Autophagy Regulates UPA Expression In Endothelial Progenitor Cells

Purpose: To investigate the regulatory role of autophagy on urokinase plasminogen activator(uPA) expressionin rat endothelial progenitor cells(EPCs) in hypoxia condition; To investigate the molecular mechanism of uPA expression regulation in EPCs by autophagy.Methods: 1. By means of Ficoll density gradient centrifugation, bone marrow-derived mononuclear cells(BMMNCs) were isolated from SD rats; Then they were cultured within special EGM-2MV medium during a period of 14 to 28 days; The characters of cultured cells were observed under the optical microscope; Flow cytometry technique and experiments of acetylated low-density lipoprotein endocytosis and ulex europaeus I lectin binding was manipulated to attain the identification. Tube formation of EPCs in matrigel in vitro was then addressed; 2. Lenti-virus system was used to overexpression and knockdown of autophagy key molecule ATG-5 in EPCs under the hypoxia condition, the infection efficacy and expression of ATG-5 was determined by fluorescence microscope and RT-PCR. Moreover, West-blot was performed to detect the expression of uPA in EPCs; 3. Western-blot was performed to detect the change of signaling pathway of mTOR-S6 K in ATG-5 overexpressed and knockdown EPCs.Results: 1.With the method of density gradient centrifugation, cultured cells were found to show EC specific surface markers such as CD 133 and CD 34, VEGFR-2 and had the ability to endocytose ac-LDL and bind UEA I lectin, displaying a high capacity of migration and vessel formation; 2. In the hypoxia condition, ATG-5 overexpression could significantly increase the expression of uPA in EPCs while ATG-5 knockdown could significantly decrease the expression of uPA in EPCs; 3. In the hypoxia condition, ATG-5 overexpression could significantly increase the expression of mTOR-S6 K in EPCs while ATG-5 knockdown could significantly decrease the expression of mTOR-S6 K in EPCsConclusion: In hypoxia condition, ATG-5 overexpression and knockdown could regulate the expression of uPA in EPCs and mTOR-S6 K signaling pathway was involved in the mechanism.