| Background and Objective Endothelial Progenitor cells(EPCs), one of the most important stem cells participated in vasculogenesis at the early stage of embryo development, were recently found in adult bone marrow and peripheral blood. As possessing ability of vigorous proliferation and angiogenic effect which independ of mature endothelial cells(ECs) in foci, EPCs are hopeful to be served as ideal donors of cell mediated neovascularization, especially for those under certain conditions, such as aging, diabetes and hypercholesterolemia, whose ECs function was impaired and with a poor response to cytokines or growth factors. However, the numberof autologous EPCs is limited though it can be slightly amplified by in vitro culture. So it is vital to enhance the biological activity of EPCs before implantation to improve its therapeutic effects. Previous studies have demonstrated that hypoxia can upregulate the expression of VEGF and VEGF receptors in multiple cell lineages, activate the angiogenesis potent of ECs and thus promote neovascularizaion in foci. However, whether dose hypoxia impose the same effect on EPCs is unclear. The aim of our investigation is to explore in which way hypoxia influence the biological activity of EPCs, such as cell proliferation, migration, paracrine function and angiogenic ability, to reveal the mechanism of those effects and to provide theoretical fundamentals of clinical application and assessment.Mehods: (l)Mononuclear cells derived from rat bone marrow were isolated by density-gradient centrifugation and were cultured on fibronectin-coated plates, supplied with bovine pituitary extract. EPCs were identified with double positive of Dil-acLDL and FITC-BS-1 lectin. (2) Hypoxia preconditioning was performed as cells cultured in a tri-gas incubator for 7 days with oxygen concentration in 2%. (3) EPCs proliferation assay was determined by cell counting. (4) EPCs migration assay was evaluated using under-agarose model. (5) EPCs paracrine function was examined by BrdU staining of ECs which were stimulated with EPCs conditional medium. (6) Angiogenic ability of EPCs was assessed with an in vitro type I collagen gel model. (7) Expression of VEGF and Flk-1 mRNA was measured by semi-quantitative RT-PCR. (8) Effect of MAPKs ERK 1/2 signal transduction pathway inducing proliferation after hypoxia was analyzed using western blot protein quantitative of phosphorylated ERK.Results (1) EPCs were cultured and identified by fluorescent staining and biological characteristics successfully. (2) Attached cells in hypoxia group were 2-fold greater than that of normoxia group, which indicated that Hypoxiacan augment proliferation of EPCs. (3) Hypoxia promoted chemotaxis and migration characteristics of EPCs responsing to VEGF, the number of migrated cells and the maximum distance of migration increased greater in hypoxia group. (4) EPCs conditional medium of hypoxia group promoted DNA synthesis of Ecs much more, which indicated that hypoxia can stimulate paracrine function of EPCs,. (5) EPCs has showen improved angiogenic ability in vitro after hypoxia preconditioning. (6) Expression of VEGF and Flk-1 mRNA was significantly enhanced by hypoxia, in accordance with the upregulated level of biological function. (7) Hypoxia preconditioning could increase the phosphorylated ERK protein expression, which coincidence with the enhanced cell proliferation, and this effect can be completely blocked by PD98059, the ERK1/2 specific inhibitor.Conclusion Hypoxia preconditioning can enhance the biological activity of EPCs in mulit-aspects, including proliferation, migration, paracrine and angiogenic ability, and those effects are in accordance with the increased expression of VEGF and VEGFR. ERK signal transduction system plays a key role in hypoxia inducible cell proliferation, but it doesn't the specific target of hypoxic stimulator. Hypoxia preconditioning can be used as a potential method to enhance the therapeutic efficiency of EPCs implantation.
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