| BackgroundAtherosclerosis?AS?is the common pathological basis of many cardiovascular and cerebrovascular diseases,which is the main cause of acute coronary syndrome?ACS?in clinic.The traditional view is that the plaque formed by lipid deposition under the intima is gradually enlarged into the lumen,resulting in stenosis of the lumen.However,recent studies have found that the severity of coronary stenosis is not parallel to the severity of ACS.About 70%of ACS occur in plaque rupture with coronary stenosis<50%.However,only less than 20%of ACS occurred in the rupture of plaque with coronary stenosis>70%.Therefore,the extent and extent of plaque vulnerability could reflect the possibility of acute coronary events in patients with coronary artery stenosis.Therefore,the mechanism of atherosclerotic plaque occurrence and vulnerability was discussed.Finding effective intervention targets to prevent the occurrence of acute cardiovascular events has become one of the hotspots in the field of cardiovascular science.AIM2 is a cytosolic double-stranded DNA?dsDNA?receptor that contributes to the host defense against bacterial and viral pathogens,such as Francisella tularensis and vaccinia virus,respectively.We previously identified that infection by the cytosolic bacterium Francisella tularensis subspecies novicida activates the transcription factor IRF1,which induces production of interferon-inducible GTPases called guanylate-binding proteins.Guanylatebinding proteins attack and kill bacteria in the cytoplasm,resulting in the liberation of bacterial DNA for recognition by AIM2.Upon binding of dsDNA in the cytoplasm of infected cells,AIM2 recruits the adaptor protein ASC to assemble an inflammasome complex that activates caspase-1,a cysteine protease that induces pyroptosis and mediates the cleavage of the inflammatory cytokines IL-1b and IL-18.Structural analysis of AIM2 revealed that the HIN200 domain binds dsDNA,whereas the pyrin domain recruits ASC.Recent studies have found that AIM2 not only plays an important role in innate immunity,but also receives multiple stimuli from PAMPs and DAMPs,and also plays an important role in many non-immune systems.Other studies have found that AIM2 plays an important role in systemic lupus erythematosus,myocarditis,psoriasis,liver ischemia-reperfusion injury and other diseases,in which AIM2 is overexpression.The lastest study found that the expression of AIM2 in human carotid plaques increases,and TNF-acan promote the expression of AIM2 in vascular smooth muscle cells.We speculate that AIM2 may influence the inflammatory response,and then affects the formation of atherosclerosis plaque.Objective?1?We established an atherosclerosis formation ApoE-/-mice model and detected the AIM2 expression levels inflammasome in plaques;?2?In order to explore the role of AIM2 in AS,we induced the AIM2 overexpression and silencing by lvAIM2 and shAIM2 virus,.?3?We further explored the mechanisms of AIM2 in atherosclerosis.MethodsAtherosclerotic plaque formation model in ApoE-/-miceEight-week-old male ApoE-/-mice?n = 100?were obtained from HFK Bioscience Company.All the mice were randomly divided into the following 5 groups?n = 20 per group?:?1?control group?chow diet?,?2?high-fat diet?HFD?group,?3?lentivirus AIM2 group?lentivector at a dose of 2X107TU/mouse?,?4?shRNA-AIM2 group?lentivector at a dose of 2X107TU/mouse?,and?5?NC group?null lentivirus at a dose of 2X107TU/mouse?.All mice were maintained under conditions of temperature was remained at 20?-22?,12:12h light-dark cycle,and humidity was kept at 50%-60%with food and water available.The control group was fed on 5%fat and no added cholesterol diet,and the other groups were fed on 16%fat and 0.25%cholesterol?high-fat?diet for 12 weeks.Lentivirus interventionAt age 8 weeks,anaesthetized mice were injected with lentivirus AIM2?1vAIM2 group,high fat diet,n=20?,shAIM2?shAIM2 group,high fat diet,n=20?and null lentivirus?NC group,high fat diet,n=20?at a total lentivector at a dose of 2X107TU/mouse.?After lentivirus intervention 2 weeks,lvAIM2,shAIM2,NC group mice were random selected to be euthanasia and detection of virus transfection efficiency by frozen sectionSerum measurementsAt the end of the experiment,the mice were hungry overnight,and their peripheral venous blood was drawn.The TG,TC,LDL were tested in the different group.Histological detectionFive aortas were chosen in each group for general oil red O staining.The remaining hearts were embedding in OCT and cut into 6um-thick sections in freezing microtome,beginning at the appearance of aortic valves.The sections were stained with hematoxylin and eosin?H&E?and Oil Red O to analyze the atherosclerosis lesion.ImmunohistochemistryThe distribution of Aim2 and IL-1 beta in tissue was observed by immunohistochemical method.Real-time RT-PCRThe expression of AIM2 expression in mouse aorta was detected by real time quantitative RT-PCR.Western blotThe expression ofAIM2?ASC?pro-caspase-1?caspase-1?GSDMD-N?MMP2?ICAM-1 were detected by Western-BlotTUNEL AssayUsing an ApopTag in situ apoptosis detection kit to explore the fragmentation of DNA was performed.ImmunofluorescenceThe distribution of Aim2 and IL-1 beta in tissue was observed by immunofluorescenceStatistical analysisAll the experimental results were presented as the mean ± standard deviation?SD?.GraphPad 5.0 and SPSS20.0 were used to analyze the data.Comparisons between different groups were analyzed by Student 's t test,and data between multiple groups were compared by one-way ANOVA.P<0.05 was considered to be statistical significance.ResultsAtherosclerotic plaque formation was induced by high-fat diet for 12 weeks in ApoE-/-miceThe atherosclerotic lesions in aortic tissues stained with Oil Red O and HE were formed in ApoE-/-mice after high fat diet.?p<0.05?AIM2 inflammasome and GSDMD-N overexpress in atherosclerosis plaqueAIM2?ASC?caspase-1 and GSDMD-N expression were increased in ApoE-/-mice fed with high fat diet compared to control?p<0.05??Besides,AIM2 mRNA level was higher than control group?p<0.05?.AIM2 can 't improve metabolic abnormal caused by hyperlipidemiaThe level of LDL-C,TC,TG in high fat diet group were higher than the control group.?p<0.05??But AIM2 overexpression and inhibition couldn't change the blood lipid level.Gene silence can inhibit the AIM2 expression and AIM2 lentivirus can promote AIM2 expressionAIM2-miRNA lentivirus was injected,the mRNA and protein level of AIM2 in plaque were less than the NC group?p<0.05?.shAIM2 lentivirus could reduce AIM2 expression in ApoE-/-mice compared with NC group.Besides ASC,caspase-1 p10 and GSDMD-N expression significant reduce compared with NC group?p<0.05?.AIM2 lentivirus could promote AIM2,ASC,caspase-1 p10 and GSDMD-N expression compared with NC group?p<0.05?.The immunohistochemical results show that the expression of AIM2 and IL-1? can be affected by gene silence and AIM2 lentivirus.?p<0.05??AIM2 affects atherosclerosis plaque formation in ApoE-/-mice.We found that the atherosclerotic lesions in aortic tissues stained with Oil Red O were larger in mice which were injected with lentivirus-AIM2 than those in NC group?P<0.05?,conversely,atherosclerotic lesion areas in shRNA-AIM2 group were smaller compared to NC group?P<0.05?.Furthermore,the cross-section stained with hematoxylin and eosin and Oil Red O revealed that atherosclerotic lesions in lentivirus-AIM2 group were significantly larger than those in NC group,while inhibition of AIM2 were accompanied with less lesion areas compared to NC group?P<0.05?.AIM2 can affects the accumulation of vascular smooth muscle cells and macrophages in plaque?p<0.05?.AIM2 accelerates cell death of smooth muscle cells in ApoE-/-miceBy using immunofluorescence double labeling,we demonstrated that the cell death was mainly concentrated in smooth muscle cells.TUNEL assay showed that the percentage of death cells in high fat diet group was higher than control group.The proportion of death cells in plaque was significantly higher more than NC group in case of AIM2 overexpression?p<0.05?.Conversely,the inhibition of AIM2 could alleviate the degree of cell death in plaque?p<0.05?.AIM2 affects the expression of MMP-2 and ICAM-1 in ApoE-/-miceWestern-blot assay showed that the expression of MMP-2 and ICAM-1 in high fat diet group was higher than control group.The expression of MMP-2 and ICAM-1 in plaque was significantly higher more than NC group in case of AIM2 overexpression?p<0.05?.Conversely,the inhibition of AIM2 could alleviate the degree of MMP-2 and ICAM-1 expression in plaque?p<0.05?.Conclusion?1?The expression of AIM2 is overexpression in atherosclerosis plaque formation;?2?AIM2 overexpression can promote the plaque formation and AIM2 inhibition can reduce the plaque formation;?3?AIM2 affects the accumulation of VSMCs and macrophages.?4?AIM2 could affects VSMCs cell death?5?AIM2 affects the expression of MMP-2 and ICAM-1Atherosclerosis?AS?is a common pathological basis of various cardiovascular and cerebrovascular diseases,leading to many chronic and acute disease in clinical practice,such as hypertension,unstable angina pectoris,myocardial infarction and sudden coronary death.The progression of AS is considered as accumulation of lipid in the vessel wall,progression in the inflammation,the structure changes and cell death.Cell death in vascular wall,especially in vascular smooth cells?VSMCs?can initiate plaque formation,thereby aggravating atherosclerosis progression.Apoptosis and pyroptosis are two forms of programmed cell death.Although a variety of researches have indicated that apoptosis is the main fashion of programmed cell death,unraveling cell death mode in atherosclerosis is complex* Pyroptosis is a novel form of programmed cell death and is uniquely dependent on caspase-1.Caspase-1 is not involved in apoptosis since caspase-1 deficient cells still respond to apoptosis signals.While caspase-1 can promote the activation and release of IL-1 p,which plays an important role in promoting inflammation.The progression of pyroptosis is characterized by the loss of cell membrane potential and the undergoing of DNA fragmentation.The DNA fragmentation in pyroptosis can be detected by positive terminal transferase-mediated deoxyuridine triphosphate-biotin nick end labeling?TUNEL?staining.Pyroptosis was initially found in immunological cells infected with bacterial or viral infection,but recent study has found ox-LDL can promote pyroptosis.Recently,absent in melanoma 2?AIM2?has been identified as a novel inflammasome-activating prcHein acting as a cytosolic DNA sensor in macrophages.AIM2 belongs to the HIN200 family of hematopoietic,interferon?IFN?-inducible and plays an important role in host defence against DNA viruses and certain cytosolic bacteria.Many prior studies focused on the role of AIM2 in inflammation on innate immune^2"41.Some researchers found that AIM2 can promote pyroptosis progression of tumor cells.AIM2 was also involved in the progression of abdominal aortic aneurysm and was further found to be overexpressed in atherosclerosis plaque in patient,which mainly affected the VSMCs.In spite of the multiple studies focusing on AIM2,little is known about the function and mechanism ofAIM2 on atherosclerosis.But,whether ox-LDL could affect vascular smooth muscle cells?VSMCs?pyroptosis has not yet been reported.Objective?1?ox-LDL stimulates the cell pyroptosis of vascular smooth muscle cells.?2?AIM2 mediates the proptosis of VSMCs stimulated by ox-LDL?3?The mechanism of ox-LDL stimulates AIM2Methods Cell culturesMouse MOVAS vascular smooth muscle cells?VSMCs?were cultured in Dulbecco's Modified Eagle Medium?DMEM??45g/L glucose?,supplemented with streptomycin?100ug/ml?,penicillin?lOOU/ml?,10%?v/v?fetal bovine serum?FBS?at37°C in a humidified atmosphere of 5% C02.Lentivirm intervenesAIM2 lentivirus and shAIM2 were transfected into SMCs for overexpression and inhibition of AIM2.Then cells were cultured for another 48 hours to detect the transfection efficiency.Then the protein was extracted from the cells when the ox-LDL?150ug/ml?was added to the medium for 12 hours.Western blot The expression of AIM2? ASC? pro-caspase-1?caspase-1 GSDMD-N were detected by Western-BlotTUNEL Assay In order to detect the fragmentation of DNA 5 an ApopTag in situ apoptosis detection kit was used.Immunofluorescence The distribution of Caspase-1 beta in VSMCs was observed by immunofluorescenceStatistical analysisAll the experimental results were presented as the mean ± standard deviation?SD?.GraphPad 5.0 and SPSS20.0 were used to analyze the data.Comparisons between different groups were analyzed by Student's t test^ and data between multiple groups were compared by one-way ANOVA.P < 0.05 was considers! to be statistical significance.Results Ox-LDL could promote the expression of AIM2 and GSDMD-NThe expression of AIM2,ASC,Capsapsel and GSDMD-N in VSMCs stimulated by ox-LDL was higher than control group?p < 0.05?.Different levels of ox-LDL increased AIM2 expression in a concentration-dependent manner.Ox-LDL could stimulate the pyroptosis in VSMCs The TUNEL positive cells and EthD-III positive cells were higher when stimulated with control group?p < 0.05?Gene silence can inhibit the AIM2 expression and AIM2 lentivirus can promote AIM2 expressionAIM2-miRNA lentivirus was intervened,the protein level of AIM2 in VSMCs were less than the NC group?p < 0.05?.AIM2 lentivirus could promote AIM2 expression in VSMCs compared with NC group?p < 0.05?.AIM2 could affects inflammation expressionAIM2-miRNA lentivirus was intervened,the protein level of ASC,Caspase 1 p 10,IL-ip in VSMCs were less than the NC group?p < 0.05?.AIM2 lentivirus could promote ASC,Caspase 1 plO,GSDMD-N expression in VSMCs compared with NC group?p<0.0?5?.AIM2 is involved in pyroptosis induced by high ox-LDLEth-III staining showed an increased proportion of cell death in AIM2 overexpression,but a decreased percentage of cell death in AIM2 inhibition?p <0.05?.Consistently,TUNEL staining confirmed the same role of AIM2 in cell death in MOVAS VSMCs.Conclusion?1?ox-LDL could stimulate the pyroptosis in VSMCs;?2?ox-LDL could improve the expression of AIM2 through NF-kB signaling?3?AIM2 mediates the pyroptosis of VSMCs when stimulated by ox-LDLAtherosclerosis can lead to fatal and non-fatal coronary artery vascular events.AS can increase the risk of cerebrovascular accidents.It can also lead to an increase in myocardial infarction?MI?and coronary death in patients with CAD.In 17 studies involving 11391 patients,it was found that more than 50 percent of patients have symptomatic carotid stenosis with 63 percent of late death events being cardiac events,and the average risk of cardiac death increases by 2.9% a year.Many studies have shown that even after adjusting for routine risk factors,Mortality of symptomatic or asymptomatic patients,Mortality and morbidity of cardiovascular diseases are also at increased risk.Ankle-brachial index?abi0.9?< 0.9 can lead to a ten-year risk of coronary events and a more than two fold increase in the overall mortality rate.Five years later,20 percent of patients will have intermittent claudication.The mortality rate of myocardial infarction or stroke will reach 10-15%.All these findings suggest that it is important to explore the occurrence of atherosclerosis and to find effective targets for prevention and treatment of cardiovascular disease.Previous studies have shown that the presence of vascular smooth muscle cells?VSMCs?is beneficial and protective in advanced atherosclerotic plaques,because it plays a key role in the formation and maintenance of fibrous caps.In contrast,the formation of atherosclerotic plaque is closely related to the percentage of VSMCs Atherosclerotic plaques are formed in many special parts of the artery,which are known as ''atherosclerotic susceptible regions”,and these sites are common to both animals and humans.When endothelial cells were exposed to these hemodynamic changes,their function was disturbed and characterized by impaired expression of endothelial nitric oxide synthase?eNOS?.The decrease of bioavailability of nitric oxide?NO?promotes multiple atherogenic effects,including increased platelet adhesion and aggregation,monocyte adhesion / infiltration,aggregation of lipoprotein oxidation and vasoconstriction.In addition,the decrease of NO level can increase the expression of matrix metalloproteinase?MMPs?and play a role in promoting the migration of VSMCs.Therefore,the study of the pathological process of vascular smooth muscle cells has become a hot research topic.Therefore,the study of the pathological process of vascular smooth muscle cells has become a hot research topic.The migration of vascular smooth muscle contributes to the early development of atherosclerosis,but it is also important to maintain the stability of plaque by maintaining a protective fibrous cap that covers the lipid nuclei of advanced lesions.Therefore,understanding the key regulatory factors of vascular smooth muscle cell growth in different stages of atherosclerosis is helpful to the strategy of regulating the disease process in the foture.Matrix xnetalloprotein?MMPs?is associated with the growth of vascular smooth muscle cells and the process of atherosclerosis.In particular,MMP-2 has been shown to play an important role in promoting the development of early lesions,and colleagues preventing the formation of late plaques.A recognized mechanism for regulating the behavior of VSMCs by MMPs is to regulate the interaction of cell to cell and cell matrix,thereby affecting the migration of smooth muscle cells.AIM2 is a cytosolic double-stranded DNA?dsDNA?receptor that contributes to the host defense against bacterial and viral pathogens,such as Francisella tularensis and vaccinia virus,respectively.We previously identified that infection by the cytosolic bacterium Francisella tularensis subspecies novicida activates the transcription factor IRF1,which induces production of interferon-inducible GTPases called guanylatebinding proteins.Guanylatebinding proteins attack and kill bacteria in the cytoplasm,resulting in the liberation of bacterial DNA for recognition by AIM2.Upon binding of dsDNA in the cytoplasm of infected cells,AIM2 recruits the adaptor protein ASC to assemble an inflammasome complex that activates caspase-1,a cysteine protease that induces pyroptosis md mediates the cleavage of the inflammatory cytokines IL-lb and IL-18.Structural analysis of AIM2 revealed that the HIN200 domain binds dsDNA,whereas the pyrin domain recruits ASC.We have suggested that AIM2 mediates the progression of atherosclerosis by promoting the MMP2 expression and the migration of VSMCs,and AIM2 inhibition may have a protective function on VSMCs in atherosclerosis.Here,we investigated the potential role and underlying mechanism of AIM2 involved in migration of VSMCs in atherosclerosis.Objective?1?ox-LDL could promote the expression of AIM2;?2?We induced the AIM2 overexpression and silencing by lvAIM2 and shAIM2 virus,and explore the role of AIM2 in VSMCs.?3?We further explored the mechanisms of the regulation of AIM2 activation in VSMCs.Cell culturesMouse MOVAS vasgular smooth muscle cells?VSMCs?were cultured-in Dulbecco's Modified Eagle Medium?DMEM??4.5g/L glucose?,supplemented with 10%?v/v?fetal bovine serum?FBS?,streptomycin?100ug/ml?,penicillin?lOOU/ml?at 37°C in a humidified atmosphere of 5% C02.Lentivirus intervene AIM2 lentivirus and shAIM2 were transfected into SMCs for overexpression and inhibition of AIM2.Then cells were cultured for another 48 hours to detect the transfection efficiency.Then the protein was extracted from the cells when the ox-LDL?150ug/ml?was added to the medium for 12 hours.Western blot The expression of A IM2 n MMP2x TGF-pN p-SMAD2,SMAD3,p-SMAD3,SMAD3 were detected by Western-BlotTrans well Assay Detection of migration of VSMCs was performed using transwell assay kit.Immunofluorescence The distribution of Aim2 in VSMCs was observed by immunofluorescenceStatistical analysis All the experimental results were presented as the mean ??? standard deviation?SD?.GraphPad 5.0 and SPSS20.0 were used to analyze the data.Comparisons between different groups were analyzed by Students t test,and data between multiple groups were compared by one-way ANOVA.P < 0.05 was considered to be statistical significance.Results Ox-LDL could promote the expression ofAIM2The expression of AIM2 in VSMCs stimulated by ox-LDL was higher than control group?p < 0.05?.ox-LDL increased AIM2 expression in a time-dependent manner.Gene silence can inhibit the AIM2 expression and AIM2 lentivirus can promote AIM2 expressionAIM2-miRNA lentivirus was intervened,the protein level of AIM2 in VSMCs were less than the NC group?p < 0.05?.AIM2 lentivirus could promote AIM2 expression in VSMCs compared with NC group.AIM2 affects MMP-2 expression induced by high ox-LDLAIM2-miRNA lentivirus was intervened,the protein level of MMP2 in VSMCs were less than the NC group?p < 0.05?.AIM2 lentiviras could promote MMP2 expression in VSMCs compared with NC group?p < 0.05?.AIM2 is involved in TGF-p/SMAD signalling induced by high ox-LDLWestern blot analysis demonstrated that AIM2 promotes ox-LDL-induced TGF-p and SMAD2/SMAD3 phosphorylation?P < 0.05?)while AIM2 inhibition reduced ox-LDL-induced TGF-(3 and SMAD2/SMAD3 phosphorylation?P < 0.05?.AIM2 is involved in migration of VSMCs induced by high ox-LDLTranswell staining showed an increased proportion of cell migration in AIM2 overexpression,but a decreased percentage of cell migration in AIM2 inhibition?p <0.05?..Conclusion?1?ox-LDL could improve the expression of AIM2 through ROS;?2?AIM2 mediates the expression of MMP2 and the migration of VSMCs when stimulated by ox-LDL;?3?AIM2 could mediates TGF-p/SMAD signaling. |
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