Mechanism Of C1q/TNF-Related Protein 9 Regulation On Reverse Cholesterol Transport

Background:Atherosclerosis is well known to be the pathological basis for triggering cardiovascular diseases such as coronary heart disease,stroke and peripheral vascular diseases.The formation of foam cells in the intima is a major mark of early atherosclerotic plaques,and plays essential role in the occurrence and development of atherosclerosis.Foam cells are potential targets for treatment and prevention of atherosclerosis.Numerous epidemiological investigations have demonstrated that HDL-C level is negatively correlated with cardiovascular disease risk.Reversed cholesterol transport(RCT)is an important anti-atherosclerotic mechanism of HDL.As a receptor and transporter,HDL can transport extra cholesterol from peripheral tissues(including foam cells in atherosclerotic plaques)to livers,where cholesterol can be synthesized into bile acid,and excrete out of body.This process is called RCT.ATP binding cassette transport A1(ABCA1)and G1(ABCG1)have complementary function in the process of RCT.At the beginning of atherosclerosis,the expression of ABCA1 and ABCG1 are decreased,which aggravates accumulation of cholesterol and formation of foam cells.ABCA1 and ABCG1 are important transporters during RCT.Upregulation of ABCA1 and ABCG1 can improve HDL regulation of RCT,and further regulate atherosclerosis.C1q/tumor necrosis factor-related protein family is a newly discovered novel adipokine family.Among 15 members,CTRP9 is the closest paralog of adiponectin.Recent studies showed that in coronary heart disease(CAD)patients and mice with atherosclerosis,serum CTRP9 levels were significantly decreased than controls,which suggests CTRP9 has a cardiovascular protective mechanism.There is a study demonstrated CTRP9 can regulate intimal hyperplasia and vascular smooth muscle cell(VSMC)proliferation and migration in atherosclerosis.There is no report on mechanism of CTRP9 regulation in RCT in atherosclerosis.Part ? The effect of CTRP9 on mice with atherosclerosis Aim:To explore the effect of CTRP9 on cholesterol efflux and lipid profile in mice with atherosclerosis.Methods:Male apoE-/-mice were fed with high fat diet for 10 weeks to set up atherosclerosis model.Mice were implanted with mini-osmotic minipumps containing different concentrations of CTRP9 for 4 weeks.Aortic atherosclerosis development was observed in vivo by oil red staining and HE staining.RAW 264.7 macrophages were radiolabeled by 3H-cholesterol,and then were injected intraperitoneally to atherosclerotic mice.Then feces were collected for 48 h.After 48 h,mice were sacrificed to collect plasma and livers.Cholesterol efflux was determined by liquid scintillation counting.Lipid profile including triglyceride,total cholesterol,low density lipoprotein cholesterol,and high density lipoprotein cholesterol levels were determined by enzymatic methods.Liver ABCA1,ABCG1,and LXR-? mRNA expressions were measured by real-time PCR.Protein levels were analyzed by western blotting.Results:CTRP9 decreased the atherosclerotic lesion size in vivo,and promoted cholesterol efflux into plasma,liver and feces.The levels of HDL-C were increased significantly and LDL-C were decreased by CTRP9.mRNA and protein expressions of ABCA1,ABCG1,LXR-? were upregulated by CTRP9.Conclusion:CTRP9 upregulates expression of ABCA1,ABCG1 and LXR-? by promoting cholesterol efflux in vivo.CTRP9 regulates lipid profile and attenuates aortic lesions in atherosclerotic mice.Part ? CTRP9 regulates reversed cholesterol transport dose-dependently and time-dependently in foam cells Aim:To investigate the effect of different CTRP9 concentrations on cholesterol efflux for different time courses in RAW 264.7 macrophage derived foam cells.Methods:RAW 264.7 macrophages were transformed into foam cells by incubating with 50mg/L ox-LDL for 24 h.First,foam cells were treated with different concentrations of CTPR9(0.3 ?g/mL,1 ?g/mL,3 ?g/mL)for 3h.Then,foam cells were treated with the best concentration of CTRP9 for different time courses(1h,3h,6h).Cholesterol efflux was determined by liquid scintillation counting.Total cholesterol,free cholesterol,and cholesterol ester levels in foam cells were examined by high performance liquid chromatography assays.mRNA and protein expressions of ABCA1 and ABCG1 were analyzed by real-time PCR and western blotting.Results:As concentrations and time courses increased,CTRP9 significantly increased cholesterol efflux,decreased cholesterol contents,and upregulated ABCA1 and ABCG1 mRNA and protein expressions.The best concentration is 3?g/mL.As time increased,the above effects peaked at 3h,and decreased after 6h.Conclusion:CTRP9 upregulated ABCA1 and ABCG1 mRNA and protein expressions,and promoted cholesterol efflux in foam cells,which inhibited foam cell formation dose-dependently and time-dependently.Part ? Cell signaling of CTRP9 on reversed cholesterol transport in foam cells Aim:To investigate CTRP9 on cholesterol efflux in RAW 264.7 macrophage derived foam cells.Methods:RAW 264.7 macrophages were transformed into foam cells by incubating with 50mg/L ox-LDL for 24 h.Foam cells were transfected with LXR-?-siRNA to knock down endogenous LXR-? expression.We observe the effect of LXR-? on CTRP9 regulating cholesterol efflux in foam cells.Then foam cells were transfected with AMPK-siRNA to knock down endogenous AMPK expression.We observe the effect of AMPK on CTRP9 regulating cholesterol efflux in foam cells.Cholesterol efflux was determined by liquid scintillation counting.Total cholesterol,free cholesterol,and cholesterol ester levels in foam cells were examined by high performance liquid chromatography assays.mRNA and protein expressions of ABCA1,ABCG1,LXR-? and phosphorylation of AMPK were analyzed by real-time PCR and western blotting.Results:When LXR-? and AMPK were knocked down by transfected with LXR-?-siRNA and AMPK-siRNA,the effect of CTRP9 increasing cholesterol efflux,decreasing cholesterol contents,attenuating foam cell formation and upregulating ABCA1 and ABCG1 was inhibited in RAW264.7 macrophage derived foam cells.Conclusion:CTRP9 promoted cholesterol efflux,decreased cellular cholesterol content,and inhibited foam cell formation through regulating AMPK-LXR-?-ABCA1/ABCG1 signaling in RAW264.7 macrophage derived foam cells.Part ? Correlation between CTRP9 and premature coronary artery disease Aim:To analyze clinical characteristic and serum CTRP9 levels in patients with premature coronary artery disease(PCAD).To investigate correlation of CTRP9 and PCAD.Methods:149 patients undergoing coronary angiography in The Second Hospital of Shanxi Medical University from May 2016 to May 2018.Patients were divided into 2 groups: Control(n=49),and PCAD(n=100)according to lesion.PCAD patients were divided into 3 groups: stable angina pectoris group(SAP,n=35),unstable angina pectoris group(UAP,n=33),acute myocardial infarction(AMI,n=32)according to different types.PCAD patients were divided into another 3 groups: single vessel lesion group(n=42),double vessels lesion group(n=33),multiple vessels lesion(n=32)according to the number of lesion vessels.PCAD patients were also divided into another 3 groups: mild lesion group(n=47),moderate lesion group(n=34),severe lesion group(n=19)according to Gensini scoring system.Clinical data and laboratory biomarkers were collected.Serum CTRP9 levels were determined by ELISA.Logistic regression analyze was used to identify risk factors and correlation between CTRP9 level and PCAD.We also analyzed correlation between CTRP9 and degree of atherosclerotic lesions.Results:Compared to control group,proportion of male patients,hypertension,family history,and smoking history were significantly increased in PCAD group.BMI,and TG level increased significantly,HDL-C and CTRP9 level decreased significantly in PCAD group(P<0.05).Male,smoking,hypertension,diabetes,and CAD family history were major risk factors of PCAD.HDL-C and CTRP9 levels were protective factors of PCAD.CTRP9 levels in SAP group,UAP group,and AMI group decreased successively compared to control group.CTRP9 level in AMI group decreased significantly compared to SAP group,while there is no difference in CTRP9 levels between SAP group and UAP group,UAP group and AMI group.CTRP9 levels in multiple vessels lesion group,double vessels lesion group,and single vessel lesion group decreased successively compared to control group.CTRP9 level in multiple vessels lesion group decreased significantly compared to single vessel lesion group,while there is no difference in CTRP9 levels between double vessels lesion group and single vessel lesion group,multiple vessels lesion group and double vessels lesion group.CTRP9 levels in mild lesion group,moderate lesion group,and severe lesion group decreased successively compared to control group.CTRP9 level in severe lesion group decreased significantly compared to mild lesion group,while there is no difference in CTRP9 levels between moderate lesion group and mild lesion group,severe lesion group and moderate lesion group.Conclusion:Male,smoking history,hypertension history,type 2 diabetes,CAD family history,decreased HDL-C level and CTRP9 level are PCAD risk factors.CTRP9 level is correlated with severity of lesion in PCAD patients.High CTRP9 level is a probably protective factor in PCAD.