| Background The hypoxia-inducible factor-1 (HIF-1) is a heterodimeric transcription factor composed of an oxygen-sensitiveαsubunit (HIF-1α) and a constitutively expressedβsubunit. HIF-1α, as a functional subunit, regulates the expression of more than 100 genes involved in cellular adaptation and survival and then plays a crucial role in the cellular response to the stress of hypoxia. HIF-1αis also a transcriptional regulator which plays a key role during the development of hypoxic pulmonary vascular remodeling (HPSR). Recently, people have found small ubiquitin-related modifier-1 (SUMO-1), which can covalently attach to HIF-1α. The process that SUMO covalently attach to proteins is known as SUMOylation. The SUMOylation is a dynamic process reversible, which can be removed from the substrate by specific protease SENP-1 as de-SUMOylation. The reversibly SUMOylational modification of HIF-1αcan change the stability and transcriptional activity of HIF-1αunder hypoxic conditions..Objective To evaluate the role of SUMO-1 and SENP-1 in regulating HIF-1αduring the development of hypoxic pulmonary hypertension(HPH)and provide the theoretical basis of mechanism for HPH. We investigated SUMOylation of HIF-1αby SUMO-1, and the expression patterns of SUMO-1, SENP-1, HIF-1α, and vascular endothelial growth factor (VEGF), a well-characterized target gene of HIF-1α, as well as their relationship to each other in the pulmonary arterioles of rats and patients at different phases of HPH development.Methods The study consisted of two parts. (1) Forty adult male Wistar rats were randomly divided into 5 groups, 8 rats in each group, and exposed to normoxia (control group) or exposed to hypoxia for 3, 7, 14 or 21 d, respectively. The mean pulmonary artery pressure (mPAP) was measured by right-heart catheterization, while right ventricular hypertrophy index (RVHI) was calculated by the ratio of right ventricle to the left ventricle plus septum, and hypoxic pulmonary vascular remodeling (HPSR) was observed with morphmetric analysis. The mRNA and protein expression of HIF-1αand VEGF in pulmonary arterioles were detected by in situ hybridization and immunohistochemistry, respectively. Reverse transcription- polymerase chain reaction (RT-PCR) and in situ hybridization were used to determine the mRNA expression of SUMO-1 and SENP-1. Immunohistochemistry and Western blot were adopted to determine the protein expression of SUMO-1 and SENP-1. Co-Immunoprecipitation were adopted to determine SUMOylation of HIF-1αby SUMO-1. (2) Thirty-six patients were divided into three groups: chronic obstructive pulmonary disease (COPD) with pulmonary pertension (PH) group (PH group), COPD without PH (COPD group) and control group. Their lung tissues were collected from surgically resected specimens. The HPSR was observed with morphmetric analysis too. The expression of HIF-1α, VEGF, SUMO-1 and SENP-1 in pulmonary arterioles was examined by in situ hybridization and immunohistochemistry. The SUMOylation of HIF-1αby SUMO-1 was examined by Co-Immunoprecipitation.Results (1) mPAP in hypoxic rats increased significantly after 7 d of hypoxia, reached its peak after 14 d of hypoxia, and then remained stable. The hypoxic rats developed HPSR after 7 d of hypoxia, and more significantly after 14 d of hypoxia. RVHI in hypoxic rats was markedly increased after 14 d of hypoxia. HIF-1αmRNA is positively stained in control and increased dramatically after 14 d of hypoxia. HIF-1α?protein was poorly positive in control, up-regulated markedly after 3 d and 7 d of hypoxia, and then declined slightly after 14 d and 21 d of hypoxia. VEGF mRNA and protein were poor positively stained in control, increased markedly after 7 d of hypoxia, and then reached their peak after 14 d of exposure to hypoxia. SUMO-1 mRNA and protein expression in pulmonary arteriole walls was markedly increased in pulmonary arteriole walls after 3 d of exposure to hypoxia, reached its peak after 14 d of hypoxia, then weakened after 21 d of hypoxia, but was still higher than that in the control. SENP-1mRNA and protein were positively stained in control. SENP-1 mRNA expression had little changes after exposure to hypoxia compared with the control, however, SENP-1 protein expression was increased Slightly after 3d of hypoxia, and declined gradually after 7 d of hypoxia. The results of Co-immunoprecipitation showed that SUMOylation of HIF-1a almost did not happen in lung tissues of control rats. But it was markedly positive after 3 and 7 d of hypoxia in lung tissues of rats, and reached its peak after 14 d of hypoxia, then remained stabilized. Linear correlation analysis showed that SUMO-1 mRNA and protein was positively correlated with SUMOylation of HIF-1αby SUMO-1. SENP-1 protein was negatively correlation with SUMOylation of HIF-1αby SUMO-1. While SUMOylation of HIF-1αby SUMO-1 was positive correlation with HIF-1αmRNA and HIF-1αprotein,HIF-1αprotein was positively correlated with VEGF mRNA and VEGF protein, HIF-1αprotein and VEGF protein were positively correlated with mPAP and pulmonary arteriole remodeling index. SUMOylation of HIF-1αby SUMO-1 was positive correlation with VEGF mRNA and VEGF protein,mPAP,RVHI,WA%,WT%.(2) The HPSR developed in patients from COPD group, and more significantly in patients from PH group. HIF-1αmRNA and protein were poorly stained in pulmonary arteriole walls in control group, increased markedly in COPD group and then HIF-1αmRNA expression increased further while HIF-1αprotein expression changed little in pulmonary arteriole walls in PH group compared with COPD group. VEGF mRNA and VEGF protein were poorly stained in pulmonary arteriole walls in control group, up-regulated markedly in COPD group, and increased further in PH group. SUMO-1 mRNA and SUMO-1 protein were poorly stained in pulmonary arteriole walls in control group, up-regulated markedly in COPD group, and increased further in PH group. SENP-1 mRNA expression remained unchanged in pulmonary arteriole walls between different groups, while SENP-1 protein was strongly stained in pulmonary arteriole walls in control group, weakened remarkably in COPD group and decreased further in PH group. The results of Co-immunoprecipitation showed that SUMOylation of HIF-1a almost did not happen in lung tissues of control group. But it was markedly positive in COPD group, and increased further in PH group. Linear correlation analysis showed that SUMO-1 mRNA and SUMO-1 protein was positively correlated with SUMOylation of HIF-1αby SUMO-1. SENP-1 protein was negative correlation with SUMOylation of HIF-1αby SUMO-1. While SUMOylation of HIF-1αby SUMO-1 was positive correlation with HIF-1αmRNA and HIF-1αprotein,HIF-1αprotein was positively correlated with VEGF mRNA and VEGF protein, HIF-1αprotein and VEGF protein were positively correlated with pulmonary arteriole remodeling index and pulmonary artery systolic pressure. SUMOylation of HIF-1αby SUMO-1 was positively correlated with VEGF mRNA and VEGF protein,pulmonary arteriole remodeling index,pulmonary artery systolic pressure.Conclusion (1) Both HIF-1αand VEGF are involved in the pathogenesis of HPH. HIF-1αmay up-regulate the expression of VEGF via transactivation, resulting in the development of HPH. (2) The dynamic expression of SUMO-1 may play a role in stabilizing protein and action of HIF-1a by SUMOylation, then being involved in the development of PH; SENP-1 protein can be degradated in hypoxia, The dynamic expression of SENP-1 protein may play a role in implicating in the development of PH.
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