Differential roles of vascular endothelial growth factor receptors 1 and 2 in dendritic cell differentiation

Impaired Ag-presenting function in dendritic cells (DCs) due to abnormal differentiation is an important mechanism of tumor escape from immune control. A major role for vascular endothelial growth factor (VEGF) and its receptors, VEGFR1/Flt-1 and VEGFR2/KDR/Flk-1, has been documented in hemopoietic development. To study the roles of each of these receptors in DC differentiation, we used an in vitro system of myeloid DC differentiation from murine embryonic stem cells. Exposure of wild-type, VEGFR1(-/-), or VEGFR2(-/-) embryonic stem cells to exogenous VEGF or the VEGFR1-specific ligand, placental growth factor, revealed distinct roles of VEGF receptors. VEGFR1 is the primary mediator of the VEGF inhibition of DC maturation, whereas VEGFR2 tyrosine kinase signaling is essential for early hemopoietic differentiation, but only marginally affects final DC maturation. SU5416, a VEGF receptor tyrosine kinase inhibitor, only partially rescued the mature DC phenotype in the presence of VEGF, suggesting the involvement of both tyrosine kinase-dependent and independent inhibitory mechanisms. VEGFR1 signaling was sufficient for blocking NF-kappaB activation in bone marrow hemopoietic progenitor cells. VEGF and placental growth factor affect the early stages of myeloid/DC differentiation. The data suggest that therapeutic strategies attempting to reverse the immunosuppressive effects of VEGF in cancer patients might be more effective if they specifically targeted VEGFR1.     

Receptor chimeras indicate that the vascular endothelial growth factor receptor-1 (VEGFR-1) modulates mitogenic activity of VEGFR-2 in endothelial cells

Vascular endothelial growth factor (VEGF) provokes angiogenesis in vivo and stimulates growth and differentiation of endothelial cells in vitro. Although VEGF receptor-1 (VEGFR-1) and VEGFR-2 are known to be high affinity receptors for VEGF, it is not clear which of the VEGFRs are responsible for the transmission of the diverse biological responses of VEGF. For this purpose we have constructed a chimeric receptor for VEGFR-1 (CTR) and VEGFR-2 (CKR) in which the extracellular domain of each receptor was replaced with the extracellular domain of human colony-stimulating factor-1 receptor (CSF-1R), and these receptors were expressed in pig aortic endothelial (PAE) cells. We show that CKR individually expressed in PAE cells is readily tyrosine-phosphorylated in vivo, autophosphorylated in vitro, and stimulates cell proliferation in a CSF-1-dependent manner. In contrast, CTR individually expressed in PAE cells showed no significant in vivo, in vitro tyrosine phosphorylation and cell growth in response to CSF-1 stimulation. The kinase activity of CKR was essential for its biological activity, since mutation of lysine 866 to arginine abolished its in vivo, in vitro tyrosine phosphorylation and mitogenic signals. Remarkably, activation of CTR repressed CKR-mediated mitogen-activate protein kinase activation and cell proliferation. Similar effects were observed for VEGFR-2 co-expressed with VEGFR-1. Collectively, these findings demonstrate that VEGFR-2 activation plays a positive role in angiogenesis by promoting endothelial cell proliferation. In contrast, activation of VEGFR-1 plays a stationary role in angiogenesis by antagonizing VEGFR-2 responses.     

Heterodimerization with vascular endothelial growth factor receptor-2 (VEGFR-2) is necessary for VEGFR-3 activity

VEGFR-3 is essential for vascular development and maintenance of lymphatic vessel's integrity. Little is known about its cooperative effect with other receptors of the same family. Contrary to VEGFR-2, stimulation of VEGFR-3 by VEGF-C and -D failed to enhance its phosphorylation either in HEK293T or in PAE cells. These ligands were unable to induce angiogenesis of PAEC expressing VEGFR-3 alone. In the presence of VEGFR-2, VEGF-C and -D induced heterodimerization of VEGFR-3 with VEGFR-2. This heterodimerization was associated with enhanced VEGFR-3 phosphorylation and subsequent cellular responses as evidenced by the formation of capillary-like structures in PAE cells and proliferation of primary human endothelial cells expressing both receptors. Taken together, these results show for the first time that VEGFR-3 needs to be associated to VEGFR-2 to induce ligand-dependent cellular responses.     

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.     

The antifungal drug itraconazole inhibits vascular endothelial growth factor receptor 2 (VEGFR2) glycosylation, trafficking, and signaling in endothelial cells

Itraconazole is a safe and widely used antifungal drug that was recently found to possess potent antiangiogenic activity. Currently, there are four active clinical trials evaluating itraconazole as a cancer therapeutic. Tumor growth is dependent on angiogenesis, which is driven by the secretion of growth factors from the tumor itself. We report here that itraconazole significantly inhibited the binding of vascular endothelial growth factor (VEGF) to VEGF receptor 2 (VEGFR2) and that both VEGFR2 and an immediate downstream substrate, phospholipase C γ1, failed to become activated after VEGF stimulation. These effects were due to a defect in VEGFR2 trafficking, leading to a decrease in cell surface expression, and were associated with the accumulation of immature N-glycans on VEGFR2. Small molecule inducers of lysosomal cholesterol accumulation and mammalian target of rapamycin (mTOR) inhibition, two previously reported itraconazole activities, failed to recapitulate itraconazole's effects on VEGFR2 glycosylation and signaling. Likewise, glycosylation inhibitors did not alter cholesterol trafficking or inhibit mTOR. Repletion of cellular cholesterol levels, which was known to rescue the effects of itraconazole on mTOR and cholesterol trafficking, was also able to restore VEGFR2 glycosylation and signaling. This suggests that the new effects of itraconazole occur in parallel to those previously reported but are downstream of a common target. We also demonstrated that itraconazole globally reduced poly-N-acetyllactosamine and tetra-antennary complex N-glycans in endothelial cells and induced hypoglycosylation of the epidermal growth factor receptor in a renal cell carcinoma line, suggesting that itraconazole's effects extend beyond VEGFR2.     

Selective inhibition of vascular endothelial growth factor receptor-2 (VEGFR-2) identifies a central role for VEGFR-2 in human aortic endothelial cell responses to VEGF

Vascular endothelial growth factor receptors (VEGFR) are considered essential for angiogenesis. The VEGFR-family proteins consist of VEGFR-1/Flt-1, VEGFR-2/KDR/Flk-1, and VEGFR-3/Flt-4. Among these, VEGFR-2 is thought to be principally responsible for angiogenesis. However, the precise role of VEGFRs1-3 in endothelial cell biology and angiogenesis remains unclear due in part to the lack of VEGFR-specific inhibitors. We used the newly described, highly selective anilinoquinazoline inhibitor of VEGFR-2 tyrosine kinase, ZM323881 (5-[[7-(benzyloxy) quinazolin-4-yl]amino]-4-fluoro-2-methylphenol), to explore the role of VEGFR-2 in endothelial cell function. Consistent with its reported effects on VEGFR-2 [IC(50) < 2 nM], ZM323881 inhibited activation of VEGFR-2, but not of VEGFR-1, epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor (PDGFR), or hepatocyte growth factor (HGF) receptor. We studied the effects of VEGF on human aortic endothelial cells (HAECs), which express VEGFR-1 and VEGFR-2, but not VEGFR-3, in the absence or presence of ZM323881. Inhibition of VEGFR-2 blocked activation of extracellular regulated-kinase, p38, Akt, and endothelial nitric oxide synthetase (eNOS) by VEGF, but did not inhibit p38 activation by the VEGFR-1-specific ligand, placental growth factor (PIGF). Inhibition of VEGFR-2 also perturbed VEGF-induced membrane extension, cell migration, and tube formation by HAECs. Vascular endothelial growth factor receptor-2 inhibition also reversed VEGF-stimulated phosphorylation of CrkII and its Src homology 2 (SH2)-binding protein p130Cas, which are known to play a pivotal role in regulating endothelial cell migration. Inhibition of VEGFR-2 thus blocked all VEGF-induced endothelial cellular responses tested, supporting that the catalytic activity of VEGFR-2 is critical for VEGF signaling and/or that VEGFR-2 may function in a heterodimer with VEGFR-1 in human vascular endothelial cells.     

Ligand-induced vascular endothelial growth factor receptor-3 (VEGFR-3) heterodimerization with VEGFR-2 in primary lymphatic endothelial cells regulates tyrosine phosphorylation sites

Vascular endothelial growth factors (VEGFs) regulate the development and growth of the blood and lymphatic vascular systems. Of the three VEGF receptors (VEGFR), VEGFR-1 and -2 are expressed on blood vessels; VEGFR-2 is found also on lymphatic vessels. VEGFR-3 is expressed mainly on lymphatic vessels but it is also up-regulated in tumor angiogenesis. Although VEGFR-3 is essential for proper lymphatic development, its signal transduction mechanisms are still incompletely understood. Trans-phosphorylation of activated, dimerized receptor tyrosine kinases is known to be critical for the regulation of kinase activity and for receptor interaction with signal transduction molecules. In this study, we have identified five tyrosyl phosphorylation sites in the VEGFR-3 carboxyl-terminal tail. These sites were used both in VEGFR-3 overexpressed in 293 cells and when the endogenous VEGFR-3 was activated in lymphatic endothelial cells. Interestingly, VEGF-C stimulation of lymphatic endothelial cells also induced the formation of VEGFR-3/VEGFR-2 heterodimers, in which VEGFR-3 was phosphorylated only at three of the five sites while the two most carboxyl-terminal tyrosine residues appeared not to be accessible for the VEGFR-2 kinase. Our data suggest that the carboxyl-terminal tail of VEGFR-3 provides important regulatory tyrosine phosphorylation sites with potential signal transduction capacity and that these sites are differentially used in ligand-induced homo- and heterodimeric receptor complexes.     

Utilization of distinct signaling pathways by receptors for vascular endothelial cell growth factor and other mitogens in the induction of endothelial cell proliferation

This study was initiated to identify signaling proteins used by the receptors for vascular endothelial cell growth factor KDR/Flk1, and Flt1. Two-hybrid cloning and immunoprecipitation from human umbilical vein endothelial cells (HUVEC) showed that KDR binds to and promotes the tyrosine phosphorylation of phospholipase Cgamma (PLCgamma). Neither placental growth factor, which activates Flt1, epidermal growth factor (EGF), or fibroblast growth factor (FGF) induced tyrosine phosphorylation of PLCgamma, indicating that KDR is uniquely important to PLCgamma activation in HUVEC. By signaling through KDR, VEGF promoted the tyrosine phosphorylation of focal adhesion kinase, induced activation of Akt, protein kinase Cepsilon (PKCepsilon), mitogen-activated protein kinase (MAPK), and promoted thymidine incorporation into DNA. VEGF activates PLCgamma, PKCepsilon, and phosphatidylinositol 3-kinase independently of one another. MEK, PLCgamma, and to a lesser extent PKC, are in the pathway through which KDR activates MAPK. PLCgamma or PKC inhibitors did not affect FGF- or EGF-mediated MAPK activation. MAPK/ERK kinase inhibition diminished VEGF-, FGF-, and EGF-promoted thymidine incorporation into DNA. However, blockade of PKC diminished thymidine incorporation into DNA induced by VEGF but not FGF or EGF. Signaling through KDR/Flk1 activates signaling pathways not utilized by other mitogens to induce proliferation of HUVEC.     

Complex formation between tissue transglutaminase II (tTG) and vascular endothelial growth factor receptor 2 (VEGFR-2): proposed mechanism for modulation of endothelial cell response to VEGF

We have recently demonstrated that thrombin-activated FXIII (FXIIIA-subunit), a plasma transglutaminase, activates VEGFR-2 by crosslinking it with the alpha(v)beta(3) integrin on the surface of endothelial cells (EC), thereby stimulating angiogenesis. Tissue transglutaminase (tTG), which is functionally and structurally related to FXIIIA, is expressed by numerous cell types, among them EC. However, its role in EC function has not been fully characterized. In the present study, we investigated the potential involvement of tTG in angiogenesis. Using co-immunoprecipitation and immunofluorescent staining experiments, we observed that tTG forms a complex with VEGFR-2 on the cell surface and within the cytoplasm of EC. Stimulation of EC with VEGF resulted in translocation of the tTG-VEGFR-2 complex from the cytoplasm to the nucleus. In VEGF-treated cells, tTG-VEGFR-2 interaction resulted in incorporation of VEGFR-2 into high molecular weight crosslinked complex (es), as revealed by an antibody against gamma-glutamyl-epsilon-lysine isopeptide bond. tTG -VEGFR-2 association was inhibited by a specific VEGFR-2 protein tyrosine kinase inhibitor (PTKI ), as well as by cystamine, inhibitor of the transglutaminase activity of tTG, but not by bacitracin which inhibits the protein-disulfide isomerase (PDI) activity of tTG. Furthermore, cystamine completely abolished the VEGF-induced nuclear translocation of the tTG-VEGFR-2 complex. Blockade of the crosslinking activity of tTG by cystamine enhanced VEGF-induced migration of EC in Boyden chamber by 31% (P < 0.02), and prolonged VEGF-induced signaling response, as demonstrated by sustained activation of the MAP kinase ERK. Taken together, our findings suggest that endothelial cell tTG might be involved in modulation of the cellular response to VEGF by forming an intracellular complex with VEGFR-2, and mediating its translocation into the nucleus upon VEGF stimulation.     

185 Vascular endothelial growth factor receptor-2 (VEGFR2): structure and functional relationship

Vascular endothelial growth factor (VEGF), is expressed in the vicinity of sprouting vessels and its receptor (VEGF-R2/Flk-1/kdr) on the angioblasts and new vessels, and both are required for vasculogenesis and angiogenesis. VEGFR2, also called as KDR or Flk-1, is identified as an early marker for endothelial cell progenitors, whose expression is restricted to endothelial cells in vivo. VEGFR2 consists of extracellular (7-Ig-like sub-domains), transmembrane and cytoplasmic domains. In order to understand the structure–functional relationship and signal transduction process of VEGFR2, we have examined their amino acid sequences from a wide range of species including mammals, birds, Zebrafish and also computed the phylogenetic tree, secondary and domain structures. Phylogeny constructed using Maximum Parsimony tree software MEGA-5 version suggested an interesting sequence similarity between Zebrafish and Gallus, closeness between human, rat, horse and pig. Strong homology in amino acids sequences was observed between the species, such as human, Macaca mulatta, gorilla, etc, and small variations in Zebrafish and zebrafinch. The Arg and Asp residues which are involved in forming salt bridges are evolutionarily conserved from Zebrafish to human in D7 domain of VEGFR2, indicating their functional importance in VEGFR activity. Amino acids, tyrosine in the extracellular loops and cysteines involved in disulphide bridges of VEGFR2, are highly conserved suggesting their importance during ligand binding, the details of which will be discussed.     

Vascular endothelial growth factor and its soluble receptors VEGFR-1 and VEGFR-2 in the serum of patients with systemic lupus erythematosus

We investigated the serum concentration of vascular endothelial growth factor (VEGF) and its two soluble receptors, sVEGFR-1 and sVEGFR-2, in a group of 60 patients with systemic lupus erythematosus (SLE), and 20 healthy controls, using an enzyme-linked immunosorbent assay. We examined a possible association between serum levels of these proteins and certain clinical and laboratory parameters as well as SLE activity. VEGF, sVEGFR-1 and sVEGFR-2 were detectable in all patients with SLE and in all normal individuals. The VEGF level was higher in active SLE (mean, 300.8 pg/ml) than in inactive SLE (mean, 165.9 pg/ml) (p < 0.05) or in the control group (mean, 124.7 pg/ml) (p < 0.04). The highest sVEGFR-1 concentrations were also detected in active SLE patients (mean, 42.2 pg/ml) and the lowest in inactive disease (mean, 32.0 pg/ml) (p < 0.01). In contrast, the levels of sVEGFR-2 were lower in SLE (mean, 12557.6 pg/ml) than in the control group (mean, 15025.3 pg/ml) (p < 0.05). We found a positive correlation between sVEGFR-1 concentration and the SLE activity score p = 0.375 (p < 0.004) and a negative, but statistically insignificant correlation between sVEGFR-2 and SLE activity (p = -0.190, p > 0.05). Treatment with steroids and cytotoxic agents did not influence VEGF or its soluble receptors levels. In conclusion, in SLE patients the levels of VEGF and sVEGFR-1 are higher in patients with active SLE than in inactive disease or healthy persons. In contrast, the level of sVEGFR-2 is lower in active SLE than in inactive disease. The imbalance between VEGF and its soluble receptors may be important in SLE pathogenesis.     

Differential roles of vascular endothelial growth factor receptor-1 and receptor-2 in angiogenesis

Vascular endothelial growth factor (VEGF)-A, a major regulator for angiogenesis, binds and activates two tyrosine kinase receptors, VEGFR1 (Flt-1) and VEGFR2 (KDR/Flk-1). These receptors regulate physiological as well as pathological angiogenesis. VEGFR2 has strong tyrosine kinase activity, and transduces the major signals for angiogenesis. However, unlike other representative tyrosine kinase receptors which use the Ras pathway, VEGFR2 mostly uses the Phospholipase-Cgamma-Protein kinase-C pathway to activate MAP-kinase and DNA synthesis. VEGFR2 is a direct signal transducer for pathological angiogenesis including cancer and diabetic retinopathy, thus, VEGFR2 itself and the signaling appear to be critical targets for the suppression of these diseases. VEGFR1 plays dual role, a negative role in angiogenesis in the embryo most likely by trapping VEGF-A, and a positive role in adulthood in a tyrosine kinase-dependent manner. VEGFR1 is expressed not only in endothelial cells but also in macrophage-lineage cells, and promotes tumor growth, metastasis, and inflammation. Furthermore, a soluble form of VEGFR1 was found to be present at abnormally high levels in the serum of preeclampsia patients, and induces proteinurea and renal dysfunction. Therefore, VEGFR1 is also an important target in the treatment of human diseases. Recently, the VEGFR2-specific ligand VEGF-E (Orf-VEGF) was extensively characterized. Interestingly, the activation of VEGFR2 via VEGF-E in vivo results in a strong angiogenic response in mice with minor side effects such as inflammation compared with VEGF-A, suggesting VEGF-E to be a novel material for pro-angiogenic therapy.     

Fibulin-5 antagonizes vascular endothelial growth factor (VEGF) signaling and angiogenic sprouting by endothelial cells

Fibulin-5 (FBLN-5) is a widely expressed, integrin-binding extracellular matrix protein that mediates endothelial cell adhesion and scaffolds cells to elastic fibers. It is also a gene target of TGF-beta in fibroblasts and endothelial cells that regulates cell proliferation and motility in a context-specific manner. Whereas FBLN-5 expression is low in adult vasculature, its expression is high in developing and injured vasculature, implicating FBLN-5 in regulating angiogenesis and endothelial cell function. We show here that TGF-beta stimulates FBLN-5 expression in endothelial cells, and that this response was inhibited by coadministration of the proangiogenic factor, VEGF. FBLN-5 expression was downregulated significantly during endothelial cell tubulogenesis, implying that FBLN-5 expression antagonizes angiogenesis. Accordingly, FBLN-5 overexpression in or recombinant FBLN-5 treatment of endothelial cells abrogated their ability to undergo angiogenic sprouting, doing so by inhibiting endothelial cell proliferation and invasion through Matrigel matrices. Moreover, FBLN-5 antagonized VEGF signaling in endothelial cells, as well as enhanced their expression of the antiangiogenic factor, thrombospondin-1. Finally, the ability of FBLN-5 to antagonize angiogenic processes was determined to be independent of its integrin-binding RGD motif. Collectively, our findings establish FBLN-5 as a novel antagonist of angiogenesis and endothelial cell activities, and offer new insights into why tumorigenesis downregulates FBLN-5 expression.     

Nephrotic syndrome unfavorable course correlates with downregulation of podocyte vascular endothelial growth factor receptor (VEGFR)-2

Idiopathic nephrotic syndrome (INS) in children is most commonly caused by primary glomerulopathies. Morphological lesions observed in INS might be secondary to inflammatory factors of mainly extra-renal origin. The vascular endothelial growth factor (VEGF) family is regarded as playing a crucial role in this pathomechanism. The aim of the present work was to analyze the possible relation between VEGF-C and VEGF receptor (VEGFR)-2 expressions at electron microscopy level in different INS cases. The study group comprised 18 children with minimal change disease (MCD), 30 patients diagnosed with diffuse mesangial proliferation (DMP) and 11 subjects with focal segmental glomerulosclerosis (FSGS). An indirect immunohistochemical assay employing monoclonal anti-VEGF-C and anti-VEGFR-2 antibodies was applied in the study. The immunohistochemical expression of VEGF-C within podocyte cytoplasm was significantly increased in DMP subjects who were resistant to steroids and in all FSGS patients compared to MCD children and controls (p 〈 0.05). VEGF-C over-expression in these cases was followed by downregulation of VEGFR-2. Nephrotic syndrome progression correlates with the downregulation of podocyte VEGFR-2. For this reason, decreased VEGFR-2 expression in the podocyte processes of children with idiopathic nephrotic syndrome might be regarded as a potent factor of unfavorable prognosis.     

Alzheimer's β-amyloid peptide blocks vascular endothelial growth factor mediated signaling via direct interaction with VEGFR-2

Beta-amyloid peptides (Aβ) are the major constituents of senile plaques and cerebrovascular deposits in the brains of Alzheimer's disease patients. We have shown previously that soluble forms of Aβ are anti-angiogenic both in vitro and in vivo . However, the mechanism of the anti-angiogenic activity of Aβ peptides is unclear. In this study, we examined the effects of Aβ1–42 on vascular endothelial growth factor receptor 2 (VEGFR-2) signaling, which plays a key role in angiogenesis. Aβ inhibited VEGF-induced migration of endothelial cells, as well as VEGF-induced permeability of an in vitro model of the blood brain barrier. Consistently, exogenous VEGF dose-dependently antagonized the anti-angiogenic activity of Aβ in a capillary network assay. Aβ1–42 also blocked VEGF-induced tyrosine phosphorylation of VEGFR-2 in two types of primary endothelial cells, suggesting an antagonistic action of Aβ toward VEGFR-2 signaling in cells. Moreover, Aβ was able to directly interact with the extracellular domain of VEGFR-2 and to compete with the binding of VEGF to its receptor in a cell-free assay. Co-immunoprecipitation experiments confirmed that Aβ can bind VEGFR-2 both in vitro and in vivo . Altogether, our data suggest that Aβ acts as an antagonist of VEGFR-2 and provide a mechanism explaining the anti-angiogenic activity of Aβ peptides.     

A novel vascular endothelial growth factor encoded by Orf virus, VEGF-E, mediates angiogenesis via signalling through VEGFR-2 (KDR) but not VEGFR-1 (Flt-1) receptor tyrosine kinases.

The different members of the vascular endothelial growth factor (VEGF) family act as key regulators of endothelial cell function controlling vasculogenesis, angiogenesis, vascular permeability and endothelial cell survival. In this study, we have functionally characterized a novel member of the VEGF family, designated VEGF-E. VEGF-E sequences are encoded by the parapoxvirus Orf virus (OV). They carry the characteristic cysteine knot motif present in all mammalian VEGFs, while forming a microheterogenic group distinct from previously described members of this family. VEGF-E was expressed as the native protein in mammalian cells or as a recombinant protein in Escherichia coli and was shown to act as a heat-stable, secreted dimer. VEGF-E and VEGF-A were found to possess similar bioactivities, i.e. both factors stimulate the release of tissue factor (TF), the proliferation, chemotaxis and sprouting of cultured vascular endothelial cells in vitro and angiogenesis in vivo. Like VEGF-A, VEGF-E was found to bind with high affinity to VEGF receptor-2 (KDR) resulting in receptor autophosphorylation and a biphasic rise in free intracellular Ca2+ concentration, whilst in contrast to VEGF-A, VEGF-E did not bind to VEGF receptor-1 (Flt-1). VEGF-E is thus a potent angiogenic factor selectively binding to VEGF receptor-2. These data strongly indicate that activation of VEGF receptor-2 alone can efficiently stimulate angiogenesis.     

Complexity in the vascular permeability factor/vascular endothelial growth factor (VPF/VEGF)-receptors signaling

The adult vasculature results from a network of vessels that is originally derived in the embryo by vasculogenesis, a process whereby vessels are formed de novo from endothelial cell (EC) precursors, known as angioblasts. During vasculogenesis, angioblasts proliferate and come together to form an initial network of vessels, also known as the primary capillary plexus. Sprouting and branching of new vessels from the preexisting vessels in the process of angiogenesis remodel the capillary plexus. Normal angiogenesis, a well-balanced process, is important in the embryo to promote primary vascular tree as well as an adequate vasculature from developing organs. On the other hand, pathological angiogenesis which frequently occurs in tumors, rheumatoid arthritis, diabetic retinopathy and other circumstances can induce their own blood supply from the preexisting vasculature in a route that is close to normal angiogenesis. Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) is perhaps the most important of pro-angiogenic cytokine because of its ability to regulate most of the steps in the angiogenic cascade. The main goal of this review article is to discuss the complex nature of the mode of action of VPF/VEGF on vascular endothelium. To this end, we conclude that more research needs to be done for completely understanding the VPF/VEGF biology with relation to angiogenesis.     

Complexity in the vascular permeability factor/vascular endothelial growth factor (VPF/VEGF)-receptors signaling

The adult vasculature results from a network of vessels that is originally derived in the embryo by vasculogenesis, a process whereby vessels are formed de novo from endothelial cell (EC) precursors, known as angioblasts. During vasculogenesis, angioblasts proliferate and come together to form an initial network of vessels, also known as the primary capillary plexus. Sprouting and branching of new vessels from the preexisting vessels in the process of angiogenesis remodel the capillary plexus. Normal angiogenesis, a well-balanced process, is important in the embryo to promote primary vascular tree as well as an adequate vasculature from developing organs. On the other hand, pathological angiogenesis which frequently occurrs in tumors, rheumatoid arthritis, diabetic retinopathy and other circumstances can induce their own blood supply from the preexisting vasculature in a route that is close to normal angiogenesis. Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) is perhaps the most important of pro-angiogenic cytokine because of its ability to regulate most of the steps in the angiogenic cascade. The main goal of this review article is to discuss the complex nature of the mode of action of VPF/VEGF on vascular endothelium. To this end, we conclude that more research needs to be done for completely understanding the VPF/VEGF biology with relation to angiogenesis. (Mol Cell Biochem 264: 51–61, 2004)     

Hypoxia differentially regulates VEGFR1 and VEGFR2 levels and alters intracellular signaling and cell migration in endothelial cells

The role of hypoxia on endothelial cell function and response to growth factors is unknown. Here, we tested the hypothesis that hypoxia re-programs endothelial function by modulating vascular endothelial growth factor receptor levels which in turn alter intracellular signaling and cell function. Hypoxia stimulated VEGF-A and VEGFR1 expression but decreased VEGFR2 levels in endothelial cells. During hypoxia, plasma membrane VEGFR1 levels were elevated whereas VEGFR2 levels were depleted. One functional consequence of hypoxia is a reduction in VEGF-A-stimulated and VEGFR2-regulated intracellular signaling including lowered endothelial nitric oxide synthase activation. Venous, arterial and capillary endothelial cells subjected to hypoxia all exhibited reduced cell migration in response to VEGF-A. A mechanistic explanation is that VEGFR1:VEGFR2 ratio is substantially increased during hypoxia to block VEGF-A-stimulated and VEGFR2-regulated endothelial responses to maximize cell viability and recovery.