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© 1999 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
Increased Secretion of Tissue Inhibitors of Metalloproteinases 1 and 2 From the Aortas of Cholesterol Fed Rabbits Partially Counterbalances Increased Metalloproteinase Activity
From the Bristol Heart Institute, University of Bristol, Bristol Royal Infirmary, Bristol, UK.
Correspondence to Dr A. Zaltsman, Bristol Heart Institute, Department of Cardiac Surgery, Bristol Royal Infirmary, Bristol BS2 8HW, UK. E-mail a.b.zaltsman{at}bristol.ac.uk
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Abstract—The balance between matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) plays an important role in extracellular matrix turnover and thereby modulates atherosclerotic plaque development. MMP-1, -2, -3, and -9 activity is increased by atherosclerosis, but the status of TIMPs is less clear. We therefore compared secretion of TIMPs-1 and -2 from cultured aortic explants derived from arch, middle, and distal portions of thoracic aortas of normal rabbits and rabbits fed a 1% cholesterol diet for 8 weeks, using reverse zymography of conditioned media. Cholesterol feeding significantly increased secretion of TIMP-1 from arch and middle portions (both 2.6-fold), accompanied by 2.0- and 2.7-fold increases in TIMP-2, respectively. Atherosclerotic aortas exhibited increased immunoreactive TIMP-1 and TIMP-2 in endothelial cells, smooth muscle cells, and macrophages. Staining of extracellular matrix was also prominent within the noncellular boundary region between fibrous cap and the lipid core, and within the lipid core. Increased TIMP-2 staining was also found in the media subjacent to the lipid core. In situ gelatin zymography demonstrated excess MMP activity within the plaque with partial inhibition in the lipid core base and subjacent media, consistent with the distribution of TIMPs. Casein zymography and in situ zymography demonstrated that increased caseinolytic activity was confined to the pericellular zones of macrophages within the lipid core, again consistent with its restriction by TIMPs. In summary, atherosclerosis increases TIMP expression, which counterbalances, in part, increased MMP activity.
Key Words: cholesterol • atherosclerosis • tissue inhibitor of metalloproteinases
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Introduction |
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Extracellular matrix turnover is integral to the pathology of atherosclerotic plaque development. Increased deposition of extracellular matrix accompanies the transition from fatty streaks to fibrous plaques and contributes significantly to intimal encroachment.1 Perhaps more important, however, erosion of the fibrous cap, plaque rupture, and hence myocardial infarction are associated with attenuation of the extracellular matrix.2 This has led to the hypothesis that an altered balance between matrix degrading enzymes and their endogenous inhibitors is a key factor in determining the stability of the plaque cap.3 4 Matrix degrading metalloproteinases (MMPs) are believed to play the primary role in turnover of the vascular extracellular matrix. Increased levels of several MMPs, including stromelysin, interstitial collagenase, and gelatinases A and B, show increased expression and/or activation in atherosclerotic plaques.3 4 5 6 7 We recently showed, furthermore, that MMP-9 expression and MMP-2 expression and activation are positively correlated with lesion severity, consistent with a pathogenetic role late in the disease process.7
The activation of MMPs and their proteolytic potential is tightly controlled by endogenous tissue inhibitors of matrix metalloproteinases (TIMPs).8 9 Four members of the TIMP family are known to date, among which only TIMP-1, -2, and -3 have been characterized in vascular tissue.3 10 11 12 13 TIMP-1 and -2 are secreted as soluble proteins, and TIMP-3 remains bound to extracellular matrix.13 14 TIMP-1, -2, and -3 appear to be approximately equally effective in binding to the catalytic site and inhibiting the active forms of stromelysin, collagenase, and gelatinases.9 TIMP-1 and -2 also interact with a separate C-terminal site differentially on the two gelatinases. TIMP-1 binds and slows activation of the latent pro-form of MMP-915 and TIMP-2 binds to and both positively and negatively regulates the activation of pro-MMP-2.16 17
In contrast to the wealth and consensus of evidence for increased MMP activity in atherosclerosis, the data for TIMPs are less abundant and conflicting. Studies with isolated smooth muscle cells (SMCs) suggest that both TIMP-1 and -2 secretion is constitutive.11 13 Consistent with this, in one immunohistochemical study of human arteries, TIMP-1 and -2 were identified at similar levels in atherosclerotic and normal tissue.3 However, in another study, immunoreactive TIMP-1 was detected at apparently higher levels in human atherosclerotic and late restenotic carotid arteries compared with normal arteries, with strong staining localized to the foam cell–rich region of plaques.12 In models of vascular injury rather than atherosclerosis, a significant increase in TIMP-1 expression was observed in rabbit aortic neointima 8 weeks after selective deendothelialization.18 An increase in TIMP-2 but not TIMP-1 was observed in a study of balloon-injured rat carotid arteries.19 However, another study did show an increase in TIMP-1 mRNA shortly after balloon injury.20
An excess of MMPs over TIMPs is presumed to promote extracellular matrix turnover. Galis et al3 21 demonstrated an excess of MMPs over TIMPs directly in tissue sections from human atherosclerotic plaques3 and plaques from balloon-injured aortas and iliac arteries from cholesterol-fed rabbits21 by using in situ zymography.
We previously used a highly reproducible explant culture model to investigate the relationship between atherosclerotic lesion severity and secretion of gelatinases in the aortas of cholesterol-fed rabbits.7 Here, we use the same model to demonstrate increased secretion of TIMP-1 and -2 in atherosclerotic aortas. Immunocytochemistry confirmed the increase in steady-state TIMP-1 and -2 expression. Furthermore, in situ zymography demonstrated that increased TIMP expression functionally modulated the increase in MMP activity.
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Methods |
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Materials
All chemicals, unless otherwise stated, were obtained from Sigma. Culture media and additives other than FCS were obtained from GibcoBRL. Human fibrosarcoma HT 1080 cell line was obtained from European Collection of Cell and Animal cultures.
Male New Zealand White rabbits (6 weeks old initially) were fed a diet
containing 1% cholesterol during 8 weeks according to an
established protocol, as detailed previously.7 Equal
numbers (8 each) of age-matched control rabbits were fed a standard
laboratory chow. Serum cholesterol levels were increased by
cholesterol feeding to 56±4 mmol/L compared with
1.3±0.1 mmol/L in control rabbits (mean±SEM,
P<0.01). After the animals were killed, the descending
thoracic aortas were removed and each aorta was divided into 3 portions
of equal length, 
2.5 cm, namely, the arch, mid, and distal (proximal
to the diaphragm) portions. Ring sections from each portion (0.5 cm)
were either fixed in 4% phosphate-buffered formaldehyde and then
wax-embedded or snap-frozen for extraction or frozen-tissue section
preparation (see below). Also, explants of aortic tunica media were
prepared from 1 cm2 segments by using the method of
McMurray et al22 as modified by Southgate and
Newby.23 As described previously,7 explants
were prepared in parallel from cholesterol-fed and control
rabbits on the same day.
Reverse Zymography
Reverse zymography was used to detect TIMP-1 and TIMP-2
activities in conditioned media essentially as described by Ward et
al.24 In brief, conditioned media from aortic medial
explants cultured for 3 days in serum-free medium was 10 times
concentrated using Centricon-10 concentrators (Amicon). Samples were
then subjected to SDS–10% polyacrylamide gel electrophoresis
on gels containing 1 mg/mL of gelatin as described
previously.13 Before gel loading, samples were normalized
to DNA concentration of explants tissue. Different amounts of each
conditioned media were mixed with 3 times strength SDS-nonreducing
sample buffer to achieve an equivalent final dilution to the least
concentrated sample (equivalent to 17.4 µg of explant tissue DNA/mL)
and then 20 µL were loaded onto the gel. After electrophoresis was
complete, gels were washed in 2.5% Triton X-100 and incubated for 1
hour at 37°C with conditioned media from phorbol 12-myristate
13-acetate (PMA)-activated skin rabbit fibroblasts, containing
a mixture of activated MMPs.24 Then gels were
incubated with 50 mmol/L Tris-HCl, 50 mmol/L NaCl, 10
mmol/L CaCl2, and 0.05% Brij for 18 hours at
37°C, stained in Coomassie Blue R250, and destained in acetic
acid/methanol. Under these conditions, TIMPs inhibit gelatin digestion
by activated MMPs and produce dark blue bands against a lighter
background. An aliquot of the same HT 1080–conditioned media was used
on each gel for standardization.
For quantification, densitometric scanning was performed by using a Bio-Rad GS 690 Image Analysis software system (Bio-Rad Laboratories). Local background subtraction was achieved for each band separately by using a nearby reference area. To validate the method, a dose–response curve with HT 1080–conditioned medium was created. Measurements were always made within the linear range according to the dose–response curve with HT 1080–conditioned media.
Casein Zymography
Four or 10 times concentrated samples of conditioned media from
cultured aortic explants were subjected to SDS–10%
polyacrylamide gel electropheresis, using 2 mg/mL ß-casein as
substrate. After electrophoresis was completed, gels were incubated
with 0.25% Triton X-100 following with incubation in buffer,
containing 50 mmol/L Tris-HCl, 50 mmol/L NaCl, 10 mmol/L
CaCl2, and 0.05% Brij for 18 hours at 37°C,
stained in Coomassie Blue R250, and destained in acetic
acid/methanol.
Immunoprecipitation
Samples of 10 times concentrated conditioned medium were
incubated with 10 µL of 50 µg/mL sheep polyclonal anti-human
stromelysin-1 (generously provided by Dr G. Murphy) or with 50 µg/mL
sheep IgG overnight at 4°C. The mixture was incubated with 50 µL of
1:4 suspension of protein G in PBS for 4 hours at 4°C. Precipitant
was removed by centrifugation at 13 000g
for 5 minutes at 4°C and washed 5 times with buffer containing
50 mmol/L Tris-HCl (pH 8.3), 45 mmol/L NaCl/0.5% Nonidet
P40. The final pellet was heated at 60°C in nonreducing SDS sample
buffer and subjected to casein zymography.
Immunohistochemistry
Immunohistochemical staining was performed on transverse 5-µm
paraffin sections for identification of cell types. Deparaffinized
section were pretreated with 3%
H2O2 followed by incubation
with 0.1% trypsin, 0.1% CaCl2 in
H2O, pH 7.8, for 10 minutes at 37°C. Slides
were blocked with 20% FCS or 20% goat serum and stained in the
presence of 0.1% BSA with the mouse monoclonal primary antibodies,
anti–
-smooth muscle cell actin (Sigma), anti-rabbit alveolar
macrophage, RAM-11, (Dako), anti–TIMP-1, or anti–TIMP-2 (Fuji
Chemical Industries Ltd). Slides were incubated with goat anti-mouse
biotinylated secondary antibodies followed by Extravidin-peroxidase
conjugate (Sigma) and developed with 0.06% diaminobenzidine in the
presence of 0.03% H2O2 and
counterstained with hematoxylin. For double-immunohistochemical
staining, slides were incubated with RAM-11 antibodies as described
above. Slides then were incubated with anti–TIMP-2 antibodies
overnight, following by incubation with secondary antibodies, then with
alkaline-phosphatase and developed with fast red. Avidin-biotin
blocking was performed before incubation with antibodies, using the
avidin-biotin blocking kit (Vector Laboratories). Controls with
nonimmune mouse IgG were conducted in parallel with each
immunostaining procedure.
In Situ Zymography
In situ gelatin and casein zymography was performed as described
by Galis et al.21 In brief for gelatin zymography, unfixed
frozen sections (8 µm) were coated with a layer of LM-1 emulsion
containing 2.3% (wt/vol) gelatin (Amersham International plc)
in the presence of incubation buffer (as shown above for reverse
zymography), and left for 8 hours at 37°C. The slides were then
developed with Kodak D-19 developer and fixed with Kodak Unifix for 8
minutes. For casein zymography, slides were uniformly coated with 1
mg/mL resorsufin-labeled universal protease substrate
(Boehringer Mannheim) in 1% (wt/vol) agarose. Frozen sections
(8 µm) were applied to coated slides. Sections were covered with
incubation buffer as above, coverslipped, and incubated overnight at
37°C. Control sections were incubated in the presence of known MMP
inhibitors, 20 mmol/L EDTA or 100 µmol/L
Ro-31-9790 (Roche Research Center).
Statistical Analysis
Data are shown as mean±SEM values. Statistical significance
between groups was established with the Wilcoxon signed rank
test, unpaired for comparison of normal diet versus
cholesterol feeding.
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Results |
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Measurement of Functional TIMP Secretion by Reverse Zymography
The secretion of TIMP-1 and TIMP-2 into conditioned media by cultured rabbit aortic explants was investigated by reverse zymography. Previous findings with gelatinase production demonstrated continuous release of enzymes from cultured rabbit aortic explants during 12 days in culture.25 We studied the production of TIMPs by cultured rabbit aortic explants daily over 7 days in culture and found that TIMP-1 and -2 were also continuously released into the media (results not shown). Figure 1A
shows a representative
reverse zymogram using 3-day, serum-free conditioned media from aortic
medial explants of cholesterol-fed rabbit aortas (lane 1).
There are 2 bands of gelatinase-inhibitory activity of 21-
and 29-kDa molecular masses (lane 1). These bands had
electrophoretic mobility equivalent to recombinant human TIMP-1 and
TIMP-2 (Figure 1A, lanes 2 and 3). Both bands were abolished by
pretreatment of explants with 100 µmol/L cycloheximide (not
shown), confirming that they arose by de novo synthesis.
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In a previous study, we demonstrated that secretion of gelatinases by
cultured explants derived from the arch, mid, or distal portions of the
aortas of cholesterol-fed rabbits is greater than from
normal rabbit aorta and parallels the severity of atheroma
formation.7 We investigated here whether this was
accompanied by a change in TIMP levels. Reverse zymography demonstrated
that although both TIMP-1 and TIMP-2 were constitutively secreted from
aortic explants derived from all portions of normal rabbits aortas,
their levels were clearly elevated by cholesterol feeding
(Figure 1B
and the Table).
Quantitative analysis of TIMP activity demonstrated that this
increase was significant for the arch and middle portions of aortas
(Table), which paralleled the previously described increases
in gelatinase A and B expression. This implies that increased
gelatinase activity is counteracted by increased TIMP activity.
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Immunocytochemical Localization of TIMP-1 and TIMP-2
In normal rabbit aortas, both the intimal
endothelial cells and the medial SMCs stained positive
for TIMP-1 and TIMP-2 (Figure 2A and 2B).
In aortic sections from cholesterol-fed rabbits, TIMP-1 was
detected almost uniformly within the neointimal and medial
layers (Figure 2C). As evident in Figure 3, which compares the location of TIMPs,
SMCs, and macrophages in serial sections, TIMP-1 was
present in all cell types, including the SMCs of the fibrous cap.
Staining was most prominent within the extracellular matrix between the
lipid core and fibrous cap (large arrows in Figures 2C and 3A). Given the available antibodies, more intense
immunocytochemical staining for TIMP-2 than TIMP-1 was obtained in
atherosclerotic plaques. TIMP-2 was found in lumenal
endothelial cells and less intensely within the SMCs of
the fibrous cap (Figures 2D and 3B). Staining was
particularly intense in the extracellular matrix surrounding
macrophage foam cells at the base of the lesions (opened arrows
in Figure 3B compared with 3C and also Figure 6B) and in
the noncellular margin between the lipid core and the fibrous cap. It
is noteworthy that SMCs in the medial layer subjacent to plaque (small
arrows in Figures 3B compared with 3D) were consistently
more intensely positive for TIMP-2 than in more distant areas or in
areas of the same section without plaque (not shown). Levels of both
TIMPs were similar in the adventitia of aortas from normal and
cholesterol-fed rabbits (Figure 2). Each feature of
this pattern of distribution was reproduced in plaques from all animals
studied, and irrespective of the site (arch, mid, or distal) from which
plaques were obtained.
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Detection of Active Gelatinases by In Situ Zymography
Given the parallel increases in expression of TIMP-1 and
TIMP-2 found here and those demonstrated earlier for
gelatinases,7 it was important to investigate, using in
situ zymography, whether enhanced production of TIMPs
counterbalances in part or entirely the proteolytic activity of
gelatinases. Gelatin in situ zymography demonstrated profound
gelatinolytic activity within the
neointima of cholesterol-fed rabbit aortas,
especially at the lumenal surface and in the fibrous cap, areas where
TIMP-2 expression was relatively weak (Figure 4). Gelatinolytic
activity within the deep layers of the lipid core and subjacent media
was clearly reduced, consistent with the high prevalence of
TIMP-2 in these areas. No significant activity was detected in the
media of normal aortas and in normal areas of media in sections with
plaques (not shown). Gelatinolytic activity
detected in the sections from cholesterol-fed rabbit aortas
was almost completely abolished by 20 mmol/L EDTA and the
synthetic MMP inhibitor Ro-31-9790, 100 µm but not
by 1 mmol/L 4-(2-aminoethyl)benzenosulfonyl fluoride
(AEBSF) (a serine protease inhibitor), or 5 mmol/L
N-ethylmaleimide (a cysteine proteases
inhibitor) (results not shown).
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Detection of Caseinolytic Activity by Gel and In Situ
Zymography
Previous studies have shown increased expression of stromelysin in
macrophages derived from atherosclerotic
plaques.3 4 5 We confirmed these data by using casein
zymography of conditioned media from cultured explants (Figure 5). Major bands of activity at 95, 72,
68, 55, 48, and 45 kDa were detected in conditioned media derived from
cholesterol-fed rabbits (Figure 5, lanes 1 and 3).
All the bands were abolished by 20 mmol/L EDTA or 100 µm
Ro-31-9790, but not by 1 mmol/L 4-(2-aminoethyl)benzenosulfonyl
fluoride (AEBSF) or 5 mmol/L N-ethylmaleimide,
confirming that they arose from MMPs. All bands were also abolished by
pretreatment of explants with 100 mmol/L cycloheximide, confirming
that they resulted from de novo synthesis (results not shown). The
ratio of the bands at 55 and 45 kDa was variable (Figure 5, compare lane 1 with lane 3). The 95-, 72-, and 68-kDa bands correspond
to the molecular masses of progelatinase B, progelatinase A, and active
gelatinase A, which also possess ß-caseinolytic
activity.26 Immunoprecipitation of the same
conditioned media with antiserum to human stromelysin-1 (MMP-3)
identified bands at 55 and 45 kDa, consistent with their
identification as the pro- and active forms of stromelysin-1 (results
not shown). The minor caseinolytic activity at 48 kDa was not
identified. To confirm the identity of the 55-kDa band as
prostromelysin-1 we treated conditioned media with aminophenylmercuric
acetate (APMA), which is known to activate pro-forms of MMPs
(Figure 5, lane 4). Treatment with APMA shifted the 55-kDa band
to one at 50 kDa, consistent with the generation of a partially
processed form. APMA did not shift the 45-kDa band, consistent
with its identification as active stromelysin-1. Only a minor 55-kDa
band corresponding to pro-stromelysin was detected in conditioned media
from explants of normal rabbit aortas (Figure 5, lane 2).
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We used in situ casein zymography to evaluate the balance between TIMPs
and stromelysin. No caseinolytic activity was revealed in normal aortas
(not shown) or in the media underlying atherosclerotic plaques (Figure 6A). There were, however, lacunar zones
of lysis in macrophage-rich regions of plaques of a size
corresponding to the dimensions of individual foam cells (Figure 6A compared with 6B), confirming the previously published
results of Galis et al.21 These lysis zones were greatly
reduced by 20 mmol/L EDTA and 100 µm Ro-31-9790, but the
treatment with 1 mmol/L 4-(2-aminoethyl)benzenosulfonyl
fluoride (AEBSF) or 5 mmol/L N-ethylmaleimide
did not change the intensity of lysis (results not shown). The absence
of lysis in the subjacent media and the confinement of lysis to the
pericellular environment of foam cells is consistent with the
location of TIMPs on the extracellular matrix in these regions
(Figure 6B).
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Discussion |
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We demonstrate here that secretion of TIMP-1 and -2 from rabbit aortic explants was increased >2-fold by cholesterol feeding. The increase was significant for the arch and middle portions of cholesterol-fed rabbit aortas, those areas showing greatest development of atherosclerotic lesions. It is a classic finding with cholesterol feeding of rabbits that plaques develop with decreasing severity along the aorta.27 Indeed the intima/media area ratio for the arch (1.1±0.3) and middle (0.8±0.3) was significantly greater than the distal portions (0.3±0.1) of the rabbits used for this study, as previously reported.7 The increases in TIMP-1 and -2 secretion parallel increases in gelatinase7 and stromelysin expression also reported previously5 and suggest that the increased expression of TIMPs restrains the activity of the MMPs. In situ zymography yielded results consistent with this hypothesis.
Our study is the first to quantify relative levels of TIMPs in atherosclerotic tissues. Previous studies in atherosclerotic and normal arteries were based on qualitative evaluation of immunocytochemistry and have yielded contradictory results. Galis et al5 found no difference in a variety of postmortem specimens, whereas Nikkari et al12 found an increase in carotid endarterectomy specimens, consistent with our findings. Knox et al28 found an increase in aortic aneurysms but not aorto-occlusive disease. One technical explanation may be the different affinities of the antibodies used.12 The different pathological specimens examined by the 3 studies may also have contributed. The lesions we examined in cholesterol-fed rabbits are in an active state of evolution and we demonstrated increased secretion of TIMP-1 and -2 in tissues derived from the most severely diseased areas. The clinical endarterectomy specimens studied by Nikkari et al12 were found to contain increased TIMP levels only in the active stage of the disease. It is possible therefore that increased TIMP expression is a characteristic, in general, of actively evolving atherosclerotic plaques. Because the secretion of TIMPs from normal isolated SMCs is constitutive,11 13 recruitment of other cells including macrophages may explain the increased levels of TIMP secretion in atherosclerotic tissues. However, there is a report that TIMP-1 mRNA levels can be increased by inflammatory cytokines in SMCs derived from aortic aneurysms,29 and hence, some of the increase might be the result of induction by cytokines.
Our immunolocalization study confirmed the increased expression of TIMP-1 and -2 in atherosclerotic plaques. In previous studies, TIMPs have been shown to be readily secreted and authors have reported the need to use monensin, an inhibitor of endosomal transport, to localize TIMPs within cells.30 In our study, staining was clearly associated with endothelial cells, SMCs, and macrophages even in the absence of monensin. However, considerable extracellular staining was also observed. This was particularly intense in the cell poor layer that divided the SMC-rich fibrous cap from the macrophage foam cell–rich lipid core. These finding imply that much of the steady-state levels of TIMPs detected in tissues represent TIMPs immobilized in the extracellular matrix. Consistent with this are our recent findings that TIMP-2, in particular, remains associated with the extracellular matrix of human saphenous veins.31
We previously reported that secretion of pro- and active MMP-2
increased 50% and 80%, respectively, in the aortas of
cholesterol-fed rabbits.7 Pro-MMP-9 secretion
also increased from undetectable values to levels that were 5% of
those of pro-MMP-2. The increases in TIMP-1 and 2 levels observed here
are therefore even greater in fold terms than those of gelatinases.
Hence, it was important to establish directly the balance between MMPs
and TIMPs by using in situ zymography. Such studies demonstrated that
gelatinase activity predominated over TIMPs, particularly in the
fibrous cap and more lumenal part of the lipid core. Even the high
levels of TIMP at the boundary between the fibrous cap and the lipid
core were more than overcome by gelatinase activity. However, the
extracellular matrix in the deeper areas of the lipid core and the
subjacent media were apparently protected from gelatinase activity,
consistent with the prevalence of TIMPs in these areas. These
data imply a role for TIMPs in stabilizing the base of the plaque and
preventing medial erosion.
Stromelysin (MMP-3) is able to digest most extracellular matrix
components, including proteoglycans, fibronectins, laminin, and
elastin. It can also activate other MMPs, further shifting the
total balance toward proteolytic activity. Henney et al4
demonstrated increase stromelysin mRNA levels in atherosclerotic plaque
macrophages. Galis et al5 demonstrated, using
endogenous labeling and immunoprecipitation, that
plaque-derived macrophages secrete MMP-3. The same group
demonstrated by immunocytochemistry that stromelysin colocalized with
macrophage-rich areas in human and rabbit atherosclerotic
plaques.5 Our zymography study clearly shows that MMP-3 is
not only secreted but also activated in the aortas from
cholesterol-fed rabbits and can therefore strongly
contribute to the net proteolytic activity. We also demonstrated, by in
situ casein zymography, lacunae of caseinolytic activity within the
macrophage-rich lipid core, similar to findings in
balloon-injured atherosclerotic iliac arteries of
hypercholesterolemic rabbits.21 From
Figure 6, it is evident that activity is confined to the
pericellular environment of foam cells with sparing of the intervening
matrix. Hence, even though the inhibitory activity of TIMPs
does not completely counterbalance stromelysin activity, it restricts
its location. Although both TIMP-1 and -2 can inhibit MMP-3, TIMP-1
binds more rapidly and may therefore be
physiologically more
important.32
In summary, our study shows that significant increases in TIMP-1 and -2 activity occur in atherosclerosis and, even if they do not completely counterbalance increased MMP activity, they significantly restrict it. From the prominent location of TIMPs on the extracellular matrix and the restriction of MMP activity to the pericellular environment, it is clear that TIMPs protect the integrity of the interstitial matrix. This implies a reduction in medial erosion and stabilization of the plaque cap, beneficial adaptations in the context of the early evolving lesions of cholesterol-fed rabbits. Indeed, it is arguable that the primary function of MMPs is to facilitate SMC migration and proliferation and hence promote fibrous cap formation.33 In the later stages of atherosclerosis in humans, however, there is clear evidence implicating MMPs in the processes of plaque destabilization and rupture.34 35 The role of TIMPs in the limitation of proteolytic activity of MMPs within atherosclerotic tissue is in accord with their protective roles in other pathologies, such as rheumatoid arthritis where excessive matrix degradation also occurs.36 Furthermore, it suggests a possible therapeutic role for synthetic MMP inhibitors or TIMP gene therapy.37
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Acknowledgments |
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This research was supported by the British Heart Foundation. We thank Jason L. Johnson for excellent technical help with the preparation of tissue sections.
Received November 2, 1998; accepted December 1, 1998.
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