Hyperhomocysteinemia-induced Pro-inflammatory and Pro- Atherosclerotic Changes in Coronary Arteries

first years in medical research, for convincing me to take the scientific road. ABBREVIATIONS Hcy: homocysteine HHcy: hyperhomocisteinemia O 2 .-: superoxide ONOO-: peroxynitrite ROS: reactive oxygen species QRT-PCR: real-time quantitative reverse transcription-polymerase chain reaction DPI: diphenyliodonium eNOS: endothelial nitric oxide synthase iNOS: inducible nitric oxide synthase SOD: superoxide dismutase nox: non-phagocytic oxidase TNFa: tumor necrosis factor alpha IL-6: interleukin 6 MCP-3: macrophage chemoattractant protein 3 MRP: multidrug resistance protein 5 ABSTRACT Hypothesis: Hyperhomocysteinemia (HHcy) is a newly recognized risk factor for myocardial infarction, however, the effect of HHcy on the function and phenotype of coronary arteries and the mechanisms responsible for HHcy-induced vascular alterations is not known. On the basis of previous studies we hypothesized that in coronary arteries HHcy-induced impairment of vascular functions is due to an increased oxidative stress and a pro-inflammatory, pro-atherosclerotic shift in gene expression profile. Methods:In isolated coronary arteries of control (C; plasma Hcy: ~6 µmol/L) and methionine diet-induced HHcy (plasma Hcy: ~40 µmol/L) rats NO-mediated flow-induced dilation was measured by videomicroscopy. Arterial O 2 .-generation was measured by lucigenin chemiluminescence and ethidium bromide fluorescence methods. Peroxynitrite generation was assessed by measuring 3-nitrotyrosine content by Western blotting and immunohistochemistry. The expression of pro-and anti-oxidant enzymes (eNOS, iNOS, NAD(P)H oxidases, xanthine oxidase, SOD isoforms) were assessed by quantitative RT-PCR and Western blotting. Gene expression profile was screened by a cDNA-based microarray technique and the results were confirmed by independent methods. Results: Impaired flow-induced NO-mediated dilation 1) Increases in intraluminal flow elicited dilations of C vessels, responses that were absent in HHcy arteries. The nitric oxide synthase inhibitor L-NAME inhibited flow-induced dilation of C coronaries, whereas it had no effect on responses of HHcy arteries. Dilations of C and HHcy arteries to the NO donor SNP were not different. Responses to flow in HHcy coronary arteries were unaffected by administration of L-arginine or the prostaglandin H 2 /thromboxane A 2 receptor antagonist SQ 29,548. 2) However, in the presence of superoxide dismutase or the superoxide scavenger Tiron increases in flow elicited L-NAME-sensitive dilations of HHcy coronaries. Increased oxidative stress (O 2 .-production, peroxynitrite generation) 3) In HHcy coronary arteries O 2 .-generation was significantly increased, which could be inhibited by SOD or the NAD(P)H oxidase inhibitors diphenyleneiodonium (DPI) and apocynin. NADPH-driven O 2 .-generation was also significantly increased in HHcy vessel homogenates. Hydroethidine staining showed that increased O 2 .-levels are present in the media of …