Research On The Role Of Circulating Extracellular Vesicles In Cardiac Remodeling During Heart Failure

The prevalence and mortality of heart failure(HF)remains high recently.The prevalence of HF has increased by 44% in the past 15 years in China,and 1.3%(about 13.7 million)of adults aged over 35 years suffer from HF,which seriously threatens the quality of life and longevity of Chinese population.Among them,cardiac ischemia and hypoxia caused by coronary heart disease is the main cause of HF.The metabolism of cardiac cells and the inflammatory changes and pathological remodeling of the local microenvironment are the main pathological manifestations of HF.If the key factors regulating the structure and function of cardiac cells can be identified from their complex pathogenesis,it will provide new therapeutic strategies for HF.Extracellular vesicles(EVs)are considered to be key mediators of intercellular and interorgan communication,reflecting the current physiological status of the body and serving as biomarkers of disease,and are closely related to pathological cardiac remodeling for HF.However,the method of isolation of circulating extracellular vesicles(CEVs)and the role in the pathogenesis of cardiac remodeling has been poorly investigated.Therefore,ultracentrifugation(UC),polyethylene glycol(PEG)precipitation and size exclusion chromatography(SEC),the most commonly used methods in the literature for the separation of EVs,are used in this study to determine the ideal isolation method for circulating extracellular vesicles from HF patients(HCEVs).The role of HCEVs in the development and progression of HF has been explored by comparative proteomic analysis.This study included 24 patients with ischemic heart disease and 12 normal subjects.The CEVs were first isolated using three methods,UC,PEG and SEC,and evaluated for quality,thus providing high purity CEVs for subsequent studies.The morphology of CEVs was mainly observed by transmission electron microscopy and the particle size and concentration of CEVs were detected by dynamic light scattering and asymmetric flow field flow separation with multi-angle light scattering.The level of unwanted protein contamination in CEVs was evaluated by Western Blotting(WB)and silver staining.After selecting the ideal isolation method,circulating extracellular vesicles from normal subjects(NCEVs)and HCEVs were isolated,and the differences in number,size,morphology,specific markers and proteomics between the two groups of CEVs were compared.Finally,the biological effects of the two groups of CEVs on human cardiomyocytes(HCM)and human cardiac fibroblasts(HCF)were evaluated in vitro based on the results of proteomic analysis.The results showed that the particle size of CEVs isolated by UC(253.02 ± 94.15 nm)was significantly larger than that of PEG(50.37 ± 14.16 nm,P < 0.001)and SEC(49.90 ± 3.74 nm,P < 0.001),and the expression levels of specific marker proteins CD63 and HSP70 were the lowest.Although CEVs isolated by PEG had the highest expression levels of specific marker proteins CD63 and TSG101,they were prone to aggregation and also had the highest expression levels of Ig G,Apo A1,Apo A4,and Apo E.Compared with PEG,CEVs isolated by SEC were less prone to aggregation and had lower expression of Albumin,Ig G,Apo A1,Apo A4,and Apo E.Among the three methods,only SEC combines high yield and low contamination,and therefore is a more ideal method for isolating CEVs and as an isolation method for downstream CEVs studies.Next,4D label-free proteomics screened 15 differential proteins in NCEVs and HCEVs,containing 9 down-regulated proteins and 6 up-regulated proteins.Of these,the up-regulated proteins mainly regulated primary metabolic processes and glycerolipid metabolic process,as well as having functions in the oxidationreduction process and positive regulation of inflammatory response.However,the down-regulated proteins mainly regulated processes such as cell development,cell differentiation,and cell population proliferation.Compared to NCEVs,HCEVs can significantly cause inflammation and triacylglycerol(TAG)accumulation in HCM in vitro and impair their activity.They can also cause endoplasmic reticulum stress and autophagy in HCM.In addition,HCEVs can induce differentiation of HCF,leading to a significant increase of pro-inflammatory and pro-fibrotic factors,as well as an increase in the extracellular matrix such as COL1A1 and FN1.Also,HCEVs can cause an increase of the HF marker protein MMP9 in HCF and negatively correlate with autophagic flux.Finally,by analyzing cytokines in patients’ plasma and cell supernatants after the action of HCEVs,we screened for significantly elevated levels of five cytokines associated with HF,including CD40 L,RESISTIN,PDGF-AA,PDGF-BB,and HGF.This study demonstrates that SEC is a more ideal method for isolating CEVs.Initially,15 differential proteins in HCEVs were screened,and their unique pro-inflammatory,pro-fibrotic and TAG accumulation-inducing characteristics in HCM were demonstrated by in vitro experiments.It reveals that HCEVs can promote HF progression through pathological cardiac remodeling,providing new insights for HF treatment.