Published ahead of print on September 4, 2003, doi:10.1164/rccm.200307-1006OC
© 2003 American Thoracic Society Cigarette Smoke Produces Airway Wall Remodeling in Rat Tracheal ExplantsDepartment of Pathology, University of British Columbia, Vancouver, British Columbia, Canada Correspondence and requests for reprints should be addressed to Andrew Churg, M.D., Department of Pathology, University of British Columbia, 2211 Wesbrook Mall, Vancouver, BC, V6T 2B5 Canada. E-mail: achurg{at}interchange.ubc.ca
Small airway remodeling ("small airways disease") is a common finding in cigarette smokers and is an important cause of airflow obstruction. Airway remodeling is usually attributed to the effects of cigarette smoke–induced inflammation in the airway wall, but little is actually known about its pathogenesis. We exposed rat tracheal explants to cigarette smoke and then maintained them in air organ culture. At 24 hours after smoke exposure, there was a dose-dependent increase in gene expression of procollagen and a significant increase in tissue hydroxyproline, a measure of collagen content. Greater increases in procollagen gene expression were found with repeated smoke exposures. Increased procollagen gene expression could be prevented with SN50, a selective inhibitor of nuclear factor–  B activation, and superoxide dismutase, catalase, and tetramethylthiourea, scavengers of active oxygen species. AG1478, an inhibitor of epidermal growth factor receptor signaling, also prevented increased procollagen gene expression, but PD98059 and SB203580, inhibitors of mitogen-activated protein kinases, did not. These findings indicate that cigarette smoke can directly induce airway remodeling, specifically airway wall fibrosis, probably through active oxygen species–dependent transactivation of the epidermal growth factor receptor and subsequent nuclear factor–B activation. Smoke-evoked inflammatory cells are not required for this process.
Key Words: cigarette smoke • airway remodeling • oxidants • epidermal growth factor receptor • nuclear factor– Chronic airflow obstruction in cigarette smokers is usually attributed to two different anatomic entities: emphysema and small airway remodeling, the latter previously referred to as "small airways disease" and more recently as "tobacco-associated bronchiolitis." Small airway remodeling in cigarette smokers affects primarily the membranous and respiratory bronchioles and morphologically appears as fibrotic and inflamed airways that have thicker walls than normal and often show mucous metaplasia of the bronchiolar epithelium as well as narrowed and distorted lumens. Hogg and coworkers (1) found that the small airways were a major site of airflow resistance in individuals with chronic obstructive lung disease, and Cosio and coworkers (2) were the first to demonstrate that pathologic abnormalities in the structure of the small airways correlated with abnormal pulmonary function tests; subsequent studies have shown that changes in these airways correlate not only with abnormalities in the so-called tests for small airways (nitrogen washout, slope of Phase III) but also with decrements in FEV1 (3–6). These changes may occur in conjunction with emphysema, but in some smokers, only small airway remodeling is seen (7–9). Although a vast body of literature has considered the pathogenesis of emphysema, little attention has been directed toward the mechanisms behind small airway remodeling. The common belief is that this process reflects longstanding smoke-induced chronic inflammation in the airway walls (10–12). In some studies, the intensity of the airway inflammatory response appears to correlate with the severity of airflow limitation (13), but, as noted by Jeffery (10), it is unclear whether the inflammatory infiltrate produces the morphologic changes or whether remodeling is a separate response to smoke inhalation. Examining the mechanisms behind smoke-induced airway remodeling is problematic because both epithelial and mesenchymal compartments are involved. Dual monolayer cultures can in theory be used but do not maintain normal epithelial–mesenchymal interactions. In vivo models of smoke exposure evoke an inflammatory response that may or may not, as noted previously, be a part of the underlying process. Tracheal explants provide an ex vivo model of the airway wall and can be maintained in air organ culture for long periods (14). Although tracheal explants, by definition, are models of the large airways, they are similar in general structure to the small airways. They consist of a single-layered epithelium overlying a mesenchymal layer that contains fibroblasts and tissue macrophages along with matrix. Tracheal explants differ from the small airways in that the explants have very little muscle (the latter a prominent feature of small airways) and that they contain a mixture of ciliated and mucous-secreting cells, whereas the small airways contain predominantly ciliated and Clara cells. Nonetheless, it is likely that the basic reactions to inhaled toxins, particularly toxins that operate through active oxygen species (see DISCUSSION), will be similar in tracheal explants and small airways, and they thus provide a reasonable, if not exact, model of reactions in the small airways. The major advantage of tracheal explants is that they contain both airway epithelium and underlying mesenchymal tissues in their normal anatomic arrangement and thus are excellent for studying processes that lead to fibrosis. In addition, explants lack exogenous inflammatory cells and thus can be used to separate direct effects of injurious agents from effects mediated by inflammatory cells. In this study, we have used the rat tracheal explant model to show that cigarette smoke in itself rapidly induces airway wall remodeling.
Preparation of Tracheal Explants and Smoke Exposure Tracheal explants were prepared from 250 g Sprague–Dawley rats as described previously by us (14). Explants were exposed to whole smoke from a high tar and nicotine (Kentucky 2R1 Research) cigarette by drawing one puff at a time into a syringe and then expelling it into a humidified chamber containing the explants (see online supplement for additional details). Control explants were exposed to room air. After smoke exposure was complete, the explants were transferred to petri dishes containing Dulbecco's modified Eagle medium in agarose with supplements (see online supplement) and maintained in air + 5% CO2 organ culture with basal feeding in an incubator at 37°C for up to 7 days.
Measurement of Hydroxyproline
Gene Expression of Procollagen (GenBank M27208)—F: 5'-CCA ATC TGG TTC CCT CCC AC-3'; R: 5'-GTA AGG TTG AAT GCA CTT-3'. Malate dehydrogenase (GenBank AF093773)—F: 5'-CAA GAA GCA TGG CGT ATA CAA-3'; R: 5'-TTT CAG CTC AGG GAT GGC CTC-3'.
Scavengers of Active Oxygen Species
Superoxide dismutase.
Catalase.
Intracellular Signaling Inhibitors
Statistical Analysis
Exposure to cigarette smoke consistently produced an increase in gene expression of procollagen by 24 hours, and this increase showed a dose response with numbers of puffs of smoke (Figure 1) . Tissue hydroxyproline, a marker of collagen content, was also increased at 24 hours (Figure 2) . If explants were exposed to smoke for 6 consecutive days and collected for analysis on Day 7, a greater increase in procollagen gene expression was seen compared with a single smoke exposure (Figure 3) .
Exposure of the explants to CAT, SOD, or tetramethylthiourea abolished the smoke-mediated increases in procollagen gene expression at 24 hours (Figure 4) ; boiled CAT or SOD was ineffective. As an additional control for these experiments, explants were exposed to bovine serum albumin to rule out nonspecific protein effects; albumin did not prevent smoke-induced fibrogenesis (data not shown). These findings indicate that enhanced production of procollagen proceeds through a pathway mediated by active oxygen species.
Treatment of the explants with the NF- B inhibitor SN50 or the EGFR inhibitor AG1478 (Figure 5)
prevented increases in procollagen gene expression, whereas the MAPK inhibitors PD98059 (a selective inhibitor of extracellular signal–related kinase 1/2) and SB203580 (a selective inhibitor of p38 kinase) did not (Figure 5). These findings suggest that smoke activates NF-B via an EGFR-dependent pathway, but one that does not involve MAPK (see DISCUSSION).
Airway remodeling is defined as an alteration in the size, mass, or number of the structural components of the airway wall (10). In humans who smoke cigarettes, a variety of changes can be seen in the large and small airways, including fibrosis, muscle hyperplasia, inflammation, and mucous metaplasia/hypersecretion. Exactly which of these components is most important in producing airflow obstruction is unclear, and different studies give somewhat different results; however, there does appear to be a consistent finding that fibrosis and thickening of the airway wall, particularly in the subepithelial compartment (often erroneously termed submucosa) in the small airways, are an important part of the pathogenesis of smoke-induced airflow limitation (16), whereas muscle hyperplasia appears to be much less important (16), as opposed to the situation in asthmatics. As noted in INTRODUCTION, little is known about the pathogenesis of these lesions in humans. Data from animal models is scanty, and most studies of animals exposed to smoke have ignored the issue. The few data that exist are somewhat contradictory. We have found that exposure of small laboratory animals to daily cigarette smoke for 4 to 6 months consistently produces a form of centrilobular emphysema and also consistently produces mucous metaplasia of the airway epithelium, but we were unable to demonstrate airway wall thickening when we specifically looked for it in guinea pigs (17). However, Rubio and coworkers (18) reported a 25% increase in the wall area of small bronchi of rats after 10 weeks of exposure to cigarette smoke; they were able to prevent this effect by administration of N-acetyl cysteine, suggesting that active oxygen species were involved. In this study, we have shown that smoke rapidly and directly induces both increased procollagen gene expression and increased hydroxyproline, i.e., actual fibrosis, in the airway walls in rat tracheal explants and that repeated exposure to smoke over several days enhances this process. Our results must be interpreted with caution because tracheal explants, as noted in INTRODUCTION, are models of the large airways, but it appears reasonable to presume that the same process will occur in the small bronchi and bronchioles, the important sites of airway remodeling. In addition, we do not know exactly how increased collagen would translate into visible structural change if the explant experiments could be continued for long periods. However, because most of the mass of the explants is cartilage, increased fibrosis must be occurring in the immediately subepithelial layer (Figure 6) and, were it continued long enough, this process presumably would result in mucosal thickening and/or lumenal distortion, the same factors that appear to be important in human smokers (16).
Cigarette smoke is a concentrated source of active oxygen species including both superoxide anion and hydrogen peroxide, and these species can interact to form hydroxyl radical using intracellular iron (19). Our data indicate that scavenging of superoxide anion with SOD, hydrogen peroxide with CAT, and hydroxyl radical with tetramethylthiourea are all effective in blocking the fibrogenic effects of smoke, indicating that active oxygen species in the smoke are crucial to, and probably the initiators of, fibrosis. It is interesting in this regard that Rahman and coworkers (20) were able to show the presence of 4-hydroxy-2-nonenal, a lipid peroxidation product, in the airway epithelial cells of smokers with chronic obstructive pulmonary disease, supporting the importance of oxidative stress in pathologic changes in the airway walls.
Cigarette smoke has been shown to activate NF-
An additional finding of interest was that the EGFR inhibitor AG1478 prevented increased procollagen gene expression, an observation indicating that cigarette smoke signaling is proceeding through EGFR activation. The EGFR is normally activated by ligand (EGF or transforming growth factor– In summary, we have demonstrated for the first time that cigarette smoke can directly produce airway wall remodeling by inducing procollagen gene expression and collagen production and that this process does not require exogenous inflammatory cells. However, because smoke-mediated fibrogenesis is driven by active oxygen species, inflammatory cells such as neutrophils and macrophages that release oxidants or inflammatory cell-mediated induction of fibrogenic growth factors might in fact enhance this process in vivo.
R.D.W. has no declared conflict of interest; H.T. has no declared conflict of interest; C.X. has no declared conflict of interest; X.W. has no declared conflict of interest; J.L.W. has no declared conflict of interest; A.C. has no declared conflict of interest.
Supported by grant MOP-53157 from the Canadian Institutes of Health Research. This article has an online supplement, which is accessible from this issue's table of contents online at www.atsjournals.org Received in original form July 22, 2003; accepted in final form September 3, 2003
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ABSTRACT
METHODS
(1)-procollagen by Reverse Transcriptase–Polymerase Chain Reaction
Ras
