Protein family review

The vascular endothelial growth factor (VEGF) family: angiogenic

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factors in health and disease
David IR Holmes*† and Ian Zachary*
Addresses: *BHF Laboratories and The Rayne Institute, Department of Medicine, University College London, 5 University Street, London
WC1E 6JJ, UK. †Ark Therapeutics Ltd, 1 Fitzroy Mews, London W1T 6DE, UK.

Correspondence: Ian Zachary. E-mail: [email protected]

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Published: 1 February 2005
Genome Biology 2005, 6:209
The electronic version of this article is the complete one and can be
found online at http://genomebiology.com/2005/6/2/209
© 2005 BioMed Central Ltd

reports
Summary

Vascular endothelial growth factors (VEGFs) are a family of secreted polypeptides with a highly
conserved receptor-binding cystine-knot structure similar to that of the platelet-derived growth
factors. VEGF-A, the founding member of the family, is highly conserved between animals as
evolutionarily distant as fish and mammals. In vertebrates, VEGFs act through a family of cognate

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receptor tyrosine kinases in endothelial cells to stimulate blood-vessel formation. VEGF-A has
important roles in mammalian vascular development and in diseases involving abnormal growth of
blood vessels; other VEGFs are also involved in the development of lymphatic vessels and
disease-related angiogenesis. Invertebrate homologs of VEGFs and VEGF receptors have been
identified in fly, nematode and jellyfish, where they function in developmental cell migration and
neurogenesis. The existence of VEGF-like molecules and their receptors in simple invertebrates
without a vascular system indicates that this family of growth factors emerged at a very early
stage in the evolution of multicellular organisms to mediate primordial developmental functions.

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The formation of a vascular system is a prerequisite for ver- known as c-Fos-induced growth factor, FIGF), and the viral
tebrate embryogenesis and involves two fundamental VEGF-Es encoded by strains D1701, NZ2 and NZ7 of the
processes: vasculogenesis, defined as the differentiation of parapoxvirus Orf (which causes pustular dermatitis) [5,6].
endothelial cell progenitors and their assembly into the The biological functions of the VEGFs are mediated by a
primary capillary plexus, and angiogenesis, the sprouting of family of cognate protein tyrosine kinase receptors

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new capillaries from pre-existing vessels [1]. In the adult, (VEGFRs) [7-9]. VEGF-A binds to VEGFR2 (also called
angiogenesis is also essential during pregnancy and in tissue KDR/Flk-1) and VEGFR1 (Flt-1); VEGF-C and VEGF-D bind
growth and repair, and is a key underlying process in the VEGFR2 and VEGFR3 (Flt4); PLGF and VEGF-B bind only
pathogenesis of several major human diseases, including to VEGFR1; and VEGF-E binds only to VEGFR2. In addition,
cancer. Since its discovery in 1983 [2] and the subsequent certain VEGF family isoforms bind to non-tyrosine kinase
cloning of the gene in 1989 [3,4], vascular endothelial receptors called neuropilins (NRPs) [10,11].
growth factor (VEGF-A, also called VEGF or vascular perme-
ability factor) has emerged as the single most important reg-
information

ulator of blood vessel formation in health and disease; it is Gene organization and evolutionary history
essential for embryonic vasculogenesis and angiogenesis, Evolution
and is a key mediator of neovascularization in cancer and VEGFs belong to the VEGF/PDGF (platelet-derived growth
other diseases [1]. VEGF-A is the prototypical member of a factor) group of the cystine-knot superfamily of hormones
family of related growth factors that includes placental and extracellular signaling molecules [12], which are all
growth factor (PLGF), VEGF-B, VEGF-C, and VEGF-D (also characterized by the presence of eight conserved cysteine

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residues forming the typical cystine-knot structure (named rerio and the pufferfish Fugu rubripes), frogs (Xenopus
after cystine, a dimer of two cysteines linked by a disulfide laevis), birds (Gallus gallus), and mammals (Table 1). The
bond). The VEGF/PDGF group is evolutionarily related to sequence and genomic organization of the vertebrate VEGF-
other groups within the cystine-knot superfamily, notably A genes is highly conserved between teleost fish and
the glycoprotein hormone and mucin-like protein families mammals, even though separation of these two groups from
and, more distantly, the transforming growth factor-␤ (TGF- their common ancestor occurred around 450 million years
␤) family. The absence of any of these proteins in unicellular ago: pufferfish VEGF-A shows 68% and 69.7% amino-acid
eukaryotes such as yeast suggests that the cystine-knot identity with human and mouse VEGF-A, respectively [13].
structure evolved to perform hormonal and extracellular-
signaling functions in multicellular organisms with tissue- VEGF-like proteins emerged relatively early in the evolution
level organization. of multicellular animal life, as indicated by their presence in
several invertebrate species. Invertebrate VEGF/VEGFR
The known members of the human VEGF family are shown systems have been identified in fly (Drosophila
in Table 1. VEGFs have been found in all vertebrate species melanogaster), nematode (Caenorhabditis elegans) and,
so far examined and are highly conserved between species. most recently, jellyfish (Podocoryne carnea). Drosophila has
VEGF-A has been found in teleost fish (the zebrafish Danio three PDGF/VEGF-like factors (PVFs), which act through a

Table 1

The human VEGF family and related proteins from Drosophila and Orf virus

Species (strain) Chromosomal Homologs‡ found in

and gene name Number of exons location* Accession number† References other species

Human VEGF-A 8 6p12 NM_003376 [59] Mus musculus

Rattus norvegicus
Sus scrofa
Bos taurus
Canis familiaris
Gallus gallus
Xenopus laevis
Danio rerio
Fugu rubripes
Human VEGF-B 7 11q13 NM_003377 [23] M. musculus
R. norvegicus
B. taurus
Human VEGF-C 7 4q34.1-q34.3 NM_005429 [60] M. musculus
R. norvegicus
B. taurus
D. rerio
Human VEGF-D 7 Xp22.31 NM_004469 [61] M. musculus
R. norvegicus
B. taurus
Human PLGF 7 14q24-q31 NM_002632 [22] M. musculus
R. norvegicus
B. taurus
Orf virus (D1701) VEGF-E - - AF106020 -
Orf virus (NZ2) VEGF-E - - S67520 -
Orf virus (NZ7) VEGF-E - - S67522 -
D. melanogaster PVF1 6 X 17E1-17E6 NM_078683 [14-16] Caenorhabditis elegans§
Podocoryne carnea¶
D. melanogaster PVF2 5 2L 27E1 NM_078775 [16] -
D. melanogaster PVF3 6 2L 27E1-27E2 NM_078776 [16] -

*Chromosome locations of human and Drosophila genes are from Entrez Gene and FlyBase. †Accession numbers are from RefSeq and GenBank.
‡Homolog data are from HomoloGene, Entrez Gene and [13]. §Putative homolog identified by survey of C. elegans genome [17]. ¶Possible homolog [18].

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single receptor, PVR [14-16]. In C. elegans, four VEGFRs, which lacks exon 6 and contains an alternative exon 8
VERs (vascular endothelial growth factor receptor related) 1, encoding a novel carboxy-terminal sequence, thereby raising
2, 3 and 4, have been identified [17]. Definitive identification the possibility of the existence of a family of sister isoforms

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of a VER ligand is awaited, although a putative homolog of containing this novel carboxyl terminus [21].
Drosophila PVF1 was revealed by a survey of the C. elegans
genome [17]. A single VEGF/VEGFR system has been found Human PLGF exists in four isoforms, PLGF-1 to PLGF-4,
in P. carnea [18], with the VEGF being a possible homolog of with PLGF-1 and PLGF-2 believed to be the major isoforms.
Drosophila PVF1. In all cases, the invertebrate ligands appear The PLGF-1 and PLGF-2 transcripts encode isoforms
to be more closely related to the VEGFs than to the PDGFs. (excluding signal peptide) of 131 and 152 amino acid
residues, respectively. PLGF-2 is able to bind heparin and
Alignment of the VEGF/PDGF homology domains (VHD) of NRP1 through an exon 6 encoded heparin-binding domain

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VEGFs, PDGFs and PVFs, encompassing the residues [22]; PLGF-1 lacks exon 6 and is thus unable to bind heparin
making up the cystine-knot structure, reveals a high degree [19]. PLGF-3 also lacks exon 6 but additionally contains a
of regional conservation (Figure 1a). The eight cysteine 216-nucleotide insertion between exons 4 and 5. PLGF-4
residues of the cystine-knot structure are highly conserved, consists of the same sequence as PLGF-3, plus the heparin-
except in Drosophila PVF2, which lacks cysteine 2, and binding domain encoded by exon 6. PLGF-3 and PLGF-4
human PDGF-C and PDGF-D, which both lack cysteine 4. may function similarly to the larger VEGF-A isoforms,
Phylogenetic analysis of these sequences reveals that the VEGF-A189 and VEGF-A206. In mice, PLGF-2 is the only
VEGF/PDGF family tree is essentially composed of two PLGF isoform identified so far.

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branches evolved from a putative common ancestor, a VEGF
branch comprising VEGFs A-D, PLGF, Orf virus encoded Alternative splicing of the human VEGF-B gene gives rise to
VEGF-Es and Drosophila PVFs 1-3, and a PDGF branch, two transcripts, encoding isoforms (excluding signal
comprising PDGFs A-D (Figure 1b). Within the human peptide) of 167 and 186 amino acid residues, differing only
VEGF family, VEGF-A is most closely related to PLGF (53% in their carboxy-terminal domains [23,24]. VEGF-B186 tran-
amino-acid identity within the VHD [19]). The Orf virus- scripts contain the entire exon 6 and encode a soluble

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encoded VEGF-Es segregate into two groups, with VEGF- isoform. In VEGF-B167 transcripts, the use of an alternative
E(D1701) and VEGF-E(NZ2) most closely related to VEGF-A and splice acceptor site in exon 6 introduces a frameshift, result-
PLGF, and VEGF-E(NZ7) more similar to VEGF-C and VEGF- ing in an alternative exon 6 (referred to as exon 6b in [23]),
D. The Drosophila PVFs are more closely related to the encoding an NRP1/heparin-binding domain similar to that
VEGFs than the PDGFs, albeit distantly, with PVF1 most encoded by exons 7 and 8 in VEGF-A165.
closely related to VEGF-C and VEGF-D (Figure 1b).
Little is known about alternative splicing of human VEGF-C
Gene structure and alternative splicing and VEGF-D, although multiple isoforms of mouse VEGF-D
The gene structures and encoded functional domains of have been described [25]. VEGF-C and VEGF-D are closely

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human and Drosophila VEGFs are shown in Figure 2. The related, both structurally and functionally. Both are ligands
human VEGF genes are characterized by a highly conserved for VEGFR2 and VEGFR3 and are initially synthesized as
seven exon structure, with the exception of VEGF-A, which disulfide-linked polypeptides containing amino- and
has eight exons. Alternative splicing of the human VEGF-A carboxy-terminal propeptide extensions not found in other
gene gives rise to at least six different transcripts (Table 2), VEGF proteins, flanking a central receptor-binding VHD.
encoding isoforms of the following lengths (in amino acids, The unprocessed full-length forms preferentially bind
excluding the signal peptide): 121 (120 in mouse), 145, 165 VEGFR3 and have low affinity for VEGFR2, whereas the
(164 in mouse), 183, 189 and 206 [20]. All transcripts fully processed forms have increased affinity for VEGFR2

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contain exons 1-5 and 8, with diversity generated through [26,27]. VEGF-C and VEGF-D lack the NRP/heparin-
the alternative splicing of exons 6 and 7. A hydrophobic binding domain found in some VEGF isoforms and appear to
signal sequence essential for secretion of VEGF-A is encoded be unable to bind NRPs.
within exon 1 and a small region of exon 2, and the VHD is
encoded by exons 3 and 4. Human VEGF-A121 and VEGF-
A165 and their equivalents in other species are the two major Characteristic structural features
isoforms in mammals; VEGF-A121 lacks exons 6 and 7, and The crystal structure of VEGF-A8-109, comprising the VHD,
VEGF-A165 lacks exon 6 (Table 2). Exon 6 encodes a heparin- has been determined [28] and subsequently refined to a res-
information

binding domain, while exons 7 and 8 encode a olution of 1.93 Å. These studies show that VEGF-A consists
NRP1/heparin-binding domain; with the exception of of two monomers, each containing a core cystine-knot struc-
VEGF-A121, all isoforms are thought to bind the polysaccha- ture held together by three intrachain disulphide bonds as in
ride heparin. VEGF-A165 binds to NRP1 and NRP2, whereas the structure of PDGF; the monomers are arranged head-to-
VEGF-A145 binds only to NRP2 [10,11]. Recently, another tail in a homodimer with two interchain disulphide bridges.
splice variant of human VEGF-A was identified, VEGF-A165b, Mutational analysis has revealed that symmetrical binding

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(a) hVEGF-A
hVEGF-B
50
45
:SYCH-PIETLVDIFQEYPD--EIEYIFKPSCVPLMRCG---GCC-----ND:
:ATCQ-PREVVVPLTVELMG--TVAKQLVPSCVTVQRCG---GCC-----PD:
89
84
hVEGF-C 129 :TQCM-PREVCIDVGKEFGV--ATNTFFKPPCVSVYRCG---GCC-----NS: 168
hVEGF-D 109 :TQCS-PRETCVEVASELGK--STNTFFKPPCVNVFRCG---GCC-----NE: 148
hPLGF 50 :SYCR-ALERLVDVVSEYPS--EVEHMFSPSCVSLLRCT---GCC-----GD: 89
ovVEGF-E(D1701) 33 :SGCK-PRPMVFRVHDEHPE--LTSQRFNPPCVTLMRCG---GCC-----ND: 72
ovVEGF-E(NZ2) 34 :SECK-PRPIVVPVSETHPE--LTSQRFNPPCVTLMRCG---GCC-----ND: 73
ovVEGF-E(NZ7) 44 :SGCK-PRDTVVYLGEEYPE--STNLQYNPRCVTVKRCS---GCC-----NG: 83
hPDGF-A 94 :AVCK-TRTVIYEIPRSQVDPTSANFLIWPPCVEVKRCT---GCC-----NT: 135
hPDGF-B 95 :AECK-TRTEVFEISRRLIDRTNANFLVWPPCVEVQRCS---GCC-----NN: 136
hPDGF-C 248 :YSCT-PRNFSVSIREELK---RTDTIFWPGCLLVKRCG---GNCACCLHNC: 291
hPDGF-D 270 :YSCT-PRNYSVNIREELK---LANVVFFPRCLLVQRCG---GNCGCGTVNW: 313
dmPVF1 140 :ASCS-PQPTIVELKPPAED--EANYYYMPACTRISRCN---GCC-----GS: 179
dmPVF2 202 :GICRVPRPEVVHITRE------TNTFYSPRATILHRCSDKVGCC-----N-: 240
dmPVF3 295 :ATCRIPQKRCQLVQQD------PSKIYTPHCTILHRCSEDSGCC-----PS: 334
* * * **

hVEGF-A 90 :EGLECVPTEESNITMQIMRIKPHQGQH--------IGEMSFLQHNKCECRP: 132

hVEGF-B 85 :DGLECVPTGQHQVRMQILMIRYPSSQ---------LGEMSLEEHSQCECRP: 126
hVEGF-C 169 :EGLQCMNTSTSYLSKTLFEITVPLSQGPK------PVTISFANHTSCRCMS: 213
hVEGF-D 149 :ESLICMNTSTSYISKQLFEISVPLTSVPE------LVPVKVANHTGCKCLP: 193
hPLGF 90 :ENLHCVPVETANVTMQLLKIRSGDRPS--------YVELTFSQHVRCECRP: 132
ovVEGF-E(D1701) 73 :ESLECVPTEEANVTMQLMGASVSGGNG--------MQHLSFVEHKKCDCKP: 115
ovVEGF-E(NZ2) 74 :ESLECVPTEEVNVSMELLGASGSGSNG--------MQRLSFVEHKKCDCRP: 116
ovVEGF-E(NZ7) 84 :DGQICTAVETRNTTVTVSVTGVSSSSGTNSGVSTNLQRISVTEHTKCDCIG: 134
hPDGF-A 136 :SSVKCQPSRVHHRSVKVAKVEYVRKKPKLK-----EVQVRLEEHLECACAT: 181
hPDGF-B 137 :RNVQCRPTQVQLRPVQVRKIEIVRKKPIFK-----KATVTLEDHLACKCET: 182
hPDGF-C 292 :NECQCVPSKVTKKYHEVLQLRP---KTGVRGLHKSLTDVALEHHEECDCVC: 339
hPDGF-D 314 :RSCTCNSGKTVKKYHEVLQFEPGHIKRRGRAKTMALVDIQLDHHERCDCIC: 364
dmPVF1 180 :TLISCQPTEVEQVQLRVRKVDRAATSGRRP-----FTIITVEQHTQCRCDC: 225
dmPVF2 241 :AGWTCQMKRNETVDRVFDKVDGRSNEP--------IVISM-ENHTECGCVK: 282
dmPVF3 335 :RSQICAAKSTHNVELHFFVKSSKHRSV--------IEKRIFVNHTECHCIE: 377
* * *

(b) hVEGF-A

hPLGF

ovVEGF-E D1701

ovVEGF-E NZ2

hVEGF-B

hVEGF-C

hVEGF-D

ovVEGF-E NZ7

dmPVF1

dmPVF2

dmPVF3

hPDGF-A

hPDGF-B

hPDGF-C

hPDGF-D

Figure 1
Comparison of human VEGFs with PDGFs and related sequences from Drosophila and Orf virus. Abbreviations: h, human; dm, Drosophila melanogaster;
ov, Orf virus. (a) An alignment of the deduced amino-acid sequences of the VEGF/PDGF homology domain (VHD) from various human, Drosophila and
Orf virus VEGFs and PGDFs. Sequence data were obtained from the GenBank and SwissProt databases; the multiple alignment was generated using
MultAlin and further optimized manually. Residues that are conserved in at least 50% of the aligned sequences are shaded in green; those fully conserved
are in yellow. The eight cysteine residues that constitute the cystine-knot structure [12] are denoted by asterisks below the sequences. (b) Predicted
evolutionary relationships between human, Drosophila and Orf virus VEGFs and PDGFs. VHD sequences from (a) were aligned using ClustalW and the
neighbor-joining method was used to construct a phylogenetic tree with TreeView. Branch lengths are proportional to the estimated evolutionary
distance between protein sequences.

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SP VHD

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ATG H N N TGA (215)
hVEGF-A 5′ 3′
Exon 1 2 3 4 5 6 7 8
Coding bp 66 52 197 77 30 72 132 19
ATG TAG (207) TGA (188)
hVEGF-B 5′ 3′

1 2 3 4 5 6 N N7
60 43 197 74 36 211
NP /135 19

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CP
ATG TAA (419)
hVEGF-C 5′ 3′
1 2 3 4 5 6 7
147 214 191 152 107 334 112
CP
ATG TGA (354)
hVEGF-D 5′ 3′
1 2 3 4 5 6 7
90 211 191 149 101 196 124

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ATG TAA (170)
hPLGF 5′ 3′
1 2 3 4 5 6 7
75 43 197 77 30 63 25
5′
ATG CP TAA (325)
dmPVF1 3′

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1 2 3 4 5 6
288 79 27 491 90
5′ CP
ATG TAA (405)
dmPVF2 3′
1 2 3 4 5
438 103 526 148
CP
ATG TAA (482)
dmPVF3 5′ 3′

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1 2 3 4 5 6
672 39 133 212 278 112

Figure 2
Gene organization and encoded functional domains of the human VEGF genes and related genes from Drosophila. Exons, represented by boxes, are
numbered and the length of coding sequence in each is marked below in base-pairs. Start (ATG) and stop (TAA, TAG, TGA) codons are marked, and the
length of each encoded unprocessed polypeptide including the signal peptide (in amino-acid residues) is indicated in parentheses. Exons are drawn to
scale, except for the last exon of hVEGF-A, which is longer than 1 kilobase (kb). Introns, represented by horizontal lines, are not drawn to scale.
Alternative exons and splicing patterns are not shown, with the exception of hVEGF-B, in which isoforms result from alternative splicing of exon 6 [23].
Arrows represent proteolytic cleavage sites. Abbreviations: 3ⴕ, 3ⴕ untranslated region (UTR); 5ⴕ, 5ⴕ UTR; CP, region encoding the carboxy-terminal

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propeptide domain; H, encodes the heparin-binding domain; N, encodes the NRP1/heparin-binding domain; NP, encodes the amino-terminal propeptide
domain; SP, signal peptide; VHD, encodes the VEGF/PDGF homology domain. Information was compiled from published literature [14-16,22,23,59-61]
and the Entrez Gene, RefSeq, GenBank and SwissProt databases.

sites for VEGFR2 are located at each pole of the homodimer The binding of VEGFs to NRP1 appears to be mediated by
and has identified key residues in each site involved in two distinct domains. In VEGF-A, these correspond to the
information

ligand-receptor interactions [28]. The crystal structure of basic heparin-binding domain encoded by exon 6 and the
PLGF19-116, comprising the VHD, bound to the second NRP1/heparin-binding domain encoded by exons 7 and 8
immunoglobulin-like loop of VEGFR1 reveals that PLGF and [10]. The nuclear magnetic resonance (NMR) structure of
VEGF-A bind to the same region of VEGFR1 in a very similar the 55 carboxy-terminal residues of VEGF-A165, containing
manner [29], despite only modest sequence conservation the NRP1/heparin-binding domain encoded by exons 7 and
(50%) between the two ligands. 8, reveals this region to be composed of two subdomains,

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Table 2

Isoforms of human VEGF-A

Isoform Size (amino acids) Coding exons* Features

VEGF-A121 121 1-5, 8 Secreted

VEGF-A145 145 1-6, 8 Binds NRP2 but not NRP1; secreted
VEGF-A165 165 1-5, 7, 8 The most abundant and biologically active isoform; secreted; binds NRP1 and NRP2
VEGF-A165b 165 1-5, 7, alternative exon 8 Secreted, endogenous inhibitory form of VEGF-A165
VEGF-A183 183 1-5, short exon 6, 7, 8 Sequestered in ECM but released by cleavage
VEGF-A189 189 1-8 Sequestered in ECM but released by cleavage
VEGF-A206 206 1-8 plus additional exon Sequestered in ECM but released by cleavage
6-encoded sequence

*All isoforms contain exons 1-5 and 8, except VEGF-A165b, which contains an alternative exon 8. Abbreviations: ECM, extracellular matrix; NRP, neuropilin.

each containing two disulphide bridges and a short two- embryonic day 7 (E7) in the extra-embryonic and embryonic
stranded antiparallel ␤ sheet, with the carboxy-terminal sub- endoderm, and by E8.5 it is present at high levels in the tro-
domain additionally containing a short ␣ helix [30]. phoblast surrounding the embryo and in the embryonic
VEGF-B167 also binds NRP1 via an NRP1/heparin-binding myocardium, gut endoderm, embryonic mesenchyme and
domain [31], encoded by an alternative exon 6 and part of amniotic ectoderm. Later in development, VEGF-A is
exon 7; this has strong similarity to the domain encoded by expressed in the mesenchyme and neuroectoderm of the
exons 7 and 8 in VEGF-A165 (Figure 2). PLGF-2 binds NRP1 head [33]. VEGF-A expression declines in most tissues in the
through its exon-6-encoded basic domain, which is similar weeks after birth and is relatively low in most adult organs,
to that encoded by exon 6 of VEGF-A. The VEGF-A145 except in a few vascular beds, including those of the brain
isoform, which lacks exon 7, binds NRP2, presumably choroid plexus, lung alveoli, kidney glomeruli and heart.
through its exon-6-encoded domain [11]. VEGF-A expression is also upregulated during specific physi-
ological processes such as development of the endocrine
corpus luteum in pregnancy, wound healing and tissue
Localization and function repair, and in diseases associated with neovascularization
Cellular localization, expression patterns and (formation of new blood vessels). VEGF-A is produced by
regulation diverse cell types, including aortic vascular smooth muscle
The VEGFs are all secreted proteins. VEGF-A121 and VEGF- cells, keratinocytes, macrophages and many tumor cells [34].
A165 are secreted as covalently linked homodimeric proteins,
whereas the larger isoforms, VEGF-A189 and VEGF-A206, Oxygen tension is a key physiological regulator of VEGF-A
although believed to be secreted, are not readily diffusible and gene expression [35]. The VEGF-A gene contains hypoxia-
may remain sequestered in the extracellular matrix (Table 2). responsive enhancer elements (HREs) in its 5⬘ and 3⬘ UTRs
VEGF bioavailability may be regulated by plasmin-mediated [36,37], the 3⬘ enhancer being similar to sequences within
proteolysis in the carboxy-terminal domains of the larger the HRE of the gene encoding the hormone erythropoietin.
matrix-bound VEGF isoforms, such as VEGF-A189, to release Transcriptional regulation of the VEGF-A gene by hypoxia is
more diffusible, biologically active species [32]. Human mediated by binding of the transcription factor HIF-1
VEGF-A165, the most abundant and biologically active form, is (hypoxia-inducible transcription factor 1) to the HRE. HIF-1
glycosylated at Asn74 and is typically expressed as a 46 kDa is a heterodimer composed of HIF-1␣ and HIF-1␤ subunits,
homodimer of 23 kDa subunits. VEGF-A121 has biological both of which are members of the basic helix-loop-helix-PAS
activity in endothelial cells, but has lower potency than VEGF- family [38]. HIF-1␣ is normally very labile, but under
A165. The amino- and carboxy-terminal propeptide domains of hypoxic conditions, it accumulates because proteasomal
VEGF-C and VEGF-D are proteolytically cleaved, possibly by degradation is inhibited: at normal oxygen tension, proline
plasmin, releasing the VHD during or after secretion to gener- hydroxylation targets HIF-1␣ for proteasomal degradation,
ate a fully processed mature form, which forms noncovalent but is inhibited by hypoxia because of the requirement of the
homodimers of approximately 21 kDa that bind VEGFR2 with responsible prolyl hydroxylases for molecular dioxygen. The
greatly increased affinity [26,27]. product of the Von Hippel-Lindau (VHL) tumor-suppressor
gene is also required for proteasomal proteolysis: a genetic
Most information on the localization and expression of deficiency of this protein causes VHL disease, a condition
VEGFs has been derived from studies on VEGF-A. During characterized by retinal and cerebellar capillary heman-
embryogenesis in the mouse, VEGF-A can be detected from gioblastomas (small, highly vascular tumors). In addition,

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VEGF-A mRNA is stabilized under conditions of low oxygen logical bypass’ for diseased arteries. Despite abundant pre-
tension as a result of binding of unidentified factors to its 3⬘ clinical data suggesting that VEGF-A protein or gene therapy
UTR. VEGF-A gene expression is also upregulated by a could be effective in treating ischemic heart disease, clinical

comment
variety of growth factors and cytokines, including PDGF-BB, trials have not so far yielded definitive evidence in support of
TGF-␤, basic fibroblast growth factor (FGF-2), interleukin- this approach [1].
1␤ and interleukin-6, some of which can act synergistically
with hypoxia [1]. VEGF-A was originally identified as vascular permeability
factor (VPF) as a result of its potent ability to increase vascu-
Function lar permeability, resulting in leakage of proteins and other
All of the vertebrate VEGFs and their cognate receptors molecules out of blood vessels [2,34]. The physiological sig-
studied so far are able to regulate angiogenesis, and several nificance of the permeability-increasing effect of VEGF-A

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have key biological roles in the formation of vascular struc- remains unclear, but it is important in mediating some path-
tures either during development or in the adult. VEGFR ogenic consequences of VEGF-A overexpression in disease,
function and signaling is reviewed extensively elsewhere an example being brain edema (swelling and build-up of
[1,39,40] and is not discussed in this article. The pivotal role fluid) following cerebral ischemia [1].
of VEGF-A in embryonic vascular development was demon-
strated by the remarkable discovery that targeted inactiva- In addition to its major role in angiogenesis, VEGF-A prob-
tion of a single VEGF-A allele in mice caused a lethal ably has functions that are independent of both endothelial
impairment of angiogenesis, resulting in death between E11 cells and blood-vessel formation. A growing body of evi-

reports
and E12 [41,42]. The importance of larger VEGF-A isoforms, dence indicates that VEGF-A has neurotrophic and neuro-
including VEGF-A165, was confirmed by the finding that protective activities in vitro and in vivo [46,47]. It has also
mice expressing only VEGF-A120 - and lacking the longer been implicated in amyotrophic lateral sclerosis (ALS), an
heparin-binding isoforms - die within 2 weeks of birth owing incurable degenerative disorder of motor neurons. Reduced
to haemorrhage and ischemic cardiomyopathy (heart failure VEGF-A expression resulting from deletion of the HRE
due to lack of blood supply to the heart muscle) [43]. A car- from the VEGF-A promoter predisposes mice to ALS-like

deposited research
diomyocyte-specific VEGF-A gene knockout generated using motor-neuron degeneration, and mice can be protected
Cre-lox technology results in reduced body weight and thin- against ALS by treatment with VEGF-A [48]. Furthermore,
walled, dilated, poorly vascularized hearts [1]. humans with particular VEGF-A promoter haplotypes have
an increased risk of ALS associated with lower circulating
Studies involving inducible VEGF-A gene inactivation or levels of VEGF-A [49].
administration of soluble (s) forms of the receptor Flt-1 to
inhibit VEGF-A function have established that VEGF-A con- The VEGFR1-specific ligand, PLGF-1, appears to be weakly
tinues to be critically important during post-natal growth angiogenic when acting alone, but VEGF-A-PLGF het-
and organ development [1]. Inducible Cre-lox-mediated dis- erodimers can bind to VEGFR2, are mitogenic for endothe-

refereed research
ruption of the VEGF-A gene in early post-natal life causes lial cells, and stimulate angiogenesis in vivo [50]. Though
increased mortality, reduced body growth, and impaired mice lacking PLGF are viable and develop normally, they
organ development, particularly of the liver. Inhibition of have reduced angiogenesis in pathophysiological situations
VEGF-A by treatment of mice with sFlt-1 between 1 and 8 such as ischemia. PLGF-deficient mice also have delayed col-
days after birth results in a more severe effect, characterized lateral artery growth following blockage of an artery, and
by growth arrest and lethality, but the effect of VEGF-A inhi- PLGF stimulates collateral vessel growth. PLGF stimulates
bition became less drastic if initiated at progressively later monocyte chemotaxis through VEGFR1, and there is
times in post-natal life. Inhibition of VEGF-A with sFlt-1 increasing evidence that the biological effects of PLGF are

interactions
shows that VEGF-A-driven vascularization is also essential mediated by mobilization of bone-marrow-derived
for endochondral bone formation and development of the haematopoietic progenitors.
corpus luteum during pregnancy [1].
A biological role for VEGF-B has not yet been clearly estab-
VEGF-A-driven angiogenesis has a major role in the patho- lished. VEGF-B knockout mice are viable, healthy and
genesis of diverse human diseases, including cancer, eye dis- fertile, but whereas Bellomo et al. [51] reported that VEGF-
orders and rheumatoid arthritis [44]. Recognition of the B-null mice have smaller hearts and recover more slowly
importance of VEGF-A for the development of several from cardiac ischemia than wild-type littermates, Aase et al.
information

important classes of cancer recently culminated in the [52] observed no effect of loss of VEGF-B on cardiac size or
approval of Avastin, a humanized monoclonal antibody to development and instead found a specific defect in atrial
VEGF-A, for the treatment of metastatic colorectal cancer conduction in the adult. VEGF-B-deficient mice also have
[45]. There has also been great interest in using VEGF-A for impaired development of pathophysiology when arthritis or
the treatment of ischemic heart disease, where the aim is to hypoxic pulmonary hypertension are experimentally
promote blood-vessel formation and thereby provide a ‘bio- induced [53].

Genome Biology 2005, 6:209

209.8 Genome Biology 2005, Volume 6, Issue 2, Article 209 Holmes and Zachary http://genomebiology.com/2005/6/2/209

VEGF-C and its receptor, VEGFR3 (Flt-4), are strongly logenesis and neurogenesis at an early evolutionary stage
implicated in the formation of the lymphatic endothelium and only later developed more specialized roles in
(lymphangiogenesis). Transgenic mice overexpressing hematopoiesis and vascular development in more complex
VEGF-C in keratinocytes of the skin epidermis develop organisms. The role of VEGFs and VEGFRs in cell migration
enlarged lymphatic vessels, while mice overexpressing appears to be fundamental to their biological functions in
VEGF-A164 in the same location show only blood-vessel invertebrate and vertebrate species.
hyperplasia [54]. VEGF-C also stimulates angiogenesis in
the mouse cornea [55], however, and also in rabbit models of
ischemia in the hindlimb. VEGF-D is mitogenic in endothe- Frontiers
lial cells and promotes angiogenesis in vitro and in several Although significant progress has been made towards eluci-
models of angiogenesis in vivo [56]. VEGF-D also stimulates dating the mechanisms mediating the angiogenic effects of
lymphangiogenesis in mice when overexpressed in skin ker- VEGF-A, several formidable challenges lie ahead. The bio-
atinocytes and tumors [57], and it induces the survival and logical and signaling roles of the VEGF receptors, particu-
migration of lymphatic endothelial cells. larly VEGFR1 and neuropilin-1, have not yet been fully
defined. Another key goal is the identification of the mecha-
The viral VEGF-Es encoded by different strains of the para- nisms underlying the role of VEGF-A in endothelial cell dif-
poxvirus Orf appear to be important for viral infection and ferentiation and early vascular development. An emergent
its associated pathology. Viruses of the Orf genus cause a area of interest is the study of VEGF and VEGFR homologs
contagious pustular dermatitis in sheep and goats, which is in invertebrates. A better understanding of how VEGF
transmissible to humans, and produces lesions characterized ligand-receptor systems function in Drosophila and C.
by extensive neovascularization, vascular dilation, and epi- elegans will shed light on the ancestral function of this
dermal proliferation. VEGF-E(NZ2) induces dermal vascular- family of molecules and may also generate novel insights
ization and epidermal proliferation in sheep, and disruption into their biological roles in vertebrates. Another major goal
of the VEGF-E(NZ2) gene resulted in a marked decrease in the in the future will be to clarify the distinct biological functions
vascularization of viral lesions without impairing viral repli- of different members of the VEGF family.
cation in the early stages of infection [58].
A key area of ongoing research will be the role of VEGFs in
Drosophila PVFs and their receptor, PVR, have key roles in human disease. As recent work on ALS demonstrates
cell migration during two developmental processes [14-16]. [48,49], it is likely that new insights into the importance of
Firstly, PVR is expressed by the border cells, a cluster of VEGFs for disease will continue to be generated. Conse-
somatic follicle cells that migrate towards the oocyte during quently, the scope for using anti-VEGF approaches thera-
oogenesis; PVF1 is produced by oocytes and acts as a guid- peutically will grow, and the challenge will be to develop
ance cue for the PVR-expressing border cells during their more effective and economic ways to prevent VEGF-driven
migration [14]. Secondly, though devoid of endothelial cells pathophysiological angiogenesis or to correct VEGF deficits.
or blood vessels, Drosophila does possess blood cells or The future use of VEGF therapy for cardiovascular disease
hemocytes, and the PVF/PVR system is involved in the remains an enticing prospect but awaits confirmatory data
migration of these cells. PVR is expressed in the developing from clinical studies.
hemocytes during Drosophila embryogenesis, whereas
PVF1, PVF2 and PVF3 are expressed along the hemocyte
migratory route; inactivating mutations in either PVR or all Acknowledgements
three PVFs arrests hemocyte movement [16]. I.Z. is supported by the British Heart Foundation.

In C. elegans, which lacks a vascular system, the VEGFR-like

References
VER proteins are localized to cells of neural origin, suggest- 1. Ferrara N, Gerber HP, LeCouter J: The biology of VEGF and its
ing a role in neurogenesis [17]. The recently identified VEGF receptors. Nat Med 2003, 9:669-676.
and VEGFR homologs in the jellyfish P. carnea [18] are A concise review of the role of VEGF-A and its receptors in biology
and disease.
expressed in tubular structures of the gastrovascular system 2. Senger DR, Galli SJ, Dvorak AM, Perruzzi CA, Harvey VS, Dvorak
and in the endoderm during development at the stage when HF: Tumor cells secrete a vascular permeability factor that
promotes accumulation of ascites fluid. Science 1983, 219:983-
undifferentiated cells migrate and differentiate into plate 985.
cells. In this process, the differentiating plate cells interact The initial discovery of a secreted VPF with the characteristics of VEGF-A.
with matrix and smooth muscle cells, a process analogous to 3. Leung DW, Cachianes G, Kuang WJ, Goeddel DV, Ferrara N: Vas-
cular endothelial growth factor is a secreted angiogenic
the interaction of endothelial and vascular smooth muscle mitogen. Science 1989, 246:1306-1309.
cells in angiogenesis. As nematodes and jellyfish lack both a This and [4] are the first reports of the cDNA cloning of VEGF-A.
vascular circulatory system and blood cells, the discovery of 4. Keck PJ, Hauser SD, Krivi G, Sanzo K, Warren T, Feder J, Connolly
DT: Vascular permeability factor, an endothelial cell
VEGF and VEGFR-like molecules in these species suggests mitogen related to PDGF. Science 1989, 246:1309-1312.
that these proteins performed primordial functions in tubu- See [3].

Genome Biology 2005, 6:209

http://genomebiology.com/2005/6/2/209 Genome Biology 2005, Volume 6, Issue 2, Article 209 Holmes and Zachary 209.9

5. Li X, Eriksson U: Novel VEGF family members: VEGF-B, related to the vascular permeability factor. Proc Natl Acad Sci
VEGF-C and VEGF-D. Int J Biochem Cell Biol 2001, 33:421-426. USA 1991, 88:9267-9271.
A review of the mammalian VEGF family. The initial identification of PLGF, a second member of the VEGF family.
6. Shibuya M: Vascular endothelial growth factor receptor-2: its 20. Robinson CJ, Stringer SE: The splice variants of vascular

comment
unique signalling and specific ligand, VEGF-E. Cancer Sci 2003, endothelial growth factor (VEGF) and their receptors. J Cell
94:751-756. Sci 2001, 114:853-865.
A review of VEGF-E. A review of the splice variants of VEGF-A and their functions.
7. Shalaby F, Rossant J, Yamaguchi TP, Gertsenstein M, Wu XF, Breit- 21. Bates DO, Cui TG, Doughty JM, Winkler M, Sugiono M, Shields JD,
man ML, Schuh AC: Failure of blood-island formation and vas- Peat D, Gillatt D, Harper SJ: VEGF165b, an inhibitory splice
culogenesis in Flk-1 deficient mice. Nature 1995, 376:62-66. variant of vascular endothelial growth factor, is down-regu-
Loss of VEGFR2 prevents endothelial cell progenitor formation and lated in renal cell carcinoma. Cancer Res 2002, 62:4123-4131.
early vascular development in mice. The discovery of a novel inhibitory VEGF-A165 variant resulting from an
8. Fong GH, Rossant J, Gertsenstein M, Breitman ML: Role of the Flt- alternative exon 8.
1 receptor tyrosine kinase in regulating the assembly of vas- 22. Maglione D, Guerriero V, Viglietto G, Ferraro MG, Aprelikova O,
cular endothelium. Nature 1995, 376:66-70. Alitalo K, Del Vecchio S, Lei KJ, Chou JY, Persico MG: Two alter-

reviews
VEGFR1 is essential for vascular development, but VEGFR1-deficient native mRNAs coding for the angiogenic factor, placenta
mice have a phenotype distinct from that of VEGFR2 knockouts. growth factor (PlGF), are transcribed from a single gene of
9. Dumont DJ, Jussila L, Taipale J, Lymboussaki A, Mustonen T, Pajusola chromosome 14. Oncogene 1993, 8:925-931.
K, Breitman M, Alitalo K: Cardiovascular failure in mouse Identification of PLGF-2, a splice variant containing an exon-6-encoded
embryos deficient in VEGF receptor-3. Science 1998, 282:946- heparin-binding domain absent from PLGF-1.
949. 23. Olofsson B, Pajusola K, von Euler G, Chilov D, Alitalo K, Eriksson U:
VEGFR3 is essential for cardiovascular development. Genomic organisation of the mouse and human genes for
10. Soker S, Takashima S, Miao HQ, Neufeld G, Klagsbrun M: Neu- vascular endothelial growth factor B (VEGF-B) and charac-
ropilin-1 is expressed by endothelial and tumor cells as an terization of a second splice isoform. J Biol Chem 1996,
isoform-specific receptor for vascular endothelial growth 271:19310-19317.
factor. Cell 1998, 92:735-745. Reports the structures of the human and mouse VEGF-B genes and the

reports
Identification of NRP1 as a non-tyrosine kinase receptor for VEGF-A165. identification of the VEGF-B186 splice variant.
11. Gluzman-Poltorak Z, Cohen T, Herzog Y, Neufeld G: Neuropilin-2 24. Olofsson B, Pajusola K, Kaipainen A, von Euler G, Joukov V, Saksela
and neuropilin-1 are receptors for the 165-amino acid form O, Orpana A, Pettersson RF, Alitalo K, Eriksson U: Vascular
of vascular endothelial growth factor (VEGF) and of pla- endothelial growth factor B, a novel growth factor for
centa growth factor-2, but only neuropilin-2 functions as a endothelial cells. Proc Natl Acad Sci USA 1996, 93:2576-2581.
receptor for the 145-amino acid form of VEGF. J Biol Chem The first identification of the VEGFR1 ligand, VEGF-B.
2000, 275:18040-18045. 25. Baldwin ME, Roufail S, Halford MM, Alitalo K, Stacker SA, Achen
This study shows that the VEGF-A145 isoform selectively recognizes MG: Multiple forms of mouse vascular endothelial growth
NRP2. factor-D are generated by RNA splicing and proteolysis. J

deposited research
12. Vitt UA, Hsu SY, Hsueh AJW: Evolution and classification of Biol Chem 2001, 276:44307-44314.
cystine knot-containing hormones and related extracellular Alternative splicing and proteolysis generates multiple isoforms of
signaling molecules. Mol Endocrinol 2001, 15:681-694. mouse VEGF-D.
General review of the cystine-knot family of extracellular proteins. 26. Joukov V, Sorsa T, Kumar V, Jeltsch M, Claesson-Welsh L, Cao Y,
13. Gong B, Liang D, Chew TG, Ge R: Characterization of the Saksela O, Kalkkinen N, Alitalo K: Proteolytic processing regu-
zebrafish vascular endothelial growth factor A gene: com- lates receptor specificity and activity of VEGF-C. EMBO J
parison with vegf-A genes in mammals and Fugu. Biochim 1997, 16:3898-3911.
Biophys Acta 2004, 1676:33-40. This paper and [27] demonstrate that VEGF-C and VEGF-D undergo
Demonstrates that human and teleost VEGF-A genes are highly con- proteolytic processing to generate mature forms with increased affinity
served and have a similar organization. for VEGFR2.
14. Duchek P, Somogyi K, Jekely G, Beccari S, Rorth P: Guidance of 27. Stacker SA, Stenvers K, Caesar C, Vitali A, Domagala T, Nice E,

refereed research
cell migration by the Drosophila PDGF/VEGF receptor. Cell Roufail S, Simpson RJ, Moritz R, Karpanen T, et al.: Biosynthesis of
2001, 107:17-26. vascular endothelial growth factor-D involves proteolytic
The first demonstration of a biological role for invertebrate processing which generates non-covalent homodimers. J Biol
VEGF/VEGFR homologs. The paper reports that Drosophila members Chem 1999, 274:32127-32136.
of the VEGF and VEGFR families play an essential role in cell migration See [26].
during oogenesis. 28. Muller YA, Li B, Christinger HW, Wells JA, Cunningham BC, de Vos
15. Heino TI, Karpanen T, Wahlstrom G, Pulkkinen M, Eriksson U, AM: Vascular endothelial growth factor: crystal structure
Alitalo K, Roos C: The Drosophila VEGF receptor homolog is and functional mapping of the kinase domain receptor
expressed in hemocytes. Mech Dev 2001, 109:69-77. binding site. Proc Natl Acad Sci USA 1997, 94:7192-7197.
Identification, characterization and expression patterns of the VEGF-like The first report of the crystal structure of the VEGF receptor-binding
Drosophila receptor PVR and its ligands, PVF1-PVF3. domain, showing that it has a structure similar to that of PDGF.
16. Cho NK, Keyes L, Johnson E, Heller J, Ryner L, Karim F, Krasnow 29. Christinger HW, Fuh G, de Vos AM, Wiesmann C: The crystal

interactions
MA: Developmental control of blood cell migration by the structure of placental growth factor in complex with
Drosophila VEGF pathway. Cell 2002, 108:865-876. domain 2 of vascular endothelial growth factor receptor-1. J
This study demonstrates a key role for VEGF and VEGFR homologs in Biol Chem 2004, 279:10382-10388.
migration of blood cells in Drosophila development. The crystal structure of the PLGF receptor-binding domain shows it is
17. Popovici C, Isnardon D, Birnbaum D, Roubin R: Caenorhabditis very similar to that of VEGF-A.
elegans receptors related to mammalian vascular endothe- 30. Fairbrother WJ, Champe MA, Christinger HW, Keyt BA, Starovasnik
lial growth factor receptors are expressed in neural cells. MA: Solution structure of the heparin-binding domain of vas-
Neurosci Lett 2002, 329:116-120. cular endothelial growth factor. Structure 1998, 6:637-648.
The first identification of VEGFR-related molecules in the nematode The NMR structure of the NRP1/heparin-binding domain of VEGF-A.
worm, a species lacking both a vascular system and blood cells. 31. Makinen T, Olofsson B, Karpanen T, Hellman U, Soker S, Klagsbrun
18. Seipel K, Eberhardt M, Muller P, Pescia E, Yanze N, Schmid V: M, Eriksson U, Alitalo K: Differential binding of vascular
information

Homologs of vascular endothelial growth factor and recep- endothelial growth factor B splice and proteolytic isoforms
tor, VEGF and VEGFR, in the jellyfish Podocoryne carnea. to neuropilin-1. J Biol Chem 1999, 274:21217-21222.
Dev Dyn 2004, 231:303-312. Demonstrates that VEGF-B167 binds NRP-1 through an exon-6-
The identification of VEGF and VEGFR homologues in Cnidaria, the encoded heparin-binding domain.
most basic phylum of the animal kingdom to have tissue organization 32. Park JE, Keller GA, Ferrara N: The vascular endothelial growth
and a nervous system. factor (VEGF) isoforms: differential deposition into the
19. Maglione D, Guerriero V, Viglietto G, Delli-Bovi P, Persico MG: Iso- subepithelial extracellular matrix and bioactivity of extra-
lation of a human placenta cDNA coding for a protein cellular matrix-bound VEGF. Mol Biol Cell 1993, 4:1317-1326.

Genome Biology 2005, 6:209

209.10 Genome Biology 2005, Volume 6, Issue 2, Article 209 Holmes and Zachary http://genomebiology.com/2005/6/2/209

Demonstration that plasmin-mediated proteolysis of VEGF-A189 bound Deletion of the HRE in the VEGF-A promoter reduces hypoxia-driven
to the extracellular matrix releases soluble and bioactive VEGF-A. expression in the spinal cord and causes adult-onset motor neuron
33. Dumont DJ, Fong GH, Puri MC, Gradwohl G, Alitalo K, Breitman degeneration in mice, reminiscent of ALS.
ML: Vascularization of the mouse embryo: a study of flk-1, 49. Lambrechts D, Storkebaum E, Morimoto M, Del-Favero J, Desmet F,
tek, tie, and vascular endothelial growth factor expression Marklund SL, Wyns S, Thijs V, Andersson J, van Marion I, et al.: VEGF
during development. Dev Dyn 1995, 203:80-92. is a modifier of amyotrophic lateral sclerosis in mice and
The localization of VEGF-A and VEGFR2 during mouse embryogenesis. humans and protects motoneurons against ischemic death.
34. Dvorak HF, Brown LF, Detmar M, Dvorak AM: Vascular perme- Nat Genet 2003, 34:383-394.
ability factor/vascular endothelial growth factor, microvas- Humans homozygous for specific haplotypes of the VEGF-A promoter
cular hyperpermeability, and angiogenesis. Am J Pathol 1995, region have reduced circulating levels of VEGF-A and greater risk of ALS.
146:1029-1039. 50. Autiero M, Luttun A, Tjwa M, Carmeliet P: Placental growth factor
A review of VEGF-A function. and its receptor, vascular endothelial growth factor receptor-
35. Shweiki D, Itin A, Soffer D, Keshet E: Vascular endothelial 1: novel targets for stimulation of ischemic tissue revascular-
growth factor induced by hypoxia may mediate hypoxia-ini- ization and inhibition of angiogenic and inflammatory
tiated angiogenesis. Nature 1992, 359:843-845. disorders. J Thromb Haemost 2003, 1:1356-1370.
A report showing that VEGF-A expression is induced by hypoxia. A review of the role of PLGF in pathophysiological angiogenesis.
36. Minchenko A, Salceda S, Bauer T, Caro J: Hypoxia regulatory ele- 51. Bellomo D, Headrick JP, Silins GU, Paterson CA, Thomas PS, Gartside
ments of the human vascular endothelial growth factor M, Mould A, Cahill MM, Tonks ID, Grimmond SM, et al.: Mice lacking
gene. Cell Mol Biol Res 1994, 40:35-39. the vascular endothelial growth factor-B gene (Vegfb) have
The identification of hypoxia regulatory elements in the 5⬘ and 3⬘ flank- smaller hearts, dysfunctional coronary vasculature, and
ing regions of the VEGF-A gene. impaired recovery from cardiac ischemia. Circ Res 2000, 86:E29-
37. Liu Y, Cox SR, Morita T, Kourembanas S: Hypoxia regulates vas- E35.
cular endothelial growth factor gene expression in endothe- VEGF-B-deficient mice have defective hearts.
lial cells: identification of a 5⬘⬘ enhancer. Circ Res 1995, 52. Aase K, von Euler G, Li X, Ponten A, Thoren P, Cao R, Cao Y, Olofs-
77:638-643. son B, Gebre-Medhin S, Pekny M, et al.: Vascular endothelial
Identification of the minimal 5ⴕ enhancer sequence in the VEGF-A pro- growth factor-B-deficient mice display an atrial conduction
moter required for hypoxia-regulated transcription. defect. Circulation 2001, 104:358-364.
38. Huang LE, Bunn HF: Hypoxia-inducible factor and its biomed- VEGF-B-deficient mice have hearts of a normal size but with a specific
ical relevance. J Biol Chem 2003, 278:19575-19578. defect in atrial conduction; this contrasts with the results shown in [51].
A review of the transcription factors mediating hypoxia-inducible gene 53. Mould AW, Tonks ID, Cahill MM, Pettit AR, Thomas R, Hayward NK,
expression. Kay GF: Vegfb gene knockout mice display reduced pathology
39. Neufeld G, Cohen T, Gengrinovitch S, Poltorak Z: Vascular and synovial angiogenesis in both antigen-induced and colla-
endothelial growth factor (VEGF) and its receptors. FASEB J gen-induced models of arthritis. Arthritis Rheum 2003, 48:2660-
1999, 13:9-22. 2669.
A review of VEGF receptors and intracellular signaling. VEGF-B is implicated in pathophysiological angiogenesis in animal models
40. Zachary I: VEGF signalling: integration and multi-tasking in of arthritis.
endothelial cell biology. Biochem Soc Trans 2003, 31:1171-1177. 54. Jeltsch M, Kaipainen A, Joukov V, Meng X, Lakso M, Rauvala H, Swartz
A review of VEGF receptor signaling. M, Fukumura D, Jain RK, Alitalo K: Hyperplasia of lymphatic
41. Carmeliet P, Ferreira V, Breier G, Pollefeyt S, Kieckens L, Gertsenstein vessels in VEGF-C transgenic mice. Science 1997, 276:1423-1425.
M, Fahrig M, Vandenhoeck A, Harpal K, Eberhardt C, et al.: Abnor- This paper indicates a role for VEGF-C in formation of the lymphatic vas-
culature.
mal blood vessel development and lethality in embryos
55. Cao Y, Linden P, Farnebo J, Cao R, Eriksson A, Kumar V, Qi JH, Claes-
lacking a single VEGF allele. Nature 1996, 380:435-439.
son-Welsh L, Alitalo K: Vascular endothelial growth factor C
This paper and [42] demonstrate that loss of a single VEGF-A allele
induces angiogenesis in vivo. Proc Natl Acad Sci USA 1998,
causes embryonic lethality.
95:14389-14394.
42. Ferrara N, Carver-Moore K, Chen H, Dowd M, Lu L, O’Shea KS, VEGF-C is angiogenic.
Powell-Braxton L, Hillan KJ, Moore MW: Heterozygous embry- 56. Marconcini L, Marchio S, Morbidelli L, Cartocci E, Albini A, Ziche M,
onic lethality induced by targeted inactivation of the VEGF Bussolino F, Oliviero S: c-fos-induced growth factor/vascular
gene. Nature 1996, 380:439-442. endothelial growth factor D induces angiogenesis in vivo and
See [41]. in vitro. Proc Natl Acad Sci USA 1999, 96:9671-9676.
43. Carmeliet P, Ng YS, Nuyens D, Theilmeier G, Brusselmans K, Cor- VEGF-D is angiogenic.
nelissen I, Ehler E, Kakkar VV, Stalmans I, Mattot V, et al.: Impaired 57. Stacker SA, Caesar C, Baldwin ME, Thornton GE, Williams RA, Prevo
myocardial angiogenesis and ischemic cardiomyopathy in R, Jackson DG, Nishikawa S, Kubo H, Achen MG: VEGF-D pro-
mice lacking the vascular endothelial growth factor iso- motes the metastatic spread of tumor cells via the lymphat-
forms VEGF164 and VEGF188. Nat Med 1999, 5:495-502. ics. Nat Med 2001, 7:186-191.
The VEGF-A164 isoform is shown to be essential for normal vascular VEGF-D-stimulated lymphangiogenesis mediates tumor metastasis.
development in the mouse. 58. Savory LJ, Stacker SA, Fleming SB, Niven BE, Mercer AA: Viral vascu-
44. Carmeliet P, Jain RK: Angiogenesis in cancer and other dis- lar endothelial growth factor plays a critical role in orf virus
eases. Nature 2000, 407:249-257. infection. J Virol 2000, 74:10699-10706.
A review of the role of angiogenesis and angiogenic factors in disease. Disruption of the VEGF-E gene results in reduced vascularization of
45. Ferrara N, Hillan KJ, Gerber HP, Novotny W: Discovery and lesions produced by Orf virus infection.
development of bevacizumab, an anti-VEGF antibody for 59. Tischer E, Mitchell R, Hartman T, Silva M, Gospodarowicz D, Fiddes
treating cancer. Nat Rev Drug Discov 2004, 3:391-400. JC, Abraham JA: The human gene for vascular endothelial
Inhibition of VEGF-A with humanized anti-VEGF-A antibody is effective growth factor. Multiple protein forms are encoded through
in treating human cancer. alternative exon splicing. J Biol Chem 1991, 266:11947-11954.
46. Sondell M, Sundler F, Kanje M: Vascular endothelial growth The first report of the gene organization and splicing of human VEGF-A.
factor is a neurotrophic factor which stimulates axonal out- 60. Chilov D, Kukk E, Taira S, Jeltsch M, Kaukonen J, Palotie A, Joukov V,
growth through the flk-1 receptor. Eur J Neurosci 2000, Alitalo K: Genomic organisation of human and mouse genes
12:4243-4254. for vascular endothelial growth factor C. J Biol Chem 1997,
This report shows that VEGF-A acts as a neurotrophic factor. 272:25176-25183.
47. Storkebaum E, Lambrechts D, Carmeliet P: VEGF: once regarded The organization of the human and mouse VEGF-C genes.
as a specific angiogenic factor, now implicated in neuropro- 61. Rocchigiani M, Lestingi M, Luddi A, Orlandini M, Franco B, Rossi E, Bal-
tection. Bioessays 2004, 26:943-954. labio A, Zuffardi O, Oliviero S: Human FIGF: Cloning, gene
A review of the role of VEGF-A in neuroprotection. structure, and mapping to chromosome Xp22.1 between
48. Oosthuyse B, Moons L, Storkebaum E, Beck H, Nuyens D, Brussel- the PIGA and the GRPR genes. Genomics 1998, 47:207-216.
mans K, Van Dorpe J, Hellings P, Gorselink M, Heymans S, et al.: Reports the organization of the human VEGF-D gene.
Deletion of the hypoxia-response element in the vascular
endothelial growth factor promoter causes motor neuron
degeneration. Nat Genet 2001, 28:131-138.

Genome Biology 2005, 6:209