Homocysteine altered ROS generation and NO accumulation in endothelial cells.
Mild hyperhomocysteinemia (HHcy) is a risk factor for vascular disease and is closely associated with endothelial dysfunction. Oxidative stress and decreased nitric oxide (NO) bioavailability were reported in HHcy-induced vascular injury; however, the exact relationship is not understood. We thus directly determine the production of reactive oxygen species (ROS) and NO in cultured endothelial cells (HUVECs) to demonstrate the correlated variation between ROS and NO induced by Hcy (homocysteine), Cys (cysteine), another thiol compound, and Met (methionine), precursor of HHcy in animal study. HUVECs were treated with Hcy, Cys, or Met for 0.5 or 22-24 h; ROS generation was detected by DCF fluorescence with flow cytometry and NO by chemiluminescence. In non-cytotoxic (<1.0 mM) concentration ranges, Met exerted no effects on either ROS production or NO concentration, Cys decreased ROS production and increased NO in both short-term (0.5 h) and long-term (22-24 h) treatments; Hcy, however, induced a biphasic effect on ROS production, i.e., inhibitory at 0.5 h but stimulatory at 24 h. The maximal stimulation by Hcy (0.25 mM) was significantly reduced by co-incubation (12 h) with estrogen (1 microM). Hcy caused an early (0.5 h) increase of medium NO which was absent in long-term Hcy treatment. The oxidative stress caused by long-term Hcy incubation could be ameliorated by estrogen, consistent with earlier in vivo observations that estrogen prevents HHcy-induced injury.
Tsen
Chih-Mei
CM
Department of Physiology and Pharmacology, Chang Gung University College of Medicine, Taoyuan, Taiwan, ROC.
Hsieh
Chien-Cheng
CC
Yen
Chia-Hung
CH
Lau
Ying-Tung
YT
D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D003545:Cysteine / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D006710:Homocysteine / Q000494:pharmacology*; D006801:Humans; D008715:Methionine / Q000494:pharmacology; D009569:Nitric Oxide / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism; D014471:Umbilical Veins / Q000166:cytology
D017382:Reactive Oxygen Species; D006710:Homocysteine; D009569:Nitric Oxide; D008715:Methionine; D003545:Cysteine