© 1997 American Heart Association, Inc.
Articles |
Thrombin Promotes Activation of Matrix Metalloproteinase-2 Produced by Cultured Vascular Smooth Muscle Cells
From the Vascular Medicine and Atherosclerosis Unit, Brigham and Women's Hospital, Boston, Mass (R.K., P.L.); Emory University School of Medicine, Cardiology Division, Atlanta, Ga (Z.S.G.); and the Wadsworth Center for Laboratories and Research, New York State Department of Health, Albany (J.W.F.).
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Abstract |
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Abstract Thrombin generated at sites of vascular injury not only participates in the coagulation cascade but can signal other events related to development and complication of atherosclerotic plaques. We investigated here a novel nonthrombotic action of thrombin: the possibility that this protease influences the expression or activation of matrix metalloproteinases (MMPs) produced by vascular smooth muscle cells (SMCs). Matrix-degrading proteinases likely contribute to several aspects of vascular lesion development. Vascular SMCs constitutively elaborate the zymogen form of gelatinase A (MMP-2), found in cell supernatants complexed with its inhibitor, the tissue inhibitor of metalloproteinases (TIMP)-2. When activated, MMP-2 digests collagens and elastin and may thus promote cell migration and vascular remodeling. Analysis of culture supernatants harvested from either human or rabbit vascular SMCs by gelatin zymography revealed that compared with supernatants of unstimulated SMCs, media conditioned by thrombin-stimulated cells contained increased amounts of proteolytically processed MMP-2, suggesting activation of this MMP. Further experiments tested whether thrombin directly activates MMP-2. In cell-free experiments, when added to medium harvested from unstimulated SMCs,
-thrombin increased in a dose- and time-dependent
manner the amount of proteolytically processed MMP-2, as shown by
zymography and by Western blotting with specific antibodies. Thrombin
cleaved pro–MMP-2 within 4 hours, even when the gelatinase was bound
with its inhibitor, TIMP-2. Thrombin treatment rendered culture media
of unstimulated SMCs able to degrade collagen type IV, consistent with
generation of active MMP-2. Addition of inhibitors of either thrombin
or MMPs decreased this type IV collagenolytic activity, but thrombin in
the absence of SMC-conditioned medium containing pro–MMP-2 exhibited
only minimal collagenolysis. Our results suggest that at sites of
vascular injury, thrombin may activate locally produced MMP-2 and
thereby facilitate cell migration and proliferation. In the case of
complicated atherosclerotic plaques, episodes of intraplaque hemorrhage
or plaque disruption with thrombosis may promote plaque instability by
increasing local matrix-degrading activity.
Key Words: thrombin • metalloproteinases • atherosclerosis • vascular remodeling • tissue repair
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Introduction |
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The MMPs, enzymes specialized in degradation of extracellular matrix, probably participate in extracellular matrix remodeling during tissue development, inflammation, and neoplasia.1 2 3 Cells secrete most known MMPs as latent zymogens that require processing leading to cleavage of their prodomain4 to become active. This activation step therefore plays a key role in the regulation of matrix degradation. Several proteases can process pro-MMPs. Plasmin may initiate a proteolytic cascade through activation of latent interstitial collagenase (pro–MMP-1) and stromelysin (pro–MMP-3).5 The broad spectrum of stromelysin's substrates includes a number of extracellular matrix components6 and zymogen forms of other members of the MMP family, such as gelatinase B (pro–MMP-9)7 and pro–MMP-1.8 9 Previous studies (summarized by Stetler-Stevenson et al10 ) regarding the capacity of plasmin and plasminogen activators to activate pro–MMP-2 have produced contradictory results. In contrast to most other MMPs, activation of pro–MMP-2 appears cell dependent.11 A newly characterized membrane-bound metalloenzyme that can activate pro–MMP-2 may serve this function.12
Recent observations have highlighted the potential contribution of MMPs produced by vascular cells13 14 15 16 in human atherogenesis,17 18 as well as in the development of arterial lesions after experimental balloon injury.19 20 Cytokines, protein mediators of inflammation, can augment MMP expression by cultured SMCs.16 By this mechanism, locally acting cytokines may promote matrix remodeling in atherosclerotic lesions. Other pathophysiologically relevant factors might also influence MMP production. For example, thrombin, in addition to its place in the coagulation cascade,21 exerts numerous other important effects on vascular cells.22 This study explored whether thrombin might also influence MMPs thought to be important in vascular biology. In particular, we studied whether thrombin induces MMP expression by SMCs, and whether this protease might activate pro–MMPs elaborated by these cells.
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Methods |
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Cell Culture
SMCs were grown from explants of either human saphenous veins obtained at bypass surgery or of rabbit aorta, in accordance with protocols approved by the Standing Committee on Animals of Harvard Medical Area. Cells from passages 2 through 5 were grown to confluence in DMEM (BioWhittaker) containing penicillin (100 U/mL), streptomycin (100 µg/mL), and amphotericin B (1.25 µg/mL) and supplemented with 5% fetal calf serum. Confluent SMCs were washed twice with Hanks' balanced salt solution, kept in serum-free medium (DMEM/F12, 1:1) supplemented with 1 mmol/L insulin and 5 µg/mL transferrin23 for 24 hours, and then incubated with fresh serum-free medium containing 0.1% pyrogen-free human serum albumin with or without addition of
-thrombin (specific activity 2687 U/mg,
final concentration 10-9 to
10-5 mol/L). For some experiments, human SMCs
were stimulated with 10 ng/mL IL-1 (Hoffmann-LaRoche).
SDS-PAGE Zymography
Culture media harvested from SMCs were analyzed for proteins
with gelatinolytic or caseinolytic activity by identification of
substrate lysis in discontinuous 10% SDS–polyacrylamide gels
containing 1 mg/mL gelatin or casein. Gels were processed to remove SDS
(two changes of 2.5% Triton X-100 for a total of 30 minutes),
incubated for 18 hours at 37°C in 50 mmol/L Tris-HCl, pH 7.4,
containing 10 mmol/L CaCl and 0.05% Brij 35, and stained with
Colloidal brilliant blue (Sigma Chemical Co). Migration of proteins
with enzymatic activity was compared with that of prestained low-MW
range markers (Bio-Rad). To compare intensity of lytic bands within a
stained gel, we scanned gel photographs by using a densitometer and
quantified lytic bands by using the National Institutes of Health IMAGE
program, version 1.55.
Cell-Free Experiments
In these experiments, culture media harvested from SMCs were
incubated with thrombin for up to 24 hours. The effect of thrombin on
proteins with gelatinolytic or caseinolytic activity released by SMCs
was assessed by SDS-PAGE zymography. Processing of pro–MMP-2 was
evaluated by immunoblotting and fluorography. Enzymatic activity of
culture media on radiolabeled collagen type IV was assayed in the
presence or absence of thrombin. Similar experiments using
SMC-conditioned media were conducted to test the effect of two other
serine proteases, plasmin (Sigma Chemical Co) and trypsin
(Worthington).
Enzymatic Assay
Aliquots (50-100 µL) of culture media harvested from 90-mm
Petri dishes of confluent, unstimulated human or rabbit SMCs were
incubated at 37°C for 18 hours with 2 µg [3H]collagen
type IV (specific activity 0.14 mCi/mg, DuPont-NEN), with or without
addition of one of the serine proteases. Enzymatic activity in the
presence of PMSF, a general serine protease inhibitor; hirudin, a
specific inhibitor of thrombin; or o-phenanthroline, an inhibitor
of MMPs, was also investigated. Degradation of collagen IV by exogenous
serine proteases alone and possible nonenzymatic degradation at 37°C
were also assessed. The reactions were stopped by addition of reducing
SDS-PAGE sample buffer and boiling for 10 minutes. Samples were then
loaded on 10% SDS–polyacrylamide gels. Degradation of radiolabeled
collagen was assessed by fluorography of gels dried after impregnation
with EN3HANCE (DuPont-NEN) and by liquid scintillation
spectroscopy of gel slices.
Immunoprecipitation
Metabolic labeling employed
Expre35S35S protein labeling mix
(DuPont-NEN).16 Newly synthesized pro–MMP-2 and TIMP-2
were immunoprecipitated from 35S-labeled culture media by
using rabbit polyclonal antibodies generously provided by Dr William
Stetler-Stevenson (NIH, Bethesda, Md) and protein A–Sepharose (Sigma
Chemical Co).16 Immunoprecipitates were incubated
overnight with or without addition of thrombin
(10-6 to 10-7 mol/L),
and proteins were analyzed by fluorography (under reducing conditions,
12% acrylamide gels).
Western Blotting
Culture media of human SMCs were separated on 10% SDS-PAGE mini
gels and transferred onto nitrocellulose (Bio-Rad Laboratories). MMP-2
was detected with rabbit polyclonal antibody, also provided by Dr
William Stetler-Stevenson, in conjunction with a chemiluminescence kit
(Amersham), used according to the manufacturer's
instructions.16 Rainbow-colored MW markers were also from
Amersham.
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Results |
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Pro–MMP-2 is the major gelatinolytic activity elaborated by unstimulated human16 or rabbit14 vascular SMCs as measured by SDS-PAGE zymography (Fig 1
).
Zymography discloses this usually latent form of the enzyme because SDS
present in the gel changes the conformation of the zymogen in a manner
that enables digestion of substrate (gelatin). We used this method of
analysis to detect changes in gelatinolytic activity secreted by
cultured rabbit (Fig 1) or human (not shown) SMCs as a result of
incubation of cells in the presence of thrombin. All samples showed a
major gelatinolytic band consistent with lysis due to the zymogen form
of MMP-2 (pro–MMP-2) and a minor band migrating at lower MW
corresponding to activated MMP-2. Culture media harvested from SMCs
incubated with -thrombin consistently produced an increase in the
lytic area corresponding to MMP-2. In some samples (Fig 1, Expt 2),
this increase was found to be due to a discrete additional lytic band
that coalesced with MMP-2. This finding reflects proteolytic processing
of the MMP-2 zymogen.11 12 24 We thus reasoned that
thrombin may either enhance the action of a recently cloned cell
membrane–bound MMP or, as thrombin is itself a protease, have a direct
enzymatic action on the latent form of MMP-2. In subsequent
experiments, we incubated culture medium collected from untreated human
SMCs containing pro–MMP-2 with -thrombin (Fig 2) at
concentrations achievable in the vascular wall. We found again an
increase in the gelatinolytic activity, presumably due to activated
MMP-2. This increase in gelatin lysis did not entail an obvious
decrease of the gelatinolytic band representing the zymogen. Saturation
of the zymogram (due to substrate depletion) or possible release of
additional zymogen from higher-MW complexes could explain this
observation. Similar thrombin concentrations did not appear to activate
latent gelatinase MMP-9, as determined by incubation with media
harvested from IL-1–stimulated SMCs, a condition that induces MMP-9
release from SMCs (Fig 2). Thrombin also failed to degrade purified
pro–MMP-9, as well as SMC-derived or recombinant human stromelysin
(MMP-3) (data not shown). The area of gelatin lysis in SDS-PAGE
zymograms produced by MMP-2 from the culture media of untreated SMCs
after incubation with thrombin increased with thrombin concentration
and time of incubation (Fig 2B). Cell-free experiments in which culture
media harvested from rabbit SMCs were incubated with -thrombin (not
shown) yielded zymographic results similar to those obtained with human
SMC-derived pro–MMP-2.
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Positive identification of MMP-2 employed Western blotting using
polyclonal anti–MMP-2 antibodies to analyze SMC-conditioned media
incubated with or without thrombin. In addition to the proenzyme
detected in untreated supernatants, antibodies reacted with several
lower- as well as higher-MW species in samples incubated with thrombin
(Fig 3). Either increasing the concentration of thrombin
or prolonging the time of incubation while keeping its concentration
constant resulted in generation of additional bands reacting with the
anti–MMP-2 (Fig 3 and other data not shown). This result supported the
interpretation that thrombin cleaves latent MMP-2 constitutively
secreted by SMCs. Anti–MMP-2 antibodies recognized additional high-MW
bands in samples incubated with thrombin, a pattern that may indicate
proteolytic processing of large complexes containing pro–MMP-2 and
possibly TIMP-2,25 that in untreated samples do not
penetrate into the resolving gel.
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Latent MMP-2 secreted in vitro by human SMCs immunoprecipitates as a
complex with its endogenous inhibitor, TIMP-2.16 Binding
of TIMP-2 interferes with cellular activation of
pro–MMP-224 and limits degradation of substrate by
activated MMP-2 forms,10 probably acting as a
posttranslational control mechanism of MMP-2 enzymatic activity. We
determined whether such pro–MMP-2/TIMP-2 complexes secreted by SMCs in
vitro are susceptible to proteolytic processing as a result of
subsequent incubation with thrombin. For this purpose, we used
anti–TIMP-2 antibodies to immunoprecipitate metabolically labeled
pro–MMP-2/TIMP-2 complexes from the culture media of human SMCs (Fig 4). Immunoprecipitates were incubated in the absence or
presence of thrombin for 18 hours, separated by SDS-PAGE, and analyzed
by fluorography. Untreated immunoprecipitates have an apparent MW
around 70 kD in nonreducing SDS-containing gels16 but
dissociate in the presence of reducing agents such as DTT (Fig 4).
Incubation of radiolabeled complexes with thrombin reduced the
radioactivity associated with pro–MMP-2 and produced additional
radiolabeled fragments with MWs between 40 kD and 20 kD, reminiscent of
the molecular species obtained by autolysis of TIMP-2–free MMP-2 after
activation initiation by using synthetic compounds.26
These experiments suggested that thrombin may overcome the usual
stabilizing effect that interaction with TIMP-2 confers on pro–MMP-2
and can thus initiate the conversion of pro–MMP-2 into smaller
molecular products.
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Taken together, these results indicate that thrombin proteolytically
processes MMP-2. However, this effect of thrombin would have biological
relevance only if the resultant MMP-2 forms manifest enzymatic
activity. We investigated this issue by using an appropriate substrate
for MMP-2, collagen type IV. Medium conditioned by SMCs (containing
pro–MMP/TIMP-2 complexes) did not by itself degrade radiolabeled
collagen type IV. Coincubation of SMC-conditioned medium with thrombin
led to degradation of this substrate (Fig 5). Inhibitors
of either thrombin or MMPs diminished collagenolysis. Thrombin alone
only minimally degraded collagen IV (Fig 5B). Other serine proteases,
such as plasmin or trypsin, themselves degraded collagen IV when tested
in this assay (not shown), obscuring a possible activating effect on
pro–MMP-2 secreted by SMCs.
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Discussion |
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Proteolytic enzymes, including members of the coagulation and fibrinolytic cascades and the MMPs, may mediate many aspects of atherogenesis. The relationship between thrombosis and atherosclerosis is rather complex, and each may act as stimulus for the other.27 Mural thrombi may serve as signals for development of atherosclerotic lesions,28 29 and in turn, structurally compromised plaques may trigger thrombosis.30 31 32 Vascular interventions cause platelet adhesion to the injured wall and activate the coagulation cascade. In such pathological situations, thrombin may achieve high local concentrations in blood vessels.21 Besides its action in hemostasis, thrombin can influence the cells and extracellular matrix of blood vessels.22 Vascular endothelial cells and SMCs express thrombin receptors.33 Thrombin promotes SMC proliferation34 35 and expression of molecules involved in SMC migration.36 37 38 Therefore, thrombin has both the opportunity and the capacity to modulate aspects of vascular tissue repair.
Several recent studies have investigated the matrix-degrading action of gelatinases, members of the MMP family, in relation to vascular pathology. Cultured human SMCs constitutively produce pro–MMP-2.16 In agreement with this in vitro observation, we detected in situ expression of immunoreactive MMP-2 in human vascular tissue.18 While SMCs of both normal and atherosclerotic arteries expressed immunoreactive MMP-2, we localized gelatinolytic activity in atheroma but not in uninvolved vessels,18 implying activation of gelatinases in the diseased arterial wall. This activity could be due to either MMP-2 or MMP-9, a gelatinase induced in cultured human SMCs by cytokines,16 which is expressed in situ in human atherosclerotic plaques.18 In addition to weakening of atheromatous tissue, matrix digestion by active MMPs may contribute to formation of hyperplastic intimal lesions after injury of previously normal vessels, a process that entails migration and proliferation of vascular SMCs. Cell locomotion requires dissolution of the extracellular matrix.39 Both proliferation and migration of SMCs in culture14 40 and in rat carotid arteries after experimental vascular injury19 20 correlate with peaks of gelatinase induction and appearance of proteolytically processed MMP-2.
The mechanism responsible for activation of pro–MMP-2 in these situations remains unknown. In vitro, membrane preparations of stimulated24 or transformed cells12 can proteolytically process latent MMP-2. Activation of pro–MMP-2 by plasmin or plasminogen activators is controversial; the source of disagreement between various reports may depend on the criteria used to assess their effect on pro–MMP-2. We also investigated the proteolytic processing of the MMP-2 zymogen by thrombin but included a functional assay of collagen type IV degradation to confirm MMP-2 activation. Coincubation of medium conditioned by SMCs with plasmin or trypsin also yielded degradation of radiolabeled collagen IV, but these two serine proteases digested this substrate in the absence of MMP-2. Thus, we were unable to assess the possible proteolytic activation of MMP-2 by plasmin or trypsin. Thrombin, which has physiological relevance in the context of atherosclerosis, by itself had little type IV collagenolytic activity but rendered pro–MMP-2 secreted by SMCs derived either from human or rabbit blood vessels able to digest exogenous radiolabeled collagen IV. We found that supernatants of SMCs stimulated in culture with thrombin could also digest this substrate (data not shown). Collagen type IV lytic activity in the culture media of confluent, unstimulated human SMCs likely derives from MMP-2, as this proteinase represents the only gelatinase secreted by SMCs under these conditions.16
Latent MMP-2 and pro–MMP-2/TIMP-2 complexes do not spontaneously
become active in vitro.25 Only inhibitor-free MMP-2 can
undergo further autocatalytic degradation to enzymatically active and
inactive smaller-MW forms.10 24 In our experiments, the
culture medium conditioned by SMCs contained pro–MMP-2 as well as
TIMP-2, both constitutively secreted by these cells. The presence of
TIMP-2 prevented neither proteolytic processing of pro–MMP-2 by
thrombin nor the subsequent digestion of exogenous collagen type IV
substrate by MMP-2. We specifically addressed the question of the
TIMP-2 effect by selecting for incubation with thrombin only pro–MMP-2
molecules already complexed to TIMP-2 (Fig 4
). All of our results agree
with the hypothesis that thrombin may partially overcome the TIMP-2
inhibition of MMP-2 activation. At present we do not know whether
thrombin acts only in the initial activation of MMP-2 zymogen, which
facilitates further autocatalysis of TIMP-2–free enzyme and production
of smaller-MW forms, or whether thrombin has a more extensive
proteolytic effect on MMP-2.
Since submission of this study, Zucker et al41 reported the capacity of thrombin to mediate the selective activation of the MMP-2 zymogen produced by cultured endothelial cells. These authors demonstrated that processing of pro–MMP-2 in culture media conditioned by endothelial cells depended neither on transduction through the cellular thrombin receptor nor induction of the membrane-associated MMP that can activate pro–MMP-2. They reported, however, that pro–MMP-2 activation by thrombin did not occur in cell-free conditions and thus concluded that endothelial cells possess a novel mechanism for activation of this gelatinase. We found, using culture media conditioned by vascular SMCs as source for pro–MMP-2, that its activation occurred after incubation with thrombin in the absence of cells. These differences may result from variations in the ratio between pro–MMP-2 and MMP inhibitors secreted by these two cell types or from other unknown causes.
A variety of cells throughout the body constitutively secrete latent MMP-2, which is likely to result in a wide extracellular distribution of this gelatinase. Basement membranes contain abundant collagen type IV, the typical substrate of MMP-2. Expression of an activator of MMP-2 on the plasma membrane of moving cells can lead to pericellular activation of latent MMP-2 deposited in the extracellular space and aid cell migration or invasion.42 However, at sites of injury to vascular and other tissues, thrombin figures prominently and may activate latent MMP-2 located in the interstitial space. Our results suggest that in addition to its previously described thromboregulatory, chemoattractant, and mitogenic functions, thrombin may contribute to the vascular response to injury by activation of latent MMP-2. These observations provide a new example of an extrahemostatic function of thrombin and a novel link between two important regulatory protease families, the coagulation cascade, and the MMPs.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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The experimental work in this study was supported by National Heart, Lung, and Blood Institute grants HL34636-10 (to Dr Libby) and HL 13160-24 (to Dr Fenton) and was performed while Zorina S. Galis was a Fellow of the American Heart Association, Massachusetts Affiliate, Inc, and while Roger Kranzhöfer was a fellow of the Deutsche Forschungsgemeinschaft, Germany (Kr 1363/1-1). Dr Galis is currently supported through a development fund from Emory University School of Medicine, Atlanta, Ga. The authors thank Dr William Stetler-Stevenson for his gift of antibodies. The assistance of Dr Maria W. Muszynski was essential for obtaining cultured SMC preparations.
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Footnotes |
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Reprint requests to Zorina S. Galis, PhD, Emory University School of Medicine, Cardiology Division, 1639 Pierce Dr, WMB #319, Atlanta, GA 30322.
Previously presented in abstract form at the Experimental Biology Meeting, Atlanta, Ga, April 1995.
Received July 6, 1995; accepted June 14, 1996.
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