Smooth muscle cells from abdominal aortic aneurysms are unique and can independently and

15 Pages·2014·1.68 MB·English

BASIC RESEARCH STUDIES
From the Society for Vascular Surgery
SVS Foundation Resident Research Prize
Smooth muscle cells from abdominal aortic
aneurysms are unique and can independently and
synergistically degrade insoluble elastin
NathanAirhart, MD,aBernard H.Brownstein, PhD,cJ.Perren Cobb, MD,a*William Schierding, MS,d
BatoolArif,MS,aTerriL.Ennis,BS,aRobertW.Thompson,MD,a,bandJohnA.Curci,MD,aStLouis,Mo;
andAuckland, NewZealand
Background:Thepurposeofthisstudywastofurtherelucidatetheroleofthevascularsmoothmusclecells(SMCs)in
abdominalaorticaneurysm(AAA)disease.WehypothesizedthatthatAAASMCsareuniqueandactivelyparticipatein
theprocessofdegradingtheaorticmatrix.
Methods: Whole-genome expression profiles of SMCs from AAAs, nondilated abdominal aorta (NAA), and carotid
endarterectomy(CEA)werecompared.WequantifiedelastolyticactivitybyculturingSMCsin[3H]elastin-coatedplates
andmeasuringsolubilizedtritiuminthemediaafter7days.Matrixmetalloproteinase(MMP)-2andMMP-9production
wasassessedusingreal-timepolymerasechainreaction,zymography,andWesternblotting.
Results:EachSMCtypeexhibitedauniquegeneexpressionpattern.AAASMCshadgreaterelastolyticactivitythanNAA-
SMCs(D68%;P<.001)andCEA-SMCs(D45%;P<.001).ZymographyshowedanincreaseofactiveMMP-2(62kD)
inmediafromAAASMCs.AAASMCsdemonstratedtwofoldgreaterexpressionofMMP-2messenger(m)RNA(P<
.05) and 7.3-fold greater MMP-9 expression (P < .01) than NAA-SMCs. Culture with U937 monocytes caused
a synergistic increase of elastolysis by AAA SMCs (41%; P < .001) but not NAA-SMCs or CEA-SMCs (P [ .99).
Coculture with U937 caused a large increase in MMP-9 mRNA in AAA-SMCs and NAA-SMCs (P < .001). MMP-2
mRNA expression was not affected. Western blots of culture media showed a fourfold increase of MMP-9 (92 kD)
proteinonlyinAAA-SMCs/U937butnotinNAA-SMCs/U937(P<.001)andalargeincreaseinactive-MMP2(62
kD),whichwaslessapparentinNAA-SMCs/U937media(P<.01).
Conclusions:AAA-SMCshaveauniquegeneexpressionprofileandaproelastolyticphenotypethatisaugmentedbymacro-
phages.Thismayoccurbyafailureofpost-transcriptionalcontrolofMMP-9synthesis.(JVascSurg2014;60:1033-42.)
Clinical Relevance: The only current therapeutic modalities for treatment of the abdominal aortic aneurysm rely on
physicalexclusionoftheaneurysmdtherapiesassociatedwithsubstantialrisksandcosts.Medicaltherapiesthatblockthe
progressivedestructionoftheaorticwallhavegreatpotentialtoreducetheneedforsurgicaltreatment.Theexperiments
inthisstudydemonstratethattheintrinsicsmoothmusclecellsinthewalloftheaneurysmalaortaareuniquelycapableof
enzymaticdestructionofelastinandpotentiatetheelastolyticcapabilityoftheinflammatorycells.Effectivetherapyfor
aneurysmsmayrequiretreatmentstargetingthedysfunctionalactivitiesoftheintrinsicsmoothmusclecells.
The aortic aneurysm remains a poorly understood wallandiscapableof(1) matrixsynthesis,(2)proteaseor
inflammatory, matrix-degenerative disease that results in proteaseinhibitorelaboration,orboth,and(3)inflamma-
significant morbidity and mortality. The vascular smooth torycellrecruitment.Assuch,itiscapableofkeyinfluence
musclecell(VSMC)istheprincipalintrinsiccelloftheaortic onthehomeostasisoftheaorticmatrix.Theeffectofthese
FromtheDepartmentofSurgery,aCellBiologyandPhysiology,bandRadi- Presented as the Resident Research Award Prize winner at the Plenary
ationOncology,cWashingtonUniversitySchoolofMedicine,St.Louis; Session(May29)ofthe2013VascularAnnualMeetingoftheSociety
andtheLigginsInstitute,UniversityofAuckland,Auckland.d forVascularSurgery,SanFrancisco,Calif,May30-June1,2013.
*Currentaffiliation:DepartmentsofSurgeryandAnesthesia,Massachusetts Additionalmaterialforthisarticlemaybefoundonlineatwww.jvascsurg.org.
GeneralHospital,Boston,Mass. Reprintrequests:JohnA.Curci,MD,AssociateProfessorofSurgery,660S
ThisstudywassupportedbytheFlightAttendantsMedicalResearchInsti- EuclidAve,CampusBox8109,5103CQueenyTower,St.Louis,MO
tute (J.A.C.), American Heart Association 0765432Z (J.A.C.), the 63110(e-mail:[email protected]).
NationalHeart,LungandBloodInstituteK08-HL-84004(J.A.C.)and Theeditorsandreviewersofthisarticlehavenorelevantfinancialrelationships
P50-HL-083762 (R.T.), the Society for Vascular Surgery Foundation todisclosepertheJVSpolicythatrequiresreviewerstodeclinereviewofany
(J.A.C.),theAmericanCollegeofSurgeons(J.A.C.),theNationalInsti- manuscriptforwhichtheymayhaveaconflictofinterest.
tuteofAgingR01-AG-037120(J.A.C.),andtheDepartmentofVeterans 0741-5214/$36.00
Affairs(J.A.C.). PublishedbyElsevierInc.onbehalfoftheSocietyforVascularSurgery.
Authorconflictofinterest:none. http://dx.doi.org/10.1016/j.jvs.2013.07.097
1033
JOURNALOFVASCULARSURGERY
1034 Airhart et al October2014
cells on the development and growth of aortic aneurysms U937 cells for use in the elastolytic assays were
may be related to the loss of synthetic capability due to cultured in Roswell Park Memorial Institute media con-
apoptosisorothermechanisms,1,2butthereisalsogrowing taining 10% fetal bovine serum and antibiotics. Before
evidence that aortic VSMCs have the potential to directly use in our experiments, the cells were differentiated in
participate in the degenerative process.3-5 In addition, the media containing 10-nM phorbol 12-myristate 13-acetate
unique predilection of aneurysms for the infrarenal aorta (Sigma-Aldrich)for24hours.
andadjacentiliacvasculaturecouldberelatedtoregionali- Expressionprofiling. Wholegenomegeneexpression
zationoftheintrinsiccellularcomponents.6-10 profiles of VSMCs were analyzed in two independent
WehypothesizedthattheVSMCsisolatedfromaortasin batches. Total RNA was extracted and analyzed on the
patients with abdominal aortic aneurysms (AAAs) would Human HT-12 v4 Expression BeadChip (Illumina, San
demonstrate a unique pattern of gene expression compared Diego,Calif),accordingtothemanufacturer’sinstructions.
with cells derived from the nondilated infrarenal aorta under The expression data were normalized in BeadStudio
identical culture conditions. We also hypothesized that this software (Illumina) using the cubic-spline method. The
expression phenotype would manifest with increased direct data were then imported into Partek Genomics Suite 6.5
andindirectelastolyticactivitycomparedwithVSMCsderived (PartekInc).Differentialgeneexpressionanalysiswasper-
fromnonaneurysmalaorticwallorevenpathologicallyaltered formedusingamixed-modelANOVA.Agenelistwasthen
VSMCsderivedfromatheroscleroticplaque.Inthisstudy,we created using a median false discovery rate of 0.05. Inclu-
compared whole genome gene expression patterns of the sion of patient age and sex did not influence the model.
explanted VSMCs from each of these tissues, directly GenesthatweredifferentiallyexpressedinVSMCsderived
measured their ability to degrade elastin, and characterized fromAAAscomparedwithVSMCsfromCEAsandNAAs
specificpathwaysandenzymesthatareinvolvedinthisprocess. weresubjectedto geneontologyanalysis.
Class prediction analysis was based on k-nearest
METHODS neighbor classification (k ¼ 1 and k ¼ 3, based on a
Human tissues were collected anonymously under an Euclidean distance measure). The misclassification rate of
exempt protocol approved by the Washington University each model was estimated using complete leave-one-out
InstitutionalReviewBoard.Detailsofthemethodsofanalysis cross-validation.
canbefoundintheSupplementaryMethods(onlineonly). Elastolytic activity. Tritiated elastin (Moravek
MicrodissectionandRNAextraction. Briefly,frozen Biochemicals,Brea,Calif)waspreparedasdescribedprevi-
sections from AAA specimens were microdissected using ously.11 Tissue culture wells (24/plate) were coated with
a PixCell IIe system (Arcturus Bioscience Inc, Mountain 200 mg [3H]elastin. Coculture experiments were per-
View,Calif).Eachhistologiclayeroftheaorticwall(intima, formed using a Transwell coculture system (Corning Inc,
media, and adventitia) was identified and separately BigFlats,NY).TheVSMCswereincubatedfor24hoursin
dissected onto “caps,” ensuring no incidental attachment serum-free Dulbecco’s modified Eagle’s medium con-
of adjacent tissue. Tissue from five serial sections was taining 20 mg/mL lipopolysaccharides (Sigma-Aldrich).
combined and RNA extracted using the PicoPure RNA Experimentswereperformedwith1(cid:2)105cellsinthewell
Kit(Arcturus).Thecomplementary(c)DNAwasamplified, orwith0.5(cid:2)105cellsinthetranswell,orboth,for7days
andtheHG-U133PlusGeneChip(Affymetrix,SantaClara, before the solubilized [3H]elastin was quantitated by
b
Calif)wasusedforthehybridizationandnormalizedacross counting an aliquot of the media in a -scintillation
microarraysusingtheRobust MultiChipAverageprogram. counter. All assays were performed with three technical
One-way analysis of variance (ANOVA) was performed replicates.Activityisreportedintotalmicrogramsofelastin
using a conservative significance cutoff to identify informa- degraded per well based on the specific activity of [3H]
tional genes that distinguished between the three cell elastin.
classesdadventitia, media, and intimadusing a microarray Degradation of mouse aortic elastin ex vivo. Serial
analysissuite(PartekInc,St.Louis,Mo;www.partek.com). sections of optimal cutting temperature-embedded in-
Cell culture. Briefly, anonymous discard tissues of frarenal aortic tissue from C57/Bl6 mice (The Jackson
nondilatedabdominalaorta(NAA),AAA,andcarotidpla- Laboratory,BarHarbor,Me)weremountedoncoverslips.
que were used. The intimal layer was removed from the The sections were examined using an Olympus BX61
aortic tissue. The tissue was digested with collagenase fluorescence microscope (Olympus, Center Valley, Pa) at
type I (Worthington Biochemical, Lakewood, NJ) and original magnification (cid:2)200. The tissue sections were
porcine pancreatic elastase (Sigma-Aldrich, Saint Louis, cultured for 10 days with VSMCs derived from AAA or
Mo). The cells were suspended in Dulbecco’s modified NAAtissue andreimaged.
Eagle’s medium containing Smooth Muscle Cell Growth Measurement of metalloproteinase production.
Supplement (ScienceCell, Carlsbad, Calif) with 10% fetal Evaluationofmatrixmetalloproteinase(MMP)production
calf serum, GlutaMAX I (Life Technologies, Grand and activity were performed as previously described.11
Island, NY), nonessential amino acids, 6 mM N-2- Briefly, the VSMCs cultured from AAA, CEA, and NAA
hydroxyethylpiperazine-N0-2-ethanesulfonate, and antibi- tissue were plated in six-well culture plates (Corning) at
otics.Samplesofcells(passagelessthanfive)wereanalyzed adensityof0.5(cid:2)106andincubatedinserum-freemedia
byflowcytometryforcelltype-specificmarkerproteins. for7days.Forcocultureexperiments,activatedU937cells
JOURNALOFVASCULARSURGERY
Volume60,Number4 Airhart et al 1035
(3(cid:2)105)weresuspendedintranswells.Equalamountsof TableI. Characteristicsofpatients from which vascular
proteinwereresolvedon4%to20%gradientgelsor12.5% smoothmusclecells(VSMCs) wereharvested for
gels containing 0.2% gelatin. The zymogram gels were microarrayanalysisa
incubated overnight in assay buffer and then rinsed with
deionized water and stained with Coomassie Blue. Age,years Sex,% Cellpassage,No.
Western blotting was performed on proteins that were
Tissuetype No. Mean(SE) Male Female Mean(SE)
transferred to nitrocellulose, blocked in 5% nonfat dried
milk in phosphate-buffered saline with Tween 20 buffer AAA 22 69.8(1.9) 76 24 2.8(0.16)
for 1 hour at room temperature, and incubated with NAA 17 45.6b(4.1) 47 53 2.2(0.12)
primary antibody toward MMP-2 or MMP-9 (ab37150, CEA 29 67.0(2.0) 53 47 2.6(0.21)
ab38898; Abcam, Cambridge, Mass) overnight. A
AAA, Abdominal aortic aneurysm; ANOVA, analysis of variance; CEA,
peroxidase-chemiluminescent system (GE Healthcare, carotidendarterectomy;SE,standarderror.
Pittsburgh, Pa) was used to detect bound primary. aVSMCswereculturedfromAAA,NAA,andCEAtissues.
ExpressionofMMP-2andMMP-9mRNAinVSMCswas bP<.001,ANOVAwithTukeypost-hoctest.
compared by quantitative real-time polymerase chain
reaction (assay ID: Hs01548727_m1, Hs00234579_m1, Wewereabletovalidatetheapplicationofgeneexpres-
and Hs02758991_g1, respectively; Applied Biosystems, sionprofilingtodiscriminateamongVSMCsderivedfrom
GrandIsland,NY).GeneexpressioninAAA-VSMCsand AAA,CEA,andNAAtissuewithhighaccuracyusingaclass
CEA-VSMCs relative to NAA-VSMCs was calculated prediction analysis based on k-nearest neighbor classifica-
using the 2(cid:3)DDCT method. tion(k¼1andk¼3).Modelswereconstructedcontain-
Statistics. For analysis of the elastolytic assay data, ing a range of predictor genes. After cross-validation with
a one-way ANOVA was performed, using SPSS 19 soft- leave-one-out cross-validation, the mean percentage of
ware (IBM Corp, Armonk, NY), comparing mean micro- correct classification of the VSMC subsets (AAA, NAA,
gramsofelastindegradedandmeanMMP-2andMMP-9 CEA) by these models ranged from 73% to 90% with the
productionbetweenexperimentalgroups.TheTukeypost- three-nearest neighbor classification containing 30
hoc test was used to compare mean micrograms of elastin predictor genes performing most accurately. Table II
degraded between the individual groups within experi- outlines the performance of this model. The model
ments. The same analysis was done to compare MMP correctly classified 20 of 22 AAA VSMC cell lines (91%),
production inWestern blotting. 14 of 18 (82%) NAA VSMC cell lines, and 27 of 29
CEAVSMCcell lines(93%).
RESULTS Expression profiles of microdissection of AAA
Characterizationofcells. Cellcultureswereobtained wall. Amplified RNA from microdissection specimens of
by enzymatic explantation techniques from fresh tissues the three principal layers of the aortic walldintima,
discarded after surgery under a Humans Studies media, and adventitiadunderwent microarray analysis.
Committee protocol. All VSMC cultures used in experi- We performed cluster analysis of these data to resolve
ments were negative by flow cytometry for cell markers whether there are consistent differences in expression
CD11c,CD20,CD3,CD31,andCD68andwerepositive that would allow us to specifically group the microdis-
fora-smoothmuscleactin. sected layers of the aortic wall. We identified 49 genes
Expression profiles of VSMCs in culture. We per- thathighlysignificantly(P<.0005)differentiatedamong
formed gene expression profile analyses from 22 AAAs, thesethreelayersoftheaorticwall(SupplementaryFig1,
29CEAs,and17NAAVSMClines,representingallavail- online only).
ablecelllinesproducedatthetimeofanalysis.Characteris- Gene ontology. Gene ontology analysis revealed that
ticsofthepatientsfromwhichthetissuewasharvestedare anumberoffunctionalclassificationgroupswereexpressed
summarized in Table I. The mean age of patients with differentiallyinAAA-derivedVSMCscomparedwithNAA
AAAs and CEAs was similar. Patients from whom NAA andCEAVSMCs.Someofthemostsignificantlyenriched
tissuewasharvestedweregenerallyyoungerthantheCEA functional categories included genes related to axis speci-
orAAApatients.MostoftheAAApatientsweremale.The fication, response to oxidative stress, proapoptotic path-
distributionofmaleandfemalepatientswasapproximately ways, the proliferation and migration of VSMCs, several
equal forNAAand CEApatients. proinflammatorypathways, andproteolysis.
Usingafalsediscoveryrateof<0.05,weidentified816 Our microarray datarevealed asignificantelevation of
genes that were differentially expressed by the VSMCs MMP-2 and MMP-9 mRNA expression in AAA-derived
derivedfromthesetissuetypes.Aprincipalcomponentanal- VSMCs relative to VSMCs cultured from NAA and CEA
ysisplottedbasedonthosegenesisshowninFig1,A.Inthe tissue.Analysisofthemicroarraydataalsosuggestedsignif-
principal component analysis, the three distinct clusters icantly increased expression of the cysteine proteases,
clearlyshowtheuniquegeneexpressionpatternofthecells cathepsin B, cathepsin L, cathepsin K, and cathepsin S.
derived from aortic aneurysmtissue, and hierarchicalclus- The data did not reveal differences in expression of the
tering further demonstrates the very distinct pattern of endogenous MMP inhibitors tissue inhibitor of metallo-
aneurysm-derivedcelltypes. proteinase(TIMP)-1andTIMP-2orthecysteineprotease
JOURNALOFVASCULARSURGERY
1036 Airhart et al October2014
Fig1. A,Principalcomponentsanalysisandhierarchicalclusteringdemonstratedistinctgeneexpressionpatternsof
culturedvascularsmoothmusclecells(VSMCs)derivedfromabdominalaorticaneurysm(AAA,n¼22;red),non-
dilatedinfrarenalaorta(NAA,n¼17;green),andcarotidendarterectomy(CEA,n¼29;blue)tissues.B,Microarray
datademonstraterelativegeneexpressionofspecificelastasesandinhibitorsbetweenVSMCsculturedfromAAA(n¼
22),NAA(n¼17),andCEA(n¼29)tissues.CandD,AAA-derivedtissuesexhibitedsignificantlyincreasedgene
expression of several elastin-degrading endopeptidases, including matrix metalloproteinase (MMP)-2, MMP-9,
cathepsin-B,cathepsin-L,andcathepsin-K.*P<.05,**P<.01,analysisofvariance(ANOVA).
inhibitor cystatin C between VSMC types. Expression of didnotresultinsignificantreductionsinelastolyticactivity
TIMP-3by AAAVSMCswasincreased (Fig 1,B). degrading, at 11.3 6 2.6 vs 18.9 6 5.5 mg (n ¼ 3; P ¼
Enhanced elastolytic activity of AAA-derived .09) and 14.9 6 2.7 vs 18.9 6 5.5 mg (n ¼ 3; P ¼ .55),
SMCs. Elastolyticactivityofthesecells(n¼11pertissue respectively (Fig 2, C and D). Elastin degradation by
type) was assayed by culturing the cells on [3H]-labeled VSMCs required direct contact because cells suspended
elastin-coated plates for 7 days. Cells derived from AAA in transwell inserts above the [3H]elastin resulted in low
consistently degraded significantly more elastin than cells levels of elastolysis in VSMCs derived from NAAs (5.0 6
derivedfromNAA(18.965.5vs6.163.9mg;P<.001) 2.9 mg), CEAs (5.8 6 4.2 mg), and AAAs (6.0 6
and CEA(10.456 5.5mg; P¼ .001;Fig 2, A). 4.8mg; n¼6).
The addition of inhibitors to the media of NAA and MMP-2andMMP-9inconditionedmedia. Aband
CEA cells, consisting of BB-94 (a nonselective MMP consistent with pro-MMP-2 was detected by zymography
m
inhibitor, 5 M), aprotinin (a serine protease inhibitor, and by Western blotting in conditioned media from the
100 mg/mL), and E64 (a cysteine protease inhibitor, VSMCs.Therewasnosignificantdifference intheVSMC
10 mM), did not have a significant effect on elastolytic production of pro-MMP-2 among the tissue of origin
activity. In AAA-derived cells, however, MMP inhibition groups.
with BB-94 inhibited elastolysis by w60% (7.6 6 0.9 vs Active MMP-2, visualized as a 62-kD band on the
18.9 6 5.5 mg, n ¼ 3; P ¼ .008). Aprotinin and E64 gelatin zymogram, was most pronounced in VSMCs
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Volume60,Number4 Airhart et al 1037
Table II. Performance ofclasspredictionanalysis (k-nearest neighbor[k ¼3]) for30predictorgenes
Tissuetype No. Proportion Correct Errors Correct,% Error,% SE,%
NAA 17 0.25 14 3 82.00 18l.00 9.25
CEA 29 0.43 27 2 93.00 7.00 4.71
AAA 22 0.32 20 2 91.00 9.00 6.13
Total 68 1 61 7 89.71 10.29 3.69
Confusionmatrix(real/predicted) NAA CEA AAA
NAA 14 1 2
CEA 2 27 0
AAA 1 1 20
AAA,Abdominalaorticaneurysm;CEA,carotidendarterectomy;NAA,nondilatedabdominalaorta;SE,standarderror.
derivedfromAAAtissue,whereasverylittleactiveMMP-2 MMP-9 and MMP-2 (Fig 3, A). The VSMC cultures
wasdetectedinVSMCsderivedfromNAAandCEAtissue demonstrated relatively little MMP-9 activity, but all cell
(Fig 3, A). A band consistent with activated MMP-2 was lines showed activity at 72 kDa, consistent with pro-
not seen on Western blotting, however, for any of the MMP-2. The AAA-derived and CEA-derived cell lines
VSMC groups (Supplementary Fig 2, online only). A low appeared to have more MMP-2 activity than the NAA-
level of MMP-9 was detectable by zymography (but not derived cells, including the presence of activated MMP-
by Western blotting) in the conditioned media from 2. Culturing the VSMCs in the presence of U937 cells
AAA-derived and CEA-derived VSMCs, but no band was intranswellsledtoincreasedamountsofactiveMMP-2in
seenat 92-kDaintheNAA-derived VSMCs. the culture media (62-kDa band) in all VSMC types
Elastolysis in VSMC-macrophage cocultures. We relative to VSMCs cultured alone. This effect was much
analyzed elastolytic activity in a coculture system where more pronounced with AAA-derived and CEA-derived
VSMCs were cultured in the presence of phorbol 12- VSMCs than with NAA-derived VSMCs (Fig 3, A).
myristate 13-acetate-activated U937 cells suspended in Significant amounts of MMP-9were detected in all wells
transwell inserts. In cocultures containing AAA-derived containing U937 cells.
VSMCs with U937 cells, a >40% increase in elastolytic Westernblottingofconditionedmediafromcocultures
activityoccurredcomparedwithculturescontainingAAA- containingAAA-derivedVSMCsdemonstratedauniformly
derivedVSMCsalone(26.6768.2vs18.965.5mg,both higher concentration of MMP-9, with a mean relative
n¼11;P<.001).Incontrast,elastolyticactivitywasnot density 3.8-fold greater than in NAA-U937 cocultures
significantly affected with the addition of U937 cells to (n¼6;P<.001;Fig3,D).Inconcordancetoourresults
NAA-derived (6.83 6 5.38 vs 6.09 6 3.89 mg, both n¼ from gelatin zymography, media from wells containing
11; P > .99) or CEA-derived (11.81 6 4.24 vs 10.45 6 cocultured U937 and VSMCs consistently demonstrated
5.40mg,bothn¼11;P¼.99)VSMCcultures(Fig2,D). the presence of active MMP-2, with a band at 62 kDa.
Elastolyticactivitywasalsomeasuredincocultureswith Once again, the increase of active MMP-2 was most
U937cellsplatedontheradiolabeled[3H]elastinwiththe pronouncedincoculturescontainingAAA-derivedVSMCs.
VSMCs suspended in transwell inserts (Fig 2, E). When The density of the band corresponding to active MMP-2
cultured alone in contact with insoluble elastin, the acti- was approximately six times that of the band observed for
vated U937 cells exhibited only a small amount of elasto- NAA-VSMCcultures(n¼6;P¼.01;Fig3,D).
lytic activity (3.3 6 2.9 mg/well, n ¼ 3). We similarly MMP-2andMMP-9expression. Underbasalcondi-
saw relatively little elastolytic activity of the VSMCs, tions,thelevelofMMP-2andMMP-9mRNAexpression
regardlessoftissueoforigin,whenculturedinthetranswell wassignificantlyhigher intheAAA-derivedVSMCs. Cells
alone and not in contact with the elastin. Coculturing cultured from AAA tissue exhibited twofold higher
NAA-derivedorCEA-derivedVSMCswiththeU937cells expression of MMP-2 compared with NAA-derived
didnotsignificantlyaugmentelastindegradationcompared VSMCs (n ¼ 11; P < .05) and 7.3-fold increased expres-
with either cell type alone. When AAA-derived VSMCs sionof MMP-9mRNA(n¼ 11;P <.01; Fig 3, B).
were suspended above the U937 cells in transwell inserts, The presence of U937 cells suspended in transwell
there was an approximate sixfold increase in elastolytic inserts did not increase VSMC expression of MMP-2
activity compared with VSMCs in the transwell alone mRNA. In fact, MMP-2 expression decreased by a small
(19.0 6 3.55 mg, n ¼ 7; P < .001) and compared with butstatisticallysignificantamount(n¼3;P<.05).Cocul-
activated macrophagesalone(P < .001). ture with U937 monocytes led to a large relative increase
MMP-2 and MMP-9 in cocultures. Zymography in the expression of MMP-9 mRNA in AAA and
using conditioned media from activated U937 monocytes NAA VSMCs (n ¼ 3; P < .001; Fig 3, C). MMP-2
demonstrated the presence of proenzyme forms of and MMP-9 mRNA production by U937 cells was
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1038 Airhart et al October2014
Fig2. A,Insoluble[3H]elastindigestion(mg6standarderror[errorbars])byvascularsmoothmusclecells(VSMCs)
from abdominal aortic aneurysm(AAA, n ¼ 11), nondilated abdominal aorta (NAA, n ¼ 11), and carotid endar-
terectomy(CEA,n¼1)plaqueculturedfor7days.TheAAA-derivedcellsdegradedsignificantlymore[3H]elastin
thanVSMCsculturedfromCEAorNAAtissue.B,VSMCsculturedfromNAAandAAAtissueswereculturedfor
10dayswithmouseaortictissueimagedwithautofluorescencebeforeandafter.Representativeimagesdemonstrate
diminishedelastininaortictissueincubatedwithAAA-derivedVSMCs.C,Theeffectofspecificproteinaseinhibitors
ontheinvitrodegradationofinsoluble[3H]elastin.VSMCsderivedfromAAA,NAA,andCEA(n¼3each)were
cultured in serum-free media in the presence or absence of BB-94 (a broad inhibitor of matrix metalloproteinases
m m m
[MMPs],5 M),aprotinin(aserineproteaseinhibitor,100 g/mL),andE64(cysteineproteaseinhibitor,10 M).
OnlyBB-94significantlyreducedtheelastindegradedbyAAA-derivedVSMC.D,TheeffectofcocultureofVSMCsin
contactwith[3H]elastinwithactivatedU937macrophagesintranswells.VSMCs(n¼11each)wereplatedin[3H]
elastin-coatedculturewellsfor7dayswithactivatedU937cellsintranswellinserts.Theshadedareaofthehistogram
represents monoculture, and the open area represents coculture conditions. The presence of activated U937 cells
significantlyincreasedelastindegradationonlywithAAA-derivedVSMCscomparedwithmonocultureofSMCs.E,
TheeffectofcocultureofactivatedU937macrophagesincontactwith[3H]elastinwithVSMCintranswellsisshown.
VSMCsderivedfromAAA(n¼7),NAA(n¼3),orCEA(n¼3)wereculturedfor7daysintranswellsabove[3H]
elastin-coatedplates,withorwithoutactivatedU937cells.Celloriginhadnoeffectonelastindegradationwhenthe
VSMCswerenotincontactwiththeelastin.However,elastindegradationwasonlysignificantlyincreasedinwellswith
AAA-derivedVSMCsintranswellcoculturewithactivatedU937macrophages.
not significantly affected by coculture with VSMC Thetraditionalhypothesishasbeenthatthedestruction
(Supplementary Fig 3, onlineonly). ofextracellularmatrixinAAAsisprimarilyaconsequenceof
anexuberantchronicinflammatoryinfiltratethatisrespon-
DISCUSSION
siblefortheelaborationofelastasesandotherproteasesthat
AAA is a progressive degenerative disease of a major damagethemedialmatrixstructureoftheaorta.6Substan-
vascular conduit with considerable impact on patient tialworkhasshownthattheintramuralmacrophagesmay
mortality and health care costs. The aneurysmal change is have a central role,1-5,7-9 but more recent studies have
frequently localized to the infrarenal segment of the also focused on the contributions of neutrophils,11,12
abdominal aorta, although it can extend into the adjacent mast cells,13,14 and other infiltrating cells.15,16 Much of
commonandinternaliliacvessels.Developmentofaweak- thesupportingevidenceforthecentralroleofinflammation
enedaorticwallisassociatedwithseveredestructionofthe in AAAs comes from mouse models, where nearly all
medialelasticfibers.Thisstudysoughttodemonstratethat manners of anti-inflammatory therapy inhibit aneurysm
theVSMCsthatpopulatetheaneurysmwallhaveaunique formation.17-19 In humans, however, anti-inflammatory
phenotype thatdirectly contributesto thepathogenesis of treatmenthasnotbeensimilarlysuccessful.20
the aneurysm. We also sought to develop a mechanistic The VSMCs of the aorta have received less attention
understandingoftheroleoftheVSMCinthedestruction than inflammation asa contributor to aneurysmal change.
ofmedial elastinthat characterizesAAAs.9 Somestudieshaveshownpotentialsecondaryparticipation
JOURNALOFVASCULARSURGERY
Volume60,Number4 Airhart et al 1039
Fig3. A,Gelatinzymographyofconditionedcellculturemedia.Vascularsmoothmusclecells(VSMCs)derivedfrom
abdominal aortic aneurysm (AAA), nondilated abdominal aorta (NAA), and carotid endarterectomy (CEA) were
culturedinserum-freemediafor7daysinthepresenceorabsenceofphorbol12-myristate13-acetate-stimulatedU937
cellsintranswellinserts.MediafromculturesofSMCsalonewasdominatedbymatrixmetalloproteinase(MMP)-2
activity (62 kDa) particularly in the AAA-derived VSMCs. Media from cocultures demonstrated consistent bands
correspondingtoMMP-9activity(92kD)andincreasedactiveMMP-2activityrelativetothemonocultures,which
was most pronounced from AAA-derived VSMCs/U937 cocultures. B, Western blot analysis of conditioned cell
culturemediaforMMP-2andMMP-9fromcocultureexperiments(representativeblotsareshownfromn¼6for
densitometryforeach).MMP-9(92kD)wasmorehighlyexpressedinthemediafromcocultureswithAAA-derived
VSMCs.Theproductionofpro-MMP2didnotdifferbySMCorigininthecoculturesetting.However,significantly
moreactiveMMP-2(62kD)waspresentinculturescontainingAAA-derivedVSMCs.C,Real-timepolymerasechain
reactioninVSMCsfrommonocultureaswellasincellsaftercoculturewithactivatedU937macrophages.Atbaseline,
MMP-2andMMP-9expressionwerebothsignificantlyelevatedinAAA-derivedVSMCsrelativetoNAAsandCEAs.
Inresponsetococulturewithactivatedmacrophages,MMP-9expressionincreaseddramaticallyinbothNAAandAAA
VSMCs,whereasexpressionofMMP-2messengerRNA(mRNA)significantlydecreasedinbothcelltypesunderthe
cocultureconditions.*P<.05,**P<.01,analysisofvariance(ANOVA)withTukeypost-hoctest.
ofthesecellsinAAApathogenesisduetoareductioninthe we also compared the profile of the aneurysm-derived
quantity or activity of medial VSMCs. The reduction in VSMC vs VSMCs derived from carotid plaque and
VSMCs has been hypothesized to limit matrix repair in confirmed that the profile of the AAA cells was distinct
the damaged aorta.21 There is also inferential evidence fromthatofatheroscleroticplaque,whichfurthersupports
that the VSMC may play a more direct role in aneurysm theconceptthattheaneurysmcellphenotypeisunique.
formation.22 ThesecrucialobservationsdemonstratethattheAAA-
Byexaminingthecompleteexpressionprofileofalarge derived cells have an intrinsic or an acquired phenotype
numberof pure, early-passage VSMC culturesfrom aneu- thatmaycontributetothedegenerationoftheaorta.These
rysmalandnonaneurysmalinfrarenalaorta,wehaveshown experiments cannot demonstrate how these AAA-derived
that VSMCs derived from AAAs exhibit a unique pattern cells developed their unique phenotype or the mechanism
of gene expression that is distinct from VSMCs derived bywhichthisphenotypepersistsrelativetootherVSMCs,
fromNAAtissue,withahighdegreeofpredictability.The evenwhenexposedtocommoninvitrocultureconditions.
microdissected aneurysm specimens suggested that the Adetaileddiscussionofthepotentialsourcesofthisunique
expression profiles of the cells in the intima were distinct phenotype has been previously reviewed in detail.9
from thatofthemediaand adventitiainaorticspecimens. Briefly, studies of the long-term growth characteristics of
Thiswouldbeconsistentwithadistinctprocessofdisease aneurysm-derived VSMCs compared with similarly
developmentofintimalatherosclerosisandaneurysmaldila- cultured VSMCs from nonaneurysmal vasculature have
tation.Therefore, tooptimize thecollection ofaneurysm- demonstrated consistent differences,21,23-25 even when
specific pathogenic cells, we grossly removed the intima derived from the same patient.26 The specific ontogeny
before cell culture of the aortic specimens Furthermore, of the distal aortic VSMC may also play an important
JOURNALOFVASCULARSURGERY
1040 Airhart et al October2014
roleintheparticularsusceptibilityofthisvascularsegment a decreased expression of MMP-2 mRNA. Somewhat
toaneurysmaldegeneration.Itisnotablethattheexternal surprisingly, we saw a nearly identical effect of coculture
iliac arteries, which are highly aneurysm-resistant, form on MMP expression in the cells derived from NAAdcells
much later in gestation and from different precursors that did not increase their elastolytic activity in coculture.
compared with the adjacent dorsal aorta-derived and There was no appreciable effect on the mRNA expression
aneurysm-prone infrarenal aorta, common iliac, and of MMP-9 or MMP-2 in the macrophages when cocul-
internal iliacvessels.27,28 turedwith VSMCs.
TounderstandhowthisnovelphenotypeoftheAAA- DespitethesimilarresponseofAAA-derivedandNAA-
derived cells may predispose to AAA development, we derivedcellstoMMPexpressioninthesettingofactivated
focusedondeterminingtheroleofthesecellswithrespect macrophages,cocultureoftheAAA-derivedcellswasasso-
totheelasticfiberdegenerationcharacteristicoftheAAA. ciated with a significantly greater increase in MMP-9
Weconfirmedthatwithintheaneurysm-specificexpression protein in the conditioned media compared with the
profile is upregulation of several proteases of metallopro- NAA-derived cells. This would suggest substantial post-
tease and cysteine classes that are capable of elastolysis. transcriptional control of MMP-9 production occurs in
Also potentially informative were the findings of the normal cells but is not effective in the AAA-derived
MMP inhibitors, including a lack of upregulation of VSMCs. We also found substantially more activated
TIMP-1 and TIMP-2, whereas there was considerable MMP-2 in the conditioned media from the AAA-derived
upregulation of TIMP-3. The lack of increased TIMP-1 VSMCscocultured with theactivatedU937.
or TIMP-2 in the setting of increased MMP activity
suggests an imbalanced proteolytic state,29,30 although CONCLUSIONS
the role of TIMP-3 is unclear. In whole aneurysm tissue, Taken together, these data demonstrate that the
others have shown significantly increased expression of VSMCsthatpopulatetheAAAhaveauniqueproelastolytic
TIMP-3 in AAAs,31 but gene expression32 and protein phenotype that is augmented in the presence of activated
production33 have both been shown to be decreased in macrophages. This effect appears to occur by a post-
thoracic aortic aneurysms. This unique role of TIMP-3 in transcriptional failure of MMP-9 synthesis control leading
AAA development is also supported by a genetic variant to increased production and activation of elastolytic
ofTIMP-3(nt-1296)thathasbeenfoundtobeassociated MMPs.Theseareuniqueandimportantfindingsregarding
with familialAAA.34 thepathobiologyofthediseasethatcreatenewopportuni-
Of the elastolytic MMPs, only MMP-2 and MMP-9 ties with respect to therapies that can result in effective
demonstrated significant upregulation in the AAA-derived aneurysminhibition.Furtherstudyisneededtodetermine
VSMCs, which we confirmed by quantitative real-time theintercellularandintracellularmechanismsthatallowfor
polymerasechainreaction.However,wealsofoundsignif- the synergistic overproduction of elastolytic proteases,
icant upregulation of several cysteine proteases that are particularly MMP-9, by aneurysm-derived VSMCs in the
capable of contributing to elastolysis. By incubating the presenceofactivated macrophages.
VSMCsonlabeledinsolubleelastinaswellasmurineaortic
sections,weconfirmedthattheAAA-derivedVSMCswere WesincerelythankStephenM.Schwartz,MD,PhD,at
able to degrade significantly more insoluble elastin than theUniversityofWashington,forallofhishelpconceptu-
alizingtheproject.
cells derived from nondilated infrarenal aorta or from
carotid plaque. The use of broad-spectrum class-selective
inhibitors showed this potent elastolytic activity appeared AUTHORCONTRIBUTIONS
to beprimarily mediatedby theactivity ofMMPs. Conceptionand design:BB,JPC, RT, JAC
This critical finding confirms our hypothesis that the Analysisand interpretation: NA,BB,WS,JAC
intrinsic VSMC found in the AAA can actively participate Datacollection:NA,BB, TE,RT
in the matrix degradation process through MMP- Writingthearticle:NA,JAC
dependent elastolytic activity. However, it left open the Criticalrevisionofthe article:NA,JPC, WS,BA, TE,RT
relative participation of aortic VSMCs and inflammatory Final approval of the article: NA, BB, JPC, WS, BA, TE,
cellsinthedegradationofelastin.Toevaluatethepotential RT, JAC
interaction of VSMCs with macrophages in the degrada- Statisticalanalysis: NA,BB,JAC
tion of elastin, we performed the coculture experiments Obtainedfunding:JAC
using activated U937 cells.35 Remarkably, the elastolytic Overallresponsibility:JAC
activity of AAA-derived VSMCs was substantially
augmented in the setting of coculture with the activated
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DISCUSSION
DrFrankLoGerfo(Boston,Mass).Whatcomesfirst?Dothe endothelial level? What causes the macrophages to get involved
macrophages somehow change the phenotype of the smooth here?
musclecells?Iguessthat’sthefirstthingthatcomestomindin Ofcourse,thiscouldallbewrong.Therecouldbeaprimary
an inflammatory process. If so, then why do the macrophages phenotypicdifferenceinthesmoothmusclecellsintheaorticwall
do that? What is the stimulus? Are there some receptors at the right from the start. But, it would seem more likely that the
JOURNALOFVASCULARSURGERY
1042 Airhart et al October2014
phenotypic change comes after this inflammatory process. I’d adaptation,wehavefoundthatassmoothmusclecellsundergo
appreciateyourthoughtsaboutthat. apoptosis, they produce a lot of proteolytic activity and
Dr Nathan Airhart. Our hypotheses is that it may be the degradethesurroundingmatrix.Itisveryremarkable.Iwould
smoothmusclecellthat’scomingfirst.AsIdiscussed,thesmooth liketoknowwhetherthemacrophagesinyourexperimentsare
musclecellsthatweareculturingfromabdominalaorticaneurysms killingthesmoothmusclecellsandwhetherthedyingsmooth
are unique. They exhibit a unique phenotype in culture which muscle cells are, in turn, secreting elastolytic and proteolytic
favors the degradation of elastin and this is a phenotype that activity.
persists,eventhroughseveralpassagesincellculture.Onepoten- Dr Airhart. Currently, we do not have evidence that co-
tial explanation for this phenomenon may be that the smooth culture with macrophages is inducing cell death in our smooth
musclecellsthatpopulatetheregionoftheinfrarenalaortahave muscle cells. We have not done objective studies to answer this
a distinct embryologic origin. Interestingly, the embryologic question, however, and it is something that we should look at
boundaries of these cells happen to match up with where more moreclosely.
than90%ofabdominalaorticaneurysmsoccur. DrB.TimothyBaxter(Omaha,Neb).Ihavetwoquestions
Thenatureofthisstudy,andvirtuallyallstudiesusingpatho- foryou.Canyoutellusaboutgrowingthesecellsout?Usuallythe
logic specimensfrom human abdominalaortic aneurysmdisease, smooth muscle cells are much more difficult to grow, and they
prevents us from answering the specific question, “which comes grow slower. And, so really you have two different populations.
first, the inflammation or the smooth muscle cell,” as our cell And,Iwonderifthereissomeselectioninthatprocess?
cultures are coming from very late-stage abdominal aortic aneu- ThesecondthingisactivationofMMP-2.Didyouhappento
rysmtissue.However,thewaythatthesecellsfromtheinfrarenal lookatMMP-14/MT1-MMPwhichistheactivatorofMMP-2?
aorta react to chronic stresses (abdominal aortic aneurysm risk DrAirhart.Wechosetofocusonmatrixmetalloproteinase-2
factors), suchas smoking,atherosclerosis, or hemodynamic pres- and matrix metalloproteinase-9 after seeing the results from our
sure, may be different than smooth muscle cells from different microarraydata.Wehaven’tdoneanyfurthervalidationwiththe
regionsoftheaortaorfromthosecellsfromindividualswhodo otherMMPs.
notdevelopaneurysms. Wehavehadexcellentsuccessculturingsmoothmusclecells
DrAlexanderClowes(Seattle,Wash). Oneof theobser- fromthepathologictissue.Ithinkthatthisreallycomesfromexten-
vationsmadeinthepastisthatthemedialsmoothmusclecells sive experience with the culture technique, careful attention to
die as aneurysms form. In our own studies of vascular tissuedissection,andmaintenanceofoptimalcultureconditions.