Abstract
In order to investigate the role of nitrative stress in vascular endothelial injury in hyperhomocysteinemia (HHcy), thirty healthy adult female Wistar rats were randomly divided into three groups: control, hyperhomocysteinemia model, and hyperhomocysteinemia with FeTMPyP (peroxynitrite scavenger) treatment. The endothelium-dependent dilatation of thoracic aorta in vitro was determined by response to acetylcholine (ACh). The histological changes in endothelium were assessed by HE staining and scanning electron microscopy (SEM). The expression of 3-nitrotyrosine (NT) in thoracic aorta was demonstrated by immunohistochemistry and immunofluorescence, and the number of circulating endothelial progenitor cells (EPCs) was quantified by flow cytometry. Hyperhomocysteinemia caused significant endothelial injury and dysfunction including vasodilative and histologic changes, associated with higher expression of NT in thoracic aorta. FeTMPyP treatment reversed these injuries significantly. Further, the effect of nitrative stress on cultured EPCs in vitro was investigated by administering peroxynitrite donor (3-morpholino-sydnonimine, SIN-1) and peroxynitrite scavenger (FeTMPyP). The roles of nitrative stress on cell viability, necrosis and apoptosis were evaluated with 3-(4,5-dimethylthiazol)-2,5-diphenyl tetrazolium (MTT) assay, lactate dehydrogenase (LDH) release assay and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay, respectively. Also, the phospho-eNOS expression and tube formation in Matrigel of cultured EPCs was detected. Our data showed that the survival of EPCs was much lower in SIN-1 group than in vehicle group, both the apoptosis and necrosis of EPCs were much more severe, and the p-eNOS expression and tube formation in Matrigel were obviously declined. Subsequent pretreatment with FeTMPyP reversed these changes. Further, pretreatment with FeTMPyP reversed homocysteine-induced EPC injury. In conclusion, this study indicates that nitrative stress plays a role in vascular endothelial injury in hyperhomocysteinemia, as well as induces endothelial progenitor cell injury directly.
Highlights
Homocysteine (Hcy) is an intermediate product of methionine metabolism in vivo
In order to probe the change in vascular function with hyperhomocysteinemia, we detected the ACh-induced vasorelaxation in the thoracic aortas
Different degrees of endothelial dysfunction have been reported in a range of experimental hyperhomocysteinemia animal models
Summary
Homocysteine (Hcy) is an intermediate product of methionine metabolism in vivo. The World Health Organization stated that the fasting plasma homocysteine levels in healthy adults range from 5 to 15 μmol/L. Hyperhomocysteinemia (HHcy) (plasma homocysteine levels more than 15 μmol/L) is an established risk factor for coronary heart disease and other cardiovascular diseases [6,7]. The amount and bioactivity of endothelial progenitor cells have became an important indicator of vascular endothelial function and risk assessment in cardiovascular disease [15,16]. What’s the effect of elevated nitrative stress on endothelial progenitor cells in hyperhomocysteinemia? By using the hyperhomocysteinemic rats model in vivo and primarily cultured endothelial progenitor cells in vitro, we would investigate the role of nitrative stress in hyperhomocysteinemia-induced endothelial dysfunction, mainly focused on the indicator of endothelial repair-endothelial progenitor cells What’s the effect of elevated nitrative stress on endothelial progenitor cells in hyperhomocysteinemia? In the current study, by using the hyperhomocysteinemic rats model in vivo and primarily cultured endothelial progenitor cells in vitro, we would investigate the role of nitrative stress in hyperhomocysteinemia-induced endothelial dysfunction, mainly focused on the indicator of endothelial repair-endothelial progenitor cells
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