Marchetti, V., Yanes, O., Aguilar, E., Wang, M., Friedlander, D.F., Moreno, S., Storm, K., Zhan, M

12 Pages·2011·5.71 MB·English

Differential Macrophage Polarization
Promotes Tissue Remodeling and Repair
SUBJECTAREAS: in a Model of Ischemic Retinopathy
ANGIOGENESIS
VASCULARBIOLOGY
ValentinaMarchetti1*,OscarYanes2*,EdithAguilar1,MatthewWang1,DavidFriedlander1,
STEMCELLS
StaceyMoreno1,KathleenStorm5,MinZhan5,SamiaNaccache3,GlenNemerow3,GarySiuzdak2,4
METABOLOMICS
&MartinFriedlander1
Received
1DepartmentofCellBiology,TheScrippsResearchInstitute,LaJolla,CA,92037,2DepartmentofMolecularBiology,TheScripps
21June2011
ResearchInstitute,LaJolla,CA,92037,3DepartmentofImmunology,TheScrippsResearchInstitute,LaJolla,CA,92037,4Scripps
Accepted CenterforMassSpectrometry,TheScrippsResearchInstitute,LaJolla,CA,92037,5SourceMDX,Boulder,CO,USA.
8August2011
Published Diabeticretinopathyistheleadingcauseofvisuallossinindividualsundertheageof55.Umbilicalcordblood
30August2011 (UCB)–derivedmyeloidprogenitorcellshavebeenshowntodecreaseneuronaldamageassociatedwith
ischemiainthecentralnervoussystem.InthisstudyweshowthatUCB-derivedCD141progenitorcells
providerescueeffectsinamousemodelofischemicretinopathybypromotingphysiologicalangiogenesisand
reducingassociatedinflammation.Weuseconfocalmicroscopytotracethefateofinjectedhuman
Correspondenceand UCB-derivedCD141cellsandPCRwithspecies-specificprobestoinvestigatetheirgeneexpressionprofile
requestsformaterials beforeandafterinjection.MetabolomicanalysismeasureschangesinducedbyCD141cells.Ourresults
shouldbeaddressedto demonstratethathumancellsdifferentiateinvivointoM2macrophagesandinducethepolarizationof
residentM2macrophages.Thisleadstostabilizationoftheischemia-injuredretinalvasculatureby
M.F.(friedlan@
modulatingtheinflammatoryresponse,reducingoxidativestressandapoptosisandpromotingtissuerepair.
scripps.edu)
Ischemicdamageisthemajorcauseofneuronaldeathinthecentralnervoussystemleadingtosignificantlossof
*Theseauthorsequally
functioninmillionsofindividualseachyear.Asadirectextensionofthecentralnervoussystem,theretina
contributedtothis suffersmanyofthesamehypoxicinjuries.Ischemicretinopathiessuchasthoseassociatedwithdiabetesand
work. prematurityaretheleadingcausesofvisuallossinindividualsundertheageof55andneonates,respectively.
Visionlossintheseindividualsoccursasaresultofabnormalitiesintheretinalvasculature,leadingtoretinal
edema,hemorrhage,gliosis(scarring)and/orneovascularizationand,insomecases,tractionalretinaldetach-
mentsandblindness.Therapeuticinterventionsfocusoneliminatingthevascularabnormalitiesthroughtheuse
oflasersorangiostaticdrugsinjectedintravitreally.Whiledramaticeffectscanbeachievedinselectgroupsof
patients,mostexperiencediseaseprogressionandassociatedvisionloss.
Stemandprogenitorcellshavebeenisolatedfrombonemarrow(BM),peripheralorhumanumbilicalcordblood
(hUCB)asaninnovativetherapyfortreatmentofischemicandneuro-degenerativeretinopathies.Theutilityofsuch
atreatmentcomesfromtheabilityofstem/progenitorcellstodifferentiateintomaturecellsandtorepairdamaged
tissueand/orprovidedirect,paracrinerescueeffects.Thesecellsandtheirdifferentiatedprogeniesmayalsorecruit
endogenousandcirculatingcellstoareasofdamagedtissue,contributingtotherescueeffect.
UCBisarichsourceofhematopoieticstemcells(HSCs)includinghighnumbersofearlyandlatemyeloid
progenitorcells1.ComparedtoadultBMandperipheralblood,UCB-derivedprogenitorcellsexhibitincreased
capacitytoproliferateandsubsequentlydifferentiateintocolonyformingunit-granulocytemacrophages(CFU-
GM)2.Inaddition,UCB-derivedcellsarelessmaturerelativetoadultstemcells;theyhavenotbeenexposedto
immunologic challenge and are unable to activate cytotoxic T-lymphocytes to synthesize pro-inflammatory
cytokines3.
Reports from several groups have shown that local transplantation or intravenously injected UCB-derived
mononuclear cells improves functionality in areas of ischemia1,4 This rescue function is associated with the
CD141 monocyte fraction since depletion ofthese cells fromthe UCBled to loss oftherescue function ina
rat model of middle cerebral artery occlusion5. In vitro, the UCB-derived CD141 cells can differentiate into
endothelial6,7,neuronal8,9andmaturemyeloid(e.g.,monocytes,macrophagesanddendritic)cells.UCB-derived
neural progenitors have also been observed to be neuroprotective in an in vitro model of ischemia10. Recent
SCIENTIFICREPORTS |1:76|DOI:10.1038/srep00076 1
www.nature.com/scientificreports
reportssuggestthatdifferenttissuemicroenvironmentsexposemye- injectedintravitreallyatpostnatalday7(P7)inC57BL/6Jpupsand
loidcellstomultiplestimuliinfluencingtheirfateintopro-,andanti- themiceweremaintainedinanatmosphereof75%oxygenfor5days
inflammatorymacrophages(M1andM2,respectively),endothelial, (P7–P12)toinducetheischemicretinopathy.AtP17,vaso-oblitera-
ordendriticcells11–15.Thus,differentsubpopulationsofmyeloidcells tionandneovascularizationwerereducedby63%and56%,respect-
couldexertoppositeeffectsduringangiogenesis;onepopulationmay ively,inretinastreatedwithCD141cellgraftscomparedtoretinas
contribute to plaque formation in experimental models of arterial treatedwiththevehicleDPBS(Fig1A,B).Nosignificantrescueeffect
occlusion16whereanothermaypromotecollateralgrowthtoalleviate wasobservedwhenCD142cellswereused(Fig1B).
ischemia17. The retina provides a good model system in which to Next,CD141cellsweretransducedwithanadenoviralvector(Ad5
studythesediscordantresults;tissue-residentandrecruitedmacro- F16)encodingenhanced-greenfluorescentprotein(eGFP)25toassist
phagepopulationshavebeenimplicatedinbothdevelopmental18and in visualizing the cell transplants. The eGFP-labelled CD141 cells
pathologicangiogenesis19,processesthatmayoccursimultaneously wereobservedtotargettheretinalvesselsfromP12andtobeprim-
inthesametissueduringresponsetoischemicinjury. arilylocalizedaroundtheretinalvasculature10daysafterintravitreal
A recent study20 has examined the role of macrophages in the injection (P17). CD141 cells targeted the vessels showing highly
establishmentofvascularnetworks.Thesecellsactas‘‘bridgecells’’ arborizingprocesseswithamacrophagecell-likeshape.EGFP1cells
byestablishingtip-cellanastomosisandreleasingangiogenicfactors, wrapped around vessels for long distances and when targeting the
suchasVEGF,thatcanleadtovesselfusionandvascularnetwork neovascularization areas maintained a round morphology as fresh
formation.Previousstudiesofthesamecellshavedemonstratedthat isolatedfromthecordblood(Fig1C,D,E).Using3Dimagerecon-
tumoursordeveloping/regeneratingtissuescanactivelyrecruitthese struction,weconfirmedthattheCD141cellswerelocalizedtothe
cellsfromthecirculation21. vascularsurfaceandnotintegratedintothevesselitself.Importantly,
InourstudywehaveusedhUCBtoobtainanenrichedpopulation intravitrealinjectionofCD141cellsinfectedwithAd5F16-eGFPdid
ofmyeloidprogenitorcells,CD141cells,whichwheninjectedineyes notreducetherescuefunctionobservedwithanon-Ad5F16-eGFP
ofmicewithoxygen-inducedretinopathy(OIR),exertedaprofound infectedCD141cells,indicatingthattransductionofthecellswith
rescueeffect.OnlyCD141cellspolarizedtoM2macrophageswere theAdvectordoesnotsignificantlyimpactrescue.Fromtheseobser-
abletocontrolthepathologicalneovascularization.Inordertobetter vations we conclude that hUCB-derived CD141 cells differentiate
understand the molecular eventsthat regulate theobserved rescue invivointoamyeloidcellwithdendritic-macrophagecell-likemor-
effectwe:(i);studiedthefateoftheCD141intheretina;(ii)quan- phology,exerttrophicrescueeffectsinthemouseOIRmodelanddo
tifiedthelevelofgeneexpressionofinjectedhUCB-derivedCD141 so by adhering to, and wrapping around, the retinal vasculature
cells before and after rescue using transcriptomic analysis with ratherthanintegratingintoit.Thisrescueeffectanddifferentiation
human specific primer probes; and (iii) studied the metabolic is not an immunological response associated with the injection of
deregulationoccurringintheretinasofOIRmicebymassspectro- human cord blood cells into the mouse eye (xenotransplant); the
metry-basedmetabolomicsbeforeandafterinjectionofCD141cells. rescueanddifferentiationeffectswerenotobservedwiththeinjec-
FromthesestudiesweconcludethathUCBCD141cellsdifferentiate tionofhumanCD142cellsintothemouseeye(Figure1B).Next,we
invivointotype2macrophagesandinitiateaseriesofeventsassoci- conductedamolecularcharacterizationofinjectedcellsandretinal
ated with modulation of the inflammatory response leading to tissueinordertobetterunderstandthemechanismbywhichCD141
reduced oxidative stress and apoptosis and, ultimately, promoting cellspromotenormalizationofretinalvasculatureandtissuerepair.
normalangiogenesisandtissuerepairintheretina.
Ourabilitytodirectlyimageandfollowthefateofhumanmono- hUCB-derivedCD141cellsreducelevelofischemiaandstimulate
cytesinvivo,togetherwiththeuseofcell/tissue-specifictranscrip- therecruitmentofendogenousmyeloidcells.InjectionofCD141
tomicandmetabolomicanalysisofthesecellsandtheirtargettissue cellssignificantlyreducedtheareaofvaso-obliteration,thuslimiting
providenovelinsightsintothebehaviourofmonocyte/macrophage retinal vascular damage and enhancing tissue repair during the
cellularsubsetsduringangiogenesisandinflammation. period of hyperoxia (P7–P12). We observed a 50% reduction of
obliterated area at P12 (one day after returning to normoxia)
(Fig2A)andsignificantrecruitmentoflectinpositivecellssuchas
Results
leukocytes and EPCs from the peripheral blood when we injected
Human umbilical cord blood-derived CD141 cells stabilize CD141cellscomparedtovehicleinjection(Fig2B,C,D).Thesecells
ischemia-injured vasculature in the retina. In the mouse OIR havebeendemonstratedtobefundamentalnotonlyforanischemic
model, a transient period of hyperoxia during retinal vascular tissuerepairbutalsoforthestabilizationofretinalvasculature26.Itis
development attenuates normal retinal vessel development and importanttonotethatinanormoxicenvironmentatP12,formation
leads to degeneration of the newly formed, immature vasculature. ofthesuperficialretinalvascularplexusisalreadycompletebutthe
Upon return to normoxia, the central retinal area where the deep and intermediate vasculature is still forming through P21.
vasculaturehasregressedbecomeshypoxicleadingtodevelopment CD141cells,bycontributingtotherecruitmentofangiogeniccells,
of pathological neovascularization (NV) at the interface between maintain vascular growth in the deep plexuses and prevent
perfused and non-perfused retina22. In this model, the retinal neovascularizationformationatP17.
vasculature of both BALB/cByJ and C57BL/6J strains of mice Next,weundertookahumanspecifictranscriptomicanalysisof
becomes obliterated to a similar extent. However, in retinas from P17mouseretinaswiththeinjectionofhUCBCD141andCD142
BALB/cByJ mice, the vaso-obliterated regions revascularize more cells to define the differentiation state of the human cells and the
rapidly and pathological, pre-laminar neovascular tufts do not mechanism of action contributing to their rescue of OIR retinas
form. In contrast, the retinas of pigmented C57BL/6J mice are duringhypoxia.
characterized by slower revascularization of the central retina and
thereisextensiveformationofpathological,pre-laminarneovascular Cordblood-derivedCD141cellsexpressmRNAsassociatedwith
tufts(SupplementaryFig1A). the oxidative stress response, cell cycle, and macrophage cell
WealsohypothesizedretinasintheBALB/cByJstrainisprotected markers. The molecular changes associated with stabilization of
fromneovascularizationbyamorerapidrecruitmentofendogenous retinal vasculature by hUCB-derived CD141 cells were assessed at
anti-inflammatory microglia during the hypoxic phase of the OIR the mRNA level by selectively detecting and quantifying the
model(unpublishedobservations). expressionlevelof50humangenesinthemouseretina.Specifically,
Freshly isolated CD141 cells derived from hUCB consist of a wemeasuredtheexpressionratioofhumangenesinthemouseretina
heterogeneous population of myeloid cells. The CD141 cells were atP17afterintravitrealinjectionatP7ofCD141,CD142cells(control
SCIENTIFICREPORTS |1:76|DOI:10.1038/srep00076 2
www.nature.com/scientificreports
Figure1|CD141cellsstabilizeandpromotenormalizationofischemia-injuredretinalvasculatureintheOIRmodel. A)CD141cellsnormalize
angiogenesisduringhyperoxiaandaccelerateretinalrevascularization.NormalretinasweredissectedfromC57BL/6JmiceandstainedwithGSlectinat
postnatalday17(P17).Theyshowacharacteristicbranchingvascularpatternradiatingoutwardfromthecentralopticnervehead(‘‘Control
Normoxia’’).Exposuretohyperoxiafor5days(fromP7toP12)leadstocentralvaso-obliteration.Oncethemicereturntonormoxia,neovascularisation
occursattheinterfacebetweenperfusedperipheral,andnon-perfusedcentral,retina.Treatmentwithvehicle(‘‘Vehicletreated’’)atP7doesnotalterthe
vaso-obliterationorneovascularisation.Incontrast,treatmentatP7withhUCB-derivedCD141cellsleadstonormalizationoftheretinalvasculature
(‘‘CD141treated’’).B)QuantificationofretinastreatedwithhUCB-derivedcellpopulations.RetinaswereanalyzedatP17usingGS-lectinstainingfor
retinalvesselobliteration(yellowbars)andtuftformation(neovascularization)(redbars)inretinalwholemounts.Nosignificantdifferencewasobserved
betweenvehicleandCD142cellswheninjectedintravitreallyatP7.Obliterationandneovascularizationarereducedby63%and56%respectively,
comparedtovehicle-treatedretinas(n557,n540,n570,n5numberofeyes)(*P,0.001Bonferronicorrectedt-test).C)D)AndE)Ad5F16-eGFP
hUCB-derivedCD141cells(green)targetanddifferentiatealongthemouseretinavasculature(red)atP17(10x,20xand40Xrespectively).
population), or vehicle in the OIR model (Fig 3). Transcriptomic andNF-kB1.TGFb1,expressedbytheleukocytelineagetocontrol
analysis of retinas treated with CD141 cells revealed increased cell differentiation, proliferation, and resolution of inflammation,
expression of human antigens of M2 macrophages. The molecule wasalsohighlyupregulatedinthemouseretinaatP17afterinjection
CD14 and other monocytic cell markers such as CD16327, CD6828, of CD141 cells. The transcription factor STAT3, responsible for
CD20929andHLA-DRA30werehighlyupregulatedaftertheinjection controlling anti-inflammatory pathways32,33, was up regulated by
ofCD141relativetoCD142(CD141/CD142.1.6).Importantly,we CD141 cells compared to the negative fraction. We also observed
alsodetected increased expression ofhuman genes (CD141/CD142 thatTNF,acytokineresponsiblefortherecruitmentofproinflam-
.1.6)associatedwiththeoxidativestressresponseincludingSOD1, matoryleukocytes,wasnotexpressedbyCD141cellsandis,infact,
encodingsuperoxidedismutase1.Interestingly,infreshlyisolatedcells slightlydownregulatedcomparedtothenegativefraction.
(day0)SOD1expressionwashigherinCD142populationcompared GenesassociatedwithcelladhesionsuchasICAM-1(alsoknown
to CD141 cells (Supplementary Fig 2). Additional oxidative stress- asCD54),humanCD44andITGAMwerealsohighlyexpressedafter
associated genes were also detected including human CAT, GPX1, injectionofCD141cellsrelativetoCD142andvehicle.Othergenes
HMOX1andALDOA. withdiversebiologicalroleswerealsoupregulatedbytheintravitreal
TheCD141cellsalsoinducedexpressionofgenes(CD141/CD142 injectionofhUCB-derivedCD141cellsrelativetotheCD142frac-
.1.6)involvedincellsignalingsuchasIFI1631,IFI6,BCL2A1,AKT1 tionandvehicle:(i)humanCCR5,encodingCCchemokinereceptor
SCIENTIFICREPORTS |1:76|DOI:10.1038/srep00076 3
www.nature.com/scientificreports
Figure2|CD141cellspromoteangiogenesisandrecruitendogenousproangiogeniccellsinhypoxicareas(A)AtP12theobliterationareainCD141
treatedeyesisreducedby50%comparedtovehicle-treatedretinas(n58,n5numberofeyesforeachcondition). (B)Lectin-positivecellsinareasof
vaso-obliterationareincreased2.2-foldafterintravitrealinjectionofCD141cellsrelativetovehicleinjection(n56,n5numberofeyesforeach
condition)(*P,0.001Bonferronicorrectedt-test).(C)10xconfocalimagesofretinastreatedwithCD141showshighrecruitmentoflectin-positivecells
withmicroglial-likemorphology.(D)Lectin-positivecellsareabsentinvehicle-injectedretinas.
5,aproteinpredominantlyexpressedonT-cells,macrophages,dend- associationwiththeexpressionofgenesthatregulateoxidativestress,
riticcellsandmicrogliathatbindscytokinesasMIP-1alphaandbeta apoptosisandcontrolofinflammationandangiogenesis.
andRantes;(ii)ALDOA,encodingaldolaseA;(iii)HIF1A,hypoxia-
induciblefactor1-alpha;(iv)GUSB,encodingb-glucuronidase;and(v) HUCB-derivedCD141cellsreducethelevelsofoxysterolsinOIR
SERPINA 1 (also alpha-1-antitrypsin), encoding a serine proteinase retinas,resemblingtheresponseofBALB/cByJmicetohypoxia.
inhibitor that protects tissues from enzymes of inflammatory cells Severe levels ofoxidative stresscandamage allcomponents ofthe
suchaselastase. Interestingly,thetissueinhibitormetalloproteinase cell, including metabolic intermediates such lipids (i.e., lipid
1(TIMP1),knowntoinhibittheactivityofMMP1andMMP13andso peroxidation). Therefore, we performed a metabolomic analysis
to block angiogenesis invivoand in vitro34 was up regulated bythe usingmassspectrometryinordertoinvestigatewhethertherescue
positivefractioncomparedtothenegativepopulation. effectsoftheCD141cellsarepartiallyexplainedbytheircapacityto
Overall, thetranscriptomic datasupports ourinitialhistological suppressmetabolicoxidativedamageinretinaltissue.
andantibodyarrayresultsindicatingthatCD141differentiatesinto We analyzed retinas of C57BL/6J mice at P12 as the first stage
a macrophage polarized M2 type and exerts rescue effects in betweenthehyperoxiaandhypoxia,P15asmiddlehypoxiaandP18
SCIENTIFICREPORTS |1:76|DOI:10.1038/srep00076 4
www.nature.com/scientificreports
Figure3|HumanUCB-derivedCD141cellsregulateoxidativestressandapoptosis,andexpressmarkersofmyeloidcelldifferentiationintheOIR
model. Severalhumangenesareup-regulatedatP17intheCD141-treatedretinascomparedtoretinasinjectedwiththenegativefraction,CD142cells
andcomparedtoretinasinjectedwiththevehicle,DPBS.Afoldincreaseof1.6orgreaterofanti-apoptoticgenesisobserved(black)(IFI6,IFI16,AKT1,
BCL2A1).Humananti-oxidativestressgenesaresignificantlyupregulatedwhenCD141cellsareinjectedcomparedtothenegativefraction(green).
NumerousgenescharacteristicofmyeloidcelldifferentiationarehighlyexpressedintheCD141-treatedretinas(blue).TheCD141cellsincrease
expressionofvascularadhesionmolecules(ICAM1)andtheextracellularmatrixreceptor(CD44)(orange).Genescharacteristicofendothelialcell
differentiation(VEGF)andinflammation(TNF,IL6)arenotsignificantlydifferentbetweentheCD141cellsandtheretinastreatedwiththenegative
fraction.(n512,n5numberofretinasindependentlyextractedandanalyzedforeachtreatment)(*P,0.01;**P,0.001).
SCIENTIFICREPORTS |1:76|DOI:10.1038/srep00076 5
www.nature.com/scientificreports
accumulationofseveralacyl-carnitinestructuresatP18inOIRret-
inas.Acyl-carnitinesarefattyacyl-CoA(coenzymeA)conjugatedto
carnitine and assist with the transport and metabolism of fatty
acids into mitochondria, where they are undergo b-oxidation.
Importantly,11-cis-retinalwasidentifiedasoneofthefewmetabo-
lites whose abundance decreased at P18 in the OIR model. The
metabolite11-cis-retinalisanendogenouschromophoreboundto
opsin; its photoisomerization is the chemical basis for vertebrate
vision. The level of oxysterols (Supplementary Fig 3A,B,C) and
11-cis-retinal (Supplementary Fig 3D) was completely normalized
atP18bythetreatmentwithCD141cellsandthelevelofacyl-carni-
tines was significantly reduced (,2-fold) compared to the non-
treatedretinas(5–9fold)(Table1).
PreviousstudieshaveshownthatBALB/cByJmiceplacedintothe
OIRmodeldonotformtuftstoanappreciabledegreeandthecentral
vaso-obliterationarearevascularizesquicklyfollowingreturntonor-
moxia26.Whenuntreated,wildtypeBALB/cByJmouseretinaswere
analyzedatP18undertheOIRconditionsusingthesameuntargeted
massspectrometry-basedmetabolomicapproach,weobservedaglo-
Figure4|GlobalmetabolomicanalysisoftheOIRmodel. Barsindicate
bal trend similar to that observed in C57BL/6J mice treated with
thepercentageofup-regulatedordown-regulatedmassspectrometry
CD141cellsunderOIRconditions(Fig4).Essentially,only,1.7%
features(*P,0.01)intheOIRretinasrelativetonormoxiaatpostnatal
ofthemetabolitesdetectedinBALB/cByJretinasappearedupregu-
day12,15and18.ResultscorrespondtoC57BL/6Jmousestrainunless
lated(.2-fold,P,0.01)atP18relativetonormoxia.Itisofinterest
otherwisestated.8–12independentlyextractedretinaltissueswere
tonote,however,thatbothBALB/cByJandC57BL/6Jmicetreated
analyzedforeachtimepointortreatment,n58–12).
withCD141cellsshowedamarkedlyhighpercentageofdown-regu-
latedmetabolitesrelativetonormoxiaatP18.
asadvancedhypoxiaandcomparedmetabolitelevelsbetweennor- Overall,themetabolomicdatarevealsthatoxidativedamageinthe
moxiaandOIR,andbetweenretinastreatedwithCD141cellsand retinadoesnotoccurduringtheperiodofhyperoxia,butdoessoas
vehicle. Untargeted metabolomic analysis (Fig 4) revealed only the result of the oxygen/reperfusion injury following the induced-
minorchangesbetween normoxiaandOIRatP12,indicating that hypoxia.Importantly,ourresultsalsodemonstratethatintravitreal
hyperoxia does not significantly affect the metabolome in the injectionofCD141cellsreducesthemetabolicoxidativedamageby
developingretina.ByP15,(after3daysofinduced-hypoxiccondi- controllingtheproductionofoxysterols.
tions)only,4%ofthemetabolitesdetectedwereupregulated(.2
fold;P,0.01)inOIRretinasrelativetonormoxia.However,after 5,6b-epoxy-cholesterol and 7-ketocholesterol induce astrocyte
6 days under hypoxic conditions (P18), and coinciding with the disorganization, gliosis, and apoptosis by activating caspase 3.
observedhistopathologicalchanges(i.e.neovasculartufts)observed Having observed a high expression level of genes that regulate
intheOIRmodel,approximately15%ofthemetabolitesdetectedin apoptosis in retinas treated with CD141 cells, we studied the role
OIRretinasweredifferentiallyregulated(.2-fold,P,0.01)relative ofoxidizedformsofcholesterolinthepathogenesisofretinaltissue.
tonormoxia.Incontrast,whentheretinasweretreatedwithCD141 Initially, astrocytes and endothelial cells, two different cell types
cells at P7, only ,2% of the metabolites remained deregulated found in the retina, were cultured in vitro and treated with the
(.2-fold,P,0.01)atP18(Fig4).Weusedtandemmassspectro- identifiedoxysterolsinadose-dependentmanner.Culturedmouse
metry (i.e. ESI-Q-TOF MS) to identify of some of the metabolites astrocytes exhibited a dose-dependent toxicity response after
deregulatedatP18underOIRconditionswhoselevelswerenormal- treatment with increasing concentrations of b-epoxy-, 7a-
ized by intravitreal injection of CD141 cells. The deregulated hydroxy-, and 7-ketocholesterol (Supplementary Fig 4A). At the
metabolites included oxysterols such as 5,6b-epoxy-cholesterol, highestdosetested,0%(P,0.001)and4.864.5%(P,0.001)of
7-ketocholesterol,and7a-hydroxycholesterol(Table1).Oxysterols the astrocytes were viable after treatment with 7-keto- and
areoxidationproductsofcholesterolthatexertcytotoxic,pro-oxid- b-epoxycholesterol, respectively, compared to 80 6 4.5% viability
antandpro-inflammatoryeffectsinvivo35–38.Wealsoobservedthe intheastrocyteculturetreatedwithvehicle(i.e.,regularmediaand
Table1|Foldchangeofoxysterols,carnitinesand11-cisretinalinOIRretinascomparedtoNormoxia
C57BL/6J(P18) BALB/cByJ(P18)
OIR/Normoxia OIR1CD141/Normoxia OIR/Normoxia
Metabolite Fold Pvalue Fold Pvalue Fold Pvalue
7-ketocholesterol 6.7 2.331026 1.3 0.2 1 0.67
5,6b-epoxy-cholesterol 5.1 2.131026 1.4 0.37 1.1 0.78
7a-hydroxycholesterol 3.9 5.831026 1 0.91 1 0.68
11-cis-retinal 0.3 5.631025 0.83 0.047 n.d n.d
Decanoylcarnitine 5.7 9.031028 2 0.002 2.4 4.2x1026
Octanoylcarnitine 5.5 5.531024 2.2 9.5831025 1.8 5.4x1025
Laurylcarnitine 9.1 2.931027 2.6 7.831024 2 1.0x1025
Tetradecenoylcarnitine 8.8 7.531029 2.1 1.131024 1.2 0.02
Hexadecenoylcarnitine 5 3.131029 1.3 0.032 0.6 0.016
Tetradecanoylcarnitine 7.2 5.931029 2 2.931024 1.4 0.0013
SCIENTIFICREPORTS |1:76|DOI:10.1038/srep00076 6
www.nature.com/scientificreports
ethanol) (Supplementary Fig 4B). In the presence of 7a- metabolite.HUVECwereexposedtohydrogenperoxide(H O )as
2 2
hydroxycholesterol, cell viability was 63.1 6 3.9% (P . 0.05). apositivecontrolofcellulartoxicity(SupplementaryFig4C).
Alternatively, when cultured human vascular endothelial cells To better understand the mechanism by which these oxidized
(HUVEC) were exposed overnight to 40 mg/ml of 5,6b-epoxy- formsofcholesterolweretoxic,westudiedthecleavageofcaspase-
cholesterol, only 4.6% of the cells remained viable (P ,0.001) 3,amarkerofapoptosis.UsingWesternblotanalysis,weobserved
(Supplementary Fig 4C). The alpha isomer (5,6a-epoxy- activation ofcaspase-3 in cellcultures ofbovine aorticendothelial
cholesterol), however, maintained HUVEC viability close to 60% cells(Fig5A)ormouseastrocytes(Fig5B)treatedwith5,6b-epoxy-,
(data not shown), indicating the functional specificity of this 7-keto-, and 7a-hydroxycholesterol. This is consistent with the
Figure5|Oxysterolsinduceapoptosisinendothelialcellsandastrocytesinvitro. WesternBlotanalysisshowsthecleavageofCaspase-3inducedby
b2epoxycholesteroland7-ketocholesterolinBovineAorticEndothelialcells(A)andC8-D1Aastrocytes(B).Staurosporin(ST)wasusedaspositivecontrol.
Actinlevelisreportedtonormalizethequantityofproteinineachsample(n53,n5numberofexperiments)(Bonferronicorrectedt-test:*P,0.01).
SCIENTIFICREPORTS |1:76|DOI:10.1038/srep00076 7
www.nature.com/scientificreports
increasedactivationofcaspase-3cleavageinwholeretinasobtained Themorphologicaleffectofintravitreallyinjectingb-epoxy-,and7-
frommiceunderOIRconditions.Weobservedanincreaseof65% ketocholesterolintoP15miceundernormaldevelopingconditions(i.e.
and 130% of caspase-3 cleavage in OIR retinas at P15 and P18, normoxia)wasevaluated3dayslateraftertreatment(P18).Compared
respectively, relative to normal developing retinas (Supplementary tocontroleyes(Fig6A,a),retinasinjectedwith5,6b-epoxy-(Fig6B,b)
Fig5).Noobservabledifferenceincaspase-3cleavagewasobserved or 7-ketocholesterol (Fig 6C, c) showed disruption ofthe astrocytes
atP12betweennormalandOIRretinas(datanotshown). associated with the superficial retinal vascular plexus and a strong
Figure6|Oxysterolsaretoxicforastrocytesandmullergliacellsinvivo. Immunohistochemicalanalysisusinglectin(red,bloodvessels)andantibody
toGFAP(green,astrocytesandactivatedMullergliacells)demonstratesthenormalretina(A)orabnormalitiesobservedwithdegenerationofastrocytes
intheinnerretinaafterintravitrealinjectionofb-epoxy-cholesterol(B)or7-ketocholesterol(C).InsetsinA,BandCrepresent20Xmagnificationviews
ofGFAPexpression.Insetfiguresa,b,cshow20Xmagnificationviewsofwholemountretinasfromthesamespecimen;astrocytes(green)appear
damagedandthemullergliacells(greendots)arevisibleinthesuperficialplexusofthevasculature(red)demonstratingseveregliosis(n55,n5number
ofeyesforeachtreatment).HUCBderivedCD141cellspreventMullerGliacellsactivationinOIRretinas.Immunohistochemicalanalysisusingan
antibodytoGFAPdemonstratesdegenerationofastrocyteslayerandgliosis(redarrows)inP17OIRretinas(D).TreatmentwithHUCBCD141cells
preventsthisdamage(E)(n56,n5numbereyesforeachcondition).
SCIENTIFICREPORTS |1:76|DOI:10.1038/srep00076 8
www.nature.com/scientificreports
activationofMullergliacellsintheouterlayer.Similarly,retinasfrom differentiateintomacrophage-likecells.TheCD141cells,oncedif-
OIRmice(notexposedtoexogenous5,6b-epoxy-,and7-ketocholes- ferentiatedinmaturemyeloidcells,recruitandactivateendogenous
terol)showedreactivegliosisofthemullergliacellsatP18(Fig6D). pro-angiogenic cells that facilitate functional angiogenesis and
ThetreatmentofOIRretinaswithCD141cellspreventeddisorgan- reduce inflammation ordinarily observed during OIR. When we
izationoftheastrocyticlayerandreactivegliosis(Fig6E). analyzed and compared the transcriptomic profile of CD141 and
Overall, our results indicate that accumulation of b-epoxy- and CD142 populations, we observed a different response of the two
7-ketocholesterolmetabolites intheOIRretinasinducesapoptosis fractions to the retinal environment. As freshly isolated cells, the
by activation of caspase-3 cleavage, leading to a disruption of the twopopulationsexhibitdifferentialexpressionformanyofthegenes
astrocyteorganizationandgliosisoftheMullergliacellsintheouter analyzed while some of the genes exhibit very similar levels of
layer.TreatmentwithCD141cellsrestoresthephenotypetothatof expression.
normaldevelopingretinas. WehavepreviouslydemonstratedthatBM-derivedhematopoietic
progenitorcellsinjectedintravitreallywilltargetactivatedglia,dif-
Polarized M2 macrophages derived from CD141 cells prevent ferentiateintoendothelialcellsandexerttrophicrescueactivityin
neovascularization in ischemic retina. The molecular charac- animalmodelsofischemicandneuro-degenerativeretinopathies45,46.
terizationoftherescueeffectsobservedinOIRretinastreatedwith InjectionofBM-derivedCD44hicellsintohypoxiceyesshowedthat
CD141 cells showed a high degree of myeloid macrophage thesecellsdifferentiateintomicrogliaandexertaparacrine-rescue
differentiation and recruitment of lectin-positive cells. We effect, stabilizing damaged retinal vasculature26. While heat shock
hypothesizedthatpolarizedactivationofmacrophages maybethe proteinsandchaperoneswereobservedtobeupregulatedinrescued
primarilymediatoroftherescueeffectsintheOIRmodel.Totestthis retinal neurons, theprecise mechanism ofrescue remains unclear.
hypothesis,weinducedtheinvitrodifferentiationoffreshlyisolated TranscriptomicanalysisofCD141cellsafterinjectionintoOIRret-
human UCB-derived CD141 cells into activated M1 and M2 inas shows a selective up-regulation of genes associated with M2-
macrophages, and each population of activated macrophages was polarizedmacrophages.
theninjectedintravitreallyinOIRmice. OurdatasuggestthatthetrophicrescueeffectoftheCD141cells
IFN-calone,orinconcertwithmicrobialstimuliorcytokines,has wasassociatedwiththeirdifferentiationintoM2cellsaswellasan
been known to induce differentiation of monocytes into activated influx of similar cells from the host circulation. Interestingly, four
M139. M2 is ageneric name for various forms of activated macro- genesthatareexpressedatverysimilarlevelsbetweenthetwocell
phages, excluding M1 cells but including cells exposed to IL-4 or populationsatday0,showsignificantlyincreasedexpressioninthe
IL-13, immune complexes, IL-10, glucocorticoids, or vitamin D3- CD141retinaltransplantincluding,TGFB1,CAT,GUSBandSOD1.
derived metabolites40. M1 macrophages were obtained by in vitro Thesegenesencodeproteinswithmultiplefunctionsincludingregu-
differentiation of CD141 cells using IFN-c, and M2 macrophages lationofproliferation,differentiation,adhesion,migrationandres-
were obtained using IL-4 as previously described41,42. After 3 days ponsetooxidativestress.Forexample,SOD1isexpressedatlower
ofinvitrodifferentiation,weusedflowcytometrytoassessthelevel levels in fresh isolated CD141 cells compared to CD142, but in
of pro- and anti-inflammatory cytokines secreted by M1 and M2 CD141 transplanted retinas, the expression of this gene was
subsets.IFN-c-stimulated CD141cells secreted high levels of pro- increasedsignificantlycomparedtoCD142transplantedretinas.
inflammatory cytokines IL-8, IL1b and IL-6, whereas IL-4-stimu- WehypothesizedthattheCD141fractioncanrespondtomicro-
lated CD141 cells secreted high levels of anti-inflammatory IL-10 environmentalcuesandregulatesinflammatorycascadesaswellas
(SupplementaryFig6). the response to hypoxia. During the hyperoxic phase of the OIR
Threedaysafterinvitrodifferentiation,M1andM2populations model, the CD141 cells induce angiogenesis, reducing the area of
derivedfromCD141cellswereinjectedintravitreallyatP7,andthe vaso-obliterationandassociatedtissuehypoxia.Vascularregression,
miceweremaintainedinanatmosphereof75%oxygenfor5days associated with endothelial cell death, is avoided by CD141 cell-
(P7–P12) to induce ischemic retinopathy. The areas of vaso- induced up-regulation of anti apoptotic and anti-oxidative genes
obliterationmeasuredatP17werereducedby75%and96%inret- and associated maintenance of retinal vasculature. Thus, the
inastreatedwithM1andM2grafts,respectively, comparedtothe CD141 cells differentiate into type 2 macrophages, acquiring an
vehicleDPBS(Fig7A,yellowbars).Importantly,onlytheM2popu- anti-inflammatoryphenotype.Recentstudieshaveshownthattissue
lationprevented tuft formation (i.e. neovascularisation) showinga macrophages exist as two polarized populations, M1 and M2 sub-
reductionof59%ofneovascularareas(Fig7A,redbars).Wedidnot sets47,48, the former being pro-inflammatory and the latter being
observe a significant decrease of tuft formation by activated M1 anti-inflammatory. The relative immunological immaturity of the
macrophages.Bothpopulationswereabletoinducerapidresolution
UCB-derived cells could explain why most monocyte populations
of obliterated areas during the hyperoxic phase, but only the M2
fromneonatalbloodbecomeM2polarizedcellsinsteadofthepro-
populationpreventedneovascularization.Thus,thedecreaseinret- inflammatory M129. The M2 cells modulate the inflammatory res-
inal neovascularization observed in association with reduction of ponse and facilitate tissue repair49 enhancing trophic rescue,
obliterationisnotsimplyaresultofadecreaseinthevascularized
enhanced removal of apoptotic cellular debris and inducing tol-
area;bothM1andM2macrophagesreducedobliteration,butonly
erance rather autoimmunity50. This idea is further supported by
theM2macrophagesalsoreducedneovascularization.Inaddition, our observation that after intravitreal injection of CD141 cells sti-
only M2 grafts induced a massive recruitment of endogenous
mulated to differentiate along M1 or M2 pathways, only the M2
MannoseReceptor-positivecells,acellshownbyotherstocontrol
fraction facilitated normalization of the retinal vasculature and
inflammationprocesses(Fig7B,C)43,44.
decreasepathologicalneovascularization.
Thesedatasupporttheconceptthatthissubsetofmacrophagesis
Theconceptthatmacrophagesplayacriticalroleduringpatho-
predominantly responsible for the rescue effects of hUCB-derived
logicalangiogenesisisstronglysupportedbyrecentwork20highlight-
CD141cells,eitherbyrecruitmentofendogenouspopulationsofM2
ingtheroleofM2mousemacrophages(Angiopoietinreceptor1and
macrophages and/or by differentiation of CD141 cells in the
Neuropilin1 positive) at the time of brain vascularization. In this
ischemicretinas.
studyM2macrophagesinteractwithendothelialtipcellstopromote
vascularanastomosisdownstreamofVEGFmediatedtipcellforma-
Discussion tionandsproutinduction.
In this study we demonstrate that hUCB-derived CD141myeloid To better understand the molecular events that promote the
progenitorcells,wheninjectedintotheischemicneonataleye,will trophicrescueeffectsoftheCD141cellsinvivo,wealsoperformed
SCIENTIFICREPORTS |1:76|DOI:10.1038/srep00076 9
www.nature.com/scientificreports
Figure7|IntravitrealinjectionofM2macrophagesderivedfromCD141cellsreducesoxygeninducedneovascularization. CD141cellswere
differentiatedintoM1orM2cellsandinjectedintothevitreousofP7micejustpriortohighoxygenexposureintheOIRmodel.AtP17theareasof
obliteration(yellow)andneovasculartuftformation(red)werequantifiedasdescribedintheexperimentalprocedures.BothM1andM2populations
significantlyreducetheareasofobliterationcomparedtothecontrol-treatedeyes(n517,15,10and10respectively,n5numberofeyesforeach
treatment)(Bonferronicorrectedt-test,*P,0.001).M2cellsaresignificantlymoreeffectivethantheM1cellsatreducingtheareaofneovascularisation
comparedtothevehicle-ornon-injectedeyes.(B)AtP17,CD141-treatedretinasshowrecruitmentofmousemacrophagesexpressingmannosereceptor
(MR)(green).GS-lectinstainingshowsmousevasculature(red).(C)MRpositivecellsarenotobservedinvehicle-treatedeyes(20X).
acomprehensivemassspectrometry-baseduntargetedmetabolomic their toxicity, protecting photoreceptors, glial components of the
analysisonnormalretinas,OIRretinasandOIReyestreatedwith retinaandstabilizingthevasculature.
CD141cellgrafts.AcomparisonbetweenBALB/cByJandC57Bl/6J Theuseofhumanumbilicalcordbloodmyeloidprogenitorcell-
mousestrainswasalsoperformedtoidentifythemetabolicfeatures derivedmonocytesandmacrophagesrepresentsapromisingalterna-
associatedwiththedifferentbehavioralresponsetothehypoxiccon- tivetoadultorembryonicstemcelltransplantationfortreatmentof
ditionscharacteristicoftheOIRmodelinthesetwostrains.Different ocular diseases. These cells have the capacity to promote physio-
oxidized metabolites of cholesterol (oxysterols) were found to be logical angiogenesis and to reduce inflammatory processes assoc-
cytotoxicandinduceapoptosisintheretinalvasculatureandastro- iated with hypoxic damage in the retina as well as to provide
cytes.OurresultsdemonstratethattreatmentwithCD141cellscon- neurotrophicandanti-apoptoticprotectiontoretinalneuronssub-
trolsthederegulationofthesemetabolitesinOIRretinasreducing jectedtoischemicdamage.Inneonates,UCBwouldbesuperiorto
SCIENTIFICREPORTS |1:76|DOI:10.1038/srep00076 10