ME減弱博來霉素在雌激素缺乏大鼠中所導(dǎo)致的PAH和肺纖維化參考模板

18/182-METHOXYESTRADIOLATTENUATESBLEOMYCIN-INDUCEDPULMONARYHYPERTENSIONANDFIBROSISINESTROGEN-DEFICIENTRATS（2ME減弱博來霉素在雌激素缺乏大鼠中所導(dǎo)致的肺動脈高壓和肺纖維化）TofovicSP,ZhangX,JacksonEK,etal.

[J].Vascularpharmacology,2009,51(2-3):190.Abstract（摘要）Pulmonaryhypertension(PH)isacommonandlife-threateningcomplicationofpulmonaryfibrosis.（肺動脈高壓（PH）是一種常見和威脅生命的肺纖維化的并發(fā)癥。）Estradiol(E2)isprotectiveinexperimentalPH,anditsnon-estrogenicmetabolite2-methoxyestradiol(2ME)preventsthedevelopmentandretardstheprogressionofmonocrotaline-inducedPHinmaleandfemalerats.（雌激素能在試驗性肺動脈高壓中提供保護并且它的非雌激素代謝物2-甲氧基雌二醇能在野百合堿誘導(dǎo)的雌性和雄性大鼠肺動脈高壓模型中阻止疾病進程的發(fā)展。）However,theeffectsofE2and2MEonpulmonaryfibrosisandassociatedPHhavenotbeenexamined.（然而，雌二醇和2-甲氧基雌二醇對肺纖維化和涉及肺動脈高壓的作用還沒有研究出來。）Therefore,wecomparedthegrowth-inhibitoryeffectsofE2and2MEinhumanlungfibroblasts(hLFs)andpulmonaryvascularsmoothmusclecells(hPASMCs),andweinvestigatedtheeffectsofestrogendeficiencyand2MEonbleomycin-inducedpulmonaryfibrosisandPH.（因此，我們對雌二醇和2-甲氧基雌二醇在人肺成纖維細(xì)胞（hlfs）和人肺血管平滑肌細(xì)胞（hpasmcs）中的生長抑制作用進行了對比，我們還調(diào)查了雌激素不足和對博萊霉素誘導(dǎo)的肺纖維化和肺動脈高壓的作用。）Intactandovariectomized(OVX)femaleSpragueDawleyratswereadministeredintratracheallyeithersalineorbleomycin(15IU/kg),andasubsetofOVXbleomycin-treatedratsreceived2ME(10μg/kg/h)for21days.（在完整和切除卵巢的雌性SD大鼠的氣管內(nèi)給予生理鹽水或者博萊霉素（15國際單位/千克），并且對切除卵巢并用博來霉素處理的部分雌性大鼠用治療21天。）EstradiolhadonlylimitedinhibitoryeffectsongrowthinhPASMCsandnoeffectinhLFs,whereas2MEexhibitedstrongandconcentration-dependent(1?10μM)antimitogeniceffectsinbothcelltypes.（雌激素對人肺血管平滑肌細(xì)胞只有有限的生長抑制作用，而對人肺成纖維細(xì)胞沒有作用，然而，2-甲氧基雌二醇對兩種類型細(xì)胞都能表現(xiàn)出很強的濃度依賴性(1?10微米)抑制有絲分裂作用。）Bleomycincausedlunginjury/PH(significantlyincreasedlungandrightventricle(RV)weights,RVpeaksystolicpressure(RVPSP),andRV/leftventricle+septumratio(RV/LV+S);causedmedialhypertrophyandadventitialwideningofpulmonaryarteries;inducedmarkedfocal/diffusefibrosiswithdiffuseinfiltrationofinflammatory(ED1+)cells;andresultedin30%mortality).（博萊霉素致肺損傷值（肺和右心室（RV）重量顯著增加，右心室收縮壓峰值（RVPSP），右心室/左心室+隔比率(RV/LV+S)；引起肺動脈內(nèi)側(cè)肥厚和外膜擴大；誘導(dǎo)明顯的病灶性或彌漫性纖維化并伴有彌漫性浸潤的炎癥（ED1+）細(xì)胞；并導(dǎo)致30%的死亡率）。）OVXexacerbatedthediseaseandincreasedmortality(to75%);whereas2MEtendedtoreducemortality(55.5%)andinsurvivinganimalsreducedRVPSPandRV/LV+Sratio,andattenuatedvascularremodeling,pulmonaryinflammationandfibrosis.（卵巢切除能使疾病加重并且死亡率上升（到75%）；然而2-甲氧基雌二醇傾向于降低死亡率（55.5%）并且能降低幸存下來的動物的右心室收縮壓峰值和右心室/左心室+隔比率，減弱血管重塑、肺部炎癥和肺纖維化）Thisstudysuggeststhat2MEmayhaveprotectiveeffectsinbleomycin-inducedPHandfibrosis.（這項研究表明2-甲氧基雌二醇，可能對博來霉素誘導(dǎo)的肺動脈高壓和纖維化有保護作用。）Furtherinvestigationof2MEinpulmonaryfibrosisandPHiswarranted.（對2-甲氧基雌二醇在肺動脈高壓和肺纖維化中作用的研究是把要的。）Keywords:

Pulmonaryfibrosis,pulmonaryhypertension,estradiol,estradiolmetabolitesINTRODUCTIONPulmonaryfibrosisisapenultimateconsequenceofchronicinterstitiallungdiseasefromvariousetiologiesanditischaracterizedbyalimitedresponsetoavailabletherapiesandpoorprognosis.Pulmonaryhypertensioniscommoninpatientswithpulmonaryfibrosisanditspresencehasasignificantadverseimpactonsurvival(Lettierietal.,2006).Mortalityratesforpulmonaryfibrosisareincreasing(34%inthelast15years),andimportantlytherateofincreaseistwiceashighinwomenthanmen(Olsonetal.,2007).However,littleisknownregardingtheeffectsofgenderandestrogensindevelopmentoflungfibrosisandassociatedpulmonaryhypertension.Amongpatientswithsystemicsclerosis(SSc,scleroderma),pulmonaryhypertensionisaseriouscomplicationandfrequentcauseofdeath(Proudmanetal.2007).（在患有系統(tǒng)性硬化癥(SSc,scleroderma)的患者中，肺動脈高壓是一種嚴(yán)重的并發(fā)癥和并且經(jīng)常導(dǎo)致死亡）Notably,inwomenwithSScmenopausesignificantlyincreasestheriskfordevelopmentofdisease(Scorzaetal.2002),whereashormonereplacementtherapypreventsthedevelopmentofPH(Berettaetal.,2006).（值得注意的是，患有系統(tǒng)性硬化癥婦女絕經(jīng)后會增加疾病發(fā)展的風(fēng)險，然而激素替代療法能防止肺動脈高壓的發(fā)展。）Itseemsalsothatpregnancyandestrogensmayinfluencethedevelopmentofdiseaseandnever-pregnantwomenwithSScareathigherriskfordevelopingPH(Arlettetal.2002).（看來懷孕和雌激素可能會影響疾病的發(fā)展并且從未懷過孕患有系統(tǒng)性硬化癥的女性有更高患肺動脈高壓的風(fēng)險）Importantly,bothestradiolanditsnon-estrogenicmetabolite2-methoxyestradiol(2ME)arepresentinhighconcentrationsinwomenduringthelasttrimesterofpregnancy(BallandKnuppen1990;Tofovicetal.,2007),suggestingthenotionthatestrogensmayattenuatethedevelopmentandretardtheprogressionofPHinpatientswithpulmonaryfibrosis.（重要的是，雌激素和它非雌激素代謝雌激素產(chǎn)物2-甲氧基雌二醇能高濃度的存在于婦女在懷孕后的最后三個月，表明了雌激素可以減輕和延緩患有系統(tǒng)性硬化癥患者肺動脈高壓的發(fā)展這一概念。）2MEisamajorE2metabolitewhichistheproductofthesequentialhydroxylationandmethylationofE2bytheenzymescytochromeP450andcatechol-O-methyltransferase.(2-甲氧基雌二醇是雌二醇的一個主要代謝產(chǎn)物，它是雌二醇在細(xì)胞色素P450酶和兒茶酚氧位甲基轉(zhuǎn)移酶作用下連續(xù)羥基化和甲基化的產(chǎn)物。)2MEisnotonlyapotentantimitogeninvariouscancercells(Pribludaetal.2000),butalsoinhibitsproliferationofcardiovascularcells,includingrataorticsmoothmusclecells,endothelialcells,cardiacfibroblastsandglomerularmesangialcells,andtheseeffectsaremediatedbyestrogenreceptor-independentmechanisms(Dubeyetal.2004).（2-甲氧基雌二醇不僅是各種癌細(xì)胞中強有力的抗細(xì)胞分裂劑，也能抑制心血管細(xì)胞增殖，包括大鼠主動脈平滑肌細(xì)胞、內(nèi)皮細(xì)胞、成纖維細(xì)胞和腎小球系膜細(xì)胞，并且這些作用的介導(dǎo)是通過雌激素受體獨立機制。）Invivo,2MEretardstheprogressionofPHinmalerats(Tofovicetal.,2005a),andinfemaleanimals2MEpreventstheexacerbationofPHandeliminatesthemortalityinovariectomizedratswithMCT-inducedPH(Tofovicetal.,2006).（在體內(nèi)，2-甲氧基雌二醇能延緩雄性大鼠肺動脈高壓的病程，在雌性動物體內(nèi)2-甲氧基雌二醇能防止肺動脈高壓的惡化和能消除切去卵巢用野百合堿誘導(dǎo)地肺動脈高壓的雌性大鼠的死亡率。）Thesebeneficialeffectsareassociatedwithmarkedinhibitionofvascularremodelingandinflammation.（這些有利的影響是與顯著地抑制血管重塑和炎癥作用聯(lián)系在一起的。）2MEalsoreducesacutelunginjuryandattenuatesthedevelopmentofPHandpulmonaryvascularremodelinginducedbytherodentocidealpha-naphthylthiourea(Tofovicetal.,2005b),andintheconstricted-aortaratmodel,2MEinhibitspressure-independentvascularremodeling(Tofovicetal.,2003),（2-甲氧基雌二醇能降低急性肺損傷和抑制肺動脈高壓的發(fā)展，通過α-萘硫脲誘導(dǎo)能降低肺血管重塑，在主動脈阻塞的大鼠模型中，2-甲氧基雌二醇能表現(xiàn)出壓力無關(guān)性的抑制血管重塑作用）Furthermore,2MEanditsmetabolicprecursor2-hydroxyestradiolhavedirect(pressure-independent)inhibitoryeffectsofcardiacremodelingandfibrosisinratswithisoproterenol-inducedcardiachypertrophy(Tofovicetal.2008).Becauseoftheaforementionedconsiderations,wehypothesizedthat2MEhasprotectiveeffectsandthatestrogendeficiencyexacerbatespulmonaryfibrosisandassociatedpulmonaryhypertension.（基于上述考慮，我們假設(shè)2-甲氧基雌二醇具有保護作用并且雌激素缺乏能使肺纖維化和肺動脈高壓加重。）Totestourhypothesisweusedbleomycin-inducedlungfibrosisandpulmonaryhypertensionratmodel.（為了測試我們的假設(shè)，我們使用博萊霉素誘導(dǎo)的肺纖維化和肺高血壓大鼠模型。）Althoughthismodelexhibitssomefeaturesofhumanformofinterstitialpulmonaryfibrosis,italsohasclearlimitations(Gharaee-Kermanietal.,2005)thatshouldbetakeninconsiderationwhenassessingtheclinicalrelevanceoftherapeuticresponsesinthismodel.（雖然這個模型能表現(xiàn)出一些人體間質(zhì)性肺纖維化的特點，它也有明顯的局限性需要被考慮，比如）Theresultsofthepresentstudysuggestthat2MEisantimitogenicinhumanlungfibroblastsandpulmonaryarteryvascularsmoothmusclecellsandhasbeneficialeffectsinexperimentalpulmonaryfibrosisandassociatedpulmonaryhypertension.（目前的研究結(jié)果表明，2-甲氧基雌二醇在人肺成纖維細(xì)胞和肺動脈血管平滑肌細(xì)胞能抑制有絲分裂并且在實驗性肺纖維化及肺動脈高壓能產(chǎn)生有益的作用。）MATERIALSANDMETHODSGrowthinhibitoryeffectsofestradioland2MECryopreservedhumanpulmonaryarterysmoothmusclecells(hPASMCs;CascadeBiologics,Inc.)wereculturedinM231supplementedwith10%FCSand2mMglutamax(Invitrogen).Cryopreservedhumanlungfibroblast(hLFs;CellApplications,Inc.,SanDiego,CA)wereculturedinDMEM(Invitrogen)supplementedwith10%FCSand2mMglutamax.Cellswereplatedina75cm2

cultureflask(Falcon)andincubatedat37°Cwith5%CO2.Twentyfour-hourslater,cellswererinsedandprovidedfreshmediumandthereafterfreshmediumwasprovidedeverythreedays.Forcellproliferationassay,hPASMCsandhLFs(5,000cells/well)weregrownin24-welltissuecultureplates(Nunc).ThegrowthofstarvedhPASMCswasstimulatedwith2.5%FCSandSmoothMuscleCellGrowthSupplement(CascadeBiologics,Inc)(containinghumanbasicfibroblastgrowthfactor(bFGF2ng/ml),humanepidermalgrowthfactor(0.5ng/ml),heparin(5ng/ml),insulin(5μg/ml)andBSA(0.2μg/ml)),andinthepresenceorabsenceofthetestedagents.ForhLFs,cellsweretreatedwithM200(phenolredfree)containing2.5%FCSand1%LowSerumGrowthSupplement(LSGS;CascadeBiologics)(containinghydrocortisone(0.5μg/ml),humanepidermalgrowthfactor,(5ng/ml),bFGF(1.5ng/ml)andheparin(5μg/ml)andbFGF(6ng/ml)),inthepresenceorabsenceofthetestedagents.Thecellsweredetachedwith0.025%trypsin/EDTA(Sigma)at5?7days.TheQuickCellProliferationAssayKit(BioVisionResearchProducts,MountainView,CA)wasusedforquantificationofcellproliferationandviability.Briefly,theessayisbasedonthecleavageofthetetrazoliumsaltWST-1toformazanbycellularmitochondrialdehydrogenase.Theactivityofdehydrogenasecorrelateswithcellproliferation,andformationofformazandyeisquantifiedbymulti-wellspectrophotometerbymeasuringtheabsorbanceofthedyeat440nm.2.AnimalstudiesForty-threefemaleSpragueDawleyratsweighing200?250gwereobtainedfromCharlesRiverLaboratories(Wilmington,MA).Animalswerefedrodentchow(ProLabRHM3000rodentdiet,PMINutrition,Inc,StLouis,MO),hadfreeaccesstowater,andwerehousedat22°C,12-hourlightcycle,and55%relativehumidity.AllexperimentswerecarriedoutinaccordancewiththeUniversityofPittsburghInstitutionalguidelinesforanimalwelfare,andtheAnimalCareandUseCommitteeapprovedexperimentalprotocols.Asubsetofanimals(n=27)underwentbilateralovariectomyusingtheflankapproach,whereastheremaininganimals(n=16)wereshamoperated.Toconfirmthattheovariesweresuccessfullyremoved,uterusweightwasmeasuredatautopsy.Toproducepulmonaryfibrosis,bothintactandOVXanimalswererandomlyassignedtoreceiveunderhalothaneanesthesiaeitheranintratrachealinjectionofbleomycin(Sigma,StLouis,MO;5mg/kg/0.3mlsaline;BleoandOVX-Bleogroups)or0.3mlsaline(ControlandOVXgroups).Asubsetofanimals(n=9)wereimplantedALZETosmoticpumps(2ML-4,DurectCorporation,Cupertino,CA)delivering2ME(10μg/kg/h;Steraloids,Inc.,Newport,RI).Theeffectsof2MEonbleomycindispositionareunknown,anditispossiblethat2MEinterfereswithbleomycindisposition.Thehalf-lifeofbleomycininrodentsrangesbetween30minutesandtwohours(LazoandPham,1984).Therefore,toavoidpossibleinterferencewithbleomycindisposition,pumpswereimplanted8hoursafterbleomycinadministration.3.AcuteHemodynamicmeasurementsTwenty-onedaysafteradministrationofbleomycin,animalsunderwentsurgeryandwereinstrumentedformeasurementofrightventricularpeaksystolicpressure(RVPSP)asdescribedpreviously(Tofovicetal.,2005a,

2006).Briefly,ratswereanesthetizedwithInactin(90mg/kgi.p.),andaPE-240polyethylenecatheterwasinsertedintothetracheatofacilitatebreathing.APE-50catheterwasinsertedintotheleftcarotidarteryandconnectedtoadigitalbloodpressureanalyzer(BPA,Micro-Med.Inc.,Louisville,KY)forcontinuousmeasurementsofsystolic,diastolicandmeanarterialbloodpressuresandheartrate.Theratswerethenmechanicallyventilated(HarvardRodentVentilator,Model683,HarvardApparatus,MA)usingconstantrespiratoryrate(50/min)andtidalvolume(1.0ml).Next,thethoraxwasopened,andtherightheartwaspuncturedwitha23-gaugeneedleattachedtoaPE-50lineandHeartPerformanceAnalyzer(HPA-200τ,Micro-Med.Inc.,Louisville,KY).Aftera30-minutestabilizationperiod,RVPSPwasrecordedfor20minutesat1-minuteintervals.4.MorphometricmeasurementsandimmunohistochemicalstudiesAnimalswereeuthanatizedbyanestheticoverdoseandheartandlungsweredissectedandweighed.Theratiosofwetweightsofheartandlungtobodyweight(BW)werecalculated(H/BWandLV/BW,respectively).Therightventricle(RV)freewallwasseparatedfromtheleftventricleandtheseptum(LV+S)todeterminethewetweight,theRVtobodyweightratio(RV/BW),theLV+Stobodyweightratio(LV+S/BW),andtheRVtoLV+Sweightratio(RV/LV+S,FultonIndex).Thelungswereremovedfromthechestcavityenblockwiththetracheaandperfused

via

thetracheawithafixativesolution(10%neutral-bufferedformalin)atapressureof25cmH2O.Perfusedtissuesampleswereimmersedinthefixativefor24to72hoursforsubsequentlightmicroscopyandimmunohistochemistry.Tissuesampleswereembeddedinparaffinblocksforlightmicroscopy.Five-micronserialtissuesectionsfromformalin-fixed,paraffin-embeddedlungsweredewaxedandstainedwithH&EandMasson'strichromeforhistologicalandmorphometricassessment(toidentifyinflammatorycells,connectivetissueandcollagendeposition).Lungtissuessectionswereexaminedbylightmicroscopyandwerescoredinblindedfashion.Thenumberofinterstitialmonocytes/macrophageswasstudiedusingapolyclonalanti-ED1antibody(Serotec,Raleigh,NC).Thisantibodyspecificallystainspositivethecytoplasmofalveolarandinterstitialmacrophages.Nonspecificstainingwasassessedbyreplacingtheprimaryantibodywithaffinity-purified,nonimmune,rabbitIgG(R&DSystems).Sectionswerewashedanddevelopedfurtheraccordingtothedirectionsofthemanufacturer(Dako,Carpentaria,CA)usinganLSAB2kit,whichcontainedasecondantibodylinkedtoavidinandperoxidase-conjugatedtobiotin.Tostudyvascularremodelingofsmallpulmonaryarteries,arterialwallsmoothmusclecellswerestainedusingamousemonoclonalanti-smoothmuscle-alphaactinantibodyatadilutionof1/100(LabVision,Fremont,CA).Themeasurementsofmediathickness,andmediaandadventitiasurfacewereconductedusinganImageAnalyzingSystem(DiagnosticInstruments,Inc.,SterlingHeights,MI)thatincludedSPOTRTCamerainstalledonNIKONEclipse50lightmicroscopeandspecializedcomputersoftwareprogram(SPOTSoftware,Version4.1).Measurementsweredone(×20magnification)ontencrosssectionedpulmonaryarterybrancheswith50?250-microndiameters.Theperipherallungfieldswerelocatedatapproximatelyequaldistancesfromthepleurallining.Onlyvesselswithanapproximatelycircularprofile(cross-sectionalcuts)werestudied.Foreachbloodvessel,tworectangulardiametersandtheirfourrespectivemediaweremeasured.Themedia%indexwascalculatedas2×media/diameter×100andispresentedasaverageoffourmediameasurementsforasinglevessel.Sincebleomycininducedasignificantwideningoftheadventitia,correctedmediaindexwascalculatedusingthediameterthatincludesonlymedia+lumenmeasurement.Foreachbloodvessel,tworectangulardiametersandtheirfourrespectivemediaweremeasured,andaveragesoffourindividualvaluesofmediathicknessandmedia%indexwerecalculated.CollagenfibrosiswasassessedonMason'sTrichromestainedlungsectionsbythesameimageanalyzingsystemon10low-powerrandomfields(×10)usingtheregionareameasurementoptionandexpressedaspercentfromacalibrated1,000,000μ2microscopicfieldarea.5.StatisticalAnalysisStatisticalanalyseswereperformedusingtheNumberCruncherStatisticalsoftwareprogram(Kaysville,Utah).Groupcomparisonswereperformedbyaone-ortwo-factoranalysisofvariance(1F-,2-FANOVA),followedbypost-hoccomparisonusingtheFisher=sLSDtest.Theprobabilityvalueofp<0.05wasconsideredstatisticallysignificant.Alldataarepresentedasmean±S.E.M.RESULTSThegrowthinhibitoryeffectsofestradioland2MEinhumanpulmonaryarterysmoothmusclecells(hPASMCs)andhumanlungfibroblasts(hLFs)arepresentedin

Figure1.InhPASMCsatphysiological(1?10nM)andlowpharmacological(100nM)concentrations,E2hadnoeffectsoncellgrowthstimulatedby2.5%FCSandgrowthfactor-supplement,butexhibitedmodest(?20and?40%)antimitogeniceffectsathighconcentrations(1and10μM;

Figure1A).NoeffectsonhLFgrowthweredetectedwhencellswereexposedfor5daystoincreasingconcentrations(1nM-10μM)ofE2(Figure1B).Incontrast,2MEexhibitedstrongandconcentration-dependentantimitogeniceffectsinbothhPASMCsandhLFs.Thedataexaminingtheeffectsof2MEonbleomycin-inducedpulmonaryhypertensionandfibrosisinestrogen-deficientratsarepresentedin

Figures2-7and

Table1.Theintratrachealinstillationofbleomycininducedseverelunginjury,substantialweightlossandprematuredeathsthatoccurredbetweendays3and16oftheexperiment.Thebodyweightofratsthatreceivedsalineintratracheallyincreasedwithtime,andweightgainwassignificantlygreaterinOVXanimals(Day21:317±11and346±7g,ContandOVXgroup,respectively).Inanimalsreceivingbleomycin,therewasnobodyweightgainduringthe3-weekstudyperiod(datanotshown),andtherewasnosignificantdifferenceinbodyweightamongthethreediseasedexperimentalgroups(Bleo,OVX-BleoandOVX-Bleo-2ME).Bleomycinincreasedlungweight,andanimalsexposedtobleomycinhadadoublingoflungweightcomparedtoanimalsthatreceivedsalineintratracheally.Importantly,ovariectomyfurtherincreasedlungweight,and2MEpreventedfurtherincreasesinlungweightinOVX-Bleorats(Figure2

upperpanel,OVX-Bleo+2MEgroup).Thedifferencesinlungweightcorrelatedwithsignsofmicrovascularleakageandseverityoffibrosis(infravide).Intratrachealinstillationofbleomycininducedpulmonaryhypertension,ovariectomyfurtherincreasedtheRVPSP(Figure2,lowerpanel)andtreatmentwith2MEpreventedfurtherincreasesinRVPSPinOVXanimalswithintratracheallyinstilledbleomycin(OVX-Bleovs.OVX-Bleo+2MEgroup,p<0.05,Fisherposthoccomparison).Bleomycin-treatedanimalsthatdiedprematurelydevelopedbothrightandleftventricularhypertrophyandOVX-Bleogrouphadthemostseverecardiachypertrophy(Figure3,Day3?16).Twenty-onedaysintotreatments,thebleomycin-inducedisolatedRVhypertrophy(i.e.,Fultonindex,RV+S/RVratio)wasexacerbatedindiseasedOVXanimals,and2MEreducedtheRVhypertrophyinOVX-Bleorats(Figure3,Day21).Animalsthatdiedinthefirstweekhadnofibrosis,butratherexpressedmarkedcongestion,edema,andinflammatory-cellinfiltration(Figure5J),andthesealterationsweremultifocalinnatureinintactfemalestreatedwithBleo.MoreseverechangesweredetectedinOVX-Bleorats,andtheseanimalshaddiffuse(ratherthanmultifocal)exudative,inflammatoryandproliferativereactions.AnimalsthatdiedbetweenDay8andDay16hadsignificantinterstitialfibrosis,andtheBleoandOvx-Bleogroupsdidnotdifferinregardtotheextensionoffibrosis(Figure4A).Incontrast,inanimalsthatsurvived,Masson-trichromestainingofformalin-fixedtissuerevealedgreateranddenseramountsofcollagendepositioninlungsfromOvx-BleoratscomparedtoBleogroup(Day21;

Figure4A,

Figures5

G&H).Treatmentwith2MEeliminatedtheexacerbationoffibrosisduetoovariectomy(Figures4Aand4I).Infibroticareassignificantnumberofcellsstainedpositiveforsmoothmuscleα-actinindicatingmyofibroblasticproliferationandsemi-quantitativeassessmentsuggestedreducedmyofibroblasticproliferationinanimalstreatedwith2ME.Bleomycin-inducedpulmonaryfibrosisandhypertensiondetectedonDay21wereaccompaniedbysignificantvascularremodeling(Figure4B,

Table1),andovariectomyincreasedmedialhypertrophyandadventitialwidening(Table1).InOVX-Bleorats,treatmentwith2MEmarkedlyinhibitedvascularremodeling,andOVX-Bleo+2MEratshadlesservascularremodelingandadventitialwideningthanevenintactdiseasedanimals(Bleogroup;

Table1).IntratrachealinstillationofbleomycinandthesubsequentlunginjurywerealsoassociatedwithsignificantinflammatoryresponsesasevidencedbythepresenceofalargenumberofED1+cells(Figures4C

and

?and5L).5L).Theinflammatoryresponsewasevenmorepronouncedinanimalsthatdiedprematurely.Importantly,OVXexacerbatedinflammationinallanimals,2MEmarkedlyreducedthenumberofED1+cells,andtheintensityofinflammationinOVX-Bleo+2MEgroupwasevensmallerthanintactfemalestreatedwithbleomycin(Bleogroup).Finally,themoreseverelunginjury(fibrosis,inflammation,PH)intheOVX-Bleogroupwasassociatedwithincreasedmortality,andtreatmentwith2MEtendedtoreducetheaugmentedmortalityduetoovariectomy(Figure6).DISCUSSIONBleomycininducesdose-dependentlungparenchymalinjuryandfibrosisinbothhumansandanimals.Inrodents,bleomycin-inducedlungfibrosisexhibitscertainfeaturesoftheinterstitialpulmonaryfibrosis(IPF)inhumans,anditisthemostcommonlyusedmodeltostudythepathogenesisandtreatmentofIPF.Evenso,italsohasclearlimitations(therapidityofitsdevelopment,itsself-limitingnature,andtheseverityoftheassociatedinflammation)thatshouldbeconsideredwhendiscussingtheclinicalrelevanceoftherapeuticinterventionsinthismodel(Gharaee-Kermanietal.,2005).Animportantfindingofthepresentstudyisthatestradiolanditsdownstreammetabolite2MEdiffersignificantlyinregardtotheirantimitigeniceffectsincelllinesinvolvedinpulmonaryfibrosisandvascularremodeling,i.e.,inhLFsandhPASMCs.ThepresentstudyshowsthatestradiolhasnoeffectsongrowthofhLFsinconcentrationsupto10μM.Toourknowledge,thisisthefirstinvestigationofE2inadulthLFs.Previousstudiesinfetallungfibroblastsdemonstrateanantimitogeniceffectofestradiolonlyathighpharmacologicalconcentrations(>5μM)(Kondoetal.,1983).Also,previousstudiesofcellulargrowthinfibroblastsfromvariousoriginsrevealbothstimulatoryandinhibitorypropertiesofE2(Dubeyetal.2004;

TomaszewskiJetal.,2003).ThevariableeffectsofE2mayreflectthephenotypicheterogeneityamongfibroblasts,includingtheirdifferentiation(particularlyduringgrowthandrepair)intomyofibroblasts.OurresultsalsoshowthatestradiolhasonlymodestantimitogeniceffectsinhPASMCs.ThisisincontrasttothewellestablishedantimitogeniceffectsofE2invascularsmoothmusclecellsfromsystemicarteries(Dubeyetal,2004;

Espinosaetal.,1996).Nonetheless,itisplausiblethatE2mayhavedifferentanti-remodelingeffectsonsystemicandpulmonarybloodvesselsthatphylogeneticallyarefromdifferentorigins.Alongtheselines,E2stimulatesproliferationofratPASMCsincellculture,hasnoeffectongrowthinintactcaninepulmonaryarterysegments,andstimulatesgrowthofPASMCsinsegmentswhentheendotheliumisremoved(Farhatetal.,1992).IncontrasttoE2,2MEinaconcentration-dependentmannerinhibitsgrowthofhLFsandhPASMCs.Thisisnotsurprisingsince2MEisamorepotentantimitogenthanE2incardiacfibroblasts,vascularsmoothmusclecellsandglomerularmesangialcells(Dubeyetal.,2004).Intheorderofpotency2ME>2HE>E2,E2anditsmetabolitesinhibittheproliferationofhepaticstellatecellsandcollagensynthesisbythesefibroblast-likecells(Liuetal.,2004).Hepaticstellatecells,togetherwithvascularsmoothmusclecells,cardiacfibroblasts,andglomerularmesangialcellsbelongtothepericytefamily.Duringgrowthortissueinjuryandrepair,thesecellsdisplaypropertiesofmyofibroblastsandarethemostimportantsourceforcollagen,fibronectin,andotherextracellularmatrixproteins.Itisnotablethatinbleomycin-treatedanimalsinfibroticareas,asignificantnumberofcellsstainedpositiveforsmoothmuscleα-actin,indicatingmyofibroblasticproliferation.Importantly,thepresentstudysuggests(usingsemi-quantitativeassessment)reducedmyofibroblasticproliferationinanimalstreatedwith2ME.Thepresentstudydemonstratesthatovariectomyexacerbatespulmonaryfibrosisandpulmonaryvascularremodelingandhypertension,suggestingthatE2isprotectiveinpulmonaryhypertensionduetolungfibrosis.Theseeffectsareconsistentwithpreviousreportsinpulmonaryhypertensiverats(Farhatetal.1993,

Tofovicetal.2006).Theeffectsofovariectomyonpulmonaryfibrosisarealsoinaccordancewithpreviousstudiesshowingthatovariectomyincreaseslungcollagen,airwaysmoothmusclethickeningandcardiachypertrophy