Acute intensive insulin therapy causes a transient worsening of diabetic retinopathy in type 1 diabetes patients and is related to VEGF expression. Reactive oxygen species (ROS) have been shown to be involved in HIF-1α and VEGF expression induced by insulin, but the role of specific ROS sources has not been fully elucidated. In this study we examined the role of NADPH oxidase subunit 4 (Nox4) in insulin-stimulated HIF-1α and VEGF expression, and angiogenic responses in human microvascular endothelial cells (HMVECs). Here we demonstrate that knockdown of Nox4 by siRNA reduced insulin-stimulated ROS generation, the tyrosine phosphorylation of IR-β and IRS-1, but did not change the serine phosphorylation of IRS-1. Nox4 gene silencing had a much greater inhibitory effect on insulin-induced AKT activation than ERK1/2 activation, whereas it had little effect on the expression of the phosphatases such as MKP-1 and SHIP. Inhibition of Nox4 expression inhibited the transcriptional activity of VEGF through HIF-1. Overexpression of wild-type Nox4 was sufficient to increase VEGF transcriptional activity, and further enhanced insulin-stimulated the activation of VEGF. Downregulation of Nox4 expression decreased insulin-stimulated mRNA and protein expression of HIF-1α, but did not change the rate of HIF-1α degradation. Inhibition of Nox4 impaired insulin-stimulated VEGF expression, cell migration, cell proliferation, and tube formation in HMVECs. Our data indicate that Nox4-derived ROS are essential for HIF-1α-dependent VEGF expression, and angiogenesis in vitro induced by insulin. Nox4 may be an attractive therapeutic target for diabetic retinopathy caused by intensive insulin treatment.

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Published in PLoS ONE, v. 7, no. 10, e48393.

© 2012 Meng et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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