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Department of Molecular Pharmacology, University Centre for Pharmacy, University of Groningen, Groningen, The Netherlands
ABSTRACT
Rationale: Recent findings have demonstrated that muscarinic M3 receptor stimulation enhances airway smooth muscle proliferation to peptide growth factors in vitro. Because both peptide growth factor expression and acetylcholine release are known to be augmented in allergic airway inflammation, it is possible that anticholinergics protect against allergen-induced airway smooth muscle remodeling in vivo. Objective: We investigated the effects of treatment with the long-acting muscarinic receptor antagonist tiotropium on airway smooth muscle changes in a guinea pig model of ongoing allergic asthma. Results: Twelve weekly repeated allergen challenges induced an increase in airway smooth muscle mass in the noncartilaginous airways. This increase was not accompanied by alterations in cell size, indicating that the allergen-induced changes were entirely from increased airway smooth muscle cell number. Morphometric analysis showed no allergen-induced changes in airway smooth muscle area in the cartilaginous airways. However, repeated ovalbumin challenge enhanced maximal contraction of open tracheal ring preparations ex vivo. This was associated with an increase in smooth muscleeCspecific myosin expression in the lung. Treatment with inhaled tiotropium considerably inhibited the increase in airway smooth muscle mass, myosin expression, and contractility. Conclusions: These results indicate a prominent role for acetylcholine in allergen-induced airway smooth muscle remodeling in vivo, a process that has been thus far considered to be primarily caused by growth factors and other mediators of inflammation. Therefore, muscarinic receptor antagonists, like the long-acting anticholinergic tiotropium bromide, could be beneficial in preventing chronic airway hyperresponsiveness and decline in lung function in allergic asthma.
Key Words: acetylcholine airway remodeling anticholinergics asthma muscarinic receptors
The use of anticholinergics in obstructive airways diseases, like asthma and chronic obstructive pulmonary disease, is primarily based on their acute bronchodilatory effects. Thus, muscarinic receptor antagonists provide acute relief from the increased levels of acetylcholine released in the airways on reflex vagal nerve stimulation during allergic airway inflammation (1). Potential effects of anticholinergics on inflammation-induced structural changes in the airways, however, have not been considered thus far. Nevertheless, it has been demonstrated that muscarinic receptor stimulation potentiates the mitogenic response of bovine tracheal smooth muscle cells to platelet-derived growth factor, the effect of which is mediated by the Gq-coupled muscarinic M3 receptor (2). In addition, muscarinic receptor stimulation augmented the mitogenic responses of human airway smooth muscle cells to epidermal growth factor (3). Because the expression levels of some peptide growth factors (e.g., epidermal growth factor and basic fibroblast growth factor) have been found to be elevated in asthma (4, 5), it may be envisaged that functional interaction of acetylcholine with growth factors during chronic airway inflammation is involved in the development of airway smooth muscle thickening, a pathologic feature observed in individuals with asthma as well as in animal models of allergic asthma (6eC8). Airway smooth muscle thickening is postulated to be involved in the development of chronic airway hyperresponsiveness in asthma (9).
In both cell culture (10) and organ culture (11) settings, growth factoreCstimulated airway smooth muscle growth has been tightly associated with phenotypic plasticity. This phenotypic plasticity allows airway smooth muscle to adapt to promitogenic environments, resulting in diminished contractility and contractile protein expression but increased proliferative and synthetic properties (12). Conversely, growth arrest can reconstitute a contractile or even a hypercontractile phenotype (13, 14). Therefore, airway smooth muscle phenotypic plasticity may contribute to airway inflammation and airway remodeling during periods of allergen exposure and to increased contractility in the periods in between. Importantly, a recent study showed the occurrence of allergen-induced airway smooth muscle phenotype switching in Brown-Norway rats, indicating that phenotypic plasticity could accompany airway smooth muscle thickening in vivo (15). The role of acetylcholine in allergen-induced airway smooth muscle phenotypic modulation, however, is presently unknown.
Therefore, in the present study, we investigated the contribution of endogenous acetylcholine to allergen-induced remodeling of airway smooth muscle in vivo. For this purpose, the effects of treatment with the long-acting muscarinic receptor antagonist tiotropium bromide (16) were evaluated after repeated allergen challenge in a guinea pig model of allergic asthma, characterized by early and late asthmatic reactions, airway hyperresponsiveness after these reactions, and airway inflammation (17). Airway smooth muscle area and cell number, contractile protein expression, and contractility were assessed ex vivo after 12 weekly repeated allergen exposures. It was demonstrated that pretreatment with tiotropium bromide considerably reduced allergen-induced airway smooth muscle remodeling in vivo.
Some of the results of these studies have been previously reported in the form of an abstract (18).
METHODS
A more detailed description of the methods is provided in the online supplement.
Animals
Outbred male, specified pathogen-free Dunkin Hartley guinea pigs (Harlan, Heathfield, UK) weighing 250 to 300 g were sensitized to ovalbumin with Al(OH)3 as adjuvant, as described previously (17). The animals were used experimentally 4 to 8 weeks later. All protocols described in this study were approved by the University of Groningen Committee for Animal Experimentation.
Provocation Procedures
Provocations were performed by inhalation of aerosolized solutions of ovalbumin (Sigma, St. Louis, MO) or saline under conscious and unrestrained conditions, as described previously (17). Allergen inhalations were discontinued when the first signs of respiratory distress were observed. No antihistaminic agent was needed to prevent anaphylactic shock.
Experimental Protocol
Guinea pigs were challenged with either ovalbumin or saline once weekly, for 12 consecutive weeks. For tiotropium treatment, animals received a nebulized dose of tiotropium bromide (Boehringer Ingelheim, Ingelheim, Germany) in saline (0.1 mM, 3 minutes), 0.5 hours before each challenge with saline or ovalbumin.
Tissue Acquisition
Twenty-four hours after the last challenge, guinea pigs were killed by a sharp blow on the head, followed by rapid exsanguination. The lungs were immediately resected and kept on ice for further processing. The trachea was removed and transferred to a Krebs-Henseleit solution (37°C), pregassed with 5% CO2, and 95% O2, pH 7.4.
Morphometric Analysis of Airway Smooth Muscle Mass
Smooth muscle area was determined in 8-e-thick cryostat lung sections. Transverse cross-sections of the main bronchi in both right and left lung lobes were used for morphometric analyses. To identify smooth muscle, sections were stained for sm--actin (Sigma, St. Louis, MO) or sm-myosin heavy chain (sm-MHC; Neomarkers, Fremont, CA). Primary antibodies were visualized using horseradish peroxidase-linked secondary antibodies and diaminobenzidine (1 mg/ml). Airways within each section were digitally photographed and classified as cartilaginous or noncartilaginous. All immunohistochemical measurements were performed digitally using quantification software (ImageJ; http://rsb.info.nih.gov/ij/index.html). For this purpose, digital photographs of lung sections were analyzed at a magnification of 40 to 100x. Measurements were performed by a single observer in a blinded fashion. In addition, hematoxylin-stained nuclei within the airway smooth muscle bundle were counted. Of each animal, four lung sections were prepared per immunohistochemical staining, in which a total of three to five airways of each classification were analyzed.
Western Analysis of Contractile Protein Expression
Lung homogenates were prepared as described in detail in the online supplement. Protein homogenates were separated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis, followed by standard immunoblotting techniques (see online supplement for details). Antibodies were visualized by enhanced chemiluminescence (Pierce, Rockford, IL). Photographs of blots were analyzed by densitometry (Totallab; Nonlinear Dynamics, Newcastle, UK). On the basis of the densitometry scans of these blots, the relative expression of myosin and actin between treatment groups was calculated, with the responses of saline-challenged animals set at 100%. Variation in the saline-challenged animals was calculated using absolute densitometry data. No protein standards were used for calibration purposes.
Isometric Tension Measurements
The trachea was prepared free of serosal connective tissue. Single, open-ring, epithelium-denuded preparations were mounted for isometric recording in organ baths, containing Krebs-Henseleit solution. After equilibration, resting tension was adjusted to 0.5 g followed by precontractions with 20 and 40 mM KCl. Following wash-outs, and another equilibration period of 30 minutes, cumulative concentration response curves were constructed to methacholine (see online supplement for more details).
Data Analysis
All data represent means ± SEM from n separate experiments. Sensitivity to methacholine was expressed as PEC50, i.e., eClog10 of the concentration causing 50% of the effect. The statistical significance of differences between data was determined by one-way analysis of variance or Student's t test with Bonferroni correction, as appropriate. Differences were considered to be statistically significant when p values were less than 0.05.
RESULTS
Effects of Ovalbumin Challenge and Tiotropium Treatment on Airway Smooth Muscle Content
Figure 1 shows representative lung sections stained for sm-MHC and sm--actin containing a main bronchus as well as blood vessels. Although the airway smooth muscle layer stained positively for contractile proteins, sm--actineC and sm-MHCeCpositive areas were slightly dissimilar (i.e., sm-MHCeCpositive area was more discontinuous and appeared to be somewhat smaller). Indeed, morphometric analysis revealed that, in saline-challenged control animals, sm--actineCpositive area was somewhat larger than sm-MHCeCpositive area, both in cartilaginous (0.100 ± 0.006 vs. 0.073 ± 0.010 mm2/mm basement membrane for sm--actin and sm-MHC, respectively; p = 0.035) and noncartilaginous airways (0.064 ± 0.008 vs. 0.042 ± 0.006 mm2/mm basement membrane for sm--actin and sm-MHC, respectively; p = 0.010; see Figures 2 and 3).
Repeated ovalbumin challenge did not change airway smooth muscle content in the larger airways, regardless of the contractile marker protein used. In noncartilaginous airways, however, ovalbumin challenge induced a significant increase in both sm--actineC and sm-MHCeCpositive area of 0.022 ± 0.006 mm2/mm basement membrane (36 ± 3% increase) and 0.024 ± 0.006 mm2/mm basement membrane (57 ± 13% increase), respectively, as compared with saline-challenged, age-matched control animals (Figures 2 and 3). This increase was largely prevented by treatment with tiotropium, for both contractile marker proteins (Figures 2B and 3B). Tiotropium bromide treatment by itself did not induce significant changes in the morphometric parameters analyzed, when compared with untreated saline-challenged control animals (Figures 2 and 3). Of note, all morphometric data shown were calculated using basement membrane length to correct for airway size. Data were also analyzed using the square of basement membrane length to correct for airway size, which produced similar results (see data shown in the online supplement: Figures E1 and E2).
To determine the nature of the changes in airway smooth muscle area within the noncartilaginous airways, the number of nuclei that comprised the airway smooth muscle layer in these airways was counted. To account for differences in airway smooth muscle content between airways as well as between treatment groups, data were expressed as the number of nuclei per mm2 of smooth muscle. With these data, the average apparent volume of the individual airway smooth muscle cell was also calculated, assuming equal thickness of all sections (8 e). For saline-challenged animals, 5,107 ± 405 nuclei were counted per square millimeter of smooth muscle, and an apparent volume of 1,650 ± 121 e3 per airway smooth muscle cell was calculated. No differences in either parameter were found between any of the treatment groups, indicating that the observed differences in airway smooth muscle content were exclusively caused by changes in cell number, not in cell size (Figure 4).
Effects of Ovalbumin Challenge and Tiotropium Treatment on Contractile Protein Expression
Changes in sm--actineC and sm-MHCeCpositive area could imply changes in contractile protein expression. Therefore, we used Western analysis to determine the relative contents of these contractile proteins in whole lung homogenates. For sm--actin expression, differences between treatment groups were small, indicating that the changes in sm--actineCpositive area in noncartilaginous airways had only little impact on total sm--actin expression in the lung (Figure 5). Surprisingly, therefore, large differences in sm-MHC expression were observed between the treatment groups. Repeated ovalbumin challenge strongly increased total sm-MHC expression in the lung to 422 ± 28% of sm-MHC content in saline-challenged controls. Pretreatment with tiotropium attenuated this increase to 300 ± 22% (p < 0.01), corresponding to 38 ± 6% inhibition of the ovalbumin-induced increase. Tiotropium by itself, however, had no effect on sm-MHC expression (Figure 6).
Effects of Ovalbumin Challenge and Tiotropium Treatment on Tracheal Smooth Muscle Contractility
Even though ovalbumin challengeeCinduced changes in airway smooth muscle content were confined to noncartilaginous airways, the observed changes in total sm-MHC expression could still allow for changes in contractility in the central airways. Indeed, repeated ovalbumin challenge enhanced methacholine-induced contraction of epithelium-denuded open-ring tracheal preparations from 2.1 ± 0.1 g in saline-challenged animals to 2.7 ± 0.2 g in ovalbumin-challenged guinea pigs (p < 0.05; Figure 7). Sensitivity to methacholine, however, was not altered (pEC50 = 6.6 ± 0.1 and 6.5 ± 0.1 for saline- and ovalbumin-challenged animals, respectively). Basal smooth muscle tone tended to be somewhat higher in ovalbumin-challenged guinea pigs, but this was not statistically significant. Pretreatment with tiotropium slightly decreased the sensitivity to methacholine in saline-challenged animals to a pEC50 value of 6.3 ± 0.1 (p < 0.05), with no significant change in maximal contraction (1.8 ± 0.2 g). However, tiotropium pretreatment completely prevented the increase in contractility induced by repeated ovalbumin challenge (1.6 ± 0.1 g; p < 0.001; Figure 7).
DISCUSSION
The most important finding of this study is that changes in airway smooth muscle content, contractile protein expression, and contractility induced by repeated allergen exposure can be partially or even fully prevented by tiotropium bromide, a long-acting muscarinic receptor antagonist shown to provide a long-lasting bronchodilating and bronchoprotective effect in patients with chronic obstructive pulmonary disease and asthma (16). In permanently instrumented, conscious, and unrestrained guinea pigs, we have previously demonstrated that the tiotropium dose used in this study provides a sustained muscarinic receptor blockade lasting more than 96 hours (19), which extends well beyond the duration of allergen-induced early- and late-phase asthmatic reactions in this animal model (17). Therefore, the effects of tiotropium described in this study are likely to represent the cumulative contribution of cholinergic activity to airway smooth muscle remodeling caused by the repeated allergen challenges. This indicates that acetylcholine could have a major impact on the progression of airway remodeling in allergic asthma, a process that has thus far primarily been associated with mediators of inflammation and growth factors (20).
Nevertheless, inflammatory mediators and growth factors are likely to play a crucial part in the observed effects of acetylcholine. Thus, tiotropium bromide was effective only in animals that were challenged with ovalbumin, indicating that acetylcholine affects airway smooth muscle mass in inflamed, but not in healthy, airways. This finding could, in part, be explained by augmented acetylcholine release after allergen challenge. Thus, eosinophilic inflammation-derived polycations, such as major basic protein, are known to cause epithelial damage, which can expose afferent sensory nerve endings to the airway lumen and increase vagal reflex activity in response to inhaled stimuli (21). In addition, cholinergic afferents can be stimulated by a variety of mediators involved in allergen-induced airway inflammation (see Reference 1 for extensive review). Eosinophil-derived major basic protein can also increase vagally induced acetylcholine release by inhibition of prejunctional autoinhibitory M2 receptors (22). Importantly, both allergen-induced M2 autoreceptor dysfunction and enhanced cholinergic reflex activity have been demonstrated in the guinea pig model of allergic asthma used in this study (23, 24). An additional mechanism that might contribute to increased levels of acetylcholine after allergen exposure is its release from inflammatory and epithelial cells. This nonneuronal release of acetylcholine may be elevated in conditions of allergic inflammation, because it was found to be increased in skin biopsies from patients with atopic dermatitis, a condition often associated with bronchial asthma (25, 26). In addition to augmented release of acetylcholine, reduced acetylcholinesterase activity, as reported in a canine model of active allergic sensitization (27), might contribute to an increased level of acetylcholine as well.
Despite the enhanced release of acetylcholine during allergic airway inflammation, it may be envisaged that acetylcholine is ineffective in airway smooth muscle remodeling by itself and that concerted action with mediators of inflammation and growth factors is required for the effect. This could explain the absence of tiotropium effect in the control animals. Furthermore, in vitro, muscarinic receptor stimulation does not or only modestly affects airway smooth muscle proliferation by itself, but effectively augments growth factoreCinduced responses (2, 3). The latter mechanism may well be responsible for the allergen-induced increase in airway smooth muscle mass observed in the noncartilaginous airways, because an increase in cell number rather than cell size was the predominant cause of allergen-induced airway smooth muscle thickening in these airways.
The mechanism of acetylcholine-induced airway smooth muscle thickening may also be relevant to airway remodeling in patients with asthma, because an increase in airway smooth muscle mass in bronchial biopsies of patients with mild to moderate asthma was accompanied by a twofold increase in cell number without a change in cell volume (28). Other studies have indicated that hypertrophy may also contribute to increased airway smooth muscle mass observed in patients with asthma (29, 30). However, because no hypertrophy was observed in our model under the applied conditions, a possible role for acetylcholine in this process remains unclear.
Airway smooth muscle content in the large pulmonary airways, including the main bronchi, did not change after repeated allergen challenge. Nevertheless, contractility of tracheal preparations was increased, suggesting a different nature of airway smooth muscle remodeling in the central airways. This is also indicated by a previous study, demonstrating increased tracheal smooth muscle contraction after repeated allergen-challenge in guinea pigs, without concomitant changes in airway smooth muscle mass (31). These observations suggest that airway smooth muscle cells in the central airways acquire a hypercontractile phenotype with repeated allergen challenge. Moreover, the inhibitory effects of tiotropium bromide indicate that endogenous acetylcholine contributes to the induction of this hypercontractile phenotype in vivo. These effects cannot be attributed to acute pharmacologic effects of putatively remaining tiotropium bromide after administration of the last dose, because previous experiments by our laboratory have shown that tiotropium bromide does not affect maximal methacholine-induced contraction of guinea pig tracheal preparations ex vivo at 24 hours after in vivo exposure (Drge and coworkers, unpublished observations). Moreover, direct pharmacologic effects of tiotropium would affect the saline-challenged, tiotropium-treated animals and the ovalbumin-challenged, tiotropium-treated animals equally.
Regulation of contractile protein expression by acetylcholine may be involved in the increased contractility, because our results demonstrate a selective increase in sm-MHC expression in the lung after allergen exposure, which was partially inhibited by tiotropium. Of note, changes in sm-MHCeCpositive area in the noncartilaginous airways may not be the sole cause of the increase in sm-MHC expression, because a 57% increase in area in these airways as such cannot result in a 322% increase in whole lung myosin expression. Together with the unchanged sm-MHCeCpositive area in the larger airways, increased sm-MHC expression per airway smooth muscle cell seems to be more likely. However, the localization of this increased sm-MHC expression is as yet unknown.
Tiotropium bromide affected the allergen-induced changes in pulmonary sm-MHC expression to a lesser extent as compared with its large effects on allergen-induced changes in airway smooth muscle mass and contractility. It is important to realize, however, that whole lung sm-MHC content also reflects vascular smooth muscle MHC, which might be regulated in a differential way. Further experiments are required to address this item.
Sm--actin expression did not increase significantly after allergen exposure. The discrepancy between the expression of sm--actin and sm-MHC may be explained by relatively high sm--actin expression by cell types other than airway smooth muscle cells (e.g., fibroblasts that express sm--actin but not sm-MHC [32]), which is supported by the observation that sm--actineCpositive area was larger than sm-MHCeCpositive area, even in the airway smooth muscle layer. The difference cannot be explained by cross-reactivity of the sm--actin antibody, however, because Western analysis revealed no staining for additional proteins. In addition, it may be envisaged that sm--actin and sm-MHC expression can be regulated independently and to different extents. Indeed, the induction of a hypercontractile canine airway smooth muscle phenotype in vitro is accompanied by a much greater increase in sm-MHC expression (± eightfold increase) as compared with sm--actin (± twofold increase) (14).
The effects of acetylcholine on contractility may at least partially be explained by activation of the RhoA/Rho-kinase pathway, because this pathway has been described to regulate both airway smooth muscle contractility (33) and smooth muscleeCspecific gene transcription (34). Moreover, muscarinic M3 receptoreCdependent activation of RhoA and Rho-kinase has been reported to induce smooth muscleeCspecific gene transcription in airway smooth muscle cells in vitro (35). These RhoA-dependent effects may even be enhanced after repeated allergen exposure, which induces an increase in RhoA expression (36). Nevertheless, prolonged (8 days) exposure of bovine tracheal smooth muscle strips to high concentrations ( 10 e) of methacholine results in a decline in contractility and contractile protein expression, caused by the prolonged elevation of [Ca2+]i (37). Therefore, Rho-dependent rather than Ca2+-dependent mechanisms are likely to regulate acetylcholine-induced alterations in contractility in vivo.
An important consideration is how the effects of tiotropium could relate to the effectiveness of anticholinergics in the long-term treatment of asthma. In asthma, 2-adrenoceptor agonists are usually more effective bronchodilators than anticholinergics (38). Nonetheless, 2-agonists appear to be at most modestly effective in inhibiting allergen-induced airway smooth muscle proliferation in vivo (39), despite their effectiveness in inhibiting airway smooth muscle proliferation in vitro (40). Moreover, chronic 2-agonist exposure has even been reported to increase airway responsiveness to acetylcholine in vivo and ex vivo (31). It appears therefore that anticholinergics could be more effective than -agonists in preventing allergen-induced airway smooth muscle remodeling. However, the effect of chronic treatment with anticholinergics on airway remodeling, responsiveness, and changes in lung function in patients with asthma is thus far unclear.
Further investigations establishing the effects of tiotropium bromide on other characteristics of airway remodeling, such as basement membrane thickening, hyperplasia of goblet cells and mucous glands, extracellular matrix deposition, and bronchial microvascular remodeling will help to determine the potential benefit that anticholinergics could have in asthma. Moreover, possible effects of tiotropium on inflammatory processes involved in airway remodeling may also be of interest. In this regard, corticosteroids have been reported to inhibit growth factoreCinduced airway smooth muscle proliferation, cytokine production, and extracellular matrix deposition in vitro (41eC43), and to inhibit but not reverse allergen-induced fibronectin deposition in rats in vivo (44). Moreover, the inhibitory effects of corticosteroids on airway smooth muscle proliferation in vitro are strongly inhibited when cells are cultured on collagen type I (45), which is increased in asthma (46). Surprisingly, however, the effect of corticosteroids on airway smooth muscle thickening in vivo has not yet been investigated. Future studies using animal models characterized by allergen-induced airway smooth muscle proliferation could therefore be useful to compare different treatment strategies.
In conclusion, we have demonstrated that tiotropium bromide inhibits allergen-induced airway remodeling in a guinea pig model of ongoing asthma. Therefore, endogenous acetylcholine appears to play an important role in airway smooth muscle remodeling, a process thus far primarily associated with mediators of inflammation and growth factors. This finding could have important implications for the use of anticholinergics in the treatment of allergic asthma, by protecting against the development of chronic airway hyperresponsiveness and decline of lung function in addition to their acute bronchodilating effects.
This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org
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