weakening of the fibrous cap owing to net degradation of extracellular matrix is believed to play an important part in plaque rupture .
foam - cell macrophages ( fcms ) are an abundant source of several matrix metalloproteinases ( mmps ) in human plaques [ 3 , 4 ] .
protective effects of certain mmps on smooth muscle migration and proliferation have been identified from experimental studies but high levels of mmp production , especially from fcms , have been implicated in plaque progression and rupture .
attention has focused recently on mmp-14 , which is an activated membrane type mmp , and a tissue inhibitor of mmps ( timp-3 ) , which binds to the extracellular matrix .
given these properties , mmp-14 and timp-3 are likely to play important divergent roles in the regulation of macrophage pericellular proteolysis .
adoptive transfer of leukocytes lacking mmp-14 into ldl - receptor null mice increased plaque collagen content , implying a harmful collagen - depleting role of mmp-14 in plaque stability .
conversely , transgenic overexpression of timp-3 in mouse macrophages reduced atherosclerosis formation and improved markers of plaque stability .
a mmp-14  timp-3 subpopulation of rabbit fcms was shown to have greater proteolytic capacity , ability to invade through synthetic extracellular matrix ( ecm ) , and greater propensity to undergo apoptosis , all of which would be expected to favour plaque rupture .
timp-3 fcms were identified in human atherosclerotic plaques   but the mechanisms responsible for generating the mmp-14  timp-3 fcms and their relationship to plaque vulnerability in man were not previously investigated .
macrophages can express a spectrum of different activation states dependent on pro- and anti - inflammatory signals [ 8 , 9 ] , although the effects on mmp-14 and timp-3 expression are not fully documented .
for example , activation by toll - like receptor agonists   and other inflammatory cytokines acting through nuclear factor-b ( nf-b ) upregulates several mmps .
such activated macrophages have been associated with unstable plaque morphology , whereas deactivated or alternatively activated macrophages are associated with increased plaque stability in most [ 1214 ] , although not all , studies .
phenotypes resulting from oxidised phospholipids   or by the action of haem at sites of thrombosis and intraplaque haemorrhage have also been recently distinguished [ 1719 ] . to clarify the nature and role of mmp-14  timp-3 fcms we investigated their activation in vitro with pro- and anti - inflammatory molecules .
we then investigated whether macrophage populations with opposing expression of mmp-14 and timp-3 colocalise with markers of macrophage activation and correlate with histological markers of vulnerability to rupture in human carotid atherosclerotic plaques .
carotid endarterectomy specimens came from the atheroexpress biobank , the details of which have been described in detail elsewhere [ 2022 ] .
briefly , patient demographics including cardiovascular risk factors , previous medication use , and presenting symptoms were recorded .
atherosclerotic plaques were dissected by a dedicated technician into 5  m thick cross - sectional segments .
histological staining ( haematoxylin and eosin ( he ) , picrosirius red ( ps ) ) were performed on the so - called culprit lesion ( segment with largest plaque area ) .
the area occupied by following parameters was scored semiquantitatively as described previously [ 2022 ] : atheroma core ( he , ps ) , calcification , ( he ) , collagen ( ps ) , smooth muscle cells ( sm - ctin ; m0851 ; dako ) , and macrophages ( cd68 ; m0814 ; dako ) ( table 1 ) .
smooth muscle cells and macrophages were also scored quantitatively by using a microscope equipped with a digital camera and using analysis 3.2 software ( soft imaging system gmbh , munster , germany ) .
the amount of microvessels was quantified by using an anti - cd34 antibody ; details have been described previously [ 2022 ] .
as specified in the atheroexpress protocol , histological sections were classified according to overall appearance into :  atheromatous lesions  containing a large lipid core ( > 40% of plaque area ) , high macrophage infiltration with low smooth muscle cell and collagen content ,  fibrous lesions  with a small ( < 40% ) or absent lipid core , low macrophage content and high smooth muscle cell and collagen content , and 
fibrous - atheromatous lesions  as an intermediate between the two other phenotypes . the inter- and intraobserver variability for this classification has been described previously .
adjacent sections were then taken for the histological analyses reported here , which were performed and analysed by 2 additional operators who were blinded as to the lesion classification .
sections were dewaxed in clearene and rehydrated in absolute alcohol . after rinsing in distilled water ,
slides were subjected to heat - induced antigen retrieval by incubation in citrate buffer ( 10  mm citric acid in 1x pbs , ph 6.0 ) and microwaved for 10 minutes at full power .
slides were allowed to cool for 30 minutes and then washed with pbs ( 3  2 minutes ) . for brightfield or fluorescence ihc , 50  l of 1% bovine serum albumin or image -
it fx signal enhancer ( invitrogen , life technologies , paisley , uk ) , respectively , was placed on sections and incubated at room temperature for 30 minutes .
the solution was then removed by tapping the slides and 50  l of the applicable primary antibody ( as detailed in table 1 ) added to the samples and incubated either overnight at 4c or for 1 hour at room temperature .
sections were then washed in pbs ( 3  2 minutes each ) and incubated in the desired secondary antibody as detailed in table 1 . for fluorescent labelling
sections were then washed in pbs ( 3  2 minutes ) and mounted with prolong gold antifade reagent containing dapi ( invitrogen , life technologies , paisley , uk ) to identify the nuclei . a negative control where the primary antibody was replaced with the relevant species igg at the same dilution was always included and the cells were counted in 6  20 magnification fields .
the percentage of cd68 positive macrophages that were also positively stained for other proteins of interest was counted by comparing serial sections or after dual - immunohistochemistry if the antibodies were suitable , by independent observers ( jlj , npj , vwps ) and the results averaged , as described previously . for dual fluorescence ,
subsequently , species - specific fluorophore conjugated secondary antibodies were used to yield either a red fluorescent product at the site of the antigen ( alexafluor 594 ) or a green fluorescent product at the site of the antigen ( alexafluor 488 ) .
sections were then mounted in prolong gold antifade reagent with 4,6-diamidino-2-phenylindole ( life technologies , paisley , uk ; p-36931 ) to fluorescently label nuclei blue .
the specificity of the immunolabeling was demonstrated by inclusion of a negative control using isotype - specific nonimmune serum or igg .
images were acquired from a fluorescence microscope using imageproplus image analysis software ( media cybernetics ) and merged if required before further analysis .
the percentage of mmp-14 positive macrophages that stained for other proteins was counted within each lesion and expressed as percentage of total mmp-14 positive macrophages .
peripheral blood mononuclear cells were isolated by differential centrifugation from whole blood of healthy donors , which were collected under south west 4 research ethics committee reference 09/h0107/22 .
blood ( 24  ml per donor ) was diluted with dulbecco 's phosphate buffered saline ( pbs ) without calcium and magnesium ( lonza ) 1x ( ratio 1  :  1 ) .
the diluted samples were subjected to density gradient separation on ficoll paque plus ( ratio 1  :  1 ) ( ge healthcare life sciences , buckinghamshire , uk ) and centrifuged .
after centrifugation the pbmc layer was collected and washed in hank 's balanced salt sodium ( hbss ) with phenol red without calcium and magnesium ( lonza ) .
monocytes were isolated by adhering the peripheral blood mononuclear cells to tissue culture plastic for 2 hours at a concentration of 2.5  10 cells / ml . monocytes were cultured in rpmi media with 2  mm l - glutamine , 100  iu / ml penicillin , 100  g / ml streptomycin , 10% fetal bovine serum ( fbs ; lonza , sigma ) , and 20  ng / ml recombinant human macrophage - colony - stimulating factor ( r & d systems ) .
the medium included 85  g / ml 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 and replaced with rpmi media supplemented with 5% fcs .
recombinant human interferon- ( 10  ng / ml r & d systems ) and lipopolysaccharide - lps ( 200  ng / ml , escherichia coli 026:b6 , sigma - aldrich ) were added for 24 hours to generate classically activated macrophages ( m1 )   or recombinant human interleukin-4 ( 20  ng / ml r & d systems ) to obtain alternatively activated macrophages ( m2 ) .
rna samples from macrophages were collected using rlt solution ( qiagen ) with -mercaptoethanol ( -me ; 1  :  100 dilution ) .
total rna was extracted using rneasy mini kit ( qiagen ) according to the manufacturer 's instructions .
rna was quantified using a nanodrop spectrophotometer and cdna generated using a quantitect reverse transcription kit ( 100200  ng rna per reaction qiagen ) .
real - time quantitative pcr was performed in a roche light cycler 1.5 to quantify the steady - state concentration of rna using a quantitect sybr green pcr kit and primers as detailed in table 2 .
denaturation for 15  min at 95c was followed by 60 cycles of denaturation ( 15 seconds at 95c ) , annealing for 20 seconds , and extension for 25 seconds at 72c .
copy numbers of mrna were calculated using standard curves constructed using the respective pcr products eluted from agarose gels .
adherent cells were washed with pbs on ice and then lysed with ice - cold 1% sds lysis buffer .
the bicinchoninic acid ( bca ) kit ( pierce ) was used to estimate protein concentration ( all in duplicate ) of cell lysates according to kit protocol .
protein concentration was read at 560  nm on a multiskan ascent ( thermo electron corporation ) plate reader .
nupage novex bis - tris mini gel system ( invitrogen , life technologies , paisley , uk ) was used for western blot experiments .
10  g of total protein was loaded on 412% bis - tris - hcl buffered ( ph 6.4 ) polyacrylamide precast gels ( invitrogen , life technologies , paisley , uk ) and run in mes - sds running buffer 1x ( invitrogen , life technologies , paisley , uk ) according to manufacturer instructions .
after electrophoresis , gels were transferred to a 0.45  m nitrocellulose membrane in novex nupage transfer buffer 1x plus 5% methanol ( v / v ) .
following transfer , the nitrocellulose membrane was blocked in 5% milk solution in tris buffered solution containing tween ( tbs - t ) ( 200  mm tris , 2% tween 20 , ph 7.6 ) . the membrane was then incubated over night at 4c using the antibodies described in table 1 diluted in 2  ml of signalboost solution 1 ( calbiochem - novabiochem ltd . ,
the membrane was incubated with 5% milk solution in tbs - t and then with the applicable horseradish peroxidase ( hpr)-labelled secondary antibody ( dako , dorset , uk ) diluted 1  :  1000 in signalboost solution 2 ( calbiochem - novabiochem ltd . ,
after incubation the membrane was washed in tbs - t . to detect the peroxidise labelled proteins , an enhanced chemiluminescence ( ecl ) detection system ( ge healthcare life sciences , buckinghamshire , uk ) was used .
the membrane was incubated with ecl reagent mix ( solution a and solution b at a ratio of 1  :  1 ) and exposed to x - ray film for the desired length of time . as a loading control 0.5  g / ml of either anti--actin antibody ( a2228 , sigma ) or anti - gapdh antibody ( mab374 , millipore ) was used .
detected bands were quantified using a bio - rad gs-690 scanning densitometer ( bio - rad , hemel hempstead , uk ) and were normalised by relevant -actin or gapdh values . for histomorphometry
, differences between categorical and continuous variables were analysed by using an independent student 's t - test in case of normally distributed data , and a mann - whitney u test in case of not normally distributed data .
differences between ordinal and continuous data were analysed by using a kruskal - wallis test , in case of not normally distributed data .
differences between dichotomous and categorical data were analysed by using a chi - square test .
differences between 2 continuous variables were analysed with a spearman correlation test in case of not normally distributed data . in all cases p < 0.05 was considered significant .
quantitative rt - pcr results normalised to the values for 18  s rna were transformed to logarithms and analysed by anova followed by student 's t - test with bonferroni correction .
human monocyte - derived macrophages were differentiated with m - csf ( mms ) and some from each batch were treated with oxidised ldl to generate foam - cell macrophages ( fcms ) , as previously validated in our laboratory .
conversion of mms to fcms did not affect mrna expression of mmp-14 or timp-3 ( figure 1(a ) ) or cox-2 and ib , markers of proinflammatory activation , or cd206 , a gene that is present constitutively but upregulated by anti - inflammatory activation [ 12 , 13 ] ( figure 1(a ) ) .
proinflammatory activation with lps and ifn ( referred to as m1 conditions ) significantly increased expression of mmp-14 by 4.5- and 7.3-fold , ib mrna ( a surrogate marker of nfb signalling ) by 7.5- and 6.7-fold , and cox-2 mrna by 146- and 187-fold , in macrophages ( mm1 ) and fcms ( fcm1 ) , respectively ( figure 1(a ) ) .
by contrast , proinflammatory activation decreased mrna levels of timp-3 by 74% and 89% and cd206 by 81% and 86% in macrophages or fcms , respectively ( figure 1(a ) ) . anti - inflammatory activation with il-4 ( referred to as m2 conditions ) had no effect on mmp-14 , cox-2 or ib mrna levels but significantly increased timp-3 mrna expression by 1.9- and 2.6-fold and cd206 expression by 7.5- and 9.9-fold in mms ( mm2 ) and fcms ( fcm2 ) , respectively ( figure 1(a ) ) .
consistent with the mrna data , proinflammatory activation significantly elevated protein levels of mmp-14 and cox-2 and significantly decreased those 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 protein levels ( figures 1(b ) and 1(d ) ) , consistent with the mrna data .
we conclude that proinflammatory macrophages are mmp-14  timp-3 whereas unactivated and anti - inflammatory stimulated macrophages are comparatively mmp-14  timp-3 .
foam - cell formation significantly increased protein expression of mmp-14 and decreased timp-3 levels ( figures 1(e ) and 1(f ) ) , despite not changing the mrna levels , in agreement with our previous findings in during rabbit macrophage foam - cell formation .
these results could be explained by the additional involvement of a posttranscriptional mechanism , such as regulation by microrna ( mir ) .
indeed , we recently showed that high levels of mir-24 in nonfoamy macrophages limit macrophage mmp-14 protein expression .
we observed a 70% decrease in mir-24 levels in fcms compared to mms ( figure 1(g ) ) , consistent with the increased mmp-14 protein levels .
interestingly , in contrast to mrna expression , pro- or anti - inflammatory activation of fcms did not affect mmp-14 protein expression ( figure 1(h ) ) , which suggests that the effect of the mir is dominant .
pro- or anti - inflammatory activation of fcms did not affect timp-3 protein expression either when compared to nonactivated fcms ( figure 1(i ) ) .
timp-3 protein expression may also be subject to mir regulation but this has not yet been demonstrated in macrophages . in summary , our results imply that nonfoamy macrophages are mmp-14  timp-3 when classically activated whereas fcms are mmp-14  timp-3 at the protein level irrespective of their activation status .
the cd68 positive cells varied widely in morphology from large , highly foamy cells to those with very few or no lipid inclusions .
positive macrophages / fcms were found predominantly in the shoulder regions ( sr ) of atheromatous carotid plaques ( figures 2(a)2(c ) ) , whereas timp-3 positive macrophages / fcms occurred predominantly within and around the fibrous cap ( fc ) of fibrous atheromatous plaques ( figures 2(d)-2(e ) ) .
the specificity of the antibodies used is demonstrated in figure 2 . in line with our previous findings
, regions of plaques tended to be either mmp-14 positive and 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 ( as described previously ) revealed that in plaque regions with abundant cd68 positive fcms , mmp-14 colocalised with nuclear - localised nf-b p65 subunit , a recognised marker of proinflammatory activation .
when counted , approximately 80% of mmp-14 positive fcms also had nuclear - localised nfb ( figures 3(a)3(d ) ) , which implies that proinflammatory activation associated with mmp-14 upregulation in macrophages / fcms in plaques . to gain further insight
, we employed arginase-1 , which is a marker for anti - inflammatory mouse macrophages   and is downregulated in a subpopulation of human fcms in advanced plaques .
arginase-1 and mmp-14 were coexpressed in more than 50% of intraplaque macrophages / fcms and the number of arginase-1 and mmp-14 positive macrophages was correlated in advanced plaques ( r = 0.960 ; p < 0.0001 , figure 4 ) .
these results are consistent with our in vitro data showing high mmp-14 protein levels in fcms irrespective of m1/m2 activation state ( figure 4 ) .
conversely to mmp-14 , less than 1% of timp-3 positive macrophages / fcms demonstrated nuclear - localised nf-b p65 ( figure 5 ) , implying that few were classically activated .
fcms that were timp-3 positive also stained for cd206 , which we showed to be expressed in unactivated ( mm ) and alternatively activated ( mm2 ) macrophages ( figures 3(f)3(i ) ) .
we therefore concluded that cd206 and timp-3 positive macrophages / fcms in plaques could be unactivated or il-4 activated .
carotid plaques entered into atheroexpress are given an initial classification into atheromatous , fibrous - atheromatous , or fibrous plaques based on a large lipid core , high abundance of macrophages , and low abundance of smooth muscle cells ( smcs ) and collagen content [ 20 , 21 , 32 ] ( table 3 ) .
of note , male and older patients are overrepresented in the group with atheromatous plaques ( table 4 ) . by comparison with these previous assignments we found that the percentage of mmp-14 ( figure 6(a ) ) and cox-2 ( figure 6(b ) ) positive macrophages / fcms was significantly greater in atheromatous or fibrous - atheromatous compared to fibrous plaques . by contrast , the percentage timp-3 ( figure 6(a ) ) and cd206 ( figure 6(b ) ) positive macrophages / fcms increased in fibrous compared to fibrous - atheromatous and atheromatous carotid plaques . despite the small number of asymptomatic patients in our cohort , significantly higher mmp-14 positivity was found in plaques from symptomatic compared to asymptomatic patients ( figure 6(c ) ) .
there was also a trend towards lower timp-3 positivity in plaques from symptomatic patients ( figure 6(c ) ) .
amplifying the relationships to overall plaque designation , we found a higher percentage of mmp-14 positive macrophages / fcms was associated with plaques showing high lipid and macrophage content or decreased smc number , which are related to plaque instability , whereas the converse was observed for timp-3 positive fcms ( figure 7 ) .
timp-3 positive macrophages / fcms were also significantly associated with increased collagen content and reduced plaque neovascularisation ( figure 7 ) , other markers of plaque stability .
furthermore , the percentage of mmp-14 positive macrophages / fcms across all the sections correlated strongly with total macrophage number and negatively with smc number ( table 5 ) .
percentage positivity for mmp-14 and cox-2 also correlated with each other ( table 5 ) .
conversely , the percentage of timp-3 positive macrophages / fcms strongly correlated negatively with macrophage number and positively with smc number ( table 5 ) .
positivity for timp-3 correlated directly with cd206 and inversely with mmp-14 ( table 5 ) .
other characteristics including the presence of calcification , intraplaque haemorrhage , and thrombus formation were also recorded .
positivity of mmp-14 or timp-3 was unrelated to plaque calcification ( data not shown ) .
however , plaques with intraplaque haemorrhage had a significantly higher percentage of mmp-14 positive macrophages / fcms and tended to have a lower percentage of timp-3 positive fcms ( table 6 ) .
our main new findings are that macrophages activated with proinflammatory stimuli or foam - cell macrophages ( fcms ) irrespective of inflammatory activation are mmp-14  timp-3 compared to unstimulated ( m - csf differentiated ; mm ) or anti - inflammatory ( il-4 activated ; mm2 ) macrophages .
furthermore , mmp-14 positive fcms are more abundant and timp-3 positive fcms are less abundant in plaques with vulnerable rather than stable characteristics .
classical macrophage activation in vitro with proinflammatory stimuli is characterised by nf-b - dependent upregulation of a variety of additional proinflammatory mediators and enzyme systems , including cox-2 . by contrast , in vitro activation with anti - inflammatory stimuli such as il-4 or il-13 mediates a distinct transcriptomic response dependent on stat-6 phosphorylation [ 26 , 33 ] .
our results demonstrate a prominent effect of classical activation on mmp-14 mrna expression in macrophages and fcm , consistent with previous work on human monocytes   and macrophages   demonstrating that nf-b activation facilitates mmp-14 expression .
formation of foam cells per se did not lead to pro- or anti - inflammatory macrophage activation ( figure 1 ) , consistent with previous studies [ 16 , 37 ] .
fcms appear resistant to m1 and m2 cytokine - stimulation but employ a posttranscriptional pathway to modulate mmp-14 protein expression and subsequent activity .
indeed we have recently identified the involvement of a microrna , mir-24 , in the direct regulation of macrophage mmp-14 protein expression .
further evidence for a novel pathway in regulating fcm mmp-14 protein expression is provided by our demonstration that mmp-14 positive macrophages / fcms in plaques correlated with classical activation markers , cox-2 and nuclear - localised nf-b , and with the well - recognised marker of alternative activation , arginase-1 . hence foam - cell formation rather than m1 or m2 macrophage polarisation appears responsible for the mmp-14 fcms detected in advanced human atherosclerotic plaques .
by contrast to mmp-14 , timp-3 mrna was decreased during proinflammatory activation of macrophages or during foam - cell formation .
timp-3 positive macrophages / fcms correlated and colocalised with the mannose receptor ( mr ) , cd206 . in agreement with other previous studies , we found that cd206 and timp-3 were highly expressed in macrophages differentiated in m - csf ( mm ) and upregulated by il-4 ( mm2 ) .
however , timp-3 expression was only doubled after il-4 treatment ( figure 1 ) and is therefore present in both phenotypes .
mr positive macrophages / fcms were previously reported to be mainly located away from the lipid core of plaques [ 12 , 13 ] , consistent with the location of timp-3 positive macrophages / fcms in our study .
although little is known about the regulation of timp-3 transcription , the striking decrease in protein expression we observed upon foam - cell formation is consistent with previous observations that posttranscriptional mechanisms can regulate timp expression during macrophage development .
studies beyond the present scope are going on in our laboratories to investigate this possibility .
injurious roles for mmp-14 and protective effects of timp-3 have been suggested from animal studies ( see the introduction ) .
consistent with these proposals , we found that mmp-14 positive fcm subpopulations were associated with histological features of plaque vulnerability in carotid plaques based primarily on correlating quantitative plaque characteristics derived from all the sections irrespective of histological appearance ( table 4 ) .
the conclusions were also confirmed in carotid atherosclerotic plaques by prior blinded assignment of lesions based on overall histological appearance ( figures 6 and 7 ) .
this second analysis can be criticised because it is based on subjective visual observation , although we have shown previously that there is good intraobserver agreement .
it is also evident that there is a gender imbalance between the differing plaque phenotypes which may act as a source of variability in the statistical analysis .
our findings show that fcms in vulnerable atherosclerotic plaques can exhibit increased mmp-14 and decreased timp-3 protein expression .
this leads to heightened invasive capability , increased proliferation , and augmented susceptibility to apoptosis , as we have previously demonstrated .
our work therefore suggests that reducing mmp-14 activity and increasing that of timp-3 could be valid therapeutic approaches to reduce plaque rupture and myocardial infarction .
 matrix metalloproteinase-14 ( mmp-14 ) promotes vulnerable plaque morphology in mice , whereas tissue inhibitor of metalloproteinases-3 ( timp-3 ) overexpression is protective . 
 mmp-14hi  timp-3lo rabbit foam cells are more invasive and more prone to apoptosis than mmp-14lo  timp-3hi cells . 
 we investigated the implications of these findings for human atherosclerosis . in vitro generated macrophages and foam - cell macrophages , together with atherosclerotic plaques characterised as unstable or stable , were examined for expression of mmp-14 , timp-3 , and inflammatory markers . 
 proinflammatory stimuli increased mmp-14 and decreased timp-3 mrna and protein expression in human macrophages . however , conversion to foam - cells with oxidized ldl increased mmp-14 and decreased timp-3 protein , independently of inflammatory mediators and partly through posttranscriptional 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 histologically designated as rupture prone . 
 we conclude that foam - cells characterised by high mmp-14 and low timp-3 expression are prevalent in rupture - prone atherosclerotic plaques , independent of pro- or anti - inflammatory activation . therefore reducing mmp-14 activity and increasing that of timp-3 could be valid therapeutic approaches to reduce plaque rupture and myocardial infarction . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion