Important role of Nox4 type NADPH oxidase in angiogenic responses in human microvascular endothelial cells in vitro

Objective-Redox signaling mediated by Nox2-containing NADPH oxidase has been implicated in angiogenic responses both in vitro and in vivo. Because Nox4 type NADPH oxidase is also highly expressed in endothelial cells, we studied the role of Nox4 in angiogenic responses in human endothelial cells in culture. Methods and Results-Inhibition of Nox4 expression by small interfering RNA reduced angiogenic responses as assessed by the tube formation and wound healing assays, in both human microvascular and umbilical vein endothelial cells. Overexpression of wild-type Nox4 enhanced, whereas expression of a dominant negative form of Nox4 suppressed the angiogenic responses in endothelial cells. These effects were mimicked by exogenous H2O2 and the antioxidant compound ebselen, respectively. Overexpression of Nox4 enhanced receptor tyrosine kinase phosphorylation and the activation of extracellular signal-regulated kinase (Erk). Inhibition of the Erk pathway reduced the endothelial angiogenic responses. Nox4 expression also promotes proliferation and migration of endothelial cells, and reduced serum deprivation-induced apoptosis. Conclusions-Nox4 type NADPH oxidase promotes endothelial angiogenic responses, at least partly, via enhanced activation of receptor tyrosine kinases and the downstream Erk pathway.