© 1998 American Heart Association, Inc.
Original Contributions |
Activation of Matrix-Degrading Metalloproteinases by Mast Cell Proteases in Atherosclerotic Plaques
From the Bristol Heart Institute, University of Bristol, Bristol Royal Infirmary, Bristol, UK.
Correspondence Mr Jason Lee Johnson, Bristol Heart Institute, University of Bristol, Bristol Royal Infirmary, Upper Maudlin Street, Bristol BS2 8HW, UK. E-mail jason.l.johnson{at}bristol.ac.uk
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Abstract—Recent studies suggest that mast cell–derived neutral proteases can activate matrix-degrading metalloproteinases (MMPs). We have investigated the role of the mast cell proteases tryptase and chymase in the activation of MMPs in human carotid endarterectomy specimens (atherosclerotic, n=32) and postmortem carotid arteries (control, n=17). In vitro degranulation of mast cells in atherosclerotic carotid arteries by compound 48/80 caused a significant increase in MMP activity. Addition of the nonselective tryptase inhibitor antipain, the specific trypsinlike protease inhibitor 4-amidinophenylmethanesulfonyl fluoride, and the chymase inhibitor chymostatin reduced this increase in MMP activity by 30±6%, 23±6%, and 9±2%, respectively. Immunocytochemistry identified significantly higher numbers of tryptase-containing cells (mast cells) and cells expressing MMP-1 and MMP-3 in the "shoulder" regions of atherosclerotic artery lesions compared with the tunica media of control arteries. Dual immunocytochemistry showed collocation of MMP-1 and MMP-3 with mast cells in the shoulder regions. Degranulation was observed in 78±5% (mean±SEM) of mast cells in this area, whereas nonactivated mast cells were observed in all other areas. In situ zymography revealed caseinolytic and gelatinolytic activity in these areas. In conclusion, in vitro mast cell degranulation, which releases mast cell proteases, in carotid arteries increases MMP activity. Furthermore, elevated MMP-1 and MMP-3 expression is collocated with increased numbers of degranulated mast cells and with greater MMP activity in the shoulder regions of atherosclerotic plaques. Activation of MMPs by mast cell–derived proteases may be an important mechanism in atherosclerotic plaque destabilization.
Key Words: atherosclerosis • mast cells • plaque rupture • matrix metalloproteinase • tryptase
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Introduction |
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Atherosclerosis is a complex disease characterized by smooth muscle cell proliferation and migration, cholesterol deposition, infiltration of mononuclear cells, and extracellular matrix degradation.1 2 Myocardial infarction and stroke are commonly caused by the rupture of atherosclerotic plaques, a process that often occurs at the "shoulder" regions of the plaque.3 4 5 Smooth muscle cells, macrophage-derived foam cells, T lymphocytes, and endothelial cells are present in the shoulder regions, and all have been shown to synthesize matrix-degrading metalloproteinases (MMPs).6 7 8 9 10 11 Recent studies have shown increased expression of interstitial collagenase (MMP-1), stromelysin-1 (MMP-3), and gelatinase B (MMP-9) in the shoulder regions of atherosclerotic plaques.12 13 14 The MMP family is capable of degrading all of the components of the extracellular matrix in blood vessels, and therefore, these enzymes are normally tightly regulated. In addition to regulation by their endogenous inhibitors, the tissue inhibitors of metalloproteinases and by transcriptional regulation, the activity of MMPs is also regulated by the necessity for proteolytic activation of secreted proenzyme forms. Proteolytic activation of pro-MMPs has been achieved in vitro by using trypsin, chymotrypsin, plasma kallikrein, plasmin, thermolysin,15 chymase, and tryptase.16 Furthermore, MMPs can activate each other; eg, activated MMP-3 can activate MMP-1.17
Mast cells play pivotal roles in many biological responses, including
hypersensitivity reactions, inflammation, tissue remodeling, and
angiogenesis, and they are involved in the pathogenesis of chronic
degenerative diseases such as asthma and rheumatoid
arthritis.18 In addition, the observation of
elevated numbers of mast cells in the shoulder regions of
atherosclerotic plaques compared with normal arteries has led to the
suggestion that mast cells may be involved in the development of
atherosclerotic lesions.19 20 21 22 Mast cells produce
a range of soluble mediators, including histamine, heparin,
leukotrienes, cytokines, growth factors, and
neutral proteases. All mast cells in blood vessels have been shown to
express the neutral protease tryptase, and 
40% of them also contain
another neutral protease, chymase.20 23 These
enzymes serve as specific markers for mast
cells.24 Chymase can directly degrade components
of the basement membrane, including collagen types IV and V, laminin,
fibronectin, and elastin.25 Tryptase can degrade
pericellular matrix proteins such as
fibronectin.26 These proteases also
activate pro-MMPs: tryptase activates pro–MMP-3 and
chymase activates both pro–MMP-1 and
pro–MMP-3.16 27 Therefore, release of tryptase
and chymase from mast cells could initiate an amplification cascade
whereby pro–MMP-1 and pro–MMP-3 are activated, which in turn
activate other MMPs, including MMP-1, MMP-2, and MMP-9, leading
to matrix degradation, plaque instability, and possibly, plaque
rupture.
Although active MMPs have been shown to be present in the shoulder regions of atherosclerotic plaques,12 13 the way in which they become activated is unknown. In these studies, we have examined the effect of mast cell degranulation on MMP activation in the human atherosclerotic internal carotid artery.
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Methods |
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Reagents
All materials were purchased from Sigma except for those stated below. Unconjugated mouse monoclonal anti-human mast cell tryptase antibody (AA1), normal goat serum, biotinylated goat anti-mouse and goat anti-rabbit IgG, and horseradish peroxidase–labeled swine anti-rabbit antibodies were purchased from Dako. Avidin/biotin blocking kit, ß-galactosidase–conjugated avidin D, and nuclear fast red were purchased from Vector Laboratories. Mouse monoclonal anti-human chymase antibody was purchased from Biogenesis. Resorufin-labeled universal protease substrate and 4-amidinophenylmethanesulfonyl fluoride (APMSF) were purchased from Boehringer Mannheim. Human MMP-1 and MMP-3 ELISA systems and LM-1 photographic emulsion were purchased from Amersham International. The bicinchoninic acid protein assay kit was obtained from Pierce & Warriner. Purified recombinant human MMP-1, MMP-2, MMP-3, and MMP-9 proteins were obtained from TCS Biologicals. Unconjugated rabbit anti-human MMP-1 and anti-human MMP-3 polyclonal antisera were generous gifts from Dr C. Long (Pfizer Ltd, Sandwich, UK), and the MMP inhibitor Ro 31-9790 was a generous gift of Dr D. Bradshaw (Roche Products, Welwyn Garden City, UK).
Sample Collection
Samples of fresh carotid artery were obtained from patients
undergoing carotid endarterectomy (atherosclerotic,
n=32; mean age ±SEM, 65±16 years) and from postmortem arteries 
24
hours after death (control, n=17; age, 62±10 years; P=NS
versus atherosclerotic). The adventitia was partially dissected from
the carotid arteries collected postmortem to mirror the
endarterectomy samples. Tissue samples were taken
from the internal carotid artery immediately above the bifurcation of
the common carotid artery. These specimens were fixed in 10%
formalin-phosphate buffer solution for 24 hours and embedded in
paraffin wax. Adjacent specimens taken for in situ zymography; ELISA;
and tryptase, chymase, and MMP assays were stored in
LN2. Ethical permission was obtained from the
United Bristol Healthcare National Health Service Trust Ethics
Committee (reference E3111).
Tryptase, Chymase, and MMP Activity Assays
Carotid endarterectomy specimens and
postmortem carotid arteries were chopped into 1-mm squares and added to
0.15 mol/L Tris-HCl, pH 7.6. To half of the samples 5 µg/mL compound
48/80, a specific mast cell degranulation
agent,28 was added, and they were incubated for
15 minutes at 37°C to induce mast cell degranulation. All samples
were microfuged at 13 000 rpm for 3 minutes and the supernatant
decanted. The supernatant was assayed in the tryptase, chymase, and MMP
assays.
Tryptase and chymase activities of the supernatant were assayed by using the chromogenic substrates N-benzoyl-D,L-arginine-p-nitroanilide and N-succinyl-L-phenylalanine-p-nitroanilide, respectively.16 In brief, 100 µL of supernatant was added to 0.9 mmol/L N-benzoyl-D,L-arginine-p-nitroanilide in tryptase buffer or 0.2 mmol/L N-succinyl-L-phenylalanine-p-nitroanilide in chymase buffer. The rate of change in absorbance was measured over 10 minutes at 410 nm for tryptase activity and overnight at 405 nm for chymase activity. With the use of a nitroaniline standard, curves of the absorbance at 405 and 410 nm were calculated, and the tryptase and chymase activities detected in the samples were expressed as millimoles per liter of nitroaniline per hour per gram wet weight. To test specificity, tryptase activity was inhibited with 100 µg/mL antipain (a nonselective tryptase inhibitor) and 40 µg/mL APMSF (a specific trypsinlike protease inhibitor), and chymase activity was inhibited with 100 µg/mL chymostatin (a chymase inhibitor). The percent inhibition of enzyme activity for each protease inhibitor was calculated.
The MMP activity of the supernatant was measured in a fluorometric assay with the use of a fluorescent substrate that is susceptible to degradation by all MMPs.29 Supernatant (300 µL) was incubated with 1 µmol/L 7-methoxycoumarin-4-acetyl-Pro-Leu-Gly-Leu-(2,4-dinitrophenylamino)-Ala-Ala-Arg-NH2 in MMP assay buffer (100 mmol/L NaCl, 100 mmol/L Tris, 10 mmol/L CaCl2, 20 µmol/L ZnCl2, and 0.05% Brij 35, pH 7.5) for up to 4 hours at 37°C. Increase in fluorescence was monitored every 30 minutes by using an excitation wavelength of 328 nm and an emission wavelength of 393 nm. Recombinant human MMP-2 was activated with 1 mmol/L p-aminophenylmercuric acetate (APMA) and used to establish standard curves of the rate of increase in fluorescence per minute (n=6). The rate of increase in fluorescence per minute in the samples was expressed as nanomoles per liter of activated MMP-2 per gram wet weight. MMP activity was inhibited by the addition of standard MMP inhibitors (20 mmol/L EDTA, 100 µmol/L Ro 31-9790). The role of proteases in MMP activation was examined by the addition of protease inhibitors: 100 µg/mL chymostatin, 100 µg/mL antipain, 40 µg/mL APMSF, 1 µg/mL aprotinin, 10 µg/mL E-64, or 1 µmol/L pepstatin. The rate of MMP activity in the presence of these inhibitors was calculated per milligram wet weight and was expressed as a percentage of the compound 48/80–degranulated control.
Immunocytochemistry
Immunocytochemistry using monoclonal anti-human mast cell
tryptase antibodies was used to identify the location of the mast cells
and determine the number of degranulated mast cells. In brief, serial
3-µm-thick paraffin sections were dewaxed and rehydrated, and
endogenous peroxidase activity was inhibited with 3%
H2O2. Sections were
permeabilized by incubation in 0.1% calf thymus
trypsin and 0.1% CaCl2 for 10 minutes at 37°C.
Sections were blocked with nonimmune goat serum and then incubated with
monoclonal anti-human mast cell tryptase antibodies (diluted 1:50),
followed by biotinylated goat anti-mouse IgG (diluted 1:200), and then
horseradish peroxidase–labeled ExtrAvidin (diluted 1:400). Color was
developed by incubation in 0.05% 3,3'-diaminobenzidine, and sections
were counterstained with hematoxylin before mounting. Tryptase-positive
cells (mast cells) were stained brown with blue nuclei, whereas
negative cells had blue nuclei only. A negative control for which the
anti-tryptase antibody was substituted with nonimmune mouse IgG was
always included. Mast cells in the fibrous cap, shoulder, and
adventitial regions of atherosclerotic arteries and in intimal, medial,
and adventitial regions of control arteries were counted in three
0.25-mm2 fields and expressed as a percentage of
the total number of nucleated cells. Mast cells with tryptase-positive
granules observed as "halos" in their immediate pericellular
vicinity were classified as degranulating. Immunocytochemistry for
tryptase and chymase protein was also carried out on 5-µm-thick
frozen sections in a similar manner to that described above. The number
of tryptase-and-chymase–containing mast cells and the number of
tryptase-only–containing mast cells were determined.
Immunocytochemistry for MMP-1 and MMP-3 was carried out in a similar
manner to that described for mast cells. The primary and secondary
antibodies were substituted with rabbit anti-human MMP-1 or MMP-3
antisera (diluted 1:500 and 1:2000, respectively) and biotinylated goat
anti-rabbit IgG (diluted 1:400). The color substrate was
3-amino-9-ethylcarbazole. Negative controls, for which the rabbit
antisera were preabsorbed with 100 ng of the appropriate peptide, were
always included. To demonstrate the specificity of the rabbit
anti–MMP-1 and –MMP-3 antisera, Western blots using purified
recombinant human MMP-1, MMP-2, MMP-3, and MMP-9 were performed. The
numbers of MMP-positive cells in the same regions as examined for mast
cells were scored on a 4-point scale, where 0=no positive cells;
1=<10% positive cells; 2=10% to 50% positive cells; and 3=>50%
positive cells.30 The reproducibility of
assessments was tested for interobserver variation by using an
unweighted 
test (=0.75).
Dual immunocytochemical labeling of mast cells and MMPs was carried out by using an indirect 2-stage method. Sections were dewaxed, rehydrated, and permeabilized, and endogenous peroxidase activity was inhibited as described above. Mast cells were detected with monoclonal anti-human mast cell tryptase antibodies as described above, with 3,3'-diaminobenzidine as the chromogenic substrate, yielding a brown product. MMP-1 and MMP-3 were detected as described above with the addition of 2 drops of avidin blocking solution per milliliter of goat serum and 2 drops of biotin blocking solution per milliliter of primary antibody. Peroxidase-labeled ExtrAvidin was substituted with ß-galactosidase avidin D, and 5-bromo-4-chloro-3-indoyl-ß-D-galactopyranosidase was used as the chromogen, yielding a green-blue product. Sections were counterstained with nuclear fast red.
In Situ Zymography
Caseinolytic and gelatinolytic activity was
located in atherosclerotic (n=12) and control (n=8) carotid arteries by
in situ zymography as described previously.31 In
brief, for caseinolytic in situ zymography, 8-µm-thick frozen
sections were applied to slides coated with 1 mg/mL Resorufin-labeled
universal protease substrate in incubation medium (50 mmol/L Tris,
50 mmol/L NaCl, 10 mmol/L CaCl2, and
0.05% [wt/vol] Brij 35, pH 7.6) supplemented with 1% (wt/vol)
agarose. Sections were covered in incubation medium and incubated for
up to 72 hours at 37°C. By fluorescence microscopy,
caseinolytic activity was identified as black "holes" of lysis on
the red background. For gelatinolytic in situ
zymography, 8-µm-thick frozen sections were applied to slides and
coated with LM-1 photographic emulsion diluted 1:2 with incubation
medium. Sections were incubated overnight at 37°C, then developed in
Kodak D-19 developer, and fixed with Kodak Unifix solution. By light
microscopy, gelatinolytic activity was identified
as white "holes" of lysis on the black background. Controls, for
which sections were incubated with incubation buffer supplemented with
20 mmol/L EDTA, 200 nmol/L of the MMP inhibitor Ro
31-9790, 100 µg/mL antipain, and 100 µg/mL chymostatin, were always
included.
Quantification of MMP-1 and MMP-3 Proteins
Frozen segments of atherosclerotic (n=14) and control (n=13)
carotid arteries were crushed under LN2, and
proteins were extracted as described
previously.32 In brief, the frozen samples
(100 mg) were crushed under LN2 with a mortar
and pestle and added to 400 µL of extraction buffer (2 mmol/L
NaCl, 100 µmol/L Tris, 1 µg/mL aprotinin, and 1 µmol/L
PMSF). Total MMP-1 and MMP-3 (the pro- form, active form, and tissue
inhibitor of metalloproteinase–bound MMP) levels were
quantified with sandwich-based ELISA systems according to the
manufacturer's instructions. Tissue extracts from atherosclerotic
(n=14 for MMP-1, n=10 for MMP-3) and control (n=13 for MMP-1, n=8 for
MMP-3) carotid arteries were diluted to fall within the linear range of
the assays. Samples were assayed in duplicate. Owing to the varying
histological composition, size, and wet weight of the
samples, the protein concentration of all tissue extract samples was
determined by using a bicinchoninic acid protein assay with BSA as the
standard. The concentrations of MMP-1 and MMP-3 were expressed per
microgram of total protein.
Statistical Analysis
Values are expressed as mean±SEM. Mean values in the MMP,
tryptase, and chymase activity assays were compared with controls by
using the 1-sample Student's t test. Mean values of MMP-1–
and MMP-3–positive cell scores of atherosclerotic and control arteries
were compared with the Mann-Whitney U test for
nonparametric data. Mean values for MMP-1 and MMP-3 protein
levels and the numbers of mast cells in atherosclerotic and control
arteries were compared by using Student's t test for
unpaired data. Differences were considered statistically significant
when P0.05.
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Results |
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Effect of Mast Cell Degranulation on Tryptase, Chymase, and MMP Activity
Incubation with compound 48/80 caused tryptase activity to increase significantly (4-fold, P=0.04) in atherosclerotic carotid arteries (n=6); however, tryptase activity was not affected in control carotid arteries (n=3) (Table 1
). The tryptase
inhibitors antipain and APMSF significantly reduced
tryptase activity in atherosclerotic carotid arteries by 92±14% and
81±18%, respectively (P<0.01). Mast cell degranulation
with compound 48/80 did not cause a detectable increase in chymase
activity in atherosclerotic (n=5) or control (n=3) carotid arteries
compared with the nonactivated controls (Table 1).
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MMP activity was significantly increased in atherosclerotic carotid
arteries (n=14) after mast cell degranulation with compound 48/80
(1.5±0.2-fold, P=0.018) (Table 1
). This increase was
blocked by standard MMP inhibitors (Table 2). Addition of antipain,
APMSF, and chymostatin significantly inhibited MMP activity by 30±6%
(P=0.001), 23±6% (P=0.034), and 9±2%
(P=0.01) (Table 2), respectively, demonstrating the
involvement of tryptase and chymase in the increased MMP activity.
Addition of aprotinin, E-64, or pepstatin A did not significantly
inhibit the increase in MMP activity caused by mast cell degranulation
(Table 2). Higher concentrations of antipain, APMSF, and chymostatin
did not increase the level of inhibition of tryptase, chymase, or MMP
activity (data not shown).
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Mast Cell Location
The distribution of mast cells in atherosclerotic and control
carotid artery specimens was examined by immunocytochemistry (Figure 1: a, b, and c, atherosclerotic; d, e,
and f, control). A significantly higher proportion of mast cells was
identified in the shoulder regions (4.9±1.3%) and fibrous caps
(2.5±1%) of atherosclerotic plaques than in the media (0±0%,
P=0.031) and intima (0.9±0.2%, P=0.048) of
control arteries. In contrast, the proportion of mast cells in the
adventitia was significantly lower in atherosclerotic arteries
(2.6±1.3%) than in control arteries (5.3±0.3%, P=0.019).
Therefore, in the whole atherosclerotic artery, there are 2-fold more
mast cells than in the controls. In the majority of cases, the mast
cells present in the shoulders of atherosclerotic plaques were
located in regions of neovascularization. Degranulation was observed in
78±5% of mast cells in the shoulder and fibrous cap regions of
atherosclerotic plaques, whereas in control arteries, few mast cells
were activated in any area. No staining was detected in the
negative control sections (data not shown). In serial frozen sections
(n=4), only 37±7% of tryptase-containing mast cells also contained
chymase (Figure 2), confirming previous
findings.20 23
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Location of MMP-1 and MMP-3 Proteins
Western blots demonstrated that the rabbit anti-human MMP-1 and
MMP-3 antisera were specific for the respective proteins and that the
antisera identified both the active and the pro- forms of the MMP
(Figure 3). Staining of serial sections
with anti–MMP-1 and anti–MMP-3 antisera showed a strikingly similar
pattern to that seen for mast cells in atherosclerotic plaques (data
not shown). The small amount of positive staining that was detected in
the control arteries was restricted to the adventitia. High levels of
MMP-1– and MMP-3–positive cells were observed in the fibrous cap and
shoulder regions of atherosclerotic arteries (n=18) compared
with control vessels (n=15) (Table 3). No signal was detected
when the antisera were preabsorbed with the appropriate peptides.
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Collocation of Mast Cells and MMP-1 and MMP-3 Proteins
Double labeling of atherosclerotic plaques showed that the mast
cells in the fibrous cap, especially in the shoulder region, were
located in zones containing cytoplasmic and extracellular MMP-1 and
MMP-3 proteins (Figure 4A and 4B,
respectively). The orange color detected after double labeling for
tryptase and MMPs develops as an artifact of combining the 2 color
substrates and is not specific (Figure 5). No staining was detected in the
negative controls (data not shown). Immunocytochemistry using serial
sections demonstrated that MMP-1 and MMP-3 expression was not detected
in mast cells (data not shown).
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Location of MMP Activity
Our immunocytochemical studies do not discriminate between the
latent and active forms of MMPs. Therefore, serial frozen sections of
freshly excised vessel segments were analyzed by in situ
zymography (Figure 4C). Areas of substrate lysis, which indicate
caseinolytic activity, were greatest in all plaques examined (n=6) in
the shoulder regions, where increased expression of MMP-1, MMP-3, and
mast cells was also detected (Figure 4A and 4B). Addition of the MMP
inhibitors Ro 31-9790 (Figure 4D) or EDTA (data not shown)
to the incubation buffer abolished the caseinolytic activity. The
addition of the mast cell protease inhibitors antipain and
chymostatin did not affect the observed caseinolytic activity (Figure 6). The control arteries (n=3) and the
uninvolved areas of the atherosclerotic specimens did not exhibit any
enzymatic activity. Gelatinolytic activity was
observed in the shoulder regions of all plaques (data not shown).
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Quantification of MMP-1 and MMP-3 Proteins
ELISA assays showed significantly higher levels of total MMP-1
(2.7±0.5 ng/mg of total protein) and MMP-3 (3.8±1.1 ng/mg of total
protein) in atherosclerotic plaques than in control arteries (MMP-1,
1.6±0.3 ng/mg of total protein, P=0.046; MMP-3, 0.9±0.2
ng/mg of total protein, P=0.050).
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Discussion |
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Matrix degradation within atherosclerotic plaques is thought to cause weakening and to lead to erosion and rupture.3 4 5 It has been suggested that mast cells contribute to this process by releasing neutral proteases such as tryptase and chymase.19 21 22 23 33 These enzymes, which are released from mast cells by degranulation, may act indirectly by activation of MMPs or directly by digestion of plaque matrix proteins. Although previous studies have found increased numbers of mast cells19 21 22 23 33 ; MMP-1–, MMP-3–, and MMP-9–immunopositive cells; and net MMP activity12 13 14 in the shoulder regions of atherosclerotic plaques, no direct collocation or involvement of mast cells in MMP activation has been reported.
We incubated carotid endarterectomy specimens with the mast cell degranulator compound 48/80, which caused a 2-fold increase in the activity of mast cell tryptase. The levels of chymase detected in the carotid arteries was much lower than that of tryptase, and no increase in chymase activity was observed after compound 48/80 activation. The addition to plaque supernatants of the tryptase inhibitors antipain and APMSF or the chymase inhibitor chymostatin abolished the activity of the enzymes. Mast cell degranulation by compound 48/80 also caused an increase in MMP activity, which was abolished by the addition of standard MMP inhibitors. This shows that mast cell degranulation leads to an increase in MMP activity. The addition of tryptase and chymase inhibitors reduced the increase in MMP activity by 30% and 10%, respectively, suggesting that chymase plays a lesser though significant role in MMP activation than tryptase. The requirement for the extended incubation to detect chymase activity and the detection of fewer chymase-and-tryptase–containing mast cells than tryptase-only–containing mast cells support the hypothesis that there are rather low concentrations of this protease in atherosclerotic plaques.
Because incubation with tryptase and chymase only reduced MMP activity
by 40%, 60% of the increase in MMP activity is not caused by
mast cell proteases. The addition of serine, cysteine, and aspartic
protease inhibitors did not affect the MMP activity,
suggesting that these classes of protease are not involved in MMP
activation and that this remaining 60% may be due to MMP
autoactivation. The increase in MMP activity detected after mast cell
degranulation with compound 48/80 is not the result of the release of
preformed MMPs from mast cells, since previous studies have shown that
MMP activity is not detectable in isolated mast
cells.16 34
Significantly higher numbers (2-fold) of mast cells were detected in atherosclerotic arteries compared with control arteries, which is correlated with the finding of 2-fold higher levels of tryptase in the atherosclerotic arteries compared with controls. Mast cells account for 5% and 2.5% of the nucleated cells in the shoulder region and fibrous cap, respectively, whereas in control arteries mast cells account for only 1% of the intimal cells. The majority of the mast cells (78±5%) in the rupture-prone regions of the atherosclerotic plaques were degranulated, suggesting that their neutral proteases were available to interact with other mediators in these regions. This finding is in accord with previous studies that have shown that the number of activated mast cells at sites of coronary artery thrombotic atheromatous erosion or rupture is considerably increased.19 20
Current knowledge suggests that mast cell degranulation is stimulated in vivo by several mechanisms. The best-understood stimulus is the binding of an allergen to mast cell–bound IgE. In addition, "histamine releasing factor," which is secreted by immunocompetent cells such as T lymphocytes35 and macrophages36 present in the shoulder regions of atherosclerotic plaques, can also stimulate mast cell degranulation. Furthermore, matrix degradation during atherosclerotic plaque development may result in the generation of breakdown products, including collagen-derived peptides, which have also been shown to induce mast cell degranulation.37 More recently, it has been suggested that oxidized LDL present in the atherosclerotic plaques may induce mast cell degranulation in atherosclerosis either directly or through recruitment of leukocytes.38 In addition, tryptase has been shown to activate mast cells, highlighting a possible amplification cascade.39
Our results show that the release of neutral proteases from mast cells in atherosclerotic plaques causes MMP activation, which may degrade the extracellular matrix and destabilize the plaque. However, there was no significant increase in MMP activity in control carotid arteries. This may be because lower numbers of mast cells are present in the control arteries. This conclusion is supported by the finding that tryptase activity is unaffected by mast cell degranulation in the control arteries. Furthermore, it may be because lower levels of MMPs are expressed in the control arteries than in the atherosclerotic plaques, and therefore there is less MMP available for activation.
In this study, activated mast cells were found in the shoulder regions in close proximity to T lymphocytes and macrophages, confirming previous findings.23 In these regions, the levels of MMP-1 and MMP-3 were significantly higher than in control vessels. Dual immunocytochemistry demonstrated directly that degranulated mast cells are collocalized with cells expressing MMPs, indicating that immunocompetent cell–induced mast cell degranulation and subsequent release of their neutral proteases, which are capable of proteolytic activation of MMPs, may be present in the vicinity of MMP proteins. The in situ zymography results clearly showed the presence of active MMPs in the regions of degranulated mast cells. The addition of chymostatin and antipain did not affect the in situ zymography results, which suggests that substrate lysis is due to mast cell protease activation of MMPs rather than direct substrate lysis. This suggests that mast cell proteases could be involved in the proteolytic activation of MMPs during plaque development and rupture.
In conclusion, our results indicate that mast cells accumulate and degranulate in the shoulder regions and fibrous caps of carotid artery atherosclerotic plaques, where expression of total MMP-1 and MMP-3 is elevated and net MMP activity is located. The in vitro assays demonstrate directly that tryptase and chymase released from mast cells activate MMPs. Together these results suggest that mast cells may play an important role in MMP activation in atherosclerotic plaques.
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Acknowledgments |
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This work was supported by the British Heart Foundation (J.L.J., G.D.A., S.J.G.) and by the Garfield Weston Trust (C.L.J., G.D.A., S.J.G.). We would like to thank Dr E.A. Sheffield and C. Irvine for providing carotid artery samples.
Received December 10, 1997; accepted April 23, 1998.
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