Cellular Signalling 19 (2007) 2003–2012www.elsevier.com/locate/cellsig

Review

Vascular endothelial growth factor receptor-2: Structure, function,intracellular signalling and therapeutic inhibition

Katherine Holmes, Owain Ll Roberts, Angharad M. Thomas, Michael J. Cross ⁎

North West Cancer Research Fund Institute, School of Biological Sciences, College of Natural Sciences, University of Wales, Bangor, LL57 2UW, United Kingdom

Received 25 April 2007; accepted 8 May 2007Available online 12 June 2007

Abstract

Vascular endothelial growth factors (VEGFs) regulate vascular development, angiogenesis and lymphangiogenesis by binding to a number ofreceptors. VEGFR-1 is required for the recruitment of haematopoietic stem cells and the migration of monocytes and macrophages, VEGFR-2regulates vascular endothelial function and VEGFR-3 regulates lymphatic endothelial cell function. Over the last decade, considerable progresshas been made in delineating the VEGFR-2 specific intracellular signalling cascades leading to proliferation, migration, survival and increasedpermeability, each of which contributes to the angiogenic response. Furthermore, therapeutic inhibition of VEGFR-2 action is now having animpact in the clinic for the treatment of a number of diseases.© 2007 Elsevier Inc. All rights reserved.

Keywords: VEGF; VEGFR-2; Angiogenesis; Endothelial; Inhibitors; Signal transduction

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20032. The VEGF ligands . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20043. The VEGF receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20054. VEGFR-2 structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20055. VEGFR-2 function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20076. VEGFR-2 intracellular signalling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2007

6.1. Proliferation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20086.2. Migration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20086.3. Survival . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20086.4. Permeability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2009

7. VEGFR-2 regulated gene expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20098. VEGFR-2 signalling in disease and therapeutic inhibition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20099. Conclusions and perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2010Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2011References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2011

⁎ Corresponding author. NWCRF Institute, University of Wales, Bangor,LL57 2UW, United Kingdom. Tel.: +44 1248 388496; fax: +44 1248 371644.

E-mail address: m.j.cross@bangor.ac.uk (M.J. Cross).

0898-6568/$ - see front matter © 2007 Elsevier Inc. All rights reserved.doi:10.1016/j.cellsig.2007.05.013

1. Introduction

Vascular endothelial growth factor (VEGF) represents afamily of homodimeric glycoproteins which are critical for theembryonic development of the blood vascular system (vascu-logenesis), lymphatic system (lymphangiogenesis) and in theformation of new blood vessels from pre-existing vessels

2004 K. Holmes et al. / Cellular Signalling 19 (2007) 2003–2012

(angiogenesis). In mammals, five different VEGF ligands havebeen identified and one parapoxvirus-encoded VEGF. Theseligands bind, in an overlapping pattern, to three different,but structurally related, VEGF-receptor (VEGFR) tyrosinekinases. VEGFR-1 is critical for haematopoietic cell develop-ment, VEGFR-2 is critical for vascular endothelial cell devel-opment and VEGFR-3 critical for lymphatic endothelial celldevelopment.

As our understanding of the role of VEGFs and VEGFRs inboth physiological vascular development and pathological dis-eases, such as tumour angiogenesis, has grown over the lastdecade, a number of researchers have identified the signallingpathways downstream of the VEGF receptors. Selective acti-vation of VEGFR-1 andVEGFR-2, coupled with gene silencing,has revealed that VEGFR-2 is the principal receptor transmittingVEGF signals in the vascular endothelium. This review willfocus on our current understanding of VEGFR-2 signalling inendothelial cells and the clinical development of inhibitors ofVEGFR-2 function.

2. The VEGF ligands

In mammals, the VEGF family consists of five members,VEGF-A, VEGF-B, VEGF-C, VEGF-D and placenta growthfactor (PLGF). Structurally related proteins include parapoxvirusOrf VEGF, denoted VEGF-E [1], and snake venom VEGFs,denoted VEGF-F [2,3]. VEGF-Awas first described in 1983, bySenger and coworkers, as a tumour-secreted vascular-permeabil-ity factor (VPF) [4]. In 1989, Ferrara and Henzel reported theisolation and amino-acid sequence of an endothelial cell mitogenthey named VEGF [5]. Subsequent molecular sequencingrevealed that VPF and VEGF were in fact the same molecule.

The biological importance of VEGF-A is highlighted by thefact that VEGF-A−/− mice exhibit severe defects in vasculardevelopment and die at E9.5–10.5 [6]. Furthermore, loss of asingle VEGF-A allele leads to vascular abnormalities and deathat E11–12 [7]. Formation of blood vessels in the VEGF-A+/−

Fig. 1. Exon structure of human VEGF-AmRNA splice variants. The gene encoding VmRNA splicing leads to the formation of several different isoforms that vary in amin

heterozygote was abnormal, but not abolished, indicating aconcentration-dependent regulation of embryonic vessel devel-opment by VEGF-A.

The human VEGF-A gene is located on chromosome 6p21.3[8]. The coding region spans approximately 14 kb and containseight exons (Fig. 1). In mammals, VEGF-A exists in a number ofdifferent isoforms following alternative splicing of a singleprecursor mRNA [9]. In humans, six VEGF-A splice variantshave been detected: VEGF-A121, VEGF-A145, VEGF-A165,VEGF-A183, VEGF-A189 and VEGF-A206. Although VEGF-A121, VEGF-A183 and VEGF-A189 are expressed in varioustissues, VEGF-A165 is the most abundantly expressed form,whereas VEGF-A145 and VEGF-A206 are relatively rare [9].Recently, a new VEGF-A splice variant termed VEGF-A165b hasbeen described, containing a different C-terminus encoded by analternative exon 8 [10]. This variant is thought to negativelyregulate VEGF-receptor activity.

In mice, VEGF-A isoforms lack a glycine residue at position8 of the mature protein and are therefore one amino acid shorterthan their corresponding human isoforms. The longer VEGF-Aisoforms, containing exons 6 and 7, show binding affinity forthe extracellular matrix (ECM) and cell surface heparansulphate proteoglycans (HSPGs). The VEGF-A206 and VEGF-A189 isoforms are basic proteins remaining bound to the ECMand are not readily diffusible. VEGF-A121, which lacks exons 6and 7, is a freely diffusible acidic protein that does not interactwith HSPGs. VEGF-A165 shows intermediate propertiesregarding HSPG binding and diffusibility. Extracellular prote-olysis appears to have a major role in regulating VEGF bio-availability. All isoforms contain a plasmin cleavage site, whichresults in the generation of a smaller, diffusible, bioactive 110amino-acid fragment [11]. Recent studies indicate that matrixmetalloproteinases may also cleave VEGF-A165 to generatenon-heparin binding bioactive proteolytic fragments [12].

The VEGFs are secreted as covalently linked homodimericproteins, stabilised by intra- and inter-chain disulphide bonds[13,14]. In humans, VEGF-A165 is the most abundant and

EGF-A comprises eight exons that encode different structural motifs. Alternativeo-acid (aa) number. In humans, VEGF165 is the most predominant form.

Fig. 2. Schematic illustration of the expression patterns, ligand specificity and cellular/physiological effects of each of the vascular endothelial growth factor receptors(VEGFRs). Each VEGF ligand (-A, -B, -C, -D, -E and PLGF) binds in a specific manner to three receptor tyrosine kinases, VEGFR-1, -2, and -3. VEGFR-1 isexpressed on haematopoietic stem cells, macrophages and monocytes as well as on the vascular endothelium. VEGFR-2 is expressed on both vascular and lymphaticendothelium, whereas expression of VEGFR-3 is generally restricted to lymphatic endothelium. The vascular endothelium is composed of a single layer of endothelialcells with tight inter-endothelial junctions, surrounded by basement membrane and stabilised by specialized smooth muscle cells, called pericytes. In contrast, thelymphatic endothelium lacks inter-endothelial junctions, basement membrane and supporting pericytes allowing for the permeability of lymphatic vessels. VEGFR-2binds all VEGF-A isoforms, VEGF-C, -D and -E. Downstream effects of VEGFR-2 activation in the vascular endothelium include cell proliferation, migration,permeability and survival, resulting in vasculogenesis and angiogenesis.

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biologically active form and is expressed as a 46 kDa homo-dimer composed of two 23 kDa subunits. VEGF-A is producedby a range of cells including vascular smooth muscle cells,macrophages and tumour cells [15]. An important regulator ofVEGF-A expression is oxygen tension [16]. The VEGF-A genecontains a hypoxia responsive element (HRE) in the 5′ and 3′UTR and hypoxia induces a rapid and sustained increase inVEGF-mRNA levels [17,18].

3. The VEGF receptors

VEGFs signal through cell surface receptor tyrosine kinases(Fig. 2). VEGFR-1 (Flt-1) is expressed on haematopoietic stemcells, monocytes, macrophages and vascular endothelial cells.VEGFR-2 (Flk-1/KDR) is expressed on vascular endothelialcells and lymphatic endothelial cells, whilst VEGFR-3, (Flt-4)expression is restricted to lymphatic endothelial cells. An alter-natively spliced soluble VEGFR-1 variant (sVEGFR-1) is alsoexpressed (for a review of VEGFR-1 and VEGFR-3, the reader

is referred to Olsson, A-K et al. [19]). VEGF-A binds to bothVEGFR-1 and VEGFR-2, whilst PLGF and VEGF-B bindexclusively to VEGFR-1. VEGF-C and VEGF-D are initiallyexpressed as pro-peptides that bind the VEGFR-3. The mature,proteolytically processed VEGF-C and VEGF-D ligands canalso bind to VEGFR-2. Viral VEGF-E binds exclusively toVEGFR-2. VEGFs also bind to co-receptors such as neuropilins(NRP). The neuropilins (NRP-1 and NRP-2) are transmembranenon-protein tyrosine kinase co-receptors for both the sema-phorin family of axonal guidance molecules and the VEGFfamily; binding of VEGF is thought to promote a complex withVEGFR-2, enhancing the function of VEGFR-2 [20,21].

4. VEGFR-2 structure

VEGFR-2 (kinase-insert domain receptor (KDR)/foetal liverkinase (Flk)-1) is a type III transmembrane kinase receptor, firstisolated in 1991 by Terman and coworkers [22]. The humanVEGFR-2 gene, located on chromosomes 4q11–q12, encodes

Fig. 3. Structure and tyrosine phosphorylation sites of human VEGFR-2 (KDR).VEGFR-2 is a type III transmembrane kinase receptor composed of 1356 aminoacids. The extracellular domain comprises seven Ig-like domains (I–VII), ofwhich the second and third domains bind VEGF-A. The intracellular domaincontains two kinase domains, which are split by a kinase-insert domain of 70amino acids. Tyrosine residues within the intracellular domain are highlighted.Five tyrosine residues have been identified as the major phosphorylation sites(Y951, Y1054, Y1059, Y1175 and Y1214). Phosphorylation of these sites, to-gether with the adjacent amino-acid sequence (shown with the tyrosine residueunderlined), creates a binding site for the SH2 domains of various signallingmolecules.

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a full-length receptor of 1356 amino acids [23]. It consists of anextracellular region composed of seven immunoglobulin (Ig)-like domains, a short transmembrane domain, and an intracel-lular region containing a tyrosine kinase domain, split by a 70-amino-acid insert (Fig. 3). Phylogenetically it belongs withthe 7-Ig/5-Ig protein tyrosine kinase superfamily, and is thusclosely related to the platelet-derived growth factor receptors

(PDGFRs), fms receptor and c-Kit receptor [24]. Within thecell, the VEGFR-2 protein is translated as a 150 kDa proteinwithout significant glycosylation. It is then processed, by aseries of glycosylations, to a mature 230 kDa form that isexpressed on the cell surface [25]. Flk-1 is the murine homo-logue of human KDR, sharing 85% sequence homology andbeing 2 amino acids shorter.

VEGF-A binds to the second and third extracellular Ig-likedomains of VEGFR-2 with a Kd of 75–125 pM [26,27], anaffinity approximately 10-fold lower than that of the VEGFR-1for VEGF-A. Ligand binding induces receptor dimerisation andautophosphorylation. Recent analysis of the VEGF/VEGFR-2complex by electron microscopy has revealed that binding of thedimeric VEGF ligand, to the Ig-like domains 2 and 3 of onereceptor monomer, increases the probability that a second recep-tor monomer binds the already tethered ligand. Once the tworeceptors are cross-linked to each other, via simultaneous inter-action with the ligand, their membrane-proximal Ig-like domain7s are held in close proximity so that low-affinity homotypicinteractions between these domains further stabilises the receptordimers [28]. This allows for the exact positioning of the intra-cellular kinase domains resulting in autophosphorylation.

The initial mapping of autophosphorylation sites in VEGFR-2used bacterial expression systems, revealing that tyrosines Y951,Y996, Y1054 and Y1059 were phosphorylated [29]. Other work,utilising baculovirus mediated expression identified Y801 andY1175 as autophosphorylation sites that bind phospholipase C-γ(PLC-γ) [30], and Y1054 and Y1059 in the activation loop of thekinase domain as critical for the catalytic activity of the receptor[31]. More recent work, using VEGF-A stimulated mammaliancells overexpressing the KDR and phosphopeptide analysis, hasrevealed that the major phosphorylation sites are Y951 in thekinase-insert domain, Y1054 and Y1059 within the kinasedomain, and Y1175 and Y1214 in the C-terminal tail of thereceptor [32,33]. Phosphorylation of specific tyrosine residuesin the receptor creates a consensus sequence for the recruitmentof specific intracellular proteins, via their Src homology 2(SH2) domains (Fig. 3). Phosphorylation of Y951 creates abinding site for VEGF-receptor-associated protein (VRAP)[34] also called T-cell-specific adapter molecule (TSAd) [33].Phosphorylation of Y1175 creates a binding site for a numberof signalling proteins. The pY–I–V–L sequence allowsbinding of PLC-γ [32], the adaptor protein Shb [35] and theadaptor protein Sck [36]. Phosphorylation of Y1214 creates abinding site for the adaptor protein Nck [37]. Phosphorylationof the tyrosine residues Y1224 [38], Y1305, Y1309 and Y1319[33] in the C-terminal tail has also been reported. However,the role of these residues in VEGFR-2 function remains to bedetermined.

In addition to possessing multiple tyrosine phosphorylationsites, the carboxyl terminus of the VEGFR-2 also contains puta-tive serine phosphorylation sites. Mutation of S1188 and S1191,in the carboxyl terminus of murine VEGFR-2 (Flk-1), has beenshown to impair ligand-dependent down regulation of VEGFR-2[39]. Recently, it has been shown that ubiquitination ofVEGFR-2,in a kinase-dependent manner, leads to endosomal sorting andlysosomal degradation of the receptor–ligand complex [40].

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5. VEGFR-2 function

In murine embryogenesis, expression of VEGFR-2/Flk-1 isfirst detected at E7.0 in mesodermal blood island progenitors.Slightly later the gene is expressed in endothelial cell precursorsand developing endothelial cells [41–43]. The critical role ofVEGFR-2 in vascular development is highlighted by the fact thatVEGFR-2−/− mice die at E8.5-9.5 due to defective developmentof blood islands, endothelial cells and haematopoietic cells [44].

Fig. 4. Schematic illustration of VEGFR-2 intracellular signalling. Binding of VEGF totyrosine residues. Activation of intracellular signalling cascades results in proliferation, mspecific phosphorylated tyrosine residues (pY) in the VEGFR-2, via their SH2 domains,pY1175 results in the hydrolysis of PIP2, generating the second messengers DAG and Iendoplasmic reticulum (E.R.) causing release of intracellular calcium (Ca2+). Entry of excertain proteins. VRAP/TSAd binds to pY951 and forms a complex with Src. Binding opY1175 regulates activation of FAK and PI3K. Abbreviations: BAD, Bcl-2 associatedeNOS, endothelial nitric oxide synthase; Erk 1/2, extracellular regulated kinases 1 and 2protein 27; IP3, inositol (1,4,5)-trisphosphate; MAPKAP 2/3, MAPK-activating protemitogen-activated protein kinase; PI3K, phosphoinositide 3-kinase; PGI2, prostacy(3,4,5)-trisphosphate; PKB, protein kinase B; PKC, protein kinase C; PLC-γ, phospVEGFR-2, VEGF receptor-2; VRAP, VEGFR-associated protein/TSAd T-cell-spec

In the adult, VEGFR-2 is expressed mostly on vascular endo-thelial cells, although expression is also detectable on neuronalcells, megakaryocytes and haematopoietic stem cells [45].

6. VEGFR-2 intracellular signalling

A number of studies have shown that VEGFR-2 is the prin-cipal mediator of several physiological and pathological effectsof VEGF-A on endothelial cells. These include proliferation,

the receptor induces dimerisation and autophosphorylation of specific intracellularigration, survival and increased permeability. Several intracellular proteins bind to

leading to the phosphorylation and activation of these proteins. Binding of PLC-γ toP3. DAG is a physiological activator of PKC, whilst IP3 acts upon receptors in thetracellular calcium, through specific channels, is also important for the activation off Nck to pY1214 results in activation of Cdc42 and p38MAPK. Binding of Shb todeath promoter; cPLA2, cytosolic phospholipase A2; DAG, sn-1,2-diacylglycerol;; FAK, focal adhesion kinase; Gab1, Grb2-associated binder-1; HSP27, heat-shockin kinases 2 and 3; MEK, MAPK/Erk kinase; NO, nitric oxide; p38 MAPK, p38clin; PIP2, phosphatidylinositol (4,5)-bisphosphate; PIP3, phosphatidylinositolholipase C-γ; Sck, Shc-like protein; VEGF, vascular endothelial growth factor;ific adaptor molecule.

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migration, survival and permeability. The intracellular signallingpathways mediating these effects downstream of VEGFR-2activation are shown schematically in Fig. 4.

6.1. Proliferation

VEGF-A is a mitogen for a variety of primary endothelialcells [46]. VEGFR-2, like most receptor tyrosine kinases (RTKs),induces proliferation via activation of the classical extracellularsignal-regulated kinases (Erk) pathway. However, unlike mostother RTKs, activation of the classical Grb2–Sos–Ras pathwayhas not been implicated in VEGFR-2 mediated Erk activation[47]. Instead, VEGFR-2 stimulates Erk phosphorylation and pro-liferation via a PKC-dependent pathway involving activation ofPLC-γ [32]. Phosphorylation of Y1175, in the C-terminal tail ofVEGFR-2, allows the binding of PLC-γ and its subsequentphosphorylation, leading to an increase in catalytic activity. PLC-γhydrolyses the membrane phospholipid phosphatidylinositol(4,5)-bisphosphate (PIP2), resulting in the generation of diacyl-glycerol (DAG) and inositol 1,4,5-trisphosphate (IP3). IP3 gen-eration results in an increase in the intracellular concentration ofcalcium [Ca2+]i, whilst DAG is a physiological activator of proteinkinase C (PKC). A number of PKC isoforms have been implicatedin regulating VEGF-mediated proliferation, these include PKCβ[47,48], PKCα and PKCζ [49]. The precise role of Ras inVEGFR-2 mediated signalling is unclear. Tyr 1214 contains thesequence pY–D–N–T (Fig. 3), which would appear to be apotential binding site for the SH2 domain of Grb2, which requiresthe consensus sequence pY–X–N–X [50]. However, binding ofGrb2 to the VEGFR-2 appears to be independent of Y1214 [37],whilst other studies have not detected any association betweenVEGFR-2 and Grb2 [47]. It remains possible, that an unidentifiedadaptor molecule is required for the VEGFR-2 to efficientlycouple to the Grb2–Sos–Ras pathway. Recent data has sug-gested that VEGF-A can stimulate Ras in human umbilicalvein endothelial cells (HUVEC) [51], via a pathway requiringPKC and sphingosine kinase [52], raising the possibility thatendothelial cells utilise a novel pathway to couple VEGFR-2to Ras activation.

The importance of PLC-γ in angiogenesis is highlighted by thefact that in mice, PLC-γ1 deficient embryos die at approximatelyE9.0 with significantly diminished vasculogenesis and erythro-poiesis [53,54]. Furthermore, recent analysis of zebrafish mutantsdemonstrated that PLC-γ1 is required for arterial development[55]. The PLC-γ1 deficient zebrafish embryos fail to respondto exogenous VEGF, highlighting the role of PLC-γ in VEGFfunction. A recent receptor knock-in approach in mice, showedthat mutation of Y1173 in Flk-1 (Y1175 in KDR), results inembryonic lethality at E8.5–9.5 due to defects in endothelial andhaematopoietic cells [56]. These defects are similar to thoseobserved in Flk-1 null mice [44]. Overall, these data suggest acritical role for Y1173 in Flk-1 in regulating angiogenesis. How-ever, since PLC-γ, Shb and Sck all bind to this site, it is not clearwhether this dependence is exclusively linked to the inability ofthe Y1173 Flk-1 mutant to activate PLC-γ1 and the subse-quent Erk cascade in vivo; further clarification awaits the pheno-typic analysis of the Shb and Sck deficient mice.

6.2. Migration

Themigration of endothelial cells is a critical component of theangiogenic response as endothelial cells move through proteasedegraded basementmembrane towards a concentration gradient ofVEGF and other growth factors. A number of intracellular sig-nalling pathways have been implicated in VEGFR-2 mediatedmigration (Fig. 4). The adaptor protein Shb is able to bind topY1175 resulting in its phosphorylation in a Src-dependent man-ner [35]. Small interfering RNA (siRNA)-mediated gene silencingof Shb resulted in an inhibition of VEGF-mediated cytoskeletalreorganisation, migration and activation of phosphoinositide3-kinase (PI3K). Shb is an adaptor protein which is able to bindto a number of other proteins such as focal adhesion kinase(FAK) [57], an enzyme important for cellular attachment andcellular migration [58]. Another receptor phosphorylation siteimplicated in migration is Y951 which is a binding site for theVRAP/TSAd [33]. Site-directed mutation of Y951 to phenyl-alanine (Y951F) in the VEGFR-2, or siRNA mediated silenc-ing of VRAP/TSAd expression, prevented VEGF-A mediatedcytoskeletal reorganisation and migration but not mitogenicity[33]. Furthermore, VEGF-A induced the formation of a com-plex between VRAP/TSAd and Src. Phosphorylation of Y1214has also been implicated in VEGF-induced actin remodelling,through the subsequent activation of Cdc42, p38 MAPK andphosphorylation of heat-shock protein-27 (HSP27) [59]. Re-cently, it has been shown that phosphorylated Y1214 binds theadaptor protein Nck [37]. Nck associates with the Src familykinase Fyn leading to the phosphorylation of p21-activatedprotein kinase-2 (PAK-2) and the subsequent activation ofCdc42 and p38 mitogen-activated protein kinase (MAPK).Interestingly, mice expressing a VEGFR-2 knock-in mutationof Y1212 in Flk-1 (Y1214 in KDR) are viable and fertile,indicating that Y1212 is not required for normal developmentin mice [56].

Activation of PI3K is known to regulate cellular migrationby a number of different growth factors. PI3K mediated gen-eration of phosphatidylinositol-3,4,5-trisphosphate (PIP3) isresponsible for activating the small molecular-weight, GTP-binding protein Rac, leading to lamellipodia/membrane rufflesand cell motility [60]. Unlike many other RTKs, the VEGFR-2does not contain the pY–X–X–M sequence recognised by theSH2 domain of the p85 regulatory subunit of PI3K [50]. How-ever, recent data from two independent research groups, hasshown that the Gab1 adaptor protein couples VEGFR-2 acti-vation to PI3K and cellular migration [61,62]. Gab1 contains abinding site for the p85 subunit of PI3K and also binds to theVEGFR-2, although the exact binding site in the receptorremains obscure.

6.3. Survival

Activation of PI3K and the generation of membrane boundPIP3 results in the membrane targeting and subsequentphosphorylation of protein kinase B (PKB/Akt) by phosphoi-nositide-dependent kinases 1 and 2 (PDK1 and PDK2) [63].VEGF-A induced cell survival in HUVEC is dependent on

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VEGFR-2, and the subsequent activation of PI3K and Akt[64]. Akt directly phosphorylates two apoptotic proteins, Bcl-2associated death promoter (BAD) and caspase 9, inhibitingtheir apoptotic activity, and thereby promoting cell survival[65,66]. Furthermore, VEGF-A also induces expression of theanti-apoptotic proteins Bcl-2 and A1 [67] and inhibitors ofapoptosis (IAP) family members XIAP and survivin, whichinhibit the terminal effector caspases 3 and 7 [68]. The survivalof endothelial cells is also influenced by the ECM. Integrinαvβ3 is required for the survival of nascent blood vesselsduring angiogenesis [69]. Cross-talk between the integrins andVEGFR-2 is indicated by the finding that the VEGFR-2 canassociate with the αvβ3 integrins, leading to enhancedVEGFR-2 activation and Akt activity [70]. The involvementof the α1β1 and α2β2 integrins in VEGF-mediated angiogen-esis has also been shown [71].

6.4. Permeability

VEGF-Awas originally discovered as a vascular-permeabil-ity factor (VPF) [4], an effect which plays a role in normalphysiology and in pathological conditions such as tumourhypermeability. Fluid and small solutes cross the endotheliumby a number of mechanisms ranging from cell–cell junctions,transcellular gaps, fenestrations and vesiculovacuolar orga-nelles (VVOs) [72,73]. The use of different VEGF ligands andVEGFR inhibitors has shown that VEGFR-2 is able to mediatevascular permeability in vivo [73]. VEGF-induced permeabilityrequires endothelial nitric oxide synthase (eNOS) mediatedgeneration of nitric oxide (NO) [74].

eNOS activity is stimulated by either PLC-γ dependentcalcium influx, or by Akt/PKB mediated phosphorylation ofeNOS on serine 1179 [75,76]. VEGF-A vascular permeability isimpaired in mice lacking the Src family members Src and Yes[77], although it is currently not clear if Src is able to bind directlyto the VEGFR-2, as well as an indirect association via bindingto VRAP/TSAd [33]. Recent data has implicated NRP-1 inVEGFR-2 mediated permeability [78], suggesting that VEGF-A mediated permeability may require co-operation betweenVEGFR-2 and neuropilins.

In addition to increasing permeability, VEGF-A also inducesvasodilation, resulting in increased blood flow to tissues. Thishypotensive effect is mediated by VEGFR-2 generation of NOand prostacyclin (PGI2), which act on vascular smooth musclecells to evoke relaxation [79]. VEGFR-2 activation results inPGI2 synthesis by a pathway requiring an increase in intra-cellular calcium and the Erk1/2 mediated phosphorylation ofcytosolic phospholipase A2 (cPLA2) [80,81]. cPLA2 hydrolysesmembrane phospholipids, to release arachidonic acid, which isfurther metabolised by cyclooxygenases (COX-1 and COX-2)and prostacyclin synthase to generate PGI2, which is released bythe endothelial cell.

7. VEGFR-2 regulated gene expression

A number of studies have utilised techniques such as cDNAmicroarrays to identify genes which are upregulated in

endothelial cells, following stimulation with VEGF [82–86].Whilst a number of genes with known roles in angiogenesis wereidentified, such as Cox-2, a number of novel genes have alsobeen identified, such as Down syndrome critical region-1(DSCR-1). DSCR-1 was identified as the most highly upregu-lated gene in one study in VEGF stimulated endothelial cells[83], and has since been found to play a role in endothelial cellmigration and angiogenesis [87]. Recently, Endocan wasidentified as a VEGF-responsive gene with increased expressionin human renal cancer [82]. Decay-accelerating factor (DAF)was identified as a VEGFR-2 responsive gene, with a potentialrole in cytoprotection during inflammatory angiogenesis [88].Similarly, VEGF induction of Egr3 and the NR3A family oftranscription factors has also been shown to be VEGFR-2specific [89]. Other VEGF-responsive genes known to bedownstream of VEGFR-2 include Ets-1, MMP-1 and Flt-1[90].

8. VEGFR-2 signalling in disease and therapeutic inhibition

Angiogenesis plays a role in a number of pathologicalconditions, with VEGFR-2 signalling implicated in both tumourangiogenesis, and diabetic retinopathy [91]. Angiogenesis iscrucial for tumour development as cancer cells have a relativelyhigh metabolic demand for oxygen and nutrients to continuegrowing. Furthermore, the capillary and vascular networkallows tumours to metastasise and spread to other sites in thebody. Tumour vasculature is highly disorganised, and tumourblood flow is chaotic. In addition, tumour vessels are ‘leaky’due to increased openings, inter-endothelial junctions and adiscontinuous or absent basement membrane.

VEGF-expression in cancer cells is induced during tumourformation by environmental stimuli such as hypoxia (Fig. 5), orby genetic mutations such as K-ras, p53 or HER2/ErbB2. Thebioavailability of VEGF also increases as monocytes and macro-phages infiltrate into the tumour, in a VEGFR-1 dependentmanner, and secrete metalloproteinase (MMP)-9. MMP-9 activ-ity releases bound VEGF from the ECM, enabling the formationof functional VEGF–VEGFR-2 complexes in the endothelium[92]. Expression of VEGFR-2 is upregulated in the tumourvasculature compared with normal vasculature [93]. Indeed,VEGFR-2 expression is a prognostic marker in the clinicaloutcome of patients with a variety of malignancies [94,95].

In 1971, Folkman first proposed the theory that inhibition ofangiogenesis may result in the arrest of tumour growth [96]. Thisvision has now become a reality, with the arrival of a number ofanti-angiogenic drugs in the clinic [97–99]. These agents can bedivided into two broad classes, namely agents targeting theVEGF ligand and agents designed to target the cell surfacereceptor (Fig. 5). Anti-angiogenic compounds are postulated tonot only reduce tumour vascularisation, but also create a morestable, or normalised, vasculature within the tumour, enablingefficient delivery of anti-tumour drugs [100]. It should be noted,that whilst this review focuses on therapeutic inhibition ofVEGFR-2 signalling, research has also been conducted intoincreasing VEGF levels as a therapeutic strategy in diseases suchas myocardial ischemia, and amyotrophic lateral sclerosis

Fig. 5. Tumour angiogenesis and inhibitors of VEGFR-2 signalling. The hypoxic state (low O2 tension) of a growing tumour allows stabilisation of hypoxia-induciblefactor (HIF-1α), which binds to the hypoxia responsive element (HRE) in the VEGF-A promoter, leading to increased VEGF-A transcription. Secreted VEGF-A bindsto VEGFR-2 expressed on the vascular endothelium, stimulating an angiogenic response, and ultimately resulting in tumour growth. VEGFR-2 signalling may beinhibited for therapeutic benefit by either sequestering the VEGF-A ligand, e.g. Bevacizumab, or by inhibiting the receptor kinase activity with a small moleculeinhibitor. A number of tyrosine kinase inhibitors are in clinical development. These are shown together with their molecular targets and IC50 concentration.

2010 K. Holmes et al. / Cellular Signalling 19 (2007) 2003–2012

(ALS). For more information on this topic, the reader is direct-ed towards recent reviews by Yla-Herttuala et al. [101], andBogaert et al. [102].

Bevacizumab (Avastin®) became the first anti-angiogenicdrug to receive FDA approval for cancer treatment, when inFebruary 2004, it was approved for use in combination with5-fluorouracil-based chemotherapy for treatment of metastaticcolorectal cancer [103,104]. Avastin is a recombinant huma-nised monoclonal IgG1 antibody which binds to, and inhibits,the biological activity of all VEGF-A isoforms [105]. It is nowin clinical trails for use against a range of different cancers incombination with chemotherapy [97].

A number of pharmaceutical companies have developedsmall molecule inhibitors of the VEGFRs. These agents targetthe ATP-binding site of the RTKs, resulting in the blockade ofdownstream intracellular signalling pathways (Fig. 5). Sorafe-nib (Nexavar®) is a bi-aryl urea multi-targeted kinase inhibitor,initially developed against the Raf-1 kinase [106,107]. Sunitinib(Sutent®) is a dual-targeted RTK inhibitor to VEGFR-2 andPDGFR-β, which exhibits anti-tumour activity [108].

Other promising tyrosine kinase inhibitors include AZD2171,PTK787/ZK 222584 and ZD6474 (Fig. 5). AZD2171 (Recen-

tin®) is a potent ATP-competitive, sub-nanomolar inhibitor ofVEGFR-2. It has been shown to inhibit VEGF-induced prolif-eration and VEGFR-2 phosphorylation in HUVEC and also toinhibit angiogenesis in vivo [109]. PTK787/ZK 222584 (Vatala-nib) is an orally active anilinophthalazine inhibitor of VEGFR-1and -2, as well as PDGFR and c-Kit at higher concentrations[110]. ZD6474 (Vandetanib/Zactima®) is a heteroaromatic-substituted anilinoquinazoline, which has been shown to inhibitVEGF signalling, tumour-induced neovascularisation and growthof xenograft tumour models in vivo [111].

Other inhibitors of VEGF functions are aimed at treating age-relatedmacular degeneration (AMD), an ocular disease that leads toeventual blindness. Pegaptanib (Macugen®) is an RNA oligo-nucleotide ligand (aptamer) that binds VEGF-A165, whilstRanibizumab (Lucentis®) is a Fab fragment that binds all VEGF-A isoforms [112]. Both these drugs have recently received FDAapproval.

9. Conclusions and perspectives

Research over the last decade has revealed the complexity ofVEGFR signalling. Three different, but related, receptor tyrosine

2011K. Holmes et al. / Cellular Signalling 19 (2007) 2003–2012

kinases regulate different aspects of vascular function. VEGFR-2signalling has been the most intensively studied, as this receptormediates the effects of VEGF-A on the vascular endothelium.This research has revealed a complex signalling mechanismregulating different aspects of physiological, and pathologicalangiogenesis. The development of anti-angiogenic drugs, thattarget the VEGFR-2 signalling pathway, is now having an impactin the clinic. However, a number of challenges remain. Firstly, theidentification of VEGFR-2 specific signalling pathways and geneexpression, leading to tubular morphogenesis in endothelial cells,remains to be determined. At the moment, the VEGFR-2 sig-nalling pathways identified are shared by a number of growthfactor receptors, which do not evoke angiogenesis, suggestingthat either unique pathways await discovery, or that thedifferential spatial and temporal activation of known pathwaysallows the VEGFR-2 its specificity in evoking an angiogenicresponse. Secondly, current anti-angiogenic therapies target eitherVEGF-A action or VEGFR-2 activity. Whilst this is a rationalapproach, evoking a clinical effect, the long-term suppression oftotal VEGFR-2 functionmay reveal a number of unexpected side-effects. Furthermore, tumours and the endothelium itself, mayrespond by upregulating other growth factors and growth factorreceptors in an attempt to overcome the VEGFR blockade.Targeting specific intracellular molecules, downstream of theVEGFR-2, may offer selectivity in regulating aberrant angiogen-esis and facilitate long-term, anti-angiogenic therapy for thecontrol of a number of diseases.

Acknowledgements

The authors are supported by grants from the Medical Re-search Council (MRC), Biotechnology and Biological SciencesResearch Council/AstraZeneca (BBSRC/AZ CASE award),Cancer Research Wales (CRW) and North West CancerResearch Fund (NWCRF).

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