Nitric oxide is an upstream signal for vascular endothelial growth factor-induced extracellular signal-regulated kinases1/2 activation in postcapillary endothelium

ABSTRACT. We recently demonstrated that nitric oxide (NO) significantly contributes to the mitogenic effect of vascular endothelial growth factor (VEGF), suggesting a role for the NO pathway in the signaling cascade following kinase-derivative receptor activation in vascular endothelium. The aim of this study was to investigate the intracellular pathways linked to VEGF/NO-induced endothelial cell proliferation. We assessed the activity of the mitogen-ctivated protein kinase (MAPK) that is specifically activated by growth factors, extracellularregulated kinase (ERK1⁄2), on cultured microvascular endothelium isolated from coronary postcapillary venules. ERK1⁄2 was immunoprecipitated, and its activity was assessed with an immunocomplex kinase assay. In endothelial cells exposed for 5 min to the NO donor drug sodium nitroprusside at a concentration of 100 mM, ERK1⁄2 activity significantly increased. VEGF produced a time- and concentration-dependent activation of ERK1⁄2. Maximal activity was obtained after 5 min of stimulation at a concentration of 10 ng/ml. The specific MAPK kinase inhibitor PD 98059 abolished ERK1⁄2 activation and endothelial cell proliferation in a concentration-dependent manner in response to VEGF and sodium nitroprusside. The NO synthase inhibitor Nv-monomethyl-Larginine, as well as the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, blocked the activation of ERK1⁄2 induced by VEGF, suggesting that NO and cGMP contributed to the VEGF-dependent ERK1⁄2 activation. These results demonstrate for the first time that kinase-derivative receptor activation triggers the NO synthase/guanylate cyclase pathway to activate the MAPK cascade and substantiates the hypothesis that the activation of ERK1⁄2 is necessary for VEGF-induced endothelial cell proliferation.