Role Of MiR-21 In Monocyte/macrophage Functions And Atherosclerosis

Part ? miR-21 deficiency attenuates atherosclerosis in part via decreased macrophage accumulationObjectives Atherosclerosis is a chronic inflammatory disorder characterized by lipid deposition and macrophage/foam cell accumulation beneath the arterial intima.Previous studies have shown the expression of miR-21 was increased in ox-LDL-treated peripheral monocytes and atherosclerotic plaques in human.However,the effect of miR-21 on atherosclerosis is still unclear.Therefore,we aimed to create the DKO mice and evaluate the role of miR-21 in the development of atherosclerotic lesions.Methods Firstly,miR-21 gene was knocked out in mice using loxP-Cre system,then DKO mice were obtained by crossing miR-21-/-mice with apoE-/-mice.The apoE-/-mice and DKO mice were fed with high-fat diet for 12 weeks to induce atherosclerosis.Plasma was collected for lipoprotein detecting and the aortas were isolated for atherosclerotic plaques assessing.Atherosclerotic lesion areas in en face aortas and aortic roots were detected by Oil Red O staining;The total collagen fibrous contents in plaques were assessed using Masson trichrome staining in sections of mice aortic roots.CD68 immunostaing was used to evaluate macrophage infiltration in atherosclerotic plaques.Besides,enzyme linked immunosorbent assay were used to analyze the production of cytokines in mouse plasma.Results miR-21 expression was significantly increased in apoE-/-mice after high-fat diet for 12 weeks.miR-21 deficiency decreased atherosclerotic lesion areas either in en face aortas or in aortic roots.Meanwhile,macrophage infiltration and collagen deposition were evidently decreased in DKO mice.However,the DKO mice showed higher LDL-C level in plasma after high-fat diet for 12 weeks.Besides,MMP-2 level in DKO mice was lower than apoE-/-mice,but the discrepancy was disappeared in the development of atherosclerosisConclusion These results demonstrate that miR-21 deficiency attenuates atherosclerosis,at least in part by impairing macrophage infiltration.Part ? miR-21 deficiency impaired monocyte/macrophage adhesion and migration in a cytokine receptors-dependent mannerObjectives Macrophages in atherosclerotic plaques drive inflammatory responses,degrade lipoproteins,and phagocytose dead cells.Our previous study showed miR-21 deficiency attenuated atherosclerosis,at least in part by impairing macrophage infiltration.However,the role of miR-21 in macrophage activation during atherogenesis is still unclear.We thus aimed to determine the influence of miR-21 deficiency on the function of macrophages in vitro.Methods The phenotype of bone marrow-derived macrophages(BMDMs)were confirmed with F4/80 and CD11b expressions.BMDMs from apoE-/-mice and miR-21-/-&apoE-/-(DKO)mice were pretreated with ox-LDL for a while,then lipid phagocytosis was evaluated by Oil Red O staining and flow cytometry analysis;meanwhile,cell supernatants were collected for cytokine detecting by ELISA.Macrophage migraion was assessed using a two-chamber migration assay and in a thioglycollate-induce peritonitis model.Macrophage-endothelial adhesion was quantified by fluorescence microscope.Besides,BMDMs apoptosis induced by ox-LDL was quantified using an annexin-V FITC apoptosis detection kit.For the probably molecular mechanism,we detected the cytokine receptors related to adhesion and migration by flow cytometry analysis.Results BMDMs from DKO mice showed weakened migration capacity towards medium lacking or containing monocyte chemoattractant protein-1(MCP-1).Macrophages migrate into peritoneal cavity in DKO mice was also markly impared compared to that of apoE-/-mice.Besides,fewer interacting BMDMs were oberserved on the endothelial cell surface after tumor necrosis factor(TNF)-a treatment in DKO mice.However,miR-21 dificiency has no effect on ox-LDL induced macrophage lipid phagocytosis,cytokines producion and apoptosis.For the cytokine receptors,BMDMs from DKO mice showed lower expressions of CCR2,CX3CR1 and CD62L.Conclusion These data demonstrated that miR-21 dificiency reduced monocyte/macrophage adhesion and migration probably in a cytokine receptors-dependent manner.Part ? Association of miR-21 levels with alterations of monocyte chemokine receptors,CCR2 and CX3CR1 in patients with coronary artery diseaseObjectives Recent data in humans and mice suggest that monocyte chemokine receptors CX3CR1 and CCR2 are involved in the pathogenesis of atherosclerosis.Our previous study showed that miR-21dificiency impaired macrophage migration with downregulating CCR2 and CX3CR1 expression.However,there is a paucity of information regarding the clinical association between miR-21 expression in circulating monocyte and alterations of monocyte chemokine receptors in humans with cardiovascular disease.Therefore,in this study,we investigated circulating monocyte heterogeneity with differential expression of CCR2 and CX3CR1 and its relevance to monocyte miR-21 expression in patients with coronary artery disease(CAD).Methods Sixty-three CAD patients with acute coronary syndrome(ACS,n=46)or stable angina pectoris(SAP,n=17)undergoing either percutaneous coronary intervention or coronary angiography and eleven non-CAD patients were enrolled in the study.Peripheral monocytes were isolated for miR-21 analysis by q-PCR and CCR2 and CX3CR1 detecting by flow cytometry analysis.Besides,ELISA was used to measure the CCL2 and CX3CL1 expression in plasma.Results It was found that miR-21 was significantly increased in ACS group in comparison to SAP or non-CAD patients and plasma CCL2 and CX3CL1 levels were substantially elevated.Furthermore,patients with ACS had significantly higher proportion of CD14+CCR2+CX3CR1+ and CD14+CCR2-CX3CR1+ monocytes but lower percentage of CD 14 CCR2 CX3CR1 monocytes than SAP or non-CAD patients did.Lastly,miR-21 expression in peripheral monocytes showed a significantly positive correlation with the proportion of CD14 CCR2 CX3CR1+monocytes,but not other monocyte subsets.Conclusions These data indicate that increased miR-21 expression in peripheral monocytes may predispose CAD patients to rupture of vulnerable plaque and thus to ACS,probably in relation to circulating monocyte phenotypic transformation with differential expression of CCR2 and CX3CR1.