Possible Dual Role of Decorin in Abdominal Aortic Aneurysm

15 Pages·2015·0.75 MB·English

RESEARCHARTICLE
Possible Dual Role of Decorin in Abdominal
Aortic Aneurysm
KoshiroUeda1,KoichiYoshimura1,2*,OsamuYamashita1,TakasukeHarada1,
NoriyasuMorikage1,KimikazuHamano1
1 DepartmentofSurgeryandClinicalScience,YamaguchiUniversityGraduateSchoolofMedicine,Ube,
755–8505,Japan,2 GraduateSchoolofHealthandWelfare,YamaguchiPrefecturalUniversity,Yamaguchi,
753–8502,Japan
* [email protected]
a11111
Abstract
Abdominalaorticaneurysm(AAA)ischaracterizedbychronicinflammation,whichleadsto
pathologicalremodelingoftheextracellularmatrix.Decorin,asmallleucine-richrepeatpro-
teoglycan,hasbeensuggestedtoregulateinflammationandstabilizetheextracellularma-
OPENACCESS
trix.Therefore,thepresentstudyinvestigatedtheroleofdecorininthepathogenesisof
Citation:UedaK,YoshimuraK,YamashitaO, AAA.Decorinwaslocalizedintheaorticadventitiaundernormalconditionsinbothmice
HaradaT,MorikageN,HamanoK(2015)Possible
andhumans.AAAwasinducedinmiceusingCaCl treatment.Initially,decorinproteinlev-
2
DualRoleofDecorininAbdominalAorticAneurysm.
elsdecreased,butasAAAprogresseddecorinlevelsincreasedinalllayers.Localadminis-
PLoSONE10(3):e0120689.doi:10.1371/journal.
pone.0120689 trationofexogenousdecorinpreventedthedevelopmentofCaCl2-inducedAAA.However,
decorinwashighlyexpressedinthedegenerativelesionsofhumanAAAwalls,andthisex-
AcademicEditor:UtakoYokoyama,YokohamaCity
UniversityGraduateSchoolofMedicine,JAPAN pressionpositivelycorrelatedwithmatrixmetalloproteinase(MMP)-9expression.Incellcul-
tureexperiments,theadditionofdecorininhibitedsecretionofMMP-9invascularsmooth
Received:September24,2014
musclecells,buthadtheoppositeeffectinmacrophages.Theresultssuggestthatdecorin
Accepted:January25,2015
playsadualroleinAAA.Adventitialdecorininnormalaortamayprotectagainstthedevel-
Published:March17,2015
opmentofAAA,butmacrophagesexpressingdecorininAAAwallsmayfacilitatethepro-
Copyright:©2015Uedaetal.Thisisanopen gressionofAAAbyup-regulatingMMP-9secretion.
accessarticledistributedunderthetermsofthe
CreativeCommonsAttributionLicense,whichpermits
unrestricteduse,distribution,andreproductioninany
medium,providedtheoriginalauthorandsourceare
credited.
DataAvailabilityStatement:Allrelevantdataare
withinthepaper. Introduction
Funding:ThisworkwassupportedbyaGrant-in-Aid Abdominalaorticaneurysm(AAA)isasegmentalexpansionoftheabdominalaorta.AAAisa
forScientificResearchfromtheJapanSocietyforthe common,fataldiseasethatcancausecatastrophicaneurysmalruptures[1].Theprevalenceof
PromotionofScience(KAKENHI24390302and
AAAisestimatedtobebetween4.0%and8.9%inoldermen[2].Aorticaneurysmswerethe
26670618toKY),theTakedaScienceFoundation(to
primarycauseof10,597deathsandacontributingcausein17,215deathsintheUnitedStates
KY),andtheUeharaMemorialFoundation(toKY).
in2009[3,4].BecausemostpatientswithAAAhavenosymptoms,themainpurposeoftreat-
Thefundershadnoroleinstudydesign,data
collectionandanalysis,decisiontopublish,or mentistoimproveprognosisbypreventinganeurysmalrupture.TherapeuticoptionsforAAA
preparationofthemanuscript. arecurrentlylimitedtoopenorendovascularsurgicalrepairtopreventrupture[5].Animpor-
tantunmetneedinthetreatmentofAAAisnon-surgicalapproaches,particularlypharmaco-
CompetingInterests:Theauthorshavedeclared
thatnocompetinginterestsexist. therapies[6–8].
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RoleofDecorininAAA
AAAischaracterizedbychronicinflammationandextracellularmatrixdegradationcaused
byproteolyticenzymes,suchasmatrixmetalloproteinase(MMP)-9[6,9].Althoughnofunda-
mentalcauseofAAAhasbeenidentified,variousproinflammatorymediatorsareactivatedin
AAAwalls,causingtheinflammatoryresponsesandshiftingthebalanceofextracellularmatrix
metabolismtowardstissuedegradation[8].Extracellularmatrixproteinsaremajorconstitu-
entsofthevascularwallandcanpotentiallyinteractwithavarietyofvascularcells.Extracellu-
larmatrixproteins,suchasperiostinandtenascinC,havebeensuggestedtoplayimportant
rolesinthepathogenesisofAAA[10–12].However,nostudyhasfullyelucidatedthesignifi-
canceofothervariousextracellularmatrixproteinsinAAA.
Decorinbelongstothefamilyofsmallleucine-richproteoglycans(SLRP)thoughttoplayes-
sentialrolesinvascularbiology[13,14].Inparticular,decoriniscapableofmodulatingcollagen
fibrillogenesis,immuneresponses,andinflammatoryresponses[14,15].Decorinisexpressed,
tosomeextent,innormalaortictissuesandaneurysmwalls.Apreviousstudyshowedthatre-
duceddecorinexpressionisassociatedwithahighriskofaorticruptureinamousemodelof
AAA[16].Thesefindingsledustohypothesizethatdecorinplaysacrucialroleinthepatho-
genesisofAAA.Inthepresentstudy,weshowthatdecorinhasbothtissue-protectiveandpro-
inflammatoryproperties,dependingonthecontextinwhichitisexpressed.
MaterialsandMethods
Animalexperiments
Six-week-oldmaleC57BL/6micewerepurchasedfromChiyodaKaihatsuCo.,Ltd.(Tokyo,
Japan).Themiceweremaintainedinplasticcages(5percage)inatemperature-andhumidi-
ty-controlledroomwitha12-hlight/12-hdarkcycle.Micewereallowedfreeaccesstostandard
foodandwaterthroughouttheexperiments.WeinducedAAAinmicewithperiaorticapplica-
tionof0.5MCaCl asdescribedpreviously[12,17,18].Moreprecisely,wetreatedtheinfrarenal
2
aortabetweentheleftrenalveinandaorticbifurcationwithCaCl .Inthepreventionstudy,we
2
placedGelfoampatches(3.5×2×2mm;Pfizer,NewYork,NY,USA)intheperiaorticspacebe-
tweentheleftrenalveinandaorticbifurcationimmediatelyafterCaCl treatment;thepatches
2
wereloadedwitheither20(gbovinedecorin(Sigma-Aldrich,St.Louis,MO,USA)dissolvedin
100(lphosphate-bufferedsaline(PBS)(CaCl +decorin,n=10)or100(lPBS(CaCl +PBS,n=
2 2
9).Untreatedmiceservedasthecontrolgroup(Control,n=6).
Forthesestudies,micewereanesthetizedwithanintraperitonealinjectionofsodiumpento-
barbital(40mg/kg)beforeundergoinglaparotomy.Theexperimentalmiceweresacrificed
withanoverdoseofsodiumpentobarbital(100mg/kg,intraperitonealinjection)42daysafter
CaCl treatmentforthepreventionstudy,or0,3,7,14,28,or42daysafterCaCl treatmentfor
2 2
thetemporalobservationstudy.Tissuewasfixedwithwhole-bodyperfusionof4%paraformal-
dehydeinPBSatphysiologicalpressureandtheabdominalaortaimmediatelyexcised,photo-
graphedformorphometricanalysis,andsectionsanalyzedhistologically.Photographsofthe
aortaswereusedtodeterminemaximumexternalaorticdiameters.
AllexperimentswereperformedinaccordancewiththeGuidefortheCareandUseofLab-
oratoryAnimalspublishedbytheUnitedStatesNationalInstitutesofHealth.Allprotocols
wereapprovedbytheYamaguchiUniversitySchoolofMedicineAnimalExperimentsReview
Board(#31–091).
Histologicalandimmunohistochemicalanalyses
Forhistologicalanalyses,paraffin-embeddedsectionswerestainedwithhematoxylinandeosin
(HE)andelastica-vanGieson(EVG).Sectionswerealsoprobedwithantibodiesraisedagainst
appropriateantigensforimmunohistochemistryasdescribedpreviously[17,19,20].We
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RoleofDecorininAAA
detecteddecorinbyprobingsectionswithanti-mousedecorinantibody(SantaCruzBiotech-
nology,Dallas,TX,USA,#sc-22753)andanti-humandecorinantibody(R&DSystems,Minne-
apolis,MN,USA,#MAB143),anddetectedMMP-9byprobingsectionswithanti-mouse
MMP-9antibody(R&DSystems,#AF909)andanti-humanMMP-9antibody(DaiichiFine
Chemical,Toyama,Japan,#F-69).Wealsousedanti-humanCD68antibody(Dako,Glostrup,
Denmark,#M0876)andanti-humansmoothmuscleactinantibody(Dako,#M0851).The
probedproteinswerevisualizedbytheavidin-biotincomplextechniqueusingtheVECTAS-
TAINABC-APkit(VectorLaboratories,Burlingame,CA,USA)orbyindirectimmunofluo-
rescencestainingusingAlexaFluor488-conjugatedanti-mouseIgGantibody(Molecular
Probes,Eugene,OR,USA)andAlexaFluor594-conjugatedanti-rabbitIgGantibody(Molecu-
larProbes).DAPI(MolecularProbes)wasusedfornuclearstaining.
Thetotalnumberofinfiltratingmononuclearcellswascountedinfivehigh-powerfields
permouseinHE-stainedsections.Wealsoevaluatedthedegreeofmediallayerelastindisrup-
tioninEVG-stainedsectionsusingthegradingmethodreportedbyHamblinetal.[21].Briefly,
thedegreeofelastindegradationwasclassifiedasmildlydisruptedwhenonlyoneelasticlamel-
lawasdisrupted(gradeI),moderatelydisruptedwhentwoelasticlayerswerebrokenordis-
rupted(gradeII),highlydisruptedwhenthreeelasticlayersexhibitedbreakageand/or
degradation(gradeIII),andseverelydisruptedwhenallfourelasticlayersexhibitedsignsof
breakageand/ordegradation(gradeIV).
Cellcultureexperiments
Rataorticvascularsmoothmusclecells(VSMCs)derivedfromthemediallayerofhealthyrat
aortawerepurchasedfromCellApplications,Inc(SanDiego,CA,USA).VSMCsweremain-
tainedinDulbecco’smodifiedEagle’smedium(DMEM)(Invitrogen,Carlsbad,CA,USA)
containing10%fetalbovineserum.Beforetheexperiments,VSMCswereseededontolaminin-
coatedplatesandserum-starvedfor48h.Thestarvedcellsweretreatedwith100ng/mllipo-
polysaccharide(LPS)(AlexisBiochemicals,SanDiego,CA,USA)for48h.Whenindicated,the
VSMCsweretreatedwith0.4,4,or40μg/mlbovinedecorin(Sigma-Aldrich)for24hpriorto
LPSadministration.
Thioglycolate-elicitedperitonealmacrophageswerecollectedfrom6-week-oldmale
C57BL/6mice(ChiyodaKaihatsu)asdescribedpreviously[22,23].Briefly,themicewerein-
jectedintraperitoneallywith2mlofthioglycolatemedium(Sigma-Aldrich).After3days,cells
wereharvestedbyperitoneallavagewith10mlPBS.Thecellswerewashedtwicewithcold
PBS,resuspendedinRPMI-1640medium(DSPharmaBiomedical,Osaka,Japan)containing
10%fetalbovineserum,andseededongelatin-coatedplates.After24h,non-adherentcells
wereremovedbywashingthecultureswithmedium.Theperitonealcellswereimmunostained
withanti-mouseMac3antibody(BDBiosciences,SanJose,CA,USA,#550292)toidentify
macrophages.Inallcases,theproportionofmacrophageswasconsistently>90%.Beforeex-
periments,macrophageswereserum-starvedfor24h,followedbytreatmentwith100ng/ml
LPSfor48h.Whenindicated,cellsweretreatedwith0.4,4,or40μg/mldecorinfor24hprior
toLPSadministration.
Gelatinzymography
Gelatinzymographywasperformedasdescribedpreviously[17,19].Briefly,equalvolumesof
conditionedmediawereelectrophoresedinthepresenceof0.2%SDSona10%polyacrylamide
gelcontaininggelatin(1mg/ml)undernon-reducingconditions.Afterelectrophoresis,thegels
werewashedin2.5%TritonX-100andincubatedat37°Cindevelopingbuffer(50mMTris
(pH7.5),200mMNaCl,5mMCaCl ,and0.02%Briji35).Thegelswerestainedwith0.5%
2
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RoleofDecorininAAA
CoomassiebrilliantblueR-250in40%methanoland10%aceticacidandtheexpressionof
MMP-9andMMP-2determinedbyquantifyingbandsatappropriatepositionsonthegel.
Humanaorticsamples
Weobtainedabdominalaorticwallspecimensfrom47patientswithAAAundergoingopen
surgicalrepair.Ascontrols,non-aneurysmalabdominalaorticwallspecimenswereobtained
fromfourautopsypatientswhodiedofunrelatedcauses.Theaortictissuespecimenswere
usedforproteinanalysesbywesternblottingandimmunohistochemistry.Allpatientsprovid-
edwritteninformedconsentinaccordancewiththeprinciplesoutlinedintheDeclarationof
Helsinki.Regardingautopsyspecimens,writteninformedconsentwasobtainedfromthenext
ofkinfortheuseofthesampleinresearch.Allexperimentalprotocolswithhumanspecimens
wereapprovedbytheInstitutionalReviewBoardatYamaguchiUniversityHospital
(#H24–26).
Proteinisolationandwesternblotting
Humanaorticwallspecimenswerehomogenizedinasolutionof25mMTris(pH7.4),150
mMNaCl,5mMEDTA,10mMsodiumpyrophosphate,10mMβ-glycerophosphate,1mM
Na VO ,1mMphenylmethanesulfonylfluoride,and10μg/mlaprotinin.Proteinswereex-
3 4
tractedbyaddingTritonX-100toafinalconcentrationof1%.Proteinconcentrationswerede-
terminedusingabicinchoninicproteinassaykit(BCAkit,Bio-Rad,Hercules,CA,USA).
Westernblottingwasperformedasdescribedpreviously[17,20].Briefly,equalamountsof
sampleproteinwereloadedontoeachlaneofanSDS-PAGEgel.Separatedproteinsweretrans-
ferredontopolyvinylidenedifluoridemembranes(Millipore,Bedford,MA,USA)andprobed
withantibodiesforhumandecorin(R&DSystems,#MAB143),glyceraldehyde3-phosphate
dehydrogenase(GAPDH)(Millipore,#MAB374),humanMMP-9(DaiichiFineChemical,
#F-69),andhumantransforminggrowthfactor(TGF)-β1(SantaCruzBiotechnology,
#sc-52893).
Enzyme-linkedimmunosorbentassay(ELISA)
TheconcentrationofTGF-βinconditionedmediawasquantifiedbyasandwichenzymeim-
munoassaytechniqueusingthemouse/rat/porcineTGF-β1ELISAKit(R&DSystems,
#SMB100)accordingtothemanufacturer’sinstructions.
Statisticalanalysis
Dataareexpressedasthemean±standarddeviation(SD).Statisticalanalyseswereperformed
withPrism5.0dsoftware(GraphPadSoftware,LaJolla,CA,USA).Theunpairedt-testoranal-
ysisofvariance(ANOVA)wasusedforcomparisonswithBonferronipost-testcorrection.As-
sociationsbetweencontinuousvariableswereassessedbythePearsoncorrelationcoefficient
test.Ap-value<0.05wasconsideredsignificant.
Results
TemporalpatternofdecorinproteinlevelsduringAAAdevelopmentin
mice
First,weanalyzedamousemodelofAAAtoevaluatedecorinproteinlevelsduringtheinitia-
tion,development,andprogressionofAAA.Themousemodelwascreatedbyperiaorticappli-
cationofCaCl intheinfrarenalaorta(Fig.1A).After3days,theCaCl treatmentinduced
2 2
infiltrationofinflammatorycells.From3to14days,theelasticlamellaebegantoexhibit
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RoleofDecorininAAA
Fig1.TemporalpatternofdecorinproteinlevelsduringAAAdevelopmentinmice.(A)Amousemodel
ofAAAwasinducedbyperiaorticapplicationofCaCl .Salineapplicationwasusedasacontrol.Themice
2
weresacrificed0,3,7,14,28,or42daysafterCaCl treatment.(B)Representativeimagesshowingaortic
2
wallsstainedwithhematoxylinandeosin(HE),elasticavan-Gieson(EVG),oranantibodyagainstdecorinat
theindicatedtimepointsaftertheapplicationofsaline(Control)orCaCl (AAAinduction).Theluminal
2
surfaceisorientedtowardthetopofeachpanel.HEandEVGstainsdepictcellnuclei(blue-black)andthe
elastinnetwork(black),respectively.Localizationofdecorinisindicatedbyredstaining.M:media,
A:adventitia.
doi:10.1371/journal.pone.0120689.g001
straighteningandfragmentation.Inflammatorycellinfiltrationandthedisruptionofelastic
layerscontinuedandgraduallyincreasedupto42days(Fig.1B).Consequently,28and42days
afterCaCl treatment,theinfrarenalaortasofAAAmiceexhibitedsignificantlylargermaxi-
2
mumdiametersthanthecontrols,whichwasconsistentwithourpreviousresults[12].
Inthecontrolmice,decorindepositionwasobservedintheadventitiaandperiaortictissues,
andproteinlevelsweresimilarbeforeandaftersalinetreatment.IntheAAAmice,adventitial
decorinwasseverelydiminished3and7daysafterCaCl treatment,butby14daysthedecorin
2
proteinlevelsreturnedtonearlybasallevelsandbegantospreadtoperiaortictissues.At
42days,weobserveddecorindepositioninalllayers,includingthethinaorticmedia,wherewe
observedmarkedincreasesininflammatorycellinfiltrationandelasticlamellaedestruction
(Fig.1B).Theseresultsindicatethatdecorinproteinisdown-regulatedattheinitiationof
AAA.However,duringtheprogressionofAAA,decorinproteinwasinverselyup-regulated
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RoleofDecorininAAA
andspreadtotheaorticwalls,whereactiveinflammationcauseddestructionofthe
elasticlayers.
EffectofdecorinadministrationonthedevelopmentofAAAinmice
ToclarifytherelationshipbetweentheinitialdeclineindecorinandtheinitiationofAAA,we
investigatedwhetherlocalapplicationofexogenousdecorininhibitspathologicalremodeling
oftheaorticwallintheAAAmouse(Fig.2A).WeappliedadecorinproteinsolutiontoGel-
foam,abiodegradableextracellularmatrixpreparationthatprovideslocaldeliveryofproteins
ofinterest[12,24],andimplantedtheGelfoampatches(CaCl +decorinorCaCl +PBS)inthe
2 2
periaorticspaceofmiceimmediatelyafterCaCl treatment(Fig.2B).AccordingtoKuhnB.
2
etal.[24],thisdeliverysystemcouldtheoreticallyenablecontinuousreleaseofdecorinduring
theexperimentalperiodanddeliverdecorintotheaorticmediainmice.Forty-twodaysafter
treatment,aortasfromtheCaCl +PBSgroupexhibitedasignificantincreaseinaorticdiameter
2
comparedtountreatedaortas(Controlgroup)(Fig.2C-D),whichwassimilartoourprevious
results[11,12,17,25].TheCaCl +PBSgroupalsohadmarkedinflammatorycellinfiltrationin
2
alllayers,andthemorphologyoftheelasticlamellaeintheaorticmediaappearedstraightened
andfragmented.WefoundabundantMMP-9expressioninthemediaandadventitia,inaddi-
tiontocellularinfiltrationandmedialdestruction,intheCaCl +PBSgroup(Fig.2E-F).Inter-
2
estingly,aortasfromtheCaCl +decoringrouphadsignificantlysmallerdiametersthanthose
2
fromtheCaCl +PBSgroup(Fig.2C-D).Histologicalanalysesclearlydemonstratedthataortas
2
fromtheCaCl +decoringrouphadfewerinfiltratingmononuclearcells,morepreservedelastic
2
lamellaemorphology,andlowerMMP-9proteinlevelscomparedtoaortasfromthe
CaCl +PBSgroup(Fig.2E-F).
2
BecauseexcessiveelastolysismediatedbyMMP-9isconsideredacriticalstepinaneurysmde-
velopment[9,20],wedeterminedthedegreeofmedialelastindisruptionintheCaCl +PBSand
2
CaCl +decoringroups(Fig.2G).Most(78%)oftheaortasintheCaCl +PBSgrouphadsevere
2 2
elastindisruption(gradeIV).Incontrast,only10%oftheaortasintheCaCl +decoringrouphad
2
gradeIVdisruption.MostoftheaortasintheCaCl +decoringroupexhibitedmoderate(grade
2
II,30%)orhighelastindisruption(gradeIII,50%)(Fig.2H).Thesefindingsindicatethattheini-
tialdeclineinadventitialdecorinpotentiallycontributestothedevelopmentofCaCl -induced
2
AAA.Preventingthisdeclineindecorinbyapplyingexogenousdecorinresultedinsignificant
suppressionofAAAdevelopment,probablybyinhibitingMMP-9-mediatedelastindisruption.
RoleofdecorininVSMCsecretionofMMP-9
BecauseourinvivofindingssuggestedthatdecorininhibitsMMP-9expressionintheaortic
wall,weinvestigatedwhetherdecorinnegativelyregulatesMMP-9proteinsecretionin
VSMCs,oneofthemajorcelltypesoftheaorticwall(Fig.3A).Underbasalconditions,MMP-
2wasreadilydetectableintheconditionedmediaofVSMCs,butMMP-9wasnearlyundetect-
able.However,theVSMCssecretedappreciablelevelsofMMP-9inresponsetoLPStreatment.
Interestingly,thisLPS-inducedup-regulationofMMP-9secretionwasabrogatedbypre-
treatmentwithexogenousdecorininadose-dependentmanner(Fig.3B-C).NeitherLPSnor
decorinaffectedthesecretionofMMP-2,whichsuggeststhatcellviabilitywaspreserveddur-
ingtheexperiments(Fig.3D).Thesedatademonstratethatdecorinplaysaroleinprotecting
VSMCsfrominflammatoryinsultsbyinhibitingtheup-regulationofMMP-9.
ExpressionofdecorininhumanAAA
Todeterminewhetherthesefindingsareapplicabletohumans,weexamineddecorinprotein
levelsinhumanAAAspecimens.BecauseweobtainedthehumanAAAspecimensfrom
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Fig2.EffectofdecorinadministrationonAAAdevelopmentinmice.(A-B)ImmediatelyafterCaCl
2
treatment,GelfoampatchesloadedwithPBS(CaCl +PBS,n=9)or20(gdecorin(CaCl +decorin,n=10)
2 2
wereplacedintotheperiaorticspacesbetweentheleftrenalvein(ltRV)andbifurcation.AA:abdominal
aorta,IVC:inferiorvenacava,ltRA:leftrenalartery.Untreatedmicewereusedascontrols(Control,n=6).
Themiceweresacrificed42daysafterCaCl treatment.(C)Representativephotographsshowaortas42
2
daysafterCaCl treatment.(D)Quantitativeanalysisofthemaximumexternaldiametersofabdominal
2
aortas.Dataaremean±SD.**p<0.01comparedtoControl;##p<0.01comparedtoCaCl +PBS.(E)
2
Representativehistologicalandimmunohistochemicalstainsofabdominalaortaspecimensfrommice
treatedasindicated.HEandEVGstainsdepictcellnuclei(blue-black)andelastinnetwork(black),
respectively.ThelocalizationofMMP-9isindicatedwithredstaining.M:media,A:adventitia.(F)Infiltrating
mononuclearcellswerecountedinfivehigh-powerfields.Dataaremean±SD.**p<0.01comparedto
Control;#p<0.05comparedtoCaCl +PBS.(G-H)Thedegreeofmediallayerelastindisruptionwasgraded
2
asmild(gradeI),moderate(gradeII),high(gradeIII),orsevere(gradeIV)basedonEVG-stainedsections.
Yellowarrowsindicatedisruptionofelasticlamellae.Whiteasterisksindicatepreservedelasticlamellae.
doi:10.1371/journal.pone.0120689.g002
patientswhohadundergoneopensurgery,thespecimensrepresentedprogressedlesionsrather
thaninitiallesions.Thus,theselesionsmostlikelycorrespondtothemouseAAAspecimens
42daysafterCaCl treatment.Asexpected,decorinexpressionwasgreatlyelevatedinthe
2
humanAAAwallscomparedtonon-aneurysmalaorticwalls(Controls)(Fig.4A-B).MMP-9
expressionwasalsohighlyelevatedinthehumanAAAwalls,asreportedpreviously[26,27].
Interestingly,decorinproteinlevelspositivelycorrelatedwithMMP-9proteinlevelsinhuman
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Fig3.RoleofdecorininVSMCsecretionofMMP-9.(A)Culturedratvascularsmoothmusclecells
(VSMCs)werepre-treatedwithorwithout0.4,4,or40μg/mldecorin,thenstimulatedwithorwithout100ng/
mllipopolysaccharide(LPS)for48h.ProteinlevelsofMMP-9andMMP-2intheconditionedmediawere
determinedbygelatinzymography.(B)Representativeresultsareshown.(C-D)CulturedratVSMCswere
pre-treatedwithorwithout40μg/mldecorin,thenstimulatedwithorwithout100ng/mlLPSfor48h.
QuantitativeanalysesforMMP-9(C)andMMP-2(D)areshown.Dataaremean±SD.**p<0.01compared
toControl;##p<0.01comparedtoLPS.
doi:10.1371/journal.pone.0120689.g003
specimens(Fig.4C).TGF-βprotein,apossiblestabilizerofextracellularmatrixinAAA[28]
alsopositivelycorrelatedwithdecorinproteinlevelsinhumanaorticspecimens(Fig.4D).
Next,wedeterminedthetissuelocalizationofdecorinandMMP-9andanalyzedtheirasso-
ciationwithpathologicaltissuearchitectureinhumanAAAwalls.TheAAAwallstypicallyex-
hibitedcellularinfiltration,fragmentation,andlossofelasticlamellae.Althoughdecorinand
MMP-9didnotstrictlyco-localize,bothdecorinandMMP-9werefoundmainlyinthemedia
andadventitia,frequentlyaccompaniedbysevereinflammationandtissuedestruction
(Fig.4E).Immunofluorescencestainingalsoshowedthatdecorinlargelycolocalizedwith
CD68+macrophagesinthemediaandadventitia,butnotwithα-SMA+smoothmusclecells
(Fig.4F-G).IncontrasttoAAAwalls,non-aneurysmalaorticwallsexhibiteddecorindeposi-
tionintheadventitia,notthemedia,andtheylackedMMP-9expression,whichisconsistent
withourobservationsincontrolmice.Takentogether,ourobservationsinhumansandmice
suggestthatdecorinplaysaprotectiveroleinAAAdevelopment.However,inprogressedAAA
walls,decorinpositivelycorrelateswithMMP-9,suggestingthatdecorinplaysastimulatory
roleinAAAprogression.
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RoleofDecorininAAA
Fig4.ExpressionofdecorininhumanAAA.(A-B)Proteinsampleswereobtainedfromtheaorticwallsof
humanAAAspecimens(n=47)andnon-aneurysmalspecimens(Control,n=4).Representativeresultsfrom
westernblotdetectionofdecorinareshown(A)withthecorrespondingquantitativeanalysis(B).GAPDH
servedasaninternalcontrol.Dataaremean±SD.*p<0.05comparedtoControl.(C)Thecorrelation
betweentheproteinlevelsofdecorinandMMP-9wasexaminedandthequantitativeanalysisisshown
(Pearsonr=0.68,n=51,p<0.001).(D)ThecorrelationbetweentheproteinlevelsofdecorinandTGF-β
wasexaminedandthequantitativeanalysisisshown(Pearsonr=0.39,n=51,p<0.01).(E)Representative
histologicalandimmunohistochemicalstainsareshownforhumanAAAwallspecimens.Theluminalsurface
isorientedtowardthetopofeachpanel.HEandEVGstainsdepictcellnuclei(blue-black)andelastin
network(black),respectively.ThelocalizationofdecorinandMMP-9isindicatedbyredstaining.M:media,A:
adventitia.(F-G)Representativeimagesofimmunofluorescencestainingfordecorin(red)andCD68
(macrophagemarker,green,F)orα-smoothmuscleactin(α-SMA)(smoothmusclecellmarker,green,G).
Yellowinthemergedimagesindicatesoverlappinglocalizationoftheredandgreensignals.
doi:10.1371/journal.pone.0120689.g004
RoleofdecorininmacrophagesecretionofMMP-9
Tounderstandtheconflictingfindingsregardingtheroleofdecorininaorticwalls,weinvesti-
gatedwhetherdecorinpositivelyregulatesMMP-9secretioninmacrophages,themajorin-
flammatorycell-typesecretingMMP-9inAAAwalls[9,17](Fig.5A).Underbasalconditions,
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RoleofDecorininAAA
MMP-9wasnearlyundetectableintheconditionedmediafromculturedmousemacrophages.
InresponsetoLPStreatment,macrophagessecreteddetectablelevelsofMMP-9.Intriguingly,
pre-treatmentwithexogenousdecorinresultedinadose-dependentincreaseinLPS-induced
MMP-9secretion(Fig.5B-C).Inaddition,LPStreatmentincreasedmacrophagesecretionof
TGF-β,andpre-treatmentwithexogenousdecorinfurtherenhancedthissecretion(Fig.5D).
ThedatademonstratethatdecorinstimulatesmacrophagesecretionofMMP-9andinhibits
VSMCsecretionofMMP-9.Thus,ourfindingsrevealthatdecorinhasoppositeeffectson
MMP-9secretiondependingonthecelltype.
Discussion
Thisstudyisthefirsttodemonstratethatadventitialdecorinproteininitiallydecreasesina
CaCl -inducedAAAmousemodelandthatlocaladministrationofdecorincaninhibitthede-
2
velopmentofAAA.Thesefindingsindicatethatadeclineinadventitialdecorinmayresultin
theinitiationofAAA.Decorinexpressioninadventitialcellsmayindirectlyresultinprotection
oftheaorticmediaagainstinflammatoryinsultsbyunknownmechanisms.However,wepre-
sentedanotherpossibility;decorin,whichisreleasedfromtheadventitiaofnormalandnon-
aneurysmalaorticwalls,canreachthemedia,affectmedialVSMCs,anddirectlyprotectthe
mediafromproteolyticdegradation.Consistentwithourresults,reducedexpressionofdecorin
intheadventitiawasassociatedwithahighriskofaorticruptureinanothermousemodelof
AAAinducedbyangiotensinII[16].Amonghumananeurysmaldiseases,deficientdecorinex-
pressionhasbeenassociatedwithlethalformsofMarfan’ssyndrome[29]andaorticdissection
[30,31].
Decorinischaracterizedbyitsinteractionswithamultitudeofstructuralcomponentswith-
intheextracellularmatrix,particularlycollagenandelastinfibers,anditisimportantforthe
regulationofcollagenfibrillogenesis.Decorinmaybeassociatedwithelastinogenesis[14,32].
SimilartootherSLRPs,decorincanhelpprotectcollagenfibrilsfromcleavagebycollagenases
[33].Thedataindicatethatstabilizationofcollagenandelastinfibersbydecorinmaycontrib-
utetothepreventionoftissuedestructioninourAAAmodel.Anotherpossibilityisthatdec-
orininhibitsAAAdevelopmentbysuppressingtheexpressionofpro-inflammatorymolecules
andcellularinfiltration.Inthepresentstudy,weshowedthatlocaladministrationofdecorin
resultsinasignificantdecreaseininflammatorycellinfiltrationandMMP-9proteinlevels.We
alsodemonstratedthatdecorincannegativelyaffectMMP-9secretioninculturedVSMCs.Sys-
temicover-expressionofdecorinwaspreviouslyreportedtoreducemacrophageinfiltration
andgelatinaseactivityinamousemodelofatherosclerosis[34].Administrationofdecorinan-
tagonizedmultiplereceptortyrosinekinases(e.g.,Met),subsequentlyleadingtothedown-
regulationofMMP-9inthecontextoftumorangiogenesis[35].Inaddition,decorindeficiency
resultsintheup-regulationofMMP-9inmousefetalmembranesatembryonicday18[36].
Incontrast,decorinwasrecentlyshowntoinducepro-inflammatorysignaling,therebylink-
inginnateimmunity,inflammation,andtumorigenesis[15,37,38].Decorinwasreportedtoin-
teractwithtoll-likereceptor(TLR)2andTLR4andstimulatetheexpressionofcytokines,
primarilypro-inflammatorytumornecrosisfactor(TNF)-αandpro-interleukin(IL)-1β,byac-
tivatingnuclearfactor(NF)-κBinmacrophages.Decorinreportedlyaugmentstheexpression
ofMMP-9incancercelllines[39].DecorinhasalsobeenshowntoenhancetheeffectsofLPS,
amajorligandforTLR4,bysignalingthroughTLR2[38].Similarly,inthisstudy,wedemon-
stratedthatdecorinenhancesLPS-inducedMMP-9secretioninmacrophages.Inaddition,
decorinblockedthebindingofTGF-βtoitsreceptor,whichreducedtheproductionofanti-
inflammatorycytokineIL-10[15,38].Thus,thesedataindicatethatdecorinsignalingstimu-
latesinflammatoryresponsesundercertainconditions.Inamousemodelofcontactdermatitis,
PLOSONE|DOI:10.1371/journal.pone.0120689 March17,2015 10/15