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ARTICLEOPEN
TGF-βpromotespericyte-myofibroblasttransitioninsubretinal
fibrosisthroughtheSmad2/3andAkt/mTORpathways
ZhenzhenZhao1,2,4,YumengZhang1,2,4,ChaoyangZhang1,2,JingtingZhang1,2,XuetingLuo1,2,QinghuaQiu1,2,3,DaweiLuo1,2and
JingfaZhang1,2✉
©TheAuthor(s)2022
Subretinalfibrosisremainsamajorobstacletothemanagementofneovascularage-
pericyteswerefoundtobeasignificantsourceofsubretinalfibrosis,buttheunderlyingmechanismsofpericyte-myofibroblast
transition(PMT)
(CNV)wasinducedbylaserphotocoagulationintransgenicmice
withthecollagen1α1-greenfluorescentprotein(Col1α1-GFP)reporter,andrecombinantadeno-associatedvirus2(rAAV2)-
mediatedTGF-β2(rAAV2-TGF-β2)-positive
pericytesweretreatedwithTGF-β2incombinationwithsiRNAstargetingSmad2/3,theAktinhibitorMK2206orthemTORinhibitor
rapamycintoexaminecellproliferation,migration,
-β2overexpressioninducedGFP-positive
pericyteinfiltrationandPMTinsubretinalfibrosis,
1234567890();,:decreasedcellproliferation,
synergisticeffectsonattenuatingα-smoothmuscleactin(α-SMA)-inducedCNV,
theadministrationoftheAkt/mTORinhibitorssuppressedpericyteproliferationandalleviatedtheseverityofsubretinalfibrosis.
OurresultsshowedthatPMTplaysapivotalroleinsubretinalfibrosis,whichwasinducedbyTGF-β2throughtheSmad2/3andAkt/
,inhibitingPMTmaybeanovelstrategyforthetreatmentofsubretinalfibrosis.
Experimental&MolecularMedicine(2022)54:673–684;/s12276-022-00778-0
INTRODUCTIONMyofibroblastsarefibroblast-likecellsthatexpressα-smooth
Age-relatedmaculardegeneration(AMD)isaleadingcauseofmuscleactin(α-SMA),depositpathologicalECM,andarethemain
individualsworldwide1,,alsoknownasmacularmyofibroblastsinmacularfibrosisoriginatefromthedifferentia-
neovascularization(MNV)3,accountsforapproximately10−15%oftion/transitionofmultiplecelltypes,suchasretinalpigment
AMDcases,ismainlycharacterizedbychoroidalneovasculariza-epithelial(RPE)cells,macrophages,andendothelialcells,although
tion(CNV)andprogressesrapidlytocauseseverevisionlossanddirectevidenceinpatientsisstilllacking10–12.
,choroid-derivedpericytesinfiltratedthe
growthfactor(VEGF)-Aplaysdistinctrolesinpathologicalsubretinalspace,contributingtoECMdepositionandfibrosis
angiogenesisandinflammationinAMD,andanti-VEGFtherapyformationinamousemodeloflaser-
hasbecomethefirst-linetreatmentofMNV4,,aorgans,includingthekidney,lungandspinalcord,pericyteshave
substantialportionofpatientsstillsufferfrompoorvisualbeenshowntofunctionastheprimaryfibrosis-formingcellsand
prognosisduetothedevelopmentofsubretinalfibrosisevenarethekeycontributorstofibrosis14–
withintensiveanti-
SubretinalfibrosisremainsamajorobstacleforMNVmanage-theformationofCNVandsubretinalfibrosisinmicewithlaser-
ment,andtherearecurrentlynoeffectivetherapeuticinterven-inducedCNV17,indicatingtheinvolvementofpericytesinfibrosis
,-induced
membranestoavariablymixedfibrovascularstructure,suchasCNVmodel,choroidalpericytesmightdifferentiateinto
fibrosiscontaininginfiltratingimmunecells,myofibroblastsandmyofibroblast-likecells,whichplayapivotalroleintheformation
excessiveamountsofextracellularmatrix(ECM)proteins,
eventuallyformsscarsandisresistanttoanti-VEGFtreatment7,-beta(TGF-β)isextensivelyimplicatedinthepathogenesis
1DepartmentofOphthalmology,ShanghaiGeneralHospital(ShanghaiFirstPeople’sHospital),ShanghaiJiaoTongUniversity,SchoolofMedicine,Shanghai,
ClinicalResearchCenterforEyeDiseases;ShanghaiKeyLaboratoryofOcularFundusDiseases;ShanghaiEngineeringCenterforVisualScienceandPhotomedicine;Shanghai
EngineeringCenterforPreciseDiagnosisandTreatmentofEyeDiseases,Shanghai,,ShigatsePeople’sHospital,Xizang,
authorscontributedequally:ZhenzhenZhao,YumengZhang.✉email:jingfa.******@
Received:28October2021Revised:20February2022Accepted:17March2022
Publishedonline:27May2022:.
.
674
ofvarioustypesoffibrosis,includingsubretinalfibrosis,whileTGF-wellplatecontainingMEMαmodification(ThermoFisherScientific),1%
β2wasshowntobemorehighlyexpressedthanotherTGF-βGlutaMAX(ThermoFisherScientific),20%FBS(Sigma–Aldrich,,
isoforms7,12,18,,USA)and1%penicillin/streptomycin(ThermoFisherScientific).The
transitionduringfibrosis,suchasthetransitionofRPEcellsintocellswereidentifiedwithappropriatemarkers,suchasPDGFRβ(+)and
myofibroblasts20,TGF-βsignalingpathways,whichconsistofTGF-CD31(−),toconfirmtheisolationofprimarypericytes(,
β/Smadcanonicalsignalingandnon-Smadsignalingpathways,).Thecellswerepassagedandculturedfor3to8
21passagesandusedforfurtherexperiments.
transdifferentiateintomyofibroblastsinresponsetoTGF-
β2stimulationinotherorgans,suchasthelung22,theCCK8assay
contributionofchoroidalpericyte-myofibroblasttransition(PMT)CellproliferationwasassessedwithCellCountingKit‐8(CCK8assay;
tosubretinalfibrosisandthedetailedmechanismsinresponsetoSigma–Aldrich)accordingtothemanufacturer’
seededin96-wellplatesatadensityof1,000cells/well,andCCK8solution
TGF-β2inalaser-inducedCNVmodelremainunknown.(10μL)
Inthisstudy,weperformedbothinvivoandinvitroexperimentscurveswereplottedaftertheabsorbancewasmeasuredat450nm.
toinvestigatetheeffectofTGF-β2onPMTandthepossible
type1promoter(Col1α1-GFPmice)asavaluabletooltodeterminewereseededatadensityof5×104cells/wellinsix-wellplatesandgrown
,thecellswerestarvedin1%FBSlow
-β2serummediumovernightandwerepretreatedbeforebeingstimulated
mediatedchoroidalpericyteproliferationandPMTviatheSmad2/3withTGF-β2(10ng/mL,R&DSystems,MN,USA)
andAkt/mTORsignalingpathways,andblockingtheAkt/mTORwasrepeatedthreetimes.
pathwayalleviatedsubretinalfibrosisinalaser-inducedCNVmodel.
EdUassay
Tofurtherevaluatecellproliferation,anEdUassaywasconductedusing
MATERIALSANDMETHODStheBeyoClick™EdUCellProliferationKitwithAlexaFluor594(Beyotime,
AnimalsShanghai,China),andHoechst33342wasusedfornuclearstaining
C57BL/6JmiceorCol1α1-GFPtransgenicmiceaged2–3monthswereaccordingtothemanufacturer’,cellswereincubated
-GFPtransgenicmiceweregeneratedaswithEdUstainingbufferfor8handfixedwith4%polyformaldehyde,and
-
InstitutesofHealth(USA)
(,revised1978)andwereapprovedbytheconsideredEdU-.
-positivecells
wasrepeatedthreetimes.
Laser-inducedCNVmousemodel13
Laser-,mice
(7–8weeksold)wereanesthetizedwithsodiumpentobarbital,andtheTranswellmigrationassay
pupilsweredilatedwith1%tropicamide(Santen,Osaka,Japan).ThefourTranswellmigrationassayswereconductedafterthecellswereharvested
injuryspotswereinducedbya532-nmlaserwithapowerof120mWforwithtrypsinandresuspended(1×105cells/mL)in1%FBSlowserum
(Visulas532S;CarlZeissMeditec,Dublin,Ireland)-β2
fashionaroundtheopticnerveusingaslit-lampsystem.(10ng/mL)
°Cwith5%CO2for24h,cellsthatdid
notmigratewereremovedfromtheuppersurfaceofthefiltersusingcotton
Intravitrealinjectionsswabs,andthecellsthathadmigratedtotheoppositesideofthetranswell
ToexaminetheeffectofTGF-β2onchoroidalpericytesinvivo,
beforelaserinjury,Col1α1-GFPtransgenicmicereceivedanintravitrealcountedinthreerandomfieldsunderaninvertedmicroscope,andthe
injectionofrAAV2-TGF-β2(1µL,1×108vg)oranequivalentvolumeof
.
-inducedCNV.
Thesizesoffibroticlesionsweredeterminedbymeasuringtheareasof
GFP-positivelesionsandα-SMA-positivelesionsonRPE-choroidcom-Woundhealingassay5
,toexcludetheeffectofrAAV2vectoradministration,Atotalof2×10cells/wellwereplatedandstarvedin1%FBSlowserum
C57BL/,scratchesonthecellmonolayersweremade
rAAV2vectoror1µLnormalsaline2weeksbeforelaserinjuryandwithasterilized1000-
:0h,6h,12h,24h,36h,48h,
determinedbymeasuringtheareasofPDGFRβ-positiveandα-SMA-
positivelesionsonRPE-,usingthepercentageofwoundclosure.
C57BL/6JmicewereinjectedintravitreallywithAkt/mTORinhibitors
immediatelyafterlaserinjuryand1weeklaterforearlyeventanalysisoratRNAinterference
()andSmad3siRNAs()weresynthesized
followinggroups:(1)theMK2206(10mM×2μL/eye,SelleckChemicals,byGenomeditechCompany(Shanghai,China).Thesequencesareshown
Houston,TX,USA)group;(2)therapamycin(500μM×2μL/eye,()waspurchased
Chemicals)group;(3)thedimethylsulfoxide(DMSO)control(2μL/eye)fromSantaCruzBiotechnology(Dallas,TX,USA).ThesiRNAswere
group;and(4)(BAIDAIBiotech,Changzhou,
sacrificed3weeksafterlaserinjury,)accordingtothemanufacturer’,toknock-
downSmad2orSmad3individuallyortogether,GFP-positiveprimary
Isolation,identificationandcultureofprimarychoroidalGFP-%RFectTransfection
RPE/choroidtissuewasincubatedwithcollagenaseA(,Roche,%RFectTransfectionReagentincombinationwithdifferent
Basel,Switzerland),dispaseII(,Roche),andDNase(µg/mL,,thecellswereculturedin
Roche)at37°Cfor15minaspreviouslydescribed24andthenincubatedin1%FBSmediafor16handthensplitintodifferentgroupsthatwere
%trypsin-EDTA(ThermoFisher
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