ABSTRACT
Background Anti-VEGF drugs are generally applied in the treatment of ocular neovascular diseases.However,the therapy effect is unsatisfactory in some patients.Studing the effect of hypoxia-inducible factor-1 (HIF-1),a upstream regulatory gene of VEGF,and its limiting enzyme prolyl-4-hydroxylase domain proteins (PHDs) is of important clinical significance.Objective This study was to investigate the negtive regulation of exogeneous PHDs on HIF-1 pathway in human RPE cells.Methods pFLAG-PHD1,pFLAG-PHD2 and pFLAG-PHD3 plasmids were constructed by extracting RNA from Hela cell line and coloning PHD1,PHD2 and PHD3 using reverse transcription PCR with restriction enzyme.The plasmids were identified by gene sequencing.ARPE-19 cells were cultured at 21％ O2 (normoxia group),1％ O2 (hypoxia group),or in hypoxia-mimicking agents (CoCl2,anoxia group),respectively,and then were transfected with plasmids encoding FLAG-tagged PHD1,PHD2,PHD3 and pFLAGCMV2 transfected cells served as blank control.The expressional intensities of PHD1,PHD2 and PHD3 in the cells were detected and compared among different groups by using Western blot assay.The transcriptional activity of HIF-1 in the cells was evaluated with dual luciierase reporter assay.Results Western blot assay showed that PHD1,PHD2 and PHD3 all were expressed in ARPE-19 cells in the normoxia group,hypoxia group and anoxia group.The expression was strong in PHD2 protein and was weak in PHD3 protein,a statistically significant difference was found between PHD2 protein expression and PHD1 or PHD3 expressions (all at P＜0.05).Endogenous HIF-1 activity was elevated in pFLAG-CMX transfected cells in the hypoxia group and anoxia group than that in the normoxia group.Compared with pFLAG-CMX transfected cells,no obvious change was seen in the endogenous HIF-1 activity in the normoxia group,however,HIF-1 activity was declined in the hypoxia group and anoxia group after pFLAG-PHD1,pFLAG-PHD2 or pFLAG-PHD3 transfection.Under the same oxygen environment,HIF-1 activity was lower in the pFLAG-PHD2 transfected cells than that in the pFLAG-PHD1 or pFLAG-PHD3 transfected cells (both at P＜0.05).Conclusions PHDs play a negative regulation to HIF-1 activating pathway in human RPE cells,especially in hypoxia and anoxia cells.Among PHDs proteins,PHD2 presents the strongest inhibition on HIF-1 activating pathway.
ABSTRACT
Objective To test the hypothesis of autophagy that silencing PHD2 gene could increase hypoxia inducible factor (HIF)-1α levels in the renal medulla and attenuate hypoxia injury in cultured human renal proximal tubular epithelial cell (HK-2) under cobalt dichloride (CoCl2) exposure.Methods HK-2 cells were harvested at hour 0,6,12,24,36 and 48 after exposure to CoC12 (200 μmol/L).The role of HIF/PHD pathway in CoCl2-induced cell apoptosis/autophagy was studied by employing small-interfering RNA (siRNA).Dynamic profiles of apoptosis markers (Bax,Bcl-xl) and autophagy marker (LC3) of HK-2 cells within 48 h after exposing to CoCl2 were recorded.Alamar Blue assay was used for quantitative analysis of cellular growth and viability.Electron microscopy analysis was employed to evaluate the changes in autophagic structures.Results The protein expressions of PHD2 were gradually increased after exposing to CoCl2 (200 μmol/L),with statistics significance at 24 h and reached the peak at 48 h (both P ＜ 0.01).PHD2 siRNA reduced PHD2 levels by ＞ 60％ and significantly increased HIF-1α protein levels (P ＜ 0.01),but had little effect on HIF-2α.The protein expression of Bcl-xl was significantly up-regulated,while the level of Bax and LC3-Ⅱ/LC3-Ⅰ were down-regulated in PHD2 siRNA group (all P ＜ 0.01),compared with the negative control group.Meanwhile,either 3-Methyladenine (an autophagy inhibitor) treatment or PHD2 knockdown rescued cell death and increased cell viability through autophagy inactivation.The ratio of LC3-Ⅱ/LC3-Ⅰ and the quantity of autophagosomes were decreased,and the cell ultrastructure was also relatively intacter than the negative control group.Of interest,co-administration of HIF-1α siRNA with PHD2 siRNA abrogated renoprotective effect conveyed by PHD2 siRNA alone,suggesting that activation of endogenous HIF-1α-dependent pathways mediated the autophagy inactivation effects of PHD2 silencing.Conclusions Direct inhibition of PHD2 promotes renal epithelia cell survival against CoCl2-induced cell apoptosis/autophagy.Activation of the HIF-1α signaling pathway is required to reduce apoptosis and autophagy via up-regulating the expression of Bcl-xl protein.
ABSTRACT
Objective To investigate the role of hypoxia inducible factor(HIF)-prolyl hydroxylase 1 (HPHl)and factor inhibiting HIF-1(FIH-1)in placentas in the pathogenesis and development of severe pre-eclampsia.Methods RT-PCR and western blot analyses were used to detect the HPH1 and FIH-1expression levels in placentas of 34 patients with severe pre-eclampsia and 24 cases of term pregnancy (normal pregnancy group)and their correlations with symptoms were analyzed.Results (1)The HPHI mRNA and protein expression levels in placentas of severe pre-eclampsia group were 0.40±0.04 and 59.5±3.4 separately,significantly lower than those of normal pregnancy group,0.84±0.12 and 71.6±1.7(P<0.01).The FIH-1 mRNA and protein expression levels in placentas of severe pre-eclampsia group wereQ 31 ±0.05 and 45.6±2.4 separately,significantly lower than those of normal pregnancy group,0.43±0.04 and 54.9±2.1(P<0.01).(2)The mRNA and protein expression levels of HPH1 and FIH-1 in severe pre-eclampsia group were all negatively correlated with mean arterial pressure(MAP)[the Spearman correlation coefficient was-0.854(P<0.01)],urinary protein per 24 hours[the Spearman correlation coefficient was-0.936(P<0.01)1 and the occurrence of fundus oculi artery spasm[the Spearman correlation coefficient was-0.854(P<0.01)].(3)rrhe expression of HPHl mRNA in placentas of all the 58 cases WBB 0.58±0.27.higher than the expression of FIH-1 mRNA,which was 0.39±0.10.There was a positive correlation between them.The pearson correlation coefficient was 0.686(P<0.01).The expression of HPH1 protein in placentas of all the 58 cases was 64.5±6.7,higher than the expression of FIH-1,which was 49.4±5.2.There was a positive correlation between them.The Pearson correlation coefficient was 0.947(P<0.01).Conclusion The expression imbalance of HPH1 and FIH-1in palcenta may play an important role in the pathogenesis and development of severe pre-eclampsia through inhibiting HIF-1a.