Abstract
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.
Highlights
Acute intensive insulin therapy causes a transient worsening of diabetic retinopathy with neovascularization in type 1 diabetes patients and is related to VEGF expression [1]
In this study we examined the role of NADPH oxidase subunit 4 (Nox4) in insulin-stimulated hypoxia-inducible factor-1a (HIF-1a) and VEGF expression, and angiogenic responses in human microvascular endothelial cells (HMVECs)
Nox4 was highly expressed in HMVECs, and the expression of Nox4 markedly exceeded that of Nox2 (Fig. 1B), which is consistent with the previous report [26]
Summary
Acute intensive insulin therapy causes a transient worsening of diabetic retinopathy with neovascularization in type 1 diabetes patients and is related to VEGF expression [1]. We showed previously that NADPH oxidase subunit 4 (Nox4)-derived ROS generation in response to insulin-like growth factor-1 (IGF-I) was linked to cell proliferation and migration in vascular smooth muscle cells (VSMCs) [15,16]. Nox mediates endothelial responses to angiotensin-II [19], transforming growth factor-b [20] and epidermal growth factor [21], which include migration and proliferation. These data suggest that Nox has a critical role in modulating endothelial cell physiology
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