The Effects And Mechanisms Of E06 On ABCA1-mediated Cholesterol Efflux And The Development Of Atherosclerosis

Echo viruse-06 associated IgM nature antibody 6（E06）is one of the monoclonal autoantibodies against oxidized phosphatidylcholine.In recent years,it has been found that E06 is closely related to atherosclerosis（As）,but the mechanism is not clear.There are some researches indicated that increasing the efficiency of reverse cholesterol transport can restrain the atherosclerosis progression.ATP binding cassette transporter A1（ABCA1）plays a key role on cholesterol efflux and is involved in the process of RCT.In this study,we firstly investigated correlation of E06 and coronary heart disease from the clinical perspective.The data showed that the levels of plasma E06 was reduced in the patients with coronary heart disease and the plasma E06was negatively correlated with HDL;then,we explored the role of E06on ABCA1 expression in macrophage-derived foam cells and the atherosclerosis progression in LDLR^-/-mice.The experiments showed that E06 promoted ABCA1-medical cholesterol efflux and reduced atherosclerotic lesion area in LDLR^-/-mice.Furthermore,E06up-regulated ABCA1 expression through the miR-203a-3p/Gli3 pathway in macrophages derived foam cell.In short,our findings provide a novel strategy to prevent and treat of As.Part Ⅰ The changes of E06 levels in the plasma of patients with CHDObjective:To detect the level of E06 in plasma of patients with coronary heart disease（CHD）and control group（non-CHD patients）,to explore the relationship between E06 and CHD,and to further analyze the correlation between E06 and HDL-C.Methods:The subjects were 204 patients who underwent coronary angiography（CAG）in the Department of Cardiology of the First Affiliated Hospital of University of South China.The degree of coronary artery stenosis was evaluated by Gensini score,and the higher the score was positively related to severity of the As lesion.According to the results of Gensini score,the subjects were divided into 4 groups:control group（Gensini=0）with no coronary stenosis,low risk group（1<Gensini≤20）with mild coronary stenosis,medium risk group（20<Gensini≤40）with moderate coronary stenosis,and high risk group（40<Gensini≤160）with severe coronary stenosis.The plasma of patients was collected and separated to detect the levels of E06 and HDL-C to determine the correlation between E06 and AS lesions and whether there was a correlation between E06 and HDL-C.Results:There was no significant difference in the general conditions（sex,age,smoking,hypertension,etc.）of the subjects in each group,and there was no significant difference in white blood cell（WBC）,monocyte,Alanine transaminase（ALT）,glutamic oxaloacetic transaminase（AST）,creatinine（Cr）,urea nitrogen（BUN）and so on.The concentration of E06 in each group was（29.63±5.82）μg/ml in Control group,（24.85±3.59）μg/ml in Low Risk group,（20.89±3.73）μg/ml in Middle Risk group,and（16.80±4.81）μg/ml in high risk group,suggesting that the concentration of E06 in patients with coronary artery stenosis decreased as the degree of coronary artery stenosis increased,and showed a downward trend as the degree of coronary artery stenosis increased,indicating that the concentration of E06 in the three groups of patients with coronary artery stenosis was lower than that in the control group,which indicated that the concentration of E06 in patients with coronary artery stenosis in the three groups was lower than that in the control group,and showed a downward trend with the increase of the degree of coronary artery stenosis.The results showed that the severity of coronary artery disease was inversely proportional to the concentration of E06.Then the correlation between the concentration of E06 and HDL-C was analyzed,which suggested that there was a positive correlation between the concentration of E06 and HDL-C（R=0.5433,P<0.001）.Summary:（1）The content of E06 in serum of patients with CHD was significantly decreased.（2）The concentration of E06 was positively correlated with the level of HDL-C.Part Ⅱ E06 regulates ABCA1 expression and cholesterol efflux through the miR-203a-3p/Gli3 pathway in macrophageObjective:To explore the effects of E06 on lipid accumulation and ABCA1-mediated cholesterol efflux in foam cells,and to explore whether E06 can regulate the expression of ABCA1 and its potential mechanism.Methods:Foam cell model was established by using THP-1monocytes.After THP-1-derived foam cells were treated with E06concentration gradient for different time,lipid accumulation in foam cells was observed by oil red O staining,cholesterol content in foam cells was analyzed by high performance liquid chromatography,ABCA1expression was detected by western blotting and real-time fluorescence quantitative PCR（q RT-PCR）,and cholesterol efflux was analyzed by liquid scintillation counter.Bioinformatics predicted the binding site of GLI zinc finger protein family protein 3（GLI3）to ABCA1 promoter region,and luciferase reporter gene and chromatin immunoprecipitation（Chip）further analyzed the binding of Gli3 to ABCA1 promoter.After the expression of Gli3 was knocked down by small interference RNA（si RNA）,the cells were treated with E06,and the expression of ABCA1was detected by q RT-PCR and WB.Through bioinformatics analysis and screening,targeted regulation of Gli3 miRNAs,and luciferase reporter gene to detect its binding.Mi R-203a-3p mimic or inhibitor was used to co-incubate THP-1 macrophage-derived foam cells,and the expression of Gli3 and ABCA1 and cholesterol efflux were observed.After THP-1macrophage-derived foam cells were treated with E06,the expression of miR-203a-3p was detected to observe the effect of E06 on miR-203a-3p,and E06 and miR-203a-3p mimic were co-treated with THP-1macrophage-derived foam cells to detect the expression of Gli3 and ABCA1 and cholesterol efflux.Results:E06 promoted the expression of ABCA1 in THP-1-derived foam cells,increased cholesterol efflux and decreased intracellular lipid accumulation.Bioinformatics predicted that there may be three Gli3binding sites in the ABCA1 promoter region,namely（-73,-60）,（-656,-642）,（-1361,-1352）.Luciferase reporter gene and Chip experiments further confirmed that there were two binding sites in the ABCA1promoter region（-73,-60）,（-1361,-1352）,and E06 could upregulate the expression of ABCA1 by promoting the binding between Gli3 and ABCA1 promoter.Bioinformatics analysis showed that there were binding sites between 3’UTR of Gli3 and miR-203a-3p;luciferase reporter gene assay showed that miR-203a-3p could interact with Gli3directly;miR-203a-3p mimic down-regulated the expression of Gli3 and ABCA1 and inhibited cholesterol efflux from macrophages;while inhibiting miR-203a-3p showed the opposite.The expression of miR-203a-3p was decreased in THP-1 macrophages treated with E06.When miR-203a-3p mimic was added to THP-1 macrophages treated with E06 at the same time,the ability of E06 to increase the expression of Gli3 and ABCA1 was limited compared with that of E06 group.Summary:（1）E06 promotes the macrophages ABCA1,increases cholesterol efflux,and inhibits cholesterol accumulation and foam cell formation,（2）E06 upregulates the expression of ABCA1 in macrophages through miR-203a-3p/Gli3 pathway.Part Ⅲ The effects of E06 on atherosclerosis and its mechanism in LDLR^-/-miceObjective:To explore the effects of E06 on blood lipid level,RCT efficiency and As plaque area in LDLR^-/-mice,and the effects of E06 on the expression of miR-203a-3p,Gli3 and ABCA1 and cholesterol efflux in vivo.Methods:40 male LDLR^-/-mice in age with 8-week-old fed with high-fat diet（HFD）were randomly divided into two groups:1,control group（n=20）:intraperitoneal injection saline,once a day until 3 day before death;2,E06 group（n=20）:E06 monoclonal antibody dissolved in saline and injection（20 mg/kg）until 3 day before death.The mice were fed with high-fat diet continuously for 12 weeks,and the mice were weighed every 2 weeks during the feeding period.The rats were killed after 12 weeks of completion of all tests.The levels of plasma TC,TG and HDL-C,liver function and renal function were measured by enzyme oxidation method.[³H]labeled cholesterol was used to detect the efficiency of RCT in mice.After the mice were killed,the aortic arch was removed and the plaques in the aorta were observed under stereomicroscope.Oil red O and HE staining were used to observe the As plaques of aortic sinus and aorta in mice.Using q RT-PCR and WB to detect the expression of miR-203a-3p,Gli3 and ABCA1 in mouse aortic tissue and peritoneal macrophages.Results:E06 had no significant effect on body weight,liver and kidney function and plasma levels of TC and TG in LDLR^-/-mice.The plaque area of aortic arch,the plaque area of aortic sinus and the lipid deposition of artery wall were significantly decreased in E06 group.It was confirmed that E06 could inhibit the development of As in vivo.At the same time,the level of HDL-C and the efficiency of RCT were significantly increased in mice treated with E06,indicating that E06 can reduce the occurrence and development of As by enhancing the efficiency of RCT.In addition,E06 can reduce the level of miR-203a-3p in plasma,aorta and peritoneal macrophages of LDLR^-/-mice,and promote the expression of Gli3 and ABCA1,indicating that E06 can also up-regulate the expression of ABCA1 through miR-203a-3p/Gli3 pathway.Summary:（1）E06 upregulates the expression of ABCA1 in LDLR^-/-mice,increases the level of plasma HDL-C,accelerates the process of RCT and inhibits the development of As.（2）E06 upregulates the expression of ABCA1 through miR-203a-3p/Gli3 pathway in LDL^-/-mice.Conclusions 1.The content of E06 in plasma of patients with CHD is significantly decreased,and it was positively correlated with the level of HDL-C.2.E06 upregulates ABCA1 expression through miR-203a-3p/Gli3 pathway to promote cholesterol efflux and inhibit lipid accumulation in macrophages.3.E06 upregulates the expression of ABCA1 to promote the plasma level of HDL-C,accelerate the RCT process and alleviate the atherosclerosis development in LDLR-/-mice.