| Background and objects:Endothelial progenitor cells(EPCs)can differentiate into endothetial cells and contribute to neovascularization after tissue ischemia.Many studies have demonstrate that statin increases the circulating level of EPCs,but the mechanisms remain poorly understood.PI3-K/AKT signal pathway is well established to play an important role in cell apoptosis,survival and proliferation.Increasing data reveal that statin induces the activation of PI3-K/AKT signal pathway. However,it remains unclear whether this mechanism mediated the effects of statin on EPCs.In this study,we estabslished a rat myocardial infarction model to investigate the effects of statin on the mobilization of EPCs.To gain insights into the mechanisms underlying,we cultured rat bone marrow-derived EPCs in vitro,and investigated the effects of statin on the expressions of AKT and eNOS,as well as the release of VEGF.We also determined whether these effects of statin could be abolished by the PI3-K/AKT blocker LY294002.Part one:Effects of simvastatin on the mobilization of EPCs in rat myocardial infarctionMethods and results:Coronary artery ligation was performed to establish the rat myocardial infarction model.The rats were divided into two groups(n=10 per group)after operation:treated group was administered 20 mg/kg simvastatin once daily,control group was gavaged with normal saline.The blood samples were taken from the tail vein before and at days 1,3,5,7,11,15,20 post-operatively.Flow cytometric analysis was performed to quantify the circulating EPCs. EPCs were defined by expression of two stem cell-related surface antigens,CD34 and CD133.There is no difference in the level of EPCs between two group before operation(CD34+:1.16±0.34‰vs 1.14±0.40‰,P＞0.05;CD133+:0.41±0.14‰vs 0.37±0.08‰,P＞0.05). In control group,circulating EPCs increased on day 1 after the onset of myocardial infarction(CD34+:5.12±3.17‰vs 1.16±0.34‰,P＜0.05; CD133+:1.32±0.27‰vs 0.41±0.14‰,P＜0.05)and peaked on day 7 (CD34+:3.51±2.70‰,CD133+:1.4±0.99‰),then gradually decreased, but the number on day 20 was still greater than that on day 1(CD34+: 1.75±0.55‰vs 1.16±0.34‰,P＜0.05;CD133+:1.23±0.95‰vs 0.41±0.14‰,P＜0.05),suggesting that myocardial infarction could induce the mobilization of EPCs.Compared with control group,the simvastatin group maintained a significantly higher number of circulating EPCs from day 3(CD34+:4.43±1.28‰vs 2.52±1.81‰,P＜0.05; CD133+:1.49±0.65‰vs 0.84±0.54‰,P＜0.05)to day 20 after myocardial infarction,suggesting that simvastatin could promote the mobilization of EPCs.Part two:Mechanisms underlying the effects of simvastatin on the mobilization of endothelial progenitor cells after rat myocardial infarctionMethods and results:In this part,we investigate the possible mechanisms underlying the effects of simvascatin on the mobilization of EPCs in both of rat myocardial infarction model and cultured bone marrow-derived EPCs.Coronary artery ligation was performed to establish the rat myocardial infarction model.The rats were divided into two groups(n=10/group)after operation:treated group was administered 20 mg/kg simvastatin once daily,control group were gavaged with normal saline.The blood samples were taken from the tail vein before and at days 1,3,5,7,11,15,20 post-operatively.Plasama concentrations of VEGF and AKT were determined by ELISA.To cultured EPCs,bone marrow mononuclear cells were collected from the femurs of SD rats by density gradient centrifugation.The cell pellet was inoculated in endothelial basal medium.Attached cells were collected and randomized into two groups:(1)simvastatin groups,simvastain was added to make a series of final concentrations:0.001,0.01,0.1 and 1μmol/L for the respective time points.(2)simvastatin+LY94002 groups,LY94002 was added to the medium and incubated for 1 h,then simvastatin at a series of concentration(0.001,0.01,0.1 and 1μmol/L)was added.The cells were collected after 24 h.Concentrations of VEGF in the medium and AKT, eNOS in cell lysate were measured by ELISA.The expression of eNOS in cultured EPCs was also detected by Western blot analysis.Result:Acute myocardial infarction led to a significant time-dependent increase of plasma VEGF.The plasma level of VEGF peaked on the day 7(81.0±7.35 pg/mL vs 57.6±4.23 pg/mL,P＜0.05),then gradually decreased,but the level on day 20 was still greater than that on day 1 (64.3±7.34 pg/mL vs 57.6±4.23 pg/mL,P＜0.05).From day 3 to day 20, VEGF levels in the simvastatin groups were significantly higher than that of control group(73.17±6.31 pg/mL vs 56.5±3.78 pg/mL,P＜0.05).In control group,plasma AKT level also increase after the onset of myocardial infarction in the control group before and after operation. From day 3 after operation,plasmal AKT level in the simvastatin groups were significantly higher than that of the control group(431.5±12.16 pg/mL vs 399.5±10.98 pg/mL,P＜0.05),indicating that simvastatin might promote the mobilization of EPCs via upregulation of plasma VEGF and AKT.In cultured EPCs,simvastatin led to dose-dependent increases in the level of VEGF in the medium,and in the expression of AKT and eNOS in EPCs,PI-3K/Akt inhibitor LY294002 could abolish these effects.Conclusion:1.Acute myocardial infarction induced the mobilization of EPCs. Simvastatin promoted the mobilization of EPCs after myocardial infarction,which might be associated with upregulation of plasma VEGF and AKT.2.In vitro,Simvastatin increased the release of VEGF and expression of eNOS in cultured EPCs via the PI3-K/AKT signaling pathway.This may explain the effect of Simvastatin on the mobilization of EPCs after myocardial infarction.
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