Viral vascular endothelial growth factor plays a critical role in orf virus infection

Infection by the parapoxvirus orf virus causes proliferative skin lesions in which extensive capillary proliferation and dilation are prominent histological features. This infective phenotype may be linked to a unique virus-encoded factor, a distinctive new member of the vascular endothelial growth factor (VEGF) family of molecules. We constructed a recombinant orf virus in which the VEGF-like gene was disrupted and show that inactivation of this gene resulted in the loss of three VEGF activities expressed by the parent virus: mitogenesis of vascular endothelial cells, induction of vascular permeability, and activation of VEGF receptor 2. We used the recombinant orf virus to assess the contribution of the viral VEGF to the vascular response seen during orf virus infection of skin. Our results demonstrate that the viral VEGF, while recognizing a unique profile of the known VEGF receptors (receptor 2 and neuropilin 1), is able to stimulate a striking proliferation of blood vessels in the dermis underlying the site of infection. Furthermore, the data demonstrate that the viral VEGF participates in promoting a distinctive pattern of epidermal proliferation. Loss of a functional viral VEGF resulted in lesions with markedly reduced clinical indications of infection. However, viral replication in the early stages of infection was not impaired, and only at later times did it appear that replication of the recombinant virus might be reduced.     

A mutant form of vascular endothelial growth factor (VEGF) that lacks VEGF receptor-2 activation retains the ability to induce vascular permeability

Vascular endothelial growth factor (VEGF) is a major mediator of vasculogenesis and angiogenesis both during development and in pathological conditions. VEGF has a variety of effects on vascular endothelium, including the ability to stimulate endothelial cell mitogenesis, and the potent induction of vascular permeability. These activities are at least in part mediated by binding to two high affinity receptors, VEGFR-1 and VEGFR-2. In this study we have made mutations of mouse VEGF in order to define the regions that are required for VEGFR-2-mediated functions. Development of a bioassay, which responds only to signals generated by cross-linking of VEGFR-2, has allowed evaluation of these mutants for their ability to activate VEGFR-2. One mutant (VEGF0), which had amino acids 83-89 of VEGF substituted with the analogous region of the related placenta growth factor, demonstrated significantly reduced VEGFR-2 binding compared with wild type VEGF, indicating that this region was required for VEGF-VEGFR-2 interaction. Intriguingly, when this mutant was evaluated in a Miles assay for its ability to induce vascular permeability, no difference was found when compared with wild type VEGF. In addition we have shown that the VEGF homology domain of the structurally related growth factor VEGF-D is capable of binding to and activating VEGFR-2 but has no vascular permeability activity, indicating that VEGFR-2 binding does not correlate with permeability activity for all VEGF family members. These data suggest different mechanisms for VEGF-mediated mitogenesis and vascular permeability and raise the possibility of an alternative receptor mediating vascular permeability.     

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.     

Expression of vascular endothelial growth factor receptors 1, 2, and 3 in quiescent endothelia.

The vascular endothelial growth factor (VEGF) family is involved in angiogenesis, and therefore VEGFs are considered as targets for anti-angiogenic therapeutic strategies against cancer. However, the physiological functions of VEGFs in quiescent tissues are unclear and may interfere with such systemic therapies. In pathological conditions, increased levels of expression of the VEGF receptors VEGFR-1, VEGFR-2, and VEGFR-3 accompany VEGF activity. In this study we investigated normal human and monkey tissues for expression patterns of these receptors. Immunohistochemical staining methods at the light and electron microscopic level were applied to normal human and monkey tissue samples, using monoclonal antibodies (MAbs) against the three VEGFRs and anti-endothelial MAbs PAL-E and anti-CD31 to identify blood and lymph vessels. In human and monkey, similar distribution patterns of the three VEGFRs were found. Co-expression of VEGFR-1, -2, and -3 was observed in microvessels adjacent to epithelia in the eye, gastrointestinal mucosa, liver, kidney, and hair follicles, which is in line with the reported preferential expression of VEGF-A in some of these epithelia. VEGFR-1, -2, and -3 expression was also observed in blood vessels and sinusoids of lymphoid tissues. Furthermore, VEGFR-1, but not VEGFR-2 and -3, was present in microvessels in brain and retina. Electron microscopy showed that VEGFR-1 expression was restricted to pericytes and VEGFR-2 to endothelial cells in normal vasculature of tonsils. These findings indicate that VEGFRs have specific distribution patterns in normal tissues, suggesting physiological functions of VEGFs that may be disturbed by systemic anti-VEGF therapy. One of these functions may be involvement of VEGF in paracrine relations between epithelia and adjacent capillaries.     

Crystal structure of the Orf virus NZ2 variant of vascular endothelial growth factor-E. Implications for receptor specificity

Mammalian vascular endothelial growth factors constitute a family of polypeptides, vascular endothelial growth factor (VEGF)-A, -B, -C, -D and placenta growth factor (PlGF), that regulate blood and lymphatic vessel development. VEGFs bind to three types of receptor tyrosine kinases, VEGF receptors 1, 2, and 3, that are predominantly expressed on endothelial and some hematopoietic cells. Pox viruses of the Orf family encode highly related proteins called VEGF-E that show only 25-35% amino acid identity with VEGF-A but bind with comparable affinity to VEGFR-2. The crystal structure of VEGF-E NZ2 described here reveals high similarity to the known structural homologs VEGF-A, PlGF, and the snake venoms Vammin and VR-1, which are all homodimers and contain the characteristic cysteine knot motif. Distinct conformational differences are observed in loop L1 and particularly in L3, which contains a highly flexible GS-rich motif that differs from all other structural homologs. Based on our structure, we created chimeric proteins by exchanging selected segments in L1 and L3 with the corresponding sequences from PlGF. Single loop mutants did not bind to either receptor, whereas a VEGF-E mutant in which both L1 and L3 were replaced gained affinity for VEGFR-1, illustrating the possibility to engineer receptor-specific chimeric VEGF molecules. In addition, changing arginine 46 to isoleucine in L1 significantly increased the affinity of VEGF-E for both VEGF receptors.     

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.     

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 biflavonoid amentoflavone inhibits neovascularization preventing the activity of proangiogenic vascular endothelial growth factors

The proangiogenic members of VEGF family and related receptors play a central role in the modulation of pathological angiogenesis. Recent insights indicate that, due to the strict biochemical and functional relationship between VEGFs and related receptors, the development of a new generation of agents able to target contemporarily more than one member of VEGFs might amplify the antiangiogenic response representing an advantage in term of therapeutic outcome. To identify molecules that are able to prevent the interaction of VEGFs with related receptors, we have screened small molecule collections consisting of >100 plant extracts. Here, we report the isolation and identification from an extract of the Malian plant Chrozophora senegalensis of the biflavonoid amentoflavone as an antiangiogenic bioactive molecule. Amentoflavone can to bind VEGFs preventing the interaction and phosphorylation of VEGF receptor 1 and 2 (VEGFR-1,VEGFR-2) and to inhibit endothelial cell migration and capillary-like tube formation induced by VEGF-A or placental growth factor 1 (PlGF-1) at low μm concentration. In vivo, amentoflavone is able to inhibit VEGF-A-induced chorioallantoic membrane neovascularization as well as tumor growth and associated neovascularization, as assessed in orthotropic melanoma and xenograft colon carcinoma models. In addition structural studies performed on the amentoflavone·PlGF-1 complex have provided evidence that this biflavonoid effectively interacts with the growth factor area crucial for VEGFR-1 receptor recognition. In conclusion, our results demonstrate that amentoflavone represents an interesting new antiangiogenic molecule that is able to prevent the activity of proangiogenic VEGF family members and that the biflavonoid structure is a new chemical scaffold to develop powerful new antiangiogenic molecules.     

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.     

Lymphangiogenesis and biological activity ov vascular endothelial growth factor-C]

Vascular endothelial growth factor (VEGF)-C is a new member of the VEGF family, a group of polypeptide growth factors which play key roles in the physiology and pathology of many aspects of the cardiovascular system, including vasculogenesis, hematopoiesis, angiogenesis and vascular permeability. VEGF signalling in endothelial cells occurs through three tyrosine kinase receptors (VEGFRs), expressed by endothelial cells and hematopoietic precursors. With respect to the first VEGF described, VEGF-A, which is an endothelial cell specific mitogen and key angiogenic factor, VEGF-C seems to play a major role in the development of the lymphatic system. This may reflect the different binding properties of VEGFs to VEGFRs, in that VEGF-A binds to VEGFR-1 and -2, whereas VEGF-C acts through VEGFR-3, whose expression becomes restricted to lymphatics and certain veins during development. However, the finding that VEGF-C also binds to and activates VEGFR-2 may explain why it induces angiogenesis under certain conditions, which makes it relevant to experimental or clinical settings in which one would wish to block or to stimulate angiogenesis. In this paper we briefly discuss current knowledge on the biological activity of VEGF-C, emphasizing that, as has already been shown for a number of other angiogenic factors, the biological effects of VEGF-C are strictly dependent on the activity of other angiogenic regulators present in the microenvironment of the responding endothelial cells.     

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.     

Lipocortin V may function as a signaling protein for vascular endothelial growth factor receptor-2/Flk-1.

Binding of vascular endothelial growth factor (VEGF) to its receptor, VEGFR-2 (Flk-1/KDR), induces dimerization and activation of the tyrosine kinase domain of the receptor, resulting in autophosphorylation of cytoplasmic tyrosine residues used as docking sites for signaling proteins that relay the signals for cell proliferation, migration, and permeability enhancement. We explored the VEGF/receptor signaling pathway by performing a two-hybrid screen of a rat lung cDNA library in yeast using the intracellular domain of rat VEGFR-2 as bait. Two clones encoding lipocortin V were isolated. Subsequent studies with the yeast two-hybrid assay showed that the complete intracellular domain of VEGFR-2 was required for the interaction. Co-immunoprecipitation of translated proteins confirmed the interaction between the VEGF receptor and lipocortin V. VEGF induced a rapid tyrosine phosphorylation of lipocortin V in human umbilical vein endothelial cells (HUVEC). Pretreatment of HUVEC with antisense oligodeoxyribonucleotide (ODN) for lipocortin V significantly inhibited VEGF-induced cell proliferation, which was accompanied by a decrease in protein synthesis and tyrosine phosphorylation of lipocortin V. Our results indicate that lipocortin V may function as a signaling protein for VEGFR-2 by directly interacting with the intracellular domain of the receptor and appears to be involved in regulation of vascular endothelial cell proliferation mediated by VEGFR-2.     

Vascular endothelial growth factor receptor-2: structure, function, intracellular signalling and therapeutic inhibition

Vascular endothelial growth factors (VEGFs) regulate vascular development, angiogenesis and lymphangiogenesis by binding to a number of receptors. VEGFR-1 is required for the recruitment of haematopoietic stem cells and the migration of monocytes and macrophages, VEGFR-2 regulates vascular endothelial function and VEGFR-3 regulates lymphatic endothelial cell function. Over the last decade, considerable progress has been made in delineating the VEGFR-2 specific intracellular signalling cascades leading to proliferation, migration, survival and increased permeability, each of which contributes to the angiogenic response. Furthermore, therapeutic inhibition of VEGFR-2 action is now having an impact in the clinic for the treatment of a number of diseases.     

Expression of VEGF-C and activation of its receptors VEGFR-2 and VEGFR-3 in trophoblast

Placental villous development requires the co-ordinated action of angiogenic factors on both endothelial and trophoblast cells. Like vascular endothelial growth factor (VEGF), VEGF-C increases vascular permeability, stimulates endothelial cell proliferation and migration. In the present study, we investigated the expression of VEGF-C and its receptors VEGFR-3 and VEGFR-2 in normal and intrauterine growth-restricted (IUGR) placenta. Immunolocalisation studies showed that like VEGF and VEGFR-1, VEGF-C, VEGFR-3 and VEGFR-2 co-localised to the syncytiotrophoblast, to cells in the maternal decidua, as well as to the endothelium of the large placental blood vessels. Western blot analysis demonstrated a significant decrease in placental VEGF-C and VEGFR-3 protein expression in severe IUGR as compared to gestationally-matched third trimester pregnancies. Conditioned medium from VEGF-C producing pancreatic carcinoma (Suit-2) and endometrial epithelial (Hec-1B) cell lines caused an increased association of the phosphorylated extracellular signal regulated kinase (ERK) in VEGFR-3 immunoprecipitates from spontaneously transformed first trimester trophoblast cells. VEGF121 caused dose-dependant phosphorylation of VEGFR-2 in trophoblast cells as well as stimulating DNA synthesis. In addition, premixing VEGF165 with heparin sulphate proteoglycan potentiated trophoblast proliferation and the association of phospho-ERK with the VEGFR-2 receptor. VEGF165-mediated DNA synthesis was inhibited by anti-VEGFR-2 neutralising antibody. The results demonstrate functional VEGFR-2 and VEGFR-3 receptors on trophoblast and suggest that the decreased expression of VEGF-C and VEGFR-3 may contribute to the abnormal villous development observed in IUGR placenta.     

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.     

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.     

Snake venom vascular endothelial growth factors (VEGFs) exhibit potent activity through their specific recognition of KDR (VEGF receptor 2)

Vascular endothelial growth factor (VEGF165) exhibits multiple effects via the activation of two distinct endothelial receptor tyrosine kinases: Flt-1 (fms-like tyrosine kinase-1) and KDR (kinase insert domain-containing receptor). KDR shows strong ligand-dependent tyrosine phosphorylation in comparison with Flt-1 and mainly mediates the mitogenic, angiogenic, and permeability-enhancing effects of VEGF165. Here we show the isolation of two VEGFs from viper venoms and the characterization of their unique biological properties. Snake venom VEGFs strongly stimulated proliferation of vascular endothelial cells in vitro. Interestingly, the maximum activities were almost twice that of VEGF165. They also induced strong hypotension on rat arterial blood pressure compared with VEGF165 in vivo. A receptor binding assay revealed that snake venom VEGFs bound to KDR-IgG with high affinity (Kd = approximately 0.1 nm) as well as to VEGF165 but did not interact with Flt-1, Flt-4, or neuropilin-1 at all. Our data clearly indicate that snake venom VEGFs act through the specific activation of KDR and show potent effects. Snake venom VEGFs are a highly specific ligand to KDR and form a new group of the VEGF family.     

血管内皮生长因子受体-2所介导信号通路的研究进展

血管新生是许多生理和病理进程发生的重要机理．在生物体内,血管新生需经过多步精细调控历程,现有研究表明,血管内皮生长因子(VEGF)及其受体蛋白酪氨酸激酶,尤其是血管内皮生长因子受体-2(VEGFR-2)所介导的信号级联通路是其中关键性的调节途径．VEGF/VEGFR-2所介导的信号级联通路可以调控血管内皮细胞的增殖、迁移、存活和通透性的改变,促进血管的新生．VEGF与VEGFR-2的胞外区特异性结合后,引起受体的二聚化和自身的交互磷酸化,使胞内特定的酪氨酸残基磷酸化．下游信号蛋白可以通过其Src同源结构域-2(SH2)与VEGFR-2结合,随后激活下游的效应蛋白,调控内皮细胞的生物学活性．此外,VEGF/VEGFR-2信号通路还可以下调树突细胞(DC)的活性．对VEGF/VEGFR-2信号通路作用的深入了解,将有助于新药的研发．     

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.     

Tyrosine residues 951 and 1059 of vascular endothelial growth factor receptor-2 (KDR) are essential for vascular permeability factor/vascular endothelial growth factor-induced endothelium migration and proliferation, respectively.

Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) exerts its multiple functions by activating two receptor tyrosine kinases, Flt-1 (VEGFR-1) and KDR (VEGFR-2), both of which are selectively expressed on primary vascular endothelium. To dissect the respective signaling pathways and biological functions mediated by these receptors in primary endothelial cells with two receptors intact, we, recently developed chimeric receptors (EGDR and EGLT) in which the extracellular domain of the epidermal growth factor receptor was fused to the transmembrane domain and intracellular domain of KDR and Flt-1, respectively. With these fusion receptors, we have shown that KDR is solely responsible for VPF/VEGF-induced human umbilical vein endothelial cell (HUVEC) proliferation and migration, whereas Flt-1 showed an inhibitory effect on KDR-mediated proliferation but not migration. To further characterize the VPF/VEGF-stimulated HUVEC proliferation and migration here, we have created several EGDR mutants by site-directed mutagenesis. We show that tyrosine residues 1059 and 951 of KDR are essential for VPF/VEGF-induced HUVEC proliferation and migration, respectively. Furthermore, the mutation of tyrosine 1059 to phenylanaline results in the complete loss of KDR/EGDR-mediated intracellular Ca(2+) mobilization and MAPK phosphorylation, but the mutation of tyrosine 951 to phenylanaline did not affect these events. Our results suggest that KDR mediates different signaling pathways for HUVEC proliferation and migration and, moreover, intracellular Ca(2+) mobilization and MAPK phosphorylation are not essential for VPF/VEGF-induced HUVEC migration.