Effects And Possible Mechanism Of Endothelial Progenitor Cells (EPCs) On Transplant Arteriosclerosis

Over the last 4 decades,although major improvements have been made in the prevention and treatment of acute transplant rejection,transplant arteriosclerosis(TA) in the arteries still limits the long-term survival of patients with solid organ transplantation.TA is characterized by diffuse,uniform,concentric narrowing of the artery owing to proliferative,fibrocellular intima that often results in transplant organ failure.A hallmark of lesions is mononuclear cell infiltration into the vessel wall of grafts at the early stage,followed by neointimal formation.Recent insights have underscored the fact that vascular lesions are the result of cumulative endothelial injuries induced both by alloimmune responses and by nonspecific insults(including ischemia-reperfusion injury,viral infections,and metabolic disorders) in the context of impaired repair mechanisms.However,the precise pathogenetic mechanisms leading to TA are largely unknown and,as a result,adequate prevention and treatment protocols are still lacking.Endothelial progenitor cells(EPCs) can differentiate into a variety of cells to replace dead cells or to repair damaged tissues and seem to be crucial in the development of cardiovascular diseases.Recent evidence indicates that EPCs are involved in the pathogenesis of TA.Although the pathogenesis of transplant arteriosclerosis is not yet fully understood,recent developments in EPCs research have suggested novel mechanisms of vascular remodeling in allografts.For example, EPCs derived from the recipient may repair damaged endothelial cells of arteries in transplant organs and TA is associated with reduction in circulating EPCs.Further evidence suggests that EPCs may be released from both bone marrow and non-bone marrow tissues and appear to replenish cells that died in donor vessels.Concomitantly, EPCs may also accumulate in the intima,where they differentiate into endothelial cells and smooth muscle cells.However,several issues concerning the contribution of EPCs to the pathogenesis of TA are controversial.So,it is highly important to clarify the role of EPCs in transplant arteriosclerosis and understand the detailed mechanisms of EPCs homing and differentiation into mature endothelial cells,and highlights the controversial issues in the field.Based on the understanding to the novel mechanisms of the pathogenesis of TA,we may be able to design a new drug or a new therapeutic strategy to direct EPCs for the prevention of transplant arteriosclerosis.In the present study,we first establish a model of TA in the mouse and investigate whether number of EPCs from peripheral blood correlate with TA severity; then,we intravenous transfusion of ex vivo-expanded bone marrow-derived EPCs to evaluate the role of EPCs in the intimal proliferation in a mouse model of transplant arteriosclerosis;Thereafter,we investigate the affects of vascular endothelial growth factor receptor tyrosine kinase inhibitors vandetanib on the number and functions of EPCs;finally,we investigate the effect of VEGF receptor tyrosine kinase inhibitors Vandetanib on the intimal proliferation in mouse aortic allografts and prospectively construct a new therapeutic strategy for the prevention of TA.PART 1.Mouse Model of Transplant Arteriosclerosis and Changes in the Number of Endothelial Progenitor Cells from peripheral bloodObjective:To establish a model of transplant arteriosclerosis in the mouse and investigate changes in the number of EPCs from peripheral blood.Methods:A segment of abdominal aorta was transplanted orthotopicaUy from C57BL/6 to BALB/c mice.The grafts were harvested at various times after the operation and studied by light and electronic microscopy.Regional changes in the lumen and intimal were measured with computer imaging analysis system.EPCs from peripheral blood were defined by the expression of Sea-1,Flk-1,and quantified by flow cytometry on days 0,1,3,7,14,28 days postoperation.Results:In this study,endothelium injury and inflammatory cells infiltration were seem in the aortic allograft at 3 days after transplantation.Neointimal lesions were observed as early as 2 weeks after surgery and had progressed at 4 and 6 weeks postoperatively.The lumen of allografts were significantly narrowed due to neointima hyperplasia.The number of circulating EPCs increased from 1 days after operation and got to summit at 3 days after the operation.Thereafter EPCs decreased rapidly and were significantly lower at 4 weeks and 6 weeks postoperation than those of pre-operation.Conclusion:Abdominal aortic transplantation from C57BL/6 to BALB/c mice present typical pathological feature of transplant arteriosclerosis.This model may become a simple and powerful tool for studying the pathogenesis and therapeutic intervention for this disease.EPCs count may be considered a novel biological marker of transplant arteriosclerosis.PART 2:Administration of ex vivo-expanded bone marrow-derived EPCs contribute to the development o neointimal thickenings in mouse aortic allograftsObjective:To investigate the effect of bone marrow-derived EPCs on the intimal proliferation in a model of transplant arteriosclerosis in the mouse.Methods:EPCs were isolated from bone marrow-derived mouse mononuclear cells (MNCs) by using a Ficoll density gradient centrifugation and cultured in endothelial basal medium.To establish a model of transplant arteriosclerosis,a segment of abdominal aorta was transplanted orthotopically from C57BL/6 to BALB/c mice. 1×10~7 EPCs or same dosage vehicle(0.9%NS) from the operation day to 2 days post operation were transplanted into the recipients by intravenous transfusion.Meanwhile EPCs were labeled by CM-DiI for cells tracking in vivo.The grafts were harvested at various times after the operation and fluorescence-labeled EPCs,endothelial regeneration rate and regional changes in the lumen and intimal were detected.Results:The adherent cells were considered EPCs that showed spindle shape and had many endothelial characteristics.Two weeks after transplantation,CM-DiI labeled EPCs were detected within the neointima and on the luminal surface of injured aortic allografts.Transplantation of EPCs compared with saline administration markedly accelerated endothelium injury of allografts.Four weeks postoperation,the neointimal thickness in transplantation group was significantly higher than that of control group.Conclusion:EPCs may participate in the formation of the vascular lesion in aortic allografts.Bone marrow-derived EPCs promote endothelial damage and contribute to the neointima formation in a murine model of transplant arteriosclerosis.PART 3:Effect and mechanism of vascular endothelial growth factor receptor tyrosine kinase inhibitors vandetanib on the number and functions of EPCsObjective:To investigate expression of Vascular endothelial growth factor receptor 2(VEGFR-2) on EPCs and effects of VEGF receptor tyrosine kinase inhibitors Vandetanib on the number and proliferation,adhesion,migration functions of EPCs and possible mechanisms associated with Akt and Erk signaling pathway.Methods:To study the effect of vandetanib on MNCs differentiation into EPCs, mononuclear cells(MNCs) were isolated from mouse bone marrow by using a Ficoll density gradient centrifugation and cultured on fibromectin-coated culture dishes with or without vandetanib for 7 days.EPCs were identified as adherent cells double positive stained for FITC-UEA-I and DiI-acLDL under fluorescence microscopy. After 7 days cultured,attached cells were treated with vandetanib(0.25～4uM) or vehicle for various time points.EPCs proliferation,migration were assayed with MTT assay and modified Boyden chamber assay respectively.EPCs adhesion assay was performed by replating them on fibronectin-coated dishes,and then adherent cells were counted.Western blotting was used to analysis the effect of vandetanib on VEGFR-2 activation and Akt and Erk phosphorylation of EPCs.Results:EPCs number differentiated from MNCs at 7 days was significantly lower in vandetanib treated cells in a dose-dependent manner than that of vehicle-treated cells.Vandetanib also significantly inhibited the proliferative,migratory and adhesive capacity of EPCs in a time and dose dependent manner.Western blotting analysis showed that VEGFR-2 were activated in EPCs after sitmulated with VEGF. Vandetanib potently inhibited phosphorylation of VEGFR-2 in a dose-dependent manner.VEGFR-2 blockade corresponded with decreased phosphorylation of downstream signaling intermediates Akt and Erk.Conclusion:Present results suggested that vandetanib could inhibit EPCs differentiation from MNCs and reduce the proliferation,migration and adhesion capacities of EPCs,possibly because it suppress Akt and Erk pathways activation by blocking the activation of VEGFR-2.PART 4:Potent inhibitory effect of Vandetanib on endothelial progenitor cells prevents Transplant Arteriosclerosis in mouse aortic allograftsObjective:To investigate the effect of VEGF receptor tyrosine kinase inhibitors Vandetanib on the number of endothelial progenitor cells(EPCs) from peripheral blood and the intimal proliferation in mouse aortic allografts and the possible mechanism on preventing transplant arteriosclerosis.Methods:To establish a model of transplant arteriosclerosis,a segment of abdominal aorta was transplanted orthotopicaUy from C57BL/6 to BALB/c mice.The recipients were divided into three groups:the allografts control group,low-dose vandetanib treated group(25mg/kg.d) and high-dose vandetanib treated group(100mg/kg.d). EPCs from peripheral blood were defined by the expression of Sea-1,Flk-1,and quantified by flow cytometry on days 0,1,3,7,14,28 days postoperation.At 2 weeks after surgery,scanning electron microscope and Evans blue staining were used to measure the reendothelialized area of aortic allografts.Animals were sacrificed and the aortic grafts were harvested at 4 weeks post operation.The aortic grafts were then processed for staining and measurements by means of pathology.Regional changes in the lumen,intimal and medial layers were measured with computer imaging analysis system.Results:The number of circulating EPCs were significantly reduced in group treated with vandetanib compared with those of control group early after the operation.At 2 weeks and 4 weeks postoperation,the level of circulating EPCs in vandetanib treated group still lower than those of pre-operation,especially in high-dose vandetanib treated group.At 2 weeks after surgery,the rate of reendothelialization in vandetanib treated group was much higher than that of control group.At 4 weeks post operation, intimal thickening was significantly reduced and inflammatory cells were suppressed in aortic allografts in high-dose vandetanib treated group,but not in low-dose vandetanib treated group.Conclusion:High-dose vandetanib can inhibit intimal proliferation in aortic allografts and prevent the transplant arteriosclerosis.The mechanisms are related to inhibit the differentiation of EPCs from the bone marrow,reduce the number and suppress function of circulating EPCs.