© 1998 American Heart Association, Inc.
Original Contribution |
Expression of Tissue Inhibitor of Metalloproteinases-3 in Human Atheroma and Regulation in Lesion-Associated Cells
A Potential Protective Mechanism in Plaque Stability
From the Vascular Medicine and Atherosclerosis Unit, Cardiovascular Division, Department of Medicine, Harvard Medical School, Brigham and Women's Hospital, Boston, Mass.
Correspondence to Peter Libby, MD, Vascular Medicine and Atherosclerosis Unit, Cardiovascular Division, Department of Medicine, Harvard Medical School, Brigham and Women's Hospital, 221 Longwood Ave, Boston, MA 02115. E-mail Plibby{at}rics.bwh.harvard.edu
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Abstract—Atherosclerotic plaque stability depends on the structural integrity of its extracellular matrix skeleton. The balance between degradation by matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) may regulate plaque stability. Although MMP expression in atheroma is well documented, localization and control of expression of TIMPs in these lesions is incomplete. Extracts of atheroma (n=14) had 5-fold higher levels of TIMP-3 than nonatherosclerotic tissue (n=10). Plaques (n=24) contained abundant TIMP-1, -2, and -3 in macrophages in plaque shoulders, intimal-medial borders, and areas overlying the lipid core, as well as in medial smooth muscle cells, albeit in lesser amounts. These observations suggested that macrophages, a cell type not heretofore known to express TIMP-3, did so in atheroma in vivo. Further studies in vitro established the human macrophage as a novel source of TIMP-3 mRNA and protein. Human smooth muscle cells constitutively expressed TIMP-1, -2 and -3 proteins; platelet-derived growth factor and transforming growth factor-ß augmented levels of TIMP-1 and TIMP-3 but not TIMP-2. These findings suggest that regulated expression of TIMP-3, in addition to the presence of TIMP-1 and TIMP-2, counteracts MMP activity in atheroma and hence influences plaque stability.
Key Words: tissue inhibitor of metalloproteinases • atherosclerosis • macrophage • smooth muscle cell
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Rupture of atherosclerotic plaques frequently causes myocardial infarction.1 2 The stability of an atherosclerotic plaque largely depends on the structural integrity of its fibrous cap, which is composed mainly of ECM components rich in collagen.3 The deposition of ECM proteins by intimal SMCs contributes to the tensile strength of the atherosclerotic plaque, rendering it resistant to rupture. Thus, dissolution of the ECM by MMPs provides a likely contributing mechanism of plaque rupture.4 In support of this hypothesis, we and others have demonstrated increased expression of MMPs such as collagenases, stromelysins, and gelatinases in atherosclerotic tissue versus normal tissue.5 6 7 8 9 Together, these MMPs can degrade all the ECM components in the plaque.
Endogenous inhibitors known as TIMPs hold MMP activities in check under usual circumstances. The TIMP family consists of TIMP-1,10 TIMP-2,11 TIMP-3,12 and a more recently described TIMP-4.13 TIMP-1 and TIMP-2 are the better-characterized members of the group and are secreted in a soluble form by cultured cells. TIMP-3, however, once secreted, binds to and forms an insoluble complex with constituents of the ECM.14 TIMP-1, -2, and -3 all have similar inhibitory activities against MMPs.15 TIMPs exhibit sequence homology and share domains of identical protein structures consisting of a highly conserved N-terminal region, considered critical for inhibition of the enzymes, and a more diverse C-terminal domain of unknown function. In addition to binding active MMPs, TIMP-1 and TIMP-2 also bind the zymogen forms of MMP-9 and MMP-2, respectively, a process that may contribute to maintaining stability of the enzymes.16 The balance between MMPs and TIMPs critically regulates ECM homeostasis. Excessive MMP activity likely contributes to a number of disease processes, including cancer invasion and metastasis, rheumatoid arthritis,17 and atherosclerosis.
TIMP-1, -2, and -3 can be secreted by SMCs from various species in vitro.18 19 20 Although a number of studies have demonstrated increased expression of MMPs in atherosclerotic tissue, only a few of these studies examined the expression of TIMPs. We have reported the constitutive expression of TIMP-1 and TIMP-2 in both normal and diseased arteries.6 Additionally, Nikkari and coworkers8 demonstrated TIMP-1 expression in diseased arteries, although they were only able to detect weak staining in normal arteries. The presence of TIMP-3 in atherosclerotic arteries in vivo, however, remains undetermined. The present study investigated whether human atheroma contain this ECM-associated form of TIMP, in addition to TIMP-1 and TIMP-2. Moreover, we examined whether growth factors and cytokines that are present in atherosclerotic tissue modulate expression of TIMPs in vitro.
We present evidence herein of increased TIMP-3 expression in atherosclerotic tissue versus normal arteries. In atheroma, TIMP-3 localized mainly with intimal macrophages in areas previously reported to be rich in MMP activities. The present study also demonstrates that fibrogenic mediators such as PDGF and TGF-ß, which are present in atherosclerotic tissue, can increase expression of TIMP-1 and TIMP-3 but not TIMP-2 in human SMCs in vitro.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Materials
Monoclonal anti-human macrophage CD68 antibody was obtained from Dako Corp, polyclonal rabbit anti-human TIMP-3 antibody was purchased from Chemicon International Inc, and polyclonal rabbit anti-human TIMP-1 and TIMP-2 antibodies were obtained from Triple Point Biologics. Human recombinant PDGF-BB and TGF-ß were purchased from Genzyme. Human recombinant IL-1
was a generous gift
from Hoffmann La Roche (Nutley, NJ). PMA was obtained from Sigma
Chemical Co.
Cell Culture
SMCs were prepared from medial explants of human saphenous vein.
Cultures were maintained in Dulbecco's Modified Eagles Medium (DMEM,
Gibco) supplemented with 10% FCS in a humidified chamber of 5%
CO2/95% air. Human aortic SMCs were isolated
enzymatically and cultured as described
previously.21 For stimulation experiments, cells
were switched to serum-free medium consisting of DMEM/F12 (1:1)
supplemented with 1 mmol/L insulin and 5 mg/mL transferrin
serum-free medium.22 After 72 hours, SMCs were
treated with the agent under investigation. Monocytes were isolated
from freshly prepared human peripheral blood mononuclear
cells obtained from plateletpheresis residues of healthy
volunteers (kindly provided by Steve K. Clinton, Dana-Faber Cancer
Institute and obtained by protocols approved by the Human Investigation
Review Committee at the Dana-Faber Cancer Institute) by Ficoll density
gradient centrifugation as previously
described.23 After adherence on plastic tissue
culture flasks (Nunc) for 2 hours in medium 199 containing 2% human
serum, the cells were washed 3 times with RPMI to remove contaminating
lymphocytes and red blood cells. Monocytes were either used immediately
or maintained in medium 199 supplemented with 2% human serum for in
vitro differentiation into macrophages for 7 to 10 days.
Preparation of ECMs and Human Arterial Extracts
Cells were washed twice in PBS and then lysed in 0.5% Triton
X-100 in phosphate buffer (pH 7.4) at room temperature for 10 minutes,
followed by a 10-minute incubation in 100 mmol/L
NH4OH to remove the nuclei and cytoskeleton. The
remaining matrix was rinsed twice in PBS, followed by several washes in
distilled water, air dried, then solubilized in 1x nonreducing Laemmli
sample buffer and stored at -20°C before further
analysis.
Specimens of human carotid atheroma and aorta were obtained by protocols approved by the Human Investigation Review Committee at Brigham and Women's Hospital.
The source of nonatherosclerotic tissue was either the thoracic aorta of transplant donors or carotid arteries of healthy individuals at autopsy. Diseased tissue was from carotid endarterectomy specimens obtained at surgery. All tissues were stored at -70°C. Specimens were ground under liquid nitrogen with a mortar and pestle, and the resulting fine tissue powder was solubilized in buffer containing 20 mmol/L NaCl, 100 mmol/L Tris-HCl, pH 7.6, and 10% SDS. Insoluble material was precipitated by centrifugation, and soluble extracts were collected and stored at -20°C before further analysis.
Gel Electrophoresis
Protein concentrations of arterial extracts were
determined with the bicinchoninic acid kit. Equivalent amounts
of protein (35 µg) were heat denatured in the presence of Laemmli
sample buffer containing 5% to 10% 2-mercaptoethanol and
electrophoresed on 12% to 15% SDS-PAGE gels. Proteins were then
transferred onto polyvinylidene difluoride membranes
(Millipore) in the presence of Towbin transfer buffer (25 mmol/L
Tris, 192 mmol/L glycine, 20% methanol, pH 8.3) by use of a
semidry electrophoretic apparatus according to the
manufacturer's recommendations (Bio-Rad). Membranes were blocked in
TBS-T containing 5% (wt/vol) nonfat dry milk for 1 hour, then
incubated in fresh buffer containing primary antibody at the desired
concentration. Membranes were washed in several changes of TBS-T
followed by incubation with TBS-T/5% dry milk containing a 1:15 000
dilution of horseradish peroxidase–conjugated donkey anti-rabbit IgG
as secondary antibody (Jackson Laboratories). Immunoreactive proteins
were visualized with the enhanced chemiluminescence detection system
(DuPont NEN) and exposure to x-ray film.
Reversed zymography was used to detect TIMP-3 activity in arterial extracts from atherosclerotic tissue as previously described.20
N-Glycosidase F Treatment
Human macrophage–conditioned media and tissue extracts
were dialyzed overnight against buffer containing 20 mmol/L
NaPO4, 0.1% SDS, and 10 mmol/L EDTA.
Duplicate aliquots of sample were denatured in the presence of 1%
2-mercaptoethanol at 90°C for 5 minutes, followed by the addition of
n-octylglucoside to a final concentration of 0.5%.
Samples were subsequently incubated at 37°C overnight either in the
presence or absence of N-glycosidase F (Boehringer
Mannheim). The inhibitors were analyzed by PAGE and
Western blotting with the specified antibody.
Immunohistochemistry
For immunostaining, cryostat sections (6
µm) were cut, air dried, and fixed in acetone at -20°C for 5
minutes. Sections were preincubated for 20 minutes with 0.3% hydrogen
peroxide in Dulbecco's PBS to reduce endogenous peroxidase
activity and with 2.5% of normal serum in PBS for 20 minutes to avoid
nonspecific binding of antibodies. Sections were incubated with primary
antibodies, diluted in PBS with 2.5% appropriate normal serum at room
temperature for 90 minutes. Species-appropriate biotinylated
secondary antibodies (Vector Laboratories) were applied for 45 minutes
at room temperature. After 30 minutes' incubation with avidin-biotin
peroxidase complex (Vectastain ABC kit, Vector Laboratories), staining
was visualized with 3-amino-9-ethyl-carbazole (Sigma Chemical).
Sections were counterstained with Gill's hematoxylin (Sigma
Diagnostics). Staining with type- and class-matched
irrelevant immunoglobulin served as a negative control.
RT-PCR and Northern Blotting
Total cellular RNA was prepared from 100-mm dishes of human
monocytes/macrophages and endarterectomy
tissue specimens with Qiagen Rneasy RNA extraction columns and RNAzol B
(Tel-test, Inc), respectively. RNA was quantified by absorbance at 260
nm, and 1 µg was reverse transcribed with random hexamers and
Superscript II reverse transcriptase according to the manufacturer's
recommendations (Gibco BRL). Identical reactions were also set up in
the absence of reverse transcriptase to eliminate the possibility of
genomic contamination. Subsequent PCR reactions were performed with
specific primers for TIMP-3 as previously
described20 and ß-actin primers as an internal
control (Clontech). Aliquots of the PCR products were
electrophoresed on 2% agarose gels and visualized by ethidium bromide
staining and UV transillumination.
For Northern analysis, 15 µg of total RNA was
fractionated on 1% agarose/2.2 mol/L formaldehyde gels, then
transferred onto nylon membranes (DuPont NEN) by capillary blotting
with 10x SSC (1.5 mol/L sodium chloride and 300 mmol/L sodium
citrate). The RNA was bound by cross-linking the membrane with UV
irradiation. Prehybridization and subsequent overnight hybridization of
the blots were performed at 42°C in buffer containing 40% formamide,
10% dextran sulfate, 4x SSC, 7 mmol/L Tris, pH 7.6, and 0.2
mg/mL salmon-sperm DNA. The probe used for hybridization was a 488-bp
fragment of the TIMP-3 cDNA20 radiolabeled with
[-32P]dATP by random hexamer priming and
Klenow enzyme (Boehringer Mannheim). Posthybridization
of blots was performed in 2x SSC, 0.1% SDS at 50°C, followed by
higher-stringency washes in 1x SSC, 0.1% SDS at 65°C.
Autoradiography of membranes was performed with
intensifying screens at -70°C for 24 to 72 hours.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Human Atheroma Contain Increased Amounts of TIMP-3
TIMP-3 expression was detected at various levels in extracts of all endarterectomy specimens examined, as demonstrated by Western blot analysis (Figure 1A
). Although most samples (12 of 14)
revealed moderate to strong expression, 2 samples (lanes 12 and 13)
contained little or no TIMP-3 protein. By contrast, extracts of normal
arteries exhibited a much weaker signal for TIMP-3 (Figure 1B).
Densitometric scanning revealed an average 5-fold increase of TIMP-3 in
endarterectomy specimens over
nonatheromatous arteries (Figure 1B). In both normal
and nonatherosclerotic tissue, 2 bands for TIMP-3 were observed on
Western blots: a major band at 24 kDa and a minor band at 
27 kDa,
which is within the predicted molecular weight of the glycosylated
species.15 Treatment with
N-glycosidase-F demonstrated that neither the 24-kDa nor the
27-kDa band converted to a lower-molecular-weight species (Figure 2A). This observation suggests that the
27-kDa band does not correspond to glycosylated TIMP-3 but indicates
that the major band at 24 kDa is the unglycosylated form of TIMP-3. The
minor 27-kDa band is also unlikely to represent unsecreted
protein in tissue extracts, because the signal peptide for TIMP-3 has a
predicted molecular weight of only 1 kDa. Human macrophage
TIMP-1 positive control sample, which is a 28-kDa protein in its native
form, yielded the expected 24-kDa deglycosylated product on
treatment with N-glycosidase F (Figure 2B).
|
|
RT-PCR analysis was used to evaluate the presence of TIMP-3
mRNA in the extracted material (Figure 3A). An expected band at 488 bp was
revealed in RNA from samples of advanced human atheroma.
The ß-actin internal control amplified the expected 800-bp
product in all the samples tested. In addition, the control
reactions (lacking reverse transcriptase) yielded the expected negative
results, demonstrating no genomic contamination (not shown).
|
Reverse zymography of protein extracts from plaques in gelatin
polyacrylamide gels revealed a 24-kDa
metalloproteinase-inhibitory activity, demonstrating that
TIMP-3 in atheroma is biologically active in blocking MMP
(Figure 3B
). A higher-molecular-weight band at 28 to 30 kDa and a weak
band at 22 kDa were also observed, corresponding to TIMP-1 and TIMP-2,
respectively (not shown).
Localization of TIMP-3 Within Nonatherosclerotic and
Atherosclerotic Specimens
Immunohistochemistry was performed to investigate further the
location of TIMP-3 expression in atherosclerotic specimens. All plaques
studied were advanced fibrolipid plaques that contained a
well-developed fibrous cap, clusters of macrophages in the
shoulder regions, and a large lipid core. All atherosclerotic specimens
contained TIMP-3. Strong staining was found in macrophages in
plaque shoulders, intimal-medial borders at the base of the plaque, and
areas overlying the necrotic core (Figure 4A, 4C, and 4E). Macrophages were
identified by use of an antibody against the macrophage
specific antigen CD68 (Figure 4B, 4D, and 4E). In addition, positive
staining was observed in the necrotic core (Figure 4A). TIMP-3 was also
found in SMCs within the media and the fibrous cap, albeit in lesser
amounts than macrophages. Staining of nonatherosclerotic
arteries (3 carotid arteries and 8 thoracic aortas) revealed little to
moderate staining of TIMP-3 in smooth muscle cells in the media (Figure 4G and 4H). Nonimmune IgG negative controls yielded no immunoreactivity
in either normal or atherosclerotic tissues (data not shown).
|
Uniform staining of TIMP-1 and TIMP-2 was found in the media and intima
of normal carotid arteries, consistent with our previous
findings (Figure 5A and 5B).6 The distribution pattern of TIMP-1 and
TIMP-2 in atherosclerotic tissue resembled that of TIMP-3; staining was
localized to plaque shoulders and macrophages overlying the
necrotic core (Figure 5, C, D, and E). In addition, staining for TIMP-1
was also found in the necrotic core (Figure 5C), in agreement with the
data of Nikkari and coworkers.8 Nonimmune IgG
negative controls yielded no staining in either normal or
atherosclerotic tissues (Figure 5F).
|
Human Macrophages but Not Monocytes Produce TIMP-3 In
Vitro
TIMP-3 colocalized mainly with macrophages in the
atherosclerotic plaque. Because no information exists on TIMP-3
expression by macrophages, we investigated whether isolated
human monocyte-derived macrophages could produce this
inhibitor. After 7 to 10 days in culture, Western blot
analysis of macrophage ECM extracts revealed a major
band at 24 kDa with TIMP-3 antibody (Figure 6A). RT-PCR analysis of
macrophages showed TIMP-3 product that comigrated with that
produced by PDGF-stimulated SMCs (Figure 6B). Northern analysis
revealed that the TIMP-3 probe hybridized to the expected transcripts
of 5, 2.8, and 1.1 kb, further demonstrating the expression of
TIMP-3 by macrophages (Figure 6C). Interestingly, freshly
isolated monocytes did not express any TIMP-3 RNA (Figure 6B). As
expected, macrophage-conditioned media contained no TIMP-3, and
little TIMP-3 was found in cell lysates. The ß-actin internal control
amplified the expected 800-bp product in all samples tested. In
addition, control reactions (minus reverse transcriptase) yielded the
expected negative results, demonstrating no genomic contamination. We
are unaware of any previous description of expression of TIMP-3 by
human macrophages. Large amounts of TIMP-1 and TIMP-2 were
found in media conditioned by macrophages, consistent
with previous reports (data not shown).
|
Cytokines and Growth Factors Regulate the Synthesis of
TIMP-1 and TIMP-3 but Not TIMP-2 in Human SMCs
Expression of TIMP-1 and TIMP-3 undergoes regulation in a number
of cell types, whereas most cells express TIMP-2 constitutively with
little variation. Growth factors such as PDGF and TGF-ß can induce
TIMP-3 in rabbit SMCs.20 To investigate further
the in vitro regulation of TIMP-1, -2, and -3 in human SMCs, cells were
exposed to various cytokines and growth factors, either alone
or in combination. Western blot analysis revealed that human
aortic SMCs express all 3 TIMP proteins constitutively (Figure 7). However, PDGF and the phorbol ester
PMA increased expression of TIMP-1 (Figure 7A), whereas PDGF and
TGF-ß further increased TIMP-3 levels (Figure 7C). TIMP-2 levels were
unaffected by the addition of exogenous stimuli (Figure 7B). An
identical pattern of regulation was observed in human saphenous vein
SMCs (not shown).
|
To ascertain whether the increase in TIMP-1 and TIMP-3 by growth
factors resulted from an increase in cell number, SMCs were dispersed
by trypsin treatment from triplicate wells of a 6-well plate and
counted with a hemocytometer (data not shown). The treatments did not
change cell number. In addition, secretion of the constitutive protein
TIMP-2 did not change, a further indication that a change in overall
protein synthesis accounts for the effect of PDGF and TGF-ß on TIMP-3
production (Figure 7B).
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Our studies demonstrate augmented TIMP-3 expression in atherosclerotic tissue compared with uninvolved arteries. Twelve of the 14 plaque samples under investigation revealed moderate to strong expression of TIMP-3, whereas 2 samples revealed little to no expression. The variation in expression levels observed is probably a reflection of the heterogeneity of human atheroma. Because the expression of TIMP-3 occurs primarily in macrophages in the plaque, it is likely that specimens from acellular plaques with fewer macrophages would have less expression of TIMP-3. However, no evident descriptor of plaque morphology distinguished the 2 specimens with low TIMP-3 levels in our sample. Further investigation by immunohistochemistry on advanced fibrolipid plaques revealed abundant expression in macrophages at rupture-prone sites of the plaque. Both medial and intimal SMCs contained lesser amounts of TIMP-3. TIMP-1 and TIMP-2 were found in nearly identical areas of the plaque to TIMP-3. Curiously, the spatial distribution of all 3 TIMPs within atheroma correlates with that previously reported for MMP expression. For example, interstitial collagenase (MMP-1), gelatinase B (MMP-9), and stromelysin-1 (MMP-3) are expressed in the fibrous cap, lesion shoulders, and the base of the lipid core of the lesion,5 6 8 and matrilysin (MMP-7) is predominantly expressed in macrophages overlying the lipid core.9 The present study localized TIMP-1, -2, and -3 in all these areas, particularly in macrophages in plaque shoulders and between the fibrous cap and necrotic core. The close proximity of enzyme and inhibitor in the plaque agrees with the hypothesis that TIMPs serve as an important counterregulatory mechanism limiting excessive MMP activity in vivo.
We also demonstrated that human monocyte-derived macrophages in vitro express TIMP-3 RNA and protein, establishing the macrophage as a novel source of TIMP-3. Moreover, the differentiation of monocytes into macrophages in culture involves induction of TIMP-3, as previously reported for MMPs.24 The present study further demonstrates that intimal and medial SMCs in normal and atherosclerotic tissue in vivo contain TIMP-3. Moreover, mediators such as PDGF and TGF-ß present in atherosclerotic tissue increased TIMP-3 expression in both human saphenous vein and arterial SMCs. Taken together, our in vitro findings suggest possible mechanisms for induction of TIMP-3 in macrophages and SMCs in vivo.
Additionally, we found constitutive expression of TIMP-1 and TIMP-2 in SMCs, which was unaffected by cytokines such as IL-1 and tumor necrosis factor (data for tumor necrosis factor not shown), consistent with previous reports.18 However, the present study found that PDGF increased TIMP-1 expression in SMCs. Together with the findings for TIMP-3, this result demonstrates that a growth factor but not the inflammatory cytokine tested can modulate production of TIMPS in SMCs. Furthermore, we demonstrate that regulation of TIMP expression is identical in human saphenous vein SMCs and human arterial SMCs in culture.
Elevated levels of MMPs may regulate SMC migration after balloon injury of the rat and pig carotid artery.25 26 27 More recently, Forough and coworkers28 demonstrated a reduction in intimal thickening after local retrovirus-mediated gene transfer of rat SMCs overexpressing TIMP-1 into balloon-injured rat carotid arteries. In addition, arterial injury in rats increases expression of TIMP-2, as well as plasminogen activator inhibitor type 1.29 These various studies suggest an important counterregulatory role for TIMP-1 and TIMP-2 in the arterial response to injury.
As mentioned above, unlike TIMP-1 and TIMP-2, secreted TIMP-3 is unique in that it is bound to components of the ECM.14 The present study confirmed this finding in SMCs and macrophages in vitro. Although it is well known that SMCs synthesize abundant ECM proteins, little is known about the ability of macrophages to produce an ECM. A recent report demonstrated that macrophages can produce an ECM layer consisting mainly of chondroitin sulfate and also heparan sulfate and dermatan sulfate. These glycosaminoglycans were shown to be involved in the binding of oxidized LDL to the ECM.30 It is possible that these glycosaminoglycans can also account for the retention of TIMP-3 in macrophage ECM. Using high-power light microscopy to visualize immunoreactive areas of TIMP-3, we could not determine with certainty whether TIMP-3 was present extracellularly in vivo. However, it is very likely that TIMP-3, once secreted by plaque cells, is sequestered within the collagenous matrix of the fibrous cap, where it can serve as a long-term inhibitory pool, resisting local proteolysis. In this manner, the matrix can serve as an active protector in lesions of fibrous character. It is unknown which of the ECM components bind TIMP-3. Advanced atherosclerotic lesions contain abundant collagen, proteoglycans, and elastin. Heparan sulfate proteoglycans can bind molecules such as fibroblast growth factor, lipoprotein lipase, and antithrombin III through specific interactions with its oligosaccharide side chains.31 Vitronectin binds another proteinase inhibitor, type 1 plasminogen activator inhibitor.32 A similar mechanism may be used for TIMP-3 binding to the matrix.
The present results suggest that increased TIMP-3, in addition to the presence of TIMP-1 and TIMP-2 in rupture-prone sites of the atherosclerotic plaque, may serve as an important protective mechanism against plaque rupture. In addition, augmentation of TIMP-1 and TIMP-3 by PDGF and TGF-ß present in atherosclerotic plaques is likely to favor decreased local proteolysis. Thus, the local balance between mediators that augment MMP expression (eg, proinflammatory cytokines) and those that also increase TIMP expression (eg, the fibrogenic mediators TGF-ß and PDGF) may help determine the "vulnerability" of a given atheroma. This model aids the understanding of the considerable variability in the lesions of atherosclerosis, even within a single coronary arterial tree.
|
Selected Abbreviations and Acronyms |
|---|
|
|
Acknowledgments |
|---|
This study was supported by Specialized Centers of Research (SCOR) grant HL-56985. We thank Eugenia Shvartz and Marysia Muszynski (Brigham and Women's Hospital, Boston, Mass) for expert technical assistance and Dr Andrew Baker (Bristol Heart Institute, University of Bristol, UK) for helpful discussions.
|
Footnotes |
|---|
This manuscript was sent to Valentin Fuster, Consulting Editor, for review by expert referees, editorial decision, and final disposition.
Received July 8, 1997; accepted April 13, 1998.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1. Davies MJ, Thomas AC. Plaque fissuring: the cause of acute myocardial infarction, sudden ischemic death, and crescendo angina. Br Heart J. 1985;53:363–373.
2. Fuster V, Badimon I, Badimon J, Chesebro J. The pathogenesis of coronary artery disease and the acute coronary syndromes. N Engl J Med. 1992;326:242–250.[Medline] [Order article via Infotrieve]
3. Richardson P, Davies M, Born G. Influence of plaque configuration and stress distribution on fissuring of coronary atherosclerotic plaques. Lancet. 1989;2:941–944.[Medline] [Order article via Infotrieve]
4.
Libby P. Molecular bases of the acute coronary
syndromes. Circulation. 1995;91:2844–2850.
5.
Henney AM, Wakeley PR, Davies MJ, Foster K, Hembry R,
Murphy G, Humphries S. Localization of stromelysin gene expression in
atherosclerotic plaques by in situ hybridization. Proc
Natl Acad Sci U S A. 1991;88:8154–8158.
6. Galis ZS, Sukhova GK, Lark MW, Libby P. Increased expression of matrix metalloproteinases and matrix degrading activity in vulnerable regions of human atherosclerotic plaques. J Clin Invest. 1994;94:2493–2503.
7.
Brown DL, Hibbs MS, Kearney M, Loushin C, Isner J.
Identification of 92-kD gelatinase in human coronary
atherosclerotic lesions: association of active enzyme synthesis with
unstable angina. Circulation. 1995;91:2125–2131.
8. Nikkari S, Geary R, Hatsukami T, Ferguson M, Forough R, Alpers C, Clowes A. Expression of collagen, interstitial collagenase, and tissue inhibitor of metalloproteinases-1 in restenosis after carotid endarterectomy. Am J Pathol. 1996;148:777–783.[Abstract]
9. Halpert I, Sires U, Roby J, Potter-Perigo S, Wight T, Shapiro S, Welgus H, Wickline S, Parks W. Matrilysin is expressed by lipid-laden macrophages at sites of potential rupture in atherosclerotic lesions and localizes to areas of versican deposition, a proteoglycan substrate for the enzyme. Proc Natl Acad Sci U S A. 1996;18:9748–9753.
10. Docherty AJP, Lyons A, Smith BJ, Wright EM, Stephens PE, Harris TJR, Murphy G, Reynolds JJ. Sequence of human tissue inhibitor of metalloproteinases and its identity to erythroid-potentiating activity. Nature. 1985;318:66–69.[Medline] [Order article via Infotrieve]
11.
Stetler-Stevenson WG, Brown PD, Onisto M, Levy AT,
Liotta LA. Tissue inhibitor of metalloproteinases-2
(TIMP-2) mRNA expression in tumor cell lines and human tumor tissues.
J Biol Chem. 1990;265:13933–13938.
12.
Uría JA, Ferrando AA, Velasco G, Freije JMP,
López-Otín C. Structure and expression in breast tumors
of human TIMP-3, a new member of the metalloproteinase
inhibitor family. Cancer Res. 1994;54:2091–2094.
13.
Greene J, Wang M, Liu Y, Raymond L, Rosen C, Ye S.
Molecular cloning and characterization of human tissue
inhibitor of metalloproteinase 4. J Biol
Chem. 1996;271:30375–30380.
14.
Pavloff N, Staskus PW, Kishnani NS, Hawkes SP. A new
inhibitor of metalloproteinases from chicken: ChIMP-3, a
third member of the TIMP family. J Biol Chem. 1992;267:17321–17326.
15.
Apte S, Olsen B, Murphy G. The gene structure of Tissue
Inhibitor of Metalloproteinases (TIMP)-3 and its
inhibitory activities define the distinct TIMP gene family.
J Biol Chem. 1995;270:14313–14318.
16. Willenbrock F, Murphy G. Structure-function relationships in the tissue inhibitors of metalloproteinases. Am J Respir Crit Care Med.. 1994;150:S165–S170.
17.
Birkedal-Hansen H, Moore WGI, Bodden MK, Windsor LJ,
Birkedal-Hansen B, DeCarlo A, Engler JA. Matrix
metalloproteinases: a review. Crit Rev Oral Biol Med. 1993;4:197–250.
18.
Galis ZS, Muszynski M, Sukhova GK, Simon-Morrissey E,
Unemori EN, Lark MW, Amento E, Libby P. Cytokine-stimulated
human vascular smooth muscle cells synthesize a complement of enzymes
required for extracellular matrix digestion. Circ Res. 1994;75:181–189.
19. Kenagy RD, Nikkari ST, Welgus HG, Clowes AW. Heparin inhibits the induction of three matrix metalloproteinases (stromelysin, 92-kD gelatinase and collagenase) in primate arterial smooth muscle cells. J Clin Invest. 1994;93:1987–1993.
20. Fabunmi R, Baker A, Murray E, Booth R, Newby A. Divergent regulation by growth factors and cytokines of 95-kDa and 72-Da gelatinases and tissue inhibitors of metalloproteinases-1, -2 and -3 in rabbit aortic smooth muscle cells. Biochem J. 1996;315:335–342.
21.
Gunther S, Alexander RW, Atkinson WJ, Gimbrone MA Jr.
Functional angiotensin II receptors in cultured vascular
smooth muscle. J Cell Biol. 1982;92:289–298.
22. Libby P, O'Brien K. Culture of quiescent arterial smooth muscle cells in a defined serum-free medium. J Cell Physiol. 1983;115:217–223.[Medline] [Order article via Infotrieve]
23.
Kranzhofer R, Clinton S, Ishii K, Coughlin S, Fenton
JPL. Thrombin potently stimulates cytokine production
in human vascular smooth muscle cells but not in mononuclear
phagocytes. Circ Res. 1996;79:286–294.
24. Welgus H, Campbell E, Cury J, Eisen A, Senior R, Wilhelm S, Goldberg G. Neutral metalloproteinases produced by human mononuclear phagocytes: enzyme profile, regulation, and expression during cellular development. J Clin Invest. 1990;86:1496–1502.
25.
Bendeck MP, Zempo N, Clowes AW, Galardy RE, Reidy MA.
Smooth muscle cell migration and matrix metalloproteinase expression
after arterial injury in the rat. Circ Res. 1994;75:539–545.
26. Zempo N, Kenagy RD, Au T, Bendeck M, Clowes MM, Reidy MA, Clowes AW. Matrix metalloproteinases of vascular wall cells are increased in balloon-injured rat carotid. J Vasc Surg. 1994;20:209–217.[Medline] [Order article via Infotrieve]
27.
Southgate KM, Fisher M, Banning AP, Thurston VJ, Baker
AH, Fabunmi RP, Groves PH, Davies M, Newby AC. Upregulation of basement
membrane degrading metalloproteinase secretion after balloon injury of
pig carotid arteries. Circ Res. 1996;79:1177–1187.
28.
Forough R, Koyama N, Hasenstab D, Lea H, Clowes M,
Nikkari S, Clowes A. Overexpression of tissue inhibitor of
matrix metalloproteinase-1 inhibits vascular smooth muscle cell
functions in vitro and in vivo. Circ Res. 1996;79:812–820.
29.
Hasenstab D, Forough R, Clowes A.
Plasminogen activator inhibitor
type 1 and tissue inhibitor of metalloproteinases-2
increase after arterial injury in rats. Circ
Res. 1997;80:490–496.
30. Kaplan M, Aviram M. Oxidized LDL binding to a macrophage-secreted extracellular matrix. Biochem Biophys Res Commun. 1997; 237:271–276.
31.
Stary H, Chandler A, Dinsmore R, Fuster V, Glagov S,
Insull W, Rosenfeld M, Schwartz C, Wagner W, Wissler R. A definition of
advanced types of atherosclerotic lesions and a
histological classification of
atherosclerosis: a report from the Committee on
Vascular Lesions of the Council on
Arteriosclerosis, American Heart Association.
Circulation. 1995;92:1355–1374.
32. Seiffert D, Mimuro J, Schleef R, Luskutoff D. Interactions between type I plasminogen activator inhibitor, extracellular matrix and vitronectin. Cell Differ Dev. 1990;32:287–292.[Medline] [Order article via Infotrieve]
This article has been cited by other articles:
 |
|
 |
|
  M. Cardellini, R. Menghini, E. Martelli, V. Casagrande, A. Marino, S. Rizza, O. Porzio, A. Mauriello, A. Solini, A. Ippoliti, et al. TIMP3 Is Reduced in Atherosclerotic Plaques From Subjects With Type 2 Diabetes and Increased by SirT1 Diabetes, October 1, 2009; 58(10): 2396 - 2401. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. C. Newby Metalloproteinase Expression in Monocytes and Macrophages and its Relationship to Atherosclerotic Plaque Instability Arterioscler Thromb Vasc Biol, December 1, 2008; 28(12): 2108 - 2114. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. L. Johnson, G. B. Sala-Newby, Y. Ismail, C. M. Aguilera, and A. C. Newby Low Tissue Inhibitor of Metalloproteinases 3 and High Matrix Metalloproteinase 14 Levels Defines a Subpopulation of Highly Invasive Foam-Cell Macrophages Arterioscler Thromb Vasc Biol, September 1, 2008; 28(9): 1647 - 1653. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. J. West, P. J. Nestel, A. C. Kirby, R. Schnabel, D. Sullivan, R. J. Simes, C. Pollicino, E. Lubos, T. F. Munzel, H. D. White, et al. The value of N-terminal fragment of brain natriuretic peptide and tissue inhibitor of metalloproteinase-1 levels as predictors of cardiovascular outcome in the LIPID study Eur. Heart J., April 1, 2008; 29(7): 923 - 931. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. D. Barish, A. R. Atkins, M. Downes, P. Olson, L.-W. Chong, M. Nelson, Y. Zou, H. Hwang, H. Kang, L. Curtiss, et al. PPAR{delta} regulates multiple proinflammatory pathways to suppress atherosclerosis PNAS, March 18, 2008; 105(11): 4271 - 4276. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. E. Vendrov, N. R. Madamanchi, Z. S. Hakim, M. Rojas, and M. S. Runge Thrombin and NAD(P)H Oxidase-Mediated Regulation of CD44 and BMP4-Id Pathway in VSMC, Restenosis, and Atherosclerosis Circ. Res., May 26, 2006; 98(10): 1254 - 1263. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. L. Johnson, A. H. Baker, K. Oka, L. Chan, A. C. Newby, C. L. Jackson, and S. J. George Suppression of Atherosclerotic Plaque Progression and Instability by Tissue Inhibitor of Metalloproteinase-2: Involvement of Macrophage Migration and Apoptosis Circulation, May 23, 2006; 113(20): 2435 - 2444. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Liang, E. Liu, Y. Yu, S. Kitajima, T. Koike, Y. Jin, M. Morimoto, K. Hatakeyama, Y. Asada, T. Watanabe, et al. Macrophage Metalloelastase Accelerates the Progression of Atherosclerosis in Transgenic Rabbits Circulation, April 25, 2006; 113(16): 1993 - 2001. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Chen, Y. H. Shen, X. Wang, J. Wang, Y. Gan, N. Chen, J. Wang, S. A. LeMaire, J. S. Coselli, and X. L. Wang Human Prolyl-4-hydroxylase {alpha}(I) Transcription Is Mediated by Upstream Stimulatory Factors J. Biol. Chem., April 21, 2006; 281(16): 10849 - 10855. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Janssens and H. R. Lijnen What has been learned about the cardiovascular effects of matrix metalloproteinases from mouse models? Cardiovasc Res, February 15, 2006; 69(3): 585 - 594. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. C. Newby Matrix metalloproteinases regulate migration, proliferation, and death of vascular smooth muscle cells by degrading matrix and non-matrix substrates Cardiovasc Res, February 15, 2006; 69(3): 614 - 624. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. M. Dollery and P. Libby Atherosclerosis and proteinase activation Cardiovasc Res, February 15, 2006; 69(3): 625 - 635. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. K. Sukhova, B. Wang, P. Libby, J.-H. Pan, Y. Zhang, A. Grubb, K. Fang, H. A. Chapman, and G.-P. Shi Cystatin C Deficiency Increases Elastic Lamina Degradation and Aortic Dilatation in Apolipoprotein E-Null Mice Circ. Res., February 18, 2005; 96(3): 368 - 375. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. C. Newby Dual Role of Matrix Metalloproteinases (Matrixins) in Intimal Thickening and Atherosclerotic Plaque Rupture Physiol Rev, January 1, 2005; 85(1): 1 - 31. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Liu, G. K. Sukhova, J.-S. Sun, W.-H. Xu, P. Libby, and G.-P. Shi Lysosomal Cysteine Proteases in Atherosclerosis Arterioscler Thromb Vasc Biol, August 1, 2004; 24(8): 1359 - 1366. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. K. Owens, M. S. Kumar, and B. R. Wamhoff Molecular Regulation of Vascular Smooth Muscle Cell Differentiation in Development and Disease Physiol Rev, July 1, 2004; 84(3): 767 - 801. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. M. Dollery, C. A. Owen, G. K. Sukhova, A. Krettek, S. D. Shapiro, and P. Libby Neutrophil Elastase in Human Atherosclerotic Plaques: Production by Macrophages Circulation, June 10, 2003; 107(22): 2829 - 2836. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z. Luan, A. J. Chase, and A. C. Newby Statins Inhibit Secretion of Metalloproteinases-1, -2, -3, and -9 From Vascular Smooth Muscle Cells and Macrophages Arterioscler Thromb Vasc Biol, May 1, 2003; 23(5): 769 - 775. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. Lemaitre, P. D. Soloway, and J. D'Armiento Increased Medial Degradation With Pseudo-Aneurysm Formation in Apolipoprotein E-Knockout Mice Deficient in Tissue Inhibitor of Metalloproteinases-1 Circulation, January 21, 2003; 107(2): 333 - 338. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Silence, D. Collen, and H.R. Lijnen Reduced Atherosclerotic Plaque but Enhanced Aneurysm Formation in Mice With Inactivation of the Tissue Inhibitor of Metalloproteinase-1 (TIMP-1) Gene Circ. Res., May 3, 2002; 90(8): 897 - 903. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. Loftus and M. Thompson The role of matrix metalloproteinases in vascular disease Vascular Medicine, May 1, 2002; 7(2): 117 - 133. [Abstract] [PDF]
|
|
|
|
 |
|
 J. L. Hunt, R. Fairman, M. E. Mitchell, J. P. Carpenter, M. Golden, T. Khalapyan, M. Wolfe, D. Neschis, R. Milner, B. Scoll, et al. Bone Formation in Carotid Plaques: A Clinicopathological Study Stroke, May 1, 2002; 33(5): 1214 - 1219. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Bond, G. Murphy, M. R. Bennett, A. C. Newby, and A. H. Baker Tissue Inhibitor of Metalloproteinase-3 Induces a Fas-associated Death Domain-dependent Type II Apoptotic Pathway J. Biol. Chem., April 12, 2002; 277(16): 13787 - 13795. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Silence, F. Lupu, D. Collen, and H. R. Lijnen Persistence of Atherosclerotic Plaque but Reduced Aneurysm Formation in Mice With Stromelysin-1 (MMP-3) Gene Inactivation Arterioscler Thromb Vasc Biol, September 1, 2001; 21(9): 1440 - 1445. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. E.J.M. Creemers, J. P.M. Cleutjens, J. F.M. Smits, and M. J.A.P. Daemen Matrix Metalloproteinase Inhibition After Myocardial Infarction: A New Approach to Prevent Heart Failure? Circ. Res., August 3, 2001; 89(3): 201 - 210. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. D. Kolodgie, J. Narula, A. P. Burke, N. Haider, A. Farb, Y. Hui-Liang, J. Smialek, and R. Virmani Localization of Apoptotic Macrophages at the Site of Plaque Rupture in Sudden Coronary Death Am. J. Pathol., October 1, 2000; 157(4): 1259 - 1268. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Saitoh, H. Kishida, Y. Tsukada, Y. Fukuma, J. Sano, M. Yasutake, N. Fukuma, Y. Kusama, and H. Hayakawa Clinical significance of increased plasma concentration of macrophage colony-stimulating factor in patients with angina pectoris J. Am. Coll. Cardiol., March 1, 2000; 35(3): 655 - 665. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. J. Pinderski Oslund, C. C. Hedrick, T. Olvera, A. Hagenbaugh, M. Territo, J. A. Berliner, and A. I. Fyfe Interleukin-10 Blocks Atherosclerotic Events In Vitro and In Vivo Arterioscler Thromb Vasc Biol, December 1, 1999; 19(12): 2847 - 2853. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Silence, D. Collen, and H.R. Lijnen Reduced Atherosclerotic Plaque but Enhanced Aneurysm Formation in Mice With Inactivation of the Tissue Inhibitor of Metalloproteinase-1 (TIMP-1) Gene Circ. Res., May 3, 2002; 90(8): 897 - 903. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||