BCL-2 EXPRESSION OR ANTIOXIDANTS PREVENT HYPERGLYCEMIA-INDUCED FORMATION OF INTRACELLULAR ADVANCED GLYCATION ENDPRODUCTS IN BOVINE ENDOTHELIAL-CELLS

Hyperglycemia rapidly induces an increase in intracellular advanced gl ycation end products (AGEs) in bovine endothelial cells, causing an al teration in bFGF activity (Giardino, I., D. Edelstein, and M. Brownlee . 1994. J, Clin. Invest. 94:110-117). Because sugar or sugar-adduct au toxidation is critical for AGE formation in vitro, we evaluated the ro le of reactive oxygen species (ROS) in intracellular AGE formation, us ing bovine aortic endothelial cells. 30 mM glucose increased intracell ular ROS formation by 250% and lipid peroxidation by 330%, while not a ffecting ROS in the media, In cells depleted of glutathione, intracell ular AGE accumulation increased linearly with ROS generation as measur ed by immunoblotting and the fluorescent probe DCFH (AGE 0.258-3.531 A U mm/5 x 10(4) cells, DCF 57-149 mean AU, r = .998, P < .002). Defero xamine, alpha-tocopherol, and dimethylsulfoxide each inhibited hypergl ycemia-induced formation of both ROS and AGE. To differentiate an effe ct of ROS generation on AGE formation from an effect of more distal ox idative processes, GM7373 endothelial cell lines were generated that s tably expressed the peroxidation-suppressing proto-oncogene bcl-2. bcl -2 had no effect on hyperglycemia-induced intracellular ROS formation. In contrast, bcl-2 expression decreased both lipid peroxidation (100% at 3 h and 29% at 168 h) and AGE formation (55% at 168 h). These data show that a ROS-dependent process plays a central role in the generat ion of intracellular AGEs, and that inhibition of oxidant pathways pre vents intracellular AGE formation.