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J. Li, J. J. Wang, M. Kalyankar, B. J. Goldstein, S. X. Zhang; NADPH Oxidase 4 Is a Key Mediator of Oxidative Stress and Blood-Retinal Barrier Breakdown in Diabetic Retinopathy. Invest. Ophthalmol. Vis. Sci. 2009;50(13):6169.
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Oxidative stress contributes to the pathogenesis of endothelial dysfunction and vascular leakage in diabetic retinopathy. However, the exact role and mechanism of reactive oxygen species (ROS) generation in retinal endothelial cells remain to be elucidated. We hypothesize that NADPH oxidase 4 (Nox4) is a major source of ROS in retinal endothelial cells and is required for diabetes-induced blood-retinal barrier breakdown through regulation of vascular endothelial growth factor (VEGF).
Expression of NADPH oxidase isoforms, pro-inflammatory factors, and vascular leakage in the retina were measured in db/db mice and oxygen-induced retinopathy (OIR). Primary human and bovine retinal capillary endothelial cells (RCECs) were exposed to hypoxia (2% O2) for 16h. Over-expression of Nox4 or depletion of Nox4 activity in RCECs was achieved by infection with adenoviruses expressing Nox4 or a dominant negative Nox4 mutant (Ad-Nox4DN) in RCECs. ROS generation was measured using carboxy-H2DCFDA probe. VEGF and tight junction protein expression was determined by Western blot analysis.
Nox4 expression was significantly up-regulated in the retina of db/db mice and the mice with OIR, coincident with increased VEGF expression and retinal vascular leakage. In cultured RCECs. Nox4 expression was markedly induced by hypoxia, in parallel with ROS generation and elevated VEGF level, which was abolished by NADPH oxidase inhibitor DPI and apocynin. Depletion of Nox4 activity using Ad-Nox4DN almost completely abolished hypoxia-induced ROS production and VEGF expression in RCECs. Moreover, over-expression of Nox4 was sufficient to induce a significant increase of VEGF and tight junction damage in RCECs. Further mechanistic study indicated that Nox4-induced VEGF expression was at least in part through HIF1-independent pathway.
Taken together, these results indicate that Nox4 is a critical contributor of ROS generation in retinal endothelial cells. Up-regulation of Nox4 in diabetic retina may represent a novel mechanism of blood-retinal breakdown in diabetic retinopathy.
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