Vascular Endothelial Growth Factor Signals Endothelial Cell Production of Nitric Oxide and Prostacyclin through Flk-1/KDR Activation of c-Src

Vascular endothelial growth factor (VEGF) is a potent endothelial cell-specific mitogen that promotes angiogenesis, vascular hyperpermeability, and vasodilation by autocrine mechanisms involving nitric oxide (NO) and prostacyclin (PGI(2)) production. These experiments used immunoprecipitation and immunoassay procedures to characterize the signaling pathways by which VEGF induces NO and PGI(2) formation in cultured endothelial cells. The data showed that VEGF stimulates complex formation of the flk-1/kinase-insert domain-containing receptor (KDR) VEGF receptor with c-Src and that Src activation is required for VEGF induction of phospholipase C gamma1 activation and inositol 1,4,5-trisphosphate formation. Reporter cell assays showed that VEGF promotes a approximately 50-fold increase in NO formation, which peaks at 5-20 min. This effect is mediated by a signaling cascade initiated by flk-1/KDR activation of c-Src, leading to phospholipase C gamma1 activation, inositol 1,4,5-trisphosphate formation, release of Ca(2+)(i) and nitric oxide synthase activation. Immunoassays of VEGF-induced 6-keto prostaglandin F(1alpha) formation as an indicator of PGI(2) production revealed a 3-4-fold increase that peaked at 45-60 min. The PGI(2) signaling pathway follows the NO pathway through release of Ca(2+)(i), but diverges prior to NOS activation and also requires activation of mitogen-activated protein kinase. These results suggest that NO and PGI(2) function in parallel in mediating the effects of VEGF.