Macrophage-stimulated MicroRNA Expression In Mural Cells Promotes Transplantation-induced Neointima Formation

Background:Restenosis of transplanted vessels,known as transplant vasculopathy(TV),happens after organ transplantation,which is the main cause of chronic graft failure.The biological mechanism of TV has not been fully elucidated.In previous studies,we found that vascular adventitial inflammation occurred in the early stage after transplantation surgery,and the infiltration of macrophages in adventitia may play a key role in TV occurrence.In this study,we used the established artery transplantation model to prove that early infiltration of macrophages is crucial in promoting the process of TV.Moreover,we studied the roles of miRNAs(using miR-21 as an example),to clarify the mechanism of macrophage-stimulated neointima formation via crosstalk with vascular mural cells by modulating their microRNA expressions.Objective:1.To establish a rat model of abdominal aorta transplantation,and to determine whether inhibiting the early adventitial infiltration and accumulation of macrophages can inhibit the intimal remodeling.2.To clarify the mechanism of macrophage stimulated microRNA expression in mural cells.3.To test whether inhibition of miRNA-21 expression by local treatment at adventitia can inhibit the occurrence and development of TV.Methods:In vivo experiment:To establish rat thoracic-abdominal aorta transplantation model,clodronate liposome were used for in vivo macrophage depletion.Transplantation rats were randomly divided into macrophage depletion group(clodronate liposomes),miR-21 inhibited group(miR-21 inhibitor)and the corresponding control group(control liposomes/control miRNA).Vascular grafts of 0、1、2、3、7 and 14 days were removed to stain with HE.The proliferation of layers of vascular grafts was measured;immunohistochemical staining detected expression of CD3,CD68,proliferating cell nuclear antigen(PCNA),monocyte chemotactic protein-1(MCP-1),vascular cell adhesion molecule-1(VCAM-I),intercellular adhesion molecule-1(ICAM-1),a-smooth muscle actin(a-SM actin).Vascular adventitia and media tissues were isolated,and the expression of miR-21,MCP-1,VCAM-1,ICAM-1 was detected by QRT-PCR at the mRNA level.In vitro experiment:Adventitia and media tissues were isolated and primarily cultured separately.RAW264.0 macrophage-conditioned mediums were prepared.The concentrations of cell factors in the conditioned medium were detected by ELISA,such as tumor necrosis factor alpha(TNF-a)and transf-orming growth factor-β1(TGF-β1).The expression of miR-21 was detected by QRT-PCR,after adventitial fibroblasts/VMSCs were stimulated by TNF-a.The expression of miR-21 was detected by QRT-PCR,after adventitial fibroblasts/VMSCs were cultured in the conditioned medium,with TNF-a neutralizing antibody added in experimental group.After adventitial fibroblasts/VMSCs were stimulated by TNF-α.the experimental group were transfected with miR-21 inhibitor,EDU and Transwell to detect the ability of cell proliferation and migration,and the expression of miR-21,MCP-1,VCAM-1,ICAM-1,CCL-5 was detected by QRT-PCR.Adventitial fibroblasts/VMSCs were transfected with miR-21 mimic,and the expression of MCP-1,VCAM-1,ICAM-1 was detected by QRT-PCR.Results:1.Macrophage depletion inhibits neointimal growth and adventitial inflammation caused by transplantation,and significantly decrease the expression of miR-21 in vascular wall.QRT-PCR results showed that macrophage depletion group and miR-21 inhibition group showed decreased expression of miR-21,MCP-1,VCAM-1,ICAM-1,and reduced neointimal growth compared with the control group.Immunohistochemistry results showed that the expression of CD68,PCNA,MCP-1,VCAM-1 and ICAM-1 in macrophage depletion group and miR-21 inhibition group was decreased compared with the control group.2.Macrophages regulate the expression of miR-21 in adventitial fibroblasts and medial smooth muscle cells by releasing TNF-α.ELISA results showed that macrophage conditioned medium contained TNF-α.QRT-PCR results showed that TNF-α can upregulate the expression of miR-21 in fibroblasts and smooth muscle cells.QRT-PCR results showed that macrophage-conditioned medium increased miR-21 expression in fibroblasts/vascular smooth muscle cells,while addtion of the TNF-α neutralizing antibody blocked this response.3.Inhibiting miR-21 suppresses TNF-induced proliferation and migration in fibroblasts and vascular smooth muscle cells(VSMCs).TNF-a stimulated proliferation and migration of both primary fibroblasts and smooth muscle cells,while transfection of miR-21 inhibitor reduced the proliferation and migration responses;QRT-PCR results showed that miR-21 inhibitor decreased the expression of MCP-1,VCAM-1,miR-21,ICAM-1 and CCL-5 stimulated by TNF.4.miR-21 has an important role of in inflammatory responses in VSMCs and fibroblasts.QRT-PCR results showed that transfection of miR-21 mimic up-regulated the expression of MCP-1 and VCAM-1 in fibroblasts.and the expression of MCP-1 in VMSCs.Conclusion:1.Macrophages play an important role in the process of transplantation-induced neointima formation.2.Macrophages regulate the expression of miR-21 in vascular mural cells via a paracrine mechanism of TNF-a release,which in turn regulates the functions and inflammatory reactions of mural cells.3.MiR-21 expression in mural cells may promote the neointimal remodeling by promoting inflammatory responses and cellular functions of mural cells in the graft.