Vascular endothelial growth factor 165 (VEGF(165)) activities are inhibited by carboxymethyl benzylamide dextran that competes for heparin binding to VEGF(165) and VEGF(165).KDR Complexes

We have previously shown that carboxymethyl dextran benzylamide (CMDB7), a heparin-like molecule, inhibits the growth of tumors xenografted in nude mice, angiogenesis, and metastasis by altering the binding of angiogenic growth factors, including platelet-derived growth factor, transforming growth factor beta, and fibroblast growth factor 2, to their specific receptors. In this study, we explore the effect of CMDB7 on the most specific angiogenic growth factor, vascular endothelial growth factor 165 (VEGF(165)). We demonstrate here that CMDB7 inhibits the mitogenic effect of VEGF(165) on human umbilical vein endothelial cells (HUV-ECs) by preventing the VEGF(165)-induced VEGF receptor-2 (KDR) autophosphorylation and consequently a specific intracellular signaling. In competition experiments, the binding of (125)I-VEGF(165) to HUV-ECs is inhibited by CMDB7 with an IC(50) of 2 microm. Accordingly, CMDB7 inhibits the cross-linking of (125)I-VEGF(165) to the surface of HUV-ECs, causing the disappearance of both labeled complexes, 170-180 and 240-250 kDa. We show that CMDB7 increases the electrophoretic mobility of VEGF(165), thus evidencing formation of a stable complex with this factor. Moreover, CMDB7 reduces the (125)I-VEGF(165) binding to coated heparin-albumin and prevents a heparin-induced increase in iodinated VEGF(165) binding to soluble (125)I-KDR-Fc chimera. Concerning KDR, CMDB7 has no effect on (125)I-KDR-Fc electrophoretic migration and does not affect labeled KDR-Fc binding to coated heparin-albumin. In the presence of VEGF(165), (125)I-KDR-Fc binding to heparin is enhanced, and under these conditions, CMDB7 interferes with KDR binding. These data indicate that CMDB7 effectively inhibits the VEGF(165) activities by interfering with heparin binding to VEGF(165) and VEGF(165).KDR complexes but not by direct interactions with KDR.     

Site-directed mutagenesis in the B-neuropilin-2 domain selectively enhances its affinity to VEGF165, but not to semaphorin 3F

Neuropilins (NRPs) are 130-kDa receptors that bind and respond to the class 3 semaphorin family of axon guidance molecules (SEMAs) and to members of the vascular endothelial growth factor (VEGF) family of angiogenic factors. Two NRPs have been reported so far, NRP1 and NRP2. Unlike NRP1, little is known about NRP2 interactions with its ligands, VEGF165 and SEMA3F. Cell binding studies reveal that VEGF165 and SEMA3F bind NRP2 with similar affinities, 5.2 and 3.9 nM, respectively, and are competitive NRP2 ligands. Immunoprecipitation studies show that the B (b1b2) extracellular domain of NRP2 is sufficient for VEGF165 binding, whereas SEMA3F requires both the A (a1a2) and B domains. To identify residues of B-NRP2 involved in VEGF165 binding, point mutations were introduced by site-directed mutagenesis. VEGF165 is a basic protein. Reduction of the electronegative potential of B-NRP2 by exchanging acidic residues for uncharged alanine (B-NRP2 E284A,E291A) in the 280-290 b1-NRP2 loop resulted in a 2-fold reduction in VEGF165 affinity. Conversely, enhancing the electronegative potential (B-NRP2 R287E,N290D and R287E,N290S) significantly increased VEGF165 affinity for B-NRP2 by 8- and 6.6-fold, respectively. The mutagenesis did not affect SEMA3F/B-NRP2 interactions. These results demonstrate that it is possible to alter VEGF165 affinity for NRP2 without affecting SEMA3F affinity. They also identify NRP2 residues involved in VEGF165 binding and suggest that modifications of B-NRP2 could lead to potentially high affinity selective inhibitors of VEGF165/NRP2 interactions.     

VEGF165 mediates formation of complexes containing VEGFR-2 and neuropilin-1 that enhance VEGF165-receptor binding

Co-expression of NRP1 and (VEGFR-2) KDR on the surface of endothelial cells (EC) enhances VEGF165 binding to KDR and EC chemotaxis in response to VEGF165. Overexpression of NRP1 by prostate tumor cells in vivo results in increased tumor angiogenesis and growth. We investigated the molecular mechanisms underlying NRP1-mediated angiogenesis by analyzing the association of NRP1 and KDR. An intracellular complex containing NRP1 and KDR was immunoprecipitated from EC by anti-NRP1 antibodies only in the presence of VEGF165. In contrast, VEGF121, which does not bind to NRP1, did not support complex formation. Complexes containing VEGF165, NRP1, and KDR were also formed in an intercellular fashion by co-culture of EC expressing KDR only, with cells expressing NRP1 only, for example, breast carcinoma cells. VEGF165 also mediated the binding of a soluble NRP1 dimer to cells expressing KDR only, confirming the formation of such complexes. Furthermore, the formation of complexes containing KDR and NRP1 markedly increased 125I-VEGF165 binding to KDR. Our results suggest that formation of a ternary complex of VEGF165, KDR, and NRP1 potentiates VEGF165 binding to KDR. These complexes are formed on the surface of EC and in a juxtacrine manner via association of tumor cell NRP1 and EC KDR.     

Glypican-1 is a VEGF165 binding proteoglycan that acts as an extracellular chaperone for VEGF165

Glypican-1 is a member of a family of glycosylphosphatidylinositol anchored cell surface heparan sulfate proteoglycans implicated in the control of cellular growth and differentiation. The 165-amino acid form of vascular endothelial growth factor (VEGF165) is a mitogen for endothelial cells and a potent angiogenic factor in vivo. Heparin binds to VEGF165 and enhances its binding to VEGF receptors. However, native HSPGs that bind VEGF165 and modulate its receptor binding have not been identified. Among the glypicans, glypican-1 is the only member that is expressed in the vascular system. We have therefore examined whether glypican-1 can interact with VEGF165. Glypican-1 from rat myoblasts binds specifically to VEGF165 but not to VEGF121. The binding has an apparent dissociation constant of 3 x 10(-10) M. The binding of glypican-1 to VEGF165 is mediated by the heparan sulfate chains of glypican-1, because heparinase treatment abolishes this interaction. Only an excess of heparin or heparan sulfates but not other types of glycosaminoglycans inhibited this interaction. VEGF165 interacts specifically not only with rat myoblast glypican-1 but also with human endothelial cell-derived glypican-1. The binding of 125I-VEGF165 to heparinase-treated human vascular endothelial cells is reduced following heparinase treatment, and addition of glypican-1 restores the binding. Glypican-1 also potentiates the binding of 125I-VEGF165 to a soluble extracellular domain of the VEGF receptor KDR/flk-1. Furthermore, we show that glypican-1 acts as an extracellular chaperone that can restore the receptor binding ability of VEGF165, which has been damaged by oxidation. Taken together, these results suggest that glypican-1 may play an important role in the control of angiogenesis by regulating the activity of VEGF165, a regulation that may be critical under conditions such as wound repair, in which oxidizing agents that can impair the activity of VEGF are produced, and in situations were the concentrations of active VEGF are limiting.     

Neuropilin-1 binds vascular endothelial growth factor 165, placenta growth factor-2, and heparin via its b1b2 domain

Neuroplin-1 (NRP1), a receptor for vascular endothelial growth factor (VEGF) family members, has three distinct extracellular domains, a1a2, b1b2, and c. To determine the VEGF(165) and placenta growth factor 2 (PlGF-2)-binding sites of NRP1, recombinant NRP1 domains were expressed in mammalian cells as Myc-tagged, soluble proteins, and used in co-precipitation experiments with 125I-VEGF165 and 125I-PlGF-2. Anti-Myc antibodies immunoprecipitated 125I-VEGF165 and 125I-PlGF-2 in the presence of the b1b2 but not of the a1a2 and c domains. Neither b1 nor b2 alone was capable of binding 125I-VEGF165. In competition experiments, VEGF165 competed PlGF-2 binding to the NRP1 b1b2 domain, suggesting that the binding sites of VEGF165 and PlGF-2 overlap. The presence of the a1a2 domain greatly enhanced VEGF165, but not PlGF-2 binding to b1b2. Heparin enhanced the binding of both 125I-VEGF165 and 125I-PlGF-2 to the b1b2 domain by 20- and 4-fold, respectively. A heparin chain of at least 20-24 monosaccharides was necessary for binding. In addition, the b1b2 domain of NRP1 could bind heparin directly, requiring heparin oligomers of at least 8 monosaccharide units. It was concluded that an intact b1b2 domain serves as the VEGF165-, PlGF-2-, and heparin-binding sites in NRP1, and that heparin is a critical component for regulating VEGF165 and PlGF-2 interactions with NRP1 by physically interacting with both receptor and ligands.     

Low molecular weight fucoidan increases VEGF165-induced endothelial cell migration by enhancing VEGF165 binding to VEGFR-2 and NRP1

Therapeutic induction of angiogenesis is a potential treatment for chronic ischemia. Heparan sulfate proteoglycans are known to play an important role by their interactions with proangiogenic growth factors such as vascular endothelial growth factor (VEGF). Low molecular weight fucoidan (LMWF), a sulfated polysaccharide from brown seaweeds that mimic some biological activities of heparin, has been shown recently to promote revascularization in rat critical hindlimb ischemia. In this report, we first used cultured human endothelial cells (ECs) to investigate the possible ability of LMWF to enhance the actions of VEGF(165). Data showed that LMWF greatly enhances EC tube formation in growth factor reduced matrigel. LMWF is a strong enhancer of VEGF(165)-induced EC chemotaxis, but not proliferation. In addition, LMWF has no effect on VEGF(121)-induced EC migration, a VEGF isoform that does not bind to heparan sulfate proteoglycans. Then, with binding studies using (125)I-VEGF(165), we observed that LMWF enhances the binding of VEGF(165) to recombinant VEGFR-2 and Neuropilin-1 (NRP1), but not to VEGFR-1. Surface plasmon resonance analysis showed that LMWF binds with high affinity to VEGF(165) (1.2 nm) and its receptors (5-20 nm), but not to VEGF(121). Pre-injection of LMWF on immobilized receptors shows that VEGF(165) has the highest affinity for VEGFR-2 and NRP1, as compared with VEGFR-1. Overall, the effects of LMWF were much more pronounced than those of LMW heparin. These findings suggested an efficient mechanism of action of LMWF by promoting VEGF(165) binding to VEGFR-2 and NRP1 on ECs that could help in stimulating therapeutic revascularization.     

VEGF189 stimulates endothelial cells proliferation and migration in vitro and up-regulates the expression of Flk-1/KDR mRNA

The vascular endothelial growth factor (VEGF) is a critical factor for development of the vascular system in physiological and pathological angiogenesis. This growth factor exists under at least three isoforms, VEGF120/121, VEGF164/165 and VEGF188/189 which are generated by alternative splicing. VEGF isoforms have different affinities for heparan sulphate as well as for VEGF receptors, and may play distinct roles in vascular development. The role of VEGF189 as an endothelial mitogen, however, remains controversial. VEGF189 is almost entirely bound to the cell surface or extracellular matrix, and is considered active after its cleavage and release from its extracellular binding site. In the present study, we demonstrate that VEGF189 induces endothelial cell proliferation and migration in vitro. The 30-60% increase observed with VEGF189 (10 ng/ml) in HUVEC proliferation was similar to that observed with VEGF165. However, the proliferative effect observed with VEGF189 appeared dependent on the origin of the endothelial cell, since the proliferation was clearly observed with HUVEC but not with BAEC or capillary endothelial cells from dermis (HMEC). The effect of VEGF189 on endothelial cell migration was also analyzed using the wound healing and the Boyden chamber assays. The migration effect was observed with BAEC which do not proliferate with VEGF189, suggesting that different mechanisms are involved in proliferation and migration. In addition, VEGF189 as well as VEGF165 induced a 2-fold increase of Flk-1/KDR expression in HUVEC, the receptor involved in proliferation and migration of endothelial cells. In the Matrigel plug assay in vivo, both VEGF189 and 165 (100 ng/ml) increased the infiltration of endothelial cells. These data suggest that VEGF189 induced endothelial cell migration and proliferation under certain circumstances.     

Combined transfer of human VEGF165 and HGF genes renders potent angiogenic effect in ischemic skeletal muscle

Increased interest in development of combined gene therapy emerges from results of recent clinical trials that indicate good safety yet unexpected low efficacy of "single-gene" administration. Multiple studies showed that vascular endothelial growth factor 165 aminoacid form (VEGF165) and hepatocyte growth factor (HGF) can be used for induction of angiogenesis in ischemic myocardium and skeletal muscle. Gene transfer system composed of a novel cytomegalovirus-based (CMV) plasmid vector and codon-optimized human VEGF165 and HGF genes combined with intramuscular low-voltage electroporation was developed and tested in vitro and in vivo. Studies in HEK293T cell culture, murine skeletal muscle explants and ELISA of tissue homogenates showed efficacy of constructed plasmids. Functional activity of angiogenic proteins secreted by HEK293T after transfection by induction of tube formation in human umbilical vein endothelial cell (HUVEC) culture. HUVEC cells were used for in vitro experiments to assay the putative signaling pathways to be responsible for combined administration effect one of which could be the ERK1/2 pathway. In vivo tests of VEGF165 and HGF genes co-transfer were conceived in mouse model of hind limb ischemia. Intramuscular administration of plasmid encoding either VEGF165 or HGF gene resulted in increased perfusion compared to empty vector administration. Mice injected with a mixture of two plasmids (VEGF165+HGF) showed significant increase in perfusion compared to single plasmid injection. These findings were supported by increased CD31+ capillary and SMA+ vessel density in animals that received combined VEGF165 and HGF gene therapy compared to single gene therapy. Results of the study suggest that co-transfer of VEGF and HGF genes renders a robust angiogenic effect in ischemic skeletal muscle and may present interest as a potential therapeutic combination for treatment of ischemic disorders.     

Facilitated diffusion of VEGF165 through descemet's membrane with sucrose octasulfate

Vascular endothelial growth factor A (VEGF-A) is a promoter of neovascularization and thus a popular therapeutic target for diseases involving excessive growth of blood vessels. In this study, we explored the potential of the disaccharide sucrose octasulfate (SOS) to alter VEGF165 diffusion through Descemet's membrane. Descemet's membranes were isolated from bovine eyes and used as a barrier between two chambers of a diffusion apparatus to measure VEGF transport. Diffusion studies revealed a dramatic increase in VEGF165 transport in the presence of SOS, with little diffusion of VEGF165 across the membrane over a 10-h time course in the absence of SOS. Diffusion studies with VEGF121, a non-heparin binding variant of VEGF, showed robust diffusion with or without SOS. To determine a possible mechanism, we measured the ability of SOS to inhibit VEGF interactions with extracellular matrix (ECM), using cell-free and cell surface binding assays. Binding studies showed SOS had no effect on VEGF165 binding to either heparin-coated plates or endothelial cell surfaces at less than mg/ml concentrations. In contrast, we show that SOS inhibited VEGF165 binding to fibronectin in a dose dependent manner and dramatically accelerated the rate of release of VEGF165 from fibronectin. SOS also inhibited the binding of VEGF165 to fibronectin-rich ECM deposited by vascular smooth muscle cells. These results suggest that fibronectin-rich extracellular matrices serve as barriers to VEGF165 diffusion by providing a network of binding sites that can trap and sequester the protein. Since the content of Descemet's membrane is typical of many basement membranes it is possible that they serve throughout the body as formidable barriers to VEGF165 diffusion and tightly regulate its bioavailability and distribution within tissues.     

Neuropilin-2 is a receptor for the vascular endothelial growth factor (VEGF) forms VEGF-145 and VEGF-165 [corrected

Neuropilin-1 (np-1) and neuropilin-2 (np-2) are receptors for axon guidance factors belonging to the class 3 semaphorins. np-1 also binds to the 165-amino acid heparin-binding form of VEGF (VEGF(165)) but not to the shorter VEGF(121) form, which lacks a heparin binding ability. We report that human umbilical vein-derived endothelial cells express the a17 and a22 splice forms of the np-2 receptor. Both np-2 forms bind VEGF(165) with high affinity in the presence of heparin (K(D) 1.3 x 10(-10) m) but not VEGF(121). np-2 also binds the heparin-binding form of placenta growth factor. These binding characteristics resemble those of np-1. VEGF(145) is a secreted heparin binding VEGF form that contains the peptide encoded by exon 6 of VEGF but not the peptide encoded by exon 7, which is present in VEGF(165). VEGF(145) binds to np-2 with high affinity (K(D) 7 x 10(-10) m). Surprisingly, VEGF(145) did not bind to np-1. Indeed, VEGF(145) does not bind to MDA-MB-231 breast cancer cells, which predominantly express np-1. By contrast, VEGF(145) binds to human umbilical vein-derived endothelial cells, which express both np-1 and np-2. The binding of VEGF(165) to porcine aortic endothelial cells expressing recombinant np-2 did not affect the proliferation or migration of the cells. Nevertheless, it is possible that VEGF-induced np-2-mediated signaling will take place only in the presence of other VEGF receptors such as VEGF receptor-1 or VEGF receptor-2.     

rhVEGF165在中国仓鼠卵巢细胞中的稳定表达、纯化及其生物学活性

利用哺乳动物细胞表达系统,稳定表达和纯化高生物学活性的人重组血管内皮生长因子 (VEGF165) 蛋白。将VEGF165克隆于表达载体pCDNA4.0,与T-GS载体共同转染CHO-S (中国仓鼠卵巢细胞) 细胞,MSX (Methionine sulphoximine) 加压筛选高表达细胞株,5 L发酵罐培养,细胞培养上清液通过三步纯化得到rhVEGF165蛋白,通过Western blotting、Biacore和人脐静脉内皮细胞增殖实验等对表达蛋白的特异性、亲和力及生物学活性等进行检测。所建立的细胞     

Covalently grafted VEGF(165) in hydrogel models upregulates the cellular pathways associated with angiogenesis

Angiogenesis is an important biological response known to be involved in many physiological and pathophysiological situations. Cellular responses involved in the formation of new blood vessels, such as increases in endothelial cell proliferation, cell migration, and the survival of apoptosis-inducing events, have been associated with vascular endothelial growth factor isoform 165 (VEGF(165)). Current research in the areas of bioengineering and biomedical science has focused on developing polyethylene glycol (PEG)-based systems capable of initiating and sustaining angiogenesis in vitro. However, a thorough understanding of how endothelial cells respond at the molecular level to VEGF(165) incorporated into these systems has not yet been established in the literature. The goal of the current study was to compare the upregulation of key intracellular proteins involved in angiogenesis in human umbilical vein endothelial cells (HUVEC) and human microvascular endothelial cells (HMEC) seeded on PEG hydrogels containing grafted VEGF(165) and adhesion peptides Arg-Gly-Asp-Ser (RGDS). Our data suggest that the covalent incorporation of VEGF(165) into PEG hydrogels encourages the upregulation of signaling proteins responsible for increases in endothelial cell proliferation, cell migration, and the survival after apoptosis-inducing events.     

Stretch induces upregulation of key tyrosine kinase receptors in microvascular endothelial cells

We previously demonstrated that cyclic stretch of cardiac myocytes activates paracrine signaling via vascular endothelial growth factor (VEGF) leading to angiogenesis. The present study tested the hypothesis that cyclic stretch upregulates tyrosine kinase receptors in rat coronary microvascular endothelial cells (RCMEC) and human umbilical vein endothelial cells (HUVEC). VEGF receptor-2 (Flk-1) protein levels increased in HUVEC and RCMEC in a time-dependent manner, but the increase occurred much earlier in RCMEC than in HUVEC. The enhancement of Flk-1 protein level was not inhibited by addition of VEGF neutralizing antibodies, indicating that VEGF is not involved in stretch-induced Flk-1 expression. VEGF receptor-1 (Flt-1) protein and mRNA were not changed by stretch. However, Tie-2 and Tie-1 protein levels increased in RCMEC. Angiopoietin-1 and -2, the ligands for Tie-2, increased in cardiac myocytes subjected to cyclic stretch but were not affected by stretch in endothelial cells (EC). Stretch or incubation of RCMEC with VEGF increased cell proliferation moderately, whereas stretch + VEGF had an additive effect on proliferation. Mechanical stretch induces upregulation of the key tyrosine kinase receptors Flk-1, Tie-2, and Tie-1 in vascular EC, which underlies the increase in sensitivity of EC to growth factors and, therefore, facilitates angiogenesis. These in vitro findings support the concept that stretch of cardiac myocytes and EC plays a key role in coronary angiogenesis.     

Vascular endothelial growth factor receptor-2 and neuropilin-1 form a receptor complex that is responsible for the differential signaling potency of VEGF(165) and VEGF(121)

The two most abundant secreted isoforms of vascular endothelial growth factor A (VEGF(165) and VEGF(121)) are formed as a result of differential splicing of the VEGF-A gene. VEGF(165) and VEGF(121) share similar affinities at the isolated VEGF receptor (VEGFR)-2 but have been previously demonstrated to have differential ability to activate VEGFR-2-mediated effects on endothelial cells. Herein we investigate whether the recently described VEGF(165) isoform-specific receptor neuropilin-1 (Npn-1) is responsible for the difference in potency observed for these ligands. We demonstrate that although VEGFR-2 and Npn-1 form a complex, this complex does not result in an increase in VEGF(165) binding affinity. Therefore, the differential activity of VEGF(165) and VEGF(121) cannot be explained by a differential binding affinity for the complex. Using an antagonist that competes for VEGF(165) binding at the VEGFR-2.Npn-1 complex, we observe specific antagonism of VEGF(165)-meditated phosphorylation of VEGFR-2 without affecting the VEGF(121) response. These data indicate that the formation of the complex is responsible for the increased potency of VEGF(165) versus VEGF(121). Taken together, these data suggest a receptor-clustering role for Npn-1, as opposed to Npn-1 behaving as an affinity-converting subunit.     

Oligonucleotide NX1838 inhibits VEGF165-mediated cellular responses in vitro

Summary VEGF (vascular endothelial growth factor) overproduction has been identified as a major factor underlying pathological angiogenesis in vivo, including such conditions as psoriasis, macular degeneration, and tumor proliferation. Endothelial cell tyrosine kinase receptors, KDR and Flt-1, have been implicated in VEGF responses including cellular migration, proliferation, and modulation of vascular permeability. Therefore, agents that limit VEGF-cellular interaction are likely therapeutic candidates for VEGF-mediated disease states (particularly agents blocking activity of VEGF₁₆₅, the most frequently occurring VEGF isoform). To that end, a nuclease-resistant, VEGF₁₆₅-specific aptamer NX1838 (2′-fluoropyrimidine, RNA-based oligonucleotide/40-kDa-PEG) was developed. We have assessed NX1838 inhibition of a variety of cellular events associated with VEGF, including cellular binding, signal transduction, calcium mobilization, and induction of cellular proliferation. Our data indicate that NX1838 inhibits binding of VEGF to HUVECs (human umbilical vein endothelial cells) and dose-dependently prevents VEGF-mediated phosphorylation of KDR and PLCγ, calcium flux, and ultimately VEGF-induced cell proliferation. NX1838-inhibition of VEGF-mediated cellular events was comparable to that observed with anti-VEGF monoclonal antibody, but was ineffective as an inhibitor of VEGF₁₂₁-induced HUVEC proliferation. These findings, coupled with nuclease stability of the molecule, suggest that NX1838 may provide therapeutic utility in vivo.     

Cellular repressor of E1A-stimulated genes regulates vascular endothelial cell migration by the ILK/AKT/mTOR/VEGF(165) signaling pathway

The migration of vascular endothelial cells plays a critical role in a variety of vascular physiological and pathological processes, such as embryonic development, angiogenesis, wound healing, re-endothelialization, and vascular remodeling. This study clarified the role and mechanism of a new vascular homeostasis regulator, Cellular repressor of E1A-stimulated genes (CREG), in the migration of primary human umbilical vein endothelial cells (HUVECs). A wound healing assay and transwell migration model showed that upregulation of CREG expression induced HUVEC migration and it was positively correlated with the expression of vascular endothelial growth factor. Furthermore, wild type integrin-linked kinase reversed the poor mobility of CREG knock-down HUVECs; in contrast, kinase-dead integrin-linked kinase weakened the migration of HUVECs. We also studied the effect of CREG on HUVEC migration by the addition of an mTOR inhibitor, recombinant vascular endothelial growth factor₁₆₅, neutralizing antibody of vascular endothelial growth factor₁₆₅ and AKT siRNA, and we concluded that CREG induces endothelial cell migration by activating the integrin-linked kinase/AKT/mTOR/VEGF₁₆₅ signaling pathway.     

Vascular endothelial growth factor induces branching morphogenesis/tubulogenesis in renal epithelial cells in a neuropilin-dependent fashion

Vascular endothelial growth factor (VEGF) is well characterized for its role in endothelial cell differentiation and vascular tube formation. Alternate splicing of the VEGF gene in mice results in various VEGF-A isoforms, including VEGF-121 and VEGF-165. VEGF-165 is the most abundant isoform in the kidney and has been implicated in glomerulogenesis. However, its role in the tubular epithelium is not known. We demonstrate that VEGF-165 but not VEGF-121 induces single-cell branching morphogenesis and multicellular tubulogenesis in mouse renal tubular epithelial cells and that these morphogenic effects require activation of the phosphatidylinositol 3-kinase (PI 3-K) and, to a lesser degree, the extracellular signal-regulated kinase and protein kinase C signaling pathways. Further, VEGF-165-stimulated sheet migration is dependent only on PI 3-K signaling. These morphogenic effects of VEGF-165 require activation of both VEGF receptor 2 (VEGFR-2) and neuropilin-1 (Nrp-1), since neutralizing antibodies to either of these receptors or the addition of semaphorin 3A (which blocks VEGF-165 binding to Nrp-1) prevents the morphogenic response and the phosphorylation of VEGFR-2 along with the downstream signaling. We thus conclude that in addition to endothelial vasculogenesis, VEGF can induce renal epithelial cell morphogenesis in a Nrp-1-dependent fashion.     

Characterization of neuropilin-1 structural features that confer binding to semaphorin 3A and vascular endothelial growth factor 165

Neuropilin-1 (Npn-1) is a receptor for both semaphorin 3A (Sema3A) and vascular endothelial growth factor 165 (VEGF(165)). To understand the role Npn-1 plays as a receptor for these structurally and functionally unrelated ligands, we set out to identify structural features of Npn-1 that confer binding to Sema3A or VEGF(165). We constructed Npn-1 variants containing deletions within the "a" and "b" domains of Npn-1. More than 16 variants were expressed in COS-1 cells and tested for alkaline phosphatase-Sema3A as well as alkaline phosphatase-VEGF(165) binding. Our results indicate that each of the two Npn-1 CUB domains and the amino-terminal coagulation factor V/VIII domain (CF V/VIII) are essential for Sema3A binding, but only the amino-terminal Npn-1 CF V/VIII domain is required for binding to VEGF(165). Guided by the structure of the bovine spermadhesin CUB domain, point mutants targeting defined surfaces of the Npn-1 a1 CUB domain were generated and tested for Sema3A and VEGF(165) binding. One Npn-1 variant, Npn-1(2ABC), exhibits complete loss of Sema3A binding while retaining normal VEGF(165) binding. Moreover, co-immunoprecipitation experiments show that Npn-1(2ABC) can form a signaling complex with the VEGF(165) signaling receptor KDR/VEGFR-2. These results establish the identity of contact sites between Npn-1 and its semaphorin ligands, and they provide a foundation for understanding how Npn-1 functions as a receptor for distinct classes of ligands in vivo.     

ADAMTS1/METH1 inhibits endothelial cell proliferation by direct binding and sequestration of VEGF165

ADAMTS1 is a metalloprotease previously shown to inhibit angiogenesis in a variety of in vitro and in vivo assays. In the present study, we demonstrate that ADAMTS1 significantly blocks VEGFR2 phosphorylation with consequent suppression of endothelial cell proliferation. The effect on VEGFR2 function was due to direct binding and sequestration of VEGF165 by ADAMTS1. Binding was confirmed by co-immunoprecipitation and cross-linking analysis. Inhibition of VEGF function was reversible, as active VEGF could be recovered from the complex. The interaction required the heparin-binding domain of the growth factor, because VEGF121 failed to bind to ADAMTS1. Structure/function analysis with independent ADAMTS1 domains indicated that binding to VEGF165 was mediated by the carboxyl-terminal (CT) region. ADAMTS1 and VEGF165 were also found in association in tumor extracts. These findings provide a mechanism for the anti-angiogenic activity of ADAMTS1 and describe a novel modulator of VEGF bioavailability.     

Matrix metalloproteinases cleave connective tissue growth factor and reactivate angiogenic activity of vascular endothelial growth factor 165

Vascular endothelial growth factor (VEGF), a potent angiogenic mitogen, plays a crucial role in angiogenesis under various pathophysiological conditions. We have recently demonstrated that VEGF(165), one of the VEGF isoforms, binds connective tissue growth factor (CTGF) and that its angiogenic activity is inhibited in the VEGF(165).CTGF complex form (Inoki, I., Shiomi, T., Hashimoto, G., Enomoto, H., Nakamura, H., Makino, K., Ikeda, E., Takata, S., Kobayashi, K. and Okada, Y. (2002) FASEB J. 16, 219-221). In the present study, we further examined the susceptibility of the VEGF(165).CTGF complex to matrix metalloproteinases (MMP-1, -2, -3, -7, -9, and -13), ADAMTS4 (aggrecanase-1), and serine proteinases, and evaluated the recovery of the angiogenic activity of VEGF(165) after the treatment. Among the MMPs, MMP-1, -3, -7, and -13 processed CTGF of the complex into the major NH(2)- and COOH-terminal fragments, whereas VEGF(165) was completely resistant to the MMPs. On the other hand, elastase and plasmin cleaved both CTGF and VEGF(165) of the complex, but they were completely resistant to ADAMTS4. By digestion of the immobilized VEGF(165).CTGF complex with MMP-3 or MMP-7, both NH(2)- and COOH-terminal fragments of CTGF were dissociated and released from the complex into the liquid phase. The in vitro angiogenic activity of VEGF(165) blocked in the VEGF(165).CTGF complex was reactivated to original levels after CTGF digestion of the complex with MMP-1, -3, and -13. Recovery of angiogenic activity was further confirmed by in vivo angiogenesis assay using a Matrigel injection model in mice. These results demonstrate for the first time that CTGF is a substrate of MMPs and that the angiogenic activity of VEGF(165) suppressed by the complex formation with CTGF is recovered through the selective degradation of CTGF by MMPs. MMPs may play a novel role through CTGF degradation in VEGF-induced angiogenesis during embryonic development, tissue maintenance, and/or pathological processes of various diseases.