| Hyperhomocysteinemia is an independent risk factor for cardiovascular disease. Most previous investigations focused on the role of homocysteine (HCY) as direct pathogenetic factor for a number of adverse vascular events. However, the underlying pathophysiological mechanism is still unknown. The endothelial cell layer displays the features of a distributed organ and has a variety of biological functions, whose damage and dysfunction plays a central role in the pathogenesis of cardiovascular disease. It is helpful in understanding the pathophysiologic mechanisms for we to study the injury of EC by HCY. Our aime was to: observe the EC structural damage and function changes induced by HCY and to understand changes in the protein spectrum of EC.The present study includes three parts:1.Induction of EC apoptosis by HCYIncreasing evidence suggests that endothelial cell apoptosis might contribute to atherogenesis, plaque erosion and acute coronary syndromes. Human umbilical vein endothelial cells (HUVEC) were cultured in vitro and identified by observing the Weibel-Palade body (W-P body) with electron microscope which only exists in endothelial cells. HUVEC damage and apoptosis, which was induced by HCY in different concentration and at different times, was investigated by the means of cell morphology, cell Typan Blue test, MTT test, LDH release test, flow cytometry and and micro-molecular weight DNA fragment electrophoresis of apoptosis. We found that HUVEC experienced low-frequency apoptosis, the apoptotic rate being 3.5% in the control medium with 10% fetal bovine serum ( FBS). When HCY in 1mmol/L concentration was added to the basic medium (IMDM+10%FBS), the apoptosis rate increased by 17.6% ( vs that of control, P<0.05), the LDH activity in released was highest and the MTT metabolic level was lowest. It was also found that, accompanied by nuclear DNA fragmentation, the ultrastructure of the nucleus and cytoplasm changed dramatically with the increase of HCY concentration or prolonging of HCY presence. These results suggested that the HCY had cytotoxic effects on HUVEC which was significant in understanding the molecular mechanism of HUVEC apoptosis.2.The effect of HCY on TAFI and TFPI expression in HUVECThrombin activatable fibrinolysis inhibitor (TAFI) and Tissue factor pathway inhibitor (TFPI) were recently discovered, which could be synthesized in the EC. TAFI provides an important link between fibrinolysis and coagulation cascade. TFPI inhibits the initial reactions of blood coagulation. Human umbilical vein endothelial cells (HUVEC) were cultured, and cells from 2 or 3 passages were used. HUVEC expression of TAFI and TFPI, in the presence of HCY in different concentration and different interfere time, was investigated by RT-PCR and Western-blot. It was found that the expression of TAFI was increased and that of TFPI was decreased when the HCY concentration increased or the HCY presence time prolonged. The results suggested that the HCY could induce an increased thrombin generation in coagulation cascade and a down regulation of fibrinolysis, changing the balance between coagulation and fibrinolysis.3.The effect of HCY on full-scale protein spectrum of human umbilical vein cell line ECV304 by two-dimensional polyacrylamide gel electrophoresis.The endothelium is a single layer of cells lining the inner surface of all blood vessels. It constitutes a major metabolic organ, which is critically involved in the regulation of multiple physiological and pathological processes. HCY can damage the EC in many ways, disturbing the balance between coagulation and fibrinolysis, influencing the expression of adhesion molecules for cells in the immune system, and interfering with the metabolism of noradrenaline and 5-hydroxytryptamine, inhibiting conversion of angiotensin I and bradykinin. In previous research, we could focus on only one aspect. With proteomics, we could analyze the detailed change in the full-scale protein spectrum of EC. Which would be very important in understanding the pathop...
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