Regulation Of Phosphatidylinositol 3-kinase/Akt In Hypoxia-induced Pulmonary Hypertensive Rat Is Dependent On Lysyl Oxidase Signaling

Background and ObjectivesPulmonary arterial hypertension(PAH)is a severe disease characterized bya progressive increase inpulmonary vascular resistance and remodeling.At cellular and molecular level,it is believed that the progressive eventsin PAH involve the participation of all cell-types and cellular components in the pulmonary arteries.The increased proliferation of pulmonary artery smooth muscle cell(PASMC),pulmonary arteryendothelial cells(PAEC),fibroblast activation and accumulation of extracellular matrix(ECM)are the main members that contribute to morphological changes in the pulmonary hypertensive vessel wall.There are increasing evidences that many growth factorsplay especially pivotal roles in the remodeling process in PAH by involving the initiation of proliferation,migration,constrictive phenotype shift and inhibition of apoptosis within PASMC.ECM is composed of two major structuralproteins,namely,collagen and elastin thatcan modulate the proliferation of vascular cells.Importantly,it revealed that the cross-linking of collagen is crucial forthe deposition of ECM and helpful for sustaining vascular tension and stiffness.Lysyl oxidase(LOX)is a copper-dependent enzyme that catalyzes covalent cross-linking of collagen.The family of LOXs includes LOX and four LOX-like proteins(LOXL-1,-2,-3,and-4)which are ubiquitously distributed inVSMCs of the lung,heart,skeletal muscle,kidney,and uterus.LOX is the main isoform responsible for 80%of LOXsenzyme activity.However,under normal condition,the expression of LOX,LOXL-1,LOXL-2,LOXL-3,and LOXL-4 appears to below in most tissues.Evidence showed that LOX,LOXL-1,LOXL-2,and LOXL-4 expression was elevated in lungs of patientswith idiopathic pulmonary arterial hypertension.In mice PASMC,LOX expression was induced after exposure to hypoxia.Moreover,administration of LOX inhibitor ?-aminopropionitrile(?-APN)attenuated hypoxia-exposed pulmonary hypertension and right ventricularhypertrophy in hypoxia-exposed mice.Current data on the modulation of LOXin hypoxia-exposed PAH are remained to be declared.Multiple mechanisms involving with many growth factors and cytokinestake part in hypoxia-mediatedLOX production.Studies have revealed that transforming growth factor-?(TGF-?),hypoxia-induciblefactor-1?(HIF-1?),and platelet-derived growth factor(PDGF)can promote LOX expression and activity,whereas tumor necrosis factor-a(TNF-?)and interferon-y(IFN-?)impair LOX expression and its activity in endothelial cells and in vascular wall.To be noted,hypoxia-induced upregulation of TGF-?,HIF-1?andPDGF is criticalto the PAH remodeling.Furthermore,the hypoxia-induced growth factors mentioned above are confirmed to activate PI3K/Akt signaling and promote PASMC proliferation and pulmonary artery thickening.Importantly,increasing evidence has supported the idea that the activation of PI3K/Aktplays a pivotal role in hypoxia-exposed PAH because its inhibitor LY294002 can amelioratethe proliferation of PASMC underhypoxic condition.Therefore,in this study we investigated expressionsof LOX and PI3K/Akt,the interaction between PI3K/Akt and LOX,and explored potential mechanism responsible for the development of hypoxia-induced PAH.Sprague-Dawley rats were exposed to hypoxia for 3-week to establish model of chronic pulmonary hypertension.The cross-linking of collagen and the expression of LOX,LOXL-1,LOXL-2,LOXL-3,LOXL-4,Akt and phospho-Akt(p-Akt)were assessed.Potent inhibitors of PI3K/Akt and LOX,wortmannin and?-APN,were administrated in rat model of hypoxia-induced PAH in order that the mechanism responsible for the development of hypoxia-induced PAH could be revealed.MethodsIn this study,we investigated the interaction between LOXs and PI3K/Akt and the mechanism responsible for the development of hypoxia-induced PAH.Firstly,male Sprague-Dawley rats were exposed to hypoxia for 3-week to establish model of chronic pulmonary hypertension.To assess the model,mean pulmonary artery pressure(mPAP)and the weight ratio of right ventricle to left ventricle plus septum weight(RV/LV+S)were measured.In addition,the ratio of wall area to total area(WA/TA)and the ratio of media thickness to external diameter(MT/ED)in pulmonary arterial vessel were observed as indicators of pulmonary arterial remodeling.Secondly,LOX inhibitor,?-APN,was concomitantly administrated to the hypoxia-exposed rat with intraperitonealinjection in order that the potential role of LOXs in the established PAH could be tested.The total collagen content was assessed based on thedetermination of hydroxyproline analysis with colorimetric method.Soluble collagen content was determined with Sircol Soluble Collagen Assay kit.Insoluble collagen equals total collagen minus soluble collagen.The degree ofcross-linking collagen was calculated as the ratio between the insoluble and soluble forms of collagen.In order to confirm that the change of collagen is relevant to LOX,we performed fluorescence spectrophotometry to detect LOX activity.We then sought to determine the expressions of LOX,LOXL-1,LOXL-2,LOXL-3 and LOXL-4 at mRNA level with quantitative real-time PCR and at protein level with western blot and immunohistochemistry.Furthermore,the expression of Akt and p-Akt was detected by western blot analysis.Finally,to understand whether PI3K/Akt is involved in the regulations of collagen cross-linking andexpression of LOX and LOXL-1-4,we applied wortmannin to inhibit PI3K/Aktsignaling.The expression of Aktand phospho-Akt(p-Akt)in pulmonary artery tissue was analyzed by western biotin order that the PI3K-LOX signaling pathway could be elucidated in hypoxia-induced pulmonary hypertensive rat.Results1.Indicators of hemodynamics were changed in hypoxia-exposed rats.After exposure to hypoxia,mPAP,RV/(LV+S),WA/TA and MT/ED in pulmonary arterial vessel were significantly increased in rat.However,after hypoxia-exposed rats were concurrently administrated with ?-APN orwortmannin,hypoxia-induced increases in mPAP,RV/LV+S,WA/TA and MT/ED were significantly reduced.2.LOX or PI3K inhibitor attenuates collagen cross-linkingin lung tissue of hypoxia-exposed rats.The contents of both insoluble and soluble collagen,collagen cross-linkingand LOX activity in hypoxia-exposed ratlung tissue were significantly elevated compared with normoxia-exposed rats,but the elevations were then reversed either by P-APN or by wortmannin,respectively.Importantly,the collagen cross-linking in hypoxia-exposed ratlung tissue was more significantly decreased than collagen content upon administration with either?-APN or wortmannin.3.LOX is upregulated in hypoxia-exposed ratpulmonary artery tissue..We performed quantitative real-time PCR and western blot todetect LOX,LOXL-1,LOXL-2,LOXL-3 and LOXL-4expressions in rat lungtissue.Interestingly,the results showed that hypoxia exposure enhanced mRNA and protein expressions of LOX and LOXL-1,whereas there was no effect of hypoxia on the expressions of LOXL-2,LOXL-3 and LOXL-4.Immunohistochemistry analysis confirmed that LOXand LOXL-1-4 proteinswere stained weakly or negatively inPASMC from normoxicrats.In contrast,LOX immunostaining was obviously strong in PASMC after exposure to hypoxia.4.Akt and phospho-Akt were upregulated in hypoxia-exposed ratpulmonary artery tissue.Importantly,western blot showed that the up-expressions of Akt and p-Akt were detected in hypoxia-exposed PAH rats.However,the upexpression of p-Akt,but not Akt in hypoxia-exposedrat pulmonary artery tissue was reduced by administration with wortmannin.5.PI3K/Akt signaling regulates LOX expressionin hypoxia-exposed rat pulmonary artery tissue.To understand whether PI3K/Akt is involved in the regulation of LOX,hypoxia-exposed rat was administrated with wortmannin to inhibit PI3K/Akt signaling.Although there was no significant change in the ratio of p-Akt to Akt(p-Akt/Akt)between hypoxic and normoxic pulmonary artery tissue,the administration with wortmannin significantly reduced the ratio of p-Akt/Akt in hypoxia-exposed rat.Notably,wortmannin effectively downregulated LOX expression at protein and mRNA levels but not LOXL-1,LOXL-2,LOXL-3 or LOXL-4 in a PI3K/Akt-dependent manner and reversed the elevated cross-linking of collagen and pulmonary arterial remodeling in hypoxia-exposed rats.Conclusions1.The present study provides evidence that the upregulation of LOX and LOXL-1 expressions in rat pulmonary artery tissue and the elevation of collagen content and cross-linking in lung tissueare involved in the hypoxia-induced PAH and pulmonary arterial remodeling.On contrast,there was no effect of hypoxia on the expression of LOXL-2,LOXL-3 and LOXL-4 in rat pulmonary artery.2.Hypoxia leads to the upregulationof Akt and p-Akt in rat pulmonary artery tissue.Wortmannin as a PI3Kinhibitor can reduce the ratio of p-Akt/Akt in hypoxia-exposed rat pulmonary artery tissue.3.LOX expression,but not LOXL-1,LOXL-2,LOXL-3,or LOXL-4 in rat pulmonary arterytissue has been demonstrated to be dependent on PI3K/Akt signaling in hypoxia-exposed PAH rat.4.Suppression of PI3K-Akt signaling pathway may contribute to the amelioration of hypoxia-induced PAH via targeting LOX.