The Effects And Mechanism Of Nrf2 On Angiogenesis Of Hypoxia Preconditioning EPCs

BackgroundIt is seriously dangerous to the elderly health of ischemic diseases caused by atherosclerosis, such as ischemic heart disease, ischemic stroke and peripheral arterial disease, etc. Although there has made great progress in the medical and interventional therapy, angiogenesis and subsequently the improvement of myocardial blood supply and heart function and prognosis will be great of significance for the patients with acute myocardial infarction and three coronary diseases not suitable for percutaneous coronary intervention. Endothelial progenitor cells(EPCs) are precursor cells of vascular endothelial cells, which has inherent advantages in angiogenesis and so it is widely used in cell transplantation in the treatment of ischemic heart disease and peripheral arterial disease, in order to promote angiogenesis and improve blood supply in ischemic tissues. However, the most of the transplanted cells undergo cell apoptosis or death for the severe microenvironment in those hypoxic-ischemic organizations or tissues and not conducive to cell survival. Therefore, it is an important significance to understand that how to regulate and promote the transplanted EPCs survival in ischemic tissues.Nuclear factor-related factor-2(Nrf2) is an important antioxidant response factor, which may induce genes transcription and proteins expression in the downstream of antioxidant genes and phase detoxifying enzyme gene after its activation, and thus play a Ⅱprotective effect against oxidative stress. Some studies shows that there is a serious inflammation and oxidative stress in hypoxic-ischemic tissues which promotes cell apoptosis and necrosis. Therefore, that whether Nrf2 is involved in the regulation of the transplanted cells to survive in hypoxic microenvironment and its specific mechanisms is unclear. It showed that hypoxic preconditioning could enhance the therapeutical efficacies and functionality in the cell transplantation and that whether Nrf2 is involved in this process is also unclear. Therefore, in the present study we will obtain EPCs by the method of density gradient centrifugation and adhere culture and then investigate the relationship between the biology of EPCs in different hypoxic conditions with Nrf2 for and subsequently explore the role and mechanism of Nrf2 in the cell survival and angiogenesis in hypoxic preconditioning by the use of molecular biology technique. Methods PartⅠ the Culture and Biological Characteristics of EPCsMarrow-derived EPCs will be obtained by the method of density gradient centrifugation and adhere culture from rat and identified by three ways including the growth morphology, endocytosis test and flow the identification of cell surface antigen markers by FACS. Part Ⅱthe Role of Mechanism of Nrf2 in the Hypoxic Preconditioning of EPCsEPCs are divided into four groups: Control group, EPCs-Nrf2-siRNA group, EPCs-Con-si RNA group and EPCs-Nrf2 group and received the hypoxic preconditioning by depending on the purpose of the experiment such as the following experiments: 1) the intracellular ROS levels was detected by the fluorescent probe DCFH-DA Kit; 2) the cell apoptosis was detected by the Annexin V / PI staining and flow cytometry; 3) Cell proliferation was detected by the MTT assay; 4) Cell migration was detected by cell scratch test; 5) The levels of HIF-1α and VEGF in the culture medium were measured by ELISA; 6) The hypoxia-induced nuclear translocation of Nrf2 was observed by the Laser Scanning Confocal; 7) The activation of signaling pathway and the m RNA transcription and protein expression of Nrf2, HO-1 and NQO1 in the hypoxic condition were detected by RT-PCR or Western Blot. PartⅢ the Role and Mechanism of Nrf2 in the angiogenesis of EPCsTube Formation Experiment was used to observe hypoxia induced angiogenesis of EPCs in the above four groups in vitro. A rat model of myocardial infarction was induced and animals were divided into four group: control group, EPCs-Nrf2-siRNA group, EPCs-Con-si RNA group and EPCs-Nrf2 group. Cell transplantation was used by the methods of direct injection into the border of myocardial infarction. The survival and apoptosis of the transplanted cells in the area of injection were observed by the immunofluorescence staining. The evaluation of angiogenesis was by the immunohistochemical staining of the CD31. Results 1) A large of quantity and high purity EPCs is obtained by the methods of density gradientcentrifugation and adhere culture. 2) EPCs were identified by the ways of endocytosis test and the identification of cellsurface antigen markers CD34, CD133, KDR and CD31 through flow cytometry. 3) The production of hypoxia-induced ROS in EPCs and subsequently the activation ofnuclear translocation of Nrf2. 4) Hypoxia-induced apoptosis and inhibition of cell proliferation and reduction of cellmigration were exacerbated by the Nrf2 knockdown with siRNA and were recoveredby high expression of Nrf2 but except for cell proliferation, which indicated that therole of Nrf2 in the maintenance of cell survival in the hypoxic microenvironment. Also,Nrf2 positively regulates the paracrine function of hypoxia-induced EPCs. 5) Hypoxia-induced EPCs increased the expression of Nrf2 through the activation of PI3K/ Akt signaling pathway and subsequently the induction the mRNA transcription andprotein expression of HO-1 and NQO1 in EPCs. 6) The structure of tube formation was generated by hypoxia-induced EPCs. The survivalsof the transplanted cells and capillary density significantly reduced after theknockdown of Nrf2 with si RNA but increase while the high expression of Nrf2 in theborder of myocardial infarction. Conclusions 1) A large of quantity and high purity EPCs is obtained by the methods of density gradientcentrifugation and adhere culture. EPCs were identified by the ways of endocytosis testand the identification of cell surface antigen markers CD34, CD133, KDR and CD31through flow cytometry. 2) Hypoxia-induced apoptosis and inhibition of cell proliferation and reduction of cellmigration were exacerbated by the Nrf2 knockdown with si RNA and were recovered byhigh expression of Nrf2 but except for cell proliferation, which indicated that the role ofNrf2 in the maintenance of cell survival in the hypoxic microenvironment. Also, Nrf2positively regulates the paracrine function of hypoxia-induced EPCs. 3) It shows that the role of Nrf2 involved in the hypoxia-induced angiogenesis effectthrough tube formation in vitro and the rat model of myocardial infarction in vivo,which may result in the improvement of heart function after myocardial infarction. 4) The production of hypoxia-induced ROS in EPCs and subsequently the activation ofnuclear translocation of Nrf2. 5) Hypoxia-induced the activation of Nrf2 and subsequently the m RNA transcription andprotein expression of HO-1 and NQO1 through the PI3K/Akt signaling pathway.