Published ahead of print on January 24, 2008, doi:10.1164/rccm.200707-977OC
© 2008 American Thoracic Society doi: 10.1164/rccm.200707-977OC
Increased Airway Smooth Muscle Mass in Children with Asthma, Cystic Fibrosis, and Non-Cystic Fibrosis Bronchiectasis1 Department of Paediatric Respiratory Medicine, Royal Brompton Hospital, London, United Kingdom; 2 Department of Gene Therapy, National Heart and Lung Institute, Imperial College London, London, United Kingdom; and 3 Institute of Anatomy, University of Berne, Berne, Switzerland Correspondence and requests for reprints should be addressed to Dr. Nicolas Regamey, M.D., Department of Gene Therapy, National Heart and Lung Institute, 1B Manresa Road, London SW3 6LR, UK. E-mail: n.regamey{at}imperial.ac.uk
Rationale: Structural alterations to airway smooth muscle (ASM) are a feature of asthma and cystic fibrosis (CF) in adults. Objectives: We investigated whether increase in ASM mass is already present in children with chronic inflammatory lung disease. Methods: Fiberoptic bronchoscopy was performed in 78 children (median age [IQR], 11.3 [8.5–13.8] yr): 24 with asthma, 27 with CF, 16 with non-CF bronchiectasis (BX), and 11 control children without lower respiratory tract disease. Endobronchial biopsy ASM content and myocyte number and size were quantified using stereology. Measurements and Main Results: The median (IQR) volume fraction of subepithelial tissue occupied by ASM was increased in the children with asthma (0.27 [0.12–0.49]; P < 0.0001), CF (0.12 [0.06–0.21]; P < 0.01), and BX (0.16 [0.04–0.21]; P < 0.01) compared with control subjects (0.04 [0.02–0.05]). ASM content was related to bronchodilator responsiveness in the asthmatic group (r = 0.66, P < 0.01). Median (IQR) myocyte number (cells per mm2 of reticular basement membrane) was 8,204 (5,270–11,749; P < 0.05) in children with asthma, 4,504 (2,838–8,962; not significant) in children with CF, 4,971 (3,476–10,057; not significant) in children with BX, and 1,944 (1,596–6,318) in control subjects. Mean (SD) myocyte size (µm3) was 3,344 (801; P < 0.01) in children with asthma, 3,264 (809; P < 0.01) in children with CF, 3,177 (873; P < 0.05) in children with BX, and 1,927 (386) in control subjects. In all disease groups, the volume fraction of ASM in subepithelial tissue was related to myocyte number (asthma: r = 0.84, P < 0.001; CF: r = 0.81, P < 0.01; BX: r = 0.95, P < 0.001), but not to myocyte size. Conclusions: Increases in ASM (both number and size) occur in children with chronic inflammatory lung diseases that include CF, asthma, and BX.
Key Words: asthma • cystic fibrosis • children • biopsy • smooth muscle
Structural changes to airway smooth muscle (ASM) are a well-known feature of chronic airway diseases, such as asthma and chronic obstructive pulmonary disease, in adults (1). In asthma, increase of smooth muscle mass in the airway wall has been linked to severity and duration of disease (2, 3), and functional consequences, such as airflow obstruction through airway wall thickening and increased airway responsiveness, have been proposed (4). There is debate as to whether the increase of smooth muscle mass in the airway wall is due to hyperplasia or hypertrophy, or both (2, 5, 6). As in asthma, patients with cystic fibrosis (CF) develop progressive airflow obstruction. A subgroup of patients with CF also have airway hyperresponsiveness and reversible airway obstruction, in which similar remodeling processes to those seen in asthma may be involved (7, 8). Indeed, an increase of ASM content has been shown in the lungs at autopsy or after transplantation or lobectomy from adults with severe CF disease (9, 10). More recently, structural changes to ASM have been described in biopsies from adults with CF and mild-to-moderate airway obstruction (11). There are, to date, only a few studies that have reported airway remodeling in children. Thickening of the reticular basement membrane (RBM), a pathological feature characteristic of adult asthma, has been shown in children with asthma and in preschool children with confirmed wheeze, and there is evidence of epithelial loss and angiogenesis in the airways of children with asthma (12–14). We have recently reported RBM thickening in children with CF and non-CF bronchiectasis (BX) (15). Thus several aspects of airway remodeling, which have been described in adults, seem to be already present in childhood. In contrast, few data are available on structural changes to ASM in children (16, 17). Our recent work has suggested that ASM mass may be increased in children with asthma and CF (18), an observation that we wished to investigate further and quantify. Because studies in asthma in particular have shown that some features of airway remodeling, such as RBM thickening, are present from early on, we hypothesized that structural changes to ASM may also be an early feature. We also hypothesized that ASM changes may be found in children with various chronic inflammatory lung diseases, as has been described in adults. We therefore studied ASM changes using endobronchial biopsies obtained from children with asthma, CF, and BX, and compared the data with those from biopsies of children with no lower respiratory tract disease. Secondary objectives in our study were to determine whether any increase in ASM mass was related to functional markers of airflow obstruction, and whether such an increase was due to myocyte hyperplasia, hypertrophy, or both. We quantified total ASM content and determined myocyte size and number on biopsies using a stereologic approach that has been previously applied and validated in the study of structural changes to ASM in endobronchial biopsies of adult patients with asthma (6). Some of the results of this study have been previously reported in abstract form (19, 20).
Subjects The present study included 78 schoolchildren (median age [interquartile range (IQR)], 11.3 [8.5–13.8] yr) who underwent a clinically indicated flexible bronchoscopy between July 2002 and June 2006, and in whom additional endobronchial biopsies were taken for the purpose of research. Twenty-four children had moderate-to-severe asthma, 27 had CF, 16 had BX, and there were 11 control subjects without lower airway disease (Table 1; and see the online supplement). Diagnoses of asthma and CF were made according to standard criteria (21, 22). Moderate-to-severe asthma was characterized by "daily symptoms despite > 400 µg/day of inhaled budesonide (or equivalent)" (23). A diagnosis of bronchiectasis was made on high-resolution computed tomography (24). Our control subjects were children who had been referred to a tertiary center for investigation of recurrent respiratory symptoms, but in whom no lower airway disease was found upon investigations that included a clinically indicated flexible bronchoscopy. Some of the children included in this study have been included in previous reports (15, 18, 25). The study was approved by the local ethics committee. Informed consent was obtained from the parents of all children.
Flexible Bronchoscopy Bronchoscopy was performed as previously described (15). Up to five (median, three; range, one to five) endobronchial biopsies were taken from a standardized site (subsegmental bronchi of the right lower lobe) and processed into paraffin wax blocks. To be included in the study, we required a priori that each child have had at least one evaluable biopsy (18). For details, see the online supplement.
Tissue Morphometry
Pulmonary Function Tests
Allergen Testing
Statistical Analysis
A total of 209 biopsies were taken from 78 children. One hundred eighty-four (88%) of these biopsies were considered evaluable, according to predefined criteria (18) (for details, see the online supplement). Fifteen (19%) children had one evaluable biopsy, 27 (35%) had two, 30 (38%) had three, 5 (6%) had four, and 1 (1%) had five. The mean (SD) number of biopsies analyzed per child was similar in patient groups: 2.3 (0.8) for asthma, 2.3 (0.9) for CF, 2.5 (1.1) for BX, and 2.4 (0.9) for control subjects. Volume fraction measurements were made on a median (IQR) of 571 (295–864) points per subject, representing a median (IQR) area of 1.00 mm2 (0.51–1.51 mm2) of tissue studied. Tissue depth was similar in patient groups: median (IQR) surface area of RBM per volume of subepithelial tissue (µm2/µm3) was 95 x 10–3 (79–113) for asthma, 108 x 10–3 (64–134) for CF, 94 x 10–3 (66–110) for BX, and 99 x 10–3 (86–122) for control subjects (Figure E3 of the online supplement). Median (IQR) volume fraction of ASM in subepithelial tissue was significantly increased in all patient groups compared with control subjects: 0.27 (0.12–0.49; P < 0.0001) in asthma, 0.12 (0.06–0.21; P < 0.01) in CF, 0.16 (0.04–0.21; P < 0.01) in BX, and 0.04 (0.02–0.05) in control subjects (Figure 1). Similarly, median (IQR) volume of ASM in subepithelial tissue was significantly increased in all patient groups compared with control subjects when indexed to the surface area of RBM (Figure E4). Analysis of covariance showed no effect of sex and age on these outcomes.
ASM content in subepithelial tissue was not related to age, FEV1, FVC, or FEV1/FVC in any of the patient groups (Table E1). However, the volume fraction of ASM in subepithelial tissue was significantly related to the improvement in FEV1 after bronchodilator inhalation in the asthma group (Figure 2). When only children with at least three evaluable biopsies or when only children with a positive bronchodilator response (FEV1 improvement of at least 12%) were considered, this relationship was even stronger and remained statistically significant (Figure 2).
There was no relationship between volume fraction of ASM in subepithelial tissue and exhaled nitric oxide, number of positive allergens on skin prick, or specific IgE testing, total serum IgE, or blood eosinophil counts in the asthma group (Table E2). There was no difference in volume fraction of ASM in subepithelial tissue between patients who used oral steroids and those who did not, and there was no relationship between volume fraction of ASM in subepithelial tissue and dose of inhaled steroids in any of the patients groups (Table E3). Compared with the control subjects, the medians (IQR) for myocyte number (cells per mm2 of RBM) were larger in all patient groups, although the difference was only significant for the asthma group: 8,204 (5,270–11,749; P < 0.05) in asthma, 4,504 (2,838–8,962) in CF, 4,971 (3,476–10,057) in BX, and 1,944 (1,596–6,318) in control subjects (Figure 3A). The means (SD) for myocyte size (µm3) were significantly increased in all patient groups compared with control subjects: 3,344 (801; P < 0.01) in asthma, 3,264 (890; P < 0.01) in CF, 3,177 (873; P < 0.05) in BX, and 1,927 (386) in control subjects (Figure 3B). The volume fraction of ASM in subepithelial tissue was related to myocyte number in all disease groups, but not to myocyte size (Figure 4).
This is the first study reporting an increase of ASM mass in chronic airway disease in childhood. We find that structural changes to ASM appear early in the course of development of chronic airway inflammatory disease in children and that this feature is not limited to children with asthma but is also present in childhood CF and in BX. We demonstrate a significant positive relationship between ASM mass and reversibility of bronchial obstruction in children with asthma and show that ASM changes include both hyperplasia and hypertrophy. Our quantitative data extend the studies of adults with chronic airway disease to the pediatric age group. Cutz and colleagues previously observed changes in peribronchial smooth muscle in lung biopsy samples from two children with asthma in remission and two children who died in status asthmaticus (16). These observations were qualitative only as were those of Jenkins and colleagues, who noted that three children with difficult-to-control asthma appeared to have an increase of ASM mass in their endobronchial biopsies (17). Thus, there has been no objective quantitative study in children with asthma and none of ASM structural changes in children with CF or BX. The ASM increases we describe here were of similar magnitude to those previously reported in bronchoscopic studies of adults. In our children with moderate-to-severe asthma, 27% of subepithelial tissue was occupied by ASM, which is similar to the values reported by Benayoun and coworkers (i.e., 18% in adults with mild-to-moderate asthma and 40% in patients with severe asthma) and Woodruff and colleagues (16% in adults with mild-to-moderate asthma) (2, 6). Also, we found that, in children with CF, 12% of subepithelial tissue was occupied by ASM, which compares with values reported by Hays and coworkers (17% in adults with CF) (11). Increase in ASM mass has previously been linked to both age and duration of disease in asthma: Bai and colleagues found that the size of ASM mass in autopsy specimens obtained from young (17–23 yr) adults with asthma was twofold increased compared with age-matched control subjects, whereas there was a fourfold increase in older (40–49 yr) subjects (3). We looked for, but did not detect, an age effect in our study, which might have been due to the small age range included (6–16 yr). Nevertheless, the fact that school-age children demonstrated an ASM increase comparable in extent to that of adults suggests that significant remodeling occurs early in life. The increase in ASM mass may result directly in an excess of airway narrowing in asthma, due to the thickening of the airway wall and/or a concomitant increase in force generation (33–35). Several studies in vitro have also demonstrated increased contractile responses (36, 37) and disturbed relaxation responses of asthmatic ASM (37), although whether these can be extrapolated to airway responses in vivo is debatable (38–40). Moreover, it is unclear whether muscle mass and muscle contractility might covary, and other factors, such as the synthetic or maturational state of the myocyte, may be important (41, 42). In the present study, we demonstrate a direct relationship between smooth muscle content in the airway wall and bronchodilatation to β2-agonists in vivo in children with asthma. This could indicate a functional association between changes to ASM and the reversible component of airflow obstruction in asthma. However, because we did not perform routine reversibility testing in the other disease groups studied, we are unable to state whether this association also applies to CF and BX. It has been suggested previously that increased ASM content could contribute to airway hyperresponsiveness in CF (9), but a recent study using endobronchial biopsies on a small number of adult patients with CF did not find a relationship between measures of ASM content and lung function that included improvement in FEV1 after bronchodilator (11). The application of stereology, in particular the disector method, to our biopsy tissue allowed us to differentiate between ASM hyperplasia (increase in cell number) and hypertrophy (increase in cell size). We found indications of both ASM hyperplasia and hypertrophy in each disease group, although the increase in myocyte number compared with controls was only statistically significant for the asthma group. Although the trend is similar, the lack of significance in the remaining groups is likely because of the small subset of subjects available for this part of the study (Figure 3). This interpretation is supported by the finding that myocyte number was related to ASM content in each disease group, whereas myocyte size was not, suggesting that hyperplasia rather than hypertrophy may be the mechanism predominantly responsible for the increase of ASM in each group. Previous studies in adults with asthma have also found evidence that both ASM hyperplasia and hypertrophy contribute to ASM mass increase (5, 43). The relative contribution of each to the pathogenesis of ASM increase may differ depending on the disease, its severity, the localization of the changes studied, and the presence and visualization of adjacent connective tissue. For instance, Ebina and colleagues performed morphometry of airway muscle cells on serial sections of autopsied lungs from 10 patients with asthma. In patients with thickened ASM mass only in the central bronchi, they observed ASM hyperplasia without hypertrophy, whereas in patients with thickened ASM in the entire airway tree, they observed the predominance of ASM hypertrophy, with mild hyperplasia localized only to the bronchi, the airways available to us for endobronchial biopsy in the children in our report (5). Benayoun and coworkers studied endobronchial biopsies obtained from adults and found myocyte hypertrophy without evidence of ASM replication in patients with severe asthma and, to a lesser extent, in patients with intermittent and mild-to-moderate asthma (2). In contrast, Woodruff and colleagues found ASM hyperplasia without significant changes in myocyte size in endobronchial biopsies from patients with mild-to-moderate asthma (6). In agreement with the findings of hyperplasia, ASM cells from adult patients with asthma have been shown to proliferate significantly faster in vitro than cells from control subjects (41). They have also been shown to produce more connective tissue growth factor in response to transforming growth factor-β, more IL-6 upon stimulation with recombinant OX40 (CD134), and less prostaglandin E2 than cells from control subjects (41, 44, 45). Woodruff and colleagues also described ASM hyperplasia without hypertrophy in bronchoscopic biopsy specimens obtained from seven adults with CF (11). Remodeling of the airway wall has been associated with chronic inflammation in adults (46), and infiltration of ASM by mast cells has been demonstrated in adult patients with asthma (47). Yet, whereas we studied three inflammatory lung disease groups, it appeared that the increase of ASM mass was most marked in the children with asthma. We now plan to investigate whether this is due to differences in the magnitude or predominant patterns of inflammation in each of these clinically distinct inflammatory conditions. We realize that the associations between inflammation and remodeling are complex and acknowledge that at least some aspects of airway remodeling may be independent of inflammation such that parallel pathways of development for inflammation and remodeling may exist (1, 15, 48, 49). Interestingly, it has been proposed in CF that the defect of the CF transmembrane conductance regulator (CFTR) itself may contribute to ASM increase (50). A novel feature of our work is the use of the BX group, also characterized by airway neutrophilia, to compare with children with CF. The finding that ASM changes were similar in the two neutrophilic groups (CF and BX) suggests that CFTR dysfunction is not specifically required, and that chronic inflammation is likely the common contributing feature responsible. Inevitably, a study of endobronchial biopsies in children has limitations. Endobronchial biopsy samples are limited in size and only reflect the morphology of the airway mucosa rather than the full airway wall thickness. Furthermore, the biopsies come mainly from relatively proximal airways (in our case, subsegmental bronchi from the right lower lobe) and from subcarinae rather than from the lateral walls, and thus may not be representative of the entire conducting airways. In addition, there is variability in structural characteristics between biopsies, and therefore multiple biopsies per subject should ideally be analyzed (51, 52). Due to time constraints during the bronchoscopic procedure and because of the difficulties of obtaining biopsies in children with adequately preserved morphology (18), we were only able to analyze between two and three biopsies of adequate morphology per child. We thus acknowledge that some relationships (e.g., between ASM content in the biopsies and number of positive allergens on skin-prick or specific IgE testing, total serum IgE, blood eosinophil counts, or exhaled nitric oxide in children with asthma) may have been missed due to inadequate sampling. In summary, our data support the initial hypothesis that the increases of ASM demonstrated previously in adults with asthma or CF are present early in the course of the development of these chronic inflammatory conditions in childhood. It is thus a much earlier feature of these airway conditions than has previously been shown. We found that the increase of ASM mass in children is not unique to asthma because it is also present in CF and BX. Thus, this aspect of remodeling is likely to be linked closely to the chronic inflammatory process. We have presented data that should prompt a search for strategies aimed at preventing changes to ASM in those who develop chronic inflammation early in life. If effective, such an approach may remove the need, in later life, to consider therapies aimed at reversing ASM changes, for instance with leukotriene modifiers (53), or even the direct removal of smooth muscle bundles from the airway wall by procedures such as bronchial thermoplasty (54, 55). These data underscore the importance of early-life events in the long-term natural history of inflammatory airway diseases.
The authors thank Chloe Dunn, Bernie Ortega, Carmen Lacruz, Eleanor Singh, Gemma Moody, and the staff of the Department of Anaesthesia, Royal Brompton Hospital, for their assistance with bronchoscopies; Marc Rosenthal and Ian Balfour-Lynn for performing some of the bronchoscopies; Donald Payne for recruiting some of the patients; Andrew Nicholson and the Department of Pathology, Royal Brompton Hospital, for their preparation of biopsy material; and Michael Roughton for statistical advice. The authors also gratefully acknowledge the patients and families that agreed to take part in the study.
N.R. is the recipient of a European Respiratory Society Fellowship (no. 64) and a grant from the Swiss National Science Foundation (no. 1172/05b). 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: 10.1164/rccm.200707-977OC on January 24, 2008 Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript. Received in original form July 3, 2007; accepted in final form January 23, 2008
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ABSTRACT
AT A GLANCE COMMENTARY
= 0.05, two-sided test) to detect a 10% difference in smooth muscle volume fraction between patient groups, with group means (SD) of 15.0 (11.4) and 5.5 (7.2). For details, see the online supplement. 
= mean myocyte size; Vv (sm/subepithelium) = volume fraction of ASM indexed to volume of subepithelial tissue. P values indicated only if significant.
