent mesenchymal
stromal cells. Exp Hematol 36:1035–1046. https://doi.org/10.1016/j.exphem.2008.03.004
Takashima Y et al (2007) Neuroepithelial cells supply an initial transient wave of MSC
differentiation. Cell 129:1377–1388. https://doi.org/10.1016/j.cell.2007.04.028
Tanaka K et al (2011) Comparison of characteristics of periodontal ligament cells obtained from
outgrowth and enzyme-digested culture methods. Arch Oral Biol 56:380–388. https://doi.
org/10.1016/j.archoralbio.2010.10.013
Tobita M, Mizuno H (2013) Adipose-derived stem cells and periodontal tissue engineering. Int
J Oral Maxillofac Implants 28:e487–e493. https://doi.org/10.11607/jomi.te29
Tomlinson JE, Topper JN (2003) New insights into endothelial diversity. Curr Atheroscler Rep
5:223–229
Tooi M et al (2016) Placenta mesenchymal stem cell derived exosomes confer plasticity on
fibroblasts. J Cell Biochem 117:1658–1670. https://doi.org/10.1002/jcb.25459
Tsumanuma Y et al (2011) Comparison of different tissue-derived stem cell sheets for periodontal
regeneration in a canine 1-wall defect model. Biomaterials 32:5819–5825. https://doi.
org/10.1016/j.biomaterials.2011.04.071
Wilde J, Yokozeki M, Terai K, Kudo A, Moriyama K (2003) The divergent expression of periostin
mRNA in the periodontal ligament during experimental tooth movement. Cell Tissue Res
312:345–351. https://doi.org/10.1007/s00441-002-0664-2
Xu J, Gong T, Heng BC, Zhang CF (2017) A systematic review: differentiation of stem cells into
functional pericytes. FASEB J 31:1775–1786. https://doi.org/10.1096/fj.201600951RRR
Xue K, Zhang X, Qi L, Zhou J, Liu K (2016) Isolation, identification, and comparison of cartilage
stem progenitor/cells from auricular cartilage and perichondrium. Am J Transl Res 8:732–741
Yamada S et al (2007) PLAP-1/asporin, a novel negative regulator of periodontal ligament
mineralization. J Biol Chem 282:23070–23080. https://doi.org/10.1074/jbc.M611181200
Yamazaki T, Mukouyama YS (2018) Tissue specific origin, development, and pathological
perspectives of Pericytes. Front Cardiovasc Med 5:78. https://doi.org/10.3389/fcvm.2018.00078
Yan XZ, Yang F, Jansen JA, de Vries RB, van den Beucken JJ (2015) Cell-based approaches in
periodontal regeneration: a systematic review and meta-analysis of periodontal defect models
in animal experimental work. Tissue Eng Part B Rev 21:411–426. https://doi.org/10.1089/ten.
TEB.2015.0049
Yang N et al (2013) Tumor necrosis factor alpha suppresses the mesenchymal stem cell osteogenesis
promoter miR-21 in estrogen deficiency-induced osteoporosis. J Bone Miner Res 28:559–573.
https://doi.org/10.1002/jbmr.1798
186 M. Komaki

Yoshida T, Owens GK (2005) Molecular determinants of vascular smooth muscle cell diversity.
Circ Res 96:280–291. https://doi.org/10.1161/01.RES.0000155951.62152.2e
Yoshizawa T et al (2004) Homeobox protein MSX2 acts as a molecular defense mechanism for
preventing ossification in ligament fibroblasts. Mol Cell Biol 24:3460–3472
Zhang X et al (2011) Isolation and characterization of mesenchymal stem cells from human
umbilical cord blood: reevaluation of critical factors for successful isolation and high ability to
proliferate and differentiate to chondrocytes as compared to mesenchymal stem cells from bone
marrow and adipose tissue. J Cell Biochem 112:1206–1218. https://doi.org/10.1002/jcb.23042
Zhang J et al (2012) The effect of aging on the pluripotential capacity and regenerative potential of
human periodontal ligament stem cells. Biomaterials 33:6974–6986. https://doi.org/10.1016/j.
biomaterials.2012.06.032
Zheng W et al (2009) Loss of proliferation and differentiation capacity of aged human periodontal
ligament stem cells and rejuvenation by exposure to the young extrinsic environment. Tissue
Eng Part A 15:2363–2371. https://doi.org/10.1089/ten.tea.2008.0562
Zhou J et al (2011) Role of bone marrow-derived progenitor cells in the maintenance and
regeneration of dental mesenchymal tissues. J Cell Physiol 226:2081–2090. https://doi.
org/10.1002/jcp.22538
Zoellner H, Chapple CC, Hunter N (2002) Microvasculature in gingivitis and chronic periodontitis:
disruption of vascular networks with protracted inflammation. Microsc Res Tech 56:15–31.
https://doi.org/10.1002/jemt.10009
Chapter 11
Pericytes in the Heart

Linda L. Lee and Vishnu Chintalgattu

Abstract Mural cells known as pericytes envelop the endothelial layer of microves-
sels throughout the body and have been described to have tissue-specific functions.
Cardiac pericytes are abundantly found in the heart, but they are relatively under-
studied. Currently, their importance is emerging in cardiovascular homeostasis and
dysfunction due to their pleiotropism. They are known to play key roles in vascular
tone and vascular integrity as well as angiogenesis. However, their dysfunctional
presence and/or absence is critical in the mechanisms that lead to cardiac patholo-
gies such as myocardial infarction, fibrosis, and thrombosis. Moreover, they are
targeted as a therapeutic potential due to their mesenchymal properties that could
allow them to repair and regenerate a damaged heart. They are also sought after as
a cell-based therapy based on their healing potential in preclinical studies of animal
models of myocardial infarction. Therefore, recognizing the importance of cardiac
pericytes and understanding their biology will lead to new therapeutic concepts.

Keywords Cardiac pericyte · Heart · Cardiovascular physiology · Cardiovascular

pathophysiology · Myocardial infarction · Myocardial ischemic disease · Vascular
stem cell · Perivascular cell · Mural cell · Vascular tone · Vascular integrity ·
Vascular biology

Abbreviations

3g5 Ganglioside 3g5

αSMA Alpha-smooth muscle actin
Alk-p Alkaline phosphatase
Angpt-1 Angiopoietin-1

L. L. Lee · V. Chintalgattu (*)

Department of CardioMetabolic Disorders, Amgen Research and Discovery,
Amgen Inc., South San Francisco, CA, USA
e-mail: [email protected]

© Springer Nature Switzerland AG 2019 187

A. Birbrair (ed.), Pericyte Biology in Different Organs,
Advances in Experimental Medicine and Biology 1122,
https://doi.org/10.1007/978-3-030-11093-2_11
188 L. L. Lee and V. Chintalgattu

Angpt-2 Angiopoietin-2
BDNF Endothelial brain-derived neurotrophic factor
CNP C-type natriuretic peptide
cPC Cardiac pericyte
CTGF/CCN2 Connective tissue growth factor
Cx43 Connexin-43
CXCR3 Chemokine receptor 3
ECM Extracellular matrix
Gli1 Glioma-associated oncogene 1
HB-EGF Heparin-binding epidermal growth factor
HGF Hepatocyte growth factor
LRP Low-density lipoprotein receptor-related protein 6
MI Myocardial infarction
MMPs Matrix metalloproteinases
MSC Mesenchymal stem cells
NANOG Homeobox transcription factor Nanog
NG2 Neural glial 2
OCT4 Octamer-binding transcription factor 4
p75 NTR Neurotrophin receptor
PDGFbb Platelet-derived growth factor bb
PDGFRβ Platelet-derived growth factor receptor beta
pro-NGF Pro-nerve growth factor
RGS5 Regulator of G protein signaling 5
S1P Sphingosine-1-phosphate
SIRT3 Sirtuin 3
SorCS2 Sortilin-related VPS10 domain-containing receptor 2
SOX2 Sex-determining region box
SVPs Saphenous vein-derived pericyte progenitor cells
Tbx18 T-box protein 18
TF Tissue factor
TGFβ Transforming growth factor beta
TrkB Tropomyosin receptor kinase B
VCAF Vascular calcification-associated factor
VEGF-A Vascular endothelial growth factor A
VEGFR2 Vascular endothelial growth factor receptor 2
vWF von Willebrand factor

11.1 Introduction

Pericytes are perivascular cells that are present throughout various vascular beds of
the body including the heart. Pericytes are found on small blood vessels including
arterioles, venules, and capillaries where they are known to have tissue-specific
functions. Pericyte number also differs in different tissue beds, and their number
appears to determine the tissue-specific endothelial barrier function. For example,
11 Pericytes in the Heart 189

the brain and retina need stringent barrier integrity as compared to other tissues like
skeletal muscle. The ratio of pericytes to endothelial cells in the brain is found as
high as 1:1 compared to places like the skeletal muscle which is found to be as low
as 1:100 (Sims 1986; Armulik et al. 2011). Moreover, pericyte dysfunction and/or
absence has been implicated in the development of fibrosis, atherosclerosis, osteo-
genesis, tumor angiogenesis, dementia, and Alzheimer’s disease (Armulik et al.
2011, 2010; Hellstrom et al. 2001; Sengillo et al. 2013; Sagare et al. 2013; Collett
and Canfield 2005; Fang and Salven 2011; Henderson et al. 2013; Matthews et al.
2016). Despite these critical roles in various tissue physiology and disease, rela-
tively little is known about cardiac pericytes. Roughly 1 in every 5000 cardiac-­
related publications mentions pericytes. Due to this gap in the literature and
emerging importance of the vascular system in pathophysiology of cardiovascular
diseases, cardiac pericyte biology has recently gained momentum and attention in
the cardiac field.
This chapter will first focus on the current known properties of cardiac pericytes
(cPC) in terms of origin, location, structure, and characterization. Next, we will
discuss their physiological function and their role in cardiac homeostasis. Through
their intimate relationship with endothelial cells, cPC participate in angiogenesis,
are important in vascular integrity and perfusion, and promote vessel stability and
vessel maturation. Cardiac pericytes have also been shown to play a part in hemo-
dynamics by altering vascular tone (O’Farrell and Attwell 2014; O’Farrell et al.
2017). We will also dive into the cardiac pericyte’s role in cardiovascular patho-
physiology. Various cardiovascular diseases such as myocardial ischemia, fibrosis,
calcification, atherosclerosis, and thrombosis all involve cPC at different stages of
disease progression. From there, we will discuss their plasticity as they have been
shown to be part of the regeneration and reparative process of a diseased heart as
well as their use in cell therapy. Understanding cPC biology and their role under
pathophysiological conditions could pave a path for development of novel therapies
for various forms of cardiovascular diseases.

11.2 Properties of Cardiac Pericytes

11.2.1 Origin

Due to the vascularization of the heart, it has been said that pericytes are the second
most abundant cell type after endothelial cells in the heart where the ratio of peri-
cyte to endothelial cells is 1:2 or 1:3 (Nees et al. 2012a). Others have said that they
occupy 5% of the total non-myocyte population (Pinto et al. 2016). Cardiac peri-
cytes are of mesothelial origin and are derived from the mesenchymal angioblast
during development (Kovacic et al. 2012; Kumar et al. 2017). Due to their origin,
they are seen as vascular stem cells (Nees et al. 2012a). Through linage studies, Cai
et al. elegantly showed that cPC originate from the epicardium (Cai et al. 2008a).
However, the mechanism to how they are recruited to the heart is currently unknown.
190 L. L. Lee and V. Chintalgattu

11.2.2 Anatomical Location

Anatomical location is one of the main properties that distinguish pericytes from the
closely related smooth muscle cells where both are mural cells derived from the
mesenchymal angioblast (Kumar et al. 2017; Feng et al. 2011). As the heart pumps
blood to feed the tissues of the body, the heart also feeds itself through its coronary
arteries, and it is here where the bulk of the cPCs lie. There are adventitial pericytes
that are found in the intima of arterioles (where the microvessel widens to the arter-
ies) and venules (where the microvessels widens to the veins), and there are peri-
cytes that are found on the capillaries (Campagnolo et al. 2010; Nees et al. 2012b;
Klein et al. 2011; Corselli et al. 2012; Avolio et al. 2015a). In the heart, endocardial
endothelial cells have been shown to be progenitors for cPCs and smooth muscle
cells (Chen et al. 2016). Also specific to the heart, smooth muscle cells derive from
pericyte progenitors (Volz et al. 2015). Smooth muscle cells sit on the higher arter-
ies and veins where they regulate blood flow through vasoconstriction and vasodila-
tion. Pericytes sit on the abluminal side of a vessel and envelop a single layer of
endothelial cells. The unique location of the pericytes allows them to be the liaison
between the endothelium and myocytes and the liaison between the intima and
media in the higher vascular branches (Nees et al. 2012b).

11.2.3 Characterization

Morphologically, pericytes have a single round nucleus with extended processes

that reach out and wrap around endothelial cells. Visually, one can spot a pericyte as
a small “bump-on-a-log” on a microvessel. In culture, they are quite flat and less
spindle-like compared to their smooth muscle cell counterpart. Their cellular archi-
tecture has been defined, but there is currently no one marker that can unequivocally
identify pericytes; rather, multiple pericytic markers must be used. Phenotypically,
the most popular and accepted markers are neural glial-2 (NG2), platelet-derived
growth factor receptor beta (PDGFRβ), and alpha-smooth muscle actin (αSMA)
(Armulik et al. 2010). These markers are expressed ten times higher in pericytes
compared to smooth muscle cells which also express the same markers (Nees et al.
2013). Microvascular pericytes are usually NG2+ αSMA−; venular pericytes are
NG2− αSMA+; arteriolar pericytes are NG2+ αSMA− (Wanjare et al. 2013). However,
a recent study showed that microvascular pericytes are also αSMA+; they were pre-
viously undetected due to the quick depolymerization of F-actin (Alarcon-­Martinez
et al. 2018). Other markers include tissue factor (TF), ganglioside 3g5 (3g5), vimen-
tin, desmin, calponin, caldesmon, and alkaline phosphatase (Alk-p) (Nees et al.
2012a; Avolio et al. 2015a; Shepro and Morel 1993; Hughes and Chan-­Ling 2004;
Crisan et al. 2008; Juchem et al. 2010; Chen et al. 2015). Most recently, glioma-
associated oncogene 1 (Gli1) and T-box protein 18 (Tbx18) have also been
11 Pericytes in the Heart 191

identified as molecular markers for pericytes (Kramann et al. 2015a, 2017;

Guimaraes-Camboa et al. 2017). They have been reported to express stemness
markers such as CD44, CD90, and CD105 (Chen et al. 2015; Covas et al. 2008) and
genetically express homeobox transcription factor Nanog (NANOG), sex-­
determining region box (SOX2), and octamer-binding transcription factor 4 (OCT4)
(Avolio et al. 2015a). They are also negative for endothelial markers such as CD31
and von Willebrand factor (vWF) as well as for hematopoietic markers such as
CD45 (Nees et al. 2012a; Avolio et al. 2015a; Juchem et al. 2010; Chen et al. 2015).
Regulator of G protein signaling 5 (RGS5) has been described as a specific marker
for brain pericytes, but the progress of research in the heart is behind (Cho et al.
2003; Mitchell et al. 2008; Nisancioglu et al. 2008).
The homogeneity of pericytes in the heart (as well as in other tissues) is under
debate. Pericytes have been isolated and characterized from different species and
tissues, and their marker expression varies (Dias Moura Prazeres et al. 2017). To
further complicate their characterization, their marker expression is also dynamic
within the same tissue as an indication of their activation state and/or maturity
(van Dijk et al. 2015). However, their functional characteristics are more homoge-
neous within the tissues. Functional characteristics of pericytes include migration
toward endothelial platelet-derived growth factor bb (PDGFbb) signaling and the
ability to mediate tubule formation in co-culture with endothelial cells (Chen et al.
2015; Zhou et al. 2016). Cardiac pericytes are pro-angiogenic and procoagulatory
(Juchem et al. 2010). Cardiac pericytes will also connect quickly with other cells
(endothelial or other pericytes) via gap junctions like connexin-43 (Cx43) as
shown by cell dye transfer studies (Nees et al. 2012a). Enzymatically, they have
been shown to have alkaline phosphatase activity (Juchem et al. 2010). However,
few groups have been able to isolate and characterize cPCs, and each group found
a different subtype. They all varied in origin, location, and molecular markers.
Nees et al. used NG2, PDGFRβ, αSMA, calponin, Cx43, and 3g5 as molecular
markers as well as functional characterization to identify their isolated pericyte
population from eight different species (Nees et al. 2012a). Couple of years later,
Chen et al. followed up with their primary isolation from human fetal and adult
hearts. They isolated a population that was NG2+ CD146+ CD34− CD45− CD56−
CD117− (Chen et al. 2015). They claim that this population can be cultivated in
homogeny by FACS. Around the same time, Avolio et al. were also able to isolate
and characterize isolated primary pericytes from human neonatal hearts. Their
unique population consisted of pericytes expressing CD34+ NG2+ CD146− cKit−.
This population was found on both the arterioles and capillaries (Avolio et al.
2015a). Table 11.1 provides all of the markers that can be used to identify cPCs.
Work remains to identify a cPC-specific marker that is homogenously expressed in
all cPC subtypes. For now, identification of pericytes requires immunogenic mark-
ers, gene and protein expression profiles, functional characteristics, and localiza-
tion in situ.
192 L. L. Lee and V. Chintalgattu

Table 11.1 Markers for cardiac pericytes (table adopted from Murray et al. 2017)
Gene
symbol Gene name References
3G5 3g5-defined ganglioside Nees et al. (2012a)
αSMA Alpha-smooth muscle actin Chen et al. (2015) and Crisan et al.
(2008)
ALPL Alkaline phosphatase Juchem et al. (2010)
CD10 Neural endopeptidase Crisan et al. (2008)
CD13 Alanine aminopeptidase Crisan et al. (2008)
CD29 Integrin beta Dar et al. (2012)
CD34 CD34 molecule Chen et al. (2013)
CD44 Receptor for hyaluronic acid Chen et al. (2015) and Crisan et al.
(2008)
CD73 5′nucleotidase, ecto Chen et al. (2015) and Crisan et al.
(2008)
CD90 Thy-1 Chen et al. (2015) and Crisan et al.
(2008)
CD105 Endoglin Chen et al. (2015) and Crisan et al.
(2008)
CD108 Sema L Chen et al. (2015) and Crisan et al.
(2008)
CD109 Platelet activation factor Crisan et al. (2008)
CD140b Platelet-derived growth factor beta Chen et al. (2015) and Crisan et al.
(PDGFRβ) (2008)
CD140a Platelet-derived growth factor alpha Psaltis et al. (2010)
(PDGFRα)
CD142 Tissue factor Juchem et al. (2010)
CD146 Melanoma cell adhesion molecule Chen et al. (2015) and Crisan et al.
(2008)
CD164 Sialomucin core protein 24 Crisan et al. (2008)
CD166 ALCAM Crisan et al. (2008)
CD318 CUB domain-containing protein 1 Crisan et al. (2008)
CD340 Human epidermal growth factor receptor 2 Crisan et al. (2008)
CD349 Frizzled-9 Crisan et al. (2008)
CNN1 Calponin Nees et al. (2012a)
Cx43 Connexin-43 Nees et al. (2012a)
Gli1 Glioma-associated oncogene 1 Kramann et al. (2015b, 2017)
HLA-CLI Human leukocyte antigen class 1 Crisan et al. (2008)
NG2 Neurol/glial antigen 2 Chen et al. (2015) and Crisan et al.
(2008)
SM-MHC Smooth muscle myosin heavy chain Chen et al. (2015)
SSEA-4 Stage-specific embryonic antigen-4 Crisan et al. (2008)
STRO-1 N/A Psaltis et al. (2010)
Tbx18 T-BOX protein 18 Guimaraes-Camboa et al. (2017)
VIM Vimentin Avolio et al. (2015a)
11 Pericytes in the Heart 193

11.3 Role in Cardiac Homeostasis

11.3.1 Vascular Integrity and Perfusion

To help maintain cardiac homeostasis, cPCs contribute to the integrity of the endo-
thelial layer by regulating vascular permeability (Armulik et al. 2005). Pericytes
and endothelial cells have an intimate relationship and together they form a func-
tional intercommunicating unit. They share a basement membrane where multiple
intracellular connections are made. The two cell types form gap junctions, tight
junctions, and peg-socket contacts to communicate and strengthen the barrier
(Fig. 11.1). These gap junctions are formed by proteins such as connexins, cadher-
ins, occludins, and claudins that are integrated into the cytoskeletal structure
(Winkler et al. 2011). In cell culture models, co-culture models of pericytes and
endothelial cells show a decrease in barrier permeability compared to endothelial
cells alone (Al Ahmad et al. 2009; Nakagawa et al. 2009). PDGFRβ is not only used
as a pericyte molecular marker, it is also part of a signaling pathway essential to
pericyte survival. As shown by Kaminski et al., PDGFRβ−/− mice are embryonically
lethal. The embryos have vascular malformations and hemorrhaging (Hellstrom
et al. 2001; Kaminski et al. 2001). In a conditional adult PDGFRβ−/− mouse, the
mice exhibit high vascular permeability and leakage, and their perfusion is compro-
mised (Bell et al. 2010; Winkler et al. 2010). Bjarnegard et al. showed that deletion
of endothelial-derived PDGFbb causes pericyte loss and cardiac deficits (Bjarnegard
et al. 2004). While studying the cardiotoxicity of cancer drugs, sunitinib malate
decreased the number of pericyte coverage of vessels on the heart, caused microvas-
cular dysfunction, decreased cardiac function (coronary flow), and induced hyper-
trophy (Chintalgattu et al. 2013). Interestingly, using an anticancer agent thalidomide
reversed the physiological effects that they found. All together, these observations
demonstrate that cPCs play a critical role which is vascular function and homeosta-
sis in the heart.

11.3.2 Angiogenesis

Another physiological aspect is that pericytes participate in is angiogenesis

(Gerhardt and Betsholtz 2003). They do so by presenting growth factors that stimulate
the formation of new vessels (Stapor et al. 2014; Matsuki et al. 2015; Caporali et al.
2017). Under angiogenic conditions, the cross talk between endothelial cells and
pericytes allows for both cell types to migrate and proliferate (Darland et al. 2003;
Bergers and Song 2005; Ribatti et al. 2011; Fuxe et al. 2011). There have been sev-
eral key processes that have been identified. First, angiopoietin-1/2 (angpt-1/2) sig-
naling from endothelial cell binding to Tie-2 receptors on pericytes causes
dissociation and aids in survival of pericytes from the vessels (Benest et al. 2006;
194 L. L. Lee and V. Chintalgattu

Fig. 11.1 Summary of the roles of cardiac pericytes in cardiac homeostasis. They are known to
play a part in hemodynamics, vascular integrity, and perfusion and angiogenesis

Wakui et al. 2006; Cai et al. 2008b; Maisonpierre et al. 1997; Dewi et al. 2018).
Matrix metalloproteinases (MMPs) and proteases alter the extracellular matrix
(ECM) to allow for pericyte migration (Stratman et al. 2010; Stratman and Davis
2012; He and Spiro 1995). Pericyte-derived vascular endothelial growth factor A
(VEGF-A) binding to endothelial VEGF receptor 2 (VEGFR2) allows for endothe-
lial proliferation and survival and leads to sprouting and formation of new vessels
(Darland et al. 2003; Benest et al. 2006; Wakui et al. 2006; Franco et al. 2011).
Next, pericyte-derived transforming growth factor beta (TGFβ) is secreted to inhibit
continuous proliferation of endothelial cells (Winkler et al. 2011; Bergers and
Song 2005). Lastly, endothelial-derived PDGFbb and heparin-binding epidermal
growth factor (HB-EGF) recruit pericytes back to stabilize the new vessels (Stratman
11 Pericytes in the Heart 195

et al. 2010; Magnusson et al. 2007; Nadal et al. 2002). Jagged1 expression and
Notch-1 signaling are required for vessel maturation for both the newly proliferated
endothelium and pericytes (Boscolo et al. 2011; Tattersall et al. 2016; Tao et al.
2017).
N-cadherin is an adhesion molecule that is required for maturation and stabiliza-
tion of the newly sprouted vessel due to its interaction with pericytes (Tillet et al.
2005). To induce the expression of adhesion molecules such as N-cadherin where its
absence would impede pericyte recruitment, pericyte sphingosine-1-phosphate
(S1P) signaling is required to bind to the S1P receptor on endothelial cells (Paik
et al. 2004; McGuire et al. 2011). Blocking N-cadherin expression decreased endo-
thelial barrier properties. Found at the edges of sprouting vessels, pericyte-derived
angpt-1 binds to the endothelial Tie-2 receptor to signal vessel stabilization, and
pericytes spatially regulate sprouting due to their expression of VEGFR1 (Zeng
et al. 2016; Eilken et al. 2017; Teichert et al. 2017). Once the vessels are stabilized,
endothelial brain-derived neurotrophic factor (BDNF) binds to tropomyosin recep-
tor kinase B (TrkB) on the pericytes for vessel maturation; however, the down-
stream signaling cascade is currently unknown. In the heart, only pericytes and
smooth muscle cells express TrkB. Anastasia et al. showed that in trk−/− mice are
embryonically lethal (Anastasia et al. 2014). The mice also had cardiac vascular
abnormalities, increased vascular permeability, and compromised vascular integ-
rity. Lastly, pericytes signal through chemokine receptor 3 (CXCR3) to regulate
angiogenesis by inhibiting vessel formation and mediate vessel dissociation (Bodnar
et al. 2013). As evidenced by the pathways discussed above, cross talk between
endothelial and pericytes is required for angiogenesis in the heart (Fig. 11.1).

11.3.3 Vascular Tone

Scientists have hypothesized that pericytes can control capillary function and act as
pre- and postcapillary sphincters due to their anatomical location and expression of
contractile proteins similar to their close relatives—the smooth muscle cells.
Multiple studies have investigated the contractile function of pericytes. Using opti-
cal and atomic force microscopy, retinal pericytes have been shown to generate
contractile forces that can wrinkle elastomeric substrata in vitro studies (Lee et al.
2010). Renal pericytes have been shown intravitally that they regulate blood flow
and pressure through endothelial-derived C-type natriuretic peptide (CNP) (Spiranec
et al. 2018). Moreover, renal smooth muscle cells and pericytes have been shown to
have calcium signals that are integrated across the vascular bed and control vascular
tone (Borysova et al. 2013). Under in vitro conditions, brain pericytes in culture
have been shown to contract by measuring their impedance changes due to dose-­
dependent responses to contraction and relaxation stimulants such as endothelin-1
and adenosine (Neuhaus et al. 2017). Calpain and talin are proteins that have been
shown to play a part in pericyte contraction and stiffness (Kotecki et al. 2010).
Multiple groups have contributed to showing that brain capillary pericytes do
196 L. L. Lee and V. Chintalgattu

contract post-ischemia (Hamilton et al. 2010; Mazzoni et al. 2015; Hall et al. 2014;
Mishra et al. 2014; Yemisci et al. 2009; Peppiatt et al. 2006; Fernandez-Klett et al.
2010). Using state-of-the-art intravital and ex vivo imaging techniques on the brain,
they showed that pericytes contract during ischemia and even after reperfusion, the
pericytes do not relax and they prevent flow back into the ischemic area (Hamilton
et al. 2010; Mazzoni et al. 2015; Hall et al. 2014; Mishra et al. 2014; Yemisci et al.
2009; Peppiatt et al. 2006; Fernandez-Klett et al. 2010). This phenomenon is known
as no-reflow (O’Farrell and Attwell 2014; Watanabe et al. 2004). Due to their tissue-­
specific properties, it was unknown if cPCs behave the same way during a myocar-
dial infraction (MI) or under ischemic conditions. Most recently, O’Farrell et al.
showed that pericytes can alternate vascular tone and cardiac hemodynamics by
constricting capillaries post-MI and reperfusion (O’Farrell et al. 2017). Adenosine
is known as a pericyte relaxant (Matsugi et al. 1997). Treating the mice with adenos-
ine showed a 30% improvement in reperfusion in the infracted area. However, the
mechanism to this no-reflow phenomenon in the heart remains to be elucidated. Due
to the evidence provided by the study from O’Farrell et al., we are left wondering if
cPCs have the ability to alter vascular tone under normal physiological conditions
and not just under ischemic conditions (Fig. 11.1).

11.4 Role in Cardiovascular Pathophysiology

We have discussed how cPC help maintain cardiac homeostasis under normal physi-
ological conditions, but what about their role in cardiac pathophysiology? Because
cPC play multiple roles in cardiac homeostasis, their dysfunction could also con-
tribute to cardiovascular pathologies. Cells respond to their environment such as
mechanical and chemical stimuli, and any perturbations and/or stress can alter their
molecular and biological functions that govern their normal physiological function.
Reformation of their cell biology can render the cell to transform into subsidies
contributing to cardiac pathogenesis. Dysfunctional cPC have been implicated in
many cardiovascular diseases and pathologies such as myocardial infarction (MI),
atherosclerosis, fibrosis, calcification, thrombosis, and inflammation (Fig. 11.2).

11.4.1 Myocardial Ischemic Disease

Myocardial ischemic disease develops when there is an occlusion to the coronary

arteries most likely due to a blockage in the lumen of the vessel that is built up over
time. The heart is deprived of oxygen, and the surrounding muscle becomes isch-
emic and eventually necrotic. Post-MI, the heart will remodel to try to compensate
for the damaged tissue and this will eventually lead to a failing heart. As discussed
earlier in their ability to control vascular tone under ischemic conditions, cPC play
a role in the no-reflow phenomena during ischemia/reperfusion injury. Pericytes
11 Pericytes in the Heart 197

Fig. 11.2 Summary of the roles and implicated roles of cardiac pericytes in cardio pathophysiology.
They have been shown to play a part in myocardial ischemic disease and have been implicated in
atherosclerosis, thrombosis, vascular calcification, and fibrosis

contract and close off the microvessels that could potentially reperfuse the oxygen-­
deprived area due to the acute MI. Because of cPC constriction, inadequate blood
flow is prevented from being reestablished (O’Farrell et al. 2017; Costa et al. 2018).
Under hypoxic conditions, hypoxia-induced long noncoding RNAs were identified
in pericytes, and HypERlnc was characterized to regulate pericyte cell proliferation,
viability, and endothelial interaction. They also found that HypERlnc expression
was significantly decreased in human cardiac tissue from heart failure patients
(Bischoff et al. 2017). Moreover, in a human and mouse study, they found that
pro-­nerve growth factor (pro-NGF), an inflammatory cytokine, accumulates at the
198 L. L. Lee and V. Chintalgattu

site of a MI secreted by cardiomyocytes and binds to neurotrophin receptor (p75

NTR) and sortilin-related VPS10 domain-containing receptor 2 (SorCS2) on cPCs
which mediates a proapoptotic pathway (Siao et al. 2012). This eventually leads to
the downstream effects of rescinded pericyte processes and increased vascular per-
meability. In mouse models of ischemia-reperfusion injury studies, genetic ablation
of Notch-3 and sirtuin 3 (SIRT3) decreased the ability of the heart to recover post-
MI because the proteins are important in microvascular homeostasis and function
(Tao et al. 2017; He et al. 2016). Notch-3 knockout mice, which have been shown
to be essential in vessel stability and maturation, showed a significant reduction in
cPC vessel coverage leading to larger infarct sizes and higher mortality rate post-
MI. The knockout mice also had a significant difference in cardiac function post-MI
recovery compared to their wild-type counterparts due to their vascular dysfunction
predisposed by their Notch-3 mutation (Tao et al. 2017). In another study utilizing
a SIRT3 knockout mouse model, they also show that the genetic manipulation
caused microvascular dysfunction where there was a decrease of cPC coverage of
capillaries and exacerbated the MI injury. This study also showed that cardiac func-
tion post-MI in the KO was decreased compared to the WT animals (He et al. 2016).
These studies in mouse model of ischemia-reperfusion injury demonstrate that cPCs
contribute to microvascular dysfunction in myocardial ischemic disease.

11.4.2 Atherosclerosis

Atherosclerosis is defined as the narrowing and hardening of the vessel walls caused
by fatty depositions in form of plaques which impede blood flow to tissues. In the
heart, complete blockage in the coronary arteries leads to a MI, and cPCs have been
implicated in the development and/or progression of atherosclerosis (Ivanova and
Orekhov 2016). The atherosclerotic process causes changes in adhesion molecule
expression during the different stages of lesion progression. T-cadherin is a surface
adhesion molecule found in endothelial cells, smooth muscle cells, and pericytes of
the aorta. The expression of T-cadherin in smooth muscle cells and pericytes is
directly correlated with the severity of disease development (Ivanov et al. 2001).
Secondly, human aortic endothelial cells and smooth muscle cells produce hepato-
cyte growth factor (HGF) (Nakamura et al. 1995). HGF has been implicated in the
atherosclerotic process and shown to mediate pericyte migration in vitro. A study in
human femoral atherosclerotic arteries shows that c-Met receptor expression on
pericytes is activated by endothelial-derived HGF (Liu et al. 2007). Activation of
c-Met by HGF triggers the PI3K/Akt pathway which mediates pericyte migration
and recruitment to atherosclerotic lesions. Moreover, microvessels within the ath-
erosclerotic lesions co-localized expression of c-Met with smooth muscle cells and
pericytes (Liu et al. 2007). Finally, a mutation in the low-density lipoprotein
receptor-­
related protein 6 (LRP) causes early atherosclerosis. This mutation
increases the proliferation of smooth muscle cells by activating the PDGFRβ
11 Pericytes in the Heart 199

pathway, whereas in the normal functioning LRP6, PDGFRβ is targeted for degra-
dation (Keramati et al. 2011). Because pericytes have high expression of PDGFRβ,
they can be targeted by the same mechanism to cause early atherosclerosis. These
studies taken together implicate cPCs in atherogenesis.

11.4.3 Calcification

Another cardiovascular pathology where dysfunctional pericytes contribute to its

calcific vasculopathy, which is also known as vascular calcification, has been well
reviewed (Collett and Canfield 2005; Canfield et al. 2000; Bardeesi et al. 2017;
Leszczynska and Murphy 2018). There are two types: intimal vascular calcification
which contributes to the complicated pathology of atherogenesis or medial vascular
calcification which contributes to vascular stiffness (Wu et al. 2013). Under patho-
logical conditions, pericytes can utilize its ability to secrete ECM proteins and its
alkaline phosphatase activity to cause calcification by mirroring what osteoblasts do
during bone formation (Murshed and McKee 2010). Under normal physiological
conditions, pericytes synthesize and release pyrophosphatase that will inhibit calci-
fication. Therefore, a dysfunctional pericyte can contribute to calcification by
becoming chondrogenic (Collett and Canfield 2005; Crisan et al. 2008; Murshed
and McKee 2010; Farrington-Rock et al. 2004). Furthermore, a mechanism has
been described for pericyte chondrogenic differentiation involving the Wnt/β-­-
catenin pathway in retinal pericytes (Kirton et al. 2007). Pericytes have been shown
to express several Wnt receptors such as LDL-related receptor 5 and 6 (Keramati
et al. 2011; Kirton et al. 2007). The TGFβ3 ligand activates the Wnt/β-catenin sig-
naling pathway and induces chondrogenic characteristics such as accumulation of
collagen and SOX-9 expression (Kirton et al. 2007). In the heart, TGFβ3 has been
implicated in aortic atherosclerotic lesion development since several of the cell
types (smooth muscle cells, foam cells, and macrophages) in the lesions have been
found to secrete high levels of the TGFβ3 ligand (Bobik et al. 1999; Toma and
McCaffrey 2012). Not only are pericytes chondrogenic, but they can also differenti-
ate into osteoclasts (Collett and Canfield 2005; Crisan et al. 2008; Farrington-Rock
et al. 2004; Kirton et al. 2006; Davaine et al. 2014). Glucocorticoid therapy such as
treatment with dexamethasone enhances osteogenic differentiation and decreases
the inhibition of mineralization in pericytes (Kirton et al. 2006). A novel gene, vas-
cular calcification-associated factor (VCAF), is found to be upregulated during vas-
cular calcification and during the osteogenic differentiation of pericytes in femoral
arteries (Alexander et al. 2005). Calcified arterial lesions contain VCAF, while their
non-calcified counterpart did not. On the other hand, knockdown of VCAF in cul-
tured pericytes increased the amount of calcified lesions and the time it took for the
mineralization to occur (Alexander et al. 2005). Together, these studies thus provide
evidence where cPC can behave similarly in the development of calcific vasculopa-
thy in the heart.
200 L. L. Lee and V. Chintalgattu

11.4.4 Cardiac Fibrosis

Cardiac fibrosis is defined as a reduction in cardiac muscle compliance due to an

excessive deposition of ECM proteins which leads to myocardial stiffness and
decreased cardiac function. Resident fibroblast maintains ECM homeostasis, but
upon injury, they convert into myofibroblast and contribute to cardiac fibrosis.
Cardiac fibrosis is necessary post-MI to help preserve cardiac structure, the scarring
process, and healing (van Amerongen et al. 2008). However, if the process is not
governed, it becomes pathogenic and consequential. How do pericytes contribute to
cardiac fibrosis? The exact role and mechanism of pericytes in fibrosis still remain
to be determined; however, recent studies in the heart and studies from other organs
such as the kidney have shed some light (Travers et al. 2016). Most recently, a
mouse model of spinal cord injury demonstrated that a subtype of pericytes was
responsible for fibrosis and scar tissue formation. Inhibiting pericyte proliferation
and their contribution to the ECM showed a reduction in fibrosis and facilitated
healing (Dias et al. 2018). Under pathological conditions, dysfunctional pericytes
can detach from the endothelium, migrate, differentiate into myofibroblasts, and
compromise the integrity of the vasculature (Schrimpf and Duffield 2011;
Greenhalgh et al. 2013). Cardiac pericytes have already been shown to secrete ECM
proteins (Nees et al. 2012a). Under culture conditions, pericytes can become
fibroblast-­like cells by induction of type-1 collagen mRNA and protein expression
as well as protein expression of connective tissue growth factor (CTGF/CCN2) and
fibroblast marker ASO2 (Shiwen et al. 2009). Shown in vitro, fibroblast-like cells
are derived from pericytes (Ivarsson et al. 1996). Moreover, studies have shown that
pericytes can differentiate into fibrotic cells that secrete collagen-1 under inflamma-
tory and wound healing conditions in vivo including the heart (Sundberg et al. 1996;
Lin et al. 2008; Weiss et al. 2013). Galectin-3 is secreted by macrophages in inflamed
myocardial tissue. This protein has been shown to stimulate pericyte proliferation
and secrete procollagen I which can thus lead to fibrosis (McCullough et al. 2011).
Conversely, type-1 pericytes accumulate at the site of injury in multiple organs, but
they do not secrete collagen-I in the heart (Birbrair et al. 2014). Moreover, Gli1+
cells were identified as a subpopulation pericytes in the mouse heart where they dif-
ferentiate into myofibroblast and are a source of fibrotic tissue (Kramann et al.
2015b, 2017; Kim and Braun 2015). Interestingly, genetic ablation of Gli+ pericytes
decreased the observed fibrosis and recovered organ function. Future studies are
required to resolve the conflicting results found in the heart.

11.4.5 Thrombosis

The development of a blood clot that can occlude and impede blood flow through
the vascular system is known as a thrombosis, and a thrombosis usually happens
when a vessel is injured. The blood clot is formed by thrombocytes to prevent fur-
ther blood loss. The role of pericytes has yet to be elucidated in not just the heart but
11 Pericytes in the Heart 201

other organs as well. In the heart, a subset of pericytes discovered by Juchem et al.
secrete high levels of TF which is a key initiator in coagulation (Juchem et al. 2010).
Interestingly, pericytes are the only cells in the vascular wall secreting TF, whereas
smooth muscle cells and monocytes that migrate to the area of plaques don’t secrete
TF unless activated (Osterud and Bjorklid 2006). Endothelial cells also do not
secrete TF. This study suggests that pericytes can secrete procoagulatory factors that
can create a thrombotic environment and can lead to plaque formation (Juchem
et al. 2010). Saphenous veins are used in the transplantation of coronary artery
bypass grafting. However, there is a high rate of acute occlusion post-surgery due to
the treatment of the grafts that leaves them de-endothelialized and causes an increase
in the release of TF (Weiss et al. 2009). Therefore, grafted vessels fail due to pro-
thrombosis environment that is created partially due to intimal pericytes.

11.5 Therapeutic Potential

11.5.1 Repair and Regeneration

During a MI, the coronary artery is blocked. This will cause a deprivation of oxygen
to the area; the myocytes and vessels downstream of the blockage will experience
ischemia and die. The heart will therefore have to work harder to compensate for the
damaged muscle. In order to heal this damage, first there needs to be repair—mean-
ing myofibroblast needs to perform fibrosis to help retain structure by replacing the
necrotic tissue. As discussed previously, pericytes can differentiate into myofibro-
blasts to aid in preserving structure (Crisan et al. 2008; Kramann et al. 2015b, 2017;
Sundberg et al. 1996; Lin et al. 2008; Kim and Braun 2015). Next, there needs to be
revascularization to perfuse the ischemic zones as the ischemic vessels are no longer
able to deliver oxygen and nutrient. As discussed above, pericytes play a central role
in angiogenesis and, in this case, even collateralization. Lastly, there needs to be
regeneration because new muscle mass needs to be formed to keep the heart from
becoming hypertrophic due to ventricular remodeling triggered by the acute
MI. Pericytes can differentiate into myoblast and provide the foundation for new
muscle development (Crisan et al. 2008; Dellavalle et al. 2007; Stallcup 2013;
Cappellari et al. 2013). Therefore, pericytes are able to address all of the above
requirements for cardiac repair and regeneration and have high therapeutic potential.

11.5.2 Mesenchymal Properties

Mesenchymal stem cells (MSCs) are at the forefront of regenerative therapy.

Pericytes share several characteristics with MSCs, and their plasticity toward other
cell phenotypes implies great healing potential. In addition to pericytes being of
mesenchymal origin and the characterized subpopulations of isolated pericytes
202 L. L. Lee and V. Chintalgattu

expressing “stemness” markers (CD44, CD73, CD90, CD105), pericytes are highly
proliferative and have multilineage capabilities as discussed in previous sections
(Volz et al. 2015; Crisan et al. 2008, 2012; Covas et al. 2008; Tintut et al. 2003).
Interestingly, pericytes are also integral to stem cell niches and hematopoietic stem
cells (Crisan et al. 2008; Sa da Bandeira et al. 2017). This brings us to the pericyte-­
MSC conundrum. Some believe that all MSCs are pericytes as it has been shown
that human pluripotent stem cells gave rise to pericytes (Dar et al. 2012). Pericytes
have also been referred to as a vascular stem cell or vascular progenitor cells.
Whether they are pluripotent or progenitors or both have yet to be elucidated, but
their potential has been rigorously reviewed and discussed (Geevarghese and
Herman 2014; Wong et al. 2015; Murray et al. 2017). Another way pericytes have
healing potential is their secretome which is also similar to stem cells. Many ways
that pericytes contribute to vascular remodeling and stabilization are through their
paracrine signaling rather than their direct physical contact (Ellison-Hughes and
Madeddu 2017). Their secretome has been found to contain pro-angiogenic factors
and anti-inflammatory cytokines as well as a plethora of other enzymes and factors
that can contribute to healing and regeneration (Juchem et al. 2010; Chen et al.
2015). More specifically, pericytes are targeted for angiogenic therapies because
they are bi-directional (Stallcup 2013; Kelly-Goss et al. 2014). In diseases such as
cancer, inhibition of angiogenesis would be beneficial, while in cases like MI, pro-
moting angiogenesis would help improve cardiac function. However, Guimaraes-­
Camboa et al. reported the opposite—pericytes in vivo do not behave like MSCs
under normal physiological nor pathophysiological conditions (Guimaraes-Camboa
et al. 2017).

11.5.3 Cell-Based Therapeutics

There have been several studies using pericyte-like cells and pericytes as cell-based
therapeutics for regenerative medicine. Many of these studies were done by a group
based in the United Kingdom led by Paolo Madeddu where they utilized leftover
human saphenous vein grafts from coronary bypass surgeries to isolate pericyte-like
progenitor cells. The first study used human saphenous vein-derived pericyte pro-
genitor cells (SVPs) injected into the ischemic limb of immunodeficient mice, and
they showed improvement in blood flow due to reparative angiogenesis (Campagnolo
et al. 2010). Several years later, they transplanted human adventitial progenitor cells
and showed improved reperfusion in the same ischemic limb model along with an
epigenetic expression profile (Gubernator et al. 2015). The same group also trans-
planted SVPs into the infract zone of mouse MI model. The study showed that the
transplantation reduced scarring, apoptosis, and fibrosis; it also increased angiogen-
esis through a mechanism involving microRNA-132 as well as improved overall
cardiac function (Katare et al. 2011; Katare and Madeddu 2013). Due to the com-
plexity of the heart, the group also did a study using a combined therapy of SVPs
and cardiac stem cells to see if they could optimize their treatment. They found the
11 Pericytes in the Heart 203

combination therapy to be complimentary by stimulating repair of vessels and mus-

cle. As previous studies, they saw improvement in cardiac function and perfusion,
increased angiogenesis, and reduced infarct size (Avolio et al. 2015b). In a study
where human pluripotent stem cells were transformed into pericytes and injected
into an ischemic limb mouse model, they found that the pericytes significantly
improved limb perfusion and vascular density (Dar et al. 2012). Next, Chen et al.
investigated the therapeutic potential of purified human skeletal pericytes on mouse
model of MI. They found that their transplantation improved cardiac contractility,
increased angiogenesis, reduced fibrosis, decreased inflammation at the infract
zones, and attenuated left ventricular dilation (Chen et al. 2013). Within that same
year, Yannarelli et al. came out with a study showing the reparative ability of human
umbilical cord perivascular cells improving cardiac function in an immunodeficient
mouse model of MI (Yannarelli et al. 2013). Most recently, a study was published
where they intramyocardially injected both human and swine allogeneic pericytes
post-acute MI in a swine model. They found that transplantation of human pericytes
only decreased fibrosis and no other endpoints they measured due to an immuno-
logical response. As an alternative, the transplantation of swine allogeneic pericytes
resulted in an increase in angiogenesis and decrease in fibrosis, but did not improve
overall cardiac function (Alvino et al. 2018). The first and only study using cPC as
a therapy was done by Chen and colleagues. They isolated human cPCs and injected
them into mouse models of MI, and they found that the cPCs demonstrated cardio-
myogenic potential (Chen et al. 2015). This could be because the cells are derived
from a cardiac source. As the popularity of using pericytes as cell-based therapies
for cardiovascular regenerative medicine continues to grow, the findings from the
studies above are encouraging and demonstrate its feasibility.

11.6 Future Directions

In the past 15–20 years, research on cPCs have accelerated. With the latest knowl-
edge gained from recent studies, the compilation of cPC biology is beginning to
unravel. We know that cPCs play a fundamental role in vascular integrity, stability,
and remodeling, and their dysfunction proves consequential to global cardiac func-
tion. However, plenty of questions remain to be answered. As we saw in Table 11.1,
there are numerous markers found in cPCs but with no specificity. The current pub-
lications that have isolated cPCs have different expression profiles. Under in vivo
conditions, this difference in expression profile could be due to their localization
along the vessel, whether they are microvascular, arteriole, or venular. This begs us
to answer “What is a cardiac pericyte?” In order to truly understand cPC biology as
a whole and differentiate among the subtypes, we need to rigorously define them.
There are plenty of functions pericytes have been described to participate in, but
do they have a main role? Most of the signaling and functions have translated well
across different tissues. Because of their heterogeneity, do the subtypes interact with
endothelial cells and influence cardiac homeostasis differently? It’s possible that
204 L. L. Lee and V. Chintalgattu

each subtype has a different role due to where they are found in the vascular bed. We
have discussed how pericyte dysfunction and/or lack of pericytes can disrupt and
decrease cardiac efficiency and function and contribute to cardiac pathophysiology.
However, different subtypes of cPCs can contribute to each pathology differently.
For example, type-1 pericytes in the heart do not secrete collagen-1 and contribute
to cardiac fibrosis, but perhaps another subpopulation does. Moreover, the pericyte-­
MSC conundrum needs to be resolved. Although studies have started on targeting
pericytes as a cell-based therapeutic agent and have seen improvements in the isch-
emic limb and MI mouse models, we wonder if the regenerative capabilities are
different among the different subtypes, tissue they are derived from, or species they
are derived from. For example, most studies have used pericytes derived from
human saphenous vein grafts and transplanted into the hearts of mice. Would we see
a bigger difference if the cells came from mice or cardiac derived? In the swine
model, there was an immunoreaction to the human cells. In the study where they
transplanted human cPCs into the heart, they showed that the pericytes had myo-
genic potential. It is possible that this subpopulation of pericytes was prepro-
grammed to gravitate toward a myogenic phenotype. We briefly mentioned that the
regenerative potential of pericytes come from their paracrine signaling. Perhaps
instead of transplanting pericytes themselves, therapies can just include the secre-
tome of pericytes. It’s possible that this could restore the signaling pathways that
were disrupted due to pericyte dysfunction or death associated with a particular
disease. There are many directions where the field of cPC biology can take, but we
believe that we must first critically and rigorously define cardiac pericytes and their
subpopulations before we are able to truly draw any conclusions on their capabili-
ties as a therapeutic agent.

References

Al Ahmad A, Gassmann M, Ogunshola OO (2009) Maintaining blood-brain barrier integrity:

pericytes perform better than astrocytes during prolonged oxygen deprivation. J Cell Physiol
218(3):612–622
Alarcon-Martinez L et al (2018) Capillary pericytes express alpha-smooth muscle actin, which
requires prevention of filamentous-actin depolymerization for detection. Elife 7
Alexander MY et al (2005) Identification and characterization of vascular calcification-associated
factor, a novel gene upregulated during vascular calcification in vitro and in vivo. Arterioscler
Thromb Vasc Biol 25(9):1851–1857
Alvino VV et al (2018) Transplantation of allogeneic pericytes improves myocardial vasculariza-
tion and reduces interstitial fibrosis in a swine model of reperfused acute myocardial infarction.
J Am Heart Assoc 7(2)
Anastasia A et al (2014) Trkb signaling in pericytes is required for cardiac microvessel stabiliza-
tion. PLoS One 9(1):e87406
Armulik A, Abramsson A, Betsholtz C (2005) Endothelial/pericyte interactions. Circ Res
97(6):512–523
Armulik A et al (2010) Pericytes regulate the blood-brain barrier. Nature 468(7323):557–561
Armulik A, Genove G, Betsholtz C (2011) Pericytes: developmental, physiological, and pathologi-
cal perspectives, problems, and promises. Dev Cell 21(2):193–215
11 Pericytes in the Heart 205

Avolio E et al (2015a) Expansion and characterization of neonatal cardiac pericytes provides a

novel cellular option for tissue engineering in congenital heart disease. J Am Heart Assoc
4(6):e002043
Avolio E et al (2015b) Combined intramyocardial delivery of human pericytes and cardiac stem
cells additively improves the healing of mouse infarcted hearts through stimulation of vascular
and muscular repair. Circ Res 116(10):e81–e94
Bardeesi ASA et al (2017) A novel role of cellular interactions in vascular calcification. J Transl
Med 15(1):95
Bell RD et al (2010) Pericytes control key neurovascular functions and neuronal phenotype in the
adult brain and during brain aging. Neuron 68(3):409–427
Benest AV et al (2006) VEGF and angiopoietin-1 stimulate different angiogenic phenotypes
that combine to enhance functional neovascularization in adult tissue. Microcirculation
13(6):423–437
Bergers G, Song S (2005) The role of pericytes in blood-vessel formation and maintenance. Neuro
Oncol 7(4):452–464
Birbrair A et al (2014) Type-1 pericytes accumulate after tissue injury and produce collagen in an
organ-dependent manner. Stem Cell Res Ther 5(6):122
Bischoff FC et al (2017) Identification and functional characterization of hypoxia-induced endo-
plasmic reticulum stress regulating lncRNA (HypERlnc) in Pericytes. Circ Res 121(4):368–375
Bjarnegard M et al (2004) Endothelium-specific ablation of PDGFB leads to pericyte loss and
glomerular, cardiac and placental abnormalities. Development 131(8):1847–1857
Bobik A et al (1999) Distinct patterns of transforming growth factor-beta isoform and receptor
expression in human atherosclerotic lesions. Colocalization implicates TGF-beta in fibrofatty
lesion development. Circulation 99(22):2883–2891
Bodnar RJ et al (2013) Pericyte regulation of vascular remodeling through the CXC receptor 3.
Arterioscler Thromb Vasc Biol 33(12):2818–2829
Borysova L et al (2013) How calcium signals in myocytes and pericytes are integrated across in
situ microvascular networks and control microvascular tone. Cell Calcium 54(3):163–174
Boscolo E et al (2011) JAGGED1 signaling regulates hemangioma stem cell-to-pericyte/vascular
smooth muscle cell differentiation. Arterioscler Thromb Vasc Biol 31(10):2181–2192
Cai CL et al (2008a) A myocardial lineage derives from Tbx18 epicardial cells. Nature
454(7200):104–108
Cai J et al (2008b) The angiopoietin/Tie-2 system regulates pericyte survival and recruitment in
diabetic retinopathy. Invest Ophthalmol Vis Sci 49(5):2163–2171
Campagnolo P et al (2010) Human adult vena saphena contains perivascular progenitor cells
endowed with clonogenic and proangiogenic potential. Circulation 121(15):1735–1745
Canfield AE et al (2000) Role of pericytes in vascular calcification: a review. Z Kardiol 89(Suppl
2):20–27
Caporali A et al (2017) Contribution of pericyte paracrine regulation of the endothelium to angio-
genesis. Pharmacol Ther 171:56–64
Cappellari O et al (2013) Dll4 and PDGF-BB convert committed skeletal myoblasts to pericytes
without erasing their myogenic memory. Dev Cell 24(6):586–599
Chen CW et al (2013) Human pericytes for ischemic heart repair. Stem Cells 31(2):305–316
Chen WC et al (2015) Human myocardial pericytes: multipotent mesodermal precursors exhibit-
ing cardiac specificity. Stem Cells 33(2):557–573
Chen Q et al (2016) Endothelial cells are progenitors of cardiac pericytes and vascular smooth
muscle cells. Nat Commun 7:12422
Chintalgattu V et al (2013) Coronary microvascular pericytes are the cellular target of sunitinib
malate-induced cardiotoxicity. Sci Transl Med 5(187):187ra69
Cho H et al (2003) Pericyte-specific expression of Rgs5: implications for PDGF and EDG receptor
signaling during vascular maturation. FASEB J 17(3):440–442
Collett GD, Canfield AE (2005) Angiogenesis and pericytes in the initiation of ectopic calcifica-
tion. Circ Res 96(9):930–938
206 L. L. Lee and V. Chintalgattu

Corselli M et al (2012) The tunica adventitia of human arteries and veins as a source of mesenchymal
stem cells. Stem Cells Dev 21(8):1299–1308
Costa MA et al (2018) Pericytes constrict blood vessels after myocardial ischemia. J Mol Cell
Cardiol 116:1–4
Covas DT et al (2008) Multipotent mesenchymal stromal cells obtained from diverse human tissues
share functional properties and gene-expression profile with CD146+ perivascular cells and
fibroblasts. Exp Hematol 36(5):642–654
Crisan M et al (2008) Purification and long-term culture of multipotent progenitor cells affiliated
with the walls of human blood vessels: myoendothelial cells and pericytes. Methods Cell Biol
86:295–309
Crisan M et al (2012) Perivascular cells for regenerative medicine. J Cell Mol Med 16(12):2851–2860
Dar A et al (2012) Multipotent vasculogenic pericytes from human pluripotent stem cells promote
recovery of murine ischemic limb. Circulation 125(1):87–99
Darland DC et al (2003) Pericyte production of cell-associated VEGF is differentiation-dependent
and is associated with endothelial survival. Dev Biol 264(1):275–288
Davaine JM et al (2014) Osteoprotegerin, pericytes and bone-like vascular calcification are associ-
ated with carotid plaque stability. PLoS One 9(9):e107642
Dellavalle A et al (2007) Pericytes of human skeletal muscle are myogenic precursors distinct from
satellite cells. Nat Cell Biol 9(3):255–267
Dewi NA et al (2018) Mechanism of retinal pericyte migration through angiopoietin/Tie-2 signaling
pathway on diabetic rats. Int J Ophthalmol 11(3):375–381
Dias Moura Prazeres PH et al (2017) Pericytes are heterogeneous in their origin within the same
tissue. Dev Biol 427(1):6–11
Dias DO et al (2018) Reducing pericyte-derived scarring promotes recovery after spinal cord
injury. Cell 173(1):153–165. e22
Eilken HM et al (2017) Pericytes regulate VEGF-induced endothelial sprouting through VEGFR1.
Nat Commun 8(1):1574
Ellison-Hughes GM, Madeddu P (2017) Exploring pericyte and cardiac stem cell secretome
unveils new tactics for drug discovery. Pharmacol Ther 171:1–12
Fang S, Salven P (2011) Stem cells in tumor angiogenesis. J Mol Cell Cardiol 50(2):290–295
Farrington-Rock C et al (2004) Chondrogenic and adipogenic potential of microvascular pericytes.
Circulation 110(15):2226–2232
Feng J et al (2011) Dual origin of mesenchymal stem cells contributing to organ growth and repair.
Proc Natl Acad Sci U S A 108(16):6503–6508
Fernandez-Klett F et al (2010) Pericytes in capillaries are contractile in vivo, but arterioles medi-
ate functional hyperemia in the mouse brain. Proc Natl Acad Sci U S A 107(51):22290–22295
Franco M et al (2011) Pericytes promote endothelial cell survival through induction of autocrine
VEGF-A signaling and Bcl-w expression. Blood 118(10):2906–2917
Fuxe J et al (2011) Pericyte requirement for anti-leak action of angiopoietin-1 and vascular remod-
eling in sustained inflammation. Am J Pathol 178(6):2897–2909
Geevarghese A, Herman IM (2014) Pericyte-endothelial crosstalk: implications and opportunities
for advanced cellular therapies. Transl Res 163(4):296–306
Gerhardt H, Betsholtz C (2003) Endothelial-pericyte interactions in angiogenesis. Cell Tissue Res
314(1):15–23
Greenhalgh SN, Iredale JP, Henderson NC (2013) Origins of fibrosis: pericytes take Centre stage.
F1000Prime Rep 5:37
Gubernator M et al (2015) Epigenetic profile of human adventitial progenitor cells correlates
with therapeutic outcomes in a mouse model of limb ischemia. Arterioscler Thromb Vasc Biol
35(3):675–688
Guimaraes-Camboa N et al (2017) Pericytes of multiple organs do not behave as mesenchymal
stem cells in vivo. Cell Stem Cell 20(3):345–359. e5
Hall CN et al (2014) Capillary pericytes regulate cerebral blood flow in health and disease. Nature
508(7494):55–60
11 Pericytes in the Heart 207

Hamilton NB, Attwell D, Hall CN (2010) Pericyte-mediated regulation of capillary diameter: a

component of neurovascular coupling in health and disease. Front Neuroenergetics 2
He Q, Spiro MJ (1995) Isolation of rat heart endothelial cells and pericytes: evaluation of their
role in the formation of extracellular matrix components. J Mol Cell Cardiol 27(5):1173–1183
He X, Zeng H, Chen JX (2016) Ablation of SIRT3 causes coronary microvascular dysfunction and
impairs cardiac recovery post myocardial ischemia. Int J Cardiol 215:349–357
Hellstrom M et al (2001) Lack of pericytes leads to endothelial hyperplasia and abnormal vascular
morphogenesis. J Cell Biol 153(3):543–553
Henderson NC et al (2013) Targeting of alphav integrin identifies a core molecular pathway that
regulates fibrosis in several organs. Nat Med 19(12):1617–1624
Hughes S, Chan-Ling T (2004) Characterization of smooth muscle cell and pericyte differentiation
in the rat retina in vivo. Invest Ophthalmol Vis Sci 45(8):2795–2806
Ivanov D et al (2001) Expression of cell adhesion molecule T-cadherin in the human vasculature.
Histochem Cell Biol 115(3):231–242
Ivanova EA, Orekhov AN (2016) Cellular model of Atherogenesis based on pluripotent vascular
wall pericytes. Stem Cells Int 2016:7321404
Ivarsson M et al (1996) Recruitment of type I collagen producing cells from the microvasculature
in vitro. Exp Cell Res 229(2):336–349
Juchem G et al (2010) Pericytes in the macrovascular intima: possible physiological and pathoge-
netic impact. Am J Physiol Heart Circ Physiol 298(3):H754–H770
Kaminski WE et al (2001) Basis of hematopoietic defects in platelet-derived growth factor
(PDGF)-B and PDGF beta-receptor null mice. Blood 97(7):1990–1998
Katare RG, Madeddu P (2013) Pericytes from human veins for treatment of myocardial ischemia.
Trends Cardiovasc Med 23(3):66–70
Katare R et al (2011) Transplantation of human pericyte progenitor cells improves the repair of
infarcted heart through activation of an angiogenic program involving micro-RNA-132. Circ
Res 109(8):894–906
Kelly-Goss MR et al (2014) Targeting pericytes for angiogenic therapies. Microcirculation
21(4):345–357
Keramati AR et al (2011) Wild-type LRP6 inhibits, whereas atherosclerosis-linked LRP6R611C
increases PDGF-dependent vascular smooth muscle cell proliferation. Proc Natl Acad Sci U S
A 108(5):1914–1918
Kim J, Braun T (2015) Targeting the cellular origin of organ fibrosis. Cell Stem Cell 16(1):3–4
Kirton JP et al (2006) Dexamethasone downregulates calcification-inhibitor molecules and accel-
erates osteogenic differentiation of vascular pericytes: implications for vascular calcification.
Circ Res 98(10):1264–1272
Kirton JP et al (2007) Wnt/beta-catenin signaling stimulates chondrogenic and inhibits adipogenic
differentiation of pericytes: potential relevance to vascular disease? Circ Res 101(6):581–589
Klein D et al (2011) Vascular wall-resident CD44+ multipotent stem cells give rise to pericytes and
smooth muscle cells and contribute to new vessel maturation. PLoS One 6(5):e20540
Kotecki M et al (2010) Calpain- and Talin-dependent control of microvascular pericyte contractil-
ity and cellular stiffness. Microvasc Res 80(3):339–348
Kovacic JC et al (2012) Epithelial-to-mesenchymal and endothelial-to-mesenchymal transition:
from cardiovascular development to disease. Circulation 125(14):1795–1808
Kramann R et al (2015a) Pharmacological GLI2 inhibition prevents myofibroblast cell-cycle pro-
gression and reduces kidney fibrosis. J Clin Invest 125(8):2935–2951
Kramann R et al (2015b) Perivascular Gli1+ progenitors are key contributors to injury-induced
organ fibrosis. Cell Stem Cell 16(1):51–66
Kramann R et al (2017) Gli1(+) Pericyte loss induces capillary rarefaction and proximal tubular
injury. J Am Soc Nephrol 28(3):776–784
Kumar A et al (2017) Specification and diversification of Pericytes and smooth muscle cells from
Mesenchymoangioblasts. Cell Rep 19(9):1902–1916
Lee S et al (2010) Pericyte actomyosin-mediated contraction at the cell-material interface can
modulate the microvascular niche. J Phys Condens Matter 22(19):194115
208 L. L. Lee and V. Chintalgattu

Leszczynska A, Murphy JM (2018) Vascular calcification: is it rather a stem/progenitor cells

driven phenomenon? Front Bioeng Biotechnol 6:10
Lin SL et al (2008) Pericytes and perivascular fibroblasts are the primary source of collagen-­
producing cells in obstructive fibrosis of the kidney. Am J Pathol 173(6):1617–1627
Liu Y et al (2007) Hepatocyte growth factor and c-met expression in pericytes: implications for
atherosclerotic plaque development. J Pathol 212(1):12–19
Magnusson PU et al (2007) Platelet-derived growth factor receptor-beta constitutive activity pro-
motes angiogenesis in vivo and in vitro. Arterioscler Thromb Vasc Biol 27(10):2142–2149
Maisonpierre PC et al (1997) Angiopoietin-2, a natural antagonist for Tie2 that disrupts in vivo
angiogenesis. Science 277(5322):55–60
Matsugi T, Chen Q, Anderson DR (1997) Adenosine-induced relaxation of cultured bovine retinal
pericytes. Invest Ophthalmol Vis Sci 38(13):2695–2701
Matsuki M et al (2015) Ninjurin1 is a novel factor to regulate angiogenesis through the function of
pericytes. Circ J 79(6):1363–1371
Matthews BG et al (2016) Osteogenic potential of alpha smooth muscle actin expressing muscle
resident progenitor cells. Bone 84:69–77
Mazzoni J, Cutforth T, Agalliu D (2015) Dissecting the role of smooth muscle cells versus Pericytes
in regulating cerebral blood flow using in vivo optical imaging. Neuron 87(1):4–6
McCullough PA, Olobatoke A, Vanhecke TE (2011) Galectin-3: a novel blood test for the evalua-
tion and management of patients with heart failure. Rev Cardiovasc Med 12(4):200–210
McGuire PG et al (2011) Pericyte-derived sphingosine 1-phosphate induces the expression of
adhesion proteins and modulates the retinal endothelial cell barrier. Arterioscler Thromb Vasc
Biol 31(12):e107–e115
Mishra A et al (2014) Imaging pericytes and capillary diameter in brain slices and isolated retinae.
Nat Protoc 9(2):323–336
Mitchell TS et al (2008) RGS5 expression is a quantitative measure of pericyte coverage of blood
vessels. Angiogenesis 11(2):141–151
Murray IR et al (2017) Skeletal and cardiac muscle pericytes: functions and therapeutic potential.
Pharmacol Ther 171:65–74
Murshed M, McKee MD (2010) Molecular determinants of extracellular matrix mineralization in
bone and blood vessels. Curr Opin Nephrol Hypertens 19(4):359–365
Nadal JA et al (2002) Angiotensin II stimulates migration of retinal microvascular pericytes:
involvement of TGF-beta and PDGF-BB. Am J Physiol Heart Circ Physiol 282(2):H739–H748
Nakagawa S et al (2009) A new blood-brain barrier model using primary rat brain endothelial cells,
pericytes and astrocytes. Neurochem Int 54(3–4):253–263
Nakamura Y et al (1995) Expression of local hepatocyte growth factor system in vascular tissues.
Biochem Biophys Res Commun 215(2):483–488
Nees S et al (2012a) Isolation, bulk cultivation, and characterization of coronary microvascular
pericytes: the second most frequent myocardial cell type in vitro. Am J Physiol Heart Circ
Physiol 302(1):H69–H84
Nees S et al (2012b) Wall structures of myocardial precapillary arterioles and postcapillary venules
reexamined and reconstructed in vitro for studies on barrier functions. Am J Physiol Heart Circ
Physiol 302(1):H51–H68
Nees S et al (2013) Abundant Pericytes in the venous intima and the vasa Venarum: evidence for
their key role in venous thrombosis. J Vasc Surg Venous Lymphat Disord 1(1):113
Neuhaus AA et al (2017) Novel method to study pericyte contractility and responses to ischaemia
in vitro using electrical impedance. J Cereb Blood Flow Metab 37(6):2013–2024
Nisancioglu MH et al (2008) Generation and characterization of rgs5 mutant mice. Mol Cell Biol
28(7):2324–2331
O’Farrell FM, Attwell D (2014) A role for pericytes in coronary no-reflow. Nat Rev Cardiol
11(7):427–432
O’Farrell FM et al (2017) Capillary pericytes mediate coronary no-reflow after myocardial
ischaemia. Elife 6
11 Pericytes in the Heart 209

Osterud B, Bjorklid E (2006) Sources of tissue factor. Semin Thromb Hemost 32(1):11–23
Paik JH et al (2004) Sphingosine 1-phosphate receptor regulation of N-cadherin mediates vascular
stabilization. Genes Dev 18(19):2392–2403
Peppiatt CM et al (2006) Bidirectional control of CNS capillary diameter by pericytes. Nature
443(7112):700–704
Pinto AR et al (2016) Revisiting cardiac cellular composition. Circ Res 118(3):400–409
Psaltis PJ et al (2010) Enrichment for STRO-1 expression enhances the cardiovascular para-
crine activity of human bone marrow-derived mesenchymal cell populations. J Cell Physiol
223(2):530–540
Ribatti D, Nico B, Crivellato E (2011) The role of pericytes in angiogenesis. Int J Dev Biol
55(3):261–268
Sa da Bandeira D, Casamitjana J, Crisan M (2017) Pericytes, integral components of adult hema-
topoietic stem cell niches. Pharmacol Ther 171:104–113
Sagare AP et al (2013) Pericyte loss influences Alzheimer-like neurodegeneration in mice. Nat
Commun 4:2932
Schrimpf C, Duffield JS (2011) Mechanisms of fibrosis: the role of the pericyte. Curr Opin Nephrol
Hypertens 20(3):297–305
Sengillo JD et al (2013) Deficiency in mural vascular cells coincides with blood-brain barrier
disruption in Alzheimer’s disease. Brain Pathol 23(3):303–310
Shepro D, Morel NM (1993) Pericyte physiology. FASEB J 7(11):1031–1038
Shiwen X et al (2009) Pericytes display increased CCN2 expression upon culturing. J Cell
Commun Signal 3(1):61–64
Siao CJ et al (2012) ProNGF, a cytokine induced after myocardial infarction in humans, targets
pericytes to promote microvascular damage and activation. J Exp Med 209(12):2291–2305
Sims DE (1986) The pericyte—a review. Tissue Cell 18(2):153–174
Spiranec K et al (2018) Endothelial C-type natriuretic peptide acts on Pericytes to regulate micro-
circulatory flow and blood pressure. Circulation 138(5):494–508
Stallcup WB (2013) Bidirectional myoblast-pericyte plasticity. Dev Cell 24(6):563–564
Stapor PC et al (2014) Pericyte dynamics during angiogenesis: new insights from new identities.
J Vasc Res 51(3):163–174
Stratman AN, Davis GE (2012) Endothelial cell-pericyte interactions stimulate basement mem-
brane matrix assembly: influence on vascular tube remodeling, maturation, and stabilization.
Microsc Microanal 18(1):68–80
Stratman AN et al (2010) Endothelial-derived PDGF-BB and HB-EGF coordinately regu-
late pericyte recruitment during vasculogenic tube assembly and stabilization. Blood
116(22):4720–4730
Sundberg C et al (1996) Pericytes as collagen-producing cells in excessive dermal scarring. Lab
Investig 74(2):452–466
Tao YK et al (2017) Notch3 deficiency impairs coronary microvascular maturation and reduces
cardiac recovery after myocardial ischemia. Int J Cardiol 236:413–422
Tattersall IW et al (2016) In vitro modeling of endothelial interaction with macrophages and peri-
cytes demonstrates notch signaling function in the vascular microenvironment. Angiogenesis
19(2):201–215
Teichert M et al (2017) Pericyte-expressed Tie2 controls angiogenesis and vessel maturation. Nat
Commun 8:16106
Tillet E et al (2005) N-cadherin deficiency impairs pericyte recruitment, and not endothelial
differentiation or sprouting, in embryonic stem cell-derived angiogenesis. Exp Cell Res
310(2):392–400
Tintut Y et al (2003) Multilineage potential of cells from the artery wall. Circulation
108(20):2505–2510
Toma I, McCaffrey TA (2012) Transforming growth factor-beta and atherosclerosis: interwoven
atherogenic and atheroprotective aspects. Cell Tissue Res 347(1):155–175
Travers JG et al (2016) Cardiac fibrosis: the fibroblast awakens. Circ Res 118(6):1021–1040
210 L. L. Lee and V. Chintalgattu

van Amerongen MJ et al (2008) Bone marrow-derived myofibroblasts contribute functionally to

scar formation after myocardial infarction. J Pathol 214(3):377–386
van Dijk CG et al (2015) The complex mural cell: pericyte function in health and disease. Int
J Cardiol 190:75–89
Volz KS et al (2015) Pericytes are progenitors for coronary artery smooth muscle. Elife 4
Wakui S et al (2006) Localization of Ang-1, −2, Tie-2, and VEGF expression at endothelial-­
pericyte interdigitation in rat angiogenesis. Lab Investig 86(11):1172–1184
Wanjare M, Kusuma S, Gerecht S (2013) Perivascular cells in blood vessel regeneration. Biotechnol
J 8(4):434–447
Watanabe N et al (2004) Three-dimensional microstructural abnormality of the coronary capillary
network after myocardial reperfusion—comparison between ‘reflow’ and ‘no-reflow’. Circ
J 68(9):868–872
Weiss DR et al (2009) Extensive deendothelialization and thrombogenicity in routinely prepared
vein grafts for coronary bypass operations: facts and remedy. Int J Clin Exp Med 2(2):95–113
Weiss RM, Miller JD, Heistad DD (2013) Fibrocalcific aortic valve disease: opportunity to under-
stand disease mechanisms using mouse models. Circ Res 113(2):209–222
Winkler EA, Bell RD, Zlokovic BV (2010) Pericyte-specific expression of PDGF beta receptor
in mouse models with normal and deficient PDGF beta receptor signaling. Mol Neurodegener
5:32
Winkler EA, Bell RD, Zlokovic BV (2011) Central nervous system pericytes in health and disease.
Nat Neurosci 14(11):1398–1405
Wong SP et al (2015) Pericytes, mesenchymal stem cells and their contributions to tissue repair.
Pharmacol Ther 151:107–120
Wu M, Rementer C, Giachelli CM (2013) Vascular calcification: an update on mechanisms and
challenges in treatment. Calcif Tissue Int 93(4):365–373
Yannarelli G et al (2013) Human umbilical cord perivascular cells exhibit enhanced cardiomyo-
cyte reprogramming and cardiac function after experimental acute myocardial infarction. Cell
Transplant 22(9):1651–1666
Yemisci M et al (2009) Pericyte contraction induced by oxidative-nitrative stress impairs capillary
reflow despite successful opening of an occluded cerebral artery. Nat Med 15(9):1031–1037
Zeng H et al (2016) LPS causes pericyte loss and microvascular dysfunction via disruption of
Sirt3/angiopoietins/Tie-2 and HIF-2alpha/Notch3 pathways. Sci Rep 6:20931
Zhou Z et al (2016) Induction of initial steps of angiogenic differentiation and maturation
of endothelial cells by pericytes in vitro and the role of collagen IV. Histochem Cell Biol
145(5):511–525
Chapter 12
Pericytes in the Umbilical Cord

Andrée Gauthier-Fisher, Peter Szaraz, and Clifford L. Librach

Abstract The structural components of the umbilical cord, including two arteries
and one vein, the stromal region/Wharton’s jelly, and amniotic epithelial membrane,
are well described at various time points of gestation. Over the last two decades,
evidence has emerged that multipotent cells sharing properties of mesenchymal
stromal cell and pericytes/mural cells can be isolated from multiple regions of the
umbilical cord, including the perivascular region of the umbilical cord arteries and
vein, Wharton’s jelly, and subamnion. These cells have increasingly gained interest
for their potential use in regenerative and immunomodulatory medicine. Recent
studies suggest that obstetrical complications including gestational diabetes melli-
tus and preeclampsia may alter the yield, properties, and potency of mesenchymal
stromal cells isolated from the umbilical cord. The role that pericytes or pericyte-­
like cells play in the development of the human umbilical cord and associated
pathologies, however, remains to be investigated.

Keywords Human umbilical cord · Perivascular cells · Fetal development ·

Gestation · Body stalk · Umbilical artery · Umbilical vein · Wharton’s jelly ·
Placenta · Obstetrical complication · Vasculature · Preeclampsia · Mesenchymal
stromal cells · Regenerative medicine

Part of “Biology of Pericytes: Development, Homeostasis and Disease”, a forthcoming volume

which will be in the Springer Nature series “Advances in Experimental Medicine and Biology
(ISSN:0065-2598)”.
A. Gauthier-Fisher (*) · P. Szaraz
CReATe Fertility Centre, University of Toronto, Toronto, ON, Canada
e-mail: [email protected]
C. L. Librach
CReATe Fertility Centre, University of Toronto, Toronto, ON, Canada
Department of Obstetrics and Gynecology, University of Toronto, Toronto, ON, Canada
Department of Physiology, University of Toronto, Toronto, ON, Canada
Institute of Medical Sciences, University of Toronto, Toronto, ON, Canada
Department of Obstetrics and Gynecology, Women’s College Hospital, Toronto, ON, Canada

© Springer Nature Switzerland AG 2019 211

A. Birbrair (ed.), Pericyte Biology in Different Organs,
Advances in Experimental Medicine and Biology 1122,
https://doi.org/10.1007/978-3-030-11093-2_12
212 A. Gauthier-Fisher et al.

12.1 Introduction

In placental mammals, the umbilical cord is a vital structure that forms early during
fetal development, allowing for oxygen- and nutrient-rich blood to pass from the
placenta to the fetus and for the return of waste and oxygen-depleted blood back to
the placenta. Normally, human umbilical cords at term are composed of one vein
and two arteries that are embedded in Wharton’s jelly, a connective tissue consisting
of a network of myofibroblasts and collagen that protect the blood vessels from
compression. Wharton’s jelly is abundant in mucopolysaccharides that provide the
jellylike substance and is covered by an amniotic epithelial cell membrane
(Fig. 12.1). An understanding of pericytes and related perivascular cells in the
human umbilical cord, the focus of this chapter, necessitates an understanding of
how this seemingly simple organ, and especially the vasculature and perivascular
tissue within it, develop and change during gestation. How mesenchymal cells or
pericyte-like cells in the umbilical cord tissue and cord blood participate in the
growth, maintenance, and pathological aspects of the umbilical cord structures and
fetal development throughout gestation remains largely unclear. However, these
cells with demonstrated plasticity have increasingly gained interest in the field of
regenerative medicine and immunomodulatory medicine, as they represent one of
the youngest and richest known sources of mesenchymal stromal cells. In this chap-
ter, we will review:
• The development of the umbilical cord, focusing on vascular structures and peri-
vascular connective tissue.
• In situ analyses of pericyte-like cells and perivascular cells in the umbilical cord.
• Analyses of pericyte-associated markers in cells isolated from various regions of
the perivascular regions of the umbilical cord as sources of stem cells for regen-
erative and immunomodulatory medicine.
• The potential involvement of pericytes/perivascular cells in umbilical cord
pathologies as well as the impact of obstetrical complications on perivascular
cells isolated from the umbilical cord.

12.2  evelopment of the Umbilical Cord, Focusing

D
on Vascular Structures and Connective Tissue

Early embryonic processes involved in the formation of the human umbilical cord
are described and illustrated in detail in eminent textbooks (Cullen 1916; Benirschke
and Kaufmann 1995) and have been summarized in recent scientific reviews
(Spurway et al. 2012; Corrao et al. 2013; Davies et al. 2017). The following is an
overview of early human umbilical cord development described in these references,
with the inclusion of occasional references to studies performed in animal models,
as these have improved our knowledge of umbilical cord vascularization (Inman
and Downs 2007). The connective tissue of the umbilical cord is derived from the
12 Pericytes in the Umbilical Cord 213

Fig. 12.1 Structural regions of the umbilical cord during the first trimester (FTM) (left panels) and
at term (right panel). In both stages, the umbilical cord is mainly comprised of two arteries (shown
for each stage in the second row) and one vein (shown for each stage in the third row). Additional
structures, including the second vein which normally atrophies during the first trimester and rem-
nants of the allantois, can also be observed, as shown here in FTM (fourth row). E epithelium, SA
subamnion, C clefts, WJ Wharton’s jelly, PV perivascular zone, M media, I tunica intima. Slides
used to illustrate umbilical cord regions were stained with OCT4 and counterstained with hema-
toxylin (Librach lab, unpublished). One can appreciate the expansion of the media (M), perivascu-
lar zone (PV), and intervascular WJ between FTM and term cords. OCT4+ cells are found in FTM,
mainly in the subendothelial regions of the arteries and vein, with occasional cells staining positive
for OCT4 in WJ, but are not observed prevalently in term cords
214 A. Gauthier-Fisher et al.

extraembryonic mesoblast, the layer of mesoderm that surrounds the amniotic ves-
icle and primary yolk sac in the 2- to 3-week-old embryo, shortly after trophoblastic
invasion of the maternal endometrium. The earliest sign of the umbilical cord can be
seen at 18 days post coitum (p.c). in the form of the connecting stalk (also referred
to as the body stalk) that is formed by the invasion of the transitory allantois, form-
ing a duct-like extension of the primary yolk sac linking the chorionic mesoderm
and the primitive embryo (Fig. 12.2). This mesenchyme tissue connection is in fact
the primitive extraembryonic urinary bladder. The cord itself forms between 28 and
40 days p.c. when the expanding amniotic cavity compresses the connecting stalk,
the allantois, and the yolk sac, thereby covering them with the amniotic epithelium.
The connecting stalk becomes supplied with fetal blood vessels originating from the
allantois around the third week p.c. The blood vessels are formed through de novo
vasculogenesis (Downs 1998; Downs et al. 1998) and invade the placenta at the
distal end of the cord, becoming connected to the villous vessels and conveying
placental blood to and from the embryo. It remains poorly understood how this is
orchestrated and how allantoic vasculature at the proximal end (at the caudal part of
embryo) fuses with the embryonic and yolk sac vessels (Downs 1998; Inman and
Downs 2007). How the primitive vasculature develops into the mature umbilical
cord vessels observed during the remainder of gestation and at birth is also poorly
described. During the first trimester, the omphalomesenteric duct (also known as the
vitelline duct), the precursor to the gastrointestinal tract derived from the yolk sac,
also passes through the cord (Fig. 12.2b). The cord lengthens as the embryo pro-
lapses backward into the amniotic sac. The full fusion of the amniotic membrane
with the chorionic membrane/mural trophoblasts takes place at approximately
12 weeks of fetal development. The allantois and vitelline duct typically recede
within the umbilical cord during this period, thinning out and losing their patency
(Fig. 12.3). The allantois and yolk sac are important structures in the formation of
the cord as they form the initial link with the body through the connecting stalk and
are the source of vascularization. However, while the allantois-derived ducts recede
in the cord as they form the intra-abdominal bladder and gut, the blood vessels
originating from the allantois persist and grow with the umbilical cord.
The umbilical cord tissue is composed of six structural and functional regions:
from outer to inner UC, we find (1) the surface epithelial membrane (amniotic epi-
thelium), (2) subamniotic stroma, (3) clefts, (4) intervascular stroma (WJ), (5) peri-
vascular stroma, and (6) blood vessels, where regions (2–5) are often, and some
would argue ambiguously, referred to collectively as the umbilical cord stromal
region or WJ (Figs. 12.1 and 12.4) (Nanaev et al. 1997; Can and Karahuseyinoglu
2007; Corrao et al. 2013; Davies et al. 2017). There are several ways in which
human umbilical cord vessels differ from major vessels in the body. First, the umbil-
ical cord vessels’ structure includes an arterial and venous tunica intima (layer of
endothelial cells) and tunica media (cell-dense layers of vascular smooth muscle
cells) but lacks a distinct tunica adventitia and external elastic lamina (Blanco et al.
2011), which provide contractile and vascular support functions in corporal blood
12 Pericytes in the Umbilical Cord 215

Fig. 12.2 Illustrations describing early development of the umbilical cord and structures. (a)
Body stalk and stem of umbilical vesicle are identified as primitive components of the developing
umbilical cord. Continuity and invagination of chorionic and amniotic cavities are illustrated.
Amniotic cavity surrounds the developing cord structure on the fetal side, while the chorionic cav-
ity surrounds it on the yolk sac side. (b) Hand-drawn illustration of early embryonic and umbilical
cord development from Cullen (1916). Illustration by Max Brödel, reprinted from Cullen, T. S.
(1916). Embryology, anatomy, and diseases of the umbilicus, together with the diseases of the
urachus. Philadelphia, and London, W. B. Saunders company
216 A. Gauthier-Fisher et al.

Fig. 12.3 Drawings and illustration of FTM umbilical cord structures. (a) Internal structure of the
developing umbilical cord during the first trimester. Amniotic cavity surrounds the developing cord
structure. Tubular structures within the cord include umbilical arteries, umbilical vein, omphalo-
mesenteric duct, and allantois, the latter two of which normally regress toward the fetus and lose
their patency by the end of the first trimester. (b) Hand-drawn illustration by Max Brödel, reprinted
from Cullen, T. S. (1916). Embryology, anatomy, and diseases of the umbilicus, together with the
diseases of the urachus. Philadelphia and London, W. B. Saunders company

Fig. 12.4 Illustration of in situ and in vitro cell phenotypes in structural regions of the umbilical
cord. Schematic representation of the umbilical cord tissue structures illustrating cell density and
localization around a blood vessel. Concentric layers starting from the umbilical cord epithelium
toward the umbilical blood vessel are as follows: E epithelium, SA subamnion, WJ Wharton’s jelly,
PV perivascular region, M tunica media, and I tunica intima. A non-exhaustive list of markers
associated with mesenchymal and pericyte-like cells (pericyte-associated markers are bolded)
from each structural region in cells in situ or in culture is presented

vessels. From placental to fetal ends, the umbilical cord is largely considered not to
be innervated (Hoyes 1969). A few controversial studies, however, have reported the
presence of neural elements supplying the umbilical arteries at the proximal end
(Pearson and Sauter 1970; Ellison 1971). Wharton’s jelly or umbilical stroma, the
perivascular mucoid connective tissue described by Thomas Wharton in 1656, and
its dispersed myofibroblasts and collagen fibers are thought to fulfill the roles of the
12 Pericytes in the Umbilical Cord 217

adventitia. This source of perivascular cells will be discussed in the next section.
Umbilical cord endothelial and perivascular cells also differ from those of typical
blood vessels, with changing characteristics throughout gestation (Parry and
Abramovich 1972; Sexton et al. 1996; Nanaev et al. 1997; Stehbens et al. 2005).
Umbilical cord vessel endothelial cells are rich in organelles such as mitochondria,
endoplasmic reticulum, and Golgi and, in the sheep at least, have been described as
resembling those of growing or of regenerating tissues (Sheppard and Bishop 1973).
Finally, unlike blood flow and exchange in other tissues, oxygen- and nutrient-rich
blood passes from the placenta to fetal circulation through one vein (which goes to
the liver), and depleted blood/waste is returned to the placenta through two large
arteries that are branches of the internal iliac arteries, with anastomosis about 3
centimeters from the placental insertion site (Cullen 1916, Benirschke and
Kaufmann 1995, Spurway et al. 2012). It is important to note that in some cords,
there are deviations from this most often described structure and that other struc-
tures can be observed in first trimester or even second trimester umbilical cords
(Figs. 12.1, 12.2 and 12.3). The human umbilical cord initially contains two veins,
the right and the left, where the left normally disappears during the second month of
pregnancy. However, two veins (four vessels) or one artery (two vessels) or, in rare
cases, five vessels (Singh et al. 2012) can be observed in about one percent of human
umbilical cords at term. In twenty percent of umbilical cords at term, short and typi-
cally non-patent remnants of allantois containing epithelial cells can be observed at
the distal end of the cord and would be expected to be located between the two
arteries during earlier stages of gestation, as it normally regresses by the eighth
week of gestation (Cullen 1916, Spurway et al. 2012). Similarly, as other umbilical
cord lengthens, the omphalomesenteric duct and the umbilical coelom regress by
the tenth and 12th week of gestation, respectively (Khati et al. 1998).
The human umbilical cord is generally described as lacking microvessels (Hoyes
1969). Transmission electron microscopy studies of developing human umbilical
arteries and veins at various time points of gestation suggest that human umbilical
cord blood vessels are devoid of vasa vasorum (Sexton et al. 1996), networks of
smaller blood vessels that supply the walls of large blood vessels such as arteries
and veins in many other tissues and where classically defined pericytes would be
found as discussed below. However, the intra-abdominal part of the umbilical cord
artery and vein do contain vasa vasorum, and these appear to increase in number
between the first trimester and term (Clarke 1965; Malas et al. 2003). It should be
noted that in many mammals, including sheep, cow, horses, and the dromedary
camel, the umbilical cord contains four blood vessels, including two umbilical
veins, two umbilical arteries, and a large allantoic duct. In the sheep umbilical cord,
vasa vasorum, including fenestrated endothelial cells and pericytes, were described
in the outer layers of the media of the artery in electron microscopy studies (Sheppard
and Bishop 1973). There have been at least two published reports of microvessels in
the umbilical cord. Montemurro et al. reported the presence of venules and arteri-
oles with diameters ranging from 10 to 100 μm in umbilical cords resected from
23–32-week preterm births but not in term umbilical cords (Montemurro et al.
2011). In addition, Blanco et al. reported the frequent finding of one or two scarcely
218 A. Gauthier-Fisher et al.

developed but erythrocyte-filled vessels in Wharton’s jelly, away from the three
major vessels, in segments of cord isolated specifically approximately 2 centimeters
from the placental insertion site (29–41 weeks gestational age) (Blanco et al. 2011).
It is important to note that our understanding of the structural organization of the
human umbilical cord has been developed from ultrastructural and histological
assessments of short segments of the cord (representing less than ten to twenty-five
percent of the total length) that are accessible after resection of the cord at birth
(from both distended and collapsed specimens) or from abortions. Furthermore, the
proximal, medial, and distal distance of these segments to the fetus are almost never
specified in publications. Specifically, vascular structures at the proximal and intra-­
abdominal regions of the umbilical cord as well as the rostral region that links to the
placenta throughout fetal development have not been extensively studied. This,
however, may be relevant to our understanding of potential sources of pericyte-like
cells throughout umbilical cord development.
In a manner that is crucial to proper fetal development and offspring health, the
length and diameter of the human umbilical cord increase from a few millimeters in
the first weeks of pregnancy to an average length of 40–65 cm and 1–2 cm girth at
birth. The steepest growth curve of the umbilical cord, including vascular structures,
occurs before the end of the second trimester (Cullen 1916; Malas et al., 2003). This
implies the presence and regulation of a rich source of active progenitors that supply
the growth of the amniotic epithelium, of endothelial cells, as well as of the suben-
dothelial smooth muscle and connective tissue, which will be the focus of the next
section. Many assumptions have been made, but not empirically tested, with regard
to the types of cells that act as progenitors and feed the growth of the perivascular
and vascular components of the cord and with regard to the source of mitogenic
factors. However, these and the mechanisms regulating progenitors are of great rel-
evance, both for the understanding of the development and pathologies of the
umbilical cord, as well as for the application of umbilical cord-derived cells in many
areas of regenerative and immunomodulatory medicine. For example, cords that are
abnormally short or too long and defects in umbilical vasculature or connective tis-
sue can each result in inadequate nutrient and oxygen delivery to the fetus, leading
to fetal demise or neurological and other severe birth defects (Benirschke and
Kaufmann 1995). As discussed at the end of this chapter, the potential association
between pericyte-like cells in the umbilical cord and these umbilical cord patholo-
gies remains largely unexplored.

12.3 I n Situ Analysis of Pericytes and Related Perivascular

Cells in the Umbilical Cord

Pericytes have been classically defined as contractile cells that wrap around endo-
thelial cells in venules, capillaries, and arterioles throughout the body. Over the last
two decades, the definition of pericytes has been extended to include a heteroge-
neous, specialized population of multipotent mural cells residing in the
12 Pericytes in the Umbilical Cord 219

subendothelial layer, adventitia, and vasa vasorum of the vasculature, including in

small, medium, and large arteries and veins (Andreeva et al. 1998). Pericytes are
now thought to function not only as hemostasis regulators but also as a source of
cells for repair and maintenance of the tissues in which they reside. For instance,
pericytes can differentiate into osteoblasts, chondrocytes, adipocytes, fibroblasts,
myofibroblasts, and smooth muscle cells (Diaz-Flores et al. 2009). Pericytes or
pericyte-like cells have thus emerged as critical components of the vascular bed in
blood vessels of all sizes, not only covering endothelial cell junctions to regulate
cellular processes such as inflammation but also as a mesenchymal progenitor cells
with metabolic, signaling, and mechanical roles to support endothelial cells and tis-
sue hemostasis as well homeostasis (Sims 2000).
In addition, many perivascular cell types across the vascular wall, including
smooth muscle cells found in the intima and myofibroblasts found in the stroma/
adventitia, are thought to have close relationships with pericytes, and as such the
reference to pericytes, mural cells, adventitial cells, and perivascular cells has
become somewhat fluid in the literature. These cells may represent a continuum of
cell phenotypes along growing and maturing vasculature (Holm et al. 2018).
Immunophenotypically, pericytes are defined by their anatomical localization in
subendothelial layers of vessels and the expression of at least one of the following
markers, neither one of which is specific to, or representative of, all pericytes and
may be associated with their quiescent or activated states: CD146 (MCAM),
platelet-­derived growth factor receptor beta (PDGRβ, CD140b), alkaline phospha-
tase (AP), regulator of G protein signaling (RGS5), alpha-smooth muscle actin
(αSMA), desmin, and CD271 and the absence of CD31, CD34, and von Willebrand
factor (VWF) and CD144 or CD45 which are endothelial and pan-hematopoietic
markers, respectively (Andreeva et al. 1998, Diaz-Flores et al. 2009). Adventitial
cells in turn are typified as CD34+, CD31−, CD146−, and CD45−. In vitro, adven-
titial cells can acquire a pericyte-like cell phenotype, suggesting that adventitial
cells may be a source of pericyte progenitors (Corselli et al. 2012).
In a groundbreaking research study, Crisan et al. showed that the perivascular
region of blood vessels in many tissues, including the umbilical cord, harbors a
reservoir of CD146+, NG2+, and PDGRβ+ progenitor cells that may be the source
of mesenchymal stromal cells (MSCs) in all vascularized bodily tissues (Crisan
et al. 2008). This has led MSC researchers to conclude that at least a subset of MSCs
are pericytes (Caplan 2008). MSCs are currently defined as plastic adherent cells
that express CD90, CD73, and CD105; do not express CD45, CD34, CD14, CD11b,
CD79a, CD19, and HLA-DR; and can differentiate into osteocytes, adipocytes, and
chondrocytes in appropriate culture conditions (Dominici et al. 2006). The co-­
expression of pericyte-associated markers in a subset of cells expressing MSC-­
associated markers is well described in various sources of MSCs including the
umbilical cord, both in vivo (as discussed in this section) and in vitro (as discussed
in the next section).
With regard to the study of perivascular cells in situ in the developing and term
human umbilical cord, ultrastructural and histological studies have been performed
on tissue sections of term human umbilical cord (both distended and collapsed) fol-
220 A. Gauthier-Fisher et al.

lowing their resection in preterm or term births (Parry 1970; Takechi et al. 1993;
Nanaev et al. 1997; Schugar et al. 2009; Coskun and Can 2015). Additional studies
have been performed on umbilical cords spanning fetal development (Parry and
Abramovich 1972; Sexton et al. 1996; Stehbens et al. 2005; Montemurro et al.
2011; Spurway et al. 2012; Hong et al. 2013).
It was noted a long time ago that cells in the subamnion, Wharton’s Jelly, and
perivascular regions of the arteries and veins differ from each other phenotypically
and that the perivascular cells had increased proliferative activity (Parry and
Abramovich 1970; Nanaev et al. 1997, Friedman et al. 2007). The umbilical cord
epithelium is characterized by epithelial-like cells expressing various cytokeratins,
while the stromal regions include mesenchymal cells expressing vimentin, desmin,
and alpha-smooth muscle actin (αSMA). Here, we will specifically discuss the
pericyte-­like properties of umbilical cord stromal cells identified in the perivascular
compartments and Wharton’s jelly.
Pericyte-associated markers NG2, CD146, and PDGFRβ have been detected
exclusively in perivascular cells of the umbilical cord arteries and vein in first tri-
mester (FTM), second trimester (STM), and term umbilical cords (Sarugaser et al.
2005; Baksh et al. 2007; Crisan et al. 2008; Schugar et al. 2009; Montemurro et al.
2011; Hong et al. 2013). CD146 (MCAM, S-Endo 1) is expressed by some peri-
cytes, but endothelial, smooth muscle cells, and stromal and perivascular stem cells
are also known to express this antigen. A subpopulation of cells surrounding the
large umbilical arteries and vein as well as small vessels in mid-gestation fetal
cords co-express CD146 and αSMA, as well as MSC-associated markers
(Montemurro et al. 2011). These cells, consisting mainly of vascular smooth mus-
cle cells, form the intima media, a layer around the endothelial cells that becomes
more prominent during the second trimester (Nanaev et al. 1997; Stehbens et al.
2005). In term cords, the two arteries present a similar expression pattern for these
markers, but only rare CD146high and αSMA-positive cells were detected around the
vein, where the vSMCs are αSMA-positive (Schugar et al. 2009; Montemurro et al.
2011). Moderate CD146 expression (relative to the high expression in endothelial
cells) was detected in perivascular zone cell (outside of media) of term but not first
trimester umbilical cord (Davies et al. 2017) (Fig. 12.5a). The multipotency-associ-
ated marker, Oct4A, however was detected in the perivascular region of FTM and
second trimester human umbilical cord perivascular cells (HUCPVCs), but not in
term (Hong et al. 2013, 2013; Montemurro et al. 2011) (Fig. 12.1). Flow cytometric
analysis of freshly isolated fetal versus term umbilical cord cells suggest that there
is a higher frequency of perivascular cell populations with pericyte-like properties
(described as either CD146+ CD34− CD45− CD56−; CD146+ PDGFRb+; or
CD146+) in early gestation (Montemurro et al. 2011; Hong et al. 2013). While the
exact proportion of such cells varies across studies based on tissue digestion and
immunophenotyping methods used, this suggests that a perivascular cell population
with increased multipotential is present in the fetal umbilical cord perivascular
region. The functional significance of these cell phenotypes during fetal develop-
ment is unclear but likely contributes to the growth of the blood vessels and expan-
12 Pericytes in the Umbilical Cord 221

Fig. 12.5 Comparison of FTM and term human umbilical cord perivascular cells (HUCPVCs).
Expression of pericyte-associated markers and physical interaction with endothelial cells in tube
formation assay. (a,b) CD146 immunostaining in first trimester (FTM) and term perivascular
regions, respectively. This image was obtained with permission from Davies, J. E., J. T. Walker and
A. Keating (2017). “Concise Review: Wharton’s Jelly: The Rich, but Enigmatic, Source of
Mesenchymal Stromal Cells.” Stem Cells Transl Med 6(7): 1620–1630. (c) Flow cytometry-based
cell surface expression levels of PDGFRβ and CD146 in perivascular cell populations isolated
from FTM and term cords (n = 4, passage 4–5). Marker levels are expressed as mean fluorescence
intensity. Both markers show higher expression in FTM-derived cell population compared to term.
(d): Aortic endothelial cell co-cultures demonstrating a pericyte-like characteristic of FTM
HUCPVC in the extent of adhesion and elongation of these cells in endothelial networks. FTM
HUCPVC exhibits higher endothelial adhesion and network coverage than term HUCPVCs, in
agreement with the pericyte-associated immunophenotype corresponding to each cell type (c)
(obtained from unpublished data from C Librach laboratory)

sion of perivascular cells (Holm et al. 2018). The properties of these HUCPVCs
when isolated and propagated in culture will be discussed in more details in the next
section.
222 A. Gauthier-Fisher et al.

The intervascular or distal perivascular stromal region or Wharton’s jelly (WJ),

which can be distinguished from the proximal perivascular region by a reduction in
cell density, with highest density near perivascular region and lower density near
the subamniotic region, is another compartmentalized source of cells with MSC-
like properties in the umbilical cord (Fig. 12.4). WJ is largely composed of myofi-
broblasts (Parry 1970; Takechi et al. 1993), and occasional mast cells dispersed in
a mucoid substance composed of water (90%), sulfated glycosaminoglycans, (HA)
proteoglycans (decorin, biglycan), and collagens in which the blood vessels are
embedded. WJ is a crucial structure, with contractile function and elasticity which
protects the vessels from compression, allowing for adequate blood flow from the
maternal placenta to fetus. Electron microscopy studies of WJ cells and smooth
vascular cells first described WJ cells as “unusual fibroblasts,” displaying partial
morphological similarities with fibroblasts and smooth muscle cells. A stellate cell
morphology and extended inter-cell connections were described in collapsed cord
segments, with cells on the outer periphery displaying longer processes (Takechi
et al. 1993; Ryu et al. 2013). In general, these cells resemble fibroblasts, with
organelle-­rich cytoplasm, with well-developed ER, abundant mitochondria, and
Golgi, suggestive of active biosynthesis and secretory activity. Myofibroblasts are
thought to produce the extracellular matrix molecules of the WJ. Small elongated
and indented nuclei, with prominent nucleoli, were observed. Gap junctions are
observed between interconnecting cell processes. Unlike fibroblasts which are not
covered by basal lamina, and smooth muscle cells which are fully covered by basal
lamina, WJ cell surfaces are partially covered by basal lamina. Unlike fibroblasts,
WJ cells express contractile proteins actin and non-muscle myosin, as well as cyto-
skeletal protein desmin and vimentin. Furthermore, unlike WJ cells, vSMCs
express muscle-associated myosin. It has been suggested, but not demonstrated,
that WJ myofibroblasts are derived from either vSMC or fibroblasts. Multiple
immunohistological studies have shown that, in the first trimester, second trimes-
ter, and term umbilical cord, the bulk of WJ myofibroblasts are CD146-negative
but do stain positive for MSC-associated markers and other pericyte-associated
markers such as αSMA, desmin, and PDGFR (Takechi et al. 1993; Baksh et al.
2007; Schugar et al. 2009; Montemurro et al. 2011; Ryu et al. 2013; Coskun and
Can 2015). In the term umbilical cord at least, CD34 and CD144 only mark the
endothelial cell layer; the perivascular and WJ regions are negative for these mark-
ers (Schugar et al. 2009).
Altogether, these data suggest that at least two cell populations expressing MSC-­
associated markers and differential expression of pericyte-associated markers are
present in the umbilical cord. The origin and function of these cells remain unclear,
and lineage tracing studies using animal models with tagged pericyte markers have
not been performed on the umbilical cord. The differential expression of pericyte
marker CD146 between perivascular and WJ cells could be attributed to the proxim-
ity of perivascular cells to endothelial cells, as it was shown that direct MSC-­
endothelial cell contact induces the upregulation of pericyte genes (CD146, NG2,
PDGFRβ) (Loibl et al. 2014).
12 Pericytes in the Umbilical Cord 223

12.4  nalysis of Pericyte-Associated Markers in Cells

A
Isolated from the Perivascular Regions of the Umbilical
Cord as Sources of Mesenchymal Stromal Cells
for Regenerative Medicine

Early microscopy studies focusing on describing the ultrastructural properties of

stromal cells in the umbilical cord have improved our understanding of the cellular
mechanisms associated with umbilical cord anomalies. Scientific interest in the cel-
lular composition of each structural region of the umbilical cord was re-ignited in
the last 20 years when it was found that they contained stem cell-like cells that could
be propagated in vitro. Cells exhibiting stem cell-like properties can be derived
from many compartments in the cord including amniotic epithelial membrane
(AM), subamnion (SA) or “cord lining”, intervascular Wharton’s jelly (WJ), and
perivascular region (PV) surrounding the three blood vessels, as well as cord blood.
It seems apparent, based on the description of cell phenotypes observed in the
defined structural regions of the umbilical cord tissue at different developmental
time points, that the compartment of umbilical cord tissue included during cell iso-
lation would yield cells with the molecular phenotypes and biological functions
associated with those regions, as described above (Fig. 12.4). MSC-like cells have
been isolated and propagated from whole umbilical cord or using protocols that
include the subamnion, WJ, perivascular region, and vessels, suggesting that there
is a close and plastic relationship, at least in culture, between myofibroblast, peri-
cytes, and smooth muscle cells in the umbilical cord. According to the description
of cells in the umbilical cord in situ, protocols which include the harvest of cells
from the whole cord or perivascular regions (and these are not consistently described
as discussed in detail in Davies et al. 2017), as opposed to removing blood vessels
and isolating cells from Wharton’s jelly, would yield corresponding proportions of
CD146+ cells. Published isolation protocols vary based on the inclusion/exclusion
of epithelial membrane, Wharton’s jelly and vessels, enzymatic digestion, or explant
culture methods. Schugar et al. demonstrated that the protocol used to isolate cells
from whole umbilical cord can have a significant impact on cell yield, morphology,
and immunophenotype. Collagenase digestion yielded an increased proportion of
freshly isolated cells expressing MSC-associated markers (CD44, CD105, CD73,
CD90) where about half of the cells expressed moderate levels of CD146 (CD146mod)
and decreased proportion of endothelial cell-associated markers (CD34, CD144,
and CD146high) when compared to dispase digestion, which yielded an increased
proportion of cells with an endothelial phenotype and decreased proportion of cells
with an MSC phenotype. These differences in immunophenotype persisted in cul-
ture for 1 to 3 weeks, and only the collagenase-isolated population showed the abil-
ity to differentiate in vitro toward the osteogenic and chondrogenic lineage (Schugar
et al. 2009). This group used fluorescence-activated cell sorting (FACS) technology
to enrich CD146+ cells in the collagenase digestion population and found that
within 48 hours, the proportion of CD146+ cells returned to fifty percent of the total
224 A. Gauthier-Fisher et al.

cells, suggesting that CD146+ cells may give rise to a CD146-negative population
of cells expressing MSC markers. This also suggests that the distinct in situ cell
phenotypes observed between Wharton’s jelly myofibroblasts and perivascular tis-
sue pericytes/vascular smooth muscle cells persist following isolation and propaga-
tion in culture (at least in the early passages). This will be discussed further below.
MSC-like cells were first isolated from the umbilical cord stroma, by focusing on
protocols that removed the blood vessels, thereby presumably varying amounts of
the perivascular tissue (McElreavey et al. 1991; Karahuseyinoglu et al. 2007;
Subramanian et al. 2015). A few of these studies have demonstrated that when WJ
cells are isolated and plated in culture, they generally maintain the ultrastructural
and molecular properties of myofibroblasts, including stellate-shaped cells with
filopodia, small nuclei in situ (relatively larger in culture), prominent nucleoli,
numerous Golgi apparatus and filaments, and cytoplasmic filopodia. Differences in
the arrangements of myofilaments (random in culture) and lipid inclusion (not
observed in culture) were however observed. The cells maintain expression of
vimentin, αSMA and PDGFR, and CD10 but were desmin-positive in early pas-
sages only (Ryu et al. 2013). Comparison of MSC derived from proximal, middle,
and distal segments of the umbilical cord WJ using explant cultures yielded cells
expressing a similar immunophenotype, morphology, and tri-lineage differentiation
potential, in addition to the ability to transdifferentiate toward osteocytes and
hepatocyte-­like cells following induction in culture, suggesting that WJ cells with
similar therapeutic potential can be isolated throughout the length of the cord (Ishige
et al. 2009). An independent study, where an enzymatic digestion protocol was
used, showed similar findings, with the exception of decreased differentiation
potential in cells derived from the middle segment when compared to those derived
from maternal and fetal ends of the cord (Lim et al. 2016). Neither of these studies
investigated pericyte-like properties in WJ-derived MSC.
When plated in culture, cells isolated from the perivascular region using a method
that excludes Wharton’s Jelly, subamnion, and epithelium have a fibroblast-like
appearance with a stellate shape and long cytoplasmic processes that extend 100–
300 μm. In vitro, HUCPVCs express alpha-actin, desmin, and vimentin and can be
stained with the 3G5 antibody (used to mark pericytes). In addition, they are posi-
tive for MSC markers but negative for hematopoietic markers, and a subpopulation
of HUCPVC expresses low levels of CD177 (c-kit) and HLA-A, HLA-B, and
HLA-C (Sarugaser et al. 2005). The bulk population of these cells shows tri-lineage
differentiation potential in vitro and in vivo. When compared to cord blood or bone
marrow, and whole cord tissue, the perivascular-targeted isolation of cells allowed
for a higher frequency of MSC-like cells and CFU-F (1:200 million vs 1:100,000 vs
1:300, respectively) (Sarugaser et al. 2005), making the perivascular region of the
umbilical cord one of the richest sources of MSC. Importantly, and in contrast to
other studies, Davies’ group used clonal experiments, starting from single cells, to
demonstrate that HUCPVCs represent a heterogeneous, hierarchical population of
self-renewing MSCs where 1:3 cells have self-renewing and multi-differentiation
capacity toward five mesenchymal lineages including myofibroblast and fibroblast
(Sarugaser et al. 2009). A comparison of in vitro passaged first trimester-derived
12 Pericytes in the Umbilical Cord 225

(FTM) umbilical cord cells isolated with collagenase (representing all cells of the
developing umbilical cord) and term HUCPVC showed that a similar proportion of
cells isolated from each of these sources expresses the pericyte-associated markers
CD146, NG2, and PDGFRβ and multipotency-associated marker SSEA-4 in cul-
ture. However, Oct4A and Sox17 expression was restricted to FTM HUCPVC, and
these cells also showed increased proliferative capacity and in vitro differentiation
capacity toward both mesenchymal and all three germ cell lineages when compared
to term HUCPVC (Hong et al. 2013; Szaraz et al. 2016; Shlush et al. 2017). In
in vitro (Iqbal et al. 2017) and in vivo (under review Iqbal et al.) angiogenesis
assays, FTM HUCPVC showed increased levels of CD146 and PDGFRβ expres-
sions and increased angiogenic activity and pericyte-like interactions with endothe-
lial cells of developing vasculature when compared to term HUCPVC or bone
marrow MSC (Fig. 12.5b, c). Altogether, the results discussed here and in the previ-
ous section, and the fact that vascular maturation and perivascular/connective tissue
growth are significant in the first and second trimester, suggest that a more primitive
source of pericyte/MSC resides in the early gestation umbilical cord and might
function as progenitors for myofibroblasts and vascular smooth muscle cells in the
cord. This could have important implications for their use in regenerative medicine.
In keeping with the idea that ontologically younger sources of MSC have increased
plasticity, it was also shown that term HUCPVCs have higher proliferative capacity
and multi-differentiation potential than bone marrow-derived MSC and that this
multi-differentiation potential is maintained in CD146-sorted HUCPVC (Baksh
et al. 2007).
Indirect evidence for a source of pericytes or pericyte progenitors in the umbili-
cal cord arterial perivascular region arises from the human aortic ring (hAR) assay.
In this angiogenic assay, angiogenic sprouts from a denuded artery form capillary-­
like structures in vitro where NG2-positive pericyte-like cells associate with endo-
thelial cell tube formations (Seano et al. 2013). Covas et al. showed that MSCs
isolated from the subendothelial layer of the umbilical cord vein (UCV) or umbili-
cal cord arteries (UCA) and passaged three to five times in culture were CD146+
and showed considerable morphological, immunophenotypical, and ultrastructural
similarity to CD146-sorted human fetal or adult retinal pericytes. This included the
observation of large nuclei with prominent nucleoli, abundant ER, and mitochon-
dria that are characteristic of metabolically active cells. In addition, gene expression
analysis of many cell types showed closest clustering between UCV-derived MSC,
bone marrow MSC, and human retinal pericytes (Covas et al. 2008). WJ cells were
not compared to perivascular cells in this study.
At least two studies systematically compared MSC isolated from various com-
partments of the cord (Ishige et al. 2009; Subramanian et al. 2015). Ishige et al.
compared the proliferative, CFU-F, immunophenotype, and in vitro tri-lineage dif-
ferentiation potential of cells isolated from umbilical cord Wharton’s jelly (UCWJ),
umbilical vein perivascular area (UCV), and umbilical artery perivascular area
(UCA) using an explant outgrowth method. While all three cell populations showed
similar immunophenotype with regard to the usual MSC and non-MSC markers,
cells derived from the UCV exhibited the highest colony-forming unit frequency
226 A. Gauthier-Fisher et al.

(CFU-F) and the slowest proliferation rate. All three cell populations were able to
differentiate toward the chondrogenic and adipogenic lineages; however, UCWJ
showed reduced effectiveness toward osteogenic differentiation in this study.
Surprisingly, UCA cells showed increasing alkaline phosphatase activity after a
week in culture, even in the absence of osteogenic induction conditions. Although
the tri-lineage differentiation capacity was not demonstrated in clonal experiments,
these results suggest that MSC-like cells can be harvested from all compartments of
the umbilical cord but that the CD146+ population in UCV/UCA may provide a
better source.
Subramanian et al. used previously established enzyme digestion and explant
protocols to isolate cells from mixed cords (MC) and three compartments includ-
ing the amnion (AM), subamnion (SA), Wharton’s jelly (WJ), and perivascular
zone (PV). They further demonstrated that, although WJ gave the highest cell num-
bers in primary culture, cells derived from each of the regions including AM and
SA, and passaged 1–10 times in culture, were positive for signature MSC markers.
WJ cells showed increased expression levels (mean fluorescent intensity) for
CD29, CD44, and CD73 and for CD24 and CD108, markers thought to distinguish
stem cell MSCs from non-stem cell mesenchymal cells in bone marrow and adi-
pose MSC cultures (Wetzig et al. 2013). Interestingly, in this study, greater than
90% of the cells isolated from all regions were positive for pericyte-associated
marker CD146, with increased expression levels in WJ and PV. WJ and PV cells
also showed increased expression of another pericyte marker CD271, while
PDGFRβ (CD140b), CD49d (integrin alpha 4), and CD40 (a marker associated
with non-stem cell MSC or contaminating cells) were detected in increased pro-
portions of SA-, AM-, and MC-derived cells (approximately 40%) and at higher
levels when compared to WJ and PV. Early passaged cells showed similar telom-
erase activity, but WJ cells showed increased levels by passage 10. While all com-
partments showed detectable mRNA expression of pluripotency-associated genes
such as OCT4A, NANOG, and SOX2 by qRT-PCR (cycle threshold values were
not specified), levels appeared significantly reduced in SA and AM when com-
pared to WJ. Fibroblast-associated genes showed the opposite pattern. Finally,
while cultures derived from all UC compartments could undergo tri-lineage dif-
ferentiation, here the authors concluded that the WJ, followed by PV, had increased
levels of osteogenic and adipogenic differentiation. Although in vitro cell pheno-
types are somewhat incongruous with the description of cell phenotypes in situ,
this study and many described here illustrate a major issue in umbilical cord MSC
research. There is very little consistency with regard to cell isolation methods,
definition, and selection of umbilical cord compartments, and there are no stan-
dardized culture conditions, immunophenotyping, and differentiation assays.
While it is difficult to make definitive conclusions as to the source of MSC and
pericyte-like cells in the umbilical cord, this study suggests that multiple subpopu-
lation of MSC/pericytes may exist in the umbilical cord and umbilical cord-derived
cell cultures (Fig. 12.4).
12 Pericytes in the Umbilical Cord 227

12.5  ericytes/Perivascular Cells and Umbilical Cord

P
Pathologies and Gestational Complications

While structural deficits in the umbilical cord have been described and associated
with obstetrical complications involving the umbilical cord, the involvement of
pericytes and perivascular mesenchymal cell progenitors in such pathologies or as
an endogenous source of cells that could participate in potential repair mechanisms
in vivo has not been explored. Common umbilical cord pathologies including
abnormally short or long cords, abnormal number of blood vessels or vessel struc-
ture, and reduced stromal tissue are reviewed in detail in other textbooks (Cullen
1916; Benirschke and Kaufmann 1995).
Reduced volume of the stromal tissue or Wharton’s jelly (WJ) around the vein
and/or absence of WJ around the umbilical cord arteries (Fig. 12.6), the latter of
which is very rare and has never been observed with regard to the umbilical vein, is
associated with poor fetal outcomes, intrauterine growth restriction, preterm deliv-
ery, and perinatal death. This is presumably because a lack of WJ protection leaves
the vessels susceptible to compression and torsion, leading to impaired placental
and fetal blood flow (Cole and Israfil-Bayli 2016). While the cellular and molecular
mechanisms for such anomalies are unclear, it has been suggested that progenitors
in the UC may play a role in the degeneration or malformation of the WJ around the
vessels as a result of improper early umbilical cord development and mesenchymal

Fig. 12.6 Image of umbilical cord pathology showing absence of Wharton’s jelly around arteries.
Lack of sufficient stromal tissue renders cord axially disintegrated. Umbilical arteries are detached
and form loops along the cord—risking entanglement and insufficient blood flow. Reprinted with
permission from Cole, J. and F. Israfil-Bayli (2016). “Wharton’s jelly: The significance of absence.”
J Obstet Gynaecol 36(4): 500–501. www.tandfonline.com
228 A. Gauthier-Fisher et al.

structures (Kulkarni et al. 2007). Progenitors in the umbilical cord, which likely
include pericyte-like cells described in previous sections and which potentially give
rise to myofibroblasts, may therefore play a role in protecting against umbilical cord
and fetal pathologies by fueling the matrix that ensures adequate placental blood
flow to the fetus.
Pericytes or pericyte progenitors in the umbilical cord may play additional roles
in protecting against pathologies. MSCs derived from WJ have been shown to have
tumoricidal properties, and, because of this, it has been suggested that WJ cells act
as a natural defense against the migration of cancer cells between fetus and mothers
with ovarian, placental, or mammary cancers (Yang and Chao 2013). As discussed
in the previous section, MSC/pericytes with in vitro and in vivo multipotential dif-
ferentiation capacity and paracrine properties can be isolated from either the peri-
vascular or WJ compartments of the umbilical cord and expanded in culture. Over
the last 15 years, MSCs derived from the umbilical cord as well as those from other
sources such as bone marrow, adipose tissue, and other fetal sources have gained
increasing interest in the field of regenerative and immunomodulatory medicine.
MSCs, including those derived from the umbilical cord, have been studied as cell
therapy candidates in many areas of regenerative medicine (Guadix et al. 2017;
Samsonraj et al. 2017). MSCs were initially isolated and characterized from the
bone marrow (BM) (Friedenstein et al. 1970), but it is now well established that
various types of MSC can be isolated from vascularized tissues throughout the body
(Crisan et al. 2008; Corselli et al. 2012). MSCs are well characterized as multipo-
tent cells that can be expanded in culture and are capable of differentiation toward
bone, cartilage, adipose, fibrous, and muscle tissue. They are best known for their
capacity to secrete bioactive molecules that promote immunomodulation, anti-­
fibrosis, angiogenesis, as well as cell survival and proliferation (Meirelles Lda et al.
2009; Shende et al. 2018). There are currently greater than 100 registered clinical
trials (www.clinicaltrials.gov) investigating umbilical cord-derived MSC for multi-
ple indications including neurological, hematological, immunological, liver, car-
diac, endocrine, musculoskeletal, pulmonary, skin, and ophthalmological diseases
(Can et al. 2017).
While the involvement of pericytes in pathologies of the umbilical cord has not
been shown empirically, there is evidence that obstetrical complications and pathol-
ogies can, in turn, impact the in vitro phenotype and behavior of mesenchymal stro-
mal cells isolated from the umbilical cord. UC-MSCs (WJ) derived from gestational
diabetes mellitus (GDM) patients show premature senescence and upregulation of
senescence genes, decreased proliferative potential, and mitochondrial dysfunction
(Kim et al. 2015). Similarly, in an independent study, decreased cell yield of and
growth of HUCPVC in early passages (but recovery in later passages under low
glucose conditions) were reported, along with altered paracrine-mediated wound
healing properties in vitro (An et al. 2017). These findings were not surprising given
that exposure to hyperglycemia causes a production and reactive oxygen species
and inflammation which, at least in other tissues, induces the accelerated loss of
pericytes in capillaries (Manea et al. 2015). Adverse effects of GDM were also
12 Pericytes in the Umbilical Cord 229

observed on the growth, angiogenic, and anticancer properties of Wharton’s jelly

MSC (Wajid et al. 2015).
Similarly, changes in gene and protein expression of Wharton’s jelly MSC have
been described in samples derived from preeclamptic patients (Bankowski et al.
1996, 2004; Jurewicz et al. 2014; Joerger-Messerli et al. 2015). Bankowski described
“aging” of Wharton’s jelly in preeclampsia (PE), but it is unclear whether changes
in perivascular and Wharton’s jelly cells reflect an adaptation to PE or participate in
the development and symptoms of PE. In addition, a thicker smooth muscle cell
layer has been reported in PE when compared to normal gestation umbilical cord
arteries. This may reflect a functional adaptation of the umbilical cord arteries to
altered hemodynamic conditions in PE (Junek et al. 2000) which may well involve
regulation of pericyte or vSMC progenitors. Altogether, these studies suggest that
obstetrical complications may impact the quality and therapeutic efficacy of umbili-
cal cord-derived MSC.

12.6 Future Trends and Directions

Our understanding of the role of umbilical cord pericyte-like cells in the develop-
ment and maintenance of the cord, umbilical cord pathologies, and as a source of
MSC is incomplete. There are currently over 100 early phase clinical trials under-
way to test the safety and efficacy of umbilical cord-derived MSC for the treatment
of various medical indications (Can et al. 2017), yet there is still no consensus on
methods to isolate and expand umbilical cord MSC, or subpopulations of MSC,
with the greatest quality and efficacy to treat each of these indications. Our under-
standing of pericyte-like cells in the umbilical cord could be improved by system-
atic histological analyses of pericyte-associated markers in all regions of normal
and abnormal cords during development and at term, and by use of inducible
pericyte-­associated gene knockout mouse models and cell fate tracing tools. To our
knowledge, this has not been done but could increase our understanding of the con-
tribution of pericyte-like cells to umbilical cord development and pathologies. The
emergence of single-cell sequencing technologies will enable researchers to com-
plement previous histological findings by fully characterizing the phenotypes and
intercellular interactions of the pericyte-like cell populations that reside in the
umbilical cord throughout gestation, both in normal pregnancies and those with
obstetrical complications.

Acknowledgments The authors would like to thank Farwah Iqbal (Librach lab) for providing
unpublished data included in Fig. 12.5 and Denis Gallagher (CReATe Fertility Centre) for his care-
ful review and edits of the manuscript. The authors would like to acknowledge the authors of previ-
ously published books and scientific articles for contributions to figures. Some images found in
Figs. 12.2, 12.3, and 12.6 were obtained with permission as described in figure legends.
230 A. Gauthier-Fisher et al.

References

An B, Kim E, Song H, Ha KS, Han ET, Park WS, Ahn TG, Yang SR, Na S, Hong SH (2017)
Gestational diabetes affects the growth and functions of perivascular stem cells. Mol Cells
40(6):434–439
Andreeva ER, Pugach IM, Gordon D, Orekhov AN (1998) Continuous subendothelial network
formed by pericyte-like cells in human vascular bed. Tissue Cell 30(1):127–135
Baksh D, Yao R, Tuan RS (2007) Comparison of proliferative and multilineage differentiation
potential of human mesenchymal stem cells derived from umbilical cord and bone marrow.
Stem Cells 25(6):1384–1392
Bankowski E, Sobolewski K, Romanowicz L, Chyczewski L, Jaworski S (1996) Collagen and
glycosaminoglycans of Wharton's jelly and their alterations in EPH-gestosis. Eur J Obstet
Gynecol Reprod Biol 66(2):109–117
Bankowski E, Sobolewski K, Palka J, Jaworski S (2004) Decreased expression of the insulin-like
growth factor-I-binding protein-1 (IGFBP-1) phosphoisoform in pre-eclamptic Wharton's jelly
and its role in the regulation of collagen biosynthesis. Clin Chem Lab Med 42(2):175–181
Benirschke K, Kaufmann P (1995) Pathology of the human placenta. Springer, New York
Blanco MV, Vega HR, Giuliano R, Grana DR, Azzato F, Lerman J, Milei J (2011) Histomorphometry
of umbilical cord blood vessels in preeclampsia. J Clin Hypertens (Greenwich) 13(1):30–34
Caplan AI (2008) All MSCs are pericytes? Cell Stem Cell 3(3):229−230
Can A, Karahuseyinoglu S (2007) Concise review: human umbilical cord stroma with regard to the
source of fetus-derived stem cells. Stem Cells 25(11):2886–2895
Can A, Celikkan FT, Cinar O (2017) Umbilical cord mesenchymal stromal cell transplantations: a
systemic analysis of clinical trials. Cytotherapy 19(12):1351–1382
Clarke JA (1965) An x-ray microscopic study of the human umbilical arteries. Z Zellforsch
Mikrosk Anat 66(2):293–299
Cole J, Israfil-Bayli F (2016) Wharton's jelly: the significance of absence. J Obstet Gynaecol
36(4):500–501
Corrao S, La Rocca G, Lo Iacono M, Corsello T, Farina F, Anzalone R (2013) Umbilical cord
revisited: from Wharton's jelly myofibroblasts to mesenchymal stem cells. Histol Histopathol
28(10):1235–1244
Corselli M, Chen CW, Sun B, Yap S, Rubin JP, Peault B (2012) The tunica adventitia of human
arteries and veins as a source of mesenchymal stem cells. Stem Cells Dev 21(8):1299–1308
Coskun H, Can A (2015) The assessment of the in vivo to in vitro cellular transition of human
umbilical cord multipotent stromal cells. Placenta 36(2):232–239
Covas DT, Panepucci RA, Fontes AM, Silva WA Jr, Orellana MD, Freitas MC, Neder L, Santos
AR, Peres LC, Jamur MC, Zago MA (2008) Multipotent mesenchymal stromal cells obtained
from diverse human tissues share functional properties and gene-expression profile with
CD146+ perivascular cells and fibroblasts. Exp Hematol 36(5):642–654
Crisan M, Yap S, Casteilla L, Chen CW, Corselli M, Park TS, Andriolo G, Sun B, Zheng B, Zhang
L, Norotte C, Teng PN, Traas J, Schugar R, Deasy BM, Badylak S, Buhring HJ, Giacobino
JP, Lazzari L, Huard J, Peault B (2008) A perivascular origin for mesenchymal stem cells in
multiple human organs. Cell Stem Cell 3(3):301–313
Cullen, T. S. (1916). Embryology, anatomy, and diseases of the umbilicus, together with the dis-
eases of the urachus. Philadelphia Saunders
Davies JE, Walker JT, Keating A (2017) Concise review: Wharton's jelly: the rich, but enigmatic,
source of mesenchymal stromal cells. Stem Cells Transl Med 6(7):1620–1630
Diaz-Flores L, Gutierrez R, Madrid JF, Varela H, Valladares F, Acosta E, Martin-Vasallo P, Diaz-­
Flores L Jr (2009) Pericytes. Morphofunction, interactions and pathology in a quiescent and
activated mesenchymal cell niche. Histol Histopathol 24(7):909–969
Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini F, Krause D, Deans R, Keating
A, Prockop D, Horwitz E (2006) Minimal criteria for defining multipotent mesenchymal stro-
mal cells. The International Society for Cellular Therapy position statement. Cytotherapy
8(4):315–317
12 Pericytes in the Umbilical Cord 231

Downs KM (1998) The murine allantois. Curr Top Dev Biol 39:1–33
Downs KM, Gifford S, Blahnik M, Gardner RL (1998) Vascularization in the murine allantois occurs
by vasculogenesis without accompanying erythropoiesis. Development 125(22):4507–4520
Ellison JP (1971) The nerves of the umbilical cord in man and the rat. Am J Anat 132(1):53–60
Friedenstein AJ, Chailakhjan RK, Lalykina KS (1970) The development of fibroblast colo-
nies in monolayer cultures of Guinea-pig bone marrow and spleen cells. Cell Tissue Kinet
3(4):393–403
Friedman R, Betancur M, Boissel L, Tuncer H, Cetrulo C, Klingemann H (2007) Umbilical
cord mesenchymal stem cells: adjuvants for human cell transplantation. Biol Blood Marrow
Transplant 13(12):1477–1486
Guadix JA, Zugaza JL, Galvez-Martin P (2017) Characteristics, applications and prospects of mes-
enchymal stem cells in cell therapy. Med Clin (Barc) 148(9):408–414
Holm A et al (2018) Microvascular mural cell Organotypic heterogeneity and functional plasticity.
Trends Cell Biol 28(4):302–316
Hong SH, Maghen L, Kenigsberg S, Teichert AM, Rammeloo AW, Shlush E, Szaraz P, Pereira S,
Lulat A, Xiao R, Yie SM, Gauthier-Fisher A, Librach CL (2013) Ontogeny of human umbilical
cord perivascular cells: molecular and fate potential changes during gestation. Stem Cells Dev
22(17):2425–2439
Hoyes AD (1969) Ultrastructure of the epithelium of the human umbilical cord. J Anat 105(Pt
1):149–162
Inman KE, Downs KM (2007) The murine allantois: emerging paradigms in development of the
mammalian umbilical cord and its relation to the fetus. Genesis 45(5):237–258
Iqbal F, Szaraz P, Librach M, Gauthier-Fisher A, Librach CL (2017) Angiogenic potency evalua-
tion of cell therapy candidates by a novel application of the in vitro aortic ring assay. Stem Cell
Res Ther 8(1):184
Ishige I, Nagamura-Inoue T, Honda MJ, Harnprasopwat R, Kido M, Sugimoto M, Nakauchi H,
Tojo A (2009) Comparison of mesenchymal stem cells derived from arterial, venous, and
Wharton’s jelly explants of human umbilical cord. Int J Hematol 90(2):261–269
Joerger-Messerli M, Bruhlmann E, Bessire A, Wagner A, Mueller M, Surbek DV, Schoeberlein A
(2015) Preeclampsia enhances neuroglial marker expression in umbilical cord Wharton’s jelly-­
derived mesenchymal stem cells. J Matern Fetal Neonatal Med 28(4):464–469
Junek T, Baum O, Lauter H, Vetter K, Matejevic D, Graf R (2000) Pre-eclampsia associated altera-
tions of the elastic fibre system in umbilical cord vessels. Anat Embryol (Berl) 201(4):291–303
Jurewicz E, Kasacka I, Bankowski E, Filipek A (2014) S100A6 and its extracellular targets in
Wharton's jelly of healthy and preeclamptic patients. Placenta 35(6):386–391
Karahuseyinoglu S, Cinar O, Kilic E, Kara F, Akay GG, Demiralp DO, Tukun A, Uckan D, Can
A (2007) Biology of stem cells in human umbilical cord stroma: in situ and in vitro surveys.
Stem Cells 25(2):319–331
Khati NJ, Enquist EG, Javitt MC (1998) Imaging of the umbilicus and periumbilical region.
Radiographics 18(2):413–431
Kim J, Piao Y, Pak YK, Chung D, Han YM, Hong JS, Jun EJ, Shim JY, Choi J, Kim CJ (2015)
Umbilical cord mesenchymal stromal cells affected by gestational diabetes mellitus display
premature aging and mitochondrial dysfunction. Stem Cells Dev 24(5):575–586
Kulkarni ML, Matadh PS, Ashok C, Pradeep N, Avinash T, Kulkarni AM (2007) Absence of
Wharton’s jelly around the umbilical arteries. Indian J Pediatr 74(8):787–789
Lim J, Razi ZR, Law J, Nawi AM, Idrus RB, Ng MH (2016) MSCs can be differentially iso-
lated from maternal, middle and fetal segments of the human umbilical cord. Cytotherapy
18(12):1493–1502
Loibl M, Binder A, Herrmann M, Duttenhoefer F, Richards RG, Nerlich M, Alini M, Verrier S
(2014) Direct cell-cell contact between mesenchymal stem cells and endothelial progenitor
cells induces a pericyte-like phenotype in vitro. Biomed Res Int 2014:395781
Malas MA, Sulak O, Gokcimen A, Sari A (2003) Morphology of umbilical vessels in human
fetuses: a quantitative light microscopy study. Eur J Morphol 41(5):167–174
232 A. Gauthier-Fisher et al.

Manea A, Manea SA, Todirita A, Albulescu IC, Raicu M, Sasson S, Simionescu M (2015) High-­
glucose-­increased expression and activation of NADPH oxidase in human vascular smooth
muscle cells is mediated by 4-hydroxynonenal-activated PPARalpha and PPARbeta/delta. Cell
Tissue Res 361(2):593–604
McElreavey KD, Irvine AI, Ennis KT, McLean WH (1991) Isolation, culture and characterisa-
tion of fibroblast-like cells derived from the Wharton’s jelly portion of human umbilical cord.
Biochem Soc Trans 19(1):29S
Meirelles Lda S, Fontes AM, Covas DT, Caplan AI (2009) Mechanisms involved in the therapeutic
properties of mesenchymal stem cells. Cytokine Growth Factor Rev 20(5–6):419–427
Montemurro T, Andriolo G, Montelatici E, Weissmann G, Crisan M, Colnaghi MR, Rebulla P,
Mosca F, Peault B, Lazzari L (2011) Differentiation and migration properties of human foetal
umbilical cord perivascular cells: potential for lung repair. J Cell Mol Med 15(4):796–808
Nanaev AK, Kohnen G, Milovanov AP, Domogatsky SP, Kaufmann P (1997) Stromal differentia-
tion and architecture of the human umbilical cord. Placenta 18(1):53–64
Parry EW (1970) Some electron microscope observations on the mesenchymal structures of full-­
term umbilical cord. J Anat 107(Pt 3):505–518
Parry EW, Abramovich DR (1970) Some observations on the surface layer of full-term human
umbilical cord epithelium. J Obstet Gynaecol Br Commonw 77(10):878–884
Parry EW, Abramovich DR (1972) The ultrastructure of human umbilical vessel endothelium from
early pregnancy to full term. J Anat 111(Pt 1):29–42
Pearson AA, Sauter RW (1970) Nerve contributions to the pelvic plexus and the umbilical cord.
Am J Anat 128(4):485–498
Ryu YJ, Seol HS, Cho TJ, Kwon TJ, Jang SJ, Cho J (2013) Comparison of the ultrastructural
and immunophenotypic characteristics of human umbilical cord-derived mesenchymal stromal
cells and in situ cells in Wharton's jelly. Ultrastruct Pathol 37(3):196–203
Samsonraj RM, Raghunath M, Nurcombe V, Hui JH, van Wijnen AJ, Cool SM (2017) Concise
review: multifaceted characterization of human mesenchymal stem cells for use in regenerative
medicine. Stem Cells Transl Med 6(12):2173–2185
Sarugaser R, Lickorish D, Baksh D, Hosseini MM, Davies JE (2005) Human umbilical cord peri-
vascular (HUCPV) cells: a source of mesenchymal progenitors. Stem Cells 23(2):220–229
Sarugaser R, Ennis J, Stanford WL, Davies JE (2009) Isolation, propagation, and characterization
of human umbilical cord perivascular cells (HUCPVCs). Methods Mol Biol 482:269–279
Schugar RC, Chirieleison SM, Wescoe KE, Schmidt BT, Askew Y, Nance JJ, Evron JM, Peault
B, Deasy BM (2009) High harvest yield, high expansion, and phenotype stability of CD146
mesenchymal stromal cells from whole primitive human umbilical cord tissue. J Biomed
Biotechnol 2009:789526
Seano G, Chiaverina G, Gagliardi PA, di Blasio L, Sessa R, Bussolino F, Primo L (2013) Modeling
human tumor angiogenesis in a three-dimensional culture system. Blood 121(21):e129–e137
Sexton AJ, Turmaine M, Cai WQ, Burnstock G (1996) A study of the ultrastructure of developing
human umbilical vessels. J Anat 188(Pt 1):75–85
Sheppard BL, Bishop AJ (1973) Electron microscopical observations on sheep umbilical vessels.
Q J Exp Physiol Cogn Med Sci 58(1):39–45
Shende P, Gupta H, Gaud RS (2018) Cytotherapy using stromal cells: current and advance multi-­
treatment approaches. Biomed Pharmacother 97:38–44
Shlush E, Maghen L, Swanson S, Kenigsberg S, Moskovtsev S, Barretto T, Gauthier-Fisher A,
Librach CL (2017) In vitro generation of Sertoli-like and haploid spermatid-like cells from
human umbilical cord perivascular cells. Stem Cell Res Ther 8(1):37
Sims DE (2000) Diversity within pericytes. Clin Exp Pharmacol Physiol 27(10):842–846
Singh N, Rao S, Sobti P, Khurana N (2012) Multiple vessels in the umbilical cord: a report of four
cases. Indian J Pathol Microbiol 55(4):597–598
Spurway J, Logan P, Pak S (2012) The development, structure and blood flow within the umbilical
cord with particular reference to the venous system. Australas J Ultrasound Med 15(3):97–102
12 Pericytes in the Umbilical Cord 233

Stehbens WE, Wakefield JS, Gilbert-Barness E, Zuccollo JM (2005) Histopathology and ultra-
structure of human umbilical blood vessels. Fetal Pediatr Pathol 24(6):297–315
Subramanian A, Fong CY, Biswas A, Bongso A (2015) Comparative characterization of cells
from the various compartments of the human umbilical cord shows that the Wharton's jelly
­compartment provides the best source of clinically utilizable mesenchymal stem cells. PLoS
One 10(6):e0127992
Szaraz P, Librach M, Maghen L, Iqbal F, Barretto TA, Kenigsberg S, Gauthier-Fisher A, Librach
CL (2016) In vitro differentiation of first trimester human umbilical cord perivascular cells into
contracting cardiomyocyte-like cells. Stem Cells Int 2016:7513252
Takechi K, Kuwabara Y, Mizuno M (1993) Ultrastructural and immunohistochemical studies of
Wharton's jelly umbilical cord cells. Placenta 14(2):235–245
Wajid N, Naseem R, Anwar SS, Awan SJ, Ali M, Javed S, Ali F (2015) The effect of gestational
diabetes on proliferation capacity and viability of human umbilical cord-derived stromal cells.
Cell Tissue Bank 16(3):389–397
Wetzig A, Alaiya A, Al-Alwan M, Pradez CB, Pulicat MS, Al-Mazrou A, Shinwari Z, Sleiman
GM, Ghebeh H, Al-Humaidan H, Gaafar A, Kanaan I, Adra C (2013) Differential marker
expression by cultures rich in mesenchymal stem cells. BMC Cell Biol 14:54
Yang HT, Chao KC (2013) Foetal defence against cancer: a hypothesis. J Cell Mol Med
17(9):1096–1098
Chapter 13
The Pluripotent Microvascular Pericytes
Are the Adult Stem Cells Even in the Testis

Michail S. Davidoff

Abstract The pericytes of the testis are part of the omnipresent population of peri-
cytes in the vertebrate body and are the only true pluripotent adult stem cells able to
produce structures typical for the tree primitive germ layers: ectoderm, mesoderm,
and endoderm. They originate very early in the embryogenesis from the pluripotent
epiblast. The pericytes become disseminated through the whole vertebrate organism
by the growing and differentiating blood vessels where they remain in specialized
periendothelial vascular niches as resting pluripotent adult stem cells for tissue gen-
eration, maintenance, repair, and regeneration. The pericytes are also the ancestors
of the perivascular multipotent stromal cells (MSCs). The variable appearance of
the pericytes and their progeny reflects the plasticity under the influence of their
own epigenetic and the local environmental factors of the host organ. In the testis
the pericytes are the ancestors of the neuroendocrine Leydig cells. After activation
the pericytes start to proliferate, migrate, and build transit-amplifying cells that
transdifferentiate into multipotent stromal cells. These represent progenitors for a
number of different cell types in an organ. Finally, it becomes evident that the peri-
cytes are a brilliant achievement of the biological nature aiming to supply every
organ with an omnipresent population of pluripotent adult stem cells. Their fasci-
nating features are prerequisites for future therapy concepts supporting cell systems
of organs.

Keywords Pericytes · Testis · Pericyte origin · Testis microvasculature · Vascular

niche · Stem cell migration · Transit amplifying cells · Transdifferentiation · Adult
stem cells · Neuroendocrine features · Peritubular cells · Neural crest cells ·
Periendothelial cells · Perivascular cells · Pericyte plasticity

M. S. Davidoff (*)
University Medical Center Hamburg-Eppendorf, Hamburg Museum of Medical History,
Hamburg, Germany
e-mail: [email protected]

© Springer Nature Switzerland AG 2019 235

A. Birbrair (ed.), Pericyte Biology in Different Organs,
Advances in Experimental Medicine and Biology 1122,
https://doi.org/10.1007/978-3-030-11093-2_13
236 M. S. Davidoff

13.1 Introduction

Until shortly the view existed that single organs or tissues possess its own tissue-­
specific (typical) adult stem cells (Soncin and Ward 2011; Rolandsson et al. 2014).
Presently three main cell populations are supposed to be adult stem cells, namely,
the stromal (mesenchymal) stem cells, the neural crest stem cells, and the
pericytes.
We already evidenced that pericytes are the ancestors of the neuroendocrine
Leydig cells of the testes (Davidoff et al. 1993, 2004, 2009; Davidoff 2017). We
applied in vivo the chemical ablation experiment with EDS (ethane dimethanesulfo-
nate) for adult Leydig cell destruction and subsequent full regeneration and suc-
ceeded to verify the individual developmental stages, to identify and localize the
Leydig stem cells, and to follow up the fate of regenerating Leydig cell progenitors
(Kerr et al. 1985, 1986, 1987a, b; Molenaar et al. 1985; Benton et al. 1995; Teerds
1996; Teerds et al. 1999, 2007; Teerds and Rijntjes 2007; Davidoff et al. 2004, 2009).
The most important conclusions obtained in our previous studies are the
following:
1. At the beginning we provided evidence that the Leydig cells are neuroendo-
crine cells (Davidoff et al. 1993).
2. We performed an in vivo experiment. This is important because as known any
in vitro manipulation of the stem cells, inclusive isolation, and putting them in
a cell culture lead to changes of their original status and move away the in vivo
features, as this is the case with the artificial embryonic stem cells (Rossant
2001; Smith 2001; Buehr and Smith 2003; Zwaka and Thomson 2005).
3. The stem cells (ancestors) of the testicular Leydig cells are the pericytes and the
smooth muscle cells of the microvasculature. Both cell types share the same
lineage.
4. The pericytes are distributed during embryogenesis throughout the organism by
the developing cardiovascular system that is the first functioning organ system
during the embryonal development.
5. The pericytes remain the whole life within the authentic microvascular niches
as reserve stem cell population for tissue and organ repair and regeneration.
6. The pericytes are pluripotent cells, such as the epiblast (De-Miguel et al. 2010).
They possess ectodermal, endodermal, and mesodermal lineage progeny which
underlines their plasticity and heterogeneity.
7. The testis pericytes behave as authentic stem cells (Davidoff et al. 2004, 2009).
After activation they start to proliferate, self-renew, and migrate out from the
vascular niche to the perivascular space and the interstitium in form of transit-­
amplifying (intermediate) cells which transdifferentiate and generate multipo-
tent stromal (mesenchymal) cells, resp., immature committed cells. The
resulting cells, under the influence of the existing local factors, differentiate
further as typical for an organ or cell culture mature cell phenotype (Leydig
cells, smooth muscle cells, peritubular myoid cells, macrophages, fibroblasts,
germ cells—DeFalco et al. 2015; Yoshida et al. 2007) characteristic for the
13 The Pluripotent Microvascular Pericytes Are the Adult Stem Cells Even in the Testis 237

organ or tissue in which they are situated. This illustrates the high plasticity of
and how the pericytes adopt their phenotype when transplanted in another place
or organ and provides evidence that the pericytes are true stem cells.
8. The impossibility to observe the fast transdifferentiation of the transit-­
amplifying stem cells caused misrecognition of some structures and misinter-
pretation of the results obtained in a lot of previous studies.
9. The pericytes are the ancestors of the multipotent adult stromal cells and the
only authentic adult stem cell (ASC) phenotype of the vertebrate organism.
Thus, the migrating transit-amplifying pericyte descendants represent the mul-
tipotent mesodermal (until shortly mesenchymal stem) stromal cells.
10. Definitively, the applied ethane dimethanesulfonate (EDS) experiment provides
evidence that the testis pericytes are authentic (adult) stem cells which are plu-
ripotent and are able to produce structures characteristic for the tree embryo-
logic germ layers, namely, ectoderm, mesoderm, and endoderm. Thus, the
pericytes of the testis are not organ-specific adult stem cells, but rather they are
part of the common for the whole organism omnipresent pericyte population.
The neuroendocrine qualities of the Leydig cells allowed the presumption that
pericytes may derive from the neural crest cells or from a common stem cell
population segregated early in embryogenesis with progeny for both the neural
crest and the mesoderm lineage.
11. The pericytes are the adult stem cells of the vertebrate organism inclusive in the
testis.
This chapter synthesizes current knowledge on the features of the microvascular
pericytes in the testes. Part of this chapter is the continuation of our earlier work
(Davidoff et al. 2004, 2009; Friedrich et al. 2012). We will mainly use the term
pericytes while it’s proven that the pericytes and the vascular smooth muscle cells
share a common lineage (Etchevers et al. 2001). Because the pericytes of the testis
belong to the whole body pericyte population, we try to present evidence that the
testis pericytes are actually member of the omnipresent (pan-organ stem cells;
Péault et al. 2007) pluripotent adult stem cell family of the vertebrate organism,
partly by comparing the data from the testis with these of other organs, especially
the pericytes of the nervous system. The testicular pericytes possess all features of
the pericytes in other organs. Their heterogeneity and differences depend on the fac-
tors existing in the microenvironment of the host organ and the stage of differentia-
tion and activity. This is why if transplanted into another organ, the pericytes
differentiate into typical for the host organ cells (Dar et al. 2012; Tang et al. 2008 b;
Bhagavati 2008; Dellavalle et al. 2011; Bouacida et al. 2012). This is a typical fea-
ture of the pericytes as adult stem cells and explains their great plasticity and ability
to transdifferentiate (Bjornson et al. 1999; Brazelton et al. 2000; Abedin et al.
2004—MSCs in vascular walls; Millner et al. 2008; Diaz-Flores et al. 2006, 2009—
in the nervous system; Wang et al. 2012; Jiang et al. 2003; Hermann et al. 2016). For
example, in a series of publications, Birbrair et al. (2013a, b, 2014) and Birbrair and
Delbono 2015) established even in the skeletal musculature the e­ xistence of two
major pericyte subpopulations: Type I and Type II, which possess differing features.
238 M. S. Davidoff

Probably this fact could be explained with the different maturation stages of the
pericytes under study.
During the last decades, evidence was provided which leads to changes of some
views and interpretations on the pericytes in the testis. These comprise the origin of
the pericytes, the relationship between pericytes and mesenchymal stem cells, and
their contribution in the development of the testicular structures as well as their
behavior during some experimental and pathological conditions (see for details,
pericytes and stem cells in the testis, Davidoff et al. 2004, 2009; Ge et al. 2006;
Chikhovskaya et al. 2012, 2014; Chen et al. 2010, 2017; Stanley et al. 2012;
Kilcoyne et al. 2014; Makala et al. 2015; Pericytes in other organs, predominantly
in the nervous system, Dore-Duffy 2008; Le Douarin et al. 2008; Krueger and
Bechmann 2010; Dore-Duffy and Cleary 2011; Sá-Pereira et al. 2012; Kurtz 2016).

13.1.1 S
 hort Repetition of the Testis Structure
and Nomenclature

The testes and epididymides are paired organs located in the scrotal sac of the exter-
nal male genital system (Fig. 13.1). The testis consists of 200–400 lobules contain-
ing seminiferous tubules and the interstitium situated between them (Brennan and
Capel 2004). The testicular interstitium contains connective tissue (fibroblasts,
tissue-­resident macrophages, and compartmentalizing co-cells), Leydig cells, nerve
fibers, blood vessels, and lymphatics (Fawcett et al. 1973; Holstein and Davidoff

Fig. 13.1 (1) Semi-schematic drawing of longitudinal sectioned structural components of the tes-
tis and epididymis (E). Arrangement of the seminiferous tubules in the human testis and of the
excurrent ductular system of the epididymis. Tunica albuginea (T), lobulus (L) of seminiferous
tubule in a space bordered by two testis septa. × 2.5. (2) Cross section of a seminiferous tubule of
a fertile man 32 years of age. Drawing of a semithin section showing the tunica albuginea (T), a
lobulus (L), and the rete testis (R), × 300 (from Holstein et al. 2003)
13 The Pluripotent Microvascular Pericytes Are the Adult Stem Cells Even in the Testis 239

1997; Holstein 1999; Holstein et al. 2003; DeFalco et al. 2014, 2015; Bhushan and
Meinhardt 2017). The seminiferous tubules are enclosed by a layer peritubular
myofibroblast cells (PMCs) and extracellular matrix produced by the cooperation
between the Sertoli cells and the testicular peritubular cells (Holstein et al. 1996;
Skinner et al. 1985) and are compartmentalized in lobules by testicular septa
(Figs. 13.1 and 13.2). The testis cords and extracellular matrix are covered by a
thick protective cap [a connective tissue testicular capsule named tunica albuginea
(Leeson and Cookson 1974)] to build an oval-shaped organ (Figs. 13.1 and 13.2).
The tunica albuginea of the human testis contains abundantly contractile elements
and is also distinguished by extraordinarily high concentrations of cyclic GMP
(cGMP)-dependent protein kinase I, known to mediate cGMP-dependent relaxation
(Leeson and Cookson 1974; Holstein et al. 1996; Middendorff et al. 2002).These
contractile elements are of importance for the movement of the non-motile sperma-
tozoa from the testis to the epididymis.
The testes have important functions in the origin, maintenance, and functions of
the male organism. They produce the sperm and the male hormones (androgens) in
vertebrates (Russell and de França 1995; Svingen and Koopman 2013). As men-
tioned, the testis cords comprise coiled seminiferous tubules with germinal epithe-
lium and peritubular tissue (lamina propria). The germinal epithelium consists of
cells that include different developmental stages of germ cells, namely, spermatogo-
nia, primary and secondary spermatocytes, and spermatids. These are located within
invaginations of Sertoli cells (Fig. 13.3).The spermatogenesis takes part within the
seminiferous tubules (Holstein 1999; Holstein et al. 2003), and the steroid hor-
mones are produced by the neuroendocrine Leydig cells (see Davidoff et al. 2009)

Fig. 13.2 (3) Cross section of a seminiferous tubule of a fertile man 32 years of age. Drawing of
a semithin section. (a) Peritubular tissue (lamina propria of seminiferous tubule). (b)
Spermatogonium. (c) Primary spermatocyte. (d) Immature spermatids. (e) Mature spermatids
×300 (from Holstein et al. 2003)
240 M. S. Davidoff

Fig. 13.3 (4 and 5) Schematic drawing of a testis segment presenting on the bottom a section of
the germinal epithelium with basal lamina propria and below a cluster of Leydig cells surrounding
a capillary. Arrows indicate different influences of secreted hormones and growth factors. On the
top the main processes of spermatogenesis are shown. (5) Demonstrates an enlarged drawing of the
top part in (4). Visible are different stages of the germ cells development. The germ cells are situ-
ated in deep invaginations of a Sertoli cell (from Holstein et al. 2003)

which are situated within the interstitium (Holstein and Davidoff 1997) surrounding
the blood vessels and the seminiferous tubules (Fig. 13.3).

13.1.2 Testis Vasculature

It must be emphasized that the pericytes of the testis, similar to the pericytes of all
vertebrate organs, show close relationships with the microvasculature. The associa-
tion of stem cell-like progenitors and blood vessels occurs early in the embryogen-
esis of the testis (cf. Brennan and Capel 2004). The relationship reaches a level in
which the microcirculatory (endothelial) cells direct the testis cord formation and
determine their course during embryonic development (Combes et al. 2009). The
pericytes themselves are structural component of the microvascular wall.
The testis is a well-vascularized organ that contains (1) “intertubular” arteries
and veins running parallel to the tubules and (2) their ramifications building a micro-
circulatory network consisting of transversally or obliquely running “peritubular”
small arterioles, precapillaries, capillaries, postcapillary venules, and small veins
(Fig. 13.4; see Müller 1957; Weerasooriya and Yamamoto 1985; Suzuki and Nagano
1986; Murakami et al. 1989; Yoshida et al. 2007; Svingen and Koopman 2013). In
13 The Pluripotent Microvascular Pericytes Are the Adult Stem Cells Even in the Testis 241

Fig. 13.4 (6) Schematic

presentation showing the
relationship between the
testicular blood vessels and
the seminiferous tubule.
ITV intertubular vessels,
PTV peritubular vessels.
The oval blood vessel
components illustrate the
pericytes. Note the close
relationship between the
blood vessels/pericytes and
the tubule wall and that
parts of the tubule surface
are devoid of blood vessels
(from Davidoff 2017)

Fig. 13.5 (7) Visualization

of Bromodeoxyuridine
(BrdU) in rat pericyte
nuclei 2 days after EDS
injection. The capillary is
designated by the arrows.
Note the close relationship
between the vessel/
pericytes and the tubules
(T) walls. In the lower
tubule, single
spermatogonia also show
BrdU immunoreactivity.
×550 (from Davidoff 2017)

the wall of the microvasculature, numerous pericytes and smooth muscle cells
(periendothelial cells, mural cells) are included (Fig. 13.4). The pericytes and the
vascular smooth muscle cells share a common lineage (Etchevers et al. 2001). The
peritubular microvessels and the seminiferous tubules are in close spatial contact
(Fig. 13.5). This fact is the reason that numerous scientists determine inaccurately
the lamina propria of the seminiferous tubules as the site of origin of the testicular
Leydig cells. These cells are in contact with the endothelial cells and are covered by
their common basal membrane that build spaces called vascular niches (Fig. 13.6,
electron micrograph; see Yoshida et al. 2007; Scadden 2006; Nikolova et al. 2007).
In the human testis, occasionally single capillaries run within the lamina propria and
242 M. S. Davidoff

Fig. 13.6 (8) This is an

electron microscopic
picture of a human
peritubular segment. At the
top the basal part of the
tubule is seen (T), followed
by the multilayered lamina
propria (LP) and at the
outside surface a part of
the interstitium with a
capillary (C),
periendothelial pericyte (P)
in the vascular niche,
surrounded by the bright
basal lamina, and a Leydig
cell (LC). ×4800 (from
Davidoff et al. 2009)

are surrounded by processes of the myofibroblasts. The intramural microvessels

within the lamina propria possess a partly fenestrated endothelial segment and a
continuous basal lamina in which pericytes are included (Ergün et al. 1994, 1996).

13.2 The Pericytes

13.2.1 Short Overview on the History of the Pericytes

The history of the pericyte discovery is presented to some extent contradictory in

the literature (Ribatti et al. 2011; Armulik et al. 2011; Attwell et al. 2016).
It was Eberth (1871) who in the small capillaries of the frog eye hyaloid mem-
brane discovered and described stellate “adventitial cells” with wall encircling pro-
cesses, whereas in the larger capillaries, arteries and veins of the human brain,
spinal cord, and retina, these cells he called “outer vessel epithelium” or
“perithelium.”
Two years later Rouget (1873, 1879) provided a more detailed description (he
observed heavily ramified cells that with their processes build perivascular rings at
the capillary walls) and observed the gradual transition of these cells into the vascu-
lar smooth muscle cells. Rouget expressed the presumption that these “perivascular
13 The Pluripotent Microvascular Pericytes Are the Adult Stem Cells Even in the Testis 243

cells” are contractile structures. In fact, he recognized two types of perivascular

cells, namely, amoeboid migratory cells and fusiform contractile cells.
Later Vimtrup (1922) offer a more detailed description of the perivascular cell
features and renamed the “adventitial cells” in “Rouget cells” with the argumenta-
tion that the first discoverer of these cells was Rouget.
In this respect, Sims (1986) wrote: “Rouget (1873, 1879) is often credited with
discovering the pericyte, although Bensley and Vimtrup (1928) acknowledge that
Eberth (1871) had previously described a capillary adventitial cell. In 1922, Vimtrup
described pericytes in somewhat greater detail. Subsequently, Krogh (1919, 1929),
who was one of the original investigators of capillary recruitment (Palm et al. 1983),
employed the name “Rouget cell” to describe what had been previously referred to
as adventitial cell” (p. 153).
That is not a precise description, because Krogh (1922) writes that he has took
over the name “Rouget cells” from Vimtrup. He wrote: “As there can be no doubt
that the richly ramified muscle cells on the capillary wall are the same as those origi-
nally found by Rouget in the hyaloid membrane, Vimtrup has named them after the
first discoverer, and we shall speak of them henceforward as Rouget cells” (p. 54).
It was Zimmermann (1923) who renamed the Rouget cells in pericytes including
their transitional forms to smooth muscle fibers. He describes tree pericyte types,
namely, precapillary pericytes, capillary pericytes, and postcapillary pericytes.
Zimmermann (1923) emphasized that the cells described by Eberth (1871)with
minor exceptions are other cells than these described by Rouget (1873). This is not
absolutely correct because as later detected, the pericyte population is heteroge-
neous and can be observed in variable morphological forms depending on the stage
of their development and activity (Attwell et al. 2016). Thus, Eberth (1871) was the
first who described and depicted these cells that he designated “adventitial cells.”
According to his description, these adventitial cells are anastomosing stellate cells
that with their ramifications wrap the wall of the blood vessels.
In the following years, different single and combined terms were used: “Rouget-­
Mayer cells” in the human skin (Günther 1917), “adventitial (Rouget) cells” (Clark
and Clark 1925a), “Rouget cells” (Dogiel 1899; Mayer 1902; Krogh 1922; Florey
and Carleton 1926; Benninghoff 1926; Bensley and Vimtrup 1928; Clark and Clark
1925b; Cervós-Navarro 1963), “Rouget cells (Pericytes)” (Michels 1936), as well
as “mural cells,” “periendothelial cells,” “histiocytes,” and “deep cells” (Zimmermann
1923; Hirschi and D’Amore 1996; Bergers and Song 2005; Dore-Duffy 2008;
Bergers 2008; Krueger and Bechmann 2010; Armulik et al. 2011; Zouani et al.
2013; van Dijk et al. 2015; see Collett and Canfield 2005, for review).

13.2.2 The Origin of the Pericytes

Early studies refer to different sources of the pericyte origin such as the mesenchy-
mal stem cells (Huhtaniemi and Pelliniemi 1992; Gondos 1980; Sbanti et al. 2007;
Dore-Duffy 2008) or, in relation to their neuroectodermal properties, the neural
244 M. S. Davidoff

crest stem cells (NCSCs; Davidoff et al. 2004, 2009; Calloni et al. 2007; Le Douarin
et al. 2008; Dore-Duffy 2008) as well as the peritubular myoid cells (Ariyaratne
et al. 2000). There is also a proposal that all pluripotent stem cells of the organism
belong to the germ lineage (Ratajczak et al. 2007). However, Brons et al. (2007)
provide evidence that the epiblast stem cells are not derivatives of the primordial
germ cells. Also the human testis-derived multipotent stem cells do not arise from
germ cells (Chikhovskaya et al. 2012, 2014). Gonzalez et al. (2009) termed cells
isolated from human testis as gonadal stem cell (GSC) which express pluripotent
stem cell markers Oct4, Nanog, and SSEA-4. These GSCs have growth kinetics and
marker expression similar to MSCs and differentiate into mesodermal lineage cells.
In the testis were found also embryonic cell-like cells (Mizrak et al. 2010). Most
likely this cell variability reflects the transit-amplifying cells of the pericyte ances-
tors in different stages of their maturation and differentiation (Nakagawa et al.
2007).
Concerning the origin of the pericytes, there is actually no consensus beside that
they originate from stem cells. However, it became strongly evident that the most
possible pericyte ancestor is the early pluripotent embryonal epiblast. In the follow-
ing some arguments for this conclusion will be presented.

13.2.2.1  elationship Between the Pericytes and the Multipotent Stromal

R
Cells

The pericytes are the ancestors of the multipotent stromal cells and the only true
adult stem cell phenotype of the vertebrate organism.
One main problem is that numerous authors do not make difference between
pluripotent pericytes and multipotent stromal cells (MSCs) (Özen et al. 2012;
Gökçinar-Yagei et al. 2015; Guimarães-Camboa et al. 2017; but see Cano et al.
2017; see Wong et al. 2015). As Skinner et al. (1985) noted, pericytes are often
mixed up with adjacent cell types of the vascular wall, the perivascular space, and
the juxtavascular parenchyma. This is important because the most experimentally
isolated and cultured mesenchymal stem cells consist of a mixture of both: pericytes
and adventitial stem/progenitor cells.
As mentioned, the pericytes have a periendothelial position, whereas the MSCs
are situated perivascularly (Rolandsson et al. 2014). Numerous studies postulate
that pericytes and multipotent stromal cells (MSCs; until now dubbed mesenchymal
stem cells) are the same cells or that all mesenchymal stem cells are pericytes
(Bianco et al. 2008; Crisan et al. 2008a; Caplan 2008; Diaz-Flores et al. 2009;
Collas 2010; da Silva Meirelles et al. 2013). This view is partially correct, but there
is now strong evidence that the pericytes are the ancestors of stromal cells (Caplan
2008; Crisan et al. 2008a; Ratajczak et al. 2008; Lindner et al. 2010; Collas 2010;
Zimmerlin et al. 2010, 2012; Murray et al. 2014; Trost et al. 2016), because espe-
13 The Pluripotent Microvascular Pericytes Are the Adult Stem Cells Even in the Testis 245

cially the earlier MSCs—descendants share close similarities with their pericyte
ancestors (Ochs et al. 2013).
Pericytes are highly undifferentiated pluripotent cells (De-Miguel et al. 2010;
Dar et al. 2012), whereas the MSCs as more differentiated are multipotent. For
example, cultured bone marrow-derived pericytes showed stem cell properties, with
higher plasticity (Kucia et al. 2005) and immaturity state than standard MSCs
(Bouacida et al. 2012).
Furthermore, a lot of authors interpret the existence or the absence of one or two
marker substances as enough to define or deny a different lineage of cells, which are
in fact pericyte daughter cells in various stages of differentiation. There are results
showing that the MSCs (APCs, adventitial progenitor cells) in cell culture condi-
tions possess the capability to dedifferentiate into pericytes: “centripetal” relation-
ship (Pacini and Petrini 2014; Rowley and Johnson 2014; Chen et al. 2017). The
reason for such events is the great plasticity of the stem cells appearing especially
during experimental or pathological conditions.
As mentioned, pericytes are also the ancestors of the adult perivascular multipo-
tent mesodermal cells that represent the migrating transit-amplifying descendants
of the periendothelial pericytes. Crisan et al. (2008b) found that pericytes and vas-
cular smooth muscle cells express mesenchymal stem cells (MSCs) markers in
human tissues and behave as MSCs in vivo as well as after heterogeneous transplan-
tation. The pericytes contribute to tissue-specific lineages in vivo through the trans-
differentiation of their transit-amplifying mesodermal progeny which is primarily
located perivascularly (Sbanti et al. 2007; Dellavalle et al. 2007; Tang et al. 2008;
Traktuev et al. 2008; Chen et al. 2015) .
Outside the vascular niche, the progeny of the pericytes behave as multipotent
stromal stem cells. These cells undergo genetic changes (Stanley et al. 2011) and
obtain different morphology and function in comparison with their ancestors. This
is an important event that caused numerous investigators to be not able to recognize
the fast transdifferentiation of the pericytes and to define the transit-amplifying cells
not as progenitor cells but as stem cells. This fact leads to the contradictory interpre-
tations of a number of results in the literature and the determination of the lineage
relationship between the ancestors and the progeny of a cell type (Davidoff et al.
2009; Davidoff 2017). Especially in the testis, the pluripotent pericytes are Leydig
stem cells, and their migrating progeny in the interstitium are the multipotent stro-
mal cells or mesodermal stromal cells with spindle-shaped form. It is also possible
that the peritubular myoid cells (PMC) of the lamina propria of the seminiferous
tubules represent differentiating multipotent PMC progenitor cells. The same is true
for the testis tissue pericyte-macrophages (Thomas 1999).
There is also strong evidence that adipocytes arise from progenitor cells (mural
cells, pericytes) of the adipose vasculature (Kahn 2008; Tang et al. 2008).
Moreover, the pericytes have the most potent adipogenic potential (Zimmerlin
et al. 2010). In addition, the combination of endothelial progenitor cells with adi-
pose stem cells in ischemic tissues shows a distinct higher neovascularization
capacity in comparison to a single stem cell application (Traktuev et al. 2008).
246 M. S. Davidoff

Wang et al. (2012) were able to perform induced differentiation of human adipose-
derived stem cells into Leydig cells.

13.2.2.2 The Pericytes and the Peritubular Myofibroblast Cells

There is strong evidence that during normal development and after EDS treatment
(between days 20 and 30), new Leydig cells were observed in peritubular position
as sheaths surrounding the seminiferous tubules (Davidoff et al. 2009; Fig. 13.7). A
number of authors presume that these Leydig cells arise from the peritubular myoid
stem cells (Maekawa et al. 1996; Ariyaratne et al. 2000; Ge et al. 2006; Stanley et al.
2012). However, some of the authors designate these cells as Leydig cell progeni-
tors (PDGF-A positive) which is more accurate (Fecteau et al. 2006). Studies con-
cerning the testis development describe that the peritubular myoid cells are
interstitial stromal cells that are attracted by the Sertoli cells, both building the basal
membrane and extracellular matrix which stabilize the differentiating seminiferous
tubules (Skinner et al. 1985; Svingen and Koopman 2013). We presume that the
peritubular myoid cells are also pericyte progeny (see Hall 2006; Diaz-Flores et al.
2012). It is well known that the activated pericytes generate myofibroblasts under
different physiological and pathological circumstances (Ribatti and Vacca 2008;
Kennedy et al. 2014; Hosaka et al. 2016; Rowley and Johnson 2014). After the
seminiferous tubules become created, developing Leydig cells in form of transit-­
amplifying cells become attracted, to build an additional fetal-type Leydig cell
sheath around the tubules. These Leydig cells provide the tubules and the peritubu-
lar myoid cells with hormones necessary for the final differentiation of the tubular
structures.
EDS treatment of adult rats also leads to damage of the seminiferous
epithelium.

Fig. 13.7 (9)

Immunoreactivity for
Cytochrome P450
cholesterol side chain
cleavage enzyme
(CytP450scc) in young rat
Leydig cells on day 21
after EDS treatment. Note
the massive peritubular
accumulation. Over some
tubules fragments a lower
number or no Leydig cell
are seen (arrow), probably
at sites where the
spermatogenesis in the
tubules became restored.
×450 (from Davidoff et al.
2004)
13 The Pluripotent Microvascular Pericytes Are the Adult Stem Cells Even in the Testis 247

The fact that early Leydig cells accumulate around tubules with impaired sper-
matogenesis suggests that Sertoli cell- or germ cell-derived factors promote the
increase in number and activity of adult-type Leydig cells (Kerr and Sharpe 1985;
Paniagua et al. 1988; Sharpe 1994; Teerds 1996). It is known that Leydig cell num-
bers can change in adult species depending on the stages of spermatogenesis
(Paniagua et al. 1988). In addition, there is strong evidence for a narrow relationship
between Leydig cells and Sertoli cells with respect to the regulation of their number
and functional activity (Sharpe 1994).
The close relationship between the blood vessels and the seminiferous tubules
provides the best conditions for exchange and influence of substances. Thus, the
migrating pericyte progeny move along the peritubular vessels and remain in con-
tact with the tubular surface after being attracted and linked by the Sertoli cells and
the peritubular myofibroblasts. In this respect, the Sertoli cells, the peritubular
myoid cells, and the Leydig progenitor cells exchange signals that regulate these
events. The most important among them are the PDGF proteins and their receptors,
the androgen receptors, and some other factors. For example, Fecteau et al. (2006)
report that in the rat, fetus PDGF-A expression corresponds with the rapid prolifera-
tion (Basciani et al. 2002—fetal and adult human testis) and the testosterone expres-
sion of the Leydig cells. PDGFs were demonstrated in rat Sertoli cells which attract
αSMA-expressing peritubular myoid cells (PMCs) in close proximity to the semi-
niferous tubules (Gnessi et al. 1995, 2000). Mariani et al. (2002) found that PDGF-­-
B/α-ß interactions may be involved in the proliferation and migration of peritubular
myoid cell (PMC) precursors from the intertubular space to the peritubular. Ergün
et al. (1994, 1999) provide evidence that endothelin produced in Leydig and Sertoli
cells can act in a paracrine manner via ET-B receptors in the human testicular micro-
vasculature and the peritubular myofibroblast. In addition, Ergün et al. (1996) found
in the lamina propria of the human testis intramural vessels which are partly fenes-
trated and possess pericytes. This seems to be partially true for the rat testis in which
accumulation of peritubular regenerating Leydig cells in capillaries closely running
peritubularly has been observed (Fig. 13.6; Davidoff et al. 2004, 2009). Earlier
study by Ge et al. (2006) provide evidence that the testicular pericytes are PDGFR-α
and PDGFR-ß positive and that at 1 week of postnatal development spindle-shaped
cells in the testicular interstitium, positive for PDGFR-α, differentiate into adult
Leydig cells which become immunoreactive for all PDGFs and their receptors.
Concerning the androgen receptors (AR), it was shown that testicular AR mRNA
exists in Leydig cells, pericytes, peritubular myoid cells, and Sertoli cells (Shan
et al. 1995). The Leydig cells are maximally sensitive to androgen during puberty,
which is consistent with the hypothesis that androgens facilitate their differentia-
tion. As mentioned, Kilcoyne et al. (2014) found that the interstitial Leydig cell
progenitors express androgen receptors (Shan and Hardy 1992). In addition, it was
established that the androgen action via testicular peritubular myoid cells (PMC) is
essential for normal testis function, spermatogenesis, and fertility in males (Welsh
et al. 2009). Moreover, the androgen action via testicular arteriole smooth muscle
cells is important for Leydig cell differentiation, function, vasomotion, and fluid
248 M. S. Davidoff

dynamics (Welsh et al. 2010, 2011). There is no convincing proof that the PMCs are
the Leydig stem cells.
In conclusion, the peritubular myofibroblasts may be also derived by the pericyte
transit-amplifying cells. In combination with the Sertoli cells, the PMC becomes
peritubular cells that are important for the differentiation of the seminiferous tubules
and the steroidogenesis of the newly arising Leydig cells as well as for the fetal
microcirculation. Important significance for these processes seems to have PDGFs
and their receptors, the androgen receptors (ARs) and the COUP-TFII, and its
receptor H3K27me3 (Haider et al. 2007; Kilcoyne et al. 2014). The PTM-AR sig-
naling is important for normal development, ultrastructure, and function of the adult
LCs (Shan and Hardy 1992; Shan et al. 1995, 1997; Welsh et al. 2009, 2010;
Takamoto 2005; Shibata et al. 2001– in human adrenals). COUP-TFII plays impor-
tant role in progenitor Leydig cells formation and early testis organogenesis. In
addition, it plays a major role in differentiation but not maintenance of Leydig cells
(You et al. 2005).
In addition, new findings established that testicular macrophages play essential
roles in normal testis homeostasis and fetal testicular development (Bhushan and
Meinhardt 2017). Testicular macrophages are closely involved in regulating ste-
roidogenesis of Leydig cells and in maintaining homeostasis within the testis
(Goluža et al. 2014). Concerning the macrophages it has to be clarified what kind of
macrophages (pericyte-macrophages; originating from pericytes, Ling and Wong
1993; Balabanov et al. 1996; Thomas 1999; Mori and Leblond 1969; Barón and
Gallego 1972) or resident tissue macrophages (mesodermal cells from the yolk sac)
are responsible for these events (DeFalco et al. 2014).
The EDS destruction of the newly formed peritubular Leydig cells leads to sig-
nificant lowering of their number, but the new building of interstitial Leydig cells
remains not affected. As Stanley et al. (2012) found that after EDS treatment, the
adult testis peritubular Leydig cells originate from stem cells that are not macro-
phages, pericytes, and smooth muscle cells of vessels. At the same time, they estab-
lished that the removal of newly differentiated, testosterone-producing LCs from
the tubule surfaces did not result in the depletion of the precursor cells. Rather, new
Leydig cells were able to form repeatedly. It is also interesting to note that when
progenitor Leydig cells acquire LH receptors, they are located partially away from
the peritubular region of the testis. In addition, they are definitely rounder in shape,
in contrast to their original spindle-shape configuration. These findings suggest that
in cells committed to the Leydig cell lineage, acquisition of LH receptors occurs
during a stage of development later than that at which they first gain the steroido-
genic enzymes. Accordingly, these findings suggest that the function of LH in
Leydig cell differentiation occurs subsequent to formation of the progenitor cells
(i.e., transformation of progenitors to immature and then mature Leydig cells;
Stanley et al. 2012). According to our hypothesis, these results confirm that the
progenitor cells of these new Leydig cells are transit-amplifying pericyte progeny
which after activation migrate toward the tubules surface. The pericyte ancestors
become activated through the lowering of the testosterone production and e­ ventually
the hypoxia arising under these conditions. The authors were unable to notice the
13 The Pluripotent Microvascular Pericytes Are the Adult Stem Cells Even in the Testis 249

fast transdifferentiation process to found the important relationship between the

pericyte stem cells and their transit-amplifying cells, the Leydig cell progenitors. As
mentioned above, the transit-amplifying cells in their further differentiation undergo
changes and start to produce factors such PDGFR-α. We would like to underline
that during testis development, the new arising Leydig cells remain probably the
whole life in direct contact with the testis vasculature through their long branches
(Figs. 13.8 and 13.9).
There is strong evidence that the pericyte progeny change the biochemical com-
position during its commitment to immature and further differentiation to adult,

Fig. 13.8 (10) Nestin immunoreactivity in activated pericytes 14 days after EDS application. Note
the spindle-shaped form of the pericytes on the capillary wall (thick arrows). The migrating peri-
cytes form aggregates in the vicinity of the vessels. Some pericytes have still contact with the
vessel wall (thin arrow). ×950 (from Davidoff et al. 2004)

Fig. 13.9 (11) Photo

montage of Rat testis
interstitium.
Immunoreactivity for
CytP450scc on day 60 of
postnatal development.
Numerous positive young
(immature) Leydig cells
possess spindle-shaped
form (large arrows). Their
outgrowths show contacts
with the vessel walls or
cover the walls over long
distances. The bodies of
cross-sectioned Leydig
cells in contact with the
vessel (asterisk) are round
or oval (small arrows). T
tubules. ×380 (from
Davidoff 2017)
250 M. S. Davidoff

mature Leydig cells (Stanley et al. 2011). The leading event is the process of fast
transdifferentiation (Tosh and Slack 2002, p. 128; Shen et al. 2004a, b; Tavazoie
et al. 2008) in which the early progeny of the pericytes become transformed into
transit-amplifying cells, immature and mature Leydig cells that lose their similari-
ties with the stem cell ancestors. This process affects also the potency of the stem/
progenitor cells. The process of transdifferentiation of the transit-amplifying stem
cell progeny, under the existing environmental factors, has led to misrecognition of
some structures and to misinterpretation of the results obtained in a lot of previous
studies (de Souza et al. 2016—MSCs produce pericytes?). For example, Klein et al.
(2011, 2014) and Chan-Ling (1997) show that CD44+ multipotent stem cells dif-
ferentiate into pericytes and smooth muscle cells. In addition Klein et al. (2014)
show that Nestin+ tissue-resident multipotent stem cells stimulate tumor progres-
sion by differentiating into pericytes and smooth muscle cells. On the other hand,
Stanley et al. (2012) informed that the peritubular cells that are immunoreactive for
PDGFR-α and negative for 3-ßHSD are stem Leydig cells. In similar cells 6 days
after EDS treatment, Kilcoyne et al. (2014) found COUP-TFII immunoreactivity. In
other studies the PDGFR-α-positive spindle-shaped interstitial cells of 1 week post-
partum rats were called putative SLCs (Ge et al. 2006) or correctly designated as
peritubular progeny of Leydig stem cells (Landreh et al. 2013, 2014) in 8, 20, 40,
and 69 dpp rats.
It seems very likely that one of the earliest changes of the pericyte progeny in the
testis is the expression of the PDGFR-α (see Brennan et al. 2003; Stanley et al.
2012), followed by the expression of other factors such as Coup—TFII, CD51,
CD90, 3ß-HSD, and additional enzymes involved in the process of steroidogenesis.
These cells are correctly to be defined as progenitor Leydig cells (Chen et al. 2017;
Ye et al. 2017) because these are certainly migrating transit-amplifying cells in pro-
cess of maturation. However, in numerous publications they are qualified as adult
Leydig stem cells produced, for example, from COUP-TFII-expressing, undifferen-
tiated, spindle-shaped interstitial cells, that do not express adult Leydig cell markers
(see Kilcoyne et al. 2014). However, the authors of these studies neglect the fact that
COUP-FMII is actively involved in the formation of the microcirculation of an
organ, and that between the stem cells and the first differentiating Leydig cells, no
genetic similarity was found. The reason for this is that the differentiated Leydig
cells appear after their transdifferentiation from the pluripotent pericytes and via
multipotent transit-amplifying cells that further develop to differentiate into adult
cells. This means that the transit-amplifying cells differentiate as progenitors of the
LCs and are descendants of the pericytes. The transit-amplifying cells develop also
as mesenchymal stem cells in the different organs. Thus, the described cells are
Leydig cell progenitors, the progeny of the stem cell pericyte that represent the
epiblast pluripotent cells in the mature organism.
It is important to note that Kilcoyne et al. (2014) established that the Coup-TFII
cells express also androgen receptors. This result provides additional evidence that
the cells under study are differentiating progenitor cells and not stem cells.
Furthermore, the spindle shape of the interstitial cells is not a solid argument, while
pericytes as well as their progeny, the transit-amplifying and immature (young)
13 The Pluripotent Microvascular Pericytes Are the Adult Stem Cells Even in the Testis 251

Leydig cells have the same shape (Figs. 13.8 and 13.9). As mentioned above, the
COUP-TFII protein appears to some extent later than the PDGFR-α, which is the
first marker that appear in the pericyte progeny.

13.2.2.3 Pericytes and the Neural Crest

The pericytes and the Leydig cells are not progeny of the neural crest (Breau et al.
2008; Weston et al. 2004; Weston and Thiery 2015; Gilbert 2000; Lee et al. 2013a, b).
The neural crest cells (NCCs) are transient appearance that originate at the neural
plate border and reside within the elevating neural folds and dorsal neural tube after
its closure (Shyamala et al. 2015). Because of its neuroectodermal (Davidoff et al.
2009) and multipotent qualities (neural and mesodermal progeny), the neural crest
was designated by a number of scientists as “the fourth germ layer” (Hall 1998,
2000; Gilbert 2000; Le Douarin et al. 2008; Shyamala et al. 2015; Bronner and
Simões-Costa 2016). In this respect Davidoff et al. (2009) presumed that the peri-
cytes may derive from neural crest cells or from a common stem cell population
segregated early in embryogenesis with progeny for both the neural crest and the
mesoderm lineage (mesectoderm, mesenchymal-neural).
However, it becomes evident that the neural crest stem cells (NCSCs) originate
later from the ectoderm than the early from the epiblast occurring pericytes
(Fig. 13.10). Already Kastschenko (1888) announced that many cells of the neural
crest in the head of the Salachian embryo do not take part in the formation of the
nervous system but give rise to reticular mesoderm or mesenchyme. New results
also show that the authentic NCSCs do not possess mesenchymal properties and are
not able to produce cells with mesenchymal features (Hill and Watson, 1958;
Nichols, 1981; Weston et al. 2004; Breau et al. 2008; Lee et al. 2013a, b; Kennedy
et al. 2014). As Donoghue et al. (2008) reported, an epithelial-to-mesenchymal tran-
sition undergoes a second distinct population of cells within the neural fold. These
cells have mesectodermal (ectomesenchyme) lineage potency and are termed meta-
blast (non-neural epithelial domain) that in the neural fold (NF) differentiate in the
vicinity of the authentic NCSCs and are responsible for the regulation of the migra-
tion and fate of the authentic cranial neural crest-derived cells (Weston et al. 2004;
Weston and Thiery 2015).The mouse cranial NF epithelium is heterogeneous, and a
sharp boundary exists between the lateral non-neural epithelium and the thickened
neural epithelium in the dorsomedial ridge (see also Thiery et al. 1984; Newgreen
et al. 1997; Denham et al. 2015). These observations raise the possibility that at
least some PDGFR-α ectomesenchyme originates from the lateral non-neural
domain of the neural fold epithelium. Previously Lee et al. (2013a, b) and Nichols
(1981, 1986) presumed that mesenchymal cells emerge precociously from an epi-
thelial neural fold domain resembling the primitive streak in the early embryonic
epiblast, and therefore Weston et al. (2004) propose the name “metablast” for this
non-neural epithelial domain to indicate that it is the site of a delayed local delami-
nation of mesenchyme similar to involution of mesoderm during gastrulation. Breau
et al. (2008) further propose the hypothesis that neural crest and ectomesenchyme
252 M. S. Davidoff

Fig. 13.10 (12) Represents the very early stages of the zygote and blastocyst differentiation. Note
that the first cell populations derived from the epiblast are the primordial germ cells, the hemato-
poietic stem cells, and the pericytes, whereas the neural crest originates a bit later from the one of
the primitive embryonic germ layers, the ectoderm (epiblast/ectoderm)

are developmentally distinct progenitor populations and that at least some ectomes-
enchyme is metablast-derived rather than neural crest-derived tissue.
To explain some events with the pericytes and keeping in mind that the Leydig
cells possess neural properties, we used information concerning the situation in the
central nervous system. As mentioned above there is a great similarity between the
biology of the developing and adult Leydig cells and the progenitors/immature/
mature nerve and glial cells. Also in the nervous system until recently, the brain
vasculature/pericytes were neglected. However, the first evidence was provided by
Palmer et al. (2000) who found that adult neurogenesis occurs within an angiogenic
niche (see Louissaint et al. 2002; Shen et al. 2004a, b). Later it becomes evident that
subventricular in the telencephalon, a very well-developed vascular plexus exists
(Tavazoie et al. 2008, Shen et al. 2008).
According to Itoh et al. (2011), pericytes and astrocytes play important role for
the maintenance of the cerebral microvasculature. In this respect, it seems very
probable that the brain microvascular pericytes possess the capability to transdif-
ferentiate into radial astroglia (Alvarez-Buylla et al. 2002; Filippov et al. 2003; Hill
et al. 2004; Sild and Rithazer 2011). Thus the conclusion can be made that the
­pericytes of the subventricular plexus are the neural stem cells (Doetsch 2003)
which generate the B progenitors (GFAP+ astrocytes; radial glia), from which the C
(transit amplifying) cells and the A (neuroblast) cells that are surrounded by an glial
13 The Pluripotent Microvascular Pericytes Are the Adult Stem Cells Even in the Testis 253

tunnel that lead them toward the olfactory bulb (see Gonzalez-Perez 2012; Jiang
et al. 2003; Morshead 2004) differentiate. This hypothesis is also supported by the
observation that Nestin and NG2-positive cells are precursors of neurons, oligoden-
drocytes, and astrocytes in the gray matter (Wei et al. 2002; Zhu et al. 2008). Similar
conditions were reported for the subgranular zone of the hippocampal dentate gyrus
(Filippov et al. 2003; Bischofberger and Schmidt-Hieber 2006; Hara et al. 2010).
The similarity between the NCSCs and the pericytes is an unequivocal fact
(Schulze et al. 1987; Angelova and Davidoff 1989; Angelova et al. 1991; Chiwakata
et al. 1991; Davidoff et al. 1993, 1996, 2001; Middendorff et al. 1993; Benton et al.
1995). This fact allowed us and others to determine the Leydig cells as neuroendo-
crine cells and lead to the presumption that the pericytes, resp., the Leydig cells are
of neural crest origin (see Davidoff et al. 2004, 2009; Diaz-Flores et al. 2009).
However, we were convinced by new results that the reason for this similarity is the
close embryologic timing in arising of these cell types (Fig. 13.8).
Consequently, the neural crest cells arise at the border of the neural plate and the
epidermis at the time of neural tube closure (Le Douarin and Dupin 2003;
Barembaum and Bronner-Fraser 2005; Shyamala et al. 2015). As new data show, in
the embryonic ectoderm of the neural folds, two different cell populations coexist:
authentic neural crest cells and mesectodermal stem cells (Weston et al. 2004; Breau
et al. 2008; Weston and Thiery 2015).The neural crest cells, derived from the epi-
blast/ectoderm, possess some restricted multipotent characteristics and represent a
migratory cell population that generates diverse cell types during vertebrate devel-
opment (Trainor et al. 2003; Trainor 2005; Crane and Trainor 2006). In comparison,
the pericytes originate something earlier direct from the epiblast in contrast to the
neural crest stem cells that arise a bit later from the primary germ layer, the
ectoderm, which is also a derivate of the epiblast (epiblast/ectoderm; Fig. 13.8). In
this respect Kojima et al. (2014) reveal that the epiblast cells possess different tran-
scriptomes in comparison with the epiblast/ectoderm cells. Thus, the NCSCs arise
indirectly from the epiblast, and as recent results show they are not pluripotent but
multipotent and are able to produce different cell types with neural qualities, but not
mesenchymal cells (Brennan et al. 2003; Ortega et al. 2004a; Yao and Barsoum
2007; Breau et al. 2008; Kennedy et al. 2014).
There is also evidence that the neural crest cells cannot produce pericytes (Brennan
et al. 2003; Joseph 2004; Weston et al. 2004; Breau et al. 2008; Griswold and
Behringer 2009; Makala et al. 2015). Therefore the only possibility that remains is
that the pericytes are the progeny of the epiblast, whereas the neural crest is the
daughter structure of the a bit later arising epiblast/ectoderm (De-Miguel et al. 2009).
It has to be noted that the neural crest cells arise and migrate after the origin of
the developing vasculature and use the blood vessels and the associated extracellu-
lar matrix as leading structures (Spence and Poole 1994). Accordingly, the pericytes
are disseminated before the migration of the neural crest stem cells and are not
produced by them. Thus, the pericytes, resp., the Leydig cells, are not neural crest
derivatives.
254 M. S. Davidoff

13.2.2.4  he Pericyte Progenitors Arise Very Early

T
During Embryogenesis from the Epiblast

The fact that the mesenchymal stem cells are progeny of the pericytes and the
authentic neural crest cells and the peritubular myofibroblasts of the lamina propria
of the seminiferous tubules are not able to produce mesectoderm allows the conclu-
sion that only the embryonic epiblast can be ancestor of the pericytes. This is sup-
ported also by the results which provide evidence that only the epiblast stem cells
are pluripotent, whereas the other two cell types are multipotent.
It becomes evident that the pericytes represent the only true pluripotent adult
stem cell in the mature vertebrate organism. In accordance, De-Miguel et al. (2009,
2010) and Nichols and Smith (2012) showed that pluripotency is confined to the
epiblast, a transient structure that persists for only few days. Only the pericytes (as
epiblast progeny) are able in vivo and in vitro to produce structures related to the
three main germ layers: ectoderm, mesoderm, and endoderm. The other candidates,
namely, the multipotent stromal cells and the neural crest stem cells are multipotent
cells (Chikhovskaya et al. 2012, 2014). They are partially committed and with lim-
ited potency. In this respect Dore-Duffy (2008) wrote: “We propose that once the
pericyte has migrated into the perivascular space it makes functional and pheno-
typic adjustments or adaptations that may include differentiation along multiple
lineages and that this is the basis for their pluripotentiality.”
Thus, in the vascular niche, the pericytes remain as resting pluripotent stem cells.
After their activation, proliferation, and migration, they become transit-amplifying
cells that transdifferentiate to give different multipotent progenitors. With the con-
tinuation of the differentiation, the progenitor cells lose their multipotency and
become oligopotent and unipotent.
There are numerous publications which presume that the adult stem cells are of
multiple origins and correspondingly show different chemical content (markers)
and other features (see Davidoff et al. 2009; Davidoff 2017). However, these differ-
ences reflect most probably the great plasticity of the pluripotent adult stem cells
depending on their position in the niches, stage of differentiation, and their behavior
under the influence of various local biophysical and biochemical factors.
As noted, recent studies provide evidence that the true adult stem cells must be
the progeny of the epiblast. The epiblast is the product of the inner cell mass at the
early blastocyst stage of embryogenesis (Kaufman 1992; Kucia et al. 2006;
Ratajczak et al. 2008, 2012a, b; Montiel-Eulefi et al. 2009), and the SSEA-1-positive
mesenchymal stem cells are thought to be the most primitive progenitors in the
murine bone marrow mesenchymal compartment (Anjos-Afonso and Bonnet 2007;
Ratajczak et al. 2008). There is strong evidence that the epiblast is also the ancestor
of the primordial germ cells (PGCs; McLaren 2003; Brons et al. 2007; Montiel-­
Eulefi et al. 2009; Ratajczak et al. 2007; De-Miguel et al. 2009) and the h­ ematopoietic
stem cells (HSCs). Brons et al. (2007) emphasized that the epiblast stem cells are
not derivatives of the PGCs. Yoshimizu et al. (2001) established that during gastru-
lation (De-Miguel et al. 2009, 2010), the epiblast cells undergo epithelial-to-­
mesenchymal transition and delaminate to become the loose mesenchyme of the
13 The Pluripotent Microvascular Pericytes Are the Adult Stem Cells Even in the Testis 255

primitive streak. Epiblast stem cells become also included as pericytes within spe-
cific vascular niches (Nikolova et al. 2007; Scadden 2006) of the developing and
growing blood vessels with which they become disseminated through the whole
body. As already mentioned the cardiovascular system is the first functional organ
system in the vertebrate embryo (Flamme et al. 1997). This is important to note
while it evidenced that pericytes as stem cells are present very early during the
embryo development in the emerging tissues and organs where they can participate
in the vascularization, origin, and organization of the corresponding structures and
in addition remain as omnipresent adult stem cells in the vascular niches for tissue
maintenance, repair, and regeneration (Dore-Duffy 2008).
The pluripotency of the pericytes is the reason for both the mesenchymal and
neural qualities of the Leydig cells of the testis. As mentioned above the pericytes
are not produced by the neural crest stem cells. By means of the pericyte ancestors,
they get their own neuroendocrine quality. This is also true for the determination
that the pericytes originate from mesoderm. The brain pericytes are stem cells
immunoreactive for αSMA, γ-glutamyl transpeptidase, alkaline phosphatase, nes-
tin, vimentin, and PDGFR-ß (Fisher 2009). These markers are also expressed in the
testicular pericytes.

13.3 Conclusion

It becomes evident that the pericytes are periendothelial pluripotent stem cells
which can differentiate into cells, tissues, and organs of all three main embryonic
germ layers. They arise in the early embryogenesis from the pluripotent epiblast,
become disseminated with the differentiating vasculature of the embryo, and stored
in true vascular niches as reserve pluripotent adult stem cells for tissue generation,
maintenance, repair, and regeneration of the structures in the vertebrate organism.
The pericytes are really the adult stem cells and the ancestors of a number of vari-
able progenitor cells. Pericytes are authentic stem cells only until the moment they
leave the vascular niche.
After the formation of the seminiferous tubules, the peritubular immature Leydig
cells reduce in number or disappear. In some publications this phenomenon is
explained with a back migration of these cells toward the interstitial space of the
testis where they accumulate in close vicinity of the blood vessels. However, a more
plausible explanation is that at this time of the fetal development, fewer hormones
are needed for the composition, maintenance, and functioning of the seminiferous
tubules which lead to the disappearance of the unnecessary cells. This event resem-
bles the involution of the fetal Leydig cells during the testis development (Orth and
Weisz 1980). As consequence the interstitial perivascular cells increase in number
and become enlarged. They remain in contact with the blood vessel wall and supply
hormones for the male design of the organism. This phenomenon is well known for
other organs, especially for the nervous system. During the creation and regenera-
tion of the nervous system for insurance, an excess number of nerve cells get
256 M. S. Davidoff

formed. With it, only cells that succeeded to make contacts with a target structure
remain alive. The other cells die and disappear.
The described features of the testicular pericytes provide enough evidence that
these cells belong to the omnipresent pericyte population of a vertebrate organism.
There is a great variability of the pericyte descendants comprising these cells in dif-
ferent migration position, developmental stages, and contacts with structures in
their environment. All this explained the great plasticity of the whole pericyte popu-
lation of an organism and the difficulties in the recognition, lineage determination,
and definition of the pericyte progeny (Hermann et al. 2016).
The pericytes are a brilliant achievement of the biological nature aiming to sup-
ply every organ with an omnipresent population of pluripotent adult stem cells.

References

Abedin M, Tintut Y, Demer LL (2004) Mesenchymal stem cells and the artery wall. Circ Res
95:671–676
Alvarez-Buylla A, Seri B, Doetsch F (2002) Identification of neural stem cells in the adult verte-
brate brain. Brain Res Bull 57:751–758
Angelova P, Davidoff MS (1989) Immunocytochemical demonstration of substance P in Hamster
Leydig cells during ontogenesis. Z Mikrosk Anat Forsch 103:560–566
Angelova P, Davidoff MS, Baleva K, Staykova M (1991) Substance P and neuron-specific enolase-­
like immunoreactivity of rodent Leydig cells in tissue section and cell culture. Acta Histochem
91:131–139
Anjos-Afonso F, Bonnet D (2007) Nonhematopoietic/endothelial SSEA-1+ cells define the most
primitive progenitors in the adult murine bone marrow mesenchymal compartment. Blood
109:1298–1306
Ariyaratne HB, Mendis-Handagama SMLC, Hales DB, Mason JI (2000) Studies on the onset of
Leydig precursor cell differentiation in the prepubertal rat testis. Biol Reprod 63:165–171
Armulik A, Genové G, Betsholtz C (2011) Pericytes: developmental, physiological, and pathologi-
cal perspectives, problems, and promises. Dev Cell 21:193–215
Attwell D, Mishra A, Hall CN, O’Farrell FM, Dalkara T (2016) What is a pericyte? J Cerebr Blood
Flow Metab 36:451–455
Balabanov R, Washington R, Wagnerova J, Dore-Duffy P (1996) CNS microvascular pericytes
express macrophage-like function, cell surface integrin αM, and macrophage marker ED-2.
Microvasc Res 52:127–142
Barón M, Gallego A (1972) The relation of the microglia with the pericytes in the cat cerebral
cortex. Z Zellforsch Mihosk Anat 128:42–57
Basciani S, Mariani S, Arizzi M, Ulisse S, Rucci N, Jannini EA, Rocca CD, Manicone A, Carani
C, Spera G, Gnessi L (2002) Expression of platelet-derived growth factor-A (PDGF-A),
PDGF-B, and PDGF receptor- a and -s during human testicular development and disease.
J Clin Endocrinol Metab 87:2310–2319
Benninghoff A (1926) Über die Formenreihe der glatten Muskulatur und die Bedeutung der
Rouget‘schen Zellen an den Kapillaren. Z Zellf Mikr Anat 4:126–170
Bensley RR, Vimtrup BJ (1928) On the nature of the Rouget cells of capillaries. Anat Rec 39:37–55
Benton L, Shan L-X, Hardy MP (1995) Differentiation of adult Leydig cells. J Steroid Biochem
Mol Biol 53:61–68
Barembaum M, Bronner-Fraser M (2005) Early steps in neural crest specification. Seminars in
Cell & Developmental Biology; 16(6): 642-646
13 The Pluripotent Microvascular Pericytes Are the Adult Stem Cells Even in the Testis 257

Bergers G (2008) Pericytes, the mural cells of the microvascular system. In: Figg WD, Folkman
J (eds) Angiogenesis. An integrative approach from science to medicine, Chapter 4. Springer,
New York, pp 45–53
Bergers G, Song S (2005) The role of pericytes in blood-vessel formation and maintenance. Neuro
Oncol 7:452–464
Bhagavati S (2008) Stem cell based therapy for skeletal muscle diseases. Curr Stem Cell Res Ther
3(3):219–228
Bhushan S, Meinhardt A (2017) The macrophages in testis function. J Repr Immun 119:107–112
Bianco P, Robey PG, Simmons PJ (2008) Mesenchymal stem cells: revisiting history, concepts,
and assays. Cell Stem Cells 2:313–319
Birbrair A, Delbono O (2015) Pericytes are essential for skeletal muscle formation. Stem Cell Rev
Rep 11:547–548
Birbrair A, Zhank T, Wang ZM, Messi ML, Enikolopov GN, Mintz A, Delbono O (2013a) Role of
pericytes in skeletal muscle regeneration and fat accumulatioin. Stem Cells Dev 22:2298–22314
Birbrair A, Zhang T, Wang Z-M, Messi ML, Enikolopov GN, Mintz A, Delbono O (2013b) Skeletal
muscle neural progenitor cells exhibit properties of NG2-glia. Exp Cell Res 319:45–63
Birbrair A, Zhang T, Files DC, Mannava S, Smith T, Wang Z-M, Messi ML, Mintz A, Delbono
O (2014) Type-1 pericytes accumulate after tissue injury and produce collagen in an organ-­
dependent manner. Stem Cell Res Ther 5:122
Bischofberger J, Schmidt-Hieber C (2006) Adulte Neurogenese im Hippocampus. e-Neuroforum
3:212–221
Bjornson CRR, Rietze RL, Reynolds BA, Magli MC, Vescovi A (1999) Turning brain into blood:
adult neural stem cells adopt a hematopoietic fate in vivo. Science 283:534–537
Bouacida A, Rosset P, Trichet V, Guilloton F, Espagnolle N, Cordonier T, Heymann D, Layrolle P,
Sensébé L, Deschaseaux F (2012) Pericyte-like progenitors show high immaturity and engraft-
ment potential as compared with mesenchymal stem cells. PLoS One 7(11):e486548
Brazelton TR, Rossi FMV, Keshet GI, Blau HM (2000) From marrow to brain: expression of
neuronal phenotypes in adult mice. Science 290:1775–1779
Breau MA, Pietri T, Stemmler MP, Thiery P, Weston JA (2008) A nonneural epithelial domain
of embryonic cranial neural folds gives rise to ectomesenchyme. Proc Natl Acad Sci U S A
105:7750–7755
Brennan J, Capel B (2004) One tissue, two fates: molecular genetic events that underlie testis
versus ovary development. Nat Rev Gen 5(7):509–521
Brennan J, Tilmann C, Capel B (2003) Pdgfr-α mediates testis cord organization and fetal Leydig
cell development in the XY gonad. Genes Dev 17:800–810
Bronner ME, Simões-Costa M (2016) The neural crest migrating into the 21st century. Curr Top
Dev Biol 116:115–134
Brons IGM, Smithers LE, Trotter MWB, Rugg-Gunn P, Sun B, Lopes SMCS, Howlett SK,
Clarkson A, Ahrlund-Richter L, Pedersen RA, Vallier L (2007) Derivation of pluripotent
epblast stem cells from mammalian embryos. Nature 448:191–196
Buehr M, Smith A (2003) Genesis of embryonic stem cells. Philos Trans R Soc Lond B Biol Sci
358:1397–1402
Calloni GW, Glavieux-Pardanaud C, Le Douarin NM, Dupin E (2007) Sonic hedgehog promotes
the development of multipotent neural crest progenitors endowed with both mesenchymal and
neural potentials. Proc Natl Acad Sci U S A 104:19879–19884
Cano E, Gebala V, Gerhardt H (2017) Pericytes or mesenchymal stem cells: is that the question?
Cell Stem Cell 20(3):296–297
Caplan AI (2008) All MSCs are pericytes? Cell Stem Cell 3:229–230
Cervós-Navarro J (1963) Electronenmicroscopische Befunden an den Capillaren der Hirnrinde.
Arch Psych Ztschr ges Neurologie 204:484–504
Chan-Ling T (1997) Glial, vascular, and neuronal cytogenesis in whole-mounted cat retina.
Microsc Res Tech 36:1–16
Chen H, Stanley E, Jin S, Zirkin BR (2010) Stem Leydig cells: From fetal to aged animals. Birth
Defects Res C Embryo Today 90:272–283
258 M. S. Davidoff

Chen WCW, Baily JE, Corselli M, Diaz ME, Sun B, Xiang G, Gray GA, Huard J, Péault B (2015)
Human myocardial pericytes: multipotent mesodermal precursors exhibiting cardiac specific-
ity. Stem Cells 33:557–573
Chen H, Wang Y, Ge R, Zirkin BR (2017) Leydig stem cells: identification, proliferation and dif-
ferentiation. Mol Cell Endocrinol 445:65–71
Chikhovskaya JV, Jonker MJ, Meissner A, Breit TM, Repping S, van Pelt AMM (2012) Human
testis-derived embryonic stem cell-like cells are not pluripotent, but possess potential of mes-
enchymal progenitors. Hum Reprod 27(1):210–221
Chikhovskaya JV, van Daalen SKM, Kover CM, Repping S, van Pelt AMM (2014) Mesenchymal
origin of multipotent human testis-derived stem cells in human testicular cell cultures. Mol
Human Reprod 20:155–167
Chiwakata C, Brackmann B, Hunt N, Davidoff M, Schulze W, Ivell R (1991) Tachykinin
(Substance P) gene expression in Leydig cells of the human and mouse testis. Endocrinology
128:2441–2448
Clark ER, Clark EL (1925a) The development of adventitial (Rouget) cells on the blood capillaries
of amphibian larvae. Amer J Anat 35:239–264
Clark ER, Clark EL (1925b) The relation of Rouget cells to capillary contractility. Amer J Anat
35: 265-282
Collas P (2010) Programming differentiation potential in mesenchymal stem cells. Epigenetics
5(6):476–482
Collett GDM, Canfield AE (2005) Angiogenesis and pericytes in the initiation of ectopic calcifica-
tion. Circ Res 96:930–938
Combes AN, Wilhelm D, Davidson T, Dejana E, Harley V, Sinclair A, Koopman P (2009)
Endothelial cell migration directs testis cord formation. Dev Biol 326:112–120
Crane JF, Trainor PA (2006) Neural Crest stem and progenitor cells. Ann Rev Cell Dev Biol 22
(1):267-286
Crisan M, Deasy B, Gavina M, Zheng B, Huard J, Lazzari L, Péault B (2008a) Purification and
long-term culture of multipotent progenitor cells affiliated with the walls of human blood ves-
sels: myoendothelial cells and pericytes. Methods Cell Biol 86:295–309
Crisan M, Yap S, Casteilla L, Chen C-W, Corselli M, Park TS, Andriolo G, Sun B, Zheng B, Zhang
L, Norotte C, Teng P-N, Trass J, Schugar R, Deasy BM, Badylak S, Bühring H-J, Giacobino
J-P, Lazzari L, Huard J, Péault B (2008b) A perivascular origin for mesenchymal stem cells in
multiple human organs. Cell Stem Cell 3:301–313
da Silva Meirelles L, Caplan AI, Nardi NB (2013) Pericytes as the source of mesenchymal stem
cells. In: Goldenberg RC d S, de Carvalho ACC (eds) Resident stem cells and regenerative ther-
apy. Elsevier, Amsterdam, pp 233–250. https://doi.org/10.1016/B978-0-12-416012-5.00012-8
Dar A, Domev H, Ben-Yosef O, Tzukerman M, Zeevi-Levin N, Novak A, Germanguz I, Amit M,
Itskovitz-Eldor J (2012) Multipotent vasculogenic pericytes from human pluripotent stem cells
promote recovery of murine ischemic limb. Circulation 125:87–99
Davidoff MS (2017) The Leydig cells of the testis originate from the microvascular pericytes.
Biomed Rev 28:5–25
Davidoff MS, Schulze W, Middendorff R, Holstein A-F (1993) The Leydig cell of the human
testis—a new member of the diffuse neuroendocrine system. Cell Tiss Res 271:429–439
Davidoff MS, Middendorff R, Holstein AF (1996) Dual nature of Leydig cells of the human testis.
Biomed Rev 6:11–41
Davidoff MS, Middendorff R, Koeva Y, Pusch W, Jezek D, Muller D (2001) Glial cell line-derived
neurotrophic factor (GDNF) and its receptors GFR a −1 and GFR a −2 in the human testis. Ital
J Anat Embryol 106(Suppl 2):p173–p180
Davidoff MS, Middendorff R, Enikolopov G, Rietmacher D, Holstein AF, Müller D (2004)
Progenitor cells of the testosterone-producing Leydig cells revealed. J Cell Biol 167:
935–944
Davidoff MS, Middendorff R, Müller D, Holstein AF (2009) The Neuroendocrine Leydig cells and
their stem cell progenitors, the pericytes. In: Sutovsky P, Clascá F, Kmiec Z, Korf H-W, Singh
13 The Pluripotent Microvascular Pericytes Are the Adult Stem Cells Even in the Testis 259

B, Timmermans J-P, Schmeisser MJ (eds) Advances in anatomy, embryology and cell biology,
vol 205. Springer, New York, pp 1–154
de Souza LEB, Malta TM, Kashima Hadat S, Covas DT (2016) Mesenchymal stem cells and peri-
cytes: to what extent are they related? Stem Cells Dev 25(24):1843–1852
DeFalco T, Bhattacharya I, Williams AV, Sams DM, Capel B (2014) Yolk-sac-derived macro-
phages regulate fetal testis vascularization and morphogenesis. Proc Natl Acad Sci U S A
111:E2384–E2393
DeFalco T, Potter SJ, Williams AV, Waller B, Kan MJ, Capel B (2015) Macrophages contribute to
the spermatogonial niche in the adult testis. Cell Rep 12:1107–1119
Dellavalle A, Sampoalesi M, Tonlorenzi R, Tagliafico E, Sacchetti B, Perani L, Innocenzi A,
Galvez BG, Messina G, Morosetti R, Li S, Belicchi M, Peretti G, Chamberlain IS, Wright
WE, Torrente Y, Ferrari S, Bianco P, Cossu G (2007) Pericytes of human skeletal muscle are
myogenic precursors distinct from satellite cells. Nat Cell Biol 9:255–267
Dellavalle A, Maroli G, Covarello D, Azzoni E, Innocenti A, Perani L, Antonioni S, Sambasivan
R, Brunelli S, Tajbakhsh S, Cossu G (2011) Pericytes resident in postnatal skeletal muscle
differentiate into muscle fibres and generate satellite cells. Nat Commun 2:499. https://doi.
org/10.1038/ncomms1508
De-Miguel MP, Arnalich-Montiel F, Lopez-Iglesias P, Blasquez-Martinez A, Nistal M (2009)
Epiblast-derived stem cells in embryonic and adult tissues. Int J Dev Biol 53:1529–1540
De-Miguel MP, Fuentis-Julian S, Alcaina Y (2010) Pluripotent stem cells: origin, maintenance and
induction. Stem Cell Rev Rep 6:633–649
Denham M, Hasegawa K, Menheniott T, Rollo B, Zhang D, Hough S, Alshawa A, Febbraro F,
Ighanian S, Leung J, Elliott DA, Newgreen DF, Pera MF, Dottori M (2015) Multipotent caudal
neural progenitors derived from humans pluripotent stem cells that give rise to lineages of the
central and peripheral nervous system. Stem Cells 33(6):1759–1770
Diaz-Flores L Jr, Madrid JF, Guitérrez R (2006) Adult stem and transit-amplifying cell location.
Histol Histopathol 21:995–1027
Diaz-Flores L, Gutiérrez R, Madrid JF, Varela H, Valladares F, Acosta E, Martin-Vasallo P, Diaz-­
Flores L Jr (2009) Pericytes. Morphofunction, interactions and pathology in a quiescent and
activated mesenchymal cell niche. Histol Histopathol 24:909–969
Diaz-Flores L, Gutiérrez R, Garcia MP, Diaz-Flores L Jr, Valladares F, Madrid JF (2012)
Ultrastructure of myopericytoma: a continuum of transitional phenotypes of myopericytes.
Ultrastr Pathol 36:189–194
Doetsch F (2003) The glial identity of neural stem cells. Nat Neurosci 6:1127–1134
Dogiel AS (1899) Über den Bau der Ganglien in den Geflechten des Darms und der Gallenblase
des Menschen und der Säugetiere. Arch Anat Physiol Anat Abt A 3–4:100–158
Donoghue PCJ, Graham A, Kelsh RN (2008) The origin and evolution of the neural crest.
BioEssays 30:530–541
Dore-Duffy P (2008) Pericytes: pluripotent cells of the blood brain barrier. Curr Pharm Des
14(16):1581–1593
Dore-Duffy P, Cleary K (2011) Morphology and properties of pericytes. Meth Mol Biol 686
(Part 1):49–68
Eberth CJ (1871) Von den Blutgefässen. Capitel VIII. In: Stricker S (ed) Handbuch der Lehre von
den Geweben des Menschen und der Tiere, Bd 1. Verlag von Wilhelm Engelmann, Leipzig,
pp 191–213
Ergün S, Stingl J, Holstein AF (1994) Microvasculature of the human testis in correlation to Leydig
cells and seminiferous tubules. Andrologia 26(5):255–262
Ergün S, Davidoff M, Holstein AF (1996) Capillaries in the lamina propria of human seminiferous
tubules are partly fenestrated. Cell Tissue Res 286(1):93–102
Ergün S, Harneit S, Paust HJ, Mukhopadhyay AK, Holstein AF (1999) Endothelin and endothelin
receptors A and B in the human testis. Anat Embryol 199:207–214
Etchevers HC, Vincent C, Le Douarin NM, Couly GF (2001) The cephalic neural crest provides
pericytes and smooth muscle cells to all blood vessels of the face and forebrain. Development
128:1059–1068
260 M. S. Davidoff

Fawcett DW, Neaves WB, Flores MN (1973) Comparative observations on intertubular lymphatics
and the organization of the interstitial tissue of the mammalian testis. Biol Reprod 9:500–532
Fecteau KA, Markonjich L, Mason JI, Mendis-Handagama SMLC (2006) Detection of platelet-­
derived growth factor-α (PDGF-A) protein in cells of Leydig lineage in the postnatal rat testis.
Histol Histopathol 21:1295–1302
Filippov V, Kronenberg G, Pivneva T, Reuter K, Steiner B, Wang L-P, Yamaguchi M, Kettenmann
H, Kempermann G (2003) Subpopulation of nestin-expressing progenitor cells in the adult
murine hippocampus shows electrophysiological and morphological characteristics of astro-
cytes. Mol Cell Neurosci 23:373–382
Fisher M, (2009) Pericyte signaling in the neurovascular unit. Stroke 40 (3, Suppl 1):S13-­S15
Flamme I, Frölich T, Risau W (1997) Molecular mechanisms of vasculogenesis and embryonic
angiogenesis. J Cell Physiol 173:206–210
Florey HW, Carleton HM (1926) Rouget cells and their function. Proc R Soc Lond B 100:23–31
Friedrich R, Holstein AF, Middendorff R, Davidoff MS (2012) Vascular wall cells contribute
to tumorigenesis in cutaneous neurofibromas of patients with Neurofibromatosis type 1. A
comparative histological, ultrastructural and immunohistochemical study. Anticancer Res
32:2139–2158
Ge R-S, Dong Q, Sottas CM, Papadopoulos V, Zirkin BR, Hardy MP (2006) In search of rat stem
Leydig cells: identification, isolation, and lineage-specific development. Proc Natl Acad Sci U
S A 103:2719–2724
Gilbert SF (2000) The Neural Crest. Developmental Biology, 6th edn. Sinauer Associates,
Sunderland, MA. Available from: https://www.ncbi.nlm.nih.gov/books/NBK10065/
Griswold SL, Behringer RR (2009) Fetal Leydig cell origin and development. Sexual Development
3 (1):1–15
Gnessi L, Emidi A, Jannini EA, Carosa E, Maroder M, Arizzi M, Ulisse S, Spera G (1995)
Testicular development involves the spatiotemporal control of PDGFs and PDGF receptors
gene expression and action. J Cell Biol 131:1105–1121
Gnessi L, Basciani S, Mariani S, Arizzi M, Spera G, Wang C, Bondjers C, Karlsson L, Betsholtz C
(2000) Leydig cell loss and spermatogenic arrest in platelet-derived growth factor (PDGF)-A-
deficient mice. J Cell Biol 149(5):1019–1025
Gökçinar-Yagei B, Uçkan-Çetinkaya D, Çelebi-Saltik B (2015) Pericytes: Properties, functions
and applications in tissue engineering. Stem Cell Rev Rep 11:549–559
Goluža T, Boscanin A, Cvetko J, Kozina V, Kosoviċ M, Bernat MM, Kasum M, Kaštelan Ž, Ježek
D (2014) Macrophages and Leydig cells in testicular biopsies of azoospermic men. Biomed
Res Int 2014:828697, 14 pages
Gondos B (1980) Development and differentiation of the testis and male reproductive tract. In:
Steinberger A, Steinberger E (eds) Testicular development structure and function. Raven,
New York, pp 3–20
Gonzalez R, Griparic L, Vargas V, Burgee K, Santacruz P, Anderson R, Schiewe M, Silva F, Patel
A (2009) A putative mesenchymal stem cells population isolated from adult human testes.
Biochem Biophys Res Commun 385:570–575
Gonzalez-Perez O (2012) Neural stem cells in the adult human brain. Biol Biomed Rep 2(1):59–69
Guimarães-Camboa N, Cattaneo P, Sun Y, Moore-Morris T, Gu Y, Dalton ND, Rockenstein E,
Masliah E, Peterson KL, Stallcup WB, Chen J, Evans SM (2017) Pericytes of multiple organs
do not behave as mesenchymal stem cells in vivo. Cell Stem Cell 20(3):345–359
Günther H (1917) Die mechanische Erregbarkeit der Hautmuskeln und Hautgefäße. In: Kraus
F et al (eds) Ergebnisse der Inneren Medizin und Kinderheilkunde, vol 15. Julius Springer,
Berlin, pp 620–714
Haider SG, Servos K, Tran N (2007) Structural and histological analysis of Leydig cell steroido-
genic function. In: Payne AH, Hardy MP (eds) Contemporary endocrinology: the Leydig cell
in health and disease. Humana Press, Totowa, NJ, pp 33–45
Hall BK (1998) Germ layers and the germ-layer theory revisited: Primary and secondary germ
layers, neural crest as a fourth germ layer, homology, demise of the germ-layer theory. Evol
Biol 30:121–186
13 The Pluripotent Microvascular Pericytes Are the Adult Stem Cells Even in the Testis 261

Hall BK (2000) The neural crest as a fourth germ layer and vertebrates as quadroblastic not trip-
loblastic. Evol Dev 2:3–5
Hall AP (2006) Review of the pericyte during angiogenesis and its role in cancer and diabetic
retinopathy. Toxicol Pathol 34:763–775
Hara Y, Nomura T, Yoshizaki K, Frisén J, Osumi N (2010) Impaired hippocampal neurogenesis
and vascular formation in ephrin-A5-deficient mice. Stem Cells 28:974–983
Hermann M, Bara JJ, Sprecher CM, Menzel U, Jalowiec JM, Osinga R, Scherberich A, Alini M,
Verrier S (2016) Pericyte plasticity—comparative investigation of the angiogenic and multilin-
eage potential of pericytes from different human tissues. Eur Cells Mater 31:236–249
Hill JP, Watson KM (1958) The early development of the brain in marsupials preliminary com-
munication. J Anat 92(Pt4):493–497
Hill WD, Hess DC, Martin-Studdard A, Carotheres JJ, Zheng J, Hale D, Maeda M, Fagan SC,
Carroll JE, Conway SJ (2004) SDF-1 (CXCL12) is upregulated in the ischemic penumbra fol-
lowing stroke: association with bone marrow cell homing to injury. J Neuropathol Exp Neurol
63:84–96
Hirschi KK, D’Amore PA (1996) Pericytes in the microvasculature. Cardiovasc Res 32:687–698
Holstein AF (1999) Spermatogenese beim Menschen: Grundlagenforschung und Klinik. Ann Anat
181:427–436
Holstein AF, Davidoff MS (1997) Organization of the intertubular tissue of the human testis. In:
Motta PM (ed) Recent advances of cells, tissues and organs. Antonio Delfino Editore, Rome,
pp 569–577
Holstein AF, Maekawa M, Nagano T, Davidoff MS (1996) Myofibroblasts in the lamina propria of
human seminiferous tubules are dynamic structures of heterogeneous phenotype. Arch Histol
Cytol 59:109–125
Holstein AF, Schulze W, Davidoff M (2003) Understanding spermatogenesis is a prerequisite for
treatment. Reprod Biol Endocrinol 1:107
Hosaka K, Yang Y, Sekia T, Fischera C, Dubeya O, Fredlundc E, Hartman J, Religa P, Morikawa
H, Ishii Y, Sasahara M, Larsson O, Cossu G, Cao R, Lim S, Cao Y (2016) Pericyte–fibro-
blast ­transition promotes tumor growth and metastasis. Proc Natl Acad Sci U S A 113(38):
E5618–E5627
Huhtaniemi I, Pelliniemi LJ (1992) Fetal Leydig cells: cellular origin, morphology, life span, and
special functional features. Proc Soc Exp Biol Med 201:125–140
Itoh Y, Toriumi H, Yamada S, Hoshino H, Suzuki N (2011) Astrocytes and pericytes coopera-
tively maintain a capillary-like structure composed of endothelial cells on gel matrix. Brain
Res 1406:74–83
Jiang Y, Hernderson D, Blackstad M, Chen A, Miller RF, Verfaillie CM (2003) Neuroectodermal
differentiation from mouse multipotent adult progenitor cells. Proc Natl Acad Sci U S A
100:11854–11860
Joseph NM (2004) Neural crest stem cells undergo multilineage differentiation in devel-
oping peripheral nerves to generate endoneurial fibroblasts in addition to Schwann cells.
Development 131 (22):5599-5612
Kahn CR (2008) Can we nip obesity in its vascular bud? Science 322:542–543
Kastschenko N (1888) Zur Entwicklungsgeschichte des Salachierembryos. Anatomischer Anzeiger
III (1–32) 16:445–467
Kaufman M (1992) The atlas of mouse development. Academic, London, p 512
Kennedy E, Mooney CJ, Hakimjavadi R, Fitzpatrick E, Guha S, Collins LE, Loscher CE, Morrow
D, Redmond EM, Cahill PA (2014) Adult vascular smooth muscle cells in culture express
neural stem cell markers typical of resident multipotent vascular stem cells. Cell Tissue Res
358:203–216
Kerr JB, Sharpe RM (1985) Stimulatory effect of follicle-stimulating hormone on rat Leydig cells.
A morphometric and ultrastructural study. Cell Tissue Res 239:405–415
Kerr JB, Donachie K, Rommerts FFG (1985) Selective destruction and regeneration of rat Leydig
cells in vivo. Cell Tiss Res 242:145–156
262 M. S. Davidoff

Kerr JB, Bartlett JMS, Donachie K (1986) Acute response of testicular interstitial tissue in rats to
the cytotoxic drug ethane dimethanesulphonate. An ultrastructural and hormonal study. Cell
Tissue Res 243:405–414
Kerr JB, Knell CM, Abbott M, Donachie K (1987a) Ultrastructural analysis of the effect of ethane
dimethanesulphonate on the testis of the rat, guinea pig, hamster and mouse. Cell Tissue Res
249:451–457
Kerr JB, Bartlett JMS, Donachie K, Sharpe RM (1987b) Origin of regenerating Leydig cells in the
testis of the adult rat. An ultrastructural, morphometric and hormonal assay study. Cell Tissue
Res 249:367–377
Kilcoyne KR, Smith LB, Atanassova N, Macpherson S, McKinell C, van den Driesche S, Jobling
MS, Ghambers TJG, De Gendt K, Verhoeven G, O’Hara L, Platts S, de Franca LR, Lara NLM,
Anderson RA, Sharpe RM (2014) Fetal programming of adult Leydig cell function by andro-
genic effects on stem/progenitor cells. Proc Natl Acad Sci U S A 111:E1924–E1932
Klein D, Weißhardt P, Kleff V, Jastrow H, Jakob HG, Ergün S (2011) Vascular wall-resident
CD44+ multipotent stem cells give rise to pericytes and smooth muscle cells and contribute to
new vessel maturation. PLoS One 6:e20540
Klein D, Meissner N, Kleff V, Jastrow H, Yamaguchi M, Ergün S, Jendrossek V (2014) Nestin(+)
tissue-resident multipotent stem cells contribute to tumor progression by differentiating into
pericytes and smooth muscle cells resulting in blood vessel remodeling. Front Oncol 4:Article
169
Kojima Y, Kaufman-Francis K, Studdert JB, Steiner KA, Power MD, Loebel DAF, Jones V, Hor
A, de Alencastro G, Logan GJ, Teber ET, Tam OH, Stutz MD, Alexander IE, Pickett HA,
Tam PPL, (2014) The transcriptional and functional properties of mouse epiblast stem cells
resemble the anterior primitive streak. Cell Stem Cell 14 (1):107–120
Krogh A (1919) The number and distribution of capillaries in muscles with calculations of the
oxygen pressure head necessary for supplying the tissue. J Physiol 52:409–415
Krogh A (1922) The anatomy and physiology of the capillaries. New Haven Yale University Press,
London, pp 1–304
Krogh A (1929) Anatomie und Physiologie der Capillaren. p.362, Springer, Berlin-Heidelberg.
Krueger M, Bechmann I (2010) CNS pericytes: concepts, misconceptions, and a way out. Glia
58:1–10
Kucia M, Reca R, Jala VR, Dawn B, Ratajczak J, Ratajczak MZ (2005) Bone marrow as a home of
heterogeneous populations of nonhematopoietic stem cells. Leukemia 19:1118–1127
Kucia M, Machalinski B, Ratajczak MZ (2006) The developmental deposition of epiblast/germ
cell-line derived cells in various organs as a hypothetical explanation of stem cell plasticity?
Acta Neurobiol Exp 66:331–341
Kurtz A (2016) Commentary for “human kidney pericytes produce renin”. Kidney Int
90:1153–1154
Landreh L, Stukenborg J-B, Söder O, Svechnikov K (2013) Phenotype and steroidogenic potential
of PDGFα-positive rat neonatal peritubular cells. Mol Cell Endocrinol 372:96–104
Landreh L, Spinner K, Schubert K, Häkkinen MR, Auriola S, Poutanern M, Söder O, Svechnikov
K, Mayerhofer A (2014) Human testicular peritubular cells host putative stem Leydig cells
with steroidogenic capacity. J Clin Endocrinol Metab 99(7):E1227–E1235
Le Douarin NM, Dupin E (2003) Multipotentiality of the neural crest. Current Opinion in Genetics
& Development 13 (5):529–536
Le Douarin NM, Callani GW, Dupin E (2008) The stem cells of the neural crest. Cell Cycle
7:1013–1019
Lee RTH, Knapik EW, Thiery JP, Carney TJ (2013a) An exclusively mesodermal origin of fin mes-
enchyme demonstrates that zebrafish trunk neural crest does not generate ectomesenchyme.
Development 140:2923–2932
Lee RTH, Negai H, Nakaya Y, Sheng G, Trainor PA, Weston JA (2013b) Cell delamination in the
mesencephalic neural fold and its implication for the origin of ectomesenchyme. Development
140:4890–4902
Leeson TS, Cookson FB (1974) The mammalian testicular capsule and its muscle elements.
J Morph 144:237–254
13 The Pluripotent Microvascular Pericytes Are the Adult Stem Cells Even in the Testis 263

Lindner U, Kramer J, Rohwedel J, Schlenke P (2010) Mesenchymal stem or stromal cells: toward
a better understanding of their biology? Transfus Med Hemother 37:75–83
Ling E-A, Wong W-C (1993) The origin and nature of ramified and amoeboid microglia: a histori-
cal review and current concepts. Glia 7:9–18
Louissaint A Jr, Rao S, Leventhal C, Goldman SA (2002) Coordinated interaction of neurogenesis
and angiogenesis in the adult songbird brain. Neuron 34(6):945–960
Maekawa M, Kamimura K, Nagano T (1996) Peritubular myoid cells in the testis: their structure
and function. Arch Histol Cytol 59:1–13
Makala H, Pothana L, Sonam S, Malla A, Goel S (2015) Regeneration of Leydig cells in ectopi-
cally autografted adult mouse testis. Reproduction 149:259–268
Mariani S, Basciani S, Arizzi M, Spera G, Gnessi L (2002) PDGF and the testis. Trends Endocrinol
Metab 13(1):11–17
Mayer S (1902) Die Muskularisierung der capillaren Blutgefäße. Nachweis des anatomischen
Substrats ihrer Kontraktilität. Anat Anz, Jena 21:442–455
McLaren A (2003) Primordial germ cells in the mouse. Dev Biol 262:1–15
Michels NA (1936) The structure of capillaries and the un-myogenic character of Rouget cells
(Pericytes) in the omentum of rabbits and the web of living frogs. Anat Rec 65:99–125
Middendorff R , Davidoff MS , Holstein AF (1993) Neuroendocrine marker substances in human
Leydig cells – changes by disturbances of testicular function. Andrologia; 25:257–262
Middendorff R, Müller D, Mewe M, Mukhopadhyay AK, Holstein A-F, Davidoff MS (2002) The
tunica albuginea of the human testis is characterized by complex contraction and relaxation
activities regulated by cyclic GMP. J Clin Endocrinol Metab 87:3486–3499
Millner R, Hung S, Erokwu B, Dore-Duffy P, LaManna JC, del Zoppo GJ (2008) Increased expres-
sion of fibronectin and the α5ß1integrin angiogenic cerebral blood vessels of mice subject to
hypobaric hypoxia. Mol Cell Neurosci 38:43–52
Mizrak SC, Chikhovskaya JV, Sadri-Ardekani H, van Daalen S, Korver CM, Hovingh SE, Roepers-­
Gajadien HL, Raya A, Fluiter K, de Reijke TM, de la Rosette JJMCH, Knegt AC, Belmonte JC,
van der Veen F, de Rooij DG, Repping S, van Pelt AMM (2010) Embryonic stem cell-like cells
derived from adult human testis. Hum Reprod 25:158–167
Molenaar R, de Rooij DG, Rommerts FFG, Reuvers PJ, van der Molen HJ (1985) Specific destruc-
tion of Leydig cells in mature rats after in vivo administration of Ethane dimethyl sulfonate.
Biol Reprod 33:1213–1222
Montiel-Eulefi E, Sánchez R, Rojas M, Bustos-Obregon E (2009) Epiblast embryo stem cells give
origin to adult pluripotent cell populations: primordial germ cell, pericytic and haematopoietic
stem cells. A review. Int J Morphol 27:1325–1333
Mori S, Leblond CP (1969) Identification of microglia in light and electron microscopy. J Comp
Neurol 135:57–79
Morshead CM (2004) Adult neural stem cells: attempting to solve the identity crisis. Dev Neurosci
26:93–199
Müller I (1957) Kanälchen—und Capillararchitektonik des Rattenhodens. Z Zellforsch 45:522–537
Murakami T, Uno Y, Ohtsuka A, Taguchi T (1989) The blood vascular architecture of the rat testis:
a scanning electron microscopic study of corrosion casts followed by light microscopy of tissue
sections. Arch Histol Cytol 52:151–172
Murray IR, West CC, Hardy WR, James AW, Park TS, Nguyen A, Tawonsawatruk T, Lazzari L,
Soo C, Péault B (2014) Natural history of mesenchymal stem cells, from vessel walls to culture
vessels. Cell Mol Life Sci 71(8):1353–1374
Nakagawa T, Nabeshima Y, Yoshida S (2007) Functional identification of the actual and potential
stem cell compartments in mouse spermatogenesis. Dev Cell 12(2):195–206
Newgreen DF, Kerr RS, Minichiello J, Warren N (1997) Changes in cell adhesion and extracel-
lular matrix molecules in spontaneous spinal neural tube defects in avian embryos. Teratology
55:195–207
Nichols DH (1981) Neural crest formation in the head of the mouse embryo as observed using a
new histological technique. J Embryol Exp Morph 64:105–120
264 M. S. Davidoff

Nichols DH (1986) Formation and distribution of neural crest mesenchyme to the first pharyngeal
arch region of the mouse embryo. Am J Anat 176:221–231
Nichols J, Smith A (2012) Pluripotency in the embryo and in culture. Cold Spring Harbor
Perspectives in Biology 4 (8):a008128-a008128
Nikolova G, Strilic B, Lammert E (2007) The vascular niche and its basement membrane. Trends
Cell Biol 17:19–25
Ochs K, Sahm F, Opitz CA, Lanz TV, Oezen I, Couraud P-O, von Deimling A, Wick W, Platten M
(2013) Immature mesenchymal stem cell-like pericytes as mediators of immunosuppression in
human malignant glioma. J Neuroimmunol 265:106–116
Ortega HH, Lorente JA, Salvetti NR (2004a) Immunohistochemical study of intermediate fila-
ments and neuroendocrine marker expression in Leydig cells of laboratory rodents. Anat Histol
Embryol 33(5):309–315
Orth J, Weisz J (1980) Development of Δ5-3ß-hydroxysteroid dehydrogenase and glucose-6-
phosphatase activity in Leydig cells of the fetal rat testis: a quantitative cytochemical study.
Biol Reprod 22:1201–1209
Özen I, Boix J, Paul G (2012) Perivascular mesenchymal stem cells in the adult human brain: a
future target for neuroregeneration? Clin Transl Med 1(1):30
Pacini S, Petrini I (2014) Are MSCs angiogenic cells? New insights on human nestin-positive bone-­
marrow-­derived multipotent cells. Front Cell Dev Biol 2:Article 20. https://doi.org/10.3389/
fcell.2014.00020
Palm T, Nielsen SL, Lassen NA (1983) Vascular recruitment in forearm muscles during exercise.
Clin Physiol 3:445–451
Palmer TD, Willhoite AR, Gage FH (2000) Vascular niche for adult hippocampal neurogenesis.
J Comp Neurol 425:479–494
Paniagua R, Rodriguez MC, Nistal M, Fraile B, Regadera J, Amat P (1988) Changes in surface
area and number of Leydig cells in relation to the 6 stages of the cycle of the human seminifer-
ous epithelium. Anat Embryol 178(5):423–427
Péault B, Rudnicki M, Torrente Y, Cossu G, Trmblay JP, Partridge T, Gussoni E, Kunkel LM,
Huard J (2007) Stem and progenitor cells in skeletal muscle development, maintenance, and
therapy. Mol Ther 15:867–877
Ratajczak MZ, Machalinski B, Wojakowski W, Ratajczak J, Kucia M (2007) A hypothesis for
embryonic origin of pluripotent Oct-4+ stem cells in adult bone marrow and other tissues.
Leukemia 21:860–867
Ratajczak MZ, Zuba-Surma EK, Wysoczynski M, Ratajczak J, Kucia M (2008) Very small
embryonic-­like stem cells: characterization, developmental origin, and biological significance.
Exp Hematol 36:742–751
Ratajczak MZ, Shin D-M, Liu R, Mierzeiewska K, Ratajczak J, Kucia M, Zuba-Surma EK (2012a)
Very small embryonic/epiblast-like stem cells (VSELs) and their potential role in aging and
organ rejuvenation—an update and comparison to other small stem cells isolated from adult
tissues. Aging 4:235–246
Ratajczak MZ, Zuba-Surma E, Kucia M, Poniewierska A, Suszynska M, Ratajchak J (2012b)
Pluripotent and multipotent stem cells in adult tissues. Adv Med Sci 57:1–17
Ribatti D, Vacca A (2008) Overview of angiogenesis during tumor growth, Chapter 14. In: Figg
WD, Folkman J (eds) Angiogenesis. An integrative approach from science to medicine.
Springer, New York, NY, pp 161–167
Ribatti D, Nico B, Crivellato E (2011) The role of pericytes in angiogenesis. Int J Dev Biol
55:261–268
Rolandsson S, Andersson Sjöland A, Brune JC, Li H, Kassem M, Martens F, Westergren A, Eriksson
L, Hanson L, Skog I, Bjermer L, Scheding S, Westergren-Thorson G (2014) Primary mesen-
chymal stem cells in human transplanted lungs are CD90/CD105 perivascularly located tissue-­
resident cells. BMJ Open Resp Res 1:e000027. https://doi.org/10.1136/bmjresp-2014-00002
Rossant J (2001) Stem cells from the mammalian blastocyst. Stem Cells 19:477–482
Rouget CMB (1873) Memoiré sur le développment, la structure et les propriétés physiologiques
des capillaires sanguins et lymphatiques. Arch Physiol 5:603–661
13 The Pluripotent Microvascular Pericytes Are the Adult Stem Cells Even in the Testis 265

Rouget CMB (1879) Sur la contractilite des capillaires sanguins. Compt Rend Acad Sci Paris
88:916–918
Rowley JE, Johnson JR (2014) Pericytes in chronic lung disease. Int Arch Allergy Immunol
164:178–188
Russell LD, de França LR (1995) Building a testis. Tissue Cell 27:129–147
Sá-Pereira I, Brites D, Brito MA (2012) Neurovascular unit: a focus on pericytes. Mol Neurobiol
45:327–347
Sbanti RM, Li W-J, Nesti LJ, Wang Y, Tuan RS (2007) Adult mesenchymal stem cells: biological
properties, characteristics, and application in maxillofacial surgery. J Oral Maxillofac Surg
65:1640–1647
Scadden DT (2006) The stem-cell niche as an entity of action. Nature 44:1075–1079
Schulze W, Davidoff MS, Holstein A-F (1987) Are Leydig cells of neural origin? Substance P-like
immunoreactivity in human testicular tissue. Acta Endocrinol 115:373–377
Shan L-X, Hardy MP (1992) Developmental changes in levels of luteinizing hormone receptor and
androgen receptor in rat Leydig cells. Endocrinology 131:1107–1114
Shan L-X, Zhu L-J, Bardin CW, Hardy MP (1995) Quantitative analysis of androgen receptor mes-
senger ribonucleic acid in developing Leydig cells and Sertoli cells by in situ hybridization.
Endocrinology 136:3856–3862
Shan L-X, Bardinb CW, Hardy MP (1997) Immunohistochemical analysis of androgen effects
on androgen receptor expression in developing Leydig and Sertoli cells. Endocrinology
138:1259–1266
Sharpe RM (1994) Regulation of spermatogenesis. In: Knobil E, Neil JD (eds) The physiology of
reproduction, vol 1, 2nd edn. Raven Press, New York, pp 1363–1434
Shen C-N, Burke ZD, Tosh D (2004a) Transdifferentiation, metaplasia and tissue regeneration.
Organogenesis 1:36–44
Shen Q, Goderie SK, Jin L, Karanth N, Sun Y, Abramova N, Vincent P, Pumiglia K, Temple S
(2004b) Endothelial cells stimulate self-renewal and expand neurogenesis of neural stem cells.
Science 304:1338–1340
Shen Q, Wang W, Kokovay E, Lin G, Chang S-M, Goderie SK, Roysam B, Temple S (2008) Adult
SVZ stem cells lie in a vascular niche: a quantitative analysis of niche-cell interactions. Cell
Stem Cell 3:289–300
Shibata H, Ikeda Y, Mukai T, K-i M, Kurihara I, Ando T, Suzuki T, Kobayashi S, Murai M, Saito
I, Saruta T (2001) Expression profiles of COUP-TF, DAX-1, and SF-1 in human adrenal
gland and adrenocortical tumors: possible implications in steroidogenesis. Mol Gen Metabol
74:206–216
Shyamala K, Yanduri S, Girish HC, Murgod S (2015) Neural crest: the fourth germ layer. J Oral
Maxillofac Pathol 19(2):221–229
Sild M, Rithazer ES (2011) Radial glía: progenitor, pathway, and partner. Neuroscience
17(3):288–302
Sims DE (1986) The pericyte—a review. Tiss Cell 18:153–174
Skinner MK, Tung PS, Fritz IB (1985) Cooperativity between Sertoli cells and testicular peritu-
bular cells in the production and deposition of extracellular matrix components. J Cell Biol
100:1941–1947
Smith AG (2001) Embryo-derived stem cells: of mice and men. Annu Rev Cell Dev Biol
17:435–462
Soncin F, Ward CM (2011) The function of E-cadherin in stem cell pluripotency and self- renewal.
Genes 2:229–259
Spence SG, Poole TJ (1994) Developing blood vessels and associated extracellular matrix as sub-
strates for neural crest migration in Japanese quail, Coturnix coturnix japonica. Int J Dev Biol
38:85–98
Stanley EL, Johanston DS, Fan J, Papadopoulos V, Chen H, Ge RS, Zirkin BR, Jelinsky SA (2011)
Stem Leydig cell differentiation: gene expression during development of the adult rat popula-
tion of Leydig cells. Biol Reprod 85:1161–1166
Stanley E, Lin C-Y, Jin S, Liu J, Sottas CM, Ge R, Zirkin BR, Chen H (2012) Identification,
prtoliferation, and differentiation of adult Leydig stem cells. Endocrinology 153:5002–5010
266 M. S. Davidoff

Suzuki F, Nagano T (1986) Microvasculature of the human testis and excurrent duct system.
Resin-casting and scanning electron-microscopic studies. Cell Tissue Res 243:79–89
Svingen T, Koopman P (2013) Building the mammalian testis: origins, differentiation, and assem-
bly of the component cell populations. Genes Dev 27:2409–2426
Takamoto N (2005) COUP-TFII is essential for radial and anteroposterior patterning of the stom-
ach. Development 132 (9):2179–2189
Tang W, Zeve D, Suh JM, Bosnakovski D, Kyba M, Hammer RE, Tallquist MD, Graff JM (2008)
White fat progenitor cells reside in the adipose vasculature. Science 322:583–586
Tavazoie M, Van der Veke L, Silva-Varga V, Louissaint M, Colonna L, Zaidi B, Garcia-Verdugo
JM, Doetsch F (2008) A specialized vascular niche for adult neural stem cells. Cell Stem Cell
3:279–288
Teerds K (1996) Regeneration of Leydig cells after depletion by EDS: a model for postnatal
Leydig cell regeneration. In: Payne AH, Hardy MP, Russel LD (eds) The Leydig cell. Cache
River, Vienna, pp 203–219
Teerds K, Rijntjes E (2007) Dynamics of Leydig cell regeneration after EDS. A model for postna-
tal Leydig cell development. In: Payne AH, Hardy MP (eds) Contemporary endocrinology: the
Leydig cell in health and disease. Humana Press, Totowa, NJ, pp 91–116
Teerds KJ, de Boer-Brouwer M, Dorrington JH, Balvers M, Ivell R (1999) Identification of mark-
ers for precursor and Leydig cell differentiation in the adult rat testis following ethane dimethyl
sulphonate administration. Biol Reprod 60:1437–1445
Teerds KJ, Rijntjes E, Veldhuizen-Tsoerkan MB, Rommerts FFG, de Boer-Brouwer M (2007)
The development of rat Leydig cell progenitors in vitro: how essential is luteinizing hormone?
J Endocrinol 194:579–593
Thiery JP, Delouvee A, Gallin W, Cunningham B, Edelman G (1984) Ontogenetic expression of
cell adhesion molecules: L-CAM is found in epithelia derived from the three primary germ
layers. Dev Biol 102:61–78
Thomas WE (1999) Brain macrophages: on the role of pericytes and perivascular cells. Brain Res
Rev 31:42–57
Tosh D, Slack JMW (2002) How cells change their phenotype. Nat Rev 3:187–194
Trainor PA, Melton KR, Manzanares M (2003) Origins and plasticity of neural crest cells and their
roles in jaw and craniofacial evolution. Int J Dev Biol 47: 541–553
Trainor PA (2005) Spezification and pattering of neural crest cells during craniofacial develop-
ment. Brain Behav Evol 66: 266–280
Traktuev DO, Merferld-Clauss S, Li J, Kolonin M, Arap W, Pasqualini R, Johnstone BH, March
KL (2008) A population of multipotent CD-34-positive adipose stromal cells share pericyte
and mesenchymal surface markers, reside in a periendothelial location, and stabilize endothe-
lial networks. Circ Res 102:77–85
Trost A, Lange S, Schroedl F, Bruckner D, Motloch KA, Bogner B, Kaser- Eichberger A,
Strohmaier C, Runge C, Aigner L, Rivera FJ, Reitsamer HA (2016) Brain and retinal pericytes:
origin, function and role. Front Cell Neurosci 10:20
van Dijk CGM, Nieuweboer FE, Pei JY, Xu YJ, Burgisser P, van Mulligen E, el Azzouzi H,
Duncker DJ, Verhaar MC, Cheng C (2015) The complex mural cell: pericyte function in health
and disease. Int J Cardiol 190:75–89
Vimtrup B (1922) Beiträge zur Anatomie der Capillaren. I. Über contractile Elemente in der
Gefäßwand der Blutcapillaren. Anat Embryol 65:150–182
Wang L, Kamath A, Frye J, Iwamoto GA, Chun JL, Berry SE (2012) Aorta-derived mesoangio-
blasts differentiate into the oligodendrocytes by inhibition of the Rho kinase signaling pathway.
Stem Cells Dev 7:1069–1189
Weerasooriya TR, Yamamoto T (1985) Three-dimensional organization of the vasculature of the
rat spermatic cord and testis. Cell Tissue Res 241:317–323
Wei LC, Shi M, Chen LW, Cao R, Zhang P, Chan YS (2002) Nestin-containing cells express glial
fibrillary acidic protein in the proliferative regions of central nervous system of postnatal devel-
oping and adult mice. Brain Res Dev Brain Res 139:9–17
Welsh M, Saunders PT, Atanassova N, Sharpe R, Smith LB (2009) Androgen action via testicular
peritubular myoid cells is essential for male fertility. FASEB J 23:4218–4230
13 The Pluripotent Microvascular Pericytes Are the Adult Stem Cells Even in the Testis 267

Welsh M, Sharpe RM, Moffat L, Atanassova N, Saunders PTK, Kilter S, Bergh A, Smith LB
(2010) Androgen action via testicular arteriole smooth muscle cells is important for Leydig cell
function, vasomotion and testicular fluid dynamics. PLoS One 5(10):e13632
Welsh M, Moffat L, Belling K, de França LR, Segatelli TM, Saunders PTK, Sharpe RM, Smith
LB (2011) Androgen receptor signaling in peritubular myoid cells is essential for normal dif-
ferentiation and function of adult Leydig cells. Int J Androl 35:25–40
Weston JA, Thiery JP (2015) Pentimento: neural crest and the origin of mesectoderm. Dev Biol
401:37–61
Weston JA, Yoshida H, Robinson V, Nishikawa S, Fraser ST, Nishikawa S (2004) Neural crest and
the origin of ectomesenchyme: neural fold heterogeneity suggests an alternative hypothesis.
Dev Dyn 229:118–130
Wong SP, Rowley JE, Redpath AN, Tilman JD, Fellous TG, Johnson JR (2015) Pericytes, mes-
enchymal stem cells and their contributions to tissue repair. Pharmacol Ther 151:107–120.
https://doi.org/10.1016/j.pharmthera.2015.03.006
Yao HH-C, Barsoum I (2007) Fetal Leydig cells. Origin, regulation, and function. In: Payne
AH, Hardy MP (eds) Contemporary endocrinology: The Leydig Cell in Health and Disease.
Humana Press, Totowa, pp. 47-54
Ye L, Li X, Li L, Chen H, Ge R-S (2017) Insights into the development of the adult Leydig cell
lineage from stem Leydig cells. Front Physiol 8:430
Yoshida S, Sukeno M, Nabeshima Y-I (2007) A vasculature-associated niche for undifferentiated
spermatogonia in the mouse testis. Science 317:1722–1726
Yoshimizu T, Obinata M, Matsui Y (2001) Stage-specific tissue and cell interactions play key roles
in mouse germ cell specification. Development 128: 481–490
You LR, Lin F-J, Lee CT, DeMayo FJ, Tsai M-J, Tsai SY (2005) Suppression of notch signaling
by COUP-TFII transcription factor regulates vein identity. Nature 435:98–104
Zhu X, Bergles DE, Nishiyama A (2008) NG2 cells generate both oligodendrocytes and gray mat-
ter astrocytes. Development 135:145–157
Zimmerlin L, Donnenberg VS, Pfeifer ME, Meyer EM, Péault B, Rubin JP, Donnenberg AD
(2010) Stromal vascular progenitors in adult human adipose tissue. Cytometry A 77A:22–30
Zimmerlin L, Donnenberg VS, Donnenberg AD (2012) Pericytes: A universal adult tissue stem
cell? Cytometry A 81A: 12-14 doi:10.1002/cyto.a21168
Zimmermann KW (1923) Der feinere Bau der Blutcapillaren. Zeitschr f d ges Anat I Abt 68:29–109
Zouani OF, Lei Y, Durrieu M-C (2013) Pericytes, stem-cell-like cells, but not mesenchymal stem
cells are recruited to support microvascular tube stabilization. Small 9:3070–3075
Zwaka TP, Thomson JA (2005) A germ cell origin of embryonic stem cells? Development
132:227–233
Index

A Arteriovenous malformations (AVM), 52

Activin-like kinase 2 (ALK2), 67 Ascorbic acid, 177
Adams-Oliver syndrome, 52 Asthma, 53
Adenosine, 33 Astrocytes, 4
Adipocytes, 173 Atherosclerosis, 198, 199
Adipogenesis, 65 ATP-binding cassette G (ABCG2), 49
Adipose-derived stem cells (ASCs), 13 Authentic adult stem cell (ASC), 237
A disintegrin and metalloproteinase 9 Autocrine signaling, 161
(ADAM9), 161 Autofluorescent lipid droplets, 155
Akt/Jagged1/Notch signaling, 49
Alcoholic steatohepatitis (ASH), 159
Alkaline phosphatase (Alk-p), 62, 190 B
Allantochorionic arterial system, 133 Basic helix-loop-helix (bHLH), 170
Alpha-smooth muscle actin (αSMA), 52, 178, β-cell dedifferentiation, 28
190, 220 Beta-cell dysfunction, 28, 34, 35
Alveolar bone regeneration, 177 Beta-cell function, 28–30
Amniotic membrane-derived MSCs β-cell maturity, 30, 31
(AMSCs), 13 insulin secretion, 32
Androgen receptors (AR), 247 Beta-cell mass, 36
Angiogenesis, 74, 75, 86, 161, 171, 193, 195 β-glycerophosphate, 177
antiangiogenic treatment, 88–90 Blood-brain barrier (BBB), 4
CPEs, 91–93 Blood retina barrier (BRB), 9, 10
endogenous angioinhibitors, 90, 91 Blood vessels, 170, 171, 173–175, 177–179, 181
PDGF, 87 endothelial cells, 74, 75
VEGF, 85, 86 functions, 75, 76
Angiopoietin-1/2 (angpt-1/2), 193 organs and vascular beds, 74
Angiopoietins (Angs), 7 vascular basement membrane, 74
Angiopoietin-Tie2 axis, 75 Body stalk, 215
Anti-angiogenic therapies, 19 Bone marrow
Anticancer niche therapy, 110, 111 arteriolar pericytes, myeloablation, 105
Anti-fibrotic strategies, 162 arterioles and sinusoids, mesenchymal
Anti-fibrotic therapies, 18, 19 pericytes, 105
Anti-inflammatory therapy, 15 cellular heterogeneity, 110
Aorta-gonad-mesonephros (AGM), 106 computational modeling, 107
Apolipoprotein E (ApoE), 156 computational simulation, 106

© Springer Nature Switzerland AG 2019 269

A. Birbrair (ed.), Pericyte Biology in Different Organs,
Advances in Experimental Medicine and Biology 1122,
https://doi.org/10.1007/978-3-030-11093-2
270 Index

Bone marrow (cont.) cardiac fibrosis, 200

developmental origins, 104 myocardial ischemic disease, 196–198
endothelial selectins, 102 thrombosis, 200, 201
hematologic tumors, 110 Cat placental vascularization, 135
HSC maintenance, 106–108 Cell outgrowth, 176
HSC niches, 105, 106 Cellular architecture, 190
HSCs, 102, 108, 110 Central nervous system (CNS), 4, 116
leukemia development and pericytes, 109 Chemokine receptor 3 (CXCR3), 195
leukemia microenvironments, 108, 109, 111 Chondrogenesis, 66, 67
leukocytes, 102 Chronic liver diseases
mesenchymal- and osteo-precursor death and liver transplantation, 73, 81
cells, 103, 104 endothelial cells, 74
metastatic bone cancer and GVB (see Gut-vascular barrier (GVB))
pericytes, 109, 110 pathological angiogenesis (see
microenvironments, 101 Angiogenesis)
niches, 102 portal hypertensive syndrome, 81–83
perivascular mesenchymal progenitors, 110 portosystemic collaterals, 84, 85
vascular structure, 102 postnatal vasculogenesis, 93, 94
vasculature remodeling, 6 splanchnic blood flow, 82, 83
vessels, 103 Chronic pulmonary diseases, 50
Bone marrow-derived cells, 174 Clonogenicity, 173
Bone morphogenetic protein 4 (BMP4), 35 Cochlear blood flow, 118–120
Bone morphogenetic proteins (BMPs), 53, 154 Cochlear blood flow regulation, 119
Bovine placental vascularization, 134 Cochlear capillary pericytes
Brain-derived neurotrophic factor (BDNF), 195 blood supply, 117
Bromodeoxyuridine (BrdU), 241 CNS, 116
morphology and cellular markers, 118
pathologies, 116
C pathophysiology, 119, 120
Caldesmon, 190 pharmacotherapy, 121
Calponin, 190 physiological functions, 118, 119
Cancer stem cells, 110 precapillary arteriolar cells, 116
Capillary blood flow regulation, 74 smooth muscle actin and tropomyosin, 116
Capillary system, 127, 130, 133, 137 stria vascularis/spiral ligament, 116, 117,
Cardiac fibrosis, 200 121
Cardiac homeostasis, 194 subpopulations, 116
angiogenesis, 193, 195 Colony-forming unit-fibroblastic (CFU-f), 172
vascular integrity and perfusion, 193 Connexin-43 (Cx43), 191
vascular tone, 195, 196 Cre-loxP technology, 46, 48
Cardiac pericytes (cPC) C-type natriuretic peptide (CNP), 195
anatomical location, 190 CXCL12, 175
biology, 203, 204 CXCL12-abundant reticular (CAR), 106
characterization, 190, 191 Cytokine niches, 108
markers, 192 Cytoplasmic polyadenylation element binding
origin, 189 proteins (CPEB), 85
subtypes, 204 Cytoplasmic polyadenylation elements
swine model, 204 (CPEs), 91–93
type-1 pericytes, 204
vascular integrity, stability and
remodeling, 203 D
Cardiovascular pathophysiology, 189 Dental pulp stem cells (DPSCs), 178
atherosclerosis, 198, 199 Desmin, 190
calcification, 199 Dexamethasone, 177
Index 271

Diabetic macular edema (DME), 15 G

Diabetic retinopathy (DR) Galectin-3, 200
angiogenic growth factors, 13 Ganglion cell layer (GCL), 5
BRB, 14, 15 Ganglioside 3g5 (3g5), 190
cell-based therapy, 15–17 Gastroesophageal varices, 84
glutamate excitation, 14 Gastrointestinal tract, 77
neovascularization, 14 Gene therapy, 89
ophthalmologic examinations, 14 Gestation, 212, 214, 217, 220
treatments, 15 Gestational diabetes mellitus (GDM), 228
vascular permeability and Gestation umbilical cord, 225
microaneurysms, 13 Glioma-associated oncogene 1 (Gli1), 190
visual impairment and blindness, 13 Gliotoxin, 156
Diphtheria toxin (DT), 48 Glucocorticoid therapy, 199
Diphtheria toxin receptor (DTR), 48 Glucose homeostasis
Duchenne muscular dystrophy (DMD), 61 β-cell maturity (see Beta-cell function)
β-cell replication, 32, 33
blood flow, 33
E endothelial, neuronal and immune
Ectomesenchyme, 251 cells, 30
Embryogenesis, 43 Glucose-stimulated insulin secretion, 28,
Embryonal development, 3 30–32, 34, 35
Endogenous angioinhibitors, 90, 91 Gonadal stem cell (GSC), 244
Endometrial stromal cells, 141 G protein-coupled receptors (GPCRs), 159
Endothelial cells, 74, 75 Gut-liver axis, 95, 96
Endothelial nitric oxide synthase (eNOS), 86 Gut-vascular barrier (GVB), 97
Endothelial progenitor cells (EPCs), 15 disruption, 97
Endothelin-1 (ET-1), 157 endothelial cells, 96
Endotheliochorial lamellar placenta, 133 gut-liver axis, 95, 96
Endotoxin, 120 lamina propria, 95
Enzymatic digestion methods, 176 morphological and functional
E-selectin, 102 characteristics, 96
Ethane dimethanesulfonate (EDS), 237 murine and human intestines, 96
Eutherian mammals, 126 peripheral tissues, 96
Exotoxin, 120 PV1, 96
Extracellular matrix (ECM), 160, 194
Extravillous trophoblast (EVT), 132, 139
H
Heart
F cardiovascular diseases, 189
Farnesoid X receptor (FXR), 160 cell-based therapeutics, 202, 203
Fetal development, 212, 214, 218, 220 cPC (see Cardiac pericytes (cPC))
Fibro/adipogenic progenitors (FAPs), 65 disease progression, 189
Fibroblast-associated genes, 226 endothelial barrier function, 188
Fibroblast growth factor receptor 1 homeostasis (see Cardiac homeostasis)
(FGFR1), 157 microvascular, arteriole/venular, 203
Fibroblast growth factors (FGFs), 154 MSCs, 201, 202
Fibroblastic colony-forming units (CFU-F), 103 pathophysiology (see Cardiovascular
Fibroblasts, 172 pathophysiology)
Fibrodysplasia ossificans progressiva pericytes, 188
(FOP), 66 repair and regeneration, 201
Fibrosis, 65, 66, 154 tissue physiology and disease, 189
5-fluorouracil (5FU), 105 Heat shock cognate (HSC) 70, 173
Follistatin, 156 Heat shock protein (HSP) 70, 173
272 Index

Hematopoietic stem cells (HSCs), 101, 102, Islet pericytes, 29, 31, 33
105–108, 110, 111, 172 Islets of Langerhans, 28, 33
Hemochorial labyrinthine placenta, 132, 133 Islet vasculature, 28, 29, 33
Hemochorial villous placenta, 130, 132 Ito cells, 153
Hemotrophic placentation, 126
Heparin-binding epidermal growth factor
(HB-EGF), 75, 194 K
Hepatic stellate cells (HSCs) Kidney fibrosis models, 18
description, 153 Kupffer cells, 157, 158, 160
developmental origin, 154
healthy liver, 155, 156
hepatic metastases, 160–161 L
steatohepatitis, 159–160 Laser photocoagulation, 15
Hepatocellular carcinoma, 157, 160 Lecithin retinol acyltransferase (LRAT), 155
Hepatocyte growth factor (HGF), 156, 198 Leptin receptor (Lepr), 106
Hepatocytes, 156–160 Leukemia cells, 108, 109
Hereditary hemorrhagic telangiectasia Leukemia development, 109
(HHT), 52, 53 Leukemic stem cell (LSC), 110
Histiotrophic placentation, 126 Leydig cells, 236, 237, 241, 246, 253, 255
House dust mite (HDM), 53 and sertoli cells, 247
Human aortic ring (hAR) assay, 225 Lipocytes, 153
Human cytomegalovirus (HCMV), 138 Lipopolysaccharide (LPS), 158
Human myelodysplastic syndrome cells, 109 Liver
Human placental vascularization, 131 HSCs (see Hepatic stellate cells (HSCs))
Human spiral arteriole, 140 Liver fibrosis, 156, 157
Human umbilical cord perivascular cells autophagy, 159
(HUCPVCs), 220 cell types, 157
Hyperglycemia, 35, 36 chronic injury, 157
Hypoxia, 171, 175 ET-1, 157
Hypoxia inducible factor-1α (HIF1α), 171 HSC activation, 157, 159
HSC migration, 157
HSCs, 158
I injured hepatocytes, 158
Immune response, 119, 120 MCP-1, 157
Immunoreactivity, 246 retinoic acid signaling, 158
Indoleamine 2,3-dioxygenase (IDO), 139 SECs, 157
Induced pluripotent stem (iPS) cells, 181 substrate stiffness, 159
Inferior mesenteric arteries (IMA), 81 Liver regeneration, 156
Inflammatory cytokine, 197 Liver sinusoidal endothelial cells (LSECs),
Injured hepatocytes, 157 153
Inner nuclear layer (INL), 3 Lung pericytes
Interferon beta (IFNβ), 159 anatomic characterization, 42, 43
Interleukin 10 (IL-10), 156 animal models
Intestines Cre recombinase activity, 48
functional considerations, 79, 80 Cre recombinase expression, 46
gastrointestinal tract, 76 DT, 48
macroscopic anatomical considerations, embryogenesis, 47
76–78 FoxD1-derived cells, 46
microscopic anatomical considerations, FoxJ1-CreER transgene, 48
78, 79 lineage tracing, 47
Intra-islet density, 33 LoxP sequences, 46
Intramuscular adipose tissue (IMAT), 65 NG2, 48
Intrastrial blood-fluid barrier, 118 oropharyngeal aspiration, 48
Islet capillaries, 29, 33, 36 pulse labeling, 47
Index 273

ROSA locus, 46 Muscle regeneration, 60, 61, 63–65, 67

tamoxifen, 48 Muscular dystrophy
asthma, 53 muscle injury, 61
cell morphology and characteristics, 44 myogenic and non-myogenic activities, 67
developmental origins, 43 pericytes, 61
gene markers, 45 SCs, 60
HHT, 52, 53 skeletal muscle (see Skeletal muscle
homeostasis and repair, 49–50 injury)
inflammation and injury, 50, 51 Myelodysplastic syndrome, 109
inflammatory response, 51 Myeloproliferative disorder model, 109
microvascular endothelium, 42 Myoblasts, 59
microvascular permeability and stability, 53 Myocardial infarction (MI), 196, 198,
molecular identification and pericyte 201–204
subtypes, 43, 44, 46 Myocardial ischemic disease, 196
myofibroblast precursors, 45 Myofiber hypotrophy, 61
PAH, 52 Myofibroblasts, 49, 51, 53, 222
pathogenesis, 53 Myogenesis, 63–65, 67
pulmonary fibrosis, 42, 51, 52 Myogenic differentiation, 60, 64, 67
pulmonary hypertension, 42
skin and gut epithelium, 49
transgenic mice, 47 N
vascular homeostasis, 42 Nanog (NANOG), 191
N-cadherin, 11, 195
Nerve growth factor (NGF), 32
M Nerve injury, 67
Mastication, 170 Nestin immunoreactivity, 249
Maternal-fetal communication, 127 Neural crest cells (NCCs), 154, 251
Matrix metalloproteinase 2 (MMP2), 161 Neural crest stem cells (NCSCs), 104, 251
Matrix metalloproteinases (MMPs), 194 Neural fold (NF), 251
Melanoma cell adhesion molecule Neural glial antigen-2 (NG2), 4, 44, 62,
(MCAM), 46 178, 190
Mesenchymal progenitor cells (MPCs), 49 Neurosensory retina, 3
Mesenchymal stem cells (MSCs), 13, 50, 102, Neurovascular unit (NVU), 9
103, 172, 173, 176–179, 181, 182, NG2 proteoglycan, 8
201, 202, 219, 245 Niches, 102, 104, 105, 110
Mesenchymoangioblast (MB), 62 Nitric oxide (NO), 157
Mesenteric arteries, 78 Nonalcoholic steatohepatitis (NASH), 159,
Mesenteric microcirculation, 81 160, 162
Mesenteric vascular bed, 82 No-reflow, 196
Mesentery, 80–81 Notch-3, 198
Mesoangioblasts, 60, 64, 65
Metablast, 251
Microangiopathy, 49 O
Microvascular dysfunction, 50 Obstetrical complications, 212, 227–229
Microvascular physiology, 74 Octamer-binding transcription factor 4
Microvessels, 76 (OCT4), 191
Monocyte chemoattractant protein-1 Optic nerve (ON), 2
(MCP-1), 156 Osler-Weber-Rendu syndrome, 52
Morphological method, 75 Osteoblasts, 173
Mouse adipose-derived stem cells (mASCs), 16 Osteogenesis/ossification, 66, 67
Multipotent stromal cells (MSCs), 244 Outer nuclear layer (ONL), 2, 3
Mural cells, 190 Outgrowth endothelial cells (OECs), 15–16
Muscle injury, 61, 63, 64, 66 Oxygen-induced retinopathy (OIR), 11
274 Index

P Peroxisome proliferator-activated receptor

Pancreas (PPAR), 160
α- and β- cells, 28 Peroxisome proliferator-activated receptor
blood glucose levels, 28 gamma (PPARγ), 159
diabetes, 28 Pigment epithelium-derived factor (PEDF), 90
exocrine and endocrine cells, 28 Placenta
Pancreatic pericytes blood flow, 128, 129
cytoplasmic processes, 29 eutherian diversification, 126
embryonic pancreatic mesenchyme, 29 hemotrophic and histiotrophic
endothelial cells, 29 bipotential, 126
exocrine pancreas, 29 pericytes
glucose homeostasis (see Glucose angiogenesis signaling, 136, 137
homeostasis) applications, 136
Paracrine, 161 cell differentiation, 141
PDGF-receptor β (PDGFRβ), 4, 43, 46, 47, 49, functions, 135
62, 137, 190 immune system, 135
Pericytes knockout/null-induced placental
HSCs (see Hepatic stellate cells (HSCs)) abnormalities, 137, 138
periodontal tissue (see Periodontal placental immune cells regulation, 139,
ligament) 140
Periodontal ligament regenerative tissue and repair
anatomy, function and distinctiveness, processes, 135–136
170–171 vascular stability, 135
bone marrow cells, 175 variability
GFP-positive cells, 174 allantois, 126
HUVECs, 180 arterial and venous levels, 128
MSCs, 176–177 carnivores, 128
pericytes classification, 126
blood vessels, 178 cotyledonary, 127
cell kinetic analysis and cytology, 178 extraembryonic membranes, 126
GFP transgenic mice, 178–179 invasive deepness, 128
H3-thymidine, 178 maternal-fetal communication units, 127
lineage tracing method, 179 models, 127
markers, 178 morphological differences, 126
microvasculature, 177 noninvasive epitheliochorial
morphometry, 178 placenta, 128
MSCs, 178, 181 rodents and lagomorphs, 128
multipotency, 178 tissue layers, 127
neural crest, 179 trophoblast layers, 128
PDLSCs, 179 vascular architecture
precaution, 179 capillary systems/microvasculature, 130
stem cells, 178 endotheliochorial lamellar placenta, 133
10T1/2 cells, 179 hemochorial labyrinthine placenta,
treatments, 181 132, 133
vascular endothelium, 179 hemochorial villous placenta, 130, 132
stem cells (see Periodontal ligament stem synepitheliochorial villous placenta, 133
cells (PDLSCs)) Placental growth factor (PGF), 136
tissue regeneration, 171–172 Placental vascularization, 126
Periodontal ligament stem cells (PDLSCs), Plasmalemma vesicle-associated protein-1
172–176 (PV1), 96
Periostin, 170, 171 Platelet-derived growth factor (PDGF), 87
Peritubular myoid cell (PMC), 247 Platelet-derived growth factor B
Perivascular cells, 102, 106, 139, 188, 203, (PDGF-B), 7, 136
242–243 Platelet-derived growth factor bb (PDGFbb), 191
Index 275

Pluripotent microvascular pericytes human eye, 2

adipocytes, 245 identification, 4, 5
astrocytes, 252 image processing, 3
COUP-TFII, 248, 250 NG2 proteoglycan, 8
EDS, 246, 248 ON, 2
embryonic ectoderm, 253 origin, 5–7
epiblast, 254 pathological retinal neovascularization, 10
genetic changes, 245 PDGFRb/PDGFB, 7, 8
history, 242 photoreceptors, 3
human testis, 239 ROP (see Retinopathy of prematurity
hypothesis, 253 (ROP))
hypoxia, 248 synapses, 2
in vivo experiment, 236 Tbx18, 9
Leydig cells, 236 tissue regeneration and wound healing,
mesenchymal stem cells, 254 17–19
NCSCs, 251, 253 vascular beds, 3, 4
PDGFR-α and PDGFR-ß, 247 Retinal angiogenesis, 49
PMCs, 248 Retinal ganglion cell (RGC), 14
seminiferous tubules, 239 Retinal pigment epithelial (RPE) cells, 3
sertoli cells, 246 Retinal vascular development, 3, 4
sources, 243 Retinols, 155
steroid hormones, 239 Retinopathy of prematurity (ROP)
testis, 236 blood vessels, 10
transdifferentiation, 250 CCN1 expression, 11
vascularized organ, 240 cell based therapies, 12, 13
vertebrate organism, 244 EC-PC communication, 11
Pneumolysin, 120 fibrous scar tissue, 11
Portal hypertensive syndrome, 81, 82 hyperoxia, 12
Portosystemic collateral vessels, 82, 84, 85 hypoxia, 10
Preeclampsia (PE), 229 IGF-1, 11
Proliferation cell nuclear antigen (PCNA), 105 laser photocoagulation and anti-VEGF
Pro-nerve growth factor (pro-NGF), 197 therapies, 11
Protease-activated receptor-2 (PAR2), 159 mural cells, 12
P-selectin, 102 neovascularization, 12
Pulmonary arterial hypertension (PAH), 52 neovascular vessels/tufts, 10
Pulmonary endothelial dysfunction, 52 OIR, 11
Pulmonary fibrosis, 42, 51, 52 oxygen-sensitive growth factors, 10–11
Pulmonary hypertension, 42 rodents, 11
vascular development, 10
Root region, 80
Q Rouget-Mayer cells, 243
Quail chick transplantations, 5

S
R Saethre-Chotzen syndrome, 170
Rat testis interstitium, 249 Satellite cells (SCs), 59–61, 63
Reactive oxygen species (ROS), 157 Scanning electron microscopy (SEM), 178
Regenerative medicine, 212, 225, 228 Scarring, 65
Regenerative therapy, 176, 177 Schwann cells, 67
Regulator of G protein signaling 5 (RGS5), Septum transversum, 154
62, 191 Sertoli cells, 239, 247
Retina Serum protein, 119
BRB, 9, 10 Sex-determining region box (SOX2), 191
DR (see Diabetic retinopathy (DR)) Sharpey’s fibers, 172
276 Index

Single nucleotide polymorphism (SNP), 34 T-cell factor/lymphoid enhancer factor (TCF/

Sinusoidal endothelial cells (SECs), 157, 158 LEF), 34
Sirtuin 3 (SIRT3), 198 Testes and epididymides, 238
Skeletal muscle Testicular interstitium, 238
limb ischemia model, 61 Testicular pericytes, 237
muscular dystrophy Thrombosis, 200, 201
adipogenesis, 65 Tissue dependency, 17
chondrogenesis and osteogenesis/ Tissue factor (TF), 190
ossification, 66, 67 Tissue inhibitor of metalloproteinase-1
fibrosis, 65, 66 (TIMP-1), 156
myogenesis, 63–65 Tissue metabolism, 177
myofibers, 59 Tissue regeneration, 17–19
nerve injury, 67 Toll-like receptors (TLRs), 160
pericyte physiology Transdifferentiation, 245, 249, 250
cellular markers, 62 Transforming growth factor beta (TGFβ), 7,
origins, 62 53, 156, 194
structure and density, 61 Transit amplifying cells, 244–246, 248–250,
pericytes, 60, 64 254
pericyte subtypes, 63 Transmission electron microscopy, 42, 217
satellite cells, 60 Trophoblast giant cells (TGC), 132
SCs, 60 Tropomyosin receptor kinase B (TrkB), 195
Smooth muscle actin (SMA), 173 Tumor growth factor (TGF)-β1, 136
Smooth muscle α-actin (α-SMA), 62 Tumor progression, 161
Smooth muscle cells (SMCs), 52 Twist, 170
Sonic hedgehog (Shh), 159 Type 2 diabetes
Sorafenib, 89 β-cell, 28
Spermatogenesis, 239 glycemic control, 31
Sphingosine-1-phosphate (S1P), 195 insulin resistance, 28
Splanchnic neovasculature, 88 pancreatic pericytes
Steatohepatitis, 157 β-cell dysfunction, 34, 35
alcoholic and nonalcoholic liver disease, 160 hyperglycemia, 35, 36
cholesterol, 160
etiologies, 159
inflammation and fibrogenesis, 159 U
liver diseases, 159 Umbilical artery, 225
neutrophils, 160 Umbilical cord
pro-inflammatory cytokines, 160 allantois and yolk sac, 214
TLRs, 160 application, 218
Stem cell factor (SCF), 106 blood vessels, 214
Streptococcus pneumoniae, 119 CD146+ cells, 223
Streptococcus pyogenes, 119 CD146 expression, 220, 222
Streptolysin, 120 cell phenotypes, 219, 223
STRO-1bright/ HSP70- cell population, 173 contractile cells, 218
Stromal-derived factor 1 (SDF-1), 157, 175, 179 cord lengthens, 214
Sudden sensorineural hearing loss (SSNHL), 119 embryonic processes, 212
Superior mesenteric arteries (SMA), 81 endothelial and perivascular cells, 217
Suppurative labyrinthitis, 120 erythrocyte-filled vessels, 218
Synepitheliochorial villous placenta, 133 gestation, 217
groundbreaking research study, 219
growth curve, 218
T HUCPVCs, 221, 225
T-box protein 18 (Tbx18), 9, 190 intra-abdominal part, 217
Tbx18-lineage cells, 50 jellylike substance, 212
T-cadherin, 198 mammals, 217
Index 277

mesenchymal progenitor cells, 219 Vascular endothelium, 179

mesenchymal stromal cells, 212 Vascular integrity, 189, 193
mesenchyme tissue connection, 214 Vascular network, 170, 171, 179
microvessels, 217 Vascular niche, 236, 245, 254, 255
MSCs, 219, 224 Vascular progenitor cells, 202
pathology, 227 Vascular smooth muscle cells
pericyte-associated markers, 212 (vSMCs), 4–5, 29
pericyte-like cells, 218 Vascular stem cells, 94, 189, 202
perivascular cells, 219 Vascular tone, 189, 195, 196
sign, 214 Vasculature, 212, 214, 218, 219, 225
stromal cells, 223 Vasoconstriction, 75
structural and functional regions, 214 Vasodilation, 75
structural organization, 218 Vasohibin-1, 91
UCWJ, 226 VEGF receptor (VEGFR), 159
vascularization, 212 VEGF receptor 2 (VEGFR2), 194
veins, 217 Villi vasculature, 79
vSMC, 222 Vimentin, 190
WJ, 222, 227, 228 Vitamin A, 155, 156
Umbilical cord Wharton’s jelly (UCWJ), 225 Vitamin D3 receptor (VDR), 160
Uterine natural killers (uNK), 132 von Willebrand factor (vWF), 191

V W
Vascular basement membrane, 32 Wharton’s jelly (WJ), 222, 226, 227
Vascular calcification, 199 Wild-type (WT), 156
Vascular calcification-associated factor Wilm’s tumor 1 (WT1), 154
(VCAF), 199 Wnt/ß-catenin pathway, 49, 199
Vascular cell adhesion molecule-1 Wnt-1-Cre mouse model, 5
(VCAM-1), 102 Wound healing, 17–19
Vascular endothelial growth factor
(VEGF), 7, 75, 85, 86, 136, 159
Vascular endothelial growth factor A Z
(VEGF-A), 194 Zygote and blastocyst differentiation, 252