Dickkopf1 Influence On The Function Of Human Vascular Smooth Muscle Cells And Its Relationship With Acute Coronary Syndrome Associated Serum Molecules

BackgroundAcute coronary syndrome(ACS)is an acute clinical event among coronary atherosclerosis heart diseases(CAD),including non-ST-segment acute coronary syndrome(NST-ACS)and ST-segment elevation myocardial infarction(STEMI).NST-ACS includes unstable angina(UA)and non-ST-segment elevation myocardialinfarction(NSTEMI).Atherosclerosis(AS)mainly causes ACS through thrombosis in coronary arteries resulting from atherosclerotic plaque rupture or erosion,and increase in coronary atherosclerotic plaque burden.Oxidized low-density lipoprotein(ox-LDL)is an important pathological factor in the development and progression of atherosclerosis,regulating several vascular cells' functions,including vascular smooth muscle cells.Dickkopfl(DKK1)is implicated in embryonic development and occurrence of various diseases through inhibiting canonical Wnt signaling pathway.Research has demonstrated patients with STEMI had higher plasma DKK1 level,compared with those with NETE-ACS and DKK1 can be a predictor of major adverse cardiovascular events in patients with ACS.DKK1 may be also significant for STEMI diagnosis.In addition,DKK1 has been proved to play a role in mediating the inflammatory reaction between platelets and vascular endothelial cells,endothelial tight junction under oscillatory shear stress,intracellular lipid accumulation in macrophages and endothelial apoptosis induced by ox-LDL.However,whether DKK1 is involved in the function change of atherosclerotic vascular smooth muscle cells remains unknown.Plasminogen activator inhibitor-1(PAI1)exerts important functions in the morbidity of AMI by inhibiting the degradation of fibrin and promoting thrombosis indirectly.Tissue factor(TF)is a key factor in initiating coagulation,and also participates in arterial thrombosis in patients with AMI.Interlukin-17(IL-17)and interlukin-1?(IL-1?)have been proved to participate in atherosclerotic inflammatory reaction.However,the relationship between DKK1 and PAI1,TF,IL-17,IL-1? in atherogenesis remains unknown.Objectives1.To observe the influence of DKK1 on the function of vascular smooth muscle cells in vitro experiments.2.To explore the relationship between DKK1 and PAI1,TF,IL-17,IL-1? in clinical research.Methods1.Vitro experimentHuman aortic smooth muscle cells(HASMCs)in good condition and in generation from 8 to 10 was used in the experiments.After adding ox-LDL into HASMCs,we collected total protein or RNA in HASMCs and detect DKK1 protein or mRNA level.The group set was Negative control(NC)group?NC+ox-LDL group?DKK1 siRNA group?DKK1 siRNA+ox-LDL group and DKKloverexpresion group.Collect cell total protein,detect the expression of DKK1,collagen III,P4Ha1,MMP-2,MMP-9 and PCNA via western blot,and detect the proliferation level of HASMCs through EdU assay.2.Clinical researchBased on criteria,we collected 27 UA patients,19 AMI patients and 15 controls in Qil Hospital,Shandong University from January 2017 to March 2017,including general information and blood samples.The plasma concentration of DKK1?PAI1,TF,IL-17 and IL-1? was tested via enzyme linked immunosorbent assay(ELISA).SPSS 17.0 was used to analyze these data.One-way ANOVA analysis was used to analyze the inter-group difference between these data.Pearson and Spearman linear correlation analysis was used to analyze the correlation between DKK1 and PAI1,TF,IL-17,IL-1?.Receiver operating characteristic(ROC)curve was used to analyze the sensitivity and specificity of the target.P<0.05 was regarded as significant statistical difference.Results1.Results in vitro experiment(1)ox-LDL upregulates the expression of DKK1 in HASMCsThe expression of DKK1 protein is upregulated in HASMCs induced by 100ug/ml ox-LDL for 12 hours(p<0.01);the expression of DKK1 mRNA isupregulated in HASMCs induced by 100ug/ml ox-LDL for 3 hours(p<0.05).(2)DKK1 influences collagen metabolism in HASMCsWestern blot shows that compared with NC group,the expression of collagen III,P4H?1,MMP-2 and MMP-9 increase in HASMCs induced by 100ug/ml ox-LDL for 12 hours(p<0.05)and DKK1 overexpression group(p<0.05);the expression of MMP-2 is downregulated when siRNA inhibits the expression of DKK1 in HASMCs(p<0.01).When stimulating by ox-LDL,the expression of collagen III,P4Hal,MMP-2 and MMP-9 decrease when DKK1 expression is inhibited by siRNA(p<0.05).(3)DKKI influences HASMCs proliferationEdU assay shows ox-LDL induction and DKK1 overexpression could both promote the proliferation of HASMCs(p<0.01).Inhibiting the expression of DKK1 by siRNA could inhibit HASMCs proliferation(p<0.01).Western blot shows that ox-LDL induction and DKK1 overexpression both promote the expression of PCNA in HASMCs(p<0.05).Inhibiting the expression of DKKI could downregulate the expression of PCNA in HASMCs(p<0.05).2.Results in clinical research(1)The general information analysis between different groupsThere is no difference in age,triglyceride(TG),creatine(Cr)between control group and ACS groups(p>0.05).Compared with control group,UA group and AMI group have lower level of serum high density lipoprotein cholesterol(HDL-C)(p<0.05);AMI group has higher level of serum alanine transaminase(ALT)(p<0.01).Compared with UA group,AMI group has higher level of total cholesterol(TC),low density lipoprotein cholesterol(LDL-C),ALT and creatine kinase isoenzyme(CK-MB)(p<0.05),and lower level HDL-C(p<0.05).(2)Plasma concentration of DKK1,PAI1,TF,IL-17 and IL-1? between different groupsCompared with control group,UA group and AMI group have higher level of plasma DKK1,PAI1,TF and IL-17(p<0.05),but there is no difference for IL-1?(p>0.05).Compared with UA group,AMI group have higher level of plasma DKK1,PAI1,TF and IL-17(p<0.05),(3)Correlation analysis of DKK1,PAI1,TF,IL-17 and IL-1?Pearson linear correlation analysis shows DKK1 positively correlates with PAI1,TF and IL-17(p<0.01).(4)The sensitivity and specificity of DKK1 in UA diagnosis and UA-AMI differential diagnosisThe area under curve(AUC)of the ROC curve for UA diagnosis is 0.956.The sensitivity is 0.963,specificity is 0.867 and cut-off value is 460.60pg/ml.The AUC of the ROC curve for UA-AMI differential diagnosis is 0.869.The sensitivity is 0.947,specificity is 0.778 and cut-off value is 1200.60pg/ml.Conclusions1.DKK1 may take part in collagen metabolism and proliferation in ox-LDL induced HASMCs.2.Compared with control group,plasma DKK1,PAI1,TF and IL-17 are higher in patients with ACS and AMI group has higher serum level of DKK1,PAI1,TF and IL-17 than UA group.DKK1 correlates with PAI1,TF and IL-17.These results show that DKK1 may participate in the pathological reaction of ACS.