The Damage Effects Of Homocysteine On The Cytoskeleton-actins Of Vascular Endothelial Cells

Objective:On the basis of dynamic observation of morphology and function of vascular endothelial cells,as well as qualitative observation and quantitative analysis of cytoskeleton-actins of ECs, to study the potential mechanism of Hcy-induced ECs damage.Methods:Human umbilical vein endothelial cells(ECV-304) cultivated in DMEM were divided randomly into control group,low-dose Hcy group(0.1mmol/L),medium-dose Hcy group(0.5mmol/L),high-dose Hcy group(1.0mmol/L).At 6 hours,12 hours,24 hours after incubation with Hcy,the morphologic changes of endothelial cells were observed with inverted phase contrast microscope.The morphology and the distribution of filamentous actin(F-actin) and globular monomeric actin(G-actin) stained with Rhodamine-Phalloidin and Alexa Flour 488-Dnaseâ… respectively were observed with laser scanning confocal microscope(LSCM).The contents of F-actin and G-actin were measured with LSCM,and counted G/F ratio.The concentration of nitric oxide(NO) and malondialdehyde(MDA) which is a lipid peroxidation metabolite in the conditioned medium were quantified by Nitrate reductase enzymes and Thibabituric acid respectively.Results:1.The morphologic changes of vascular endothelial cells(ECs)The shape of ECs of control group were fusiform,arranging like the typical cobblestone appearance.The cells of Hcy groups contracted,the width of intercellular spaces increased with increase of dose and/or the prolongation of times,even necrosis and defluvium of ECs appeared.2.The changes of cytoskeleton-actins(1) The morphologic changes of cytoskeleton-actins:parts of F-actin of ECs in control group fonn dense periphery band,the other parts appeared in stress fibers in the cytoplasm.In Hcy groups,dense periphery band gradually showed fragmentation,the stress fibers got decurtated and thickened in the cytoplasm,and disappeared in the high-dose Hcy group.With the prolongation of times,dense periphery band showed fragmentation and stress fibers got increasingly thick.Compared with control group,G-actin of ECs in Hcy groups were limited close to the nucleus,G-actin gradually dispersed,and fluorescence intensity increased when Hcy concentration increased or incubation time prolonged.(2) The content changes of G-actin:At 6h and 24h,G-actin contents dose-dependently increased in Hcy groups compared with the control group of the corresponding period.At 12h, the overall G-actin content of the Hcy groups increased,but didn't show conspicious dose-dependent feature.G-actin contents of the control group and 0.1mmol/L Hcy increased, then reduced with the prologation of times;At 0.5mmol/L Hcy and 1.0mmol/L Hcy,G-actin contents increased time-dependently.(3) The content changes of F-actin:At 6h and 12h,compared with the control group, F-actin contents of ECs increased in Hcy groups,F-actin contents were up to the highest in the low-dose group;At 24h,the F-actin contents rised with dose-dependent.In different Hcy groups, F-actin contents increased with prolongation of times in the high-dose Hcy group,in other Hcy groups increased first,then decreased,but the overall content was increasing.(4) The changes of G/F ratio:At 6h,G/F ratio increased in Hcy groups compared with the control group of the corresponding period,G/F ratio was the highest in 0.5 mmol/L Hcy group, at 12h,24h,G/F ratio increased in dose-dependent way.G/F ratio time-dependently increased in every Hcy group.3.The changes of endothelium function(1) The content changes of NO:The overall trends of NO production in Hcy groups reduced with increase of dose in different times,but at 0.5 mmol/L Hcy group,there was a slight trend of turning point.NO contents showed a downward trend with prologation of times;At 12h,NO contents increased in every Hcy group compared with 6h and 24h.(2) The content changes of MDA:MDA contents increased with increase of Hcy dose and/or the prolongation of times.Conclusion:1.Hcy not only injures ECs morphology,and reduces endothelial NO production,but also promotes the increase of lipid peroxidation MDA,thereby,Hcy has a damage effect on the function of ECs. 2.Hcy leads to the morphologic changes and rearrangements of endothelial cytoskeletal-actins, and injures morphology and function of endothelial by changing the contents of G-actin and F-actin,and G/F ratio.