Study On The Role Of Small Extracellular Vesicles In Heart Failure

BackgroundHeart failure is the end stage of various cardiovascular diseases such as hypertension and coronary heart disease,characterized by high incidence and high mortality rates.Heart failure is a systemic syndrome,and its development process often involves functional impairment of multiple organ systems,making the treatment of heart failure extremely difficult.Therefore,exploring the interactions between the heart and other organs from a systemic perspective is of great significance for exploring new strategies for the prevention and treatment of heart failure.Small extracellular vesicles（sEV）are nanoscale（60-200nm）extracellular microvesicles that contain various biomolecular information such as proteins,mRNAs,and microRNAs.They can be synthesized and secreted by most cells in the body and transported through the blood circulation as carriers,participating in the regulation of physiological functions.In heart failure,sEV play an important role by mediating processes such as oxidative stress and inflammation in cardiomyocytes.Micro RNAs（miRNAs）are abundant biomolecular information molecules contained in sEV and can exert biological effects between cells and organs through sEV transport.In heart failure,studies have found that miRNAs in serum sEV play an important role in cardiac remodeling and can serve as biomarkers for the diagnosis of heart failure.The sources of sEV and their contained miRNAs that influence the occurrence and development of heart failure remain to be further investigated.sEV exert different biological effects primarily through changes in their quantity and the types of contents.The quantity of sEV changes in many pathological conditions,and the reasons for the changes in sEV quantity in heart failure are still unclear.Sympathetic hyperactivity is an important pathophysiological mechanism in the progression of heart failure,and blocking sympathetic nerve activity has become a major strategy and drug target for current heart failure treatment.Studies have shown that sympathetic nerves can mediate biological effects in osteoblasts by inducing an increase in sEV release.In heart failure,the mechanism of action of sympathetic hyperactivity has not been fully elucidated,and its relationship with sEV needs further investigation.Based on the above theoretical background,our research will focus on the theme of"the role of sEV in heart failure"and investigate the regulation of sEV release in heart failure,the relationship between sEV and sympathetic hyperactivity,and the role of miRNAs in sEV.MethodsFor the first part of the research,we first investigated the changes in the quantity of cardiac-derived sEV in heart failure through methods such as nanoparticle tracking analysis（NTA）.Secondly,we screened and investigated the main cell types that were the sources of cardiac-derived sEV through single-cell analysis,immunofluorescence,and other methods.Next,we explored the main receptors mediating the sympathetic-induced increase in cardiac-derived sEV using receptor agonists and inhibitors,as well as the combination of fibroblast-specific Cre mice and adeno-associated viruses（AAV）.Finally,we investigated the pathways involved in sympathetic regulation of cardiac-derived sEV release through transcriptome sequencing and related validations.For the second part of the research,we first screened for co-change miRNAs in circulating sEV and the rostral ventrolateral medulla（RVLM）through miRNA chip sequencing,and validated them through qPCR.Secondly,we verified the effects of co-change miRNAs on inflammatory responses using miRNA mimics.Finally,we screened for target genes of co-change miRNAs through bioinformatic analyses such as GO and KEGG,and validated them through dual-luciferase reporter assays.For the third part of the research,we first detected the expression levels of miR-214-3p in circulating sEV and the heart in heart failure through qPCR,and clarified the role of miR-214-3p in cardiac fibrosis by overexpressing miR-214-3p using AAV in mice and treating cardiac fibroblasts with miR-214-3p mimics.Secondly,we verified the main sources of miR-214-3p in circulating sEV through methods such as administering exogenous sEV,treating with sEV release inhibitors,and in vivo imaging.ResultsPart 1 ResultsⅠ.The number of cardiac-derived sEV is increased in heart failureDetection of sEV numbers in mouse heart tissue showed that the sEV particle concentration in the heart failure（HF）group was significantly higher than that of sham surgery（Sham）group（Sham group:3.5±0.5 E+11/ml;HF group:10.4±0.8 E+11/ml;P<0.001）.Detection of serum sEV numbers in heart transplant patients showed that the sEV particle concentration was significantly higher before transplantation than after transplantation（before transplantation:5.6±0.2 E+11/ml;after transplantation:3.8±0.3 E+11/ml;P<0.01）.Ⅱ.Cardiac fibroblasts are the main source of increased cardiac-derived sEV in heart failureThe results of co-localization of the sEV marker CD63 with markers of three different cell types in failing hearts showed that CD63 expression was highest in cardiac fibroblasts.After stimulating cardiac fibroblasts,cardiomyocytes,and cardiac endothelial cells with norepinephrine,the sEV particle concentration released from cardiac fibroblasts was significantly higher than that of cardiomyocytes and cardiac endothelial cells（H9C2 E group:4.0±0.4 E+11/ml VS RCF E group:8.7±0.1 E+11/ml,P<0.001;MCECs E group:3.7±0.1E+11/ml VS RCF E group:8.7±0.1 E+11/ml,P<0.001）.Ⅲ.β₂-AR is the main receptor mediating increased sEV release from cardiac fibroblasts via the sympathetic nerveThe results of administration ofα-AR,β₁-AR,β₂-AR agonists to mice and cardiac fibroblasts showed that theβ₂-AR agonist group had a significantly higher sEV particle concentration than the other agonist groups（P<0.05）.Immunofluorescence showed that compared to the Sham group,expression ofβ₂-AR was increased on the cardiac fibroblast marker PDGFRαin the HF group.Inhibition ofβ₂-AR in heart failure mice significantly reduced sEV particle concentration（P<0.05）.Cardiac fibroblast-specific overexpression ofβ₂-AR in mice significantly increased cardiac-derived sEV particle concentration（control group:3.8±0.6 E+11/ml;cardiac fibroblast-specificβ₂-AR overexpression group:7.9±0.5 E+11/ml;P<0.01）.Cardiac fibroblast-specific knockdown ofβ₂-AR in heart failure mice significantly reduced cardiac-derived sEV particle concentration（heart failure group:9.5±0.7 E+11/ml,heart failure+cardiac fibroblast-specificβ₂-AR knockdown group:5.0±0.6 E+11/ml;P<0.01）.Ⅳ.β₂-AR upregulates Rab27b expression to increase sEV release from cardiac fibroblastsSequencing results showed that stimulation ofβ₂-AR significantly upregulated Rab27b expression（P<0.05）.In cardiac fibroblasts,stimulation ofβ₂-AR significantly upregulated Rab27b expression（P<0.05）,and knockdown of Rab27b significantly reduced sEV particle concentration（β₂-AR agonist group:5.8±0.3 E+11/ml,β₂-AR agonist+Rab27b si RNA group:1.6±0.2 E+11/ml;P<0.001）.Part 2 ResultsⅠ.Screening and validation of co-change miRNAs in serum sEV and RVLMMicroarray analysis showed that the co-change miRNAs between RVLM and serum sEV in HF rats were miR-214-3p,miR-139-3p,miR-874-3p,let-7i-5p,let-7g-5p.qPCR verification showed that the co-change miRNAs were miR-214-3p,let-7g-5p,let-7i-5p.Ⅱ.Effects of co-change miRNAs on inflammationThe miR-214-3p mimics significantly increased the levels of TNF-α,IL-1β,and IL-6 in PC12 cells（P<0.05）,while the let-7g-5p and let-7i-5p mimics significantly decreased the levels of TNF-α,IL-1β,and IL-6（P<0.05）.Ⅲ.Bioinformatics analysis and verification of target genes of co-change miRNAsThrough GO,KEGG,and other bioinformatics analyses,Traf3,Smad2,and Mapk6 were identified as inflammation-related target genes of miR-214-3p,let-7g-5p,and let-7i-5p,respectively.Dual-luciferase reporter gene assays found that miR-214-3p,let-7g-5p,and let-7i-5p significantly reduced the luciferase activity of wild-type Traf3,Smad2,and Mapk6（P<0.01）,but did not significantly change the luciferase activity of MUT Traf3,MUT Smad2,and MUT Mapk6,indicating that miR-214-3p,let-7g-5p,and let-7i-5p can respectively target and downregulate the expression of Traf3,Smad2,and Mapk6（P<0.05）.Part 3 ResultsⅠ.miR-214-3p increases cardiac fibrosisCompared with the Sham group,the expression levels of miR-214-3p in serum sEV and heart tissues were significantly increased in the HF group of mice（P<0.01）.The level of miR-214-3p in serum sEV of heart transplant patients was significantly decreased after heart transplantation（P<0.001）.After overexpressing miR-214-3p in normal mouse hearts,the expression levels of TGF-β,Col1a1,and Col3a1 were significantly increased（P<0.05）,and the fibrotic area was significantly increased（NC group:4.7±0.6%,OE-miR-214-3p group:8.9±0.5%,P<0.001）.The miR-214-3p mimics significantly increased the expression levels of TGF-β,Col1a1,and Col3a1 in cardiac fibroblasts（P<0.01）.Ⅱ.miR-214-3p increases cardiac fibrosis by negatively regulating Traf3After overexpressing miR-214-3p in mouse hearts and transfecting miR-214-3p mimics into cardiac fibroblasts,the expression levels of Traf3 were significantly decreased（P<0.05）.After overexpressing Traf3 in the hearts of heart failure mice,the expression levels of TGF-β,Col1a1,and Col3a1 were significantly decreased（P<0.05）,and the fibrotic area was significantly reduced（HF group:15.0±1.4%,HF+OE-Traf3 group:8.5±0.4%,P<0.01）.When overexpressing both miR-214-3p and Traf3 in normal mice simultaneously,there were no significant changes in the expression levels of TGF-β,Col1a1,Col3a1,or the fibrotic area.Ⅲ.Increased miR-214-3p in circulating sEV during heart failure originates from the lungsCompared with the Sham group,the expression level of miR-214-3p in lung-derived sEV was significantly increased in the HF group of mice（P<0.05）.The lung-derived sEV of heart failure mice significantly increased the expression levels of TGF-β,Col1a1,and Col3a1 in normal mouse hearts（P<0.05）and significantly increased the cardiac fibrotic area(sEV_Shamgroup:4.6±0.4%,sEV_HF group:11.6±1.3%,P<0.01).Compared with sEV from the control group,the sEV released from bronchial epithelial cells after angiotensin II（Ang II）stimulation and added to cardiac fibroblasts significantly increased the expression levels of TGF-β,Col1a1,and Col3a1（P<0.001）.ConclusionThis study first discovered that sympathetic hyperactivity in heart failure activatesβ₂-AR in cardiac fibroblasts,leading to an increased release of cardiac-derived sEV.It is then clarified that the miRNAs in peripheral circulating sEV during heart failure can mediate the regulation of the periphery on the central nervous system,enhancing neuroinflammation in the RVLM.Finally,it is demonstrated that miR-214-3p in lung-derived sEV can increase cardiac fibrosis in heart failure.From the perspectives of both sEV quantity and contents,this study focused on the interrelationship between sympathetic hyperactivity and sEV,elucidating the roles of sEV in heart failure.The present study provides new insights into further investigating the pathogenic mechanisms of heart failure and exploring novel therapeutic strategies.