Loss of Timp3 Gene Leads to Abdominal Aortic Aneurysm Formation in Response to Angiotensin II

15 Pages·2012·6.09 MB·English

THEJOURNALOFBIOLOGICALCHEMISTRYVOL.287,NO.53,pp.44083–44096,December28,2012
©2012byTheAmericanSocietyforBiochemistryandMolecularBiology,Inc. PublishedintheU.S.A.
Loss of Timp3 Gene Leads to Abdominal Aortic Aneurysm
Formation in Response to Angiotensin II*
Receivedforpublication,October5,2012,andinrevisedform,October30,2012Published,JBCPapersinPress,November9,2012,DOI10.1074/jbc.M112.425652
RatnadeepBasu‡§1,DongFan‡§,VijayKandalam‡§1,JiwonLee‡§,SubhashK.Das§¶,XiuhuaWang‡§,
TroyA.Baldwin(cid:1)2,GavinY.Oudit§¶3,andZamanehKassiri‡§4
FromtheDepartmentsof‡Physiologyand(cid:1)MedicalMicrobiologyandImmunologyand¶DepartmentofMedicine,Divisionof
Cardiology,UniversityofAlberta,Edmonton,AlbertaT6G2S2,Canadaand§CardiovascularResearchCenter,Mazankowski
AlbertaHeartInstitute,Edmonton,AlbertaT6G2S2,Canada
Background:TIMP3isECM-boundandisimplicatedinpatientswithabdominalaorticaneurysm(AAA).
Results:Timp3deficiencytriggersAAAinresponsetoAngII.AdditionaldeletionofMmp2exacerbatedAAAwithenhanced
inflammation.BroadspectrumMMPinhibitionpreventedAAAinbothgenotypes.
Conclusion:TIMP3isprotectiveagainstAngII-mediatedadverseremodeling.
Significance:ReplenishmentofTIMP3inaneurysmalaortacouldpreventaneurysmexpansionandrupture.
Aortic aneurysm is dilation of the aorta primarily due to Abdominalaorticaneurysm(AAA)5isadegenerativevascu-
degradation of the aortic wall extracellular matrix (ECM). lar disorder characterized by dilation of the aorta due to
Tissue inhibitors of metalloproteinases (TIMPs) inhibit destructiveremodelingoftheaorticwallanddegradationofthe
matrix metalloproteinases (MMPs), the proteases that fibrillar proteins of the vascular extracellular matrix (ECM).
degradetheECM.Timp3istheonlyECM-boundTimp,and AAA has remained an unresolved clinical problem because
itslevelsarealteredintheaortafrompatientswithabdomi- (cid:1)-blockers(1,2),statintherapy(3),andangiotensin-converting
nalaorticaneurysm(AAA).Weinvestigatedthecausalroleof enzyme inhibitors have been ineffective in controlling AAA
Timp3inAAAformation.InfusionofangiotensinII(AngII) expansion,andAAAremainsasthe13thleadingcauseofdeath
usingmicro-osmotic(Alzet)pumpsinTimp3(cid:1)/(cid:1)malemice, inWesterncountries(4–6).Surgicalandmechanicalinterven-
tionsaretheonlyeffectivetreatmentstopreventAAArupture,
butnotinwildtypecontrolmice,ledtoadverseremodelingof
butthatisonlyrecommendedinsevereAAAcaseswherethe
the abdominal aorta, reduced collagen and elastin proteins
inherentriskofsurgeryislessthantheriskofaorticrupture(1,
butnotmRNA,andelevatedproteolyticactivities,suggesting
6).The10-yearsurvivalforpatientsdeemedunfitforsurgical
excessproteindegradationwithin2weeksthatledtoforma-
repair is less than 25% (7). Additionally, the recent Tromsø
tionofAAAby4weeks.Intriguingly,despiteearlyup-regu-
study indicates that the incident of AAA is greater in males
lation of MMP2 in Timp3(cid:1)/(cid:1)Ang II aortas, additional dele-
comparedwithfemalepatients(8,9),andsimilarfindingshave
tion of Mmp2 in these mice (Timp3(cid:1)/(cid:1)/Mmp2(cid:1)/(cid:1)) resulted
beenreportedinanimalmodels(10).Assuch,understanding
inexacerbatedAAA,compromisedsurvivalduetoaorticrup-
the molecular mechanism underlying AAA development,
ture, and inflammation in the abdominal aorta. Reconstitu-
expansion,andruptureiscriticalindevelopingeffectivethera-
tion of WT bone marrow in Timp3(cid:1)/(cid:1)/Mmp2(cid:1)/(cid:1) mice
piesforthisdisease.
reduced inflammation and prevented AAA in these animals ArterialECMisprimarilycomposedofcollagenandelastin
followingAngIIinfusion.Treatmentwithabroadspectrum fibers that provide significant structural support and recoil
MMP inhibitor (PD166793) prevented the Ang II-induced propertiesforthearteries(11,12).Matrixmetalloproteinases
AAAinTimp3(cid:1)/(cid:1)andTimp3(cid:1)/(cid:1)/Mmp2(cid:1)/(cid:1)mice.Ourstudy (MMPs) degrade the ECM structural proteins, whereas their
demonstratesthattheregulatoryfunctionofTIMP3iscriti- inhibitors, tissue inhibitors of metalloproteinases (TIMPs),
cal in preventing adverse vascular remodeling and AAA. keep their activity in check. An imbalance in the interaction
Hence, replenishing TIMP3, a physiological inhibitor of a betweenMMPsandTIMPshasbeenreportedintheaortaof
number of metalloproteinases, could serve as a therapeutic patients with abdominal aneurysm (13). Among the four
approachinlimitingAAAdevelopmentorexpansion. TIMPsidentifiedinmammals,TIMP3isECM-boundwhereby
itcanexerttissue-specificeffects(14,15).TIMP3proteinlevels
arereducedintheaortaofpatientswithMarfansyndromewith
increasedrateofaorticrupture(16).Timp3mRNAlevelsare
increased in dilated aorta from patients with aortic aneu-
*ThisworkwassupportedbyCanadianInstituteofHealthResearchGrant
84279andaHeartandStrokeFoundationofCanadagrant(toZ.K.). rysm,whereasotherTIMPswerenotaltered(17),However,
1SupportedbyanAlbertaInnovates-HealthSolutions(AI-HS)studentship. TIMP3 protein levels were not measured in this study. In
2AnAI-HSscholar.
3AnAI-HSclinician-scientist.
4AnewinvestigatoroftheHeartandStrokeFoundationofCanadaandan 5Theabbreviationsusedare:AAA,abdominalaorticaneurysm;AngII,angio-
AI-HSscholar.Towhomcorrespondenceshouldbeaddressed:Dept.of tensinII;ECM,extracellularmatrix;GT,Gomoritrichrome;MMP,matrix
Physiology,UniversityofAlberta,474HMRC,Edmonton,AlbertaT6G2S2, metalloproteinase;TIMP,tissueinhibitorofmetalloproteinase;VVG,Ver-
Canada.Tel.:[phone];Fax:[phone];E-mail:[email protected]. hoff-VanGieson;MMPi,MMPinhibitor;BM,bonemarrow.
This is an open access article under the CC BY license.
DECEMBER28,2012•VOLUME287•NUMBER53 JOURNALOFBIOLOGICALCHEMISTRY 44083
TIMP3andAbdominalAorticAneurysm
addition, a significant interaction between polymorphisms expansion index ((Systolic aortic diameter (cid:1) Diastolic aortic
ofTimp3,butnot Timp1orTimp2,occursinpatientswith diameter)/Systolic diameter (cid:2) 100) (29). Three independent
AAAandwithapositivefamilyhistoryofAAA(18).Hence, measurementsweremadeforeachmouse.
thecausalroleofTIMP3inAAAdevelopmentremainstobe Histological Analyses and Scanning Electron Microscopy—
determined. Abdominalaortaswerefixedin10%formalin,paraffin-embed-
OurstudyexaminedtheroleofTimp3inangiotensinII(Ang ded, and used for Gomori trichrome (GT) and Verhoeff-Van
II)-induced vascular remodeling and formation of AAA. We Gieson (VVG) staining. Tissue processing and scanning elec-
foundthatAngIIinfusionledtoAAAinmicelackingTimp3 tron microscopy were done as before (25). Neutrophil and
butnotinWTmice.AlthoughdeletionofMmp2inTimp3(cid:1)/(cid:1) macrophage staining was performed as described previously
mice resulted in heightened inflammation and more severe (30) using anti-mouse neutrophil antibody with Cy3-labeled
AAA, broad spectrum inhibition of MMPs prevented AAA anti-rat secondary antibody and F4/80 macrophage antibody
formation. withCy3-labeledanti-ratsecondaryantibodythatwaspseudo-
coloredtoappeargreenforvisualdifferentiationfromneutro-
EXPERIMENTALPROCEDURES
philstaining.WeusednuclearstainingbyDAPItoconfirmthe
Experimental Animals and Procedures—Wild type (WT) presence of infiltrating cells in positively stained regions.
micewerepurchasedfromTheJacksonLaboratory.Wecross- Superimposed staining for neutrophils or macrophages with
bred Timp3(cid:1)/(cid:1) (19) and Mmp2(cid:1)/(cid:1) (20) mice to generate DAPIispresented.
Timp3(cid:1)/(cid:1)/Mmp2(cid:1)/(cid:1) mice. Timp3(cid:1)/(cid:1)/Mmp2(cid:1)/(cid:1) mice are ProteinExtraction,WesternBlotAnalysis,GelatinZymogra-
healthyandfertile.AllmiceareinaC57BL/6background.Alzet phy, and mRNA Analysis—Flash frozen abdominal aorta was
micro-osmotic pumps (Model 1002, Durect Corp.) were usedforproteinorRNAextraction.Westernblottingwasper-
implantedsubcutaneouslyinmalemiceoftheindicatedgen- formed to detect collagen type I (COL1A1) (Santa Cruz Bio-
otypes to deliver 1.5 mg/kg/day Ang II (21–23) or saline technology) and (cid:3)-elastin (Abcam Inc.), and in vitro gelatin
(control). Broad spectrum MMP inhibitor (MMPi; zymographywascarriedoutasbefore(30).ACoomassieBlue-
PD166793) was administered orally by daily gavage (30 stainedSDS-PAGEgel(afterproteinsweretransferredtoPVDF
mg/kg/day) as before (24, 25). All experiments were con- membrane)wasusedasaloadingcontrol.Insitugelatinzymog-
ductedinaccordancewiththeguidelinesoftheUniversityof raphywasperformedon14-(cid:2)mcryosectionsasdescribed(31).
AlbertaAnimalCareandUseCommitteeandtheCanadian Totalcollagenaseandgelatinaseactivitiesweremeasuredusing
CouncilofAnimalCare. Enzchek collagenase (catalog number D-12060) and elastase
BoneMarrowReconstitution—Bonemarrow(BM)reconsti- (catalognumberE-12056)activityassaykits(fromMillipore)as
tutionwasperformedtogeneratechimericmiceasdescribed before(30).Briefly,50(cid:2)gofproteinextractwasincubatedwith
previously (26). Donor WT and Timp3(cid:1)/(cid:1)/Mmp2(cid:1)/(cid:1) mice fluorescentlylabeledcollagenorelastinsubstrate.Thegener-
received 100 (cid:2)g of purified anti-CD8 antibody (clone 2.43) atedfluorescencesignalwasrecordedfor4h(excitation,485
intraperitoneally on days (cid:1)2 and (cid:1)1 before BM harvest to nm;emission,515nm),andtheslopesofthecurveswereused
depleteCD8T-cells.BMwasharvestedfromthefemur,tibia, to calculate the collagenase or elastase activities. mRNA
andhumerusinEasySepmedium(PBS,2%FCS,2mMEDTA) expressionwasmeasuredusingTaqManrealtimePCR(Table
andpassedthrough70-(cid:2)mnyloncellstrainers(Fisherbrand). 1)(30,32).
RecipientWTandTimp3(cid:1)/(cid:1)/Mmp2(cid:1)/(cid:1)miceweresublethally StatisticalAnalyses—Thestatisticalanalyseswereperformed
irradiated(1,000grays)andreceived10(cid:2)106BMcellsofthe usingSPSSsoftware(Version10.1;Chicago,IL).Weperformed
oppositegenotypeviatailveininjection.WTBMwasreconsti- normality (Shapiro-Wilk) and homogeneity of variance tests
tuted in Timp3(cid:1)/(cid:1)/Mmp2(cid:1)/(cid:1) mice (Timp3(cid:1)/(cid:1)/Mmp2(cid:1)/(cid:1)- (Levene)toconfirmnormaldistributionofalldata.Tocompare
chimera mice), and Timp3(cid:1)/(cid:1)/Mmp2(cid:1)/(cid:1) BM was reconsti- data sets with two factors (genotype and treatment), we per-
tuted in WT mice (WT-chimera mice). Mice were provided formedtwo-wayanalysisofvariance(Figs.1–6).InFigs.5–8,to
withantibioticwater(40mgofneomycinand15mgofpoly- comparethreelevelsofthegenotype,two-wayanalysisofvari-
myxin/1 liter) for 4 weeks postinjection, and 7 weeks were ancewasfollowedbyBonferroniposthoctestingiftheresults
allowedforreconstitutionbeforeAlzetpumpswereimplanted attainedstatisticalsignificance(p(cid:3)0.05).InFig.7B,theeffects
forAngIIinfusion. ofMMPiineachgenotypeweretestedusinganunpairedttest.
UltrasonicImagingofAbdominalAorta—Ultrasonicimages Survivaldata(Fig.4B)werecomparedusingKaplan-Meiersur-
oftheabdominalaortawereobtainedinmiceanesthetizedwith vival curves followed by the log rank test adjusted with the
isoflurane using a Vevo 770 high resolution imaging system Bonferroni method. Averaged values represent mean (cid:4) S.E.
equippedwitharealtimemicrovisualizationscanhead(RMV Statisticalsignificancewasrecognizedatp(cid:3)0.05.
704,VisualSonics,Toronto,Canada)(27).Theaorticdiameters
RESULTS
were measured by M-mode at the suprarenal region where
aneurysm was detected (in Timp3(cid:1)/(cid:1) and Timp3(cid:1)/(cid:1)/ Timp3-deficient Mice Exhibit Adverse Aortic Remodeling
Mmp2(cid:1)/(cid:1) mice) (28). The maximum aortic lumen diameter after2WeeksofAngiotensinIIInfusion—InWTmice,2weeks
(aorticsystolicdiametercorrespondingtocardiacsystole)and of Ang II infusion significantly elevated Timp3 mRNA and
theminimumaorticlumendiameter(aorticdiastolicdiameter TIMP3proteinlevelsintheabdominalaorta(Fig.1A,panelsi
correspondingtocardiacdiastole)monitoredbysimultaneous andii).ToexaminetheroleofTIMP3inAngII-inducedaortic
ECGrecordingsweremeasuredandusedtocalculatetheaortic remodeling,wesubjectedmicelackingTimp3toAngIIinfu-
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TIMP3andAbdominalAorticAneurysm
TABLE1
TaqManprimerandprobesequencesfortheindicatedgenes
Hprt,hypoxanthinephosphoribosyltransferase;Mcp-1,monocytechemotacticprotein-1.Theprimer/probemixtureforelastinwaspurchasedfromAppliedBiosystems
(Mm00514670_m1).TAMRA,tetramethylrhodamine;FAM,6-carboxyfluorescein.
Gene Primer/probe Sequence
ProcollagenI-(cid:3)1 Forward 5(cid:5)-CTTCACCTACAGCACCCTTGTG-3(cid:5)
Reverse 5(cid:5)-TGACTGTCTTGCCCCAAGTTC-3(cid:5)
Probe 5(cid:5)-FAM-CTGCACGAGTCACACC-TAMRA-3(cid:5)
Mmp2 Forward 5(cid:5)-AACTACGATGATGACCGGAAGTG-3(cid:5)
Reverse 5(cid:5)-TGGCATGGCCGAACTCA-3(cid:5)
Probe 5(cid:5)-FAM-TCTGTCCTGACCAAGGATATAGCCTATTCCTCG-TAMRA-3(cid:5)
Mmp9 Forward 5(cid:5)-CGAACTTCGACACTGACAAGAAGT-3(cid:5)
Reverse 5(cid:5)-GCACGCTGGAATGATCTAAGC-3(cid:5)
Probe 5(cid:5)-FAM-TCTGTCCAGACCAAGGGTACAGCCTGTTC-TAMRA-3(cid:5)
IL-1(cid:1) Forward 5(cid:5)-AACCTGCTGGTGTGTGACGTTC-3(cid:5)
Reverse 5(cid:5)-CAGCACGAGGCTTTTTTGTTGT-3(cid:5)
Probe 5(cid:5)-FAM-TTAGACAGCTGCACTACAGGCTCCGAGATG-TAMRA-3(cid:5)
IL-6 Forward 5(cid:5)-ACAACCACGGCCTTCCCTACTT-3(cid:5)
Reverse 5(cid:5)-CACGATTTCCCAGAGAACATGTG-3(cid:5)
Probe 5(cid:5)-FAM-TTCACAGAGGATACCACTCCCAACAGACCT-TAMRA-3(cid:5)
Mcp-1 Forward 5(cid:5)-GTTGGCTCAGCCAGATGCA-3(cid:5)
Reverse 5(cid:5)-AGCCTACTCATTGGGATCATCTTG-3(cid:5)
Probe 5(cid:5)-FAM-TTAACGCCCCACTCACCTGCTGCTACT-TAMRA-3(cid:5)
Hprt Forward 5(cid:5)-AGCTTGCTGGTGAAAAGGAC-3(cid:5)
Reverse 5(cid:5)-CAACTTGCGCTCATCTTAGG-3(cid:5)
Probe 5(cid:5)-FAM-CAACAAAGTCTGGCCTGTATCCAAC-TAMRA-3(cid:5)
sion,andweassessedthepredominantarterialstructuralpro- Timp3-deficientMiceDevelopedAAAafter4WeeksofAngII
teins, elastin and fibrillar collagen type I (12). Verhoeff-Van Infusion—Next,weexaminedthelongtermoutcomeofthese
Gieson staining showed disorganized and disrupted elastin structuralremodelingintheTimp3(cid:1)/(cid:1)aorta.After4weeksof
fiberswithagreaterfrequencyofelastinfiberdisruptioninthe AngIIinfusion,Timp3(cid:1)/(cid:1)mice,butnotWTmice,developed
abdominalaortaofTimp3(cid:1)/(cid:1)-AngIIcomparedwithWT-Ang ananeurysmintheabdominalaortaandnotintheascendingor
IImice(Fig.1,BandC).TheAngII-inducedreductioninelas- descendingthoracicaorta(Fig.3A).Aneurysmisdefinedasa
tinproteincontentwasgreaterinTimp3(cid:1)/(cid:1)abdominalaorta dilation of 50% or more in the abdominal aortic diameter (1,
(Fig. 1D, panels i and ii) despite a larger increase in mRNA 35), measured by ultrasound imaging. In WT mice, Ang II-
synthesis of elastin in these mice (Fig. 1D, panel iii). Gomori induceddilationoftheabdominalaortadidnotexceed20%of
trichrome staining similarly revealed disorganized collagen the original aortic diameter, whereas 60% of Timp3(cid:1)/(cid:1) mice
structures(Fig.1E,greenishblue)andadrasticdecreaseincol- exhibited greater than 50% aortic dilation in the suprarenal
lagen type I protein levels (Fig. 1F, panels i and ii) despite a region.Representativeultrasonicimages(Fig.3B)andaveraged
marked rise in its mRNA synthesis (Fig. 1F, panel iii) in diameteroftheabdominalaorta(Fig.3C,panelsiandii)dem-
Timp3(cid:1)/(cid:1)-AngIIcomparedwithWT-AngIIabdominalaor- onstratemarkedaorticdilationatthesuprarenalregioninAng
tas.Thesedatasuggestthatthedecreaseinelastinandcollagen II-infused Timp3(cid:1)/(cid:1) compared with the parallel WT group.
proteinlevelsinTimp3-deficientaortaisduetopost-transla- Theaorticsystolicexpansionindex,ameasureofaorticelastic-
tionaldegradationoftheseproteinsandnotreducedsynthesis. ity and recoil property during systole and diastole (29), was
EnhancedMMP2ActivationinTimp3(cid:1)/(cid:1)Micefollowing2 significantlysuppressedintheaneurysmalaortainTimp3(cid:1)/(cid:1)-
WeeksofAngiotensinIIInfusion—TIMP3isapotentinhibitor AngIImice(Fig.3C,paneliii),confirmingthecompromised
ofanumberofactiveMMPsandcaninhibitcellsurfaceactiva- structuralandfunctionalintegrityoftheTimp3-deficientaorta.
tionofMMP2(33);hence,itsabsencecanleadtouncontrolled Deletion of Mmp2 in Timp3(cid:1)/(cid:1) Mice Led to Exacerbated
proteolytic activities (34). In situ gelatin zymography showed AAA—We observed that after 2 weeks of Ang II infusion
thatAngIIinfusionresultedinastrongergelatinaseactivityin MMP9 levels increased similarly in both genotypes, whereas
Timp3(cid:1)/(cid:1) compared with WT aorta (Fig. 2A). To determine theincreaseinMMP2levelswasgreaterintheTimp3(cid:1)/(cid:1)com-
thecontributionofindividualgelatinases,namelyMMP2and pared with WT abdominal aorta (Fig. 2B). To determine the
MMP9, we performed in vitro gelatin zymography, which contributionoftheelevatedMMP2activationindevelopment
revealed a markedly greater MMP2 activation in Timp3(cid:1)/(cid:1)- of AAA in Timp3(cid:1)/(cid:1)-Ang II mice, we generated Timp3(cid:1)/(cid:1)/
AngIIabdominalaortaasindicatedbyastronger64-kDaband, Mmp2(cid:1)/(cid:1)doubledeficientmice.Saline-infusedTimp3(cid:1)/(cid:1)and
whereasMMP9levelsrosesimilarlyinbothgenotypes(Fig.2B, Timp3(cid:1)/(cid:1)/Mmp2(cid:1)/(cid:1) mice showed a similar aortic structure
panelsiandii).TheincreaseinMMP2activationwasalsoasso- and expansion index compared with WT mice (data not
ciated with increased mRNA expression (Fig. 2C, panel i), shown). Intriguingly, Timp3(cid:1)/(cid:1)/Mmp2(cid:1)/(cid:1) mice exhibited
whereas Mmp9 mRNA did not increase (Fig. 2C, panel ii). more severe AAA after 4 weeks of Ang II infusion compared
ExpressionanalysisofotherMmpsshowedagreaterincreasein with Timp3(cid:1)/(cid:1) mice (Fig. 4A) as dilation of the abdominal
Mmp13levelsintheTimp3(cid:1)/(cid:1)-AngIIgroup(Fig.2C,paneliii), aorta exceeded 50% of the original aortic diameter in 75% of
whereasmembranetypeMmp(MT1-MMP)waselevatedsim- these mice. In addition, whereas Timp3(cid:1)/(cid:1) mice exhibited
ilarlyinbothgenotypes(Fig.2C,paneliv). compromised survival post-Ang II infusion, mortality was
DECEMBER28,2012•VOLUME287•NUMBER53 JOURNALOFBIOLOGICALCHEMISTRY 44085
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increasedfurtherinTimp3(cid:1)/(cid:1)/Mmp2(cid:1)/(cid:1)mice(Fig.4B,panel wasdetected(Fig.4B,panelii)wasthecauseoftheincreased
i).Routineautopsyexaminationrevealedthataorticruptureat mortality in these mice. Morphometric analysis of the full
the suprarenal region of the aorta where aneurysmal dilation cross-section of abdominal aorta in saline-infused WT com-
44086 JOURNALOFBIOLOGICALCHEMISTRY VOLUME287•NUMBER53•DECEMBER28,2012
TIMP3andAbdominalAorticAneurysm
FIGURE2.ElevatedproteaseactivityinTimp3(cid:1)/(cid:1)abdominalaortaafter2weeksofAngIIinfusion.A,insitugelatinzymographyinaortafromsaline-or
AngII-infusedWTandTimp3(cid:1)/(cid:1)mice.B,invitrogelatinzymography(paneli)andbandintensityforMMP9,pro-MMP2,andactiveMMP2(panelii).C,mRNA
levelsofMmp2(paneli),Mmp9(panelii),Mmp13(paneliii),andmembranetypeMmp(MT1-MMP)(paneliv)inabdominalaortaofsaline-orAngII-infusedWT
andTimp3(cid:1)/(cid:1)mice.ACoomassieBlue-stainedgelwasusedastheloadingcontrol.n(cid:6)5/group/genotype.A.U.,arbitraryunits;R.E,relativeexpression.*,p(cid:3)
0.05forthemaineffect;#,p(cid:3)0.05fortheinteraction.Averageddatarepresentmean(cid:4)S.E(errorbars).(cid:7)vecon,positivecontrol.
pared with Ang II-infused WT, Timp3(cid:1)/(cid:1), and Timp3(cid:1)/(cid:1)/ abdominalaorticwalls.AngIIinfusionledtoremodelingof
Mmp2(cid:1)/(cid:1)miceclearlyshowedthedilationinabdominalaorta theaorticwallinWTmiceasindicatedbycompactlyfolded
in Timp3(cid:1)/(cid:1) mice and a strikingly greater dilation in the elastinlamellae(Fig.4D,paneli)thatweremoreevidentata
Timp3(cid:1)/(cid:1)/Mmp2(cid:1)/(cid:1) mice (Fig. 4C). Histological analyses at higher magnification (Fig. 4D, panel ii). Timp3-deficient
highermagnificationsfurtherrevealeddeteriorationoftheaor- mice on the other hand exhibited disrupted medial elastic
ticwallatthesiteofaneurysmasindicatedbydisruptedelastin lamellae and disrupted fibrillar structures (Fig. 4D, panel i,
fibers(VVGstaining)andcollagenstructure(GTstaining)that openarrows),andthisstructuraldeteriorationwasworsened
weremoresevereinTimp3(cid:1)/(cid:1)/Mmp2(cid:1)/(cid:1)mice(Fig.4C).Aor- in Timp3(cid:1)/(cid:1)/Mmp2(cid:1)/(cid:1)-Ang II mice (Fig. 4D, panels i and
ticwallstructuresinsaline-infusedmiceofthethreegenotypes ii). The structure and diameter of the abdominal aorta in
werecomparable(datanotshown). saline-infused mice were comparable among the genotypes
Scanning electron microscopy allowed us to perform a (data not shown). These findings demonstrate that despite
moredetailedevaluationofthestructuralremodelingofthe themarkedincreaseinMMP2activationinTimp3(cid:1)/(cid:1)aortas
FIGURE1.AdverseremodelingofECMstructureinTimp3(cid:1)/(cid:1)abdominalaortafollowing2weeksofAngIIinfusion.A,mRNAexpression(paneli)and
representativeWesternblotandaveragedproteinlevels(panelii)ofTIMP3intheabdominalaortafromWTmiceafter2weeksofAngIIorsalineinfusion.B–D,
Verhoeff-VanGiesonstaining(B),numberofelastinbreaksperfieldunder40(cid:2)magnification(C),representativeWesternblotsandaveragedelastinprotein
levels(D,panelsiandii),andelastinmRNAlevels(D,paneliii)inabdominalaortafromsaline-andAngII-infusedWTandTimp3(cid:1)/(cid:1)mice.EandF,collagen
stainingbyGomoritrichrome(E),representativeWesternblotandaveragedcollagenproteincontent(F,panelsiandii),andmRNAexpressionlevels(F,panel
iii)insaline-andAngII-infusedWTandTimp3(cid:1)/(cid:1)mice.CoomassieBlue-stainedgelswereusedastheloadingcontrolfortheWesternblots.A.U.,arbitraryunits;
R.E,relativeexpression.n(cid:6)5/group(protein)and5/group(mRNA).*,p(cid:3)0.05comparedwithsaline;#,p(cid:3)0.05forthemaineffect;flower,p(cid:3)0.05forthe
interaction.Arrowspointtodisruptedcollagenorelastinstructure.Averageddatarepresentmean(cid:4)S.E(errorbars).
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FIGURE3.Timp3-deficientmicedevelopedAAAafter4weeksofAngIIinfusion.A,representativephotographsoftheentireaortashowingmacroscopic
featuresofaneurysminducedbyAngIIinTimp3(cid:1)/(cid:1)mice.Enlargedpicturesoftheaneurysmalaortaareshownintheinset.B,ultrasonographicB-mode(panel
i)andM-mode(panelii)imagesoftheabdominalaortainsaline-andAngII-infusedWTandTimp3(cid:1)/(cid:1)mice.“K”indicatesthetopoftheleftkidneyasreference.
Theredlinesshowwheremeasurementsofaorticdiameterwereobtained(suprarenal),andthewhitelineshighlighttheaorticdiameterineachgroup.
C,averagedaorticsystolic(paneli)anddiastolic(panelii)diametersandaorticsystolicexpansionindex(paneliii)ofabdominalaortainsaline-orAngII-infused
WTandTimp3(cid:1)/(cid:1)mice.Arrowpointstotheaneurysmalregion.n(cid:6)9/group/genotype.*,p(cid:3)0.05forthemaineffect;#,p(cid:3)0.05fortheinteractions.Averaged
datarepresentmean(cid:4)S.E(errorbars).
earlyinthepost-AngIIinfusionperiod,itsdeletionindeed greater in Timp3(cid:1)/(cid:1)/Mmp2(cid:1)/(cid:1) compared with Timp3(cid:1)/(cid:1)
aggravated the structural deterioration and the adverse abdominalaortadespitetheabsenceofMMP2(Fig.5A,panel
remodeling in the abdominal aorta resulting in worsened ii).MMP2andMMP9aretheclassicallyknowngelatinases,and
AAA. as such, the rise in total gelatinase activity in the absence of
Timp3(cid:1)/(cid:1)/Mmp2(cid:1)/(cid:1)-Ang II Mice Exhibited Heightened MMP2impliedanincreaseinMMP9activity.Invitrogelatin
Inflammation in Abdominal Aorta—Further examination of zymographyconfirmedastrikingup-regulationinMMP9lev-
the Timp3(cid:1)/(cid:1)/Mmp2(cid:1)/(cid:1) mice revealed that Ang II-induced els in Ang II-infused Timp3(cid:1)/(cid:1)/Mmp2(cid:1)/(cid:1) abdominal aortas
totalcollagenaseactivitywasthelargestintheabdominalaorta comparedwithallothergroups(Fig.5B,panelsiandii).This
of Timp3(cid:1)/(cid:1) mice and was reduced significantly in the wasalsoaccompaniedbyelevatedmRNAexpressionofMmp9
Timp3(cid:1)/(cid:1)/Mmp2(cid:1)/(cid:1)group(Fig.5A,paneli).Thisisconsis- (Fig. 5B, panel iii). The increase in MMP9 levels in WT and
tentwithMMP2alsobeingapotentcollagenase(36).However, Timp3(cid:1)/(cid:1)aortasafter2weeksofAngIIinfusion(Fig.2B)was
the Ang II-induced rise in gelatinase activity was markedly dissipated by 4 weeks. This could perhaps be due to an early
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FIGURE4.MMP2deletioninTimp3-deficientmiceledtoexacerbatedAAAafter4weeksofAngIIinfusion.A,photographsoffullaortasfromAngII-infused
Timp3(cid:1)/(cid:1)andTimp3(cid:1)/(cid:1)/Mmp2(cid:1)/(cid:1)mice.Arrowspointtotheaneurysmalarea.n(cid:6)20/WT,n(cid:6)30/Timp3(cid:1)/(cid:1),andn(cid:6)30/Timp3(cid:1)/(cid:1)/Mmp2(cid:1)/(cid:1).B,paneli,survivalcurve
reflectingmortalitiesduetoaorticrupture.Panelii,representativeimageofanaorticruptureatthesuprarenalregion(arrow).Thekidneyswereleftinplaceasapoint
ofreference.*,p(cid:3)0.05comparedwithWT-AngII.C,microscopicimagesoffullcross-sectionsofabdominalaorta(scalebar,100mm;notethedifferentscalebarsize
inthedoubledeficientgroup)andVVG-andGT-stainedimagesatahighermagnification.Arrowspointtoareasofstructuraldegradation.*indicatestheareaof
aneurysm.D,scanningelectronmicroscopyofabdominalaortainsaline-andAngII-infusedmiceofdifferentgenotypesattwodifferentmagnifications.Openarrows
pointtothedisruptedelastinstructures(D,paneli);solidarrowspointtothemagnifiedelastinlamellastructures(D,panelii).
inflammatoryresponsethatoccurredirrespectiveofthegeno- ofluorescent staining for inflammatory cells, neutrophils and
type.MMP9isawellknownelastasethatcandegradevascular macrophages (superimposed with DAPI nucleus staining),
elastin lamella (37), leading to invasion of inflammatory cells showedenhancedinfiltrationofneutrophilsandmacrophages
andinflammation(38)andexacerbationofAAA(22).Immun- intheabdominalaortaofAngII-infusedTimp3(cid:1)/(cid:1)/Mmp2(cid:1)/(cid:1)
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FIGURE5.AneurysmalabdominalaortafromTimp3(cid:1)/(cid:1)/Mmp2(cid:1)/(cid:1)miceexhibitselevatedMmp9levelsandinflammation.A,totalcollagenase(paneli)
andgelatinase(panelii)activitiesafter4weeksofsalineorAngIIinfusionintheindicatedgenotypes(normalizedtoWT-salinegroup).n(cid:6)6/group/genotype.
B,representativeinvitrogelatinzymography(paneli)andaveragedbandintensitiesforMMP9,pro-andMMP2(panelsiandii),andMmp9mRNAexpression
levels(paneliii)inallgroups.ACoomassieBlue-stainedgelwasusedastheproteinloadingcontrol.n(cid:6)5/group/genotype.C,immunofluorescentstainingfor
neutrophil(paneli;red)andmacrophage(panelii;green)superimposedwithDAPInucleusstaining(blue)inabdominalaortaoftheindicatedgroups.,“M”
indicatesthemedialthickness.Arrowspointtopositivelystainedneutrophilsormacrophages.D,mRNAexpressionlevelsofinflammatorymarkersIL-1(cid:1),IL-6,
andmonocytechemotacticprotein-1(Mcp-1)intheabdominalaortaoftheindicatedgroupsafter4weeksofsalineorAngIIinfusion.n(cid:6)6/group/genotype.
A.U.,arbitraryunits;R.E.,relativeexpression;N.D.,notdetectable.*,p(cid:3)0.05comparedwithsaline;flower,p(cid:3)0.05comparedwithotherAngII-infusedgroups.
Averageddatarepresentmean(cid:4)S.E(errorbars).
mice (Fig. 5C). The Timp3(cid:1)/(cid:1)-Ang II group showed a less (IL-1(cid:1)),IL-6,andmonocytechemotacticprotein-1weresignif-
severe macrophage infiltration, whereas no infiltrating cells icantly elevated in the aneurysmal aorta of Timp3(cid:1)/(cid:1)/
weredetectedinWT-AngIIabdominalaorta(Fig.5C).Consis- Mmp2(cid:1)/(cid:1) mice compared with parallel WT and Timp3(cid:1)/(cid:1)
tently, mRNA levels of inflammatory markers interleukin-1(cid:1) groups(Fig.5D,panelsi–iii).
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Inflammation Is a Key Factor in Exacerbated AAA in tivelywithimagingandcontrollingofriskfactorssuchassmok-
Timp3(cid:1)/(cid:1)/Mmp2(cid:1)/(cid:1)-AngIIMice—Todeterminethemecha- ingandhypertension,patientscontinuetoexperiencesignifi-
nismunderlyingtheworseningofAAAinTimp3(cid:1)/(cid:1)/Mmp2(cid:1)/(cid:1)- cantmorbidityandmortalityfromrupturedaneurysms(4,6).
AngIImiceandthecontributionoftheobservedheightened DevelopmentandexpansionofAAAresultfromdisruptionof
inflammationintheseaortas,wereconstitutedWTbonemar- the orderly structure of the aortic wall and ECM. Elastin and
row in Timp3(cid:1)/(cid:1)/Mmp2(cid:1)/(cid:1) mice and vice versa. We gener- collagen fibers, which are the main structural proteins of the
ated Timp3(cid:1)/(cid:1)/Mmp2(cid:1)/(cid:1)-chimera mice by reconstituting aorticECM,underlietherecoilpropertiesandimpartstrength
WT bone marrow in these mice and WT-chimera mice by tothevesselwall,respectively(11,12).TIMP3istheonlyECM-
reconstituting Timp3(cid:1)/(cid:1)/Mmp2(cid:1)/(cid:1) bone marrow in WT boundTIMP,anditsalteredlevelshavebeenlinkedtoaortic
mice. After 7 weeks of reconstitution followed by 4 weeks of rupture(16)andaorticaneurysm(17),anditspolymorphism
AngIIinfusion,wefoundthatTimp3(cid:1)/(cid:1)/Mmp2(cid:1)/(cid:1)-chimera showedstrongassociationwithAAAinpatientswithafamily
micedidnotdevelopaorticaneurysm(Fig.6A)asthediameter historyofAAA(18).Inthisstudy,weprovideevidenceforthe
ofabdominalaorta(Fig.6B,panelsiandii)andaorticexpan- causalroleofTimp3deficiencyinthedevelopmentofAAAby
sion index (Fig. 6B, panel iii) in these mice were comparable demonstratingthatmicelackingTimp3aremoresusceptibleto
withsaline-infusedanimals.Consistently,minimalinfiltration AngII-inducedAAA.WefoundariseinTIMP3levelsinthe
of neutrophils and macrophages was detected (Fig. 6C, panel abdominalaortaofWTmicefollowingAngIIinfusion.Thisis
ii),whereasMMP9levelsweresignificantlyreduced(Fig.6D)in consistent with the finding in patients with aortic aneurysm
theTimp3(cid:1)/(cid:1)/Mmp2(cid:1)/(cid:1)-chimera-AngIImice.WT-chimera that the increase in Timp3 mRNA was a compensatory
miceexhibitedneutrophilandmacrophageinfiltrationthatwas responsetotheaugmentedMMPactivity(17).
notdetectedinAngII-inducedintactWTmice(Fig.6C,panel We used the Ang II-infused model of aortic aneurysm for-
i).InvitrogelatinzymographyshowedincreasedMMP9anda mation in the absence of dyslipidemia and/or metabolic syn-
strikingincreaseinMMP2levelsintheWT-chimeramice(Fig. drome(40).AngIIisaphysiologicalhormonethatiselevatedin
6D).Thesedataindicatethatinflammationwastheunderlying patients with cardiovascular diseases (41–43) and has been
cause of exacerbated AAA in Timp3(cid:1)/(cid:1)/Mmp2(cid:1)/(cid:1)-Ang II showntoexertdirecteffectsonvascularremodelingandfunc-
mice. tioninnumerousstudies(22,44,45).TheAngIIinfusionmodel
MultipleMMPInhibitionPreventedAAAinTimp3(cid:1)/(cid:1)and allowedustoexaminetheroleofTIMP3intheadverseremod-
Timp3(cid:1)/(cid:1)/Mmp2(cid:1)/(cid:1)Mice—Tofurtherinvestigatethecontri- elingoftheaorticwallleadingtoaorticaneurysmformation.In
butionoftheelevatedproteaseactivityastheunderlyingmech- thisstudy,wereportthefollowing.First,theregulatoryfunc-
anismforAAAinTimp3(cid:1)/(cid:1)andTimp3(cid:1)/(cid:1)/Mmp2(cid:1)/(cid:1)mice, tion of TIMP3 is essential in preventing AAA development.
wetreatedthesemicewithabroadspectrumMMPi,PD166793 Second,despitethegreaterearlyriseinMMP2activationinthe
(24, 39), during the course of Ang II infusion. Interestingly, Timp3(cid:1)/(cid:1)abdominalaorta,specificdeletionofMMP2ledto
simultaneousinhibitionofanumberofMMPswiththisinhib- inflammation, elevated MMP9 levels, and exacerbated AAA.
itorblockedAAAdevelopmentinTimp3(cid:1)/(cid:1)andsignificantly Third,WTbonemarrowreconstitutionpreventedinflamma-
suppressedAAAformationinTimp3(cid:1)/(cid:1)/Mmp2(cid:1)/(cid:1)mice(Fig. tion, MMP9 up-regulation, and AAA in the double deficient
7A). Ultrasonic measurement of abdominal aortic diameter mice. Fourth, simultaneous inhibition of a number of ECM-
showedthatMMPitreatmentwasstronglyeffectiveinprevent- degrading proteinases (MMPs) could be a more effective
ing the Ang II-induced dilation of the abdominal aorta in approachintreatingAAA.
Timp3(cid:1)/(cid:1) and Timp3(cid:1)/(cid:1)/Mmp2(cid:1)/(cid:1) mice (Fig. 7B, panels i Thesaline-infusedTimp3(cid:1)/(cid:1)miceshowedMMPactivities
andii),andtheaorticrecoilpropertyasmeasuredbysystolic andaorticstructurecomparablewithWT-salinegroupanddid
expansion index was significantly restored in MMPi-treated notdevelopAAA,suggestingthatundernormalconditionsthe
Timp3(cid:1)/(cid:1)-Ang II mice and to a lesser extent in Timp3(cid:1)/(cid:1)/ remainingTIMPs(-1,-2,and-4)aresufficienttokeeptheactiv-
Mmp2(cid:1)/(cid:1)-AngIImice(Fig.7B,paneliii).Theseimprovements ityofMMPsundercontrolintheabsenceofTIMP3.However,
were well reflected in the abdominal aortic wall structures as in the presence of a pathological stimulus such as Ang II,
shown by intact elastin (VVG) and collagen (GT) structures TIMP3iscriticalinregulatingtheproteolyticactivitiesforopti-
(Fig.8A)andpreservedelastinlamellaorganizationasseenin malremodelingintheabdominalaorticwall.TIMP3hasbeen
the scanning electron microscopy images (Fig. 8B). Consis- reportedtohindertheactivationofpro-MMP2(72kDa)intoits
tently,MMPitreatmentpreventedtheelevationofcollagenase cleaved(64-kDa)form(33),andconsistently,wefoundthatthe
and gelatinase activities in the abdominal aorta of Ang II- absenceofTIMP3promotedactivationofpro-MMP2intoits
infused Timp3(cid:1)/(cid:1) and Timp3(cid:1)/(cid:1)/Mmp2(cid:1)/(cid:1) mice (Fig. 8C). 64-kDa form in the Timp3(cid:1)/(cid:1)-Ang II aortas. However,
These data demonstrate the protective function of MMPi Timp3(cid:1)/(cid:1)/Mmp2(cid:1)/(cid:1) mice revealed that MMP2 activation is
againstAngII-inducedaorticwalldeteriorationthatother- not the deriving factor in AAA formation in these mice. The
wise leads to AAA in Timp3(cid:1)/(cid:1) and Timp3(cid:1)/(cid:1)/Mmp2(cid:1)/(cid:1) moresevereAAAinTimp3(cid:1)/(cid:1)/Mmp2(cid:1)/(cid:1)-AngIImiceispar-
mice. ticularlyinterestingbecauseMMP2hasbeenstronglylinkedto
AAA in patients (46, 47), and Mmp2(cid:1)/(cid:1) mice are protected
DISCUSSION
againstCaCl -inducedAAA(48).However,intheabsenceof
2
Abdominalaorticaneurysmisacommonandlethalvascular TIMP3,MMP2deletionresultedinadverseoutcomessuchas
disorderasabout6–9%oftheelderlypopulationhaveanAAA inflammation and MMP9 up-regulation. MMP2 has been
(6,40).Althoughsmallaneurysmscanbemanagedconserva- showntoplayaroleinearlystagesofvascularremodelingsuch
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