Mechanisms of homocysteine-induced oxidative stress.
Hyperhomocysteinemia decreases vascular reactivity and is associated with cardiovascular morbidity and mortality. However, pathogenic mechanisms that increase oxidative stress by homocysteine (Hcy) are unsubstantiated. The aim of this study was to examine the molecular mechanism by which Hcy triggers oxidative stress and reduces bioavailability of nitric oxide (NO) in cardiac microvascular endothelial cells (MVEC). MVEC were cultured for 0-24 h with 0-100 microM Hcy. Differential expression of protease-activated receptors (PARs), thioredoxin, NADPH oxidase, endothelial NO synthase, inducible NO synthase, neuronal NO synthase, and dimethylarginine-dimethylaminohydrolase (DDAH) were measured by real-time quantitative RT-PCR. Reactive oxygen species were measured by using a fluorescent probe, 2',7'-dichlorofluorescein diacetate. Levels of asymmetric dimethylarginine (ADMA) were measured by ELISA and NO levels by the Griess method in the cultured MVEC. There were no alterations in the basal NO levels with 0-100 microM Hcy and 0-24 h of treatment. However, Hcy significantly induced inducible NO synthase and decreased endothelial NO synthase without altering neuronal NO synthase levels. There was significant accumulation of ADMA, in part because of reduced DDAH expression by Hcy in MVEC. Nitrotyrosine expression was increased significantly by Hcy. The results suggest that Hcy activates PAR-4, which induces production of reactive oxygen species by increasing NADPH oxidase and decreasing thioredoxin expression and reduces NO bioavailability in cultured MVEC by 1) increasing NO2-tyrosine formation and 2) accumulating ADMA by decreasing DDAH expression.
Tyagi
Neetu
N
Dept. of Physiology and Biophysics, School of Medicine, 500 S. Preston St., 1115-A, Univ. of Louisville, Louisville, KY 40202, USA.
Sedoris
Kara C
KC
Steed
Mesia
M
Ovechkin
Alexander V
AV
Moshal
Karni S
KS
Tyagi
Suresh C
SC
D000818:Animals; D002478:Cells, Cultured; D003326:Coronary Circulation / Q000187:drug effects / Q000502:physiology; D004305:Dose-Response Relationship, Drug; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology; D006710:Homocysteine / Q000008:administration & dosage*; D008833:Microcirculation / Q000187:drug effects / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D018384:Oxidative Stress / Q000187:drug effects / Q000502:physiology*; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism; D044462:Receptors, Proteinase-Activated / Q000378:metabolism*
D017382:Reactive Oxygen Species; D044462:Receptors, Proteinase-Activated; D006710:Homocysteine; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase