Simvastatin Improves The Homing Of BMSCs Via The PI3K/AKT/miR-9 Pathway

Background and objective:Coronary artery bypass grafting is the most effective treatment of serious coronary heart disease, autogenous vein is the most wildly used vessel graft, but about 20%-40% of the vein graft goes stenotic or occlusive two years after the operation, which seriously affects its curative effect, how to prevent or slow down the stenosis of vein graft needs to be solved urgently. Endothelial lesion and dysfunction initiates restenosis of vein graft which is caused by injury in operation, ischemia-reperfusion lesion, changes of blood flow and intraluminal pressure. The formation of neointima can be effectively alleviated by restoration of the injured endothelium, which is significant for the prevention and treatment of restenosis. Bone marrow mesenchymal stem cells (BMSCs) can differentiate into endothelial cells, promote the repair of injured vascular endothelium and alleviate the formation of neointima. Stromal cell derived factor 1 alpha (SDF-1)/chemokine receptors (CXCR4) axis plays a very important role in homing of BMSCs to the injuried tissue. Surface expression of CXCR4 on BMSCs decreases after several passages in vitro, which results in decreased homing ability of BMSCs, thereby affects the efficacy of cell therapy. Therefore, up-regulation of CXCR4 expression in BMSCs can improve the therapeutic effect of cell transplantation therapy. Simvastatin is widely used in atherosclerotic diseases as it can regulate the level of serum lipid. In addition to this effect, simvastatin can also activate PI3K/AKT signaling pathway in a variety of cells, such as endothelial progenitor cells, endothelial cells, foot cells, etc. Upregulation of AKT phosphorylation can promote the expression of CXCR4 in BMSCs. Therefore, simvastatin may activate the PI3K/AKT pathway in BMSCs and promote the CXCR4 expression of BMSCs. However, there has been no report which proves that Simvastatin can improve CXCR4 expression of BMSCs via PI3K/AKT pathway. Based on this, we will study the effect of simvastatin on CXCR4 expression of BMSCs and whether phosphorylation of AKT is involved in this process. MicroRNA (miRNA) is a kind of single stranded RNA molecules which does not encode any protein. But it is involved in the regulation of gene tranlation, and then regulates the biological characteristics of the cell. A variety of miRNAs have been proved to regulate the expression of CXCR4. Therefore, it will be further studied whether there is one or more miRNAs participate in the CXCR4 expression of BMSCs. The purpose of this research is to study the effects of simvastatin on CXCR4 expression of BMSCs and the homing ability of BMSCs to the injuried tissue and organs. The role of PI3K/AKT pathway and miRNA in this process will be discussed, too.This study can be divided into three parts.Part one:Simvastatin improves homing of BMSCs and their role in alleviating restenosis of vein graftsObjective:To study the effect of simvastatin on the expression of CXCR4 in BMSCs and its effect on the migration of BMSCs in vitro and in vivo.Methods:BMSCs were cultured under different concentrations of simvastatin.48h later, the expression of CXCR4 was detected by Western blot. The expression of CXCR4 on cell surface was detected by flow cytometry. Transwell assay was carried out to observe the migration of BMSCs to SDF-1 alpha under the effect of simvastatin and /or AMD 3100 (an inhibitor of the CXCR4 chemokine receptor antagonist). A vein grafting model of rat was established, and then CM-Dil labeled BMSCs was transplanted via the tail vein injection. The vein grafts were observed 7 days after cell transplantation and the number of CM-Dil labeled cells was counted. The histological examination of vein grafts was performed observed 28 days after cell transplantation or operation.Results:The expression of CXCR4 in BMSCs increased with the concentration of simvastatin. Simvastatin can increase surface expression CXCR4, including the positive rate, average fluorescence intensity, which was proved by flow cytometry. Under the effect of SDF-1α; the migration of BMSCs was significantly increased after 48h exposure to simvastatin. This increase could be blocked by AMD 3100. After 7 days of cell transplantation, a large number of CM-Dil labeled transplanted cells were observed in the intima of the vein grafts. Simvastatin can significantly increase the number of the migrated cells. This trend was weakened by AMD 3100.28 days later, compared with the BMSCs transplantation group, the formation of neointima in the simvastatin group was significantly alleviated.Conclusion:Under the effect of simvastatin, the CXCR4 expression of BMSCs was increased, especially the surface expression and the migration to SDF-1 a was enhanced. In the vein grafting model, simvastatin could promote the BMSCs migrating to the intima of vein grafts, thereby inhibit neointimal hyperplasia.Part Two:The PI3K/AKT pathway participates in the process of GXCR4 overexpression caused by simvastatinObjective:To study the role of PI3K/AKT pathway in the process of over-expression of CXCR4 caused by simvastatin.Methods:BMSCs were exposed to different concentrations of simvastatin.48h later, the expression levels of AKT and phosphorylated AKT were detected by Western blot and then compared with the changes of CXCR4 expression. After pretreatment with LY294002, a PI3K/AKT pathway inhibitor, BMSCs were then treated with simvastatin, and the expression levels of CXCR4, AKT and phosphorylated AKT were studied. Migration to SDF-1 a was also observed.Results:Western blot results showed that as the concentration of simvastatin increased, the phosphorylation of Akt gradually increased, and this rising trend was similar with that of CXCR4 expression. However, AKT expression showed no significant change. LY293004 pretreatment can inhibit the phosphorylation of AKT, and the expression of CXCR4; Upregulation of CXCR4 expression caused by simvastatin was also weakened by LY294002 pretreatment. LY294002 pretreatment could also significantly inhibit the effect of simvastatin on the migration of BMSCs to SDF-1a.Conclusion:After exposed to simvastatin, the PI3K/AKT signaling pathway is activated in BMSCs, and the phosphorylation of AKT promotes the expression of CXCR4. Inhibition of this pathway can reduce the effect of simvastatin on the expression of CXCR4, thus inhibit the migration of BMSCs.Part Three:miR-9 participates in the process of CXCR4 overexpression caused by simvastatin and its expression can be affected by PI3K/AKT pathwayObjective:To study the the expression of miRNAs after pretreatment of simvastatin, which may target CXCR4 according to the prediction of miRanda and to verify their function.Methods:According to the prediction of TargetScan 5.2, PicTar, and miRanda, miRNAs related to CXCR4 with a high degree were found out, and these miRNAs was studied. The expression changes of these miRNAs were detected by RT-qPCR after BMSCs were exposed to simvastatin. One or more miRNAs were studied according to the expression changes. After transient transfection of mimics and inhibitors of the studied miRNAs, CXCR4 expression and migration of BMSCs was observed. Then, the dual luciferase reporter gene system was used to investigate the mechanism through which the studied miRNAs regulated CXCR4 expression.The expression of the studied miRNA was observed after the BMSCs was exposed to LY294002 and different concentrations of simvastatin. And the expression trend was compared with the expression trend of the phosphorylated AKT, which may speculated the relationship between the miRNA expression and PI3K/AKT pathwayResults:According to targetscan 5.2, pictar, and Miranda’s prediction,40 kinds of miRNA expression were analyzed after stimulation BMSCs with simvastatin, in which expression of miR-21, miR-369-3p, miR-132 and miR-9 decreased. MiR-9 decreased the most significantly. Transient transfection of mimics of miR-9 to BMSCs inhibited CXCR4 expression, and its inhibitors promoted CXCR4 expression. Flow cytometry showed that inhibitor of miR-9 could promote the expression of CXCR4 on the surface of BMSCs. Transwell experiments showed that the migration of BMSCs to SDF-1 a was also enhanced. CXCR4 was validated as a target gene of miR-9 by luciferase assay. As the concentration of simvastatin increased, the expression of miR-9 decreased. This trend was opposite to that of AKT phosphorylation. The expression of miR-9 was significantly increased compared with the control group after the inhibition of PI3K/AKT pathway by LY294002.Conclusion:CXCR4 is the target gene of miR-9, and inhibition of miR-9 can promote the expression of CXCR4, and then promote the homing of BMSCs to SDF-1 a. Simvastatin promotes the expression of CXCR4 by inhibiting the expression of miR-9. The expression of miR-9 may be related to the PI3K/AKT pathway.According to the results and conclusions mentioned above, it is not difficult to draw that total and cell surface CXCR4 expression levels of BMSCs are increased under the influence of simvastatin. Homing of BMSCs to intima of vein grafts is improved, too. There is involvement of PI3K/AKT pathway and miR-9 in this progress. Simvastatin activates PI3K/AKT signaling pathway, and phosphorylation of AKT promotes the expression of CXCR4. Simvastatin also inhibits the expression of miR-9; while CXCR4 is the target gene of miR-9 and down regulation of miR-9 can promote the expression of CXCR4. In addition, the expression of miR-9 may be related to the PI3K/AKT pathway. Therefore, the positive effect of simvastatin on the expression of CXCR4 in BMSCs is likely to be achieved by activating the PI3K/AKT pathway and then inhibiting the expression of miR-9.