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Published ahead of print on June 30, 2005, doi:10.1165/rcmb.2005-0203OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 33, pp. 387-393, 2005
© 2005 American Thoracic Society
DOI: 10.1165/rcmb.2005-0203OC

Transforming Growth Factor-{beta}1 Drives Airway Remodeling in Cigarette Smoke–Exposed Tracheal Explants

Rong D. Wang, Joanne L. Wright and Andrew Churg

Department 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


   Abstract
Top
 Abstract
Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Small airway remodeling (SAR) is an important cause of airflow obstruction in cigarette smokers, but whether SAR represents a response to smoke-evoked inflammation or is directly mediated by smoke-induced growth factor production is disputed. To examine this process, we exposed rat tracheal explants, a model free of exogenous inflammatory cells, to cigarette smoke in vitro. Cigarette smoke caused release of active transforming growth factor (TGF)-{beta}1, and this was prevented by the oxidant scavenger tetramethythiourea. Nuclear immunostaining for phospho-Smad2, a TGF-{beta} downstream signaling molecule, was present in epithelial and interstitial cells within 1 h after exposure. Smoke caused upregulation of gene expression of connective tissue growth factor (CTGF), a mediator of TGF-{beta} fibrogenic effects, within 2 h, and upregulation of procollagen gene expression at 24 h; both changes could be prevented by the TGF-{beta} antagonist fetuin ({alpha}2-HS-glycoprotein). In a cell-free system, recombinant human TGF-{beta} latency-associated peptide was oxidized by cigarette smoke, and smoke released active TGF-{beta}1 from recombinant latent TGF-{beta}1 via an oxidant mechanism. These experiments suggest that SAR in cigarette smokers may be caused by direct, smoke-mediated, oxidant-driven induction of growth factor signaling in the airway wall, and that SAR does not necessarily require exogenous inflammatory cells.

Key Words: cigarette smoke • chronic obstructive pulmonary disease • connective tissue growth factor • oxidants • transforming growth factor-{beta}


   Introduction
Top
 Abstract
 Introduction
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
There are two distinct anatomic causes of airflow obstruction in cigarette smokers with chronic obstructive pulmonary disease (COPD): emphysema and small airway remodeling (SAR), also referred to as "small airways disease" and "tobacco-associated bronchiolitis." Anatomically, SAR consists of a variable combination of increased fibrous tissue, smooth muscle, and inflammatory cells in the walls of the membranous and respiratory bronchioles, such that the airway lumen becomes narrowed and/or distorted (1–3). The epithelium frequently shows goblet cell and squamous metaplasia, along with intralumenal mucus. The consequence of this process is a marked increase in resistance to airflow in the small airways. Cosio and coworkers (4) were the first to show that structural abnormalities in the small airways in cigarette smokers correlated with abnormalities of pulmonary function, and this has been confirmed in a variety of subsequent studies (5–8).

The pathogenesis of SAR is an area of intense investigation. There are two general schools of thought (9). The most widely accepted theory is that SAR represents a response to repeated inflammatory insults caused by cigarette smoke–evoked inflammatory cells, or by a propensity to develop abnormal inflammatory reactions to minor stimuli (8–10). In this view the resulting changes in airway structure are a manifestation of aberrant healing induced by inflammatory cells, implying that anti-inflammatory agents can be used to prevent SAR. The importance of inflammation has been emphasized in several reports that demonstrate a correlation between numbers of inflammatory cells in the walls of small airways and the severity of airflow obstruction (8, 11).

An alternative theory is that smoke causes excessive production of growth factors that lead to increased muscle and fibrous tissue in the airway wall, and that growth factor production is a direct response to the smoke, possibly mediated through chronic injury or repair of the airway epithelium, but is independent of the inflammatory response (9). This scenario implies that interference with inflammation may not be of much benefit, whereas interference with growth factor production/signaling would be the most effective approach to therapy.

We have previously shown that rat tracheal explants briefly exposed to cigarette smoke show evidence of increased gene expression of procollagen and increased collagen production within 24 h (12); that is, early biochemical evidence of airway remodeling. Because tracheal explants are free of the usual smoke-evoked inflammatory cells, they provide a useful model for examining direct effects of smoke on airway remodeling. In this study we use the tracheal explant model to show that smoke drives release of the strongly profibrotic growth factor, transforming growth factor (TGF)-{beta}1, from tracheal explants, and that TGF-{beta}1 in turn causes autocrine downstream signaling in the form of phospho-Smad2 nuclear translocation, and connective tissue growth factor (CTGF) and procollagen gene expression, thus supporting the direct growth factor induction hypothesis of airway remodeling.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Preparation of Explants and Smoke Exposure
Tracheal explants were prepared from 250-g Sprague-Dawley rats. Tracheas were isolated, opened along the membranous posterior portion, adventitial fat removed, and explants created by making multiple transverse cuts along the length of the isolated trachea, creating segments of ~ 2-mm length. The explants were then placed, serosal side down, on filter paper moistened with culture medium and exposed to whole smoke from 2R1 Kentucky Research Cigarettes (University of Kentucky) in a humidified chamber. Exposures were done at 21°C. We used a dose of 10 puffs of smoke, as we have previously demonstrated that this dose produces distinct increases in procollagen gene expression and collagen production in this system (12). Total smoke exposure time for 10 puffs of smoke (~ 2/3 of a cigarette) was 15 min. The total particulate matter content of 2R1 cigarettes is 44.6 mg/cigarette; tar 36.8 mg/cigarette; and nicotine 2.45 mg/cigarette.

Gene Expression of CTGF, Type I Procollagen, and TGF-{beta}1 in Explants
After smoke exposure, explants were transferred, serosal side down, to Petri dishes containing Dulbecco's modified Eagle's medium (DMEM) in agarose supplemented with 1% glutamine, 1% penicillin–streptomycin–fungizone, 1 µg/ml insulin, 0.1 µg/ml hydrocortisone, 1.5x amino acids, and 10% chicken serum, and maintained in air plus 5% CO2 organ culture with basal feeding in an incubator at 37°C.

For CTGF, expression explants were harvested at 2 and 6 h after the start of smoke exposure; for procollagen and TGF-{beta}1, expression explants were harvested at 24 h. RT-PCR was run as described by us (14). Five explants were combined for each data point, and three data points were generated for each treatment. GAPDH expression was used as a housekeeper. Primer sequences were as follows. TGF-{beta}1 (GenBank X52498): F, 5'-CGA GGT GAC CTG GGC ACC ATC CAT GAC-3'; R, 5'-CTG CTC CAC CTT GGG CTT GCG ACC CAC-3'; Type I Procollagen: F, 5'-CCA ATC TGG TTC CCT CCC AC-3' (GenBank M27208); R, 5'-GTA AGG TTG AAT GCA CTT-3'; CTGF: F, 5'-CGA CTC CTT CCA AAG CAG-3' (GenBank NM022266); R,5'-CGG TAG GCA GCT AGG G-3'; GAPDH: F, 5'-TCT ACC CAC GGA AGT-3' (GenBank NM017008); R, 5'-CCA CCC TTC AGG TGA G-3'.

Inhibitors
In some experiments, explants were first exposed to the oxidant scavenger, tetramethylthiourea (TMTU; Sigma) at a concentration of 10 mM for 2 h before smoke exposure; TMTU was also included in the agarose medium in these experiments. Similarly, in some experiments explants were first exposed to the TGF-{beta} antagonist fetuin (also known as {alpha}2-HS-glycoprotein [13], obtained from Sigma) at concentrations of 25 or 100 µM for 2 h, and fetuin was included in the agarose culture medium.

Release of Active TGF-{beta}1 from Explants
After smoke exposure, explants were transferred to plates containing a small amount of liquid culture medium, sufficient to submerge the explants. Explants were incubated 18 h at 37°C with rocking, and the supernatant then collected and assayed for active TGF-{beta}1 using the mouse TGF-{beta}1 Quantikine Kit (R&D Systems, Minneapolis, MN), according to the manufacturer's instructions.

Immunohistochemical Staining for Phospho-Smad2 and Macrophages
Deparaffinized 5-µm sections were treated with citrate and heat to unmask antigens, then quenched with 3% hydrogen peroxide, blocked with 5% normal goat or horse serum in PBS-T (0.1% Tween-20), and incubated for 12–16 h at 4°C with primary anti–phospho-Smad2 (Cell Signaling Technology, Beverly, MA) at a dilution of 1:200. Negative control slides were incubated with nonimmune IgG instead of the primary antibody. Sections were then incubated sequentially with Dako Biotin Blocking Systems and biotinylated second antibody (DakoCytomation A/S, Copenhagen, Denmark) at a dilution of 1:500–1:1,000, followed by Vector NovaRED for color development (Vector Laboratories, Burlingame, CA). To identify tissue macrophages, similar procedures were performed using anti-rat macrophage antibody CBL260 (Calbiochem, Temecula, CA).

To obtain a quantitative estimate of the changes in phospho-Smad2 staining over time, numbers of stained and unstained nuclei in the epithelial and interstitial compartments were counted per mm of basement membrane length at 1 and 2 h. No nuclear staining was seen in the controls, so these were not counted.

Smoke-Conditioned Medium
The whole smoke from five 2R1 cigarettes was bubbled through 10 ml PBS, the solution filtered to remove particulates, and the pH adjusted to 7.4.

Smoke-Mediated Oxidation of Latency-Associated Peptide
Recombinant human latency-associated peptide (LAP) was purchased from R&D Systems. LAP (30 ng/ml) was exposed to smoke-conditioned medium for 1 h at 37°C; as a control, LAP was exposed to medium that had not been conditioned with smoke. Oxidation of LAP was detected using the Oxyblot Protein Oxidation Detection Kit (Chemicon, Temecula, CA) which detects protein carbonyls. Briefly, proteins were denatured with 6% SDS. Samples were then derivatized by adding dinitrophenylhydrazine (DNPH) 1:1 (vol:vol) and incubated at room temperature for 15 min, followed by the addition of neutralization solution to stop the reaction. Proteins were loaded on a SDS-PAGE 12% Tris-HCl gel and run at 200 V for 1 h. Proteins were transferred onto PVDF membranes. 1% bovine serum albumin in PBS-T was used as a blocking solution throughout. Membranes were blocked for 1 h, followed by incubation with rabbit anti-DNPH (1:150, 1 h; Chemicon International). After incubation, membranes were washed 3x in PBS-T, and incubated with appropriate secondary antibody (1:300) conjugated to horseradish peroxidase for 1 h followed by three washes in PBS-T and three washes in PBS. Proteins were detected with chemiluminescence (Amersham, Piscataway, NJ).

Release of Active TGF-{beta}1 from Recombinant Latent TGF-{beta}1
Recombinant human latent TGF-{beta}1 (R&D Systems) was exposed to smoke-conditioned medium for 1 h at 37°C. Active TGF-{beta}1 was measured immediately with the mouse TGF-{beta}1 Quantikine Kit. For some experiments 100 mM TMTU was added to the smoke-conditioned medium before addition of the protein.

Statistics
Differences among groups were evaluated by ANOVA. Levels of P < 0.05 or less were considered significant. All experiments were repeated at least once. Representative data are shown.


   RESULTS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
Figure 1 shows release of active TGF-{beta}1 from smoke-exposed and cultured explants. Exposure to 10 puffs of smoke roughly tripled TGF-{beta}1 release, and this effect was completely prevented by treatment with TMTU, indicating that the process is oxidant driven.



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  		Figure 1. Release of active TGF-{beta}1 from tracheal explants after smoke exposure. Smoke causes a marked increase in TGF-{beta} release, and this is entirely prevented by TMTU, indicating that the process is oxidant driven. Values are mean ± SD from three different preparations (*P < 0.05 compared with control).

 
Figure 2 shows immunohistochemical staining for phospho-Smad2, a mediator of TGF-{beta} signaling. Nuclear staining in this context indicates active TGF-{beta}–driven signaling (see DISCUSSION). At 1 h after starting smoke exposure, there was nuclear and some cytoplasmic staining for phospho-Smad2 in the epithelial cells, and staining of scattered nuclei in the interstitium. At 2 h nuclear staining persisted in the epithelial cells, although the relative proportion of stained to unstained nuclei decreased (Figure 2D), and there was an increase in the relative proportion of stained to unstained nuclei in the interstitial compartment (Figure 2D).



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  		Figure 2. Immunohistochemical staining for phospho-Smad2. (A) Control explant not exposed to smoke shows no staining. (B) Explant harvested 1 h after the beginning of smoke exposure. Note nuclear and some cytoplasmic staining in the epithelial cells, and nuclear staining in the interstitial compartment. (C) Explant harvested 2 h after the beginning of smoke exposure. Nuclear staining persists in the epithelial cells, although somewhat fewer nuclei stain compared with total epithelial nuclei, and staining has increased in the interstitial cells. These images indicate that smoke rapidly induces TGF-{beta} signaling. E indicates epithelium; I indicates subepithelial interstitium. All magnifications x400. (D) Graphical representation of ratio of phosphor-Smad2–stained to unstained nuclei in the epithelial and interstitial compartment/mm of basement membrane length. The ratio decreases in the epithelial cells from 1–2 h and increases in the interstitial compartment. Data are from three different preparations. Values are mean ± SD (*P < 0.05 compared with 1 h time point for epithelial cells or 1 h time point for interstitial cells).

 
Immunohistochemical staining for macrophages showed small numbers of cells in the adventitial tissues of the explants, but no macrophages were detected in the subepithelial interstitial compartment (data not shown). Thus the staining cells in Figure 2 do not appear to be macrophages.

Figure 3 shows gene expression of CTGF, a downstream mediator of TGF-{beta}–induced fibrosis that is believed to be the proximate driver of collagen production (see DISCUSSION). By 2 h after the beginning of smoke exposure, CTGF gene expression was increased by ~ 2.5-fold, and this effect was abolished in a dose–response fashion by increasing concentrations of fetuin. Increased expression, but at a lower level, was also seen at 6 h after smoke exposure and again was abrogated by fetuin (data not shown).



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  		Figure 3. Gene expression of CTGF in rat tracheal explants 2 h after starting smoke exposure. Smoke induces a 2.5-fold increase in gene expression, and the TGF-{beta} antagonist fetuin prevents this increase in a dose–response fashion, indicating that the response is TGF-{beta} driven. Each treatment is represented by three different preparations. Values are mean ± SD (*P < 0.05 compared with control).

 
Figure 4 shows gene expression of procollagen and Figure 5 expression of TGF-{beta}1. At 24 h after smoke exposure, there was a significant increase in procollagen expression, and this was prevented by fetuin in a dose–response fashion. TGF-{beta}1 expression was also increased, but not to as great an extent.



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  		Figure 4. Gene expression of procollagen 24 h after smoke exposure. Smoke increases procollagen production by ~ 2-fold, and this process is inhibited by fetuin, indicating that it is driven by TGF-{beta}. Each treatment is represented by three different preparations. Values are mean ± SD (*P < 0.05 compared with control).

 


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  		Figure 5. Gene expression of TGF-{beta}1 24 h after smoke exposure. Smoke increases expression of TGF-{beta}1. Each treatment is represented by three different preparations. Values are mean ± SD (*P < 0.05 compared with control).

 
Figure 6 shows a Western blot for oxidized TGF-{beta} LAP analyzed by derivatization with DNPH to detect oxidized proteins as carbonyls, and visualized with an antibody against DNPH. In a cell-free system, recombinant human LAP exposed to medium alone did not produce any signal, whereas LAP exposed to smoke-conditioned medium showed up as a band, indicating that cigarette smoke had oxidized the peptide.



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  		Figure 6. Smoke oxidizes recombinant TGF-{beta} latency– associated peptide in a cell-free system. Latency-associated peptide was exposed to smoke-conditioned medium and then derivatized with DNPH and visualized with antibody to DNPH. Top image: Western blot against DNPH. C, control peptide not exposed to smoke; S1, peptide exposed to smoke and then derivatized; S2, peptide exposed to smoke but not derivatized. Bottom image: Western blot was stopped before complete transfer of protein from from the get to the blotting membrane, and the remaining protein in the gel was visualized by Comassie blue staining (shown here) as a loading control.

 
Figure 7 shows the effects of exposing recombinant human latent TGF-{beta}1 to smoke-conditioned medium in a cell-free system. Smoke caused release of active TGF-{beta}1, and this effect was prevented by pretreating the smoke-conditioned medium with TMTU.



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  		Figure 7. Smoke releases active TGF-{beta}1 from recombinant latent TGF-{beta}1 in a cell-free system. The process is inhibited by TMTU, confirming that release is oxidant driven. Each treatment is represented by three different preparations. Values are mean ± SD (*P < 0.05 compared with control).

 

   DISCUSSION
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
In these experiments we have shown that cigarette smoke induced release of active TGF-{beta}1 from rat tracheal explants via an oxidant-mediated mechanism, and also produced manifestations of downstream signaling of TGF-{beta}, resulting in increased procollagen gene expression. Fetuin/{alpha}2-HS-glycoprotein, a serum protein that mimics the structure of the TGF-{beta} type II receptor and antagonizes the action of TGF-{beta} by binding to the cytokine and preventing it from activating the receptor (13) (see below), abolished the increases in procollagen gene expression, indicating that TGF-{beta} is driving the fibrogenic response. Since we have previously shown that smoke exposure also causes increases in actual collagen content in tracheal explants, and that this response increases with repeated smoke exposures (12), these experiments suggest that cigarette smoke can directly induce fibrogenic growth factors and SAR.

TGF-{beta} is a powerful profibrotic cytokine that causes smooth muscle cell hyperplasia, collagen production by fibroblasts, and differentiation of fibroblasts to myofibroblasts, cells that are important in both matrix production and extracellular matrix contraction (15, 16). A role for TGF-{beta} in the small airway remodeling seen in both cigarette smokers and in individuals with asthma has long been postulated, and Kenyon and colleagues (16) showed that intratracheal instillation of recombinant TGF-{beta}1 in Balb/C mice resulted in increases in subepithelial collagen, gene expression of type I and type III collagen, and total collagen content in lobar bronchi and more distal airways. Studies on biopsies or resected lung specimens from humans have consistently shown the presence of TGF-{beta}1 in small airway epithelial cells and airway wall cells as assessed by RT-PCR or in situ hybridization to detect gene expression, or immunochemistry to detect protein (17–20), and Takizawa and coworkers (19) found that cultured small airway cells spontaneously release TGF-{beta}1.

Nonetheless, the role of TGF-{beta} in particular, and growth factors in general, in SAR in human smokers is uncertain. de Boer and colleagues (17) examined TGF-{beta} immunostaining and in situ RT-PCR in bronchial biopsies, and concluded that there was a significant negative correlation between TGF-{beta} levels and FEV1. Vignola and coworkers (18) reported a correlation between number of airway cells expressing TGF-{beta} and basement membrane thickness in chronic bronchitis. Takizawa and colleagues (19) evaluated TGF-{beta}1 gene expression and protein in small airway epithelial cells; gene expression levels correlated with pack-years of smoking and severity of airflow obstruction, but protein levels apparently did not differ between smokers with and without COPD. Moreover, active TGF-{beta} release from cultured airway epithelial cells was not different between smokers of any kind and nonsmokers. Aubert and coworkers (20) detected TGF-{beta}1 in airway epithelial and wall cells by immunochemistry and in situ hybridization, but there were no differences seen among individuals with asthma, smokers with COPD, and smokers without COPD.

These findings are difficult to interpret because TGF-{beta} is produced with the TGF-{beta} protein complexed to an LAP that prevents biologic activity, and this complex needs to be dissociated to liberate active TGF-{beta} (15, 21). For that reason, gene expression studies, while providing useful information, do not necessarily correlate with TGF-{beta} activity. Similarly, most TGF-{beta} detected immunochemically is latent protein, but measurements of latent TGF-{beta} indicate only the potential for TGF-{beta}–mediated effects. Actually implicating TGF-{beta} in SAR requires information about release of active protein and/or evidence of downstream TGF-{beta} signaling effects. Moreover, the lungs of human smokers always show a smoke-evoked inflammatory response, so that separating direct smoke effects from effects secondary to inflammation is not simple.

Our experiments were designed to specifically address these issues and to show evidence of downstream TGF-{beta} effects. TGF-{beta} signals through binding to the TGF-{beta} type II receptor at the cell surface, which in turn binds to the type I receptor, leading to phosphorylation of a set of cytoplasmic signaling proteins known as Smads (15, 22). Phospho-Smad2 and 3 are effector Smads that complex with Smad4 and are translocated to the cell nucleus, where they activate the promoters of genes driven by TGF-{beta}. One of the genes activated is CTGF. CTGF is believed to be the proximate mediator of increased TGF-{beta}–driven collagen production, and there is evidence for a CTGF response element in the promoter of type I procollagen (15).

Our results in the present experiments not only show liberation of active TGF-{beta}1, but immunohistochemical evidence of TGF-{beta} signaling in the form of visible nuclear phospho-Smad2 in both the epithelial and interstitial compartments, and rapid induction of CTGF gene expression. The presence of nuclear staining for phospho-Smad2 in interstitial, presumably fibroblast (given the elongated shape and location), nuclei shows that TGF-{beta} signaling affects the anatomic compartment that is crucial to the development of airway wall fibrosis. Induction of CTGF gene expression was inhibited by fetuin, confirming that it is TGF-{beta}–driven.

Of note, phospho-Smad2 staining was seen by 1 h after beginning smoke exposure and CTGF gene expression was elevated by 2 h. This set of events is much too rapid for new gene transcription of TGF-{beta} and protein production, and implies that smoke must directly liberate active TGF-{beta}1 from preformed latent TGF-{beta}1. In addition, in the tracheal explants there are no exogenous inflammatory cells and no immunochemically detectable interstitial macrophages; thus our data also imply that, in vivo, smoke-evoked inflammatory cells may not be required for release of TGF-{beta}.

There are a variety of mediators/processes that release active TGF-{beta} from the LAP (21). Heat and low pH, which probably are only encountered in the laboratory, denature the LAP, but not TGF-{beta} itself. Plasmin and other proteases appear to proteo lytically cleave the LAP, whereas thrombospondin-1 and the integrin {alpha}v{beta}6 are believed to interact directly with the LAP to disrupt the noncovalent binding between the LAP and TGF-{beta} (21).

Cigarette smoke is a highly concentrated source of oxidants in the form of both reactive oxygen and reactive nitrogen species (23). There is considerable evidence that smoke produces oxidative stress in the lower respiratory tract (reviewed in Refs. 24, 25); in addition to biochemical markers of oxidative damage, immunohistochemical staining for the smoke-induced lipid peroxidation product 4-hydroxynonenal can be found in the lungs in human smokers and experimental animals exposed to smoke (26, 27). These findings suggested to us that smoke might oxidatively activate latent TGF-{beta}.

There is relatively little information available about oxidative activation of TGF-{beta}. Barcellos-Hoff and colleagues (28) showed that, in a cell-free system, radiation releases active TGF-{beta} via an oxidant mechanism, and that this could be reproduced using a combination of transition metals and ascorbate, a system that generates superoxide anion, hydrogen peroxide, and hydroxyl radical. The importance of oxidant-mediated TGF-{beta} release in fibrotic diseases has been documented by Pociask and coworkers (29), who showed that reactive oxygen species generated by asbestos fibers oxidized the LAP, but not TGF-{beta} itself, and that the oxidized LAP no longer bound TGF-{beta}, thus separating the peptide from the active TGF-{beta} molecule. Again, this system required ascorbate, and could be inhibited by oxidant scavengers.

Our data are similar. Release of active TGF-{beta}1 from the explants after smoke exposure could be prevented by TMTU, implicating an oxidant mechanism. Using recombinant human LAP and recombinant latent TGF-{beta}1 in a cell-free system, we found that, in a fashion quite analogous to that seen with asbestos fibers, smoke oxidized the LAP and also liberated active TGF-{beta} from the latent protein; the latter effect could be prevented by oxidant scavenging with TMTU. However, in contrast to the asbestos model, neither ascorbate nor transition metals were necessary, indicating that cigarette smoke can, by itself, oxidize latent TGF-{beta} and release active TGF-{beta}.

The limitations of our model must be recognized. The major advantage of the tracheal explant system is that it allows one to examine airway wall fibrosis in a situation in which the anatomic components (epithelium, interstitial cells) are maintained in their normal anatomic arrangement—an important point, since direct epithelium/mesenchymal contacts are probably important in fibrogenesis. In addition, as noted, the explants have no exogenous inflammatory cells, nor do they have obvious macrophages in the subepithelial interstitial compartment. They do not normally have mural lymphocytes or neutrophils.

However, our results here must be interpreted with caution because tracheal explants are, obviously, models of large airways rather than the small airways that are the site of increased flow resistance and SAR in humans. There are clear anatomic differences between the two sites. The cartilage, which is irrelevant in this context, can probably be ignored, as can the very sparse bronchial glands present in rat trachea. The epithelial cells are somewhat different, since the small airways, particularly in rodents, have a large proportion of Clara cells. But if one considers only the tissues from epithelium to perichondrium in the explants, which is where we observe the changes of interest, the overall structure is similar to that of the membranous bronchioles. Moreover, there is considerable evidence from human studies that TGF-{beta}1 is produced in the small airways, as it is in the explants. These observations suggest that the alterations occurring in the tracheal explant system will most likely also occur in the small airways, but this needs to be confirmed using in vivo models.

Our results do provide a clear-cut demonstration that cigarette smoke can, in and of itself, very rapidly release a profibrotic growth factor, TGF-{beta}, and cause autocrine downstream signaling by that growth factor, and imply that, with continued smoking, direct growth factor induction will produce airway remodeling manifest as visibly increased collagen levels and possibly increased smooth muscle. It is also interesting that smoke exposure increased gene expression of TGF-{beta}1. This observation suggests not only that smoke releases active TGF-{beta}, but also that it causes increased production of TGF-{beta}1, thus potentially creating a positive feedback loop in which continuing smoke exposure leads to progressively increasing growth factor production.

Our results thus imply that smoke can directly induce remodeling without any need for exogenous inflammatory cells. However, it is entirely possible that the inflammatory cells normally evoked by smoke exposure augment this process, particularly because these cells themselves produce oxidants that could cause additional TGF-{beta} release, and also secrete proteases that may in themselves affect airway wall structure, and that also have the potential to proteolytically liberate additional TGF-{beta} from matrix.


   Footnotes
 
Supported by grant MOP 42539 from the Canadian Institutes of Health Research Explant TGF{beta} May 30, 2005.

Conflict of Interest Statement: None of the authors have a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in final form May 30, 2005


   References
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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