, . 185: 10–17 (1998)

CALCIFICATION IN ATHEROSCLEROTIC PLAQUE OFHUMAN CAROTID ARTERIES: ASSOCIATIONS WITH

MAST CELLS AND MACROPHAGES

1, c2 . 1*1University Department of Medicine, Manchester Royal Infirmary, Oxford Road, Manchester M13 9WL, U.K.

2University Department of Surgery, Withington Hospital, Manchester M20 8LR, U.K.

SUMMARY

Calcification has been examined in 250 samples of atherosclerotic lesions (types II to VI) of human carotid arteries using von Kossaand haematoxylin staining. Early calcification described as ‘stippling’ was first noted in stage III specimens, with intermediate and solidcalcifications becoming increasingly prominent within advanced plaques, especially stages Vb and VI. Although the relative frequenciesof stippling, intermediate and large calcified deposits varied between plaques of the same stage, the prevalent sites of calcification wererecognized as the deeper regions of the intima and the atheroma. Immunolocalization and histochemical techniques were used to identifythe associations of mast cells (MCs), macrophages, smooth muscle cells (SMCs), and elastin with the different stages of calcification.Early, dispersed stippling was commonly associated with local accumulations of macrophages (HAM56 and CD68-positive), MCs andextracellular MC tryptase, the presence of immunoreactive elastin, but the relative absence of SMCs. Intermediate stages ofcalcification described as ‘morula’ deposits were also associated with local increases in the numbers of macrophages and MCs. Largercalcified deposits, even within the same plaque specimen, showed no regular pattern of cellular or elastin associations. However, in thevast majority of specimens, macrophages represented the predominant cell type associated with different phases of calcification. Bycontrast, the calcification less frequently observed in the media beneath advanced plaques was commonly associated with SMCs andelastin; only rarely were macrophages or MCs present. These studies are the first to demonstrate that macrophages, MCs, andextracellular tryptase frequently occupy micro-environmental loci showing the first stages of calcification within the atheroscleroticplaque; similar associations with more advanced mineral deposits are discussed in relation to plaque rupture. ? 1998 John Wiley &Sons, Ltd.

J. Pathol. 185: 10–17, 1998.

KEY WORDS—atherosclerosis; calcification; macrophages; mast cells; smooth muscle cells; elastin; immunolocalization

INTRODUCTION

Atheromatous plaques are composed predominantlyof macrophages, smooth muscle cells (SMCs), endo-thelial cells, and extracellular matrix,1,2 but little isknown of the processes that lead to calcification of thearteries.3 Calcification is a prominent feature of athero-sclerotic lesions, especially in advanced stages of plaquedevelopment where it is implicated in the biomechanicalchanges of the artery wall and clinical complicationsassociated with cardiovascular disease. Recent studiesreport that atherosclerotic calcification is an organized,regulated process, rather than a passive ageingphenomenon.4–6 This view is based on the morphologi-cal similarities between advanced arterial calcificationand trabecular bone, and the demonstration withinplaques of several factors related to bone and mineralformation, such as osteocalcin, osteopontin, osteo-nectin, and bone morphogenetic protein-2a.3,6–8 More-over, in vitro studies have demonstrated the osteogenicproperties of pericyte-like cells derived from aortas,9and ultrastructural evidence supports the hypothesis

that hydroxyapatite, the major component of calciumdeposits, is formed within matrix vesicles produced fromarterial wall cells.3,10,11 Indeed, vesicles and cellulardebris associated with extracellular lipid-rich or choles-terol accumulations10,12 and elastin13 are also recognizedas sites of early calcium deposition. Such observationssuggest various mechanisms of calcification in athero-sclerosis, but the cell biology and micro-environmentalconditions required for calcification remain poorlyunderstood. Similarly, the role of calcification inthe pathogenesis of atherosclerotic plaque remainsuncertain.

Most studies of atherosclerotic plaque calcificationhave used coronary arteries or human aortas.3,10 Thepresent study has examined atherosclerotic carotidarteries and has demonstrated various stages of calcifi-cation, from early ‘stippling’ to larger solid deposits; wereport here the prevalent tissue sites of calcification inlate stage disease (stages IV–VI).10 Although macro-phages, foam cells, and SMCs are strongly implicated inthe pathogenesis of atherosclerosis,1,2 and mast cellshave recently been demonstrated in all developmentalstages of atherosclerotic plaque of carotid arteries,14 theassociation and potential contributions of these celltypes to the calcification process have not been investi-gated. We have therefore examined the distribution ofthese specific cell types in relation to the microzones

*Correspondence to: Dr David Woolley, University Department ofMedicine, Manchester Royal Infirmary, Oxford Road, ManchesterM13 9WL, U.K.

Contract grant sponsor: British Heart Foundation; Contract grantnumber: PG/94148.

CCC 0022–3417/98/050010–08 $17.50? 1998 John Wiley & Sons, Ltd.

Received 8 May 1997Accepted 3 December 1997

of calcification which contain either early, intermediate,or advanced calcified deposits. Since ultrastructuralstudies have reported that unesterified cholesterol andaltered elastin fibres are also important for the calcifi-cation process,13 we have similarly examined the distri-bution of elastin in these carotid plaque specimens.Our observations of the specific cellular associationswith various stages and sites of calcification show differ-ences between the intima and media of atheroscleroticcarotid arteries and point to a prominent association ofmacrophages, mast cells, and extracellular tryptase withearly stages of calcification.

MATERIALS AND METHODS

Materials

Samples of carotid arteries were collected from 101patients (61 males aged 49–81 years and 40 females aged58–85 years) undergoing therapeutic interventions foratherosclerotic changes in the University Departmentof Surgery, Withington Hospital, Manchester, U.K.Material consisted of endarterectomy specimens (n=90)and full thickness resections (n=11) of common carotidartery, frequently with bifurcation and an adjacentportion of the internal branch. This source of materialhas provided more than 250 tissue samples whichrepresented all stages of atherosclerotic plaquedevelopment.10

Processing and histology

Immediately after resection, the specimens wereplaced in Dulbecco’s modified Eagles’ medium(DMEM) and within minutes they were macroscopicallyassessed for atherosclerotic lesions under sterile con-ditions. Each specimen was cross-sectioned in 3–4 mmsegments and alternate fragments were fixed either inCarnoy’s fixative for 4–6 h or in 10 per cent neutralbuffered formalin for 18–24 h and processed to paraffinblocks. Specimens were sectioned at 3 ìm, or 5 ìm inheavily calcified material, placed on glass slides treatedwith poly--lysine (Sigma), dewaxed in Histoclear,rehydrated in a graded series of ethanol, and stainedwith haematoxylin and eosin. Each specimen wasassessed for the stage of plaque development, deter-mined by the most advanced portion of each lesionexamined, according to criteria described by Staryet al.10 The numbers of plaques for the different stageswere as follows: 30 for stages I–III and 50, 48, and 52 forstages IV, V, and VI, respectively.

Each specimen was examined for calcification usingeither haematoxylin or the von Kossa staining tech-nique,15 followed by counterstaining with Safranin O.Specimens which showed signs of calcification,from early ‘stippling’ through to macroscopiccalcified deposits, were subsequently examined for thedistributions of the following:

(i) mast cells, using immunolocalization with themonoclonal antibody to mast cell tryptase(Biogenesis, Poole, U.K.);

(ii) macrophages, using immunolocalization with themonoclonal antibodies to CD68 (KP1) andHAM56 (DAKO, Glostrup, Denmark);

(iii) smooth muscle cells, using immunolocalizationwith antibodies to muscle actin HHF35and smooth muscle actin clone 1A4 (DAKO,Glostrup, Denmark); and

(iv) elastin, immunolocalized with monoclonal anti-elastin antibody (Sigma Chemical Co. Ltd, Poole,U.K.).

All the secondary antibodies of biotinylated rabbit anti-mouse IgG and goat anti-mouse IgG were purchasedfrom DAKO.

Immunolocalization procedures

Mast cell tryptase—Tissue sections were incubatedwith 10 per cent (v/v) non-immune rabbit serum for30 min, followed by primary anti-tryptase antibody for2 h at room temperature. After washing in Tris-bufferedsaline (TBS, 3#10 min), the sections were incubatedwith secondary biotinylated rabbit anti-mouse antibodyfor 45 min. After further washing in TBS, StreptAB-Complex alkaline phosphatase (AP) conjugate (ABC-AP) was applied for 45 min, the sections were washedagain with TBS, and AP was developed using NewFuchsin.14

Macrophages—These were identified using two differ-ent monoclonal antibodies, CD68(KP1) and HAM56,each followed by biotinylated goat anti-mouse IgG assecondary antibody. Tissue sections from formalin-fixedmaterial needed pretreatment with trypsin (0·1 mg/mlTBS containing 0·1 per cent CaCl2 at 37)C for 15 min).After washes in TBS, the sections were treated withABC-AP and subsequently developed with NewFuchsin. Pretreatment of tissue sections and all washeswere as described for the tryptase immunolocalizationprocedure above.

Smooth muscle cells—These were identified using themonoclonal antibodies HHF35 and 1A4; the latterrecognizes á-actin of smooth muscle and myoepithelialcells. Procedures were essentially as described for thetryptase method. Formalin-fixed tissue sections requiredpretreatment with trypsin as described above, whereasthis procedure only slightly improved the stainingobtained in Carnoy’s fixed material.

Elastin—Tissue sections were first treated with trypsinas described above, followed by 10 per cent normal goatserum in TBS for 30 min at room temperature. Sectionswere then treated with monoclonal antibody to humanelastin for 2 h at room temperature, followed by theprocedure described above for tryptase.

Control tissue sections were incubated with eitherTBS or an appropriate isotypic normal mouse immu-noglobulin fraction at matched protein concentrationsinstead of the primary antibody, or with TBS instead ofthe secondary antibody. Controls were included for

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the examination of each specimen and consistentlyproduced negative results.

All tissue sections were counterstained with Mayer’shaematoxylin, mounted in Histomount (MensuraTechnology, Wigan, U.K.), examined with a ZeissPhotomicroscope III, and photographed usingEktachrome 160 tungsten film.

RESULTS

Calcification

Each of 250 tissue samples derived from 101 carotidartery specimens was examined initially with haema-toxylin and eosin staining. This provided information onboth the stage of atherosclerotic plaque developmentand the extent of calcification. Haematoxylin proveduseful for the demonstration of large calcified depositsbut was less effective for the early stages of calcification,visualization of these being improved by Nomarskiinterference microscopy. Von Kossa staining was usedto assess the extent and nature of calcification in speci-mens from all stages of plaque development. Most latestage plaques showed extensive calcification with soliddeposits of variable size. The different stages of calcifi-cation are described here as dispersed ‘stippling’,‘morula’, and ‘solid’ deposits, representing a progressiveincrease in size (Fig. 1).

Early stippling was observed in a proportion of speci-mens of stage III plaque development, especially in areasperipheral to the small pools of extracellular lipid. Theformation of intermediate or ‘morula’ calcifications wasincreasingly evident in stages IV and V, these being morecommonly located in the deeper aspect of atheroma.Some also showed intermediate calcifications within thecore of extracellular lipid, with larger calcified depositsnormally being observed at the periphery of the lipidcore at stage V. More extensive solid calcifications werea common feature of stages Vb and VI, these usuallybeing located in the deeper aspect of the atheroma, aswell as in the deeper layers of the intima. In some cases,calcified deposits were observed within the media imme-diately subjacent to the intima. In most cases of stageVI, all three phases of calcification could be observed,with stippling and intermediate forms often associatedwith the larger macroscopic calcified deposits (Fig. 1eand 1f). Early calcification, as judged by stippling, wasalso observed in the fibrous cap and shoulder regions oflate stage, complicated atherosclerotic lesions, usually inassociation with focal accumulations of inflammatorycells.

Figure 2 provides a summary illustration of theprevalent calcification sites observed in plaques repre-senting developmental stages IV to VI. Although theextent of calcification in a few of these specimenswas minimal, most showed intermediate and/or solidcalcifications in the later stages (Vb–VI). However,whereas the three stages of calcification (Fig. 1) wereobserved for the majority of these samples, some didnot contain large solid deposits. By contrast, in stageIV the main type of calcification was stippling andintermediate.

Although von Kossa staining effectively demonstratedall stages of calcification in plaque specimens, it wasnot compatible with the immunohistochemical tech-niques used to identify specific cell types. Thus, inorder to assess the distributions of specific cell types inrelation to sites of calcification, we have used immuno-localization procedures combined with haematoxylincounterstaining.

Calcification and mast cells

Immunolocalization of mast cell tryptase demon-strated the presence of mast cells in all developmentalstages of atherosclerotic plaque, especially their focalaccumulations in the shoulder regions of late stagedisease. Mast cells and extracellular tryptase stainingwere often associated with areas of early calcification,such as the stippling and morula-type calcificationsshown in Fig. 3a, 3b, and 3c, this being observed in mostspecimens representing stages III to VI. Although mastcells were also observed in association with larger calci-fied deposits, this was a less consistent finding (Fig. 3d).However, some late-stage complicated plaque specimensdemonstrated prominent solid calcifications with closelyassociated mast cells and extensive extracellular tryp-tase, this often being related to local matrix disruptionand oedema at the matrix–calcification interface.

Calcification and macrophages

Two monoclonal antibody markers, CD68 (KP1) andHAM56, were used to identify macrophagic cells in allstages of plaque development (Fig. 3e–3h). A compari-son of their staining properties revealed a wider speci-ficity for the CD68 marker, this being useful for theidentification of large foam cells, giant cells, and cellularremnants within atheroma. By contrast, HAM56showed specificity for monocyte-macrophages and smallfoam cells, this being preferred for the demonstration ofmacrophages associated with calcification in Fig. 3e–3g.Macrophages were commonly associated with all areasof calcification, especially microzones showing dispersedstippling and small morula deposits, and were morefrequently observed in association with solid calcifieddeposits than mast cells.

Calcification and smooth muscle cells (SMCs)

Two monoclonal antibodies, HHF35 and anti-humaná-actin, were used to identify SMCs and myoepithelialcells. Both markers revealed a relatively small number ofSMCs in the region of atheroma and most areas ofcalcification. Whereas SMCs of the media showedstrong staining for actin, relatively few of these cellswere observed in the intima or calcification sites of earlystages of plaque development (II to IV). Advancedatherosclerotic lesions (V to VI) showed SMCs in thefibrous cap, shoulder regions, and around the atheroma,but the number of cells stained with these antibodies inthe intima was much reduced compared to macrophages(Fig. 3k), and they were rarely associated with calcifi-cations. By contrast, calcified deposits within themedia were invariably associated with SMCs.

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Calcification and elastin

The demonstration of elastin within the intima wasvariable; it was often observed in the sites associatedwith early stippling, but only rarely with intermediate orsolid calcified deposits. On the other hand, elastin,together with SMCs, was commonly associated withcalcifications of the media.

Comparative analyses on the relative distributions ofmast cells, macrophages, SMCs, and elastin in relationto calcification were carried out on consecutive tissue

sections of 15 specimens representing stages V and VI.Figure 3i–3l provide an example of these studies wheremast cells, macrophages, and elastin are closely associ-ated with a solid calcification from stage Vb plaque, butSMCs appear remote from the calcification. Such obser-vations are in accord with the overall summary for thefrequencies of specific cell types associated with thedifferent stages of calcification provided by Table I.Macrophages were the predominant cell type associatedwith early stippling and intermediate stages of calcifica-tion within the intima, with mast cells and the presence

Fig. 1—Different stages of carotid artery calcification demonstrated by von Kossa staining. (a, b) Very early calcifications (black staining)in the intima adjacent to atherosclerotic lesion type III (a) which at higher magnification (b) shows disperse ‘stippling’ calcification. (c, d)Intermediate stage, described as ‘morula’ calcification, from stage III of atherosclerotic plaque. (e, f) Large calcification deposits inatherosclerotic lesion type VI at low (e) and higher magnification (f). Note that the large calcified deposits are adjacent to areas of smaller,dispersed calcifications. Such observations were commonly encountered in the deep layer of the intima at the base of atheromatous lesionsof stages V and VI. All microphotographs show von Kossa staining with Safranin O counterstain. (a, c, e) #150; (b, d, f) #270

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of elastin also prominent in those sites. SMCs wereinfrequently associated with early stippling, and variableobservations for all three cell types and elastin werenoted in regions of advanced calcified deposits. Bycontrast, calcification sites in the media were almostalways devoid of macrophages or mast cells.

DISCUSSION

Calcification of atherosclerotic plaque is consideredto be an organized and regulated process with manysimilarities to the mechanisms associated with boneformation.4–6,16 Hydroxyapatite is the predominantcrystalline form, appearing initially as very smalldispersed crystals described as ‘stippling’ in variousmicrozones of the intima. This early calcification wasseen in some specimens of stage III plaque development,becoming more frequent in the more advanced stages ofV and VI.10 Intermediate (morula-type) and solid calci-fied deposits were more frequently observed in theselate-stage plaque specimens. The more prevalent siteswere recognized both within and at the periphery of thelipid core, and in the deeper regions of the intimasubjacent to the atheroma. Whether these locationsrepresent the original sites of calcification is uncertain. Itseems possible that some of the larger calcified depositsobserved within the lipid core or atheroma could haverelocated in response to the haemodynamic pulsatileforces of the arterial blood flow, or to the progressive

reorganization of the cells and matrix around the largerdeposits. By contrast, the histological observations ofearly, stippled calcification almost certainly representthe authentic sites of the initial calcification process.It therefore seems likely that these are the locationsin which the cells, osteogenic factors, and matrixcomponents responsible for the calcifying process are tobe found.

The demonstration of macrophages and mast cells inthe micro-environments containing early stippling was acommon feature of most plaque specimens. Both celltypes contain or may express numerous proinflamma-tory and reparative mediators, such as cytokines, pros-tanoids, and growth factors.17,18 The observation ofdiffuse extracellular tryptase associated with such micro-zones of calcification suggests that mast cell activationcould potentially contribute to the calcifying process.Tryptase is a tetrameric proteinase with numerousproperties; it has been implicated in tissue remodellingbecause it can degrade matrix components such asfibronectin and type VI collagen, can activate precursorsof the matrix metalloproteinases, is mitogenic for fibro-blasts and epithelial cells, stimulates collagen synthesis,and acts directly as a chemoattractant for neutrophilsand eosinophils.18,19 The demonstration of mast celltryptase within the extracellular matrix of neocalcifica-tion sites also provides a marker for other potentmast cell mediators released in parallel with tryptase,such as histamine, heparin, TNFá, and TGFâ.14,19,20

However, at present it is not known whether mast cell or

Fig. 2—Summary illustration of the prevalent calcification sites in late stage atherosclerotic plaque of carotid arteries

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macrophage ‘activation’ either precedes or results fromthe initial deposition of hydroxyapatite in the tissue.Similarly, it is not known whether macrophages or mast

cells are recruited to such sites before or after calcifica-tion commences. Both cell types have different mecha-nisms for recruitment and maturation within tissues; for

Fig. 3—Sites of calcification in atherosclerotic lesions of human carotid arteries and their associations with mast cells, macrophages, smooth musclecells, and elastin. (a–d) Mast cells and calcifications visualized by staining for anti-tryptase (red) and haematoxylin. Note the extracellular stainingfor MC tryptase in areas of calcified stippling (a and b) and the presence of MCs and tryptase around intermediate and large calcifications (c andd). (e–g) Macrophages and calcification visualized by staining for HAM56 (red) and haematoxylin. Note the close spatial relationship betweenmacrophages and early stippling calcifications (e and f) and the intimate association of macrophages with more advanced calcifications (g).Calcifications are identified by arrows. (h) CD68 staining showing a positively stained giant cell (arrow) and other monocyte-macrophages in closeassociation with a solid calcification. (i–l) Consecutive tissue sections from atherosclerotic lesion stage Vb stained for mast cell tryptase (i),macrophage marker HAM56 (j), smooth muscle cell actin (k), and elastin (l). Note the proportions and distributions of each cell type, and the localconcentration of immunoreactive elastin, in relation to the site of calcification (haematoxylin). All positively stained cells and elastin are red. (a, b,c, d, e, f, g, h) #270; (i, j, k, l) #100

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example, mast cells require the c-kit ligand or stem cellfactor,21 whereas elevated extracellular calcium concen-trations are known to recruit macrophages effectivelyand facilitate foam cell accumulations.22 It is uncertainalso whether macrophages or mast cells are involved,either directly or indirectly, in the regulation of severalmatrix proteins associated with mineralization pro-cesses. These include the osteogenic proteins osteo-pontin, osteonectin, and osteocalcin, whose mRNAexpression has been demonstrated in association withareas of mineralization in coronary arteries,6 and bonemorphogenetic protein-2a, a potent osteoblastic differ-entiation factor which has been identified in calcifiedatherosclerotic plaque.7

It is recognized that vascular SMCs may assumeseveral phenotypes within atherosclerotic arteries, fromcontractile cells in the media with strong actin-stainingproperties to proliferative, matrix-secreting cells ofthe intima which contain few contractile elements.23,24 Itis therefore possible that the immunotechniques forSMC actin used in this study may have under-estimated the contribution of the transformed, intimalSMC to the calcification process. Nevertheless, itwas clear that calcification sites within the mediawere almost exclusively associated with SMC andelastin, in contradistinction to the high frequencies ofmacrophage and mast cell associations with intimalcalcifications.

In contrast to the possible contributions of macro-phages and mast cells to early calcification processes,these same cells may assume quite different functions inrelation to well-established mineral deposits found inlate stage atherosclerosis. The clinical complications ofplaque rupture and subsequent thrombosis are com-monly associated with the lateral margins of lipid poolsor where there is significant calcification within theintima.25 At such sites, the interface between rigidcalcium mineral and compliant structures of the intimalcomponents may become destabilized by haemodynamicforces and/or by the inflammatory response. The obser-vations reported here of local concentrations of macro-phages and mast cells around some of the larger calcifieddeposits may well contribute to plaque destabilization.For example, it is possible that macrophage activationwould lead to matrix disruption by the expression ofproinflammatory cytokines and subsequent stimulationof matrix metalloproteinase production by macrophagesor SMCs.26–28 Similarly, mast cell activation would

inevitably bring about local oedematous and proteolyticeffects which could favour matrix disruption.14,29

Thus, the interaction of either macrophages or mastcells with the larger mineralized deposits of late stagedisease might well result in their activation, not onlyperpetuating the inflammatory response, but also bring-ing about damage to the extracellular matrix andincreasing the risk of plaque destabilization and localthrombosis.

Several recent studies have described the cell typespurportedly responsible for calcification of atheroscle-rotic plaque in vivo. These include the calcifying vascularcells, described from in vitro studies as microvascular‘pericyte-like’,7,9 and vascular dendritic cells which pro-duce the calcium-binding S-100 protein.30 Moreover,vascular SMCs and macrophage-derived foam cells wererecently shown to express the two bone-associatedproteins osteopontin and matrix Gla protein, especiallyin association with necrotic lipid cores and areas ofcalcification.24 However, the in vivo distributions andpotential interactions of these ‘calcifying’ cells relative tomacrophages, mast cells, and calcification sites remainuncertain. By contrast, some neocalcification sites areobserved to be relatively acellular, a realization whichprompted Bobryshev et al.13 to propose that somecalcified deposits may form independently of cellularmechanisms. Examples of this include the physico-chemical impregnation of extracellular structures suchas elastin and cholesterol esters by calcium salts,13 andthe observation that lipids or cholesterol may act asnucleation sites for hydroxyapatite deposition.12,31

Elastin has also been reported to act as a calcifyingprotein, and vesicles probably derived from dead cellsmay also serve as calcification sites.3,10 Such reportsemphasize the disparate nature of the calcificationprocess in atherosclerotic plaque.

From our studies and those of others, it seemsapparent that the dynamic recruitment of specific celltypes, the expression of osteogenic factors, the compos-ition of the matrix components, and the local calciumconcentrations may all provide the specific micro-environmental conditions which promote calcification.As yet, the sequence of cellular events responsible for thecalcification process remains uncertain, but differencesobserved between the intimal and medial sites of calcifi-cation strongly suggest that there may be more than aunitary explanation for calcification in atheroscleroticarteries.

Table I—Summary table to illustrate the association of specific cell types and elastin with different stagesof calcification in atherosclerotic carotid arteries

Intima

MediaStippling Morula Deposits

Macrophages + + + + + + + + +/+ + "/+Mast cells + + + + "/+ + "/+Smooth muscle cells "/+ +/+ + "/+ + + + +Elastin +/+ + + + + "/+ + + + +

" =Absent; + to + + + + =increasing frequency of cellular or elastin associations with microzones of calcification.

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ACKNOWLEDGEMENTS

We thank Anne Farrell and Andrew Picton for theirassistance in the provision of the surgical material. Thiswork was supported by the British Heart FoundationProject Grant No. PG/94148.

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