EXPRESSION OF APOPTOSIS REGULATORY GENES BY RETINAL PERICYTES AFTER RAPID GLUCOSE REDUCTION

PURPOSE. Retinal capillary pericytes underwent apoptosis in vitro afte r they had been exposed long-term to high levels of glucose followed b y a rapid glucose reduction. The present work was designed to study th e expression of bcl-2 family members and apoptosis regulatory genes an d to determine the status of oxidative stress induced by high concentr ations of glucose in this in vitro apoptosis model. METHODS. Pericytes were grown in normal or high glucose concentrations (5, 20, 30, and 4 0 mM) for 10 days and then exposed to a rapid reduction of glucose to 0.5 mM or 5 mM. Pericyte cell death was evaluated by determining the l oss of cell viability and the fragmentation of DNA using agarose gel e lectrophoresis. In parallel, the quantitative reverse transcription-po lymerase chain reaction technique was used to determine the expression of bcl-2, bax, p53, and glutathione peroxidase (GSH-Px) genes. The in tracellular level of glutathione (GSH) and the DNA fragmentation were determined simultaneously for pericytes treated with or without exogen ous GSH monoethylester. Retinal capillary endothelial cells, experienc ing the same glucose variation, were studied as a comparison. RESULTS. For pericytes, downregulation of bcl-2 was observed as early as 24 ho urs after rapid glucose reduction, whereas DNA fragmentation was not d etectable at that time. After 72 hours, a decreased protein ratio of B cl-2 to Bax was concomitant to evident loss of pericyte viability. Dur ing the period of high glucose and the following glucose reduction, p5 3 expression essentially was unchanged. Decreased levels of GSH induce d by high concentrations of glucose (>30 mM) became further depleted w hen the glucose levels were rapidly reduced. Addition of GSH monoethyl ester to the medium restored the level of GSH in pericytes and prevent ed pericyte apoptosis induced by glucose variation. Moreover, the mRNA levels of GSH;Px were significantly elevated. By contrast, with the s ame glucose reduction endothelial cells did not undergo apoptosis. The ir mRNA levels of bcl-2, bax, and GSH-Px essentially were unchanged. C ONCLUSIONS. High levels of glucose and rapid reduction of glucose modu late the expression of bcl-2 family genes in retinal pericytes. Upregu lation of GSH-Px and depletion of GSH indicate a reparative process of accelerated elimination of reactive oxygen species following rapid gl ucose reduction. These findings indicate that the aggravated oxidative stress and the weakened antioxidant defense induced by the combined e ffects of high levels of glucose and subsequent rapid glucose reductio n cause pericyte apoptosis. Prevention of DNA fragmentation of pericyt es by exogenous GSH further supports this notion. Because endothelial cells did not show similar pathologic changes, this proposed mechanism seems to be specific to pericytes.