Study Of Novel Biomarkers And Non-classical Risk Factors For Poor Prognosis In Older Men With Chronic Coronary Syndrome

Background: Chronic coronary syndrome(CCS)is a newly proposed concept.Compared with traditional stable coronary heart disease,it has a more detailed classification of the disease and a new understanding of the pathological mechanism.CCS tends to be characterized by more long-term major adverse cardiovascular events(MACEs),thus requiring accurate and reliable prognostic evaluation methods for initial risk stratification.This study was carried out from two aspects of novel biomarkers and non-classical risk factors.Despite the Human Genome Project’s mapped human genes,genomic data alone still cannot predict disease progression for most diseases.Proteins are the ultimate executors of gene function activities and the direct embodiment of the complexity and variability of life phenomena.Therefore,in order to clarify the law of life activity as a whole,it is necessary to study proteins that are the products of genes.Proteomes contain information more closely related to phenotypic variation than genomic or transcriptomic data,and are important in life science research.The discovery of new biomarkers has gradually become possible through the development of proteomic technology.By analyzing and comparing the proteome differences between normal and disease states,some "disease-specific" proteins can be found,which may become the basis for new drug design.Targets will also provide new biomarkers for the diagnosis and treatment of diseases.Non-classical risk factors for smoking can lead to various pathophysiological disorders such as inflammation,impaired endothelial function,abnormal lipid metabolism,myocardial mitochondrial damage,and accelerated atherosclerosis progression.The effect of smoking on pharmacokinetics is thought to be mainly by changing the activities of cytochrome enzymes CYP1A2,CYP2E1,etc.,resulting in accelerated drug metabolism and reduced absorption and bioavailability.Clopidogrel and aspirin are the most commonly used antiplatelet drugs in CCS patients,but smoking has different effects on these two drugs.Smoking may be a protective factor for patients with coronary heart disease receiving clopidogrel,but a risk factor for aspirin-treated patients,increasing aspirin resistance.Therefore,whether this paradox occurs in CCS patients and whether it is related to drugs has caused new thinking,and whether the single-nucleotide polymorphism of CYP1A2 plays a role in it is also worth exploring.Frailty is a result of age-related decline with multiple organ systems and is characterized by an increased risk of adverse health outcomes from relatively minor stressful events,as demonstrated in numerous chronic diseases.The combination of chronic coronary syndrome and frailty can further affect the life quality of these older people and increase the risk of major adverse events.Evidence from longitudinal studies linking frailty and coronary heart disease is currently inconsistent,so studying frailty status in predicting adverse events in elderly CCS patients will help in early intervention and treatment management.Purpose: From two aspects of novel biomarker discovery and non-classical clinical risk factors,we explored the factors affecting the prognosis of elderly chronic coronary syndrome.Methods: A proteomic study was conducted in the first part of the study,and 38 CCS patients were included in the discovery cohort,including 19 in the CCS event group and 19 in the non-event group as controls.Liquid chromatography tandem mass spectrometry(DIA,LC-MS/MS)quantitative proteomics with data independent acquisition was performed to identify proteins with significant expression differences.After that,we performed bioinformatics analysis on the omics data.In addition,we have developed an integrated machine learning-based approach to identify proteins as potential biomarkers for further scientifically rigorous screening of omics results.Finally,we combined omics results,pathways derived from bioinformatics analysis,machine learning,and verified target proteins by enzyme-linked immunosorbent assay(ELISA)in an independent prospective validation cohort(n = 352),and afterwards,further correlation analysis,Cox survival analysis and comparative analysis among various models and scores were performed.Secondly,in the second part of the study,the target protein endothelin C receptor(EPCR)was selected for basic experimental research to explore its function in human umbilical vein endothelial cells(HUVECs).It mainly involved the following methods,including immunofluorescence detection,inflammatory factor detection,endothelial barrier function detection,apoptosis detection and neovascularization experiments.Finally,the third part of the clinical study was conducted to explore the relationship between non-classical risk factors smoking and frailty and the adverse prognosis of older men with chronic coronary syndrome.The participants were 668 elderly patients with CCS(inclusion and exclusion criteria were the same as before),detecting platelet aggregation function and associated SNPs.Smoking was calculated on pack-year,and frailty was assessed on the FRAIL scale.Results:1.Fifty-seven differentially expressed proteins were identified by quantitative proteomics,and three final biomarkers were preliminarily selected from a comprehensive machine learning-based approach: endothelin C receptor(EPCR),cholesteryl ester transfer protein(CETP)and carboxypeptidase B2(CPB2).Further validation in a prospective cohort showed that the EPCR and CETP levels at admission were significantly higher in the CCS event group than in the non-event group,whereas CPB2 levels were not significantly different between the two groups.Correlation analysis showed that CETP was positively correlated with high-density lipoprotein cholesterol(HDL-C),triglyceride(TG)and the ratio of TG/HDL-C,and EPCR was positively correlated with fibrinogen.In the Cox survival analysis,EPCR and CETP were independent risk factors for MACEs.Patients with high EPCR and CETP levels had significantly shorter cumulative risk durations than patients with low EPCR and CETP levels.We constructed a new prognostic model by combining the Framingham coronary heart disease(CHD)risk model with EPCR and CETP levels.Compared to the Framingham CHD risk model alone,this new model significantly improved the ability to predict MACEs in CCS patients(AUC:0.732 vs 0.684,p<0.05).2.On the other hand,after functional verification in cell experiments,we found that adding lipopolysaccharide to normal cultured HUVEC would affect the integrity of endothelial cells and cause endothelial dysfunction.The specific blocking antibody RCR252 of activated protein C(APC)can competitively bind to APC,thereby indirectly affecting the function of EPCR,resulting in increased expression of inflammatory factors on the surface of HUVEC,destruction of endothelial barrier function,increased apoptosis,and decreased neovascularization ability.As the ligand of EPCR,APC can play a protective role and reduce a series of reactions caused by lipopolysaccharide.3.In the study of the relationship between smoking and prognosis,we found that in patients with chronic coronary syndrome treated with clopidogrel,the frequency of CYP1A2*1F gene homozygous carriers(AA)(rs762551,163C>A)in smokers was significantly higher than that of non-smokers.Compared with non-smokers,smokers had a reduced risk of major adverse cardiovascular and cerebrovascular events [HR=0.466,95%CI(0.262,0.829),p<0.05].In the aspirin subgroup,we found a significant increase in adverse clinical events in the smoking group compared with the non-smoking group [HR=1.90,95%CI(1.128,3.225),p<0.05].In this study,the incidence of frailty in elderly patients with chronic coronary syndrome was26.9%,and after adjustment for risk factors such as age,BMI,hypertension,diabetes,and hyperlipidemia,frailty was an independent risk factors for adverse events in CCS patients[HR=1.862,95%CI(1.096,3.162),p<0.05].Conclusions:1.Plasma proteomics can be used to find biomarkers that predict poor prognosis in patients with chronic coronary syndromes.2.EPCR and CETP were discovered and validated as promising novel biomarkers.3.The combination of EPCR and ligand APC can inhibit inflammatory response,inhibit apoptosis,protect cell barrier and promote neovascularization.LPS induces the shedding of EPCRs on the endothelial cell surface into soluble s EPCR,leading to the possible binding of APCs by competing s EPCR in the circulation,thereby affecting the function of the endothelial cell APC/EPCR pathway itself.The poor prognosis of patients with coronary heart disease may be due to the imbalance of vascular homeostasis,which is related to the increase of circulating s EPCR and the decrease of vascular endothelial EPCR.4.In elderly patients with chronic coronary syndrome,smokers benefit from clopidogrel therapy more than aspirin.Long-term smoking may lead to increased variants of CYP1A2*1F,which may partially explain the "smoking paradox" that smoking is a protective factor in patients treated with clopidogrel.Additionally,cigarette smoking is an independent risk factor for aspirin-treated patients.5.Frailty significantly increases the risk of adverse events in elderly patients with chronic coronary syndrome.