Clinical Evaluation Of The Relationship Between PCSK9 And The Development Of Coronary Heart Disease

Objectives:Proprotein convertase subtilisin kexin type 9(PCSK9)is of great importance to cholesterol metabolism via degrading hepatic low density lipoprotein receptor(LDLR).It also have been proved that PCSK9 gain-of-function mutations are associated with increased cardiovascular risk.Therefore,we hypothesized that plasma PCSK9 levels were significantly associated with lipids and chronic inflammatory indices,the two kinds of critical parameters for atherosclerosis;while there was discordance between PCSK9 and current lipid measurement,providing additional information for determining dyslipidemia.Methods:The present study comprised two parts of contents:(1)evaluation of associations between plasma PCSK9 levels and atherosclerosis-related lipids or inflammatory indices in patients with stable coronary artery disease(CAD);(2)investigation of associations between PCSK9 levels and current classification of dyslipidemias aimed to elucidate the potential value of PCSK9 in determining lipid disorder.In the first part,a small sample of 251 consecutive,stable CAD patients who were not treated with lipid-lowering drugs were studied.The baseline clinical characteristics were collected and the plasma PCSK9 levels were determined using ELISA.The associations of plasma PCSK9 levels with atherosclerosis-related lipids including triglyceride(TG),total cholesterol(TC),high density lipoprotein cholesterol(HDL-C),low density lipoprotein cholesterol(LDL-C),apolipoprotein A1(apoAl),apoB,lipoprotein(a)[Lp(a)],free fatty acid(FFA),and chronic inflammatory parameters including white blood cell and its subsets were investigated.In the second part,a group of 1605 consecutive,none-lipid-lowering-drug-treated patients undergoing coronary angiography were analyzed.Patients were divided into 5 groups according to their dyslipidemia status:hypertriglyceridemia,hypercholesterolemia,combined hyperlipidemia,hypo HDL cholesterolemia and normalipidemia.The collections and measurements of clinical characteristics were as described above.The study was carried out as follows:firstly,the associations of PCSK9 levels with current classification of dyslipidemias and its cut-off level for predicting the dyslipidemias were evaluated;secondly,the interactions between PCSK9 and lipid indices in the classification were explored;finally,the discordance between PCSK9 and the current lipid measurement were examined.Results:(1)In patients with stable CAD,plasma PCSK9 levels were significantly associated with lipids including TC(r =0.294,p<0.001),HDL-C(r =0.128,p =0.042),LDL-C(r =0.256,p<0.001),apoA 1(r =0.229,p<0.001),apoB(r =0.278,p<0.001)and inflammatory parameters including white blood cell(r =0.167;p =0.008)and its subset lymphocyte count(r =0.219,p<0.001).Multivariable regression analysis with adjustment for gender,age,body mass index,smoking,alcohol intake,hypertension,diabetes,family history of CAD and lipid indices revealed that the associations of PCSK9 levels with white blood cell and lymphocyte still existed.(2)Compared to the PCSK9 levels in patients with normalipidemia(228.87 ± 67.24ng/ml),those with hypercholesterolemia(255.30 ± 70.82ng/ml)and combined hyperlipidemia(251.99 ±65.63ng/ml)had the higher PCSK9 levels while there was no difference in patients with hypertriglyceridemia(231.10 ± 68.58ng/ml)and hypo HDL cholesterolemia(221.30 ±67.03ng/ml).Furthermore,PCSK9 significantly conferred prediction of both hypercholesterolemia and combined hyperlipidemia at a level of 235 ng/ml with the areas under curve 0.609[0.564-0.654],p<0.001 and 0.606[0.563-0.648],p<0.001,respectively.In a polytomous logistic model comparing increasing LDL-C categories or severity of hypercholesterolemia,the interactions between high PCSK9 with LDL-C elevated gradually.Interestingly,discordances of PCSK9 with LDL-C status were observed.Conclusion:These data demonstrated that the plasma PCSK9 levels were positively associated with the atherosclerosis-related lipids and chronic inflammatory indices and PCSK9 levels might provide important clinical information in determining dyslipidemia and lipid management.Objectives:Proprotein convertase subtilisin/kexin type 9(PCSK9)has emerged as a novel modulator of cholesterol metabolism.A growing number of studies have demonstrated that gain-of-function mutations of PCSK9 are associated with hypercholesterolemia and increased cardiovascular risk whereas loss-of-function mutations are linked to low plasma levels of low density lipoprotein cholesterol(LDL-C)and a reduction risk for coronary artery disease(CAD).The identification of PCSK9 has resulted in substantial revision to previous knowledge regarding dyslipidemia researches,presented a new compelling therapeutic target to reduce LDL-C in clinical practice.Recently,it seems relevant to measure circulating levels of PCSK9 from a clinical perspective or a research insight.However,few studies have comprehensively evaluated plasma PCSK9 levels with severity and prognosis of CAD.Hence,the present study,as the first from China,aimed to investigate the association between baseline PCSK9 levels and cardiovascular risk graded with number of risk factors(RFs),coronary severity and outcomes in patients with stable CAD.Methods:A total of 616 consecutive,non-treated patients with stable CAD were enrolled from October 2012 to January 2015 in our lipid clinic.Baseline clinical characteristics and laboratory examinations were collected.Plasma PCSK9 levels were measured by ELISA.Coronary severity was measured using number of diseased arteries and SYNTAX,Gensini or Jeopardy scoring systems.Patients were then received treatment and followed to April 2015 for a median of 17 months.The primary endpoints were cardiac death,stroke,myocardial infarction(MI),post-discharge revascularization or unstable angina(UA).Results:At baseline,plasma PCSK9 levels were increased with an increasing number of RFs(number 0-2,n=66,222.12[118.40-273.91]ng/ml;number 3-5,n=348,230.19[190.38-277.77]ng/ml;number>6,n=202,271.06[211.33-327.19]ng/ml).Also,we also found that PCSK9 levels were positively associated with coronary severity:patients with the greater number of diseased arteries had higher PCSK9 levels(single vessel,222.55[185.65-278,31]ng/ml,two vessels,226.61[191.63-271.01]ng/ml,multiple vessels,236.58[191.37-292.69]ng/ml);the higher coronary severity scores evaluated by SYNTAX,Gensini or Jeopardy scoring systems,respectively,the higher PCSK9 levels were detected(p<0.05).Overall,follow-up data were obtained from 603 patients and 13 patients were defaulters.As a result,72(11.9%)patients presented with at least one major adverse cardiovascular event(MACE)(4 cardiac deaths,4 strokes,6 MIs,28 revascularizations,and 30 UAs).Multivariate Cox regression analysis showed that PCSK9 and coronary scores were independently predictive for MACEs after the adjustment for age,sex,body mass index,hypertension,diabetes,hypercholesterolemia,low high density lipoprotein cholesterolemia,smoking,family history of CAD,uric acid and high sensitivity C reactive protein(p<0.05).Interestingly,significant predictive values of PCSK9 in medical-alone-treated sub-population were found(p<0.05).Conclusion:Together,these data demonstrated that plasma PCSK9 levels were positively associated with coronary severity and could independently predict MACEs in patients with stable CAD.Objectives:Dyslipidemia and inflammation have central roles in the pathogenesis ofatherosclerotic cardiovascular disease,but their mechanistic links,especially the interactions of dyslipidemia with inflammation to atherogenisis remain unclear.Recently,a novel protein named proprotein convertase subtilisin/kexin type 9(PCSK9)is indentified as a key regulator of lipid metabolism.The best-described function of PCSK9 is directed toward low density lipoprotein(LDL)receptor(LDLR)degradation thus affecting plasma LDL-cholesterol(LDL-C)levels.We hypothesized PCSK9 as a critical regulator between inflammation and lipid metabolism.Hence,we using C-reactive protein(CRP)as inflammatory stimulators aimed to reveal the mechanistic links between inflammation and lipid metabolism in terms of PCSK9 in cellular studies.Methods:The present study consisted of two parts.First,experiments were carried out in the presence of human CRP(10?g/mL)on the protein and mRNA expression of PCSK9 and the underlying pathways were investigated in human hepatoma cell line HepG2 cells.Observation indexes included:(1)the mRNA and protein levels of PCSK9 induced by CRP treatment;(2)the related inflammatory signal transduction proteins;(3)the upstream transcription factors of PCSK9,sterol regulatory element binding protein-2(SREBP-2)and hepatocyte nuclear factor-la(HNF-1?);(4)the gene-protein binding activity between PCSK9 gene and the activated transcription protein(s).Second,studies were designed to investigate the role of PCSK9 in CRP-induced changes in LDLR expression and LDL uptake in HepG2 cells.Observation indexes included:(1)the mRNA and protein levels of LDLR,LDL uptake in the status of CRP treatment;(2)the mRNA and protein levels of LDLR,LDL uptake in the status of CRP treatment(10?g/mL)and PCSK9 related inhibitors.Results:Treatment with CRP(10?g/mL)enhanced significantly the mRNA and protein expression of PCSK9 and suppressed the expression of LDLR.Of note,an late return of LDLR mRNA levels occurred at 24h,while the LDLR protein continued to decrease,suggesting the late decrease in LDLR protein levels was unlikely to be accounted for by the decrease of LDL mRNA.Secondly,the role of PCSK9 in CRP-induced LDLR decrease and the underlying pathways were investigated.As a result,inhibition of PCSK9 expression by small interfering RNA(siRNA)returned partly the level of LDLR protein and LDL uptake during the CRP treatment;CRP-induced PCSK9 increase was inhibited by the p38 mitogen-activated protein kinase(p38MAPK)inhibitor,SB203580,resulting in a significant rescue of LDLR protein expression and LDL uptake;the pathway was involved in HNFla but not SREBPs preceded by the phosphorylation of p38MAPK.Conclusion:These findings indicated that CRP increased PCSK9 expression by activating p38MAPK-HNF1? pathway,with a certain downstream impairment in LDL metabolism in HepG2 cells.