The Effects Of Endothelial Progenitor Cells In Preventing Thrombosis Formation And Repairing Injured Arteries After Microsurgical Vascular Anastomosis

Objective: This study aims to study the process of thrombosis formation and vascular repair after microsurgical vascular anastomosis.Furthermore,the effects of endothelial progenitor cells(EPCs)in thrombosis formation and vascular repair were also evaluated.Methods: There models of microsurgical vascular anastomosis were established.Vascular patency was checked using milk test.The anastomotic arteries were harvested for histological detection and scanning electron microscope.Microsurgical anastomosis of crushed femoral artery was established in rats.Local transfer of SDF-1? gene was performed to evaluate the antithrombotic effect.Immunohistofluorescence was performed to identify endogenous EPCs recruited to the anastomotic carotid arteries.AMD3100 was used to block the axis of SDF-1/CXCR4.The effect of SDF-1/CXCR4 axis in EPCs recruitment and vascular repair was studied.Results: Loss of endothelial cells and thrombosis formation were recorded after vascular anastomosis.Thrombosis formation peaked in 2 hours after anastomosis.Intimal hyperplasia was evident at 2 weeks after surgery.Local transfer of SDF-1? inhibited thrombosis formation,enhanced vascular patency,and increased the expression of endothelial nitric oxide synthase and plasma level of NO.EPCs were identified in the intima of anastomotic arteries.Administration of AMD3100 inhibited EPCs recruitment,delayed the process of re-endothelialization and stimulated intimal hyperplasia.Conclusions: The procedure of microsurgical vascular anastomosis inevitably caused loss of endothelial cells,leading to subsequent thrombosis formation and intimal hyperplasia.SDF-1? increases vascular patency and inhibits microsurgical anastomotic thrombosis in a crush model of femoral artery in rat,which may be mediated through up-regulation of endothelial nitric oxide synthase and increased production of NO.Endogenous EPCs participated in the process of re-endothelialization via SDF-1/CXCR4 axis and inhibited intimal hyperplasia after end-to-end arterial anastomosis.Part One Establishment of microsurgical vascular anastomosis in ratsObjective: Animal models of microsurgical vascular anastomosis were established in rats and the histopathologic changes after microsurgical vascular anastomosis were detected.Methods: There models of microsurgical vascular anastomosis were established through anastomosis of carotid artery,femoral artery,and crushed femoral artery.Vascular patency after vascular anastomosis was checked using milk test.The anastomosised arteries were harvested for histological detection and scanning electron microscope.Results: Loss of endothelial cells and thrombosis formation were recorded after vascular anastomosis,which differed in different models of microsurgical vascular anastomosis in rats.At 2 hours after anastomosis,all vessels were patent and little thrombosis was observed after anastomosis of carotid artery.More thrombosis was observed with all vessels patent after anastomosis of femoral artery.Most thrombosis was observed after anastomosis of crushed femoral artery and only 20% vessels were patent.Intimal hyperplasia appeared at 1 week and was evident at 2 weeks after surgery.Conclusions: The procedure of microsurgical vascular anastomosis inevitably caused loss of endothelial cells,leading to subsequent thrombosis formation and intimal hyperplasia.Part Two Mobilization of endothelial progenitor cells using stromal cell-derived factor-1? for preventing microsurgical anastomotic thrombosisObjective: Intimal injury plays a critical role in initiating the pathogenesis of thrombosis formation after microsurgical anastomosis.Application of stromal cell-derived factor-1?(SDF-1?)is reported to promote early regeneration of injured intima through migration of endothelial cells and mobilization of endothelial progenitor cells.We therefore hypothesized that local transfer of SDF-1? gene would inhibit microsurgical anastomotic thrombosis.Methods: Sixty Sprague-Dawley rats were used and divided randomly into three groups(SDF-1? group,plasmid group,and saline group)in this study.Plasmid DNA encoding SDF-1?,empty plasmid and saline were injected into the left femoral muscles of rats from each group respectively.Seven days after injection,peripheral blood samples were obtained to measure the plasma levels of SDF-1? and nitric oxide(NO).The left femoral artery of each rat was crushed,transected,and repaired by end-to-end microsurgical anastomosis.Vascular patency was assessed at 15,30 and 120 minutes after reperfusion using milk test.Thrombosis formation was assessed with H&E staining and scanning electron microscopy at 120 minutes postoperatively.RT-PCR was performed to measure the expression changes of endothelial nitric oxide synthase,tissue type plasminogen activator,thrombomodulin,and von Willebrand factor.Results: The plasma levels of SDF-1? and NO in SDF-1? group were significantly higher than that in plasmid group and saline group(p < 0.01).The patency rate in SDF-1? group was significantly higher than that in control groups at 120 minutes after reperfusion(p < 0.05).Treatment of SDF-1? significantly reduced the size of thrombotic occlusion when compared with controls(p < 0.01).All SDF-1? recipients exhibited decreased thrombosis under scanning electron microscopy.RT-PCR results showed that the expression of endothelial nitric oxide synthase was up-regulated in SDF-1? group when compared with control groups(P < 0.05).Conclusions: This study demonstrated that SDF-1? increases vascular patency and inhibits microsurgical anastomotic thrombosis in a crush model of femoral artery in rat.The antithrombotic effect of SDF-1? may be mediated through up-regulation of endothelial nitric oxide synthase and increased production of endogenous NO.These findings offer a new way in preventing thrombosis formation after microsurgical vascular anastomosis.Part Three The effect and mechanism of endogenous endothelial progenitor cells in vascular repair after vascular anastomosisObjective: Early regeneration of injured intima is critical for vascular remodeling.Endothelial progenitor cells(EPCs)mobilized or transfused facilitate the process of re-endothelialization after vascular intima injury.Here,we investigated the role of endogenous EPCs in repair of whole layer arterial injury induced by anastomosis.Methods: Adult male Sprague-Dawley rats were randomly assigned to the AMD3100 group and control group.The left common carotid artery of each rat was transected and repaired with end-to-end anastomosis.Each rat received intraperitoneal injection of AMD3100 or saline consecutively for 7 days after arterial anastomosis.Progress of re-endothelialization was assessed using Evans blue staining and scanning electron microscope.Intimal hyperplasia was detected by H&E staining.Immunohistofluorescence was performed to identify endogenous EPCs recruited to the anastomotic arteries.Expression of stromal cell-derived factor-1?,vascular endothelial growth factor,and endothelial nitric oxide synthase were measured using ELISA and quantitative reverse-transcriptase polymerase chain reaction.Results: The percentages of endothelium-denuded area in control group increased,peaked at days 3,and reduced to preoperative level at days 14 after anastomosis.CD34+/Flk-1+ and CD34+/VE-cadherin+ EPCs were identified in the intima of anastomotic arteries.Less CD34+/Flk-1+ and CD34+/VE-cadherin+ EPCs were recruited to the anastomotic arteries in AMD3100 group.Administration of AMD3100 also delayed the process of re-endothelialization and stimulated intimal hyperplasia.Moreover,AMD3100 treatment significantly inhibited m RNA levels of vascular endothelial growth factor,and endothelial nitric oxide synthase.Conclusions: Endogenous EPCs participated in the process of re-endothelialization via SDF-1/CXCR4 axis and inhibited intimal hyperplasia after end-to-end arterial anastomosis.These findings provide a novel target to enhance the results of vascular anastomosis.