Androgen Modulates Functions Of Endothelial Progenitor Cells Through Activated Egr1 Signaling

Objective: Study the VEGF(vascular endothelial growth factor)expression,tube formation capacity and the migratory ability in DHT(dihydrotestosterone)-treated endothelial progenitor cells(EPC)in vitro,and explore the angiogenesis ability of DHT-treated EPC in mouse hind limb ischemia model.Furthermore,we explore the mechanism and possible pathway in this process.Method: We isolated MNC from bone marrow.The BM-MNC were induced differentiated into EPC and characterized by DiI-ac-LDL uptake and quantitative fluorescence-activated cell sorting(FACS).On the culture day 2,EPC were treated with various concentration of DHT(0,1,10,100 nmol/L).Total RNA was isolated from EPC on the culture day 4,5,7,9 and 11.Then RT-PCR(reverse transcription polymerase chain reaction)was performed to evaluate the expression of VEGF mRNA.On the culture day 7,the in vitro incorporation assay and Transwell assay were performed to determine the tube formation capacity and the migratory ability of DHTtreated EPC.We made the mouse hind limb ischemia model,and transplanted the DHTtreated or non-treated EPC.Laser Doppler Flowmetry(LDF)was used to determine the limb perfusion recovery of each group,and immunofluorescence to evaluate the density of capillary and the survival of transplanted EPC.We designed the Egr1-siRNA,and then performed the in vitro incorporation assay and Transwell assay again to determine the role of Egr1 in the modulation of EPC functions by DHT.Results: The DiI-ac-LDL uptake assay and FACS showed the EPC characters of the isolated cells.The result of RT-PCR showed DHT can increase the expression of VEGF in the EPC in a dose-dependent manner with the maximum effect at the concentration of 10 nmol/L and day 7(P<0.05).Our results showed that DHT can dramatically enhance the embedding of EPC into tube structures and the migratory ability in a dosedependent manner with the maximum effect at the concentration of 10 nmol/L(P<0.05).The mouse hind limb ischemia model was established successfully.Compared with the non-treated EPC group and the control group,DHT-treated EPC group showed a significantly increased ischemic limb perfusion recovery(P<0.05).The immunofluorescence showed an enhanced density of capillary and survival of transplanted EPC in the DHT-treated EPC transplanted group.We designed the Egr1(Early growth response protein 1)-siRNA successfully(P<0.05).The siRNA transfection efficiency can be achieved up to 90%,and the Egr1-siRNA group showed an inhibited expression of Egr-1,and the inhibition efficiencies can be up to 60%(P<0.05).After transfection with Egr1-siRNA,the angiogenesis and migration abilities of DHT-treated EPC were significantly decreased compared to those treated with DHT alone(P<0.05).Conclusion: Our findings suggest that DHT markedly enhances the vessel forming ability and migration capacity of EPC in a dose dependent manner peaking at the concentration of 10 nmol/L DHT.DHT can also enhance the angiogenesis capacity of EPC in vivo study.Egr1 signaling may be a possible pathway in this process.