PROTEIN-KINASE G MEDIATES VASCULAR ENDOTHELIAL GROWTH FACTOR-INDUCED RAF-1 ACTIVATION AND PROLIFERATION IN HUMAN ENDOTHELIAL-CELLS

Vascular endothelial growth factor (VEGF) is an endothelium-specific, secreted protein that acts as a vasodilator, angiogenic peptide, and h yperpermeability factor. Recent reports have shown that nitric oxide s ynthase inhibitors block proliferation and microvascular hyperpermeabi lity induced by VEGF. This study examined the mechanisms by which nitr ic oxide and its downstream signals mediate the VEGF-induced prolifera tive response in human umbilical vein endothelial cells (HUVECs), Nitr ic oxide synthase blockade by N-G-nitro-L-arginine methyl ester preven ted both the proliferative effect of VEGF and Raf-1 activation by VEGF as measured by cell counting and the capacity of immunoprecipitated R af-1 to phosphorylate syntide 2, a Raf-1-specific synthetic substrate. VEGF-induced proliferation and Raf-1 kinase activity were also inhibi ted by Rp-8-pCPT-cGMPs and KT5823, inhibitors of the regulatory and ca talytic subunits of cGMP-dependent protein kinase (PKG), respectively. The ability of PKG to stimulate proliferation was verified by the obs ervation that the PKG activator, 8-pCPT-cGMPs, stimulated both Raf-1 k inase activity and endothelial proliferation in a dose-dependent manne r. Furthermore, recombinant catalytically active PKG phosphorylated an d activated Raf-1 in a reconstituted system. Finally, Raf-1 immunoprec ipitated from VEGF-stimulated endothelial cells coprecipitated with PK G, indicating a direct protein-protein interaction in activated cells. We conclude that VEGF induces increases in both proliferation and Raf -1 kinase activity in HUVECs and these activities are dependent on NO and its downstream effector, PKG.