© 1995 American Heart Association, Inc.
Articles |
Plasminogen Activator Expression in Human Atherosclerotic Lesions
From the Thrombosis Research Institute (F.L., F.B., V.V.K.), London, UK, and the Division of Hematology, Department of Medicine (F.L., D.A.H., F.B., E.K.O.K.) and Cardiovascular Surgery Service (M.H.), University Hospital Center (CHUV), Lausanne, Switzerland.
Correspondence to Florea Lupu, Vascular Biology Laboratory, Thrombosis Research Institute, Emmanuel Kaye Bldg, Manresa Rd, Chelsea, London SW3 6LR, UK.
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Abstract The plasminogen activator (PA) system may participate in the pathogenesis of atherosclerosis by modulating the turnover of intimal fibrin and extracellular matrix deposits and by contributing to intimal cell migration. We present an analysis of tissue-type PA (tPA) and urokinase-type PA (uPA) expression at three levels: mRNA by in situ hybridization, antigen by immunohistochemistry, and enzymatic activity by histoenzymology and zymography. For PA colocalization with cellular or matrix components, we used double immunofluorescence labeling in conjunction with confocal microscopy. In normal arteries, tPA antigen and mRNA were detected in endothelial cells and smooth muscle cells (SMCs). In atherosclerotic arteries, tPA antigen and mRNA were increased in intimal SMCs and in macrophage-derived foam cells of fibrofatty lesions. Part of the tPA was detected in the extracellular space and colocalized with fibrin deposits. uPA antigen and mRNA were detected in association with the intimal macrophages and SMCs. A particularly high uPA expression was noted on macrophages localized on the rims of the necrotic core. Moreover, using a novel histoenzymological assay as well as classic zymography, we revealed uPA-dependent lytic activity in the advanced lesions, whereas in normal arteries, only tPA-dependent activity was detected, mainly over the vasa vasorum. Also, strong tPA and uPA staining was detected in neomicrovessels of the plaques, suggesting that PAs may play a role in plaque angiogenesis. Our results suggest a local dynamic process of PA-dependent proteolysis in lesion areas that is associated with macrophages and SMCs. A better comprehension of these proteolytic mechanisms in advanced atherosclerotic plaques may provide the basis for therapeutic approaches for plaque stabilization.
Key Words: atherosclerosis • tissue-type plasminogen activator • urokinase-type plasminogen activator • smooth muscle cells • macrophages
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The PA system is an enzyme cascade system that generates localized proteolysis in a highly regulated fashion. The zymogen plasminogen is activated to plasmin, a trypsin-like protease, by the action of PAs, of which there are two types, tPA and uPA.1 PAI-1 and PAI-2, as well as specialized cell surface binding proteins such as the uPA and plasminogen receptors, ensure that proteolytic activity is confined in space and limited in time.
The PA system may participate in the development of atherosclerotic lesions by a variety of mechanisms: the extent of PA expression and consequently of plasmin generation not only affects the rate of fibrin degradation on or within advanced plaques but also may influence, directly or by activation of metalloproteinases, the turnover of ECM deposits. Furthermore, plasmin can activate cytokines, such as transforming growth factor-ß and basic fibroblast growth factor, that are known to participate in atherogenic processes.2 3 4
Fibrinolytic dysfunctions, in particular elevated plasma levels of PAI-1, have been associated with thromboembolic disease and may represent a risk factor for acute coronary thrombosis5 or postoperative deep vein thrombosis in elective hip surgery.6 An increased expression of PAI-1 antigen and mRNA occurs in atherosclerotic lesions in SMCs and in macrophages located at the periphery of the necrotic core.7 8 In pathological situations, PA expression may also be modified. Thus, in a rat model of injured carotid artery, an increase of both uPA and tPA activity and mRNA was detected in extracts of the media.9 Atherosclerotic lesion areas contain fibrin as well as fibrin degradation products, suggesting a continuous formation of fibrin coupled with a fibrinolytic process in the arterial intima.10
In the present study, we investigated in human atherosclerotic arteries the localization of the activity, antigen, and mRNA of both tPA and uPA. Cell type–specific antibodies were used to identify the producer cells. Our results demonstrate that in atherosclerotic lesions, both PAs are expressed by macrophages and SMCs.
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Preparation of Vascular Tissue
All samples were obtained during cardiovascular surgery, immediately embedded in optimal cutting temperature compound (OCT compound, Miles Scientific), frozen in cold methyl butane (Merck), and stored at -70°C. The aorta samples for this study were small pieces cut from the ascending aorta before the proximal anastomosis of an aortocoronary bypass was constructed. Three specimens had advanced atherosclerotic lesions with a necrotic core and a fibrous cap, four showed a lipid-rich thickened intima that was invaded by numerous macrophages, and three showed a thickened intima with no or minimal macrophage invasion. Ten carotid specimens showing advanced atherosclerosis were obtained after endarterectomy surgery. Segments of normal internal mammary or popliteal arteries were used as control tissues. The tissue samples were cryosectioned at 7 µm and thaw-mounted onto poly-L-lysine–coated slides for in situ hybridization and on gelatin-coated slides for immunohistochemistry. For in situ hybridization, the sections were air-dried, fixed in 4% paraformaldehyde (Sigma Chemical Co) in PBS composed of (in g/L) NaCl 8, KCl 0.2, Na2HPO4 · 2H2O 1.44, and KH2PO4 0.2, pH 7.4, dehydrated, and stored at -20°C with desiccant. Slides for immunohistochemistry were air-dried overnight and fixed in cold acetone.
Antibodies
In addition to the MAbs listed in the Table
, two
polyclonal goat anti–human melanoma tPA antibodies (American
Diagnostica, No. 387, and Biopool) and a rabbit anti–human
uPA antibody (American Diagnostica, No. 389) were used at a
concentration of 20 µg/mL for immunohistochemical purposes or to
block PA activity for zymographic analysis.
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An isotype-matched MAb that did not react with human tissue or serum proteins (Ms IgG1, Coulter Immunology), mouse ascites fluid, and nonimmune serum from goat (Sigma) were used as controls.
cRNA Probes
A 1974-bp fragment from the human tPA cDNA and a 600-bp fragment
from the human uPA cDNA were subcloned in the Bluescript M13+ vector
(Stratagene Inc) and labeled by run-on transcription with
35S-labeled UTP (specific activity, 1300 Ci/mmol; New
England Nuclear). Sense and antisense probes were prepared by
linearizing the constructs with the appropriate restriction
endonucleases and the use of either T7 or T3 RNA polymerase
(Boehringer Mannheim).
Immunohistochemistry
For bright-field immunohistochemical staining, either the
alkaline phosphatase/anti–alkaline phosphatase double bridge
(Dako-APAAP Kit) or the avidin–biotinylated peroxidase complex
(Vectastain ABC Kit, Vector Laboratories) technique was applied.
Incubation with the first antibody was done for 60 minutes at room
temperature. All further steps were performed according to the
manufacturer's instructions.
Immunofluorescence and Confocal
Microscopy
To establish the PA distribution in different cell types of
normal and atherosclerotic tissues, we used a double
immunofluorescence labeling approach. PAs were
identified by specific polyclonal antibodies raised in rabbits against
recombinant tPA or human uPA (both from American
Diagnostica). As cell-specific markers, we used MAbs as
listed in the Table
. After the proper blocking treatments, the frozen
tissue sections were incubated overnight at 4°C with cocktails
containing a PA-specific polyclonal antibody and a cell type–specific
MAb. As secondary antibodies, a mixture of horse anti-mouse IgG–Texas
Red and goat anti-rabbit IgG–FITC was used. The sections were studied
with a Bio-Rad MRC 600 confocal laser scanning unit attached to Nikon
Diaphot inverted microscope (Bio-Rad Microscience Ltd). The light
source was a krypton/argon laser (Ion Laser Technology) with principal
lines at 488, 568, and 674 nm. For simultaneous
visualization of FITC and Texas Red staining, the K1 and K2 filter
blocks were used.
In Situ Hybridization
The procedure was performed according to the method of
Holland.11 The slides were brought to room temperature at
least 2 hours before use, acid-treated in 0.2 mol/L HCl for 20 minutes,
washed, and fixed in 4% (wt/vol) paraformaldehyde for
10 minutes. This was followed by two wash steps in PBS and 10 minutes
of incubation in 0.25% acetic anhydride in 0.1 mol/L Tris HCl, pH 8.
After three wash steps in PBS, the slides were dehydrated and
air-dried. The cRNA probe (in 50% formamide [BRL], 0.3 mol/L NaCl,
1x Denhardt's solution, 0.02 mol/L Tris-HCl, pH 8.0, 5 mmol/L EDTA,
5% dextrane sulfate, 50 mmol/L dithiothreitol, and 500 µg/mL yeast
total RNA [Boehringer Mannheim]) was used at
106 cpm per slide. Sections were covered with
siliconized coverslips and hybridized at 50°C overnight (12 hours) in
a chamber humidified with 50% formamide, 0.3 mol/L NaCl, and 1x
Denhardt's solution. To remove coverslips, sections were immersed in
4xSSC (1xSSC is 0.15 mol/L NaCl and 0.015 mol/L trisodium citrate, pH
7.0) at 37°C and then washed in 4xSSC at 37°C. After an RNase A
(Boehringer Mannheim) treatment (20 mg/mL) for 30 minutes at
37°C, the slides were washed in 0.5 mol/L NaCl, 10 mmol/L Tris HCl,
pH 7.5, and 1 mmol/L EDTA, followed by 1 hour of incubation at 50°C
in 1xSSC and 2x1 hour at 50°C in 0.1xSSC. All solutions of the
posthybridization wash steps contained 2 mmol/L dithiothreitol. The
graded alcohol series for the final dehydration contained 300 mmol/L
ammonium acetate. The sections were air-dried and covered with an
autoradiographic emulsion (NTB-2, Kodak) according to
the manufacturer's instructions, stored in black airtight boxes at
4°C, and developed after 10 to 20 days. The slides were viewed with a
Nikon Optiphot 2 microscope equipped with a mercury UV lamp and
epipolarization filters.
Localization and Quantification of PA Proteolytic
Activity
Enzyme Histochemistry
We developed a novel histoenzymological method using a synthetic
peptide coupled to 4-methoxy-ß-naphthylamide (4MßNA) as a
substrate to localize and quantify the proteolytic activity of uPA.
Similar methods have already been developed for other proteases, such
as elastase and cathepsin B.12 To demonstrate
urokinase activity, unfixed cryostat sections were prepared as
described above. The incubation medium (1 mL 0.1 mol/L Tris-HCl buffer,
pH 7.2, 30.5 mg NaCl [final concentration 0.5 mol/L], 10 µL
2-hydroxy-5-nitrobenzaldehyde [5' nitrosalicyl aldehyde; 17 mg
dissolved in 200 µL ether and 800 µL dimethylformamide, final
concentration 1 mmol/L], and 1 mg of H-Gly-Arg-4MßNA [Bachem] as a
specific substrate13 ) was applied to the section, onto
which a coverslip was placed. The development of yellow
fluorescence as an indicator of protease activity was monitored
with the CLSM equipment. A semiquantitative evaluation of the
fluorescent reaction product was done with pseudocolor
bands. Samples incubated in substrate-free medium were used as
controls.
Zymography
The zymography technique, which is not confined in space and
does not provide quantifiable results, was used simply to confirm the
histoenzymological data. The protocol is based on the method of
Todd.14 Briefly, cryosections of vascular tissue were
overlaid with 100 µL of a mixture consisting of 2% casein, 0.8%
agarose, and 40 µg/mL Glu-plasminogen at 50°C. The
slides were incubated at 37°C in a humidified chamber for 1 to 3
hours. Controls were performed with a plasminogen-free
overlay or a mixture to which specific neutralizing antibodies such as
goat anti–human melanoma tPA IgG or specific rabbit anti–human uPA
IgG (both from American Diagnostica) were added.
Photographs were taken under dark-field illumination.
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PAs in Normal Arterial Tissue
tPA
A panel of monoclonal and polyclonal antibodies specific for human tPA and directed against different epitopes was used to determine the localization of this protein in normal arterial tissue. In apparently healthy human mammary arteries, tPA antigen was detected both in endothelial cells and in SMCs (Fig 1a
) identified by anti–von Willebrand factor
(Fig 1c through 1e) or anti–
-actin (not shown) antibodies,
respectively. In situ hybridization analysis revealed that the
same cells were also positive for tPA mRNA (Fig 1f). In the adventitia,
expression of tPA antigen (Fig 1b) and mRNA (Fig 1g) was restricted to
SMCs and endothelial cells of small arteries and
venules, whereas no tPA was detected in cells of the adventitial
connective tissue. Controls for immunostaining using
isotype-matched MAbs, mouse ascites fluid, and nonimmune goat serum
(not shown) or for in situ hybridization using sense cRNA (Fig 1h) were
consistently negative.
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uPA
Immunocytochemical analysis for uPA revealed that this
protein was present in luminal endothelial cells
and in medial SMCs (Fig 1i). The distribution of uPA in the media was
discontinuous, which is in contrast to the more homogeneous
distribution of tPA. In the adventitia, a positive reaction for uPA was
detected in association with SMCs and endothelial cells
of small arteries and venules (Fig 1j).
PAs in Atherosclerotic Tissue
tPA
Successive sections of human aortas with fibrous lesions were
analyzed for tPA mRNA expression by in situ hybridization. The
cell types present in the lesion areas were identified by use of
specific MAbs. A particularly strong tPA mRNA signal was detected over
the thickened intima (Fig 2a and 2b) compared with the
subjacent media. The silver grains were associated with cells positive
for the macrophage-specific marker (Fig 2c) or with
-actin–positive SMCs (Fig 2d). Medial SMCs appear to express lower
tPA levels.
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tPA antigen was detected both in the thickened intima and in the media
(Fig 3). Double immunofluorescence
staining for tPA (Fig 3a) and von Willebrand factor (Fig 3b)
revealed the colocalization of the two antigens over luminal
endothelial cells (Fig 3c). Serial frozen sections
stained for tPA (Fig 3d) or -actin (Fig 3e) suggest that SMCs
located both in the intima and in the media contain tPA.
Simultaneous immunofluorescence
staining on the same section of tPA (Fig 3f) and -actin (Fig 3g)
revealed a high degree of coincident labeling (Fig 3h). In fibrofatty
lesions of human aortas and carotid arteries, tPA antigen was detected
also in foam cells positive for macrophage-specific
markers, as was revealed by immunolabeling for the two antigens on
successive sections (Fig 3i and 3j) and double
immunofluorescence labeling on the same section
(Fig 3k through 3m). By means of confocal imaging, integrating Z-serial
optical sections (0.5 µm thick) through a double-labeled tissue
cryosection (20 µm), it was possible to follow the track of
tPA-immunostained microvessels in the thickness of the
section. By use of this method, a very strong tPA labeling was observed
in neomicrovessels located in the fibrous cap of the plaque and
identified by von Willebrand factor staining (Fig 3n).
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0.5 µm thick)
through a frozen section were "stacked" in the same plane by use
of the "extended focus" command of the COMOS
program. This procedure allows us to follow the track of the von
Willebrand–stained neomicrovessels through the thickness of
the section. Note that on the merged image (yellow), a high degree of
tPA colocalization with the endothelial cells of the
microvessels can be observed. o, Double immunolabeling of tPA (green)
with fibrin (red) in a carotid atherosclerotic plaque; areas of
coincident staining (yellow, arrows) suggest that part of the tPA is
bound to fibrin deposits. Specimens were photographed with
fluorescence CLSM (a through c, f through h, and k through o)
or bright-field illumination (d and e, i and j). mv
indicates microvessels. Magnifications: a through c, f through h, and k
through o, x175; d and e, x133; i and j, x268. Bars=100
µm.
tPA localization was mainly cell-associated, but in advanced
atherosclerotic plaques, we could detect some extracellular tPA, mainly
in association with fibrin deposits (Fig 3o).
uPA
In atherosclerotic aortas, uPA mRNA and antigen were detected in
the thickened intima and in the media (Fig 4a and 4b).
Immunohistochemical analysis using cell type–specific
antibodies revealed that uPA is expressed both in
macrophage-rich intimal areas (Fig 4c) and in SMC-rich areas
(Fig 4d). uPA antigen was detected in association with von
Willebrand–positive microvessels located within the cap of the
plaque (Fig 4g and 4h). Particularly strong uPA-specific mRNA signals
were seen over macrophages located in the cap of the necrotic
core (Fig 5a and 5b). To colocalize uPA antigen and
activity with macrophages within the atherosclerotic plaque,
serial frozen sections were analyzed both by
immunocytochemistry and/or in situ histoenzymology and zymography. The
double immunofluorescence labeling for uPA and
macrophage marker confirmed the colocalization of uPA antigen
and macrophages within the shoulders of the plaque surrounding
or located within the plaque core (Fig 5c through 5e). The
histoenzymological localization of uPA activity by the method described
above showed the fluorescent reaction product in large
amounts in the areas of the plaque shoulders and core. Image
analysis by pseudocolor banding demonstrated high-intensity
fluorescent staining (Fig 5g, magenta) in the areas stained by
uPA- and macrophage-specific
antibodies, revealing the presence of active uPA associated with
macrophages (Fig 5f and 5g). The fluorescent
precipitate was found in low amounts or was absent over these areas
when the substrate was omitted (Fig 5h) or in the presence of
uPA-neutralizing antibodies (not shown), proving the specificity of the
method. However, some unspecific fluorescent staining was found
over calcified regions.
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The localization of PA activity in association with macrophages
was established by tissue zymography also. Zymographs performed in the
presence of plasminogen showed PA-dependent lytic activity
over cell-rich areas of atherosclerotic lesions (Fig 6).
Polyclonal antibodies to uPA abolished the lytic zone (Fig 6b), whereas
anti-tPA had no effect (Fig 6a). No caseinolysis was detected in the
absence of plasminogen (not shown). On zymographs it is
very difficult to determine the cell type expressing proteolytic
activity. However, by studying the time course of caseinolysis, one can
observe that the lytic process started in the macrophage-rich
areas (Fig 6a and 6d). In normal tissue, only tPA-dependent activity
was detected, mainly in association with blood vessels of the
adventitia (not shown).
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Proteolytic events participate in many aspects of atherogenesis and its major complications, myocardial infarction and stroke. They are involved in (1) intimal migration of macrophages and SMCs; (2) activation of basic fibroblast growth factor and transforming growth factor-ß, which play a role in atherogenesis2 3 ; (3) turnover of intimal fibrin deposits; (4) degradation of ECM proteins, leading to weakening of the fibrous cap of the plaque; and (5) lysis of occlusive thrombi in stenosed coronary arteries, which is the cause of myocardial infarction.
Previously an increase of PAI-1 expression was reported in atherosclerotic areas.7 8 The aim of the present work was to determine to what extent and by which cells PAs are expressed in normal and atherosclerotic arterial tissue. In normal human arteries, we found that endothelial cells and SMCs express not only tPA antigen and protein but also uPA antigen. The presence of tPA immunoreactivity in all three layers of rat aorta was recently reported by Padro et al.15 Signals for uPA mRNA were weak over SMCs and undetectable over endothelial cells. The absence of a signal over endothelial cells may be due to insufficient sensitivity of the in situ hybridization technique. However, it is also possible that intimal uPA is derived from the blood circulation and becomes bound to the endothelial uPA receptor.16
The role of the various factors of the PA system in the normal blood vessel wall is at present only partially understood. Endothelial cells in vivo contain tPA,17 which is present to some extent in storage granules18 and can be released at high levels after a thrombogenic stimulus.19 The presence of tPA in and of uPA on endothelial cells may contribute to the antithrombotic properties of the blood vessel wall. The role of the PA system in the arterial media is less obvious. SMCs located in the media showed a strong staining for tPA mRNA and antigen, suggesting a constant tPA production. It has not yet been established whether SMCs, like endothelial cells, contain tPA storage vesicles. Also, uPA was detected in the media. By zymographic analysis we detected tPA-dependent activity associated with the vasa vasorum, but we detected no PA activity over the media, which is consistent with the presence of a high local concentration of PAI-1. These observations do not exclude PA-dependent proteolysis in the media, which could take place at the level of cell surfaces. Such a mechanism could provide for a regulated, steady turnover of ECM, which would contribute to the plasticity of the vascular tissue.
In atherosclerotic tissue, both tPA and uPA were detected. These proteins were expressed by SMCs in the intima and in the adjacent media. Particularly strong tPA and uPA antigen and mRNA signals were detected in macrophage-rich lesion areas, in macrophage-derived foam cells, and in macrophages located in the cap of the necrotic core. Recently, increased fibrinolytic activity was reported in extracts of the intima of atheromatous arteries.20 In accordance with these findings, we observed increased uPA-mediated activity over the macrophage-rich area of an advanced lesion. The increased expression of tPA and of uPA in intimal SMCs may contribute to the migration of these cells into and within the intima. Indeed, in rats, balloon catheter injury of the carotid artery resulted within days in an increased expression of uPA and tPA in intimal SMCs.9 Inhibition of the PA system by orally administered tranexamic acid led to a significant reduction of the number of cells that had migrated into the intima.21 In this report, we show an increase of tPA mRNA expression in SMCs located in active lesions, possibly mediated by platelet-derived growth factor,21 which suggests that, in humans, tPA is produced by SMCs and may facilitate their migration into an atherosclerotic intima.
Macrophages constitute a regular component of an atherosclerotic lesion and locally produce relatively high amounts of tPA, uPA, and PAI-1, resulting in detectable uPA-mediated lytic activity. Most likely, macrophage-associated PAs contribute to their migration into and within the lesion areas. The simultaneous expression of the PAIs and the uPA receptor would confine PA activity at the cell surface.22 The mechanism of tPA expression by the intimal macrophages is not clear. Resting monocytes do not normally produce tPA, but under certain conditions, eg, after stimulation with lipopolysaccharide or interleukin-4,23 they are able to do so. Which of the cytokines or growth factors in lesion areas are responsible for the induction of tPA production by macrophages remains to be established.
Plaque disruption with subsequent platelet aggregation and thrombosis is the most important mechanism by which atherosclerosis leads to acute cardiovascular disease, such as myocardial infarction and sudden cardiac death.24 25 Plaques that are disrupted and cause thrombotic disease contain higher concentrations of lipids and macrophages than nondisrupted plaques.26 27 The detection of uPA within the advanced, macrophage-rich atherosclerotic plaques gives new insights into the mechanisms governing the proteolytic weakening of the plaque.28 The key enzymes that regulate the extracellular proteolysis are uPA and plasmin. Both bind to specific surface receptors and thus focus the proteolytic activity on the cell surface.29 30 31 Plasmin may activate the metalloproteinases released by macrophages, such as collagenases, gelatinases, and stromelysin, with subsequent degradation of collagen, elastin, and proteoglycans of the fibrous cap.32 33 34 Recently, Galis et al35 showed that plaque shoulders and regions of foam cell accumulation display locally increased expression of 92-kD gelatinase, stromelysin, and interstitial collagenase, suggesting that focal overexpression of activated matrix metalloproteinases may promote destabilization and complication of atherosclerotic plaques. Macrophage foam cells are frequently found at the rupture site, where the cap is usually thin, probably because of preceding tissue destruction causing progressive plaque weakening.36 Ultimately, the cap may disintegrate or rupture whether it is stressed or not. Macrophages may be "bad," producing proteolytic enzymes34 37 and other tissue-destroying substances, but they may also constitute an important defense mechanism, attempting to clear lipids from the plaque. Transforming growth factor-ß is an interesting example of the complexities of local regulation and participates in a double-negative feedback regulation. Local activation of the PA system leads to enhanced ECM degradation but also to activation of transforming growth factor-ß. This growth factor is a powerful inducer of PAI-1 biosynthesis in endothelial cells and SMCs38 and thereby induces a decrease of PA activity. In addition, it is a powerful inducer of the biosynthesis of ECM components.
The overexpression of plasminogen activators in the atherosclerotic plaques, especially in the advanced ones, may have important functions in their destabilization and rupture and provide novel targets for therapeutic interventions.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This work was supported by stipends from the Roche Foundation (Dr Heim), the Sandoz Foundation, the Foundation for Research on Atherosclerosis and Thrombosis (Dr Bachmann), the Swiss National Fund for Scientific Research (grant 31.26302.89), and in part by the British Heart Foundation grant PG 94188 (Drs Lupu and Kakkar). We thank Elisabeth Cousin for preparing the plasmids used for in situ hybridization and Patricia Harley for her expert technical assistance in the immunofluorescence work.
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