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

There is now increasing evidence suggesting that non-enzymatic glycation (NEG) of proteins is involved in the pathogenesis of chronic diabetic complication. In this study we demonstrate that chronic exposure to high-glucose concentration leads to intracellular protein glycation in cultured bovine retinal capillary pericytes and endothelial cells. The level of intracellular protein glycation, as measured using a competitive enzyme-linked immunoabsorbant assay (ELISA), was found to increase in both pericytes and endothelial cells as function of time. As expected products of NEG were only detected when the Schiff base and the Amadori products were chemically reduced to glucitollysine by sodium borohydride. Despite the accumulation of early glycation products on cellular proteins there was no further rearrangement reaction into advanced glycation endproducts (AGEs), even after 12 days of incubation in high-glucose medium. Immunofluorescence microscopy demonstrated that the monoclonal antibody reacting with glucitollysine stains the cytoplasm of both pericytes and endothelial cells in a finely punctate pattern. Further studies using Western blot analysis suggested that a number of cellular proteins, including smooth muscle actin in pericytes, become rapidly glycated. The results from this in vitro study suggest that excessive accumulation of early products of non-enzymatic glycation in pericytes and endothelial cells may play an important role in the pathogenesis of diabetic retinopathy.