生技一文献翻译作业4.第四组

题Hypoxia-InducedPulmonaryVascularRemodelingRequiresRecruitmentofCirculatingMesenchymalPrecursorsofaMonocyte/MacrophageLineage低氧诱导下肺血管重需要补充的循环间质单核/巨噬细胞系的前MariaG.Frid,*JacquelineA.Brunetti,*DanielleL.Burke,*ToddC.Carpenter,*NeilJ.Davie,*JohnT.Reeves,*MarkT.Roedersheimer,*NicovanRooijen,†andKurtR.FromtheDepartmentofPediatrics,*UniversityofColoradoHealthSciencesCenter,Denver,Colorado;andtheDepartmentofMolecularCellBiology,†VrijeUniversiteit,VrijeUniversiteitMedicalCenter,Amsterdam,TheNetherlandsfibroproliferativechangesinthepulmonaryartery(PA)adventitia.AlthoughresidentPAfibroblastshavelongbeenconsideredtheprimarycontributorstotheseprocesses,wetestedthehypothesisthathypoxia-inducedpulmonaryvascularremodelingrequiresrecruitmentofcirculatingmesenchymalprecursorsofamonocyte/macrophagelineage,termedfibrocytes.Usingtwoneonatalanimalmodels(ratsandcalves)ofchronichypoxicpulmonaryhypertension,wedemonstratedadramaticperivascularaccumulationofmcononuclearcellsofamonocyte/macrophagelineage(expressingCD45,CD11b,CD14,CD68,ED1,ED2).ManyofthesecellsproducedtypeIcollagen,expresseda-smoothmuscleactin,andproliferated,thusexhibitingmesenchymalcellcharacteristicsattributedtofibrocytes.Theblood-borneoriginofthesecellswasconfirmedinexperimentswhereincirculatingmonocytes/macrophagesofchronicallyhypoxicratswereinvivo-labeledwithDiIfluorochromevialiposomearterialadventitia.TheDiI-labeledcellsthatappearedinthevesselwallexpressedmonocyte/macrophagemarkersandprocollagen.Selectivedepletionofthismonocyticcellpopulation,usingeitherclodronate-liposomesorgadoliniumchloride,preventedpulmonaryadventitialremodeling(ie,productionofcollagen,fibronectin,andtenascin-Candaccumulationofmyofibroblasts).Weconcludethatcirculatingmesenchyprecursorsofamonocyte/macrophagelineage,includingfibrocytes,areessentialcontributorstohypoxia-inducedpulmonaryvascularremodeling.(AmJPathol2006,168:659–669; 慢性缺氧性高压肺血管重建包括肺动脉（PA的外膜纤维显着的增生变化虽然在这些过程中PA者,我们测试低氧性肺动脉血管重塑需要循环间质补充单核细胞/巨噬细胞系的前体的假说使用两个患慢性低氧性肺动脉高压新生儿动物模型（大鼠和小牛，我们证明单核细胞/巨噬细胞系的单核细胞的剧烈的血管周围积累（表达CD11bCD14CD68ED1ED2许多这些细胞产生I型胶原蛋白的表达和α内标记通过的DiI荧光脂递以及随后在所识别的改造肺但不是全身性的，动脉所述的DiI记的细胞中出现的血管壁表示单核细胞/巨噬细胞标记和前胶肺外膜重塑（即生产胶原蛋白，纤连蛋白，腱生蛋白和-C和肌纤维母细胞的积是必不可少的贡献者缺氧引起的肺血管重构。（AM病理学2006年，168：659-669;：10.2353/ajpath.2006.050599Significantfibroproliferativechangesinthepulmonaryartery(PA)adventitiaareprominentcharacteristicsofchronicpulmonaryhypertensionofmanycauses,includinghypoxia.TheseprocessesoccurinbothlargeandsmallPAsasaresultofadventitialcellproliferation,production/depositionofextracellularmatrixproteins,especiallycollagens,andaccumulationofmyofibroblasts[α-smoothmuscleactin(α-SMA)-expressingfibroblasts].1–5Thesefibroproliferativechangeshavelongbeenassumedtobeduetoactivation,proliferation,anddifferentiationofresidentadventitialfibroblasts.However,thepossibilitythatnonresidentcellsmaycontributedirectlytothehypoxia-inducedpulmonaryremodelingprocessemergesfromrecentreportsinvariousdiseasemodels(includingthoseofthelung)thatdemonstraterecruitmentofcirculating,bonemarrow-derivedcellscapableofassumingamesenchymal/fibroblastphenotypeatthesiteoftissueinjury.6–9Amongdifferenttypesofcirculatingmesenchymalprogenitors,mononuclearcells(MNCs)ofamonocyte/macrophagelineage,specificallyasubpopulationofMNCstermed“fibrocytes,”haverecentlyreceivedsignificantattentionwithregardtotheirpotentialroleinvariousfibroproliferativediseases.10–13FibrocytescompriseasubpopulationofcirculatingMNCsofamonocyte/macrophagelineage(CD11b,CD13,CD14)thatcanexhibitfibroblastpropertiesatthesiteoftissueinjury(collagenproduction,differentiationintomyofibroblasts).10–15Fibrocyteshavebeenshowntobeanimportantsourceoftissuefibroblastsinasthmaandlungfibrosis.12,13,16However,toourknowledge,therearenostudiesdemonstratingthecontributionofcirculatingfibrocytesorothertypesofmesenchymalprecursorsofamononuclearorigintopulmonaryvascularremodelinginthesettingofhypoxicpulmonaryhypertension.Wethustestedthehypothesisthatchronichypoxicexposureinducesrecruitmentofcirculatingfibrocytestothepulmonarycirculation,andthatthesecellscontributedirectlyandsubstantiallytopulmonaryvascularremodeling.Twoneonatalanimalmodels(ratandcalf)ofchronichypoxia-inducedpulmonaryhypertensionwereusedtotestthishypothesisinanefforttodeterminehowgeneralizedandconsistenttheresponsewasacrossspecies.Inthesemodelswesoughtto1)identifythecontributionofMNCsandfibrocytestoPAadventitialthickeningusingimmunofluorescenceysiswithapanelofcelltypespecificantibodies;2)delineatethedirectcontributionofthesecellstofibroproliferativeprocesses(collagenproduction,proliferation,accumulationofmyofibroblasts)inhypoxicpulmonaryvascularremodelingusingdoublelabelimmunofluorescentstainingforleukocyte,markersoffibrosis(procollagen),cellreplication[5bromo-2-deoxyuridine(BrdU)],andmyofibroblasts(α-SMA);3)assesstheblood-borneoriginofthesecellsintheremodeledpulmonaryadventitiaofchronicallyhypoxicanimalsbyinvivolabelingofthesecellsinthecirculationandsubsequentdeterminationoftheirlocalizationinthelungandsystemicvasculature;and4)evaluatetheroleofthesecellsinhypoxicpulmonaryvascularremodelingprocessbyselectivelydepletingthemandsubsequentlyassessingtheimpactonpulmonaryadventitialfibrosisandmyofibroblastaccumulation.在肺显著纤维增生性变化动脉（PA）外膜是突出特点原因很多，其中包括慢缺氧性肺动脉高压。这些过程发生在大和小功率放大器为外膜细胞增殖，生产/α平滑（α-S的成纤维细胞-xping]0.15这些纤维增生性改变长被接向缺氧诱发的肺动脉重塑过程贡献的可能性出现从各种疾病最近的报告模型（包括那些肺细胞在组织间的不同类型的循环间充质祖细胞，单核细胞单核细胞（外周血单个核细胞潜在作用纤维细胞包括循环的亚群单核细胞（细胞1b的外周血单个核细胞？13?,14？，可以在表现出成纤维细胞特性组织损伤的部位.（鼠和牛是用来测试这个假说以确定响应的一般性与一致性是跨物种的。在这些模型下我们应该1明确外周血单个核细胞的贡献和成纤维细胞A外膜增厚是使用型特异性抗体的免疫荧光分析技术2描述这些细胞对于纤维增生流程（胶原蛋白的生产，扩撒，肌成纤维细胞的积累的直接贡献这个贡献在于利荧光免疫检验法的白细胞抗原染色方法来研究缺氧性肺血管重建，纤维化的标志（元骨胶原，细胞复制[5-bom-2doxyuidined尿嘧啶和肌成纤维细胞(α-S3评估这些改制的肺部动脉外膜慢性缺氧动物的细胞，这是通过标记这些细胞在肺癌和系统性的脉管系统方面的循环和随后的决心4通过选择性的消耗细胞和评估在肺外膜的纤维化的肌成纤维细胞的积累过程中的影响来评估这些细胞在缺氧性肺血管重建过程中的作用。MaterialsandAnimalTwoanimalspecieswereusedasmodelsofhypoxiainducedneonatalpulmonaryhypertension:weanlingratsand,toconfirmthefindingsinalargem species,calves.Theadvantagesofusingthehypoxiccalfmodelhavebeendescribedbefore1andincluderemarkablethickeningofPAadventitia,whichresemblesthepathologicalpictureinhumanneonatalpulmonary4Ratswereusedinthisstudyasananimalmodelofhypoxicpulmonaryhypertensionbecausetheserodents,incontrasttomice,developmarkedPAadventitialthickening17andbecausecertainexperimentalmanipulationscanbeperformedinratsbutarenoteconomicallyfeasibleincalves.TheWistar-KyotoratstrainwaschosenbecauseitdevelopsmoreseverehypoxicpulmonaryhypertensionthantheSprague-Dawleystrain.18Weanling(4weeksold)maleWistar-KyotoratswerepurchasedfromCharlesRiverLaboratories(Wilmington,MA).Experimentalhypoxicgroupsofrats(=4to6,eachtimepoint)wereexposedtohypobarichypoxia(PB=380mmHg)for24hoursto4weeks.Age-matchedcontrolswerekeptatambientaltitude.One-day-oldmaleHolsteincalveswerepurchasedfromLalunadairyfarm(FortCollins,CO).Theexperimentalhypoxicgroup(n=7)wasexposedfor2weekstohypobarichypoxia(PB=445mmHg),whileage-matchedcontrols(n=6)werekeptatambientaltitude(Denver,CO;=640mmHg).1Animalsofbothspecieswereeuthanizedbyoverdoseofsodiumpentobarbital(160mg/kgbodyweight).Standardveterinarycarewasusedfollowinginstitutionalguidelines:forrats,attheUniversityofColoradoHealthSciencesCenterforLaboratoryAnimalCare(Denver,CO)incompliancewithInstitutionalAnimalCareandUseCommittee-approvedprotocols;forcalves,attheDepartmentofPhysiology,SchoolofVeterinaryMedicine,ColoradoStateUniversity(FortCollins,CO).牛是为了在大型哺乳动物中来证实这项发现。采用缺氧的牛模型的优势在之前A，大鼠被用作这项研究中的缺氧性肺动脉高压的一个动物模型是因为因为这些啮齿动物与小鼠相比,发展的A外膜增厚更显著，也因为已确定的的实验操作可以在大鼠上进行但在牛身上进行时不经济的方式选择ita-Kyoto鼠种是因为与Spagu-ley鼠种相比它可以发展成更为严重的缺氧型肺动脉高压。刚断奶的（4周大）雄性ita-Kyo大鼠是从查尔斯河(ilminton,)得到的。缺氧型实验组大鼠(n=4to每个时点t)在4周的每个小时里都被在低压缺氧P=380mmHg)的环境中。 组被置于正常环境下。一天大的雄性牛从aluna奶牛场Fotlin,) 得到。缺氧型实验（n被在低压缺氧PB445mmH的环境中2周组被置于正常环境下(nv,;PB640mmg)。，两种物种中的动物都被用过量戊巴比妥钠（160mg/kg）处以。兽医护理(Denver,CO)与动物制度和使用协议相符合；对于牛来说，在科罗拉多州立大学的生理学部门和兽医学院(FortCollins,CO)。TissueSamplesandFreshlyobtainedtissuesampleswereembeddedinO.C.T.(SakuraFinetek,Torrance,CA)andfrozenat-70°Cuntiluse.Thefollowingantibodiesagainstleukocytewereusedinthestudy:bovine-specificmonoclonalantibodies(mAbs)againstCD45,CD11b,CD14(15ug/ml;VMRDInc.,Pullman,WA),CD68(EBM11,1:100;DakoCytomationCorp.,Carpinteria,CA);rat-specificmAbs (OX- 1:100;ChemiconInt.,Temecula,CA),mAbsagainstED1,ED2(1:10,1:50;BDPharmingen,SanDiego,CA),andanti-granulocytemAbs(RK-4,1:50;CedarlineLaboratories,Ontario,Canada).Thefollowingantibodiesagainstmarkersoffibrosiswereused:mAbsagainsttypeIprocollagen(SP1.D8,1:10;DevelopmentalStudiesHybridomaBank,UniversityofIowa,IowaCity,IA),mAbsagainstcollagen-prolyl-4-hydroxylasea(6–6H2,1:50;Medicorp,Montreal,Canada),mAbsagainstED-A-FN(ist-9,1:10;OxfordBiotechnology,Oxfordshire,UK),andrabbitpolyclonalanti-tenascin-CAbs(1:100,ChemiconInt.).Todefinetheexpressionofasmoothmusclemarkerα-SMA,mAb1A4wasused(1:100;SigmaChemicalCo.,St.Louis,MO).Proliferatingcellswereidentifiedusingfluoresceinisothiocyanate-conjugatedanti-BrdUmAbs(1:3,BDPharmingen).刚获得的组织样本被嵌入在O.C.TSakuraFinetek,TorranceCA)直到使用冻结在-70C研究中使用了以下的白细胞抗原抗体:牛特异性单克隆抗体(单抗)CD45,CD11b,CD14(15ug/ml;VMRDInc.,Pullman,WA)，CD68(1:100;DakoCytomationCorp.,Carpinteria,CA);大鼠特异性的单克隆抗体CD45，CD11b(OX-1,OX-42,1:100;ChemiconInt.,Temecula,CA)，mAb对抗ED1,ED2(1:10,1:50;BDPharmingen,SanDiego,CA)，和抗粒细胞单克隆抗体(RK-4,1:50;CedarlineLaboratoriesOntarioCanada)。使用了以下抗体的纤维化标志物:单克隆抗体ⅰ型前胶原(SP1.D8,1:10;DevelopmentalStudiesHybridomaBankUniversityofIowa,IowaCityIA)，单克隆抗体胶原-脯氨酰-4-羟化酶(6–6H2,1:50;Medicorp,Montreal,Canada)的单克隆抗体ED-A-FN(ist-9,1:10;OxfordBiotechnology,Oxfordshire,UK)，和兔多克隆抗-腱-CAbs(1:100,ChemiconInt.)。若要定义的平滑肌markerα-SMA表达，单克隆抗体1A4是用(1:100;SigmaChemicalCo.,St.Louis,MO)荧光素异硫酸酯共轭抗BrdU单克隆抗体(1:3，BDPharmingen)，确定了增殖细胞。ImmunofluorescentTissuecryosections(5μm)werefixedinmethanol:acetone(1:1).Nonspecificbindingwasblockedwithfetalbovineserum:phosphate-bufferedsaline(PBS)(1:1),andsectionswereincubatedovernightwithprimaryAbsat4°C.SecondarybiotinylatedAbs(VectorLaboratories,Burlingame,CA),streptavidin-Alexa-594(red)andstreptavidin-Alexa-488(green)(MolecularProbes,Eugene,OR)wereusedatdilutions mendedbythemanufacturers.ImmunolabeledsectionsweremountedinVe hield/DAPI(VectorLaboratories)andexaminedunderaZeissfluorescentmicroscopewithanAxioVisiondigitalimagingsystem(CarlZeissMicroImaging,Inc.,Thornwood,NY).Fordouble-labelconfocalmicroscopy,labelingwithantibodiesagainstCD45,CD11b,CD14,CD68,andED1wasplishedfirst.Next,anti-procollagenorα-SMAmAbs,directlylabeledwithZenon-Alexa-fluorkit(MolecularProbes),wereappliedfor1to2hours.Imageswere yzedonDeltaVisiondigitaldeconvolutionconfocalmicroscope(OlympusAmericaInc.,Melville,NY).PhotographsweretakenwithaPhotometricsxcooleddigitalcharge-coupleddevicecamera(Olympus). ysisofthepercentageofmonocyticcellscoexpressingprocollagen,BrdU,andα-SMAwasperformedon12to18stainedlungthatwereobtainedfromfourtosixanimals(ratsandcalves,免疫荧光标（11儿bovineserum：磷酸盐缓冲盐水（PBS（1：1，和切片过夜性绝对值在亚州，链亲和Alexa-594（红）和链霉的Alexa-488（绿色（分子探针，Eugene，OR）被用来在制造商推荐的稀释液。免疫标记切片装入的Ve/DAPI（矢量）和蔡司荧光显微镜用的AxioVision数字成像系统（卡尔CD45，CD11b的，CD14，CD68，和ED1标记首次完成的。接着，抗原胶原-SMAmAb，直接标记与泽农-Alexa的-氟试剂盒（分子探针施加12小时。图像上DeltaVision数字反卷积共聚焦显微镜（奥林巴斯公司，梅尔维尔，纽约州）进行分析。的是用Photometrics的x冷却数字电荷耦尿嘧啶，和α-SMA被从四到六只动物（大鼠和肉牛，分别）获得的1218个TimeCourseRatswereexposedtohypoxiafor24hours,48hours,72hours,96hours,and1,2,3,and4weeks.Lungcryosectionswereimmunolabeledforamonocyte/macrophagemarkerCD11b,andtheperimeterofthePAwall(contiguoustoalveoli)wasexaminedwithintheradialdistanceof1501moutwardfromtheexternalelasticlamina.CD11b1cellnumberwasassessedperunitvolume(10 mradialdistanceby500mperimeterlength).时间进程分肺泡）为150μm的径向距离内进行了检查向外从外弹性膜。CD11b的numberwasassessedperunitvolume（由500米周长长度10米半径距离BrdURats(72-hourhypoxicornormoxic)wereinjectedwithBrdU(1001g/kgbodyweight)at24,18,and2hoursbeforeeuthanization.尿嘧啶注大鼠（72小时低氧或含氧量正常的）均在之前24，18，和2个小时注射用尿嘧啶（100克/千克体重。DiI-Liposome,Clodronate-Liposome,andGadoliniumChlorideInjectionsLiposomeswerepreparedaspreviouslydescribed.19Forliposome-mediatedinvivoDiI-labelingofcirculatingmonocytes/macrophages,liposomescontainingredfluorochrome,DiI,wereintroducedintothecirculationviaaseriesofintravenousinjections(seebelow).TheseliposomesarereadilytakenupviaphagocytosisbycirculatingphagocyticMNCsofamonocyte/macrophagelineage(butnotbyothersubsetsofMNCs),andDiIfluorochromeisincorporatedintothecellmembrane,resultinginselectivelabelingofcirculatingmonocytes/macrophages.19,20DiIliposomeswereintravenouslyinjected(0.1ml/10gbodyweight)intoanesthetizedrats(n=5)everythirddayduringthe3-weekhypoxic(ornormoxic)exposure,aswellason3consecutivedaysbeforetheendoftheexperimentbeforeeuthanasia.TissueswerefrozeninO.C.T.,andunfixedcryosectionswereexaminedfortheredDiIfluorescence.AftercapturingtheimagedemonstratingDiIfluorescence,thecryosectionwasfixedinacetone:methanol,whichcomple ybleachedDiIfluorescence,andthenlabeledwithantibodiesagainstCD11b,and/orED1,and/orTodepletecirculatingMNCsofamonocyte/macrophagelineage,weusedtwoseparateapproaches:serialintravenousinjectionsofliposomescontainingclodronate(Cl2MBP;giftofRocheDiagnosticsGmbH,Mannheim,Germany)orintravenousinjectionsofgadoliniumchloride(GdCl3,SigmaChemicalCo.).Thesemethodsarecommonlyusedtotargetanddepletephagocyticcells.Clodronatehasanextremelyshorthalf-lifeinthecirculationandthereforeisnottoxic.19However,whendeliveredintophagocyticcellsusingliposomesasvehicles,clodronateaccumulatesinthecellandinducesapoptosisafterexceedingathresholdconcentration.19Importantly,Cl2MBPliposomesdonotcrossvascularendothelialbarriersandthuswouldnotbetakenupbyresidenttissuephagocyticcells.TheselectiveactionofGdCl3onmonocytes/macrophagesisbasedonthefactthatthiscompoundisreadilydissolvedinnormalsaline;however,wheninjectedintothebloodstream,itrapidlyaggregatesintorelativelylargecolloidalparticlesatneutralpH.Similartoliposomes,theparticlesofGdCl3aretakenupexclusivelybycirculatingphagocyticMNCsbutnotbyothercells.Onceinsidethephagocyticcellandafterexceedingthethresholdconcentration,GdCl3causescellExperimentalhypoxicandnormoxicrats(n=8,eachgroup)wereintravenouslywithCl2MBP-liposomesbi-weeklyfor4weeks(firstinjectionwasat0.1ml/10gbodyweight,subsequentinjectionswereat0.05ml/10gbodyweight).ControlhypoxicandnormoxicratswereintravenouslyinjectedwithPBSliposomes(n=6)onthesameschedule.GdCl3(2mg/ml,10mg/kgbodyweight)wasadministeredasintravenousinjectionsbiweeklyfor4weeksofhypoxicornormoxicexposure(n=6,each),andcontrolratswereinjectedwithPBS(n=8).DiI-脂，氯膦酸盐- ，和氯化钆注脂 如先前所述对于脂 介导的循环含有红色荧光色素的单核细胞/巨噬细胞，脂 的体内的I标记，的，分别经由一系列静脉内注射（见下文）引入到循环 。这些脂可以容易地吸收通过吞噬作用通过循环单核细胞/巨噬细胞谱系的细胞吞噬外周血单个核细胞（但不是以外周血单个核细胞的其它子集，并且fluoohoe掺入细胞膜，从而导致循环单核细胞的选择性标记/maophag.1,20的I脂 静脉内注射（.1毫升/10克体重）到麻醉（n=3周低（或常氧） 期间每三天以及连续3天的实验结束前前组织冷冻在OCT中，未固定的冷冻切片检查红色的I荧光。拍摄图像展示的iI荧光后将冷冻切片固定在甲醇后者完全漂白的I荧光，然后标记抗1b的，和或，和或。为了减少循环的单核细胞/巨噬细胞系的外周血单个核细胞，我们使用了两种不同的方法含有氯膦酸盐脂的连续静脉注（P罗氏诊断，曼海姆，德国的）或氯化钆静脉注射（氯化钆，西格玛化工。这些方法通常用于靶向和耗尽吞噬细胞。r苷具有极短的半衰期，在流通，因此不会toxic.9但是当输送到使用脂作为载体氯膦酸盐积聚在细胞和超过阈onntatin.9Plipooms不胞pH进入吞噬细胞和超过阈值后的浓度，氯化钆导致细胞凋亡。实验性缺氧和含氧量正常鼠（n=8每组）静脉注射Cl2MBP脂每两周进行4周（第一次注射0.1毫升/10克体重，随后的注射0.05毫升/10g体（N=6（2毫克/毫升，10毫克/公斤体重）给予静脉内注射双周4周低氧或含氧量正常的（每组6，各自，和对照组注射用PBS（N=8 Dataarepresentedasmean±SEM.StatisticalysisusedStudent’st-testandone-wayysisofvariance;significanceacceptedatP<0.5数据分数据表示为平均值±SEM表示。统计分析使用斯氏t检验和单向方差分析;意义在接受P<0.5HypoxiaInducesRobustAccumulationofMNCsofaMonocyte/MacrophageLineageinthePAAdventitia.结低氧诱导单核/巨噬细胞系中单个核细胞的PA外膜跨的大量积Innormoxicanimalsofbothspecies(ratsandcalves),thePAadventitiawasthin[Figure1,A(rat)andC(calf)],whereasinanimalsexposedtochronichypobarichypoxia(ratsfor4weeksandcalvesfor2weeks),thePAadventitiawasremarkablythickened[Figure1,B(rat)andD(calf)].WefirstexaminedwhetherMNCsofamonocyte/macrophagelineagewererecruitedtothePAinresponsetochronichypoxicexposureusingapanelofantibodiestomonocyte/macrophage(inrats,CD45,CD11b,ED1,ED2;incalves,CD45,CD11b,CD14,CD68).Innormoxicanimals(bothratsandcalves),theadventitiacontainedveryfewcellsexpressingthese[Figure2,normoxiacolumns,AandC(rat)andE(calf)].However,inchronicallyhypoxicanimalsofbothspecies,numerouscellsexpressingmonocyte/macrophagewereobservedinthethickenedadventitiaofbothlargeandsmallPAs[Figure2,hypoxiacolumns,BandD(rat)andF(calf)].Accumulationofthesecellswasspecifictothelungvasculature,becausetheaorta,carotid,andfemoralarteriesofhypoxicanimalsweredevoidofsuchcells(datanotshown).Moreover,neutrophilswerenotidentifiedinthePAadventitiaofeitherhypoxiccalves(at2weeksofhypoxicexposure)orhypoxicrats(startingfrom24hoursandupto4weeksofhypoxicexposure)(datanotshown).Akinetic ysisdemonstratedaprogressiveaccumulationofCD11bmonocytes/macrophagesinthepulmonaryperivascularspaceofhypoxic(24hoursto4weeks)ratswithapeakaccumulationindexof11at4weeksofhypoxicexposurecomparedto2.5innormoxicrats(Figure3).Furthermore,aprogressiveincreaseinthedistancefromtheexternalelasticlamellae(xaxis)oftheCD11bcellpeakwasobserved,whichcorrelatedwiththeprogressiveincreaseinthethickeningofPAadventitiainresponsetochronicexposuretohypoxia.（大鼠和肉牛的nnomoicA的外膜是薄图1（鼠）和（小牛），而在动物于慢性低压缺（大鼠4周和肉牛2周中，的外膜被显着地增稠图，（大鼠）和（小牛。我们首先检查是否单核巨噬细胞系的外周血单个核细胞使用抗体单核巨噬细胞的抗原组成的小组被招募巴勒斯坦权力机构应对慢性低氧（大鼠451b的犊牛，45，1b的，14，68。在含氧量正常的动物（大鼠和小牛，外膜含有很少细胞表达这些抗原[图，常氧列，A和（大鼠）和（小牛。然而，在这两个物种的慢性缺氧动物，很多细胞表达单核细胞/巨噬细胞抗原，观察在大型和小型的A图2B和（大鼠和（小牛的加厚外（数据未显示。此外，嗜中性粒细胞不在任缺氧牛犊的A的外膜识别（2周低氧的）或低氧大鼠（由24小时开始和最多4周性低氧）（数据未显示动力学分析表明CD11b的单核细胞/巨噬细胞在低氧的肺动脉血管周围空间中的渐进累积（24小时至4周）的大鼠为11的峰积累指数在4周时缺氧相2.5在含氧量正常大鼠（Figure3。此外，在距离外弹性薄片的CD11b的细胞的峰值逐渐增加（x轴）观察，其与响应于慢性于缺氧逐步增加PA的外膜的Figure1.chronichypoxiainducesPAadventitialthickening.H&estainingofpulmonaryarteriesfromrats(A,B)andcalves(C,D).Theadventitiallayer(Adv)isverythininnormoxicrats(A)andcalves(C).Inchronicallyhypoxicanimalsofbothspecies,thePAadventitiaisremarkablythickened(B,rats;D,calves).Smallarrowspointtonewlyformedcapillariesofthevasavasorum.M,tunicamedia.Scalebars,20μm.图1.长期缺氧诱导PA外膜增厚。肺动脉大鼠H＆E染色（A，B）和小（C，D（C在这两个物种的慢性缺氧动物，PA的外膜显着增厚（B，大鼠D，小牛。小Figure2.HypoxiainducesarobustappearanceofMNCsinthePAadventitia.CryosectionsofPAsfromrats(A–D)andcalves(E,F)werelabeledwithantibodiesagainstMNC/macrophage(red)andcellnuclei(DAPI,blue).FewMNCsarepresentintheadventitiaofnormoxicratsandcalves(singlelargearrowheadsinthenormoxiacolumns).Intheadventitiaofchronicallyhypoxicratsandcalves,abundantMNCsofamonocyte/macrophagephenotypearepresent(triplelargearrowheadsinthehypoxiacolumns).Forrats,micrographsAandBshowmainPAs,whereasCandDshowdistalPAs(80to1001mindiameter).GreenautofluorescenceoftheelasticlamellaeinA--DdefinestheboundariesofthePAtunicamedia.ThePAlumensaremarkedwithsmallgreenarrows.M,media;A,adventitia.Scalebars:1001m(A,B);201m(C–F).图2.缺氧诱导强大的外表外周血单个核细胞在PA外膜。PA的大鼠（A-D）小牛（，）冷冻切片标记抗巨噬细胞抗原（红色）和细胞核（P，蓝色（单个大箭头在常氧列的外膜。在慢性低氧大鼠和牛犊的外膜，单核/巨噬细胞的表型的丰富的外周血（在缺氧列三大箭头A和B显示主功率放大器，而CD示出远侧功率放大器（80100微米直径。在一个弹性薄片的青自体荧光-Ð定义了PA的界限该PA流明标有绿色的小箭头。男，;A，外膜。比例尺：100米（A，B）;小于20m（C-MNCs,AccumulatingintheRemodeledPAAdventitia,ExhibitaFibrocyteExtensivecollagendeposition,accumulationofmyofibroblasts,andcellproliferationinthePAadventitiaareprominentfeaturesofhypoxia-inducedpulmonaryvascularremodeling.1–5,22WethusevaluatedwhetheranyoftheMNCsofamonocyte/macrophagelineageaccumulatinginthepulmonaryperivascularspaceinresponsetochronichypoxiaexhibitedaphenotypecharacteristicofafibrocyte(ie,producedcollagen,expressedα-SMA,单个核细胞，积聚在重建PA外膜，表现出纤维细胞表A肺动脉血管重塑.突出的特征因而我们评价是否任何一个单核细胞/巨噬细胞谱系的积聚在肺血管周围的外周血单个核细胞响应于慢性缺氧空间表现出纤维细胞的表型特性（即，产生的胶原，αSA表达，增殖）CollagenToidentifyfibrocytesinthePAadventitia,double-labelimmunostainingwasperformedforMNC-macrophage(inrat,CD45,CD11b,ED1,ED2;incalf,CD45,CD11b,CD14,CD68)andintracellularcollagenprecursormolecules[inrat,collagen-prolyl-4-hydroxylase-(cP4H);incalf,-IprocollagenRoutineandconfocalfluorescencemicroscopydemonstratedthatasubstantialproportionofMNCs,accumulatingintheremodeledPAadventitiaofhypoxicanimals,wascomprisedofcollagen-expressingfibrocytes(64.34.3%inrats,and38.13.6%incalves)[Figure4,A–C(rat)andD–E(calf)].NofibrocyteswereidentifiedinthePAadventitiaofnormoxiccalvesand/orrats(notshown)胶原蛋白生以识别的PA外膜成纤维细胞MNC巨噬细胞抗原进行双标记免疫染（在和胞内胶原前体分子[大鼠，胶原脯氨酰-4-羟化酶（cP4H）;在小牛，-I型前胶（pA（大鼠.％图4的（鼠和-（小牛正常牛犊和或大鼠（未示出）的A的外膜识别α-SMAWedeterminedifanyMNCsco-expressedα-SMAandthuscontributeddirectlytotheaccumulationofmyofibroblasts(α-SMA-expressingfibroblasts)inthePAadventitiaofchronicallyhypoxicanimals. ysisofdouble-labelimmunostainingdemonstratedthat,inhypoxicanimalsofbothanimalspecies,numerousadventitialcellsco-expressedmacrophage(CD11binratsandCD68incalves)andα-SMA[Figure5,AandB(rat)andCandD(calf)].Deconvolutionconfocalmicroscopyshowedthat,amongallMNCswithintheremodeledadventitia,19.24.3%MNCs(inrats)and7.83.2%MNCs(incalves)co-expressedα-SMA.Noα-SMA-expressingMNCswereidentifiedinthePAadventitiaofnormoxiccalvesandrats(notshown).α-SMA的表胞在慢性低氧动物的PA外膜的积累（α-SMA表达成纤维细胞。双重标记的胞的抗原（CD11b的大鼠和CD68在小牛）和α-SMA的[5，AB（大鼠）和CD（小牛显微镜显示改装的外膜内的所有单个核细胞中，19.24.3％的单个核细胞（大鼠）和7.83.2％的单个核细胞（犊牛）共表达α-SMA。无α-SMA的表达单个核细胞是在常氧牛犊和大鼠的PA的外膜标识（未示出incorporation(Figure6B),whereasnoproliferatingadventitialcellswereobservedinnormoxicrats(Figure6A).ysisofdouble-labelimmunofluorescentstainingoflungtissuesfromchronicallyhypoxicratsdemonstratedthatasignificantnumber(15.42.3%)ofMNCsexpressingCD45and/orED2hadincorporatedBrdU(Figure6B,arrows;CD45isshown).CellsotherthanMNCs,potentiallyresidentfibroblasts,werealsoobservedtoincorporatedBrdU(Figure6B,arrowheads).TherelativecontributionofcellsexpressingMNCmarkerstotheoverallBrdUlabelingindexinthePAadventitiaat72hoursofhypoxicexposurewas28.64.1%.Thus,theMNCs,accumulatingwithintheremodeledPAadventitia,contributedsignificantlytotheoverallincreasesincellproliferationobservedinthePAadventitiaofhypoxicanimals.细胞增低氧大鼠的A（图6膜细胞含氧量正常大鼠（图6）观察。从慢性低氧大鼠肺组织的双标记免疫荧光分析表明，外周血单个核细胞的显著数量（15.42.％）表达45和或已将尿嘧啶（图6，箭头;45如图。细胞比单个核细胞，可能驻留的成纤（图6C标记来中外膜整体尿嘧啶标记指数在72小时时缺氧的相对贡献为28.6.1％。因AA外膜观察做出了贡献。Fibrocytes,AccumulatinginthePAAdventitia,OriginatefromtheCirculationBasedonexpressionofhematopoieticmarkers(includingCD45),wehypothesizedthatfibrocytes,accumulatingintheremodeledPAadventitia,wererecruitedfromthecirculation.Wesoughttodeterminetheblood-borneoriginoffibrocytesusingaliposomemediatedinvivolabelingapproach,whereinphagocyticMNCscouldbeselectivelylabeledvialiposomedeliveryofDiIfluorochromedirectlyinthecirculationofliverats(MaterialsandMethods).19,20Figure7,A–C,demonstratesthatperipheralbloodMNCs,exclusivelyofamonocyticorigin(ED1),takeupliposomesloadedwithredDiIfluorochrome.TheDiI-labeledcirculatingcellsweresubsequentlyidentifiedinthePAadventitiaofchronicallyhypoxic(Figure7,D–G)butnotnormoxic(Figure7H)ratsasfollows.Lungandsystemicvasculartissuesfromhypoxicandnormoxicrats,whichwereintravenouslyinjectedwithDiIliposomes,wereexaminedforthepresenceofDiI-labeledcellswithinthevesselwall.Inhypoxicanimals,numerousDiI-labeledcellswereidentifiedinthePAadventitia(Figure7D).NoDiI-labeledcellswereobservedaroundaortasorcarotidarteriesofhypoxicrats(Figure7H,aortaisshown)oraroundPAofnormoxicrats(notshown).TodeterminethephenotypeofDiI-labeledcells,immunofluorescentlabelingofmonocyte/macrophage(CD11b,ED1,ED2)wasperformedonthesametissuesectionthatwasfirstbleachedoffluorescence(Figure7E)andthenlabeledwithantiMNCantibodies(Figure7F,alsoseeMaterialsandMethods).MostDiI-labeledcellsexpressedMNC(Figure7F,ED2immunoreactivityisshown).Importantly,manyoftheDiI-labeledMNCsintheadventitiaalsoexpressedtypeIprocollagen(Figure8),aphenotypeconsistentwiththatofafibrocyte.纤维细胞，积累了PA外膜的流通发基于对造血标志物（包括CD45）的表达，我们推测，纤维细胞，积聚在改造的PA外膜，从循环中被吸收。我们试图确定所述血源性原点使用脂介导的I荧光的脂递送直接在活老鼠的循环（材料和方法）.19,20图标记7，-，表明外周血单个核，完全是单核细胞的（）的，占用脂载有红色的I荧光。所述的I标记的循环细胞后来被定在慢性缺氧（图7中的-G（图7H大鼠如下的A的外膜。肺和从低氧和含氧量正常大鼠其中静脉内注射的脂的I全身血管组织被检查的I标记的细胞在血管壁内的存在。在缺氧的动物，众多的I标记的细胞进行鉴定，在（图7I（图7H（未示出I标记的细胞，单核细胞的免疫荧光标记（细胞1b的表型上相（图7antiMC（图进行也见材料和方法I标记的细胞表达的C（图7免疫反应性示出。重要的是，许多在外膜的的I标记的单个核细胞也表达型胶原（图8，与一纤维细胞的一致的表型Figure4.MonocyticcellsintheremodeledPAadventitiaexpressprocollagen.A:SerialtissuesectionsoftheratPAlabeledwithantibodiesagainstamonocyticcellmarkerCD11b(red,left)andcollagenprecursormolecule,collagen-prolyl-hydroxylase-(cP4H)(green,right).Elasticlamellaeofthetunicamedia(left)exhibitgreenautofluorescence,whichisshowntobetterdefinethetunicamediaboundaries.B:MicrographoftheadventitialareashownwithintheframeinA.Double-labelimmunofluorescentstainingfollowedbydeconvolutionconfocalmicroscopydemonstratesthattheremodeledPAadventitiaofchronicallyhypoxicratscontainsnumerouscellsthatco-express(arrowheads)CD11bfluorescence)andcP4H(greenfluorescence).Thereisacell(smallsinglearrow)thatexpressesamonocyticantigenCD11b(red)butnotcP4H,andanothercell(smalldoublearrow)expressescP4HbutnotCD11b.DAPI(blue)labelsallcellnuclei.C:RoutineimmunofluorescencemicroscopyofthesmalldistalPAofchronicallyhypoxicratshowsadventitialcellsthatalsoco-expressCD11b(red)andcP4H(green)(arrows).D:Serialsectio