Over–Expression of Aldose Reductase Gene Induces Apoptosis in Bovine Pericyte

Abstract

Abstract: : Purpose: Diabetic retinopathy (DR) is a major complication in diabetic patients. Several factors and pathways are involved in diabetic complication. One of major contributory factors is aldose reductase (AR). Activation of AR generates osmotic and oxidative stress via polyol pathway. We studied the effect of AR–overexpression in the cell survival and the expression and role of VEGF in pericyte–survival in vitro and explored the association between increased polyol pathway and cell death. Methods: cDNA of human AR gene was isolated from human lens cDNA library using PCR and a construct was prepared using eukaryotic expression vector (pEGFP–C). Bovine and pig pericyte were transfected using lipofectamin (Gibco). Western analysis was performed to monitor AR, GFP–AR and GFP protein expressions. MTS and TUNEL assays were used to detect apoptotic cell death. VEGF mRNA and protein expression was examined using real–time PCR and western analysis, respectively. Results: Cytoplasmic localization of GFP–AR was detected in 80% cells. A band of 90 kDa was monitored in over–expressing cells using anti–human AR antibody. Cell survival gradually decreased in AR over–expressing cells from 72 to 120 hrs incubated with 50 mM glucose (p<0.001) and number of TUNEL positive cells increased significantly. Expression of VEGF was up–regulated in AR–over–expressing pericytes under high glucose culture. Conclusions: Present findings reveal that over expression of AR increases apoptosis in pericyte during elevated glucose level. Furthermore, modulation of polyol pathway in the cultured cells over– expressed with AR may induce VEGF expression in pericyte and promote the progression of diabetic retinopathy.

Keywords: apoptosis/cell death • retina • diabetic retinopathy