Oxidative damage of vascular smooth muscle cells by the glycated protein–cupric ion system

• Published in: Atherosclerosis Vol. 136; no. 2; pp. 263 - 274
• Main Authors: Sakata, Noriyuki, Miyamoto, Koji, Meng, Jing, Tachikawa, Yutaka, Imanaga, Yoshinobu, Takebayashi, Shigeo, Furukawa, Tatsuo
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier Ireland Ltd 01.02.1998; Elsevier
• Subjects: 1,2-Dihydroxybenzene-3,5-Disulfonic Acid Disodium Salt - pharmacology; Animals; Atherogenesis; Atherosclerosis (general aspects, experimental research); Biological and medical sciences; Blood and lymphatic vessels; Cardiology. Vascular system; Catalase - pharmacology; Cell injury; Cells, Cultured; Copper - pharmacology; Dose-Response Relationship, Drug; Flow Cytometry; Glycation; Glycation End Products, Advanced - metabolism; Glycosylation; Mannitol - pharmacology; Medical sciences; Muscle, Smooth, Vascular - metabolism; Oxidants - metabolism; Oxidation; Reactive Oxygen Species; Serum Albumin, Bovine - pharmacology; Smooth muscle cells; Spectrometry, Fluorescence; Superoxide Dismutase - pharmacology; Swine; Atherogenesis; Reactive oxygen species; g-BSA, glycated bovine serum albumin; DCFH-DA, 2′, 7′-dichlorofluorescein diacetate; AGEs, advanced glycation end products; SMCs, cultured smooth muscle cells; BSA, unmodified bovine serum albumin; Smooth muscle cells; Glycation; SOD, superoxide dismutase; Cell injury; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; CAT, catalase; DHR 123, dihydrorhodamine 123; Oxidation; tiron, 4,5-dihydroxy-1,3-benzenedisulfonic acid; Vascular disease; Human; Cell culture; Pathogenesis; Atherosclerosis; Cardiovascular disease; Aorta; Cytotoxicity; Smooth muscle; Cupric ion
• ISSN: 0021-9150; 1879-1484
• DOI: 10.1016/S0021-9150(97)00219-0

To clarify the mechanism of cellular injury through the nonenzymatic reaction of glucose with proteins, we studied the cytotoxic effect of glycated bovine serum albumin on cultured smooth muscle cells in the presence of cupric ion. Glycated proteins were prepared by incubating bovine serum albumin with 0.5 M d-glucose in 0.3 M sodium phosphate buffer at 37°C for 2, 4 and 16 weeks (g-BSA-2, g-BSA-4 and g-BSA-16, respectively). Early glycation products, such as fructosamine, were formed more than two weeks after incubation. However, the immunoreactivity 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 concentration-dependent cytotoxicity in smooth muscle cells in the presence of 80 μM cupric ion by an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) dye reduction assay and dye exclusion test. Flow cytometry and spectrofluorophotometry using dihydrorhodamine 123 showed 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 intracellular 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 catalase and tiron, but not by superoxide dismutase or mannitol, a hydroxyl radical scavenger. These results indicate that smooth muscle cells may be damaged by reactive oxygen species which are produced extracellularly by the interaction with the early glycation products and cupric ion, and suggest that hydrogen peroxide may be a candidate for reactive oxygen species which contribute to such oxidative damage of smooth muscle cells.