Abstract
Hyperhomocysteinemia is believed to induce endothelial dysfunction and promote atherosclerosis; however, the pathogenic mechanism has not been clearly elucidated. In this study, we examined the molecular mechanism by which homocysteine (HCy) causes endothelial cell apoptosis and by which nitric oxide (NO) affects HCy-induced apoptosis. Our data demonstrated that HCy caused caspase-dependent apoptosis in cultured human umbilical vein endothelial cells, as determined by cell viability, nuclear condensation, and caspase-3 activation and activity. These apoptotic characteristics were correlated with reactive oxygen species (ROS) production, lipid peroxidation, p53 and Noxa expression, and mitochondrial cytochrome c release following HCy treatment. HCy also induced p53 and Noxa expression and apoptosis in endothelial cells from wild type mice but not in the p53-deficient cells. The NO donor S-nitroso-N-acetylpenicillamine, adenoviral transfer of inducible NO synthase gene, and antioxidants (alpha-tocopherol and superoxide dismutase plus catalase) but not oxidized SNAP, 8-Br-cGMP, nitrite, and nitrate, suppressed ROS production, p53-dependent Noxa expression, and apoptosis induced by HCy. The cytotoxic effect of HCy was decreased by small interfering RNA-mediated suppression of Noxa expression, indicating that Noxa up-regulation plays an important role in HCy-induced endothelial cell apoptosis. Overexpression of inducible NO synthase increased the formation of S-nitroso-HCy, which was inhibited by the NO synthase inhibitor N-monomethyl-l-arginine. Moreover, S-nitroso-HCy did not increase ROS generation, p53-dependent Noxa expression, and apoptosis. These results suggest that up-regulation of p53-dependent Noxa expression may play an important role in the pathogenesis of atherosclerosis induced by HCy and that an increase in vascular NO production may prevent HCy-induced endothelial dysfunction by S-nitrosylation.
Highlights
Hyperhomocysteinemia is believed to induce endothelial dysfunction and promote atherosclerosis; the pathogenic mechanism has not been clearly elucidated
To examine the roles of nitric oxide (NO) and antioxidants in endothelial cell apoptosis induced by HCy, HUVECs were treated with 1 mM HCy in the presence or absence of the chemical NO donor SNAP, the NOS inhibitor NMA, and antioxidants ␣-tocopherol and SOD plus catalase, and cell viability was measured
We examined the effect of oxidized SNAP (OxiSNAP, which has exhausted all of the NO and does not liberate NO), nitrite, nitrate, and 8-Br-cGMP on HCy-induced HUVEC apoptosis
Summary
Homocysteine; HUVEC, human umbilical vein endothelial cell; NO, nitric oxide; NOS, nitric-oxide synthase; SNAP, S-nitroso-N-acetyl-DL-penicillamine; OxiSNAP, oxidized SNAP, Z-VAD-fmk, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone; NMA, N-monomethyl-L-arginine; ROS, reactive oxygen species; SOD, superoxide dismutase; pNA, p-nitroanilide; Ac, N-acetyl; AdiNOS, adenoviral iNOS; DCF-DA, dichlorofluorescein diacetate; S-NOHCy, S-nitrosohomocysteine; MDA, malondialdehyde; FBS, fetal bovine serum; DAPI, 4Ј,6-diamidino-2-phenylindole; RT, reverse transcription; PBS, phosphate-buffered saline; iNOS, inducible NOS; cho, aldehyde. Crease in the bioavailability of NO is associated with an increase in the vascular inflammation and the proliferation of smooth muscle cells, all of which have been shown to play an important role in the development of atherosclerosis. These evidences indicate that NO is a key molecule in preventing endothelial dysfunction and atherosclerosis as well as promoting vascular relaxation. Extensive studies have indicated that HCy can cause endothelial dysfunction and vascular lesion formation; the cellular and molecular mechanisms between hyperhomocystemia and vascular disease are poorly understood.
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