Repair Mechanisms Of Endothelial Progenitor Cells In Intermittent Hypoxia Rat Model

Objectives: OSA was a major public healthy burden and an independent risk factor of hypertension,atherosclerosis and coronary heart disease as well as all kinds of cardiovascular diseases.The major pathophysiologic characteristic from OSA was IH and the OSA pattern of intermittent hypoxia bring the vascular damage.There was a balance between damage mediated process of OSA activated and recruitment and homing of EPC during repair process.And this balance would decide to the blood vessel function of OSA patients.So we mimicked IH rat model to study the repair mechanism of EPC for injury of vascular endothelial.Methods: Respire simulation system was used for intermittent hypoxia(IH)exposure to mimick OSA.C57BL/6J rats(male)weighing 26~30g at the age of 7 age weeks were divided into intermittent normal oxygen group,Mild IH group,Moderate IH group and Severe IH group randomly.We obtained the mononuclear cells from the peripheral blood with density gradient centrifugation,on the basis of aldehyde dehydrogenase(ALDH)activity and combined CD133,CD34 and containing insert fragment receptor kinase domain(KDR)of EPC,and using flow cytometric instrument to measure the levels of different classify EPC which contained CD133+KDR+EPC and CD133+CD34+EPC,CD34+KDR+EPC,ALDHlowCD34+KDR+EPC.We measured the concentration levels of stromal cell-derived factor-1alpha(SDF-1?),vascular endothelial growth factor(VEGF)and hypoxia-inducible factor-1alpha(HIF-1?)from serum of rats through Enzyme Linked Immunosorbnent Assay(ELISA).We cultured mononuclear cells of peripheral blood(PBMCs)to count the number of spindle cells of double-stained and cells of double-stained by acetylated low density lipoprotein(Dil-ac LDL)uptake and lectin Ulex europeaus agglutinin-1(UEA-I)binding in order to assess the proliferative capacities of EPC and identified EPC.We counted the number of transmigrated EPC through Transwell model in order to assess the migration ability of EPC.To measure the CXCR4 and VEGFR2 of the EPC surface protein by Western Blot.We observed and evaluated the influence of EPC to new vein endothelial ability after cultured endothelial cells(EC)on Matrigel,which come from EPC medium from different levels of IH.The datas were analyed through SPSS 18.0 software.Results:(1)Respire simulation system was used for intermittent hypoxia(IH)exposure to mimick OSA successful and low oxygen scheme as flowed,Intermittent normal oxygen group(21% O2),Mild IH group(15% O2 30s),Moderate IH group(10% O2 30s),Severe IH group(5% O2 30s).(2)Flow cytometric instrument quantification levels of CD133+KDR+ EPC,CD133+CD34+ EPC and CD34+ KDR+EPC had significant difference(F=1163.164 ?50.383 ?676.123,P =0.000)and the results were described as Severe IH group > Moderate IH group > Mild IH group > Intermittent normal oxygen group.And ALDHlowCD34+KDR+ EPC levels were higher in Mild IH group than Intermittent normal oxygen group while dropped in Moderate IH and Severe IH group.(3)The concentration levels of SDF-1?,HIF-1? and VEGF had significant difference and the results were described as the Severe IH group > Moderate IH group > Mild IH group > Intermittent normal oxygen group.(4)The number of spindle cells of double-stained and cells of double-stained had significant difference and the results were described as the Mild IH group > Intermittent normal oxygen group > Moderate IH group > Severe IH group.(5)The number of transmigrated EPC had significant difference and the results were described as Mild IH group > Intermittent normal oxygen group > Moderate IH group > Severe IH group.(6)The level of VEGFR2 and CXCR4 proteins expression from surface of EPC had significant difference and the results were described as the Mild IH group > Intermittent normal oxygen group > Moderate IH group > Severe IH group.(7)The mean tube length of endothelial cells had significant difference and the results were described the as Mild IH group > Intermittent normal oxygen group > Moderate IH group > Severe IH group.Conclusion:(1)After Mild IH,the blood cytokines were activated,EPC mobilization were increased and migrated to the damaged endothelium.The number of new endothelial vascular formation were increased and the repair capacity of endothelial progenitor cells was enhanced.In Moderate IH and Severe IH group,although EPC mobilization was increased and the number of cells were increased,recruit a large number of invalid EPC(ALDHhiEPC)and the EPC(ALDHlowCD34+KDR+EPC)which participated in the repair of endothelial were not increased even decreased.So the number of new endothelial vascular formation were decreased and the Repair capacity of endothelial progenitor cell was weakened.(2)After Mild IH,the ability of activation,mobilization,recruitment,proliferation,homing and angiogenesis were increased.But,the ability of mobilization,recruitment,proliferation,homing,angiogenesis were decreased largely as the degree aggravating of IH(Moderate IH and Severe IH).A balance would be destroy between endothelial injure and the repairing by endothelial progenitor cell(EPC),and the repair capacity for the damaged endothelium was weakened,so the risk of cardiovascular disease would also increased greatly.