ME减弱博来霉素在雌激素缺乏大鼠中所导致的PAH和肺纤维化参考模板

18/182-METHOXYESTRADIOLATTENUATESBLEOMYCIN-INDUCEDPULMONARYHYPERTENSIONANDFIBROSISINESTROGEN-DEFICIENTRATS（2ME减弱博来霉素在雌激素缺乏大鼠中所导致的肺动脉高压和肺纤维化）TofovicSP,ZhangX,JacksonEK,etal.

[J].Vascularpharmacology,2009,51(2-3):190.Abstract（摘要）Pulmonaryhypertension(PH)isacommonandlife-threateningcomplicationofpulmonaryfibrosis.（肺动脉高压（PH）是一种常见和威胁生命的肺纤维化的并发症。）Estradiol(E2)isprotectiveinexperimentalPH,anditsnon-estrogenicmetabolite2-methoxyestradiol(2ME)preventsthedevelopmentandretardstheprogressionofmonocrotaline-inducedPHinmaleandfemalerats.（雌激素能在试验性肺动脉高压中提供保护并且它的非雌激素代谢物2-甲氧基雌二醇能在野百合碱诱导的雌性和雄性大鼠肺动脉高压模型中阻止疾病进程的发展。）However,theeffectsofE2and2MEonpulmonaryfibrosisandassociatedPHhavenotbeenexamined.（然而，雌二醇和2-甲氧基雌二醇对肺纤维化和涉及肺动脉高压的作用还没有研究出来。）Therefore,wecomparedthegrowth-inhibitoryeffectsofE2and2MEinhumanlungfibroblasts(hLFs)andpulmonaryvascularsmoothmusclecells(hPASMCs),andweinvestigatedtheeffectsofestrogendeficiencyand2MEonbleomycin-inducedpulmonaryfibrosisandPH.（因此，我们对雌二醇和2-甲氧基雌二醇在人肺成纤维细胞（hlfs）和人肺血管平滑肌细胞（hpasmcs）中的生长抑制作用进行了对比，我们还调查了雌激素不足和对博莱霉素诱导的肺纤维化和肺动脉高压的作用。）Intactandovariectomized(OVX)femaleSpragueDawleyratswereadministeredintratracheallyeithersalineorbleomycin(15IU/kg),andasubsetofOVXbleomycin-treatedratsreceived2ME(10μg/kg/h)for21days.（在完整和切除卵巢的雌性SD大鼠的气管内给予生理盐水或者博莱霉素（15国际单位/千克），并且对切除卵巢并用博来霉素处理的部分雌性大鼠用治疗21天。）EstradiolhadonlylimitedinhibitoryeffectsongrowthinhPASMCsandnoeffectinhLFs,whereas2MEexhibitedstrongandconcentration-dependent(1−10μM)antimitogeniceffectsinbothcelltypes.（雌激素对人肺血管平滑肌细胞只有有限的生长抑制作用，而对人肺成纤维细胞没有作用，然而，2-甲氧基雌二醇对两种类型细胞都能表现出很强的浓度依赖性(1−10微米)抑制有丝分裂作用。）Bleomycincausedlunginjury/PH(significantlyincreasedlungandrightventricle(RV)weights,RVpeaksystolicpressure(RVPSP),andRV/leftventricle+septumratio(RV/LV+S);causedmedialhypertrophyandadventitialwideningofpulmonaryarteries;inducedmarkedfocal/diffusefibrosiswithdiffuseinfiltrationofinflammatory(ED1+)cells;andresultedin30%mortality).（博莱霉素致肺损伤值（肺和右心室（RV）重量显著增加，右心室收缩压峰值（RVPSP），右心室/左心室+隔比率(RV/LV+S)；引起肺动脉内侧肥厚和外膜扩大；诱导明显的病灶性或弥漫性纤维化并伴有弥漫性浸润的炎症（ED1+）细胞；并导致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-甲氧基雌二醇倾向于降低死亡率（55.5%）并且能降低幸存下来的动物的右心室收缩压峰值和右心室/左心室+隔比率，减弱血管重塑、肺部炎症和肺纤维化）Thisstudysuggeststhat2MEmayhaveprotectiveeffectsinbleomycin-inducedPHandfibrosis.（这项研究表明2-甲氧基雌二醇，可能对博来霉素诱导的肺动脉高压和纤维化有保护作用。）Furtherinvestigationof2MEinpulmonaryfibrosisandPHiswarranted.（对2-甲氧基雌二醇在肺动脉高压和肺纤维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).（在患有系统性硬化症(SSc,scleroderma)的患者中，肺动脉高压是一种严重的并发症和并且经常导致死亡）Notably,inwomenwithSScmenopausesignificantlyincreasestheriskfordevelopmentofdisease(Scorzaetal.2002),whereashormonereplacementtherapypreventsthedevelopmentofPH(Berettaetal.,2006).（值得注意的是，患有系统性硬化症妇女绝经后会增加疾病发展的风险，然而激素替代疗法能防止肺动脉高压的发展。）Itseemsalsothatpregnancyandestrogensmayinfluencethedevelopmentofdiseaseandnever-pregnantwomenwithSScareathigherriskfordevelopingPH(Arlettetal.2002).（看来怀孕和雌激素可能会影响疾病的发展并且从未怀过孕患有系统性硬化症的女性有更高患肺动脉高压的风险）Importantly,bothestradiolanditsnon-estrogenicmetabolite2-methoxyestradiol(2ME)arepresentinhighconcentrationsinwomenduringthelasttrimesterofpregnancy(BallandKnuppen1990;Tofovicetal.,2007),suggestingthenotionthatestrogensmayattenuatethedevelopmentandretardtheprogressionofPHinpatientswithpulmonaryfibrosis.（重要的是，雌激素和它非雌激素代谢雌激素产物2-甲氧基雌二醇能高浓度的存在于妇女在怀孕后的最后三个月，表明了雌激素可以减轻和延缓患有系统性硬化症患者肺动脉高压的发展这一概念。）2MEisamajorE2metabolitewhichistheproductofthesequentialhydroxylationandmethylationofE2bytheenzymescytochromeP450andcatechol-O-methyltransferase.(2-甲氧基雌二醇是雌二醇的一个主要代谢产物，它是雌二醇在细胞色素P450酶和儿茶酚氧位甲基转移酶作用下连续羟基化和甲基化的产物。)2MEisnotonlyapotentantimitogeninvariouscancercells(Pribludaetal.2000),butalsoinhibitsproliferationofcardiovascularcells,includingrataorticsmoothmusclecells,endothelialcells,cardiacfibroblastsandglomerularmesangialcells,andtheseeffectsaremediatedbyestrogenreceptor-independentmechanisms(Dubeyetal.2004).（2-甲氧基雌二醇不仅是各种癌细胞中强有力的抗细胞分裂剂，也能抑制心血管细胞增殖，包括大鼠主动脉平滑肌细胞、内皮细胞、成纤维细胞和肾小球系膜细胞，并且这些作用的介导是通过雌激素受体独立机制。）Invivo,2MEretardstheprogressionofPHinmalerats(Tofovicetal.,2005a),andinfemaleanimals2MEpreventstheexacerbationofPHandeliminatesthemortalityinovariectomizedratswithMCT-inducedPH(Tofovicetal.,2006).（在体内，2-甲氧基雌二醇能延缓雄性大鼠肺动脉高压的病程，在雌性动物体内2-甲氧基雌二醇能防止肺动脉高压的恶化和能消除切去卵巢用野百合碱诱导地肺动脉高压的雌性大鼠的死亡率。）Thesebeneficialeffectsareassociatedwithmarkedinhibitionofvascularremodelingandinflammation.（这些有利的影响是与显著地抑制血管重塑和炎症作用联系在一起的。）2MEalsoreducesacutelunginjuryandattenuatesthedevelopmentofPHandpulmonaryvascularremodelinginducedbytherodentocidealpha-naphthylthiourea(Tofovicetal.,2005b),andintheconstricted-aortaratmodel,2MEinhibitspressure-independentvascularremodeling(Tofovicetal.,2003),（2-甲氧基雌二醇能降低急性肺损伤和抑制肺动脉高压的发展，通过α-萘硫脲诱导能降低肺血管重塑，在主动脉阻塞的大鼠模型中，2-甲氧基雌二醇能表现出压力无关性的抑制血管重塑作用）Furthermore,2MEanditsmetabolicprecursor2-hydroxyestradiolhavedirect(pressure-independent)inhibitoryeffectsofcardiacremodelingandfibrosisinratswithisoproterenol-inducedcardiachypertrophy(Tofovicetal.2008).Becauseoftheaforementionedconsiderations,wehypothesizedthat2MEhasprotectiveeffectsandthatestrogendeficiencyexacerbatespulmonaryfibrosisandassociatedpulmonaryhypertension.（基于上述考虑，我们假设2-甲氧基雌二醇具有保护作用并且雌激素缺乏能使肺纤维化和肺动脉高压加重。）Totestourhypothesisweusedbleomycin-inducedlungfibrosisandpulmonaryhypertensionratmodel.（为了测试我们的假设，我们使用博莱霉素诱导的肺纤维化和肺高血压大鼠模型。）Althoughthismodelexhibitssomefeaturesofhumanformofinterstitialpulmonaryfibrosis,italsohasclearlimitations(Gharaee-Kermanietal.,2005)thatshouldbetakeninconsiderationwhenassessingtheclinicalrelevanceoftherapeuticresponsesinthismodel.（虽然这个模型能表现出一些人体间质性肺纤维化的特点，它也有明显的局限性需要被考虑，比如）Theresultsofthepresentstudysuggestthat2MEisantimitogenicinhumanlungfibroblastsandpulmonaryarteryvascularsmoothmusclecellsandhasbeneficialeffectsinexperimentalpulmonaryfibrosisandassociatedpulmonaryhypertension.（目前的研究结果表明，2-甲氧基雌二醇在人肺成纤维细胞和肺动脉血管平滑肌细胞能抑制有丝分裂并且在实验性肺纤维化及肺动脉高压能产生有益的作用。）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