Study On The Clinical Phenotype Of Familial Hypercholesterolemia And The Mechanism Of SORBS2 Gene Promoting Atherosclerosi

Part Ⅰ:Impact of diabetes on coronary severity and cardiovascular outcomes in patients with HeFHBackground:Type 2 diabetes mellitus(T2DM)is an independent risk factor for cardiovascular disease.A large cross-sectional study from the Netherlands indicated that the incidence of T2DM was significantly lower in patients with familial hypercholesterolemia(FH)than in the non-FH population.However,the association between T2DM and coronary artery disease(CAD)in patients with heterozygous familial hypercholesterolemia(HeFH)has not been thoroughly evaluated.Whether T2DM affects the HeFH population in the same way as the general population is unclear.Based on these facts,our study aimed to assess the effect of T2DM on CAD severity and hard cardiovascular endpoints in a HeFH cohort.Method:Dutch Lipid Clinic Network(DLCN)criteria were used for clinical diagnosis of selected patients,and three FH causative genes:low-density lipoprotein receptor(LDLR),apolipoprotein B(APOB),and proprotein convertase substilisin/kexin type 9(PCSK9)were detected.A total of 432 patients with HeFH with a molecular and/or clinical Dutch Lipid Clinic Network score≥6(definite and probable)was enrolled.HeFH patients were divided into T2DM group(n=99)and non-T2DM group(n=333).The coronary vessels of the patients were examined by contrast-enhanced CT and coronary angiography.The severity of coronary stenosis was assessed by the number of diseased vessels and Gensini,SYNTAX,and Jeopardy scores.Cardiovascular hard endpoints included a composite of non-fatal myocardial infarction,non-fatal stroke,and cardiac death.Multivariate Cox regression analysis and Kaplan-Meier analysis were used to evaluate the effect of T2DM on cardiovascular hard endpoints.Hazard ratios(HR)and 95%confidence intervals(CI)were calculated.Results:A total of 432 HeFH patients were included in this study.The prevalence of CAD in T2DM patients was higher than that in non-T2DM patients(96.0%in the T2DM group vs.77.5%in the non-T2DM group,P<0.001).Patients with T2DM have more diseased blood vessels and more severe coronary artery disease.Meanwhile,T2DM patients had high Gensini(unadjusted odds ratio[OR]=1.97,95%CI 1.22-3.17,P=0.005)and Jeopardy score(unadjusted OR=1.81,95%CI 1.11-2.97,P=0.018)tertile.During a median follow-up of 3.75 years,13 hard cardiovascular endpoints were recorded in 99 patients with T2DM and 16 in 333 patients without T2DM at baseline.Compared with patients without T2DM,patients with T2DM had a significantly increased risk of cardiovascular hard endpoints(HR=2.33,95%CI 1.02-4.85,P=0.024).After multivariate adjustment for other variables,the significant trend for cardiovascular hard endpoints did not change(adjusted hazard ratio[HR]=2.32,95%CI 1.11-4.84,P=0.025).Additionally,patients with T2DM and good glucose control(HbA1c<7.0%)were at a lower risk of hard endpoints compared with those with poor glucose control(HbA1c≥ 7.0%,HR=0.08,95%CI 0.01-0.56,P=0.011).Conclusions:T2DM was associated with severity of coronary lesions in HeFH patients assessed by number of diseased vessels,Gensini,SYNTAX and Jeopardy scores.The study also concluded that T2DM is an independent predictor of cardiovascular events in HeFH patients,suggesting that T2DM could be further used for risk stratification in HeFH patients.Part Ⅱ:Relations of physical signs to genotype,lipid and inflammatory markers,coronary stenosis or calcification,and outcomes in patients with HeFHBackground:Familial hypercholesterolemia(FH)is clinically characterized by elevated plasma low density lipoprotein cholesterol(LDL-C),tendon xanthomas(TX),corneal arcus(CA)and premature atherosclerotic cardiovascular disease.Although the presence of physical signs,TX/CA,are associated with the risk of coronary artery disease in patients with heterozygous familial hypercholesterolemia(HeFH),their relationship with genotypes and clinical biomarkers have not been fully determined.This study aimed to examine the association of TX/CA with genetic mutation,lipid-and inflammation-related markers,the severity of coronary stenosis or calcification,and cardiovascular events(CVEs)in patients with HeFH.Methods:Three causative genes:low-density lipoprotein receptor(LDLR),apolipoprotein B(APOB),proprotein convertase subtilisin 9(PCSK9)were screened in 523 HeFH patients.Patients with TX/CA(n=50)were 1:4 propensity score-matched to patients without TX/CA(n=200)to adjust for age and sex.Lipid and inflammatory markers,including PCSK9,lipoprotein(a)[Lp(a)]and high-sensitivity C-reactive protein(hsCRP)were measured.Gensini,SYNTAX and Jeopardy scores were used for evaluating coronary severity.Coronary artery calcium(CAC)score was used for evaluating the severity of coronary artery calcification.Coronary angiography and follow-up for CVEs were also performed.Results:The study included 250 patients with HeFH.Patients in the TX/CA group had significantly higher LDL-C levels compared to the non-TX/CA group.Besides,patients with physical signs(TX/CA)had higher PCSK9 or hsCRP concentrations.The proportion of patients with LDLR-positive mutations in the TX/CA group was higher than that in the non-TX/CA group.The study further assessed coronary severity using three scores,and found higher prevalence of high tertiles of Gensini,SYNTAX and Jeopardy scores in the TX/CA group(all P<0.05).During the mean follow-up of 3.7 years,the incidence of CVE in the TX/CA group was significantly higher than that in the non-TX/CA group(Log rank,P<0.001).After adjustment for multiple confounding factors,the risk of CVE in the TX/CA group was still 2-fold higher than that in the non-TX/CA group and was statistically significant(hazard ratio[HR]=2.75,95%confidence interval(CI)]1.04-7.26,P=0.024].Further subgroup analysis found that the risk of CVE in patients with TX/CA signs and mutation-positive was 3 times that of patients without TX/CA signs and mutation-negative(HR=3.34,95%CI 1.04-10.72,P=0.024).Conclusions:Physical signs(TX/CA)were positively correlated with positive genetic mutation,especially LDLR positive mutations in HeFH patients.Besides,TX/CA signs were associated with higher PCSK9 or hsCRP concentrations,coronary artery severity,coronary calcification,and cardiovascular outcomes,suggesting that identification of TX/CA signs may help in risk stratification of cardiovascular events in HeFH patients.Part Ⅲ:SORBS2 as a molecular target for atherosclerosis in patients with familial hypercholesterolemiaBackground:Familial hypercholesterolemia(FH)is a metabolic disease in which patients are prone to develop premature atherosclerosis(AS).The hallmark of AS is thrombosis caused by rupture of unstable plaques,resulting in acute coronary syndrome(ACS),which is the leading cause of global mortality and morbidity.Multiple factors are involved in the formation of unstable plaques in AS,including inflammatory responses,lipid metabolism disorders and other factors.Current studies have found that Sorbin and SH3 Domain Containing 2(SORBS2)plays a role in coronary heart disease(CHD).However,the mechanism underlying SORBS2 involvement in the development of hypercholesterolemia remains unknown.Here,we used the human monocyte cell line THP-1 to establish the foam cell model and investigated the effects of SORBS2 on inflammation and foam cell formation and its underlying mechanisms.Methods:Using bioinformatics analysis,we established that SORBS2 is upregulated in patients with FH.Circulating SORBS2 concentrations in FH patients were measured using an enzyme-linked immune sorbent assay(ELISA)(n=30).The association between circulating SORBS2 levels and inflammatory factors or lipid indexes were conducted using Spearman correlation analysis.We induced the differentiation of THP-1 cells into macrophages with phorbol 12-myristate 13-acetate(PMA),and then added oxidized low density lipoprotein(Ox-LDL)to stimulate the foam model.We further conducted in vitro experiments that the expression of SORBS2 were analyzed,and small interfering RNA(siRNA)of SORBS2 were transfected into Ox-LDL-induced macrophages.The expression of pro-inflammatory factors in the cell supernatant was detected by ELISA,and the Nod like receptor family pyrin domain-containing 3 protein(NLRP3),Caspase-1,nuclear factor κB(NF-κB),ATP-binding cassette transporter G1/peroxisome proliferator activated receptor gamma(ABCG1-PPAR)signaling pathway-related proteins were detected by Western Blot.Immunofluorescence and Oil Red O staining were used to detect the expression of corresponding substances in cells.Results:Circulating SORBS2 levels in FH patients were higher than those in non-FH patients,and circulating SORBS2 levels were positively correlated with inflammatory factors and lipid indexes,including total cholesterol(TC)and low density lipoprotein cholesterol(LDL-C).We also observed that high in vitro expression of SORBS2 in oxidized LDL(Ox-LDL)-induced macrophages.After SORBS2 silencing,Nod like receptor family pyrin domain-containing 3 protein(NLRP3)-Caspase1 activation and NF-κB activation were attenuated,and secretion of pro-inflammatory cytokines(IL-1βand IL-18)was decreased.Moreover,SORBS2 silencing blocked reactive oxygen species(ROS)production and lipid accumulation in macrophages,and promoted cholesterol efflux by inhibiting the ABCG1-PPARγ pathway.Conclusions:SORBS2 can regulate lipid-induced inflammation and foam cell formation,and its mechanism may be related to the activation of the NLRP3/Caspase-1 pathway through NF-κB activation,which may affect the cholesterol efflux process by affecting the ABCG1-PPARγ pathway.Therefore,SORBS2 may be a potential therapeutic target for hypercholesterolemia.