The effect of chronic intermittent hypoxia on angiogenesis functions of platelets and localization of multipotent epithelial stem/progenitor cells in the lung

Introduction: The granules of platelets contain proangiogenic stromal cell-derived factor-1 (SDF-1/CXCL12), which mediates neo-alveolarization. It has also been shown that platelet-derived SDF-1 mobilizes stem cells from the bone marrow and recruits them by forming a hemo-vascular niche. SDF-1 is also a potent stimulator of stem cells to transform into endothelial progenitor cells (EPCs). Platelets promote angiogenesis by releasing molecules besides SDF-1, such as vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and matrix metalloproteinases. Platelets also contain antiangiogenic factors such as plasminogen activator inhibitor-1 (PAI-1), endostatin and thrombospondin. Since platelets contain both proangiogenic and antiangiogenic regulatory proteins, it is not known exactly how platelets can stimulate or inhibit angiogenesis and by which environmental factors this is regulated. Aim: The aim of this study was to investigate the effect of chronic intermittent hypoxia on platelet-derived proangiogenic and antiangiogenic molecules, whether it improves endothelial progenitor cell placement in tissue after lung injury, and whether the placement of endothelial progenitor cells at the tissue damage site is affected by a possible change in platelet-derived SDF-1 level. Materials and methods: Male Wistar Albino rats of 10 weeks of age were allocated into Control, Lung Injury (AH), Chronic Intermittent Hypoxia (KH), Chronic Intermittent Hypoxia + Lung Injury (KH+AH) groups. Hypoxia was applied in the hypobaric hypoxic chamber for 42 days, 6 hours a day, at an altitude of 3,000 meters, in an atmospheric pressure (69.3 kPa, 520 mmHg, ≈14% O2) environment. Lipopolysaccharide was administered intratracheally at a dose of 100 μg/100g to induce lung injury. Animals were sacrificed 3 days after lung injury and blood and lung samples were taken. Platelets were separated from blood samples, counted and washed via a previously described three-step procedure. VEGF, SDF-1, PDGF, endostatin, thrombospondin-1, PAI-1 concentrations were measured in the supernatant samples obtained after the lysis of platelets. Presence of EPC in lung tissue was evaluated by immunofluorescence method. Results: There was no significant difference in the weight of the rats before and after hypoxia. The number of cells per milliliter (p<0,001) and the total number of cells obtained in the KH+AH group (p<0,001) were higher than the other groups. SDF-1 concentrations of KH group were lower than the control (p<0,01) and AH (p<0,05) groups. PAI-1 concentration in the KH group was higher than the control group (p<0,05). Endostatin concentration was significantly higher in the AH group than the control (p<0,01) and KH+AH (p<0,01) groups. Thrombospondin concentration was higher in the KH group than control (p<0,01), AH(p<0,01) and KH+AH (p<0,05) groups. SDF-1 concentration per cell was lower in the KH+AH groups compared to the control (p<0,001) and AH (p<0,01) groups. VEGF, PDGF, PAI-1, endostatin, and thrombospondin concentrations per cell were lower in the KH+AH group than control (p<0,001), AH (p<0,001) and KH (p<0,05) groups. In immunofluorescent imaging, EPCs were observed more intensely in the AH group compared to the control group, while EPCs were not observed in the KH and KH+AH groups. Conclusions: Our results demonstrated that SDF-1, one of the proangiogenic factors, decreased after to hypoxia, and accordingly, the placement of EPCs to the damaged tissue decreased after lung injury. No significant changes were observed in VEGF and PDGF concentrations after hypoxia. The concentrations of antiangiogenic factors, including PAI-1 and thrombospondin-1, increased due to chronic hypoxia, while the concentration of endostatin increased due to lung injury. In the KH+AH group, all factor concentrations per cell were found to be considerably lower than other groups. This might be related to the effect of chronic hypoxia on megakaryocytes. Our results show that chronic hypoxia may alter platelet functions, however, more studies are needed in this area.