The Role Of Adventitia Vascular Smooth Muscle Progenitor Cells In The Pathogenesis Of Transplant Arteriosclerosis

Part Ⅰ Identification and induced differentiation of vascular adventitial smooth muscle progenitor cellsObjective: To determine the source of the neointima cells of transplant arteriosclerosis and to investigate their biological characteristics.Methods:The eGFP transgenic mice with C57BL/6background and wild type C57BL/6mice who received each other’s bone marrow transplantation acted as recipients of aortic transplantation. The grafts were analysed by immune fluorescence at1,2,4,6,8, and10weeks after the transplantation. The flow cytometry technology was used to sort out the eGFP positive cells in the grafts and these cells were cultured in vitro. To investigate whether the smooth muscle progenitor cells could migrate into the intima, the smooth muscle progenitor cells labelled with eGFP were seeded around the wild type C57BL/6grafts during the transplanting operation.Results: The bone marrow-derived mononuclear cells could not express the smooth muscle markers SM-MHC, but they expressed monocyte/macrophage marker CD68. In two week after operation, non bone marrow-derived mononuclear cells began to appear at the adventitia of the grafts, they expressed the stem cell markers Sca-1and the smooth muscle differentiation marker SM-22alphas. This group of cells could migrate into the intima and accounted for most of cells in the neointima. The smooth progenitor cells could express SM-MHC if induced by PDGF-BB or TGF-beta in vitro. In vivo experiments the cultured smooth progenitor cells could migrate into the intima from adventitia and promote the intimal hyperplasia.Conclusion:The non bone marrow derived smooth muscle progenitor cells can migrate into the intima and differentiate into smooth muscle like cells and promote the neointima hyperplasia.Part II MicroRNA-155promotes resident smooth progenitor cells directional migration by regulating MCP-1concentration gradient between noeintima andadventitiaObjective:To investigate the role of miR-155in the pathogenesis of transplant arteriosclerosis.Methods:MiR-155knockout mice and the wild type C57BL/6mice were transplanted with each other’s bone marrow cells, and then they acted as aortic transplantation recipients. The bone marrow-derived mononuclear cells were sorted by flow cytometry and the mRNA level of more than10kinds of cytokine expression was determined by RT-PCR. After transfection of miR-155mimics and inhibitor, the level of the cytokines in the supernatant was mersured by ELISA. The chemokine concentration gradient between the intima and adventitia was determined by RT-PCR at different time after the transplantation. The transwell experiment was used to determine the migration of smooth muscle progenitor cells.Results:Eight weeks after operation we found that the intimal hyperplasia was mildest in the recipients harboring miR-155-/-bone marrow cells, and the miR-155-/-mice received miR-155+/+bone marrow cells developed more significantly intimal hyperplasia. MiR-155increased the expression of monocyte chemotactic protein-1(MCP-1) in the bone marrow derived cells and promoted the migration of smooth muscle progenitor cells. When miR-155gene was knocked out in the bone marrow derived cells, the MCP-1concentration gradient between intima and adventitia is greatly reduced.Conclusion:MicroRNA-155can promote the advantitial smooth progenitor cells directional migration by regulating MCP-1concentration gradient between noeintima and adventitia.Part Ⅲ Inhibition of intimal hyperplasia in murine aortic allografts by the oral administration of the TGF-beta-RI kinase inhibitor SD-208Objective:Transforming growth factor (TGF)-β plays a significant role in the pathogenesis of the intimal hyperplasia of transplant arteriosclerosis (TA). The aim of this study was to evaluate the efficacy of an oral inhibitor of TGF-β receptor I kinase (SD-208) on the development of TA.Methods:Fully major histocompatibility complex-mismatched BALB/C donor aortas were transplanted into C57BL/6recipients, and the mice then received different doses (40or60mg/kg) of SD-208or control vehicle by daily gavage for8weeks. The grafts were analyzed by histology and morphometry1,2,4,6and8weeks after transplantation. The effects of TGF-β and SD-208on neointimal smooth muscle-like cell (SMLC) and vascular smooth muscle cell (VSMC) proliferation and migration were evaluated, and the expression levels of Smad3, p-Smad3, CTGF and collagen I were determined by in vitro experiments.Results:When mice were treated daily with40or60mg/kg of SD-208in the absence of any other immunosuppression, the intimal hyperplasia of the graft was significantly reduced compared with the vehicle-treated control group (32%and48%reduction for40mg/kg and60mg/kg SD-208compared with the controls, respectively, n=5, P<0.05). SD-208also significantly reduced SMLC proliferation and the production of intimal collagen. However, no additional inhibitory effect on VSMC proliferation was observed. Connective tissue growth factor, a protein downstream of TGF-P, was down-regulated with the inhibition of Smad3phosphorylation.Conclusion:These results demonstrate that SD-208can effectively reduce the formation of intimal hyperplasia of TA in the murine aortic allograft model.