Clinical And Mechanistic Study On The Relationship Between Lipoprotein (a) And Vascular Calcificatio

Abstract 1Associations between lipoprotein(a),coronary artery calcification and the risk of cardiovascular eventsBackground and aim:Lipoprotein(a)[Lp(a)],a LDL-like particle,covalently bonds to apolipoprotein(a)with a disulfide bond,which has the characteristics of proatherosclerosis,pro-thrombosis and pro-inflammation.Currently,Lp(a)has been considered as a risk factor of cardiovascular diseases and its role in coronary artery disease(CAD)is well-established.Coronary artery calcification(CAC)is an important predictor of CAD,which has been demonstrated to better predict cardiovascular risks.There have been some clinical studies about the relation between Lp(a)and CAC.but these results are contradictory.Meanwhile,the study of associations of Lp(a)and CAC alone and their combination with major adverse cardiovascular events(MACEs)is few.Therefore,the current study is aimed to investigate the relationship of Lp(a)with CAC and assess the predictive value of Lp(a)and CAC alone and their combination in patients admitted to hospital for the first time with chest pain.Methods:This study consecutively enrolled 2806 patients with chest pain who received coronary computed tomography(CT)from May 2012 to April 2018 in the division of dyslipidemia at Fuwai hospital.Coronary artery calcification score(CACS)was calculated by Agatston methods and patients were divided into CAC and non-CAC groups according to CACS>0 and=0.The plasma levels of Lp(a)were measured by an immunoturbidimetry and<10,10-30 and>30 mg/dL of Lp(a)were defined as low,mild and high Lp(a)groups.All patients were followed for the development of MACEs including unstable angina,myocardial infarction,ischemic stroke,coronary revascularization and cardiac death.Logistic regression and linear regression models were used to evaluate the relationship of Lp(a)with CAC and CACS.The predictive values of Lp(a)and CAC alone and their combination in MACEs were assessed by Kaplan-Meier survival curves and Cox regression analysis.Results:A total of 581 patients(20.7%)diagnosed with CAC by coronary CT,and results showed that CAC group had higher Lp(a)concentration than those without CAC(p<0.05).The levels of CACS were significantly higher in the mild and high Lp(a)groups compared with the low Lp(a)group(all p<0.05).Multivariate Logistic regression analysis indicated that the elevated Lp(a)levels was an independent predictor for CAC[odd ratio:1.110,95%confidence interval(CI):1.013-1.217,p=0.025].Multivariate linear regression analysis suggested that Lp(a)was positively related to CACS(β=0.132,p<0.001).During a mean of 38.2 months,201(7.2%)MACEs were developed.Our results suggested that patients with MACEs had higher Lp(a)levels and percentage of CAC than that without MACEs(all p<0.05).Multivariable Cox regression analysis revealed that the high Lp(a)group and CAC were remarkably and independently associated with MACE risks,respectively[hazard ratio(HR):1.612,95%CI:1.1292.303,p=0.009;HR:2.404,95%CI:1.802-3.208,p<0.001,respectively].When combined Lp(a)levels with CAC status,CAC patients in high Lp(a)levels had increased incidence of MACEs compared to non-CAC patients in low Lp(a)levels(HR:4.13,95%CI:2.689-6.342,p<0.001).Conclusions:The present study found that the plasma concentrations of Lp(a)was an independent predictor for CAC and were associated with CAC severity.Furthermore,Lp(a)and CAC were related to cardiovascular events and the combination of Lp(a)with CAC have a better predictive ability of cardiovascular outcomes,prompting that Lp(a)and CAC contribute to further cardiovascular risks stratification in patients with chest pain.Abstract 2The molecular mechanisms of lipoprotein(a)-mediated vascular calcification Background and aim:Lipoprotein(a)[Lp(a)]has been recognized as a causal risk factor of cardiovascular diseases and previous studies including ours have found a significant association of Lp(a)and vascular calcification.However,the role of Lp(a)in vascular calcification has not been clarified.Notch signal is a key intercellular communication connection that controls cell fate involving embryonic development and postnatal proliferation,self-renewal,and differentiation.Furthermore,canonical Notch1 signal is involved in osteogenic conversion and calcification of vascular smooth muscle cells within atheroma.Therefore,the current study hypothesizes that Lp(a)can mediate human aortic smooth muscle cells(HASMCs)calcification and further explores its potential mechanisms.Methods:HASMCs were stimulated with different concentrations of Lp(a)(0,2.5 and 5μg/mL)at different time points to observe cells calcification.Western blot was used to record the expression of calcific proteins including osteopontin(OPN)and bone morphogenetic protein 2(BMP2);von kossa staining was used to observe cell calcification deposition,alkaline phosphatase(ALP)activity staining was used for measuring cells ALP activity and FLIPR Calcium 6 assay kit was applied to determine the levels of intercellular calcium.Furthermore,co-treatment of Lp(a)with the Notch1 pathway inhibitor DAPT,the BMP2 pathway inhibitor Noggin or nuclear factor-κBspecific small interfering RNA(siNF-κB)in HASMCs were used to explore the specific mechanisms of Lp(a)-induced vascular calcification.Meanwhile,57 randomly selected patients with coronary artery calcification(CAC)and 57 age-and sex-matched patients without CAC as controls from CAC study cohort were measured plasma levels of Notch1 and OPN by enzyme-linked immunosorbent assay.The differences of plasma levels of Nochl and OPN between the two groups were compared and the associations of Notch1 and OPN levels with CAC score(CACS)were assessed.Results:Lp(a)significantly increased cellular calcification deposition(14 days)and ALP activity(7 days)and up-regulated the expression of calcification protein markers including BMP2 and OPN(24 hours)at a concentration of 5 μg/mL in HASMCs.Moreover,Lp(a)could up-regulate the expression of Notch1 pathway-related proteins to induce HASMCs calcification,which was inhibited by DAPT.Additionally,Lp(a)mediated activation of Notch1 also activated the BMP2-Smad 1/5/9 pathway,while Noggin significantly attenuated Lp(a)-induced vascular calcification.Interestingly,activation of Notchl translocated NF-κB,simultaneously up-regulated the expression of OPN and increased downstream inflammatory factors including interleukin-1β and interleukin-6.However,siNF-κB remarkedly weakened Lp(a)-induced vascular calcification.In addition,circulating concentrations of Notchl and OPN were markedly higher in patients with CAC than those without(p<0.05).Moreover,multivariate linear regression analysis found that elevated levels of Notchl and OPN were independently and significantly positive correlated with Log(CACS+1),respectively(p<0.05).Conclusions:This study found that Lp(a)was closely related to vascular calcification and its mechanism involved in Notchl-BMP2-Smad1/5/9 and Notchl-NF-κB pathways,which provided valuable scientific evidence for the possible mechanisms of Lp(a)in vascular calcification and future clinical interventions for Lp(a).