| BackgroundHypoxia-inducible factor (HIF) functions as a master regulater of O2 homeostasis by playing critical roles in organism, it is also a transcriptional regulator which plays a key role during the development of hypoxia-induced pulmonary hypertension (HPH). The heterodimeric HIF complex is regulated by three HIF prolyl hydroxylases, PHD1, PHD2, PHD3, through proteolysis of itsα-subunits, following oxygendependent hydroxylation of specific prolyl residues. OS-9 interacts with both HIF-1αand PHD2 and promotes PHD-mediated hydroxylation of HIF-1αin cultured cells. The abundance of PHD1 and PHD3 can also be regulated via their targeting for proteasome-dependent degradation by the E3 ubiquitin ligases Siah.ObjectiveTo investigate the dynamic levels of Siah and OS-9 in HPH rats and COPD patients. To study the correlation relationship between Siah,OS-9,PHDs,HIFs-αand the role of Siah and OS-9 in regulating PHDs during the development of HPH. To investigate the dynamic expression of HIF-1αDNA binding activity and protein levels in pulmonary of rats with HPH and provide more theoretical basis of HIF-1αin HPH development.MethodsThe study consisted of two parts. 1) Models of chronic HPH rats were duplicated by anoxia (respired mixted gases containing 10% O2 , 8 hours per day for 21 days intermittently). After anoxia for 3d, 7d, 14d and 21d, mean pulmonary artery pressure (mPAP), was measured by right-heart catheterization, right ventricular hypertrophy index (RVHI) was calculated by the ratio of right ventricle to the left ventricle plus septum, and hypoxic pulmonary vascular remodeling (HPVR) was observed with morphmetric analysis. RT-PCR and in situ hybridization were used to determine the expression of mRNA levels. Immunohistochemistry and western blot were adopted to determine the expression of protein levels. Electrophoretic mobility shift assay(EMSA) were used to determine the DNA binding activity of HIF-1α. 2) Small pulmonary arterial remodeling was observed in COPD and the control patients by morphometric analysis. The expression of Siah1,Siah2,OS-9,PHDs and HIFs-αin lung tissue was examined in COPD patients and the control by in situ hybridization and immunohistochemistry.ResultsmPAP increased significantly after 7d of hypoxia (P<0.05, compared with group Control), reaching its peak after 14d of hypoxia, and then remained stable. Pulmonary artery remodeling developed significantly after 14 d of hypoxia. HIF-1α?protein was poorly positive in control, markedly up-regulated after 3d and 7d of hypoxia(P<0.05, compared with group Control), and then declined slightly after 14d and 21d of hypoxia. HIF-1αmRNA increased dramatically after 14d of hypoxia (P<0.05, compared with group Control). PHD1, PHD2 mRNA and protein was positive in group Control. PHD2 mRNA and protein were up-regulated after 3d of hypoxia(P<0.05, compared with group Control), reaching its peak after 14d of hypoxia while PHD1 protein declined after 14d of hypoxia(P<0.05, compared with group Control) without statistic mRNA changing. PHD3 mRNA and protein were detected at a low level in control, markedly up-regulated after 3d of hypoxia(P<0.05, compared with group Control), and then PHD3 mRNA kept at high level while PHD3 protein declined after 14d of hypoxia(P<0.05, compared with H7). The DNA binding activity of HIF-1α? was weak in control rats, increased dramatically after 3d of hypoxia, and reaching its peak after 14d of hypoxia. Siah1 mRNA and protein were poor positively stained in control, increased markedly after 7d of hypoxia(P<0.05, compared with group Control) and reaching its peak after 14d of hypoxia, and then declined a little after 21d of hypoxia. Siah2 showed the same dynamic tendency with Siah1. OS-9 mRNA was positively in control, markedly decreased after 3d of hypoxia(P<0.05, compared with group Control), reaching its lowest lever after 14 d of hypoxia. Linear correlation analysis showed that Siah protein were positively correlated with mPAP,RVHI,WA %,HIF-2αprotein and HIF-3αprotein while OS-9 protein negatively correlated with mPAP,RVHI,WA %,LA %,HIF-2αprotein and HIF-3αprotein. In COPD subject, HIFs-αmRNA and proteins increased significantly, PHD1 protein decreased without mRNA change. PHD2 mRNA and protein increased significantly. PHD3 mRNA increased without protein changes.Siah1 mRNA and protein increased . Siah2 showed the same dynamic tendency with Siah1. OS-9 mRNA and protein decreased in COPD. Linear correlation analysis showed that Siah protein were positively correlated with HIFs-αprotein while OS-9 protein negatively correlated with HIFs-αprotein.ConclusionAlterlation of HIF-1αDNA binding activity may have an important mechanism during the development of hypoxic pulmonary hypertension in rat HPH model . Siah1 and Siah2 may target PHDs for proteasome-dependent degradation in rat lung after hypoxia exposure and therefore resulted in HIFs-αaccumulation. OS-9 may interact with PHDs and HIFs-αand accelerate HIFs-αdegradation via ubiquitination and proteasome destruction in rat hypoxia model. COPD subject may have the same mechanism.
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