| BackgroundHypoxic pulmonary arterial hypertension(HPH)is a clinical disease caused by the continuous hypoxia,and it is characterized by pulmonary vasoconstriction,pulmonary remodeling as well as pulmonary embolism formation,eventually result in abnormal hemodynamic and right heart failure.HPH is a species of clinical familiar disease,but these lake of useful drugs to cure it.The pathological process is complicated,which is involving multiple pathways and many kinds of vascular molecules.It is clear that the pulmonary vasoconstriction and pulmonary revascularization are the important pathological characteristics in HPH,more importantly,the pulmonary smooth muscle cells(PASMCs)are involved in this process.The excessive proliferation and migration,as well as the phenotype transformed from differentiated to dedifferentiated types of PASMCs could induce thicken of the pulmonary vascular wall,increase of pulmonary arterial pressure and pulmonary vascular resistance.Thus,the excessive proliferation and migration of PASMCs is research hot spot presently because that has been considered to be the key process of pulmonary vascular remodeling.The molecular mechanism of PASMCs hypertrophy may involve genetic factors,molecular medium,cells abnormal,etc.Impaired endothelial cells,inflammatory reaction and abnormal immune participate in this process,moreover,imbalance of a variety of vascular active substances promote it,eventually leading to pulmonary vasoconstriction and pulmonary vascular structure reconstruction.DJ-1,also as known as park7,is expressed ubiquitously in various tissues,although it is cytoplasmic protein,but it is expressed in the nucleus and cytoplasm.It was well known to be the mutant gene which could cause familial Parkinson's disease.In addition,DJ-1 has biological function in many cells,commonly involving in transcriptional regulation,anti-oxidative stress and tumor formation,and DJ-1 was indicated to protect against cardiovascular disease through mitochondrial pathway.Previously study indicated that DJ-1 could regulate the expression of caveolin-1(Cav-1),however the effects of DJ-1 in HPH has not been reported.Caveolae is a special vesicular depression on the cell membrane,which is composed to caveolin protein.Cav-1 is expressed in various cells,and plays important roles in the signal transduction pathways.Studies have showed that the level of Cav-1 was decreased in patients with idiopathic pulmonary arterial hypertension(IPAH),and it has been used as a biological marker of IPAH.So Cav-1 has become an Indispensable molecules during the pathological process of IPAH.Therefore,we speculate that DJ-1 and Cav-1 may affect the pathological process of PAH by interacting with each other.AimThe subject intends to use continuous hypoxia environment to construct HPH rats model,detesting the hemodynamic characteristics and the protein expression of Cav-1 and DJ-1 in the tissues of HPH rats.Transfected the overexpression vector and DJ-1 and silencing vector of Cav-1 into hypoxia induced PASMCs respectively demonstrated their effects to PASMCs proliferation,migration,Ca2+ concentration and transformation cell phenotype.Our subject will explore the role of DJ-1 and Cav-1 in the HPH and its potential molecular mechanisms,provide new targets genes and theoretical basis for the clinical treatment of HPH.Methods1.The protein expression of DJ-1 and Cav-1 in rats model of PAH.Male wild-type(WT)rats and DJ-1 knockout(KO)rats were exposed to hypoxia(10%O2 environment),after 4 weeks the rats models of HPH were constructed successfully;the normoxia group were maintained under normoxic conditions with normal diet during the same period.Then the hemodynamic indexes and expression of DJ-1 and Cav-1 were detected by multi-function physiologic recorder and western blot,respectively.2.Effects of DJ-1 overexpression or Cav-1 silence on hypoxic induced PASMCs.PASMCs were exposed in the hypoxic condition,transfected with DJ-1 overexpression vector or Cav-1 silencing vector and then detected the cell proliferation,migration,intracellular Ca2+ concentration and protein levels of contractile phenotype markers by MTT assay,Transwell filter assay,Fluo3-AM and western blot respectively,in order to determine the effects of DJ-1 on the hypoxia induced PASMCs.3.Regulation of DJ-1 overexpression on the protein levels of ERK1/2 and TGF?1/Smad pathways.PASMCs were transfected with DJ-1 overexpression vector or Cav-1 silencing vector after induced by hypoxia,then the levels of molecules in ERK1/2 and TGF?1/Smad were detected by western blot to investigate the molecular mechanism of DJ-1 and Cav-1 on HPH.Results1.The expression of DJ-1 and Cav-1 protein were abnormal in HPH rats.The rats model of HPH were constructed successfully,and the protein levels of DJ-1 and Cav-1 in the tissues of HPH rats were decreased.Additionally,the symptoms of HPH rats in DJ-1 KO rats were worse than WT HPH rats,suggesting that DJ-1 may play important role in the pathological process of HPH.2.DJ-1 may regulate the biological function of PASMCs through Cav-1.PASMCs were transfected with DJ-1 overexpression vector or Cav-1 silencing vector after induced by hypoxia,the results showed that the cell proliferation,cell migration,intracellular calcium concentration were increased significantly,as well as the levels of contractile phenotype markers were decreased in hypoxic PASMCs.However,overexpression of DJ-1 inhibited the effects of DJ-1 on hypoxic PASMCs,additionally,effects of DJ-1 on hypoxic PASMCs were reversed by silence of Cav-1.Thus we suspected that DJ-1 could protect against PASMCs injure through Cav-1.3.DJ-1 may inhibit the activation of ERK1/2 and TGFj31/Smad by regulating Cav-1.PASMCs were transfected with DJ-1 overexpression vector or Cav-1 silencing vector after induced by hypoxia to investigated the molecular mechanism of effects of DJ-1 on hypoxic PASMCs proliferation and migration.The results showed that ERK1/2 and TGF?1/Smad were activated in hypoxia induced PASMCs,which were inhibited after transfected DJ-1 overexpression vector.On the contrary,the Cav-1 silence reversed the effects of DJ-1,suggesting that DJ-1 may inhibit the activation of ERK1/2 and TGF?1/Smad pathway,as well as the effects of hypoxia induced on the PASMCs by regulating Cav-1.Conclusion1.Expression of DJ-1 and Cav-1 protein were reduced in pulmonary tissues of HPH rats compared with normal rats;expression of DJ-1 and Cav-1 were reduced in hypoxia induced PASMCs compared with normal PASMCs.2.Overexpression of DJ-1 inhibited the cell proliferation,migration,intracellular calcium concentration and cell phenotype conversion induced by hypoxia.Moreover,the silence of Cav-1 improved the cell proliferation,migration,intracellular calcium concentration and cell phenotype conversion of PASMCs induced by hypoxia.Effects of DJ-1 were reversed after PASMCs were co-transfected with DJ-1 overexpression vector and Cav-1 silence vector.3.Overexpression of DJ-1 inhibited the activation of ERK1/2 and TGF?1/Smad pathway,and these effects were reversed after Cav-1 silence vector transfection.In conclusion,DJ-1 may inhibit the activation of ERK1/2 and TGF?1/Smad pathway,cell proliferation,migration,intracellular calcium concentration and cell phenotype conversion of PASMCs induced by hypoxia. |
PDF Full Text Request