The Effect Of HIF-1α/SDF-1 Axis On Hypoxia-induced Progenitor Cell Migration And Adhesion

Purpose:Preliminarily study the role of HIF-1α/SDF-1 axis in hypoxia-induced progenitor cell migration and adhesion to pulmonary artery endothelial cells which leads to pulmonary vascular remodeling. For further understanding of the biological mechanism of pulmonary vascular remodeling, thus to provide new ideas for treatment of hypoxic pulmonary hypertension.Methods:The peripheral blood progenitor cells and pulmonary artery endothelial cells of SD rats were used and the experimentation is divided into following parts:1. Extracting peripheral blood progenitor cell of SD rat: G-CSF mobilized peripheral blood progenitor cells, Immunomagnetic bead was used to separate and purify SD rat CD34/CXCR4-positive peripheral blood progenitor cells, FCM was used to detect the purity CD34/CXCR4-positive peripheral blood progenitor cells2. Cultivating and purificating SD rat pulmonary artery endothelial cells: The mothod of tissue adherence was used.3. Collecting the pulmonary artery endothelial cell of nomoxic and different hypoxia time points respectively at 6h, 12h and 24h.1) Immunofluorescence was used to detect the expression of HIF-1αand SDF-1;2) Western-blot detecting the expression of HIF-1α;3) ELISA detecting the expression of SDF-1 in cell supernatant: ; 4) ELISA detecting SDF-1 expression after used HIF-1αinhibitor 2ME2.4.progenitor cell migration experiments1) experiment is divided into the following groups:①normoxic group: 21% O2, 5% CO2, 74% N2②hypoxia 12h group: 1% O2, 5% CO2, 94% N2③hypoxia 12h + HIF-1αinhibitor(2ME2) group④hypoxia 12h + anti-SDF-1 group⑤hypoxia 12h + HIF-1αinhibitor(2ME2) + anti-SDF-1 group2) CXCR4-positive progenitor cells were inoculated into the upper chamber which have been put on a chemotactic membranes and each group of pulmonary artery endothelial cells conditioned medium was placed in the next room, incubated 6h, removed the cells of membrane positive (above), methanol fixed , counting the the cells number of back membrane (below). Migration Index=average cells number of experimental group per 10HPF/ average cells number of control group per 10HPF 5. progenitor cell adhesion experiments:1) experiment was divided into the following groups:①normoxic group: 21% O2, 5% CO2, 74% N2②hypoxia 12h group: 1% O2, 5% CO2, 94% N2③hypoxia 12h + HIF-1αinhibitor(2ME2) group④hypoxia 12h + anti-SDF-1 group⑤hypoxia 12h + HIF-1αinhibitor(2ME2) + anti-SDF-1 group2)Pulmonary artery endothelial cells monolayer cultured in each group with the fluorescent labeling of CD34/CXCR4-positive cells inoculated into 96-well plates, incubated 6h, washing cells which did not adhere and detect the fluorescence intensity (expressed by OD values), calculating the cell adhesion rate. Pulmonary artery endothelial cells located hole and progenitor cells hole as control holes. Formula is: Adhesion rate =( OD value of experimental group - OD value of pulmonary artery endothelial cells control group )/ OD value of progenitor cells control group×100%Results1. Microscope showed SD rat peripheral blood progenitor cells almost all adhere to the beads, the shape of cell size was a diameter of about 7-10μm; immunofluorescent showed CD34/CXCR4-positive, FCM was used to detect the purity of CD34/CXCR4-positive peripheral blood progenitor cells was 91%;2. Pulmonary artery endothelial cells which purified and cultured showed the characteristic of polygon,Ⅷfactor positive expression in cytoplasm, CD34 positive expression in the membrane;3. HIF-1αexpression in Hypoxia-induced pulmonary artery endothelial cells group was expressed in the nuclear and no obvious expression in normoxic group; SDF-1 expression of hypoxia-induced pulmonary artery endothelial cells was expressed in the cytoplasm and membrane and no obvious expression in normoxic group;4. Hypoxia-induced pulmonary artery endothelial cells expressed HIF-1αprotein: HIF-1αprotein in the hypoxia 12h group (0.791±0.027) was significantly higher than normoxic group (0.035±0.001, P <0.01) detected by Western-blot; 12h group (0.791±0.027) was higher than 6h(0.568±0.032) and 24h(0.432±0.016) group (P <0.01);5. Hypoxia-induced pulmonary artery endothelial cells expressed SDF-1 protein: SDF-1 protein in the hypoxia 12h group (331.874±20.989pg/ml) was significantly higher than normoxic group (18.621±3.323pg/ml, P <0.01) detected by ELISA; 6h group and 24h group(125.267±25.341pg/ml) was higher than normoxic group (18.621±3.323pg/ml, P <0.05) detected by ELISA;6. The HIF-1αinhibitor(2ME2) may reduced expression of SDF-1: hypoxia 12h group with the HIF-1αinhibitor SDF-1 expression (41.053±9.770pg/ml) was significantly lower than no HIF-1αinhibitor group (P < 0.05); 7. With HIF-1αinhibitor or SDF-1 corresponding antibodies both enabled the migration index and adhesion rate of CD34/CXCR4-positive progenitor cells to the pulmonary artery endothelial cell decreased .ConclusionHypoxia can induce HIF-1αand its downstream regulatory factor SDF-1 expression. HIF-1α/SDF-1 axis may play an important role in progenitor cell-mediated migration and adhesion to the pulmonary artery endothelial cells and suggests it may be an important mechanism in hypoxic pulmonary vascular remodeling in oxygen deficiency pulmonary hypertension.