Published online before print April 25, 2002, doi: 10.1161/01.RES.0000019240.72809.76
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© 2002 American Heart Association, Inc.
Integrative Physiology |
Factor Xa Releases Matrix Metalloproteinase-2 (MMP-2) From Human Vascular Smooth Muscle Cells and Stimulates the Conversion of Pro–MMP-2 to MMP-2
Role of MMP-2 in Factor Xa–Induced DNA Synthesis and Matrix Invasion
From the Institut für Pharmakologie und Klinische Pharmakologie (B.H.R., M.B., K.S.), UniversitätsKlinikum Düsseldorf, Heinrich-Heine-Universität, Düsseldorf; and Zentrum für Vaskuläre Biologie und Medizin Erfurt (E.B.), Friedrich-Schiller-Universität Jena, Germany.
Correspondence to Karsten Schrör, Institut für Pharmakologie und Klinische Pharmakologie, Moorenstr. 5, D-40225 Düsseldorf, Germany. E-mail kschroer{at}uni-duesseldorf.de
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Abstract |
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Pro–matrix metalloproteinase-2 (pro–MMP-2) is expressed in vascular smooth muscle cells (SMCs). We report that activated coagulation factor X (FXa) induces the release of MMP-2 (65 kDa) from human SMCs. In addition, FXa cleaves pro–MMP-2 (72 kDa) into MMP-2. Pro–MMP-2 and MMP-2 were determined by gelatin zymography. MMP-2 was generated in conditioned medium containing pro–MMP-2 in a concentration-dependent fashion by FXa (3 to 100 nmol/L). FX at concentrations up to 300 nmol/L was ineffective. The conversion of pro–MMP-2 to MMP-2 was inhibited by a selective FXa inhibitor (DX-9065a) at 3 to 10 µmol/L. There was a concentration-dependent induction of an intermediate MMP-2 form (68 kDa) in lysates of FXa-treated cells. This indicates that cellular mechanisms are involved in FXa-induced conversion of pro–MMP-2. As a possible biological consequence of MMP-2 activation by FXa, DNA synthesis and matrix invasion of SMCs were determined. Both were stimulated by FXa and inhibited by the selective FXa inhibitor DX-9065a and the MMP inhibitor GM 6001 but not by hirudin or aprotinin. It is concluded that stimulation of SMCs by FXa increases the levels of MMP-2 in the extracellular space and that two different mechanisms are involved: release of active MMP-2 and cleavage of secreted pro–MMP-2. Both might contribute to the mitogenic potency of FXa and FXa-stimulated matrix invasion of SMCs.
Key Words: matrix metalloproteinase-2 • factor Xa • vascular smooth muscle cells • extracellular matrix invasion • mitogenesis
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Introduction |
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Matrix metalloproteinases (MMPs) are a family of structurally related zinc-endopeptidases. MMPs are thought to play an important role in the physiological turnover of extracellular matrix (ECM) components. This includes embryonic tissue morphogenesis, tissue repair, and angiogenesis. In pathological conditions such as atherosclerosis, arthritis, glomerulonephritis, gastric ulcer, tumor invasion, and metastasis, MMPs are also involved in ECM degradation.1–5 MMPs are synthesized intracellularly and secreted into the extracellular space as proenzymes. The propeptide domain keeps the proenzyme inactive by covalent binding of the catalytic zinc ion. Cell surface–associated urokinase-type plasminogen activator (uPA)/plasmin complex and other MMPs can activate it after proteolytic cleavage.1,2,6 MMP activity is controlled by tissue inhibitors of metalloproteinases (TIMPs).3 Recently, it has been found that membrane-type MMPs (MT-MMPs) can cause MMP activation, leading to the hypothesis of a predominantly pericellular MMP activation cascade.6
Recent research has also shown that proliferation and migration of smooth muscle cells (SMCs) are linked to coagulation and fibrinolysis.7,8 Generation of plasmin causes MMP activation and subsequent ECM breakdown.9 This is considered as a prerequisite for cell migration into damaged tissues, for example, tumor invasion and tissue remodeling.4 It has been demonstrated that MMP-2 contributes to cell proliferation, migration, and matrix invasion in a number of cell types such as tumor cells, fibroblasts, and SMCs.10–13
In addition to the activation of MMPs by plasmin, activation of MMP-2 by thrombin is also well established.14,15 However, little is known about the effects of other coagulation factors, such as factor Xa (FXa), on the activation of MMPs. Both, thrombin and FXa are not only key enzymes in blood coagulation but also mitogens in vascular SMCs.16 Because MMP-2 and MMP-9 are dominant MMPs in the vascular tissue,5 we have investigated the effects of FXa on these enzymes in vascular SMCs.
Known activation mechanisms for MMP-2 are the cleavage of pro–MMP-2 by MT1-MMP2,17 or thrombin.18 Others have demonstrated the cleavage of MMP-2 in the presence of the coagulation factors II, Va, VIIa, and Xa in human umbilical vein endothelial cells.19 MMP-2 can be induced by platelet-derived growth factor (PDGF) in rat SMCs.12 In addition, it has been shown that MMP-9 can be induced by inflammatory cytokines, such as interleukin (IL)-1
and tumor necrosis factor (TNF)- in rabbit and human fibroblasts. These effects were enhanced by simultaneous stimulation with PDGF-BB.20
We report in the present study that FXa releases MMP-2 from cultured human SMCs. Furthermore, we demonstrate that FXa converts pro–MMP-2 into active MMP-2 in conditioned, cell-free medium. This elevation of extracellular MMP-2 levels by FXa might contribute to its mitogenic potency as well as matrix invasion of SMCs.
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Materials and Methods |
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Materials
Gelatin (porcine skin, 300 bloom), trypsin, ethylenediamine-tetraacetic acid (EDTA), sodium dodecyl sulfate (SDS), Triton X-100 was obtained from Sigma. Dulbecco’s modified Eagle’s medium (DMEM), antibiotics, and fetal calf serum were obtained from Life Technologies (Karlsruhe, Germany). MMP-2 zymography standard, MMP inhibitor GM 6001, and monoclonal anti–MMP-2-antibodies (clone 42-5D11) were purchased from Calbiochem. Coomassie Brilliant Blue R-250 was obtained from Bio-Rad. Human activated and inactive coagulation factors (FXa and FX) were from Kordia (Leiden, The Netherlands). Factor Xa inhibitor DX-9065a was kindly provided by Dr S. Kunitada (Daiichi Pharmaceutical Co, Ltd, Tokyo, Japan).
-Thrombin was kindly provided by Dr J. Stürzebecher (Zentrum für Vaskuläre Biologie und Medizin, Friedrich-Schiller-Universität Jena, Germany). Cell culture materials were purchased from Becton Dickinson.
Cell Culture
Vascular SMCs were isolated from human saphenous veins or human mammary arteries by the explant technique and cultured as previously described.21 SMCs from passages 4 to 9 were serum-deprived for 72 hours and then stimulated with FX or FXa.
Zymography
SMCs seeded in 24-well plates were harvested with serum-free medium for 72 hours. Media were collected and centrifuged for 10 minutes at 14 000g at room temperature to remove detached cells and debris. These conditioned media from unstimulated cells were used to study the actions of FXa and FX in a cell-free system. Alternatively, cultured cells were stimulated with these compounds, and the medium was collected afterward for zymography.
Zymography was performed using 7% SDS/polyacrylamide gels (SDS-PAGE), containing 0.7 mg/mL gelatin. Samples of cell culture medium were resolved in nonreducing Laemmli-buffer (final concentrations: 2% wt/vol SDS, 10% glycerol, 0.0625 mol/L sodium dihydrogen phosphate/disodium hydrogen phosphate, pH 7.0, and 0.01% bromphenol blue). To obtain cell lysates, after stimulation of the cells, they were washed 3 times with phosphate-buffered saline (PBS) and lysed in Laemmli-buffer. Samples were separated by electrophoresis. Then, gels were washed 3 times for 10 minutes at room temperature (50 mmol/L Tris-HCl, pH 7.5, 10 mmol/L CaCl2, 1 µmol/L ZnCl2, 2.5% Triton X-100, 0.02% NaN3) to remove SDS from the gels. Using a modified buffer (1% Triton X-100 instead of 2.5%) gels were incubated for 18 to 36 hours at 37°C. To visualize lytic bands, gels were stained with Coomassie Brilliant Blue R-250 (0.2%) in 40% methanol and 10% acetic acid. Intensity of pro–MMP-2 and MMP-2 bands was quantified using Gel Doc 1000 and software Quantity One, version 4.1.1 (Bio-Rad). After background subtraction, intensity of MMP-2 bands was related to the respective pro–MMP-2 band. This quotient of pro–MMP-2 and MMP-2 signal from unstimulated controls was set to 100% and stimulated cells were referred to control.
Western Blotting
SMCs were seeded in 6-well plates and serum-deprived for 72 hours. Cells were stimulated with FX, FXa, or thrombin for further 24 hours. Media were centrifuged for 10 minutes at 14 000g and then lyophilized (freeze dryer Beta I, Christ GmbH) to concentrate MMPs. Electrophoresis (7% SDS-PAGE), blotting of proteins onto polyvinylidene difluoride membranes (Immobilon-P, Millipore), and blocking of membranes in Blotto (Tris-buffered saline, 0.1% Tween-20, 5% wt/vol nonfat dry milk) was carried out as previously described.21 Membranes were probed with monoclonal MMP-2 antibodies (1:100 in Blotto) and incubated with peroxidase-conjugated secondary antibodies (1:3,000 in Blotto). Bands were visualized by enhanced chemiluminescence (Amersham-Pharmacia Biotech) and quantified by the Gel Doc 1000 system. Quantification was performed in the same way as described above for zymography.
[3H]Thymidine Incorporation
Subconfluent cells were treated with serum-free medium for 24 hours. Cells were labeled with [3H]thymidine (2 µCi/mL) and stimulated with FXa in the absence or presence of a MMP inhibitor, GM 6001 (100 nmol/L), for 24 hours. Media were removed and cells were washed with cold PBS and HClO4 (0.3 mol/L) as previously described.16 Cells were solubilized by addition of 0.3 mL NaOH (0.1 mol/L) for 30 minutes at 37°C. Aliquots (0.2 mL) were added to 3 mL of scintillant. [3H]Thymidine incorporation was determined by liquid scintillation spectrometry.
Cell Invasion Assay
To determine SMC invasion, a commercially available cell invasion assay kit (Chemicon International) was used.22 This kit possesses 2 chambers: 1 inner chamber for cell seeding and an outer chamber for cell culture medium. An 8-µm pore size polycarbonate membrane separates the chambers. Invasive cells are able to dissolve the matrix and to migrate through it to the lower surface of the polycarbonate membrane. According to the manufacturer’s protocol, 3x105 cells were seeded into the inner chamber in serum-free medium. Cells were stimulated with FXa (100 nmol/L) in the absence or presence of the FXa inhibitor DX-9065a (10 µmol/L) or the MMP inhibitor GM 6001 (100 nmol/L). After an incubation period of 6 days, cells from the inner chamber were removed, and the lower surface of the polycarbonate membrane was stained with the solution provided. Cells were photographed and counted using an Olympus Optical microscope BX50 F (Olympus Optical). To standardize the cell count, cells were counted in the central and 4 peripheral microscope fields.
Statistics
Data represent the mean±SEM of n experiments. Statistical analysis was performed using a paired 2-tailed t test. Values of P
0.05 were considered significant.
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Pro–MMP-2 and Pro–MMP-9 in Cultured Human SMCs
Under basal conditions, cultured human SMCs expressed pro–MMP-2, which was released into the cell culture medium. Zymography of culture medium from SMCs stimulated with FXa (100 nmol/L) revealed a band of MMP-2. This indicates a cleavage of pro–MMP-2 by FXa. In contrast, FX (100 nmol/L) had no effect on the cleavage of MMP-2 (Figure 1A).
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Cells treated with FXa did not express pro–MMP-9 or MMP-9. This indicates that FXa promotes conversion of MMP-2, but not of MMP-9, in cultured human SMCs.
Generation of MMP-2 by FXa was confirmed by Western blotting (Figure 1B). Media of FXa-stimulated cells were subjected to immunoblotting using a monoclonal antibody specific for pro–MMP-2 and MMP-2. Blots are demonstrating the presence of MMP-2 in the medium of FXa-stimulated cells. Media from thrombin-stimulated cells were used as positive controls.
Conversion of Pro–MMP-2 by FXa: Effects of Cellular Stimulation With FXa Compared With Addition of FXa to Pro–MMP-2 Containing Cell-Free Culture Medium
To investigate whether pro–MMP-2 is cleaved directly by FXa into MMP-2, experiments were carried out in cell-free conditioned medium, containing pro–MMP-2 and compared with cell-containing medium after stimulation with FXa. Samples were analyzed on the same zymography gels (Figures 2A through 2D). After 1 hour of stimulation with FXa (10 to 100 nmol/L), there was a significant MMP-2 generation in the presence of cells but not in conditioned medium in the absence of cells (Figures 2A and 2B). When cell-free medium and cell-containing medium were incubated with FXa (3 to 100 nmol/L) for a longer period of time (72 hours), levels of MMP-2 in the presence of cells were similar. However, there was a significantly increased, although lower, generation of MMP-2 by FXa in conditioned medium without cells (Figures 2C and 2D).
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Conversion of Pro–MMP-2 Into MMP-2 and Release of MMP-2 Is Specific for FXa
To investigate whether the conversion of pro–MMP-2 into MMP-2 and its release from SMCs is specific for FXa, the selective FXa inhibitor DX-9065a was used. DX-9065a (0.3 to 10 µmol/L) inhibited the release of MMP-2 by FXa (100 nmol/L) in a concentration-dependent fashion (Figure 3). To exclude the possible involvement of thrombin or plasmin in FXa effects on MMP-2, hirudin and aprotinin were used. Neither hirudin (10 to 300 nmol/L) nor aprotinin (0.1 to 10 µmol/L) did affect FXa-mediated conversion of pro–MMP-2 into MMP-2 (data not shown). Additionally, when the effects of FXa were studied in cell-free medium, DX-9065a inhibited the conversion of pro–MMP-2 into MMP-2 in a concentration-dependent fashion (data not shown). These data demonstrate that FXa specifically releases MMP-2 and converts pro–MMP-2 into MMP-2, and that this action does not involve the serine proteases thrombin or plasmin.
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Effects of FXa on Arterial SMCs
To establish that the stimulatory effects of FXa on MMP-2 generation are not restricted to SMCs from venous tissue, additional experiments were carried out in arterial SMCs. Stimulation of human mammary artery SMCs with FXa (30 to 100 nmol/L) also resulted in generation of MMP-2. This conversion of pro–MMP-2 into MMP-2 was inhibited by DX-9065a (1 µmol/L), but not by hirudin (1 µmol/L) or aprotinin (1 µmol/L) (Figure 4). This confirmed the findings on venous SMCs.
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Effects of FXa on MMP-2 in SMC Lysates
SMCs were stimulated with FXa (3 to 100 nmol/L) and afterward the lysates were analyzed by zymography. Data show various bands of cell-bound gelatinolytic activity (Figure 5). A strong band of pro–MMP-2 was determined. Furthermore, a weaker band of an intermediate form of MMP-2 (68 kDa) in addition to active MMP-2 (65 kDa) was seen. On stimulation with FXa, an increased formation of intermediate MMP-2 was observed at 30 to 100 nmol/L FXa (Figure 5). This indicates the contribution of cellular mechanisms to the activation of MMP-2 in cultured human vascular SMCs by FXa.
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FXa-Induced DNA Synthesis Is Reduced by MMP Inhibition
To investigate whether FXa-induced MMP-2 activation contributes to proliferation of human vascular SMCs,14 mitogenesis was measured by [3H]thymidine incorporation into cellular DNA. FXa (100 nmol/L) increased the incorporation of [3H]thymidine 4- to 5-fold above control. This strong mitogenic effect of FXa was significantly inhibited by the specific FXa inhibitor DX-9065a.16 Inhibition was also seen by preincubation of cells with the MMP inhibitor GM 6001 (100 nmol/L) (Figure 6). Neither DX-9065a16 nor GM 6001 alone affected cellular [3H]thymidine incorporation.
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FXa-Induced Extracellular Matrix Invasion Is Reduced by MMP Inhibition
A cell invasion assay was used to investigate whether FXa-induced release of active MMP-2 mediates ECM invasion of SMCs. Cells were stimulated for 6 days with FXa (100 nmol/L) in the absence or presence of either DX-9065a (10 µmol/L) or GM 6001 (100 nmol/L). Microscopy revealed that FXa stimulated SMC migration through the matrix gel. Pretreatment of cells with either the FXa inhibitor DX-9065a or the MMP inhibitor GM 6001 reduced these effects back to baseline. Treatment of cells with the inhibitors alone had no effect on cell invasion (Figure 7).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The activation of MMPs plays a significant role in both physiological and pathophysiological conditions.1–4 Therefore, mechanisms of activation are of great interest in the understanding of ECM regulation. In the present study, we report that the coagulation factor Xa stimulates the release of MMP-2 in human SMCs. In addition, conversion of pro–MMP-2 into MMP-2 is also increased in cell-free medium containing pro–MMP. Similar results were obtained on human arterial SMCs and human fibroblasts (Figure 4, data for fibroblasts are not shown). These findings provide new insights into the mechanism(s) SMC proliferation and matrix invasion. In addition to the role of FXa in blood coagulation, this represents a novel function of FXa, which has not been described in SMCs so far.
It is well established that most MMPs are secreted as inactive proenzymes and are activated in the extracellular space. Activation is caused by disruption of the Zn2+-blockade in the catalytic domain, excreted by the cysteine residue in the propeptide domain.2,4 Our findings indicate that the serine protease FXa initiates the process of pro–MMP-2 cleavage, eventually resulting in its release into the extracellular space. Cleavage of pro–MMP-2 and generation of MMP-2 was demonstrated by gelatin zymography (Figure 1A) and Western blotting using monoclonal antibodies specific for pro–MMP-2 (72 kDa) and active MMP-2 (65 kDa) (Figure 1B). There was no detectable pro–MMP-9 or MMP-9 by zymography after stimulation of the cells with FXa. These findings suggest that FXa preferentially modifies the MMP-2 pathway but does not interfere with MMP-9 in human SMCs.
Next, it was investigated whether the active MMP-2 in the medium was derived from extracellular cleavage of the secreted proenzyme and/or whether cellular mechanisms were involved. In these experiments, FXa was added either to the SMCs with subsequent collection of the medium or directly to the cell-free supernatants of SMCs containing secreted pro–MMP-2. After short-term incubation (1 hour), there was only generation of MMP-2 after stimulation of cells. At longer incubation periods (72 hours), this effect was maintained. In cell-free medium, stimulated with FXa, a significantly increased level of MMP-2 was now detected (Figure 2). This amount of MMP-2 could be considerably further enhanced by increasing the concentration of FXa in the medium above 100 nmol/L (data not shown). This phenomenon might be explained by the ability of active MMPs to stimulate additional lytic enzymes1,4 or simply a longer duration of action might explain the stronger effects seen after longer incubation periods. In any case, the conversion of pro–MMP-2 into MMP-2 by FXa was specific for FXa because it was concentration-dependently inhibited by DX-9065a, a specific active-site inhibitor of FXa23,24 (Figure 3). Furthermore, the possibility that thrombin or plasmin may have contributed to FXa-induced activation of MMP-2 was excluded because incubation of SMCs with hirudin and aprotinin had no effect (Figure 4).
In SMC lysates, we detected pro–MMP-2 (72 kDa), an intermediate form (68 kDa), and active MMP-2 (65 kDa) after stimulation with FXa (Figure 5). These findings are in concert with previous reports of multimer forms of MMP-2 in lysates from various cell types.18,26–28 At 30 to 100 nmol/L FXa, we observed the appearance of an intermediate form of MMP-2 (Figure 5), indicating a FXa-dependent pro–MMP-2 cleavage via an intermediate cell-associated complex. Although these data are probably due to complex mechanisms, they might suggest that FXa-stimulated conversion of pro–MMP-2 to MMP-2 is a membrane-related process.26,29 Preliminary studies with separated membrane and cytosolic fractions of SMCs appear to support this conclusion (data not shown).
It has been reported that MMP-2 is activated on the cell surface by MT1-MMP1,29 and the uPA/plasmin system.6,30 Thrombin can MT-MMP-dependently activate pro–MMP-2.18 It is possible that FXa initiates the release of active MMP-2 from the cell surface by altering the MT1-MMP/TIMP-2/MMP-2 complex. This hypothesis is supported by the findings that TIMP-2 binds and inhibits active MMP-2 and that the MMP-2/TIMP-2 complex is then released from the cell surface.25 Another study reported the release of active MMP-2 from the cell surface and the control of integrin-mediated MMP-2 activation by collagen.26 The FXa-induced release of MMP-2 was not affected in cells pretreated with TIMP-2 (2 to 20 nmol/L) (not shown).
Our observations raise the question whether metalloproteinases contain cleavage sites for FXa, which could be located in the propeptide domain to induce MMP activation. A potential FXa cleavage site31 in the amino acid sequence of MMP-232 is present at the border between the propeptide domain and the first catalytic domain and might be responsible for pro–MMP-2 activation. However, the exact mechanism by which pro–MMP-2 is converted to active MMP-2 is still unknown.26
The functional significance of MMP-2, aside from degrading ECM, is the regulation of cell proliferation, migration, and tumor invasion.10,13,33,34 In addition, an important role of MMP-2 in the regulation of SMC proliferation, migration, and matrix invasion has been reported.11,12,35,36 It has been demonstrated that FXa stimulates SMC proliferation16 and that the synthetic FXa inhibitor DX-9065a reduces SMC proliferation in vitro16 and in vivo.24 We hypothesize that FXa-induced MMP-2 activation may contribute to mitogenesis of SMCs and matrix invasion. Both effects were are reduced by inhibitors of FXa and MMPs, respectively. Therefore, we conclude that FXa contributes via MMP activation to both cell proliferation and SMC matrix invasion.
These findings might be of clinical importance for newly developed FXa inhibitors. Several compounds are currently subject of clinical trials, for example in patients with acute coronary syndrome.37 In addition to the anticoagulatory effect, an inhibition of FXa-induced MMP activation by these compounds might contribute to the patients‘ benefit.
In summary, we demonstrate for the first time that FXa generates significant levels of MMP-2 in the environment of SMCs by stimulation of MMP-2 release and conversion of pro–MMP-2 to MMP-2. These increased local levels of MMP-2 may play a role in FXa-induced cell proliferation and matrix invasion.
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Acknowledgments |
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This study was supported by a grant from the Forschungsgruppe Herz-Kreislauf e.V. (Düsseldorf) and a grant from the IZKF der Friedrich-Schiller-Universität Jena to E.B. (B307-0139). The authors are grateful to Dr Artur-Aron Weber for a critical review of the manuscript and helpful suggestions. They also thank Kerstin Freidel and Beate Weyrauther for competent technical support and Erika Lohmann for expert secretarial assistance.
Received November 28, 2001; revision received April 10, 2002; accepted April 11, 2002.
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