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

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1 Clinical Science (2005) 109, (Printed in Great Britain) doi: /cs R E V I E W The vascular endothelial growth factor (VEGF)/ VEGF receptor system and its role under physiological and pathological conditions Hiroyuki TAKAHASHI and Masabumi SHIBUYA Division of Genetics, Institute of Medical Science, University of Tokyo, Shirokane-dai, Minato-ku, Tokyo, , Japan, and Department of Internal Medicine, Graduate School of Medicine, University of Tokyo, Hongo, Bunkyo-ku, Tokyo, , Japan A B S T R A C T The VEGF (vascular endothelial growth factor) family and its receptors are essential regulators of 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-A has at least nine subtypes due to the alternative splicing of a single gene. Although the VEGF 165 isoform plays a central role in vascular development, recent studies have demonstrated that each VEGF isoform plays distinct roles in vascular patterning and arterial development. VEGF-A binds to and activates two tyrosine kinase receptors, VEGFR (VEGF receptor)-1 and VEGFR-2. VEGFR-2 mediates most of the endothelial growth and survival signals, but VEGFR-1-mediated signalling plays important roles in pathological conditions such as cancer, ischaemia and inflammation. In solid tumours, VEGF-A and its receptor are involved in carcinogenesis, invasion and distant metastasis as well as tumour angiogenesis. VEGF-A also has a neuroprotective effect on hypoxic motor neurons, and is a modifier of ALS (amyotrophic lateral sclerosis). Recent progress in the molecular and biological understanding of the VEGF/VEGFR system provides us with novel and promising therapeutic strategies and target proteins for overcoming a variety of diseases. INTRODUCTION Angiogenesis and vasculogenesis are regulated predominantly by several different growth factors and their associated RTKs (receptor tyrosine kinases). Foremost among these is the VEGF (vascular endothelial growth factor) 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), vascular permeability. 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 response element; 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, nitric oxide; NOS; NO synthase; enos, endothelial NOS; NRP, neuropilin; PAIP2, polyadenylated-binding protein-interacting protein 2; PDK1, phosphoinositide-dependent kinase 1; PI3K, phosphoinositide 3-kinase; PILSAP, puromycin-intensive leucyl-specific aminopeptidase; PKC, protein kinase C; PLC, phospholipase C; PlGF, placenta growth factor; pvhl, von Hippel Lindau tumour suppressor protein; RA, rheumatoid arthritis; RTK, receptor tyrosine kinase; S6K, S6 kinase; Tag, T antigen; T H 2, 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 ( shibuya@ims.u-tokyo.ac.jp).

2 228 H. Takahashi and M. Shibuya recognized as the major growth factor that is relatively specific for endothelial cells. VEGF-A is a dimeric glycoprotein essential for many angiogenic processes in normal and abnormal states, such as tumour vascularization, mainly by interacting with two tyrosine kinase receptors, VEGFR-1 [also known as Flt-1 (Fms-like tyrosine kinase-1)] and VEGFR-2 [also known as Flk-1 (fetal liver kinase-1) and, in humans, as KDR (kinase insert domaincontaining receptor)] [3 5]. VEGF-A exhibits two major biological activities: one is the capacity to stimulate vascular endothelial cell proliferation [1,6,7], and the other is the ability to increase vascular permeability [2,8]. VEGF-A also promotes the survival and migration of endothelial cells. In addition, recent studies have revealed a variety of biological functions and the precise molecular mechanisms of the VEGF/VEGFR system. In this review, we will discuss the recent advances in the basic biology of the VEGF/VEGFR system, which give insight into many physiological 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 molecular and biological functions of each ligand have been well characterized. VEGF-A Structurally, VEGF belongs to the VEGF/PDGF (platelet-derived growth factor) supergene family. Among the gene products of this family, eight cysteine residues are conserved at the same positions. These products function as a dimer, since two out of eight cysteine residues generate intermolecular cross-linked S S bonds [9]. The human 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 the alternative splicing of a single gene: VEGF 121,VEGF 145, VEGF 148, VEGF 162, VEGF 165, VEGF 165 b, VEGF 183, VEGF 189 and VEGF 206 [13,14] (Figure 1). VEGF 165 bisan endogenous inhibitory form of VEGF, which binds VEGFR-2 with the same affinity as VEGF 165, but does not activate it or stimulate downstream signalling pathways [15]. VEGF is produced in endothelial cells, macrophages, activated T-cells and a variety of other cell types [16 18]. Although virtually nothing is known about how VEGF isoform levels are regulated, most VEGFproducing cells appear to preferentially express VEGF 121, VEGF 165 and VEGF 189.VEGF 165, the predominant isoform, is secreted as an approx. 46 kda homodimer, which has 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, VEGF 121, which lacks the residues encoded by exons 6 and 7, does not bind heparin and is freely released from the cell. VEGF 189, which contains an additional sequence encoded by exon 6, binds heparin strongly and is completely sequestered in the ECM (extracellular matrix) and to a lesser extent at the cell surface [16]. The ECMbound isoforms can be released slowly by exposure to heparin and heparinases, or more rapidly released as bioactive fragments through cleavage by plasmin or urokinase at the C-terminus [19]. VEGF 165 binds the coreceptors NRP-1 (neuropilin-1) [20] and NRP-2 (neuropilin-2), whereas VEGF 145 binds only NRP-2 [21] (Figure 2). Approx. 50 % of mice expressing exclusively the VEGF 120 isoform (murine VEGF is shorter by one amino acid) die within a few hours after birth and the rest die within 14 days due to impaired myocardial angiogenesis and ischaemic cardiomyopathy [22]. VEGF 120/120 mice also exhibit a specific decrease in capillary branch formation and the impairment of the directed extension of endothelial cell filopodia during embryogenesis [23] as well as severe defects in retinal vascular outgrowth and patterning [24], suggesting that the heparin-binding VEGF isoforms provide spatially restricted stimulatory cues to initiate vascular branch formation. VEGF 164/164 mice are normal and healthy, and have a normal retinal angiogenesis, whereas VEGF 188/188 mice display normal venular outgrowth but impaired arterial development in retinas as well as dwarfism, disrupted development of growth plates and secondary ossification centres, and knee joint dysplasia [25]. These findings suggest that the various VEGF isoforms play distinct roles in vascular patterning and arterial development, although the VEGF 164 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-stimulating hormone), tumour promoters and NO (nitric oxide). Although all of the stimuli responsible for the upregulation of the VEGF gene are quite interesting, hypoxia has been of particular interest because of its importance and the unique transcriptional regulation involved. It is now well established that HIF-1 (hypoxiainducible factor-1) is a key mediator of hypoxic responses. HIF-1 is a transcriptional activator composed of HIF-1α and HIF-1β subunits. Both HIF-1α and HIF-1β are constitutively expressed in various types of tumour. Under normal oxygenation conditions, HIF- 1α is scarcely detectable because it is targeted for rapid destruction by an E3 ubiquitin ligase containing pvhl (von Hippel Lindau tumour suppressor protein). The interaction between pvhl and a specific domain of the HIF-1α subunit is regulated through hydroxylation of a proline residue (Pro 564 in HIF-1α) by prolyl- 4-hydroxylase, which requires molecular oxygen and

3 VEGF/VEGFR system in health and disease 229 Figure 1 Comparison of structures of the VEGF family Alternative 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 with VEGF 165 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 prolyl hydroxylation of HIF-1α and decreased ubiquitination and degradation [26,27]. Furthermore, hypoxia inhibits the oxygen-dependent hydroxylation of an asparagine residue (Asn 803 in HIF-1α) in the C-terminal transactivation domain of HIF-1α to promote interaction with the p300/cbp [CREB (camp-response-elementbinding protein)-binding protein] co-activator and induce a HRE (hypoxia response element)-driven transcription of the VEGF gene [28]. Very recently, Gerald et al. [29] have demonstrated that JunD, a member of the AP-1 family of transcription factors, is involved in the regulation of prolyl hydroxylase activity. Deletion of JunD increases H 2 O 2 levels, and thus inhibits prolyl hydroxylase enzymatic activity by limiting FeII levels. Consequently, HIF-1α protein accumulates under normoxic conditions, and the transcription of VEGF-A is increased [29]. VEGF is also regulated at the level of mrna stability. The 5 -and3 -UTRs (untranslated regions) of the VEGF gene 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 mrna stabilization [30,31]. Furthermore, VEGF expression can be regulated at the translational level. It has been shown that the 5 -UTR of VEGF mrna contains two functional internal ribosome entry sites that maintain efficient cap-independent translation and ensure efficient production of VEGF, even under unfavourable stress conditions such as hypoxia [32]. PlGF PlGF was originally discovered in human placenta in 1991 [33]. The PlGF gene is highly expressed in placenta at all stages of human gestation. PlGF transcripts have also

4 230 H. Takahashi and M. Shibuya Figure 2 Schematic diagram illustrating the receptor-binding specificity of VEGF family members and the VEGFR-2 signalling pathways The 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 the bottom. Tyr 1175 (Y1175) and Tyr 1214 (Y1214) are the two major autophosphorylation sites in VEGFR-2. PLC-γ binds to Y1175, leading to the phosphorylation and activation 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 through an unknown mechanism, including FAK, PI3K and Src. The activation of downstream signal transduction molecules leads to several different endothelial cell functions such as migration, vascular permeability, survival and proliferation. been detected in the heart, lung, thyroid gland and skeletal muscle [34]. PlGF binds VEGFR-1, but not VEGFR-2 [35,36]. Alternative splicing of the human PlGF gene generates four isoforms which differ in size and binding properties: PlGF-1 (PlGF 131 ), PlGF-2 (PlGF 152 ), PlGF-3 (PlGF 203 ) and PlGF-4 (PlGF 224 ) [37 39] (Figure 1). PlGF-1 is the shortest isoform and a non-heparin binding protein. PlGF-2 is able to bind heparin and the coreceptors NRP-1 and NRP-2 due to the insertion of a highly basic 21-amino acid sequence encoded by exon VI near the C-terminus [37] (Figure 2). PlGF-3, which contains an insertion of 216 nucleotides coding for a 72- amino acid sequence between exons 4 and 5 of the PlGF gene but lacks the coding sequence of exon 6, is unable to bind heparin [38]. PlGF-4 consists of the same sequence of PlGF-3, plus a heparin-binding domain previously thought to be present only in PlGF-2 [39]. The crystal structure of human PlGF-1 has shown that this protein is structurally similar to VEGF-A [40]. Furthermore, despite this moderate sequence conservation, PlGF and VEGF-A bind to the same binding interface of VEGFR-1 in a very similar fashion [41]. However, recent studies have reported that, unlike in VEGF-A, N-glycosylation in PlGF plays an important role in VEGFR-1 binding [42]. Carmeliet et al. [43] have shown that a deficiency in PlGF (PlGF / ) does not affect embryonic angiogenesis in mice. However, loss of PlGF impairs angiogenesis, plasma extravasation and collateral growth during ischaemia, inflammation, wound healing and cancer,

5 VEGF/VEGFR system in health and disease 231 indicating the importance of VEGFR-1 signalling in pathological conditions. VEGF-B VEGF-B has a wide tissue distribution, but is particularly abundant in the heart and skeletal muscle [44]. Human VEGF-B has two isoforms generated by alternative splicing: VEGF-B 167 and VEGF-B 186 (Figure 1). The VEGF-B isoforms bind and activate VEGFR-1 and can also bind to NRP-1 [44] (Figure 2). Studies using VEGF-B knockout (VEGF-B / ) mice have yielded slightly conflicting results regarding the role of VEGF-B in angiogenesis and the development of the cardiovascular system. VEGF-B / mice are viable and fertile; however, although Bellomo et al. [45] demonstrated that VEGF-B / mice had smaller hearts, dysfunctional coronary arteries and an impaired recovery from experimentally induced myocardial ischaemia, Aase et al. [46] claimed that these mice showed a subtle cardiac phenotype such as an atrial conduction abnormality characterized by a prolonged PQ interval, and that VEGF-B was not required for proper development of the cardiovascular system either during development or angiogenesis in adults. Recent studies using VEGF- B / mice have demonstrated the role of VEGF-B in pathological vascular remodelling in inflammatory arthritis [47] and protection of the brain from ischaemic injury [48]. VEGF-C and VEGF-D VEGF-C contains a region sharing approx. 30 % amino acid identity with VEGF 165 ; however, it is more closely related to VEGF-D by virtue of the presence of N- and C-terminal extensions that are not found in other VEGF family members [49] (Figure 1). Both VEGF-C and VEGF-D bind and activate VEGFR-3 (Flt-4; a member of the VEGFR family that does not bind VEGF-A) as well as VEGFR-2, and are mitogenic for cultured endothelial cells. VEGF-C also binds to NRP-2 [49] (Figure 2). Both VEGF-C and VEGF-D are produced as a preproprotein with long N- and C-terminal propeptides flanking the VEGF homology domain. Initial proteolytic cleavage of the precursor generates a form with a moderate affinity for VEGFR-3, but a second proteolytic step is required to produce the fully processed form with a high affinity for both VEGFR-2 and VEGFR-3 [49]. This activation of VEGF-C and VEGF-D by proteolytic cleavage is at least partly regulated by the serine protease plasmin [50]. Overexpression of VEGF-C in the epidermis of transgenic mice results in the development of a hyperplastic lymphatic vessel network [51]. In vitro, VEGF- C and VEGF-D stimulate the migration and mitogenesis of cultured endothelial cells [49]. A recent study using VEGF-C / mice has demonstrated that VEGF-C is required for the initial steps in lymphatic development and that both VEGF-C alleles are required for normal lymphatic development [52]. Thus VEGF-C is the paracrine factor essential for lymphangiogenesis. Less is known of the function of VEGF-D, but Stacker et al. [53] have revealed that VEGF-D induces the formation of lymphatics within tumours and promotes the metastasis of tumour cells. VEGF-E Homologues of VEGF have also been identified in the genome of the parapoxvirus Orf virus [54] and have been shown to have VEGF-A-like activities. VEGF-E is the collective term for a group of these proteins, including VEGF-E NZ-2 (VEGF from Orf virus strain NZ-2) [55], VEGF-E NZ-7 (VEGF from Orf virus strain NZ-7) [56], VEGF-E NZ-10 (VEGF from Orf virus strain NZ- 10) [57], VEGF-E D1701 (VEGF from Orf virus strain D1701) [58] and VEGF-E VR634 (VEGF from Pseudocowpox virus strain VR634) [57]. All VEGF-E variants studied bind and activate VEGFR-2, but not VEGFR-1 or VEGFR-3. VEGF-E NZ-2, VEGF-E NZ-10 and VEGF- E D1701 can bind NRP-1. VEGF-E NZ-7 and VEGF-E VR634, however, are unable to bind NRP-1 (Figure 2). VEGF-E seems to be as potent as VEGF 165 at stimulating endothelial cell proliferation despite lacking a heparin-binding basic domain. K14-driven VEGF-E NZ-7 transgenic mice have shown a significant increase in angiogenesis at subcutaneous tissue without clear side effects [59]. svvegf Recently, VEGF family proteins have been identified in snake venom, including svvegf from Bothrops insularis [60] and Tf svvegf (Trimeresurus flavoviridis svvegf) [61] from pit vipers in addition to HF (hypotensive factor) [62], ICPP (increasing capillary permeability protein) [63] and vammin [64] from vipers. Takahashi et al. [61] have shown that snakes utilize these venomspecific VEGFs in addition to VEGF-A. svvegfs function as dimers and each chain comprises approx amino acid residues. The cysteine knot motif, a characteristic of the VEGF family of proteins, is completely conserved in svvegfs and the sequence identity with human VEGF 165 is approx. 50 % (Figure 1). Vammin does not bind VEGFR-1 but binds VEGFR-2 with high affinity as well as VEGF 165 [64]. However, Tf svvegf binds VEGFR-1 with high affinity and VEGFR-2 with low affinity compared with VEGF 165, leading to a strong enhancement of vascular permeability but weak stimulation of endothelial cell proliferation [61] (Figure 2). Both vammin and Tf svvegf are unable to bind VEGFR-3 or NRP-1, but Tf svvegf binds heparin. svvegfs may contribute to the enhancement of toxicity in envenomation, but they seem to have individual biological characteristics reflecting divergence in the classification of the host snake.

6 232 H. Takahashi and M. Shibuya VEGFRs VEGFR-1 VEGFR-1 is a 180 kda high-affinity receptor for VEGF- A, VEGF-B, PlGF and Tf svvegf. It is expressed in vascular endothelial cells and a range of non-endothelial cells, including macrophages and monocytes [65], and haematopoietic stem cells [66]. The second Ig domain of VEGFR-1 is the major binding site for VEGF-A and PlGF [16,41,67]. VEGFR-1 binds VEGF-A with at least 10-fold higher affinity than VEGFR-2 (K d = pm) [16]; however, ligand binding results in a maximal 2-fold increase in kinase activity. In many cases, the effects of VEGFR-2 on endothelial cells, such as those on cell survival and proliferation, can be induced only weakly or slightly by treatment with VEGFR-1-specific ligands. VEGFR-1 is a negative regulator of angiogenesis during early development, but plays an important role in angiogenesis under pathological conditions (as described below). VEGFR-1-blocking antibodies prevent the migration but not proliferation of HUVECs (human umbilical vein endothelial cells) in response to VEGF-A, indicating the involvement of VEGFR-1 in endothelial cell migration [68]. VEGFR-1-mediated signalling appears to preferentially modulate the reorganization of actin via p38 MAPK (mitogen-activated protein kinase), whereas VEGFR-2 contributes to the re-organization of the cytoskeleton by phosphorylating FAK (focal adhesion kinase) and paxillin (Figure 2), suggesting a different contribution of the two receptors to the chemotactic response. VEGFR-1 signalling is also involved in the migration of monocytes/macrophages [65] and in the reconstitution of haematopoiesis by recruiting haematopoietic stem cells [66]. An alternatively spliced form of VEGFR-1 that encodes a soluble truncated form of the receptor, containing only the first six Ig domains, has been cloned from a HUVEC cdna library [16]. svegfr-1 (soluble VEGFR-1) inhibits VEGF-A activity by sequestering VEGF-A from signalling receptors and by forming non-signalling heterodimers with VEGFR-2 [69]. Plasma levels of svegfr-1 are elevated in individuals with cancer, ischaemia and pre-eclampsia [70 72]. A recent study has demonstrated that elevated levels of svegfr-1 play an important role in pre-eclampsia [73]. Increased circulating levels of svegfr-1 in patients with preeclampsia are associated with decreased circulating levels of free VEGF and PlGF, resulting in general endothelial dysfunction [73]. VEGFR-2 VEGFR-2 is a kda high-affinity receptor for VEGF-A (K d = pm), VEGF-E and svvegfs as well 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 expressed in vascular and lymphatic endothelial cells, and other cell types such as megakaryocytes and haematopoietic stem cells [75]. Tyrosine phosphorylation sites in human VEGFR-2 bound to VEGF-A are Tyr 951 and Tyr 996 in the kinase-insert domain, Tyr 1054 and Tyr 1059 in the kinase domain, and Tyr 1175 and Tyr 1214 in the C-terminal tail. Among them, Tyr 1175 and Tyr 1214 are the two major VEGF-A-dependent autophosphorylation sites [76]. Tyr 951 creates a binding site for the VEGFRassociated protein [77] and Tyr 1175 creates a binding site for 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 that the activation of VEGFR-2 also promotes lymphangiogenesis [80,81]. Survival signalling for endothelial cells from VEGFR-2 is reported to involve the PI3K (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 usually not very strong. Byzova et al. [84] have reported that the activation of VEGFR-2 by VEGF-A results in the PI3K/Akt-dependent activation of several integrins, leading to enhanced cell adhesion and migration. This synergic interaction with integrins is required for productive signalling from VEGFR-2. Very recently, a naturally occurring soluble truncated form of VEGFR-2 has been detected in mouse and human plasma [85]. Similar to svegfr-1, svegfr-2 (soluble VEGFR-2) may have regulatory consequences with respect to VEGF-mediated angiogenesis. VEGFR-3 VEGFR-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 fifth extracellular Ig loop into a 120 kda and a 75 kda form during synthesis, and the two forms are linked by a disulphide bridge [49]. Overexpression of a soluble VEGFR-3 in the skin of mice inhibits fetal lymphangiogenesis and induces a regression of already formed lymphatic vessels [86]. Furthermore, overexpression of a VEGFR-3-specific mutant of VEGF-C (VEGF-C 156S) in the skin induces the growth of lymphatic vessels without an influence on the blood vessel architecture [87], indicating that stimulation of VEGFR-3 alone is sufficient to induce lymphangiogenesis. The stimulation of VEGFR-3 also protects the lymphatic endothelial cells from serum deprivation-induced apoptosis. The phosphorylation of VEGFR-3 has been shown to lead to a PI3K-dependent activation of Akt and PKC (protein kinase C)-dependent activation of p42/p44 MAPK [88]. A recent study [89] has demonstrated that blockade of VEGFR-3 signalling significantly suppresses corneal

7 VEGF/VEGFR system in health and disease 233 dendritic cell trafficking to draining lymph nodes as well as the induction of delayed-type hypersensitivity and rejection of corneal transplants, suggesting a role for VEGFR-3 in adaptive immunity. NRP-1 and NRP-2 NRP-1 is a kda cell-surface glycoprotein first identified as a semaphorin receptor involved in neuronal guidance [90] and subsequently found as an isoformspecific receptor for VEGF-A [20]. NRP-2 was identified by virtue of its sequence homology with NRP-1 and shares 44 % identity at the amino acid level with NRP-1 [90]. NRP-1 is able to bind VEGF 165, VEGF-B, PlGF-2 and some VEGF-E variants, whereas NRP-2 can bind VEGF 145,VEGF 165, PlGF-2 and VEGF-C. The intracellular domains of NRPs are short and do not suffice for the independent transduction of biological signals subsequent to semaphorin or VEGF binding. It has been shown that both NRPs can join with receptors belonging to the plexin family, and such plexin/nrp complexes are able to transduce signals as the physiological receptor of class-3 semaphorins [91,92]. The VEGF 165 -induced proliferation and migration of cells that express VEGFR-2 are enhanced in the presence of NRP-1. Thus NRP-1 also seems to function as an enhancer of VEGFR-2 activity in the presence of VEGF 165. Recent studies have demonstrated that this effect is the result of the formation of a complex between VEGFR-2 and NRP-1 [93,94]. An in vivo study with transgenic mice has shown that NRP-1 is important not only for neuronal development, but also for vascular formation [95]. NRP-1 / mice suffer from severe defects in the cardiovascular system in addition to a disorganized neural development, resulting in the death of homozygous embryos by embryonic day 14 [96]. Defects in vessel formation include a failure of capillary ingrowth into the brain and the abnormal formation of aortic arches and the yolk-sac vasculature, suggesting the importance of NRP-1 in embryonic vessel formation. In contrast, NRP-2 / mice show an absence or severe reduction of small lymphatic vessels and capillaries during development [97]. Arteries, veins and larger collecting lymphatic vessels develop normally, suggesting that NRP-2 is selectively required for the formation of small lymphatic vessels and capillaries. VEGF/VEGFR SYSTEM IN PHYSIOLOGICAL AND PATHOLOGICAL CONDITIONS Physiological angiogenesis The loss of a single VEGF allele is lethal in the mouse embryo between days 11 and 12 [98,99]. VEGF +/ embryos exhibit significant defects in the vasculature of several organs and a markedly reduced number of nucleated red 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 abnormalities in heart development and lethality at embryonic days 12.5 and 14 [100]. These results demonstrate the importance of tightly regulating VEGF-A expression during embryonic development. Homozygous loss of the VEGFR-1 or VEGFR-2 gene results in embryonic lethality between days 8.5 and 9.5, indicating that these VEGFRs play important roles in vasculogenesis and angiogenesis [101,102]. VEGFR-2 / mice die due to a lack of endothelial cell growth and blood vessel formation as well as extremely poor haematopoiesis. On the other hand, VEGFR-1 / mice die due to an overgrowth of endothelial cells and disorganization of blood vessels. Furthermore, normal vascular development in mice lacking the tyrosine kinase domain of VEGFR-1 [103] has indicated that VEGFR-2 is the major positive signal transducer, whereas VEGFR-1 has a negative regulatory role in angiogenesis early in embryogenesis. Takahashi et al. [76] have shown that Tyr 1175 and Tyr 1214 are two major VEGF-A-dependent autophosphorylation sites in VEGFR-2. However, only autophosphorylation of Tyr 1175 is crucial for VEGF-dependent endothelial cell proliferation via the PLC-γ /PKC/Raf/MEK [MAPK/ ERK (extracellular-signal-regulated kinase) kinase]/erk pathway. An unusual feature of mitogenic signalling from VEGFR-2 is the requirement for PKC but not Ras [104]. Our recent study [105] using knockin mice substituting Tyr 1173 (corresponding to Tyr 1175 in human VEGFR-2) and Tyr 1212 (Tyr 1214 in human) of the VEGFR-2 gene with phenylalanine has revealed that the signalling via Tyr 1173 of VEGFR-2 is essential for endothelial and haematopoietic development during embryogenesis. In contrast, the phosphorylation of Tyr 1214 appears to be required to trigger the sequential activation of Cdc42 and p38 MAPK and to drive p38 MAPK-mediated actin remodelling in stress fibres in endothelial cells exposed to VEGF-A [106]. The activation of the PI3K/p70 S6K (S6 kinase) pathway by VEGFR-2 is also involved in VEGF-A-induced endothelial cell proliferation [107] (Figure 2). PILSAP (puromycin-intensive leucyl-specific aminopeptidase) plays a crucial role in the activation of this pathway via the binding and modification of PDK1 (phosphoinositide-dependent kinase 1) [108]. In addition, recent studies have revealed various downstream mediators of VEGF-induced angiogenic signalling, such as diacylglycerol kinase α [109], SRF (serum response factor) [110], SREBP (sterol-regulatory-element-binding protein) [111] and IQGAP1 [112]. Studies using DNA microarrays have reported possible endogenous feedback inhibitors for VEGF-induced angiogenesis. Vasohibin and DSCR1 (Down syndrome critical region protein 1) are significantly induced by VEGF in endothelial cells [113,114]. Up-regulation of DSCR1 in endothelial cells inhibits the nuclear localization of NFAT (nuclear factor of activated T-cells), proliferation and tube formation [115].

8 234 H. Takahashi and M. Shibuya Vascular permeability VEGF-A is known to increase the vascular permeability of microvessels to circulating macromolecules [14]. Increased vascular permeability is often observed in areas of pathological angiogenesis in solid tumours, wounds and chronic inflammation. VEGF-A significantly accumulates in malignant ascites [116] and pleural effusion [117], suggesting that it plays a fundamental role in the accumulation of malignant fluid through the enhancement of vascular permeability. Consistent with a role in the regulation of vascular permeability, VEGF-A induces endothelial fenestration in some vascular beds and in cultured adrenal endothelial cells, the extravasation of ferritin by way of the VVO (vesiculo-vacuolar organelle) [14], and disorganization of endothelial junctional proteins such as VE-cadherin and occludin [118]. VEGF-A increases vascular permeability in mesenteric microvessels by activation of VEGFR-2 on endothelial cells and subsequent activation of PLC. This causes increased production of diacylglycerol that results in influx of calcium [14]. Other studies have also demonstrated the crucial role of VEGFR-2 signalling in the enhancement of vascular permeability; however, our recent study [61] using Tf svvegf has shown that the enhancement of vascular permeability is intensified by the activation of VEGFR-1 more than the proliferation of endothelial cells under some active signalling from VEGFR-2. This finding indicates the importance of VEGFR-1 signalling in vascular permeability. An analysis of mice deficient in specific Src family kinases has demonstrated no decrease in VEGF-dependent neovascularization, but a complete ablation of vascular permeability in Src / or Yes / mice, whereas Fyn / mice show no such defect [119]. In addition, blockade of Src prevents the disassociation of a complex comprising VEGFR-2, VE-cadherin and β-catenin with the same kinetics with which it prevents VEGF-mediated vascular permeability and oedema [120]. These findings indicate that the activity of specific Src family kinases is essential for the VEGF-induced enhancement of vascular permeability through the disruption of the VEGFR-2/ cadherin/catenin complex. VEGF-A can induce production of NO and endogenous NO can increase vascular permeability [121]. Among the three isoforms of NOS (NO synthase), enos (endothelial NOS) plays a predominant role in VEGF-induced angiogenesis and vascular permeability [122]. Furthermore, the activation of enos is regulated by the PI3K/Akt pathway [123,124]. The small GTPbinding protein Rac, which is also activated by PI3K, has been implicated in the regulation of vascular permeability [125]. A recent study [126] has shown that inhibition of p38 MAPK activity abrogated VEGF-induced vascular permeability in vivo and in vitro, suggesting the involvement of p38 MAPK in the control of vascular permeability (Figure 2). Solid tumours Numerous studies have established VEGF-A as a key angiogenic player in cancer. VEGF-A is expressed in most tumours and its expression correlates with tumour progression. In addition to tumour cells, tumour-associated stroma is also an important source of VEGF-A [127]. In the absence of access to an adequate vasculature, tumour cells become necrotic and apoptotic, restraining the increase in tumour volume that should result from continuous cell proliferation [128]. The expression of VEGF-A mrna is highest in hypoxic tumour cells adjacent to necrotic areas [16], indicating that the induction of VEGF-A by hypoxia in growing tumours can change the balance of inhibitors and activators of angiogenesis, leading to the growth of new blood vessels into tumour. Consistent with this hypothesis, capturing of VEGF or blocking of its signalling receptor VEGFR-2 by a VEGFR tyrosine kinase inhibitor, antisense oligonucleotides, vaccination or neutralizing antibodies reduced tumour angiogenesis and growth in preclinical studies [129]. Unlike in physiological angiogenesis, VEGFR-1 signalling plays an important role in angiogenesis under pathological conditions [43,130]. Autiero et al. [131] have proposed that PlGF regulates inter- and intra-molecular cross-talk between VEGFR-1 and VEGFR-2, amplifying VEGF-driven angiogenesis through VEGFR-2. Several studies also describe the role of VEGF in carcinogenesis [132]. Rip1 Tag2 (T-antigen 2) mice develop islet tumours of the pancreas by weeks of age as a result of expression of the SV40 Tag oncogene in insulin-producing β-cells. In this mouse, angiogenic activity first appears in a subset of hyperplastic islets before the onset of tumour formation. VEGF-A and VEGFRs are constitutively expressed in the islet vasculature before and after the initiation of angiogenesis (angiogenic switch) [133]; however, when VEGF-A is absent from islet β-cells of Rip1 Tag2 mice, both angiogenic switching and carcinogenesis as well as tumour growth are severely disrupted [134], indicating that VEGF-A plays a critical role in angiogenic switching and carcinogenesis. Bergers et al. [135] have revealed that MMP (matrix metalloproteinase)-9 is also a component of the angiogenic switch, as this proteinase makes VEGF-A available for the interaction with its receptors by releasing sequestered VEGF-A. VEGF-A impairs the endothelial barrier by disrupting a VE-cadherin/β-catenin complex via the activation of Src and facilitates tumour cell extravasation and metastasis [136]. VEGF-A also induces the disruption of hepatocellular tight junctions, which may promote tumour invasion [137]. Pharmacological blockade of VEGFR-2 stabilizes the endothelial barrier function and suppresses tumour cell extravasation in vivo [136], suggesting the importance of VEGFR-2 signalling in this kind of tumour invasion and metastasis. Hiratsuka et al.

9 VEGF/VEGFR system in health and disease 235 [138] have shown that VEGFR-1 signalling is also involved in tumour metastasis, being linked to the induction of MMP-9 in lung endothelial cells and to the facilitation of lung-specific metastasis. Recently, Hurwitz et al. [139] have shown that the addition of bevacizumab (a humanized anti-vegf monoclonal antibody) to fluorouracil-based combination chemotherapy results in statistically significant and clinically meaningful improvement in survival among patients with metastatic colorectal cancer. Based on this result, bevacizumab (Avastin) was approved by the FDA (Food and Drug Administration) in February 2004 as a first-line treatment for metastatic colorectal carcinoma. Besides bevacizumab, many other VEGF inhibitors are being pursued clinically. These inhibitors include small-molecule RTK inhibitors such as PTK787, soluble receptors such as VEGF-Trap and anti-vegfr-2 mabs (monoclonal antibodies) [129]. Inflammatory diseases VEGF acts as a pro-inflammatory cytokine by increasing the permeability of endothelial cells, inducing the expression of endothelial adhesion molecules and via its ability to act as a monocyte chemoattractant [ ]. VEGF is strongly expressed by epidermal keratinocytes in wound healing and psoriasis, conditions that are characterized by increased microvascular permeability and angiogenesis [16]. Transgenic mice that overexpress VEGF-A specifically in the epidermis exhibit an increased density of tortuous cutaneous blood capillaries as well as highly increased leucocyte rolling and adhesion in postcapillary skin venules, suggesting that enhanced expression of VEGF-A in epidermal keratinocytes is sufficient to develop psoriasis-like inflammatory skin lesions [143]. Moreover, heterozygous VEGF-A transgenic mice, which do not spontaneously develop inflammatory skin lesions, are unable to down-regulate experimentally induced inflammation and exhibit a psoriasis-like phenotype characterized by epidermal hyperplasia, the accumulation of lymphocytes, and lymphatic vessel proliferation and enlargement [144]. Transgenic overexpression of PlGF-2 in epidermal keratinocytes also results in a significantly increased inflammatory response, whereas a deficiency of PlGF results in a diminished and abbreviated inflammatory response [145], suggesting the importance of VEGFR-1 signalling in chronic skin inflammation. Local production of VEGF-A in arthritic synovial tissue has been documented [16] and appears to correlate with disease activity in humans. Subsequently, VEGF-A has been shown to be important in the pathogenesis of RA (rheumatoid arthritis) in animal models [ ]. Treatment with anti-vegfr-1 mabs, but not anti-vegfr-2 mabs, significantly reduces the arthritic destruction of joints by suppressing synovial inflammation and neovascularization, emphasizing the importance of VEGFR-1 signalling in the destruction. The anti-inflammatory effects of anti-vegfr-1 are attributable to a reduced mobilization of bone-marrowderived myeloid progenitors into peripheral blood [147]. The reduction of synovial inflammation in VEGF-B / mice [47] also implies a critical role for VEGFR-1 signalling in RA. Exaggerated levels of VEGF-A have been detected in tissues and biological samples from people with asthma, where these levels correlate directly with disease [149] and inversely with airway function [150]. VEGF has been postulated to contribute to asthmatic tissue oedema through its effect on vascular permeability. A recent study using lung-targeted VEGF 165 transgenic mice has revealed a novel function of VEGF-A in allergic responses. In these mice, VEGF-A induces asthmalike inflammation, airway and vascular remodelling, and airway hyper-responsiveness. VEGF-A also enhances respiratory sensitization to antigen as well as T H 2 (T-helper type 2) cell-mediated inflammation and increases the number of activated dendritic cells [151]. Thus VEGF-A has a critical role in pulmonary T H 2 inflammation. Other studies have provided evidence for a role for VEGF-A as a pro-inflammatory mediator in allograft rejection [152] and neointimal formation [153]. Other pathological conditions VEGF-A mrna expression, not normally found in the adult mouse brain, is up-regulated after cerebral ischaemia, and elevated VEGF-A levels can be detected as early as 3 h after stroke with a peak between 12 and 48 h [154]. Previous studies have demonstrated that the antagonism of VEGF-A results in reduced oedema and tissue damage after ischaemia implicating VEGF-A in the pathophysiology of stroke [155]. Paul et al. [156] have reported that Src / mice are resistant to VEGF- A-induced vascular permeability and show decreased infarct volumes after stroke. Systemic application of a Src inhibitor suppresses vascular permeability, protecting wild-type mice from ischaemia-induced brain damage without influencing VEGF-A expression. However, Sun et al. [157] have reported that intracerebroventricular administration of VEGF-A reduces infarct size, improves neurological performance and enhances the delayed survival of newborn neurons. These conflicting results appear to reflect dual roles of VEGF-A in stroke: neuroprotective and pro-inflammatory effects. In this context, when infused through the internal carotid artery, low and intermediate doses of VEGF-A significantly promote neuroprotection of the ischaemic brain, whereas a high dose of VEGF-A offers no neuroprotection to the ischaemic brain or the damaged neurons of normal brain [158]. Further studies are required for the therapeutic application of VEGF-A against stroke. Extensive evidence has suggested a causal role of VEGF in several diseases of the human eye in which

10 236 H. Takahashi and M. Shibuya neovascularization and increased vascular permeability occur. VEGF levels are increased in the vitreous and retina of patients and laboratory animals with active neovascularization from ischaemic retinopathies such as proliferative diabetic retinopathy, central retinal vein occlusion and retinopathy of prematurity. Subsequent studies using various VEGF inhibitors have confirmed that VEGF plays a central role in ischaemia-induced intraocular neovascularization [159]. An anti-vegf aptamer, pegaptanib (Macugen), has produced a statistically significant and clinically meaningful benefit in the treatment of neovascular AMD (age-related macular degeneration) [160], which is the leading cause of irreversible severe loss of vision in people 50 years of age and older in the developed world, and was approved by the FDA in December Oosthuyse et al. [161] have reported that deletion of the HRE in the VEGF promoter reduces hypoxic VEGF expression in the spinal cord and causes adult-onset progressive motor neuron degeneration, reminiscent of ALS (amyotrophic lateral sclerosis). VEGF 165 promotes survival of motor neurons during hypoxia through binding VEGFR-2 and NRP-1 [161]. A subsequent study has revealed that VEGF-A is a modifier associated with motor neuron degeneration in human ALS and in a mouse model of ALS [162]. VEGF-A treatment increases the life expectancy of ALS mice without causing toxic side effects [163,164], indicating that VEGF-A has neuroprotective effects on motor neurons, and treatment with VEGF-A could be one of the most effective therapies for ALS reportedsofar. LeCouter et al. [165] recently provided evidence for a novel function of VEGFR-1 in LSECs (liver sinusoidal endothelial cells). The activation of VEGFR-1 results in the paracrine release of HGF (hepatocyte growth factor), IL-6 (interleukin-6) and other hepatotrophic molecules by LSECs to the extent that hepatocytes are stimulated to proliferate when co-cultured with LSECs. VEGF-A has no direct mitogenic effect on hepatocytes. A VEGFR-1 agonist protected the liver from CCl 4 -induced damage, in spite of its inability to induce the proliferation of LSECs. CONCLUSION VEGF was originally described as a specific angiogenic and permeability-inducing factor and its function was considered to be specific for endothelial cells. However, emerging evidence has revealed that the role of the VEGF/VEGFR system extends far beyond previous expectations. First, a wide variety of VEGF family proteins and numerous splicing variants have been identified and found to play distinct but critical roles in various conditions, including lymphangiogenesis. VEGF family proteins have been utilized even in snake venoms and some viruses. Secondly, several different VEGFRs have been shown to be essential, but the interaction between these receptors has appeared to be complicated. VEGFR-1 has a negative regulatory role in embryonic angiogenesis, but functions as a positive signal transducer in some cases individually and sometimes synergistically with VEGFR-2 via the intra- and inter-molecular crosstalk between these two receptors. An association between VEGFR-2 and VEGFR-3 has also been reported [166]. Thirdly, it has been shown that the VEGF/VEGFR system has multiple functions, such as the induction of tumour metastasis, inflammation, neuroprotection, protection of liver and mobilization of marrow-derived stem cells, as well as lymphangiogenesis. VEGF is also important for memory and learning [167]. Fourthly, numerous other molecules have been found to associate with the VEGF/VEGFR system. 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