Journal
CARDIOVASCULAR TOXICOLOGY
Volume 5, Issue 1, Pages 9-20Publisher
HUMANA PRESS INC
DOI: 10.1385/CT:5:1:009
Keywords
homocysteine; oxidative stress; apoptosis; NADPH oxidase; stress signaling
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Funding
- NATIONAL CENTER FOR RESEARCH RESOURCES [P20RR016474, P20RR015640] Funding Source: NIH RePORTER
- NATIONAL INSTITUTE ON AGING [R03AG021324] Funding Source: NIH RePORTER
- NCRR NIH HHS [P20 RR15640, RR-16474] Funding Source: Medline
- NIA NIH HHS [NIH R03 AG21324-01] Funding Source: Medline
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Hyperhomocysteinemia is an independent risk factor for cardiovascular diseases, although the mechanism leading to vascular dysfunction is not clear. The aim of this study was to examine the effect of homocysteine (Hcy) on oxidative stress and apoptosis in human umbilical vein endothelial cells (HUVECs). HUVECs were challenged for 24 h with Hcy (10 mu M-3 mM) in the presence of various stress signaling inhibitors, including the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor apocynin (100 mu M), the p38 mitogen-activated protein kinase inhibitor SB203580 (2.5 mu M), the extracellular signal-regulated kinase inhibitor UO126 (2.5 mu M), the stress-activated protein kinase (SAPK)/c-Jun NH2-terminal kinase (JNK) inhibitor JNK inhibitor II (10 mu M), and antioxidants alpha-tocopherol (5 mu g/mL) and N-acetyl cysteine (NAC, 2 mM). Reactive oxygen species (ROS) were detected using 5-(6)-chloromethyl2',7'-dichlorodiliydrofluorescein diacetate. Apoptosis was evaluated by 4',6'diamidino-2'-phenylindoladihydrochloride staining, annexin-V phosphatidylserine/propidium iodide, and caspase-3 assay. NADPH oxidase and SAPK/JNK signal were evaluated with immunoblotting. Hcy significantly enhanced ROS generation and apoptosis after 24-h incubation. Apocynin prevented Hcy-induced ROS generation but only partially restored Hcy-induced apoptosis. JNK inhibitor II, (alpha-tocopherol, and NAC partially reduced Hcy-induced apoptosis, although SB203580 and U0126 had no effect. immunoblotting analysis confirmed upregulation of NADPH oxidase and SAPK/JNK signaling. Collectively, our results suggested that Hcy may induce oxidative stress and apoptosis through an NADPH oxidase and/or JNK-dependent mechanism(s).
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