| Background:Endothelial injury and recovery play an important role in the development of restenosis. Deendothelialization has been recognized a trigger point for the formation of restenosis, and reendothelialization is presumed to be the next target for restenosis prevention. Yet the mechanisms about atrovastatin's effects on reendothelialization and restenosis have not been elucidated clearly. Based on the importance of EPCs in reendothelialization, we suppose that statins, the classic restenosis preventing drugs, accelerate reendothelialization by promoting EPCs' mobilization and homing, and accordingly prevent restenosis.Objective:To investigate the correlation of atrovastatin's effects on restenosis to reendothelialization, and to investigate whether atrovastatin can accelerate reendothelialization by promoting EPCs' mobilization and homing.Methods: 40 SD rats were randomly divided into equal four groups: shamoperation group, immediate sarefice (after balloon injury) group, modelgroup and atrovastatin group. Except sham operation group, each rat'sleft common carotid artery was denuded by balloon. 7days after ballooninjure, each rat was drawn 5 ml blood, from which PBMCs wereisolated and cultured for a week in M199 medium. The EPCs werecharacterized as adherent cells double positive for Dil-acLDL-uptakingand lectin-binding by laser scanning confocal microscope with directfluorescent staining. EPCs proliferation and migration were measuredby MTT assay and modified Boyden chamber assay, respectively. EPCsadhesion assay was performed by counting the adherent cells numberwhen EPCs were replated on gelatin-coated dishes. 4 weeks afterballoon injury, the intima/media ratio and reendothelialization ratio ofeach subject were measured. And except the two transplantation groups,VEGF immunohistochemistry and VEGF mRNA expression in injuredartery were detected.Results:1. Compared with controls, atrovastatin treatment alleviated theintima/media ratio (1.70±0.05 to 0.90±0.04, P＜0.001), increased thereendothelialization ratio (15.05±2.61% to 65.88±2.86%, P＜0.001).2. Compared with controls, atrovastatin raised EPCs' number(32.70±3.53 to 43.40±4.93, P＜0.001), enhanced EPCs' proliferation (0.13±0.02 to 0.17±0.03, P＜0.01), adhesion (34.80±2.86 to75.40±2.22, P＜0.001), migration (9.40±2.07 to 33.80±2.53, P＜0.001)functions, and promoted VEGF protein (IHC staining score: 9.50±1.51to 15.70±2.71, P＜0.001) and mRNA expression (0.86±0.06 to 1.30±0.04, P＜0.001) in balloon injured carotid artery.3. In atrovastatin group, intima/media ratio is negatively linearlycorrelated to reendothelialization ratio (Pearson coefficient-0.85,P＜0.01), and the latter was not only linearly correlated to EPCs numbers(Pearson coefficient 0.74, P＜0.05), but also linearly correlated toVEGF mRNA level (Pearson coefficient 0.66, P＜0.05).Conclusions:These data suggest that atrovastatin's effects on restenosis is highlylinearly correlated to accelerating endothelial recovery, the possiblemechanisms for atrovastatin accelerating endothelialization include:increasing EPCs level, promoting EPCs' proliferation, adhesion,migration functions, and enhancing local VEGF expression so as tofacilitate EPCs homing to balloon-injured carotid artery. Background:Deenothelialization has been recognized a trigger point for theformation of restenosis, and reendothelialization is presumed to be thenext target for restenosis prevention. Although EPCs mobilization hasbeen witnessed during the treatment of endothelial injury withatrovastatin, It's not clear whether the EPCs mobilization is aprecipitating cause for endothelial recovery indeed, or it's merely asecondary effect without significant consequence. To answer thisquestion, it may be helpful to observe the effects of granulocytecolony-stimulating factor (G-CSF), a single leucocyte mobilizing drugwithout anti-inflammation, lipid-lowering and so on effects that statinspossess, in accelerating reendothelialization and inhibiting restenosis.Moreover, it's interesting whether combination of G-CSF andatrovastatin is more effective in mobilizing EPCs, acceleratingreendothelialization and preventing restenosis than either of the twodrugs. Objective:To investigate the difference and combination effects of G-CSF andatrovastatin in mobilizing EPCs, Accelerating Reendothelialization andpreventing restenosis.Methods:50 SD rats were randomly divided equally to sham operation group,model group, atrovastatin group, G-CSF group and drugs combination(of atrovastatin and G-CSF) group. In the latter 4 groups each rat's leftcommon carotid artery was deendothelialized by balloon. 7days afterballoon injure, each rat was drawn 5 ml blood, from which PBMCs wereisolated and cultured for a week in M199 medium, then the EPCs werecharacterized as adherent cells double positive for Dil-acLDL-uptakingand lectin-binding by laser scanning confocal microscope with directfluorescent staining. EPCs proliferation and migration were measured byMTT assay and modified Boyden chamber assay, respectively. EPCsadhesion assay was performed by counting the adherent cells numberwhen EPCs were replated on gelatin-coated dishes. 4 weeks afterballoon injury, the intima/media ratio and reendothelialization ratio ofeach subject were measured, besides, VEGF antigen and VEGF mRNAexpression in injured artery were detected.Results:1. Compared with controls, G-CSF raised EPCs' number (32.70±3.53 to 96.70±3.40, P＜0.001), promoted EPCs' proliferation(0.13±0.02 to 0.22±0.03, P＜0.001), adhesion (34.8±2.86 to39.00±2.21, P＜0.01), migration (9.40±2.07 to 13.30±2.06, P＜0.01)effects, accelerated reendothelialization (15.05±2.61% to 80.47±2.55%,P＜0.001) and alleviated intima/media ratio (1.70±0.05 to 0.89±0.04,P＜0.001). In G-CSF group, intima/media ratio was inversely linearlycorrelated to reendothelialization ratio (Pearson coefficient-0.86,P＜0.01), and the reendothelialization ratio is positively linearlycorrelated to EPCs number level (Pearson coefficient 0.80, P＜0.01).2. Compared with atrovastatin of equal restenosis prevention effect,G-CSF was more effective in raising EPCs' number (43.40±4.93 to96.70±3.40, P＜0.001) and proliferation functions (0.17±0.03 to0.22±0.03, P＜0.01), but less effective in promoting EPCs' adhesion(75.40±2.22 to 39.00±2.21, P＜0.001) and migration functions(33.80±2.53 to 13.30±2.06, P＜0.001).3. Compared with controls, G-CSF has on influence in VEGFprotein (9.50±1.51 to 8.80±1.55, P＞0.05) and mRNA expression ininjured artery (0.86±0.06 to 0.86±0.05, P＞0.05).4. Moreover, compared with 50μg/kg.d G-CSF with equal restenosisprevention effects (0.89±0.04 to 0.90±0.04, P＞0.05), 10mg/kg/datrovastatin is less effective in accelerating reendothelialization (80.47±2.55%to 65.88±2.86%, P＜0.001). 5. Compared with G-CSF and atrovastatin, drugs combinationgroup was more effective in raising EPCs' number (data of G-CSFgroup, atrovastatin group to drugs combination group are 96.70±3.40,43.40±4.93 to 148.40±4.65 respectively, both P＜0.001), promotingEPCs' proliferation (0.22±0.03, 0.17±0.03 to 0.25±0.02, both P＜0.01),adhesion (39.00±2.21, 75.40±2.22 to 82.3±3.59, both P＜0.001),migration (12.20±2.15, 33.80±2.53 to 37.00±3.74, both P＜0.05)functions, accelerating Reendothelialization (80.47±2.55%,65.88±2.86% to 95.29±1.80%, both P＜0.001) and preventing restenosis(0.89±0.04, 0.90±0.04 to 0.32±0.03, both P＜0.001). In drugscombination group, intima/media ratio was inversely linearly correlatedto reendothelialization ratio (Pearson coefficient-0.85, P＜0.01), and thelatter is positively linearly correlated to EPCs number (Pearsoncoefficient 0.72, P＜0.05).Conclusions:These data suggest that G-CSF's effects on restenosis are highlylinearly correlated to reendothelialization, and the possible associatedmechanisms include mobilizing EPCs, promoting EPCs' proliferation,adhesion and migration functions, but do not include enhancing localVEGF mRNA expression to facilitate EPCs' homing to balloon-injuredcarotid artery as does atrovastatin. In addition to accelerating endothelialrecovery, atrovastatin inhibits restenosis by other mechanisms. Drugs combination of G-CSF and atrovastatin is more effective in mobilizingEPCs, Accelerating Reendothelialization and preventing restenosis thaneither of the two drugs. Background:Endothelial injury and recovery play an important role in thedevelopment of restenosis. EPCs interfere in reendothelialization,therefore mobilization and autologous transplantation of EPCs may beable to inhibit restenosis. Effects of EPCs mobilization has beenconformed in researches in G-CSF's restenosis prevention effects. Incontrast, researches in autologous EPCs transplantation are seldom.Based on the cognition that statins can promote EPCs' proliferation,adhesion and migration functions,atrovastatin-pretreated EPCs may bemore effective in restenosis prevention than simple EPCs transplantationdoes, yet it has not been reported up to now.Objective:To investigate whether autologous EPCs transplantation canaccelerate reendothelialization and inhibit restenosis, and whetherautologous atrovastatin-pretreated EPCs transplantation is moreeffective in accelerating restenosis and preventing restenosis than simpleEPCs transplantation is. Methods:50 SD rats were randomly divided into equal 5 groups: shamoperation group, model group, autologous EPCs transplantation group,sacrifice 1 week after transplantation group, and autologousatrovastatin-pretreated EPCs transplantation group. Except shamoperation group, each rat's left common carotid artery wasdeendothelialized by balloon. And 3 weeks before balloon injure, all ratsready to undergoing transplantation were drawn 5 ml blood, from whichPBMCs were isolated and cultured in M199 medium. From day 7th, thespindle shape cells were passaged every 4 days. Part EPCs wereidentified, characterized as adherent cells double positive forDil-acLDL-uptaking and lectin-bindlng by laser scanning confocalmicroscope with direct fluorescent staining. In sacrifice 1 week aftertransplantation group, the EPCs for transplantation were labeled with 10μM Brdu one day before transplantation, for detecting the offspring cellsof previous transplanted Brdu-labeled EPCs by Brduimmunohistochemistry after 1 week. Immediately after balloonimpairment, 1×106 EPCs were autologously transplanted by caudal veininjection in three transplantation groups. 4 weeks after balloon injury,except the sacrifice 1 week after transplantation group, the intima/mediaratio and reendothelialization ratio of each group were measured.Results:1. Brdu-labeled cells can be detected in the carotid artery intima 1 week after Brdu-labeled EPCs transplantation.2. Compared with model group, autologous EPCs transplantationincreased the reendothelialization ratio (15.05±2.61% to 84.47±2.56%,P＜0.001) and alleviated the intima/media ratio (1.70±0.05 to 0.84±0.03,P＜0.001).3. Compared with EPCs transplantation, atrovastatin pretreatedEPCs transplantation was more effective in acceleratingreendothelialization (84.47±2.56% to 97.15±2.50%, P＜0.001) andpreventing restenosis (intima/media ratio 0.84±0.03 to 0.29±0.03,P＜0.001).4.Intima/media ratio was inversely linearly correlated toreendothelialization ratio in EPCs transplantation group (Pearsoncoefficient-0.89, P＜0.01) and atrovastatin pretreated EPCstransplantation group (Pearson coefficient-0.91, P＜0.001).Conclusions:Autologous EPCs transplantation can accelerate endothelialrecovery and inhibit restenosis, and atrovastatin-pretreated EPCs aremore effective in accelerating endothelial recovery and inhibitingrestenosis than EPCs are. Background:Endothelial impairment is not only one of the earliest pathologicalchanges of coronary heart disease (CHD), but also the trigger point ofrestenosis after PTCA. The circulating endothelial progenitor cells(EPCs) may participate in endothelial repair and endothelial functionsimprovement, and accordingly play an important role in the pathologicalprocess of CHD and restenosis. Recent data indicate that during thecourse of treatment with statins in patients with CHD (including thoseundergoing PTCA), improvements in EPCs' number and functionswere significant. Yet if we want to conclude whether theseimprovements in EPCs are really the precipitating cause of statins'clinical benefit in CHD and restenosis, of are merely a secondary effectof consequence, and to elucidate the possible related mechanisms onsignaling pathway, experiments in vitro are needed. At present, reportson the effects of statins on EPCs' number and functions and the relatedmechanisms on signaling pathways are rare. Objective:To study whether atrovastatin augments the number andproliferation, adhesion, migration functions of PBMCs-derived EPCsisolated from patients with coronary heart disease, and the possiblemechanisms on PI3/Akt and ERK signaling pathway.Methods:21 patients with coronary heart disease (CI-ID) and 21 matchedcontrol subjects were enrolled. In both groups 5 subjects were randomlychosen out, whose PBMCs-derived EPCs were used only for detectingVEGFR2,CD34 and AC133 expression by FCS. In the remained 16CHD patients, total PBMCs of each subject were divided into 6 equalshare, randomly enrolled in the following 6 groups: control group, 0.1μM AT (atrovastatin) group, 1μM AT group, 10μM AT group, 10μMAT plus 10μM LY (LY294002) group, and 10μM AT plus 10μM PD(PD98059) group. Then the PBMCs of each subject were plated ongelatin-coated culture bottles. After cultured for 7 days in M199, theattached cells were stimulated with different drugs according tocorresponding groups. Then EPCs were characterized as adherent cellsdouble positive for Dil-acLDL-uptaking and lectin-binding. EPCsproliferation and migration were measured by MTT assay and modifiedBoyden chamber assay, respectively. EPCs adhesion assay wasperformed by counting the adherent cells number when EPCs were replated on gelatin-coated dishes.Results:1. The cultured EPCs is not only double positive forDil-acLDL-uptaking and lectin-binding, but also positive for VEGFR2,CD34 and AC133.2. Compared with normal subjects, the number (61.50±2.68 to29.63±1.96, P＜0.001) and proliferation (0.23±0.02 to 0.14±0.02, P＜0.001), adhesion (32.25±1.84 to 15.75±1.65, P＜0.001), migration(14.69±2.18 to 8.19±1.47, P＜0.001) functions of EPCs derived frompatients with CHD were significantly impaired.3. Incubated with 0.1μM atrovastatin, EPCs' number (29.63±1.96to 39.00±2.37, P＜0.001) and proliferating (0.14±0.02 to 0.16±0.02, P＜0.05), adhering (15.75±1.65 to 18.25±1.44, P＜0.001), migrating(8.19±1.47 to 9.75±1.13, P＜0.01) functions significantly improved, and when the drug concentration rised to 1 and 10μM, the effects onEPCs' number (corresponding data are 46.19±1.60 and 56.13±3.40respectively) and proliferating (0.18±0.02 and 0.20±0.02), adhering(23.75±2.05 and 31.50±2.00), migrating (11.75±1.61 and 15.13±2.09)functions were promoted step by step, correspondingly (all P＜0.05).4. Treated with LY294002, effects of atrovastatin on EPCs' number(56.13±3.40 to 30.81±3.41, P＜0.001) and proliferation (0.20±0.02 to0.16±0.02, P＜0.001), other than effects on adhesion and migration function (P＞0.05) were hampered. In contrast, PD98059 inhibited theeffects of atrovastatin on EPCs' adhesion (31.50±2.00 to 16.13±2.00, P＜0.001) and migration (15.13±2.09 to 10.06±1.84, P＜0.001), butdid not affect the number and proliferation of EPCs (P＞0.05).Conclusion:This study shows that the number and proliferation, adhesion, migration functions of PBMCs-derived EPCs of CHD patients wereseriously impaired. Statins can increase EPCs' number and proliferationvia PI3/Akt signaling pathway, and promote EPCs' adhering andmigrating functions via ERK signaling pathway.
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