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Objective Oxidative stress plays an important role in the pathogenesis of neovascular retinal diseases,such as retinopathy of prematurity (ROP) and diabetic retinopathy (DR).Our previous study demonstrates that NADPH oxidase 4 (Nox4),a major isoform of NADPH oxidase in retinal endothelial cells,is responsible for retinal vascular leakage in type 2 diabetes.However,the role of Nox4 in retinal neovascularization (NV) remains largely unknown.The purpose of this study is to investigate the function and mechanisms of Nox4 in the development of retinal NV.Methods A mouse model of oxygen-induced retinopathy (OIR) was used to study retina NV.Nox4 expression was examined by western-blot analysis,real-time RT-PCR and immunohistochemistry in normal and OIR retinas.Cultured human retinal microvascular endothelial cells (HREC) were used for in vitro study.Over-expression or knockdown of Nox4 expression in mouse retinas or HREC was achieved by adenoviral infection.Generation of intracellular and extracellular H2O2 was measured by DCF assay and Amplex red assay respectively.Matrigel angiogenesis assay and transwell migration assay or wound healing assay were used to evaluate endothelial angiogenic capacity and migration.Phosphorylation of VEGF receptor2 (VEGFR2) and ERK were also determined.Results Nox4 was mainly localized in the vasculature of mouse retina and its expression was markedly increased in OIR,in parallel with enhanced phosphorylation of ERK.Over-expression of Nox4 by adenoviral transduction of wild-type Nox4 gene significantly increased extracellular H2O2 generation,potentiated VEGF-stimulated VEGFR2 activation,and promoted endothelial tube formation.In contrast,the knockdown of Nox4 by siRNA or scavenging H2O2 by overexpression of catalase inhibited endothelial migration,VEGF-induced VEGFR2 phosphorylation and tube formation.Importantly,reducing retinal Nox4 expression by siRNA suppressed ERK phosphorylation and remarkably attenuated retinal NV formation in OIR.Conclusion Upregulation of Nox4 contributed to retinal NV formation in OIR.Modulation of retinal Nox4 expression might present a promising therapeutic approach for neovascular retinal diseases.