Novel Zinc Finger Gene ZFP580 Promotes EPCs Differentiation Via ENOS/NO Pathway

Objective : The differentiation of endothelial progenitor cells(EPCs) plays a pivotal role in endothelial repair and re-endothelialization after vascular injury.However, the underlying mechanisms still remain largely elusive. Here, we investigated the role of the novel C2H2 zinc finger transcription factor ZFP580 in EPC differentiation and the molecular mechanisms behind EPCs-mediated endothelial repair.Methods:Bone marrow-derived EPCs were isolated by density gradient centrifugation, cultured, and identified. Firstly we observed the cell morphology on0 d, 4d, 7d and 14 d, and fluorescence-activated cell sorting(FACS) was performed using antibodies against CD133, and CD31. Additionally, we tested whether ZFP580 was expressed during the differentiation of EPCs by performing QPCR to detect m RNA expression and Western blotting to detect protein expression during EPCs differentiation. Then, EPCs were infected with an adenovirus encoding ZFP580 or Ad-si ZFP580 to silence ZFP580. QPCR and Western blotting to detect mature endothelial phenotypes CD31 and v WF m RNA and protein expression during EPCs differentiation. FACS analysis was performed to analyze EPC surface makers. Further more, L-NAME, an inhibitor of e NOS, was used to elucidate the possible molecular mechanism. Tube formation in vitro and in vivo were also used in this study.Results: After 7 days of culture, adherent EPCs were characterized by immunofluorescence and FACS. The majority of cells(87.39±2.98%) stained positive for Di I-Ac LDL and lectin, and expressed endothelial/stem cell markers, including CD34(30.21%) and VEGFR-2(82.69%), confirming the cell type of EPCs. Moreover,endothelial phenotypes were confirmed by additional immunostaining of VEGFR-2and CD31. We analyzed the expression of ZFP580 in differentiating EPCs and found that ZFP580 expression was low during the early stages of differentiation but had increased significantly by day 14 of culture. Both ZFP580 and e NOS were displayed dynamic expression during EPCs differentiation. Overexpression of ZFP580 up-regulated the expression of endothelial nitric oxide synthase(eNOS) and markers of mature ECs in EPCs, while knockdown suppressed it. Upregulation/knockdown of ZFP580 also enhanced/reduced endothelial tube formation from EPC in vitro and in vivo. The effects of ZFP580 could be blocked by treatment with L-NAME, an inhibitor of e NOS.Conclusions:ZFP580 promotes not only the differentiation of EPCs into ECs by increasing the expression of e NOS and the availability of nitric oxide, it also promotes the vessel formation in vitro and in vivo. This might represent a novel mechanism of ZFP580 in EPCs differentiation and its therapeutic value in the treatment of vascular disease.