A Mutant Form of Vascular Endothelial Growth Factor (VEGF) That Lacks VEGF Receptor-2 Activation Retains the Ability to Induce Vascular Permeability

Abstract

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.

Viral Vascular Endothelial Growth Factor Plays a Critical Role in Orf Virus Infection

“…It has been shown that VEGF family members that bind VEGFR-2 can stimulate the growth of this cell line in a specific and dose-dependent fashion (37). Figure 2B shows that CM from WT-infected BT cells was active in the bioassay but that CM from BT cells infected with ovVEGF-⌬ was not.…”

“…The Ba/F3-derived cell line Ba/F3-VEGFR-2-EpoR, expressing a chimeric receptor consisting of the extracellular domain of mouse VEGFR-2 and the transmembrane and cytoplasmic domains of the mouse erythropoietin receptor, was used to determine if CM was capable of binding and activating VEGFR-2 (37). Cells were washed free of interleukin 3 and then resuspended in dilutions of CM from virus-infected cells or from COS-7 cells transiently expressing the mouse VEGF isoform consisting of 164 amino acids, VEGF 164 (37). Cells were incubated for 48 h, and DNA synthesis was quantified by measuring [ 3 H]thymidine incorporation with a beta counter.…”

“…The family members are all structurally similar and share a central VEGF homology domain, which contains eight cysteine residues that form part of a cysteine knot motif. The VEGF family members are ligands for a set of mammalian tyrosine kinase receptors (37). VEGF binds and activates both VEGF receptor 1 (VEGFR-1) (Flt-1) and VEGFR-2 (Flk-1 or KDR), while PlGF and VEGF-B bind only VEGFR-1.…”

A gene therapy for cancer based on the angiogenesis inhibitor, vasostatin

“…36 In vitro biological assay for vasostatin Biological activity of vasostatin was determined using conditioned medium from transfected cells in endothelial cell proliferation assay. 36,38 Briefly, 3000 endothelial cells (HUVECs and SVEC4-10), non-endothelial cells (NIH/3T3 fibroblast and T/G HA-VSMCs) or tumor cells (Meth A and LL/2c) were plated on to 96-well culture plates in triplicate and incubated (37°C, 5% CO 2 ) for 24 h in 100 l medium containing bFGF. The medium was replaced with 20 or 40 l of vasostatin-containing supernatant from transfected COS cells mentioned above or control cells.…”

“…After 72 h, the cell number was determined by a trypan blue dye exclusion test, and the percentage inhibition was calculated by the following formula as described: inhibition % = [(NϪN T )/(NϪNo)] × 100, where N is the number of untreated cells cultured for n days, No is the cell number on day 0, and N T is the number of treated cells cultured for n days. 3,36,38 …”