The Experimental Study Of Bone Marrow-Derived Mesenchymal Stem Cells Transplantation Inhibiting Neointimal Formation In Autologous Vein Grafts

Background and objective:Vascular bypass surgery using autologous vein grafts has become an established procedure for treating many conditions,most notably myocardial infarction(e.g.coronary bypass)and limb claudication(e.g. femoral-popliteal bypass grafting),but obliterative stenosis often follows.About 20%-40%of grafts become stenotic and require intervention within 2 years because of hemodynamically significant neointimal hyperplasia.More extensive research using vein grafting models have demonstrated that immune system-mediated injury to endothelial cells,and then smooth muscle cell(SMC)migration and proliferation are thought to be central to the impaired reendothelialization and formation of neointima. Currently,there is a lack of effective preventive measures for the restenosis of autologous vein grafts.Theoretically,neointimal formation of vein grafts could be potentially prevented if the original damaged endothelium could be replaced.Recently more and more attention has been paid to the study about stem cells. The defining characteristics of stem cells are self-renewal and the ability to differentiate into one or more specialized cell types.According to their capacity of differentiation,stem cells have been divided into three major groups:totipotential stem cells,multipotential stem cells and unipotential stem cells.It was believed that only embryonic stem cells derived from the inner cell mass could differentiate into any cell type,but unipotential stem cells,also called adult stem cells or tissue-specific stem cells,could only differentiate into cells of the tissue of origin. However,several studies now provide evidence that tissue-specific stem cells may be able to give rise to a variety of differentiated cell types found in embryonic germ layers.This founding is important for the developing of cytobiology theory and the treatment for many diseases.The two most attractive tissue-specific stem cells are:1) hematopoietic stem cells,which must provide a continuous source of progenitors for red cells,platelets,monocytes,granulocytes,lymphocytes;and 2)bone marrow-derived mesenchymal stem cells(BMSCs),which arise from the complex array of supporting structures in marrow.Recent work has shown that BMSCs have the capacity to differentiate along a number of lineages under defined conditions in vitro and in vivo.Because BMSCs can be harvested easily from bone marrow with standardized isolation techniques and have a high proliferative potential in culture, also BMSCs are capable of differentiation to a wide variety of cell types,and when autologous transplanted,there exist no immunorejection.Therefore,BMSCs seem to be the ideal candidate for tissue repair.Several studies have also shown that BMSCs transplantation can implicate in neovascularization,resulting in adult blood vessel formation.Despite the anticipated stem cell plasticity,the role of BMSCs in reendothelialization and neointimal formation has not been clarified in vein grafting model.The current study used a direct approach to test the hypothesis that,after autologous vein grafting in the rat model,BMSCs have potential effects on reendothelialization and neointimal formation.Methods:Male adult Wistar rats(weighing 250 to 300 g)were used in all vein grafting and BMSCs transplantation experiments.Male young Wistar rats(weighing 100g)were used for the isolation of BMSCs.This protocol resulted in the creation of 3 groups(26 per group):(1)Control group(without vein grafting and BMSCs transplantation),(2)Vein grafting group(vein grafting without BMSCs transplantation),and(3)BMSCs transplantation group(vein grafting with BMSCs transplantation).BMSCs were isolated from adult rats using density gradient centrifugation and anchoring culture,then cultured in low-glucose DMEM supplement with 10%FBS for expanding.The morphology of BMSCs was observed at different time and FACS was used to determine the phenotypic character of BMSCs.To study the plasticity of BMSCs toward ECs,the passaged BMSCs were induced by medium containing vascular endothelial growth factor(VEGF),bFGF and endothelial cell growth supplement(ECGS).Immunocytochemistry of CD31 was used to identify the phenotype of induced cells.Autologously interpositioning left external jugular vein(LEJV)to left common carotid artery-induced vein grafting model of rat was utilized.Vascular lesion formation after transplantation of BMSCs labeled with 4',6-diamidino-2-phenylindole(DAPI)was investigated.After 2 weeks, grafted cells were visualized in sections of vein grafts by fluorescent double-staining for DAPI and endothelial marker CD31 and endothelial nitric oxide synthase(eNOS). Moreover,proliferation activity was examined by proliferating cell nuclear antigen (PCNA)immunohistochemistry and endothelial function was assessed by measuring the expression level of eNOS and inducible nitric oxide synthase(iNOS)through real-time reverse transcription-PCR and western blot analysis.Four weeks posttransplantation,hematoxylin and eosin staining was performed and the neointima and medial thicknesses was measured.Results:Colonies of BMSCs formed at 4th day at primary culture,at about 7th day,BMSCs got together and the number of division cells decreased,then were subcultured by 0.25%trypsine-0.02%EDTA.BMSCs were purified at 3rd generation. The passage BMSCs proliferated fast,and could be subculture about every 5 days. Examination of BMSCs revealed that in passage 3 of initial culture almost all adherent cells(95%)took on typical morphologies of fibroblastoid cells in vitro and maintained similar morphology with passages.Expression of cell surface antigens on BMSCs in early passages(up to passage 3)was analyzed by flow cytometry and cells were défined as CD34-/CD45-/ CD90+/CD44+.Cytokines can induce BMSCs to express CD31 after cultured for 5 days later.After labeling,DAPI stains specifically 100%of the BMSCs nuclei.Vein grafting was successfully performed in rats by using left external jugular vein(LEJV)and autologously interpositioning to left common carotid artery with cuff technique and all survived.The mean operative time was 40min.The cold ischemia time and warm ischemia time of the donor was 10-15min and 3-5min,respectively.Two weeks after BMSCs implantation,DAPI-labeled BMSCs were frequently observed in the vein grafts and these transplanted cells were mostly positive for CD31.Furthermore,DAN-positive cells showed extensive eNOS labeling.No grafted cells were detected to differentiate intoα-SM-actin positive smooth muscle cells.With BMSCs treatment,vascular wall presented less PCNA-positive proliferating cells compared with vein grafting group(P＜0.05).Both real-time reverse transcription-PCR and western blot showed a rise of eNOS in the BMSCs group compared with the vein grafting group(P＜0.05),however only Real-time reverse transcription-PCR show a rise of iNOS(P＜0.05).The neointima at four weeks was significantly thicker in vein grafting group(262.20±36.91μm)in comparison to BMSCs group(30.93±8.07μm)(P＜0.05).The media thickness increased to 80.42±15.20μm in vein grafting group and this achieved statistical significance contrasted with 54.27±9.31μm in BMSCs group(P＜0.05).Conclusions:Engrafted BMSCs appeared to differentiate into endothelial cells, diminish the neointima formation and contribute to the improvement on endothelial function,which indicates that BMSCs may exert an important function as repair mechanism in vascular injury after vein grafting.This discovery raises the possibility that treatment involving BMSCs might help to prevent advanced vascular lesion formation.