The role of IL-6 in pathogenesis of abdominal aortic aneurysm in mice

19 Pages·2017·14.32 MB·English

RESEARCHARTICLE
The role of IL-6 in pathogenesis of abdominal
aortic aneurysm in mice
MichihideNishihara1,HirokiAoki2*,SatokoOhno1,AyaFurusho1,SakiHirakata1,
NorifumiNishida1,SoheiIto1,MakikoHayashi1,TsutomuImaizumi3,YoshihiroFukumoto1
1 DivisionofCardiovascularMedicine,DepartmentofInternalMedicine,KurumeUniversitySchoolof
Medicine,Kurume,Japan,2 CardiovascularResearchInstitute,KurumeUniversity,Kurume,Japan,
3 InternationalUniversityofHealthandWelfare,Fukuoka,Japan
*[email protected]
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Abstract
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Althoughthepathogenesisofabdominalaorticaneurysm(AAA)remainsunclear,evidence
isaccumulatingtosupportacentralroleforinflammation.Inflammatoryresponsesarecoor-
dinatedbyvarioussolublecytokinesofwhichIL-6isoneofthemajorproinflammatorycyto-
kines.InthisstudyweexaminedtheroleofIL-6inthepathogenesisofexperimentalAAA
OPENACCESS
inducedbyaperiaorticexposuretoCaCl inmice.Wenowreportthattheadministrationof
2
Citation:NishiharaM,AokiH,OhnoS,FurushoA, MR16-1,aneutralizingmonoclonalantibodyspecificforthemouseIL-6receptor,mildlysup-
HirakataS,NishidaN,etal.(2017)TheroleofIL-6
pressedthedevelopmentofAAA.TheinhibitionofIL-6signalingprovokedbyMR16-1also
inpathogenesisofabdominalaorticaneurysmin
resultedinasuppressionofStat3activity.Conversely,nosignificantchangesineither
mice.PLoSONE12(10):e0185923.https://doi.org/
10.1371/journal.pone.0185923 NFκBactivity,Jnkactivityortheexpressionofmatrixmetalloproteinases(Mmp)-2and-9
wereidentified.TranscriptomeanalysesrevealedthatMR16-1-sensitivegenesencodeche-
Editor:ElenaAikawa,BrighamandWomen’s
Hospital,HarvardMedicalSchool,UNITEDSTATES mokinesandtheirreceptors,aswellasfactorsthatregulatevascularpermeabilityandcell
migration.Imagingcytometricanalysesthenconsistentlydemonstratedreducedcellular
Received:June7,2017
infiltrationforMR16-1-treatedAAA.TheseresultssuggestthatIL-6playsanimportantbut
Accepted:September21,2017
limitedroleinAAApathogenesis,andprimarilyregulatescellmigrationandinfiltration.
Published:October5,2017
ThesedatawouldalsosuggestthatIL-6activitymayplayanimportantroleinscenariosof
Copyright:©2017Nishiharaetal.Thisisanopen continuouscellularinfiltration,possiblyincludinghumanAAA.
accessarticledistributedunderthetermsofthe
CreativeCommonsAttributionLicense,which
permitsunrestricteduse,distribution,and
reproductioninanymedium,providedtheoriginal
authorandsourcearecredited.
Introduction
DataAvailabilityStatement:Allrelevantdataare
withinthepaper. Abdominalaorticaorta(AAA)isalocalexpansionoftheaorticdiameterduetoaweakened
aorticwall[1].Theabsenceofaprecisemechanismforthispathologyhashamperedthedevel-
Funding:ThisworkwassupportedbytheJapan
opmentofeffectivetherapeuticstrategies.Consequently,surgicalintervention(withagraft)is
SocietyforthePromotionofScience(http://www.
jsps.go.jp/index.html)25861238(MN),21390367 currentlytheonlytreatmentoption.Recentstudieshavehighlightedtheimportanceoftissue
(HA),24390334(HA),24659640(HA),26670621 destructiveinflammationinAAApathogenesis[2–4].Indeed,weandothershavedemon-
(HA),16H05428(HA);DaiichiSankyoFoundation stratedthatpharmacologicinterventionagainstmediatorsofpro-inflammatorysignaling,
ofLifeScience(http://www.ds-fdn.or.jp)(HA);
includingc-JunN-terminalkinase(Jnk)[5]andnuclearfactorkappaB(NFκB)[6]areeffec-
UeharaMemorialFoundation(http://www.
tiveinsuppressingtissuedestructioninamousemodelofAAA.Further,thereisnowanaccu-
ueharazaidan.or.jp)(HA);VehicleRacing
mulatingbodyofevidencetosupporttheideathatregulatinginflammationisapromising
CommemorativeFoundation(http://www.vecof.or.
jp)(HA).Thefundershadnoroleinstudydesign, strategywithwhichtocontroltheprogressionofAAA[7].
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IL-6inmurineabdominalaorticaneurysm
datacollectionandanalysis,decisiontopublish,or Whileinflammationisanessentialdefensemechanisminallowinganorganismtocombat
preparationofthemanuscript. tissuedamageandexogenouspathogens,inflammationcanbeharmfulwhenitfailstoself-
Competinginterests:Theauthorshavedeclared limit,asexemplifiedbyAAA.InadditiontoAAA,autoimmunediseasessuchasrheumatoid
thatnocompetinginterestsexist. arthritisarealsocausedbynonself-limitinginflammation.Recently,aseriesofbiological
agentshavebeenintroducedintoclinicalpracticethattargetproinflammatorycytokinesin
autoimmunediseaseanddramaticallyimproveclinicaloutcomes[8].Thesebiologicalagents,
thatincludeantibodiesanddecoyreceptorsforTNFα,IL-1β,andIL-6,areeffectiveinsup-
pressingotherwiseuncontrolledinflammationinthesediseases.
Theimprovementinclinicaloutcomeprovokedbyinhibitingproinflammatorycytokines
inautoimmunedisorderssuggeststhatthisstrategymayalsoprovetobeeffectiveincontrol-
linginflammationprovokedinAAA.Indeed,severalreportshaveshownthattheinhibitionof
TNFα[9,10]orIL-1β[11]areeffectiveinsuppressingAAAdevelopmentinanimalmodels.
IL-6hasalsobeenimplicatedinthemolecularcircuitryforvascularinflammationinaortic
diseases,includingaorticdissectionandAAA[12].
DespiteanabundanceofIL-6inAAAtissue,exactlyhowIL-6participatesinAAApatho-
genesis,andwhetheritssuppressionwouldbeofbenefitincontrollinginflammationremains
unknown[13].WethereforeinvestigatedtheeffectsofMR16-1,aratmonoclonalantibody
specificforthemouseIL-6receptor[14],inamurinemodelofAAA.Ourfindingsindicate
thatdespiteasuppresseddevelopmentofAAA,MR16-1’seffectsaremodest.MR16-1sup-
pressedgeneexpressionforchemokines,theirreceptors,andthepeptidasesthatcontrolvascu-
larpermeabilityandcellularinfiltration.Theseeffectswereallachievedintheabsenceofa
majorimpactontissuedegradingmatrixmetalloproteinases.Therefore,IL-6wouldappearto
playalimitedroleinAAAdevelopment,andprimarilyregulatescellularinfiltration.
Materialsandmethods
MousemodelofAAA
AllanimalexperimentalprotocolswereapprovedbytheAnimalExperimentsReviewBoards
ofKurumeUniversity.ThemouseAAAmodelwascreatedinmaleC57BL/6Jmice(Charles
RiverLaboratoriesJapan)attheageof10–12weeksbyperiaorticapplicationof0.5MCaCl ,
2
asdescribedpreviously[5,15].Briefly,themouseinfrarenalaortawasexposedbylaparotomy
undergeneralanesthesiawith2%isoflurane.ExposuretoCaCl wasachievedusingsmall
2
piecesofcottonsoakedin0.5MCaCl for20minutes.Wealsoperformedshamoperation
2
withtheexposuretonormalsalineinsteadofCaCl ,whichservedasanegativecontrolfor
2
CaCl exposure.Weperformedexperimentseitherwith1weekofobservationalperiodmainly
2
toassesstheshorttermresponseofinflammatorysignaling,orwith6weeksofobservational
periodmainlytoassessthemorphologyofAAA.Oneor6weeksafterCaCl exposure,the
2
micewereeuthanizedbyanoverdoseofpentobarbitalandtissueandbloodsamplesharvested.
TheaortawasimmediatelyexcisedtoobtainRNAorproteinsamples.Excisionformorpho-
metricandhistologicalanalyseswasafterperfusionfixationatphysiologicalpressurewith4%
paraformaldehyde.WeassessedAAAdiameterbymeasuringthemaximaldiameterofinfrare-
nalaorta.Wealsotookthereferencediameteroftheabdominalaortabetweentherightand
leftrenalarteries,whichwasnotexposedtoCaCl tocalculatetheratioofAAAdiameterto
2
leftsuprarenalaorta.
IL-6receptorneutralizingantibody
MR16-1,aratmonoclonalantibodyraisedagainstthemouseIL-6receptor,wasprovidedby
ChugaiPharmaceuticalCo.Ltd;MR16-1administrationwasusedtoneutralizethefunctionof
IL-6[14].Weadministered2mgMR16-1bytailveininjectiononedaybeforeexposureto
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IL-6inmurineabdominalaorticaneurysm
CaCl tosuppressIL-6signalingandtoinducetolerancetotheratantibody[16].For6-week
2
experiments,theinitialtailveininjectionbeforeCaCl exposurewasfollowedbyintraperito-
2
nealinjectionof0.25mgMR16-1everyweektoensurethesuppressionofIL-6effectthrough-
outtheobservationalperiod.For1-weekexperiments,onlytailveininjectionof2mgMR16-1
wasperformed.Administrationofphysiologicalsaline(NaCl)andnon-specificratIgG(MP
Biomedicals#0855951)servedasnegativecontrolstoMR16-1.
Histology,immunostaining,andimagingcytometricanalyses
Murineaortaewereembeddedinparaffinand5μmsectionspreparedforstaining.Hematoxy-
linandeosin(H&E),elasticavanGieson(EVG)orVonKossastainswereperformed.Immu-
nohistochemicalstainingofaortictissuewasperformedformacrophages(Iba1;WakoPure
ChemicalIndustries#019–19741)andMcp-1(Abcam#ab25124).TheareaofVonKossaor
Mcp-1stainingandthetissueareawasmeasuredbyBZAnalyzersoftware(Keyence).
Weperformedimagingcytometricanalysesforaorticsampleswithimmunofluorescence
stainingusingArrayScanXTI(ThermoFisherScientific)andtheFlowJoSoftware(FlowJo
LLC).Briefly,tissuesectionsfromfourmouseaortaeineachexperimentalgroupwerepro-
cessedfor3-colorfluorescencestaining;DAPIfornuclei,smoothmuscleα-actin(SMA)anti-
body(clone1A4,Sigma-Aldrich#A2547)forsmoothmusclecells,andantibodiesspecificfor
eitherphospho-Stat3(cloneD3A7,CellSignalingTechnology#9145),phospho-Smad2(clone
138D4,CellSignalingTechnology#3108),orNFκB(cloneD14E12,CellSignalingTechnology
#8242).Stainingconditionsweredeterminedbysinglestainingwithagivenantibodyandvali-
datedtoshowidenticalresultsin3-colorstaining.Allsamplesinagivensetofanalysiswere
processedwiththesamestainingconditionatthesametimeandphotomicrographswere
takenwiththesameexposureconditiontoobtainconsistentresults.Intheimagingcytometric
analyseswithArrayScanXTI,eachcellwasidentifiedbynuclearDAPIstainingandarbitrarily
gatedtoseparatethepositive(smoothmusclecells)andnegative(non-smoothmusclecells)
populationsforSMAsignalsurroundingthenucleus.Thegatesforthenuclearsignalintensity
ofphospho-Stat3,phospho-Smad2andNFκBwerealsodeterminedarbitrarily.Thesegates
weresetconstantthroughouttheexperimentalgroupsinagivensetofanalysis.
Immunoblottingandgelatinzymography
ImmunoblotsforJnk(rabbitpolyclonalantibody,Abcam),phospho-Jnk(clone98F2,CellSig-
nalingTechnology),Stat3(rabbitpolyclonalantibody,CellSignalingTechnology),phospho-
Stat3(cloneD3A7,CellSignalingTechnology),andLox(rabbitpolyclonalantibody,Abcam)
wereperformedusingtheNuPAGEsystem(ThermoFisherScientific).Gelatinzymographywas
performedusingtheNovexzymogramsystem(ThermoFisherScientific)accordingtotheman-
ufacturer’sinstruction.SerumcytokineconcentrationswerequantifiedusingaBio-Plexmulti-
plexbeadarraysystemwiththemousecytokineTh17panelA(#M6000007NY,Bio-Rad).
Transcriptomeanalysis
Theaortictissuewassnap-frozeninliquidnitrogenimmediatelyafterkillingthemice
andobtainingthetissue.TheaortictissuewashomogenizedinTRIzolreagent(Thermo-
fisherScientific),andtotalRNAwasisolatedusingRNeasykit(Qiagen)accordingtothe
instructionbythemanufacturer.TranscriptomeanalyseswereperformedusingaSure-
PrintG3MouseGEmicroarray8x60K(AgilentTechnologies).Functionalannotation
clusterswereobtainedusingtheDatabaseforAnnotation,Visualization,andIntegrated
Discovery(DAVID,https://david.ncifcrf.gov/)[17]withtheGeneOntologytermssetto
GOTERM_BP_FAT,GOTERM_CC_FATandGOTERM_MF_FAT.
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Fig1.TheeffectofMR16-1onthedevelopmentofAAA.(A)MorphometricanalysesofmouseAAA,(B)maximalaorticdiametersinmillimeters,
and(C)relativetoaninternalreferencediameterattheleveloftheleftrenalartery.InpanelA,CaCl indicatestheAAAmodel6weeksafterCaCl
2 2
exposure.NaClandIgGindicatetheAAAmodeltreatedwithphysiologicalsalineandnon-specificratIgG,respectively,asnegativecontrolsforMR16-1
(denotedMR).Thewhitebardenotes1mm.InpanelsBandC,symbolsindicateindividualdataandbarsindicatemeans±standarderrors.The
numbersofmiceforobservationareindicatedinparenthesis.*p<0.05and***p<0.001.
https://doi.org/10.1371/journal.pone.0185923.g001
Statisticalanalyses
StatisticalanalyseswereperformedusingGraphPadPRISM5(GraphPadSoftware).Oneway
ANOVAwasperformedtocompare3ormoregroupsfollowedbyBonferroni’smultiplecom-
parisontest,whenthedatapassedD’AgostinoandPearsonnormalitytestandBartlett’stest
forequalvariances.Otherwise,Kruskal-WallistestwasperformedfollowedbyDunn’smulti-
plecomparisontest.Statisticalsignificancewasindicatedbyapvalueoflessthan0.05.
Results
TheeffectofIL-6receptorneutralizationonthedevelopmentofAAA
Weusedawell-establishedmousemodelofAAAthatemploysaperiaorticapplicationof0.5
MCaCl for20min(CaCl exposure)totheexposedaorta[5,15,18].Thisprocedureinduces
2 2
chronicinflammationoftheaorticwallthatresultsintissuedestructionandthedevelopment
ofAAA.AsshowninFig1,CaCl exposureprovokedanincreaseinthemaximumdiameter
2
oftheaortafrom0.55±0.01mmto1.05±0.04mm,6weeksafterCaCl exposure.Wethen
2
administeredtheMR16-1reagenttomice,whichisaratmonoclonalantibodydirectedagainst
themouseIL-6receptor,inordertoneutralizethefunctionofIL-6[14].AsshowninFig1B,
administrationofMR16-1resultedinasignificantreductioninthemaximalaorticdiameter6
weeksafterCaCl exposure(0.92±0.03mm).Ontheotherhand,acontrolratIgGorsaline
2
injectionfailedtoalterthemaximalaorticdiameterinthesametimeframe(i.e.6weeksafter
theCaCl exposure)(1.08±0.03mm).Whenwecomparedthemaximaldiametertotheaortic
2
diameterjustabovetheleftrenalarteryasaninternalreference,thedatawereessentiallyiden-
tical(Fig1C).TheseresultsindicatethatinthismousemodelofAAA,IL-6signalingis
requiredforthefulldevelopmentofAAA.
MicroscopicfindingsintheAAAmodel
Histologicalexaminationrevealedprominentdegenerationofthemediallayerswithsubstantial
cellularinfiltrationintheadventitiallayersoftheaorticwall1weekafterCaCl exposure(Fig
2
2).Immunohistochemicalstainingsdemonstratedabundantinfiltrationofcellsthatwereposi-
tiveforIba1,amarkerofmonocytes/macrophagesintotheaortictissueatthistimepoint.Aor-
tictissuewithMR16-1administrationshowedlessamountofIba1positivecells.Sixweeksafter
CaCl exposure,medialdegenerationwasfurtherprogressedwithflattenedandelongatedelas-
2
ticlamellae.Atthistimepoint,theadventitiallayerdemonstratedlesscellularinfiltrationcom-
paredtothatobserved1weekafterCaCl exposure.TheadministrationofMR16-1wasfound
2
tomodestlysuppressadventitialcellularinfiltrationat1weekandmedialdegeneration6weeks
afterCaCl exposure,althoughsuppressionwasincomplete.Thesefindingsareconsistentwith
2
themodestdecreaseinAAAdiameterprovokedbyMR16-1asdemonstratedmacroscopically.
Asweobservedinfiltrationofmonocytes/macrophagesinAAAtissue,weexaminedthe
expressionofmonocytechemoattractantprotein-1(Mcp-1)thathasbeenreportedtopartici-
pateinAAApathogenesis[19,20]byimmunohistochemistry(Fig3A).Atthebaseline,we
observedweakMcp-1staininginthemediallayeroftheaorta.OneweekaftertheCaCl
2
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Fig2.HistologicalstudyofAAA.Histologicalfindingsareshownforperfusion-fixedaortaeattheirmaximum
diameterbyhematoxylin-eosin(H&E),andelasticavanGieson(EVG)staining.Tissuesampleswereobtainedprior
to,1weekand6weeksafterCaCl exposure,withorwithoutMR16-1administration.Immunohistochemicalstaining
2
ofIba1,amarkerformonocyte/macrophages,wasalsoperformed1weekafterCaCl exposure.RectanglesinH&E
2
stainingindicatetheareaforIba1staining.Theblackandgraybarsdenote200μmand5μm,respectively.Arrow-
headsindicateIba1-positivecells.
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exposurefollowedbythesalineinjection,Mcp-1stainingbecamemoreprominentinthe
mediaandadventitiaoftheaortictissueOntheotherhand,administrationofMR16-1signifi-
cantlysuppressedtheMcp-1positiveareaintheaortictissue,indicatingthatIL-6wasrespon-
sibleatleastinpartforMcp-1expressionintheaortictissue.
BecausecalcificationisacommonfeatureoftheCaCl -inducedAAAmodelandhuman
2
AAA[4],andIL-6maybecausallyrelatedtovascularcalcification[21],wealsoevaluatedthe
effectofMR16-1ontheextentofaorticwallcalcification(Fig3B).VonKossastainingshowed
nocalcificationoftheaorticwallatthebaseline,butprominentaorticwallcalcification6
weeksaftertheCaCl exposure.AdministrationofMR16-1didnotcausesignificantchanges
2
intheratioofthecalcifiedareatothetissuearea,suggestingthatIL-6maynotplayamajor
roleinaorticcalcificationinthisexperimentalsetting.
SerumcytokineprofilinginAAAmodel
Weexaminedtheserumcytokineprofilesofmice1weekafterCaCl exposure(Fig4),when
2
theinflammatoryresponseisatitspeak[5].FollowingCaCl exposurewithphysiological
2
saline(NaCl)injectionorcontrolratIgGinjection,noneofthecytokinesshowedsignificant
changes,whichisconsistentwithourpreviousreportofCaCl exposurenotprovokingasys-
2
temiccytokineresponse[22].AdministrationofMR16-1resultedinasignificantincrease
comparedtosham-operatedgroupintheserumconcentrationofIL-6,whichmostlikely
reflectsareleaseofreceptor-boundIL-6byMR16-1,aspreviouslyreported[23].Thiseffectis
compatiblewiththenotionofMR16-1inhibitingIL-6bindingtoitsreceptor.
InflammatorysignalinginAAAtissue
ThefundamentalmechanismofAAAappearstoinvolveaninflammatoryresponsethatpro-
motestissuedegradation.WethereforeexaminedtheactivationstatusofJnk,acriticalmediator
ofAAApathogenesis[5]andStat3,awell-establishedmediatorofIL-6signalingbyimmuno-
blotanalysis1weekafterCaCl exposure(Fig5).CaCl exposurecausedsignificantincreasesin
2 2
theproteinexpressionsofJnkandStat3andtheiractivities.AlthoughadministrationofMR16-
1seemedtoresultinlesssignificantactivationofJnk,theeffectwassimilartocontrolratIgG.
Ontheotherhand,MR16-1,butnotcontrolratIgG,diminishedStat3activationbyCaCl
2
exposure.Wenextexaminedeffectorsthatareinvolvedinthemetabolismoftheextracellular
matrix(ECM).CaCl treatmentcausedsignificantincreasesinMmp-9andMmp-2expressions,
2
asshownbythegelatinzymography,andlysyloxidase(Lox)asshownbyimmunoblotsofaortic
samples.TheadministrationofMR16-1provokednosignificantchangeinthelevelsofMmp-9,
Mmp-2,orLox.TheseresultswereconsistentwiththenotionthatStat3isthemainmediatorof
IL-6signaling.SuppressiveeffectofMR16-1onAAAexpansiondidnotseemtoinvolvethe
changesinJnkactivityorECMmetabolicenzymesasexaminedinthisstudy.
SignalingatthecellularlevelinAAAtissue
TobetterunderstandtheroleofIL-6signalinginAAApathogenesisatthecellularlevel,we
examinedthenuclearlocalizationofNFκB,amediatorofAAApathogenesis[6],pStat3,a
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Fig3.EffectofMR16-1onMcp-1expressionandcalcification.(A)PhotomicrographsareshownforMcp-1
stainingofaortictissuewithshamoperation,1weekafterCaCl exposurewithsalinetreatment(CaCl +NaCl),and
2 2
1weekafterCaCl exposurewithMR16-1treatment(CaCl +MR).ExpressionofMcp-1wasevaluatedby
2 2
calculatingtheratioofMcp-1-positiveareatothetissuearea(Mcp-1area).(B)VonKossastainingwasperformed
foraortictissuewithshamoperation(sham),6weeksafterCaCl exposurewithsalinetreatment(CaCl +NaCl),
2 2
and6weeksafterCaCl exposurewithMR16-1treatment(CaCl +MR).Theextentofaortictissuecalcificationwas
2 2
evaluatedbycalculatingtheratioofthecalcified(brown)areatothetissuearea(Calcifiedarea).Symbolsingraphs
indicateindividualdataandbarsindicatemeans±standarderrors.Thenumbersofmiceforobservationare
indicatedinparentheses.*p<0.05,**p<0.01,and***p<0.001.Barsdenote200μm.
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mediatorofIL-6signaling,andpSmad2,amediatorofTGFβsignalingthatisinvolvedininflam-
mation,ECMmetabolism,andregulationofsmoothmusclecellfunction[24],togetherwith
stainingforsmoothmuscleα-actin(SMA),amarkerofsmoothmusclecells(SMCs)(Fig6A).In
aortafromsham-operatedmice,nuclearlocalizationofNFκBandpStat3werenegligible,indica-
tiveoftheabsenceoftheiractivitiesatthebaseline.Ontheotherhand,pSmad2showedstrong
signalmainlyinmedialSMCs.CaCl exposureresultedintheincreaseinadventitialcellswith
2
nuclearNFκB,concomitantwiththedecreaseinSMAstaining.ItalsocausedincreaseinpStat3
inpartofadventitialandintimalcells,increaseinnuclearpSmad2inadventitia,butdecreasein
nuclearpSmad2inmedialcells.
Weperformedimagingcytometricanalysisoftheseaortictissue(Fig6B).Whengateswere
settoarbitrarilyseparatetheSMA-positiveand-negativepopulations,approximately55%of
thetotalcellpopulationwasSMA-positive,andapproximately45%SMA-negative,presum-
ablyindicatingSMCsandnon-SMCs,respectively,inNFκB,pStat3andpSmad2analyses.
CaCl treatmentcausedadramaticincreaseinnon-SMCstoapproximately80%(Fig6B).
2
AdministrationofMR16-1resultedinthedecreaseinSMA-negativepopulationto70%and
increaseinSMA-positivepopulationto30%,whereascontrolratIgGdidnotshowsuchan
effect.CaCl exposurecausedanincreaseinnuclearNFκBofbothSMA-negativeand-positive
2
populationsfrom3.5%to34%oftotalpopulation,forwhichMR16-1didnotshowdiscernible
effect.CaCl exposurealsocausedanincreaseinnuclearpStat3from6.6%to10.9%mainlyin
2
SMA-negativepopulation,whichwasreducedbyMR16-1administrationto7%whereascon-
trolratIgGshowednoeffect.CaCl exposurecausedincreaseinpSmad2mainlyinSMA-neg-
2
ativepopulationandreductioninSMA-pSmad2doublepositivepopulationfrom19.9%to
9.5%.MR16-1administrationprovokedarecoveryoftheSMA-pSmad2doublepositivepopu-
lationto29.9%.
TheseresultssuggestedthatCaCl exposurecausedthereductionofSMA-positivepopu-
2
lationpossiblyduetothecellularinfiltration,reductioninSMAexpressioninSMCsorloss
ofSMCs,whichwasassociatedwiththeactivationofNFkB,Stat3andSmad2mainlyin
SMA-negativepopulation.MR16-1causedapartialrecoveryofSMA-positivepopulation,
whichwasassociatedwiththedecreaseinStat3activitybutnotwiththechangesinNFκB
activity.
TranscriptomeanalysisduringAAAdevelopment
TobetterunderstandtheroleofIL-6inthepathogenesisofAAA,weperformedatranscrip-
tomeanalysis1weekafterexposuretoCaCl ,withorwithoutMR16-1administration.Func-
2
tionalannotationclusterswereobtainedusingtheDatabaseforAnnotation,Visualization,
andIntegratedDiscovery(DAVID).Ofthe55,681probesontheDNAmicroarray,5,940and
4,694probeswithmatchedDAVIDIDswereup-anddown-regulated,respectively,byCaCl
2
treatment,asdefinedbyafoldchangeofmorethan1.5xandp<0.05forup-regulatedprobes,
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