Intracellular protein glycation in cultured retinal capillary pericytes and endothelial cells exposed to high-glucose concentration

There is now increasing evidence suggesting that non-enzymatic glycation (N EG) of proteins is involved in the pathogenesis of chronic diabetic complic ation. In this study we demonstrate that chronic exposure to high-glucose c oncentration leads to intracellular protein glycation in cultured bovine re tinal capillary pericytes and endothelial cells. The level of intracellular protein glycation, as measured using a competitive enzyme-linked immunoabs orbant assay (ELISA), was found to increase in both pericytes and endotheli al cells as function of time. As expected products of NEG were only detecte d when the Schiff base and the Amadori products were chemically reduced to glucitollysine by sodium borohydride. Despite the accumulation of early gly cation products on cellular proteins there was no further rearrangement rea ction into advanced glycation endproducts (AGEs), even after 12 days of inc ubation in high-glucose medium. Immunofluorescence microscopy demonstrated that the monoclonal antibody reacting with glucitollysine stains the cytopl asm of both pericytes and endothelial cells in a finely punctate pattern. F urther studies using Western blot analysis suggested that a number of cellu lar proteins, including smooth muscle actin in pericytes, become rapidly gl ycated. The results from this in vitro study suggest that excessive accumul ation of early products of non-enzymatic glycation in pericytes and endothe lial cells may play an important role in the pathogenesis of diabetic retin opathy.