Role of Endothelial Nitric Oxide Synthase in Endothelial Cell Migration

Abstract

Abstract —Endothelium-derived nitric oxide (NO) and its precursor l -arginine have been implied to promote angiogenesis, but little is known about the precise mechanism. The inhibition of endogenous NO formation by N ω -nitro -l -arginine methyl ester ( l -NAME) (1 mmol/L) but not its inactive enantiomer d -NAME (1 mmol/L) inhibited endothelial cell sprouting from the scratched edge of the cultured bovine aortic endothelial cell monolayer. Inhibition of endogenous NO release by l -NAME was confirmed by amperometric measurement using an NO-specific electrode. In the modified Boyden chamber, l -NAME (1 mmol/L) significantly inhibited endothelial cell migration, whereas l -NAME did not affect endothelial DNA synthesis as assessed by analysis of [ 3 H]thymidine incorporation. We then examined alteration of endothelial cell adhesion molecule expression after the inhibition of NO by l -NAME in cultured human umbilical vein endothelial cells. In both normoxic and hypoxic conditions, l -NAME (1 mmol/L) inhibited surface expression of integrin αvβ3, which is an important integrin facilitating endothelial cell survival and angiogenesis. However, l -NAME did not affect the expression of platelet endothelial cell adhesion molecule-1, intercellular adhesion molecule-1, vascular endothelial adhesion molecule-1, gap junction protein connexin 43, and VE-cadherin, which have been reported to potentially affect angiogenesis. In summary, inhibition of endothelial NO synthase by l -NAME attenuated endothelial cell migration but not proliferation in vitro. Furthermore, endogenous endothelium-derived NO maintains the functional expression of integrin αvβ3, a mediator for endothelial migration, survival, and angiogenesis. Endothelium-derived NO, thus, may play an important role in mediating angiogenesis by supporting endothelial cell migration, at least partly, via an integrin-dependent mechanism.