VEGF’s Angiogenic Function Involves Peroxynitrite–Mediated Activation of VEGFR2/Src/FAK.

Abstract

Abstract: : Purpose: We have previously shown that VEGF causes rapid increases in the formation of superoxide anion, nitric oxide and their combination product peroxynitrite. We have shown also that treatment with physiological levels of peroxynitrite mimicks the effects of VEGF in increasing endothelial cell growth and their alignment in capillary–like structures in three–dimensional cultures. These effects were blocked by inhibiting peroxynitrite or by reducing its formation. The purpose of this study was to further characterize the angiogenic action of peroxynitrite in vivo and to define the signaling pathway underlying these effects. Methods: The chick chorioallantoic membrane (CAM) assay was used to compare the effects of VEGF and peroxynitrite on angiogenesis in vivo. Western blot and immunoprecipitation techniques were used to compare the effects of VEGF and peroxynitrite on the in vitro activation of three signaling molecules known to be involved in VEGF’s angiogenic function, VEGFR2, Src and focal adhesion kinase (FAK). Results: These studies showed that peroxynitrite (1 µM) mimicked the action of VEGF (20 ng/ml) in stimulating angiogenesis in vivo and inducing time dependent increases in the phosphorylation of VEGFR2, Src and FAK in cultured retinal endothelial cells in vitro. The specific peroxynitrite inhibitor FETPPS (2.5 µm) blocked the effects of VEGF in activating VEGFR2, Src and FAK. Conclusions: Taken together, these data indicate that peroxynitrite is a critical mediator in VEGF’s angiogenic signaling pathway. Treatments targeting peroxynitrite may be effective in anti–angiogenic therapy.

Keywords: oxidation/oxidative or free radical damage • retinal neovascularization • signal transduction