Journal of Neurotrauma

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© Mary Ann Liebert, Inc.
DOI: 10.1089/neu.2020.7042
1
Expression of Angiopoietins and Angiogenic Signaling Pathway
Molecules in Chronic Subdural Hematomas
This paper has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.

Taiki Isaji, MD, PhD,a Koji Osuka, MD, PhD,a Yusuke Ohmichi, PhD,b Mika Ohmichi, PhD,b
Munekazu Naito, MD, PhD,b Takashi Nakano, MD, PhD,b Kenichiro Iwami, MD, PhD,a and
Shigeru Miyachi, MD, PhD,a
Expression of Angiopoietins and Angiogenic Signaling Pathway Molecules in Chronic Subdural Hematomas (DOI: 10.1089/neu.2020.7042)

a
Department of Neurosurgery and bDepartment of Anatomy, Aichi Medical University,

1-1 Yazakokarimata, Nagakute, Aichi, 480-1195, Japan

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Running title:

ANGIOPOIETIN AND TIE2 IN CHRONIC SUBDURAL HEMATOMA

*Corresponding Author

Koji Osuka, MD, PhD

Journal of Neurotrauma

1-1 Yazakokarimata, Nagakute, Aichi, 480-1195, Japan

Tel: +81-561-62-3311, Fax: +81-561-62-2879

E-mail: [email protected]

Full mailing addresses and contact information

Taiki Isaji, M.D., Ph.D.

Department of Neurological Surgery, Aichi Medical University

1-1 Yazakokarimata, Nagakute, Aichi 480-1195, Japan

Tel.: +81-561-62-3311, Fax: +81-561-63-2879

E-mail: [email protected]
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Koji Osuka, M.D., Ph.D.

Department of Neurological Surgery, Aichi Medical University

This paper has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.

1-1 Yazakokarimata, Nagakute, Aichi 480-1195, Japan

Tel.: +81-561-62-3311, Fax: +81-561-63-2879

Expression of Angiopoietins and Angiogenic Signaling Pathway Molecules in Chronic Subdural Hematomas (DOI: 10.1089/neu.2020.7042)

E-mail: [email protected]

Yusuke Ohmichi, Ph.D.

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Department of Anatomy, Aichi Medical University

1-1 Yazakokarimata, Nagakute, Aichi 480-1195, Japan

Tel.: +81-561-62-3311, Fax: +81-561-61-0324

E-mail: [email protected]
Journal of Neurotrauma

Mika Ohmichi, Ph.D.

Department of Anatomy, Aichi Medical University

1-1 Yazakokarimata, Nagakute, Aichi 480-1195, Japan

Tel.: +81-561-62-3311, Fax: +81-561-61-0324

E-mail: [email protected]

Munekazu Naito, M.D., Ph.D.

Department of Anatomy, Aichi Medical University

1-1 Yazakokarimata, Nagakute, Aichi 480-1195, Japan

Tel.: +81-561-62-3311, Fax: +81-561-61-0324

E-mail: [email protected]
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Takashi Nakano, M.D., Ph.D.

Department of Anatomy, Aichi Medical University

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1-1 Yazakokarimata, Nagakute, Aichi 480-1195, Japan

Tel.: +81-561-62-3311, Fax: +81-561-61-0324

Expression of Angiopoietins and Angiogenic Signaling Pathway Molecules in Chronic Subdural Hematomas (DOI: 10.1089/neu.2020.7042)

E-mail: [email protected]

Kenichiro Iwami, M.D., Ph.D.

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Department of Neurological Surgery, Aichi Medical University

1-1 Yazakokarimata, Nagakute, Aichi 480-1195, Japan

Tel.: +81-561-62-3311, Fax: +81-561-63-2879

E-mail: [email protected]
Journal of Neurotrauma

Shigeru Miyachi, M.D., Ph.D.

Department of Neurological Surgery, Aichi Medical University,

1-1 Yazakokarimata, Nagakute, Aichi 480-1195, Japan

Tel.: +81-561-62-3311; Fax: +81-561-63-2879

E-mail: [email protected]
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Abstract

Chronic subdural hematoma (CSDH) is an angiogenic disease that is involved with

This paper has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.

many inflammatory mediators. Tie2 is predominantly expressed in the embryonic

endothelium and plays an important role in the maturation and stabilization of the
vasculature. Angiopoietin1 (Ang1) and Ang2 are well-known ligands of the Tie2 receptor.
Expression of Angiopoietins and Angiogenic Signaling Pathway Molecules in Chronic Subdural Hematomas (DOI: 10.1089/neu.2020.7042)

We examined the expression of Ang1 and Ang2 in the CSDH fluid and the expression of Tie-
2 receptor and components of the angiogenic signaling pathways in the outer membrane
of CSDH. Twenty-five samples of CSDH fluid and 8 samples of outer membrane of CSDH
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were included. The concentrations of Ang1 and Ang2 in the CSDH fluid were measured
using ELISA kits. The expression of Tie2, phosphoinositide 3-kinase (PI3-kinase), protein
kinase B (Akt) mechanistic target of rapamycin (mTOR), GβL, 70kDa ribosomal protein S6
kinase (p70S6K), eukaryotic initiation factor 4E (eIF-4E) and β-actin was examined by a
Western blot analysis. The expression of Tie2, Akt and mTOR was also examined by
Journal of Neurotrauma

immunohistochemistry. The concentration of Ang2 in CSDH fluid was significantly higher

than that in the serum or CSF and also higher than that of Ang1 in CSDH fluid. Tie2, PI3-
kinase, Akt, mTOR, GβL, p70S6K, eIF-4E were detected in all cases. In addition, Tie2, Akt
and mTOR were localized in the endothelial cells of vessels within CSDH outer membrane.
Our data suggest that Ang2, while not Ang1, in CSDH fluid promotes angiogenesis in
endothelial cells through the Tie2 receptor. The Ang2/Tie2 signaling pathway might
therefore be a useful therapeutic target for treating the growth of intractable CSDH.

Key words: angiogenesis, angiopoietin, chronic subdural hematoma, Tie2

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Introduction

Chronic subdural hematomas (CSDHs) occur in elderly people who have suffered
This paper has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.

mild head trauma. Patients with CSDH present with symptoms such as headache,
hemiparesis, gait disturbance and a decreased cognitive function. The treatment for
symptomatic CSDH is trepanation surgery, even in elderly people. However, recurrence of
Expression of Angiopoietins and Angiogenic Signaling Pathway Molecules in Chronic Subdural Hematomas (DOI: 10.1089/neu.2020.7042)

CSDH after the surgery has been observed at a rate of 5%-17%. 1, 2 The precise mechanism
underlying the development and recurrence of CSDH still remains to be elucidated.

Vascular endothelial growth factor (VEGF) is a potent angiogenic factor, and high
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concentrations of VEGF in CSDH fluid have been reported,3-5 suggesting that VEGF might
be involved in the angiogenesis of the CSDH outer membrane. VEGF in CSDH fluid activates
the NF-κB and Ras/MEK/ERK pathways in endothelial cells, resulting in angiogenesis in the
outer membrane of CSDH.6, 7 The inflammatory cytokine interleukin-6 (IL-6) has been
reported to be elevated in CSDH fluids and significantly correlated with recurrence. 8 IL-6
Journal of Neurotrauma

activates the janus kinase (JAK)/signal transducer and activator of transcription (STAT)
signaling pathway in fibroblasts and endothelial cells within CSDH outer membranes and
plays a role in the growth of CSDH.9, 10 These data suggest that angiogenesis and
inflammation play key roles in the development of CSDH. 11

Angiopoietins are another family of growth factors that are involved in blood vessel
formation during developmental and pathological angiogenesis. Ang1-deficient mice die
between E11.5 and E12.5 due to dilated vessels and a decreased complexity of the
vascular network.12 A previous study showed that mRNA species encoding Ang1, Ang2 and
Tie-2 were detected in outer membrane samples, suggesting that angiogenic mechanisms
induced by angiopoietins play an important role in the pathophysiology of CSDH. 13
However, the protein levels of these molecules are still unknown, as the expression may
differ between mRNA and protein.

Therefore, we explored whether or not angiopoietins exist in CSDH fluid and

investigated the angiogenic signaling pathway in the outer membrane of CSDH using
immunoblotting and immunohistochemical analyses.
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Material and Methods

This study was approved by Certified Clinical Research Review Board of Aichi
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Medical University Hospital (17-H047). Informed consent was obtained from each patient
or patient’s family. Burr hole irrigation and drainage surgery were performed on all
patients under local anesthesia at Aichi University Hospital.
Expression of Angiopoietins and Angiogenic Signaling Pathway Molecules in Chronic Subdural Hematomas (DOI: 10.1089/neu.2020.7042)

Materials

All chemicals were obtained from Sigma Chemical (St. Louis, MO) unless otherwise
specified.
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The analysis of angiopoietins in CSDH fluid

Fluid from 25 consecutive CSDHs were obtained during trepanation surgery. As a

control, CSF samples were obtained from 10 patients undergoing neck clipping for
Journal of Neurotrauma

unruptured cerebral aneurysm and serum samples were obtained from 6 patients
suffering from CSDH. After collection, all samples were immediately centrifuged, and the
supernatant was stored at -80 °C until the analysis.

We measured the concentrations of angiopoietin1 (Ang1) and Ang2 using enzyme-

linked immunosorbent assay (ELISA) kits (R&D Systems, Minneapolis, MN), according to
the manufacture’s instructions. The mean minimum detectable doses of these assays were
3.5 pg/mL for Ang1 and 8.3 pg/mL for Ang2.

Western blotting analyses

Eight samples of the outer membrane of CSDH were obtained during surgery. The
membranes were homogenized in 75 μL of homogenization buffer containing 50 mM Tris
base/HCL (pH 7.5), 0.1 mM dithiothreitol, 0.2 mM ethylenediaminetetraacetate, 0.2 mM
ethylene glycol bis(aminoethyl ether)tetraacetate, 0.2 mM phenylmethylsulfonyl fluoride,
1.25 μg/mL of pepstatin A, 0.2 μg/mL of aprotinin, 1 mM sodium orthovanadate, 50 nM
sodium fluoride, 2 mM sodium pyrophosphate and 1% Nonidet P-40. The homogenates
were centrifuged at 12000×g for 10 minutes at 4 ℃. The protein concentrations of the
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supernatants were separated using 7.5% or 10% sodium dodecyl sulfate (SDS)-
polyacrylamide gel electrophoresis.
This paper has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.

The proteins in the gel were transferred to polyvinylidene difluoride membrane

and incubated with primary polyclonal antibodies against Tie2 (R&D Systems),
phosphoinositide 3-kinase (PI3 kinase, Cell Signaling Technology, Danvers, MA), protein
Expression of Angiopoietins and Angiogenic Signaling Pathway Molecules in Chronic Subdural Hematomas (DOI: 10.1089/neu.2020.7042)

kinase B (Akt; Cell Signaling Technology), mechanistic target of rapamycin (mTOR; Cell
Signaling Technology), GβL (Cell Signaling Technology), 70kDa ribosomal protein S6 kinase
(p70S6K; Cell Signaling Technology), eukaryotic initiation factor 4E (eIF4E; R&D Systems)
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and β-actin (Sigma). Akt and mTOR antibody were diluted 1:1,000, and all the others were
used at a 1:500 dilution and incubated overnight at 4 °C. After washing away unbound
antibodies, the membranes were incubated in secondary antibodies with horseradish
peroxidase (Sigma) at a 1:3000 dilution for 30 minutes at room temperature. The reactions
were developed with ECL Plus (GE Healthcare, Buckinghamshire, UK). Positive controls
Journal of Neurotrauma

were Jurkat whole cell lysate (Cell Signaling Technology) and NIH/3T3 whole cell lysate
(Santa Cruz biotechnology, Dallas, TX).

Histological examinations

For the analysis of the cellular localization of Tie2, Akt and mTOR,
immunohistochemical staining was performed on samples from three patients at room
temperature using the avidin-biotinylated peroxidase complex (ABC) technique. To
preserve the outer membrane of the CSDH samples, the membranes were incubated in 10
mL of ice-cold 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 3 h and then
embedded in paraffin.

In this study, 10-μm-thick sections were prepared on a microtome and mounted

onto MAS-coated glass slides (Matsunami Glass, Kishiwada Japan). The sections were
deparaffinized in xylene, immersed in decreasing concentrations of ethanol, and
rehydrated in water. Endogenous peroxidase activity was blocked with 0.3% H 2O2 in 100%
methanol for 20 min. All sections for immunostaining were processed for microwave-
enhanced antigen retrieval. Slide-mounted sections immersed in 0.01 M sodium citrate
buffer (pH 6.0) were placed for 15 min in a 700-W microwave oven at maximum power.
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Non-specific immunoreactivity was blocked by incubation with goat or donkey
serum for 30 min, depending on the primary antibody. The samples were treated with
This paper has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.

primary antibodies against Tie2 (R&D Systems) at a dilution of 1:150, Akt (Cell Signaling
Technology) at a dilution of 1:300 and mTOR (Cell Signaling Technology) at a dilution of
1:100 over 2 nights at 4 °C. After several rinses in PBS, the samples were incubated with
Expression of Angiopoietins and Angiogenic Signaling Pathway Molecules in Chronic Subdural Hematomas (DOI: 10.1089/neu.2020.7042)

secondary biotinylated antibodies (anti-goat IgG 1:200, anti-rabbit IgG 1:200; Santa Cruz
Biotechnology) at room temperature for 2 h. After several more rinses in PBS, they were
incubated with the Vectastain ABC reagent (Vectastain ABC Kit; Vector Laboratories,
Burlingame, CA) for 1 h. After several more rinses in PBS, the bound peroxidase was
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visualized by incubating the sections with a solution containing 0.05% 3,3'-

diaminobenzidine tetrahydrochloride (Sigma Aldrich) and 0.01% H2O2 in 0.05 M Tris-HCl
(pH 7.4) for 10 min. After several rinses in water, the immunostained sections were
dehydrated and cover-slipped with Entellan new (Merk, Kenilworth, NJ).
Journal of Neurotrauma

Statistical analyses

Data are expressed as the mean ± standard error. Significant differences between
groups were assessed using a one-way analysis of variance (ANOVA) followed by
Bonferroni’s/Dunn’s tests for multiple comparisons. The Mann-Whitney U test was used
for the analysis between two groups. Significance was indicated when p < 0.05.

Results

Clinical data

Clinical data are presented in Table 1. There were 18 men and 7 women in this
study, ranging in age from 51-92 years, with an average age of 78.1 years. In total, 25 CSDH
fluid samples were obtained and included two recurrent cases. One patient (No. 16) died
from a pancreatic cancer as the comorbidity. Hematoma type of CT images are divided
according to the classification into four types grouped by Nakaguchi et al. 1 There were 4
homogenous types, 7 laminar types, 6 separated types and 8 trabecular types.
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Concentrations of Ang1 and Ang2 in CSDH fluid

The concentration of Ang1 in serum (29,327.3 ± 5,282.6 pg/ml) was significantly

This paper has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.

higher than that in CSDH fluid or CSF (1,614.9 ± 184.3 pg/ml and 294.3 ± 37.7 pg/ml,
respectively) according to a one-way ANOVA followed by Bonferroni’s/Dunn’s test (p <
0.05, Figs. 1A). In contrast, the concentration of Ang2 in CSDH fluid (97,523.8 ± 14,455.5
Expression of Angiopoietins and Angiogenic Signaling Pathway Molecules in Chronic Subdural Hematomas (DOI: 10.1089/neu.2020.7042)

pg/ml) was significantly higher than that in the serum or CSF (1,203.2 ± 162.2 pg/ml and
1,596.3 ± 272.1 pg/ml, respectively) according to a one-way ANOVA followed by
Bonferroni’s/Dunn’s test (p < 0.05, Figs. 1B). The concentration of Ang2 in CSDH fluid was
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significantly much more higher than that of Ang1in the CSDH fluid according to a Mann-
Whitney U test (p < 0.01, Fig. 1C).

Western blotting analyses of Tie2 and the angiogenic signaling pathway

Fig. 2 shows the results of Western blotting analyses of Tie2 and the angiogenic
Journal of Neurotrauma

signaling pathway. Nearly constant β-actin levels were detected in CSDH outer membrane
samples. Tie2, PI3 kinase, Akt, mTOR, GβL, p70S6 kinase and eIF-4E were detected in
almost all samples; however, in some cases, the signals were weak. Positive controls
revealed that these molecules had been correctly detected.

Historical observations

Tie2 (Figs. 3A and B), Akt (Figs. 3C and D) and mTOR (Figs. 3E and F) were localized
in the endothelial cells of vessels within the outer membrane. Note that vascular
longitudinal cross-sections in 10 µm consecutive slices at a higher magnification clearly
demonstrate that these molecules are expressed in the endothelium (Figs. 3B, D and F). In
the negative controls examined without primary antibodies, the endothelial cells were
consistently negative for the markers listed above (Fig. 3G).

Discussion

The expression of Ang2 in CSDH fluid was significantly higher than that of Ang1 in
CSDH fluid. Molecules of the angiogenic pathway, including Tie2, were detected in the
outer membrane of CSDH. Tie2, Akt and mTOR were expressed in the endothelium of
vessels of the CSDH outer membrane.
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Tie2 was identified as a receptor tyrosine kinase expressed principally on the
vascular endothelium.10 Ang1 phosphorylates Tie2 and induces vessel maturation. Ang1
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mediates migration, adhesion and the survival of endothelial cells. Both Ang1 and Ang2
bind to the Tie2 receptor. In general, Ang2 antagonize Ang1 by keeping Ang1 from binding
to Tie2 receptors. Ang2 disrupts the connections between the endothelium and
Expression of Angiopoietins and Angiogenic Signaling Pathway Molecules in Chronic Subdural Hematomas (DOI: 10.1089/neu.2020.7042)

perivascular cells, resulting in vascular regression. 14, 15 However, some reports have shown
that Ang2 is a Tie2 agonist, suggesting that the action of Ang2 as a Tie2 agonist or
antagonist is context-dependent.16 The increased expression of Ang2 resulted in increased
tumor vascular density, and the increased expression of Ang2 relative to Ang1 in tumors
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correlates with a poor prognosis.17 Glioblastomas are characterized by prominent

vascularization. Ang1 mRNA was expressed in tumor cells, while Ang2 mRNA was
confirmed in endothelial cells of glioblastoma blood vessels, especially in small capillaries
with few periendothelial support cells.18, 19 Hepatocellular carcinoma (HCC) is also a tumor
frequently associated with increased vascularity. The Ang2 expression may play an
Journal of Neurotrauma

important role in vascular remodeling and angiogenesis in HCC tumors. 20, 21 In systemic
sclerosis patients, the serum levels of Ang1 were shown to be significantly lower and those
of Ang2 significantly higher than in the control group. 22 Altered Ang1/Ang2 levels might
underlie the aberrant angiogenesis in systemic sclerosis. These data are agreement with
our own finding that the level of Ang1 in CSDH fluids was lower while that of Ang2 was
higher than the relevant serum levels in CSDH patients. A previous study revealed that the
mean ratio of the Ang1/Ang2 mRNA expression in CSDH membrane was 0.48,13 while the
mean ratio of the Ang1/Ang2 protein level in CSDH fluid was 0.02 in our data. Ang2 can act
as an agonist of the Tie2 receptor in the absence of Ang1. 16 Recently, Ang2 was shown to
be upregulated in multiple inflammatory diseases and was implicated in the mediation of
inflammatory processes as well.23 Taken together, these findings suggest that the high
concentration of Ang2 in CSDH fluid might be involved in the regulation of angiogenesis
and inflammation within CSDH.

The expression of PI3K/Akt signaling molecules was confirmed by Western blot

analyses. PI3K/AKT constitutes an important pathway regulating the signaling of multiple
biological processes, such as apoptosis, metabolism and cell proliferation. Dysregulation of
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the PI3/Akt pathway leads to diseases, such as cancer, diabetic mellitus and cardiovascular
disease.24 Ang2 induced the phosphorylation of Tie2, PI3 kinase and Akt in human
This paper has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.

umbilical vein endothelial cells, resulting in the endothelial cell survival.25 A previous study
showed that activated Akt was expressed in the endothelial cells of vessels in CSDH, which
is in agreement with our immunohistochemical result. 26 mTOR, which is a downstream
Expression of Angiopoietins and Angiogenic Signaling Pathway Molecules in Chronic Subdural Hematomas (DOI: 10.1089/neu.2020.7042)

kinase of the PI3K/Akt signaling pathway, plays an important regulatory role in the cell
survival, vascular permeability, migration and proliferation, as well as in angiogenesis. 27, 28
mTOR exists in two protein complexes: mTOR complex 1 (mTORC1) and mTOR complex 2
(mTORC2). mTORC1 consists of mTOR, Raptor and GβL. Activated mTORC1 catalyzes
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p70S6K and eukaryotic initiation factor 4E-bindign protein-1 (4E-BP1) phosphorylation.29, 30

p70S6K plays an important role in regulating tumor angiogenesis in endothelial cells,31
while 4E-BP1 is a translational repressor. The phosphorylation of 4E-BP1 leads to the
release of eIF-4E from the 4EBP1/eIF4E complex. eIF-4E is a regulatory protein that binds
to the mRNA cap-binding32 and induces tumor vascularization in vivo.33 HIF1 is a
Journal of Neurotrauma

heterodimeric transcriptional factor composed of α and β subunits. Accumulated HIF1α

translocates to the nucleus and dimerises with HIF1β to form a functional transcription
factor capable of DNA binding at hypoxia response elements (HREs) and activating the
transcription of target genes (Fig. 4).34 HIF1 plays a central role in tumor progression and
angiogenesis in vivo. The PI3K/Akt signaling pathway mediates the upregulation of HIF1α
by increasing HIF1α synthesis or HIF1α stability, resulting in the transcriptional activity of
HIF1.35 HIF1α was confirmed to be positive in the outer membrane of CSDH and induced
the excessive development of fragile microvessels through VEGF.3 Both p70S6K and HIF-1
are essential for tumor growth and angiogenesis.31 Vanillic acid, an active compound
isolated from green tea, inhibits angiogenesis in human colon cancer cells by suppressing
the HIF-1α expression and mTOR/p70S6K/4EBP-1 pathway.36 Given our findings that Tie2,
Akt and mTOR were expressed in endothelial cells, the PI3 kinase/Akt/mTOR/eIF-4E
signaling pathway activated by Ang2/Tie2 receptor may play an important role in
angiogenesis in the CSDH outer membrane.

Several limitations associated with the present study warrant mention. First, from
our limited case number, we were unable to detect the correlation between the level of
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angiopoietin in CSDH fluid, the data from Western blot analyses and the growth stage of
CSDH. Further studies, including more patients, will be needed in order to clarify this point.
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Second, we only detected the existence of these angiogenic signaling molecules in the
outer membrane of CSDH. Whether or not this cascade is activated remains to be
elucidated. We also need control cases, such as cases of chronic subdural effusion, in order
Expression of Angiopoietins and Angiogenic Signaling Pathway Molecules in Chronic Subdural Hematomas (DOI: 10.1089/neu.2020.7042)

to explore whether or not this Ang/Tie2 signaling pathway plays an important role in
angiogenesis of CSDH.

Conclusion
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In the present study, we detected the expression of Tie2 and the PI3
kinase/Akt/mTOR angiogenic signaling pathway in the CSDH outer membrane for the first
time. High concentrations of Ang2 in CSDH fluid might play an important role in
angiogenesis and inflammation in CSDH, resulting in the growth of the hematoma. This
Ang2/Tie2 signaling pathway might be a therapeutically attractive target for the treatment
Journal of Neurotrauma

of intractable CSDH.

Acknowledgments

This work was supported in part by a Japanese Grant-in-Aid for Scientific Research
(C), grant number 17K10853 (K.O.).

Author Disclosure Statement

No competing financial interests exist.

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chronic subdural hematomas that influence their postoperative recurrence. J

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Expression of Angiopoietins and Angiogenic Signaling Pathway Molecules in Chronic Subdural Hematomas (DOI: 10.1089/neu.2020.7042)

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6. Osuka, K., Watanabe, Y., Usuda, N., Aoyama, M., Kawaguchi, R., Takeuchi, M., and
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9. Osuka, K., Watanabe, Y., Usuda, N., Aoyama, M., Kawaguchi, R., Watabe, T., and
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19. Zagzag, D., Hooper, A., Friedlander, D.R., Chan, W., Holash, J., Wiegand, S.J.,
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Table 1. Characteristics of 25 patients with chronic subdural hematoma.

Case age sex side hematoma size midline symptoms GCS GOS N.B.
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type (mm) shift (mm)

1 89 W lt H 20 4 gait disturbance 15 5
ngiopoietins and Angiogenic Signaling Pathway Molecules in Chronic Subdural Hematomas (DOI: 10.1089/neu.2020.7042)

2 77 M rt H 28 7 headache, dizziness 15 5

3 70 M lt H 15 5 hemiparesis 14 5

4 72 M rt H 21 8 gait disturbance 15 5

5 65 M rt L 23 7 hemiparesis 14 5

6 83 M lt L 18 5 headache 15 5

7 64 M rt L 30 5 headache 15 3 recurrence
Journal of Neurotrauma

8 62 W lt L 17 2 headache 14 5

9 78 W lt L 20 13 hemiparesis 14 3

10 73 M lt L 25 10 hemiparesis 13 5

11 84 M lt L 28 9 hemiparesis 15 4 recurrence
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12 84 W lt S 23 4 hemiparesis 15 5

13 81 M rt S 20 1 gait disturbance 14 3
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14 92 W rt S 15 4 hemiparesis 11 3

15 82 M lt S 24 5 hemiparesis 13 3
ngiopoietins and Angiogenic Signaling Pathway Molecules in Chronic Subdural Hematomas (DOI: 10.1089/neu.2020.7042)

16 76 W lt S 18 15 headache 14 1 pancreatic cancer

17 91 M rt S 23 5 gait disturbance 14 3

18 82 M lt T 18 5 hemiparesis 15 5

19 84 M rt T 15 1 hemiparesis 13 3

20 90 M lt T 18 6 gait disturbance 14 5

21 76 M rt T 18 5 hemiparesis 15 5
Journal of Neurotrauma

22 51 W lt T 26 13 hemiparesis 15 5

23 90 M lt T 32 18 hemiparesis 14 5

24 78 M rt T 17 5 hemiparesis 15 4

25 79 M rt T 30 7 hemiparesis 15 5
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Journal of Neurotrauma
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and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.

GCS, Glasgow Coma Scale at admission; GOS, Glasgow Outcome Scale at discharge
M, man; W, woman, lt, left; rt, right; H, homogeneous type; L, laminar type; S, separated type; T, trabecular type.

No. 7 and 11 are recurrent cases. The size refers to the largest extent measured in pre-operative CT slices. Shift refers to the midline sift.
20
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Figure legends
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Journal of Neurotrauma

FIG. 1.

Concentrations of angiopoietin-1 (A and angiopoietin-2 (B) in chronic subdural

hematoma (CSDH, n = 25), serum from CSDH patients (serum, n = 6) and control
cerebrospinal fluid (CSF, n = 10). The concentrations of angiopoetin-1 in serum and
angiopoetin-2 in CSDH fluid were significantly higher than those values in other groups
according to a one-way ANOVA, followed by Bonferroni’s/Dunn’s test. Furthemore,
the concentration of angiopoietin-2 (Ang2) in CSDH fluid was significantly much more
higher than that of angiopoietin-1 (Ang1) by a Mann-Whitney U-test (C). Data
represent the median values, 25th and 75th percentiles with maximum/minimum
whiskers. * p < 0.05 denotes a significant difference between the groups.
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FIG. 2.

Western blots showing the expression of angiogenic pathway molecules in the outer
Journal of Neurotrauma

membrane of chronic subdural hematomas from eight patients. Tie2,

phosphoinositide 3-kinase (PI3 kinase), protein kinase B (Akt), mechanistic target of
rapamycin (mTOR), GβL, 70kDa ribosomal protein S6 kinase (p70S6K) and eukaryotic
initiation factor 4E (eIF4E) were detected in almost all cases. Positive controls are
shown in the right two lanes and suggest that these molecules were correctly
detected. Jurkat, Jurkat whole cell lysate; 3T3/NIH, 3T3/NIH whole cell lysate
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Journal of Neurotrauma

FIG. 3.

Ten-micrometer consecutive slices were immunostained with polyclonal antibodies

against Tie 2 (A and B), protein kinase B (Akt, C and D) and mechanistic target of
rapamycin (mTOR, E and F) using the ABC method. The areas within the rectangle,
labeled A, C and E, are shown at a higher magnification in panels B, D and F,
respectively. Note that these molecules were expressed in endothelial cells within the
vascular longitudinal cross-sections in 10 µm consecutive slices (B, D and F).
Consecutive slices immunostained without primary antibodies are shown in (G). Scale
bars = 50 µm.
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Journal of Neurotrauma

FIG. 4.

The signal transduction of angiopoietin in the outer membrane of chronic subdural

hematoma (CSDH). Angiopoietins binds to Tie 2 receptor. mTOR, which is a
downstream kinase of the PI3K/Akt signaling pathway, plays an important regulatory
role in the cell survival, vascular permeability, migration and proliferation. mTORC1
consists of mTOR, Raptor and GβL. Activated mTORC1 catalyzes p70S6K and 4E-BP1
phosphorylation, resulting in the release of eIf-4E from the 4EBP1/eIF-4E complex. eIF-
4E is a regulatory protein of HIF-1α synthesis that dimerizes with HIF1α and HIF1β and
translocates into the nucleus, where it regulates transcription factors. The expression
of molecules bordered by a solid line was confirmed by Western blot analyses. HRE,
hypoxia response elements.