Mechanism Of Mitochondrial Dysfunction In Endothelial Cells Induced By Homocysteine

Background:Cardiovascular disease is a serious threat to human health and ranks first amongthe world’s deadliest diseases.Atherosclerosis(AS)is the common pathological basis of many cardiovascular diseases.Endothelial cells are an important part of the blood vessel wall and play anindispensable role in progresses such as regulating blood flow,platelet activation and leukocyte adhesion.While the vascular endothelial cells got damaged,the balance between vasoconstriction and vasodilatation is broken and endothelial function is affected.Moreover,endothelial dysfunction is the initiating or contributing factor in the development of various cardiovascular diseases.Therefore,early prevention and treatment of endothelial dysfunction is the key to the prevention and therapy of cardiovascular diseases.Clinical studies have shown that homocysteine(Hey)is an independent and significant risk factor for cardiovascular diseases,especially in AS.Hey is an intermediate produced in the methionine metabolism.Fasting plasma in which Hey is greater than 15 μM is known as hyperhomocysteinemia(HHcy).Reports have shown that HHcy is associated with endothelial cell damage and may be associated with oxidative stress,inflammatory response,and down-regulation of nitrosylation levels.However,the specific mechanism has not yet been fully elucidated.Therefore,it is urgent to search for the targets of Hcy-related cardiovascular diseases.Mitochondria are not only the main site for cellular ATP synthesis,but also participate in plenty of biological processes such as calcium homeostasis,signal transduction and autophagy.It has been reported that changes in mitochondrial dynamics have been found in the endothelium of patients with cardiovascular diseases.The pathological changes of mitochondrial dynamics are related to endothelial cell dysfunction.At present,the mitochondrial damage induced by Hcy in endothelial cells and its association with endothelial dysfunction are rarely reported.Therefore,it is critically needed to further investigate the mechanism of mitochondrial damage and the underlying mechanisms of Hcy and endothelial cells.Ca2+ signals play an indispensable role in cell survival through affecting mitochondrial-related biological processes.Unbalanced Ca2+ homeostasis could lead to cell dysfunction and metabolic disorders.Mitochondria-associated endoplasmic reticulum membranes(MAM)are close contact sites formed between mitochondria and endoplasmic reticulum(ER)which play a central role in biological processes like lipid synthesis,Ca2+ signaling,mitochondrial biosynthesis and dynamics,autophagy and cell survival,sepecially for the Ca2+ shuttling between ER and mitochondria.There are a variety of mitochondria-ER tethering proteins in MAM structures,such as PACS-2,Mfn2-Mfnl/2 and IP3R-Grp75-VDAC.It was found that PACS-2 plays an important role in maintaining the mitochondrial-endoplasmic reticulum coupling,endoplasmic reticulum homeostasis,apoptosis and participates in maintaining the integrity of the MAM structure.Mfn2-Mfn1/2 physically connected the mitochondrial and endoplasmic reticulum,but its maintenance of the MAM structure in the positive or negative direction is still in dispute.Grp75 connects ER membrane protein IP3R and mitochondrial outer membrane protein VDAC,which is an important channel for releasing Ca2+ from ER to mitochondria.In addition,MAM structures have been found to be changed in many diseases like neurodegenerative diseases,metabolic diseases,and tumors.Up to now,the effect of Hcy on the MAM structure in endothelial cells has not yet been reported.Therefore,the structure of MAM and Ca2+ signal have become the focus of our research in order to find the potential targets of the endothelial injury,especially the mitochondrial damage induced by Hey.Consequently,human umbilical vein vascular endothelial cells were selected to investigate the effect of Hcy on the mitochondrial function,and we are aimed to explore the underlying mechanisms,especially focus on the MAM structures and the role of Ca2+ signal in the mitochondrial dysfunction induced by Hcy,hoping to provide experimental evidence and novel idea for the treatment of Hcy-related cardiovascular diseases.Objective:To investigate the effect of Hcy on the mitochondrial function in endothelial cells and the underlying mechanisms.Methods:1.Human umbilical vein endothelial cells EA.hy926 cell line was treated with exogenous Hcy.MTT was used to detect cell viability in endothelial cells treated with different concentrations of Hcy for 24 h.Time course of cell viability upon treatment with Hcy(200,400 and 800 μM)at 3,6,12,24 h was then detected.2.The expression level of endothelial markers was determined by Western-blot and the total cellular NO concentration was examined using the total NO detection kit.3.The effect of Hcy on the subcellular structure of endothelial cells was observed through transmission electron microscopy(TEM).JC-1 staining was used to detect the effect of Hcy on the mitochondrial membrane potential in endothelial cells.The ATP test kit was used to investigate the effect of Hey on the ATP content.The effect of Hey on the ROS level was detected with DCFH-DA probe.4.Protein co-immunoprecipitation(co-IP)was used to investigate the effect of Hey on the interaction of Grp75 with IP3R and VDAC in endothelial cels.Western-blot was used to detect the effect of Hey on the expression of PACS-2,the major tethering protein for MAM.5.The effect of Hey on the expression level of mitochondrial dynamics,apoptosis and autophagy-related proteins in endothelial cells was assessed by Western-blot.Immunofluorescence was used to detect the effect of Hey on the mitochondrial translocation of Drpl and Parkin.6.Mitochondrial Ca2+ was labeled with Rhod 2 AM probe.Flow cytometry was used to detect the effect of Hey on the concentration of mitochondrial Ca2+ in endothelial cells.7.Cells were co-treated with the IP3R inhibitor Xestospongin C(XeC)and Hey to investigate the role of MAM-mediated Ca2+ signaling in Hcy-induced changed mitochondrial dynamics and mitochondrial autophagy in endothelial cells.Results:1.Hey caused damage to EA.hy926 endothelial cells in a concentration-dependent manner:The IC50 of D,L-Hcy on the viability of EA.hy926 endothelial cell was 6.03 mM.Cells were treated with different concentrations of Hey for 24 h and cell viability was reduced concentration-dependently.After treatment with Hey(800 μM)for 3,6,12,24 h,the cell viability was reduced in a time-dependent manner.In the later period,endothelial cells were treated with 200,400,800μM Hey for 24 h.Results of Western-blot showed that compared with the control group,the expression level of the markers of endothelial cells,CD31 and VEGF,were reduced after treatment with Hey(800 μM)for 24 h significantly.The phosphorylation level of the eNOS active site Ser1177 was down-regulated while the inhibitory phosphorylation site Thr495 was up-regulated,the level of ET-1 was increased notably.The concentration of the total intracellular NO was decreased.2.Hcy caused mitochondrial dysfunction and mitophagy without activation of endoplasmic reticulum stress in endothelial cell:Transmission electron microscopy revealed that the degree of mitochondrial damage was aggravated by Hcy in a concentration-dependent manner.The results of JC-1 staining showed that the mitochondrial membrane potential of endothelial cells was decreased upon the treatment of Hcy.ATP level was reduced,and ROS level was increased.There were no significant changes in the ratio of apoptosis-related proteins Bax/Bcl-2 and Cytochrome C in the control and Hey treatment groups,indicating that Hcy did not activate the mitochondria-dependent apoptosis in endothelial cells.Further studies found that the autophagosomes were increased significantly in endothelial cells after treated with Hcy.Results of Western-blot and immunofluorescence showed that Hcy increased the expression of LC3 II,the autophagy marker,and caused accumulation of Parkin on the mitochondrial outer membrane.Moreover,results of TEM showed no significant changes in the morphology of endoplasmic reticulum,and Western-blot results showed that there was no obvious change in the expression of Grp78 and p-eIF2α(Ser51)compared to the control group.Therefore,Hcy did not induce endothelial endoplasmic reticulum stress,indicating that the damage to endothelial mitochondria induced by Hey preceded endoplasmic reticulum.3.Hcy enhanced MAM structure and increased mitochondrial Ca2+ concentration:Results of TEM showed that the number and the length of the mitochondria-ER coupling were significantly increased after treated with Hcy,suggesting that Hey caused an increased MAM structure in endothelial cells.Results of Western-blot showed that after Hcy treatment,the major tethering protein for MAM structures PACS-2 was up-regulated significantly,the level of Mfn2 was down-regulated and Mfnl was no obvious effect.Results of protein immunoprecipitation showed that the interaction between Grp75,IP3R and VDAC was enhanced,suggesting that Hcy caused an enhanced mitochondria-ER coupling.The expression of MCU was also increased in the Hcy treatment group,and mNCX was not affected.Using Rhod 2 AM probe to label the mitochondrial Ca2+,combined with flow cytometry and fluorescence imaging,we found that Hcy caused an increase in mitochondrial Ca2+ concentration in endothelial cells.Using the IP3R inhibitor Xestospongin C(XeC)to inhibit the IP3R-mediated Ca2+ flow,it was found that the mitochondrial Ca2+ concentration in the Hcy + XeC co-treatment group was lower than that in the Hey treatment group.These results suggested that Hey induced an increased mitochondrial Ca2+ concentration and may be related to the increase of the formation of IP3R-Grp75-VDAC complex.4.Hcy affected mitochondrial dynamics through GSK3β activation and MAM Ca2+alteration:Hcy caused disturbance of mitochondrial dynamics imbalance,manifested as decreased mitochondrial fusion and increased fission.The expression of mitochondrial fusion protein Mfn2 was significantly decreased,Mfnl was not affected,and the level of mitochondrial fission protein Drp1 was significantly increased.Further studies revealed that the level of p-mTOR(Ser2481),p-Akt(Ser473)and p-GSK3β(Ser9)in the mitochondrial fraction were significantly decreased upon the treatment of Hcy,and the interaction between GSK3β and Drpl was enhanced,indicating that mTORC2/Akt/GSK3β pathway was invoved in the excessive mitochondrial fission induced by Hcy.In addition,Drp1 expression and its mitochondrial translocation were both significantly reduced in the Hcy + XeC co-treatment group compared with the Hcy treatment group,suggesting that the excessive mitochondrial fission induced by Hcy was also related to the increase of mitochondrial Ca2+ concentration.5.Increased concentration of mitochondrial Ca2+ induced by Hcy was involved in the mitochondrial dysfunction and mitophagy activation:Compared to the Hcy treatment group,the intracellular content of ATP and the mitochondrial membrane potential in the Hcy + XeC co-treatment group were both increased.And the expression of autophagy marker LC3 II was decreased.The colocalization of Parkin with mitochondrial membrane was also weakened.These results indicated that the increase of mitochondrial Ca2+ in endothelial cells induced by Hcy was involved in the mitochondrial dysfunction and mitophagy activation.Conclusions:1.During the cytotoxicity of Hcy on endothelial cells,mitochondrial damage preceded the endoplasmic reticulum.Hcy had a significant effect on mitochondria in a concentration-dependent manner,manifested as the decreased mitochondrial membrane potential,reduced ATP content and increased ROS level,whereas did not activate the mitochondria-dependent apoptosis.2.Hey up-regulated the level of MCU and did not affect the level of mNCX,resulting in an increased mitochondrial Ca2+ concentration,thereby increased mitochondrial fission and caused mitochondrial dysfunction as well as mitophagy activation.The increased concentration of mitochondrial Ca2+ induced by Hcy may be related to its enhancement of Grp75 and IP3R,VDAC interactions,upregulation of PACS-2 expression,and enhancement of MAM structure in endothelial cells.In addition,excessive mitochondrial fission induced by Hcy was also associated with the inhibition of the mTORC2/Akt/GSK3β signaling pathway and the finally activation of GSK3β.