KLF5 Mediated The Expression And Secretion Of MiR-155 And Tongxinlou Intervented It

Atherosclerosis(Atherosclerosis,AS)is the basis of the pathogenesis of many cardiovascular and cerebrovascular disease,which is the leading cause of death of coronary heart disease,myocardial infarction and angina.Its mechanism is complex.Various cell types within the arterial wall,including endothelial cells,vascular smooth muscle cells(VSMCs)and macrophages,are substantially involved in the progression of atherosclerosis.These cells all secrete growth factors,cytokines/chemokines,and extracellular matrix,which regulate complex interactions between inflammation,lipid deposition,endothelial dysfunction,and extracellular matrix remodeling and lead to the formation of atherosclerotic plaque.TXL(Tongxinluo,TXL)as a traditional Chinese medicine compound preparation is composed of twelve animals and plants herbal ingredients,which has been used in clinical twenty years.It is also the compound extract of ginseng,semen,peony,sandalwood,dalbergia,leech,cicada,centipede,scorpion,eupolyphaga,frankincense,borneol and other 32 Chinese herbal medicines.A number of studies have demonstrated that TXL could significantly improve endothelial function,decrease the formation of atherosclerosis and stabilize vulnerable plaque.Exosomes have been proposed as vehicles for microRNA(miRNA)-based intercellular communication and a source of miRNA biomarkers in bodily fluids.Exosome-mediated miRNA transfer between cells has been proposed to be a mechanism for intercellular signaling.Although the possibility of exosome-mediated miRNA transfer as a mode of intercellular communication is an attractive concept,current mechanistic models lack detail,and the physiologic significance of this paradigm is not yet established.In this study,we focus on the study of exosome communication and drug interventionon atherosclerotic disease.PartⅠ KLF5 participated the atherosclerosis by regulating the secretion of miR-155Objective: To investigate the effect of KLF5 on the formation of atherosclerosis.Methods: Expression of miRNA was determined by microarray and Real-time PCR.Expression of KLF5 was determined by Real-time PCR and Western blot.Secretion of miRNA was detected by microarray and Real-time PCR.miR-155 promoter activation was detected by the dual luciferase reporter gene assay.CD31 exprssion was observed by immunofluorescence.Plaque formation was detected by oil red O staining.Expression of miR-155 was determined by Immunofluorescence in situ hybridization.Endothelial permeability was observed by Evans blue staining.Endothelial tight junction proteins were detected by Western blot.The proliferation and migration of endothelial cells were observed by MTS and Wound-healing assay.Results:1 Overexpressing KLF5 regulated the expression of miRNAs in smooth muscle cells.Microarray expression profile showed that overexpression of pAd-KLF5 significantly induced 43 kinds of miRNA expression in smooth muscle cells compared with overexpression of pAd-GFP.qRT-PCR analysis revealed that overexpression of pAd-KLF5 significantly strengthened the expression of miR-155,miR-146 a and reduced the expression of miR-143,miR-145,miR-221,and miR-222 compared with overexpression of pAd-GFP,which consistent with the microarray results.2 oxLDL regulated the expression of miRNAs.Western blot and Real-time PCR showed that oxLDL significantly induced the expression of KLF5 in smooth muscle cells.Meanwhile,oxLDL significantly induced the expression of miR-155,miR-146 a,reduced the expression ofmiR-143,miR-145,miR-221 and miR-222.3 Overexpressing KLF5 promoted the expression and secretion of miR-155 insmooth muscle cells.Quantitative analysis of electron micrographs showed that the diameter of the vesicles is heterogeneous,but most are between 60 and 130 nm with a median size of 100 nm,indicating that most of the isolated vesicles comprise exosomes but not larger apoptotic bodies.Chip screening results showed miR-155 increased significantly in the exosomes by overexpressing pAd-KLF5 in smooth muscle cells compared to control group.Using qRT-PCR,we confirmed that the miR-155 expression level was significantly higher in the KLF5-overexpressing or oxLDL-overexpressing exosomes compared with control group.While knockdown the expression of KLF5,miR-155 level was decreased in the exosomes.Reporter gene analysis further identified that KLF5 could directly activate the miR-155 promoter activity.4 miR-155 transported into the endothelial cells by exosomes.The smooth muscle cells and endothelial cells were co-cultured using transwell system.Fluorescence microscopy revealed that FITC-labeled-miR-155 in endothelial cells was significantly increased in the KLF5-overexpressing group,especially with oxLDL treatment.KLF5 silencing abrogated the effect of oxLDL treatment on miR-155 expression,suggesting a major contribution of KLF5-induced exosomes to the transfer of miR-155.Analysis of exosome uptake performed in the endothelial cells by confocal microscopy revealed that an increasing uptake of labeled exosomes(PKH67-labeled)in a time-dependent manner,especially in pAd-KLF5 tranfected HASMCs-derived exosomes.5 p Ad-KLF5 transfected HASMCs-derived exosomes promoted the formation of atherosclerosis.ApoE-/-mice were injected with pAd-KLF5-derived SMC exosomes and fed with high fat diet for 8 weeks.Oil Red O staining showed that plaque in pAd-KLF5 group was significantly increased than that of pAd group,suggesting that p Ad-KLF5-derived exosomes promoted the formation of atherosclerosis.6 miR-155 promoted atherosclerosis formation by inhibiting the endothelialtight junction protein expression.Evans blue staining showed that endothelial permeability of apoE-/-mice was increased than that of apoE-/-miR-155-/-mice,suggesting lack of miR-155 has a protective effect on endothelial cells.Endothelial proliferation,migration and angiogenic ability significantly decreased in pAd-KLF5-derived exosome group.Western blot showed that endothelial tight junction proteins,such as beta-catenin,ve-cadherin,claudin1 and ZO-1 in pAd HASMCs-derived exosome group were significantly higher than pAd-KLF5 HASMCs-derived exosome group.Accordingly,the permeability activity of endothelial cells in pAd-KLF5 HASMCs-derived exosome group significantly increased compared with p Ad permeability exosome group.These results siggest that miR-155 promoted atherosclerosis formation by inhibiting the endothelial tight junction protein expression.PartⅡ TXL inhibited miR-155 expression and secretion in endothelial cellsObjective:To observe the effect of TXL on atherosclerotic plaque and its mechanism.Method: In situ hybridization was performed to locate the distribution of miR-155.Wound-healing and MTS assays were performed to examine the migration and proliferation of macrophages.The expression of LRP1,RAB27 a and RAB22 a were detected by Western blot.Results:1 miR-155 was highly expressed in atherosclerotic plaques.miR-155 expression was mainly detected in human or mouse atherosclerotic vascular tissue.2 miR-155 transported to macrophages from the endothelial cellsFluorescence microscopy revealed that FITC-labeled-miR-155 in macrophages in the lower chamber was significantly increased in the KLF5-overexpressing group,especially with oxLDL treatment.KLF5 silencing abrogated the effect of oxLDL treatment on miR-155 expression,suggesting a major contribution of KLF5-induced exosomes to the transfer ofmiR-155.Overexpression of miR-155 increased the proliferation and migration of macrophages.Accordingly,Knockdown the expression of miR-155 decreased the proliferation and migration of macrophages.3 TXL inhibited the expression and secretion of miR-155 mediated by KLF5.apoE-/-mice fed with high fat diet were randomly divided into two groups:apoE-/-mice,apo E-/-+TXL treated mice.Oil Red O staining showed that plaque in apoE-/-mice was significantly increased than that of TXL treated group.In vitro experiments further confirmed that pre-incubation TXL could significantly inhibit miR-155 expression induced by overexpressing KLF5.Western blot results showed that TXL pre-incubation could significantly induce the expression of LRP1,RAB27 a and RAB22 a,which are exocrine packaging protein.Conlusion:1 Overexpressing KLF5 could regulate the expression of various miRNA in smooth muscle cells.2 Overexpressing KLF5 could promote the expression and secretion of miR-155 in smooth muscle cells.3 miR-155 was highly expressed in atherosclerotic plaques.4 miR-155 in smooth muscle cell-derived exosomes could transprot into the endothelial cells and promote atherosclerosis formation by inhibiting the endothelial tight junction protein expression.5 miR-155 increased the proliferation and migration of macrophages.6 TXL inhibited the expression and secretion of miR-155 mediated by KLF5.