Altered mRNA levels of antioxidant enzymes in pre-apoptotic pericytes fromhuman diabetic retinas

Because programmed cell death (PCD) is an important mode of pericyte dropou t in human diabetic retinopathy, whether increased oxidative stress in cell s with diminished antioxidant defenses plays a causative role in the PCD pr ocess in diabetic pericytes has been studied. Ten diabetic and eight non-di abetic eye-bank eyes from 5 diabetic and 4 non-diabetic patients were inclu ded in this study. From individual neural retinas pericytes were isolated b y a newly developed immunomagnetic technique. Total mRNA of the purified pe ricytes was isolated for quantitative reverse transcriptase (RT)-PCR assay, mRNA levels of a death protease (CPP32), the major enzyme that initiates t he proteolytic cascade leading to cell death, were determined in associatio n with the expression of antioxidative enzymes including glutathione peroxi dase (GSH-Px), glutathione reductase, CuZn superoxide dismutase (SOD), MnSO D and catalase genes in pericytes. In comparison with pericytes from non-di abetic retinas, pericytes from diabetic retinas highly expressed CPP32 gene s (4 +/- 0.6 fold increase, p<0.01, n=9). In diabetic pericytes, up-regulat ion of glutathione peroxidase (GSH-Px) (8.2 +/- 0.9 fold increase, p<0.01, n=9) and down-regulation of glutathione reductase (Gr) (4.1 +/- 0.4 fold de crease, p<0.05, n=9) and CuZnSOD (2.1 +/- 0.7 fold decrease, p<0.05, n=9) w ere observed, mRNA levels of MnSOD and catalase of diabetic pericytes did n ot differ significantly from those of non-diabetic pericytes. Overexpressio n of a member of interleukin-1 beta-convertin, enzyme (ICE) family, CPP32, indicated that the pericytes from diabetic retinas are in a "pre-PCD" state . This is the first evidence that the ICE family of death proteases is invo lved in pericyte dropout in diabetes. In these pre-PCD cells, the expressio n of antioxidant enzyme genes also was changed. Up-regulation of GSH-Px ind icates a compensation mechanism to meet the demand of excessive glutathione in reduced form. Decreased levels of both glutathione reductase and CuZnSO D, despite the oxidative stress in the diabetic condition, suggest the brea kdown of the antioxidant defense in pericytes. Most importantly, the altere d gene profile of scavenging enzymes under diabetic conditions, correlating with overexpression of the cell death protease gene, together suggest incr eased oxidative stress as an etiological agent of pericyte dropout in diabe tic retinopathy.