| BackgroundAcute coronary syndrome(ACS),including acute myocardial infarction and unstable angina pectoris,is a common high-risk disease in the field of emergency medicine and the main cause of death of atherosclerotic cardiovascular disease.Over the years,a series of important advances in drugs,interventional therapies and surgery have greatly reduced the mortality rate and improved the prognosis of patients with ACS,but coronary artery disease still kills 19 million people each year,and many people with asymptomatic heart disease die suddenly.Therefore,how to identify highrisk individuals early,achieve accurate prevention and treatment,and reduce the occurrence of ACS is still a major issue in the field of ACS,and its urgency is becoming increasingly prominent.ACS has the pathological features of coronary atherosclerosis plaque rupture or erosion(named vulnerable plaques).One of the characteristics of vulnerable plaques is the enlarged necrotic core.In recent years,more and more studies have shown that dampened efferocytosis is the main driving factor for the enlarged necrotic core of atherosclerotic plaques.Efferocytosis refers to the process of removing apoptotic cell debris by professional and non-professional phagocytes.The body can effectively remove apoptotic cells,prevent secondary necrosis of apoptotic cells,reduce the release of proinflammatory mediators,and maintain tissue and organ homeostasis through the process of efferocytosis.Plaque enlargement and plaque vulnerability result from the accumulation of apoptotic clasts,which are cleared by macrophage phagocytosis(efferocytosis).Necrotic core is a marker of atherosclerotic plaque vulnerability,and plaque type is closely related to heart attack and stroke.In recent years,it has been found that reduced efferocytosis is the main driving factor of atherosclerotic necrotic core formation,which can lead to plaque rupture and acute thrombotic cardiovascular events.Therefore,effective clearance of apoptotic cells plays a key role in preventing the development of atherosclerotic plaques.Clinical studies have shown that mutations of the aldehyde dehydrogenase 2(ALDH2)gene increase the risk of ACS to nearly two-fold and significantly increase the incidence of adverse outcomes of ACS.ALDH2 is a key enzyme in the metabolic pathway of alcohol(ethanol)and many endogenous and exogenous toxic aldehydes in human body.Human mitochondrial ALDH2 protein,encoded by ALDH2 gene,is widely distributed in all organs and tissues of the human body,most abundant in liver,and also abundant in other organs that need to consume a lot of ATP,such as heart and brain.Glu504Lys polymorphism locus(rs671)of ALDH2 in exon 12 is widely found in Chinese,Japanese,Korean and other East Asian populations,with mutation rate up to 30%-50%,but rare in Caucasians of Europe and America.This ALDH2 gene polymorphism is closely associated with many diseases,such as alcohol-related diseases(hepatitis,cirrhosis,etc.),pulmonary hypertension,myocardial infarction,stroke,etc.Exploring the potential therapeutic targets for the occurrence and development of atherosclerotic plaques(especially vulnerable plaques)is of great clinical value and social significance for the accurate prevention and treatment of ACS patients,further reducing their incidence,and improving their survival rate and quality of life.The results of clinical studies suggest that ALDH2 may be an important breakthrough point for the precision prevention and treatment of ACS patients in China.Considering that the high prevalence of ALDH2 rs671 mutation in China,it is particularly important to explore the mechanism of ALDH2 rs671 mutation increasing the incidence of ACS and adverse prognosis,as the mutations appear in 30%-50%of Chinese.In order to further clarify the specific mechanism of ALDH2 regulating the progression of atherosclerosis,we designed and carried out this experimental subject.ObjectivesThis experiment aims to investigate:1.Whether macrophage ALDH2 gene defect can promote the progression of atherosclerosis2.Whether the macrophage ALDH2 gene defect can reduce its efferocytosis ability3.How does ALDH2 regulate macrophage efferocytosisMethodsWe constructed myeloid ALDH2 gene knockout mice under APOE-/-background by bone marrow transplantation,and constructed atherosclerosis model by high-fat feeding for 8 weeks to explore the effect of myeloid ALDH2 gene knockout on atherosclerosis.Then,primary peritoneal macrophages and bone-marrow induced macrophages of ALDH2-/-mice were extracted,and efferocytosis was detected by flow cytometry,and downstream efferocytosis related proteins affected by ALDH2 were screened by RNA-seq and proteomics.Then,HEK293T cells were transfected with exogenous plasmids to further explore the regulatory mechanism of ALDH2 on efferocytosis.(Ⅰ)For vivo study1.Animal experiment protocol:Myeloid cells ALDH2 gene knockout mice were constructed as follows:APOE-/-mice aged 6-8 weeks were randomly divided into two groups:APOE-/-myeloid cell receptor group and APOE-/-ALDH2-/-myeloid cell receptor group.Bone marrow cells from APOE-/-mice and APOE-/-ALDH2-/mice aged 6-8 weeks were then extracted for bone marrow transplantation through caudal vein injection.Primary peritoneal macrophages of mice were extracted 15 days after bone marrow transplantation to detect ALDH2 protein content and verify the efficiency of bone marrow transplantation.2.Establishment of mouse atherosclerosis model:After bone marrow transplantation,mice were fed with high fat and high cholesterol for 8 weeks after recovery feeding for 4 weeks.After model construction,mice were sampled,and their serum,aorta and heart were collected for subsequent testing.3.Aortic gross staining for atherosclerotic plaque area detection:The aorta of mice was stained with gross oil red O,and the oil red O stain was used as the plaque,and the ratio of gross stained plaque area to aortic area was used as the index of gross plaque area.4.Detection of atherosclerotic plaque area and plaque contents in aortic root section:After the heart was harvested,the aortic root was taken into continuous frozen sections,and the sections were stained with HE,oil red O,α-SMA,momA2 and Sirius red.The ratio of the plaque area to the heart cavity area in HE staining results was used as the indicator of atherosclerotic plaque area.The ratio of positive area to plaque area was used as the area index of plaque contents.5.Detection of apoptotic cell content and in vivo efferocytosis ability in atherosclerotic plaque area of mice:Heart out after the frozen section in the aortic root in mice TUNEL staining marked apoptosis cells,as well as the MOMA-2 immunofluorescence stain marks macrophages,in average fluorescence optical density TUNEL staining plaques as indicators of apoptotic cell content,with positioning with macrophage apoptosis cells/free the ratio of apoptosis cells as the target cell was buried.6.Determination of blood lipid content in mice:when the mice were sampled,the blood was collected in the way of heart tip blood,and the blood was centrifuged at 4℃ at 2500g for 15 minutes.The serum was collected and the serum cholesterol,triglyceride,high-density lipoprotein and low-density lipoprotein were detected using the corresponding kit.7.Statistical analysis:When the measurement data conforms to normal distribution and the variance is uniform,Mean±SEM is used to represent the data,and t test is used for statistics.When the data does not conform to normal distribution or conforms to normal distribution but the variance is uneven,median(second quartile,third quartile)is used to represent the data,and Mann-Whitney test is used.GraphPad Prism 8 software was used for statistical analysis of the data,including normal distribution test,homogeneity of variance test,T test and Mann-Whitney test,etc.,P<0.05 was considered statistically significant.(Ⅱ)For vitro study1.Cell experiment:In order to explore whether ALDH2 gene knockout can inhibit macrophages’ in vitro efferocytosis,primary peritoneal macrophages and bonemarrow induced macrophages of mice were extracted and co-cultured with apoptoses-inducing smooth muscle cells,and efferocytosis was detected by flow cytometry.In order to explore downstream efferocytosis related genes affected by ALDH2 gene knockout,primary peritoneal macrophages stimulated by apoptotic cells were collected for RNA-seq and proteomics detection,and bioinformatics analysis was conducted to screen out downstream efferocytosis protein Rac2 affected by ALDH2.Subsequently,wild-type mouse peritoneal primary macrophages and ALDH2-/-mouse peritoneal primary macrophages were extracted,and the expression of downstream burial-related protein Rac2 was detected after apoptotic cell stimulation.To explore how ALDH2 regulates Rac2,we constructed ALDH2 wild-type plasmid,ALDH2 rs671 mutant plasmid and Rac2 plasmid,and detected the binding effect of ALDH2 wild-type or ALDH2 mutant to Rac2 by immunoprecipitation.Subsequently,the specific mechanism of ALDH2 regulating Rac2 protein was explored by using ubiquitin binding assay.2.Extraction of primary peritoneal macrophages:the mice were intraperitoneally injected with starch broth,and the mice were sacrificed 3 days later after being removed from the cervical cavity.The mice were then drained of serum-free highglucose DMEM medium using a 10ml sterile syringe.3.Marrow induced macrophage extraction:The bone marrow cells in the medullary cavity were washed out with PBS,and the broken bone was removed by cell sieve.After centrifugation at 4℃ for 15 min at 1500g,the precipitate was suspended again.The cultured mice were cultured in DMEM medium containing 25ng/ml mouse m-csf and 10%fetal bovine serum for 7 days.Induce differentiation of macrophages.4.Efferocytosis ability detection:cytosinin was used to induce apoptosis of smooth muscle cells,apoptotic smooth muscle cells were collected and counted.Apoptosis cells and macrophages were co-cultured for 1.5 hours according to the ratio of apoptotic cells:macrophages=1:1,and efferocytosis ability was detected by flow cytometry.5.HEK293T cell plasmid transfection:Lipo 2000 was used as the co-transfection reagent to transfect the corresponding plasmid into HEK293T cells.6.Protein detection:After the protein was extracted by primary macrophages,it was separated by 10%-15%SDS-PAGE electrophoresis,membrane transfer,primary antibody and secondary antibody were incubated and detected.7.mRNA detection:mRNA was extracted from primary macrophages and detected by RT-qPCR.8.Statistical analysis:same as animal experiments.Results1.Myeloid cell ALDH2 gene knockout promotes the progression of atherosclerosis Myeloid ALDH2 gene knockout mice were constructed by bone marrow transplantation under APOE-/-background.After 8 weeks of high-fat feeding,the results showed that the myeloid ALDH2 gene knockout mice significantly increased the area of atherosclerotic plaque,plaque infiltration and lipid deposition,and necrotic core area without affecting blood lipid.The content of collagen fiber was decreased,but the content of smooth muscle cell was not affected.2.ALDH2 gene knockout in macrophages inhibits efferocytosis in vitro and in vivo TUNEL staining and TUNEL+ MOMA-2 staining showed that ALDH2 gene knockout in myeloid cells did not affect the total number of apoptotic cells in plaque,but significantly reduced the efferocytosis capacity of macrophages in plaque.Primary macrophages and BMDM were extracted for in vitro efferocytosis experiments,and it was found that ALDH2 gene knockout significantly inhibited the efferocytosis ability of macrophages.3.ALDH2 gene knockout inhibited the expression of downstream burial-related gene Rac2The macrophages stimulated by apoptotic cells were collected and sent for mRNA sequencing.Bioinformatics analysis showed that ALDH2 gene knockout inhibited the expression of genes related to efferocytosis pathway.Combined analysis of sequencing results with single cell sequencing results from GEO database showed that Rac2 gene expression was most obviously up-regulated in efferocytosis among ALDH2-regulated genes related to efferocytosis.At the same time,the proteins of macrophages stimulated by apoptotic cells were collected,and the proteins bound to ALDH2 were enriched by ALDH2 antibody,and then sent for protein spectrum analysis.The results showed that ALDH2 protein could bind Rac2 protein.Subsequent validation experiments also confirmed that ALDH2 knockdown inhibited Rac2 mRNA and protein level expression.4.ALDH2 gene knockout inhibits macrophage cytoburial by inhibiting Rac2 expressionInhibition of Rac2 expression in macrophages by siRNA significantly inhibited the efferocytosis ability of primary peritoneal macrophages and bone-marrow induced macrophages.In bone marrow induced macrophages with ALDH2 gene knockout,adenovirus overexpression of Rac2 protein significantly saved the impaired efferocytosis capacity.5.ALDH2 protein can directly bind to Rac2 proteinConfocal microscopy was used to double stain ALDH2 and Rac2 proteins in primary macrophages.The results showed that ALDH2 and Rac2 proteins had obvious colocalization.HEK293T cells were transfected with ALDH2-WT plasmid,AlDH2-rs671 plasmid and Rac2 plasmid.Protein immunoprecipitation assay showed that ALDH2 protein could bind Rac2 protein.Aldh2-wt protein,ALDH2-rs671 protein and Rac2 protein were expressed in vitro by in vitro translation system.In vitro binding experiments were carried out,and it was found that ALDH2-WT protein and ALDH2rs671 protein could bind to Rac2 protein through direct binding.By constructing Rac2 truncation,exogenous binding experiments showed that the binding region of ALDH2 protein and Rac2 protein was within the 1-136 amino acid peptide segment of Rac2 protein.6.ALDH2 inhibits Rac2 ubiquitination and stabilizes Rac2 proteinMice peritoneal macrophages were extracted and protein synthase inhibitor CHX was added to inhibit protein synthesis.Proteins were collected at 0,2,4 and 8 hours after stimulation to detect the degradation of Rac2 protein.The results showed that the degradation rate of Rac2 protein was significantly increased in ALDH2 knockout macrophages compared with wild macrophages.ALDH2-wt and ALDH2-rs671 were supplemented in ALDH2 knockout macrophages,and it was found that ALDH2-WT could rescue Rac2 protein degradation,while ALDH2-rs671 could not.Macrophages in the ALDH2 wild group and the ALDH2 gene knockout group were treated with CHX stimulation and MG-132 or chloroquine stimulation respectively to detect Rac2 protein degradation.The results showed that the addition of MG-132 inhibited the proteasome degradation pathway significantly inhibited Rac2 degradation in both groups.Rac2 degradation was not inhibited by adding chloroquine,indicating that Rac2 degradation mainly occurred through the proteasome pathway.Subsequently,ALDH2-WT and ALDH2-rs671 plasmids were co-transfected into HEK293T cells with ubiquitinated plasmids and Rac2 plasmids,respectively.Immunoprecipitation results showed that compared with ALDH2-WT,ALDH2-rs671 mutation or lack of ALDH2 gene significantly increased Rac2 protein ubiquitination.Subsequently,we constructed a single amino acid mutation at the lysine site on Rac2 protein and co-transferred it,ubiquitinated plasmid and ALDH2 plasmid into HEK293T cells.It was found that the ALDH2-regulated Rac2 ubiquitination site was located at lysine 123.Meanwhile,through the ubiquitin binding experiment.The ubiquitination form of Rac2 regulated by ALDH2 was found to be ubiquitination of K48 connection.7.The mutation of ALDH2 rs671 in human macrophages inhibits the upregulation of Rac2 in efferocytosis,and thus inhibits its efferocytosisCompared with ALDH2-WT,ALDH2-rs671 did not affect Rac2 expression at baseline,but inhibited the up-regulation of Rac2 expression induced by apoptotic cells by human mononuclear macrophages and apoptotic Jurkat cells.At the same time,flow cytometry was used to detect the efferocytosis capacity of mononuclear macrophages in the two groups,and it was found that the efferocytosis capacity of ALDH2-rs671 mutant macrophages was significantly decreased compared with that of ALDH2-WT macrophages.Aldh2-wt virus and ALDH2-rs671 virus were injected into ALDH2-/macrophages,respectively,and it was found that ALDH2-WT could save the impaired efferocytosis,while ALDH2-rs671 could not.Conclusion1.Absence/mutation of ALDH2 gene in macrophages promotes the progression of atherosclerosis by inhibiting efferocytosis.2.By directly binding to Rac2 protein,ALDH2 protein in macrophages inhibits the ubiquitination of Rac2 protein,thus stabilizing Rac2 protein and promoting efferocytosis.3.ALDH2 or Rac2 is a promising therapeutic target for atherosclerosis. |
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