Abstract
Airway remodeling decreases lung function in chronic obstructive pulmonary disease (COPD). Extracellular matrix (ECM) deposition is increased in remodeled airways and drives cellular processes of proliferation, migration, and inflammation. We investigated the role of cigarette smoke in altering the ECM deposited from human lung fibroblasts. Lung fibroblasts isolated from patients with COPD or other lung disease were exposed to cigarette smoke extract (CSE) and 5 ng/ml transforming growth factor-β1 for 72 hours; in some experiments, inhibitors of signaling molecules were added. Deposition of perlecan, fibronectin, and elastin were measured by ELISA, as was release of IL-8 and IL-13. Unstimulated fibroblast cells were reseeded onto deposited matrix and assessed for proliferation and cytokine release. CSE (5%) increased deposition of fibronectin and perlecan from only COPD fibroblasts. Fibronectin and perlecan deposition was attenuated by addition of the NF-κB inhibitor, BMS-345541, and the signal transduction and activator of transcription-1/3 inhibitor, pyridone 6, respectively. CSE (5%) increased IL-8 release from COPD fibroblasts more than non-COPD fibroblasts. This increase was attenuated by BMS-345541. Matrix deposited after 5% CSE stimulation increased proliferation of fibroblasts, but did not alter cytokine release. ECM produced from COPD fibroblasts after CSE exposure has proproliferative effects. Thus, the ECM in patients with COPD may create an environment that promotes airway remodeling.
This research demonstrates a direct effect of cigarette smoke exposure on initiating changes in vitro which may underlie the development of chronic obstructive pulmonary disease (COPD). In addition this work highlights the possible role of the extracellular matrix in driving airway remodeling in COPD.
Chronic obstructive pulmonary disease (COPD) is a preventable obstructive disease of the lung caused by inhalation of noxious particles (1). Prevalence of COPD is increasing and is projected to be the third leading cause of death worldwide by 2020 (2). COPD involves remodeling of the lungs, characterized by emphysematous destruction of the alveoli coupled with airway wall thickening. The extent of airway wall thickening is associated with disease progression (3), and this thickening is the major cause of decreased lung function in COPD as remodeling reduces airflow and distensibility (4). Thickening of the epithelium, greater airway smooth muscle bulk, and increased extracellular matrix (ECM) deposition are key structural changes of the remodeled airway wall (4).
The ECM is an acellular scaffold that surrounds cells and tissues, and influences cellular processes, such as proliferation, migration, repair, and inflammation (5). As the ECM is involved in many functional processes, any alterations in lung ECM may precipitate changes resulting in airway remodeling.
Lung matrix is predominantly deposited by fibroblasts, which have been shown to contribute to airway remodeling in other airway diseases, such as asthma, by up-regulating matrix deposition (6) and increasing cytokine release (7).
The main cause of COPD is chronic particulate exposure—most commonly, cigarette smoke (8). The emphysematous destruction seen in the lungs of smokers is most likely due to the cytotoxic and proinflammatory activity of cigarette smoke (9); however, whether cigarette smoke can directly cause remodeling is unknown.
We aimed to examine whether cigarette smoke extract (CSE) alters the ECM deposited by primary human lung fibroblasts, and if smoke-induced ECM can alter proliferation and cytokine release. We also investigated whether the release of profibrotic cytokines from fibroblasts was increased by CSE.
Exploring the process by which cigarette smoke may cause airway remodeling may yield new therapeutic targets by which airway remodeling may be prevented in COPD and other chronic diseases of the lung.
The following chemicals were obtained from the companies indicated: Dulbecco's modified Eagle medium (DMEM), DMSO, BSA, ammonium hydroxide, Direct Red 80, picric acid (Sigma, St. Louis, MO), PBS, penicillin, streptomycin, amphotericin B (Invitrogen, Carlsbad, CA), LY294002, BMS-345541, pyridone-6 (Calbiochem, San Diego, CA), SP60012 (A.G. Scientific, San Deigo, CA), and FBS (JRH Biosciences, Melbourne, VIC, Australia).
Approval for all experiments with human lung was provided by the Human Ethics Committees of the University of Sydney and the Sydney South West Area Health Service. Human lung fibroblasts were isolated from lung tissue obtained from donors undergoing resection for thoracic malignancies or lung transplantation, who gave written, informed consent. Comparisons of available donor characteristics from the COPD (n = 14) and non-COPD (n = 19) groups are provided in Table 1. The available clinical characteristics of all donors, including age, number of smokers, pack-years, and FEV1, are provided in Table E1. Methods for isolation of human lung fibroblasts are also provided in the online supplement.
| COPD | Non-COPD | |
| n | 15 | 22 |
| Mean age, yr | 55 (± 2.56) | 55.45 (± 3.89) |
| Number of males | 11 | 18 |
| Number of females | 4 | 4 |
| % of donors with a history of smoking | 100 | 53 |
CSE was prepared fresh by bubbling smoke from one filtered, high-tar, commercial cigarette at a constant rate through 25 ml DMEM (10). This solution (100% CSE) was then diluted in 0.1% (vol/vol) FBS/antibiotic/DMEM and applied to cells within 30 minutes of preparation.
Fibroblasts were incubated with 0.5 and 5% CSE in 0.1% (vol/vol) FBS/antibiotic/DMEM for 72 hours before supernatants were collected and ECM was exposed. Smoke-exposed and smoke-naïve plates were incubated in separate, isolated incubators to prevent smoke extract from “leaching” across into naive plates.
In addition to CSE exposure, in some experiments cells were stimulated with 200 pM recombinant human transforming growth factor (TGF)-β1 (R&D Systems, Minneapolis, MN) as a positive control.
Deposition of proteins into the ECM was measured by ELISA using mouse anti–fibronectin C–terminal (Chemicon, Billerica, MA), mouse anti-perlecan (Zymed, Carlsbad, CA), rat anti–laminin β1, mouse anti-collagen V (Abcam, Cambridge, MA), mouse anti-tenascin, mouse anti–collagen IV, mouse anti-elastin, mouse anti–collagen I, mouse anti–collagen III (Sigma), and mouse anti-versican (R&D Systems) antibodies at 1:500 dilution in 1% BSA/PBS. Full details of this method are available in the online supplement.
Deposition of total fibrillar collagen was measured using a modified picroscirius red assay (11). Full details of this method are available in the online supplement.
Quiesced fibroblasts were incubated in the presence of SP600125 (10 μM), BMS-345541 (30 μM), pyridone-6 (65 nM), or LY294002 (3 μM) in appropriate concentrations of DMSO in 0.1% FBS/antibiotics/DMEM. After 1 hour, media were aspirated before stimulation with 0.5%, 5% CSE or TGF-β1 (5 ng/ml) in the presence of inhibitors for 72 hours.
Levels of IL-6, IL-8, and IL-13 released into the supernatant were assessed using commercial antibody kits according to the manufacturer's instructions (R&D Systems).
To determine if ECM deposited by fibroblasts under different stimulation altered cellular proliferation, fibroblasts were reseeded on top of exposed matrix at a density of 0.5 × 104 cells/ml in 0.1% FBS/antibiotics/DMEM and incubated without stimulation for 72 hours, whereby supernatant was collected, and viable cells were manually counted.
All analysis was performed using GraphPad Prism 5.0 software (GraphPad Software, La Jolla, CA). Full details are available in the online supplement. Differences were considered significant at P ≤ 0.05.
To ensure that our model of CSE was functional, we assessed fibronectin deposition by primary human lung fibroblasts after exposure to CSE, using TGF-β1 as a positive control.
Exposure to CSE significantly up-regulated fibronectin deposition from fibroblasts obtained from lung samples from donors with COPD (P < 0.05; n = 6) (Figure 1). In contrast, fibroblasts obtained from donors with non-COPD lung disease did not increase fibronectin deposition after 5% CSE exposure (Figure 1).
Figure 1. Deposition of fibronectin from human lung fibroblasts from patients with chronic obstructive pulmonary disease (COPD) (n = 6) or non-COPD lung disease (n = 7), as measured by ELISA after 72-hour stimulation with 0.5 or 5% cigarette smoke extract (CSE). Transforming growth factor (TGF)-β1 (5 ng/ml) was used as a positive control. Data are expressed as absorbance at 405 nm. Bar graphs represent mean values (±SEM). *P < 0.05 versus unstimulated, two-way, repeated-measures ANOVA.
[More] [Minimize]The increase of fibronectin deposition by CSE in COPD fibroblasts was significantly attenuated by the addition of the NF-κB inhibitor, BMS-345541 (P < 0.01; n = 5) (Figure 2). TGF-β1–induced fibronectin deposition was not significantly altered by BMS-345541 (Figure E3A).
Figure 2. Attenuation of 5% CSE induced fibronectin deposition from COPD human lung fibroblasts by the addition of NF-κB inhibitor BMS-345541 (BMS) as compared with 0.006% (vol/vol) DMSO (vehicle control), after 72-hour stimulation with 5% CSE in the presence of 30 mM BMS-345541. Data are expressed as percentage of 5% CSE–induced fibronectin deposition. Bar graphs represent mean values (±SEM). **P < 0.01 versus vehicle control. Student's paired t test (n = 5).
[More] [Minimize]The addition of the c-Jun N-terminal kinase inhibitor, SP600125, signal transduction and activator of transcription (STAT)-1/3 inhibitor, pyridone-6, and phosphoinositide 3-kinase inhibitor, LY294002, did not alter TGF-β1– or CSE-induced fibronectin deposition (Figure E3A).
To examine if other ECM proteins were altered by CSE, we also assessed the expression of perlecan in the deposited matrices, and found that TGF-β1 and CSE significantly up-regulated perlecan deposition in fibroblasts obtained from COPD donors (P < 0.05; n = 5) (Figure 3A). Surprisingly, whereas TGF-β1 induced the deposition of perlecan in non-COPD fibroblasts (P < 0.05; n = 4) (Figure 3B), CSE did not. CSE induced perlecan deposition was partially attenuated by the addition of pyridone 6 (P < 0.05; n = 5) (Figure 4A). TGF-β1–induced perlecan deposition was also partially attenuated by pyridone-6; however, this difference was not statistically significant (Figure E3B). In comparison to fibronectin (Figure 2), the addition of BMS-345541 did not alter CSE-induced deposition of perlecan (Figure 4B). The addition of LY294002 or SP600125 had no effect on CSE- or TGF-β1–induced perlecan deposition (Figure E3B).
Figure 3. Deposition of perlecan from human lung fibroblasts from (A) patients with COPD (n = 5) or (B) non-COPD lung disease (n = 4), as measured by ELISA after 72-hour stimulation with 0.5 or 5% CSE. The profibrotic cytokine, TGF-β1 (5 ng/ml), was used as a positive control. Data are expressed as absorbance at 405 nm. Bar graphs represent mean values (±SEM). *P < 0.05 versus unstimulated (repeated-measures one-way ANOVA with Dunnet's post test).
[More] [Minimize]
Figure 4. Attenuation of 5% CSE induced perlecan deposition from COPD human lung fibroblasts by pyridone-6 (A), but not BMS-345541 (B), as compared with 0.006% (vol/vol) DMSO (vehicle control). Data are expressed as percentage of 5% CSE–induced perlecan deposition. Bar graphs represent mean values (±SEM). *P < 0.01 versus vehicle control. Student's paired t test (n = 5).
[More] [Minimize]We did not observe any changes in the deposition of laminin, elastin, tenascin, collagen I, collagen III, collagen IV, collagen V, or versican (data not shown).
To demonstrate that the fibroblasts used in this study could indeed synthesize collagens, we then measured total fibrillar collagen using a modified picroscirius red assay.
Although we did not observe an increase in collagen I deposition, TGF-β significantly up-regulated deposition of total fibrillar collagen from both COPD and non-COPD fibroblasts (Figure 5). Exposure to CSE did not alter total fibrillar collagen in either group.
Figure 5. Deposition of total collagen from human lung fibroblasts after stimulation with 0.5 or 5% CSE; 5 ng/ml TGF-β, as measured by picroscirius red assay. Data expressed as percentage of unstimulated control. Bar graphs represent mean values (±SEM). *P < 0.05 versus unstimulated control (n = 4 for each disease type).
[More] [Minimize]To investigate the effect of CSE on cytokine release, supernatants were collected after 72-hour stimulation, and cytokine levels were analyzed by ELISA. Production of IL-8 was increased after 5% CSE stimulation. Interestingly, fibroblasts obtained from COPD donors expressed a significantly greater amount of IL-8 after 5% CSE stimulation (versus non COPD; P < 0.01; n = 5 COPD, 7 non-COPD) (Figure 6). This increase in IL-8 production was attenuated by the addition of BMS-345541 (versus vehicle control; P < 0.05; n = 4) (Figure E1).
Figure 6. Release of IL-8 from human lung fibroblasts in response to stimulation with 0.5 or 5% CSE, as compared with the positive control, TGF-β1. Bar graphs represent mean values (±SEM). **P < 0.01 COPD (n = 5) versus non-COPD (n = 7); two-way repeated-measures ANOVA.
[More] [Minimize]IL-13 was not detected in the supernatants of fibroblasts at baseline or after stimulation (n = 12) (data not shown).
To see if there was any differential matrix production from fibroblasts obtained from different sites in the lungs, we obtained fibroblasts from central and peripheral small airways of the same donors, which were then stimulated for 72 hours with 5 ng/ml TGF-β1 and 5% CSE.
Deposition of the ECM proteins, fibronectin and perlecan, was up-regulated by both TGF-β1 and 5% CSE to the same extent in both cell types (Figure E2).
We examined the effect of reseeding smoke-naive fibroblasts onto matrix deposited by fibroblasts under CSE stimulation to assess whether the matrix was functionally altered. There was a significantly greater number of fibroblasts after 72-hour incubation on matrix deposited under 5% CSE stimulation (Figure 7A) (n = 12 matrix, 28 fibroblasts; P < 0.05), whereby the number of fibroblasts was roughly twofold greater when compared with those seeded upon matrix deposited under control conditions.
Figure 7. Effect of deposited matrix on proliferation of (A) reseeded primary human lung fibroblasts (n = 28 fibroblast lines reseeded on 12 matrices). Data are separated into COPD or non-COPD matrix. (B) Stimuli-naive cells were plated on top of matrix deposited from fibroblasts stimulated with 0.5 or 5% CSE for 72 hours, and viable cells were counted manually. Data were normalized to percent of control. Bar graphs represent mean values (±SEM). *P < 0.05 versus unstimulated (n = 28 fibroblasts reseeded on 12 matrices).
[More] [Minimize]When data were separated into disease state of the donor cells responsible for depositing the matrix, there was an apparent, but nonsignificant, trend for higher cell counts on matrices deposited by smoke-stimulated COPD fibroblasts (Figure 7B) as compared with smoke-stimulated non-COPD fibroblasts.
After determining that deposited matrix affected proliferation, we examined whether matrix deposited under stimulation would alter the production of the cytokines, IL-6 and IL-8, from reseeded fibroblasts. Matrix deposited by fibroblasts under 5% CSE stimulation appeared to reduce release of IL-6 from reseeded stimuli-naive fibroblasts (Figure 8A) when compared with matrix deposited from unstimulated, non-COPD fibroblasts. COPD matrices had no effect on IL-6 release, regardless of stimuli conditions.
Figure 8. Effect of deposited matrix on (A) IL-6 and (B) IL-8 release from reseeded primary human lung fibroblasts. Stimuli-naive cells were plated on top of matrix deposited from fibroblasts stimulated with 0.5 or 5% CSE for 72 hours, and supernatants were collected and analyzed for IL-6 and IL-8 release via ELISA. Bar graphs represent mean values (±SEM) (n = 28 fibroblasts reseeded on 6 COPD or 6 non-COPD matrices).
[More] [Minimize]There was no significant alteration in IL-8 release from stimuli-naive fibroblasts reseeded on matrices from either COPD or non-COPD matrices (Figure 8B). Stimuli-naive fibroblasts tended to have a 200-pg/ml greater IL-8 release when seeded on COPD matrices as compared with when seeded on non-COPD matrices; however, this difference was not statistically significant.
This study has demonstrated that human lung fibroblasts obtained from donors with COPD are more responsive to CSE and produce profibrotic cytokines more readily than non-COPD donors. In addition, we have shown that CSE directly activates the NF-κB pathway. We have also demonstrated that the ECM produced by lung fibroblasts after exposure to CSE is functionally altered, having proproliferative characteristics.
We demonstrated in vitro that, by up-regulating deposition of ECM proteins and cytokines from lung fibroblasts, CSE exposure may directly influence airway remodeling. As the ECM is involved in processes of cellular proliferation and angiogenesis, and various ECM proteins have been demonstrated to be up-regulated in lungs of patients with COPD (12, 13), it is rational to conclude that the functional alterations of the ECM may be relevant in vivo.
Matrix produced by fibroblasts after CSE exposure is proproliferative. The proproliferative effects of the CSE-induced matrix were unexpected, as previous research has shown that direct exposure of CSE to fibroblasts inhibits proliferation (14). The role of the ECM in affecting cellular proliferation, migration, and differentiation is well known (5). Fibronectin can have proproliferative activity on human lung carcinoma cell growth via suppression of the p21 pathway (15). As 5% CSE increased fibronectin deposition into the matrix, we speculate that the proproliferative actions of smoke-induced matrix on human lung fibroblasts may be due to this increased fibronectin.
Perlecan, a heparan sulfate proteoglycan, can store and protect growth factors, such as members of the fibroblast growth factor family (16). Perlecan expression has been linked to tumor growth and angiogenesis (17). Reducing perlecan synthesis with a perlecan antisense cDNA construct decreases proliferation and migration of cancer cells in vitro (18). Thus, the observed increase in perlecan and fibronectin deposition from COPD fibroblasts after CSE exposure may increase cellular proliferation and angiogenesis in the lungs, which, in turn, may cause irreversible airway remodeling. In addition, the presence of perlecan is increased in damaged tissue that undergoes mechanical strain, such as cardiac tissue (19), and the presence of perlecan is also involved in the formation of proper basement membranes (20), so the up-regulation of perlecan from lung tissue may also combat strain induced by hyperinflation of the alveolar airspaces in emphysema.
Thus, we hypothesize that a functionally altered ECM may perpetuate the pathophysiology of COPD in the absence of stimulation. The extent and duration for which the matrix remains functionally altered requires investigation, but may provide insight into the mechanisms by which decreased lung function never completely recovers in patients with COPD who have quit smoking (21, 22).
It has been previously described that fibronectin deposition from human lung fibroblasts after cigarette smoke exposure is due to the c-Jun N-terminal kinase and mitogen-activated protein kinase pathways (23). Although the addition of inhibitors to these signaling molecules did not result in a statistically significant attenuation of CSE effects, a trend was observed. Our data compliment and extend previous work by demonstrating the role of the NF-κB pathway in smoke-induced fibronectin deposition. This pathway may also be more readily activated in COPD, as seen by increased IL-8 release in response to CSE exposure. We also demonstrated that perlecan and fibronectin are produced by different signaling pathways in response to CSE stimulation. This was not unexpected, as CSE is composed of many different chemicals that can activate multiple pathways; however, these findings highlight the fact that fibroblasts may produce different matrix proteins, depending on external stimulation.
We are the first to show that smoke-induced deposition of perlecan from COPD fibroblasts may be due to STAT-1/3 activation. It is reasonable to suggest that activation of this pathway may occur at a lower threshold/more easily in cells from donors with COPD; thus, we see deposition of ECM proteins in response to smoke exposure, as well as changes in proliferation and increased IL-8 release. This is supported by recent work showing increased activation of mitogen-activated protein kinase in the lungs of patients with COPD (24). Further investigation of the role of STAT activation in COPD is warranted.
As fibroblasts are situated in the submucosal layer of the lungs, it is appropriate to use CSE, as any chemicals that diffuse across the epithelial layer will be in a soluble form. Our study demonstrates that the location from which the fibroblasts were derived did not affect the response to cigarette smoke.
Cigarette smoke is composed of over 4,000 different chemicals, and the exact mediator for the changes in ECM in both composition and function is not known. Nicotine has been shown to induce fibronectin deposition in a dose-dependent manner (23), and it is known that molecules that cause oxidative stress, such as hydrogen peroxide, are found in greater levels in the lungs of patients with COPD (25), and can activate intracellular signaling molecules (26). It is likely that a combination of smoke-derived molecules and activation of multiple signaling pathways underlie processes by which ECM deposition is up-regulated. As inhibiting signaling molecules had different effects on CSE- and TGF-β1–induced ECM deposition, it is likely that CSE and TGF-β1 involve different signaling mechanisms to up-regulate ECM protein deposition.
The COPD fibroblasts came from donors undergoing transplantation for severe, end-stage COPD, and required a body mass index, obstruction, dyspnoea, exercise capacity index score of 7–10 to be eligible for transplantation (27). Fibroblasts from donors with COPD had increased deposition of fibronectin and perlecan in response to CSE, whereas fibroblasts obtained from donors with non-COPD lung disease did not. This suggests that the lungs of donors with COPD may be primed for remodeling by cigarette smoke exposure. Further studies comparing smokers without COPD, nonsmokers, and smokers with COPD would aid in answering whether altered matrix deposition is a result of prior chronic exposure to cigarette smoke or an underlying phenotype characteristic of COPD.
The non-COPD donor group came from a diverse range of lung diseases, such as lung cancer and asthma (see Table E1), the majority of which, can involve airway obstruction and areas of fibrosis. Although cells from these donors responded to TGF-β1 stimulation, they did not up-regulate ECM deposition after CSE stimulation. Thus, we can conclude that COPD fibroblasts are altered in such a way as to deposit ECM proteins after CSE stimulation. Pathogenesis of other smoking-induced diseases suggests that, although there is a genetic component, the disease pathogenesis is most likely due to alterations resulting from prolonged exposure to cigarette smoke. Genetic variability of individuals may affect the sensitivity of an individual to cigarette smoke–induced pathogenesis, but we believe that, given enough time and enough exposure, various hallmark characteristics of COPD will appear, to some extent, in all persistent smokers. Therefore, we believe that the differential response of the COPD fibroblasts is most likely due to epigenetic reprogramming of genetically susceptible individuals by cigarette smoke.
In conclusion, we have demonstrated, for the first time, that the ECM produced by fibroblasts after stimulation with CSE is functionally different, and cigarette smoke may prime the airways in such a way as to create an environment whereby airway remodeling is encouraged. We have also added data to the growing pool of knowledge, whereby differences between cells from donors with COPD and other lung diseases are known. Further research on these differences may result in viable therapeutic targets for reducing the detrimental airway changes underlying COPD.
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This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org
Originally Published in Press as DOI: 10.1165/rcmb.2010-0426OC on July 21, 2011
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