The Effects And Underlying Mechanisms Of CTRP9 On Atherosclerotic Plaque Progression

1 BackgroundCardiovascular and cerebrovascular diseases are the leading cause of morbidity and mortality worldwide and their main pathological basis is atherosclerosis.Numerous studies have revealed that atherosclerosis is a chronic inflammatory and metabolic disease which occurs in the large-and medium-sized arteries.The pathophysiological mechanism of atherosclerosis remains to be further elucidated.A growing body of evidence has highlighted that autophagy plays a critical role in the development of atherosclerosis and several atherosclerotic factors may lead to dysregulation of autophagy.As major cell types involved in the formation of atherosclerosis,smooth muscle cells(SMCs),endothelial cells(ECs)and macrophages can present characteristics of autophagy.It has been revealed that in atherosclerotic plaques,autophagy is involved in regulating oxidative stress,metabolism,inflammation and apoptosis.The mammalian target of rapamycin(mTOR)is the target molecule of rapamycin in mammalian and consists of mTORC1 and mTORC2.Of the two complexes,mTORC1 plays a key role at the interface of pathways that coordinates the balance between cell growth and autophagy.Inhibition of mTORC1 initiates a cluster of antiatherosclerotic properties including reducing endothelial dysfunction,monocyte adhesion and migration,foam cell formation,macrophage inflammatory reaction and SMCs proliferation.As an energy sensor in cellular energy homeostasis,adenosine monophosphate-activated protein kinase(AMPK)negatively regulates mTORC1 activity,thereby upregulating autophagy.Accordingly,activation of AMPK and repression of mTOR may be promising therapeutic targets in the prevention and treatment of atherosclerosis.C1q-TNF-related protein-9(CTRP9)is a novel adipokine that shares the most similar structure which belongs to the CTRPs family.Studies have showed that CTRP9 plays important roles in regulating metabolism,protecting myocardium,ameliorating endothelial function and resisting cardiovascular damage of diabetes.Several studies have demonstrated that CTRP9 exerts protective effects in cardiovascular system.CTRP9 modulated the behavior of SMCs to delay the progression of pathological vascular remodeling and attenuated cardiac damage by targeting apoptosis and inflammation in cardiac myocytes.Serum CTRP9 was a protective factor of coronary atherosclerotic heart disease which was independent of adiponectin.Our previous study found that the serum levels of CTRP9 and APN were significantly reduced in acute coronary syndrome(ACS)patients compared with control group,and serum level of CTRP9 and APN was associated with the severity of ACS.Importantly,our previous study demonstrated that CTRP9 could enhance carotid plaque stability in advanced atherosclerotic mice.Additionally,CTRP9 induced autophagy in human primary hepatocytes and its effects on ER stress,apoptosis and hepatic steatosis were canceled when the autophagy process was suppressed by 3 methyladenine,an autophagy inhibitor.In the present study,we aim to investigate the effects of CTRP9 on the development of atherosclerosis in ApoE-/-mice and the underlying mechanism.2 Objective(1)To investigate the effects of CTRP9 on the progression of atherosclerosis.(2)To elucidate the mechanism of CTRP9 in the development of atherosclerosis.3 Methods3.1 Animal modelMale ApoE-/-mice(8 weeks old)were obtained from Beijing Wei Tong Li Hua Experimental Animal Technology Co.LTD(Beijing,China).All animals were fed with a high-fat diet(0.25%cholesterol and 15%fat)until sacrifice.All mice were randomly assigned to three groups:Lv-CTRP9 group,Lv-eGFP group and Control group.The Lv-CTRP9 group was given 2×107TU CTRP9 lentiviral,the Lv-eGFP group was given 2×107 TU empty lentiviral vector and the Control group were given the same volume of PBS.3.2 Biological measurementsAt the end of the experiment,blood samples were taken from the left ventricle and all mice were sacrificed.Serum levels of monocyte chemoattractant protein 1(MCP-1)and tumor necrosis factor alpha(TNF-a)were measured by an enzyme-linked immunosorbent assay(ELISA)according to the manufacturer's instructions.Serum levels of total cholesterol(TC),triglyceride(TG),high-density lipoprotein cholesterol(HDL-C)and low-density lipoprotein cholesterol(LDL-C)were detected using an automatic biochemist lyzer.3.3 Histopathological analysisThe aortic roots were harvested and placed in 4%formaldehyde for at least 24 h.The cryosections with thickness of 5 ?m were obtained for hematoxylin and eosin staining and oil red O staining.In addition,the whole aortas were stained for oil red O.The severity of the lesions was represented by the ratio of red stained lipid to aortic areas.3.4 Immunohistochemical analysisImmunohistochemical analysis of MOMA-2 and a-SMA was used to evaluate the content of macrophages and SMCs.Data was analyzed by Image Pro-Plus software.The expression of MOMA-2 and a-SMA was measured by the ratio of the positive component areas to the total plaque areas.3.5 Western blotTotal proteins were extracted from arota.The protein expression of MCP-1,TNF-?,LC3B,SQSTM1/p62,p-AMPK,p-mTOR was analyzed by western blot.3.6 Statistical AnalysisSPSS software 18.0 was used for statistical analysis.Data was presented as mean± SD.The differences were determined by one-way ANOVA.P<0.05 was considered to be statistically significant.4 Results4.1 Lentiviral transfection enhanced the expression of CTRP9 in serumOne week after transfection,aortic root plaques had obvious GFP fluorescence,which showed a successful transfection of lentivirus.Also,the protein expression of CTRP9 measured by Western blot was significantly increased in serum of Lv-CTRP9 group compared with Lv-eGFP group or Control group(p<0.05).There was no significant difference between Lv-eGFP group and Control group(p>0.05).4.2 Effect of CTRP9 on serum lipid profileSerum levels of TG,TC,HDL-C,LDL-C did not significantly change among the three groups(p>0.05).4.3 CTRP9 reduced the atherosclerotic plaque areas of miceCompared with Lv-eGFP group and Control group,the Lv-CTRP9 group showed larger lesion areas by using en face analysis of the aorta(p<0.05).Atherogenesis severity at the sinus was evaluated by H&E staining and oil red O staining by ratio of total atherosclerotic lesion areas to aortic lumen areas.The mean lesion size at the aortic sinus was smaller in Lv-CTRP9 group than the other two groups(p<0.05).These results showed that overexpression of CTRP9 ameliorated the progression of atherosclerotic lesions.4.4 CTRP9 suppressed the infiltration of macrophages in plaquesImmunohistochemistry showed that the content of macrophages in Lv-CTRP9 group was reduced when compared with Lv-eGFP group and Control group(p<0.05).We did not see difference between the other two groups(p>0.05).4.5 CTRP9 ameliorated the accumulation of smooth muscle cells in plaquesImmunohistochemistry demonstrated that the content of SMCs was decreased in atherosclerotic lesions of CTRP9-treated mice.There was no difference between the Lv-eGFP group and Control group(p>0.05).4.6 CTRP9 alleviated the expression of pro-inflammatory cytokines in miceWestern blot and ELISA were used to evaluate the expression of pro-inflammatory cytokines.The expression of MCP-1 and TNF-a were decreased in Lv-CTRP9 group compared with Lv-eGFP group and Control group(p<0.05).There was no difference between the other two groups(p>0.05).4.7 The effects of CTRP9 on autophagy in ApoE-/-miceMicrotubule-associated protein light chain 3B(LC3B)and sequestosome 1(SQSTM1/p62)were regarded as autophagic biomarkers.Western blot showed that CTRP9 overexpression increased the expression of LC3B-? and reduced the expression of SQSTM1/p62 in atherosclerotic lesions of ApoE-/-mice(p<0.05).There was no difference between the Lv-eGFP group and Control group(p>0.05).4.8 AMPK/mTOR pathway participated in CTRP9-mediated autophagy in vivoWestern blot showed that CTRP9 significantly activated the phosphorylation level of AMPK and downregulated the phosphorylation level of mTOR in Lv-CTRP9 group when compared with Lv-eGFP group and Control group(p<0.05).5 Conclusions(1)CTRP9 treatment significantly ameliorated the progression of atherosclerosis in ApoE-/-mice.(2)CTRP9 administration could enhance the autophagy level of ApoE-/-mice.(3)The antiatherosclerotic effect of CTRP9 was associated with autophagy induction via AMPK/mTOR pathway.