Selective Inhibition of Rac-1 Prevents Hypoxia and High Glucose-Induced VEGF Expression in Retinal Endothelial Cells

Abstract

Purpose:: Increased expression of VEGF is a critical event in the etiology of diabetic and other ischemic retinopathies. We have previously shown that treatment with statin can prevent both hyperglycemia and ischemia-induced retinal expression of VEGF. Statin’s effects involve blockade of the small G protein Rac-1. This latter is a critical regulator of important cellular functions including activation of NAD(P)H oxidase which we have previously shown to stimulate VEGF expression through activation of the transcription factor STAT3. Here we studied the effects of direct and selective inhibition of Rac-1 on VEGF expression induced by hypoxia and elevated glucose levels in retinal microvascular endothelial cells.

Methods:: Bovine retinal microvascular endothelial cells (BREC) were exposed to high glucose levels (25mM D-glucose) for 48 hr or to hypoxia (pO₂=1%, for 1,2,3, 6 hr). BREC cultured in normoxic and normal glucose conditions (pO₂=23%, 5mM, D-glucose) were used as controls. Selective Rac-1 inhibition was achieved by RNA interference (siRac-1) or by adenovirus-mediated gene transduction of a Rac-1 inactive mutant (Rac-1N17). Specific controls were represented by BREC transfected with scramble siRNAs (siSCR) or with adenoviruses carrying the reporter gene GFP (GFP). Levels of VEGF protein, tyrosine phosphorylated STAT3 (PYSTAT3) and active Rac-1 (GTP-bound) were measured by Western blotting. VEGF’s mRNA expression was assessed by quantitative RT-PCR.

Results:: Exposure of BREC to HG stimulated increases in VEGF protein, PYSTAT3 and active Rac-1 as compared to the controls. Over-expression of the dominant negative Rac-1N17 or transfection with siRac-1 significantly blocked the HG-induced -PYSTAT3 formation and normalized VEGF protein. BREC exposed to hypoxic conditions also showed increases in PYSTAT3 formation and VEGF mRNA. These effects correlated with increases in active Rac-1 and in STAT3 protein. Transfection of BREC with siRac-1 or transduction with Rac-1N17 blocked these effects whereas the siSCR or adGFP did not change the effects of hypoxia on PYSTAT3 or VEGF levels.

Conclusions:: In conclusion, selective inhibition of Rac-1 prevents hypoxia- and high glucose-induced increases of VEGF expression through blockade of STAT3-activation. These data suggest a role for Rac-1 in the induction of ischemic retinopathies that may explain the beneficial effects of statin treatment.

Keywords: diabetic retinopathy • signal transduction • vascular cells