Observation Of Anti-atherosclerotic Effects Of PRMT2 And Investigation Of The Possible Mechanism

The morbidity and mortality of cardiovascular disease(CVD)are on the rise.The economic and social burden of CVD is becoming more and more heavy,which has seriously reduced the living standard and quality of CVD patients and their families.Coronary atherosclerotic heart disease(CHD),referred to as coronary heart disease,is the most important component of CVD,and is also the common cause of death in patients with CVD.Atherosclerosis(AS)is the common soil of almost all CHD,and it is also an important pathological basis and characteristic of CHD.The pathogenesis of AS is very complex and has not been fully elucidated.Many factors such as heredity,environment,stress,lipid metabolism disorder and infection are involved in the pathogenesis of AS.Therefore,it is of great significance to explore the pathogenesis of as and find new targets for the prevention and treatment of ASThe generation of foam cells is an important pathological feature of AS in the early stage.Vascular smooth muscle cells(VSMC)and macrophages,which engulf lipoprotein derived cholesterol,especially oxidized low density lipoprotein(ox-LDL),under the endocardium,cause lipid accumulation in macrophages and VSMC,and then foam cells.LDL is mainly recognized by CD36 and SR-A receptors on the membrane of macrophages and VSMC.After ingestion,the lipid can be metabolized and degraded by macrophages and VSMC,and then transported to the liver,and finally excreted to the intestinal tract in the form of bile secreted by the liver.This process is called reverse cholesterol transport(RCT).There are three main pathways of RCT in macrophages and VSMC: ABCA1,ATP binding cassette transporter G1(ABCG1)and scavenger receptor B type I(SR-B1).The abnormality of RCT is an important reason for foam cell production and final AS formation.Protein arginine methyltransferases(PRMTs)are ubiquitous methylases in eukaryotic cells,which are mainly involved in protein arginine methylation modification.Nine PRMTs have been found to be expressed in mammalian cells,and prmt2 is one of the important family members of PRMTs.PRMT2 was expressed in all tissues of human body,but the expression level of PRMT2 in heart,blood vessel,ovary,mammary gland and nervous system was relatively high.The biological functions of PRMT2 include participating in RNA metabolism,interacting with various nuclear proteins and participating in gene transcription regulation through methylation histone as co activator.Recent studies have found that compared with non-diabetic mice,the expression of PRMT2 in monocytes of diabetic mice is decreased,and the expression of PRMT2 is down regulated under hyperglycemia.After knockout of PRMT2 gene,the expression of ABCA1 in primary bone marrow-derived macrophages was significantly down regulated,and the cholesterol efflux rate of bone marrow-derived macrophages mediated by ABCA1 was also significantly reduced.These studies also indicate that PRMT2 can participate in the regulation of RCT in macrophages,which may affect the production of foam cells.Therefore,PRMT2 may be a potential target for anti AS and has an anti AS effect.However,the anti as effect of prmt2 has not been clearly confirmed,and its detailed mechanism is still unclear.Therefore,in order to elucidate the possible mechanism of PRMT2 against AS,we first explored the relationship between the level of serum PRMT2 and CHD in clinical cases of CHD,and further observed the effect of high expression of PRMT2 on the formation of high fat induced AS in apo E-/-mice and the expression of ABCA1,ABCG1 and SR-B1,and the expression of SR-A1 and SR-B1 of cholesterol uptake key proteins in the foam cells.Chapter 1:The relationships between level of serum protein arginine methyltransferase 2 and coronary atherosclerotic heart disease.Objective:To investigate the relationships between level of serum PRMT2 in CHD patients and the risk factors of CHD,and the severity of CHD.Methods:In this study,CHD group included 110 patients who diagnosed as CHD by coronary angiography(CAG)examination,and hospitalized in the First Affiliated Hospital of University of South China(USC)from September 2017 to September 2019.The control group were 110 healthy volunteers who contemporarily taken physical examination in the First Affiliated Hospital of USC,for the control group,their age and sex matched with the CHD group.Questionnaire survey and physical examination were used to collect basic information and data for both groups.Blood samples were collected from both groups to detect the levels of fasting blood glucose(FBG),blood lipid and serum PRMT2.Correlation analysis and regression analysis were conducted between level of serum PRMT2 and classical risk factors of CHD and the severity of CHD.Results:1.There were no significant differences between the control group and CHD group about the percentage of people older than or equal to 60 years old,the percentage of male population and the heart rate(all P>0.05).Compared with the control group,the percentage of people with history of drinking,smoking,hypertension and diabetes in CHD group was significantly increased(P<0.05).2.Compared with the control group,the levels of FBG,body mass index(BMI),systolic blood pressure(SBP),diastolic blood pressure(DBP),total cholesterol(TC),triglycerides(TG),low density lipoprotein cholesterol(LDL)in CHD group were all significantly increased(all P<0.05).Compared with the control group,the serum levels of high-density lipoprotein cholesterol(HDL-C)and PRMT2 in CHD group were significantly lower(all P<0.05).3.level of Serum PRMT2 was significantly negatively correlated with levels of BMI,serum TG,serum TC and serum LDL-C(all P<0.05),but was significantly positively correlated with level of serum HDL-C(P<0.05).There was no significant correlation between level of serum PRMT2 and DBP,SBP and FBG(all P>0.05).4.The serum PRMT2 level in CHD patients was significantly negatively correlated with Gensini score of CHD(r=-0.63,P<0.05),with the increase of Gensini score,the serum PRMT2 level of CHD patients was significantly decreased(all P<0.05).5.Univariate logistic regression analysis showed that the decrease of level of serum PRMT2 might be a risk factor for CHD(OR=0.51,95% CI:0.28-0.62,P<0.05).Multivariate logistic regression analysis showed that after adjustment for classic risk factors such as hypertension,dyslipidemia and type 2 diabetes,as well as age and gender,the risk of CHD was reduced by 36% for increase of each one standard deviation of level of serum PRMT2 in CHD patients(OR=0.47,95% CI: 0.37-0.83,P<0.05).The decrease of level of serum PRMT2 in CHD patients was independently correlated with the increased risk of CHD,and the level of serum PRMT2 was negatively correlated with the severity of CHD.Level of serum PRMT2 was expected to be an indicator for the risk assessment and severity assessment of CHD.Conclusion:Chapter 2:Observation of effect of overexpression of PRMT2 on macrophage derived foam cells and investigation of its mechanismObjective:To observe the effect of overexpression of PRMT2 on the formation of macrophage derived foam cells induced by ox-LDL and to explore the possible mechanism.Methods:1.The lentiviral vector LV-PRMT2 and the control lentiviral vector GV492 were transfected into RAW 264.7 macrophages respectively.The macrophages were treated with 4.0 μg/m L puromycin for 4 weeks to select the positive clone cells.The macrophage which stably over expressing PRMT2 was established.2.The macrophages were treated with ox-LDL(50 μg /m L)for 48 h to induce the formation of foam cells.3.The cells were divided into six groups: the normal control group,the ox-LDL group,the empty vector group,the PRMT2 overexpression group,the empty vector + ox-LDL group and the PRMT2 overexpression+ ox-LDL group.4.Lipid accumulation in cells was observed by oil red O staining.The levels of total cholesterol(TC),free cholesterol(FC)and cholesteryl ester(CE)in cells were determined by high performance liquidchromatography(HPLC).The cholesterol efflux rate of macrophages was detected by [3H] labeled cholesterol.5.Real-time PCR was used to detect the expression of PRMT2 m RNA,and Western blot was used to detect the expression of ABCA1,ABCG1,SR-B1,CD36 and SR-A1 in macrophages.Results:1.Compared with the normal control group,the expression of m RNA and protein of PRMT2 in the ox-LDL group were significantly decreased(all P<0.05).2.Compared with the empty vector group,the expression of m RNA and protein of PRMT2 in macrophages in the PRMT2 overexpression group were significantly increased(all P<0.05).3.A large number of lipid droplets were observed in the macrophages in the ox-LDL group and empty vector + ox-LDL group.While compared with the empty vector + ox-LDL group,the number of lipid droplets in macrophages in the PRMT2 over-expression + ox-LDL group were significantly reduced,and the lipid loading was significantly alleviated.4.Compared with the empty vector + ox-LDL group,the levels of TC,FC,CE and ratio of CE/TC in macrophages in the PRMT2over-expression + ox-LDL group were significantly decreased(all P<0.05).5.Compared with the empty vector + ox-LDL group,the cholesterol efflux rate of macrophages was significantly increased in the PRMT2over-expression + ox-LDL group(P<0.05).6.Compared with the empty vector + ox-LDL group,the level of protein expression of ABCA1 in macrophages was significantly increased in the PRMT2 over-expression + ox-LDL group(P<0.05).There were no significant differences in the expression of ABCG1,CD36 and SR-A1 between the PRMT2 over-expression + ox-LDL group and the empty vector + ox-LDL group(all P>0.05).Conclusion:Overexpression of PRMT2 inhibited the formation of foam cells induced by treatment of ox-LDL in macrophages,of which the mechanism might be an increase of the cholesterol efflux in macrophages through upregulating the expression of ABCA1.Chapter 3:Observation of effects of PRMT2 on AS formation in Apo E-/-mice and investigation of its mechanism.Objective:To investigate the effects of over expression of PRMT2 on the formation of AS in Apo E-/-mice and to explore the mechanism.Methods:1.Male Apo E-/-mice,10 weeks old,were randomly divided into three groups with 10 mice in each one(n = 10).The AS group: the mice were fed with high-fat diet for 12 weeks.The model and empty vector group: the mice were injected with lentivirus vector GV492,and 24 hours later the mice were continued to feed with high-fat diet for 12 weeks.The second injection of lentivirus vector GV492 through tail vein was on the14 th day days after the first injection.The model and PRMT2 group: the mice were injected with over expression of PRMT2 lentiviral vector LV-PRMT2 and 24 hours later the mice were continued to feed with high-fat diet for 12 weeks.The second injection of lentivirus vector LV-PRMT2 through tail vein was on the 14 th day days after the first injection.2.At the end of the experiment,the mice were anesthetized and fixed,and then were killed by heart bleeding,all blood samples were collected.The serum levels of TG,TC,HDL-C and LDL-C were detected.3.The peritoneal macrophages(PM)of Apo E-/-mice were collected and cultured,and the cholesterol efflux rate of PM was measured by [3H]labeled cholesterol.4.The aortic root of Apo E-/-mice was taken for serial frozen sections.The morphological changes of aortic sinus were observed by oil red O staining,H-E staining and Masson staining.5.Real-time PCR and Western blot were used to detect the m RNA and protein expression of PRMT2 in vascular tissue and PM of mice.The protein expression of ABCA1,ATP binding cassette transporter G1(ABCG1),scavenger receptor B1(SR-B1),scavenger receptor A1(SR-A1)and CD36 in vascular tissue and PM were detected by Western blot.Results:1.Construction of the over expression of PRMT2 lentiviral vector LV-PRMT2 and the control lentiviral vector GV492.2.After the first injection of lentivirus vector,the levels of m RNA and protein expression of PRMT2 in mice derived PM were detected a remarkably up-regulation compared with those of the mice without injection(0 day),from the 3rd day to the 84 th day,and the differences were statistically significant(all p<0.05).3.Compared with the AS group,the level of m RNA and protein expression of PRMT2 in the aorta in model and PRMT2 group were significantly up-regulated(P<0.05).However,there was no significant difference in the level of m RNA and protein expression of PRMT2 between the model and empty vector group and the AS group(P>0.05).4.Compared with the AS group,the model + empty vector group and the model + PRMT2 group both showed no significant difference in body weight at both the beginning and end point of the experiment(P>0.05).5.Compared with the AS group,there was no significant difference in the levels of serum TC,TG,LDL-C and HDL-C in the model + empty vector group(P>0.05).Compared with the model + empty vector group,the levels of serum TG,TC and LDL-C in model + PRMT2 group were significantly decreased(P<0.05),and the level of serum HDL-C was significantly increased(P<0.05).6.The results of H-E staining showed remarkably AS lesions in the aortic sinus of mice in both the model control group and model + empty vector group,while the AS lesions in the model + PRMT2 group were significantly alleviated.The results of oil red O staining showed obvious AS lesions in the aortic sinus of mice in the AS group and the model +empty vector group,while the AS lesions in the model + PRMT2 group were significantly reduced.Compared with the model + empty vector group,the percentage of AS lesions area in the aortic sinus of mice in the model + PRMT2 group was significantly decreased(P<0.05).7.Compared with model + empty vector group,Masson staining results showed that the level of collagen in aortic sinus plaque in model +PRMT2 group was significantly increased(p<0.05).8.Compared with model + empty vector group,cholesterol efflux rate of PM in model +PRMT2 group was significantly increased(P<0.05).9.Compared with model + empty vector group,the expression of ABCA1 in aorta and PM in model + expression PRMT2 group were significantly increased(P<0.05).Compared with model + empty vector group,the expression of ABCG1,SR-B1,SR-A1 and CD36 in aorta and PM in model + PRMT2 group showed no significant difference(all P>0.05).Conclusion:Overexpression of PRMT2 inhibited the formation of AS in Apo E-/-mice fed with high fat diet,of which the mechanism might be the promotion of cholesterol efflux through the up regulation of expression of ABCA1.In conclusion,our results suggest that the decrease of serum PRMT2 level is independently correlated with the increased risk of CHD,and the serum PRMT2 level is negatively correlated with the severity of CHD.Serum PRMT2 level is expected to be an indicator for the risk assessment and severity assessment of CHD.Overexpression of PRMT2 inhibited the formation of AS in Apo E-/-mice fed high-fat diet and the formation of macrophage derived foam cells induced by ox-LDL.The mechanism may be related to upregulating ABCA1 expression and promoting cholesterol efflux in macrophages.