A novel snake venom vascular endothelial growth factor (VEGF) predominantly induces vascular permeability through preferential signaling via VEGF receptor-1

Vascular endothelial growth factor (VEGF)/vascular permeability factor induces both angiogenesis and vascular permeability mainly through VEGF receptor (VEGFR)-2 activation. VEGF binds VEGFR-1 as well, but the importance of VEGFR-1 signaling in vascular permeability has been largely neglected. Here, we report the purification and characterization of a novel VEGF-like protein from Trimeresurus flavoviridis Habu snake venom. The Habu snake has a venom-specific VEGF-like molecule, T. flavoviridis snake venom VEGF (TfsvVEGF), in addition to VEGF-A. TfsvVEGF has almost 10-fold less mitotic activity than VEGF(165), a predominant isoform of human VEGF-A, but a similar effect on vascular permeability. TfsvVEGF bound VEGFR-1 and induced its autophosphorylation to almost the same extent as VEGF(165), but bound VEGFR-2 weakly and induced its autophosphorylation almost 10-fold less effectively than VEGF(165). This unique binding affinity for VEGFR-1 and VEGFR-2 leads to the vascular permeability-dominant activity of TfsvVEGF. These results suggest that Habu snakes have acquired a highly purposive molecule for a toxin, which enhances the toxicity in envenomation without inducing effective angiogenesis and the following regeneration of damaged tissues, taking advantage of the difference in signaling properties involving VEGFR-1 and VEGFR-2 between vascular permeability and angiogenesis. TfsvVEGF is thus a potent inducing factor selective for vascular permeability through preferential signaling via VEGFR-1. These data strongly indicate the importance of VEGFR-1 signaling in vascular permeability.     

The C-terminus of viral vascular endothelial growth factor-E partially blocks binding to VEGF receptor-1

Vascular endothelial growth factor (VEGF) family members play important roles in embryonic development and angiogenesis during wound healing and in pathological conditions such as tumor formation. Parapoxviruses express a new member of the VEGF family which is a functional mitogen that specifically activates VEGF receptor (VEGFR)-2 but not VEGFR-1. In this study, we show that deletion from the viral VEGF of a unique C-terminal region increases both VEGFR-1 binding and VEGFR-1-mediated monocyte migration. Enzymatic removal of O-linked glycosylation from the C-terminus also increased VEGFR-1 binding and migration of THP-1 monocytes indicating that both the C-terminal residues and O-linked sugars contribute to blocking viral VEGF binding to VEGFR-1. The data suggest that conservation of the C-terminal residues throughout the viral VEGF subfamily may represent a means of reducing the immunostimulatory activities associated with VEGFR-1 activation while maintaining the ability to induce angiogenesis via VEGFR-2.     

Analysis of biological effects and signaling properties of Flt-1 (VEGFR-1) and KDR (VEGFR-2). A reassessment using novel receptor-specific vascular endothelial growth factor mutants

Endothelial cells express two related vascular endothelial growth factor (VEGF) receptor tyrosine kinases, KDR (kinase-insert domain containing receptor, or VEGFR-2) and Flt-1 (fms-like tyrosine kinase, or VEGFR-1). Although considerable experimental evidence links KDR activation to endothelial cell mitogenesis, there is still significant uncertainty concerning the role of individual VEGF receptors for other biological effects such as vascular permeability. VEGF mutants that bind to either KDR or Flt-1 with high selectivity were used to determine which of the two receptors serves to mediate different VEGF functions. In addition to mediating mitogenic signaling, selective KDR activation was sufficient for the activation of intracellular signaling pathways implicated in cell migration. KDR stimulation caused tyrosine phosphorylation of both phosphatidylinositol 3-kinase and phospholipase Cgamma in primary endothelial cells and stimulated cell migration. KDR-selective VEGF was also able to induce angiogenesis in the rat cornea to an extent indistinguishable from wild type VEGF. We also demonstrate that KDR, but not Flt-1, stimulation is responsible for the induction of vascular permeability by VEGF.     

Viral vascular endothelial growth factors vary extensively in amino acid sequence,receptor-binding specificities,and the ability to induce vascular permeability yet are uniformly active mitogens

Infections of humans and ungulates by parapoxviruses result in skin lesions characterized by extensive vascular changes that have been linked to viral-encoded homologues of vascular endothelial growth factor (VEGF). VEGF acts via a family of receptors (VEGFRs) to mediate endothelial cell proliferation, vascular permeability, and angiogenesis. The VEGF genes from independent parapoxvirus isolates show an extraordinary degree of inter-strain sequence variation. We conducted functional comparisons of five representatives of the divergent viral VEGFs. These revealed that despite the sequence divergence, all were equally active mitogens, stimulating proliferation of human endothelial cells in vitro and vascularization of sheep skin in vivo with potencies equivalent to VEGF. This was achieved even though the viral VEGFs bound VEGFR-2 less avidly than did VEGF. Surprisingly the viral VEGFs varied in their ability to cross-link VEGFR-2, induce vascular permeability and bind neuropilin-1. Correlations between these three activities were detected. In addition it was possible to correlate these functional variations with certain sequence and structural motifs specific to the viral VEGFs. In contrast to the conserved ability to bind human VEGFR-2, the viral growth factors did not bind either VEGFR-1 or VEGFR-3. We propose that the extensive sequence divergence seen in the viral VEGFs was generated primarily by selection against VEGFR-1 binding.     

Signal transduction by VEGF receptor-1 wild type and mutant proteins

The role of the vascular endothelial growth factor receptor-1 (VEGFR-1) in endothelial cell function is unclear. We have previously identified four tyrosine phosphorylation sites in the C-terminal tail of this receptor. We now show that the wild type VEGFR-1 expressed in porcine aortic endothelial (PAE/VEGFR-1) cells was able to transduce signals for increased DNA synthesis and proliferation. Tyrosine phosphorylation of phospholipase Cgamma (PLCgamma), tyrosine phosphatase SHP-2, Crk, and extracellular regulated kinases 1 and 2 (Erk1/2) was registered in response to VEGF-A treatment of the PAE/VEGFR-1 cells. VEGFR-1 mutated at Y1213, Y1242, and Y1333 were constructed and expressed in PAE cells, to the same level as that of PAE/VEGFR-1 cells. The affinities of the wild type and mutated receptors for VEGF-A(165) binding were similar. The mutated VEGFR-1 Y1213F expressed in PAE cells was kinase inactive. PAE cells expressing the mutated VEGFR-1 Y1242F and Y1333F receptors mediated increased tyrosine phosphorylation of PLCgamma in response to VEGF-A stimulation. However, these two mutant VEGFR-1 failed to mediate increased mitogenesis and were unable to stimulate increased tyrosine phosphorylation of SHP-2, Crk, and Erk1/2, indicating that the mutations lead to a perturbation in VEGF-A-induced signal transduction.     

Contact inhibition of VEGF-induced proliferation requires vascular endothelial cadherin,beta-catenin,and the phosphatase DEP-1/CD148

Confluent endothelial cells respond poorly to the proliferative signals of VEGF. Comparing isogenic endothelial cells differing for vascular endothelial cadherin (VE-cadherin) expression only, we found that the presence of this protein attenuates VEGF-induced VEGF receptor (VEGFR) 2 phosphorylation in tyrosine, p44/p42 MAP kinase phosphorylation, and cell proliferation. VE-cadherin truncated in beta-catenin but not p120 binding domain is unable to associate with VEGFR-2 and to induce its inactivation. beta-Catenin-null endothelial cells are not contact inhibited by VE-cadherin and are still responsive to VEGF, indicating that this protein is required to restrain growth factor signaling. A dominant-negative mutant of high cell density-enhanced PTP 1 (DEP-1)//CD148 as well as reduction of its expression by RNA interference partially restore VEGFR-2 phosphorylation and MAP kinase activation. Overall the data indicate that VE-cadherin-beta-catenin complex participates in contact inhibition of VEGF signaling. Upon stimulation with VEGF, VEGFR-2 associates with the complex and concentrates at cell-cell contacts, where it may be inactivated by junctional phosphatases such as DEP-1. In sparse cells or in VE-cadherin-null cells, this phenomenon cannot occur and the receptor is fully activated by the growth factor.     

Monoclonal antibodies to vascular endothelial growth factor-D block its interactions with both VEGF receptor-2 and VEGF receptor-3.

Vascular endothelial growth factor-D (VEGF-D), the most recently discovered mammalian member of the VEGF family, is an angiogenic protein that activates VEGF receptor-2 (VEGFR-2/Flk1/KDR) and VEGFR-3 (Flt4). These receptor tyrosine kinases, localized on vascular and lymphatic endothelial cells, signal for angiogenesis and lymphangiogenesis. VEGF-D consists of a central receptor-binding VEGF homology domain (VHD) and N-terminal and C-terminal propeptides that are cleaved from the VHD to generate a mature, bioactive form consisting of dimers of the VHD. Here we report characterization of mAbs raised to the VHD of human VEGF-D in order to generate VEGF-D antagonists. The mAbs bind the fully processed VHD with high affinity and also bind unprocessed VEGF-D. We demonstrate, using bioassays for the binding and cross-linking of VEGFR-2 and VEGFR-3 and biosensor analysis with immobilized receptors, that one of the mAbs, designated VD1, is able to compete potently with mature VEGF-D for binding to both VEGFR-2 and VEGFR-3 for binding to mature VEGF-D. This indicates that the binding epitopes on VEGF-D for these two receptors may be in close proximity. Furthermore, VD1 blocks the mitogenic response of human microvascular endothelial cells to VEGF-D. The anti-(VEGF-D) mAbs raised to the bioactive region of this growth factor will be powerful tools for analysis of the biological functions of VEGF-D.     

Upregulation of neuropilin-1 by basic fibroblast growth factor enhances vascular smooth muscle cell migration in response to VEGF

Neuropilin-1 (NRP-1) is a co-receptor for vascular endothelial growth factor (VEGF). During neovascularization, vascular smooth muscle cells (VSMCs) and pericytes modulate the function of endothelial cells. Factors that mediate NRP-1 in human VSMCs (hVSMCs) remain to be elucidated. We studied various angiogenic cytokines to identify factors that increase NRP-1 expression in hVSMCs. Treatment of hVSMCs with basic fibroblast growth factor (b-FGF) induced expressions of NRP-1 mRNA and protein whereas epidermal growth factor, insulin-like growth factor-1, and interleukin-1beta did not. b-FGF induced phosphorylation of Erk-1/2 and JNK. MEK1/2 and nuclear factor kappa B (NF-kappaB) inhibitors (U0126 and TLCK, respectively) blocked the ability of b-FGF to induce NRP-1 mRNA expression, but inhibition of JNK (SP600125) or PI3-kinase activity (wortmannin) did not. Further, the increase in NRP-1 expression by b-FGF enhanced hVSMCs migration in response to VEGF(165). This effect was dependent on the binding of VEGF(165) to VEGFR-2, as blocking antibodies to VEGFR-2, but not VEGFR-1, inhibited VEGF(165)-induced migration. In conclusion, b-FGF increased NRP-1 expression in hVSMCs that in turn enhance the effect of VEGF(165) on cell migration. The enhanced migration of hVSMCs was mediated through binding of VEGF(165) to both NRP-1 and VEGFR-2, as inhibition of VEGFR-2 on these cells blocked the effect of VEGF-mediated cell migration.     

Ganglioside GM3 inhibits VEGF/VEGFR-2-mediated angiogenesis: direct interaction of GM3 with VEGFR-2

Angiogenesis is associated with growth, invasion, and metastasis of human solid tumors. Aberrant activation of endothelial cells and induction of microvascular permeability by a vascular endothelial growth factor (VEGF) receptor-2 (VEGFR-2) signaling pathway is observed in pathological angiogenesis including tumor, wound healing, arthritis, psoriasis, diabetic retinopathy, and others. Here, we show that GM3 regulated the activity of various downstream signaling pathways and biological events through the inhibition of VEGF-stimulated VEGFR-2 activation in vascular endothelial cells in vitro. Furthermore, GM3 strongly blocked VEGF-induced neovascularization in vivo, in models including the chick chorioallantoic membrane and Matrigel plug assay. Interestingly, GM3 suppressed VEGF-induced VEGFR-2 activation by blocking its dimerization and also blocked the binding of VEGF to VEGFR-2 through a GM3-specific interaction with the extracellular domain of VEGFR-2, but not with VEGF. Primary tumor growth in mice was inhibited by subcutaneous injection of GM3. Immunohistochemical analyses showed GM3 inhibition of angiogenesis and tumor cell proliferation. GM3 also resulted in the suppression of VEGF-stimulated microvessel permeability in mouse skin capillaries. These results suggest that GM3 inhibits VEGFR-2-mediated changes in vascular endothelial cell function and angiogenesis, and might be of value in anti-angiogenic therapy.     

Neuropilin-1-mediated vascular permeability factor/vascular endothelial growth factor-dependent endothelial cell migration

Neuropilin-1 (NRP-1) has been found to be expressed by endothelial cells and tumor cells as an isoform-specific receptor for vascular permeability factor/vascular endothelial growth factor (VEGF). Previous studies were mainly focused on the extracellular domain of NRP-1 that can bind to VEGF165 and, thus, enables NRP-1 to act as a co-receptor for VEGF165, which enhances its binding to VEGFR-2 and its bioactivity. However, the exact functional roles and related signaling mechanisms of NRP-1 in angiogenesis are not well understood. In this study we constructed a chimeric receptor, EGNP-1, by fusing the extracellular domain of epidermal growth factor receptor to the transmembrane and intracellular domains of NRP-1 and transduced it into HUVECs with a retroviral expression vector. We observed that NRP-1/EGNP-1 mediates ligand-stimulated migration of human umbilical vein endothelial cells (HUVECs) but not proliferation. Our results show that NRP-1 alone can mediate HUVEC migration through its intracellular domain, and its C-terminal three amino acids (SEA-COOH) are essential for the process. We demonstrate that phosphatidylinositol 3-kinase inhibitor Ly294002 and the p85 dominant negative mutant can block NRP-1-mediated HUVEC migration. NRP-1-mediated migration can be significantly reduced by overexpression of the dominant negative mutant of RhoA (RhoA-19N). In addition, Gq family proteins and Gbetagamma subunits are also required for NRP-1-mediated HUVEC migration. These results show for the first time that NRP-1 can independently promote cell signaling in endothelial cells and also demonstrate the importance of last three amino acids of NRP-1 for its function.     

A set of loop-1 and -3 structures in the novel vascular endothelial growth factor (VEGF) family member,VEGF-ENZ-7, is essential for the activation of VEGFR-2 signaling

The vascular endothelial growth factor (VEGF) family plays important roles in angiogenesis and vascular permeability. Novel members of the VEGF family encoded in the Orf virus genome, VEGF-E, function as potent angiogenic factors by specifically binding and activating VEGFR-2 (KDR). VEGF-E is about 45% homologous to VEGF-A at amino acid levels, however, the amino acid residues in VEGF-A crucial for the VEGFR-2-binding are not conserved in VEGF-E. To understand the molecular basis of the biological activity of VEGF-E, we have functionally mapped residues important for interaction of VEGF-E with VEGFR-2 by exchanging the domains between VEGF-E(NZ-7) and PlGF, which binds only to VEGFR-1 (Flt-1). Exchange on the amino- and carboxyl-terminal regions had no suppressive effect on biological activity. However, exchange on either the loop-1 or -3 region of VEGF-E(NZ-7) significantly reduced activities. On the other hand, introduction of the loop-1 and -3 of VEGF-E(NZ-7) to placenta growth factor rescued the biological activities. The chimera between VEGF-A and VEGF-E(NZ-7) gave essentially the same results. These findings strongly suggest that a common rule exists for VEGFR-2 ligands (VEGF-E(NZ-7) and VEGF-A) that they build up the binding structure for VEGFR-2 through the appropriate interaction between loop-1 and -3 regions.     

Autocrine VEGF signaling is required for vascular homeostasis

Vascular endothelial growth factor (VEGF) is essential for developmental and pathological angiogenesis. Here we show that in the absence of any pathological insult, autocrine VEGF is required for the homeostasis of blood vessels in the adult. Genetic deletion of vegf specifically in the endothelial lineage leads to progressive endothelial degeneration and sudden death in 55% of mutant mice by 25 weeks of age. The phenotype is manifested without detectable changes in the total levels of VEGF mRNA or protein, indicating that paracrine VEGF could not compensate for the absence of endothelial VEGF. Furthermore, wild-type, but not VEGF null, endothelial cells showed phosphorylation of VEGFR2 in the absence of exogenous VEGF. Activation of the receptor in wild-type cells was suppressed by small molecule antagonists but not by extracellular blockade of VEGF. These results reveal a cell-autonomous VEGF signaling pathway that holds significance for vascular homeostasis but is dispensable for the angiogenic cascade.     

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.     

Administration of moderate and high doses of gonadotropins to female rats increases ovarian vascular endothelial growth factor (VEGF) and VEGF receptor-2 expression that is associated to vascular hyperpermeability

Convincing evidence supports the role of ovarian-origin vascular endothelial growth factor (VEGF) in inducing vascular permeability (VP) and ascites associated with ovarian hyperstimulation syndrome (OHSS) in mammals, including humans. A circulatory dysfunction has been described in every woman treated with gonadotropins for in vitro fertilization. It is not known, however, whether the action of gonadotropins also includes up-regulation of the VEGF receptor-2 (VEGFR-2) and whether increased VP is also found when milder stimulation is used. Thus, we applied an OHSS animal model to answer these questions. Immature female rats were stimulated with saline (control group) or with high (10 IU of eCG x 4 days + 30 IU hCG, OHSS group) or mild (10 IU of eCG + 10 IU of hCG, mild-stimulation group) doses of gonadotropins. The VP and the expression of whole-VEGF and VEGFR-2 mRNAs were analyzed through time-course experiments (0, 24, 48, and 96 h after hCG). Although eCG increased VP and the expression of VEGF and VEGFR-2 mRNAs in the ovaries of both mild- and OHSS-stimulated animals, hCG further augmented these parameters and produced the highest values after 48 h. A linear correlation was found between increased expression of VEGF and VEGFR-2 mRNAs and enhanced VP in both mild and OHSS groups. Immunohistochemistry showed the presence of VEGF and VEGFR-2 in the granulosa-lutein and endothelial cells of the entire corpus luteum. These studies confirm that in hyperstimulated animals as well as in mildly treated rats, VEGF and VEGFR-2 are overexpressed and associated with an increase in VP, which may be responsible for the accumulation of ascitic fluid in the syndrome.     

Heterotrimeric G alpha q/G alpha 11 proteins function upstream of vascular endothelial growth factor (VEGF) receptor-2 (KDR) phosphorylation in vascular permeability factor/VEGF signaling

Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) functions by activating two receptor-tyrosine kinases, Flt-1 (VEGF receptor (VEGFR)-1) and KDR (VEGFR-2), both of which are selectively expressed on primary vascular endothelium. KDR is responsible for VPF/VEGF-stimulated endothelial cell proliferation and migration, whereas Flt-1 down-modulates KDR-mediated endothelial cell proliferation. Our most recent works show that pertussis toxin-sensitive G proteins and Gbetagamma subunits are required for Flt-1-mediated down-regulation of human umbilical vein endothelial cell (HUVEC) proliferation and that Gq/11 proteins are required for KDR-mediated RhoA activation and HUVEC migration. In this study, we demonstrate that Gq/11 proteins are also required for VPF/VEGF-stimulated HUVEC proliferation. Our results further indicate that Gq/11 proteins specifically mediate KDR signaling such as intracellular Ca2+ mobilization rather than Flt-1-induced CDC42 activation and that a Gq/11 antisense oligonucleotide completely inhibits MAPK phosphorylation induced by KDR but has no effect on Flt-1-induced MAPK activation. More importantly, we demonstrate that Gq/11 proteins interact with KDR in vivo, and the interaction of Gq/11 proteins with KDR does not require KDR tyrosine phosphorylation. Surprisingly, the Gq/11 antisense oligonucleotide completely inhibits VPF/VEGF-stimulated KDR phosphorylation. Expression of a constitutively active mutant of G11 but not Gq can cause phosphorylation of KDR and MAPK. In addition, a Gbetagamma minigene, hbetaARK1(495), inhibits VPF/VEGF-stimulated HUVEC proliferation, MAPK phosphorylation, and intracellular Ca2+ mobilization but has no effect on KDR phosphorylation. Taken together, this study demonstrates that Gq/11 proteins mediate KDR tyrosine phosphorylation and KDR-mediated HUVEC proliferation through interaction with KDR.     

Ultrastructural localization of the vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) receptor-2 (FLK-1, KDR) in normal mouse kidney and in the hyperpermeable vessels induced by VPF/VEGF-expressing tumors and adenoviral vectors.

Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) interacts with two high-affinity tyrosine kinase receptors, VEGFR-1 and VEGFR-2, to increase microvascular permeability and induce angiogenesis. Both receptors are selectively expressed by vascular endothelial cells and are strikingly increased in tumor vessels. We used a specific antibody to localize VEGFR-2 (FLK-1, KDR) in microvascular endothelium of normal mouse kidneys and in the microvessels induced by the TA3/St mammary tumor or by infection with an adenoviral vector engineered to express VPF/VEGF. A pre-embedding method was employed at the light and electron microscopic levels using either nanogold or peroxidase as reporters. Equivalent staining was observed on both the luminal and abluminal surfaces of tumor- and adenovirus-induced vascular endothelium, but plasma membranes at interendothelial junctions were spared except at sites connected to vesiculovacuolar organelles (VVOs). VEGFR-2 was also localized to the membranes and stomatal diaphragms of some VVOs. This staining distribution is consistent with a model in which VPF/VEGF increases microvascular permeability by opening VVOs to allow the transendothelial cell passage of plasma and plasma proteins.     

Tuftsin binds neuropilin-1 through a sequence similar to that encoded by exon 8 of vascular endothelial growth factor

Tuftsin, Thr-Lys-Pro-Arg (TKPR), is an immunostimulatory peptide with reported nervous system effects as well. We unexpectedly found that tuftsin and a higher affinity antagonist, TKPPR, bind selectively to neuropilin-1 and block vascular endothelial growth factor (VEGF) binding to that receptor. Dimeric and tetrameric forms of TKPPR had greatly increased affinity for neuropilin-1 based on competition binding experiments. On endothelial cells tetrameric TKPPR inhibited the VEGF(165)-induced autophosphorylation of vascular endothelial growth factor receptor-2 (VEGFR-2) even though it did not directly inhibit VEGF binding to VEGFR-2. Homology between exon 8 of VEGF and TKPPR suggests that the sequence coded for by exon 8 may stabilize VEGF binding to neuropilin-1 to facilitate signaling through VEGFR-2. Given the overlap between processes involving neuropilin-1 and tuftsin, we propose that at least some of the previously reported effects of tuftsin are mediated through neuropilin-1.     

The vascular endothelial growth factor VEGF165 induces perlecan synthesis via VEGF receptor-2 in cultured human brain microvascular endothelial cells

A member of the vascular endothelial growth factor (VEGF) family, VEGF165, regulates vascular endothelial cell functions in autocrine and paracrine fashions in microvessels. Proteoglycans are highly glycosylated poly-anionic macromolecules that influence cellular behaviors such as proliferation and migration by interacting with cytokines/growth factors. In the present study, we investigated the regulation of proteoglycan synthesis by VEGF165 in cultured human brain microvascular endothelial cells. The cells were exposed to recombinant human VEGF165, and the proteoglycans were then characterized using biochemical techniques. VEGF165 treatment increased the accumulation of proteoglycans 1.4- and 1.6-fold in the cell layer and conditioned medium, respectively. This effect resulted from the activation of VEGFR-2, and was mimicked by vammin, a VEGFR-2 ligand from snake venom but not placenta growth factor, which binds specifically to VEGFR-1. VEGF165 stimulated the production and secretion of perlecan, substituted with shorter heparan sulfate side chains, but with unaltered sulfated disaccharide composition. The perlecan secreted by VEGF165-stimulated endothelial cells may be involved in the regulation of cellular behavior during angiogenesis, in diseases of the brain microvessels, and in the maintenance of the endothelial cell monolayer.     

VEGF in biological control

Vascular endothelial growth factor A (VEGF-A) belongs to a family of heparin binding growth factors that include VEGF-B, VEGF-C, VEGF-D, and placental-like growth factor (PLGF). First discovered for its ability to regulate vascular endothelial cell permeability, VEGF is a well-known angiogenic factor that is important for vascular development and maintenance in all mammalian organs. The development of molecular tools and pharmacological agents to selectively inhibit VEGF function and block angiogenesis and/or vascular permeability has led to great promise in the treatment of various cancers, macular degeneration, and wound healing. However, VEGF is also important in animals for the regulation of angiogenesis, stem cell and monocyte/macrophage recruitment, maintenance of kidney and lung barrier functions and neuroprotection. In addition to its role in regulating endothelial cell proliferation, migration, and cell survival, VEGF receptors are also located on many non-endothelial cells and act through autrocrine pathways to regulate cell survival and function. The following review will discuss the role of VEGF in physiological angiogenesis as well as its role in non-angiogenic processes that take place in adult organs.