Division of Respiratory Medicine, City Hospital, University of Nottingham, Nottingham NG5 1PB, United Kingdom
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ABSTRACT |
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Airway smooth muscle proliferation is important in asthma and is dependent on pro- and antimitogenic factors and cell-matrix interactions. Here we show an antiproliferative effect of protease inhibitors on human airway smooth muscle due to inhibition of autocrine-derived matrix metalloproteinase (MMP)-2. Proliferation in response to fetal bovine serum, thrombin, and platelet-derived growth factor was inhibited by the broad-spectrum protease inhibitor Complete and the MMP inhibitors EDTA and Ro-31-9790 but not by cysteine or serine protease inhibitors. Conditioned medium from airway smooth muscle cells contained 72-kDa gelatinase that was secreted by growth-arrested cells and increased by fetal bovine serum but not by thrombin or platelet-derived growth factor. Immunostaining of cultured human airway smooth muscle cells and normal lung biopsies confirmed this gelatinase to be MMP-2. Our results suggest a novel role for MMP-2 as an important autocrine factor required for airway smooth muscle proliferation. Inhibition of MMPs could provide a target for the prevention of smooth muscle hyperplasia and airway remodeling in asthma.
serine protease; cysteine protease; protease inhibitors; extracellular matrix; asthma
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INTRODUCTION |
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AIRWAY SMOOTH MUSCLE hyperplasia and hypertrophy are important processes in the pathogenesis of asthma (15). The use of cultured cells has allowed analysis of the mechanisms underlying these processes (37). Airway smooth muscle cells from a number of species proliferate in response to inflammatory mediators such as histamine (25) and endothelin enzymes (9) including mast cell tryptase (2); cytokines (36); and growth factors including platelet-derived growth factor (PDGF) (12), epidermal growth factor (35), and insulin-like growth factors I and II (23). Less is known about the factors limiting airway smooth muscle proliferation, although heparin, PGE2 (13), vasoactive intestinal polypeptide (19), and interleukin-4 (11) can inhibit proliferation of airway smooth muscle cells in culture. In addition to pro- and antimitogenic stimuli, cell-matrix interactions are important regulators of cellular proliferation.
Proteases are enzymes capable of hydrolyzing peptide bonds and have been categorized according to the composition of their active sites. The major groups of proteases include serine, cysteine, aspartate, and matrix metalloproteinases (MMPs). Proteolytic enzymes were initially described in the breakdown of protein in the gastrointestinal tract but are now recognized as important in a huge range of intra- and extracellular functions including complement activation, the coagulation cascade, apoptosis (16, 42), major histocompatibility complex class II responses (5), antigen presentation (17), receptor processing (1), enzyme activation (21), receptor activation (38), and turnover of the extracellular matrix (21). In the airways, the extracellular matrix is composed of fibronectin, glycosaminoglycans, laminin, type IV collagen, tenascin, entactin, integrins, and metalloproteinases. Bronchial epithelial cells secrete fibronectin (31), MMP-2 and MMP-9 (40), and probably other matrix components, whereas in the pulmonary interstitium, fibroblasts secrete most components of the extracellular matrix (3, 4, 10, 39). In addition to proliferation, cell-matrix interactions are also important in organogenesis, development, wound healing, and tumor growth and spread. Many other proteases contribute to the turnover of the extracellular matrix, including other MMPs [collagenases, stromelysins, and matrilysin (39)], plasmin, and the plasminogen activators (urokinase-type and tissue-type plasminogen activators) (20). The capacity of matrix components to regulate proliferation in airway smooth muscle has not been extensively studied.
We hypothesized that the widespread effects of proteases, particularly on the extracellular matrix and cell surface receptors, would be likely to result in changes in airway smooth muscle proliferation, which may be important in regulating airway smooth muscle in asthma. We therefore studied the effect of a broad-spectrum protease inhibitor on the proliferation of primary cultures of human airway smooth muscle cells and then went on to further categorize the inhibitory effect seen with the specific inhibitors of cysteine, serine, and metalloproteinases. We found an antiproliferative effect of protease inhibition, which was predominantly due to an effect on metalloproteases, and went on to show that this antiproliferative effect may be due to a specific action on MMP-2 secreted by human airway smooth muscle in vitro and in vivo.
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METHODS |
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Cell Culture
Human tracheae that were pathologically normal were obtained from cadavers <24 h after death from patients with no history of airway or lung disease. Primary cultures of human airway smooth muscle cells were prepared as previously described (26). Cells from three separate donors were used at passages 3-4. Pang and Knox (27) have previously demonstrated that these cells have the light-microscopic and immunocytochemical characteristics of smooth muscle cells.Thymidine Incorporation
Thymidine incorporation was performed as previously described (13) with the following modifications. Cells were grown in 96-well plates in DMEM supplemented with 10% fetal bovine serum (FBS), 4 mM L-glutamine, 50 U/ml of penicillin, 0.05 mg/ml of streptomycin, and 2.5 µg/ml of amphotericin B until 90-100% confluent and then growth arrested in serum-free medium for 24 h. The cells were then incubated with serum-free medium plus the appropriate mitogen (either FBS, thrombin, PDGF-AB, or control vehicle) and, in some experiments, in the presence of one of the protease inhibitors E-64 (10 µg/ml), aprotinin (2 µg/ml), EDTA (5 mg/ml), Complete (full strength), Ro-31-9790 (100 µM), or vehicle for 48 h. We have previously found these cells to show a maximal increase in thymidine incorporation at 48 h under these conditions. For the final 6 h, the cells were pulsed with tritiated thymidine at a final concentration of 4 µCi/ml. DNA was isolated with a cell harvester (Automash 2000, Dynatec, West Sussex, UK) and adsorbed onto filter paper, treated with 200 µl of 0.01 M potassium hydroxide, and immersed in 4 ml of Ready Protein scintillation fluid. The vials were shaken, and the radioactivity was quantified with a scintillation counter (Minaxi 4000, Packard) with a counting efficiency in Ready Protein of 40% and was compared with the control value.MTT Assay
Cell viability was measured with the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay (28). Cells were grown in 96-well plates and incubated with the protease inhibitors for 48 h as described for the thymidine incorporation experiments. After incubation, 20 µl of 5 mg/ml MTT were added to each well and incubated at 37°C for 1 h, the medium was removed, 200 µl of dimethyl sulfoxide were added to solubilize the blue formazan product, and the plates were shaken for 5 min. The optical density at 570 nM was compared with the control value with a Dynatech MR 5000 plate reader.Cell Counts
Cells were seeded in 24-well plates in DMEM and 10% FBS. After 24 h (day 0), the medium was changed to DMEM and 10% FBS plus either EDTA (0.5 mg/ml), Complete (half strength), or control (DMEM and 10% FBS alone). The culture medium and protease inhibitors were changed daily. After 2 days, the cells were removed from the wells with trypsin-EDTA and centrifuged, and the supernatant was discarded. The pellets were resuspended in 20 µl of trypan blue and counted with a hemacytometer. Samples were run in triplicate and counted twice.Zymography
Cells were grown in 12-well plates until confluent, growth arrested for 24-48 h, and treated with either FBS (10%), thrombin (1 IU/ml), or PDGF (50 ng/ml) according to the experimental protocol. Experiments were repeated on at least three occasions. Conditioned medium was harvested at the appropriate time points and stored atImmunohistochemical Staining
Tissue sections. Lung tissue was obtained from archive sections of tissue distant from the tumor from patients who had undergone resection for bronchial carcinoma. Paraffin sections were stained by the streptavidin-biotin complex method with the DAKO Duet kit. Tissue was dewaxed for 5 min in xylene. Endogenous peroxidase activity was blocked by immersion in 0.5% hydrogen peroxide in methanol for 10 min, and the sections were washed in tap water and then in Tris-buffered saline (TBS; pH 7.6). Nonspecific binding was blocked with 1:5 normal swine serum (NSS) in TBS. The primary antibody diluted 1:10 in blocking solution was applied for 1 h, the section was washed in TBS, and the secondary biotinylated goat anti-mouse or anti-rabbit antibody was applied for 30 min at a dilution of 1:100 in NSS. The biotinylated goat anti-mouse or anti-rabbit antibody and the streptavidin-biotinylated peroxidase complex were diluted 1:100 in NSS and applied for 1 h. The slides were then washed in TBS, and the peroxidase was developed with diaminobenzidine for 10 min; the color was further developed by washing in copper sulfate (0.5% in 0.8% sodium chloride) and then in water, counterstained with hematoxylin, dehydrated in alcohol, and mounted.Cultured cells. Cultured cells were
grown on eight-well glass chamber slides, fixed in situ for 20 min with
methanol at 20°C, and stained as described in
Tissue sections with the
following modifications. Primary antibodies diluted 1:17 in NSS-TBS
were applied for 30 min, and a secondary peroxidase-conjugated rabbit anti-mouse antibody was applied for 30 min at a dilution of 1:100 in NSS.
Materials
Tritiated thymidine was obtained from Amersham Life Science (Little Chalfont, UK); Ready Protein scintillation fluid was from Beckman; and aprotinin, E-64, and Complete were from Boehringer Mannheim (Mannheim, Germany). Ro-31-9790 and Ro-31-4767 were kindly donated by Dr. William Johnson (Roche Pharmaceuticals, Welwyn, UK). Zymogram gels, sample buffer, renaturing buffer, and developing buffer were purchased from Novex (San Diego, CA). Mouse monoclonal anti-MMP-2 and anti-MMP-9 antibodies were obtained from R&D Systems (Oxford, UK). The DAKO Duet kit was obtained from DAKO (Ely, UK). Eight-well chamber slides were obtained from Life Technologies (Glasgow, UK). All other chemicals and cell culture reagents were obtained from Sigma (Poole, UK).Ro-31-9790 and Ro-31-4767 were dissolved in 0.5% ethanol and compared with the vehicle control; all other compounds were dissolved in culture medium.
Analyses
Because different batches of the primary airway smooth muscle cells gave responses of differing magnitudes to the mitogens, each separate thymidine incorporation and zymography experiment incorporated its own control, and the results were calculated as a ratio of this control to best compare separate experiments. Results were compared with control experiments by Student's t-test, with a P value of <0.05 regarded as significant. ![]() |
RESULTS |
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Effect of Mitogens on Airway Smooth Muscle Proliferation
There was a concentration-dependent increase in thymidine incorporation in response to 0-10% FBS, 0-5 IU/ml of thrombin, or 0-30 ng/ml of PDGF-AB. The maximal proliferative responses were 90-, 9-, and 11-fold increases from baseline for 10% FBS, 1 IU/ml of thrombin, and 30 ng/ml of PDGF, respectively (Fig. 1).
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Effect of Protease Inhibitors on Airway Smooth Muscle Cell Proliferation
Complete (at full strength according to the manufacturer's instructions) inhibited FBS-, thrombin-, and PDGF-induced thymidine incorporation by 97 (P < 0.001), 64, and 56% (P < 0.05), respectively. EDTA (0.5 mg/ml) inhibited FBS-, thrombin-, and PDGF-induced thymidine incorporation by 87, 82, and 30%, respectively (all P < 0.001; Fig. 2). To confirm that changes in thymidine incorporation truly reflected cell proliferation, we performed manual cell counts in some experiments. FBS (10%) increased the cell number by 196% after 2 days; this was reduced to a rise of 154% by EDTA (0.5 mg/ml) and abolished by Complete (full strength; Fig. 3).
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We then performed concentration-response curves for Complete and EDTA
in cells stimulated by the three mitogens studied. There was a
concentration-dependent inhibition of thymidine incorporation stimulated by FBS (10%), thrombin (1 IU/ml), or PDGF-AB (30 ng/ml) by
both EDTA (0-0.5 mg/ml) and Complete (made from twice the
manufacturer's recommended concentration to a 1:100 dilution of full
strength; Fig. 4).
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E-64 (10 µg/ml) and aprotinin (2 µg/ml) had no effect on thymidine incorporation stimulated by FBS (10%), thrombin (1 IU/ml), or PDGF-AB (30 ng/ml).
Effect of a Selective MMP Inhibitor on Proliferation
To assess whether the antiproliferative effects of EDTA and Complete were due to the inhibition of MMPs, we also studied the effect of the specific MMP inhibitor Ro-31-9790 on proliferation. Ro-31-9790 concentration dependently inhibited the increase in thymidine incorporation induced by FBS by a maximum of 51% of the control value at 100 µM Ro-31-9790 (Fig. 5A). The rise in cell number induced by FBS was almost completely abrogated over 8 days in culture when compared with the vehicle control (Fig. 5B).
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Toxicity
Complete, EDTA, E-64, aprotinin, and Ro-31-9790 had no effect on MTT cleavage under unstimulated conditions at the concentrations we used. In preliminary studies, we tried to use 1,10-phenanthroline and Ro-31-4767 as alternative MMP inhibitors. These were both toxic to airway smooth muscle cells and were not studied further.Zymography
Zymograms of airway smooth muscle-conditioned medium showed that cultured cells secrete 72-kDa gelatinase when growth arrested. FBS alone had some intrinsic gelatinase activity. Incubation of airway smooth muscle cells with FBS resulted in a twofold increase in gelatinase activity (P = 0.02) when corrected for cell number and after subtraction of the intrinsic gelatinase activity of FBS. Thrombin and PDGF did not alter the secretion of 72-kDa gelatinase. A representative zymogram is seen in Fig. 6A.
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Addition of the protease inhibitors EDTA (0.5 mg/ml), Complete (full strength), and Ro-31-9790 (100 µM) to the zymogram incubation buffer caused complete inhibition of 72-kDa gelatinase activity in the gel slabs. E-64 (10 µg/ml) and aprotinin (2 µg/ml), however, had no effect on gelatinase activity (Fig. 6B).
Immunohistochemistry
Staining for MMP-2 at a low level was consistently present in normal smooth muscle surrounding the small airways of lung sections (Fig. 7, A and B). Some sections showed weaker staining for MMP-9 (data not shown). Positive staining for MMP-2 and MMP-9 was observed at a higher level in alveolar macrophages in the tissue samples. The cultured human airway smooth muscle cells were weakly positive for MMP-2 (Fig. 7, C and D), and, again, weaker staining was seen for MMP-9.
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DISCUSSION |
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We have shown that protease inhibitors concentration dependently inhibited human airway smooth muscle cell proliferation in response to several mitogens. This effect was seen with Complete, EDTA, and Ro-31-9790 but not with the cysteine protease inhibitor E-64 or the serine protease inhibitor aprotinin, suggesting that the effect was due to inhibition of MMPs. Furthermore, we have provided evidence that human airway smooth muscle cells produce MMP-2, suggesting that metalloproteinases may play a critical role in regulating airway smooth muscle proliferation.
We used thymidine uptake as a sensitive index of proliferation and, in key experiments, confirmed that thymidine uptake correlated with changes in cell number. Serum, PDGF, and thrombin are well-characterized airway smooth muscle mitogens (12, 24, 30) and demonstrated clear concentration-dependent effects on thymidine incorporation in our cells. We can therefore be confident that the changes in thymidine incorporation truly reflected cell proliferation. Under unstimulated conditions in serum-free medium, there was no significant fall in MTT cleavage with any of the protease inhibitors we used, making an effect on mitochondrial function and cell viability unlikely.
The fact that EDTA and Complete caused a fall in thymidine incorporation to all mitogens (serum, thrombin, and PDGF) suggests that the antiproliferative effects were due to inhibition of a pathway common to all three stimuli. Complete is a broad-spectrum protease inhibitor (with activities against serine, cysteine, and metalloproteinases), and the inhibitory effects of EDTA on all three mitogens were similar to those of Complete. EDTA chelates the active-site zinc ion of metalloproteases but can also inhibit other metal ion-dependent enzymes such as calcium-dependent cysteine proteases (29). To confirm that the actions of EDTA and Complete were mainly due to inhibition of MMPs, we studied the effect of selective MMP inhibitors. One hundred micromolar Ro-31-9790, a synthetic hydroxamate-based compound, caused a 50% inhibition of thymidine incorporation in response to FBS and the complete abrogation of cell growth over 8 days as judged by cell number. The apparent discrepancy between the two techniques suggests that in the short term, the cells are able to synthesize DNA to some degree (as measured by thymidine incorporation), but over longer periods, they are unable to grow across the culture plates (as measured by the change in cell number). MMP-2 may act to inhibit airway smooth muscle cell-extracellular matrix interaction downstream from the mitogenic stimuli employed, acting as a permissive factor allowing proliferation to occur once the cells have been stimulated. Ro-31-9790 is a competitive antagonist of MMP-2; 100 µM produced 75% inhibition of MMP-2 in a fluorescence assay (34), the magnitude of which is in keeping with our findings. Our findings suggest that inhibition of MMP-2 was involved in the action of EDTA and Complete. However, the fact that the magnitude of the effect of Ro-31-9790 was less than that of EDTA or Complete suggests that EDTA and Complete use additional mechanisms of MMP inhibition to produce their antiproliferative effects. In airway smooth muscle cells, thrombin and other mitogens increase intracellular calcium, and although this is not the primary growth signal in these cells (24, 35), calcium is involved in the proliferation in several cell types (18), and its chelation leading to a fall in intracellular calcium may contribute to the antiproliferative effect of Complete and EDTA.
Aprotinin and E-64 had no effect on proliferation at doses previously shown to cause irreversible inhibition of serine and cysteine proteases, respectively (29). Although cysteine proteases take part in the regulation of the extracellular matrix, such as in the breakdown of elastic fibers, these results suggest that cysteine proteases are not important in the regulation of airway smooth muscle proliferation in culture. Similarly, serine proteases such as plasmin, which breaks down fibrin, fibronectin, and vitronectin, and urokinase- and tissue-type plasminogen activators, which convert inactive plasminogen to plasmin, are involved in extracellular matrix processing. In airway smooth muscle cell culture, these enzymes may not be present and in vivo may be derived from other cell types.
Having shown that proliferation was inhibited by MMP inhibitors, we used zymography to measure MMP activity in conditioned medium. We showed that airway smooth muscle cells constitutively secrete a 72-kDa gelatinase and were able to confirm this to be MMP-2 with immunostaining in cultured cells and lung biopsies. The fact that MMP-2 was only expressed at low levels in both cultured cells and paraffin sections (and was not enhanced by microwave antigen retrieval in the latter) suggests either that the majority of MMP-2 produced by the cells is secreted or that the rate of production is low. MMP-9 staining was also weakly present, although in our experiments, no gelatinolytic activity was seen at 92 kDa, which would correspond to MMP-9. Significant expression of MMP-2 and MMP-9 by alveolar macrophages is consistent with a previous report (7). Airway smooth muscle has important secretory functions and participates in pro- and anti-inflammatory responses in addition to its contractile role (14). Although the production of MMP-2 has previously been documented in vascular smooth muscle (32), production of MMP-2 by airway smooth muscle suggests that airway smooth muscle contributes to extracellular matrix turnover and airway remodeling and represents an important extension to what is known about the role of airway smooth muscle in inflammatory diseases such as asthma. Furthermore, in vivo MMP-2 may be produced by other airway cells including fibroblasts and macrophages; thus other airway cells may interact to support airway smooth muscle proliferation.
Secretion of MMP-2 was increased twofold by serum but not by thrombin or PDGF. The constitutive nature of MMP-2 secretion suggests that there is a constant turnover of the basement membrane even when the cells are not proliferating. Although a selective MMP inhibitor potently inhibited proliferation and MMP-2 was secreted constitutively by our cells, proliferation in response to the airway smooth muscle mitogens was not uniformly accompanied by an increase in MMP-2 synthesis. This suggests that although MMP-2 secretion is necessary for proliferation, it acts as a permissive factor that is required for proliferation to proceed rather than as an autocrine growth factor in its own right. Inhibition of proliferation in response to a range of mitogens suggests that MMP-2 may be acting to inhibit airway smooth muscle cell-extracellular matrix interaction downstream from the mitogenic stimuli employed and need not be specifically upregulated by these stimuli. The rise in gelatinase activity in response to serum rather than to thrombin or PDGF may contribute to the larger mitogenic effect of serum compared with thrombin or PDGF in airway smooth muscle.
Evidence from other biological systems also suggests that MMPs may be important in cellular growth. In cultured rabbit and sheep aortic smooth muscle and rat mesangial cells, proliferation is associated with the secretion of gelatinases that could be inhibited by the synthetic MMP inhibitors Ro-31-7476 and Ro-31-4724 (32, 34). Gelatinase mRNA and protein secretion are increased in vascular smooth muscle in response to thrombin (8) and arterial injury (a proliferative stimulus), where they parallel arterial migration and proliferation (33, 41). In light of these studies, it has been postulated that degradation of the basement membrane by gelatinase is required for proliferation and outgrowth of some cell types (22). In our study, FBS but not thrombin or PDGF upregulated MMP-2 secretion; other studies have shown that growth factors can activate MMP-2, although there is both organ and species variation. MMP-2 from human umbilical vein endothelial cells (43) and vascular smooth muscle (8) is activated by thrombin, whereas in rabbit aortic smooth muscle cells, thrombin and FBS do not affect synthesis of MMP-2, although synthesis of MMP-9 is increased (6), suggesting cell-specific differences in the regulation of gelatinase activity. Further categorization of the production of MMPs by airway smooth muscle and the factors governing secretion, activation, and inhibition by the tissue inhibitors of metalloproteinase is required.
In summary, we have shown that human airway smooth muscle cells secrete MMP-2 and that inhibition of MMP-2 reduces airway smooth muscle proliferation in culture. These data suggest that airway smooth muscle contributes to extracellular matrix turnover and remodeling in the airways and that MMP activity is required for airway smooth muscle proliferation. Inhibition of MMPs may provide a new therapeutic target for the prevention of irreversible airflow obstruction in asthma.
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ACKNOWLEDGEMENTS |
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We are grateful to Hilary Collins and Dr. Sue Watson (Department of Cancer Studies, University of Nottingham, Nottingham, UK) for assistance with zymography; Dr. Colin Clelland, John Ronan, Jane Bell, and Heather Key (Department of Histopathology, City Hospital, Nottingham) for assistance with immunohistochemistry; and Dr. Sharon Crouch (Lumitech, Nottingham, UK) for technical advice.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: S. Johnson, Division of Respiratory Medicine, University of Nottingham, City Hospital, Hucknall Rd., Nottingham NG5 1PB, UK.
Received 23 December 1998; accepted in final form 26 August 1999.
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