Hyperhomocysteinemia Promoting The Formation Of Unstable Plaque:A Role Of Endoplasmic Reticulum Stress And Histone Methylation

Objective Atherosclerosis is a chronic inflammatory disease of the arterial wall,arising from an imbalance in lipid metabolism and a maladaptive inflammatory response.In recent decades,atherosclerosis-related arterial diseases have become the leading cause of death and morbidity worldwide.In China,atherosclerosis and related diseases are increased sharply and becaming a major cause of mortality with the economic development and lifestyle improvement.Clinic trials indicate that the sudden rupture of coronary unstable plaque and secondery formation of thromnbus are the cause of acute coronary syndrome.Hence,a more detailed understanding of the mechanisms underlying plaque destabilization may help us to improve detection and treatment of plaque vulnerability.Hundreds of clinical and observational studies consistently indicate that Hcy strongly associates with atherosclerosis.Importantly,two clinical studies demonstrated that vulnerable plaques in coronary and carotid arteries were associated with hyperhomocysteinemia(Hhcy).Furthermore,advanced atherosclerosis has been confirmed in atherosclerosis-prone apolipoprotein E-deficient(ApoE-/-)mice with Hhcy.Lesional macrophage apoptosis and plaque necrosis are two key hallmarks of advanced atherosclerosis.Lesional macrophage apoptosis and necrotic core formation contribute greatly to the progression of plaques.However,the effects of Hcy on plaque progression mediated by macrophage apoptosis and their underlying mechanisms are not well defined.Macrophage apoptosis occurs during all stages of atherosclerosis and influences early lesion formation,plaque progression,and plaque stability.Chronic or unresolved ER stress with the activation of different branches of the UPR eventually initiates apoptosis.Several studies have verified that advanced lesional macrophage apoptosis is associated with the development of a key feature of so-called ‘‘vulnerable plaques’ ’.But the role of endoplasmic reticulum stress in the unstable plaque formation meditated by homocysteine still is an open issue.The aetiology of atherosclerosis involves the interplay of genetic and environmental factors,where even the strongest genetic risk factors can explain just above 10% of the observed inheritance of atherosclerosis.Consequently,exploring epigenetic regulation is critical for our understanding of the implications of Hhcy in atherosclerosis.Hcy is an important intermediate of the methyltransferase reaction,which contributes to epigenetic gene regulation by donating methyl groups for DNA and/or protein methylation.The influences of homocysteine on global and gene promoter-specific DNA methylation have been identified in vivo and in vitro.Recently,emerging evidence indicates alternation of histone methylation is involved in the pathogenesis of advanced plaques.However,the exact mechanisms underlying the deleterious effect of Hhcy in the formation of advanced plaques and whether the modification of histone methylation is involved in this process is still unexplored.Therefore,in this study,we sought to explore the following issuesthrough in vivo and in vitro experiments:(1)The role of the macrophage apoptosis in the formation of Hcy promoting the formation of unstable plaque;(2)The effect and mechanisms of endoplasmic reticulum stress underlying Hcy promoting macrophage apoptosis and unstable plaque formation;(3)Whether histone methylation involving in Hcy promoting macrophage apoptosis and unstable plaque formation.We hope that the results obtained from the study could help clarify the mechanisms underlying Hcy activating macrophage-derived foam cell formation and macrophage apoptosis to promote the formation of vulnerable atherosclerotic plaques,thus providing a useful potential drug target for the treatment of HHcy-indcued AS..Methods Part one : Six-week-old Male ApoE-/-mice(n=36)and C57BL/6 mice(n=36)were were randomly assigned to three diet(Keaoxieli,Beijing)groups with similar mean body weights.The control group(n=12)was maintained on a standard rodent maintenance diet as recommended by the American Institute of Nutrition-93 purified diet(AIN-93G);the methionine supplemented group(n=12)was maintained on a control diet that contained 17 g methionine/kg food;the folic acid treatment group was maintained on a control diet that contained 17 g methionine/kg and 0.008 g folic acid food.After twenty weeks intervention,all the mice were sacrificed.The fasting blood samples were withdrawn from the orbital vein.Homocysteine and lipid profiling was performed using an automatic biochemistry analyser.Lesion preparation and analysis was conducted by the en face method and aortic sinus cross section method.Lesion area and necrotic core size were analysed by HE staining and Oil Red O.The stability of plaque was evaluated using multiple special tissue staining.We explored the apoptosis cell in the plaque by TUNEL staining.Furthermore,after cultured macrophage Raw 264.7 cells lines were challenged with homocysteine alone or in combination with folic acid in the presence of ox-LDL,we detected the cell apoptosis using AVPI and TUNEL staining.Lipid deposition of macrophage was assessed by ORO staining.Finally,we detected the expression of mitochondrial apoptotic pathway by western blot.Part two: Six-week-old Male ApoE-/-mice(n=36)were randomly assigned to three groups with similar mean body weights.The control group(n=12)was maintained on a standard rodent maintenance diet as same as part one;the methionine supplemented group(n=12)was maintained on a control diet that contained 17 g methionine/kg food;the 4-PBA intervention group was maintained on a control diet that contained 17 g methionine/kg for 12 weeks.This group were intraperitoneal injected with the chemical chaperone 4-PBA(100 mg/kg/time)twice a week for 8 weeks.After twenty weeks intervention,all the mice were sacrificed.The fasting blood samples were withdrawn from the orbital vein.Homocysteine and lipid profiling was performed using an automatic biochemistry analyser.Lesion preparation and analysis was conducted by the en face method and aortic sinus cross section method.Lesion area and necrotic core size were analysed by HE staining and Oil Red O.The stability of plaque was evaluated using multiple special tissue staining.We explored the apoptosis cell in the plaque by TUNEL staining.Furthermore,after cultured macrophage Raw 264.7 cells lines were challenged with homocysteine alone or in combination with 4-PBA in the presence of ox-LDL,we detected the cell apoptosis using AVPI and TUNEL staining.Lipid depositions of macrophage were assessed by ORO staining.Finally,we detected the expression of endoplasmic reticulum stress sensors and mitochondrial apoptotic pathway by western blot.Part three: The expression of H3K4m2 and H3K9m2 were detected by IHC and western blot in the aortic issue of the first part one.Involvement of histone methylation in macrophage apoptosis and foam cell formation were assessed in macrophage Raw 264.7 cells after being challenged with homocysteine alone or in combination with the histone methylation inhibitor BIX 01294.Rusults The following were found: part one(1)Methionine diet induces Hhcy without aggravated lipid disorder in C57bl/6c and ApoE-/-mice;(2)Hhcy induced by methionine supplementation does not independently cause atherosclerosis in C57BL/6J mice;(3)Methionine diet induces Hhcy exacerbated atherosclerosis development and necrotic core size in ApoE-/-mice;(4)The methionine diet enhanced plaque instability in ApoE-/-mice;(5)Lesional apoptotic cells are increased in ApoE-/-mice supplemented with the methionine diet;(6)Hcy promotes foam cell formation in vitro;(7)Hcy accelerates macrophage apoptosis in vitro;(8)Hcy upregulated macrophage apoptosis gene Cleaved caspase3 and Bax expression and inhibited the expression of Bcl-2 in vitro;Part two:(9)4-PBA(ERS inhibitor)alleviated the lesion and necrotic core area enlargement and unstable plaque formation induced by Methionine diet-induced Hhcy in ApoE-/-mice;(10)4-PBA intervention inhibited the increased of apoptotic cell in ApoE-/-mice supplemented with a methionine diet;(11)4-PBA abolished the macrophage apoptosis and macrophage lipid deposition in Raw 264.7 cell lines induced by Hcy;(12)he expression of ERS markers are increased in ApoE-/-mice supplemented with a methionine diet.4-PBA abolished the increase of ERS markers in ApoE-/-mice supplemented with a methionine diet;(13)Hcy increased the expression of ERS markers in vitro.4-PBA abolished the increase of ERS markers in vitro;(14)4-PBA abolished the increase of Bcl-2 pathway apoptotic genes in vitro;(15)Methionine diet-supplemented ApoE-/-mice showed a significant decrease in H3K9m2,but not H3K4m2,expression in the plaques of the aortic sinus;(16)Hcy modulates H3K9m2 and histone methyltransferases G9 a in Raw 264.7 cells;(17)H3K9m2 is involved in Hcy promoting foam cell formation in vitro;(18)H3K9m2 is involved in Hcy accelerating macrophage apoptosis in vitro;(19)H3K9m2 is involved in the expression modification induced by Hcy in cultured Raw 264.7 cells.Conclusions(1)Macrophage apoptosis plays a role in homocysteine accelerating unstable plaque formation;(2)Endoplasmic reticulum stress plays a crucial role in homocysteine promoting macrophage apoptosis and unstable plaque formation;(3)Histone methylation H3K9m2 might be involved in macrophage apoptosis and unstable plaque formation in methionine induced hyperhomocysteinemic ApoE-/-mice.