© 1999 American Heart Association, Inc.
Vascular Biology |
Noncollagenous Bone Matrix Proteins, Calcification, and Thrombosis in Carotid Artery Atherosclerosis
From the Laboratory of Blood Coagulation Biochemistry (A.B., B.J.K.), Lindsley F. Kimball Research Institute, New York Blood Center, New York, NY; the Department of Biochemistry (K.G.M.), University of Vermont, Burlington; and the Department of Pathology (F.J.S.), Brigham and Women's Hospital and Harvard Medical School, Boston, Mass.
Correspondence to Alessandra Bini, PhD, Laboratory of Blood Coagulation Biochemistry, Lindsley F. Kimball Research Institute, New York Blood Center, 310 E 67th St, New York, NY 10021. E-mail abini{at}server.nybc.org
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Abstract |
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Abstract—Advanced atherosclerosis is often associated with dystrophic calcification, which may contribute to plaque rupture and thrombosis. In this work, the localization and association of the noncollagenous bone matrix proteins osteonectin, osteopontin, and osteocalcin with calcification, lipoproteins, thrombus/hemorrhage (T/H), and matrix metalloproteinases (MMPs) in human carotid arteries from endarterectomy samples have been determined. According to the recent American Heart Association classification, 6 of the advanced lesions studied were type V (fibroatheroma) and 16 type VI (complicated). Osteonectin, osteocalcin, and osteopontin were identified by monoclonal antibodies IIIA3A8, G12, and MPIIIB101 and antiserum LF-123. Apolipoprotein (apo) AI, B, and E; lipoprotein(a); fibrinogen; fibrin; fragment D/D-dimer; MMP-2 (gelatinase A); and MMP-3 (stromelysin-1) were identified with previously characterized antibodies. Calcium phosphate deposits (von Kossa's stain) were present in 82% of samples (3 type V and 15 type VI). Osteonectin was localized in endothelial cells, SMCs, and macrophages and was associated with calcium deposits in 33% of type V and 88% of type VI lesions. Osteopontin was distributed similarly to osteonectin and was associated with calcium deposits in 50% of type V and 94% of type VI lesions. Osteocalcin was localized in large calcified areas only (in 17% of type V and 38% of type VI lesions). ApoB colocalized with cholesterol crystals and calcium deposits. Lipoprotein(a) was localized in the intima, subintima, and plaque shoulder. Fibrin (T/H) colocalized with bone matrix proteins in 33% of type V and 69% of type VI lesions. MMP-3 was cytoplasmic in most cells and colocalized with calcium and fibrin deposits. MMP-2 was less often associated with calcification. The results of this study show that osteonectin, osteopontin, and osteocalcin colocalized with calcium deposits with apoB, fibrin, and MMP-3 in advanced, symptomatic carotid lesions. These data suggest that the occurrence of T/H might contribute to dystrophic arterial calcification in the progression and complications of atherosclerosis.
Key Words: bone matrix proteins • calcification • matrix metalloproteinases • fibrin(ogen) • atherosclerosis
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Introduction |
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Dystrophic mineralization of the arterial wall increases the risk of myocardial infarction and ischemia in peripheral vascular lesions.1 More than 80% of coronary lesions are calcified,2 and calcium deposits are often associated with unesterified cholesterol.3 Two very recent studies, on the in vivo4 and ex vivo5 detection of calcium deposits in coronary arteries, have shown that calcification is correlated with atherosclerotic plaque burden and that noninvasive detection of calcification may have predictive value in the development of subsequent coronary events.5
The molecular determinants regulating extracellular matrix calcification have yet to be identified. Recent studies have shown that noncollagenous bone matrix proteins such as osteonectin, osteocalcin, and osteopontin are also found in atherosclerotic vessels and may regulate dystrophic calcification. For example, osteonectin (SPARC) has been identified in vessel wall cells6 and platelets7 and participates in the regulation of bone mineralization,8 cell migration/proliferation,9 and remodeling of extracellular matrix.10 11 12 Recently, it has been shown that osteonectin binds to plasminogen,13 increases its activation and binding to collagen,13 and induces the expression of type 1 plasminogen activator inhibitor in endothelial cells (ECs).14 That suggests a role for osteonectin in both the degradation of extracellular matrix and the regulation of fibrinolysis. Moreover, osteonectin upregulates the expression of matrix metalloproteinases (MMPs) in cultured fibroblasts.15 Previous studies have implicated MMPs in destabilization and rupture of atherosclerotic plaques,16 17 18 19 and we recently showed that both fibrinogen and cross-linked fibrin, proteins known to be associated with both early and complicated plaques, can be degraded by MMP-2, MMP-3,20 and MMP-7.21
Osteopontin is synthesized by most vascular cells,22 and its distribution in coronary arteries has been shown to be associated mainly with macrophages23 and foam cells24 in the lipid core, adjacent to23 or colocalized with25 the calcification front and in calcified areas in carotid arteries.26 Expression of both osteopontin and osteonectin was previously studied in a limited number of aortas (8 autopsy cases).6 Osteopontin, together with osteocalcin and bone syaloprotein II, is also found in platelets.27 28 29 Osteocalcin is the most abundant of the noncollagenous proteins of bone produced by osteoblasts that promotes adhesion and chemotaxis in osteoclasts, but it has not been described in other cells.30 Additionally, although osteocalcin has been extracted from atherosclerotic plaques,31 32 data on its distribution are not available. Recent studies have shown that bovine osteoblast-like vascular cells can synthesize osteocalcin in vitro.33
In this study, the cellular and extracellular localization of bone matrix proteins in clinical carotid artery atherosclerosis suggests the existence of a potentially important and complex interaction among noncollagenous bone proteins, calcific deposits, plaque growth, and matrix degradation that might further contribute to plaque disruption and thrombosis.
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Methods |
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Human carotid endarterectomy specimens were collected at surgery and immediately rinsed in Tris-buffered saline containing benzamidine and aprotinin as described,34 fixed in Histochoice (Amresco), and subsequently embedded in paraffin. All samples, with the exception of 1 (No. 7641), were embedded undecalcified, as in previous studies.34 The use of undecalcified tissue allows observation of the earliest deposits, including cell association, therefore offering the possibility of linking descriptive morphology to mechanism.35 Sample No. 7641 was heavily calcified and brittle and therefore was decalcified by chelation.36 It has been shown that such procedure maintains the antigenicity of monoclonal antibody (MoAb) MPIIIB101 to osteopontin,37 also used in this study.
Lesions were classified according to American Heart Association criteria.38 Six lesions were type V (defined as type Va: fibroatheroma with lipid core and fibrotic layer; type Vb: multiple lipid cores and fibrotic layers; or type Vc: mainly calcific, or mainly fibrotic), and 16 were type VI (defined as complicated lesion, with surface defect, hematoma, hemorrhage, and/or thrombotic deposits). Tissues used as controls (for antibodies to osteonectin and osteocalcin only, because all other antibodies used in the study were previously characterized) included normal arteries34 and normal-term human placenta.39 Fresh specimens were processed as above. All samples were part of different Institutional Review Board protocols approved by our own and other collaborating institutions.
Immunohistochemistry
Previously characterized MoAbs and polyclonal antibodies were
used with the avidin-biotin complex immunoperoxidase technique,
essentially as described,34 39 with use of the Vectastain
Elite ABC kit (Vector Laboratories Inc). The end product of the
reaction with diaminobenzidine forms an insoluble brown precipitate.
The MoAbs used were as follows: IIIA3A8 (10
µg/mL) to osteonectin and G12 (2 µg/mL) to
osteocalcin.40 In normal vessels and placental tissue,
osteonectin was detected in ECs, smooth muscle cells (SMCs), and
decidua cells as previously described.11 Osteocalcin was
not detected in any cell type.
In previous studies, the 2 antibodies used to detect osteopontin, MPIIIB101 and antiserum LF-7 (Dr Larry W. Fisher, National Institutes of Health, Bethesda, Md), showed a different distribution. Therefore, in this study we used both antibodies. MoAb MPIIIB101 (clone culture fluid 1/20) to osteopontin was obtained from Dr Karen Jansen, Developmental Studies Hybridoma Bank, Department of Biological Sciences, University of Iowa, Iowa City,41 and rabbit antisera LF-123 (to the recombinant carboxyl half of human osteopontin, 1/8000) and LF-124 (to the recombinant amino half of human osteopontin, 1/8000) were a kind gift of Dr Larry W. Fisher.42 43 Because the 2 antibodies reacted similarly in serial sections from the same vessel specimens, LF-123 only was used throughout the study.
MoAb 18C6 to fibrinogen/fibrin I, MoAb T2G1 to fibrin II, and MoAb GC4
to fibrin(ogen) degradation product fragments D and D-dimer have
been previously described.34 39 To detect intact
fibrinogen, we used a recently developed MoAb (FPA 19/7), specific for
the human fibrinopeptide A sequence (A
1-16), which
reacts significantly better with intact fibrinogen than it does with
the free peptide.44
MoAbs to human matrix MMP-2 (gelatinase A) and MMP-3 (stromelysin 1, 1 to 3 µg/mL) were purchased from Oncogene Science. Goat antisera to human apoAI, apoE, and apoB were generously provided by Dr Paul S. Roheim, Louisiana State Medical Center, New Orleans, and to Lp(a) were kindly provided by Dr Angelo Scanu, University of Chicago, Chicago, Ill. MoAbs HAM56 and HHF35 (Enzo Diagnostics) were used to identify macrophages and SMC populations, respectively. Ulex europaeus agglutinin and rabbit antiserum to ulex europaeus lectin (Dako Corp) were used to identify ECs. A selected number of samples were double-stained with MoAb IIIA3A8 (osteonectin) and alternatively with antibodies to macrophages, SMCs, and ECs to identify in which cells osteonectin was cytoplasmic. The Vector VIP substrate kit produces a purple precipitate that is distinguishable from the brown precipitate produced by diaminobenzidine.) All samples were routinely stained with HPS (hematoxylin, phloxine B, and safranin O) and with von Kossa's stain, which detects the presence of calcium phosphates in mineralized tissues.45
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Results |
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Calcification in Endarterectomy Specimens
Calcium deposits, identified by von Kossa's reaction for calcium phosphate, were present in 18 of 22 samples (3 of 6 type V and 15 of 16 type VI). These deposits were distinguished as large masses of calcification, mainly central to the lesion (7 samples) or as smaller clusters of needle-shaped apatite crystals (21 samples).46
Distribution of Bone Matrix Proteins, Apolipoproteins,
Fibrin(ogen), and Calcification
All of these antigens were present in both type V and type VI
lesions, with a cellular and/or extracellular localization. Therefore,
a table summarizing all scores would convey little information.
Similarly, because endarterectomy samples are only
an incomplete fraction of the atherosclerotic plaque, a traditional
morphometric analysis could not be completed. For the same
reason, the site of plaque rupture was rarely identified. In fact, it
is possible that during the course of
endarterectomy, the actual site of plaque rupture
is not excised. However, whereas all type V lesions were of types Vb
and Vc (ie, there were no type Va "vulnerable" plaques), in 12 of
16 type VI lesions (75%), a central core with calcification,
hemorrhage, thrombus, and/or cholesterol crystals
could be identified. These data are summarized in Figure 1
as the colocalization of calcium
deposits, cholesterol crystals, and
thrombus/hemorrhage (T/H) identified by their major protein
components.
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The areas immunostained with MoAb T2G1 have been subdivided as areas of either organizing thrombus and intraplaque hemorrhage (ie, T/H) or fibrin II. Fibrin II is used to indicate fibrin that is localized in parallel bundles and threads in the intima, subintima, and media and in association with vascular wall cells (mainly macrophages and macrophage-derived foam cells)34 as distinct from fibrin that appears colocalized with T/H.
Type V Lesions
Fibrinogen/fibrin I was diffuse from the lumen to the intact
portion of the media (Figure 2a), similar
to apoB (Figure 2e). Fibrin II, fragment D/D-dimer, and Lp(a)
colocalized in the intima and neointima in small focal
deposits, along the calcification front, and were associated with
macrophages, foam cells, and SMCs (Figures 2b through
2f). Osteonectin was cytoplasmic in ECs, SMCs, macrophages, and
macrophage-derived foam cells, as identified by their
respective antibodies (Figures 2d and 2g). Small calcifications,
detected with von Kossa's stain, were present in most lesions,
along the calcification front and in degenerative areas (Figure 2h). Osteopontin was cytoplasmic in macrophages and SMCs
in the intima and neointima (Figures 3a and 3b) and along the calcification
front (with antiserum LF-123 only; Figure 3a). MMP-3 was
cytoplasmic in most cells: in the extracellular matrix, along the
calcification front, and in the fibrotic portion of the lesions in a
granular pattern (Figure 3c). Slightly diffuse staining for
MMP-2 was observed at the calcification front only (Figure 3d).
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Type VI Lesions
Calcification was present in all but 1 type VI lesion studied.
A calcified lipid core and smaller areas of sparse calcification are
shown with von Kossa's stain (Figures 4a and 5a). Fibrinogen/fibrin I was
distributed along and within the intima and in the plaque shoulder
associated with macrophages, SMCs, cholesterol
crystals, and calcium deposits (Figures 4b and 5b).
Fibrin II (Figures 4c and 5c) and fragment D/D-dimer
(Figures 4d and 5d) showed similar associations, were
more focal, and also colocalized with areas of intramural T/H. In
addition to the MoAbs to fibrin(ogen) used in our previous studies, we
recently developed a MoAb, FPA 19/7, that reacts only with intact
fibrinogen and is specifically directed to the intact amino terminus of
the A-chain. Immunolocalization of fibrinogen with intact A-chain
colocalized with that of intact fibrinogen Bß-chain (MoAb I8C6) but
usually involved a smaller area (not shown).
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The distribution of apoB extended from the normal portion of the media
into the lipid, calcified core (Figure 4e
), similar to that of
apoAI and apoE (not shown). ApoB colocalized with fibrinogen, fibrin
II, and fragment D/D-dimer in the intima, plaque shoulder, and lipid
core (Figures 4b through 4f). The distribution of Lp(a) was
different from that of the other apolipoproteins. Lp(a) was mainly
localized along the margins of the lipid, calcified core and was
colocalized extracellularly with fibrin II and fragment D/D-dimer along
the intima, in the subintima, and in association with
macrophages (Figures 4e and 5e).
Fibrinogen, fibrin II, and fragment D/D-dimer colocalized with
osteonectin, osteopontin, osteocalcin, and apolipoproteins in the
calcified core (Figures 4b through 4d, 4g through 4i, and
5b through 5h). Osteocalcin occurred as both large calcium
deposits (Figures 4i and 5g) and as smaller calcification
foci (Figure 5g), similar to osteonectin and osteopontin
(Figures 4g, 4h, 5f, and 5h). Osteopontin colocalized in
the plaque shoulder with fibrinogen, fibrin II, fragment D/D-dimer,
Lp(a), apoB, and MMP-3 (Figure 4h).
A higher-magnification view of the calcification front area in this
lesion (demarcated area in Figure 5i) showed cytoplasmic
colocalization of osteonectin, osteopontin, and MMP-3 in
macrophage, foam cells, and calcium deposits (Figures 6a through 6e). Osteopontin was seen
mainly in macrophages, similar to MMP-2 (Figures 6b and 6d). MMP-3 was cytoplasmic in most cells (Figure 6c), similar to
osteonectin and osteopontin, and colocalized with fibrin II,
apolipoproteins, and calcium deposits (Figures 4j and 6c). MMP-2 was heterogeneously localized in various
cell types and, when in association with calcium deposits, was found
mainly at the calcification front (Figures 4l and 6d).
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In summary (Figure 1), osteonectin was associated with
calcium deposits in 33% of type V and in 88% of type VI lesions.
Osteopontin (antiserum LF-123) colocalized with calcium phosphate
crystals in 50% of type V lesions and 94% of type VI lesions. No
association of osteopontin with calcium deposits was detected with MoAb
MPIIIB101. Osteocalcin was detected in large calcium
deposits only, in 17% of type V and in 38% of type VI lesions. MMP-3
was present in 50% of type V lesions and in 93.6% of type VI
lesions, whereas MMP-2 was in 50% of type V and in 53% of type VI
lesions.
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Role of Bone-Related, Noncollagenous Proteins in Atherosclerosis Calcification
This work has identified the cellular and extracellular localization of the major bone proteins osteonectin, osteocalcin, and osteopontin with calcium deposits in undecalcified carotid atherosclerotic plaques to determine their association with major protein components of atherosclerotic plaques, including apolipoproteins, fibrin(ogen)-related antigens, and MMPs. As shown in Figure 1
, all antigens studied were increasingly associated with
calcification, progressing from type V to type VI lesions, including
the bone matrix proteins osteonectin, osteopontin, and osteocalcin.
MMP-3 also strongly colocalized with calcium deposits, whereas MMP-2
seemed to be less associated with it. Previous work showed that a number of bone-related, noncollagenous matrix proteins, including osteonectin, osteocalcin, and osteopontin, may also play a role in plaque calcification by regulating the growth of hydroxyapatite crystals.47 In-vitro studies have shown that these proteins also function in cell migration and tissue healing.9 30 48 However, their role in the regulation of bone mineralization (ie, favoring or inhibiting bone mineralization) has not yet been completely elucidated.30 In fact, osteocalcin-knockout mice show increased bone formation.49 Vascular SMCs have the ability to calcify in vitro, expressing higher levels of matrix Gla protein and low levels of osteopontin.50 However, mice lacking matrix Gla protein incur spontaneous calcification of arteries and cartilage.51 Surprisingly, mice lacking osteoprotegerin, a protein that regulates bone resorption, also develop arterial calcification.52 Osteonectin has been shown to promote binding of calcium to collagen,53 thus suggesting a role in promoting calcium deposition. Additional work has indicated that osteonectin, and to a lesser extent osteocalcin, could inhibit hydroxyapatite crystal formation.54 This role seems to prevent excessive mineralization in bone. Osteonectin and osteocalcin are detectable in normal plasma and serum40 55 and together with osteopontin are also present in human platelets.7 27 28
The results on the distribution of these 3 bone-related proteins, osteonectin, osteopontin, and osteocalcin, in carotid artery atherosclerotic plaques have shown that they colocalize with large calcium deposits in clinically significant type V and type VI lesions. Moreover, osteonectin and osteopontin colocalized with areas of small calcification, with and without degeneration,56 and were cytoplasmic in most cells, whereas osteocalcin was not detected in any cell type. Previous studies localized osteonectin in arterial medial SMCs,6 in tumoral and vascular cells,11 and in a variety of other cell types in addition to mineralized tissue.8 57 In all type V and VI lesions, osteonectin was cytoplasmic in ECs, macrophages, foam cells, selected SMCs, and a few unidentified cells, possibly pericyte-like cells. These have also been shown to synthesize osteopontin and osteonectin in vitro33 and bring about local formation of calcification nodules.58 59
Antibodies to osteopontin, MoAb MPIIIB10123 48 and antiserum LF-7,25 26 47 were used in previous studies and showed a different distribution. In our study, MoAb MPIIIB101 was mainly associated with macrophages,23 48 although both ECs and SMCs stained lightly. Antiserum LF-123 (raised against the carboxy-terminal portion of human osteonectin, whereas LF-7 was raised against full-length osteonectin) exhibited a stronger association with calcium deposits and gave a granular staining pattern to the extracellular matrix, in addition to the macrophage staining as shown by others.25 26 47 Results obtained with both antibodies can explain the cellular versus cellular/extracellular localization shown in previous work.23 25 26 47 48 In those studies and in this work, larger numbers of cells containing osteopontin were observed in atherosclerotic versus normal vessels.
This study confirms and extends previous data on bone matrix proteins in atherosclerotic plaques and suggests that more than 1 cell type secretes several of these proteins (bone morphogenetic protein-2a, osteopontin, osteonectin, and matrix Gla protein) and thereby may participate in the calcification process that occurs in the development of atherosclerosis.6 23 25 26 60 Although osteocalcin has been extracted from atherosclerotic plaques,31 32 we did not detect it associated with any cell types in any of the atheromas examined. Because in this work we have shown that osteocalcin colocalized with fibrin II/calcium deposits, it might be derived from plasma and/or platelets. The presence of osteonectin, osteocalcin, and osteopontin in plasma and platelets may serve as a concentrated source of noncollagenous matrix proteins that can contribute to vessel wall calcification after a thrombotic or hemorrhagic event.61
Relationships Among Lipids, Apolipoproteins, and
Thrombosis
The distribution of apolipoproteins and fibrin(ogen) in these
lesions was similar to that in previous studies.34 62
ApoAI, B, and E codistributed with fibrin II and
cholesterol crystal deposits. Lp(a) was observed mainly at
the margins of the cholesterol crystal deposits. In the
intima and subintima, Lp(a) was closer to the lumen, similar to what
was formerly described for the aorta and coronary
arteries,63 64 65 and often colocalized with fibrin II
deposits. That might interfere with the assembly of fibrinolytic
proteins on the fibrin surfaces, thereby hampering
fibrinolysis.66 67
The main morphological difference between type V and type VI lesions
was that in all type VI lesions, there was either intraplaque
hemorrhage or mural thrombus. However, in all type V and VI
lesions, there was also cell-associated or extracellular fibrin II. In
type V lesions, fibrin II was localized in bundles and threads in the
intima and subintima, associated with SMCs and macrophages,
possibly due to the localized formation of intraplaque fibrin as
previously described39 and more recently
shown.68 In addition, with a new antibody to the intact
amino terminus of the A-chain of fibrinogen that was not available
in previous studies, we confirmed that part of the fibrin(ogen)-related
antigen was intact, possibly transglutaminase–cross-linked
fibrinogen.69
Bone proteins such as osteonectin and osteopontin can be cross-linked by tissue transglutaminase and factor XIIIa.70 These proteins might become cross-linked to fibrin(ogen) in the progression of an atherosclerotic plaque, particularly during or after formation of intraplaque hemorrhage or thrombus, where they are abundant and factor XIII is in its active form (factor XIIIa). Additionally, because fibronectin is present in both the vessel wall and the clot, bone proteins might be indirectly bound to fibrinogen via fibronectin cross-linking and therefore might participate in the formation of matrix that will evolve in dystrophic calcification.
Role of Calcification in Plaque Progression and Instability:
Possible Upregulation of MMPs
No major differences in cellularity were observed between
calcified and noncalcified lesions. Both in the current and previous
studies, calcium hydroxyapatite crystals were seen within organizing
thrombi. Several factors likely play a role in the calcification of the
vessel wall, including (1) calcification of thrombus; (2) calcification
of degenerate(d) SMCs and macrophages; (3) local synthesis of
calcification proteins; and (4) local bone neoformation, such as that
seen occasionally in atherosclerotic vessels.
The main questions about bone matrix proteins are how, when, and which of these proteins favors or limits the calcification process. Because osteonectin has been shown to upregulate MMPs in cultured fibroblasts,15 it might be suggested that osteonectin can upregulate the synthesis of MMPs in both SMCs and macrophages in atherosclerotic plaques. Therefore, calcification and degradation of the extracellular matrix in atherosclerotic plaques might be synergistically regulated.
Recently, we have shown that MMP-2 can degrade fibrinogen and that MMP-3 and MMP-7 can degrade both fibrinogen and cross-linked fibrin.20 21 Different classes of MMPs might exert different roles in plaque progression and rupture according to their preferred substrates. The possible role of such a mechanism in atherosclerosis requires further investigation.
Early work suggested that lipids might be involved in biological mineral formation.71 72 73 More recently, it has been found that mineral deposits in the human aorta contain a relatively high concentration of protein (12% to 18%), in addition to calcium apatite and calcium carbonate, and most likely include some glycoprotein.74 Some of these may be bone proteins such as osteonectin and osteopontin, which were identified in the atherosclerotic lesions examined in the present study.
Recently, it was hypothesized that calcium deposits form after plaque rupture as part of complicated lesions, a phenomenon that is rarely seen in small, "soft," cholesterol-rich plaques. In our study, 69% of complicated type VI lesions showed a central core that was necrotic, calcified, thrombosed, and/or hemorrhaged and that contained bone proteins, apolipoproteins, fibrin, and MMPs (particularly MMP-3), suggesting that these lesions were derived from type Va lesions (according to the recent American Heart Association classification).
In conclusion, the present study establishes the colocalization of dystrophic calcification with intraplaque T/H, cholesterol, and their corresponding protein components in human carotid arterial atherosclerosis. These results suggest that intraplaque T/H might contribute to arterial calcification as a source of osteocalcin and that bone matrix proteins, apolipoproteins, fibrin(ogen), and MMPs might interact in the formation of dystrophic calcification, progression, and complications of atherosclerotic lesions. The results of this study also suggest potential targets for early and noninvasive detection and characterization of lesions.
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Acknowledgments |
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This study was supported in part by grants HL-48743 (to F.J.S. and Peter Libby [Principal Investigator]), HL-38118 (to Robert J. Levy [Principal Investigator]), and AG-08777-05S1 (to K.G.M.) from the National Institute of Health, Bethesda, Md. The authors would like to thank Norie Joson-Gonzales for excellent technical assistance and Elena Rabkin for preliminary staining of samples with the MoAb to osteopontin. Our thanks are also extended to Drs Peter Libby for help in obtaining samples; Paul S. Roheim, Angelo Scanu, and Larry W. Fisher for their generous gift of antibodies; and G. Fantini for providing some of the control tissues. Alfred T. Lammé (FPBA) provided expert photographic assistance.
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Footnotes |
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Parts of this work were presented at the Joint Conference on Arteriosclerosis, Thrombosis, and Vascular Biology of the American Heart Association, Salt Lake City, Utah, February 18–20, 1996.
Received November 12, 1998; accepted January 25, 1999.
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