Glyc-oxidized LDL impair endothelial function more potently than oxidized LDL: role of enhanced oxidative stress

Hypercholesterolemia is associated with impairment of endothelial function due to increased levels of LDL. In diabetic patients, however, attenuation of endothelial function occurs even under normocholesterolemic conditions. Here we assessed whether glycation of LDL potentiates their influence on endothelial function, with particular emphasis on the oxidizability of LDL and the role of O2-. Human LDL was glycated by dialyzation for 7 days against buffer containing 200 mmol/l glucose, or sham-treated without glucose, and oxidized by incubation with Cu2+. Glycation significantly enhanced the oxidizability of LDL, as detected by diene formation and by electrophoretic mobility (27.5 mm for oxidized LDL vs. 34 mm for oxidized glycated LDL at 20 h of oxidation). Isolated rings of rabbit aorta were superfused with physiological salt solution, and isometric tension was recorded. Incubation of the aortic rings with sham-treated or with glycated LDL, not oxidized, had no influence on acetylcholine-induced, endothelium-dependent relaxation. Exposure of the aortic rings to oxidized non-glycated LDL caused a significant inhibition (30% at 1 microM acetylcholine) of the endothelium-dependent relaxation only in the presence of diethyl-dithiocarbamate (DDC), an inhibitor of the endogenous superoxide dismutase (SOD). Incubation of aortic rings with oxidized glycated LDL attenuated endothelium-dependent relaxation even in the absence of DDC (by 31% at 1 microM acetylcholine). The presence of DDC potentiated the inhibition of relaxation (65% inhibition at 1 microM acetylcholine), and co-incubation with exogenous SOD and catalase prevented the inhibition of relaxation, indicating a mediator role of O2-. Endothelium-independent relaxation induced by forskolin was unaffected by any of the lipoproteins. Using a chemiluminescence assay, significantly increased O2- production of aortic rings pretreated with oxidized glycated LDL (4101 +/- 360 counts/s) in comparison to control rings (753 +/- 81 counts/s) or arteries pretreated with oxidized non-glycated LDL (2358 +/- 169 counts/s) could be detected, suggesting that enhanced NO-inactivation by O2- could be the underlying mechanism for the stronger impairment of endothelium-dependent dilations by oxidized glycated LDL. Glycation increases the oxidizability of LDL and potentiates its endothelium-damaging influence. The likely mechanism for attenuation of endothelium-dependent dilations is increased formation of O2-, resulting in inactivation of nitric oxide. This mechanism may play an important role in diabetic patients and may contribute to disturbed organ perfusion.