Abstract

Introduction/Aim: Previously we reported the therapeutic effect of BMPR2 augmented endothelial progenitor cell (EPC) therapy in a rat PH model. EPCs are have a role in vascular repair, angiogenesis and are known to mobilise in response to hypoxia. In this study, we have assessed the functional differences between PAH patient and control peripheral blood derived outgrowth EPCs (OECs) in response to hypoxia. Methods: OECs were isolated and cultured from both PAH and control patient’s peripheral blood (15mL). Following characterisation and expansion, cells were exposed to either normoxia (5%CO2/ atmosO2 and N2) or hypoxia (1%O2/5% CO2/94% N2). Proliferation and apoptosis were assessed using MTT colourimetric assays and flow cytometry. Immunofluorescence (IF) was used to assess the effect of hypoxia on cell matrix changes and reversal of these changes with BMP7 treatment. Protein analysis of cell lysates was conducted via western blots. Results: In hypoxia the proliferation rate of control OECs decreased significantly while the PAH OECs continued to proliferate. This proliferation could be due to apoptosis resistance in PAH OECs as apoptosis assays revealed a lower level of apoptosis after hypoxia exposure for 5 days in PAH lines than in control lines. IF showed significant cellular remodelling via fibronectin staining in all cells in hypoxia, as well as the PAH OECs in normoxia. This was reversed with BMP7 treatment. (Figure) Western blot analysis has shown significant decreses in VEGFR2 and pSmad1/5/8 with a significant increase in p-p38MAPK.  Conclusion: PAH derived OECs display functional differences under hypoxic conditions when compared to control OECs.