The epicardium as a source of multipotent adult cardiac progenitor cells: Their origin, role and fate

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Pharmacological Research

jo u r n al ho me p a g e :ww w . e l s e v i e r . c o m / l o c a t e / y p h r s

Review

The epicardium as a source of multipotent adult cardiac progenitor cells: Their origin, role and fate

Anke M. Smits

, Esther Dronkers, Marie-José Goumans

DepartmentofMolecularCellBiology,LeidenUniversityMedicalCenter,P.O.Box9600,PostzoneS-1-P,2300RCLeiden,TheNetherlands

a r t i c l e i n f o

Articlehistory:

Received24March2017

Receivedinrevisedform12June2017 Accepted21July2017

Availableonline24July2017

Keywords:

Cardiacprogenitorcell Epicardium

Myocardialinfarction Cardiacdevelopment Cardiacrepair

a b s t r a c t

Sincetheregenerativecapacityoftheadultmammalianheartislimited,cardiacinjuryleadstothefor- mationofscartissueandtherebyincreasestheriskofdevelopingcompensatoryheartfailure.Stemcell therapyisapromisingtherapeuticapproachbutisfacingproblemswithengraftmentandclinicalfeasibil- ity.Targetinganendogenousstemcellpopulationcouldcircumventtheselimitations.Theepicardium, amembranouslayercoveringtheoutsideofthemyocardium,isanaccessiblecellpopulationwhich playsakeyroleinthedevelopingheart.Epicardialcellsundergoepithelialtomesenchymaltransition (EMT),thusprovidingepicardialderivedcells(EPDCs)thatmigrateintothemyocardiumandcooperate inmyocardialvascularisationandcompaction.Intheadultheart,injuryactivatestheepicardium,andan embryonic-likeresponseisobservedwhichincludesEMTanddifferentiationoftheEPDCsintocardiac celltypes.Furthermore,paracrinecommunicationbetweentheepicardiumandmyocardiumimproves theregenerativeresponse.Thesignificantroleoftheepicardiumhasbeenshowninboththedeveloping andtheregeneratingheart.Interestingly,theepicardialcontributiontocardiacrepaircanbeimproved inseveralways.Inthisreview,anoverviewoftheepicardialoriginandfatewillbegivenandpotential therapeuticapproacheswillbediscussed.

©2017TheAuthors.PublishedbyElsevierLtd.ThisisanopenaccessarticleundertheCCBYlicense (http://creativecommons.org/licenses/by/4.0/).

Contents

1. Introduction...130

2. Theepicardium...130

2.1. Theproepicardium:developmentaloriginoftheepicardium...130

2.2. Theheterogeneouscompositionoftheepicardium...131

2.3. Theepicardiumintheformationofthemyocardium...131

2.3.1. Epithelialtomesenchymaltransitionandmigrationofepicardial(-derived)cells...131

2.3.2. DifferentiationofEPDCsintocardiaccelltypes...131

2.3.3. Contributionofepicardialparacrinesignallingtotheformationofthemyocardium...132

3. Theepicardiumintheadultmammalianheart...132

3.1. Thequiescentepicardiumintheadultintactheart...132

3.2. Theactivatedepicardiumintheinfarctedadultheart...133

3.2.1. Activationoftheembryonicgeneprogrammeintheepicardiumpost-MI...133

3.2.2. ProliferationandEMTofepicardialcellspost-MI...133

3.2.3. MigrationofEPDCsintotheinjuredadultheart ... 133

3.3. Differentiationofadultepicardialcellsintocardiaccelltypesinvivo...134

3.3.1. Fibroblasts...134

3.3.2. Endothelialandsmoothmusclecells...134

3.3.3. Cardiomyocytes...134

3.4. Paracrinecontributionofepicardialcellstocardiacrepair...135

4. Theepicardiumasasourceofmultipotentprogenitorcells?...135

5. Futureperspectives...136

∗ Correspondingauthor.

E-mailaddress:a.m.smits@lumc.nl(A.M.Smits).

https://doi.org/10.1016/j.phrs.2017.07.020

1043-6618/©2017TheAuthors.PublishedbyElsevierLtd.ThisisanopenaccessarticleundertheCCBYlicense(http://creativecommons.org/licenses/by/4.0/).

Conflictofinterest ... 137

Funding...137

Acknowledgement...137

References...137

1. Introduction

Myocardialinfarction(MI)isoneofthemostfrequentlyoccur- ringconsequencesofcoronaryheartdisease.Ithasbeentheleading causeofdeathinthewesternworldformanydecades,anditis expectedtoremainsoforyears tocome[1].Theobstructionof bloodflowinacoronaryarteryandtheensuingsuddencessationof oxygensupplytoregionsofthemuscleresultsinmassivecelldeath, followedbyaninfluxofinflammatorycellsandcollagenproducing myofibroblasts[2].Althoughadultcardiomyocytesmaystillpos- sesssomeresidualproliferativeability[3],itisevidentthattheir contributionisinsufficienttorepairtheheart.Asaresult,injured tissueisreplacedbyarigidnon-contractilescar.Thisscartissue providestensilestrengththatpotentiallypreventstheruptureof thedamagedmyocardialwall.Importantly,scartissuealsoseverely impairscardiaccontractionwhichwillresultincardiacdysfunction becauseofpathologicalcompensatoryremodelling.Eventually,this combinationoffactorswillprogressintoheartfailure.

CurrenttherapiesforMI mainlyaimatreopeningtheculprit bloodvessel,therebyreinstatingperfusionofthedamaged area.

Theseapproachesareveryeffective,andhavegreatlyreducedthe numberof patientsthat acutelydieafterMI. Conversely,ithas resultedinanincreaseinthenumberofpeoplepronetodevel- opingheartfailure,forwhichahearttransplantistheonlyoption fortreatment.Torepairtheheart,itisnecessarytofindotherways toincreasethenumberofcardiacmyocytesafterdamage.

Overthelasttwodecades,extensiveresearchhasbeeninvested inidentifyingcellswiththeabilitytogeneratenewcardiactissue (reviewedin[4,5]).Severalofthesecellsources,whichincludethe bonemarrow,blood,andadiposetissue,havebeentestedinclini- caltrialsbuttheinfluenceoncardiacfunctionhasbeenlowerthan wasanticipated[6].However,oneofthemostimportantobser- vationsinthiseraofcell-basedtherapyisthefactthattheadult heartitselfharbourscardiacprogenitorcells.Thesecellsbearstem cell-likefeaturesandhavetheabilitytodifferentiateintonewcar- diaccellsinvitroandinvivo[7–11].Thedirecttransplantationof culturedhumancardiacprogenitorcellsintotheischemicmouse heartpositivelyinfluencedcardiacfunction[12],butonlyresulted inamarginalincreaseinnewlyformedcardiactissue[7].Never- thelessithasopenednewavenuestoexplorefortreatingcardiac damage.Thecurrentcell-basedtherapyapproachwouldrequire theisolationofprogenitorcellsfrombiopsiesofcardiactissue,and asubsequentlengthycultureprotocoltoincreasethenumberof theserarecellstoobtainsufficientmaterialtotransplant.Ifitis possibletolocallystimulateprogenitorcells,thiscouldbeavalu- ableadditiontocurrenttherapies.Assuch,theepicardiumisan intriguingcellpopulationtostudysinceitisaneasilyaccessible sourceofcellsasitislocatedontheoutsideoftheheart.Inthis reviewwewillhighlighttheoriginandfateoftheepicardium,and whyitcanpotentiallybeusedintherapeuticapproachestocure thedamagedheart.

2. Theepicardium

Anatomicallytheepicardiumispartofthepericardium,which isadoublelayeredmembranoussaccoveringtheheartandthe rootofthelargevessels.Theouter,mostsuperficiallayerofthe pericardiumisknownasthefibrouspericardiumandconsistsof connectivetissue.Itanchorsthehearttothediaphragm,thepleura,

and thesternum.Theinner fibrouspericardium is linedwitha serousmembranethatfoldsandcoverstheheart,therebyforming thepericardialsac.Theserouslayerliningthefibrouspericardium iscalledtheparietalpericardium,whilethelayerliningtheheart isknownasthevisceralpericardiumorepicardium.

2.1. Theproepicardium:developmentaloriginoftheepicardium

Earlyindevelopment,theembryonicheartconsistsofonlytwo layers:themyocardium and theendocardium.The epicardium, whichisnotyetpresentatthistubularheartstage,derivesfroman extra-cardiacmesothelialcellclusterknownastheproepicardium (PE,Fig. 1).This structure arises from thepericardial coelomic mesotheliumincloseproximityoftheheartandtheliver(Reviewed in[13]).Theliverprovidesadditionalinductivesignalsthataidthe developmentofthePE[14].Inmouse,thePEbecomesvisiblefrom embryonicday8.5(E8.5)asatransientcauliflower-likeclusterof cellslocatedatthebaseofthevenousinflowtractofthedeveloping heart.ThecellsofthePEarelocatedadjacenttotheprimitiveheart tube,butdonotdirectlyinteractwiththecardiogenicmesoderm thatwillformthefuturemyocardium[15,16].

ThePEiscomposedofdifferentcellpopulations,withanouter layerofcuboidalepithelialcellsexpressingWilms’tumour1(WT1) thatoverliesaninnercoreofextracellularmatrixharbouringsev- eralmesenchymalcelltypes,aswellasendothelialcells[17].The WT1positivecellswithinthePEoriginatefromtheearlycardiac progenitorfieldsthatexpressNkx2.5andIsl-1[18].Togetherwith theT-boxtranscriptionfactorTbx18andthebasichelix-loop-helix transcriptionfactorTcf21,WT1isusedtoidentifyPEand(laterin development)epicardial(-derived)cells[19,20].Adistinctsubset ofPEcellsexpressScleraxis(Scx)andSemaphorin3D(Sema3D) andbothproteinsonlypartiallyoverlapwithWT1andTbx18[21], therebyemphasisingtheheterogeneityofthecellcluster.

Oncetheembryonichearthaslooped,thebarehearttubewill becoveredwithanepicardial layerofcellsderivedfromthePE thattranslocatetoheartviadifferentmechanisms.Forinstancein chick,villousprotrusionsofthePEextendanddirectlyattachto themyocardialsurface,generatinga“bridge”forcellstocross[22], whileinmiceislandsofPEcellsareformedastheyareeitherpulled offfromthevilli[23]orarereleasedasfree-floatingvesicles[24].

Recently,anothermechanismwasreportedwherePEcellsreach theventriclebymigratingalongthesurfaceoftheinflowtract[25].

Next,theadheringclustersofPEcellsstarttoproliferateandspread tocoverthedevelopingmyocardiumwithasquamousepithelial layer.EpicardialcoverageoccursinthemousebetweenE9.5and E10.5inadorsaltoventralpattern.Inaddition,theleftventricleis coveredfirst,andwithamoredenselayerascomparedtotheright ventricle[26].

TounraveltheroleofthePEintheformationoftheepicardium andmyocardialwall,studieswereconductedwherethePEwaspre- ventedfromoutgrowth.Bothmicrosurgicalandgeneticinhibition ofthePEcells’migrationandadhesionreducedtheproliferationof cardiomyocytes,resultinginathin-walledcompactmyocardium [20,27,28]. Moreover, genetic mouse models causing disturbed epicardial developmentareassociated withdefectsin endocar- dial valve development and heart looping [29], cardiomyocyte proliferationandalignment[30],developmentofthecoronaryvas- culature[27,31,32],andthecardiacconductionsystem[33].Given thecrucialroleofthe(pro)epicardiumintheformationofafully

Fig.1. Developmentoftheepicardiumandepicardiallineages.

Theepicardiumoriginatesfromtheproepicardium(PE).Proepicardialcellsgothroughepithelialtomesenchymaltransition(EMT)andtranslocatetothemyocardialsurface oftheloopingheartwheretheyadhere,migrate,proliferateandundergomesenchymaltoepithelialtransition(MET)toformasquamousepitheliallayer;theepicardium.

Whiletheepicardiumremainsanintactlayer,someoftheepithelialcellswillundergoasecondroundofEMTandmigrateintothematrixrichsub-epicardiallayer(pink).

Fatemappingandgeneticlineagetracinghasdemonstratedthemulti-lineagepotentialoftheseepicardiumderivedcells(EPDCs).EPDCsdifferentiateintosmoothmuscle cells(SMCs),contributingtothecoronaryvasculatureandcardiacfibroblasts(CF)ofthematureheart.ThecontributionofEPDCstocardiacendothelialcells(ECs)and cardiomyocytes(CMs)hasbeendescribedbutismatterofanongoingdebate.

functioningheart,itisimportanttounderstanditscontributionin developmentanddisease.

2.2. Theheterogeneouscompositionoftheepicardium

WhentheepicardiumcompletelycoverstheheartaroundE11.5 inmouse [26],and week5duringhumancardiac development [34],ithasestablishedamulti-cellularepitheliumliningtheven- tricles[35].Thedetailedcompositionoftheepicardiumisnotfully known,butseveralepicardialcellspecificproteinshavebeensug- gested,althoughnotnecessarilyexpressedinthesamecell.These includeWT1[36],Tbx18[37],Tcf21[38],Gata5[39] andcytok- eratin [40]. Additionally, Semad3D, and Scx are expressed in a subsetofcells[21].Furthermore,withintheepicardium,clusters withbonemarrow-derivedCD45+cellsarepresent,demonstrat- ingthattheepicardiumisnotsolelycomposedofPE-derivedcells [41].WhetherthecellpopulationswithinthePEarerelated,i.e.if theyrepresentdistinctstagesofacontinuumofdifferentiation,or iftheyaredistinctpopulationswithspecificabilities,remainstobe investigated.

2.3. Theepicardiumintheformationofthemyocardium

2.3.1. Epithelialtomesenchymaltransitionandmigrationof epicardial(-derived)cells

Oncetheepicardiumisestablished,epicardialcellswilldirectly starttoparticipatein theformationofthecellularcomposition ofthemyocardium.Asubsetoftheepicardialcellswillundergo aprocessknownasepithelial-to-mesenchymaltransition(EMT) (reviewedin[42];Fig.1).EMTstartswithepicardialcellslosing epithelialcharacteristicsliketheirapical-basalpolarity,andtheir cell–cellcontacts,byreducingtheexpressionofthetransmem- braneadhesionproteinsE-cadherinandzonulaoccludens-1(ZO-1).

Subsequentlytheepicardialcellsacquiremesenchymalcellcharac- teristics;theygainaspindleshapemorphologyandupregulatethe expressionoffibronectin,N-cadherinandmatrixmetalloproteases (MMPs).ThisendowstheEPDCswiththeabilitytomigrateand populatethesubepicardialspace:anamorphousmatrix-richlayer which ispresent betweentheepicardiumand themyocardium (Fig.1).Fromthesubepicardium,EPDCsmigrateintothemyocar- dialinterstitiumwhere theycandifferentiateintodifferentcell

typesandcontributetothedevelopmentandmaturationofthe myocardium[43](Fig.1).

Epicardial EMT and migration are controlled via various myocardial-andepicardial-derivedgrowthfactorslikeTGF␤[44], PDGF[45],andFGF[46](reviewedin[42]).

EMTisregulatedbytranscriptionfactorssuchasWT1,TCF21, aswellasthesnailfamilymembers Snail1andSnail2 [42].For instance,WT1stimulatesEMTviadownregulationofE-cadherin expression[47],byincreasingtheexpressionofSnail,byenhanc- ing Wnt signalling, and via upregulation of Raldh2 resulting in enhanced retinoic acid signalling [48]. Furthermore, loss of neurofibromin(encodedby Nf1)resultedin increasedEMTand EPDCproliferation[49].Interestingly,whenTCF21isspecifically deletedfromtheepicardium,migrationofepicardialcellsintothe myocardiumishamperedduetoadefectinEMT[50].

Migrationofepicardialcellsintothemyocardiumisimportant forpropermyocardialdevelopmentanditiscontrolledbyasetof transcriptionfactorslikeNfatc1andMRTFs.Nfatc1isexpressedin asubsetofepicardialcellsandwhendeleteditresultedinreduced levelsofmatrix degradingenzymesand a subsequentimpaired migration of EPDCs into the myocardium [51]. The myocardin relatedtranscriptionfactors(MRTF)-Aand−Bareinducedwhen EPDCsaretreatedwithTGF␤invitro,resultinginenhancedmigra- tion.Invivo,whenMRTF-A/Bareknockedoutspecificallyinthe epicardium, migration ofEPDCs intothesub epicardial layeris impaired[52].

Theinvolvementofallthesefactorsinatemporalandspatial controlledmannerto regulateepicardial EMTand migration of EPDCsintothemyocardiumiscrucialforproperdevelopmentof themyocardialwall.

2.3.2. DifferentiationofEPDCsintocardiaccelltypes

OnceEPDCshaveinvadedthemyocardium,theywillstartto differentiateintoseveralcardiaccelltypes(Fig.1).Thepredomi- nantcelltypesareinterstitialfibroblaststhatproducethecardiac extracellular matrix, smooth musclecells (SMCs), and adventi- tialfibroblaststhatsustainthecoronaryvasculature[36,53,54].In contrast,contributiontoendothelialcellsandcardiomyocytesis subjecttodebate[55,56](Fig.1).

ToexplorethedifferentiationcapacityofEPDCs,invitromod- elshavebeendevelopedtocultureEPDCsfrommouseembryonic heartexplants.Theseculturesystemshaveconfirmeddifferenti-

ationofEPDCsintofibroblasts andSMCsafteradditionofTGF␤ [44,57,58].Furthermore,additionofthymosin␤4,combinedwith VEGF/FGF7,resultedinahighlysignificantincreaseinTie2-positive endothelialcells[59].Additionally,cardiomyocytedifferentiation invitrowasobservedusingembryonicWT1⁺cells[36].Whilethese invitromodelsmaypredicttheabilityofepicardialcellstodiffer- entiateintomanycelltypesthatcomposetheheart,itisimportant torealisethattheseconditionsareartificialandmaynotrepresent theactualinvivosituationwhereinteractionwithothercellsand signalsoccurs.

Invivo,thefirststudiesinvestigatingthedifferentiationpoten- tialof epicardium were performed usingretroviral labelling in quail-chickchimeraexperiments.Theseexperimentsindicatedan ability of EPDCs to contribute to fibroblast, SMCand coronary endothelial lineages [60–62]. Subsequent genetic fate-mapping experimentsin micewhere the expression of Cre-recombinase (Cre) is driven by epicardial-cellspecific promoters (e.g. WT1, Tbx18,Tcf21,Gata5,Sema3dandScx)generallyconfirmedtheepi- cardialoriginoffibroblastsandSMCs(seeTable1,andTianetal.

[63])

Concerningtheoriginofendothelialcells,lineagetracingstud- ieshavedemonstrateddifferentresultsdependingonthepromoter thatwasusedtofollowthecells.Tcf21andTbx18 lineagetrac- ingstudieswereunabletoestablishepicardialderivedendothelial cells[37,50,64],whileWT1,GATA4,SEMA3DandScxfate-mapping studieshaveshownco-localizationofgeneticallylabelledcellswith ECproteins(Table1) [21,32,36,65]. Controversybetweenthese studiescouldbeexplainedbythefactthatthePEandepicardium containheterogeneouscellpopulationsthatcouldreflectdistinct populationswitha diversedifferentiationpotential(seeSection 2.2).ThiswasfurtherdemonstratedbyKatzetal.,showingthata TBX18/WT1negativeandSEMA3D/Scleraxispositivecellpopula- tioninthePEgivesrisetoendothelialcells[21].Furthermore,some resultsshouldbeinterpretedcautiously.Chick-quailchimerascan becontaminatedwithcellsofthesinusvenosus;astructureknown tocontributetothecoronaryvasculature[66].Additionally,WT1 wasshowntobeexpressedincardiacendothelialcells[34,56,67], whichmayobscuretheresultsfromlineagetracingexperiments.

Therefore,althoughsomestudiesconcludethatendothelialcells canderivefromtheepicardium,itremainsanongoingdebateto whatextendthePE/epicardiumcontributestothecoronaryvas- culatureinthedevelopingheart.Overallthedirectcontributionof epicardialcells,ifany,appearstobelowandboththesinusveno- susandendocardium[21,68]areconsideredthemajorcontributors (reviewedin[63]).

With respect to epicardial-derived cardiomyocytes, lineage tracingstudiesusingScleraxis,WT1andTBX18totracecellshave demonstrated epicardial-derived functionally active cardiomy- ocytes(Table1).Howeveritmustbenotedthatthesefindingsare controversial,sincesomeofthegenesdrivingtheexpressionofCre recombinaseinthesestudies,e.g.Tbx18[37],andWT1[36],have beenshowntobepresentwithincardiomyocytesatcertainstages duringdevelopment[55,67].

Althoughthefulldifferentiationpotentialinvivoisyettobecon- firmed,itishoweverclearthatembryonicEPDCshaveanessential impactoncardiacdevelopmentviadirectcontributionofcellsto themyocardium(Fig.1).

2.3.3. Contributionofepicardialparacrinesignallingtothe formationofthemyocardium

Anotherprocessthatoccursoncetheepicardiumhascovered the developing heart is that epicardial cells will start to pro- duceparacrinefactorsthatsupportmyocardialgrowth[69–71].

Thesefactorsprovide anessential exchangeof signalsbetween the myocardium and the epicardium that are crucially impor- tant for the developmentof the coronary vessels (reviewed in

[72]), as well as the growth and differentiation of the heart muscle[73].For instance, Kolanderet al. showedthat disrupt- ing GATA4and GATA6 signalling specifically in theepicardium resultsindefectivecoronaryvasculardevelopmentbyregulation ofthenumberofsubepicardialendothelialcellsviasecretedfac- tors[74].Anotherexampleofepicardial-myocardialsignallingis retinoic acid (RA), a known contributor tocardiomyocyte pro- liferation. Retinoid X receptor ␣ (RXR␣) knock out mice are lethalasaresultofhypoplasticmyocardium,anddemonstratea poorlyattachedepicardium[75].Interestingly,thisphenotypewas demonstratedtobeepicardial-relatedsincedeletionofRXR␣in GATA5expressingcellsalsodemonstratedimpairedcardiaccom- paction,asaconsequenceofadecreasedcyclingofcardiomyocytes [76].Furthermore,multipleFibroblastGrowthFactors(FGFs)are expressedin theepicardium andare shown tobeessential for propermyocardialformation[46,77,78].Forexample,depletionof theepicardiallyexpressedFibroblastGrowthFactor9(FGF9),or depletionoftheconcomitantFGFreceptorspresentonpremature cardiacmyoblasts,resultsinembryoniclethalityandreducedcar- diomyocyteproliferationinthedevelopingheart[77].Alsoother producedfactors,suchasHedgehog[78,79]andCXCL12[80]were showntobeinvolvedinmyocardialmorphogenesisandcoronary development.Thisemphasisestheimportanceoftheepicardium incardiacdevelopment,bycontributingcellsaswellasessential cytokinesandgrowthfactorstoinducethemyocardialdevelop- ment.

3. Theepicardiumintheadultmammalianheart 3.1. Thequiescentepicardiumintheadultintactheart

Contrarytodevelopment,thepostnatalmammalianepicardium is a dormant single-cell layer. This was shown via the analy- sisofgenesinvolvedinepicardialactivationsuchasWT1,Tbx18 andRaldh2.Thesegenesareabundantlypresentintheembryonic heart,butarerapidlydownregulatedpostnatally,onlytobebarely detectableat3monthsofageinthemouse[81].Thesamecon- clusiononthedormancyoftheepicardiumwasdrawnbyusing amousemodelwheretheGreenFluorescentProtein(GFP)gene wasknocked-inintotheWT1locus(WT1^GFPCre).Wheninvestigat- ingtheWT1-drivenexpressionofGFPindifferentstagesofheart development,Zhouetal.observedthatintheembryo,itsexpres- sionwasrestrictedtotheepicardiumandlabelledapproximately 90%oftheepicardialcells[82].Incontrast,intheadultheart,GFP expressionwasobservedinfewerthan25%ofthecellswithinthe epicardium;therebyrevealingthatWT1ispresentatstageswhere theepicardiumactivelyplaysaroleinheartdevelopment.Thislack ofactivationintheadultheartwasfurtherunderscoredbyusinga tamoxifeninducibleWT1^CreERT2micecrossedontoaCrereporter line(Rosa^mTmG).Inthismodel,theactivationofWT1onlyresultsin translocationofCrerecombinasetothenucleuswhentamoxifenis administered.BycrossingthesemiceontotheRosa^mTmGreporter line,nucleartranslocationofCreresultsintheirreversibleexchange ofexpressionofredfluorescentTomatointotheindefiniteexpres- sionofgreenfluorescentGFP.Thismodel,whichisextensivelyused inthefieldofepicardialresearch,therebyprovidestheopportunity tolabelWT1⁺cellsatanygiventimepoint,andtotracktheirfate basedonthecontinuousexpressionofGFP.Investigatinguninjured adultheartsusingthismodeluptoeightweeksaftertamoxifen injectionrevealednomigrationofWT1/GFPexpressingcellsinto themyocardium[82].Together,thesedatashowthatintheadult uninjuredheart,theepicardiumisaquiescentcelllayer,asshown bythedownregulationofepicardialspecificgenesandthelackof migrationofcellsaftertheformationoftheheartiscompleted.

Table1

Overviewofinvivoepicardiallineagetracinginmouseembryos.

Mousemodel Reportergene EPDCdifferentiation Identifiedmarkers Reference

SEMA3D^GFPCre/+ R26R^LacZ ECSMCCM^$Fibro cTnT,SM22␣,Vimentin,Flk-1,PECAM [21]

Scx^GFPCre R26R^LacZ ECSMC^$CMFibroEndocardial^$ cTnT,Cx43,SMM,FSP,Flk-1,PECAM,Nfatc1,NRP-1,EphrinB2 [21]

G2-Gata4^Cre Rosa26^YFP ECSMC* PECAM,IB4,␣SMA,NOTCH1 [32]

Wt1^CreYFP+ Rosa26^YFP ECSMC* PECAM,␣SMA [32]

Wt1^CreERT2 Rosa26^YFP EC* PECAM [32]

Wt1^GFPCre Rosa26^fsLzandZ/Red SMCECCM PDGFR␤,SM22,SM-MHC,Flk-1,PECAM,Tnnt2,Actn1,GATA4,

Nkx2.5,Cx43,functionalcoupling

[36]

Wt1^CreERT2 Rosa26^fsLzandZ/Red SMCECCM Actn1,PECAM,PDGFR,Tnnt [36]

Tbx18^Cre R26R^LacZ CMSMCFibro Tnnt,cTnI,MF20,GATA4,Nkx2.5,SM-MHC,NRP-1,PDGFrb,

Col1a2

[37]

Tcf21^iCre/+ R26R^YFPandR26R^tdT Fibro PDGFR␣,Col1 [50]

Wt1^Cre R26R^mTmG FibroCM* FlnA,Vimentin,Col1a1,MF20 [54]

Tbx18^cre/+ Rosa26^mTmG FibroSMC# Nos3,Emcn,SMaActin,Notch3,Postn,Tnni3,Myhc [64]

Wt1^CreER Rosa26^RFP SMCPericyteEC* SM22,SM-MHC,SMA,PECAM [65]

Tbx18^Cre Rosa26^RFP SMC* SMA [65]

Wt1^CreERT2 Rosa^mTmG Pericyte* CSPG4,PDGFR␤ [115]

Onlystudiesaimingtousethemousemodelforepicardiallineagetracingwereincluded.

*Didnotinvestigateothercelltypes,$Sporadic,#investigatedCMsonlyinrightventricle.

Abbreviations:Actn1:Alpha-actinin-1,CM:Cardiomyocyte,Col:Collagen,CSPG4:ChondroitinSulfateProteoglycan4,cTnI:cardiacTroponinI,cTnT:cardiacmuscletroponin T,Cx43:Connexin43,EC:EndothelialCell,Emcn:Endomucin,Fibro:Fibroblast,Flk-1:Fetalliverkinase1,FlnA:FilaminA,FSP:Fibroblast-specificProtein,IB4:IsolectinB4, MF20:MyosinHeavyChain,Myhc:Myosinheavychaincardiacmuscle,Nfatc1:NuclearfactorofactivatedT-cells,cytoplasmic1,Nkx2.5:NK2Homeobox5,Nos3:Nitric oxidesynthase3,Notch:Neurogeniclocusnotchhomologprotein,NRP-1:Neuropilin-1,Pdgfr:Platelet-derivedgrowthfactorreceptor,PECAM:PlateletEndothelialCell AdhesionMolecule,Postn:Periostin,SM22:SmoothMuscleProtein22,SMaActin:Actin␣2SmoothMuscleaorta,SMC:SmoothMuscleCell,SMM:Smoothmusclemyosin, SM-MHC:SmoothMuscle-MyosinHeavyChain,Tnni3:TroponinIcardiac3,Tnnt:TroponinT,␣SMA:␣SmoothMuscleActin.

3.2. Theactivatedepicardiumintheinfarctedadultheart

3.2.1. Activationoftheembryonicgeneprogrammeinthe epicardiumpost-MI

In the adult heart, the epicardium can be awakened from its dormant state. Several studies have shown that MI or ischemia/reperfusioncanresultin reactivationof theepicardial layer,includingitsproliferationandexpansion,EMT,andmigra- tionofepicardialderivedcells (reviewedin[42,83]).Withinthe firstdaysafterMI,theepicardiallayerdisplaysapartialrecapit- ulationoftheembryonicgene programme,which isevidentby theupregulationoftheepicardial genesWt1,Raldh2 andTbx18 [82,84,85].Atfivedayspost-MI,theexpressionofthesegenespeak, andtheyremainpresentinapproximately75%oftheepicardial cellsat14daysafterinjury,afterwhichtheexpressiondecreases gradually[82].Interestingly,theupregulationofgenesisobserved notonlyatthesiteofinjury,butthroughouttheepicardium.One theoryisthatthisoccursviafactorssecretedintothepericardial fluid.StudiesinducingMIwithanintactpericardiumhaveshown anattenuatedcardiacfunctionpost-MIcomparedtoMIwherethe pericardiumwasopenedpriortoligation[84,86].

Howtheactivationoftheepicardiumisregulatedonatran- scriptionallevelisnotfullyunderstood,howeveritappearstobe partiallycontrolledviatheC/EBPtranscriptionfactorfamily[85].

Interestingly,incontrasttoMI,miceundergoingtransverseaor- ticconstriction(TAC)toinducehypertrophyandfibrosisdisplay noactivationofWT1⁺cellswithintheepicardium[87],suggesting thatcardiacdamagethroughischemiaislikelyapotentactivator ofthislayer.

3.2.2. ProliferationandEMTofepicardialcellspost-MI

FollowingMI,theepicardiumcoveringtheinjuredareaisini- tiallycompletelylostandisrebuiltfromthesurvivingepicardium within three days post-injury [88]. The tamoxifen inducible WT1^CreERT2Rosa26^mTmGlineagetracingmodelwasabletoconfirm thatthisisduetoproliferationofpre-existingepicardialcells.At twodayspost-MI,phospho-histoneH3andBrdUstainingrevealed manyproliferatingWT1⁺/GFP⁺epicardialcells[82].Thisresultedin atransitionfromasinglecellepitheliumintoamulti-layeredsheet, inanorganwidefashion,butmostpronouncedneartheinjured area[82,88].Thisprocessisspecifictoacuteischemicinjurieslike

MIandischemiareperfusion,asthickeningoftheepicardiumis absentinmodelsforhypertrophylikeTAC[89].

Furtherrecapitulationoftheembryonicactivationprocessinthe ischemicheartisshownbytheupregulationofseveralEMT-related transcriptionfactorsincludingSmad1,Snail,SlugandTwistinEPDCs [82,88]. Interestingly,BrdU incorporation experiments revealed thattheepicardialcellspositiveforEMT-markersareresponsible forthenewlygeneratedsubepicardialmesenchyme[88].

Theactivationandexpansionoftheepicardiallayeriscrucialfor thepost-injuryresponse,aswasillustratedbyDuanandcolleagues [90].ThespecificdisruptionofWntsignallingwithinepicardialcells inamousemodelofcardiacischemiareperfusioninjuryrevealed lessepicardialEMT,andareducedsubepicardialcollagendeposi- tion.Asaresult,ventriculardilatationandadecreasedfractional shorteningwereobserved[90].Equally,inastudyweretheepi- cardiumwasprimedbysystemicinjectionofthymosin␤4prior toMI,athicker layerof activatedepicardium,and moremigra- tionwasobservedandresultedinasubsequentpositiveeffecton cardiacfunction[81].Thesestudiesemphasizetheimportanceof understandingandoptimisingtheepicardialactivationandEMT toenhancethepost-injuryresponse[42].Ourrecentinvitrodata showthatbothhumanadultandfoetalepicardialcells undergo activationandEMT[91].Interestingly,foetalcellswereactivated moreefficiently,andunderwentEMTspontaneously.Thispoten- tiallyreflectsthedifferencesinefficiencyofactivationinvivoduring developmentandintheadultheart.Assuchthefoetalprocesses mayserveasablueprintforanoptimaladultpost-injuryresponse.

3.2.3. MigrationofEPDCsintotheinjuredadultheart

Whether adult EPDCs retain the ability to migrate into the damaged area is subject to debate. Two studies using the WT1^CreERT2Rosa26^mTmGinjectedwithtamoxifentotracetheepi- cardiumwithGFPindeedconfirmedtheexpansionofthis layer afterMI,butlabelledWT1⁺cellsappearedtoberetainedsubepi- cardially showing noindicationof migrating into thedamaged myocardium [82,92]. However,in a later studyusing thesame mousemodel,theinjectionofmodified-RNAencodingforVEGF- Aprotein(modRNAVEGF-A)afterinjurydidrevealmigrationof cellsintotheheart[93].

Severalalternativemethodstomark cellswithintheepicar- diallayerhavebeenapplied.Thefirstoneistoinjectfluorescent

protein-producinglentiviruses into the pericardial fluid, which isin directcontact withtheepicardiallayer,allowinginfection and labelling of the epicardium [53,94]. A second approach is toapplyabiocompatiblegelcontainingamodified RNAforCre on the epicardium of the Rosa^mTmG reporter mouse [93]. The patch wasapplied two weeks prior to MI to prevent labelling ofnon-epicardialcells in thecomplicated injuredenvironment.

Bothmethods revealedthatepicardial-derivedcellswerefound withintheinfarctedmyocardiumat7and21dayspost-MI[93,94].

Althoughthesestudiesrelyonepicardiallabellingbasedonlocation ratherthanusingepicardialspecificmarkers,theycoincidewith theresultsofageneticlabellingstudywhereintheBacterialArti- ficialChromosome(BAC)-WT1^Cre;R26Rmice,beta-galactosidase expressingcellswereobservedintheinfarctedareaafter1month [88].

Itischallengingtocorrelatethesedifferentfindingsinmouse lineagetracemodels.Inthenon-inducibleBACmodel,background labelling can occurif cells within themyocardial wall start to expressWT1[56],whichislesslikelytohappenintheinducible lineagetracemodelsincethelabellingoccursduringashortperiod oftime.However,adownsideofthetamoxifenlabellingisthatthe shortdurationoflabellingmayresultinmissingofmanycellsand thereforeanunderestimationoftheepicardialcontribution.

Althoughthesedifferentconclusionsregardingmigrationmay betheresultoftechnicalissues,itappearsthat,atleastpartially, migration isa component of theadultreactivating responseto injury.

3.3. Differentiationofadultepicardialcellsintocardiaccelltypes invivo

3.3.1. Fibroblasts

Asmentionedabove,themostprominentcontributionofepi- cardialcellstotheformationoftheembryonicmyocardiumisvia differentiationintocoronarySMCs,andinterstitialandadventitial fibroblasts.Intheadultinjuredheart,asimilardifferentiationpro- fileisobserved(Table2).LineagetracingmodelsusingWT1asa Credriver,showedthatamajorityoftracedcells transitioninto fibroblasts[82,88,92].Additionally,afterischemicinjury,theepi- cardiumthickness increasesupto6-fold,and thedepositionof collageninthesubepicardialspaceisalsodramaticallyincreased [89]indicatingariseinfibroblastactivity.

Russell et al. [95] used a Notch reporter mouse line which revealedsignificantactivationofthereporterintheepicardium.

Notch reporter positive epicardial cells wereisolated and sub- jectedtomicroarrayanalysiswhichshowedthatthesecellshave afibroblastsignaturewithhighexpressionofcollagen-I,elastin andfibronectin.Therefore,fibroblastslikelyrepresentthedefault programme inthe activatedepicardial layer[95]. Althoughtoo muchactivityofcardiacfibroblastscouldresultinexcessivescar formation,theyrepresent a vital cellularcomponent in cardiac homeostasis and wound healing [96]. Reducing Wnt/␤catenin specifically in cardiac fibroblasts resulted in reduced collagen depositionand a worsecardiacfunctionpost-injuryrevealing a beneficialroleofcardiacfibroblastsincardiacrepair[90].

3.3.2. Endothelialandsmoothmusclecells

Withrespecttoneovascularisationpost-injury,manystudies haveidentifiedlineage-tracedEPDCsthatdifferentiateintosmooth musclecellspost-MI[82,88,92,93,97,98](Table2),whichwasantic- ipatedbasedonthedescribedembryonicfateofEPDCs.Incontrast, most analyses failed to identify EPDCs that differentiated into endothelialcells[81,82,92](Table2).However,astudyusingthe BACWT1^Cre;R26Rmouselinetodeterminethefateofepicardial cellsbasedon␤-galactosidase(␤-gal)expressionrevealed␤-gal⁺ cellsco-expressingPECAMwerefoundtolinevesselswithinthe

infarctedarea[88].Althoughintriguing,thisislikely duetothe factthatWT1canbere-expressedinendothelialcellspost-injury [56]andcouldthereforerepresentanartefact,makingitunlikely thatthereisadirectcontributiontoendothelialcellsinthenormal responseofthehearttoMI.

Interestingly, injection of modRNA VEGF-A into the mouse myocardiumafter MIresulted in anincreasedcapillary density andasmallerinfarctsize.Thiscoincidedwithanenhancedacti- vationof epicardialcells, which showedanupregulationofthe VEGFreceptorKDRinWT1-GFPexpressingcellsintheWT1^GFPCre mouseline.Afterisolationandsortingofepicardialcellspost-MI, culturedWT1+cellsrespondedtoVEGF-Abyincreasingtheirpro- liferation.Moreover,VEGF-Awasabletoshiftthedifferentiation ofepicardialprogenitorcells towardstheendotheliallineageas shownbyanincreaseofVE-Cadherin,KDRandPECAM1expression.

Thiswasfurthercorroboratedbyinvitroclonalassaysandlineage traceexperimentsusingeithertheWT-1^CreRT2xROSA^mTmGandor amodRNACreexpressingpatchontheepicardium[93].Thisstudy emphasisestheplasticityofepicardialcells,providedtherightcues aredeliveredattherighttime(Table2).

3.3.3. Cardiomyocytes

ThedifferentiationofEPDCsintocardiomyocytesintheembryo iscontroversial,andthesameholdstrueintheadult(Table2).In astudybyLimanaetal.,theepicardiumwaslabelled viainjec- tionof aGFP producinglentivirus3daysbeforeexperimentally induced MI, which revealedthat somecells thatmigrated into the infarcted myocardium expressed ␣-sarcomeric actin, albeit inverylownumbers[94].Theobservationthat EPDCshavethe abilitytodifferentiateintocardiomyocyteswasconfirmedinthe BAC-WT1^Cre;R26Rmouseline.Epicardialderived␤-gal⁺cellsco- expressingtroponinIwerefoundwithinthemyocardiumbutonly afteroneandthreemonthspost-MI.TheseEPDC-derivedcardiomy- ocytesmaintainedanimmaturephenotype,beingsmallroundcells lackingsarcomericorganisation,andnumberswerestillverylow [88].

An enhanced post-injury activation of the epicardium was observed when animals were treated with thymosin ␤4 prior to myocardial infarction. Interestingly, in this study using the WT1^CreERT2;R26R^EYFPline,differentiationofepicardialderivedcells intotroponinTand␣-sarcomericactinexpressingYFP⁺cellsthat coupledtothesurroundingmyocardial tissuewasobserved.To counterthattheseresultsareduetoextra-epicardialexpressionof WT1,YFP⁺epicardialderivedcellswereisolatedfromthymosin␤4 treatedmice4daysaftermyocardialinfarctionandtransplanted into the infarcted myocardium of a recipient wildtype mouse;

a small number of the YFP⁺ donor cells differentiated into ␣- sarcomericactinexpressingcells. Asimilareffectwasobserved whenmodRNAencodingVEGF-Awasinjectedintotheperi-infarct zonetoenhancetheepicardialresponse;besidesendothelialcells,a slightincreaseincardiomyocyteswasfoundbasedonTNN3expres- sioninlineagetracedepicardialcells[93].Althoughtheoccurrence ofcardiomyocytedifferentiationisstillextremelylowinthesetwo studies[81,93],itprovidesaproof-of–conceptthatcardiomyocyte formationfromEPDCscouldbeapossibility.

In contrast,there are severalstudiesusing a similarlineage tracingapproach(WT1^CreERT2;R26^mTmG)thatdidnotobservedif- ferentiation into cardiomyocytes [82,98]. Of note is that these studiesalsodidnotobservemigrationofEPDCsfromthesubepicar- dialtothemyocardiumafterinjury,whetherthisisduetotechnical issuesneedstobeaddressed.

Thedataarguethatintheadult,theepicardialpotentialforcar- diomyocyteformationiscomparabletotheembryonicheart:its abilitytodelivercardiomyocytesdirectlyislowtonon-existentbut canbeenhancedbyprimingtheepicardiumusingspecificfactors.

Table2

Overviewofinvivoepicardiallineagetracingfollowingmyocardialinfarctioninmice.

Mousemodel Reportergene EPDCdifferentiation Identifiedmarkers Treatment Reference

WT1^CreERT2/+ R26R^EYFP CM* cTnT,s␣Actin,Cx43,N-Cad,Ca2⁺transients,Functional

coupling

T␤4 [81]

WT1^CreERT2/+ R26R^mTmG SMCFibroEC^$ SM-MHC,␣SMA,SM22␣,FN1,ColIII,FSP1,ProCol1,PECAM – [82]

Ad:Msln-Cre R26R^mTmG Fibro FSP1 – [82]

(BAC)WT1^EGFPCre R26R SMCECCM^$Fibro ␣SMA,PECAM+locationinvesselwall,cTnI,SERCA2,via exclusionofothermarkers

– [88]

WT1^CreERT2/+ R26R^mTmG SMCFibro ␣SMA,DDR2,ProCol1,desmin,FSP1,ColIII T␤4 [92]

WT1^CreERT2 R26R^mTmG ECCM* PECAM1,Kdr,Tnnt2,TNNI3 VEGF-AmodRNA [93]

CremodRNAgel R26R^mTmG SMCECCM SM-MHC,PECAM1,TNNI3 VEGF-AmodRNA [93]

LV-CMVGFP – CM* s␣Actin,morphology – [94]

Gata5-Cre R26R^EYFP SMC^ˆEC^ˆFibro ␣SMA,PECAM,ProCol1 T␤4 [97]

Onlystudiesaimingtousethemousemodelforepicardiallineagetracingwereincluded.

*Othercelltypesnotreported,$Sporadic, ˆDisplayednomaturecharacteristics,remainedrounded.

Abbreviations:Ad:Adenovirus,CM:Cardiomyocyte,ColIII:CollagentypeIII,cTnI:cardiacTroponinI,cTnT:cardiacmuscleTroponinT,Cx43:Connexin43,DDR2:Discoidin DomainReceptor2,EC:EndothelialCell,Fibro:Fibroblast,FN1:Fibronectin1,FSP1:Fibroblast-specificprotein1,Kdr:Kinaseinsertdomainreceptor,Msln:Mesothelin,N-Cad:

N-Cadherin,PECAM:PlateletEndothelialCellAdhesionMolecule,proCol1:procollagen1,SERCA2:SarcoplasmicReticulumCa2+ATPase2,SM22:SmoothMuscleProtein22, SMC:SmoothMuscleCell,SM-MHC:SmoothMuscle-MyosinHeavyChain,s␣Actin:sarcomeric␣Actin,Tnni3:TroponinIcardiac3,Tnnt:TroponinT,T␤4:Thymosin␤4,

␣SMA:␣SmoothMuscleActin.

3.4. Paracrinecontributionofepicardialcellstocardiacrepair

Besidescontributingdirectlytotheformation ofcardiaccell types,epicardial cells canparticipate in repair of theheart via paracrineprocesses.

Zhouetal.observedmanybloodvesselswithinthethickened subepicardiumafterMI.Thesenewlyformedvesselswereoften locatedincloseproximitytoEPDCs.Invitroexperimentswiththe GFP⁺lineage-tracedEPDCsconfirmedthatthesecellsindeedhave proangiogenicproperties[82].Interestingly,theinjectionofcon- ditioned mediumfrom cultured EPDCs intothe infarcted heart resultedinanincreaseinvesseldensityandreducedtheadverse remodellingoftheheartpost-MIinshortandlong-termfollow- up[82].A similarfindingwas observedwhen injecting human EPDCsintotheinfarctedheartofNOD-SCIDmice.Althoughhuman cellsurvivalwasminimalat6weekspost-MI,asignificanteffect onvascularisationwasapparent,againemphasisinganangiogenic effect of EPDCs [99] (and our own unpublished observations).

Interestingly,EPDCsmayalsoinfluencecardiomyocyteswithinthe myocardiumviaparacrinemechanisms.Co-cultureexperimentsof EPDCsandcardiomyocytesresultedinincreasedmyocyteprolifera- tionandenhancedlevelsofcardiomyocytedifferentiation[30,100].

Besides using conditioned medium or cells, a single factor excretedbyepicardialcellswasidentifiedthatsignificantlyaidsin cardiacrepair.Follistatin-like1wasidentifiedasahighlyenriched factor produced by epicardial cells with the ability to induce cardiomyocyteproliferation[101]andtoincreasecardiomyocyte survival[102,103].Bydeliveringthisfactordirectlytotheinfarcted myocardiumviaanepicardiallyappliedpatchWeietal.showed anincreaseinproliferationoflocalcardiomyocytesresultinginan increasedsurvival,areductionoffibrosisandpreventeddeterio- rationofcardiacfunction[101].Follistatin-like1didnothavean effectontheepicardiumitself.

Conversely,epicardialcellscanbeinfluencedtopartakeincar- diacrepair byadditionofa singlefactor.Foglioetal.identified clusterinas highlyenriched proteinwithinthepericardialfluid ofMIpatients[86].Invitro,clusterinwasshowntoinducepro- liferationandEMTofepicardialcells.Invivo,theinjectionofthis singleproteinintothepericardialfluidoftheinfarctedmouseheart wassufficienttoenhanceepicardialEMT,andinducecellsurvival, arterioledensity,andresultedinanamelioratedcardiacfunction [86].

Thesestudiesstressthatepicardialcellsdonotonlyparticipate incardiac repairbyproviding cells, buthave amajorcontribu- tionto therepair mechanisms withintheheart via production ofessentialproteins.Moreover,sinceepicardialcellsthemselves

aresensitivetoinductionviacytokinesandgrowthfactors,one canimagineafeedforwardloopweresingleproteinsenhancethe numberofepicardialcellsthatproduceparacrinefactors,thereby furtherstimulatingcardiacrepairafterinjury.

4. Theepicardiumasasourceofmultipotentprogenitor cells?

Fromthe datadescribed above,thequestion ariseswhether EPDCsshouldbeconsideredcardiacprogenitorcells.Itbecomes clearthattheadultepicardiumintheinfarctedheartisactivated, undergoesEMTandcontributestoseveralcardiaccelltypes.Epi- cardialEMThasbeenpostulatedtobeinvolvedintheformationof residentcardiacprogenitorcells[104].Theadultepicardiummay thereforefunctionasareservoirofmesenchymalprogenitorcells [94].

Classically,stemor progenitorcells canbedefinedasaself- maintainingpopulationofrelativelyundifferentiated,proliferative cells that can produce a variety of differentiated progeny with theabilitytoregeneratetissue ofparts thereof[105].Based on theexpressionofanembryonicgeneprogramme,theirabilityto displayproliferationupondamage,andtheirdifferentiationinto severallineages,EPDCspartiallyfulfilthesecriteria.However,if theyfulfilallcriteriatobetruecardiacprogenitorcellsremains tobeestablished.Tothisenditisimportanttodefinewhichpro- genitorsarenecessarytodeliverthecellsofthedevelopingheart [106].

Withintheadultheart,cellshavebeendiscoveredexpressing stemcellmarkerslikethetyrosinekinasereceptorc-Kit[11],and stemcellantigen(Sca)-1[10]ontheircellsurface.Thesec-Kitand Sca-1expressingcellsareconsideredtobestem-orprogenitorcells basedontheirabilitytoformcolonies,todisplaytelomeraseactiv- ityandtoshowlong-termlabelretention.Thesecellularabilities aregenerally linkedtostem-cell features.Moreover,thesecells havebeenshowntodifferentiateintocardiomyocytes,endothe- lialcells,smoothmusclecellsandfibroblasts,therebyindicating theirabilitytodifferentiateintoallcelltypesrequiredtogenerate cardiactissueandparticipateincardiacregeneration(pleaserefer tothereviewsincludedinthisspecialeditionofPharmacological Research).

Thehumanfoetalandadult(sub)epicardiumishighlyheteroge- neous[107]andwasshowntoharbouraminorpopulationofc-Kit andCD34expressingcells[84,94].Invitro,thesec-Kitexpressing epicardialcellsdifferentiatedintotheendothelialandsmoothmus- clecelllineage[94].Asimilarpopulationwasobservedintheadult mouseheart.Interestingly,thepercentageofproliferativec-Kit⁺

Fig.2.Potentialmechanismsofepicardial-derivedcontributiontocardiacrepair.

ActivatedepicardialcellscanhaveadirectcellularcontributiontocardiacregenerationasepicardialcellsundergoEMT,migrateintothemyocardiumanddifferentiate towardscardiaccells,suchascardiacfibroblasts,smoothmusclecellsandpotentiallyothercelltypes.Asecondepicardialcontributiontocardiacrepairactsviaparacrine signalling,inducing(1)proliferationofcardiomyocytes,(2)angiogenesisand(3)survivalofcardiomyocytesintheinfarctedarea.

cellsincreasedafterinductionofMIandthesecellsappearedtopar- ticipateintheformationofsubepicardialbloodvessels[94].Bollini andcolleagues[108]performedadirectcomparisonbetweenthe expressionprofilesofmouseembryonicEPDCs,andadultEPDCs isolatedafterthymosin␤4stimulationandMI[108].Theyobserved alowexpressionofSca-1intheWT1⁺embryoniccells,whilein theadult,Sca-1waspresentonapproximately60%oftheWT1⁺ EPDCs.Moreover,the mesenchymalmarkers endoglin (CD105), thehyaluronanreceptorCD44,majorT-cellantigen 1(Thy-1or CD90)andPlateletDerivedGrowthFactorReceptor(PDGFR)␤were observedintheactivatedadultEPDCs.Theycontinuedtoshowthat withintheadultepicardiumpost-MI,theWT1⁺,Sca-1⁺,CD90^hi, CD44^hi populationsretaincardiovascularmultipotency,asbased ontheexpressionofcardiacprogenitormarkersinthispopulation [108].Thesedataindicatethatthymosin␤4incombinationwithMI canstimulateacardiacprogenitorcelltypewithintheepicardium.

Cells with mesenchymal-like properties have been isolated andculturedfromtheadultmouse[109]andadulthumanheart [110,111].Thesecells,termedcardiaccolony-formingunitsfibro- blast(cCFU-F),originatedfromtheepicardium,andweredefinedas mesenchymalstemcellsbecausetheyshowedclonogenicpropa- gation,long-termgrowthforover40passageswithoutsenescence, andmulti-lineagedifferentiation.Furthermore,thesemesenchy- mal progenitors express mesenchymal cell markers including CD105,CD44,CD90,andthestemcellmarkersOct4andc-Myc.

Additionally,theyareMSC-likeintheirtranscriptomeprofile[112].

AlthoughcCFU-FsexpressSca-1andPDGFR␣,lineagetracingusing theNkx2.5^Crefatemappingmouselinesuggestedthattheydonot arisefromtheNkx2.5cardiacprogenitorlineage[109].

A comparable cell culture population of mesenchymal-like EPDCswasisolated fromthehumanepicardium. Thesespindle EPDCs(sEPDCs)areabundantlydecoratedwiththemesenchymal stemcellmarkersCD44,CD90andCD105,expresstheearlycardiac transcriptionfactor GATA-4and showmulti-lineage differentia- tion.WhenstimulatedinvitrowithTGF-␤,sEPDCsexpressproteins ofthesmooth musclecelllineageand calcifywhencultured in osteogenicmedium[58].Interestingly,whilesEPDCsonlyexpress GATA-4,freshlyisolatedcCFU-Fsarealsoreportedtoexpressthe earlycardiac markerNkx2.5. Furthermore, sEPDCsdifferentiate intothemesenchymallineage, butcCFU-Fshave theadditional abilitytoacquireanendothelialphenotype,whichhasnotbeen

convincinglyestablishedforEPDCs.sEPDCsandcCFU-Fsarethere- foreveryalike,butwhethertheyaresimilarisnotcleartodate.

Russelletal.isolatedapopulationofnotch-reporterpositivecells, thatwererelatedtotheepicardium.Thesecellswerenotonlyable todifferentiateintofibroblasts,butundertherightculturecon- ditionsformedcardiomyocytes[95].Additionally,invitrostudies usingVEGF-AonWT-1positiveisolatedcellsshowedthatthesecell couldbeinstructedtoproliferateandbecomecardiovascularcell types[93].

Overall,epicardialcellsandEPDCsappeartobehavelikemulti- potentprogenitorcellsintheembryoproper.Howeverintheadult, itappearsthatthispotentialisnotfullyexploitedyet,butcanbe enhancedbyprovidingtherightcues.

5. Futureperspectives

Intheembryo,thecontributionoftheepicardiumtothefor- mationoftheheartisessentialviathecontributionofcardiaccell typesaswellastheproductionofgrowthfactorsandcytokinesthat influencemyocardialgrowth[113](Fig.1).Aswehavedescribedin thisreview,manyoftheseabilitiesareretainedintheadultinjured heartbutappeartooccurlessefficientcomparedtotheembryo.

Therefore,thequestionariseswhethertheepicardialresponsecan beoptimisedtomoreefficientlypartakeintherepairoftheinjured heart.

Possibleapproachesincludeinducingproliferationandsubse- quentEMToftheepicardium.Thiswould providealargerpool ofcellsthathastheabilitytomigrateintotheheart(Fig.2,left).

Thismethodappearstobefeasiblebasedonthefindingthatthy- mosin␤4treatmentpriortoMIincreasesthenumberofactivated epicardialcells,andenhancescardiacfunctionanddifferentiation intocardiaccelltypes[81].Unfortunately,thistreatmentisonly successfulwhenappliedpriortoinjury[92],makingitdifficultto imaginethymosin␤4treatmentasaclinicalapproach.Promising resultscamefromapplyingmodRNAforVEGF-Awhichresulted inanincreaseintheproliferationandmigrationofepicardialcells anddirecttheirdifferentiationcapacityintoendothelialaswellas cardiomyocytes.Importantly,thisapproachincreasedcardiacfunc- tionpost-injury[93].Identifyingthemostpotentactivatorsofthe epicardiumwillthereforebeanimportantgoaltopursue.