The Roles And Mechanisms Of Aquaporin 1 In Hypoxia-induced Pulmonary Hypertension

BackgroundHypoxia-induced pulmonary hypertension?HPH?is a serious respiratory disease caused by longstanding chronic hypoxia exposure.HPH is characterized by hypoxia-induced pulmonary vasoconstriction,pulmonary arterial remodeling and subsequent pulmonary artery pressure increasing,and ultimately leads to right ventricular failure and premature death.Although the important advances have been achieved on the disease over the recent years,no specific treatment for HPH exists yet.Hypoxia-induced excessive proliferation and migration of PASMCs are key factors lead to pulmonary arterial remodeling and vascular resistance,in the development of HPH.However,the exact molecular mechanism under the process of PASMC dysfunction in the disease is rarely known.A better understanding of the mechanisms and novel effective treatments for HPH are therefore urgent.The aquaporins?AQPs?,a family of 13?AQP0-AQP12?small water-transporting membrane proteins,are selectively expressed in cells of various organs.It has been widely reported that aquaporin1?AQP1?is up-regulated in several different types of cancers,which involved in cancer cell proliferation,migration and tumor angiogenesis.In lung,AQP1 is expressed in alveolar type II cell and microvascular endothelia associated with airways,and recent evidence indicate that it is also expressed in PASMCs.Interestingly,hypoxia exposure markedly up-regulates AQP1expression and down-regulates AQP5 expression in animal lungs,whereas other AQPs levels were not changed.It has been reported that AQP1 is required for PASMCs proliferation and migration in response to hypoxia in vitro.Thus,we hypothesized that AQP1 may participate in mediating pulmonary vascular remodeling in the development of HPH.Therefore,the primary objective of this study is to investigate the roles of AQP1 in the pathogenesis of HPH,and explore the possible mechanisms.Methods1.Animals and Experimental DesignFourteen male adult wild type CD1 mice,fourteen male adult AQP1-nullmice,and ten male Sprague-Dawley rats?200-250 g?were used.The wild type mice were randomly divided into two groups?seven mice per group?:?1?Wild type normoxia group and?2?Wild type hypoxia group;The AQP1-nullmice were randomly divided into two groups?seven mice per group?:?3?AQP1-nullnormoxia group and?4?AQP1-null hypoxia group.The rats were randomly divided into two groups?five mice per group?:?5?normoxia group and?6?hypoxia group.For normoxia groups,the mice and rats were housed at room air with normobaric normoxia?FIO₂ of 0.21?,and for chronic hypoxia groups,the mice and rats were exposed to normobaric hypoxia?FIO₂of 0.10?for 4 weeks.2.Analysis of lung AQP1 expression after chronic hypoxia exposureAfter chronic hypoxia exposure for four weeks,the left lung tissues were homogenized and AQP1 expression in lung was analyzed by Western Blot.The right lungs were fixed and processed for paraffin embedding and then the sections were cut.?-SMA mouse antibody and anti-AQP1 rabbit antibody were used for double immunofluorescence staining,to analysis AQP1 expression in pulmonary arterial vascular.3.Hemodynamic experiments and morphological investigationAfter the mice were anesthetized,the right ventricle systolic pressure?RVSP?was then recorded using Power Lab Software.The right ventricle?RV?and left ventricle plus septum?LV+S?were collected,and the weight ratio of?RV/LV+S?was calculated as an index of RV hypertrophy.The lung sections were stained with hematoxylin and eosin.The medial wall area and total vessel area of pulmonary arteries were measured.The percent medial wall area?WA%?were calculated to present pulmonary vascular structure remodeling.The medial wall thickness of pulmonary vascular was examined by elastic Van Gieson.4.Immunohistochemical staining of pulmonary arteriolar remodeling inhypoxia-induced pulmonary hypertension miceThe lung sections were stained with anti-?-SMA antibody,anti-PCNA antibody and anti-CD31 antibody.Immunoreactivity was visualized using diaminobenzidine.5.Hypoxia-inducible factor?HIF?expression in lungs of hypoxia-inducedpulmonary hypertension miceThe lung tissues from 4 groups were homogenized and then HIF-1?and HIF-2?expression in lungs was analyzed by Western Blot.6.Primary culture of pulmonary arterial smooth muscle cells?PASMCs?,and cellproliferation,migration,cell cycle and apoptosis detection?1?Primary PASMCs were cultured by collagenase digestion method and used for experiments between passages 3 and 5.For all experiments,cells were divided into 4groups:wide type normoxia group,wide type hypoxia group,AQP1-null normoxia group and AQP1-null hypoxia group.?2?AQP1 expression in PASMC was analyzed by western blot and immunofluorescence staining with anti-AQP1 antibody.?3?The role of AQP1 on hypoxia-induced PASMC proliferation was detected by MTT analysis,anti-Ki67 antibody immunofluorescence staining and anti-PCNA antibody western blot.?4?Cell cycle and apoptosis relevant protein was analyzed by western blot to explore the effect of AQP1 deficiency on PASMC cell cycle and apoptosis.?5?Wound healing assay and Rhodamine phalloidin staining for cytoskeleton were used to explore the effect of AQP1 deficiency on hypoxia-induced PASMC migration and cytoskeleton reorganization.Results1.Chronic hypoxia up-graduates AQP1 expression in lungs and pulmonary arteries of mice and rats.2.The RVSP of wide type hypoxia group was increased significantly compared with the normoxia groups.However,the RVSP of AQP1 deficiency hypoxia group was much lower than that of wide type hypoxia group.In accordance with the RVSP,hypoxia-induced elevation of the ratio of RV/LV+S was inhibited by AQP1deficiency.3.Hypoxia markedly increased the WA%,number of PCNA-positive cells,?-SMA optical density value,and CD31 positive area in pulmonary arterioles of wild-type mice,as well as vascular muscularization,compared with normoxia control mice,whereas these indexes of AQP1-null mice under hypoxia was much lower.4.AQP1 deficiency inhibited hypoxia-induced HIF-1?and HIF-2?up-regulation in lung.5.Hypoxia up-regulated AQP1 expression in PASMCs cytoplasm.The viability of AQP1 deficient PASMCs was significantly inhibited compared with that of the wide-type cells in normoxia and hypoxia.6.Consistent with viability assay,AQP1 deficiency inhibited the up-regulation of Ki67 induced by hypoxia.PCNA and c-Myc expression in PASMCs,detected by Western Blot,exhibited similar results.7.Hypoxia down-regulated the protein level of cleaved-PARP,cleaved-caspase 9 and cleaved-caspase 3 in wide-type PASMCs.AQP1 deficient up-regulated the expression of the apoptosis related protein compared with that of wide-type PASMCs.For cell cycle detection,in wide-type PASMCs hypoxia remarkably enhanced the expression of cyclin D and decreased that of p27.However,the expression of cyclin D and p27were obviously reversed in AQP1 deficient PASMCs compared with wide-type PASMCs under hypoxia.8.AQP1 deficiency attenuated hypoxia-induced migration and inhibited cytoskeleton reorganization of PASMCs.Conclusion1.Chronic hypoxia exposure up-graduated AQP1 expression in lungs and pulmonary arteries of mice and rats.2.AQP1 deficiency attenuated hypoxia-induced pulmonary hypertension and vascular remodeling.3.AQP1 deficiency reversed hypoxia-induced HIF accumulation in lung.4.AQP1 deficiency reduced hypoxia-induced PASMCs abnormal proliferation by arresting cell cycle at G0/G1 phase.5.AQP1 deficiency reversed hypoxia-induced PASMCs apoptosis resistance.6.AQP1 deficiency attenuates hypoxia-induced migration of PASMCs.