Inhibition of reactive oxygen species by lovastatin down-regulates VEGF expression and ameliorates blood-retinal barrier breakdown in db/db mice: role of NADPH oxidase 4

Abstract Objective Oxidative stress is a key pathogenic factor in diabetic retinopathy (DR). We previously showed that lovastatin mitigates blood-retinal barrier (BRB) breakdown in db/db mice. The purpose of this study is to determine the mechanisms underlying the salutary effects of lovastatin in DR. Research Design and Methods Expression of NADPH oxidase 4 (Nox4), vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1a (HIF-1α), production of reactive oxygen species (ROS), retinal vascular permeability were measured in cultured retinal capillary endothelial cells (RCEC) and in db/db mice treated with lovastatin. Results Expressions of Nox4 and VEGF were significantly increased in retinas of db/db mice and reduced by lovastatin treatment. In cultured RCEC, hypoxia and high glucose up-regulated mRNA and protein expression of Nox4, ROS generation and VEGF level. These changes were abrogated by pre-treatment with lovastatin or NADPH oxidase inhibitor DPI. Over-expression of Nox4 increased basal level of ROS generation, HIF-1α and VEGF expression in RCEC. In contrast, blockade of Nox4 activity using adenovirus expressing dominant negative Nox4 abolished hypoxia- and high glucose-induced ROS production and VEGF expression. Moreover, inhibition of Nox4 attenuated hypoxia-induced up-regulation of HIF-1α and high glucose-elicited phosphorylation of STAT3. Finally, depletion of Nox4 by adenovirus-delivered Nox4 siRNA significantly decreased retinal NADPH oxidase activity and VEGF expression and reduced retinal vascular premeability in db/db mice. Conclusions Activation of Nox4 plays an important role in high glucose- and hypoxia-mediated VEGF expression and diabetes-induced BRB breakdown. Inhibition of Nox4 at least in part contributes to the protective effects of lovastatin in DR.