Role Of ERO1-alpha In Homocysteine-induced Macrophage Apoptosis And Atherosclerotic Plaque Formation

BackgroundAtherosclerosis,especially the ruptures of plaques and acute cardiovascular complications,which is the consequent of plaque rupture,are still the leading causes of morbidity and mortality worldwide.Macrophage,the main ingredient in the plaque,and macrophage apoptosis occurs throughout atherogenesis.Increasing evidence suggests that in advanced lesional,macrophage apoptosis is associated with the development of plaque necrosis,which is a key feature of vulnerable plaques.Regulation of macrophage apoptosis is particularly crucial for development of atherosclerotic plaques.Studies have shown that hyperhomocysteinemia(HHcy,plasma homocysteine?15?M)is an independent risk factor for atherosclerosis.Importantly,clinical studies have also found that HHcy is associated with the development of atherosclerotic plaques.The effect of HHcy accelerates atherosclerosis may because of Hcy can induce endothelial dysfunction,promote inflammation,contributes to smooth muscle cell proliferation,and promote lipid accumulation or macrophage apoptosis in plaque.During all the stages of atherosclerosis development,endoplasmic reticulum(ER)stress plays an important role.The ER is a compartment of the eukaryotic cell,with complex membrane,is responsible for maturation and folding of secreted proteins,many of which are rich in disulfide bonds.However,accumulation of unfolded proteins is accompanied by abnormal formation of disulfide bond in the ER lumen causing ER stress,which is an important driver for macrophage and endothelial cell apoptosis in advanced lesions.Proteins folding process is regulated by a series of enzymes,endoplasmic reticulum oxidoreductase 1?(ERO1?),is an ER-resident sulfhydryl oxidase,responsible for catalyzing disulfide bond formation in nascent polypeptide substrates.It has been found that increased ERO1?expression is involved in the development of ER stress-induced vascular endothelial dysfunction and cardiomyopathy.ERO1?can exacerbate ER stress,and leads to cell apoptosis eventually.Studies had shown that ERO1?is critical for ER stress–induced apoptosis in insulin-resistant obese mice,and macrophages apoptosis occurs in advanced plaque induced by HHcy.Therefore,we hypothesize that ERO1?may paticpate in ER-stress induced macrophage apoptosis and plaque stability in atherosclerotic lesions induced by HHcy.In order to test the hypothesis,we use Apo E^-/-mice and mouse peritoneal macrophages treated by high homocysteine(Hcy)with or without lentivirus to knockdown or overexpress ERO1?to check ERO1?expression,the ER stress,macrophages apoptosis and atherosclerotic plaque development.Objective1.To investigate the effect of HHcy on vulnerable plaque and apoptosis of macrophages in AS.2.To investigate the effect of ER stress on macrophage apoptosis in the presence of Hcy.3.To investigate the role of ERO1?on the vulnerability of atherosclerotic plaques in the presence of HHcy and the underlying mechanism.4.To investigate the role of ERO1?on Hcy-induced apoptosis of macrophages.Methods1.Adding Hcy in drinking water of Apo E^-/-mice to make HHcy AS mouse model,HE staining and Masson staining,ORO staining,immunohistochemistry and immunofluorescence method were used to measure plaque lesion area,necrotic core area ratio and indicators such AS plaque vulnerability index.2.The effect of HHcy on the apoptosis of macrophages in AS plaques was evaluated by TUNEL staining combined with F4/80 immunofluorescence staining,and TUNEL+F4/80+was used to represent the apoptotic macrophages in plaques.The effect of HHcy on endoplasmic reticulum stress of macrophages in AS plaques was evaluated by double staining with GRP78 and F4/80 immunofluorescence markers,and GRP78⁺F4/80⁺was used to represent the macrophages with ER stress in plaques.3.Isolating mouse peritoneal macrophages,and cells were treated with different concentrations of Hcy for 24 h,or with the same concentration of Hcy for different times points(12 h 24 h 48 h),to screen the appropriate concentration and time point of Hcy promoting the apoptosis of macrophages.The effect of Hcy on promoting apoptosis of macrophages was detected by flow cytometry,4-PBA were used to explore the role of ER stress in Hcy induced macrophage apoptosis.ER stress pathway and the expression of ERO1?in macrophages was detected Western blot.4.The expression level of ERO1?in Apo E^-/-mice was decreased or increased by lent-virus injection through tail vein,and the effects of changing the expression level of ERO1?on the progression and stability of Apo E^-/-mice AS plaques,macrophage apoptosis and ER stress in the plaques were detected with immunofluorescent staining in the presence of HHcy.5.ERO1?expression in mouse peritoneal macrophages was modulated by lentovirus transfection,and the effects on apoptosis of macrophages and ER stress-related signaling pathways in vitro were detected under the stimulation of Hcy.Results1.Exogenous Hcy supplementation can increase the plasma Hcy level of mice and meet the diagnostic criteria of HHcy.Plaque lesional area and vulnerability index of HHcy were higher than that in control group.2.The rate of apoptosis of macrophages and the positive area of endoplasmic reticulum stress markers in HHcy group were higher than those in control group.3.Hcy promoted the apoptosis of mouse peritoneal macrophages,and the expression of ERO1?and ER stress related markers were changed in a Hcy dose-dependent and time-dependent manner.The intervention of 200?M Hcy for 24 h was selected as the intervention condition for subsequent experiments.Inhibiting ER stress by 4-PBA can reverse the effect of Hcy on apoptosis of macrophages.4.The expression level of ERO1?in Apo E^-/-mice was decreased,and the lesion area and vulnerability index of AS plaques in mice with HHcy Apo E^-/-mice were decreased,and the overexpression of ERO1?was aggravated.5.In Vitro,knocking down ERO1?alleviates the Hcy-stimulated endoplasmic reticulum stress of macrophages,while overexpression of ERO1?aggravates it.Knocking down ERO1?reduces Hcy-stimulated macrophage apoptosis and vice versa.ConclusionHcy via upregulating ERO1?expression activates ER stress-dependent macrophage apoptosis,so as to promoting plaque formation and development.ERO1?may be a potential therapeutic target for atherosclerosis.