State of the Art. Remodeling in Asthma SUBEPITHELIAL FIBROSIS EPITHELIAL ALTERATION. Céline Bergeron 1, Wisam Al-Ramli 2, and Qutayba Hamid 2

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1 State of the Art Remodeling in Asthma Céline Bergeron 1, Wisam Al-Ramli 2, and Qutayba Hamid 2 1 Hotel-Dieu Hospital, Centre Hospitalier de l Université de Montréal, University of Montreal, Montréal, Quebec, Canada; and 2 Meakins-Christie Laboratories, McGilll University, Health Centre Research Institute, Montréal, Canada Airway remodeling refers to the structural changes that occur in both the large and the small airways of miscellaneous diseases, including asthma. In asthma, airway structural changes include subepithelial fibrosis, increased smooth muscle mass, enlargement of glands, neovascularization, and epithelial alterations. Although controversial, airway remodeling is commonly attributed to the underlying chronic inflammatory process. These remodeling changes contribute to thickening of airway walls and consequently lead to airway narrowing, bronchial hyperresponsiveness, airway edema, and mucous hypersecretion. Airway remodeling is associated with poorer clinical outcome among patients with asthma. Early diagnosis and prevention of airway remodeling has the potential to decrease disease severity, to improve control, and to prevent disease expression. In this article, we briefly provide an update on the characteristic features of airway remodeling observed in asthma and their clinical consequences. Keywords: remodeling; asthma; inflammation Airway remodeling in asthma was first described in 1922 by Hubert and Koessler in cases of fatal asthma (reviewed in Reference 1). Airway remodeling has been documented in all degrees of asthma severities and in both large and small airways (2). Airway remodeling refers to structural changes in airways of subjects with asthma which are not seen in healthy subjects. Figure 1 summarizes airway remodeling features. Structural changes include loss of epithelial integrity (3), thickening of basement membrane (4), subepithelial fibrosis (5), goblet cell and submucosal gland enlargement (6, 7), increased smooth muscle mass (6), decreased cartilage integrity (8), and increased airway vascularity (9, 10). Figure 2 shows remodeling features in asthma. It is believed that these changes largely stem from an ongoing chronic inflammatory process that involves activation of inflammatory cells including CD4 1 T cells, eosinophils, neutrophils, and mast cells (11 15). Duration of asthma has been associated with reduced lung function, increased hyperresponsiveness and asthma symptoms, as well as greater use of medication (5, 16, 17), and the remodeling process has been proposed to explain these features. EPITHELIAL ALTERATION Epithelial alterations in asthma include epithelial shedding, loss of ciliated cells, goblet cell hyperplasia, up-regulation of growthfactor release, and the overexpression of receptors, such as epidermal growth factor receptors (EGFR) (3, 6, 18 20). The (Received in original form August 19, 2008; accepted in final form February 27, 2009) Correspondence and requests for reprints should be addressed to Qutayba Hamid, M.D., Ph.D., Meakins-Christie Laboratories, McGill University, 3626 St Urbain Street, Montréal, PQ, H2X 2P2 Canada. qutayba.hamid@mcgill.ca Proc Am Thorac Soc Vol 6. pp , 2009 DOI: /pats RM Internet address: process of epithelial shedding is currently controversial and still a matter of debate, as some propose the detachment to be simply an artifact of physical tissue sampling of the asthmatic airways (20, 21). Studies suggest that either the asthmatic epithelium has a weakened attachment to the basement membrane or that asthmatic epithelial cells have a higher turnover compared with epithelial cells from healthy control subjects. Clinically, the extent of epithelial injury is correlated to airway hyperresponsiveness (17, 22), clearly suggesting that the degree of epithelial loss and/or turnover is related to asthma development and severity. The intact airway epithelium normally provides a physical protective barrier against inhaled small particles such as allergens. The loss of epithelium surface and the resultant denudation of the basement membrane may decrease this protective effect and increase the propensity for allergic insult on the airway. SUBEPITHELIAL FIBROSIS A second important feature of airway remodeling is the subepithelial fibrosis. Since it was first observed in 1922 (reviewed in Reference 1) in cases of fatal asthma, subepithelial fibrosis has been reported in all severities of asthma (5, 17) as well as in subjects with atopic rhinitis (23, 24), and even in children with difficult-to-treat asthma (25, 26). The subepithelial fibrosis occurs in the lamina reticularis layer just below the basement membrane, which leads to thickening of the basement membrane just underneath the epithelium. Fibrosis is a result of increased deposition of extracellular matrix (ECM) proteins, including collagens I, III, and V; fibronectin; tenascin; lumican; and biglycan (4, 27 30) by fibroblasts. Some variable correlations have been found between the severity of asthma, airway hyperresponsiveness or attack score, and subepithelial collagen types I and III deposition in the airways (31 34). Subepithelial fibrosis has been associated with the severity of asthma, and in severe asthma, increased airway wall thickness is observed (35). Proteoglycan deposition in the ECM and bronchial fibroblast production of proteoglycans also correlate with airway responsiveness in subjects with asthma (27, 36). An imbalance between ECM proteins production and degradation has also been found in asthmatic airways. The level of proteases and antiproteases may favor a profibrotic balance. Interstitial cells, macrophages, and neutrophils are the major sources of proteases and antiproteases. Matrix metalloproteinases (MMPs) are a family of proteases implicated in collagen degradation. MMP-2, MMP-3, MMP-8, and MMP-9 are the MMPs related to asthma (37 40). Among these, MMP-9 levels are reported to be significantly higher in the sputum of patients with asthma compared with control subjects (39). MMPs are implicated in airway inflammation through their influence on eosinophil trafficking (41) and in airway remodeling, not only by matrix reorganization but also by its effects on angiogenesis (42) and smooth muscle hyperplasia (43, 44). Elevated sputum

2 302 PROCEEDINGS OF THE AMERICAN THORACIC SOCIETY VOL Figure 1. Main characteristics of airway remodeling. Airway remodeling refers to structural changes in airways of individuals with asthma; these changes include (1) epithelial alteration, (2) subepithelial fibrosis, (3) increased smooth muscle mass, (4) goblet and mucous gland hyperplasia, (5) angiogenesis, (6) loss of cartilage integrity, and (7) inflammation. MMP-9 levels are associated with a fall in FEV 1 after allergen challenge and are linked to asthma severity (41, 45). Bronchoalveolar lavage level of MMP-8 inversely correlates with FEV 1 in patients with asthma (38). It has also been proposed that the remodeling process might be beneficial in airway diseases. Lambert and colleagues (46) have suggested that airway wall thickening may protect against bronchoconstriction. Collagen deposition in subepithelial matrix may inhibit narrowing by making the airway wall stiffer, representing an additional load on airway smooth muscle (24). Hyaluronan and versican deposition in and around the smooth muscle also counteracts airway narrowing and smooth muscle shortening. Experiments conducted on rat models of allergeninduced asthma suggested that airway responsiveness may increase after airway inflammation, but may decrease with airway deposition of fibronectin and collagen (47). INCREASED SMOOTH MUSCLE MASS Respiratory airway smooth muscle is the critical effector cell modulating airway tone. In asthmatic airways the smooth muscle mass is increased due to coordinated increase in size (hypertrophy) and number (hyperplasia) of airway smooth muscle cells. Importantly, the asthmatic smooth muscle cells take on not only the secretory and proliferative phenotype, but can also migrate to the subepithelial area of the asthmatic airways (6, 48, 49). Smooth muscle cells are known to actively participate in the inflammatory and remodeling processes though their release of proinflammatory cytokines, chemokines, and ECM proteins (50 52), and may therefore contribute to the pathogenesis of asthma. Migration of smooth muscle cells is a recently described feature of airway remodeling. We have shown that chemokines have the ability to induce human airway smooth muscle cell migration and to increase their contractility Figure 2. (A) Endobronchial biopsy stained with hematoxylin and eosin of a subject with asthma showing goblet (G) cell hyperplasia. (B) Endobronchial biopsy stained with hematoxylin and eosin of a subject with asthma showing extensive total subepithelial fibrosis (F) and increased smooth muscle (SMC) mass. (C) Lung biopsy showing a small airway from a subject with asthma showing increased smooth muscle mass and submucosal fibrosis. Reprinted by permission from Reference 65. in vitro, implementing another avenue which may significantly contribute to the overall airflow obstruction in these patients (49). The importance of smooth muscle mass has been correlated to asthma severity (53). GOBLET AND MUCOUS GLAND HYPERPLASIA Goblet cells hyperplasia and submucosal gland hyperplasia are seen in asthmatic airways of both adults and children; it is a feature particularly evident in fatal asthma (6, 7, 26). Functional consequences of these abnormalities mostly refer to increased

3 Bergeron, Al-Ramli, and Hamid: Remodeling in Asthma 303 sputum production, airway narrowing from sputum secretion, and increased airway wall thickness (6). ANGIOGENESIS Vascular alterations include increased size of airway wall vessels and angiogenesis (9). Changes in the airway wall microvasculature can contribute to airway wall edema and result from angiogenesis. Increased airway vascularity is seen in asthma (9, 10) in association with a greater expression of the vascular endothelial growth factor (VEGF) (54). Clinical consequences of airway wall angiogenesis are reduced airway caliber via airway wall edema, and increased inflammatory and remodeling mediators delivery into the airway wall having subsequent influence on structural and inflammatory cells. LOSS OF CARTILAGE INTEGRITY Cartilage is an important determinant of airway wall stiffness and integrity. Decreased cartilage volume and increased cartilage proteoglycan degradation are seen in asthmatic airways (8). Reduced cartilage integrity may result in a more powerful bronchoconstriction from airway smooth muscle bundles load reduction. Cartilage degradation can contribute to chronic airway obstruction, and allow more powerful bronchoconstriction for a given degree of airway smooth muscle contraction (55). INFLAMMATION Inflammation is another component of airway remodeling. There is good evidence that asthma is a heterogeneous disease. Airways of individuals with mild and moderate asthma are characterized by a Th2 profile inflammation, in which there is an overabundance of eosinophils, mast cells, and Th2 lymphocytes. These inflammatory cells release mediators which then trigger bronchoconstriction, mucus secretion, and, possibly, remodeling. The number of infiltrating leukocytes such as mast cells, eosinophils, and CD8 1 and CD45 1 T cells correlates with airway hyperresponsiveness in patients treated with inhaled corticosteroids (ICS) (56). The inflammatory mediators that drive this process include Th2 cytokines (IL-4, IL-5, IL-9, and IL-13), transforming growth factor (TGF)-b, granulocyte/macrophagecolony stimulating factor (GM-CSF), lipid mediators, and histamine. Some of these mediators have potent remodeling properties, such as TGF-b, IL-11, and IL-17 (31, 57, 58). Recently, a new subtype of T cells has been implicated in the pathogenesis of asthma, particularly in severe asthma, where the importance of noneosinophilic or neutrophilic inflammation is well described. This subtype of T cells are called Th-17, which secrete IL-17 A and F and might be involved in airway remodeling. TOOLS USED TO MEASURE AIRWAY REMODELING Airway remodeling is clinically defined as persistent airflow obstruction despite aggressive antiinflammatory therapies. The standard assessment of remodeling is obtained by surgical lung specimen or airway tissues sampled through flexible bronchoscopy. Flexible bronchoscopy is a minimally invasive technique, but requires specialist expertise; tools have been developed to bypass the biopsy sampling. Among these tools, indirect analysis of blood, urine, or sputum remodeling markers have been developed. However, we can only gain an insight into the ongoing fibrotic process, and we do not know if the fluid variations in remodeling markers have significant consequences in the diseased airway walls. Other alternative tools, including high-resolution computed tomography, endobronchial ultrasounds, and lung function measurement can also be used as screening tools, but modulation of airway remodeling will need to be confirmed on airway wall specimen. Clinicians should consider airway remodeling in all subjects with asthma and rhinitis. Fixed airflow obstruction is regarded as a late and irreversible manifestation of airway remodeling. For this reason, even without good and easily available tools to confirm the presence of remodeling, clinicians should use controlling medications to prevent development or worsening of airway and tissue remodeling. REMODELING IN ALLERGIC DISEASES IS NOT RESTRICTED TO THE AIRWAYS Allergen exposure triggers in sensitized subjects an inflammatory response that will be expressed in the targeted organ, such as the nasal mucosa, the skin, and the airway mucosa. Allergic rhinitis, atopic dermatitis, and asthma share many pathologic features. In fact, the same profile of inflammation, mediators, and adhesion molecules are observed in upper and lower allergic airway diseases as well as skin allergic disease. There is a common cellular inflammation pattern characterized by eosinophil, mast cell, and CD4 1 T cell influx (59, 60). Mediators, including histamine, cysteinyl-leukotrienes, interleukin (IL)-4, IL-5, IL-13, RANTES, and eotaxin are expressed in both upper and lower airways (61, 62). Although initial inflammation induced by allergens is similar in upper and lower airways, long-term structural consequences differ. In allergic rhinitis, minimal epithelial shedding is observed with less subsequent degree of basement membrane thickening (reviewed in Reference 63). Atopic dermatitis is also characterized by remodeling with increased expression of profibrotic cytokines (including TGF-b, IL-11, and IL-17) and increased subepithelial deposition of collagen (64). Remodeling is observed in all atopic diseases, which reinforces the hypothesis that remodeling is an inflammatory driven process. CONCLUSIONS The features of airway remodeling include subepithelial fibrosis, elevated numbers and volume of mucous cells in the epithelium, increased amounts of airway smooth muscle, and increased vascularization of the airway wall. It is important to understand the etiology of airway remodeling in asthma to develop therapies that inhibit or reverse it. 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