The Study Of HIF-α/EPO And VEGF Signal Pathway In The Bone Marrow Cells With Chronic Mountain Sickness And The Regulation Of EPO By The MiR-122 In Chronic Hypoxia

Chronic mountain sickness(CMS)is a clinical synrome caused by maladaptation to chronic exposure to a high-altitude hypoxic environment and occurs among some individuals in high-altitude regions worldwide.CMS is characterized by excessive erythrocytosis(EE)and hypoxemia.Given the EE in CMS,previous research was focused on the changes in serum EPO levels.Hypoxic induction of EPO depends in large part on the transcription factor hypoxia-inducible factors(HIFs),and HIFs control a wide spectrum of tissue specific and systemic hypoxia responses.HIF-1α and HIF-2α play divergent,but complementary roles during hypoxic response in tissues under both physiological and pathophysiological conditions.It is well known that HIF-α is the master regulator of EPO and VEGF gene expression,both of them are essential for adaptive erythropoiesis and angiogenesis.The bone marrow niche is known to be one of the primary sites of erythropoiesis,and the erythrocytosis depends on the support and regulation of the hematopoietic microenvironment.The expression and role of HIF-α and its target genes in the bone marrow of CMS have not been studied previously.Micro RNAs(mi RNAs)are one of the most studied noncoding RNA molecules and recent studies have evidenced an increased role of mi RNA in the regulation of erythropoiesis and proliferation of erythroblasts.Our previous study found the level of mi R-122 was reduced in plasma of CMS patients than in the controls.Mi R-122 is the liver-specific micro RNA,while EPO is produced mainly in the kidney and liver.We hypothesized that mi R-122 might be involved in EE through the regulation EPO production with CMS.Part Ⅰ The change of HIF-α/EPO and VEGF in the bone marrow cells with chronic mountain sicknessObjective:It is well recognized that EPO and VEGF may act as an important regulator of erythrocytosis and vasculogenesis through autocrine or/and paracrine mechanisms in the bone marrow nich,and both of them are required to conjoincorresponding receptor on the cell membrane in order to realize biological effects.To the best of our knowledge,no research on the EPO/EPOR and VEGF/VEGFRs in relation to CMS have been previously reported.This study was aimed at investigating the associations of the levels of HIFs,EPO/EPOR and VEGF/VEGFR as well as microvessel density(MVD)in the bone marrow with CMS.Methods:A total of 34 patients with CMS and 30 control subjects residing in areas at altitudes of 3000–4500 m were recruited for this study.The levels of HIF-1α,HIF-2α,EPO,EPOR,VEGF,VEGFR-1 and VEGFR-2 in bone marrow mononuclear cells(BMMNCs)were detected by flow cytometry technique(FCM)and Real-time quantitative PCR(RT-q PCR).Immunohistochemical evaluation(ICH)of HIF-1α,HIF-2α,EPO,EPOR,VEGF,VEGFR-1,VEGFR-2 and MVD were examined in the bone marrow tissue.The concentration of EPO and VEGF in the serum and bone marrow supernatant was measured using an enzyme-linked immu-nosorbent assay(ELISA).Results 1.The m RNA levels3.87%(1.92%,6.32%)of HIF-2α,EPO and VEGFR-2 were higher in the BMMNCs of CMS than in the controls(P <0.05),while HIF-1α,EPOR,VEGF and VEGFR-1 m RNA levels were similar between the two groups(P>0.05).2.The percentage of HIF-2α,EPO and VEGFR-2 positive cells in the BMMNCs of CMS were higher than that of the controls [ M(Q1,Q3)] [3.87%(1.92%,6.32%)vs 1.86%(0.84%,3.81%);22.65%(13.38%,36.72%)vs 0.52%(0.28%,1.05%);20.67%(8.06%,67.61%)vs 8.13%(2.21%,14.85%),respectively](P<0.05),but the percentage of HIF-1α,EPOR,VEGF and VEGFR-1 positive cells were similar between the two groups(P>0.05).Furthermore,hemoglobin(Hb),the percentage of EPO positive cells and HIF-1α positive cells were correlated with the percentage of HIF-2α positive cells in the CMS BMMNCs(r=0.462,r=0.342,r=0.351,respectively,P<0.05);Both of the percentage of EPO positive cells and the percentage of VEGFR-2 positive cells were positively correlated with Hb(r=0.347,r=0.453,respectively,P<0.05);There was the significant positive correlation between the percentage of VEGFR-2 and VEGF positive cells(r=0.373,P<0.05)3.The expression of HIF-2α,EPO,VEGF and VEGFR-2 was significantlyhigher in the CMS patients than in the controls(P<0.05),while there was no difference in the HIF-1α,EPOR and VEGFR-1expression between the two groups(P>0.05).The MVD values in the bone marrow tissues from CMS patients were significantly higher than those in the controls(14.54±8.84 vs.8.45±2.98,P= 0.004)Spearman analyses indicated that the expression of HIF-2α,EPO,VEGFand MVD were correlated with Hb levels(ρ=0.560,ρ=0.545,ρ=0.362,respectively,P<0.05)in the CMS tissues;The expression of HIF-1α,HIF-2α,EPO,VEGF and VEGFR-2 were correlated with MVD(ρ= 0.439,ρ=0.540,ρ= 0.545,ρ= 0.371,ρ= 0.439,respectively,P<0.05)4.EPO concentration in the serum was similar between the two groups [M(Q1,Q3)][62.44pg/ml(42.46,88.13)v.s.52.05pg/ml(42.50,87.42)](P=0.068),whereas it was significantly higher in the bone marrow supernatant of the CMS patients than in the controls [285.75pg/ml(185.07,330.02)vs 198.00pg/ml(137.70,266.62)](P= 0.027).The concentration gradient of EPO was found to be significantly higher in the CMS patients than in the controls [219.46pg/ml(138.65,317.40)vs 154.89 pg/ml(55.63,246.90)](P= 0.014);Both VEGF concentration in the serum and in the bone marrow supernatant were higher in CMS group than the controls [564.80 pg/ml(396.68,705.39)vs 309.72 pg/ml(171.70,599.52)](P=0.002)and [3961.96 pg/ml(1167.95,5081.64)vs 817.01 pg/ml(604.90,1184.54)](P=0.001).The concentration gradient of VEGF was significantly higher in the CMS patients than in the controls [1797.46 pg/ml(184.44,4445.02)vs 205.11 pg/ml(12.76,415.96)](P=0.012).Furthermore,either EPO concentrations in the bone marrow supernatants or the concentration gradient of EPO correlated with Hb levels in the CMS patients(r = 0.397,r=0.460,respectively,P<0.05).Significant negative correlations were found between VEGF in bone marrow and oxygen saturation(Sa O2)(r =-0.418,P=0.014).Conclusions:1.The upregulation of the HIF-2α/EPO pathway within the bone marrow niche of CMS patients may regulate EE and angiogenesis.2.Bone marrow nich of CMS may show enhanced activity of the VEGFR2 and it appears to be involved in the pathogenesis of CMS.3.Increased levels of EPO and VEGF in the bone marrow may be involved regulator of EE and vasculogenesis in CMS through autocrineor/andparacrine mechanisms.Part Ⅱ The regulation of EPO by mi R122 in chronic hypoxiaObjective MiR-122 is the liver-specific microRNA,while the circulating miR-122 could come from liver.EPO is produced mainly in the kidney and liver.Although the variation in serum EPO levels does not explain the striking variation in hemoglobin at high altitudes,EPO in the serum usually shows high expression in patients with elevated hemoglobin levels.Our previous study found the level of mi R-122 was reduced in plasma of CMS patients than in the controls.This study was aimed at regulation of EPO,hematopoietic stem cells and erythroid progenitor cell by mi R-122 in the animal model of CMS.Methods 1.Mi R-122 were detected by RT-q PCR after overexpression of HIF-1α and HIF-2α in hepatocyte cells with lentiviral transfection;2.Thirty-two healthy SD rats were randomly divided four groups and placed the low pressure oxygen chamber,that stimulated 5000 m altitude,including normoxia control group(n=8),seven days after hypoxia group(n=8),fourteen days after hypoxia group(n=8),thirty days after hypoxia group(n=8)and circulating mi R-122 were detected with RT-q PCR;3.Balb/c mice were injected with mi R-122-mimics(n=6),mi R-122-inhibitor(n=6),a negative control(NC)(n=6)from tail vein and were stimulated 5000 m altitude for thirty days.Six mice were included normoxic control group(n=6).The concentration of EPO in the serum was measured using ELISA and cells positive for CD34 and Ter119 in BMMNCs were detected by FCM.Results 1、Mi R122 levels were similar between the group of overexpression of HIF-1α and controls,Mi R122 were also similar in overexpression of HIF-2α and controls(1.097-and 1.060-fold higher,respectively;P > 0.05)2、The mi R122 levels in SD rats of hypoxia 7d,14 d,30d were significantly decreased(0.3915-,0.3403-and 0.2358-fold,respectively;P<0.05)than in the normoxia controls.3、The concentration of EPO in Balb/c mice after hypoxia 30 d in mi R-122-mimics group(308.02±8.47 pg/m L)were significantly decreased than NC group(354.81±13.03 pg/m L)(P<0.05),and in mi R-122-inhibitors group(366.03 ±13.64 pg/m L)were significantly increased than normoxic control group(284.43±9.35 pg/m L)(P<0.05).4、The percentage of CD34-positive cells were similar in the BMMNCs of Balb/c mice after hypoxia 30 d in hypoxic controls(15.60%±3.60%),in mi R-122-mimics group(11.75%±4.77%),in mi R-122-inhibitors group(12.08%±4.54%),in NC group(15.00%±2.16%)than that in the normoxic control group(12.55%±3.62%)(P>0.05);The percentage of Ter119-positive cells were significantly decreased in mi R-122-mimics group(12.23%±3.12%)than mi R-122-inhibitors group,hypoxic controls group and NC group(17.52%±2.32%,19.03%±1.40%,19.50%±0.73% respectively;P<0.01),while it was similar between mi R-122-mimics group and the normoxic control group(11.55%±1.97%)(P>0.05).Conclusions The decreased circulating mi R122 in animal model of CMS,which enhanced expression of EPO and proliferation of erythroblasts without depending on HIF-α.