mineral surface in calcified plaque is like that of bone: further evidence for regulated...

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DOI: 10.1161/ATVBAHA.108.172387 14, 2008;

2008;28;2030-2034; originally published online AugArterioscler Thromb Vasc BiolSchoppet, Catherine M. Shanahan, Jeremy N. Skepper and Erica R. Wise

Melinda J. Duer, Tomislav Friscic, Diane Proudfoot, David G. Reid, MichaelRegulated Mineralization

Mineral Surface in Calcified Plaque Is Like That of Bone: Further Evidence for

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Mineral Surface in Calcified Plaque Is Like That of BoneFurther Evidence for Regulated Mineralization

Melinda J. Duer, Tomislav Friscic, Diane Proudfoot, David G. Reid, Michael Schoppet,Catherine M. Shanahan, Jeremy N. Skepper, Erica R. Wise

Objectives—Cell biological studies demonstrate remarkable similarities between mineralization processes in bone andvasculature, but knowledge of the components acting to initiate mineralization in atherosclerosis is limited. Themolecular level microenvironment at the organic–inorganic interface holds a record of the mechanisms controllingmineral nucleation. This study was undertaken to compare the poorly understood interface in mineralized plaque withthat of bone, which is considerably better characterized.

Methods and Results—Solid state nuclear magnetic resonance (SSNMR) spectroscopy provides powerful tools forstudying the organic–inorganic interface in calcium phosphate biominerals. The rotational echo double resonance(REDOR) technique, applied to calcified human plaque, shows that this interface predominantly comprises sugars, mostlikely glycosaminoglycans (GAGs). In this respect, and in the pattern of secondary effects seen to protein (mainlycollagen), calcified plaque strongly resembles bone.

Conclusion—The similarity between biomineral formed under highly controlled (bone) and pathological (plaque)conditions suggests that the control mechanisms are more similar than previously thought, and may be adaptive. It isstrong further evidence for regulation of plaque mineralization by osteo/chondrocytic vascular smooth muscle cells.(Arterioscler Thromb Vasc Biol. 2008;28:2030-2034)

Key Words: atherosclerosis � biomineralization � glycosaminoglycans � nuclear magnetic resonance spectroscopy� vascular calcification

Calcification1 is prevalent in numerous vascular patholo-gies and is associated with adverse clinical outcomes.

Parallels have frequently been drawn between ectopic calci-fication processes in vessels and normal developmental os-teogenesis. In fact vascular calcified material is by manycriteria indistinguishable from bone, and its formation in-volves the participation of many cellular and molecularsignaling processes underlying normal osteogenesis.2 Theseinclude matrix vesicle release, expression of mineralization-regulating proteins by vascular smooth muscle cells(VSMCs) of the vessel wall, and the association of calcifica-tion with lipid and chronic inflammation, a hallmark ofatherosclerosis.3 Indeed vascular calcification is recognizedas regulated biomineralization instead of merely passiveprecipitation. However questions are still unanswered con-cerning the mechanisms leading to the initiation of calcifica-tion in the vessel wall particularly as it occurs in differentvascular environments (ie, media and intima), which do notmimic that of bone. Moreover, VSMCs can overexpressmineralization inhibitors such as matrix Gla protein (MGP) toeffectively block mineralization.4

Aspects of the molecular structure of mineralized tissues5

can be studied by the solid state NMR (SSNMR) technique13C{31P} rotational echo double resonance (REDOR).6 13C isa stable, nonradioactive, NMR-receptive isotope of carbonoccurring naturally at about 1.1% abundance; similarly 31P isa stable NMR-receptive isotope of phosphorus which how-ever is naturally present at 100% abundance. A 13C NMRspectrum records a signal for each chemically distinct carbonsite in the sample. In a REDOR experiment a 13C NMRspectrum is recorded with and without a train of radiofre-quency pulses applied to the 31P spins before acquisition ofthe spectrum, forming the so-called nondephased anddephased 13C spectra, respectively. The nondephased 13Cspectrum recorded acts as a reference, whereas in the spec-trum recorded with the 31P pulse train, signals attributable to13C spins in close proximity to 31P spins in the material willshow a reduction in intensity. Thus, those 13C sites which arespatially close to 31P are immediately identified from theresonance frequency of those signals showing reduction inintensity. By following the reduction in intensity of a given13C signal as a function of the time for which the 31P pulse

Original received April 21, 2008; final version accepted August 4, 2008.From the Departments of Chemistry (M.J.D., T.F., D.G.R., E.R.W.), Medicine (D.P.), and Physiology, Development, & Neuroscience (J.N.S.),

University of Cambridge, UK; the Department of Internal Medicine and Cardiology (M.S.), Philipps-University, Marburg, Germany; and theCardiovascular Division (C.M.S.), Kings College London, UK.

Correspondence to Melinda J. Duer, Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, CB2 1EW, UK. [email protected]

© 2008 American Heart Association, Inc.

Arterioscler Thromb Vasc Biol is available at http://atvb.ahajournals.org DOI: 10.1161/ATVBAHA.108.172387

2030

Cell Biology/Signaling



train is applied, quantitative determination of the 13C-31Pdistance is possible. The REDOR effect is only transmittedover interatomic distances of less than 10 Ångstroms (1 nm),so it is a conclusive proof of a true molecular compositematerial in which individual components are bound in asso-ciations mediated by intermolecular interatomic forces (forinstance electrostatic and hydrogen bonding) and not merelymixed together. The interface between the organic matrix andcalcium phosphate biomineral particles in plaques and bone isparticularly amenable, because practically all 13C is in theformer and all 31P in the latter. This makes the experimenthighly selective for the interface itself. In bone it shows thatthe molecular constituents of the organic-mineral interfaceare rich in polysaccharides, most likely glycosaminoglycan(GAG) sugars, rather than proteins.7 Therefore, a comparativeanalysis of this interface may provide clues to the exact natureof similarities and differences between mineralization pro-cesses in the vasculature, and bone, that cannot be revealed bycell biological studies alone.

Materials and MethodsCalcified Atherosclerotic PlaqueMaterial was collected with appropriate ethical approval duringcarotid endarterectomy procedures. The human tissue (n�4) was putinto sterile flasks with 15 mL medium (M199 with penicillin andstreptomycin) and 1 mL elastase (from a stock solution of 5 mg/mLporcine pancreatic Type IV elastase, Sigma) and 5 mL collagenase(from a stock solution of 9 mg/mL C. hemolyticum Type I collage-nase, Sigma) added, shaken for 6 hours in a waterbath at 37°C,centrifuged at 800 r.p.m. for 5 minutes, washed with cold sterilephosphate buffered saline, spun again, and the resultant pellet frozenand stored for analysis. The procedure is necessary as SSNMR is arelatively insensitive technique, so it is highly desirable to removefrom the sample as much extraneous material as possible in order toconcentrate the sites of interest. To determine whether this pretreat-ment of the material interfered with the SSNMR measurements, itwas compared with untreated tissue that was frozen before grinding.For all SSNMR measurements, thawed material was lightly groundwith a pestle and mortar and packed into 4-mm outer diameterzirconia magic angle spinning (MAS) rotors (Bruker).

A bone from an adult horse used for general purpose exercise andeuthanized for humanitarian reasons unconnected with this study wasused for comparison. Several bone specimens obtained at surgerywere found to be identical to those obtained at autopsy, indicatingthat the difference in harvesting between plaque (surgery) and bone(autopsy) does not introduce noticeable differences. In the cases ofboth mineralized plaque and bone, the grinding produced particlesthe order of several tens of micrometers in size; as typical biomineralparticles are much smaller than this and the interatomic interactionswe are exploring operate over subnanometer length scales, webelieve the grinding will not affect our observations of theseinteractions significantly, if at all. In previous studies on bone, wehave explored a variety of ways of preparing the sample material,including slicing/chopping and grating, as well as grinding todifferent particle sizes and saw no difference in spectra betweendifferent preparation methods.

NMR SpectroscopyThis was performed on a Bruker Avance-400 triple channel spec-trometer at a polarizing magnetic field strength of 9.4 T (Tesla), withsamples packed into 4-mm outer diameter zirconia rotors. Theseenable the samples to be spun rapidly (at a rotational frequency of12.5 kHz in these experiments) at an angle of 54.7° to the polarizingmagnetic field, the so called “magic angle.” This “magic anglespinning” (MAS) is necessary to remove certain magnetic interac-tions between the NMR active nuclei in the sample which would

otherwise broaden the resonances and result in featureless spectra.Where there was insufficient material to fill the rotor, the dead spacewas packed out with Teflon tape which gives no NMR signal withthe data acquisition methods we have used. The following spectralacquisition parameters were used: Resonance frequencies: 1H 400.4MHz, 13C 100.7 MHz, 31P 162.1 MHz; 1H �/2 pulse 2.5 �s, meanramped cross polarization (CP) field strength 70 kHz, CP time 2.5ms, 1H decoupling field 100 kHz, recycle delay 2 s, MAS rate 12.5kHz. For the 31P spectra 128 signal accumulations were averaged forsatisfactory signal to noise; 13C spectra required averaging ofbetween 20 k and 40 k signal accumulations. This difference reflectsthe 100% and 1.1% abundance of the 31P and 13C isotopes, respec-tively. 13C{31P} REDOR was performed with 1H-13C CP, 31P � pulsesof 8.4 �s, the separation between the center of the 31P pulses pulsesbeing 80 �s and synchronous with the MAS, and a 13C refocusing �pulse of 10 �s inserted at the middle of the recoupling 31P pulse train.By convention, the notation “13C{31P}” signifies that it is the signalfrom 13C magnetic nuclear spins which is observed directly (at 100.7MHz), whereas the 31P magnetic nuclear spins are caused to interactwith them by a suitable combination of nuclear spin energy levelexcitations applied at 162.1 MHz.

X-Ray Powder Diffraction (XRPD)This was performed on a Philips X’Pert Pro powder diffractometerequipped with an X’celerator RTMS detector, using Ni-filtered CuK�

radiation. Data collection was performed in a 5 to 80° range usingsamples on a flat plate, with a scanning step size of 0.008°, time perstep of 10.8 seconds and scan speed of 0.0985°/s.

Scanning Electron Microscopy (SEM)Extracted mineral was dusted onto spectroscopically pure double-sided carbon tabs mounted on Cambridge SEM stubs. It was viewedin a FEI Phillips XL30 FEGSEM operated at 5kv.

Figure 1. Comparison of 13C spectra of mineralized plaque andbone. Some putative glycosaminoglycan (GAG) signals are high-lighted in red and related to the generic GAG structural formulashown. Some collagen protein signals are also assigned (A, Ala-nine; G, Glycine; P, Proline; O, Hydroxyproline).

Duer et al Glycosaminoglycans in Plaque Calcification 2031



ResultsFigure 1 compares typical 13C SSNMR spectra of calcifiedplaque, and bone; supplemental Figure I (available online athttp://atvb.ahajournals.org) shows data from all samples andclearly shows a high degree of reproducibility. Collagenaccounts for most signals.8 Notable exceptions are a broadsignal at about 103 ppm, weak but present in all samples, andanother at 76 ppm, which tends to be more prominent inmineralized plaque than in bone. These are consistent, respec-tively, with the anomeric carbons, and some of the otherpyranose 6-membered sugar ring carbons, of �-linked poly-saccharides like GAGs.9 To highlight the relationship be-tween GAG signals and GAG chemical structure, a chemicalrepresentation of the repeat subunit (-glucuronic acid � (1–3)N-acetylgalactosamine-4-sulfate � (1–4)-) of the widely oc-curring GAG chondroitin-4-sulfate is included. The signalattributable to most of the GAG sugar ring carbon atoms, thesignal attributable to the GAG sugar anomeric carbons(marked * in the chemical structural formula and the spectra),and the signal attributable to the GAG acidic carboxylatecarbons (marked # in the chemical structural formula andspectra) are identified. The latter also contains signal fromprotein acidic carboxylate carbons; it presents a prominentshoulder on the high frequency edge of the signal envelopecantred at about 175 ppm, itself attributable to overlap ofnumerous protein (and GAG) carbonyl signals. Spectra oftreated and untreated human carotid endarterectomy materialare shown in supplemental Figure II. This clearly shows thatcollagenase/elastase pretreatment did not affect the GAGcomposition or structure in the material.

Figure 2 shows X-Ray powder diffraction (XRPD) and31P SSNMR data proving the mineral is essentially hy-

droxyapatite- and bone-like.10 However the XRPD trace ofmicrocrystalline hydroxyapatite contains numerous sharp re-flections consistent with a highly ordered crystalline environ-ment; in consequence of this order the 31P NMR spectrum isalso narrow, showing that all phosphorus atoms experiencevery similar environments. The XRPD trace and 31P spectrumof bone show much broader features consistent with lessperfectly crystalline mineral, and greater environmental het-erogeneity attributable to molecular disorder and mineralsurface effects. The XRPD trace and 31P NMR spectrum ofcalcified plaque is intermediate between these extremes,probably reflecting greater mineral crystallinity and largeraverage mineral crystallite size. This is borne out by SEMillustrating the typical heterogeneous micro/nanocrystallinenature of the calcifications.

Figure 3 presents typical 13C{31P} REDOR results reflect-ing the mineral-organic interface composition; more compre-hensive data on other samples is presented in supplementalFigure III. The black trace is the reference spectrum, which inthis case is largely due to the various components of collagen.As described in the Introduction, this spectrum containssignals from all 13C sites in the sample. The dephasedspectrum in red is obtained after the application of radiofre-quency pulses to the 31P nuclei in the sample. As alreadyexplained, the effect of these pulses is to reduce in intensityany signal due to a 13C nucleus close in space to a 31P; closein space in this context is less than 10 Å. As in bone, the mostaffected signals, which can therefore be associated withmolecules in intimate association with mineral, are the 76ppm polysaccharide peak, and signals at 175 and 181 ppmwhich are probably from amide carbonyl, and carboxylate,groups, which are abundant in GAG polysaccharides (as well

Figure 2. Comparison of X-ray powder diffraction (XRPD) patterns and 31P solid state NMR spectra, of synthetic microcrystalline hydroxyap-atite, bone, and mineralized plaque. SEM illustrates the typical heterogeneous micro/nanocrystalline nature of the calcifications.

2032 Arterioscler Thromb Vasc Biol November 2008



as in some proteins, of course). The intensity reductionshould increase with increasing time for which the radiofre-quency pulses are applied to the 31P nuclei; this is illustratedin supplemental Figure IV, and compared with that of bone insupplemental Figure V. Also in common with bone, othersignals suffer a smaller amount of intensity loss; some ofthese are from collagen but others may be from exocyclic6-carbons (ca. 65 to 70 ppm), aminated or amidated ringcarbons (ca. 54 ppm), or N-acetyl methyl groups (ca. 25 ppm)of GAGs and other polysaccharides. Thus, the macromole-cules most strongly associated with the mineral phase incalcified plaque are polysaccharides, probably GAGs. Wehave also examined samples histologically after staining withvon Kossa stain, specific for calcified deposits, and alcianblue, which recognizes polyanions; the Inset shows typicaldata and more appears in the supplemental material. We findthat calcification (dark brown) always occurs in alcian bluestaining regions which are probably GAG-rich (blue), al-though not all alcian blue staining regions calcify. Moreover,GAG-rich calcified regions were often found surroundinglipid pools—areas previously described as colocalizing withmineralization.11 This suggests that calcification tends tooccur in polyanion-rich regions, where the polyanions aremost likely GAGs as these are the predominant polyanionspresent in these tissues. Coincidence of GAG and calcifica-tion at the micron resolution length scale of light microscopy

is not synonymous with the subnanometer atomic length scaleintermolecular bonding revealed by the NMR, of course, thoughit is consistent with the hypothesis this article presents.

DiscussionWe have shown that the matrix-mineral atomic interface incalcified atherosclerotic plaque is very similar to that in boneand, surprisingly, is characterized by a predominance ofGAGs. Colocalization of GAGs and mineralization has beendetected histologically in calcifying tissue,12–14 and impor-tantly there is accumulating evidence that the type of GAG inthe matrix may exert tight control over mineralization,particularly during its initiation.15,16 An early event in bonemineralization is release of membrane-bound matrix vesicleswhich act as the nidus for mineral nucleation by concentrat-ing calcium and phosphate. Matrix vesicles bind GAGs, andthis binding is increased in mineralization-competent hyper-trophic chondrocyte-derived matrix vesicles, further suggest-ing that GAGs play a direct role in initiating physiologicalmineralization.17 However, as mineralization progresses,GAGs may also act to prevent uncontrolled propagation ofcalcium phosphate crystallization. Further evidence for thiscomes from human disease where dysregulated expression ofGAGs leads to bone defects, whereas a lower incidence ofplaque calcification in chronic asthma sufferers, who showelevated levels of circulating GAGs like heparin, has beenreported.18

Atherosclerotic lesions contain proteoglycan, intermixedwith loosely scattered collagen fibrils;19 as plaques developsecretion and accumulation of GAGs occurs within theextracellular matrix. Also, GAGs bind low-density lipopro-tein,20 retaining cholesterol in the plaque and enhancing localproinflammatory signals. Lipid and mineralization are coin-cident in plaques,4,11 and inflammatory signals at lipid accu-mulation sites initiate calcification by inducing vascularsmooth muscle cell (VSMC) death and vesicle release.21–24

Vesicles contain hydroxyapatitic nanocrystals representingthe initial calcification seed. Thereafter, VSMCs that haveundergone osteo/chondrocytic conversion are found abuttingthe microcalcifications.21 Taken together with this study,showing the remarkable similarities between the resultantbiomineral found in plaques, and bone, it seems reasonable topostulate that GAG binding to lipid may initiate VSMCinjury and death. This leads to an initial calcification nidus,followed by osteogenic phenotypic conversion of VSMCs,although the mineralization process may also trigger GAGproduction.

Indeed, the similarities between bone and carotid arterialmineralization which we show support the contention thatthis may be protective, by rapidly dampening unregulateddeposition of mineral initiated by injury. Part of this responsemay be to produce biomineral that is inert, rather thanproinflammatory such as in synovial fluid.25 It is plausiblethat this is a common feature of all vascular calcification butthis requires more evidence to establish.

As in bone, GAGs have attracted less attention in plaquethan proteins,26 such as matrix Gla protein and osteocalcin,27

decorin,28 osteopontin,29 bone sialoprotein,4 and collagenitself,30 which have all been proposed as important mineral-

Figure 3. 13C{31P} rotational echo double resonance (REDOR)NMR spectra of mineralized plaque at 2 different 31P-13C dipolarrecoupling times. Inset: adjacent histology sections from miner-alized plaque, one stained for polyanions with alcian blue, theother for polyanions with alcian blue and calcification with vonKossa.

Duer et al Glycosaminoglycans in Plaque Calcification 2033



ization modulators. Nonetheless, many of these are oftenheavily glycosylated, and it may be part of their role to targetGAGs to specific sites in developing, and mineralizing,matrices. However, it is particularly interesting that theseproteins were not more abundant in atherosclerotic mineraland that other vascular specific extracellular matrix proteinssuch as elastin, often postulated to be a nucleator of hydrox-apatite in the vessel wall, were not present in close associa-tion with mineral. This further supports the notion that somevessel wall biomineralization may be adaptive, and this hasimplications for its treatment.

AcknowledgmentsWe thank Nikki Figg for histological preparations.

Sources of FundingThis work was supported by UK BBSRC & EPSRC and the BritishHeart Foundation.

DisclosuresNone.

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Duer: Glycosaminoglycans in plaque calcificationOnline material



Figure I. Intersample variability. 13C solid state NMR spectra of the four samples studied, all shown after collagenase and elastase digestion. The similarity between samples is not as marked as what we observe for tissue which mineralizes normally, like bone. Although other macromolecules, and lipid in the case of sample D, probably contribute, the signals characteristic of collagen and GAGs are strongly conserved (cf. Figure 1 of the main paper). In particular the “signature” signals of the GAG sugar ring carbons, and of the γ-carbon of hydroxyproline, an amino acid which is distinctive of collagen, are highlighted by red and black arrows respectively in each dataset.



Figure II. Effects of collagenase/elastase digestion. Comparison between 13C solid state NMR spectra of calcified plaque (designated sample B in remaining online material) before (bottom) and after (top) collagenase and elastase digestion. Not surprisingly the procedure does change the overall appearance of the spectrum, in particular several sharp signals become much less prominent or disappear. These are probably due to metabolites which are more mobile (on a molecular motional scale) than the extracellular matrix structural macromolecules, most likely lipids. However all the prominent features ascribable to GAG and collagen protein are retained. Some of these are indicated and assignments, corresponding to those in Figure 1 of the main paper, are shown.

Another factor in the decision to study digested material is dictated by the practicalities of the solid state NMR experiment. This involves spinning the sample rapidly in a small rotor at a rotation frequency (for our experiments) of 12.5 kHz. This places great demands on rotor stability, and sample homogeneity is paramount in ensuring this. Before digestion samples are heterogeneous, with calcified bodies intermixed with lipid rich organic material, which is not conducive to efficient sample spinning, particularly for the REDOR experiments which require control of the spinning frequency to within +/- 1 Hz. We believe however that the material removed by the brief digestion we used is incidental to mineralization, and that the organic material left behind is that which is truly composited with mineral and therefore the focus of this study.



Figure III. 13C{31P} REDOR intersample variability. A comparison of the REDOR effects seen for three of the samples at a single phosphorus-carbon magnetic coupling time of 5.6 ms. The interpretation of each pair of spectra is explained in the legend to Figure 3 in the main paper; the REDOR effect is conveyed by the difference between the black reference trace (without phosphorus-carbon magnetic coupling) and the red trace (with phosphorus-carbon magnetic coupling). In all cases there are significant effects to GAG, as well as to some protein signals. As NMR is an insensitive technique acquiring satisfactory REDOR data is time consuming, especially when the volume of sample is limited as it usually is for clinical tissue. Data at a single magnetic coupling time point can take several days to acquire and, while desirable, it is often impractical to collect data at several time points.

Quantification In simpler samples, such as pure crystalline organic compounds, it is customary to quantify the REDOR effect by integrating the intensity of each signal in the reference and the corresponding REDOR spectrum. The ratio of signal intensity in the REDOR spectrum to signal intensity in the reference spectrum is then plotted against the coupling time. This time dependence is proportional to the inverse cube power of the distance between coupled atoms, and becomes negligible for atoms more than about 1 nm apart. In favourable cases, usually where each atom is coupling with only a single other



magnetically active atom, the so-called two spin case, absolute values of the interatomic distances can be extracted, and a detailed structure for the molecule calculated.

We have not quantified our REDOR effects for two reasons. Firstly, because of signal overlap meaningful quantitation is difficult. Secondly, because of the 100% natural abundance of the magnetically active 31P isotope of phosphorus and its high density in the biomineral, each carbon atom will be coupled to at least two, probably several, phosphorus sites, presenting a situation difficult or impossible to analyze. Moreover each biopolymer will adopt a variety of (perhaps similar) conformations and spatial relationships with the mineral surface, so extracting any better than approximate information representative of an ensemble-average structure cannot be done. However there is relatively more phosphorus-carbon coupling to protein signals than we observe at similar coupling times in bone (see Fig. V), probably reflecting a less organised organic matrix in the mineralized plaque.



Figure IV. Mineralized plaque: Dependence of 13C{31P} REDOR on phosphorus-carbon magnetic coupling time. The traces show REDOR data acquired with four different phosphorus-carbon magnetic coupling times. Note that it is the GAG signal at 76 ppm which begins losing intensity at the shortest magnetic coupling times along with the high frequency carboxylate shoulder which also contains a GAG contribution. As we increase the magnetic coupling time the REDOR demagnetization is driven further out from the mineral surface, and its effects become more generalized and embrace numerous collagen signals. Also, the GAG signals are the only ones to decrease to baseline at the longest coupling times showing that all detectable GAG is close to, that is within about 1 nm of, the mineral surface. The bulk of the protein is further away.



Figure V. Bone: Dependence of 13C{31P} REDOR on phosphorus-carbon magnetic coupling time. The traces show REDOR data acquired under the same phosphorus-carbon magnetic coupling times as Figure IV. Note the same initial response of the GAG signals, and their ultimate loss of intensity down to baseline. Also note that effects to collagen are less widespread than in calcified plaque, possibly reflecting a more ordered structure with average mineral-collagen separations greater than those of plaque.

In the course of a major project we have studied a large number of bone samples, harvested at autopsy and at surgery, from horses, humans, rats, and mice. Samples have been powdered for solid state NMR by grinding in a mechanical ball mill, or by filing using a metal file. When examined under identical NMR experimental conditions there are no significant differences between any material or preparation method. Both treatments produce particles which are many microns in size, whereas the mineral particles in bone are sub-micron. Thus each powder particle contains numerous mineral nanoparticles in what must be an essentially native state. By this token we also believe that the pre-treatments we used on the plaque samples – digestion, centrifugation, and grinding in a pestle and mortar (under much lower energy conditions than we have to use to prepare bone samples) – will not have disturbed the atomic level structures and relationships we are interested in.



Blue staining is proteoglycans/GAG’s and brown staining is calcification. There is a lot of GAGstaining throughout the vessel wall particularly in association with vascular smooth muscle cells (VSMCs). In general calcification occurs in GAG-rich areas but not all GAG-rich areas are calcified - as in the lesion above. Calcification occurs in this vessel in the media in association with VSMCs (arrowed).

Examples of calcification in a GAG richarea just bordering on an area of the intimathat is almost devoid of GAG staining (In).

In

In



Calcified remnants of lipid pools in a GAG-rich area. Note lack of brown calcification in the surrounding GAG-poor areas (arrowed).

Alcian blue

Alcian blue + von Kossa Kossa Kossa

Serial sections showing a calcified area that is rich in GAG’s (arrowed on left) surrounded byan area poor in GAG’s that is not calcified). Co-staining of this same area is shown on the rightto reveal calcification.

Med In

Calcified areas very rich in GAG’s. Med, media; In, intima.



mineral surface in calcified plaque is like that of bone: further evidence for regulated...

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