OXIDATIVE DAMAGE OF VASCULAR SMOOTH-MUSCLE CELLS BY THE GLYCATED PROTEIN-CUPRIC ION SYSTEM

To clarify the mechanism of cellular injury through the nonenzymatic r eaction of glucose with proteins, we studied the cytotoxic effect of g lycated bovine serum albumin on cultured smooth muscle cells in the pr esence of cupric ion. Glycated proteins were prepared by incubating bo vine serum albumin with 0.5 M D-glucose in 0.3 M sodium phosphate buff er at 37 degrees C for 2, 4 and 16 weeks (g-BSA-2, g-BSA-4 and g-BSA-1 6, respectively). Early glycation products, such as fructosamine, were formed more than two weeks after incubation. However, the immunoreact ivity of glycated proteins to anti-AGE antibody was 12-fold higher in g-BSA-16 than in g-BSA-2. Both g-BSA-2 and g-BSA-16 showed a concentra tion-dependent cytotoxicity in smooth muscle cells in the presence of 80 mu M cupric ion by an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-dipheny l tetrazolium bromide) dye reduction assay and dye exclusion test. Flo w cytometry and spectrofluorophotometry using dihydrorhodamine 123 sho wed that the extracellular generation of oxidants was dose-dependently enhanced with increasing concentrations of g-BSA-2 or g-BSA-16 in the presence of cupric ion. However, no difference was observed in the in tracellular generation of oxidants between the presence and absence of glycated proteins by flow cytometry using 2', 7'-dichlorofluorescein diacetate. Cytotoxicity and oxidant generation were prevented by catal ase and tiron, but not by superoxide dismutase or mannitol, a hydroxyl radical scavenger. These results indicate that smooth muscle cells ma y be damaged by reactive oxygen species which are produced extracellul arly by the interaction with the early glycation products and cupric i on, and suggest that hydrogen peroxide may be a candidate for reactive oxygen species which contribute to such oxidative damage of smooth mu scle cells. (C) 1998 Elsevier Science Ireland Ltd.