Exosomes Derived From M2 Macrophages Inhibit The Progression Of Atherosclerosis By Regulating Smooth Muscle Phenotype Conversion And Screening Of Biomarkers Related To Immune Infiltration In Atherosclerosi

Part 1 M2 macrophage derived exosomes inhibit the progression of atherosclerosis by regulating the phenotype conversion of smooth muscle cellsBackground and objectiveVascular smooth muscle cells(VSMCs)play an important role in the development of atherosclerosis(AS).During the development stage of AS,a large number of VSMCs migrate from the middle membrane to the inner membrane,enhancing their proliferative ability,and transitioning from the contractile phenotype to the secretory phenotype,promoting the continuous enlargement of plaques,narrowing the lumen,and faster blood flow velocity at the narrowed area.Plaques are prone to detachment and formation of thrombi,which is life-threatening.Macrophages are also very important in atherosclerosis.M2 macrophages,as anti-inflammatory cell,are abundant in stable plaques.Exosomes are a kind of intercellular communication carrier that can transfer other cell molecules to receptor cells and play an important role in intercellular information exchange.Therefore,this study was based on in vivo and in vitro experiments to explore the role of M2 macrophage-derived exosomes(M2-exo)on phenotype conversion of smooth muscle cells and atherosclerosis.Methods1.RAW264.7 macrophages were polarized to M2 by interleukin-4(IL-4).M2-exo was obtained by ultracentrifugation,and the particle size,purity and surface specific markers of exosomes were analyzed by nanoparticle tracking,transmission electron microscopy,and Western Blot(WB).2.The phenotypic transition cell model of smooth muscle was established by using platelet-derived growth factor BB(PDGF-BB).The effect of M2-exo on phenotypic transformation of smooth muscle cells was evaluated by CCK8 proliferation test,cell scratch,and WB.3.ApoE-/-mice were fed with high fat for 16 weeks to establish AS animal model.M2exo was injected into tail vein from the 12th week.The effect of M2-exo on AS disease was elucidated by blood lipid level,oil red O,Hype staining and Masson staining in aortic root and arch of mice.The expression of phenotypic markers of contraction and secretion of mouse aortic smooth muscle cells was observed by immunohistochemical staining to clarify the effect of M2-exo on phenotypic transformation of mouse aortic smooth muscle cells.Results1.The expression of CD206,a marker protein of M2-type macrophages were observed and CD206 was strongly positive under fluorescence microscope.2.Exosomes derived from M2 macrophages,nanoparticle tracking results show that 95.7% of them have a diameter of 139nm,transmission electron microscopy shows that they have double-layer membrane lipid results,and WB results show that they express exosome marker protein Cluster of differentiation 63(CD63)and tumor susceptibility gene 101 protein(TSG101).3.The CCK8 assay of MOVAs smooth muscle induced by PDGF-BB showed that the proliferation activity was significantly enhanced,and the cell scratch test showed that the relative mobility was significantly increased.WB imaging showed that the expression of smooth muscle contraction phenotypic marker a smooth muscle actin(αSMA)decreased and the expression of secretory phenotypic marker Osteopontin(OPN)increased,which accorded with the characteristics of contractile phenotype to secretory phenotype transition model.The above experiments were repeated after the addition of M2-exo,and it was found that the cell proliferation activity decreased,the relative mobility decreased,the expression of α-SMA increased,and the expression of OPN decreased.4.Compared with the Ctrl group,there was no difference in blood lipid level and collagen content in the aortic root of ApoE-/-mice in M2-exo group.The area of oil red O in the aortic root and arch decreased significantly,the area of the vacuole in aortic root decreased,the amount of α-SMA immunohistochemical staining increased,and the amount of OPN decreased.Conclusions1.RAW264.7 can be polarized by IL-4 successfully,and the substance obtained by ultracentrifugation in M2 macrophage culture medium is M2-exo.2.PDGF-BB can successfully induce the phenotypic transition of smooth muscle cells,and M2-exo can inhibit the phenotypic transition of MOVAs smooth muscle induced by PDGF-BB in vitro.3.M2-exo had no significant effect on blood lipid level and aortic root plaque stability in mice,but could reduce the area of aortic plaque and necrotic nucleus,increase the expression of VSMCs α-SMA and decrease the expression of OPN,and inhibit phenotypic transformation.Part 2 The Integrated Analysis Identifies Three Critical Genes as Novel Diagnostic Biomarkers Involved in Immune Infiltration in AtherosclerosisBackground and objectiveAtherosclerosis(AS),a chronic inflammatory disease of the blood vessels,is the primary cause of cardiovascular disease,the leading cause of death worldwide.This study aimed to identify possible diagnostic markers for AS and determine their correlation with the infiltration of immune cells in AS.MethodsIn total,10 serum samples from AS patients and 10 samples from healthy subjects were collected.The original gene expression profiles of GSE43292 and GSE57691 were downloaded from the Gene Expression Omnibus database.Least absolute shrinkage and selection operator regression model and support vector machine recursive feature elimination analyses were carried out to identify candidate markers.The diagnostic values of the identified biomarkers were determined using receiver operating characteristic assays.The compositional patterns of the 22 types of immune cell fraction in AS were estimated using CIBERSORT.RT-PCR was performed to further determine the expression of the critical genes.ResultsThis study identified 17 differentially expressed genes(DEGs)in AS samples.The identified DEGs were mainly involved in non-small cell lung carcinoma,pulmonary fibrosis,polycystic ovary syndrome,glucose intolerance,and T-cell leukemia.FHL5,IBSP,and SCRG1 have been identified as the diagnostic genes in AS.The expression of SCRG1 and FHL5 was distinctly downregulated in AS samples,and the expression of IBSP was distinctly upregulated in AS samples,which was further confirmed using our cohort by RT-PCR.Moreover,immune assays revealed that FHL5,IBSP,and SCRG1 were associated with several immune cells,such as CD8 T cells,na?ve B cells,macrophage M0,activated memory CD4 T cells,and activated NK cells.ConclusionsOverall,future investigations into the occurrence and molecular mechanisms of AS may benefit from using the genes FHL5,IBSP,and SCRG1 as diagnostic markers for the condition.