Possible involvement of NADPH oxidase and JNK in homocysteine-induced oxidative stress and apoptosis in human umbilical vein endothelial cells.
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 oxi-dative stress and apoptosis in human umbilical vein endothelial cells (HUVECs). HUVECs were challenged for 24 h with Hcy (10 microM-3 mM) in the presence of various stress signaling inhibitors, including the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor apocynin (100 microM), the p38 mito-gen-activated protein kinase inhibitor SB203580 (2.5 microM), the extracellular signal-regulated kinase inhibitor U0126 (2.5 microM), the stress-activated protein kinase (SAPK)/c-Jun NH2-terminal kinase (JNK) inhibitor JNK inhibitor II (10 microM), and antioxidants alpha-tocopherol (5 microg/mL) and N-acetyl cysteine (NAC, 2 mM). Reactive oxygen species (ROS) were detected using 5-(6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate. Apoptosis was evaluated by 4',6'-diamidino-2'-phenylindoladihydrochloride staining, annexin-V phosphatidyl- serine/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 apopto-sis through an NADPH oxidase and/or JNK-dependent mechanism(s).
Dong
Feng
F
Division of Pharmaceutical Sciences and Center for Cardiovascular Research and Alternative Medicine, University of Wyoming, Laramie, WY 82071, USA.
Zhang
Xiaochun
X
Li
Shi-Yan
SY
Zhang
Zhaojie
Z
Ren
Qun
Q
Culver
Bruce
B
Ren
Jun
J
D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004730:Endothelium, Vascular / Q000187:drug effects / Q000201:enzymology*; D006710:Homocysteine / Q000633:toxicity*; D006801:Humans; D019255:NADPH Oxidases / Q000502:physiology*; D018384:Oxidative Stress / Q000187:drug effects / Q000502:physiology*; D014471:Umbilical Veins / Q000187:drug effects / Q000201:enzymology
D006710:Homocysteine; D019255:NADPH Oxidases