The Therapeutic Effect Of AMD3100 And SDF-1 To Promote Reendothelialization In Carotid Artery Injury Model Of Diabetic Rat And The Underlying Mechanism

PART ? THE THERAPEUTIC EFFECT OF AMD3100 AND SDF-1 TO PROMOTE REENDOTHELIALIZATION IN RAT CAROTID ARTERY INJURY MODEL AND THE UNDERLYING MECHANISMBackground:Vascular restenosis after interventional therapy is still a serious problem.Endothelial progenitor cell play a key role in reendothelialization which can attenuate neointimal hyperplasia effectively.Thus,In this study,we considered the role of AMD3100 and SDF-1 in biological function of endothelial progenitors and the underlying mechanism.We further evaluated the efficacy of AMD 3100and SDF-1 to promote reendothelialization and inhibit neointimal hyperplasia.Methods:The proliferation and adhesion capacity of endothelial progenitor cells(EPCs)was assessed after treated with AMD 3100 and SDF-1.Confocal immunofluorescence,western blotting and flow cytometry were conducted to reveal the expression and distribution of CXCR4 and CXCR7 proteins.The numbers of EPCs in peripheral blood at 1 and 3 days after treatment were evaluated with flow cytometry.To assess the role of AMD 3100 and SDF-1 in intimal repair in vivo,immunofluorescence was used to evaluate the numbers of EPCs homing to the site of artery injury 1 day after treatment with AMD 3100 and SDF-1.Reendothelialization and neointimal hyperplasia were assessed via Evans blue and HE staining 7 and 14 days after treatment.Results:Treatment with SDF-1 alone or AMD 3100 along with SDF-1enhanced EPCs biological function in vitro.The expression of CXCR4and CXCR7 receptors at cell surface was up-regulated after treated with AMD 3100 and SDF-1,indicating both CXCR4 and CXCR7 were the contributor involved in enhancement of EPCs biological function.Though administration of AMD 3100 rapidly increased CD34~+KDR~+EPC numbers in peripheral blood(101.5±7.8 vs 81±7.1,p=0.036),it impaired endothelial reparation(7 days:54±5%vs 30±7%,p=0.014;14 days:89±6%VS 81±1%,p=0.039).In contrast,AMD 3100 in combination with local injection of SDF-1 promoted reendothelialization(7 days:54±5%vs 88±6%,p<0.001;14 days:89±6%vs 97±3%,p=0.031)and attenuated neointimal hyperplasia(7 days:56±4%vs 67±7%,p=0.036;14 days:59±6%VS 75±5%,p=0.001)by recruiting more CD34~+KDR~+EPCs to the injured site(15.3±5.97 vs 9.0±3.92,p=0.031).Conclusions:AMD 3100 in combination with local injection of SDF-1accelerated reendothelialization and inhibited neointimal hyperplasia after endothelial injury.Up-regulation of CXCR4 and CXCR7 receptor at cell surface is involved in enhancement of EPCs biological function after treated with AMD 3100 and SDF-1.PART ? THE THERAPEUTIC EFFECT OF AMD3100 AND SDF-1 TO PROMOTE REENDOTHELIALIZATION IN CAROTID ARTERY INJURY MODEL OF DIABETIC RATBackground : Reendothelialization is attenuated in diabetic patients due to dysfunction or decreasing of EPCs in circulation.Thus we evaluate the biological function of endothelial progenitor cells isolated from diabetic or normal rat.In combined with the results of part ?,we further considered the role of AMD3100 and SDF-1 in biological function of endothelial progenitors.We also evaluated the efficacy of different treatment regimens,SDF-1 alone or AMD3100 plus SDF-1,to promote reendothelialization and inhibit neointimal hyperplasia.Material and methods: The proliferation and migration capacity of EPCs was assessed by CCK-8 and transwell assay.Adhesion assay was conducted to evaluated the EPCs numbers adhering to fibronectin or endothelial cells.The proliferation and adhesion capacity of endothelial progenitor cells(EPCs)was also assessed after treated with AMD 3100 and SDF-1.To assess the role of AMD 3100 and SDF-1 in intimal repair in vivo,flow cytometry was conducted to reveal the numbers of EPCs in peripheral blood at 1 and 3 days after treatment.Immunofluorescence was used to evaluate the numbers of EPCs homing to the site of artery injury 1 day after treatment with AMD 3100 and SDF-1.Reendothelialization and neointimal hyperplasia were assessed via Evans blue and HE staining 7 and 14 days after treatment respectively.Results:the biological function of endothelial progenitor cells,including proliferation,migration and adhesion capacity was attenuated in diabetic rats compared with control group.Treatment with SDF-1 or AMD 3100 plus SDF-1 enhanced biological function of EPCs in vitro.AMD 3100 plus SDF-1 rapidly increased CD34+KDR+ EPC numbers in peripheral blood(76.1±14.6 VS 37.4±12.5,P<0.001)and recruit more EPCs into injury site(8.7±2.4 VS 3.1±1.2,p=0.006),which contribute to reendothelialization(7 days :60.3±14.9 % VS 33.5%±3.4%,P<0.001,14 days : 92.0 ±15.2% VS 88.6±13.1%,P=0.517)and attenuated neointimal hyperplasia(7 days: 0.46 ± 0.02 VS 0.67±0.03,P=0.016,14days: 0.51±0.03 VS 0.78±0.04,P<0.001).Conclusion: the biological function,including proliferation,migration and adhesion capacity,was attenuated in ?type diabetic rats.AMD3100 plus SDF-1 can effectively enhance EPCs biological function.Furthermore,AMD 3100 in combination with local injection of SDF-1accelerated reendothelialization and inhibited neointimal hyperplasia after endothelial injury,indicated this treatment regimens may be an useful practice for treatment of vascular restenosis after interventional therapy.