Study On The Role Of Hypoxia In Radiation Damage And Repair Of Hematopoietic Stem Cells

Our China has the biggest population in the plateau over the world,and the Qinghai-Tibet Plateau,which has an average altitude of over 4,000 meters and covers a quarter of the land,is unique and huge geopolitically significant for our country.Meanwhile,with the extensive applications of nuclear energy and nuclear technologies in industry,agriculture,environmental protection,medical treatment and other fields,the risk of injuries caused by ionizing radiation（IR）in plateau areas will increase gradually.Because the main environmental feature of the plateau is low oxygen or hypoxia,the research on the radiation-casused damages and repairs in hypoxic environment will have great important practical meanings for radiation protection and wounded treatment in plateau areas.Induced apoptosis of hematopoietic stem cells and vascular niche injuries are the main causes of bone marrow damage and hematopoietic failure due to moderate to severe myeloid acute radiculopathy;therefore,how to protect hematopoietic stem cells and hematopoietic niches from ionizing radiation is the main goal of developing new anti-radiation medical countermeasures.Hematopoietic stem cells and their progenies,such as multi-pluripotent progenitor cells（MPP）and hematopoietic progenitor cells（HPC）,are collectively referred to as LSK（Lin^-Sca1⁺c-Kit⁺）cells because they do not express surface markers of mature hematopoietic cells but express the stem cell factor receptor c-Kit and the stem cell antigen Sca-1.Hematopoietic stem cells are randomly distributed in the vicinity of physiologically hypoperfused and hypoxic vascular niches.Vascular endothelial cells and perivascular stromal cells expressing leptin receptor（Lep R⁺）can secrete factors to promote the maintenance of hematopoietic stem cells.Under homeostasis,hematopoietic stem cells tend to use anaerobic glycolysis to fulfil the energy requirements,but they quickly shift to aerobic metabolism due to a surge in energy demand as a result of proliferation and differentiation,when the body has to face various stresses,such as infections,radiations and chemotherapies.At the same time,large amounts of reactive oxygengenspecies（ROS）are generated as the consequent production.Studies have confirmed that physiological levels of ROS promote the differentiation and proliferation of hematopoietic stem cells,while excessive ROS will damage the long-term proliferation of hematopoietic stem cells.Hypoxia inducible factor-1α（HIF-1α）is known to be a hypoxic stress-sensitive protein that is stable under hypoxic conditions.It’s expressed highly in hematopoietic stem cells,especially in long-term hematopoietic stem cells,and its downstream signals maintain the static,survival and metabolic dynamic balance of hematopoietic stem cells through cellular autonomic and involuntary mechanisms.So it plays an important role in the resistance regulation when hematopoietic stem cells make responses to various stresses.We speculate that the role of hypoxia in the repairs of hematopoietic stem cell radiation damages may be related to HIF-1α,however,the mechanisms involved and the effects on hematopoietic stem cells and the vascular niche still need to be confirmed by further research.Vascular endothelial growth factor A（VEGFA）,a hypoxia-induced angiogenic factor and mainly derived from LSK cells,maintains the characteristics of hematopoietic stem cells by autocrine of hematopoietic stem cells,and it’s indispensible for embryonic and adult hematopoiesis.Studies have shown that increased HIF-1αand VEGFA expression in bone marrow are consistent with decreased oxygen concentration in bone marrow.The body promotes survival and function of hematopoietic stem cells by inducing up-regulation of VEGFA through HIF-1α,and the vascular permeability increased meanwhile.Nevertherless,the changing trend of VEGFA as well as its effects on hematopoietic stem cells and vascular niches are unclear after long-term exposure to hypoxia and radiationAngiopoietin-1（ANGPT1）,a kind of vascular stimulating factor which is not expressed by osteoblasts,and low expressed in c-Kit⁺hematopoietic progenitor cells,megakaryocytes and Lep R⁺stromal cells,but most highly expressed in hematopoietic stem cells.By ANGPT1-Ti E2 signal transduction,overexpression of ANGPT1 induces LSK cells to be more stationary,promoting the maintenance of LT-HSC’s stemness and its adhesion to vascular niche.So it’s very important for hematopoietic differentiation and functional regulation of LT-HSCs.In addition,ANGPT1 reduces vascular leakage by promoting the chemical attraction of smooth muscle cells to newly formed blood vessels,inhibiting the internalization of vascular endothelial cadherin（VE-Cadherin）,and enhancing the tight connection between endothelial cells.However,whether ANGPT1 is involved in hypoxia regulation of hematopoietic stem cell function and influence the radiation damages and repairs on hematopoietic stem cell has not been determined.Around those above problems,we first designed the atmospheric hypoxia animal feeding chamber and portable low oxygen radiation transfer device.Then,hypoxic experimental platform and hypoxic acclimation animal model were established.By using one-time radiation of⁶⁰Co to simulate an acute radiation event,we carried out blood routine test,flow cytometry analysis,immunofluorescence staining,histopathology,enzyme-linked immunosorbent assay et.al.We observed and compared the effects of hypoxic acclimation before radiation and hypoxic treatment after radiation on the survival rate of mice,and the impacts of acute radiation injuries and repairs of hematopoietic stem cells were also involved.Based on this study,we explored the related mechanisms.Main results and conclusions we’ve got are as follows:1.The self-designed hypoxic experimental equipments,which can maintain the hypoxic environment continuously and stably,are certificated with the national new utility patent.2.Pre-irradiation hypoxia acclimation had a radiation protective effect,improving the survival rate of mice after radiation.Moreover,the protective effect was enhanced with the extention of hypoxic acclimation time,and 14 days group had the highest survival rate,comparing with other time periods.Hypoxic acclimation could increase the number and proportion of LSK cells and LT-HSC,remodel the vascular niche,reduce the injuries of hematopoietic stem cells and the vascular niche,and accelerate the recovery of peripheral blood cells.3.Post-radiation hypoxia treatment significantly improved the survival rate of irradiated mice,especially the 12h hypoxia treatment group had the best effect,while returning to normal oxygen immediately after radiation deteriorated the injuries of hematopoietic stem cells and the vascular niche.Post-irradiation hypoxia treatment blocked the cell cycle of hematopoietic stem cells,inhibited excessive proliferation of hematopoietic stem cells,reduced excessive production of Reactive Oxygen Species（ROS）and early apoptosis of hematopoietic stem cells,alleviated vascular niche damages,and promoted recovery of peripheral blood cells.4.Hypoxia induced the stabilization of HIF-1αin hematopoietic stem cells and promoted the synthesis of VEGFA and ANGPT1 in hematopoietic stem cells,which not only regulated the quiescence of hematopoietic stem cells,but also regulated endothelial integrity,by regulating the vascular endothelial calmodulin VE-cadherin,and the endothelial integrity was essential for the function of hematopoietic stem cells.In conclusion,we designed and established an experimental platform of atmospheric hypoxia,clarified that hypoxic habituation before radiation and hypoxic treatment after radiation can reduce hematopoietic stem cell radiation damages and promote hematopoietic recoveries,revealed that hypoxia can be used as an independent or auxiliary method to reduce radiation-related hematopoietic stem cell damages,and initially elucidated the the mechanisms of HIF-1α,VEGFA and ANGPT1 in the regulation of hematopoietic stem cell and the vascular niche function by hypoxia,providing new ideas for radiation injury protection and medical treatment in highland hypoxic areas.