The Effects And Mechanisms Of Apoptosis On The Reversal Of Hypoxic Pulmonary Arterial Remodeling During Reoxygenation

Hypoxic pulmonary hypertension（HPH）,a chronic progressive disease mainly characterized by pulmonary vasoconstriction and vascular remodeling,could result from chronic lung diseases or occur as an idiopathic case at high altitude,which is a common type of pulmonary hypertension（PH）.Generally,long-term and sustained pulmonary hypertension could lead to serious heart failure and the death of the patients.As the most significant pathological changes,hypoxic pulmonary arterial remodeling is the key factor contributing to the prolonged pulmonary hypertension.Although there have been many kinds of medications for the treatment of pulmonary hypertension,most of them could not effectively inhibit and ameliorate the occurrence of pulmonary arterial remodeling.Recently,large amounts of studies demonstrated that hypoxic pulmonary arterial remodeling,especially thickened medial layers,could gradually regress once reoxygenated.During the reversal of pulmonary arterial remodeling,namely,the thinning of thickened pulmonary arterial wall,we hypothesized it must be associated with the outnumbering of dying cells to newborn ones.Only in this case,the increased cells in pulmonary arteries could be effectively eliminated,resulting the reversal of pulmonary arterial remodeling.Therefore,the dynamic changes of the balance between “cellular death” and “cellular proliferation” will be the crucial influence factor.Recently,the important modes of cell death include apoptosis,autophagy,necrosis and necroptosis.During the reoxygenation-induced reversal of hypoxic pulmonary arterial remodeling,which one results in the reversal process remains unknown.Generally,for aerobic cells,hypoxia and reoxygenation are considered as the common stress stimuli,which could influence normal cellular metabolism and lead to their functional disorders even cell death.For example,during the myocardial ischemia-reperfusion（hypoxia-reoxygenation）injury,there are always oxidative respiration burst and increased ROS.The increased ROS,regarded as a serious damage factor,could seriously trigger some damages for the intracellular macromolecular proteins and organelles,for instance mitochondrial damage,which finally induced myocardial apoptosis.During the reoxygenation-induced reversal of hypoxic pulmonary arterial remodeling,whether there was oxidative stress-induced damages required further studies.【Aims】 1.To determine the changes of hypoxic pulmonary arterial remodeling during reoxygenation and its related mechanisms.2.To further investigate the reversal mechanism of hypoxic pulmonary arterial remodeling in the terms of oxidative damages.【Methods】 1.Adult male SD rats were randomly divides into: normoxia for 4 weeks group（N）,hypoxia for 4 weeks group（H）,reoxygenation for 1 week after hypoxia for 4 weeks（R1）,reoxygenation for 6 weeks after hypoxia for 4 weeks（R6）.Rats in N group were maintained in normal environment closed to the hypoxic chamber for 4 weeks.Rats in H group were intermittently housed in a hypobaric hypoxia chamber,with fractional inspired oxygen（FiO₂）0.10,8 h/d for 4 weeks.Rats in R1 and R6 groups were first exposed to hypoxia for 4 weeks and then transferred to normoxia environment（Fi O₂ 0.21）for 1 week and 6 weeks,respectively.After all interventions,examine the changes of right ventricle systolic pressure（RVSP）,right ventricle（RV）hypertrophy（RV/（LV+Sep））,medial thickness（MT）and medial area（MA）during hypoxia-reoxygenation.2.Immunohistochemical staining against α-SMA,cleaved-caspase3,Bax,Bcl-2,LC3 as well as P62 and TUNEL staining were performed in the lung sections of different groups.Moreover,the expression of cleaved-caspase3/PARP,Bcl-2,Bax,PCNA,LC3 and P62 were determined through Western blotting analysis.The amount of H₂O₂ in fresh lung tissues was immediately examined using a commercially available Hydrogen Peroxide Assay Kit.3.Rat primary PASMCs were prepared through tissue explant method.PASMCs from passages 3 to 5 were exposed to hypoxia（5% O₂）for 0,24,36,48,60 and 72 h to determine the most significant point for the PASMCs growth through MTT assay.On this basis,PASMCs were then transferred to normoxia for 0,24,36,48 and 60 h to observe the changes of PASMCs growth rates during reoxygenation.4.The levels of apoptosis and proliferation of PASMCs during hypoxia-reoxygenation were examined through Annexin V-PI staining,Brd U assay and Western blotting analysis.Moreover,the expressions of LC3 and P62 in PASMCs during hypoxia-reoxygenation were investigated through Western blotting analysis to determine the level of PASMCs autophagy.5.The total intracellular ROS and mitochondrial ROS in cultured PASMCs during hypoxia-reoxygenation were determined using DCFH-DA and Mito SOX Red fluorescent dyes.Moreover,after the clearance of ROS by NAC and Mito TEMPO,the total intracellular ROS and m ROS were examined again to determine the resource of increased ROS.The level of m ROS was further tested in the isolated mitochondria.Finally,after the clearance of ROS,the levels of PASMCs apoptosis in different groups were examined to investigate the effect of ROS on the apoptosis of PASMCs.6.The mitochondrial membrane potential（?Ψ）was examined using JC-1 dye.The mitochondrial function was further determined according to the production of ATP.Moreover,the expression of Bax and Bcl-2 as well as the distribution of cytochrome C between mitochondria and cytoplasma was analyzed through Western blotting to investigate the changes of mitochondrial permeability and PASMCs apoptosis during hypoxia-reoxygenation.【Results】 1.RVSP,RV/（LV+Sep）,MT% and MA%,associated with the functional and structural characters of HPH,gradually ameliorated when HPH rats were transferred to normal condition.2.The results demonstrated that the apoptosis of PASMCs increased significantly during reoxygenation,however that the PASMCs autophagy reversed to normal level.Moreover,the amount of H₂O₂ increased significantly during reoxygenation.3.The results of MTT assay demonstrated that hypoxia accelerated the growth of PASMCs,especially hypoxia for 48 h,and that reoxygenation following hypoxia decelerated its growth rates,particularly reoxygenation for 24 h.4.During reoxygenation,the apoptosis and proliferation of PASMCs increased obviously as compared with normoxia group,especially the apoptosis.However,the PASMCs autophagy reversed to normoxia level.5.During reoxygenation,both total intracellular ROS and m ROS increased significantly as compared with normoxia group,which were inhibited equally by the intervention of NAC and Mito TEMPO.The results of isolated mitochondria also indicated an increased m ROS during reoxygenation.The clearance of ROS/m ROS could result in a significant inhibition of PASMCs apoptosis.6.Reoxygenation significantly decreased the mitochondrial membrane potential,damaged the mitochondrial synthetic function of ATP and increased the release of cytochrome C from mitochondria to cytoplasm,which was effectively ameliorated by the clearance of ROS/m ROS.【Conclusions】 1.Hypoxic pulmonary arterial remodeling,the characteristic pathological change of hypoxic pulmonary arterial hypertension,could gradually reverse during reoxygenation,and the PASMCs-specific apoptosis participated in this reversal.2.Reoxygenation-induced large amounts of ROS in PASMCs,especially mitochondrial ROS,resulted in the mitochondrial damages,which finally initiated the internal apoptotic programs of PASMCs.