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Clinical Science (2005) 109, 227–241 (Printed in Great Britain) doi:10.1042/CS20040370 227

R E V I E W

The vascular endothelial growth factor (VEGF)/VEGF receptor system and its role underphysiological and pathological conditions

Hiroyuki TAKAHASHI∗† and Masabumi SHIBUYA∗∗Division of Genetics, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokane-dai, Minato-ku, Tokyo, 108-8639,Japan, and †Department of Internal Medicine, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku,Tokyo, 113-0033, Japan

A B S T R A C T

The VEGF (vascular endothelial growth factor) family and its receptors are essential regulatorsof angiogenesis and vascular permeability. Currently, the VEGF family consists of VEGF-A, PlGF(placenta growth factor), VEGF-B, VEGF-C, VEGF-D, VEGF-E and snake venom VEGF. VEGF-Ahas at least nine subtypes due to the alternative splicing of a single gene. Although the VEGF165

isoform plays a central role in vascular development, recent studies have demonstrated that eachVEGF isoform plays distinct roles in vascular patterning and arterial development. VEGF-A binds toand activates two tyrosine kinase receptors, VEGFR (VEGF receptor)-1 and VEGFR-2. VEGFR-2mediates most of the endothelial growth and survival signals, but VEGFR-1-mediated signallingplays important roles in pathological conditions such as cancer, ischaemia and inflammation. Insolid tumours, VEGF-A and its receptor are involved in carcinogenesis, invasion and distantmetastasis as well as tumour angiogenesis. VEGF-A also has a neuroprotective effect on hypoxicmotor neurons, and is a modifier of ALS (amyotrophic lateral sclerosis). Recent progress in themolecular and biological understanding of the VEGF/VEGFR system provides us with novel andpromising therapeutic strategies and target proteins for overcoming a variety of diseases.

INTRODUCTION

Angiogenesis and vasculogenesis are regulated pre-dominantly by several different growth factors and theirassociated RTKs (receptor tyrosine kinases). Foremost

among these is the VEGF (vascular endothelial growthfactor) family and VEGFRs (VEGF receptors). VEGF-A, also referred to as VPF (vascular permeability factor),an important regulator of endothelial cell physiology,was identified approx. 15 years ago [1,2] and has been

Key words: angiogenesis, inflammation, signal transduction, tumour, vascular endothelial growth factor (VEGF), vascularpermeability.Abbreviations: ALS, amyotrophic lateral sclerosis; DSCR1, Down syndrome critical region protein 1; ECM, extracellular matrix;ERK, extracellular signal-regulated kinase; FAK, focal adhesion kinase; HIF-1, hypoxia-inducible factor-1; HRE, hypoxia responseelement; HUVEC, human umbilical vein endothelial cell; LSEC, liver sinusoidal endothelial cell; mAb, monoclonal antibody;MAPK, mitogen-activated protein kinase; MMP, matrix metalloproteinase; NFATc, nuclear factor of activated T-cell; NO, nitricoxide; NOS; NO synthase; eNOS, endothelial NOS; NRP, neuropilin; PAIP2, polyadenylated-binding protein-interacting pro-tein 2; PDK1, phosphoinositide-dependent kinase 1; PI3K, phosphoinositide 3-kinase; PILSAP, puromycin-intensive leucyl-specificaminopeptidase; PKC, protein kinase C; PLC, phospholipase C; PlGF, placenta growth factor; pVHL, von Hippel–Lindau tumoursuppressor protein; RA, rheumatoid arthritis; RTK, receptor tyrosine kinase; S6K, S6 kinase; Tag, T antigen; TH2, T-helper type 2;UTR, untranslated region; VEGF, vascular endothelial growth factor; VEGFR, VEGF receptor; sVEGFR-1, soluble VEGFR-1;svVEGF, snake venom VEGF; Tf svVEGF, Trimeresurus flavoviridis svVEGF; VPF, vascular permeability factor.Correspondence: Professor Masabumi Shibuya (email [email protected]).

C© 2005 The Biochemical Society

228 H. Takahashi and M. Shibuya

recognized as the major growth factor that is relativelyspecific for endothelial cells. VEGF-A is a dimeric glyco-protein essential for many angiogenic processes in normaland abnormal states, such as tumour vascularization,mainly by interacting with two tyrosine kinase receptors,VEGFR-1 [also known as Flt-1 (Fms-like tyrosine kin-ase-1)] and VEGFR-2 [also known as Flk-1 (fetal liverkinase-1) and, in humans, as KDR (kinase insert domain-containing receptor)] [3–5]. VEGF-A exhibits two majorbiological activities: one is the capacity to stimulatevascular endothelial cell proliferation [1,6,7], and theother is the ability to increase vascular permeability [2,8].VEGF-A also promotes the survival and migration ofendothelial cells. In addition, recent studies have revealeda variety of biological functions and the precise molecularmechanisms of the VEGF/VEGFR system. In this review,we will discuss the recent advances in the basic biology ofthe VEGF/VEGFR system, which give insight into manyphysiological and pathological conditions.

VEGF AND VEGF FAMILY PROTEINS

Currently, the VEGF family includes VEGF-A, PlGF(placenta growth factor), VEGF-B, VEGF-C, VEGF-D,VEGF-E and svVEGF (snake venom VEGF). The mol-ecular and biological functions of each ligand have beenwell characterized.

VEGF-AStructurally, VEGF belongs to the VEGF/PDGF (plate-let-derived growth factor) supergene family. Among thegene products of this family, eight cysteine residues areconserved at the same positions. These products functionas a dimer, since two out of eight cysteine residues gen-erate intermolecular cross-linked S–S bonds [9]. Thehuman VEGF-A gene is organized into eight exons,separated by seven introns [10,11] and is located at 6p21.3[12].

Human VEGF-A has at least nine subtypes due to thealternative splicing of a single gene: VEGF121, VEGF145,VEGF148, VEGF162, VEGF165, VEGF165b, VEGF183,VEGF189 and VEGF206 [13,14] (Figure 1). VEGF165b is anendogenous inhibitory form of VEGF, which bindsVEGFR-2 with the same affinity as VEGF165, but doesnot activate it or stimulate downstream signalling path-ways [15]. VEGF is produced in endothelial cells, macro-phages, activated T-cells and a variety of other cell types[16–18]. Although virtually nothing is known abouthow VEGF isoform levels are regulated, most VEGF-producing cells appear to preferentially express VEGF121,VEGF165 and VEGF189. VEGF165, the predominant iso-form, is secreted as an approx. 46 kDa homodimer, whichhas a basic character and moderate affinity for heparin,owing to the presence of 15 basic amino acids within

the 44 residues encoded by exon 7 [1,2,7]. In contrast,VEGF121, which lacks the residues encoded by exons6 and 7, does not bind heparin and is freely releasedfrom the cell. VEGF189, which contains an additionalsequence encoded by exon 6, binds heparin strongly and iscompletely sequestered in the ECM (extracellular matrix)and to a lesser extent at the cell surface [16]. The ECM-bound isoforms can be released slowly by exposure toheparin and heparinases, or more rapidly released asbioactive fragments through cleavage by plasmin or uro-kinase at the C-terminus [19]. VEGF165 binds the co-receptors NRP-1 (neuropilin-1) [20] and NRP-2 (neuro-pilin-2), whereas VEGF145 binds only NRP-2 [21](Figure 2).

Approx. 50 % of mice expressing exclusively theVEGF120 isoform (murine VEGF is shorter by one aminoacid) die within a few hours after birth and the rest diewithin 14 days due to impaired myocardial angiogenesisand ischaemic cardiomyopathy [22]. VEGF120/120 micealso exhibit a specific decrease in capillary branch form-ation and the impairment of the directed extension ofendothelial cell filopodia during embryogenesis [23] aswell as severe defects in retinal vascular outgrowthand patterning [24], suggesting that the heparin-bindingVEGF isoforms provide spatially restricted stimulatorycues to initiate vascular branch formation. VEGF164/164

mice are normal and healthy, and have a normal retinalangiogenesis, whereas VEGF188/188 mice display normalvenular outgrowth but impaired arterial development inretinas as well as dwarfism, disrupted developmentof growth plates and secondary ossification centres, andknee joint dysplasia [25]. These findings suggest that thevarious VEGF isoforms play distinct roles in vascular pat-terning and arterial development, although the VEGF164

isoform plays a central role in vascular development.Gene expression of VEGF is regulated by a variety of

stimuli such as hypoxia, growth factors, transformation,p53 mutation, oestrogen, TSH (thyroid-stimulatinghormone), tumour promoters and NO (nitric oxide).Although all of the stimuli responsible for the up-regulation of the VEGF gene are quite interesting, hyp-oxia has been of particular interest because of its import-ance and the unique transcriptional regulation involved.It is now well established that HIF-1 (hypoxia-inducible factor-1) is a key mediator of hypoxic res-ponses. HIF-1 is a transcriptional activator composedof HIF-1α and HIF-1β subunits. Both HIF-1α andHIF-1β are constitutively expressed in various types oftumour. Under normal oxygenation conditions, HIF-1α is scarcely detectable because it is targeted for rapiddestruction by an E3 ubiquitin ligase containing pVHL(von Hippel–Lindau tumour suppressor protein). Theinteraction between pVHL and a specific domain ofthe HIF-1α subunit is regulated through hydroxylationof a proline residue (Pro564 in HIF-1α) by prolyl-4-hydroxylase, which requires molecular oxygen and


VEGF/VEGFR system in health and disease 229

Figure 1 Comparison of structures of the VEGF familyAlternative exon splicing results in the generation of several isoforms of VEGF-A, PlGF and VEGF-B. Numbers on the right side of structures indicate identities withVEGF165 at the amino acid level. Arrows denote positions of proteolytic cleavage that give rise to mature VEGF-C or VEGF-D.

iron for its activity. Under hypoxic conditions, HIF-1α expression increases as a result of suppressed prolylhydroxylation of HIF-1α and decreased ubiquitinationand degradation [26,27]. Furthermore, hypoxia inhibitsthe oxygen-dependent hydroxylation of an asparagineresidue (Asn803 in HIF-1α) in the C-terminal trans-activation domain of HIF-1α to promote interactionwith the p300/CBP [CREB (cAMP-response-element-binding protein)-binding protein] co-activator and in-duce a HRE (hypoxia response element)-driven tran-scription of the VEGF gene [28]. Very recently, Geraldet al. [29] have demonstrated that JunD, a member ofthe AP-1 family of transcription factors, is involvedin the regulation of prolyl hydroxylase activity. Deletionof JunD increases H2O2 levels, and thus inhibits prolylhydroxylase enzymatic activity by limiting FeII levels.Consequently, HIF-1α protein accumulates under nor-moxic conditions, and the transcription of VEGF-A isincreased [29].

VEGF is also regulated at the level of mRNA stability.The 5′- and 3′-UTRs (untranslated regions) of the VEGFgene confer increased mRNA stability during hypoxia.HuR, an AU-rich element binding protein, and PAIP2[polyadenylated-binding protein-interacting protein 2]have been identified as crucial proteins for VEGF mRNAstabilization [30,31]. Furthermore, VEGF expressioncan be regulated at the translational level. It has beenshown that the 5′-UTR of VEGF mRNA contains twofunctional internal ribosome entry sites that maintainefficient cap-independent translation and ensure efficientproduction of VEGF, even under unfavourable stressconditions such as hypoxia [32].

PlGFPlGF was originally discovered in human placenta in 1991[33]. The PlGF gene is highly expressed in placenta atall stages of human gestation. PlGF transcripts have also



Figure 2 Schematic diagram illustrating the receptor-binding specificity of VEGF family members and the VEGFR-2 signallingpathwaysThe VEGF family of ligands and their receptor-binding patterns are shown at the top. Downstream VEGFR signalling pathways focusing on VEGFR-2 are shown at thebottom. Tyr1175 (Y1175) and Tyr1214 (Y1214) are the two major autophosphorylation sites in VEGFR-2. PLC-γ binds to Y1175, leading to the phosphorylation andactivation of this protein. Y1214 appears to be required to trigger the sequential activation of Cdc42 and p38 MAPK. Many proteins are activated by VEGFR-2 throughan unknown mechanism, including FAK, PI3K and Src. The activation of downstream signal transduction molecules leads to several different endothelial cell functionssuch as migration, vascular permeability, survival and proliferation.

been detected in the heart, lung, thyroid gland and skeletalmuscle [34]. PlGF binds VEGFR-1, but not VEGFR-2[35,36]. Alternative splicing of the human PlGF genegenerates four isoforms which differ in size and bindingproperties: PlGF-1 (PlGF131), PlGF-2 (PlGF152), PlGF-3(PlGF203) and PlGF-4 (PlGF224) [37–39] (Figure 1).PlGF-1 is the shortest isoform and a non-heparin bindingprotein. PlGF-2 is able to bind heparin and the co-receptors NRP-1 and NRP-2 due to the insertion of ahighly basic 21-amino acid sequence encoded by exonVI near the C-terminus [37] (Figure 2). PlGF-3, whichcontains an insertion of 216 nucleotides coding for a 72-amino acid sequence between exons 4 and 5 of the PlGFgene but lacks the coding sequence of exon 6, is unable tobind heparin [38]. PlGF-4 consists of the same sequence

of PlGF-3, plus a heparin-binding domain previouslythought to be present only in PlGF-2 [39].

The crystal structure of human PlGF-1 has shownthat this protein is structurally similar to VEGF-A [40].Furthermore, despite this moderate sequence conser-vation, PlGF and VEGF-A bind to the same bindinginterface of VEGFR-1 in a very similar fashion [41].However, recent studies have reported that, unlike inVEGF-A, N-glycosylation in PlGF plays an importantrole in VEGFR-1 binding [42].

Carmeliet et al. [43] have shown that a deficiency inPlGF (PlGF−/−) does not affect embryonic angiogenesisin mice. However, loss of PlGF impairs angio-genesis, plasma extravasation and collateral growth du-ring ischaemia, inflammation, wound healing and cancer,



indicating the importance of VEGFR-1 signalling inpathological conditions.

VEGF-BVEGF-B has a wide tissue distribution, but is particularlyabundant in the heart and skeletal muscle [44]. HumanVEGF-B has two isoforms generated by alternativesplicing: VEGF-B167 and VEGF-B186 (Figure 1). TheVEGF-B isoforms bind and activate VEGFR-1 and canalso bind to NRP-1 [44] (Figure 2).

Studies using VEGF-B knockout (VEGF-B−/−) micehave yielded slightly conflicting results regarding therole of VEGF-B in angiogenesis and the developmentof the cardiovascular system. VEGF-B−/− mice areviable and fertile; however, although Bellomo et al. [45]demonstrated that VEGF-B−/− mice had smaller hearts,dysfunctional coronary arteries and an impaired recoveryfrom experimentally induced myocardial ischaemia, Aaseet al. [46] claimed that these mice showed a subtle cardiacphenotype such as an atrial conduction abnormalitycharacterized by a prolonged PQ interval, and thatVEGF-B was not required for proper development ofthe cardiovascular system either during developmentor angiogenesis in adults. Recent studies using VEGF-B−/− mice have demonstrated the role of VEGF-Bin pathological vascular remodelling in inflammatoryarthritis [47] and protection of the brain from ischaemicinjury [48].

VEGF-C and VEGF-DVEGF-C contains a region sharing approx. 30 % aminoacid identity with VEGF165; however, it is more closelyrelated to VEGF-D by virtue of the presence of N- andC-terminal extensions that are not found in other VEGFfamily members [49] (Figure 1). Both VEGF-C andVEGF-D bind and activate VEGFR-3 (Flt-4; a member ofthe VEGFR family that does not bind VEGF-A) as wellas VEGFR-2, and are mitogenic for cultured endothelialcells. VEGF-C also binds to NRP-2 [49] (Figure 2). BothVEGF-C and VEGF-D are produced as a preproproteinwith long N- and C-terminal propeptides flanking theVEGF homology domain. Initial proteolytic cleavage ofthe precursor generates a form with a moderate affinityfor VEGFR-3, but a second proteolytic step is requiredto produce the fully processed form with a high affinityfor both VEGFR-2 and VEGFR-3 [49]. This activationof VEGF-C and VEGF-D by proteolytic cleavage is atleast partly regulated by the serine protease plasmin [50].

Overexpression of VEGF-C in the epidermis oftransgenic mice results in the development of a hyper-plastic lymphatic vessel network [51]. In vitro, VEGF-C and VEGF-D stimulate the migration and mitogenesisof cultured endothelial cells [49]. A recent study usingVEGF-C−/− mice has demonstrated that VEGF-C isrequired for the initial steps in lymphatic developmentand that both VEGF-C alleles are required for normal

lymphatic development [52]. Thus VEGF-C is the para-crine factor essential for lymphangiogenesis. Less isknown of the function of VEGF-D, but Stacker et al.[53] have revealed that VEGF-D induces the formation oflymphatics within tumours and promotes the metastasisof tumour cells.

VEGF-EHomologues of VEGF have also been identified in thegenome of the parapoxvirus Orf virus [54] and have beenshown to have VEGF-A-like activities. VEGF-E is thecollective term for a group of these proteins, includingVEGF-ENZ-2 (VEGF from Orf virus strain NZ-2)[55], VEGF-ENZ-7 (VEGF from Orf virus strain NZ-7)[56], VEGF-ENZ-10 (VEGF from Orf virus strain NZ-10) [57], VEGF-ED1701 (VEGF from Orf virus strainD1701) [58] and VEGF-EVR634 (VEGF from Pseudo-cowpox virus strain VR634) [57]. All VEGF-E variantsstudied bind and activate VEGFR-2, but not VEGFR-1or VEGFR-3. VEGF-ENZ-2, VEGF-ENZ-10 and VEGF-ED1701 can bind NRP-1. VEGF-ENZ-7 and VEGF-EVR634,however, are unable to bind NRP-1 (Figure 2). VEGF-Eseems to be as potent as VEGF165 at stimulating endo-thelial cell proliferation despite lacking a heparin-bindingbasic domain. K14-driven VEGF-ENZ-7 transgenic micehave shown a significant increase in angiogenesis at sub-cutaneous tissue without clear side effects [59].

svVEGFRecently, VEGF family proteins have been identified insnake venom, including svVEGF from Bothrops insularis[60] and Tf svVEGF (Trimeresurus flavoviridis svVEGF)[61] from pit vipers in addition to HF (hypotensivefactor) [62], ICPP (increasing capillary permeabilityprotein) [63] and vammin [64] from vipers. Takahashiet al. [61] have shown that snakes utilize these venom-specific VEGFs in addition to VEGF-A. svVEGFs func-tion as dimers and each chain comprises approx. 110–122 amino acid residues. The cysteine knot motif, acharacteristic of the VEGF family of proteins, is com-pletely conserved in svVEGFs and the sequence identitywith human VEGF165 is approx. 50 % (Figure 1).Vammin does not bind VEGFR-1 but binds VEGFR-2with high affinity as well as VEGF165 [64]. However,Tf svVEGF binds VEGFR-1 with high affinity andVEGFR-2 with low affinity compared with VEGF165,leading to a strong enhancement of vascular permeabilitybut weak stimulation of endothelial cell proliferation[61] (Figure 2). Both vammin and Tf svVEGF are unableto bind VEGFR-3 or NRP-1, but Tf svVEGF bindsheparin. svVEGFs may contribute to the enhancementof toxicity in envenomation, but they seem to have indi-vidual biological characteristics reflecting divergence inthe classification of the host snake.



VEGFRs

VEGFR-1VEGFR-1 is a 180 kDa high-affinity receptor for VEGF-A, VEGF-B, PlGF and Tf svVEGF. It is expressed invascular endothelial cells and a range of non-endo-thelial cells, including macrophages and monocytes [65],and haematopoietic stem cells [66]. The second Ig domainof VEGFR-1 is the major binding site for VEGF-Aand PlGF [16,41,67]. VEGFR-1 binds VEGF-A with atleast 10-fold higher affinity than VEGFR-2 (Kd = 10–30 pM) [16]; however, ligand binding results in a maximal2-fold increase in kinase activity. In many cases, theeffects of VEGFR-2 on endothelial cells, such as thoseon cell survival and proliferation, can be induced onlyweakly or slightly by treatment with VEGFR-1-specificligands. VEGFR-1 is a negative regulator of angiogenesisduring early development, but plays an important role inangiogenesis under pathological conditions (as describedbelow). VEGFR-1-blocking antibodies prevent themigration but not proliferation of HUVECs (humanumbilical vein endothelial cells) in response to VEGF-A,indicating the involvement of VEGFR-1 in endothelialcell migration [68]. VEGFR-1-mediated signalling ap-pears to preferentially modulate the reorganization ofactin via p38 MAPK (mitogen-activated protein kinase),whereas VEGFR-2 contributes to the re-organizationof the cytoskeleton by phosphorylating FAK (focaladhesion kinase) and paxillin (Figure 2), suggesting adifferent contribution of the two receptors to the chemo-tactic response. VEGFR-1 signalling is also involved inthe migration of monocytes/macrophages [65] and in thereconstitution of haematopoiesis by recruiting haemato-poietic stem cells [66].

An alternatively spliced form of VEGFR-1 that en-codes a soluble truncated form of the receptor, containingonly the first six Ig domains, has been cloned froma HUVEC cDNA library [16]. sVEGFR-1 (solubleVEGFR-1) inhibits VEGF-A activity by sequester-ing VEGF-A from signalling receptors and by formingnon-signalling heterodimers with VEGFR-2 [69]. Plasmalevels of sVEGFR-1 are elevated in individuals withcancer, ischaemia and pre-eclampsia [70–72]. A recentstudy has demonstrated that elevated levels of sVEGFR-1play an important role in pre-eclampsia [73]. Increasedcirculating levels of sVEGFR-1 in patients with pre-eclampsia are associated with decreased circulating levelsof free VEGF and PlGF, resulting in general endothelialdysfunction [73].

VEGFR-2VEGFR-2 is a 200–230 kDa high-affinity receptor forVEGF-A (Kd = 75–760 pM), VEGF-E and svVEGFs aswell as the processed form of VEGF-C and VEGF-D.The binding site for VEGF-A has been mapped to the

second and third Ig domains [74]. VEGFR-2 is ex-pressed in vascular and lymphatic endothelial cells, andother cell types such as megakaryocytes and haemato-poietic stem cells [75]. Tyrosine phosphorylation sitesin human VEGFR-2 bound to VEGF-A are Tyr951 andTyr996 in the kinase-insert domain, Tyr1054 and Tyr1059

in the kinase domain, and Tyr1175 and Tyr1214 in theC-terminal tail. Among them, Tyr1175 and Tyr1214 arethe two major VEGF-A-dependent autophosphorylationsites [76]. Tyr951 creates a binding site for the VEGFR-associated protein [77] and Tyr1175 creates a binding sitefor Sck [78], Shb [79] and PLC (phospholipase C)-γ [76].

VEGFR-2 is the major mediator of the mitogenic,angiogenic and permeability-enhancing effects of VEGF-A. Furthermore, recent studies have indicated thatthe activation of VEGFR-2 also promotes lymphan-giogenesis [80,81]. Survival signalling for endothelialcells from VEGFR-2 is reported to involve thePI3K (phosphoinositide 3-kinase)/Akt pathway [82,83](Figure 2). However, another pathway may be involved,since the signal to activate PI3K by VEGFR-2 is usuallynot very strong. Byzova et al. [84] have reported thatthe activation of VEGFR-2 by VEGF-A results in thePI3K/Akt-dependent activation of several integrins,leading to enhanced cell adhesion and migration. Thissynergic interaction with integrins is required forproductive signalling from VEGFR-2.

Very recently, a naturally occurring soluble truncatedform of VEGFR-2 has been detected in mouse andhuman plasma [85]. Similar to sVEGFR-1, sVEGFR-2(soluble VEGFR-2) may have regulatory consequenceswith respect to VEGF-mediated angiogenesis.

VEGFR-3VEGFR-3 is a 195 kDa high-affinity receptor for VEGF-C and VEGF-D. Unlike VEGFR-1 and VEGFR-2,VEGFR-3 is proteolytically cleaved within the fifthextracellular Ig loop into a 120 kDa and a 75 kDa formduring synthesis, and the two forms are linked bya disulphide bridge [49]. Overexpression of a solubleVEGFR-3 in the skin of mice inhibits fetal lymphan-giogenesis and induces a regression of already formedlymphatic vessels [86]. Furthermore, overexpression of aVEGFR-3-specific mutant of VEGF-C (VEGF-C 156S)in the skin induces the growth of lymphatic vesselswithout an influence on the blood vessel architecture[87], indicating that stimulation of VEGFR-3 alone issufficient to induce lymphangiogenesis. The stimulationof VEGFR-3 also protects the lymphatic endothelialcells from serum deprivation-induced apoptosis. Thephosphorylation of VEGFR-3 has been shown to leadto a PI3K-dependent activation of Akt and PKC (proteinkinase C)-dependent activation of p42/p44 MAPK [88].A recent study [89] has demonstrated that blockadeof VEGFR-3 signalling significantly suppresses corneal



dendritic cell trafficking to draining lymph nodes as wellas the induction of delayed-type hypersensitivity andrejection of corneal transplants, suggesting a role forVEGFR-3 in adaptive immunity.

NRP-1 and NRP-2NRP-1 is a 130–140 kDa cell-surface glycoprotein firstidentified as a semaphorin receptor involved in neuronalguidance [90] and subsequently found as an isoform-specific receptor for VEGF-A [20]. NRP-2 was identifiedby virtue of its sequence homology with NRP-1 andshares 44 % identity at the amino acid level with NRP-1[90]. NRP-1 is able to bind VEGF165, VEGF-B, PlGF-2and some VEGF-E variants, whereas NRP-2 can bindVEGF145, VEGF165, PlGF-2 and VEGF-C. The intra-cellular domains of NRPs are short and do not sufficefor the independent transduction of biological signalssubsequent to semaphorin or VEGF binding. It has beenshown that both NRPs can join with receptors belongingto the plexin family, and such plexin/NRP complexes areable to transduce signals as the physiological receptor ofclass-3 semaphorins [91,92]. The VEGF165-induced pro-liferation and migration of cells that express VEGFR-2are enhanced in the presence of NRP-1. Thus NRP-1also seems to function as an enhancer of VEGFR-2activity in the presence of VEGF165. Recent studies havedemonstrated that this effect is the result of the formationof a complex between VEGFR-2 and NRP-1 [93,94].

An in vivo study with transgenic mice has shown thatNRP-1 is important not only for neuronal development,but also for vascular formation [95]. NRP-1−/− micesuffer from severe defects in the cardiovascular system inaddition to a disorganized neural development, resultingin the death of homozygous embryos by embryonic day14 [96]. Defects in vessel formation include a failureof capillary ingrowth into the brain and the abnormalformation of aortic arches and the yolk-sac vasculature,suggesting the importance of NRP-1 in embryonic vesselformation. In contrast, NRP-2−/− mice show an absenceor severe reduction of small lymphatic vessels and capil-laries during development [97]. Arteries, veins and largercollecting lymphatic vessels develop normally, suggestingthat NRP-2 is selectively required for the formation ofsmall lymphatic vessels and capillaries.

VEGF/VEGFR SYSTEM IN PHYSIOLOGICALAND PATHOLOGICAL CONDITIONS

Physiological angiogenesisThe loss of a single VEGF allele is lethal in the mouse em-bryo between days 11 and 12 [98,99]. VEGF+/− embryosexhibit significant defects in the vasculature of severalorgans and a markedly reduced number of nucleatedred blood cells within the blood islands in the yolk sac.In addition, a 2- to 3-fold overexpression of VEGF-A

from its endogenous locus results in severe abnormalitiesin heart development and lethality at embryonic days12.5 and 14 [100]. These results demonstrate the im-portance of tightly regulating VEGF-A expressionduring embryonic development. Homozygous loss ofthe VEGFR-1 or VEGFR-2 gene results in embryoniclethality between days 8.5 and 9.5, indicating that theseVEGFRs play important roles in vasculogenesis andangiogenesis [101,102]. VEGFR-2−/− mice die due to alack of endothelial cell growth and blood vessel formationas well as extremely poor haematopoiesis. On the otherhand, VEGFR-1−/− mice die due to an overgrowth ofendothelial cells and disorganization of blood vessels.Furthermore, normal vascular development in micelacking the tyrosine kinase domain of VEGFR-1 [103]has indicated that VEGFR-2 is the major positive signaltransducer, whereas VEGFR-1 has a negative regulatoryrole in angiogenesis early in embryogenesis.

Takahashi et al. [76] have shown that Tyr1175 and Tyr1214

are two major VEGF-A-dependent autophosphorylationsites in VEGFR-2. However, only autophosphorylationof Tyr1175 is crucial for VEGF-dependent endothelial cellproliferation via the PLC-γ /PKC/Raf/MEK [MAPK/ERK (extracellular-signal-regulated kinase) kinase]/ERKpathway. An unusual feature of mitogenic signalling fromVEGFR-2 is the requirement for PKC but not Ras [104].Our recent study [105] using knockin mice substitutingTyr1173 (corresponding to Tyr1175 in human VEGFR-2)and Tyr1212 (Tyr1214 in human) of the VEGFR-2 genewith phenylalanine has revealed that the signalling viaTyr1173 of VEGFR-2 is essential for endothelial andhaematopoietic development during embryogenesis. Incontrast, the phosphorylation of Tyr1214 appears to berequired to trigger the sequential activation of Cdc42and p38 MAPK and to drive p38 MAPK-mediated actinremodelling in stress fibres in endothelial cells exposedto VEGF-A [106]. The activation of the PI3K/p70 S6K(S6 kinase) pathway by VEGFR-2 is also involved inVEGF-A-induced endothelial cell proliferation [107](Figure 2). PILSAP (puromycin-intensive leucyl-specificaminopeptidase) plays a crucial role in the activationof this pathway via the binding and modification ofPDK1 (phosphoinositide-dependent kinase 1) [108]. Inaddition, recent studies have revealed various down-stream mediators of VEGF-induced angiogenic signal-ling, such as diacylglycerol kinase α [109], SRF (serumresponse factor) [110], SREBP (sterol-regulatory-ele-ment-binding protein) [111] and IQGAP1 [112].

Studies using DNA microarrays have reported pos-sible endogenous feedback inhibitors for VEGF-inducedangiogenesis. Vasohibin and DSCR1 (Down syndromecritical region protein 1) are significantly induced byVEGF in endothelial cells [113,114]. Up-regulationof DSCR1 in endothelial cells inhibits the nuclearlocalization of NFAT (nuclear factor of activated T-cells),proliferation and tube formation [115].



Vascular permeabilityVEGF-A is known to increase the vascular permeabilityof microvessels to circulating macromolecules [14]. In-creased vascular permeability is often observed in areasof pathological angiogenesis in solid tumours, woundsand chronic inflammation. VEGF-A significantly accu-mulates in malignant ascites [116] and pleural effusion[117], suggesting that it plays a fundamental role in theaccumulation of malignant fluid through the enhance-ment of vascular permeability. Consistent with a role inthe regulation of vascular permeability, VEGF-A inducesendothelial fenestration in some vascular beds and incultured adrenal endothelial cells, the extravasation offerritin by way of the VVO (vesiculo-vacuolar organelle)[14], and disorganization of endothelial junctional pro-teins such as VE-cadherin and occludin [118]. VEGF-Aincreases vascular permeability in mesenteric microvess-els by activation of VEGFR-2 on endothelial cells andsubsequent activation of PLC. This causes increasedproduction of diacylglycerol that results in influx ofcalcium [14]. Other studies have also demonstrated thecrucial role of VEGFR-2 signalling in the enhancementof vascular permeability; however, our recent study [61]using Tf svVEGF has shown that the enhancement ofvascular permeability is intensified by the activationof VEGFR-1 more than the proliferation of endothelialcells under some active signalling from VEGFR-2. Thisfinding indicates the importance of VEGFR-1 signallingin vascular permeability.

An analysis of mice deficient in specific Src familykinases has demonstrated no decrease in VEGF-depen-dent neovascularization, but a complete ablation ofvascular permeability in Src−/− or Yes−/− mice, whereasFyn−/− mice show no such defect [119]. In addition,blockade of Src prevents the disassociation of a complexcomprising VEGFR-2, VE-cadherin and β-catenin withthe same kinetics with which it prevents VEGF-mediatedvascular permeability and oedema [120]. These findingsindicate that the activity of specific Src family kinases isessential for the VEGF-induced enhancement of vascularpermeability through the disruption of the VEGFR-2/cadherin/catenin complex.

VEGF-A can induce production of NO and endo-genous NO can increase vascular permeability [121].Among the three isoforms of NOS (NO synthase),eNOS (endothelial NOS) plays a predominant role inVEGF-induced angiogenesis and vascular permeability[122]. Furthermore, the activation of eNOS is regulatedby the PI3K/Akt pathway [123,124]. The small GTP-binding protein Rac, which is also activated by PI3K, hasbeen implicated in the regulation of vascular permeability[125]. A recent study [126] has shown that inhibition ofp38 MAPK activity abrogated VEGF-induced vascularpermeability in vivo and in vitro, suggesting the in-volvement of p38 MAPK in the control of vascularpermeability (Figure 2).

Solid tumoursNumerous studies have established VEGF-A as a keyangiogenic player in cancer. VEGF-A is expressed inmost tumours and its expression correlates with tumourprogression. In addition to tumour cells, tumour-asso-ciated stroma is also an important source of VEGF-A[127]. In the absence of access to an adequate vas-culature, tumour cells become necrotic and apoptotic,restraining the increase in tumour volume that shouldresult from continuous cell proliferation [128]. Theexpression of VEGF-A mRNA is highest in hypoxictumour cells adjacent to necrotic areas [16], indicatingthat the induction of VEGF-A by hypoxia in growingtumours can change the balance of inhibitors and activat-ors of angiogenesis, leading to the growth of new bloodvessels into tumour. Consistent with this hypothesis,capturing of VEGF or blocking of its signalling recep-tor VEGFR-2 by a VEGFR tyrosine kinase inhibitor,antisense oligonucleotides, vaccination or neutralizingantibodies reduced tumour angiogenesis and growth inpreclinical studies [129]. Unlike in physiological angio-genesis, VEGFR-1 signalling plays an important rolein angiogenesis under pathological conditions [43,130].Autiero et al. [131] have proposed that PlGF regulatesinter- and intra-molecular cross-talk between VEGFR-1and VEGFR-2, amplifying VEGF-driven angiogenesisthrough VEGFR-2.

Several studies also describe the role of VEGF incarcinogenesis [132]. Rip1–Tag2 (T-antigen 2) mice de-velop islet tumours of the pancreas by 12–14 weeks ofage as a result of expression of the SV40 Tag oncogenein insulin-producing β-cells. In this mouse, angiogenicactivity first appears in a subset of hyperplastic isletsbefore the onset of tumour formation. VEGF-A andVEGFRs are constitutively expressed in the islet vascu-lature before and after the initiation of angiogenesis(angiogenic switch) [133]; however, when VEGF-A isabsent from islet β-cells of Rip1–Tag2 mice, both angio-genic switching and carcinogenesis as well as tumourgrowth are severely disrupted [134], indicating thatVEGF-A plays a critical role in angiogenic switchingand carcinogenesis. Bergers et al. [135] have revealed thatMMP (matrix metalloproteinase)-9 is also a component ofthe angiogenic switch, as this proteinase makes VEGF-Aavailable for the interaction with its receptors by releasingsequestered VEGF-A.

VEGF-A impairs the endothelial barrier by disruptinga VE-cadherin/β-catenin complex via the activation ofSrc and facilitates tumour cell extravasation and meta-stasis [136]. VEGF-A also induces the disruption ofhepatocellular tight junctions, which may promotetumour invasion [137]. Pharmacological blockade ofVEGFR-2 stabilizes the endothelial barrier functionand suppresses tumour cell extravasation in vivo [136],suggesting the importance of VEGFR-2 signalling in thiskind of tumour invasion and metastasis. Hiratsuka et al.



[138] have shown that VEGFR-1 signalling is alsoinvolved in tumour metastasis, being linked to the induc-tion of MMP-9 in lung endothelial cells and to thefacilitation of lung-specific metastasis.

Recently, Hurwitz et al. [139] have shown that the ad-dition of bevacizumab (a humanized anti-VEGF mono-clonal antibody) to fluorouracil-based combinationchemotherapy results in statistically significant and clini-cally meaningful improvement in survival among patientswith metastatic colorectal cancer. Based on this result,bevacizumab (Avastin) was approved by the FDA(Food and Drug Administration) in February 2004 as afirst-line treatment for metastatic colorectal carcinoma.Besides bevacizumab, many other VEGF inhibitorsare being pursued clinically. These inhibitors includesmall-molecule RTK inhibitors such as PTK787, solublereceptors such as VEGF-Trap and anti-VEGFR-2 mAbs(monoclonal antibodies) [129].

Inflammatory diseasesVEGF acts as a pro-inflammatory cytokine by increasingthe permeability of endothelial cells, inducing the ex-pression of endothelial adhesion molecules and via itsability to act as a monocyte chemoattractant [140–142].VEGF is strongly expressed by epidermal keratinocytesin wound healing and psoriasis, conditions that arecharacterized by increased microvascular permeabilityand angiogenesis [16]. Transgenic mice that overexpressVEGF-A specifically in the epidermis exhibit an increaseddensity of tortuous cutaneous blood capillaries as wellas highly increased leucocyte rolling and adhesion inpostcapillary skin venules, suggesting that enhanced ex-pression of VEGF-A in epidermal keratinocytes is suffi-cient to develop psoriasis-like inflammatory skin lesions[143]. Moreover, heterozygous VEGF-A transgenic mice,which do not spontaneously develop inflammatoryskin lesions, are unable to down-regulate experimentallyinduced inflammation and exhibit a psoriasis-like pheno-type characterized by epidermal hyperplasia, the accumu-lation of lymphocytes, and lymphatic vessel proliferationand enlargement [144]. Transgenic overexpression ofPlGF-2 in epidermal keratinocytes also results in asignificantly increased inflammatory response, whereasa deficiency of PlGF results in a diminished and abbre-viated inflammatory response [145], suggesting the im-portance of VEGFR-1 signalling in chronic skin inflam-mation.

Local production of VEGF-A in arthritic synovialtissue has been documented [16] and appears to cor-relate with disease activity in humans. Subsequently,VEGF-A has been shown to be important in the patho-genesis of RA (rheumatoid arthritis) in animal models[146–148]. Treatment with anti-VEGFR-1 mAbs, butnot anti-VEGFR-2 mAbs, significantly reduces thearthritic destruction of joints by suppressing synovialinflammation and neovascularization, emphasizing the

importance of VEGFR-1 signalling in the destruction.The anti-inflammatory effects of anti-VEGFR-1 areattributable to a reduced mobilization of bone-marrow-derived myeloid progenitors into peripheral blood [147].The reduction of synovial inflammation in VEGF-B−/−

mice [47] also implies a critical role for VEGFR-1signalling in RA.

Exaggerated levels of VEGF-A have been detectedin tissues and biological samples from people withasthma, where these levels correlate directly with disease[149] and inversely with airway function [150]. VEGFhas been postulated to contribute to asthmatic tissueoedema through its effect on vascular permeability. Arecent study using lung-targeted VEGF165 transgenicmice has revealed a novel function of VEGF-A in allergicresponses. In these mice, VEGF-A induces asthma-like inflammation, airway and vascular remodelling, andairway hyper-responsiveness. VEGF-A also enhancesrespiratory sensitization to antigen as well as TH2(T-helper type 2) cell-mediated inflammation and in-creases the number of activated dendritic cells [151].Thus VEGF-A has a critical role in pulmonary TH2inflammation. Other studies have provided evidence fora role for VEGF-A as a pro-inflammatory mediator inallograft rejection [152] and neointimal formation [153].

Other pathological conditionsVEGF-A mRNA expression, not normally found in theadult mouse brain, is up-regulated after cerebral isch-aemia, and elevated VEGF-A levels can be detected asearly as 3 h after stroke with a peak between 12 and48 h [154]. Previous studies have demonstrated that theantagonism of VEGF-A results in reduced oedema andtissue damage after ischaemia implicating VEGF-A inthe pathophysiology of stroke [155]. Paul et al. [156]have reported that Src−/− mice are resistant to VEGF-A-induced vascular permeability and show decreasedinfarct volumes after stroke. Systemic application of aSrc inhibitor suppresses vascular permeability, protectingwild-type mice from ischaemia-induced brain damagewithout influencing VEGF-A expression. However, Sunet al. [157] have reported that intracerebroventricularadministration of VEGF-A reduces infarct size, improvesneurological performance and enhances the delayedsurvival of newborn neurons. These conflicting resultsappear to reflect dual roles of VEGF-A in stroke: neuro-protective and pro-inflammatory effects. In this context,when infused through the internal carotid artery, lowand intermediate doses of VEGF-A significantly promoteneuroprotection of the ischaemic brain, whereas a highdose of VEGF-A offers no neuroprotection to theischaemic brain or the damaged neurons of normal brain[158]. Further studies are required for the therapeuticapplication of VEGF-A against stroke.

Extensive evidence has suggested a causal role ofVEGF in several diseases of the human eye in which



neovascularization and increased vascular permeabilityoccur. VEGF levels are increased in the vitreous andretina of patients and laboratory animals with active neo-vascularization from ischaemic retinopathies such asproliferative diabetic retinopathy, central retinal veinocclusion and retinopathy of prematurity. Subsequentstudies using various VEGF inhibitors have confirmedthat VEGF plays a central role in ischaemia-inducedintraocular neovascularization [159]. An anti-VEGFaptamer, pegaptanib (Macugen), has produced a stat-istically significant and clinically meaningful benefit inthe treatment of neovascular AMD (age-related maculardegeneration) [160], which is the leading cause of irre-versible severe loss of vision in people 50 years of age andolder in the developed world, and was approved by theFDA in December 2004.

Oosthuyse et al. [161] have reported that deletion ofthe HRE in the VEGF promoter reduces hypoxic VEGFexpression in the spinal cord and causes adult-onsetprogressive motor neuron degeneration, reminiscent ofALS (amyotrophic lateral sclerosis). VEGF165 promotessurvival of motor neurons during hypoxia throughbinding VEGFR-2 and NRP-1 [161]. A subsequent studyhas revealed that VEGF-A is a modifier associated withmotor neuron degeneration in human ALS and in a mousemodel of ALS [162]. VEGF-A treatment increases the lifeexpectancy of ALS mice without causing toxic side effects[163,164], indicating that VEGF-A has neuroprotectiveeffects on motor neurons, and treatment with VEGF-Acould be one of the most effective therapies for ALSreported so far.

LeCouter et al. [165] recently provided evidence for anovel function of VEGFR-1 in LSECs (liver sinusoidalendothelial cells). The activation of VEGFR-1 results inthe paracrine release of HGF (hepatocyte growth factor),IL-6 (interleukin-6) and other hepatotrophic moleculesby LSECs to the extent that hepatocytes are stimulated toproliferate when co-cultured with LSECs. VEGF-A hasno direct mitogenic effect on hepatocytes. A VEGFR-1agonist protected the liver from CCl4-induced damage, inspite of its inability to induce the proliferation of LSECs.

CONCLUSIONVEGF was originally described as a specific angiogenicand permeability-inducing factor and its function wasconsidered to be specific for endothelial cells. However,emerging evidence has revealed that the role of theVEGF/VEGFR system extends far beyond previousexpectations. First, a wide variety of VEGF family pro-teins and numerous splicing variants have been identifiedand found to play distinct but critical roles in variousconditions, including lymphangiogenesis. VEGF familyproteins have been utilized even in snake venoms andsome viruses. Secondly, several different VEGFRs havebeen shown to be essential, but the interaction between

these receptors has appeared to be complicated.VEGFR-1 has a negative regulatory role in embryonicangiogenesis, but functions as a positive signal transducerin some cases individually and sometimes synergisticallywith VEGFR-2 via the intra- and inter-molecular cross-talk between these two receptors. An association betweenVEGFR-2 and VEGFR-3 has also been reported [166].Thirdly, it has been shown that the VEGF/VEGFRsystem has multiple functions, such as the inductionof tumour metastasis, inflammation, neuroprotection,protection of liver and mobilization of marrow-derivedstem cells, as well as lymphangiogenesis. VEGF is alsoimportant for memory and learning [167]. Fourthly,numerous other molecules have been found to associatewith the VEGF/VEGFR system. Further studies arerequired to achieve a comprehensive understanding of theVEGF/VEGFR system; however, the recent progress inthe molecular and biological study of this system providesus with novel and promising therapeutic strategies forovercoming a variety of diseases.

ACKNOWLEDGMENTS

The authors’ work was supported by Grant-in-AidSpecial Project Research on Cancer-Bioscience 12215024from the Ministry of Education, Culture, Sports, Scienceand Technology of Japan, and grants for the program‘Research for the Future’ from the Japan Society forPromotion of Science, and for the program ‘Promotionof Fundamental Research in Health Sciences’ from theOrganization for Pharmaceutical Safety and Research.

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Received 23 December 2004/5 April 2005; accepted 9 May 2005Published on the Internet 24 August 2005, doi:10.1042/CS20040370


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