Signaling via Vascular Endothelial Growth Factor Receptors

Experimental Cell Research 253, 117–130 (1999) Article ID excr.1999.4707, available online at http://www.idealibrary.com on

Signaling via Vascular Endothelial Growth Factor Receptors Tatiana V. Petrova, Taija Makinen, and Kari Alitalo 1 Molecular/Cancer Biology Laboratory, Haartman Institute, University of Helsinki, Helsinki, Finland

Angiogenesis, or development of blood vessels from preexisting vasculature, has important functions under both normal and pathophysiological conditions. Vascular endothelial growth factor receptors 1–3, also known as flt-1, KDR, and flt-4, are endothelial cellspecific receptor tyrosine kinases which serve as key mediators of the angiogenic responses. The review focuses on the signaling pathways that are initiated from these receptors and the recently identified VEGF coreceptor neuroplilin-1. © 1999 Academic Press Key Words: angiogenesis; endothelial cells; receptor tyrosine kinases; signal transduction; VEGF receptors; VEGF.

INTRODUCTION

Angiogenesis, or formation of new blood capillaries from preexisting vessels, plays both beneficial and detrimental roles in the organism. Physiological angiogenesis occurs during the female reproductive cycle and in wound healing. Abnormally enhanced angiogenic response is observed in rheumatoid arthritis, diabetic retinopathy, and during tumor development [1]. Inhibition of the neovasculature was shown to abolish or slow tumor growth in various experimental models (e.g., [2, 3]). On the other hand, promotion of the angiogenic response can prove beneficial in the treatment of ischemic conditions, such as myocardial ischemia/ infarction [4]. It is therefore obvious that our ability to prevent and treat many of these diseases relies on our understanding of the molecular mechanisms underlying the angiogenic response. Angiogenesis is a result of complex interplay of positive and negative regulators. Molecules that serve as inducers of angiogenesis include VEGF, fibroblast growth factors-1 and -2, transforming growth factors-a and b, tumor necrosis factor a, angiogenin, and angiopoietins [5–9]. The negative regulators identified to date comprise angiostatin [10], endostatin [11], the 1

To whom correspondence and reprint requests should be addressed at Molecular/Cancer Biology Laboratory, Haartman Institute, University of Helsinki, P.O. Box 21(Haartmaninkatu 3), SF00014 Helsinki, Finland. Fax: 358 9 191 26448. E-mail: [email protected].

16-kDa N-terminal fragment of prolactin [12], trombospondin [13], and platelet factor-4 [14]. VEGF occupies a particular place among the positive regulators of angiogenesis due to its potency and specificity for endothelial cells and a multitude of responses it can elicit in these cells (for recent reviews see [15, 16]). During the past few years, several other members of the VEGF family have been discovered, including placenta growth factor (PlGF) [17], VEGF-B [18], VEGF-C [19, 20], VEGF-D [21, 22], and viral homologues of VEGF, collectively termed here VEGF-E [23–25]. The action of VEGF and other family members is mediated by a particular family of receptor tyrosine kinases (RTKs), VEGFR-1 (Flt-1), VEGFR-2 (KDR), and VEGFR-3 (Flt4),which are expressed almost exclusively on endothelial cells. In addition, neuropilin-1, a transmembrane protein involved in regulation of axonal guidance in neurons, has been recently described as a coreceptor for VEGF 165 [26]. The focus of the present review is on the molecular characteristics of VEGFR-1–3 and neuropilin-1 and on signal transduction pathways which are initiated in endothelial cells upon their activation. Other issues, such as biology of VEGF family factors and roles of VEGFRs during development and in pathophysiological situations, are discussed in more detail in several recent reviews [15, 16, 27–29]. LIGANDS

All VEGF family members possess a homology domain belonging to the cysteine knot family and containing six distinctly spaced cysteine residues. In addition, VEGF-C and VEGF-D have long N- and C-terminal extensions [19, 21, 22]. The N-terminal parts of VEGF-C and VEGF-D do not display sequence homology to any known protein, whereas their C-terminal portions contain several repeats of cysteine-rich motifs Cys-X10-Cys-X-Cys-X-Cys which are similar to motifs in Balbiani ring 3 protein, a major component of silk produced by the midge Chironomus tentans. VEGF, which is a ligand for VEGFR-1 and VEGFR-2, is expressed as several isoforms consisting of polypeptides with 121, 145, 165, 189, and 206 amino acid residues. The isoforms differ in their ability to interact with extracellular matrix components, such as

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heparan sulfate proteoglycans (reviewed in [15]). The most abundant and presumably major biologically active form is VEGF 165, which binds VEGFR-1 and VEGFR-2 with K d values of 10 –20 and 75–125 pM, respectively [30, 31]. VEGF 165 also binds with high affinity to neuropilin-1 [26]. PlGF and VEGF-B are two members of the VEGF family which interact exclusively with VEGFR-1 and neuropilin-1 [26, 32–34]. PlGF exists as isoforms of 131, 203, and 152 amino acid residues, the latter containing a stretch of 21 basic residues, which confers heparin- and neuropilin-1-binding ability [17, 35, 36]. VEGF-B has two isoforms of 167 and 186 residues which share their 115 N-terminal amino acid residues, but have distinct C-termini [37]. VEGF-C and VEGF-D are produced as long precursor proteins which undergo proteolytic processing and generate several forms that differ in their receptor binding properties [38, 70]. Unprocessed or partially processed VEGF-C and VEGF-D are able to bind and activate exclusively VEGFR-3, whereas processed forms stimulate both VEGFR-2 and VEGFR-3. The mature form of VEGF-C, containing only the VEGF homology domain, binds to VEGFR-2 and VEGFR-3 with K d values of 410 and 135 pM, respectively. Finally, viral homologues of VEGF, termed collectively VEGF-E, activate exclusively VEGFR-2 [23–25] and at least NZ2-VEGF-E can use neuropilin-1 as a coreceptor [24]. VEGF RECEPTORS 1–3

General Characteristics VEGFR-1, VEGFR-2, and VEGFR-3 belong to the class III receptor tyrosine kinases of the platelet-derived growth factor (PDGF) receptor subfamily [39 – 41] which is characterized by the presence of seven immunoglobulin homology domains (Ig domains) in their extracellular, ligand-binding part and an intracellular tyrosine kinase domain split by a kinase insert (Fig. 1). The kinase insert represents a stretch of 65–97 hydrophilic residues and it is important for substrate recognition [39]. As is the case for many other RTKs, ligand binding induces homodimerization of the VEGF receptors and transphosphorylation of their intracellular domains, which allows the activation of complex intracellular signaling pathways. Human VEGFR-1 was first cloned from a placental cDNA library [42]; mouse and rat homologues were subsequently identified [20, 43– 45]. VEGFR-2 was isolated from a human endothelial cell cDNA library [31, 46]. Mouse [47– 49], rat [50], quail [51], and zebrafish [52] homologues have also been isolated. VEGFR-3 was cloned from human erythroleukemia cell and placental cDNA libraries [53, 54]. Mouse [43] and quail (desig-

nated Quek2) [51]) homologues of VEGFR-3 were subsequently identified from embryonic cDNA libraries. Human genes encoding VEGFR-1, -2, and -3 are located in chromosomal regions 13q12-q13, 4q11-q13, and 5q33-q35, respectively ([31, 53–57]. Mouse homologues for VEGFR-1 and -2 have been mapped to chromosome 5 [47, 56] and VEGFR-3 to chromosome 11 [58]. VEGFR-1 is a 180-kDa transmembrane glycoprotein, but alternative splicing can also produce a shorter soluble protein containing only six first extracellular immunoglobulin homology domains followed by 31 unique amino acid residues [42, 59, 60]. VEGFR-2 is a 230-kDa protein and no splice variants have been reported for this receptor [46]. VEGFR-3 is produced as 4.5- and 5.8-kb transcripts due to alternative 39 polyadenylation signals [61]. The longer form encodes 65 additional amino acid residues and is the major form detected in tissues. After its biosynthesis, the glycosylated 195-kDa VEGFR-3 is proteolytically cleaved in the fifth Ig domain, but the resulting 120- and 75-kDa chains remain linked by disulfide bonds. Structure and Binding Epitopes Domain deletion studies of VEGFR-1 and VEGFR-2 have shown that ligand binding function is localized to the second Ig domain but the presence of the first and third domain is necessary for full affinity binding of VEGF [62– 65]. Deletion of domain 3 leads to a 20- and 1000-fold decrease in the affinity of VEGF for VEGFR-1 and VEGFR-2, respectively, thus suggesting that domain 3 plays different roles in the VEGFR-1 and VEGFR-2 interaction with VEGF [65]. Domain 4 was suggested to be important for ligand-induced dimerization of VEGFR-1 [63]. Crystal structures of VEGF alone and in the complex with domain 2 of VEGFR-1 have been elucidated [66 – 68] (Fig. 2). The VEGF molecule represents an antiparallel disulfide-linked homodimer which interacts with the receptor via binding sites at the poles of each molecule. The interface between VEGF and domain 2 of VEGFR-1 is composed predominantly of hydrophobic residues, the only direct polar interaction existing between Asp-63 of VEGF and Arg-224 of VEGFR-1. These data are in contrast with earlier alanine-scanning mutagenesis studies that implicated a number of other polar residues in the interaction of VEGF with its receptors [69]. VEGF competes with PlGF and VEGF-B for VEGFR-1 binding, suggesting that they interact with the same or closely related contact sites on VEGFR-1 [32, 63]. Both the full-length and the mature forms of VEGF-C bind VEGFR-3; however, only the mature VEGF-C lacking a C-terminal extension is able to bind to and activate VEGFR-2 [19, 38]. VEGF-C shares re-

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FIG. 1. Schematic representation of VEGFRs and their ligands. VEGFs are shown in red, VEGFR-1, VEGFR-2, and VEGFR-3 in blue, and neuroplilin-1 in green. The different structural elements of the receptors are illustrated as follows: blue lozenge, immunoglobulin domain; blue hexagon, tyrosine kinase domain; green oval, CUB domain; green rectangle, domain homologous to coagulation factors V and VIII; green octagon, MAM domain.

ceptor specificity with its closest homologue, VEGF-D [22], which is also processed in a similar fashion [70]. The receptor binding affinity of recombinant mature VEGF-C for VEGFR-3 is approximately threefold higher than for VEGFR-2 [38]. Replacement of cysteine 156 with a serine residue generates a protein that specifically activates VEGFR-3 but not VEGFR-2, providing a valuable tool for investigation of VEGFR-3 function [71]. Interestingly, in contrast to the wild-type protein, the mutant is unable to induce vascular permeability, thus suggesting that VEGFR-3 is not involved in this process [71]. Expression Pattern All VEGFRs are expressed early in development; they display overlapping expression profiles, mostly confined to endothelial cells [72, 73]. Interestingly, in fetal human heart only VEGFR-1 expression could be

detected in coronary endothelium [72, 73]. Among nonendothelial cells, VEGFR-1 expression could be detected in macrophages and monocytes [74, 75], trophoblasts [76], and renal mesangial cells [77], whereas VEGFR-2 is produced in hematopoietic stem cells, megacaryocytes and platelets [78, 79], and retinal progenitor cells [80]. VEGF is expressed in all tumor cells studied thus far, whereas VEGFR-2 is expressed by the tumor-associated vasculature, suggesting a paracrine interaction (reviewed in [28]). VEGFR-3 is expressed initially in all embryonic endothelial cells, but in the course of development its expression in large blood vessels decreases, and it becomes largely restricted to the lymphatic endothelium in adult tissues [81, 82] with an additional weak expression in fenestrated endothelia of some tissues [83, and Taina Partanen, unpublished observations]. VEGFR-3 becomes upregulated in angiogenic blood

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tron-containing fragment of mouse VEGFR-2 is necessary to confer endothelial-specific expression of LacZ in transgenic mice, whereas a 59 flanking sequence alone already conferred endothelium-specific expression in vitro. An Ets motif and a cyclic AMP response element were found to be important for the transcriptional regulation of VEGFR-1 [93]. In vivo, targeted inactivation of JunB, a member of the AP-1 transcription factor family, leads to defective yolk sac vascularization as a result of reduced levels of VEGFR-1 but not of VEGFR-2 in yolk sac mesoderm [94]. SIGNALING

FIG. 2. Crystal structure of VEGF 8 –109 in the complex with domain 2 of VEGFR-1 [67]. VEGF molecules are shown in green and blue. They form an antiparallel homodimer, with two cysteines covalently linking VEGF polypeptides and remaining six cysteines forming a cysteine knot (yellow). Two molecules of Ig domain 2 of VEGFR-1 are shown in yellow. VEGF interacts with VEGFR-1 via sites at the poles of the homodimer (red). The N- and C-termini of one VEGF and one VEGFR-1 molecule are indicated. (A) Front view looking from the membrane. (B) Lateral view of the complex.

vessels in breast cancer, in the lymphatic endothelium of metastatic lymph nodes, and in lymphangiomas, vascular skin tumors, and Kaposi’s sarcoma spindle cells [81, 83– 85], whereas various brain tumors were found to be VEGFR-3 negative [86, 87]. Regulation of Expression Hypoxia is a major regulator of VEGF production under both physiological and pathological conditions. Regulation of VEGFRs expression appears to be less straightforward. Systemic hypoxia was shown to upregulate VEGFR-1 but not VEGFR-2 in mice in vivo [88]; however, in other experimental settings both VEGFR-1 and VEGFR-2 were induced under hypoxic conditions [89, 90]. In HUVE cells hypoxia stimulated VEGFR-1 but not VEGFR-2 production, an observation that could be attributed to the presence of HIF-1a consensus binding sites in the promoter of VEGFR-1 [91]. In contrast, VEGFR-2 expression is regulated by HIF-2a [92]. Interestingly, a 59 flanking and first-in-

Most signal transduction studies have been carried out using endothelial cells that express more than one type of VEGFR. It is therefore difficult to attribute the effect to the activation of a specific receptor. This complication could be circumvented in some cases by using receptor-specific mutants of the ligands (e.g., [69, 71, 95]) or transfected cells expressing the receptor of interest. Although in the latter case the results may be compromised by the lack of appropriate cellular background, elucidation of the cellular mechanisms regulating angiogenic responses is of significant biomedical importance since it may provide new targets for therapeutic intervention. Veger-1 and Verger-2 The role of VEGFR-1 as a signal-transducing molecule remains less well defined than that of VEGFR-2. Indeed, it is rather difficult to detect VEGFR-1 autophosphorylation upon ligand binding [30, 96] and overexpression of VEGFR-1 in both endothelial and nonendothelial cells failed to induce mitogenesis in response to VEGF [97, 98]. Nevertheless, it was recently shown that in VEGFR-1-transfected endothelial cells PlGF but not VEGF could activate the MAPK pathway to induce a mitogenic response and plasminogen activator production [99]. Two major and two minor phosphorylation sites of VEGFR-1 have been identified as Tyr1213/Tyr1242 and Tyr1327/Tyr1333, respectively [100]. They are located in the C-terminal tail domain and may serve as docking sites for such signaling molecules as PLCg, Nck, SHP-1, and the p85 subunit of PI3 kinase. Activation of PLCg and RasGAP has been reported in VEGFR-1-transfected NIH3T3 fibroblasts [97] while the p85 subunit of PI3 kinase, tyrosine phosphatase SHP-2, and adaptor protein Nck were shown to bind VEGFR-1 in a yeast two-hybrid assay [101, 102]. Mice expressing a truncated form of VEGFR-1 which lacks the tyrosine kinase domain possess normal vasculature [103], in contrast to the full VEGFR-1 knockout animals (see below). Taken together with the weak

VEGF RECEPTORS AND SIGNAL TRANSDUCTION

signaling abilities of VEGFR-1, these data suggest that endothelial VEGFR-1 might act as a decoy receptor rather than as a signal-transducing molecule per se. Among nonendothelial cells, monocytes/macrophages which express exclusively VEGFR-1 respond to stimulation with PlGF and VEGF with increased intracellular calcium levels, production of tissue factor, and enhanced migration [74, 75, 104, 105], suggesting that at least in these cells VEGFR-1-mediated signaling is taking place. Accordingly, macrophage migration in response to VEGF or PlGF is suppressed in mice having the kinase-deleted VEGFR-1 [103]. Although the affinity of VEGF is about 10-fold higher for VEGFR-1 than for VEGFR-2, the latter appears to be the major receptor that conveys VEGF-induced signals in endothelial cells. In many endothelial cell types VEGF stimulation leads to robust VEGFR-2 autophosphorylation, activation of the MAP kinase cascade, cell proliferation, chemotaxis, and changes in the biosynthesis of different proteins. Activation of VEGFR-2 also protects endothelial cells against starvation- and tumor necrosis a-induced apoptosis [95, 106]. Additional confirmation of the essential role of VEGFR-2 in the generation of angiogenic response comes from studies of viral homologues of VEGF, which bind exclusively to VEGFR-2. Similar to VEGF, VEGF-E can stimulate the release of tissue factor, proliferation, chemotaxis, and sprouting of cultured vascular endothelial cells in vitro and angiogenesis in vivo [24, 25]. Interestingly, in contrast to endothelial cells, activation of VEGFR-2 in fibroblasts induces only a weak mitogenic response [97, 107], suggesting the existence of endothelial cell-specific signaling pathways. Four major autophosphorylation sites, Tyr951, 996, 1054, and 1059, have been identified in VEGFR-2 [108]. They are located in the kinase insert domain (Tyr951/996) and tyrosine kinase catalytic domain (Tyr1054/1059). In endothelial cells overexpressing VEGFR-2, activation of the receptor leads to a rapid recruitment of adaptor proteins Shc, Grb2, and Nck and protein tyrosine phosphatases SHP-1 and -2 [98]. The Shc homologue Sck has also been shown to interact with VEGFR-2 via its SH2 domain in the yeast twohybrid assay [109]. On the other hand, in VEGF-stimulated endothelial cells phosphorylation of Shc was barely detectable in spite of activation of the guanine nucleotide exchange factor Sos and stimulation of a mitogenic response [97]. Activation of the Raf-MEK-MAP kinase pathway in response to VEGF has been observed in many types of endothelial cells [97–99, 107, 110 –112]. Although many RTKs, such as receptors for fibroblast growth factors and platelet-derived growth factors, transmit their signals through activated Ras [113, 114], it may not be the case for VEGFR-2, since it was shown recently that in liver sinusoidal endothelial cells the ma-

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jor growth signal is transduced mostly through PLCgPKC and not Ras [112]. Phosphorylation and activation of PLCg in VEGF-treated endothelial cells have been reported by several groups [96, 107, 110, 115, 116]. PLCg activation results in hydrolysis of a cell membrane phospholipid, phosphatidyl inositol 4,5bisphosphate, to sn-1,2-diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3). IP3 is likely to mediate the release of Ca 21 from intracellular stores following VEGF stimulation [30, 117, 118], while DAG activates some PKC isoforms. VEGF has been shown to selectively activate the Ca 21-sensitive PKC isoforms g and b2 in bovine aortic endothelial cells, and the mitogenic effect of VEGF in endothelial cells can be suppressed by PKC-b and -g-selective inhibitors [112, 116]. Endothelial nitric oxide synthase becomes activated upon VEGF treatment; a subsequent increase in cGMP may contribute to activation of the MAPK cascade and proliferation, since these responses can be partially prevented in HUVE cells by treatment with nitric oxide synthase inhibitors [119]. Increased production of nitric oxide upon VEGF treatment is likely a result of both the rise in intracellular calcium levels which stimulates eNOS directly and the activation of serine kinase Akt/PKB (see below) which can phosphorylate and activate eNOS in a calcium-independent manner [120, 121]. Interestingly, VEGF-induced activation of MAPK and mitogenic response in brain capillary endothelial cells could be selectively inhibited by an antiangiogenic 16-kDa N-terminal fragment of prolactin [110, 122, 123]. VEGF induces tyrosine phosphorylation and activation of phosphoinositol-3 kinase (PI3-K) in endothelial cells [116]. PI3-K activation does not seem to be necessary for endothelial cell proliferation, but instead it is implicated in VEGF-induced endothelial cell survival via activation of its downstream target serine kinase Akt/PKB [95]. Akt becomes activated in HUVE cells stimulated with a VEGFR-2-selective VEGF mutant, whereas no activation was observed in cells stimulated with PlGF or a VEGFR-1-selective mutant. Recently it was shown that VEGFR-2 participates in a multimeric complex together with the adherens junction protein VE-cadherin, b-catenin, and PI3-K. Targeted inactivation or truncation of VE-cadherin (which suppresses the interaction with b-catenin) prevented activation of PI3-K and Akt in response to VEGF and abolished VEGF-induced cell survival demonstrating the crucial role of VE-cadherin and b-catenin in regulation of endothelial cell apoptosis [124]. The a vb 3 integrins are specifically expressed by the angiogenic endothelium and have been implicated in the regulation of endothelial cell adhesion, cell cycle, and survival [125–128]. VEGFR-2 tyrosine phosphorylation and VEGF-induced mitogenicity were enhanced in endothelial cells plated on the a vb 3 ligand vitronec-

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TABLE 1 List of Proteins Upregulated in Endothelial Cells upon Treatment with VEGF Name A1 (Bfl-1) [136] a1 and a2 integrins [135] Angiopoietin-2 [166] Bcl-2 [136] Endothelial nitric oxide synthase [167–169], inducible nitric oxide synthase [168] Ets-1 [170] Heparin-binding epidermal growth factor-like growth factor [171] Interstitial collagenase [132] PAI-1 [131] PDGF-BB [171] Tissue factor [25, 75, 134, 137, 138] VEGFR-2 [172] Urokinase-type plasminogen activator, tissue-type plasminogen activator [99, 131, 173], urokinase plasminogen activator receptor [173]

Function

Cell type

Receptor

Antiapoptotic Adhesion, collagen receptors Ligand for Tie-2 Antiapoptotic Production of nitric oxide

HUVEC a HDMEC b BMEC c HUVEC HUVEC, VEGFR-2 expressing PAEC d HUVEC, HMEC e (lung) HUVEC, HCAEC f

Not determined (n.d.) n.d. n.d. n.d. VEGFR-2

HUVEC HUVEC HUVEC, HCAEC HUVEC

n.d. n.d. n.d. VEGFR-2, VEGFR-1

ACE cells g HUVEC, VEGFR-2- and VEGFR-1-expressing PAEC

VEGFR-2 VEGFR-2, VEGFR-1

Transcription factor Growth factor Protease Protease inhibitor Growth factor Initiation of blood coagulation cascade Receptor for VEGF Protease, protease receptor

n.d. n.d.

a

HUVEC, human umbilical vein EC. HDMEC, human dermal microvascular EC. c BMEC, bovine microvascular EC. d PAEC, porcine aortic EC. e HMEC, human microvascular EC. f HCAEC, human carotid artery EC. g ACE cells, adrenal cortex capillary EC. b

tin, demonstrating that specific integrins can contribute to the full activation of VEGFR-2 triggered by VEGF [128]. VEGF induces actin stress fiber formation and migration of endothelial cells [111, 129]. Activation of the p38 stress kinase pathway in VEGF-treated HUVE cells has been implicated in these events [111]. Rapid phosphorylation of the p125 focal adhesion kinase and focal adhesion-associated protein paxillin was observed upon VEGF stimulation in HUVE cells [129], and related adhesion focal tyrosine kinase (RAFTK) becomes activated in the bone marrow endothelial cell line stimulated with VEGF [130], suggesting that these proteins may link the signaling from VEGF receptors to the cytoskeleton. Some VEGF target genes have been identified in endothelial cells (see Table 1). Increased production of proteases, tissue factor, and integrins [105, 131–135] is required for degradation of basement membrane and cell migration, whereas expression of another set of genes such as Bcl-2 and A1 can be responsible for enhanced endothelial cell survival in the presence of VEGF [136]. Tissue factor expression in HUVE cells in response to VEGF treatment is mediated by transcription factors Egr-1 and NFAT [137, 138]. Recently, all three VEGF receptors were shown to be strong activators of STAT3 and STAT5, while STAT1 was not activated by the VEGFRs [139]. STAT proteins were thus

identified as novel targets for the VEGFRs, suggesting that they may be involved in the regulation of endothelial function. VEGFR-3

In comparison to VEGFR-2, relatively little is known about the signal transduction pathways initiated from VEGFR-3. VEGF-C and VEGF-D, two recently isolated members of the VEGF family, bind VEGFR-3 and induce its phosphorylation [19, 22]. Long and short isoforms of VEGFR-3 differ in their signaling properties, only the long isoform being able to mediate cell growth in soft agar and tumorigenicity in nude mice [140]. The longer form also exhibited higher autophosphorylation levels [140], probably because of the presence of additional autophosphorylation sites in its C-terminus at Tyr1333, Tyr1337, and Tyr1363 [61]. Similar to VEGFR-2, activation of VEGFR-3 is rapidly followed by Shc tyrosine phosphorylation and increased cell proliferation [141–143]. Shc phosphorylation levels are higher in cells expressing the long isoform of VEGFR-3, and mutation of Tyr1377 to phenylalanine reduces Shc phosphorylation and prevents tumorigenic cell transformation by VEGFR-3. Shc appears to serve as a negative regulator of VEGFR-3 activity, because mutations of Shc phosphorylation sites lead to increased transforming activity of VEGFR-3 [144]. Both

VEGF RECEPTORS AND SIGNAL TRANSDUCTION

VEGFR-3 isoforms bind in a ligand-dependent manner the SH2 domains of Grb2 and PLCg but not that of PI3-K [140 –142]. VEGFR-3-tranfected endothelial cells respond to VEGF-C stimulation by increased cell motility, actin reorganization, and proliferation, thus suggesting that at least in these cells VEGFR-2 and VEGFR-3 activate similar or overlapping signaling pathways [143]. A human erythroleukemia cell line expressing VEGFR-3 at high levels responds to VEGF-C stimulation by recruitment of Shc, Grb2, and Sos to the activated receptor and increased cell growth [145]. In these cells VEGF-C also induced tyrosine phosphorylation of the cytoskeletal protein paxillin by RAFTK, a member of the focal adhesion kinase family. A mutation in VEGFR-3 has recently been linked to hereditary lymphedema, a developmental disorder of the lymphatic system, which leads to a disabling swelling of the extremities [146]. The mutation, which converts proline 1114 to leucine, occurs in the VEGFR-3 tyrosine kinase domain (see Fig. 1), indicating that a disturbance in VEGFR-3 signaling may play a part in the development of this disease. These data together with specific VEGFR-3 expression in lymphatic endothelium and some data from transgenic mice (see below) indicate that VEGFR-3 is specifically involved in development and maintenance of lymphatic vessels. Future challenges lie in deciphering pathways that are specific for VEGFR-3 signaling and in identifying the proteins whose expression is modulated by VEGF-C and VEGF-D. Roles of VEGFR-1–3 in Vascular Development The importance of VEGFRs for vascular development has been demonstrated using gene-targeting approaches. Disruption of any VEGFR leads to embryonic lethality. Targeted inactivation of VEGFR-2 blocks the early differentiation of both endothelial and primitive hematopoietic cells and prevents blood vessel formation [147], suggesting that VEGFR-2 can be necessary for generation of hemoangioblasts, hypothetical stem cells for both the hematolymphopoietic and endothelial lineages. Recently, VEGFR-2 expression has been shown in postnatal human hematopoietic stem cells (HSCs) but not in lineage-committed hematopoietic progenitor cells [79], thus designating VEGFR-2 as a positive functional marker for HSCs. The VEGFR-1-deficient mice show enhanced production of endothelial cells in both embryonic and extraembryonic locations, which fail to organize into functional vessels [148]. Vascular defects in VEGFR-1deficient animals were further explained by an increased number of endothelial progenitors due to an increased mesenchymal– hemangioblast transition [149].

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VEGFR-3-deficient embryos die as a result of a defect in the remodeling of the primary vascular network and cardiovascular failure at E9.5 [150]. Interestingly, differentiation of endothelial cells, formation of primary vascular networks, and vascular sprouting were not disturbed in VEGFR-3 knockout embryos. The phenotype of mice homozygous for a disrupted VEGFR-3 allele suggests that VEGFR-3 signaling plays a general role in the early cardiovascular development and only later, after generation of the lymphatic vascular system, does VEGFR-3 function become gradually restricted to the lymphatic endothelium. The importance of VEGFR-3 for lymphatic vessel development is illustrated by the results from transgenic mice expressing VEGF-C under the keratin 14 promoter, which directs transgene expression to the skin epidermis. These mice display a selective hyperplasia of the superficial lymphatic vasculature [151], whereas overexpression of VEGF 164 under the same promoter leads to increased blood vessel proliferation [152, 153]. NEUROPILIN-1

General Characteristics and Expression Pattern Neuropilin-1 is a 140-kDa type 1 membrane protein. Its extracellular component can be subdivided into three domains: the CUB domain homologous to complement components C1r/C1s, a domain similar to coagulation factors V and VII, and the MAM domain, displaying similarity to protease meprin and receptor tyrosine phosphatases m and k [154]. The short cytoplasmic C-terminal domain does not display significant homology to known proteins. Neuropilin-1 has been identified as a receptor expressed in the tips of actively growing axons of particular classes of neurons [155–157]. By binding to semaphorins/collapsins, NRP1 controls axon growth and guidance during embryonic development. In addition to neuronal cells, NRP1 expression is detected in the endothelial cells of capillaries and blood vessels and in mesenchymal cells surrounding blood vessels [158]. Although the expression of NRP1 in neural tissues diminishes after birth, expression persists in many adult nonneuronal tissues, being especially strong in the placenta and heart [26]. High NRP1 expression is detected also in some tumor cell lines that do not express VEGFR-1 and VEGFR-2. This suggests that NRP1 could directly mediate yet unidentified signals upon VEGF 165 binding to these cells [26]. The observations that homozygous NRP1 knockout embryos die of cardiovascular failure at E10.5–E12.5 and that ectopic overexpression of NRP1 leads to excess dilated blood vessels and hemorrhaging resulting in embryonic death gave the first suggestions of the role of NRP1 in the regulation of angiogenesis [158,

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159]. The direct evidence that NRP1 is a mediator of angiogenesis came when NRP1 was shown to act as an isoform-specific receptor for VEGF 165 [26]. Thereafter, NRP1 has been shown also to bind other members of the VEGF family, such as PlGF-2, VEGF-B, and NZ2VEGF-E [24, 33, 34]. Functions of Neuropilin-1 in Neuronal and Endothelial Cells In neuronal cells, collapsin-1 (Sema III) binding to NRP1 inhibits the motility of axons and results in growth cone collapse. In a similar fashion, via NRP1 interaction, collapsin-1 can inhibit endothelial cell motility and disorganize the cytoskeleton by preventing formation of lamellipodia and depolymerizing F-actin [160]. Furthermore, collapsin-1 inhibits endothelial cell sprouting and tube formation. It is however controversial if there is any intrinsic signaling function via NRP1 itself. Although the high conservation of the short cytoplasmic domain of NRP1 suggests the importance of this part for NRP1 function, no obvious protein homology domains or binding partners have been reported for this portion. In fact, the transmembrane and cytoplasmic portions of NRP1 are not required for Sema III-induced growth cone collapse, which suggest the role of NRP1 only as a coreceptor for some other signal-transducing receptor(s) [161–163]. The exact role of NRP1 binding in the biology of VEGFs is unknown, but coexpression of NRP1 with VEGFR-2 in transfected cells resulted in increased VEGF 165 binding and enhanced chemotaxis and mitogenicity in response to VEGF 165 [26]. VEGF 165 binding to NRP1 thus results in an opposite effect on cell motility when compared to collapsin-1 binding. Interestingly, recently it was reported that VEGFR-2 is expressed in some classes of neurons, for which VEGF acts a neurotrophic and mitogenic factor [164], therefore demonstrating an overlap in signaling systems used by neurons and endothelial cells. CONCLUDING REMARKS

In addition to the VEGF–VEGFR system, two other receptor tyrosine kinase systems that act in an endothelial cell-specific manner have been described, namely Tie-1, Tie-2/angiopoietins and EphB2-B4/ephrin-B1-B2 (reviewed in [165]). While the importance of all three systems for vascular development in vivo has been shown using gene-targeting approaches, the intracellular mechanisms and especially interactions between different signaling pathways in the regulation of endothelial cell functions are only beginning to be elucidated. Understanding how signaling pathways are integrated within the cell and identification of their endothelial cell-specific molecular targets are among

the challenges vascular biologists will face in the nearest future. The studies in the authors’ laboratory were supported by the Finnish Academy, the Sigrid Jiselius Foundation, the Finnish State Technology Development Centre, the Helsinki University Hospital Research Fund, the Novo Nordisk Foundation, and the Finnish Cancer Research Foundation. We thank Michael Jeltsch for help in the preparation of Fig. 2, Tanja Veikkola and Marika Karkkainen for critical reading of the manuscript, and Taina Partanen for sharing results prior to publication.

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