Hindawi Publishing Corporation - Research Article …downloads.hindawi.com/journals/mi/2014/276457.pdfResearch Article Relationship of MMP-14 and TIMP-3 Expression with Macrophage

Research ArticleRelationship of MMP-14 and TIMP-3 Expression withMacrophage Activation and Human AtheroscleroticPlaque Vulnerability

Jason L Johnson12 Nicholas P Jenkins12 Wei-Chun Huang2

Karina Di Gregoli12 Graciela B Sala-Newby2 Vincent P W Scholtes3

Frans L Moll3 Gerard Pasterkamp3 and Andrew C Newby2

1 Laboratory of Cardiovascular Pathology School of Clinical Sciences Level 7 Bristol Royal Infirmary Bristol BS2 8HW UK2 School of Clinical Sciences University of Bristol Bristol BS2 8HW UK3University Medical Centre 3584 Utrecht The Netherlands

Correspondence should be addressed to Jason L Johnson jasonljohnsonbrisacuk

Received 31 March 2014 Revised 9 July 2014 Accepted 21 July 2014 Published 24 August 2014

Academic Editor Beatriz De las Heras

Copyright copy 2014 Jason L Johnson et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Matrix metalloproteinase-14 (MMP-14) promotes vulnerable plaque morphology in mice whereas tissue inhibitor ofmetalloproteinases-3 (TIMP-3) overexpression is protective MMP-14hi TIMP-3lo rabbit foam cells are more invasive and moreprone to apoptosis than MMP-14lo TIMP-3hi cells We investigated the implications of these findings for human atherosclerosisIn vitro generated macrophages and foam-cell macrophages together with atherosclerotic plaques characterised as unstableor stable were examined for expression of MMP-14 TIMP-3 and inflammatory markers Proinflammatory stimuli increasedMMP-14 and decreased TIMP-3 mRNA and protein expression in human macrophages However conversion to foam-cells withoxidized LDL increased MMP-14 and decreased TIMP-3 protein independently of inflammatory mediators and partly throughposttranscriptional mechanisms Within atherosclerotic plaques MMP-14 was prominent in foam-cells with either pro- or anti-inflammatory macrophage markers whereas TIMP-3 was present in less foamy macrophages and colocalised with CD206 MMP-14 positive macrophages were more abundant whereas TIMP-3 positive macrophages were less abundant in plaques histologicallydesignated as rupture prone We conclude that foam-cells characterised by highMMP-14 and low TIMP-3 expression are prevalentin rupture-prone atherosclerotic plaques independent of pro- or anti-inflammatory activationTherefore reducingMMP-14 activityand increasing that of TIMP-3 could be valid therapeutic approaches to reduce plaque rupture and myocardial infarction

1 Introduction

Plaque rupture accounts for three quarters of myocardialinfarctions [1] Weakening of the fibrous cap owing to netdegradation of extracellular matrix is believed to play animportant part in plaque rupture [2] Foam-cell macrophages(FCMs) are an abundant source of several matrixmetallopro-teinases (MMPs) in human plaques [3 4] Protective effects ofcertainMMPs on smoothmusclemigration and proliferationhave been identified from experimental studies but highlevels of MMP production especially from FCMs have beenimplicated in plaque progression and rupture [2] Attention

has focused recently onMMP-14 which is an activatedmem-brane type MMP and a tissue inhibitor of MMPs (TIMP-3) which binds to the extracellular matrix Given theseproperties MMP-14 and TIMP-3 are likely to play importantdivergent roles in the regulation of macrophage pericellularproteolysis Adoptive transfer of leukocytes lacking MMP-14into LDL-receptor null mice increased plaque collagen con-tent implying a harmful collagen-depleting role of MMP-14in plaque stability [5] Conversely transgenic overexpressionof TIMP-3 in mouse macrophages reduced atherosclerosisformation and improved markers of plaque stability [6]A MMP-14hi TIMP-3lo subpopulation of rabbit FCMs was

Hindawi Publishing CorporationMediators of InflammationVolume 2014 Article ID 276457 17 pageshttpdxdoiorg1011552014276457

2 Mediators of Inflammation

shown to have greater proteolytic capacity ability to invadethrough synthetic extracellular matrix (ECM) and greaterpropensity to undergo apoptosis all of which would beexpected to favour plaque rupture [7] MMP-14hi TIMP-3loFCMs were identified in human atherosclerotic plaques[7] but the mechanisms responsible for generating theMMP-14hi TIMP-3lo FCMs and their relationship to plaquevulnerability in man were not previously investigated

Macrophages can express a spectrum of different acti-vation states dependent on pro- and anti-inflammatory sig-nals [8 9] although the effects on MMP-14 and TIMP-3expression are not fully documented For example activationby Toll-like receptor agonists [10] and other inflamma-tory cytokines acting through nuclear factor-120581B (NF-120581B)upregulates several MMPs [11] Such activated macrophageshave been associated with unstable plaque morphologywhereas deactivated or alternatively activated macrophagesare associated with increased plaque stability in most [12ndash14] although not all studies [15] Phenotypes resulting fromoxidised phospholipids [16] or by the action of haem at sitesof thrombosis and intraplaque haemorrhage have also beenrecently distinguished [17ndash19]

To clarify the nature and role of MMP-14hi TIMP-3loFCMs we investigated their activation in vitro with pro- andanti-inflammatory molecules We then investigated whethermacrophage populations with opposing expression of MMP-14 and TIMP-3 colocalise with markers of macrophage acti-vation and correlatewith histologicalmarkers of vulnerabilityto rupture in human carotid atherosclerotic plaques

2 Materials and Methods

21 Carotid Artery Specimens Carotid endarterectomy spec-imens came from the AtheroExpress Biobank the detailsof which have been described in detail elsewhere [20ndash22]Briefly patient demographics including cardiovascular riskfactors previous medication use and presenting symptomswere recorded Atherosclerotic plaques were dissected bya dedicated technician into 5120583m thick cross-sectional seg-ments Histological staining (haematoxylin and eosin (HE)Picrosirius Red (PS)) were performed on the so-called culpritlesion (segment with largest plaque area) The area occu-pied by following parameters was scored semiquantitativelyas described previously [20ndash22] atheroma core (HE PS)calcification (HE) collagen (PS) smoothmuscle cells (120572SM-ctin M0851 Dako) and macrophages (CD68 M0814 Dako)(Table 1) Smooth muscle cells and macrophages were alsoscored quantitatively by using a microscope equipped witha digital camera and using AnalySIS 32 software (SoftImaging System GmbH Munster Germany) The amount ofmicrovessels was quantified by using an anti-CD34 antibodydetails have been described previously [20ndash22] As specifiedin the AtheroExpress protocol histological sections wereclassified according to overall appearance into ldquoatheroma-tous lesionsrdquo containing a large lipid core (gt40 of plaquearea) high macrophage infiltration with low smooth musclecell and collagen content ldquofibrous lesionsrdquo with a small(lt40) or absent lipid core low macrophage content and

high smooth muscle cell and collagen content and ldquofibrous-atheromatous lesionsrdquo as an intermediate between the twoother phenotypes The inter- and intraobserver variabilityfor this classification has been described previously [23]Adjacent sections were then taken for the histological anal-yses reported here which were performed and analysed by2 additional operators who were blinded as to the lesionclassification

22 Immunohistochemistry Sections were dewaxed in Clear-ene and rehydrated in absolute alcohol After rinsing indistilled water slides were subjected to heat-induced antigenretrieval by incubation in citrate buffer (10mM citric acidin 1x PBS pH 60) and microwaved for 10 minutes at fullpower Slides were allowed to cool for 30 minutes andthen washed with PBS (3 times 2 minutes) For brightfield orfluorescence IHC 50 120583L of 1 bovine serum albumin orImage-iT FX Signal Enhancer (Invitrogen Life Technolo-gies Paisley UK) respectively was placed on sections andincubated at room temperature for 30 minutes The solutionwas then removed by tapping the slides and 50 120583L of theapplicable primary antibody (as detailed in Table 1) addedto the samples and incubated either overnight at 4∘C orfor 1 hour at room temperature Sections were then washedin PBS (3 times 2 minutes each) and incubated in the desiredsecondary antibody as detailed in Table 1 For fluorescentlabelling sections were incubated in the dark for 1 hour atroom temperature Sections were then washed in PBS (3 times 2minutes) and mounted with ProLong Gold antifade reagentcontaining DAPI (Invitrogen Life Technologies Paisley UK)to identify the nuclei A negative control where the primaryantibody was replaced with the relevant species IgG atthe same dilution was always included and the cells werecounted in 6 times 20 magnification fields Positive cells werecounted and expressed as a percentage of total nucleatedcells The percentage of CD68 positive macrophages thatwere also positively stained for other proteins of interestwas counted by comparing serial sections or after dual-immunohistochemistry if the antibodies were suitable byindependent observers (JLJ NPJ VWPS) and the resultsaveraged as described previously [24]

For dual fluorescence primary antibodies from differentspecies were used and incubated together Subsequentlyspecies-specific fluorophore conjugated secondary antibodieswere used to yield either a red fluorescent product at the siteof the antigen (AlexaFluor 594) or a green fluorescent productat the site of the antigen (AlexaFluor 488) Sections werethen mounted in ProLong Gold Antifade Reagent with 410158406-diamidino-2-phenylindole (Life Technologies Paisley UKP-36931) to fluorescently label nuclei blue The specificityof the immunolabeling was demonstrated by inclusion of anegative control using isotype-specific nonimmune serum orIgG Images were acquired from a fluorescence microscopeusing ImageProPlus image analysis software (Media Cyber-netics) and merged if required before further analysis Thepercentage of MMP-14 positive macrophages that stained forother proteins was counted within each lesion and expressedas percentage of total MMP-14 positive macrophages

Mediators of Inflammation 3

Table 1 Antibodies used for immunohistochemistry

Antibody Supplier Cat no Species Dilution (Western) Dilution (IHC) Secondary antibodyArginase-1 Santa Cruz sc-20150 Rabbit na 150 Goat anti-rabbitCD206 RampD AF2534 Goat 1100 150 Chicken anti-goatCD68 Dako M0876 Mouse na 1100 Chicken anti-mouseCOX-2 Abcam AB15191 Rabbit 12000 1200 Goat anti-rabbitMMP-14 Millipore MAB3317 Mouse 11000 1200 Chicken anti-mouseNF-kB (p65) Abcam AB7970 Rabbit 1500 150 Goat anti-rabbitTIMP-3 Millipore MAB3318 Mouse 11000 1200 Chicken anti-mouse

23 Studies with Isolated PrimaryHumanMacrophages Peri-pheral blood mononuclear cells were isolated by differentialcentrifugation from whole blood of healthy donors whichwere collected under South West 4 Research Ethics Com-mittee reference 09H010722 Blood (24mL per donor) wasdiluted with Dulbeccorsquos Phosphate Buffered Saline (PBS)without calcium and magnesium (Lonza) 1x (ratio 1 1)The diluted samples were subjected to density gradientseparation on Ficoll Paque Plus (ratio 1 1) (GE HealthcareLife Sciences Buckinghamshire UK) and centrifuged Aftercentrifugation the PBMC layer was collected and washedin Hankrsquos Balanced Salt Sodium (HBSS) with phenol redwithout calcium and magnesium (Lonza) Monocytes wereisolated by adhering the peripheral blood mononuclear cellsto tissue culture plastic for 2 hours at a concentration of25 times 10

6 cellsmL Monocytes were cultured in RPMI mediawith 2mM L-glutamine 100 IUmL penicillin 100 120583gmLstreptomycin 10 fetal bovine serum (FBS Lonza Sigma)and 20 ngmL recombinant human macrophage-colony-stimulating factor (R amp D systems) The medium included85 120583gmL of fully oxidised low-density lipoprotein oxLDL(Intracel) from day 5 when foam-cell macrophages (FCM)were generated At Day 7 the medium was removed andreplaced with RPMI media supplemented with 5 FCSRecombinant human interferon-120574 (10 ngmL R amp D systems)and lipopolysaccharide-LPS (200 ngmL Escherichia coli026B6 Sigma-Aldrich) were added for 24 hours to generateclassically activated macrophages (M1) [25] or recombinanthuman interleukin-4 (20 ngmL R amp D systems) to obtainalternatively activated macrophages (M2) [26]

24 Reverse Transcription-Polymerase Chain Reaction (RT-PCR) RNA samples frommacrophages were collected usingRLT solution (Qiagen) with 120573-mercaptoethanol (120573-ME1 100 dilution) Total RNA was extracted using RNeasy Minikit (Qiagen) according to the manufacturerrsquos instructionsRNA was quantified using a Nanodrop Spectrophotometerand cDNA generated using a QuantiTect Reverse Transcrip-tion Kit (100ndash200 ng RNA per reaction Qiagen) Real-timequantitative PCR was performed in a Roche Light Cycler 15to quantify the steady-state concentration of RNA using aQuantiTect SYBR Green PCR Kit and primers as detailed inTable 2 The reaction contained 36ndash73 ng RNA and 05120583Mprimers Denaturation for 15min at 95∘C was followed by 60cycles of denaturation (15 seconds at 95∘C) annealing for 20

seconds and extension for 25 seconds at 72∘C Copy numbersof mRNA were calculated using standard curves constructedusing the respective PCR products eluted from agarose gelsAll fragments were sequenced to confirm identity (CogenicsTakeley UK) Primers used and their annealing temperaturesare in Table 2

25 Western Blotting Adherent cells were washed with PBSon ice and then lysed with ice-cold 1 SDS lysis buffer TheBicinchoninic acid (BCA) Kit (Pierce) was used to estimateprotein concentration (all in duplicate) of cell lysates accord-ing to kit protocol Protein concentration was read at 560 nmon a Multiskan Ascent (Thermo Electron Corporation) platereader NuPAGENovex Bis-TrisMini Gel system (InvitrogenLife Technologies Paisley UK) was used for western blotexperiments 10 120583g of total protein was loaded on 4ndash12Bis-Tris-HCl buffered (pH 64) polyacrylamide precast gels(Invitrogen Life Technologies Paisley UK) and run inMES-SDS Running Buffer 1x (Invitrogen Life TechnologiesPaisley UK) according to manufacturer instructions Afterelectrophoresis gels were transferred to a 045 120583m nitrocel-lulose membrane in NOVEX NuPAGE Transfer Buffer 1xplus 5 Methanol (vv) Following transfer the nitrocellu-lose membrane was blocked in 5 milk solution in Trisbuffered solution containing Tween (TBS-T) (200mM Tris2 Tween 20 pH 76) The membrane was then incubatedover night at 4∘C using the antibodies described in Table 1diluted in 2mL of SignalBoost Solution 1 (Calbiochem-Novabiochem Ltd Nottingham UK) The following day themembrane was incubated with 5 milk solution in TBS-Tand then with the applicable horseradish peroxidase (HPR)-labelled secondary antibody (Dako Dorset UK) diluted1 1000 in SignalBoost Solution 2 (Calbiochem-NovabiochemLtd Nottingham UK) After incubation the membrane waswashed in TBS-T To detect the peroxidise labelled proteinsan enhanced chemiluminescence (ECL) detection system(GE Healthcare Life Sciences Buckinghamshire UK) wasused The membrane was incubated with ECL reagent mix(solution A and solution B at a ratio of 1 1) and exposed toX-ray film for the desired length of time As a loading control05 120583gmL of either anti-120573-actin antibody (A2228 Sigma)or anti-GAPDH antibody (MAB374 Millipore) was usedDetected bands were quantified using a Bio-Rad GS-690scanning densitometer (Bio-Rad Hemel Hempstead UK)and were normalised by relevant 120573-actin or GAPDH values


Table 2 Primers for quantitative RT-PCR

Primer Sequence Annealing temp (∘C) Fragment size (bp)

MMP-14 Forward GGAGACAAGCATTGGGTGTT 60 343Reverse GGTAGCCCGGTTCTACCTTC

TIMP-3 Forward CTTCCGAGAGTCTCTGTGGCCTTA 60 230Reverse CTCGTTCTTGGAAGTCACAAAGCA

COX-2 Forward GCCATGGGGTGGACTTAAATCATA 60 168Reverse CAGGGACTTGAGGAGGGTAGATCA

CD206 Forward CGGTGACCTCACAAGTATCCACAC 58 216Reverse TTCATCACCACACAATCCTCCTGT

IkB120572 Forward CTACTGGACGACCGCCACGACAGC 60 58Reverse CGAGGCGGATCTCCTGCAGCTCCTTG

36B4 Forward GCCAGCGAAGCCACGCTGCTGAAC 60 76Reverse CGAACACCTGCTGGATGACCAGCCC

26 Statistical Methods For histomorphometry differencesbetween categorical and continuous variables were analysedby using an independent Studentrsquos 119905-test in case of normallydistributed data and a Mann-Whitney 119880 test in case of notnormally distributed data Differences between ordinal andcontinuous data were analysed by using a Kruskal-Wallistest in case of not normally distributed data Differencesbetween dichotomous and categorical data were analysed byusing a Chi-square test Differences between 2 continuousvariables were analysed with a Spearman correlation testin case of not normally distributed data In all cases 119875 lt005 was considered significant All statistical analysis wasperformed with SPSS version 17 (SPSS Inc Chicago ILUSA) Quantitative RT-PCR results normalised to the valuesfor 18 s RNA were transformed to logarithms and analysedby ANOVA followed by Studentrsquos 119905-test with Bonferronicorrection

3 Results

31 MMP-14 and TIMP-3 mRNA Expression in Human Pri-mary Macrophages and FCMs In Vitro Human monocyte-derivedmacrophages were differentiated withM-CSF (MMs)and some from each batch were treated with oxidised LDLto generate foam-cell macrophages (FCMs) as previouslyvalidated in our laboratory [27] Conversion of MMs toFCMs did not affect mRNA expression of MMP-14 or TIMP-3 (Figure 1(a)) or COX-2 and I120581B120572 markers of proinflamma-tory activation or CD206 a gene that is present constitutivelybut upregulated by anti-inflammatory activation [12 13](Figure 1(a)) Proinflammatory activation with LPS and IFN120574(referred to as M1 conditions [26]) significantly increasedexpression of MMP-14 by 45- and 73-fold I120581B120572 mRNA (asurrogate marker of NF120581B signalling [28]) by 75- and 67-fold andCOX-2mRNAby 146- and 187-fold inmacrophages(MM1) and FCMs (FCM1) respectively (Figure 1(a)) Bycontrast proinflammatory activation decreasedmRNA levelsof TIMP-3 by 74 and 89 and CD206 by 81 and 86in macrophages or FCMs respectively (Figure 1(a)) Anti-inflammatory activation with IL-4 (referred to as M2 condi-tions [26]) had no effect on MMP-14 COX-2 or I120581B120572mRNA

levels but significantly increased TIMP-3 mRNA expressionby 19- and 26-fold and CD206 expression by 75- and99-fold in MMs (MM2) and FCMs (FCM2) respectively(Figure 1(a))

32 MMP-14 and TIMP-3 Protein Expression in Macrophagesand FCMs In Vitro Consistent with the mRNA dataproinflammatory activation significantly elevated proteinlevels of MMP-14 and COX-2 and significantly decreasedthose of TIMP-3 and CD206 in nonfoamy macrophages(Figures 1(b) and 1(c)) Anti-inflammatory activation (MM2)also increased macrophage TIMP-3 and CD206 proteinlevels (Figures 1(b) and 1(d)) consistent with the mRNAdata We conclude that proinflammatory macrophagesare MMP-14hi TIMP-3lo whereas unactivated and anti-inflammatory stimulated macrophages are comparativelyMMP-14lo TIMP-3hi Foam-cell formation significantlyincreased protein expression of MMP-14 and decreasedTIMP-3 levels (Figures 1(e) and 1(f)) despite not changingthe mRNA levels in agreement with our previous findingsin during rabbit macrophage foam-cell formation [7] Theseresults could be explained by the additional involvementof a posttranscriptional mechanism such as regulationby microRNA (miR) Indeed we recently showed thathigh levels of miR-24 in nonfoamy macrophages limitmacrophage MMP-14 protein expression [29] We observeda 70 decrease in miR-24 levels in FCMs compared to MMs(Figure 1(g)) consistent with the increased MMP-14 proteinlevels Interestingly in contrast to mRNA expression pro- oranti-inflammatory activation of FCMs did not affectMMP-14protein expression (Figure 1(h)) which suggests that theeffect of the miR is dominant Pro- or anti-inflammatoryactivation of FCMs did not affect TIMP-3 protein expressioneither when compared to nonactivated FCMs (Figure 1(i))TIMP-3 protein expression may also be subject to miRregulation but this has not yet been demonstrated inmacrophages In summary our results imply that nonfoamymacrophages are MMP-14hi TIMP-3lo when classicallyactivated whereas FCMs are MMP-14hi TIMP-3lo at theprotein level irrespective of their activation status


10

100

1000

10000

100000

Cop

y nu

mbe

rng

MMMM1MM2

FCMFCM1FCM2

MMP-14 COX-2 TIMP-3 CD206I120581B120572

lowastlowast

lowast

lowast

lowast

$ $ DaggerDagger

RNA

(a)

0001001

011

10100

1000

Fold

chan

ge

MM1MM2

MMP-14 COX-2 TIMP-3 CD206

lowast

lowastlowast

lowast

lowast

lowast

com

pare

d to

MM

(b)

MM MM1 MM2

GAPDH

GAPDH

MMP-14

COX-2

(c)

CD206

GAPDH

GAPDH

TIMP-3

MM MM1 MM2

(d)

MMP-14

0

1

2

3

4

5

6

Fold

chan

ge co

mpa

red

to M

M

MM FCM

lowast

120573-Actin

(e)

MM FCM

TIMP-3

0

02

04

06

08

1

12

Fold

chan

ge co

mpa

red

to M

M

lowastlowastlowast

120573-Actin

(f)

0

05

1

15

MM FCM

lowast

Fold

chan

ge in

miR

-24

com

pare

d to

MM

(g)

0

02

04

06

08

1

12

14

FCM FCM1 FCM2

Fold

chan

ge co

mpa

red

to F

CM

MMP-14

120573-Actin

(h)

Figure 1 Continued


0

1

2

3

4

FCM FCM1 FCM2

Fold

chan

ge co

mpa

red

to F

CM

TIMP-3

120573-Actin

(i)

Figure 1 (a) mRNA expression of M-CSF differentiated macrophages (MM) and foam-cell macrophages (FCM) after activation with IFN120574and LPS (MM1 and FCM1) or IL-4 (MM2 and FCM2) Data are mean plusmn SEM 119899 = 7 lowast = 119875 lt 005 compared to MM and MM2 =119875 lt 005 compared to FCM and FCM2 $ = 119875 lt 005 compared to MM and MM1 and Dagger = 119875 lt 005 compared to FCM and FCM1((b)ndash(d)) Densitometric quantification and representative Western blots for MMP-14 COX-2 TIMP-3 and CD206 in M-CSF differentiatedmacrophages (MM) or after activation with IFN120574 and LPS (MM1) or IL-4 (MM2) Data are meanplusmn SEM lowast119875 lt 005 compared to MM 119899 = 8((e)-(f)) Densitometric quantification and representativeWestern blots forMMP-14 (e) and TIMP-3 (f) inM-CSF differentiatedmacrophages(MM) and foam-cell macrophages (FCM) Data are mean plusmn SEM lowast119875 lt 005 compared to MM lowast119875 lt 0001 compared to MM 119899 = 4(g) QPCR expression of miR-24 in M-CSF differentiated macrophages (MM) and foam-cell macrophages (FCM) Data are mean plusmn SEMlowast

119875 lt 005 compared to MM ((h)-(i)) Densitometric quantification and representative Western blots for MMP-14 (h) and TIMP-3 (i) infoam-cell macrophages (FCM) after activation with IFN120574 and LPS (FCM1) or IL-4 (FCM2) Data are mean plusmn SEM 119899 = 4

33 Localisation of MMP-14 and TIMP-3 in Human CarotidAtherosclerotic Plaques Macrophages and FCMs were iden-tified in plaques using immunohistochemistry for CD68The CD68 positive cells varied widely in morphology fromlarge highly foamy cells to those with very few or nolipid inclusions We counted all cells irrespective of mor-phology MMP-14 positive macrophagesFCMs were foundpredominantly in the shoulder regions (SR) of atheromatouscarotid plaques (Figures 2(a)ndash2(c)) whereas TIMP-3 posi-tive macrophagesFCMs occurred predominantly within andaround the fibrous cap (FC) of fibrous atheromatous plaques(Figures 2(d)-2(e)) The specificity of the antibodies used isdemonstrated in Figure 2 In line with our previous findings[7] regions of plaques tended to be either MMP-14 positiveand TIMP-3 negative (Figure 2(g) compared to Figure 2(h))or MMP-14 negative and TIMP-3 positive (Figure 2(i)compared to Figure 2(j)) Dual Immunohistochemistry (asdescribed previously [24]) revealed that in plaque regionswith abundant CD68 positive FCMs MMP-14 colocalisedwith nuclear-localised NF-120581B p65 subunit a recognisedmarker of proinflammatory activation When countedapproximately 80 of MMP-14 positive FCMs also hadnuclear-localised NF120581B (Figures 3(a)ndash3(d)) which impliesthat proinflammatory activation associated with MMP-14upregulation in macrophagesFCMs in plaques To gainfurther insight we employed arginase-1 which is a markerfor anti-inflammatorymousemacrophages [30] and is down-regulated in a subpopulation of human FCMs in advancedplaques [31] Arginase-1 and MMP-14 were coexpressedin more than 50 of intraplaque macrophagesFCMs and

the number of arginase-1 and MMP-14 positive macrophageswas correlated in advanced plaques (1199032 = 0960 119875 lt 00001Figure 4) These results are consistent with our in vitro datashowing high MMP-14 protein levels in FCMs irrespectiveof M1M2 activation state (Figure 4) Conversely to MMP-14 less than 1 of TIMP-3 positive macrophagesFCMsdemonstrated nuclear-localisedNF-120581Bp65 (Figure 5) imply-ing that few were classically activated Approximately 80 ofmacrophagesFCMs that were TIMP-3 positive also stainedfor CD206 which we showed to be expressed in unacti-vated (MM) and alternatively activated (MM2) macrophages(Figures 3(f)ndash3(i)) We therefore concluded that CD206 andTIMP-3 positive macrophagesFCMs in plaques could beunactivated or IL-4 activated

34 Relationship of MMP-14 and TIMP-3 Staining to Histo-logical Features of Plaque Stability and Occurrence of Symp-toms in Carotid Atherosclerotic Plaques We next investigatedthe association between MMP-14 and TIMP-3 staining andplaque composition Carotid plaques entered into AtheroEx-press are given an initial classification into atheromatousfibrous-atheromatous or fibrous plaques based on a largelipid core high abundance of macrophages and low abun-dance of smooth muscle cells (SMCs) and collagen content[20 21 32] (Table 3) Of note male and older patients areoverrepresented in the group with atheromatous plaques(Table 4) By comparison with these previous assignmentswe found that the percentage of MMP-14 (Figure 6(a)) andCOX-2 (Figure 6(b)) positive macrophagesFCMs was sig-nificantly greater in atheromatous or fibrous-atheromatous


NC

SR

CD68 250120583m

(a)

CD68 50120583m

(b)

MMP-14

(c)

CD68

FC

(d)

CD68CD68

(e)

TIMP-3

(f)

MMP-14

50120583m

(g)

TIMP-3

(h)

MMP-14

(i)

TIMP-3

(j)

Figure 2 Continued


MMP-14

(k)

COX-2

(l)

NF120581B

(m)

TIMP-3

(n)

CD206

(o)

IgG

(p)

IgG

(q)

IgG

(r)

IgG

(s)

IgG

(t)

Figure 2 ((a)ndash(f)) Immunolocalisation of macrophages (CD68) MMP-14 and TIMP-3 in human atheromatous ((a)ndash(c)) and fibrous-atheromatous ((d)-(e)) carotid plaques Panels (b)-(c) and (e)-(f) are higher magnification fields of boxes depicted in panels (a) and(d) respectively SR denotes shoulder region NC denotes necrotic core and FC denotes fibrous cap ((g)ndash(j)) Immunohistochemistry ofatheromatous carotid plaques demonstrating MMP-14hi TIMP-3lo ((g) and (h) serial sections) and MMP-14lo TIMP-3hi ((i) and (j) serialsections) foam-cell macrophage regions Arrows indicate same cells in adjoining serial sections ((k)ndash(t)) Immunohistochemical staining(brown product colour) of carotid plaque sections for (k) MMP-14 (l) COX-2 (m) NF120581B (n) TIMP-3 and (o) CD206 and their relevantcontrol IgG on serial sections ((p)ndash(t) resp)

compared to fibrous plaques By contrast the percent-age TIMP-3 (Figure 6(a)) and CD206 (Figure 6(b)) pos-itive macrophagesFCMs increased in fibrous comparedto fibrous-atheromatous and atheromatous carotid plaquesDespite the small number of asymptomatic patients in ourcohort significantly higher MMP-14 positivity was foundin plaques from symptomatic compared to asymptomaticpatients (Figure 6(c)) There was also a trend towards lowerTIMP-3 positivity in plaques from symptomatic patients(Figure 6(c))

Amplifying the relationships to overall plaque designa-tion we found a higher percentage of MMP-14 positivemacrophagesFCMs was associated with plaques showinghigh lipid and macrophage content or decreased SMCnumber which are related to plaque instability whereasthe converse was observed for TIMP-3 positive FCMs(Figure 7) TIMP-3 positive macrophagesFCMs were alsosignificantly associated with increased collagen contentand reduced plaque neovascularisation (Figure 7) othermarkers of plaque stability Furthermore the percentageof MMP-14 positive macrophagesFCMs across all thesections correlated strongly with total macrophage num-ber and negatively with SMC number (Table 5) Percent-age positivity for MMP-14 and COX-2 also correlatedwith each other (Table 5) Conversely the percentage ofTIMP-3 positive macrophagesFCMs strongly correlatednegatively with macrophage number and positively withSMC number (Table 5) Positivity for TIMP-3 correlateddirectly with CD206 and inversely with MMP-14 (Table 5)Other characteristics including the presence of calcificationintraplaque haemorrhage and thrombus formation were alsorecorded Positivity of MMP-14 or TIMP-3 was unrelatedto plaque calcification (data not shown) However plaqueswith intraplaque haemorrhage had a significantly higher

percentage of MMP-14 positive macrophagesFCMs andtended to have a lower percentage of TIMP-3 positive FCMs(Table 6)

4 Discussion

Our main new findings are that macrophages activatedwith proinflammatory stimuli or foam-cell macrophages(FCMs) irrespective of inflammatory activation are MMP-14

hi TIMP-3lo compared to unstimulated (M-CSF differen-tiated MM) or anti-inflammatory (IL-4 activated MM2)macrophages Furthermore MMP-14 positive FCMs aremore abundant and TIMP-3 positive FCMs are less abundantin plaques with vulnerable rather than stable characteristics

Multiple macrophage and FCM phenotypes have beenpreviously described [33 34] Classical macrophage acti-vation in vitro with proinflammatory stimuli is charac-terised by NF-120581B-dependent upregulation of a variety ofadditional proinflammatory mediators and enzyme systemsincluding COX-2 By contrast in vitro activation with anti-inflammatory stimuli such as IL-4 or IL-13mediates a distincttranscriptomic response dependent on STAT-6 phosphory-lation [26 33] Our results demonstrate a prominent effectof classical activation on MMP-14 mRNA expression inmacrophages and FCM [35] consistent with previous workon human monocytes [36] and macrophages [35] demon-strating that NF-120581B activation facilitatesMMP-14 expressionFormation of foam cells per se did not lead to pro- or anti-inflammatory macrophage activation (Figure 1) consistentwith previous studies [16 37] However transformation toFCM increased MMP-14 protein expression regardless ofproinflammatory activation FCMs appear resistant to M1


CD68

CD206

Merged

Rabbit IgG Mouse IgG

CD68

Merged

MMP-14 TIMP-3

(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

(i)

(j)

NF-120581B

Figure 3 Continued


0102030405060708090

100

DualTIMP3CD206

TIMP3 CD2060

102030405060708090

Cytoplasmic Negative

100

(k) (l)

MM

P-14

posit

ive F

CMs (

)

Posit

ive F

CMs (

)

NF120581BNuclear NF120581B

Figure 3 (a)ndash(j) Representative images of immunohistochemical labelling of unstable ((a)ndash(e)) and stable ((f)ndash(j)) human carotid arteryatherosclerotic plaques for macrophages (CD68 panels (a) and (f)) and colocalisation of nuclear-localised NF120581B with MMP-14 ((b)ndash(d))and CD206 with TIMP-3 ((h)ndash(j)) with nuclei counterstained with DAPI (blue) Box in panels (a) and (f) represents higher magnificationin panels (b)ndash(e) and (g)ndash(j) respectively Scale bar in (a) represents 250120583m and is applicable to panels (a) and (f) whereas scale bar in (b)represents 100 120583m and is applicable to panels (b)ndash(e) and (g)ndash(j) Arrows in panels (b)ndash(d) indicate MMP-14 +ve FCMs with nuclear NF120581BPanels (e) and (j) represent negative controls where the primary antibodies were replaced with the relevant species IgG (k)The percentage ofMMP-14 positive FCMs that also stained for nuclear-localised NF120581B was significantly greater (lowastlowastlowast119875 lt 0001) compared to only cytoplasmicNF120581B or no staining (l) The percentage of TIMP-3 positive FCMs that stained also for CD206 was significantly greater (lowastlowastlowast119875 lt 0001)compared to those stained for TIMP-3 or CD206 alone

Table 3 Atherosclerotic plaque characteristics

Plaque characteristics Plaque phenotype Gradation 119875 value

fatatheromaNolt40gt40

Fibrous 15 (50) 15 (50) 0 (0)00001Fibrous-atheromatous 0 (0) 11 (92) 1 (8)

Atheromatous 0 (0) 2 (5) 35 (95)

Collagen semiquantitative(minormoderateheavy)


Atheromatous 17 (46) 19 (51) 1 (3)

SMC semiquantitative(nominormoderateheavy)

Fibrous 0 (0) 0 (0) 10 (33) 20 (67)00001Fibrous-atheromatous 0 (0) 3 (25) 7 (58) 2 (17)

Atheromatous 2 (5) 30 (81) 5 (14) 0 (0)

SMC quantitative plaque area median [IQR]

Fibrous 431 [173ndash569]00001Fibrous-atheromatous 131 [039ndash462]

Atheromatous 053 [031ndash107]

Macrophagesemiquantitative(nominormoderateheavy)


Atheromatous 0 (0) 0 (0) 19 (51) 18 (49)

Macrophage quantitative plaque area median [IQR]



Thrombus present(nominormoderateheavy)


Atheromatous 4 (11) 10 (27) 16 (43) 7 (19)

CD34 stainingNumber of vessels median[IQR]


Atheromatous 70 [43ndash110]119875 values for differences among the three groups of semiquantitative histological markers and percentage of macrophages and smooth muscle cells weredetermined using the Kruskal-Wallis test


MMP-14

(a)

Arg-1

(b)

DAPI

(c)

Merge

(d)

MMP-14 MMP-14Arg-1

Arg-10

10

20

30

40

50

60

70

Posit

ive F

CMs (

)

lowast

(e)

0

20

40

60

80

100

120

140

160

0 50 100 150 200 250

Arg

-1 p

ositi

ve F

CMs (

)

MMP-14 positive FCMs ()

r2 = 0960

P lt 00001

(f)

Figure 4 ((a)ndash(d)) Representative images of immunohistochemical labelling of advanced human carotid artery atherosclerotic plaques forcolocalisation of MMP-14 (a) with arginase-1 (b) with nuclei counterstained with DAPI (blue (c)) Scale bar in (a) represents 100 120583m and isapplicable to panels (a)ndash(d) (e)The percentage of MMP-14 positive FCMs that also stained for arginase-1 was significantly greater comparedto only MMP-14 or arginase-1 (lowast119875 lt 005 119899 = 10 per group data expressed as mean plusmn SEM) (f) Correlation of arginase-1 and MMP-14positive macrophages in human carotid artery atherosclerotic plaques (1199032 = 0960 119875 lt 00001 119899 = 10)

and M2 cytokine-stimulation but employ a posttranscrip-tional pathway to modulate MMP-14 protein expression andsubsequent activity Indeed we have recently identified theinvolvement of a microRNA miR-24 in the direct regulationof macrophage MMP-14 protein expression [29] Further

evidence for a novel pathway in regulating FCM MMP-14 protein expression is provided by our demonstrationthat MMP-14 positive macrophagesFCMs in plaques corre-lated with classical activation markers COX-2 and nuclear-localised NF-120581B and with the well-recognised marker of


DAPI50120583M

(a)

NF120581B

(b)

TIMP-3

(c)

Merged

(d)

0102030405060708090

100

Negative

TIM

P-3

posit

ive F

CMs (

)

lowast

Cytoplasmic NF120581BNuclear NF120581B

(e)

Figure 5 Immunohistochemical staining of carotid plaque sections for (a) 410158406-diamidino-2-phenylindole (DAPI) (b) NF120581B (c) TIMP-3 and (d) merged demonstrating that the percentage of TIMP-3 positive FCMs with no nuclear NF120581B was significantly greater (119899 = 20lowast

119875 lt 00001) compared to cytoplasmic or nuclear NF120581B staining as depicted in adjoining graph (e)

alternative activation arginase-1 Hence foam-cell formationrather than M1 or M2 macrophage polarisation appearsresponsible for the MMP-14hi FCMs detected in advancedhuman atherosclerotic plaques

By contrast to MMP-14 TIMP-3 mRNA was decreasedduring proinflammatory activation of macrophages or dur-ing foam-cell formation A combination of these factorsprobably accounts for the TIMP-3lo populations seen inplaques TIMP-3 positive macrophagesFCMs correlated and

colocalised with the mannose receptor (MR) CD206 Inagreement with other previous studies [38] we found thatCD206 and TIMP-3 were highly expressed in macrophagesdifferentiated in M-CSF (MM) and upregulated by IL-4(MM2) However TIMP-3 expression was only doubledafter IL-4 treatment (Figure 1) and is therefore present inboth phenotypes MR positive macrophagesFCMs werepreviously reported to be mainly located away from thelipid core of plaques [12 13] consistent with the location of


Posit

ive F

CM (

)

Atheromatous 37Fibrous-atheromatous 12 11

MMP-14 TIMP-3

0

20

40

60

80

100

Fibrous 30 21

Overall plaque phenotype35

MMP-14lowastlowastlowast TIMP-3lowastlowastlowast

(a)Po

sitiv

e FCM

()

MMP-14 TIMP-3

0

20

40

60

80

100

Atheromatous 19Fibrous 13 9

Overall plaque phenotype

COX-2lowastlowastlowast CD206lowast

18

(b)

Asymptomatic 10 6Symptomatic 69 61

0

20

40

60

80

100

Posit

ive F

CM (

)

MMP-14 TIMP-3

MMP-14lowastlowast TIMP-3

(c)

Figure 6 Relationship betweenMMP-14 and TIMP-3 staining and plaquemorphology or symptomsThe percentage of CD68 positive FCMsthat stained also for (a) MMP-14 and TIMP-3 or (b) COX-2 and CD206 was related to atheromatous fibrous-atheromatous and fibrouscarotid plaques (c) Association of FCMs that stained also for MMP-14 and TIMP-3 was assessed in symptomatic and asymptomatic patientsData analyzed using a Kruskal-Wallis test (lowast119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001) Patient numbers within each group are depicted below eachbar graph

TIMP-3 positive macrophagesFCMs in our study Althoughlittle is known about the regulation of TIMP-3 transcrip-tion [39] the striking decrease in protein expression weobserved upon foam-cell formation is consistent with pre-vious observations that posttranscriptional mechanisms canregulate TIMP expression during macrophage development[40] Studies beyond the present scope are going on in ourlaboratories to investigate this possibility

Injurious roles for MMP-14 and protective effects ofTIMP-3 have been suggested from animal studies (see theIntroduction) Consistent with these proposals we found thatMMP-14 positive FCM subpopulations were associated with

histological features of plaque vulnerability in carotid plaquesbased primarily on correlating quantitative plaque character-istics derived from all the sections irrespective of histologicalappearance (Table 4)The conclusions were also confirmed incarotid atherosclerotic plaques by prior blinded assignmentof lesions based on overall histological appearance (Figures6 and 7) This second analysis can be criticised because itis based on subjective visual observation although we haveshown previously that there is good intraobserver agreement[23] It is also evident that there is a gender imbalancebetween the differing plaque phenotypes which may act asa source of variability in the statistical analysis Nonetheless


0

20

40

60

80

100

Lipid content

No fat

Posit

ive F

CM (

)

lowastlowastlowast lowastlowast

lt40

gt40

MMP-14 TIMP-3

(a)

Macrophages

MinorModerateHeavy

0

20

40

60

80

100

Posit

ive F

CM (

)


(b)

MinorModerateHeavy

Smooth muscle cells

0

20

40

60

80

100

Posit

ive F

CM (

)


MMP-14 TIMP-3

(c)

MinorModerateHeavy

0

20

40

60

80

100

Collagen

Posit

ive F

CM (

)lowast

MMP-14 TIMP-3

(d)

0

20

40

60

80

100

Vessel density (tertiles)

Posit

ive F

CM (

)

lowastlowast

1st2nd3rd

MMP-14 TIMP-3

(e)

Figure 7 The percentage of MMP-14 and TIMP-3 positive foam-cell macrophages (median [95 confidence limits]) was compared withsemiquantitative plaque features and 119875 values computed with a Kruskal-Wallis test (lowast119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001)


Table 4 Patient characteristics

Patient characteristics Fibrous plaques(119899 = 30)

Fibrous-atheromatous(119899 = 12)

Atheromatous plaques(119899 = 37) 119875 value

Symptomatic patients 119899 () 22 (73) 11 (92) 36 (97) 0012lowast

Amaurosis fugax 119899 () 6 (20) 5 (42) 1 (3)

0010lowastTIA 119899 () 12 (40) 4 (33) 25 (68)Stroke 119899 () 4 (13) 2 (17) 10 (27)

Asymptomatic 119899 () 8 (27) 1 (8) 1 (3)M (D)F (C) 11D 19C 11D 1C 34D 3C 0007lowast

Age mean (sd) 65 (96) 73 (81) 70 (77) 0016lowast

Hypertension 119899 () 22 (73) 10 (83) 31 (84) 0540Smoking 119899 () 9 (30) 5 (42) 13 (35) 0761Diabetes 119899 () 5 (17) 0 (0) 4 (11) 0304Hypercholesterolemia 119899 () 17 (57) 6 (50) 20 (54) 0924Positive family history of heart disease 7 (23) 2 (17) 10 (27) 0761History of coronary intervention 119899 () 7 (23) 3 (25) 8 (22) 0967History of peripheral intervention 119899 () 6 (20) 1 (8) 10 (27) 0379History of myocardial infarction 4 (13) 3 (25) 1 (3) 0064Statins 16 (53) 5 (42) 26 (70) 0147Oral anticoagulants 6 (20) 4 (33) 3 (8) 0098Aspirin 18 (60) 3 (25) 17 (46) 0114Carbasalate calcium 8 (27) 8 (67) 20 (54) 0023lowast

Aspirin or Carbasalate calcium 24 (80) 9 (75) 34 (92) 0237Dipyridamole 18 (60) 4 (33) 22 (60) 0238Clopidogrel 3 (10) 0 (0) 7 (19) 0198Diuretics 9 (30) 2 (17) 11 (30) 0881Beta blockers 13 (43) 3 (25) 18 (49) 0355Calcium antagonists 6 (20) 3 (25) 12 (32) 0514ACE inhibitors 11 (37) 3 (25) 12 (33) 0764Angiotensin II inhibitors 5 (17) 2 (17) 4 (11) 0755Insulin 2 (7) 0 (0) 0 (0) 0187Oral glucose inhibitors 3 (10) 0 (0) 4 (11) 1000lowast

119875 lt 005

Table 5 Correlations of immunostains with histological parameters for plaque vulnerability in human carotid plaques

Plaque characteristic of MMP-14hi FCMs of TIMP-3hi FCMs119899 rho 119875 value 119899 rho 119875 value

Macrophage density 79 0453 lt0001 68 minus0316 0009SMC density 76 minus0466 lt0001 66 0283 0021 of MMP-14hi FCMs x x x 71 minus0366 0002 of COX-2+ FCMs 32 0726 lt0001 28 minus0185 0345 of CD206+ FCMs 27 minus0013 0948 27 0606 0001Correlations are shown between the percentage of CD68 positive foam-cell macrophages (FCMs) also positive for MMP-14 and TIMP-3 with quantitativehistological markers and percentage of CD68 positive for COX-2 and CD206 using Spearman correlation tests Negative correlations are shown in italics

collectively our results demonstrate that MMP-14 positiveFCMs associate with plaque vulnerability Whereas TIMP-3positive macrophages correlate with plaque stability

In summary our findings show that FCMs in vulner-able atherosclerotic plaques can exhibit increased MMP-14 and decreased TIMP-3 protein expression This leads to

heightened invasive capability increased proliferation andaugmented susceptibility to apoptosis as we have previouslydemonstrated [7] Our work therefore suggests that reducingMMP-14 activity and increasing that of TIMP-3 could bevalid therapeutic approaches to reduce plaque rupture andmyocardial infarction


Table 6 MMP-14 and TIMP-3 expression and plaque characteristics

Plaque feature Absent 119899 Present 119899 119875 valueMMP-14 ( of positive foam cell macrophages)

Intraplaque haemorrhage 24 [0ndash64] 51 67 [28ndash81] 28 0007Any thrombus or haemorrhage 11 [1ndash48] 24 59 [6ndash81] 55 0026

TIMP-3 ( of positive foam cell macrophages)Intraplaque haemorrhage 24 [8ndash43] 44 11 [5ndash25] 24 0095Any thrombus or haemorrhage 28 [8ndash77] 22 13 [5ndash34] 46 0067The percentage ofMMP-14 and TIMP-3 positive foam cell macrophages (median [95 confidence limits]) was compared with semiquantitative plaque featuresand P values computed with a Kruskal-Wallis test

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Jason L Johnson and Nicholas P Jenkins contributed equallyto this study Jason L Johnson and Andrew C Newbydesigned the study Jason L Johnson Wei-Chun HuangKarina Di Gregoli and Graciela B Sala-Newby performedthe cell culture studies and qPCR analysis Jason L JohnsonNicholas P Jenkins and Vincent P W Scholtes performedthe immunohistochemical staining and analysis Jason LJohnson Nicholas P Jenkins Vincent P W Scholtes Frans LMoll and Gerard Pasterkamp participated in the collectionof patient details handling of human tissues pathologicalevaluation and phenotyping Jason L Johnson Nicholas PJenkins and Andrew C Newby wrote the paper All authorsread and approved the final paper

Acknowledgments

The authors acknowledge the skillful technical assistanceof Ms Michelle Sommerville and Mrs Laura Bevan Theauthorsrsquo work is supported by the British Heart Foundation(CH95001 RG04009 FS0705324069) and the NationalHealth Research Institute (UK) Bristol Biomedical ResearchUnit in Cardiovascular Medicine W-C Huang held Fellow-ships from the Yen Tjing Ling Medical Foundation andMedical Foundation in Memory of Dr Deh-Lin ChengTaiwan

References

[1] R Virmani A P Burke A Farb and F D Kolodgie ldquoPathologyof the vulnerable plaquerdquo Journal of the American College ofCardiology vol 47 no 8 pp C13ndashC18 2006

[2] A C Newby ldquoDual role of matrix metalloproteinases (matrix-ins) in intimal thickening and atherosclerotic plaque rupturerdquoPhysiological Reviews vol 85 no 1 pp 1ndash31 2005

[3] C M Dollery and P Libby ldquoAtherosclerosis and proteinaseactivationrdquo Cardiovascular Research vol 69 no 3 pp 625ndash6352006

[4] A C Newby ldquoMatrix metalloproteinase inhibition therapy forvascular diseasesrdquo Vascular pharmacology vol 56 no 5-6 pp232ndash244 2012

[5] F Schneider G K Sukhova M Aikawa et al ldquoMatrix metallo-proteinase-14 deficiency in bone marrow-derived cells pro-motes collagen accumulation inmouse atherosclerotic plaquesrdquoCirculation vol 117 no 7 pp 931ndash939 2008

[6] VCasagrande RMenghini SMenini et al ldquoOverexpression oftissue inhibitor of metalloproteinase 3 in macrophages reducesatherosclerosis in low-density lipoprotein receptor knockoutmicerdquoArteriosclerosisThrombosis andVascular Biology vol 32no 1 pp 74ndash81 2012

[7] J L Johnson G B Sala-Newby Y Ismail C M Aguilera andA C Newby ldquoLow tissue inhibitor of metalloproteinases 3 andhighmatrix metalloproteinase 14 levels defines a subpopulationof highly invasive foam-cell macrophagesrdquo ArteriosclerosisThrombosis and Vascular Biology vol 28 no 9 pp 1647ndash16532008

[8] F O Martinez L Helming and S Gordon ldquoAlternative activa-tion of macrophages an immunologic functional perspectiverdquoAnnual Review of Immunology vol 27 pp 451ndash483 2009

[9] D M Mosser and J P Edwards ldquoExploring the full spectrumof macrophage activationrdquo Nature Reviews Immunology vol 8no 12 pp 958ndash969 2008

[10] C Monaco S M Gregan T J Navin B M J Foxwell AH Davies and M Feldmann ldquoToll-like receptor-2 mediatesinflammation and matrix degradation in human atherosclero-sisrdquo Circulation vol 120 no 24 pp 2462ndash2469 2009

[11] A C Newby S J George Y Ismail J L Johnson G B Sala-Newby and A C Thomas ldquoVulnerable atherosclerotic plaquemetalloproteinases and foam cell phenotypesrdquoThrombosis andHaemostasis vol 101 no 6 pp 1006ndash1011 2009

[12] M A Bouhlel B Derudas E Rigamonti et al ldquoPPAR120574activation primes humanmonocytes into alternativem2macro-phages with anti-inflammatory propertiesrdquo Cell Metabolismvol 6 no 2 pp 137ndash143 2007

[13] G Chinetti-Gbaguidi M Baron M A Bouhlel et al ldquoHumanatherosclerotic plaque alternative macrophages display lowcholesterol handling but high phagocytosis because of distinctactivities of the PPAR120574 and LXR120572 pathwaysrdquo CirculationResearch vol 108 no 8 pp 985ndash995 2011

[14] J L Stoger M J J Gijbels S van der Velden et al ldquoDistributionof macrophage polarization markers in human atherosclerosisrdquoAtherosclerosis vol 225 no 2 pp 461ndash468 2012

[15] A V Finn O Saeed and R Virmani ldquoMacrophage subsetsin human atherosclerosisrdquo Circulation Research vol 110 articlee64 2012

[16] A Kadl A K Meher P R Sharma et al ldquoIdentification ofa novel macrophage phenotype that develops in response toatherogenic phospholipids via Nrf2rdquo Circulation Research vol107 no 6 pp 737ndash746 2010


[17] J J Boyle H A Harrington E Piper et al ldquoCoronaryintraplaque hemorrhage evokes a novel atheroprotectivemacrophage phenotyperdquo The American Journal of Pathologyvol 174 no 3 pp 1097ndash1108 2009

[18] J J Boyle M Johns T Kampfer et al ldquoActivating transcriptionfactor 1 directs Mhem atheroprotective macrophages throughcoordinated iron handling and foam cell protectionrdquo Circula-tion Research vol 110 no 1 pp 20ndash33 2012

[19] A V Finn M Nakano R Polavarapu et al ldquoHemoglobindirects macrophage differentiation and prevents foam cellformation in human atherosclerotic plaquesrdquo Journal of theAmerican College of Cardiology vol 59 no 2 pp 166ndash177 2012

[20] B A N Verhoeven E Velema A H Schoneveld et alldquoAthero-express differential atherosclerotic plaque expressionof mRNA and protein in relation to cardiovascular events andpatient characteristics Rationale and designrdquo European Journalof Epidemiology vol 19 no 12 pp 1127ndash1133 2004

[21] J P G SluijterW P C Pulskens AH Schoneveld et al ldquoMatrixmetalloproteinase 2 is associated with stable and matrix met-alloproteinases 8 and 9 with vulnerable carotid atheroscleroticlesionsmdasha study in human endarterectomy specimen pointingto a role for different extracellular matrix metalloproteinaseinducer glycosylation formsrdquo Stroke vol 37 no 1 pp 235ndash2392006

[22] D P V de Kleijn F L Moll W E Hellings et al ldquoLocalatherosclerotic plaques are a source of prognostic biomarkersfor adverse cardiovascular eventsrdquo Arteriosclerosis Thrombosisand Vascular Biology vol 30 no 3 pp 612ndash619 2010

[23] W E Hellings G Pasterkamp A Vollebregt et al ldquoIntraob-server and interobserver variability and spatial differences inhistologic examination of carotid endarterectomy specimensrdquoJournal of Vascular Surgery vol 46 no 6 pp 1147ndash1154 2007

[24] V P W Scholtes J L Johnson N Jenkins et al ldquoCarotidatherosclerotic plaque matrix metalloproteinase-12-positivemacrophage subpopulation predicts adverse outcome afterendarterectomyrdquo Journal of the AmericanHeart Association vol1 no 6 Article ID e001040 2012

[25] E Monsalve A Ruiz-Garcıa V Baladron et al ldquoNotch1upregulates LPS-induced macrophage activation by increasingNF-120581B activityrdquo European Journal of Immunology vol 39 no 9pp 2556ndash2570 2009

[26] F O Martinez S Gordon M Locati and A Mantovani ldquoTran-scriptional profiling of the human monocyte-to-macrophagedifferentiation and polarization new molecules and patterns ofgene expressionrdquo Journal of Immunology vol 177 no 10 pp7303ndash7311 2006

[27] D Shiffman T Mikita J T N Tai et al ldquoLarge scale geneexpression analysis of cholesterol-loaded macrophagesrdquo TheJournal of Biological Chemistry vol 275 no 48 pp 37324ndash37332 2000

[28] S-C Sun PAGanchiDWBallard andWCGreene ldquoNF-120581Bcontrols expression of inhibitor I120581B120572 evidence for an inducibleautoregulatory pathwayrdquo Science vol 259 no 5103 pp 1912ndash1915 1993

[29] K di Gregoli N Jenkins R Salter S White A C Newbyand J L Johnson ldquoMicrorna-24 regulates macrophage behaviorand retards atherosclerosisrdquo Arteriosclerosis Thrombosis andVascular Biology 2014

[30] A Mantovani C Garlanda andM Locati ldquoMacrophage diver-sity and polarization in atherosclerosis a question of balancerdquoArteriosclerosis Thrombosis and Vascular Biology vol 29 no10 pp 1419ndash1423 2009

[31] A C Thomas G B Sala-Newby Y Ismail J L Johnson GPasterkamp and A C Newby ldquoGenomics of foam cells andnonfoamy macrophages from rabbits identifies arginase-I as adifferential regulator of nitric oxide productionrdquo Arteriosclero-sis Thrombosis and Vascular Biology vol 27 no 3 pp 571ndash5772007

[32] W Peeters W E Hellings D P V de Kleijn et al ldquoCarotidatherosclerotic plaques stabilize after stroke insights into thenatural process of atherosclerotic plaque stabilizationrdquo Arte-riosclerosis Thrombosis and Vascular Biology vol 29 no 1 pp128ndash133 2009

[33] G Chinetti-Gbaguidi and B Staels ldquoMacrophage polarizationin metabolic disorders functions and regulationrdquo CurrentOpinion in Lipidology vol 22 no 5 pp 365ndash372 2011

[34] J L Johnson and A C Newby ldquoMacrophage heterogeneity inatherosclerotic plaquesrdquo Current Opinion in Lipidology vol 20no 5 pp 370ndash378 2009

[35] W-C Huang G B Sala-Newby A Susana J L Johnson andA C Newby ldquoClassical macrophage activation up-regulatesseveral matrix metalloproteinases through mitogen activatedprotein kinases and nuclear factor-120581Brdquo PLoS ONE vol 7 no8 Article ID e42507 2012

[36] B Reel G B Sala-Newby W Huang and A C NewbyldquoDiverse patterns of cyclooxygenase-independent metallopro-teinase gene regulation in humanmonocytesrdquo British Journal ofPharmacology vol 163 no 8 pp 1679ndash1690 2011

[37] J SpannNathanael XGarmire LanaGMcDonald Jeffrey et alldquoRegulated accumulation of desmosterol integratesmacrophagelipidmetabolism and inflammatory responsesrdquoCell vol 151 pp138ndash152 2012

[38] R P Fabunmi G K Sukhova S Sugiyama and P LibbyldquoExpression of tissue inhibitor of metalloproteinases-3 inhuman atheroma and regulation in lesion-associated cells apotential protective mechanism in plaque stabilityrdquo CirculationResearch vol 83 no 3 pp 270ndash278 1998

[39] M Wick R Haronen D Mumberg et al ldquoStructure of thehuman TIMP-3 gene and its cell cycle-regulated promoterrdquoBiochemical Journal vol 311 no 2 pp 549ndash554 1995

[40] G A R Doyle U K Saarialho-Kere andW C Parks ldquoDistinctmechanisms regulate TIMP-1 expression at different stages ofphorbol ester-mediated differentiation of U937 cellsrdquo Biochem-istry vol 36 no 9 pp 2492ndash2500 1997

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


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Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


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The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

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Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


shown to have greater proteolytic capacity ability to invadethrough synthetic extracellular matrix (ECM) and greaterpropensity to undergo apoptosis all of which would beexpected to favour plaque rupture [7] MMP-14hi TIMP-3loFCMs were identified in human atherosclerotic plaques[7] but the mechanisms responsible for generating theMMP-14hi TIMP-3lo FCMs and their relationship to plaquevulnerability in man were not previously investigated

Macrophages can express a spectrum of different acti-vation states dependent on pro- and anti-inflammatory sig-nals [8 9] although the effects on MMP-14 and TIMP-3expression are not fully documented For example activationby Toll-like receptor agonists [10] and other inflamma-tory cytokines acting through nuclear factor-120581B (NF-120581B)upregulates several MMPs [11] Such activated macrophageshave been associated with unstable plaque morphologywhereas deactivated or alternatively activated macrophagesare associated with increased plaque stability in most [12ndash14] although not all studies [15] Phenotypes resulting fromoxidised phospholipids [16] or by the action of haem at sitesof thrombosis and intraplaque haemorrhage have also beenrecently distinguished [17ndash19]

To clarify the nature and role of MMP-14hi TIMP-3loFCMs we investigated their activation in vitro with pro- andanti-inflammatory molecules We then investigated whethermacrophage populations with opposing expression of MMP-14 and TIMP-3 colocalise with markers of macrophage acti-vation and correlatewith histologicalmarkers of vulnerabilityto rupture in human carotid atherosclerotic plaques

2 Materials and Methods

21 Carotid Artery Specimens Carotid endarterectomy spec-imens came from the AtheroExpress Biobank the detailsof which have been described in detail elsewhere [20ndash22]Briefly patient demographics including cardiovascular riskfactors previous medication use and presenting symptomswere recorded Atherosclerotic plaques were dissected bya dedicated technician into 5120583m thick cross-sectional seg-ments Histological staining (haematoxylin and eosin (HE)Picrosirius Red (PS)) were performed on the so-called culpritlesion (segment with largest plaque area) The area occu-pied by following parameters was scored semiquantitativelyas described previously [20ndash22] atheroma core (HE PS)calcification (HE) collagen (PS) smoothmuscle cells (120572SM-ctin M0851 Dako) and macrophages (CD68 M0814 Dako)(Table 1) Smooth muscle cells and macrophages were alsoscored quantitatively by using a microscope equipped witha digital camera and using AnalySIS 32 software (SoftImaging System GmbH Munster Germany) The amount ofmicrovessels was quantified by using an anti-CD34 antibodydetails have been described previously [20ndash22] As specifiedin the AtheroExpress protocol histological sections wereclassified according to overall appearance into ldquoatheroma-tous lesionsrdquo containing a large lipid core (gt40 of plaquearea) high macrophage infiltration with low smooth musclecell and collagen content ldquofibrous lesionsrdquo with a small(lt40) or absent lipid core low macrophage content and

high smooth muscle cell and collagen content and ldquofibrous-atheromatous lesionsrdquo as an intermediate between the twoother phenotypes The inter- and intraobserver variabilityfor this classification has been described previously [23]Adjacent sections were then taken for the histological anal-yses reported here which were performed and analysed by2 additional operators who were blinded as to the lesionclassification

22 Immunohistochemistry Sections were dewaxed in Clear-ene and rehydrated in absolute alcohol After rinsing indistilled water slides were subjected to heat-induced antigenretrieval by incubation in citrate buffer (10mM citric acidin 1x PBS pH 60) and microwaved for 10 minutes at fullpower Slides were allowed to cool for 30 minutes andthen washed with PBS (3 times 2 minutes) For brightfield orfluorescence IHC 50 120583L of 1 bovine serum albumin orImage-iT FX Signal Enhancer (Invitrogen Life Technolo-gies Paisley UK) respectively was placed on sections andincubated at room temperature for 30 minutes The solutionwas then removed by tapping the slides and 50 120583L of theapplicable primary antibody (as detailed in Table 1) addedto the samples and incubated either overnight at 4∘C orfor 1 hour at room temperature Sections were then washedin PBS (3 times 2 minutes each) and incubated in the desiredsecondary antibody as detailed in Table 1 For fluorescentlabelling sections were incubated in the dark for 1 hour atroom temperature Sections were then washed in PBS (3 times 2minutes) and mounted with ProLong Gold antifade reagentcontaining DAPI (Invitrogen Life Technologies Paisley UK)to identify the nuclei A negative control where the primaryantibody was replaced with the relevant species IgG atthe same dilution was always included and the cells werecounted in 6 times 20 magnification fields Positive cells werecounted and expressed as a percentage of total nucleatedcells The percentage of CD68 positive macrophages thatwere also positively stained for other proteins of interestwas counted by comparing serial sections or after dual-immunohistochemistry if the antibodies were suitable byindependent observers (JLJ NPJ VWPS) and the resultsaveraged as described previously [24]

For dual fluorescence primary antibodies from differentspecies were used and incubated together Subsequentlyspecies-specific fluorophore conjugated secondary antibodieswere used to yield either a red fluorescent product at the siteof the antigen (AlexaFluor 594) or a green fluorescent productat the site of the antigen (AlexaFluor 488) Sections werethen mounted in ProLong Gold Antifade Reagent with 410158406-diamidino-2-phenylindole (Life Technologies Paisley UKP-36931) to fluorescently label nuclei blue The specificityof the immunolabeling was demonstrated by inclusion of anegative control using isotype-specific nonimmune serum orIgG Images were acquired from a fluorescence microscopeusing ImageProPlus image analysis software (Media Cyber-netics) and merged if required before further analysis Thepercentage of MMP-14 positive macrophages that stained forother proteins was counted within each lesion and expressedas percentage of total MMP-14 positive macrophages



















3 Results





10

100

1000

10000

100000

Cop

y nu

mbe

rng

MMMM1MM2

FCMFCM1FCM2

MMP-14 COX-2 TIMP-3 CD206I120581B120572

lowastlowast

lowast

lowast

lowast

$ $ DaggerDagger

RNA

(a)

0001001

011

10100

1000

Fold

chan

ge

MM1MM2


lowast

lowastlowast

lowast

lowast

lowast

com

pare

d to

MM

(b)

MM MM1 MM2

GAPDH

GAPDH

MMP-14

COX-2

(c)

CD206

GAPDH

GAPDH

TIMP-3

MM MM1 MM2

(d)

MMP-14

0

1

2

3

4

5

6

Fold

chan

ge co

mpa

red

to M

M

MM FCM

lowast

120573-Actin

(e)

MM FCM

TIMP-3

0

02

04

06

08

1

12

Fold

chan

ge co

mpa

red

to M

M

lowastlowastlowast

120573-Actin

(f)

0

05

1

15

MM FCM

lowast

Fold

chan

ge in

miR

-24

com

pare

d to

MM

(g)

0

02

04

06

08

1

12

14

FCM FCM1 FCM2

Fold

chan

ge co

mpa

red

to F

CM

MMP-14

120573-Actin

(h)

Figure 1 Continued


0

1

2

3

4

FCM FCM1 FCM2

Fold

chan

ge co

mpa

red

to F

CM

TIMP-3

120573-Actin

(i)







NC

SR

CD68 250120583m

(a)

CD68 50120583m

(b)

MMP-14

(c)

CD68

FC

(d)

CD68CD68

(e)

TIMP-3

(f)

MMP-14

50120583m

(g)

TIMP-3

(h)

MMP-14

(i)

TIMP-3

(j)

Figure 2 Continued


MMP-14

(k)

COX-2

(l)

NF120581B

(m)

TIMP-3

(n)

CD206

(o)

IgG

(p)

IgG

(q)

IgG

(r)

IgG

(s)

IgG

(t)





4 Discussion





CD68

CD206

Merged


CD68

Merged

MMP-14 TIMP-3

(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

(i)

(j)

NF-120581B

Figure 3 Continued


0102030405060708090

100

DualTIMP3CD206

TIMP3 CD2060

102030405060708090


100

(k) (l)

MM

P-14

posit

ive F

CMs (

)

Posit

ive F

CMs (

)







Atheromatous 0 (0) 2 (5) 35 (95)



Atheromatous 17 (46) 19 (51) 1 (3)



Atheromatous 2 (5) 30 (81) 5 (14) 0 (0)






Atheromatous 0 (0) 0 (0) 19 (51) 18 (49)






Atheromatous 4 (11) 10 (27) 16 (43) 7 (19)





MMP-14

(a)

Arg-1

(b)

DAPI

(c)

Merge

(d)

MMP-14 MMP-14Arg-1

Arg-10

10

20

30

40

50

60

70

Posit

ive F

CMs (

)

lowast

(e)

0

20

40

60

80

100

120

140

160

0 50 100 150 200 250

Arg

-1 p

ositi

ve F

CMs (

)


r2 = 0960

P lt 00001

(f)





DAPI50120583M

(a)

NF120581B

(b)

TIMP-3

(c)

Merged

(d)

0102030405060708090

100

Negative

TIM

P-3

posit

ive F

CMs (

)

lowast


(e)







Posit

ive F

CM (

)


MMP-14 TIMP-3

0

20

40

60

80

100

Fibrous 30 21



(a)Po

sitiv

e FCM

()

MMP-14 TIMP-3

0

20

40

60

80

100




18

(b)


0

20

40

60

80

100

Posit

ive F

CM (

)

MMP-14 TIMP-3


(c)






0

20

40

60

80

100

Lipid content

No fat

Posit

ive F

CM (

)


lt40

gt40

MMP-14 TIMP-3

(a)

Macrophages

MinorModerateHeavy

0

20

40

60

80

100

Posit

ive F

CM (

)


(b)

MinorModerateHeavy

Smooth muscle cells

0

20

40

60

80

100

Posit

ive F

CM (

)


MMP-14 TIMP-3

(c)

MinorModerateHeavy

0

20

40

60

80

100

Collagen

Posit

ive F

CM (

)lowast

MMP-14 TIMP-3

(d)

0

20

40

60

80

100


Posit

ive F

CM (

)

lowastlowast

1st2nd3rd

MMP-14 TIMP-3

(e)








Amaurosis fugax 119899 () 6 (20) 5 (42) 1 (3)






119875 lt 005
















Acknowledgments


References















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


































3 Results





10

100

1000

10000

100000

Cop

y nu

mbe

rng

MMMM1MM2

FCMFCM1FCM2

MMP-14 COX-2 TIMP-3 CD206I120581B120572

lowastlowast

lowast

lowast

lowast

$ $ DaggerDagger

RNA

(a)

0001001

011

10100

1000

Fold

chan

ge

MM1MM2


lowast

lowastlowast

lowast

lowast

lowast

com

pare

d to

MM

(b)

MM MM1 MM2

GAPDH

GAPDH

MMP-14

COX-2

(c)

CD206

GAPDH

GAPDH

TIMP-3

MM MM1 MM2

(d)

MMP-14

0

1

2

3

4

5

6

Fold

chan

ge co

mpa

red

to M

M

MM FCM

lowast

120573-Actin

(e)

MM FCM

TIMP-3

0

02

04

06

08

1

12

Fold

chan

ge co

mpa

red

to M

M

lowastlowastlowast

120573-Actin

(f)

0

05

1

15

MM FCM

lowast

Fold

chan

ge in

miR

-24

com

pare

d to

MM

(g)

0

02

04

06

08

1

12

14

FCM FCM1 FCM2

Fold

chan

ge co

mpa

red

to F

CM

MMP-14

120573-Actin

(h)

Figure 1 Continued


0

1

2

3

4

FCM FCM1 FCM2

Fold

chan

ge co

mpa

red

to F

CM

TIMP-3

120573-Actin

(i)







NC

SR

CD68 250120583m

(a)

CD68 50120583m

(b)

MMP-14

(c)

CD68

FC

(d)

CD68CD68

(e)

TIMP-3

(f)

MMP-14

50120583m

(g)

TIMP-3

(h)

MMP-14

(i)

TIMP-3

(j)

Figure 2 Continued


MMP-14

(k)

COX-2

(l)

NF120581B

(m)

TIMP-3

(n)

CD206

(o)

IgG

(p)

IgG

(q)

IgG

(r)

IgG

(s)

IgG

(t)





4 Discussion





CD68

CD206

Merged


CD68

Merged

MMP-14 TIMP-3

(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

(i)

(j)

NF-120581B

Figure 3 Continued


0102030405060708090

100

DualTIMP3CD206

TIMP3 CD2060

102030405060708090


100

(k) (l)

MM

P-14

posit

ive F

CMs (

)

Posit

ive F

CMs (

)







Atheromatous 0 (0) 2 (5) 35 (95)



Atheromatous 17 (46) 19 (51) 1 (3)



Atheromatous 2 (5) 30 (81) 5 (14) 0 (0)






Atheromatous 0 (0) 0 (0) 19 (51) 18 (49)






Atheromatous 4 (11) 10 (27) 16 (43) 7 (19)





MMP-14

(a)

Arg-1

(b)

DAPI

(c)

Merge

(d)

MMP-14 MMP-14Arg-1

Arg-10

10

20

30

40

50

60

70

Posit

ive F

CMs (

)

lowast

(e)

0

20

40

60

80

100

120

140

160

0 50 100 150 200 250

Arg

-1 p

ositi

ve F

CMs (

)


r2 = 0960

P lt 00001

(f)





DAPI50120583M

(a)

NF120581B

(b)

TIMP-3

(c)

Merged

(d)

0102030405060708090

100

Negative

TIM

P-3

posit

ive F

CMs (

)

lowast


(e)







Posit

ive F

CM (

)


MMP-14 TIMP-3

0

20

40

60

80

100

Fibrous 30 21



(a)Po

sitiv

e FCM

()

MMP-14 TIMP-3

0

20

40

60

80

100




18

(b)


0

20

40

60

80

100

Posit

ive F

CM (

)

MMP-14 TIMP-3


(c)






0

20

40

60

80

100

Lipid content

No fat

Posit

ive F

CM (

)


lt40

gt40

MMP-14 TIMP-3

(a)

Macrophages

MinorModerateHeavy

0

20

40

60

80

100

Posit

ive F

CM (

)


(b)

MinorModerateHeavy

Smooth muscle cells

0

20

40

60

80

100

Posit

ive F

CM (

)


MMP-14 TIMP-3

(c)

MinorModerateHeavy

0

20

40

60

80

100

Collagen

Posit

ive F

CM (

)lowast

MMP-14 TIMP-3

(d)

0

20

40

60

80

100


Posit

ive F

CM (

)

lowastlowast

1st2nd3rd

MMP-14 TIMP-3

(e)








Amaurosis fugax 119899 () 6 (20) 5 (42) 1 (3)






119875 lt 005
















Acknowledgments


References















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


























3 Results





10

100

1000

10000

100000

Cop

y nu

mbe

rng

MMMM1MM2

FCMFCM1FCM2

MMP-14 COX-2 TIMP-3 CD206I120581B120572

lowastlowast

lowast

lowast

lowast

$ $ DaggerDagger

RNA

(a)

0001001

011

10100

1000

Fold

chan

ge

MM1MM2


lowast

lowastlowast

lowast

lowast

lowast

com

pare

d to

MM

(b)

MM MM1 MM2

GAPDH

GAPDH

MMP-14

COX-2

(c)

CD206

GAPDH

GAPDH

TIMP-3

MM MM1 MM2

(d)

MMP-14

0

1

2

3

4

5

6

Fold

chan

ge co

mpa

red

to M

M

MM FCM

lowast

120573-Actin

(e)

MM FCM

TIMP-3

0

02

04

06

08

1

12

Fold

chan

ge co

mpa

red

to M

M

lowastlowastlowast

120573-Actin

(f)

0

05

1

15

MM FCM

lowast

Fold

chan

ge in

miR

-24

com

pare

d to

MM

(g)

0

02

04

06

08

1

12

14

FCM FCM1 FCM2

Fold

chan

ge co

mpa

red

to F

CM

MMP-14

120573-Actin

(h)

Figure 1 Continued


0

1

2

3

4

FCM FCM1 FCM2

Fold

chan

ge co

mpa

red

to F

CM

TIMP-3

120573-Actin

(i)







NC

SR

CD68 250120583m

(a)

CD68 50120583m

(b)

MMP-14

(c)

CD68

FC

(d)

CD68CD68

(e)

TIMP-3

(f)

MMP-14

50120583m

(g)

TIMP-3

(h)

MMP-14

(i)

TIMP-3

(j)

Figure 2 Continued


MMP-14

(k)

COX-2

(l)

NF120581B

(m)

TIMP-3

(n)

CD206

(o)

IgG

(p)

IgG

(q)

IgG

(r)

IgG

(s)

IgG

(t)





4 Discussion





CD68

CD206

Merged


CD68

Merged

MMP-14 TIMP-3

(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

(i)

(j)

NF-120581B

Figure 3 Continued


0102030405060708090

100

DualTIMP3CD206

TIMP3 CD2060

102030405060708090


100

(k) (l)

MM

P-14

posit

ive F

CMs (

)

Posit

ive F

CMs (

)







Atheromatous 0 (0) 2 (5) 35 (95)



Atheromatous 17 (46) 19 (51) 1 (3)



Atheromatous 2 (5) 30 (81) 5 (14) 0 (0)






Atheromatous 0 (0) 0 (0) 19 (51) 18 (49)






Atheromatous 4 (11) 10 (27) 16 (43) 7 (19)





MMP-14

(a)

Arg-1

(b)

DAPI

(c)

Merge

(d)

MMP-14 MMP-14Arg-1

Arg-10

10

20

30

40

50

60

70

Posit

ive F

CMs (

)

lowast

(e)

0

20

40

60

80

100

120

140

160

0 50 100 150 200 250

Arg

-1 p

ositi

ve F

CMs (

)


r2 = 0960

P lt 00001

(f)





DAPI50120583M

(a)

NF120581B

(b)

TIMP-3

(c)

Merged

(d)

0102030405060708090

100

Negative

TIM

P-3

posit

ive F

CMs (

)

lowast


(e)







Posit

ive F

CM (

)


MMP-14 TIMP-3

0

20

40

60

80

100

Fibrous 30 21



(a)Po

sitiv

e FCM

()

MMP-14 TIMP-3

0

20

40

60

80

100




18

(b)


0

20

40

60

80

100

Posit

ive F

CM (

)

MMP-14 TIMP-3


(c)






0

20

40

60

80

100

Lipid content

No fat

Posit

ive F

CM (

)


lt40

gt40

MMP-14 TIMP-3

(a)

Macrophages

MinorModerateHeavy

0

20

40

60

80

100

Posit

ive F

CM (

)


(b)

MinorModerateHeavy

Smooth muscle cells

0

20

40

60

80

100

Posit

ive F

CM (

)


MMP-14 TIMP-3

(c)

MinorModerateHeavy

0

20

40

60

80

100

Collagen

Posit

ive F

CM (

)lowast

MMP-14 TIMP-3

(d)

0

20

40

60

80

100


Posit

ive F

CM (

)

lowastlowast

1st2nd3rd

MMP-14 TIMP-3

(e)








Amaurosis fugax 119899 () 6 (20) 5 (42) 1 (3)






119875 lt 005
















Acknowledgments


References















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















10

100

1000

10000

100000

Cop

y nu

mbe

rng

MMMM1MM2

FCMFCM1FCM2

MMP-14 COX-2 TIMP-3 CD206I120581B120572

lowastlowast

lowast

lowast

lowast

$ $ DaggerDagger

RNA

(a)

0001001

011

10100

1000

Fold

chan

ge

MM1MM2


lowast

lowastlowast

lowast

lowast

lowast

com

pare

d to

MM

(b)

MM MM1 MM2

GAPDH

GAPDH

MMP-14

COX-2

(c)

CD206

GAPDH

GAPDH

TIMP-3

MM MM1 MM2

(d)

MMP-14

0

1

2

3

4

5

6

Fold

chan

ge co

mpa

red

to M

M

MM FCM

lowast

120573-Actin

(e)

MM FCM

TIMP-3

0

02

04

06

08

1

12

Fold

chan

ge co

mpa

red

to M

M

lowastlowastlowast

120573-Actin

(f)

0

05

1

15

MM FCM

lowast

Fold

chan

ge in

miR

-24

com

pare

d to

MM

(g)

0

02

04

06

08

1

12

14

FCM FCM1 FCM2

Fold

chan

ge co

mpa

red

to F

CM

MMP-14

120573-Actin

(h)

Figure 1 Continued


0

1

2

3

4

FCM FCM1 FCM2

Fold

chan

ge co

mpa

red

to F

CM

TIMP-3

120573-Actin

(i)







NC

SR

CD68 250120583m

(a)

CD68 50120583m

(b)

MMP-14

(c)

CD68

FC

(d)

CD68CD68

(e)

TIMP-3

(f)

MMP-14

50120583m

(g)

TIMP-3

(h)

MMP-14

(i)

TIMP-3

(j)

Figure 2 Continued


MMP-14

(k)

COX-2

(l)

NF120581B

(m)

TIMP-3

(n)

CD206

(o)

IgG

(p)

IgG

(q)

IgG

(r)

IgG

(s)

IgG

(t)





4 Discussion





CD68

CD206

Merged


CD68

Merged

MMP-14 TIMP-3

(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

(i)

(j)

NF-120581B

Figure 3 Continued


0102030405060708090

100

DualTIMP3CD206

TIMP3 CD2060

102030405060708090


100

(k) (l)

MM

P-14

posit

ive F

CMs (

)

Posit

ive F

CMs (

)







Atheromatous 0 (0) 2 (5) 35 (95)



Atheromatous 17 (46) 19 (51) 1 (3)



Atheromatous 2 (5) 30 (81) 5 (14) 0 (0)






Atheromatous 0 (0) 0 (0) 19 (51) 18 (49)






Atheromatous 4 (11) 10 (27) 16 (43) 7 (19)





MMP-14

(a)

Arg-1

(b)

DAPI

(c)

Merge

(d)

MMP-14 MMP-14Arg-1

Arg-10

10

20

30

40

50

60

70

Posit

ive F

CMs (

)

lowast

(e)

0

20

40

60

80

100

120

140

160

0 50 100 150 200 250

Arg

-1 p

ositi

ve F

CMs (

)


r2 = 0960

P lt 00001

(f)





DAPI50120583M

(a)

NF120581B

(b)

TIMP-3

(c)

Merged

(d)

0102030405060708090

100

Negative

TIM

P-3

posit

ive F

CMs (

)

lowast


(e)







Posit

ive F

CM (

)


MMP-14 TIMP-3

0

20

40

60

80

100

Fibrous 30 21



(a)Po

sitiv

e FCM

()

MMP-14 TIMP-3

0

20

40

60

80

100




18

(b)


0

20

40

60

80

100

Posit

ive F

CM (

)

MMP-14 TIMP-3


(c)






0

20

40

60

80

100

Lipid content

No fat

Posit

ive F

CM (

)


lt40

gt40

MMP-14 TIMP-3

(a)

Macrophages

MinorModerateHeavy

0

20

40

60

80

100

Posit

ive F

CM (

)


(b)

MinorModerateHeavy

Smooth muscle cells

0

20

40

60

80

100

Posit

ive F

CM (

)


MMP-14 TIMP-3

(c)

MinorModerateHeavy

0

20

40

60

80

100

Collagen

Posit

ive F

CM (

)lowast

MMP-14 TIMP-3

(d)

0

20

40

60

80

100


Posit

ive F

CM (

)

lowastlowast

1st2nd3rd

MMP-14 TIMP-3

(e)








Amaurosis fugax 119899 () 6 (20) 5 (42) 1 (3)






119875 lt 005
















Acknowledgments


References















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

1

2

3

4

FCM FCM1 FCM2

Fold

chan

ge co

mpa

red

to F

CM

TIMP-3

120573-Actin

(i)







NC

SR

CD68 250120583m

(a)

CD68 50120583m

(b)

MMP-14

(c)

CD68

FC

(d)

CD68CD68

(e)

TIMP-3

(f)

MMP-14

50120583m

(g)

TIMP-3

(h)

MMP-14

(i)

TIMP-3

(j)

Figure 2 Continued


MMP-14

(k)

COX-2

(l)

NF120581B

(m)

TIMP-3

(n)

CD206

(o)

IgG

(p)

IgG

(q)

IgG

(r)

IgG

(s)

IgG

(t)





4 Discussion





CD68

CD206

Merged


CD68

Merged

MMP-14 TIMP-3

(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

(i)

(j)

NF-120581B

Figure 3 Continued


0102030405060708090

100

DualTIMP3CD206

TIMP3 CD2060

102030405060708090


100

(k) (l)

MM

P-14

posit

ive F

CMs (

)

Posit

ive F

CMs (

)







Atheromatous 0 (0) 2 (5) 35 (95)



Atheromatous 17 (46) 19 (51) 1 (3)



Atheromatous 2 (5) 30 (81) 5 (14) 0 (0)






Atheromatous 0 (0) 0 (0) 19 (51) 18 (49)






Atheromatous 4 (11) 10 (27) 16 (43) 7 (19)





MMP-14

(a)

Arg-1

(b)

DAPI

(c)

Merge

(d)

MMP-14 MMP-14Arg-1

Arg-10

10

20

30

40

50

60

70

Posit

ive F

CMs (

)

lowast

(e)

0

20

40

60

80

100

120

140

160

0 50 100 150 200 250

Arg

-1 p

ositi

ve F

CMs (

)


r2 = 0960

P lt 00001

(f)





DAPI50120583M

(a)

NF120581B

(b)

TIMP-3

(c)

Merged

(d)

0102030405060708090

100

Negative

TIM

P-3

posit

ive F

CMs (

)

lowast


(e)







Posit

ive F

CM (

)


MMP-14 TIMP-3

0

20

40

60

80

100

Fibrous 30 21



(a)Po

sitiv

e FCM

()

MMP-14 TIMP-3

0

20

40

60

80

100




18

(b)


0

20

40

60

80

100

Posit

ive F

CM (

)

MMP-14 TIMP-3


(c)






0

20

40

60

80

100

Lipid content

No fat

Posit

ive F

CM (

)


lt40

gt40

MMP-14 TIMP-3

(a)

Macrophages

MinorModerateHeavy

0

20

40

60

80

100

Posit

ive F

CM (

)


(b)

MinorModerateHeavy

Smooth muscle cells

0

20

40

60

80

100

Posit

ive F

CM (

)


MMP-14 TIMP-3

(c)

MinorModerateHeavy

0

20

40

60

80

100

Collagen

Posit

ive F

CM (

)lowast

MMP-14 TIMP-3

(d)

0

20

40

60

80

100


Posit

ive F

CM (

)

lowastlowast

1st2nd3rd

MMP-14 TIMP-3

(e)








Amaurosis fugax 119899 () 6 (20) 5 (42) 1 (3)






119875 lt 005
















Acknowledgments


References















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















NC

SR

CD68 250120583m

(a)

CD68 50120583m

(b)

MMP-14

(c)

CD68

FC

(d)

CD68CD68

(e)

TIMP-3

(f)

MMP-14

50120583m

(g)

TIMP-3

(h)

MMP-14

(i)

TIMP-3

(j)

Figure 2 Continued


MMP-14

(k)

COX-2

(l)

NF120581B

(m)

TIMP-3

(n)

CD206

(o)

IgG

(p)

IgG

(q)

IgG

(r)

IgG

(s)

IgG

(t)





4 Discussion





CD68

CD206

Merged


CD68

Merged

MMP-14 TIMP-3

(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

(i)

(j)

NF-120581B

Figure 3 Continued


0102030405060708090

100

DualTIMP3CD206

TIMP3 CD2060

102030405060708090


100

(k) (l)

MM

P-14

posit

ive F

CMs (

)

Posit

ive F

CMs (

)







Atheromatous 0 (0) 2 (5) 35 (95)



Atheromatous 17 (46) 19 (51) 1 (3)



Atheromatous 2 (5) 30 (81) 5 (14) 0 (0)






Atheromatous 0 (0) 0 (0) 19 (51) 18 (49)






Atheromatous 4 (11) 10 (27) 16 (43) 7 (19)





MMP-14

(a)

Arg-1

(b)

DAPI

(c)

Merge

(d)

MMP-14 MMP-14Arg-1

Arg-10

10

20

30

40

50

60

70

Posit

ive F

CMs (

)

lowast

(e)

0

20

40

60

80

100

120

140

160

0 50 100 150 200 250

Arg

-1 p

ositi

ve F

CMs (

)


r2 = 0960

P lt 00001

(f)





DAPI50120583M

(a)

NF120581B

(b)

TIMP-3

(c)

Merged

(d)

0102030405060708090

100

Negative

TIM

P-3

posit

ive F

CMs (

)

lowast


(e)







Posit

ive F

CM (

)


MMP-14 TIMP-3

0

20

40

60

80

100

Fibrous 30 21



(a)Po

sitiv

e FCM

()

MMP-14 TIMP-3

0

20

40

60

80

100




18

(b)


0

20

40

60

80

100

Posit

ive F

CM (

)

MMP-14 TIMP-3


(c)






0

20

40

60

80

100

Lipid content

No fat

Posit

ive F

CM (

)


lt40

gt40

MMP-14 TIMP-3

(a)

Macrophages

MinorModerateHeavy

0

20

40

60

80

100

Posit

ive F

CM (

)


(b)

MinorModerateHeavy

Smooth muscle cells

0

20

40

60

80

100

Posit

ive F

CM (

)


MMP-14 TIMP-3

(c)

MinorModerateHeavy

0

20

40

60

80

100

Collagen

Posit

ive F

CM (

)lowast

MMP-14 TIMP-3

(d)

0

20

40

60

80

100


Posit

ive F

CM (

)

lowastlowast

1st2nd3rd

MMP-14 TIMP-3

(e)








Amaurosis fugax 119899 () 6 (20) 5 (42) 1 (3)






119875 lt 005
















Acknowledgments


References















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















MMP-14

(k)

COX-2

(l)

NF120581B

(m)

TIMP-3

(n)

CD206

(o)

IgG

(p)

IgG

(q)

IgG

(r)

IgG

(s)

IgG

(t)





4 Discussion





CD68

CD206

Merged


CD68

Merged

MMP-14 TIMP-3

(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

(i)

(j)

NF-120581B

Figure 3 Continued


0102030405060708090

100

DualTIMP3CD206

TIMP3 CD2060

102030405060708090


100

(k) (l)

MM

P-14

posit

ive F

CMs (

)

Posit

ive F

CMs (

)







Atheromatous 0 (0) 2 (5) 35 (95)



Atheromatous 17 (46) 19 (51) 1 (3)



Atheromatous 2 (5) 30 (81) 5 (14) 0 (0)






Atheromatous 0 (0) 0 (0) 19 (51) 18 (49)






Atheromatous 4 (11) 10 (27) 16 (43) 7 (19)





MMP-14

(a)

Arg-1

(b)

DAPI

(c)

Merge

(d)

MMP-14 MMP-14Arg-1

Arg-10

10

20

30

40

50

60

70

Posit

ive F

CMs (

)

lowast

(e)

0

20

40

60

80

100

120

140

160

0 50 100 150 200 250

Arg

-1 p

ositi

ve F

CMs (

)


r2 = 0960

P lt 00001

(f)





DAPI50120583M

(a)

NF120581B

(b)

TIMP-3

(c)

Merged

(d)

0102030405060708090

100

Negative

TIM

P-3

posit

ive F

CMs (

)

lowast


(e)







Posit

ive F

CM (

)


MMP-14 TIMP-3

0

20

40

60

80

100

Fibrous 30 21



(a)Po

sitiv

e FCM

()

MMP-14 TIMP-3

0

20

40

60

80

100




18

(b)


0

20

40

60

80

100

Posit

ive F

CM (

)

MMP-14 TIMP-3


(c)






0

20

40

60

80

100

Lipid content

No fat

Posit

ive F

CM (

)


lt40

gt40

MMP-14 TIMP-3

(a)

Macrophages

MinorModerateHeavy

0

20

40

60

80

100

Posit

ive F

CM (

)


(b)

MinorModerateHeavy

Smooth muscle cells

0

20

40

60

80

100

Posit

ive F

CM (

)


MMP-14 TIMP-3

(c)

MinorModerateHeavy

0

20

40

60

80

100

Collagen

Posit

ive F

CM (

)lowast

MMP-14 TIMP-3

(d)

0

20

40

60

80

100


Posit

ive F

CM (

)

lowastlowast

1st2nd3rd

MMP-14 TIMP-3

(e)








Amaurosis fugax 119899 () 6 (20) 5 (42) 1 (3)






119875 lt 005
















Acknowledgments


References















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















CD68

CD206

Merged


CD68

Merged

MMP-14 TIMP-3

(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

(i)

(j)

NF-120581B

Figure 3 Continued


0102030405060708090

100

DualTIMP3CD206

TIMP3 CD2060

102030405060708090


100

(k) (l)

MM

P-14

posit

ive F

CMs (

)

Posit

ive F

CMs (

)







Atheromatous 0 (0) 2 (5) 35 (95)



Atheromatous 17 (46) 19 (51) 1 (3)



Atheromatous 2 (5) 30 (81) 5 (14) 0 (0)






Atheromatous 0 (0) 0 (0) 19 (51) 18 (49)






Atheromatous 4 (11) 10 (27) 16 (43) 7 (19)





MMP-14

(a)

Arg-1

(b)

DAPI

(c)

Merge

(d)

MMP-14 MMP-14Arg-1

Arg-10

10

20

30

40

50

60

70

Posit

ive F

CMs (

)

lowast

(e)

0

20

40

60

80

100

120

140

160

0 50 100 150 200 250

Arg

-1 p

ositi

ve F

CMs (

)


r2 = 0960

P lt 00001

(f)





DAPI50120583M

(a)

NF120581B

(b)

TIMP-3

(c)

Merged

(d)

0102030405060708090

100

Negative

TIM

P-3

posit

ive F

CMs (

)

lowast


(e)







Posit

ive F

CM (

)


MMP-14 TIMP-3

0

20

40

60

80

100

Fibrous 30 21



(a)Po

sitiv

e FCM

()

MMP-14 TIMP-3

0

20

40

60

80

100




18

(b)


0

20

40

60

80

100

Posit

ive F

CM (

)

MMP-14 TIMP-3


(c)






0

20

40

60

80

100

Lipid content

No fat

Posit

ive F

CM (

)


lt40

gt40

MMP-14 TIMP-3

(a)

Macrophages

MinorModerateHeavy

0

20

40

60

80

100

Posit

ive F

CM (

)


(b)

MinorModerateHeavy

Smooth muscle cells

0

20

40

60

80

100

Posit

ive F

CM (

)


MMP-14 TIMP-3

(c)

MinorModerateHeavy

0

20

40

60

80

100

Collagen

Posit

ive F

CM (

)lowast

MMP-14 TIMP-3

(d)

0

20

40

60

80

100


Posit

ive F

CM (

)

lowastlowast

1st2nd3rd

MMP-14 TIMP-3

(e)








Amaurosis fugax 119899 () 6 (20) 5 (42) 1 (3)






119875 lt 005
















Acknowledgments


References















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0102030405060708090

100

DualTIMP3CD206

TIMP3 CD2060

102030405060708090


100

(k) (l)

MM

P-14

posit

ive F

CMs (

)

Posit

ive F

CMs (

)







Atheromatous 0 (0) 2 (5) 35 (95)



Atheromatous 17 (46) 19 (51) 1 (3)



Atheromatous 2 (5) 30 (81) 5 (14) 0 (0)






Atheromatous 0 (0) 0 (0) 19 (51) 18 (49)






Atheromatous 4 (11) 10 (27) 16 (43) 7 (19)





MMP-14

(a)

Arg-1

(b)

DAPI

(c)

Merge

(d)

MMP-14 MMP-14Arg-1

Arg-10

10

20

30

40

50

60

70

Posit

ive F

CMs (

)

lowast

(e)

0

20

40

60

80

100

120

140

160

0 50 100 150 200 250

Arg

-1 p

ositi

ve F

CMs (

)


r2 = 0960

P lt 00001

(f)





DAPI50120583M

(a)

NF120581B

(b)

TIMP-3

(c)

Merged

(d)

0102030405060708090

100

Negative

TIM

P-3

posit

ive F

CMs (

)

lowast


(e)







Posit

ive F

CM (

)


MMP-14 TIMP-3

0

20

40

60

80

100

Fibrous 30 21



(a)Po

sitiv

e FCM

()

MMP-14 TIMP-3

0

20

40

60

80

100




18

(b)


0

20

40

60

80

100

Posit

ive F

CM (

)

MMP-14 TIMP-3


(c)






0

20

40

60

80

100

Lipid content

No fat

Posit

ive F

CM (

)


lt40

gt40

MMP-14 TIMP-3

(a)

Macrophages

MinorModerateHeavy

0

20

40

60

80

100

Posit

ive F

CM (

)


(b)

MinorModerateHeavy

Smooth muscle cells

0

20

40

60

80

100

Posit

ive F

CM (

)


MMP-14 TIMP-3

(c)

MinorModerateHeavy

0

20

40

60

80

100

Collagen

Posit

ive F

CM (

)lowast

MMP-14 TIMP-3

(d)

0

20

40

60

80

100


Posit

ive F

CM (

)

lowastlowast

1st2nd3rd

MMP-14 TIMP-3

(e)








Amaurosis fugax 119899 () 6 (20) 5 (42) 1 (3)






119875 lt 005
















Acknowledgments


References















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















MMP-14

(a)

Arg-1

(b)

DAPI

(c)

Merge

(d)

MMP-14 MMP-14Arg-1

Arg-10

10

20

30

40

50

60

70

Posit

ive F

CMs (

)

lowast

(e)

0

20

40

60

80

100

120

140

160

0 50 100 150 200 250

Arg

-1 p

ositi

ve F

CMs (

)


r2 = 0960

P lt 00001

(f)





DAPI50120583M

(a)

NF120581B

(b)

TIMP-3

(c)

Merged

(d)

0102030405060708090

100

Negative

TIM

P-3

posit

ive F

CMs (

)

lowast


(e)







Posit

ive F

CM (

)


MMP-14 TIMP-3

0

20

40

60

80

100

Fibrous 30 21



(a)Po

sitiv

e FCM

()

MMP-14 TIMP-3

0

20

40

60

80

100




18

(b)


0

20

40

60

80

100

Posit

ive F

CM (

)

MMP-14 TIMP-3


(c)






0

20

40

60

80

100

Lipid content

No fat

Posit

ive F

CM (

)


lt40

gt40

MMP-14 TIMP-3

(a)

Macrophages

MinorModerateHeavy

0

20

40

60

80

100

Posit

ive F

CM (

)


(b)

MinorModerateHeavy

Smooth muscle cells

0

20

40

60

80

100

Posit

ive F

CM (

)


MMP-14 TIMP-3

(c)

MinorModerateHeavy

0

20

40

60

80

100

Collagen

Posit

ive F

CM (

)lowast

MMP-14 TIMP-3

(d)

0

20

40

60

80

100


Posit

ive F

CM (

)

lowastlowast

1st2nd3rd

MMP-14 TIMP-3

(e)








Amaurosis fugax 119899 () 6 (20) 5 (42) 1 (3)






119875 lt 005
















Acknowledgments


References















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















DAPI50120583M

(a)

NF120581B

(b)

TIMP-3

(c)

Merged

(d)

0102030405060708090

100

Negative

TIM

P-3

posit

ive F

CMs (

)

lowast


(e)







Posit

ive F

CM (

)


MMP-14 TIMP-3

0

20

40

60

80

100

Fibrous 30 21



(a)Po

sitiv

e FCM

()

MMP-14 TIMP-3

0

20

40

60

80

100




18

(b)


0

20

40

60

80

100

Posit

ive F

CM (

)

MMP-14 TIMP-3


(c)






0

20

40

60

80

100

Lipid content

No fat

Posit

ive F

CM (

)


lt40

gt40

MMP-14 TIMP-3

(a)

Macrophages

MinorModerateHeavy

0

20

40

60

80

100

Posit

ive F

CM (

)


(b)

MinorModerateHeavy

Smooth muscle cells

0

20

40

60

80

100

Posit

ive F

CM (

)


MMP-14 TIMP-3

(c)

MinorModerateHeavy

0

20

40

60

80

100

Collagen

Posit

ive F

CM (

)lowast

MMP-14 TIMP-3

(d)

0

20

40

60

80

100


Posit

ive F

CM (

)

lowastlowast

1st2nd3rd

MMP-14 TIMP-3

(e)








Amaurosis fugax 119899 () 6 (20) 5 (42) 1 (3)






119875 lt 005
















Acknowledgments


References















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Posit

ive F

CM (

)


MMP-14 TIMP-3

0

20

40

60

80

100

Fibrous 30 21



(a)Po

sitiv

e FCM

()

MMP-14 TIMP-3

0

20

40

60

80

100




18

(b)


0

20

40

60

80

100

Posit

ive F

CM (

)

MMP-14 TIMP-3


(c)






0

20

40

60

80

100

Lipid content

No fat

Posit

ive F

CM (

)


lt40

gt40

MMP-14 TIMP-3

(a)

Macrophages

MinorModerateHeavy

0

20

40

60

80

100

Posit

ive F

CM (

)


(b)

MinorModerateHeavy

Smooth muscle cells

0

20

40

60

80

100

Posit

ive F

CM (

)


MMP-14 TIMP-3

(c)

MinorModerateHeavy

0

20

40

60

80

100

Collagen

Posit

ive F

CM (

)lowast

MMP-14 TIMP-3

(d)

0

20

40

60

80

100


Posit

ive F

CM (

)

lowastlowast

1st2nd3rd

MMP-14 TIMP-3

(e)








Amaurosis fugax 119899 () 6 (20) 5 (42) 1 (3)






119875 lt 005
















Acknowledgments


References















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

20

40

60

80

100

Lipid content

No fat

Posit

ive F

CM (

)


lt40

gt40

MMP-14 TIMP-3

(a)

Macrophages

MinorModerateHeavy

0

20

40

60

80

100

Posit

ive F

CM (

)


(b)

MinorModerateHeavy

Smooth muscle cells

0

20

40

60

80

100

Posit

ive F

CM (

)


MMP-14 TIMP-3

(c)

MinorModerateHeavy

0

20

40

60

80

100

Collagen

Posit

ive F

CM (

)lowast

MMP-14 TIMP-3

(d)

0

20

40

60

80

100


Posit

ive F

CM (

)

lowastlowast

1st2nd3rd

MMP-14 TIMP-3

(e)








Amaurosis fugax 119899 () 6 (20) 5 (42) 1 (3)






119875 lt 005
















Acknowledgments


References















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















Amaurosis fugax 119899 () 6 (20) 5 (42) 1 (3)






119875 lt 005
















Acknowledgments


References















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

























Acknowledgments


References















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of














































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Documents

Hindawi Publishing Corporation - Research Article …downloads.hindawi.com/journals/mi/2014/276457.pdfResearch Article Relationship of MMP-14 and TIMP-3 Expression with Macrophage