The Research Of Mechanism In Androgens Enhances Angiogenesis Of Endothelial Progenitor Cells

Objective:Explore the mechanism of the effect of mouse endothelial progenitor cells on the angiogenesis while the intervention of androgen, and confirm the androgen’s target regulation in endothelial progenitor cells. Provide the theoretical basis for the treatment of ischemic heart disease based on endothelial progenitor cells.Methods:Firstly, BM-MNCs were isolated from bones of C57/BL mouse through density gradient centrifugation. Then BM-MNCs were cultured in EBM-2 culture medium to obtain EPCs. The EPCs were identified in vitro by phagocytosis test of Dil-ac-LDL and analyzed by FACS for the expression of CD31, CD34, CD11b, CD133, CD105 and CD45. Different concentration gradient of DHT were add to co-culture of EPCs and HUVEC while the in vitro incorporation assay. Accroding to the number of Dil-labeled EPCs, the optimum concentration of DHT was confirmed as 10 nmol/L. The selection of altered expression genes of EPCs which was effected by DHT through gene expression profiling and pathway analysis software. We choose the Efnb2 as the target gene after checking the related genes expression using realtime-PCR. While shRNA-Enfb2 was inserted into the lentiviral vector, the expression of Efnb2 was detected by realtime-PCR after lentiviral infection of EPCs was carried out according to manufacturer’s instruction. The in vitro incorporation assay of EPCs were divided into four groups:①EPCs as control group;② 10nmol/L DHT-EPCs group; ③10nmol/L DHT+NC-shRNA-EPCs group;④10nmol/L DHT+Efnb2-shRNA-EPCs group. The biological effect of angiogenesis in every group was evaluated by the number of DiI-labeled EPCs. Based on the model of hind limb ischemia in mice, studies were divided into five groups in vivo:① blank as control group; ②EPCs group; ③10nmol/L DHT-EPCs group; ④10nmol/L DHT+NC-shRNA-EPCs group; ⑤10nmol/L DHT+Efnb2-shRNA-EPCs group. Second days after operation four kinds of EPCs (25μl) were injected in infarctive region of four groups mouse except of blank group. The lower limb blood perfusion was evaluated by laser Doppler while seventh days and fourteenth days after operation. The lower limb muscle tissue specimens were obtained on fourteenth days after operation. Capillary density and the number of GFP+/Lectin1+ cells was detected by immunofluorescence staining technique. All above experiments were repeated 3 times, results are expressed as mean±standard error or mean fold changes in count and analyzed using a general linear model. Statistical analysis was performed with GraphPad PRISM version 5.01. Differences were considered significant at P<0.05.Result:Mouses BM-MNCs were successfully isolated, also EPCs were obtained in EBM-2 culture medium favourably. Of these cultured EPCs, both of FACS analysis and DiI-ac-LDL phagocytosis test showed that most cells expressed characteristics of EPCs. The in vitro incorporation assay displayed 10 nmol/L DHT as the appropriate concentration. Eleven altered expression genes of EPCs were obtained by Gene expression profiling and realtime-PCR, including Egrl, Vcan, Fgf3, Cxcl 10, Cxcl 2, Hdac4, Bmp7, Stfa211, Cldnl, Cdk2apl and Efnb2. We choose the Efnb2 as the target gene and then EPCs were infected by lentiviral vectors which carried shRNA-Efnb2. The expression of Efnb2 in infected EPCs was depressed remarkably while detected by fluorescence and realtime-PCR. Compared with the DHT-treated EPCs, the capillary density per tube length in vitro which differentiation of EPCs were attenuated significantly due to the biological effect of Efnb2-shRNA. So we consider that Efnb2 is a key role while DHT regulates EPCs pathway in the process of angiogensis. In vivo based on laser Doppler detection, well blood reperfusion in hind limb was showed both in lOnmol/L DHT-EPCs group and lOnmol/L DHT+NC-shRNA-EPCs group. Moreover, the GFP+ cells and Lectinl+ cells showed a same tendencies.Conclusions:DHT can significantly improve the angiogenic ability of endothelial progenitor cells to form the vascular like structures, the appropriate concentration was 10nmol/L. Efnb2 as a key role while DHT regulates EPCs pathway in the process of angiogensis. The elevated blood reperfusion in hind limb infarctive region depend on proper concentration of DHT and high expression of Efnb2. This study to provide theoretical basis for the treatment of ischemic heart disease based on endothelial progenitor cells.