Study On The Injury Mechanism In The Vascular Endothelial Cells Caused By Homocysteine

Objective: To observe whether homocysteine(HCY) plus copper could induce apoptosis in human vascular endothelial cell and explore its probable molecular mechanism.Method: Human umbilical venous endothelial cells were cultured, and were exposed to different concerntrations of HCY with/or not with CuSO4; Cell counting of detached cells were performed with a hematocytometer; the cell survival was monitored by MTT assay and the cellular injury was evaluated by LDH release; Hoechst 33258 staining was used to observe nuclear morphological changes and the apoptosis was measured by DNA agarose gel electrophoresis ;the percentage of apoptosis was quantified by flow cytometry. It was also observed that the influence of HCY on the content of cellular MDA and the changes in the activity of major antioxidant enzymes(SOD GSH-Px).Moreover, the phosphorylation of p38MAPK was detected by Western blot.Results: When endothelial cells were exposed to different concerntrations of HCY(100~100011 mol/L) plus 10 μ mol/L CuSO4 for different time, the value of cellular A570 decreased but detached cells and LDH release were increase gradually with the HCY concentration increasing and action time prolonging. By Hoechst 33258 staining used to observe nuclear morphological changes, we found that after the endothelial cells were exposed 1000 μ mol/L plus 10μ mol/L CuSO4 for 48h, the typical apoptosis phenomenon of condensed and/or fragment nuclei appeared and DNA ladder of typical apoptosis could be seen by DNA agarose gel electrophoresis. The percentage of apoptosis was increased by higher concentration of HCY and longer time. But we also observed that these detective indexes did not change when the endothelial cells were exposed to the different concentrations of HCY alone. Besides, the study indicated that when copper existing, the activities of antioxidant enzymes(SOD. GSH-Px) of these groups decreased and the content of MDA increased with the HCY concentration increasing and action timeprolonging.For studying the probable signal transmitting pathway of HCY-induced apoptosis furthermore, the changes of pSSMAPK were detected that when the endothelial cells were exposed to HCY plus copper. This experiment showed that the whole expression of p38MAPK didn't change but the phosphorylation of pSSMAPK was enhanced in a dose-dependent manner when the cells were treated HCY and this effect of HCY on cells were antagonized by SB203580, a specific inhibitor of p38MAPK;the percentage of apoptosis was decreased when SB203580 added.Conclusions:1 -, With the physiologic concentration of copper, the lower concentration of HCY (100~1000 u mol/L) could induce apoptosis in vascular endothelial cells.2 The apoptosis in vascular endothelial cells induced by HCY plus copper was involved in the mechanism of oxidative stress-mediated injury.3, With the physiologic concentration of copper, HCY could induce apoptosis in vascular endothelial cells via activation of p38MAPK pathway.