Differential Effects of Extracellular Matrix Proteins

Differential Effects of Extracellular Matrix Proteins on Human Airway Smooth Muscle Cell Proliferation and Phenotype Stuart J. Hirst, Charles H. C. Twort, and Tak H. Lee Department of Respiratory Medicine and Allergy, The Guy’s, King’s, and St. Thomas’ School of Medicine, King’s College London, Guy’s Hospital Campus, London, United Kingdom

Mature airway smooth muscle cells are characterized by a low proliferative index and expression of contractile marker proteins such as smooth muscle ␣-actin (sm-␣-actin), calponin, and smooth muscle myosin heavy chain (sm-MHC). In the present study, defined extracellular matrix (ECM) components were examined on the proliferative and phenotypic status of mitogenstimulated, cultured human airway smooth muscle cells. The results demonstrate that although cells adhered and spread on plates precoated with (1 to 100 ␮g/ml) of fibronectin (FN), collagen I (Col I), laminin (LN), or Matrigel, their subsequent proliferative response varied qualitatively. FN and Col I enhanced proliferation in response to either platelet-derived growth factor (PDGF)-BB or ␣-thrombin, compared with cells on plastic. LN, however, reduced mitogen-stimulated proliferation. A similar reduction was found in cells cultured on Matrigel. The effect of ECM substrates on contractile phenotype was determined by examining cellular expression of sm-␣-actin, smMHC, and calponin using immunocytochemical and flow cytometric methods. Approximately 75% of PDGF-BB–stimulated cells, cultured on LN or Matrigel, expressed sm-␣-actin, calponin, and sm-MHC, but only 8 to 10% stained for the Ki67 nuclear antigen proliferation marker. In contrast, more than 75% of cells cultured on FN or Col I were positive for Ki67 antigen, but only 20% were positive for contractile proteins. Flow cytometric analysis of sm-␣-actin and DNA content confirmed the immunocytochemical findings and showed that the observed reduction in sm-␣-actin content after culture on FN or Col I, compared with LN and Matrigel, occurred in the majority of the cell population, supporting bidirectional phenotype modulation. Overall, the data suggest that ECM substrates modulate both proliferation and phenotype of human airway smooth muscle cells in culture.

Chronic persistent asthma is often characterized by poorly reversible airway obstruction and is associated with intense inflammation and structural remodeling of the airway wall (1, 2). One of the most prominent structural features associated with these changes is an increase in the content of airway wall smooth muscle. This is believed to involve cellular hyperplasia and/or hypertrophy of the smooth muscle (3). (Received in original form October 21, 1999 and in revised form April 13, 2000) Address correspondence to: Dr. Stuart J. Hirst, Dept. of Respiratory Medicine and Allergy, The Guy’s, King’s and St. Thomas’ School of Medicine, Thomas Guy House, Guy’s Hospital Campus, London SE1 9RT, UK. E-mail: [email protected] Abbreviations: analysis of variance, ANOVA; bovine serum albumin, BSA; collagen, Col; Dulbecco’s modified Eagle’s medium, DMEM; ethylenediaminetetraacetic acid, EDTA; extracellular matrix, ECM; fetal bovine serum, FBS; fluorescein isothiocyanate, FITC; fibronectin, FN; immunoglobulin, Ig; laminin, LN; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, MTT; optical density, OD; phosphate-buffered saline, PBS; PBS containing 0.1% Tween 20 and 0.1% BSA, PBT; platelet-derived growth factor, PDGF; smooth muscle ␣-actin, sm-␣-actin; smooth muscle myosin heavy chain, sm-MHC. Am. J. Respir. Cell Mol. Biol. Vol. 23, pp. 335–344, 2000 Internet address: www.atsjournals.org

This apparent functional diversity of the smooth muscle response in airway remodeling has prompted interest in the possibility that there is plasticity in its function which may be related to the severity of the tissue remodeling process during chronic inflammation of the airway wall (4, 5). Another prominent feature of airway wall remodeling is an abnormally thickened basement membrane (1, 2). This is thought to involve increased deposition of extracellular matrix (ECM) proteins in the subepithelial “basement membrane” (lamina reticularis), though more subtle changes may also occur in the basal lamina. Together, these changes in the airway wall structure are associated with the development of persistent airway obstruction and increased, nonspecific airway hyperresponsiveness (1, 6), and may contribute to the accelerated decline in airway function in chronic severe asthma (7). The precise cellular and molecular mechanisms, however, that are responsible for these changes in airway structure and function remain poorly characterized. The ECM that surrounds cells both in vivo and in vitro is a highly organized and dynamic structure composed of several macromolecule groups. It contributes to the control of cellular function and is involved in the maintenance of the cell’s state of differentiation (8). Abnormalities in the ECM have been proposed to contribute to airway structural remodeling in asthma (2), although the nature and source of the ECM elements involved have been little studied. Biopsy studies of asthmatic airways report deposition of collagen (Col) types I, III, and V, and fibronectin (FN) in the subepithelium basement membrane but not of Col IV or laminin (LN) (9, 10). Other studies confirm that in asthma, subepithelial fibrosis is associated with increased deposition of Col III and V, FN, LN (2, 11), and tenascin (11, 12) as well as other components such as decorin, hyaluranon, and versican (13). Increased collagen deposition is also reported in and around the airway smooth muscle bundles (14) and between the smooth muscle and basement membrane (15). In severe asthma, there are changes in the molecular structure of LN where upregulation of LN ␣2 and ␤2 chains is present, possibly reflecting a shift in the molecular phenotype of LN toward that normally associated with lung morphogenesis (11). Increased levels of FN, hyaluronate, and LN products, consistent with increased ECM turnover, are found in the bronchoalveolar lavage fluid of asthmatics (2), and the observed increases in basement membrane thickness (16) have been linked with asthma severity (17). Such changes are believed to be related to the inflammation and altered tissue repair mechanisms thought to contribute to the airway wall remodeling process (11), but their impact on airway smooth muscle has not been previously investigated. In the wall of the intact, healthy mature airway, the ma-

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jority of airway smooth muscle cells are thought to exist in a quiescent and fully differentiated state with a low proliferative index. Based on their functional, biochemical, and ultrastructural properties, the differentiated state of smooth muscle cells is characterized in part by well-developed contractile elements and a stable ECM environment (18). What distinguishes smooth muscle from the airways and other tissues (e.g., the vascular wall and gastrointestinal tract) from other muscle cell types (e.g., skeletal myocytes and cardiomyocytes) is their capacity to re-enter the cell cycle and divide. This remarkable functional plasticity is thought to be necessary for growth and repair during injury when additional functions such as proliferation and secretion are apparent (5, 18). While it is generally well accepted that in the airway wall the function of the contractile smooth muscle phenotype is dedicated to the regulation of airway caliber, the existence and function of the proliferative-synthetic phenotype are less clear. In systemic vascular disease, the synthetic smooth muscle cell is believed to be central to the pathogenesis of the repair injury response of an atherosclerotic lesion (18) and is responsible for the production of paracrine and autocrine factors, and the synthesis and deposition of ECM components (18). With increasing recognition that changes in the functional and biochemical diversity of airway smooth muscle may be important for the disease progression in asthma, some investigators have explored possible similarities in the behavior of smooth muscle in the structural remodeling process of both the vascular and airway walls (4, 5). In the present study, we tested the hypothesis that specific components of the ECM, which are reported to be altered in chronic severe asthma, can regulate both the proliferation and contractile protein expression of human airway smooth muscle in cell culture. Our results indicate that interstitial ECM proteins such as FN and Col I promote proliferation of human airway smooth muscle cells and suppress contractile protein expression. In contrast, basement membrane elements such as LN inhibit proliferation and support a more contractile phenotype.

Materials and Methods Airway Smooth Muscle Cell Isolation and Culture Human bronchial smooth muscle was obtained from the lobar or main bronchus of 19 patients of either sex (mean age, 62 ⫾ 5 yr; range, 25 to 79 yr) undergoing lung resection for carcinoma of the bronchus, as previously described in detail (19). After removal of the epithelium, portions of the smooth muscle not invaded by the carcinoma were dissected free of adherent connective and parenchymal tissue under aseptic conditions in Hanks’ balanced salt solution. The smooth muscle was digested in 2 ml Dulbecco’s modified Eagle’s medium (DMEM) (supplemented with 1 mM sodium pyruvate, 2 mM L-glutamine, 1:100 nonessential amino acid mixture, 50 ␮g/ml gentamicin, and 1.5 ␮g/ml amphotericin B) containing 1 ␮M insulin, 5 ␮g/ml transferrin, 100 ␮M ascorbate, 1 mg/ ml bovine serum albumin (BSA), and 3 mg/ml collagenase. After 30 min incubation at 37⬚C, the tissue was transferred to a similar enzyme mixture containing 3 U/ml elastase and further dissected under a microscope to remove any unwanted fibrous tissue from within the muscle itself. The tissue was chopped finely (approximately 1 mm3) and returned to the incubator for a further 18 h until fully digested. The resulting cell suspension was centrifuged (200 ⫻ g for 5 min) and the pellet washed in supplemented DMEM

containing 10% fetal bovine serum (FBS). Cells were seeded at 5 ⫻ 105 viable cells in 25 cm2 culture flasks and maintained in a humidified atmosphere at 37⬚C in 5% CO2/95% air. Fresh medium was replaced every 72 h. After 14 to 18 d, human airway smooth muscle cells in primary culture grew to confluence and were subcultured by incubating each flask with 3.5 ml trypsin/ethylenediaminetetraacetic acid (EDTA) (0.1 mg/ml in phosphate-buffered saline [PBS]) solution for 5 min before adding an equal volume of supplemented DMEM containing 10% FBS and mechanically dispersing the cells by repeated gentle pipetting. Cells were centrifuged (200 ⫻ g for 5 min) and resuspended in supplemented DMEM containing 10% FBS and seeded either into 24-well plates for experimental work or into a 75-cm2 culture flask to continue each cell line. Cells at passages 2 to 5 were used for all experiments during which the proliferative response to FBS and growth factors was unchanged (20).

Surface Coating for Smooth Muscle Cell Culture Mouse LN, purified from Engelbreth-Holm-Swarm sarcoma, and bovine plasma FN were reconstituted in sterile PBS without Ca2⫹ and Mg2⫹, and diluted in PBS to concentrations between 0 and 100 ␮g/ml. Diluted ECM proteins (0.5 ml) were adsorbed to 24well plates for 4 to 6 h at room temperature. Lyophilized Col I, reconstituted at 3 mg/ml in 0.01 M hydrochloric acid before dilution in PBS, was applied to the culture plates and air-dried at room temperature. Unoccupied protein-binding sites were blocked by a further 30-min incubation with 0.1% BSA after which coated surfaces were washed twice with unsupplemented, FBS-free DMEM. For cells grown on Matrigel, the growth factor–reduced Matrigel preparation was used. After thawing overnight at 4⬚C, it was mixed to homogeneity using cooled pipettes. Culture dishes were coated with 50 ␮l/cm2 of undiluted Matrigel at 4⬚C and then placed in the incubator for 1 h at 37⬚C before equilibration with FBS-free DMEM for 1 h at 37⬚C before addition of cells.

Attachment and Proliferation of Human Airway Smooth Muscle Cells Human airway smooth muscle cells were harvested from 75-cm2 flasks during passages 2 to 4, by treatment with 0.01% trypsin/ EDTA in Ca2⫹/Mg2⫹-free PBS. Cells were washed twice in PBS and transferred in DMEM containing 0.5% FBS into 24-well plates precoated with ECM protein at a seeding density of 2 ⫻ 104 cells/well. At varying time intervals, plates were removed from the incubator and the overlying medium removed by gentle aspiration. After washing in 0.5 ml PBS, the remaining adherent cells were removed by trypsin/EDTA (0.25 ml/well) and counted using a hemocytometer. For proliferation studies, cells were left undisturbed for 8 h. Thereafter, the medium was replaced with FBS-free DMEM, supplemented with insulin (1 ␮M), transferrin (5 ␮g/ml), ascorbate (100 ␮M), and BSA (0.1% wt/vol). Growth was initiated after 72 h by replacement of the FBS-free medium with fresh FBS-free DMEM containing either platelet-derived growth factor (PDGF)-BB or ␣-thrombin. Fresh medium containing mitogens was replaced every 48 h until confluence was reached. Proliferation of human airway smooth muscle cells was assessed on Days 4, 7, 9, and 11 after initiation of growth, by mitochondrial-dependent reduction of 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide (MTT) to formazan (21). Cell monolayers were washed twice in 500 ␮l DMEM containing 10% FBS. MTT (100 ␮l of 5 mg/ml in PBS; final concentration, 0.8 mg/ ml) was then added to each well and the cells incubated at 37⬚C for 6 h before overnight solubilization in a further 500-␮l sodium dodecyl sulfate (10% wt/vol in 0.01 M HCl). A sample (150 ␮l) from each duplicate well was then transferred to a 96-well microplate and the optical density determined by automated dual

Hirst, Twort, and Lee: Matrix and Airway Smooth Muscle Proliferation

wavelength spectrophotometry (model HTII; Anthos, Salzburg, Austria) against a reagent blank (i.e., no cells) at a test wavelength of 570 nm and a reference of 630 nm. The application of this method to human airway smooth muscle cell proliferation has already been described in detail (20). The resultant increase in optical density correlates directly with cell number, as determined by hemacytometry in parallel experiments (20).

Cell Morphology and Immunofluorescence Microscopy Cells on Lab-Tek 4-well multichamber plastic microscope slides, coated with ECM substrates, were allowed to proliferate in response to peptide mitogens for 5 d. Cells were then washed in icecold Ca2⫹/Mg2⫹-free PBS and immediately fixed in ice-cold 70% ethanol for 20 min. Fixed cells were either prepared for morphologic examination using phase contrast or differential interference contrast (Nomarski) microscopy, or prepared for epifluorescent immunocytochemical examination. For the latter, monolayers of ethanol-fixed cells were washed in ice-cold PBS containing 0.1% vol/vol Tween 20 for 5 min and then blocked in 1% wt/vol normal goat serum for 30 min. Mouse antihuman monoclonal primary antibodies to smooth muscle ␣-actin (sm-␣-actin) (1:200 dilution in 0.1% wt/vol BSA in PBS), smooth muscle myosin heavy chain (sm-MHC) (1:100), calponin (1:10,000), vimentin (1:50), and Ki67 (undiluted) were incubated with cells (200 ␮l/well) for 3 h at 37⬚C, followed by goat antimouse fluorescein isothiocyanate (FITC)– conjugated secondary antibody (1:100 dilution in 0.1% wt/vol BSA in PBS; 1 h at 37⬚C) after washing in PBS (twice for 5 min). In control experiments, primary and/or secondary antibodies were omitted from the protocol and cells were incubated in either 200 ␮l PBS containing 0.1% wt/vol BSA alone or in the presence of concentration-matched, isotype-matched controls (immunoglobulin [Ig] G1 and IgG2a). After immunostaining, cells were washed twice in distilled, deionized water, mounted in PBS/glycerol (1:9 vol/vol), and viewed with an Olympus BX-50 microscope (Olympus Optical Co., London, UK) equipped with reflected ultraviolet light illumination. The number of strongly positive staining cells in 10 separate, random photographed fields was counted (three Lab-Tek chambers for each experimental condition) and expressed as a percentage of the total number of cells present in each field using phase-contrast microscopy.

Flow Cytometric Analysis Cells were grown in 25-cm2 flasks, either directly on the plastic or after coating with ECM proteins (10 ␮g/ml), and were harvested using trypsin/EDTA after stimulation for 5 d with peptide mitogens. Cells grown on Matrigel were isolated by incubation with undiluted Matrisperse solution and placed on ice for 30 min to disperse the Matrigel substrate. Cells were washed in ice-cold PBS (200 ⫻ g for 5 min) and fixed dropwise in 70% ethanol at 4⬚C for at least 30 min before overnight storage at ⫺20⬚C. Fixed cells were then placed on ice for 30 min before centrifugation (300 ⫻ g for 5 min). The resultant pellets were resuspended in 3 ml ice-cold PBS containing 0.1% Tween 20 and 0.1% BSA (PBT) by vigorous stirring using a vortex mixer. Cell suspensions were filtered through a disposable cell strainer (70-␮m nylon mesh) to remove cell aggregates and cell numbers were determined by hemocytometry. Aliquots of 1 ⫻ 106 cells were centrifuged (200 ⫻ g for 5 min) and the pellets resuspended in 0.5 ml PBT containing primary monoclonal antibodies to either contractile phenotype marker proteins or their isotype-matched, monoclonal antibody control proteins. Antibody titers were the same as those used in the immunocytochemical studies. Cells were incubated with antibodies for 16 to 18 h at 4⬚C using constant agitation, after which cell suspensions were diluted with 10 ml ice-cold PBT and washed twice by centrifugation before resuspension in PBT containing a complementary FITC-conjugated, goat antimouse IgG secondary anti-

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body. After 3 to 4 h, the cell suspension was diluted with 10 ml PBT and then centrifuged. For DNA ploidy determinations, the final pellet of immunolabeled cells was resuspended at room temperature in 1 ml PBS containing 20 mM EDTA, 25 ␮g/ml DNase-free RNase, and 15 ␮g/ml propidium iodide. Suspensions of dual-labeled cells were returned to ice for at least 1 h before flow cytometric analysis. After flow cytometry, cells were confirmed microscopically to be intact. Simultaneous analysis of sm-␣-actin immunofluorescence and DNA fluorescent labeling was determined on a flow cytometer (FACScan; Becton Dickinson, Oxford, UK) using an argon laser (488 nm). For detection of FITC fluorescence, a 530-nm bandpass filter was placed in the light path. Forward versus 90⬚ light scatter histograms were used to gate on intact cells and eliminate debris. Detection of propidium iodide fluorescence was determined through a 585-nm band-pass filter. Fluorescence histograms of 1,024 channel resolution were collected for at least 10,000 events, which satisfied the light scatter gating criteria at a flow rate of 60 ␮l/min. The distribution of cells in G0/G1, S, and G2/M of the cell cycle was determined using the LYSIS (TM II) analysis program (Becton Dickinson).

Statistical Analysis Data in the text and figure legends are expressed as mean ⫾ standard error of the mean (SEM) of observations obtained from airway smooth muscle cells cultured from n patients. Results were compared using one-way analysis of variance (ANOVA) followed by Bonferroni’s t test to determine statistical differences after multiple comparisons (SigmaStat; Jandel Scientific, Erkrath, Germany). A probability value of less than 0.05 was considered significant.

Materials All chemicals were of analytical grade or higher. Recombinant human PDGF-BB, cell culture medium, FBS, and cell culture reagents were obtained from GIBCO BRL Life Technologies (Paisley, UK). Collagenase (type CLS 1) and elastase (type 1) were obtained from Worthington Biochemical Corporation (Freehold, NJ). Matrigel growth factor reduced (GFR) and Matrisperse were obtained from Collaborative Research (Becton Dickinson). Mouse monoclonal antihuman Ki67 antigen (clone no. 7b11) was obtained from Zymed Laboratories (San Francisco, CA). Propidium iodide was purchased from Molecular Probes (Leiden, The Netherlands). Human reactive, mouse monoclonal primary antibodies to smMHC (clone no. hSM-V), sm-␣-actin (clone no. 1A4), calponin (clone no. hCP), and vimentin (clone no. V9), mouse IgG1 and IgG2a isotype controls, and goat antimouse FITC-conjugated IgG secondary antibody were purchased from Sigma (Poole, UK). All other chemical reagents, including human plasma ␣-thrombin, human plasma FN, rat-tail Col I, and mouse LN-1 (extracted from the basement membrane of Engelbreth-Holm-Swarm mouse sarcoma) were also obtained from Sigma. Lab-Tek four-well multichamber plastic microscope slides were purchased from NUNC Laboratories (Life Technologies). All other cell culture plasticware was purchased from Falcon Labware (Becton Dickinson).

Results Effects of Extracellular Adhesive Proteins on Airway Smooth Muscle Cell Attachment Early passage human airway smooth muscle cells (1.25 ⫻ 104 cells/cm2), removed from their plastic-based culture flasks using trypsin/EDTA (0.01%), adhered to various ECM substrates within 2 to 5 h after seeding. Attachment onto FN-coated (10 ␮g/ml) plates was significantly greater during the first 3 h than with plastic or any other ECM

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tically significant at 2 and 3 h (P ⬍ 0.001 by Bonferroni’s t test; n ⫽ 4). Cellular adherence directly onto plastic was initially (1 to 5 h) significantly less compared with any of the ECM substrates examined, excepting Matrigel (P ⬍ 0.001 by Bonferroni’s t test; n ⫽ 4). However, by 6 h no statistical difference in the extent of adherence to any substrate was detected (Figure 1) (P ⬎ 0.05 by ANOVA; n ⫽ 4).

Figure 1. Attachment human airway smooth muscle cells to varying ECM substrates. Near confluent cells (2 ⫻ 104 cells/well) were allowed to settle either directly onto the plastic surface of culture plates (circles) or after coating with 10 ␮g/ml FN (squares), Col I (triangles), LN (diamonds), or 50 ␮l/cm2 undiluted Matrigel (inverted triangles). Points represent mean ⫾ SEM of duplicate values from independent experiments using cells cultured from four patients, cell passages 2 to 4. ***P ⬍ 0.001 FN versus all other substrates by Bonferroni’s t test. NS, P ⬎ 0.05 by ANOVA.

substrate investigated (P ⬍ 0.001 by Bonferroni’s t test; n ⫽ 4). Attachment was apparent within 5 to 15 min of seeding, and by 2 h more than 90% of the cells were adhered (Figure 1). Attachment to 10 ␮g/ml Col I was also rapid, occurring within 30 min of plating, with all cells being attached by 2 to 3 h. Adherence onto 10 ␮g/ml LN was similar to Col I. In contrast, attachment to Matrigel was much reduced compared with the other matrices. This was statis-

Effects of Extracellular Adhesive Proteins on Airway Smooth Muscle Cell Morphology On FN, cell spreading was visible by phase-contrast microscopy within 1 h of plating, and all cells were elongated (i.e., bipolar) by 4 h (data not shown). Similarly, all cells that attached to Col I were bipolar at 4 h. In contrast, cells on LN remained rounded for up to 4 h with complete elongation occurring only after 5 to 7 h. Likewise, on Matrigel, though most cells were firmly attached by 6 h, they remained rounded (data not shown). After stimulation for 5 d with PDGF-BB (5 ng/ml), cells grown on plastic adopted the characteristic “hill and valley” pattern (Figure 2A). On FN (10 ␮g/ml) and Col I (10 ␮g/ml) (data for Col I not shown), though many were elongated and arranged more or less in parallel, cells were less organized and possessed multiple processes (Figure 2B), features that are more often associated with later passage cells. The characteristic “hill and valley” appearance was less apparent. Growth on Matrigel (undiluted) revealed a grossly different morphology compared with cells grown on plastic. Cells grown on Matrigel were characterized by aggregation into multilayered nodules with spindle-like networks or cables of elongated cells extending to neighboring nodules (Figure 2C). Cells on LN were also markedly

Figure 2. Morphologic comparison of human airway smooth muscle cells cultured on plastic or ECM protein– coated microscope slides, examined using phase-contrast microscopy. Cells were grown in FBS-free DMEM in the presence of PDGF-BB (5 ng/ ml) for 5 d on (A) uncoated plastic slides or after coating with (B) 10 ␮g/ml FN, (C) 50 ␮l/cm2 undiluted Matrigel, or (D) 10 ␮g/ml LN. Bars represent 200 ␮m. Photomicrographs are representative of cells cultured from five separate patients, cell passages 2 to 3.


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Figure 3. Effect of FN, Col I, and LN on proliferation of human airway smooth muscle induced either by (a) PDGF-BB (5 ng/ ml) or (b) ␣-thrombin (0.5 U/ml). Cells were cultured in the absence (open circles) or presence (open squares) of peptide mitogen on plastic or in the presence of mitogen on 1 ␮g/ml (solid circles), 3 ␮g/ml (solid squares), 10 ␮g/ml (solid triangles), 30 ␮g/ml (solid diamonds), or 100 ␮g/ml (solid inverted trianges) of each ECM protein substrate. Points represent mean of duplicate optical density (OD) values obtained from five independent experiments using cells cultured from five patients, cell passages 2 to 4. Error bars have been omitted for clarity but were within 12% of the mean. See text for details of statistical comparisons.

different in their morphology compared with plastic, FN, or Col I. Cells formed loose clumps with interconnecting radial processes (Figure 2D) but were more flattened and spread compared with cells on Matrigel. Effects of Extracellular Adhesive Proteins on Airway Smooth Muscle Cell Proliferation After attachment and spreading on different matrices for 8 h, human airway smooth muscle cells were growth-arrested for 72 h and then stimulated to divide by replacement of the medium with supplemented FBS-free DMEM containing either the B-chain homodimer of PDGF-BB or the serine proteinase ␣-thrombin. When attached to varying concentrations of FN (1 to 100 ␮g/ml) and stimulated to divide by PDGF-BB (5 ng/ml), there was a concentration-dependent increase in the responsiveness of cells over 11 d compared with cells growing directly on the plastic (Day 11: plastic versus 100 ␮g/ml FN; P ⬍ 0.001 by Bonferroni’s t test; n ⫽ 5) (Figure 3A). The concentration of FN, which enhanced this effect by 50% (EC50) on Day 9, was 6 ⫾ 0.5 ␮g/ml. Similarly, when coated with increasing concentrations of Col I, proliferation in response to PDGF-BB was enhanced (Day 11: plastic versus 100 ␮g/ml Col I; P ⬍ 0.001 by Bonferroni’s t test; n ⫽ 5). The EC50 for the effect of Col I was 9 ⫾ 1 ␮g/ml. In contrast, LN (IC50, 4 ⫾ 0.5 ␮g/ml) reduced the responsiveness of cells to PDGF-BB. Maximal inhibition occurred at 30 ␮g/ml where the response was not significantly different from proliferation on Day 11 on plastic in the absence of mitogen (P ⬎ 0.05 by ANOVA; n ⫽ 5). When ␣-thrombin (0.5 U/ml) was substituted for PDGF-BB as a smooth muscle mitogen, the same profile of responsiveness to each of the ECM substrates was observed. Both FN (EC50, 2.4 ⫾ 0.7 ␮g/ml) and Col I (EC50, 2 ⫾ 0.4 ␮g/ml) increased the responsiveness to ␣-thrombin (Day 10: plastic versus 100 ␮g/ml FN or Col I; P ⬍ 0.001 by Bonferroni’s t test; n ⫽ 5), whereas LN (IC50, 7 ⫾ 0.6 ␮g/ml) inhibited the response back to unstimulated levels (Figure 3B). In four experiments, the effect of each ECM substrate

was compared on the concentration-relationship for each peptide mitogen. Coating of the culture plates with either FN (30 ␮g/ml) or Col I (30 ␮g/ml) enhanced the maximum proliferative response of airway smooth muscle cells to either PDGF-BB (P ⬍ 0.005 by Bonferroni’s t test) or ␣-thrombin (P ⬍ 0.005 by Bonferroni’s t test), when compared with cells proliferating directly on the plastic. In contrast, coating the plasticware with LN (30 ␮g/ml) significantly attenuated the maximal proliferation induced by either mitogen (P ⬍ 0.005 by Bonferroni’s t test) (Figure 4). A similar effect was seen on Matrigel (P ⬍ 0.001 by Bonferroni’s t test). In the absence of mitogen, however, no differences in cellular proliferation were detected for any of the substrates investigated (P ⬎ 0.05 by ANOVA). Effects of Extracellular Adhesive Proteins on Airway Smooth Muscle Cell Contractile Protein Expression and Proliferative Status Immunocytochemical detection. After 5 d in culture, approximately 65 to 75% of early passage (1 to 3), unstimu-

Figure 4. Proliferation induced by (a) PDGF-BB and (b) ␣-thrombin in human airway smooth muscle cells cultured for 7 d on plastic (open circles) or on the ECM substrates (30 ␮g/ml) FN (solid circles), Col I (solid squares), LN (solid triangles), or 50 ␮l/cm2 undiluted Matrigel (solid diamonds). Points represent mean ⫾ SEM of duplicate optical density (OD) values obtained from four independent experiments using cells cultured from four patients, cell passages 2 to 4. **P ⬍ 0.05, ***P ⬍ 0.001 compared with plastic by Bonferroni’s t test.

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lated human airway smooth muscle cells on plastic expressed sm-MHC, calponin, and sm-␣-actin myofilament immunoreactivity in their cytoplasm (Table 1). Positive nuclear staining for the Ki67 nuclear antigen, a proliferative index marker, was detected in less than 5% of the total cell population. After stimulation with PDGF-BB (5 ng/ml), however, positive Ki67 nuclear staining was detected in more than 60% of the total population (P ⬍ 0.005 by Bonferroni’s t test; n ⫽ 6), but positive staining for all three contractile marker proteins was significantly reduced (P ⬍ 0.05 by Bonferroni’s t test; n ⫽ 6) (Table 1). In contrast, expression of contractile marker proteins in PDGF-stimulated cells grown on LN (10 ␮g/ml) remained high (Figures 5A, 5C, and 5E). The number of cells with positive nuclear staining for the proliferative marker Ki67 was significantly reduced (Figure 5G) (P ⬍ 0.005 by Bonferroni’s t test; n ⫽ 6) compared with PDGF-stimulated cells growing on plastic. Similar data were obtained for PDGF-stimulated cells grown on Matrigel (summarized in Table 1). In contrast, the majority of cells grown on FN (10 ␮g/ml) showed significantly less immunoreactivity for contractile marker proteins (Figure 5B, 5D, and 5F) (sm-MHC and sm-␣-actin, P ⬍ 0.005; calponin, P ⬍ 0.05 by Bonferroni’s t test; n ⫽ 6) compared with cells grown on LN but were significantly more positive for the Ki67 nuclear antigen (P ⬍ 0.005 by Bonferroni’s t test; n ⫽ 6) (Figure 5H). Similar data were obtained for cells grown on Col I (10 ␮g/ml) and are summarized in Table 1. Simultaneous detection by flow cytometry. In separate experiments using cells (passages 2 to 3) cultured from four patients, flow cytometric analysis of the sm-␣-actin content detected in unstimulated cells after culture on plastic for 5 d showed a uniform sharp peak of fluorescence compared with the negative IgG2a isotype control that showed relatively dull uniform staining (Figure 6). Under these culture conditions, the distribution of propidium iodide fluorescence in immunolabeled cells revealed

TABLE 1

Immunocytochemical detection of contractile phenotype marker proteins in human airway smooth muscle cells cultured for 5 d in the presence of PDGF-BB on plastic or varying ECM substrates Percentage of Cells Staining sm-MHC Calponin sm-␣-actin

Plastic Plastic ⫹ PDGF-BB FN ⫹ PDGF-BB Col I ⫹ PDGF-BB LN ⫹ PDGF-BB Matrigel ⫹ PDGF-BB

65 ⫾ 4 25 ⫾ 7 8⫾2 7⫾3 69 ⫾ 7 73 ⫾ 8

76 ⫾ 5 31 ⫾ 3 19 ⫾ 4 17 ⫾ 6 74 ⫾ 3 68 ⫾ 6

71 ⫾ 6 30 ⫾ 7 12 ⫾ 5 13 ⫾ 3 77 ⫾ 6 81 ⫾ 10

Ki67

Vimentin*

5⫾1 62 ⫾ 5 76 ⫾ 6 82 ⫾ 7 10 ⫾ 4 8⫾4

⫹ ⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹ ⫹(⫹)

Cellular proliferative status was identified by positive immunoreactivity for the Ki67 nuclear antigen. The number of strongly positive staining cells in 10 separate random fields was counted under epifluorescent illumination and expressed as a percentage of the total number of cells present in each field using phase-contrast microscopy. *For vimentin, where all cells were found to stain, cells were classified into positive (⫹), strongly positive (⫹⫹), or intermediate (⫹[⫹]). Data are representative of five independent experiments using cells cultured from six separate patients, cell passages 2 to 3. See legend to Figure 5 for explanation of abbreviations and culture conditions.

that 76% of gated total cells were in the G0 and/or G1 phase of the cell cycle. When PDGF-BB (5 ng/ml) was present, the mean fluorescence intensity for the sm-␣-actin content of this cell population was significantly reduced (P ⬍ 0.05 by Bonferroni’s t test; n ⫽ 4) (Figures 6a and 6b) and the proportion of cells outside the G0 and/or G1 phase of the cell cycle increased from 24 to 46% (P ⬍ 0.05 by Bonferroni’s t test; n ⫽ 4), consistent with phenotypic modulation from a contractile to a more synthetic-proliferative state. Similar data were obtained for cells cultured in the presence of PDGF-BB for 5 d on either FN or Col I (10 ␮g/ml). When cultured on LN (10 ␮g/ml) (Figures 6a and 6b) or undiluted Matrigel (Figure 6b), however, sm-␣-actin content in human airway smooth muscle cells was relatively high (P ⬍ 0.05 by Bonferroni’s t test; n ⫽ 4) compared with cells grown on Col I, FN, or plastic, despite the presence of PDGF-BB, and up to 75% of the cells remained in the G0 and/or G1 phase of the cell cycle (Figure 6b).

Discussion The major finding of this study was that specific components of the ECM differentially regulated the responsiveness of subcultured human airway smooth muscle cells to peptide mitogens and that this effect was accompanied by reciprocal changes in the biochemical contractile phenotype of these cells. To investigate these phenomena, several complementary approaches were used. In the first, proliferation of early-passage human airway smooth muscle cells grown on defined ECM proteins was assessed using the MTT reduction colorimetric assay. To determine whether the observed proliferative changes were accompanied by changes in cellular phenotype, expression of smooth muscle contractile proteins was examined using immunocytochemistry and bivariate flow cytometric analysis. Likewise, positive nuclear staining for the Ki67 antigen and the ploidy marker propidium iodide were used to identify the proliferative status of cells maintained on different ECM substrates in order to corroborate the growth curve data obtained from the MTT assay. In cell culture–based studies of vascular smooth muscle, ECM components have been shown to influence the proliferative response differentially (22–24). In keeping with these studies, we found that human airway smooth muscle cells showed little or no proliferation in the absence of mitogen, indicating that the majority were in a quiescent state. After stimulation with PDGF-BB or ␣-thrombin, however, large numbers of cells entered the cell cycle (determined by Ki67 nuclear antigen immunoreactivity and propidium iodide content) and replicated (determined by reduction of MTT). When seeded on substrates of either FN or Col I, cells were distinctly more responsive to peptide mitogens than were cells seeded on LN or plastic. Proliferation was not induced by the ECM proteins alone, indicating that the presence of mitogens was required before the growth-modulating effects could be manifest. The differential effects observed with the ECM proteins were unlikely to result from variable attachment to the matrices (causing varying cell densities at the beginning of the proliferation studies) because by 6 h complete attachment to each of the substrates investigated had occurred, and only


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Figure 5. Fluorescent immunocytochemistry of human airway smooth muscle cells showing differential regulation of smooth muscle contractile proteins by the ECM proteins LN (A, C, E, G) and FN (B, D, F, H) after culture for 5 d in the presence of PDGF-BB (5 ng/ml). Cells were stained for sm-MHC (A, B), calponin (C, D), and sm-␣-actin (E, F). The proliferative status of cells growing on either LN or FN substrates was confirmed by positive staining for the Ki67 nuclear antigen (G, H). Bars represent 50 ␮m. Photomicrographs are representative of five independent experiments using cells cultured from six separate patients, cell passages 2 to 3.

thereafter was the period of growth arrest and proliferation begun. Likewise, the initial proliferative response in the absence of either mitogen was similar regardless of the ECM environment conferred on the cells. Cells seeded on Matrigel, a solubilized basement membrane matrix extracted from the Engelbreth-Holm-Swarm mouse sarcoma (25), were very different in their morphology compared with cells growing on plastic and were also less responsive to peptide mitogens, confirming a previous report by Li and colleagues (26) showing attenuation of serum-stimulated proliferation of rat aortic smooth muscle cells by Matrigel. The main molecular species component thought to be responsible for this effect is LN-1, and, indeed, the source of LN used in this study was also purified from the Engelbreth-Holm-Swarm mouse sarcoma. Other factors that retard smooth muscle cell proliferation are also present in the Matrigel formulation (22, 27). These include heparan sulfate proteoglycans, Col IV, and entactin/nidogen. Naturally occurring growth factors are also present, but in the present study, a growth factor–reduced preparation was used to limit their contribution to altered smooth muscle behavior. We noted that the growth inhibitory effect of Matrigel and LN persisted only for 6 to 7 d after stimulation. At later time points (Days 9 and 11), the higher

concentrations of LN (⬎ 10 to 30 ␮g/ml) appeared less effective than lower concentrations (⬍ 10 ␮g/ml). One explanation for this effect is that the principal integrin receptors that mediate the growth-retarding effects of LN, namely ␣1␤1 and ␣2␤1 (28), also interact with other ligands such as Col I, which may be produced by these cells, thereby counteracting the growth retardation effect of LN. In addition, loss in growth retardation by LN at late time points may also reflect elaboration of FN by airway smooth muscle cells, as has been reported elsewhere (29) for smooth muscle cells cultured from rat aorta. Future studies will address the effectiveness of FN-blocking peptides to prolong the growth-retarding activity of LN in cultures of human airway smooth muscle. To examine whether the observed effects of ECM proteins on airway smooth muscle proliferation were associated with changes in cellular phenotype, their effect on expression of smooth muscle contractile phenotype marker proteins was examined. Halayko and colleagues (30) identified markers of smooth muscle contractile phenotype in freshly dispersed canine tracheal smooth muscle cells and in their cultured proliferating counterparts. They found that the content of sm-␣-actin, sm-MHC, and calponin diminished by more than 75% when cells became prolifera-

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Figure 6. Flow cytometric analysis of sm-␣-actin content and ploidy in human airway smooth muscle cells cultured for 5 d in the presence of PDGF-BB (5 ng/ml) on plastic or on varying ECM substrates. Representative histograms (a) show the effect of FN and LN on the frequency distribution of cells stained for sm-␣-actin compared with cells growing directly on the plastic (shown in gray). Changes in mean fluorescence intensities (b) due to each ECM substrate are also depicted. Numbers above bars represent the proportion (%) of immunolabeled cells stained with propidium iodide in S⫹G2⫹M phase of the cell cycle. Data in both panels are representative of four independent experiments using cells cultured from four patients, cell passages 2 to 4. See text for details of statistical comparisons.

tive on Day 5 of culture but increased again at confluence. Based on these observations and those of Wong and colleagues (31), who reported a similar reduction in contractile protein expression in proliferating rat tracheal smooth muscle cells, we identified modulation of contractile phenotype toward a synthetic-proliferative phenotype in human cultured airway smooth muscle cells as the relative loss in expression of sm-␣-actin, sm-MHC, and calponin, together with increased expression of the intermediate filament protein vimentin. Although sm-␣-actin is the most widespread, used smooth muscle cell marker, its specificity is not restricted to smooth muscle cells (32). For this reason, expression of calponin and sm-MHC was also evaluated. A similar profile and interpretation have been reported by investigators examining the regulation of vascular smooth muscle phenotype (26–29). The monoclonal antibody we used to detect sm-MHC recognizes both the SM-1 and SM-2 myosin isoforms. These isoforms, particularly the SM-2 variant, are believed to be among the most rigorous

markers for identification of differentiated smooth muscle because they show the highest degree of cell specificity of any known marker for differentiated myocytes, including those derived from the airways (33). In the present study, more than 65 to 70% of unstimulated secondary and tertiary passage human airway smooth muscle cells grown on plastic were positive for sm-␣-actin, calponin, and sm-MHC, confirming their contractile phenotype status after mitogen withdrawal. These cells also possessed a low proliferative index with less than 3% of the total cells present showing positive nuclear immunoreactivity for the Ki67 antigen, consistent with the MTT cell proliferation data. In the presence of mitogen, however, expression of each of the contractile phenotype marker proteins was reduced to approximately 25%, with around 54 to 62% of cells entering the cell cycle, judged by the increased propidium iodide content and Ki67 staining. When seeded on a LN or Matrigel substrate, immunocytochemical analysis revealed that cells possessed a distinctly higher sm-␣-actin, calponin, and sm-MHC content than did cells seeded on FN or Col I. Flow cytometric analysis confirmed this observation with cells growing on LN and Matrigel being predominantly diploid (2n) and expressing sm-␣-actin, whereas those grown on FN or Col I substrates showed a higher tetraploid (4n) or intermediate DNA content, indicating their transition through S phase to the G2 and M phases of the cell cycle, and possessed less than half the sm-␣-actin content, judged by the reduction in mean fluorescent intensity (Figure 6). Although the observed trend was unchanged, the extent of the reduction in contractile marker protein expression was less than in the immunocytochemical study (Table 1). The likely explanation for this discrepancy is that in our immunocytochemical analysis we did not distinguish poorly stained “positive” cells from the “negative” isotype-matched controls, though from the pattern of myofilament staining (compared with the isotype controls) specific marker protein expression was evident. Clearly, with the greater sensitivity and more quantitative nature of the flow cytometric analysis, these less intensely but nevertheless specifically stained cells could be resolved, and this was reflected, for example, in the separation of the FN and IgG2a histograms (Figure 6). While the initial ECM substrate is undoubtedly modified by the cells themselves during the time course of the experiments performed, particularly by deposition of endogenously synthesized ECM proteins, our results indicate that the initial ECM protein composition is an important determinant of the subsequent expression of biochemical contractile phenotype marker proteins in early passage human airway smooth muscle cells and that this is associated with a reciprocal effect on the proliferative response of these cells to PDGF-BB and ␣-thrombin. Clearly, some degree of heterogeneity exists among the smooth muscle cells maintained on the various ECM substrates examined. This was particularly evident for the immunocytochemical detection of contractile phenotype markers, where approximately 25% of the total cells grown on LN or Matrigel could not be induced to express contractile marker proteins. Similarly, in both the immunocytochemical and flow cytometry studies, approximately 10% of the cells grown on FN or Col I expressed contractile marker proteins despite the presence of


peptide mitogens, as has been reported elsewhere for human vascular smooth muscle cells (34). It remains to be determined whether this involves variations within a single population of cells or the existence of a subpopulation of cells that respond differently upon interaction with the various ECM substrates, reflecting the presence of primordial or smooth muscle precursor cells. However, it is known during vascular and airway development that smooth muscle cells sequentially express specific contractile proteins (18, 32, 35) after their commitment to a smooth muscle lineage. Thus, it seems more likely that the observed heterogeneity of adult human airway smooth muscle cells is the result, not of the presence of primordial cells, but of cells that are some way advanced along their differentiation program, existing at different points of a maturation continuum in which, in vivo, differentiated smooth muscle cells would normally predominate. This view is supported by our flow cytometric observations, where changes in the sm-␣-actin content of the whole population were observed rather than the egression of a discrete subpopulation. With the emergence of increasing evidence to support the heterogeneity of airway smooth muscle in freshly isolated cells (36) or its induction in cell culture (37, 38), our data suggest that the ECM may be an important determinant in controlling this process. Consistent with this possibility, in the mouse developing lung, a possible role for LN ␣1 chain synthesis has been identified for development and maturation of airway smooth muscle (39). The findings of this study using smooth muscle cells isolated from human airways are in general agreement with earlier observations on the behavior of early-passage vascular smooth muscle cells cultured on ECM substrates of FN, Col I, LN, and Matrigel (22–25, 27, 29). Based on the expression of smooth muscle–specific biochemical marker proteins, we conclude that a substrate of basement membrane proteins stimulates the expression in vitro of a differentiated contractile phenotype and retards proliferation. In contrast, interstitial ECM proteins promote the expression of a less contractile phenotype that is more responsive to the effects of smooth muscle mitogens. This study provides new information on the proliferation and modulation of contractile protein expression in cultures of human airway smooth muscle and their regulation by defined components of the ECM. The data raise the notion that in airway disease in which the proportions of Col I and FN are increased, the content of smooth muscle and levels of contractile proteins may change, subtly altering the biochemical and mechanical properties of airway smooth muscle, and that basement membrane ECM proteins such as LN may function to hold this process in check. Acknowledgments: The authors thank Prof. Newman L. Stephens and Dr. Edward Rector (University of Manitoba, Winnipeg, Manitoba, Canada) for their generous and informative input during the writing of this manuscript. S.J.H. is the recipient of a Wellcome Trust Research Career Development Fellowship. This study was supported by grant no. 051435 from the Wellcome Trust (UK).

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