| Hyperhomocysteinemia is an independent risk factor for the development of atherosclerosis and is associated with endothelial dysfunction manifested by impaired nitric oxide-dependent vasodilation. Production of reactive oxygen species (ROS), which can inactivate nitric oxide, may contribute to the pathophysiology of hyperhomocysteinemia. In order to determine whether ROS production might result from intracellular or extracellular pro-oxidant processes, we loaded bovine aortic endothelial cells (EC) and fibroblasts from hyperhomocysteinemic patients deficient in cystathionine β-synthase (CBS−/−FB) with the di(acetoxymethyl ester) of 6-carboxy-2′,7′dichlorodihydrofluorescein diacetate (DCF; fluorescent after reaction with ROS) and exposed the cells to conditions that alter intracellular or extracellular homocysteine levels. Exposure of EC and CBS−/−FB to exogenous homocysteine in concentrations from 0.01 to 1mM in the presence of 5 μM Cu2+ significantly and dose-dependently increased DCF fluorescence compared to controls (P < 0.05 by 1-way ANOVA, N = 3). Homocysteine alone, methionine (to increase endogenous intracellular homocysteine production), Cu 2+, or methionine with Cu2+ produced no increase in DCF fluorescence. Inhibiting re-methylation of homocysteine in CBS −/−FB by 24 hour treatment with aminopterin in the presence or absence of methionine, followed by challenge with either media, homocysteine, methionine, or H2O2 caused no increase in DCF fluorescence levels compared with pretreatment controls. In pure serum, which may contain 2 μM redox-active copper, no increase in DCF fluorescence was observed in EC treated with 500 μM homocysteine alone or with 5 μM copper added as CuCl2. Cyclic GMP accumulation in EC stimulated with 100 μM S-nitrosoglutathione (GSNO) was unaffected by 500 μM homocysteine. Inhibition of ADP-induced platelet aggregation in platelet-rich plasma by nitric oxide donors (GSNO, 1 μM; diethylamine-NONOate, 0.5 μM; and Angeli's salt, 10 μM) was unaffected by 500 μM homocysteine. Thus, although homocysteine supports the extracellular, but not intracellular generation of ROS in the presence of copper, in serum this effect is undetectable by DCF. Further, homocysteine alone has no effect on the bioavailability of exogenously delivered nitric oxide in EC or platelets. Endothelial dysfunction in hyperhomocysteinemia may be caused by deficient nitric oxide release from EC rather than by direct inactivation of nitric oxide by homocysteine or by homocysteine-derived ROS. |
