Mimi L.K. Tang , John W. Wilson , Alastair G. Stewart , Simon G. Royce

n the airway wall in asthma. These include epithelial hyperplasia and

of lung function. The precise sequence of events that take place during the

growth factor; HRCT, high-resolution computed tomography; ICS, inhaled corticosteroid; IL, interleukin; LRA, leukotriene receptor antagonist; MAPK, mitogen-activated protein kinase; MMP, matrix metalloprotease; OVA, ovalbumin; PAI1, plasminogen activator inhibitor-1; PDE, phosphodiesterase; RBM, reticular

te/pharmtherabasement membrane; TIMP, tissue inhibitor of matrix metalloprotease; TGF, transforming growth factor ; TNF-, tumour necrosis factor ; VEGF, vascularendothelial growth factor; VEGFR, vascular endothelial growth factor receptor.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4752. Consequences of airway remodelling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4763. Overlapping aetiologies for airway remodelling in asthma . . . . . . . . . . . . . . . . . . . . . . . . 477

3.1. Repeated episodes of allergic inflammation . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4773.2. Defective epithelial repair . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 477remodelling process and the mechanisms regulating these changes remain poorly understood. It is thought that airway remodelling is initiated andpromoted by repeated episodes of allergic inflammation that damage the surface epithelium of the airway. However, other mechanisms are alsolikely to contribute to this process. Moreover, the interrelationship between airway remodelling, inflammation and AHR has not been clearlydefined. Currently, there are no effective treatments that halt or reverse the changes of airway remodelling and its effects on lung function.Glucocorticoids have been unable to eliminate the progression of remodelling changes and there is limited evidence of a beneficial effect fromother available therapies. The search for novel therapies that can directly target individual components of the remodelling process should be madea priority. In this review, we describe the current understanding of the airway remodelling process and the mechanisms regulating its development.The impact of currently available asthma therapies on airway remodelling is also discussed. 2006 Elsevier Inc. All rights reserved.

Keywords: Airway remodelling; Asthma; Fibrosis; Therapy; Airway wall thickening; Allergic inflammation

Abbreviations: AAD, allergic airways disease; ADAM33, a disintegrin and metalloprotease 33; AHR, airway hyperresponsiveness; ASM, airway smooth muscle;BALF, bronchoalveolar lavage fluid; BM, basement membrane; C/EBP, CCAAT/enhancer binding protein ; COPD, chronic obstructive pulmonary disease; ECM,extracellular matrix; EGFR, epidermal growth factor receptor; EMTU, epithelial mesenchymal trophic unit; FEV1, forced expiratory volume in 1sec; FGF, fibroblastairway hyperresponsiveness (AHR), and a progressive irreversible loss

metaplasia, subepithelial fibrosis, muscle cell hyperplasia and angiogenesis. These structural changes result in thickening of the airway wall,Airway remodelling refers to the structural changes that occur ia Department of Immunology, Murdoch Children's Research Institute, The Royal Children's Hospital, Melbourne 3052, Australiab Department of Allergy Clinical Immunology and Respiratory Medicine, The Alfred Hospital, Melbourne, Australia

c Department of Pharmacology, The University of Melbourne, Australia

Abstract CorrespondE-mail add

0163-7258/$ -doi:10.1016/j.pAssociate editor: P.S. Foster

Airway remodelling in asthma: Current understandingand implications for future therapies

a, b c a

Pharmacology & Therapeutics 112 (2006) 474488www.elsevier.com/loca3.3. Mechanical stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4793.4. Cigarette smoke . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 479

ing author. Tel.: +61 3 9345 5733; fax: +61 3 9345 6348.ress: mimi.tang@rch.org.au (M.L.K. Tang).

see front matter 2006 Elsevier Inc. All rights reserved.harmthera.2006.05.001

. .

. .deptein. .ayer. .way. .. .. .. .. .. .. .

and the mechanisms by which these changes may be regulated.

prevent or reThe majo

epithelial mecell hyperpladeposition oin the basemand in theincreased thmyofibroblaepithelial cesignificant pprocess. Epgoblet cellssynthesisingmuscle cellsproteins po

asthma shortly after diagnosis when exposure to the sensitising

akir et al.,. Likewise,ng can bef diagnosisis thereforedelling andtion from a, it has beenobstructionasthma, theal., 1999)elly et al.,

&to directly target airway remodelling and henceverse deterioration in lung function.r components of airway remodelling are (1) surfacetaplasia with increased epithelial thickness, gobletsia and increased mucus secretion, (2) fibrosis withf abnormal extracellular matrix (ECM) componentsent membrane (BM) layer beneath the epitheliumdeeper submucosa (Belleguic et al., 2002), (3)ickness of smooth muscle due to muscle cell andst hyperplasia and (4) angiogenesis. The airwaylls, fibroblasts and smooth muscle cells undergohenotypic differentiation during the remodellingithelial cells differentiate into mucus producing, fibroblasts acquire a contractile and collagen-

patients and those with atopy prior to the devesymptomatic asthma (Djukanovic et al., 1992; Ch1996; Warner et al., 2000; Pohunek et al., 2005)significant epithelial and subepithelial remodellidocumented early in disease even at the time o(Djukanovic et al., 1992; Chetta et al., 1997). Itlikely that many asthmatics develop changes of remoprogressive bronchodilator-resistant airway obstrucvery early stage in their disease. Consistent with thisdemonstrated that although irreversible airwaycorrelates strongly with the duration and severity ofhighest rate of loss of airway function (Orsida etoccurs early after diagnosis (Ulrik & Lange, 1994; K1988).Understanding the molecular events leading to structuralchanges may facilitate the development of novel therapeuticapproaches

agent has been relatively brief (Saetta et al., 1992). Unexpect-edly, there is BM thickening in infants and children, rhinitis

lopment of4. The components of airway remodelling . . . . .4.1. Fibrosis . . . . . . . . . . . . . . . . . .

4.1.1. Regulation of extracellular matrix4.1.2. Effects of extracellular matrix pro

4.2. Epithelial changes . . . . . . . . . . . . .4.3. Thickening of the airway smooth muscle l4.4. Angiogenesis and neovascularization . . .

5. Current asthma treatments and their effects on air5.1. Glucocorticoids . . . . . . . . . . . . . .5.2. agonists . . . . . . . . . . . . . . . . .5.3. Leukotriene modifiers . . . . . . . . . . .

6. Genetic susceptibility to airway remodelling . . .7. Conclusion . . . . . . . . . . . . . . . . . . . .Acknowledgment . . . . . . . . . . . . . . . . . . .References . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction

The term remodelling refers to structural changes that occurin organs and tissues following disease pathogenesis. Airwayremodelling pertains specifically to the changes that occur inand around the trachea, bronchi and bronchioles. Airwayremodelling is a central feature of asthma, linking inflammationwith airway hyperresponsiveness (AHR). The structuralchanges observed in asthma involve all components of theairway wall including the epithelial, submucosal, and smoothmuscle layers as well as vascular structures. The consequencesof airway remodelling are increased AHR, fixed airflowobstruction, and irreversible loss of lung function. Themechanisms regulating airway remodelling changes and theorder in which these changes develop remain poorly under-stood. Although structural changes in the airway are stronglycorrelated with inflammation, aspects of airway remodellingmay occur independently of airway inflammation and mayprecede the presentation of symptomatic asthma. In this review,we discuss each of the structural changes observed in asthma

M.L.K. Tang et al. / Pharmacologyphenotype known as the myofibroblast and smoothare thought to decrease expression of contractilessibly as a prelude to acquiring proliferative. . . . . . . . . . . . . . . . . . . . . . . . . . 479

. . . . . . . . . . . . . . . . . . . . . . . . . . 479osition . . . . . . . . . . . . . . . . . . . . . . 479s. . . . . . . . . . . . . . . . . . . . . . . . . . 480. . . . . . . . . . . . . . . . . . . . . . . . . . 480. . . . . . . . . . . . . . . . . . . . . . . . . . 481. . . . . . . . . . . . . . . . . . . . . . . . . . 482remodelling . . . . . . . . . . . . . . . . . . . 482. . . . . . . . . . . . . . . . . . . . . . . . . . 482. . . . . . . . . . . . . . . . . . . . . . . . . . 483. . . . . . . . . . . . . . . . . . . . . . . . . . 483. . . . . . . . . . . . . . . . . . . . . . . . . . 483. . . . . . . . . . . . . . . . . . . . . . . . . . 483. . . . . . . . . . . . . . . . . . . . . . . . . . 484. . . . . . . . . . . . . . . . . . . . . . . . . . 484

capacity, although this remains controversial. As a result ofthese remodelling changes, there is thickening of the airwaywall, which makes the airways stiffer and less distensible, aswell as narrowing of the airway lumen.

There is little information on the specific sequence of eventsduring the remodelling process. The current understanding ofairway remodelling changes is predominantly based uponfindings from cross-sectional studies and there is a paucity ofasthmatic biopsy material available over the course of thedisease. Furthermore, the histopathological analysis of bron-chial biopsy samples provides a relatively superficial evaluationof changes within the airway, and more detailed examination ofremodelling changes is only possible with post-mortem analysisof lung tissue in fatal asthma.

Studies of human asthma have shown that elements ofremodelling, including epithelial metaplasia and subepithelialfibrosis, can occur early. Remodelling changes have beendocumented in patients with mild intermittent asthma (Beasleyet al., 1989; Jeffery et al., 1989; Roche et al., 1989), in youngchildren with asthma (Cutz et al., 1978), and in occupational

475Therapeutics 112 (2006) 474488Established animal models of allergic airways disease(AAD) may assist with understanding the sequence of airwayremodelling changes. The most cited of these are murine

&ovalbumin (OVA) sensitization models in which the challengeperiod can be varied to simulate acute or chronic asthma (Fosteret al., 1996; Bani et al., 1997; Keramidaris et al., 2001; Kumar& Foster, 2001; Leigh et al., 2002; McMillan & Lloyd, 2004).Although these and other models vary from human asthma(Pabst, 2002; Kannan & Deshpande, 2003; Epstein, 2004) theycan provide some evidence for early and late events in theairway remodelling process. For example, goblet cell hyper-plasia is observed after only a few days of OVA challenge andpersists for weeks (Wills-Karp et al., 1998). Similarly, in acomparison of OVA models of AAD, we found that epithelialthickening and goblet cell hyperplasia were early changes thatoccurred after only 4days of challenge and persisted followinglonger term exposure to OVA (unpublished observation). Incontrast, we and others have found that subepithelial collagendeposition developed progressively and was most pronouncedafter long term exposure to OVA (unpublished observation;Foster et al., 2000). Greater understanding of the precisesequence of events in the progression of airway remodellingwill provide insight into the regulation these processes.

2. Consequences of airway remodelling

Reduced forced expiratory volume in 1sec (FEV1) andincreased AHR are used routinely in the clinical diagnosis ofasthma. FEV1 is the most frequently used test for assessment ofpulmonary function in asthma. A deep breath is taken tomaximally fill the lungs and expired as forcefully as possibleinto a spirometer. FEV1 is reduced with bronchoconstriction.Airway responsiveness is assessed by measuring changes inFEV1 following exposure to increasing doses of bronchocon-strictor (such as methacholine). Results are expressed as theconcentration of bronchoconstrictor which provokes a 20%decline in FEV1 sustained for 3min (PD or PC20%). IncreasedAHR is seen as a reduced PC20%.

In mice, lung function can be measured by invasiveplethysmography in anaesthetized animals or by whole body(non-invasive) plethysmography in unrestrained mice. In theinvasive approach, animals are tracheotomised and airwayresistance and compliance are calculated from flow and pressuremeasurements. In the non-invasive whole body plethysmogra-phy approach, a measure of respiratory effort known asenhanced pause (Penh) is used as a correlate of airwayresistance in non-anaesthetized unrestrained mice.

The original studies reporting the use of Penh showed that itcorrelated closely with airway resistance; and that bronchocon-strictors caused increases in Penh that were greater in OVA-challenged animals (Hamelmann et al., 1997; Chong et al.,1998; Dohi et al., 1999; Henderson et al., 2002). More recently,a number of potential confounding factors for Penh measure-ment have been recognised including plethysmograph temper-ature, respiratory pattern, and the fact that the airflow signalused to calculate Penh is comprised of both nasal and thoracicflow. Many investigators have found that there are discordances

476 M.L.K. Tang et al. / Pharmacologybetween data obtained with standard pulmonary mechanics andthat obtained with Penh (Adler et al., 2004). It has also beenreported that the time-course of pathological changes in a modelof lung injury are discordant with Penh changes, with Penhchanges apparently correlated to breathing pattern rather thanlung mechanics (Petak et al., 2001).

The single most important objection to the use of Penh as ameasure of airway obstruction and airway resistance comes fromthe absence of any physiological correlate of this parameter(Mitzner & Tankersley, 1998, 2003; Bates et al., 2004). Theseconfounding factors have highlighted important limitations inthe methodology. Nevertheless, despite widespread knowledgeof these discussions, papers continue to be published in whichPenh is the sole measure of airway obstruction, most probablybecause this method is more convenient, less time consuming,and allows evaluation of live mice.

The clinical consequences of airway remodelling includefixed airway obstruction and increased AHR (Fiscus et al.,2001). It is not known which specific structural changescontribute to these losses in lung function and the relationshipbetween individual components of airway remodelling andairway function is complex, particularly when assessed inunivariate models. Many investigators have reported a correla-tion between subepithelial fibrosis and AHR or FEV1 (Chetta etal., 1997; Hoshino et al., 1998a, 1998b), however, this was notfound in all studies (Brewster et al., 1990). Total airway wallthickness as measured by high-resolution computed tomography(HRCT) (Boulet et al., 1995) is increased after methacholinechallenge (Okazawa et al., 1996) but did not correlate with AHRor FEV1 (Boulet et al., 1995). Furthermore, airway remodellingchanges may be seen in children, independent of inflammatoryor lung function changes (Jenkins et al., 2003a, 2003b).

While remodelling may theoretically have some beneficialphysiological effects, these are outweighed by the deleteriouseffects contributing to airway narrowing (McParland et al.,2003). The potential beneficial effects include, firstly, thatincreased airway wall thickness could enable the airways toresist dynamic compression as a result of increased airway wallstiffness; secondly, that the deposition of collagen fibres aroundthe smooth muscle could provide an elastic impedance tocontraction; and thirdly, that the distortion and folding ofthickened remodelled tissue that occurs with smooth musclecontraction may provide a greater than normal elastic load onthe muscle. Conversely, the structural changes of remodellingcan have significant undesirable effects on airway resistance.Hyperplasia of the mucus-secreting goblet cells will contributeto luminal narrowing. The ability of the airway wall to respondto bronchodilators may be considerably impaired by theremodelling sequence (Wilson et al., 1993). A smooth musclelayer of greater volume will narrow the airway lumen more thana lesser volume (Moreno et al., 1986; Wiggs et al., 1992).Furthermore, mathematical models have predicted that anincrease in the volume of the airway wall on the luminal sideof the smooth muscle layer will greatly increase the effect ofsmooth muscle contraction in narrowing the lumen of an airway(Moreno et al., 1986; James et al., 1989a, 1989b; Wiggs et al.,1992). Thickening of the adventitial side will have a similar

Therapeutics 112 (2006) 474488effect (Macklem, 1995). An increase in the adventitial wall areawill act to reduce airway smooth muscle (ASM) parenchymalload by acting as a buffer from recoil caused by stretching of the

&parenchyma and reducing the load per unit area (Pare & Bai,1995; McParland et al., 2003). This will allow an increasedvelocity and degree of smooth muscle shortening that couldcause airway closure before the end of the expiration cycle. Sothe combined effect of ASM hyperplasia and airway wallthickening is the generation of a greater degree of airwaynarrowing and airway resistance for a given level of smoothmuscle shortening (James et al., 1989a, 1989b). This mayexplain the failure of deep inspiration in asthmatics to elicit amaintained dilatation of the airways and the loss of the plateauresponse in moderate to severe asthma (Fernandes et al., 2003).Finally, it has been suggested that smooth muscle adapts to ashorter length during the acute and chronic smooth muscleshortening in airway remodelling (McParland et al., 2003).

Taken together, these findings suggest that the sum effect ofairway remodelling changes is a greater degree of airwaynarrowing that results in increased airway resistance andhyperresponsiveness.

3. Overlapping aetiologiesfor airway remodelling in asthma

3.1. Repeated episodes of allergic inflammation

Allergic inflammation in asthma is thought to be animportant primary cause of airway remodelling and maycontribute to all aspects of the remodelling process. Repeatedcycles of acute allergic inflammation not only contribute toAHR and reduced lung function by inducing mucosal oedemaand inflammatory cell migration to the lumen, but also stimulateirreversible components of long-term airway structural change.

A characteristic feature of allergic inflammation is thepredominance of Th2 lymphocytes and their products. Activat-ed CD4+ T lymphocytes recruit effector inflammatory cells(mast cells, eosinophils and lymphocytes) to the airway andcontrol release of inflammatory mediators from these cells. Th2lymphocytes release interleukin5 (IL) 5, IL4 and IL13. IL4 andIL13 induce IgM class switching to IgE. Crosslinking of IgEmolecules on the mast cell surface causes degranulation andrelease of additional inflammatory mediators. Mast cell tryptasestimulates myocyte proliferation, type I collagen production byfibroblasts (Akers et al., 2000) and also release of plasminogenactivator inhibitor 1 (PAI1) (Cho et al., 2000). PAI1 inhibitsmembers of the plasmin system that convert plasminogen andpro-MMP into their active forms (Kucharewicz et al., 2003).PAI1, therefore, is a profibrotic factor and mouse models haverevealed it to have an important role in lung fibrosis (Eitzman etal., 1996), as well as airway remodelling in AAD (Oh et al.,2002).

Remodelling changes can be modulated by a number ofproducts of the Th2 pathway. Murine studies have shown thatIL-9, -4 and -13 all have a role in goblet cell hyperplasia andmucin gene expression (Wills-Karp, 2000). IL13 is also a keyfibrogenic effector in asthma and shows an ability to upregulate

M.L.K. Tang et al. / Pharmacologyand activate transforming growth factor (TGF) 1 (Elias etal., 2003; Wills-Karp & Chiaramonte, 2003). A neutralizingantibody to IL13 was effective in reducing AHR andremodelling in a murine model of AAD (Yang et al., 2004).However this effect on AHR was not confirmed in anotherstudy (Kumar et al., 2004). Polymorphisms in the IL13 genehave been shown to modulate the expression of other genesimplicated in asthma and remodelling of smooth muscle such asvascular cellular adhesion molecule (Lobb et al., 1996), IL-13R2, tenascin C and histamine receptor H1 (Syed et al.,2005). IL5 from Th2 cells is important for the recruitment ofeosinophils, a major effector cell in asthma that causes cellulardamage and airway structural change. IL5 is necessary for themigration, differentiation and activation of eosinophils (Rothen-berg et al., 1989; Sanderson, 1992; Sehmi et al., 1992).Antibodies against IL5 decreased eosinophil numbers in thebronchoalveolar lavage fluid (BALF) and blood (Leckie et al.,2000) as well as in peribronchial tissues (Flood-Page et al.,2003), and this treatment resulted in reduced reticular basementlayer thickening (Braunstahl et al., 2003). In general, structuralchanges associated with airway remodelling are reduced ineosinophil-deficient mice. However, some aspects of airwayremodelling such as goblet cell metaplasia and AHR aredependent on the strain of mouse used (Humbles et al., 2004;Lee et al., 2004a, 2004b; Wills-Karp & Karp, 2004).

TGF1 is a potent profibrogenic cytokine that is mainlyexpressed in the epithelium of the normal airway (Magnan et al.,1994) and is also secreted by fibroblasts, epithelial cells,eosinophils and macrophages. TGF1 promotes the differen-tiation of fibroblasts to myofibroblasts in vitro (Malmstrom etal., 2001). It can also suppress apoptosis of myofibroblasts invitro (Zhang & Phan, 1999). TGF1 mRNA and proteinexpression is increased in asthmatic airways compared toairways from normal individuals and expression levels correlatewith severity of disease (Minshall et al., 1997). Lee et al.targeted TGF1 to the murine lung with a triple transgenicsystem which resulted in tissue fibrosis, myofibroblast andsmooth muscle cell hyperplasia, and epithelial apoptosis (Lee etal., 2004a, 2004b). Mice instilled with TGF1 showedincreased collagen I and III mRNA expression, increasedsubepithelial fibrosis and AHR after 4 weeks of treatment(Kenyon et al., 2003). TGF2 may have additional effects suchas increasing bronchial epithelial cell mucin through increasedMUC5AC expression (Chu et al., 2004).

The significance of the eosinophil in airway remodellingcomes primarily from its secretory profile. It is able to produce arange of cytokines, including TGF, IL4 and IL13, as well asvascular endothelial growth factor (VEGF) and fibroblastgrowth factor (FGF) 2, which have all been shown to promotevarious aspects of remodelling (Kay et al., 2004). Otherinflammatory cells such as the neutrophil may also contributeto remodelling changes. Neutrophil elastase, a serine protease,can induce mucous cell hypertrophy and hyperplasia (Stockleyet al., 1994) and stimulate mucin gene expression by an oxidant-dependent mechanism (Fischer & Voynow, 2002, 2000).

3.2. Defective epithelial repair

477Therapeutics 112 (2006) 474488The airway epithelium is made up of more than 10 residentcell types as well as numerous migratory leukocytes (Ayers &

Jeffery, 1988; Djukanovic et al., 1990), and is now recognisedto be actively involved in the regulation of airway structure.The epithelial mesenchymal trophic unit (EMTU) is a usefulconcept that describes the interplay between the epitheliumand subepithelial tissues involved in remodeling (Holgate,1999). It describes the interactions between the bronchialepithelium and mesenchymally derived cells immediatelybelow it in the bronchial wall (Brewster et al., 1990). Thisinterplay is essential for lung branching and morphogenesisduring ontogeny, but is also important for lung developmentthroughout life as the lung may remodel postnatally in diseasestates (Fig. 1).

It has been proposed that airway remodelling in asthmamay result from a primary defect of epithelial repair. Thisdefect of the epithelial repair process may be present frombirth in asthma, as structural changes have been noted in earlylife before the clinical manifestation of disease (Pohunek etal., 2005). In the setting of an epithelial repair defect,repeated episodes of epithelial injury caused by airwayinflammation can result in prolonged activation of theEMTU that in turn leads to tissue remodelling (Phipps etal., 2004). In the asthmatic airway, there is normalupregulation of CD44 and epidermal growth factor receptor(EGFR) expression by damaged airway epithelium (Holgateet al., 1999). However this increased EGFR expression doesnot result in appropriate epithelial repair (Holgate et al.,

example, activated epithelial cells secrete fibrogenic growthfactors including TGF1, TGF2, basic fibroblast growthfactor (bFGF), epidermal growth factor (EGF), endothelin,and insulin growth factor (Holgate et al., 1999) that act topromote proliferation of myofibroblasts and increase theirproduction of matrix proteins. Activated lung epithelial cellsand fibroblasts also show increased expression of theintegrins 51 and v6 (Sheppard, 1998), which facilitatesubepithelial accumulation of fibronectin and tenascin, as wellas promoting the migration and differentiation of fibroblasts(Knight, 2001). During the repair process, epithelial andsubepithelial cells express IL11 which correlated with theextent of types I and III collagen deposition (Minshall et al.,2000). There is also upregulation and hypersecretion of theMUC family mucins (MUC5B and MUC5AC over the moretypical MUC2) that can cause mucous cell hyperplasia,metaplasia and epithelial thickening. These findings supportthe concept of a functional EMTU and highlight the importantlink between airway epithelium and the development ofremodelling changes both within the epithelium and deeper inthe submucosal layer (Fedorov et al., 2005). Understandingthese pathways reveals a range of candidates for therapeuticintervention. Of particular interest is EGFR. Intratrachealinstillation of TGF, an EGFR ligand, induced goblet cellhyperplasia, and treatment with a selective kinase inhibitor ofEGFR prevented this hyperplasia and also prevented goblet

aysu

478 M.L.K. Tang et al. / Pharmacology & Therapeutics 112 (2006) 4744881999). As a result, secondary repair processes are initiatedthat lead to increased deposition of ECM proteins. For

Fig. 1. Sections of normal bronchial wall and that from an asthmatic with airwmagnification 40). (B) H&E stained airway in severe asthma. Note thickened

between the smooth muscle layer (SM) and goblet cell hyperplasia in epitheliumdemonstrate basement membrane (original magnification 40). (D) Collagen IV staiand vessels (VV) in submucosa (original magnification 60).cell hyperplasia in an OVA model of AAD (Takeyama et al.,1999).

remodelling changes. (A) H&E stained airway from normal control (originalbepithelial collagen below the basement membrane (BM), inflammatory cells

(original magnification 40). (C) Collagen IV staining of a control airway toning of asthmatic airway showing true basement membrane beneath epithelium

&3.3. Mechanical stress

Using co-cultures of human bronchial epithelial cells andlung fibroblasts, Swartz et al. have demonstrated thatmechanical stress in the form of transmembrane pressure cancause epithelial cells to communicate a remodelling response tofibroblasts (Swartz et al., 2001). Increased mechanical stress ledto increased fibronectin protein expression, increased collagentypes III and V, and increased MMP-9 relative to TIMP1, butnot increased fibroblast proliferation (Swartz et al., 2001).These studies performed in the absence of inflammatory cellssuggest that airway remodelling may be directly promoted bymechanical stress on epithelial cells occurring during repeatedepisodes of airway obstruction.

3.4. Cigarette smoke

There is limited evidence from animal models and in vitroexperiments that cigarette smoke can induce airway remodel-ling in the absence of other mechanisms. Wang et al. showedincreased total tissue collagen and procollagen gene expressionin rat tracheal explants exposed to cigarette smoke (Wang et al.,2003). They reported that blocking EGFR could suppress thiseffect. Transactivation of EGFR by cigarette smoke andhydrogen peroxide can also cause mucin gene expression(Takeyama et al., 2001). Tobacco smoke may also havedetrimental effects on lung development in utero. The distancebetween alveolar attachments to the airways is increased inneonates exposed to cigarette smoke in utero and this has beensuggested to lessen the opposing force of the parenchyma onairway narrowing (Elliot et al., 2003).

4. The components of airway remodelling

4.1. Fibrosis

Fibrosis in the airway wall is a central feature of airwayremodelling in asthma. This fibrosis is prominent in the laminareticularis below the true BM (Roche et al., 1989) and may alsooccur in the deeper submucosa, around smooth muscle andarterioles (Wilson & Li, 1997). The development of fibrosis inthe lamina reticularis is specific to asthma and does not occur inother airway disease (Roche et al., 1989). The ECM proteinsdeposited in the lamina reticularis include collagen types I andIII, tenascin, and to a lesser extent, collagen V and fibronectin(Roche et al., 1989, Laitinen et al., 1997), whereas the BMpredominantly comprises collagen IV, fibronectin and elastin(Merker, 1994). During remodelling, the true BM becomessupported by this less ordered excess of ECM proteins andtogether they constitute the reticular basement membrane(RBM) layer (Chakir et al., 1996). The thickness of the RBMcan be used as a surrogate marker for other changes in theairway wall, including smooth muscle area in small and largecartilaginous airways, as well as submucosal gland area in the

M.L.K. Tang et al. / Pharmacologylarge airways (James et al., 2001) and entire wall thickness(Kasahara et al., 2002). Thickening of the RBM layer has beenfound to correlate with airflow limitation and AHR in mildasthma (Shiba et al., 2002) and to be negatively correlated withairway distensibility (Ward et al., 2001). RBM layer thickeningis 1 of the changes that can be used to differentiate asthma fromchronic obstructive pulmonary disease (COPD) (Fabbri et al.,2003) as the RBM is thicker in asthma and rhinitis compared toCOPD (Milanese et al., 2001) but does not necessarily correlatewith asthma severity (Chu et al., 1998).

4.1.1. Regulation of extracellular matrix depositionECM deposition in tissues is determined by the balance

between production and degradation of fibrillar proteins.Production of ECM proteins is regulated at the level of geneexpression. In the airway, collagen is produced by myofibro-blasts, a specialised network of fibroblastic cells with longcytoplasmic extensions that reside in the subepithelial layer ofthe airway wall (Brewster et al., 1990). Fibronectin is producedby epithelial cells (Lobb et al., 1996). The TGF family ofproteins, specifically TGF1 and TGF2, are the major factorspromoting collagen and fibronectin production by myofibro-blasts and epithelial cells, respectively, in the lung (Ignotz &Massague, 1986). TGF is generated by airway epithelial cellsin response to injury and by inflammatory cells, particularlyeosinophils, entering the airway. ECM degradation is regulatedby 2 families of molecules, the matrix metalloproteases (MMP)and the tissue inhibitors of matrix metalloproteases (TIMP).MMP degrade fibrillar proteins while TIMP inhibit MMPactivity. Collagenases (MMP-1, MMP-13) degrade interstitialcollagens, the gelatinases (MMP-2, MMP-9) degrade BMcomponents (fibronectin, elastin) and the stromelysins (MMP-3, MMP-10, MMP-11) have a broad spectrum of ECM targets.Most MMP are secreted in pro-MMP form by a variety of celltypes and are activated by cleavage of the propeptide sequence.TGF can inhibit the synthesis of MMP, and stimulate therelease of the antiproteinases TIMP (Edwards et al., 1987).Therefore, TGF is a key inducer of ECM deposition thatcontrols both the production and degradation of ECM proteins.Conversely, the peptide hormone relaxin has been shown to be amajor inhibitor of ECM deposition in tissues. Its role as aninhibitor of ECM deposition in the lung is supported by severalobservations. Relaxin inhibits TGF induced upregulation ofprocollagen type I and type III in human lung fibroblasts(Unemori et al., 1996). Relaxin also acts directly on human lungfibroblasts to increase the expression of the ECM degradingenzymes, proMMP-1, proMMP-2 and proMMP-3 and decreasethe production of TIMP (Unemori et al., 1996; Goldsmith et al.,2004). Hence, the relative amounts of TGF to relaxin, andconsequently TIMP to MMP, that are present within a tissuemay determine the balance between collagen deposition andbreakdown. Manipulation of these factors may provide aneffective means of regulating ECM deposition in asthma.

MMP-9 is likely to be the major MMP relevant to asthma andairway remodelling (Lee et al., 2001; Ohbayashi & Shimokata,2005). It is localized to inflammatory cells (Dahlen et al., 1999),the bronchial epithelium and submucosa (Han et al., 2003). In

479Therapeutics 112 (2006) 474488healthy states it is balanced in a 1:1 molar ratio by TIMP1(Nagase, 1996). In chronic asthma, TIMP1 is increased overMMP-9 (Mautino et al., 1999). An increased ratio of TIMP1/

&MMP-9 provides a signal to increase tissue collagen depositionand has been shown to correlate with airway wall thickening(Vignola et al., 2004; Matsumoto et al., 2005). TIMP1 has alsobeen shown to promote the growth of bronchial fibroblasts invitro (Hayakawa, 1994). Paradoxically, following acute allergenchallenge in asthma, there is increased MMP-9 in sputum(Cataldo et al., 2002a, 2002b; Boulay et al., 2004) and plasma(Belleguic et al., 2002), while TIMP1 remains unchanged(Cataldo et al., 2002a, 2002b), resulting in a decreased TIMP1/MMP-9 ratio. Increased MMP-9 activity may weaken the BMthereby contributing to tissue injury and remodelling (Tanaka etal., 2000). Tumour necrosis factor (TNF-) has been shown tostimulate MMP-9 release from eosinophils (Schwingshackl etal., 1999), which may account for the increased MMP-9expression in the setting of acute inflammation. Other MMPsare also likely to play important roles in airway remodelling.MMP-2 is released from epithelial cells in vitro and stimulatesfibroblast proliferation (Xu et al., 2002). Activated ASM cellshave been shown to secrete MMP-3, MMP-9 and pro-MMP-2 invitro, while unstimulated smooth muscle cells produce TIMP1and 2 (Elshaw et al., 2004). The significance of altered MMPlevels in asthma is complicated by the other effects of MMPsincluding promotion of eosinophil and inflammatory cellmigration into the airways (Cataldo et al., 2002a, 2002b;Corry et al., 2004). The true significance ofMMPs and TIMPs inasthma will only be elucidated by temporal and spatiallocalization of these proteins in the asthmatic lung. There isalso evidence for a reduced ability of fibroblasts to degradecollagen by phagocytosis (Laliberte et al., 2001).

TGF1 mRNA and protein expression is increased inasthmatic airways (Minshall et al., 1997), however, relaxinexpression in asthma has not been reported. TGF inducesdifferentiation of fibroblasts into myofibroblasts, which isassociated with expression of SMA, and increased expressionof collagen types I and III (Morishima et al., 2001). Baselineprocollagen I and III synthesis is the same in fibroblasts fromasthmatics and normal individuals (Dube et al., 1998), butincreased deposition may be determined by increased conver-sion of procollagen to collagen. Differentiation of myofibro-blasts is part of the normal wound healing process and is usuallydownregulated upon completion of the repair process byvitronectin. However, in airway remodelling in asthma thereis a failure of this downregulation and myofibroblasts maypersist (Scaffidi et al., 2001). There is also evidence that inasthma circulating fibrocytes are recruited to the airwayswhereupon they differentiate and contribute to the myofibro-blast population (Schmidt et al., 2003). The persistence ofmyofibroblasts is thought to contribute to increased rigidity inthe airway wall (Gabbrielli et al., 1994), and the number ofmyofibroblasts correlates with the degree of subepithelialcollagen deposition in asthmatics (Brewster et al., 1990;Gizycki et al., 1997). Myofibroblasts produce a wide array ofgrowth factors and inflammatory mediators that are mitogenicfor fibroblasts and ASM including bFGF and EGF, respectively

480 M.L.K. Tang et al. / Pharmacology(Evans et al., 1999). They may also promote inflammation asthey have been shown to inhibit eosinophil apoptosis in vitro(Zhang et al., 1996).4.1.2. Effects of extracellular matrix proteinsFibronectin is a glycoprotein that is deposited in the ECM in

an insoluble fibrillar form and is involved in cell adhesion toECM collagens. It is chemotactic for epithelial cells andfibroblasts, and has been shown to induce fibroblast prolifer-ation in vitro (Shoji et al., 1989). Fibronectin may therefore actin a positive feedback loop to perpetuate localized subepithelialfibrosis in the remodelling process. Fibronectin is produced byepithelial cells at a site of injury, and its production is stimulatedby epithelial-derived TGF (Sacco et al., 1992). Levels offibronectin are increased in the BALF of asthmatics (Mattoli etal., 1991; Vignola et al., 1993; Meerschaert et al., 1999). It hasbeen found at increased levels in the plasma of asthmatics(Ohke et al., 2001), although this was not supported in anotherstudy (Laitinen et al., 1997). Fibronectin receptor expression onbronchial epithelial cells is also increased in asthma (Lobb et al.,1996).

Tenascin is an ECM glycoprotein normally expressed duringontogeny and repair. Tenascin expression is increased in theRBM layer in chronic asthma (Laitinen et al., 1997) and duringallergen challenge in mild asthma (Phipps et al., 2004).Tenascin thickness is dependent on the inflammatory response(Karjalainen et al., 2003). Hence tenascin may represent anacute form of structural change in the airway and may be 1 ofthe earliest remodelling changes observed in asthma (Laitinen etal., 1996).

Studies suggest that RBM fibrosis per se may drive theprogression of remodelling by contributing significantly toother structural changes of remodelling such as ASMhyperplasia. ECM proteins deposited in the RBMmay stimulatesmooth muscle proliferation by several mechanisms. Collagentypes I and III and fibronectin have been reported to have directmitogenic effects on ASM (Hirst et al., 2000). In addition, oneconsequence of airway fibrosis is decreased distensibility of theairway, and decreased stretch on ASM cells has beendemonstrated to further enhance the proliferative effects ofcollagen on ASM in vitro (Bonacci et al., 2003a, 2003b). ECMproteins can also bind a number of pro-inflammatory growthfactors that may act to exacerbate allergic inflammation.

In summary, fibrosis in the BM region of the airway inasthma is a characteristic feature of asthma that develops as anaberrant response to epithelial damage in the context of aprimary defect of epithelial repair. Fibrosis in the RBM canoccur early in the course of disease and may be an importantevent that drives the progression of remodelling by promotingother structural changes in the airway. Furthermore, ECMproteins and myofibroblasts can act in a positive feedback loopto perpetuate the signals for fibrosis.

4.2. Epithelial changes

Observations in human asthma and from animal modelssuggest that epithelial metaplasia and hyperplasia represent anearly expression of airway remodelling in asthma. As in the

Therapeutics 112 (2006) 474488gastrointestinal and biliary tracts, airway epithelium will react toinjurious stimuli in the lumen with cell proliferation andphenotypic changes. In asthma, there is epithelial shedding and

&metaplasia, as well as goblet cell hyperplasia and increasedmucus production.

The airway epithelium in asthmatics is more susceptible todamage than airway epithelium in control subjects (Knight &Holgate, 2003). A key feature of asthma observed in airwaybiopsies and post-mortem specimens is epithelial shedding(Jeffery, 2000, Jeffery et al., 2000, Wilson & Bamford, 2001),and this can be used to differentiate asthma from COPD andother lung diseases (Jeffery, 2000). Epithelial loss has beenshown to correlate with AHR (Jeffery et al., 1989). The resultsof an ultrastructural study suggest that altered desmosomecontact may play a part in epithelial shedding as desmosomelength was decreased in the columnar and the basal cells ofallergic and non-allergic asthmatics (Shahana et al., 2005).Another factor that may contribute to epithelial shedding is thehigh shear stress that occurs at the epithelium when ASMcontracts and the mucosa buckles and folds (Shahana et al.,2005).

In asthma there is an increase in goblet cell numbersresulting in increased mucus secretion (Ordonez et al., 2001).This hyperplasia of goblet cells may also contribute tothickening of the epithelial layer in turn contributing to AHRand fixed airway obstruction. In mouse models of AAD gobletcell hyperplasia is a significant contributor to the thickness ofthe surface epithelium of the airway (Temelkovski et al., 1998).In the human literature there is little reference to thecontribution of goblet cell hyperplasia to epithelial thicknesspresumably because of the overriding effect of epithelialshedding on mean airway epithelial thickness.

The mucus of asthmatics is also abnormal and contributes toairway obstruction in fatal asthma. Curschmann's spirals (twistsof mucus) are characteristically found in post-mortem lungsfrom these cases. A greater number of goblet cells are likely tocontribute to these observations (Fahy, 2001). The majormucins secreted in the lung are MUC5AC and MUC5B. Bothare gel-forming (Moniaux et al., 2001). A recent immunohis-tochemical study found no change in the distribution of thesemucins between fatal and mild asthma or in individuals treatedwith corticosteroids compared to normal controls (Groneberg etal., 2002) although a separate RT PCR study found increasedMUC5AC, reduced MUC5B, as well as detectable expressionof MUC2 the major intestinal mucin in asthma as compared tonormals (Ordonez et al., 2001).

The submucosal glands of the human airway are mixedserous-mucous glands. Large submucosal gland area ischaracteristic of fatal asthma (Chen et al., 2004) and is afeature of airway remodelling in both childhood and adultasthma (Jenkins et al., 2003a, 2003b; Rogers, 2003). Submu-cosal gland area was significantly higher in patients with mild tomoderate and severe persistent asthma than controls (Benayounet al., 2003), but this did not correlate with severity (Hogg,1997). Another study found no association between mucousgland area and airflow obstruction (Cho et al., 1996).

These structural changes affecting the epithelium develop in

M.L.K. Tang et al. / Pharmacologyresponse to epithelial injury, which may occur by severalmechanisms. Allergic airway inflammation is a common causeof damage to the epithelial layer. Epithelial integrity may also becompromised directly by allergens themselves. A single pollengrain consists of hundreds of starch granules that are smallenough (0.62.5 m) to bypass the passive defenses of theproximal airway to reach the small airways. Pollen grains haveprojecting spikes that adhere to the stigma of a flower andcontain proteolytic enzymes for the entry of the tube nucleus.Preparations of these enzymes from pollen have been shown todetach mouse epithelial cells from ECM in a dose dependentmanner (Hassim et al., 1998). There is evidence that otherdomestic allergens, such as house dust mite allergen and catallergen Felis domesticus 1 (Feld1) may also have proteolyticactions (Ring et al., 2000). Cellular damage to epithelial cellsmay also arise through enzymatic secretions from theeosinophil. Eosinophil peroxidase (EPO) is an eosinophilicbasic protein that causes increased permeability and damage tobronchial epithelial cells in asthma. Eosinophils and activatedT-cells also release interferon (IFN-) and TNF- that induceapoptosis of epithelial cells. Chemical insult may also causeepithelial damage and the breath condensate in acute asthma hasbeen shown to be acidic in nature (Agarwal et al., 2003).

4.3. Thickening of the airway smooth muscle layer

Thickening of the ASM layer is one of the changes observedduring airway remodelling in asthma. Smooth muscle volume inthe bronchial wall is higher in fatal (James et al., 1989a, 1989b;Ebina et al., 1993), and in non-fatal cases of asthma (Benayounet al., 2003) than in controls. Both hypertrophy and hyperplasiahave been suggested as possible causes for increased ASMvolume in asthma. There is evidence for ASM hypertrophy(Ebina et al., 1993; Benayoun et al., 2003), however, manystereological studies emphasize the importance of hyperplasia(Woodruff et al., 2004).

The mechanisms that regulate hyperplasia of the ASM inasthma are not well understood. Hyperplasia may be caused byproliferation, reduced apoptosis of resident smooth musclecells, or migration of interstitial mesenchymal cells orcirculating stem cells that then differentiate into fibroblastswithin the ASM layer. There is ample evidence for vascular SMproliferation in the analogous setting of arterial wall remodel-ling in atherosclerosis or restenosis following angioplasty. Inaddition, it has been shown that the inherent proliferative rate ofasthmatic ASM is increased (Johnson et al., 2001). However,contrary to this in vitro observation, we have failed to detect anyincrease in proliferation markers within muscle bundles in ASM(Mast et al., 2003), suggesting that other mechanisms may alsocontribute to the hyperplasia of ASM in asthma.

There is evidence that ASM apoptosis is reduced in AAD.Martin et al. have reported markedly decreased rates of ASMapoptosis as measured by TUNEL, and more modest prolifer-ation of cells as measured by bromodeoxyuridine incorporation,in antigen-challenged rats and naturally allergic horses (Martin& Ramos-Barbon, 2003). There is as yet no data on the rates ofASM apoptosis in asthma or in healthy airways. In cultured

481Therapeutics 112 (2006) 474488ASM, extreme conditions such as inhibition of protein synthesisare required to induce apoptosis, suggesting that human ASMmay be relatively resistant to apoptosis.

&Migration of (myo)fibroblasts derived locally from sub-epithelial fibroblasts or of fibrocyte stem cells recruited from thecirculation/bone marrow may also contribute to ASM hyper-plasia in asthma. We have found increased proliferation inmesenchymal cells within the subepithelial space but not in themuscle bundles, which would support this (unpublishedobservation). Nevertheless, the contention that migration ofresident fibroblasts or circulating stem cells can contribute tothe development of ASM hyperplasia remains speculative andmore extensive investigation of these processes is required.

A further mechanism by which thickening of the ASM layermay occur is through the deposition of ECM. Using astereological approach, Schellenberg et al. found an increaseddeposition of ECM around the ASM and suggested that thismay be a more important contributor to ASM thickening thanASM hyperplasia or hypertrophy (Thomson et al., 1996).However, the findings of Schellenberg et al. are difficult toreconcile with those of Ebina et al. who also used a stereologicalapproach but identified ASM hypertrophy as the major factorleading to ASM thickening (Ebina et al., 1993). Theseconflicting findings may reflect the large number of smokersin the control group in Schellenberg's study. Nevertheless, thereis evidence that cultured human ASM can make a variety ofECM components and that ASM derived from asthmatic donorsdemonstrates an increased capacity to synthesise ECM(Johnson et al., 2000). It is not clear whether such peri-muscular deposition would be of net benefit or detriment toASM function, but it seems likely that increased ECM wouldprovide additional resistance to smooth muscle shortening. Thefinding that muscle from asthmatic patients shows lessequilibrium tension development than that from non-asthmaticscontrols would support this.

There are studies suggesting that airway inflammation canlead to increased ASM proliferation in both animals and inhuman airways, although there is no direct evidence of this inasthma. The levels of a large number of potential ASMmitogens are increased by inflammation in the asthmatic airway.Those most extensively investigated include platelet-derivedgrowth factor (PDGF), bFGF, thrombin and serum. Some ofthese mitogenic factors activate receptors with intrinsic kinaseactivity (i.e. PDGF and EGF) and others are contractile agonistswhich act via G-protein-coupled receptors (e.g. thrombin).Proinflammatory cytokines that are increased in asthmatics alsoappear to have modest effects on ASM proliferation (De et al.,1995; Stewart et al., 1995). In turn, ASM cells themselves canproduce a wide range of inflammatory mediators andchemokines (Panettieri, 2004).

Mechanical factors and ECM proteins have also beenimplicated as potential mitogenic influences. Increased me-chanical strain has been reported to increase ASM proliferation(Smith et al., 1994). Conversely, we have shown that a decreasein strain from a resting physiological level of 4% to zero canalso increase proliferation when cells are exposed to a collagen-rich ECM (Bonacci et al., 2003a, 2003b). However, our studies

482 M.L.K. Tang et al. / Pharmacologywere performed using a monomeric form of collagen rather thanthe fibrillar form that may dominate in vivo, and the fibrillarform of collagen has been shown to be anti-mitogenic (Ichii etal., 2001). We speculate that MMP released in response toairway inflammation may lead to remodelling of collagenwithin the airway by increasing the ratio of monomeric tofibrillar collagen which would have a net mitogenic action onthe ASM. We and others have also reported that laminin andseveral other ECM components influence ASM proliferation.Laminin is anti-proliferative, whereas fibronectin and vitronec-tin are mitogenic. The monomeric form of collagen inducesresistance to the growth regulatory actions of GCS but not 2-adrenoceptor agonists.

4.4. Angiogenesis and neovascularization

Increased vascularity is commonly seen in the airway wall ofasthmatics (Li & Wilson, 1997) and may correlate with severityof the disease (Vrugt et al., 2000; Salvato, 2001). Changes in thevascularity of the airway wall have traditionally been detectedusing histochemical staining against vessel wall markers such asendothelial cell antigen CD31 (Braunstahl et al., 2003) or EN4(Vrugt et al., 2000). Tissue components associated with thevessel wall such as collagen IV and factor VIII can also be usedas surrogate markers of vascularity (Kuwano et al., 1993; Li &Wilson, 1997). Numerous image analysis methods have beenused to quantitate blood vessel density, number and size inparaffin sections. Vascularity may vary between 7.313.5% incontrols and 8.024.3% in mild asthmatics. This variation islikely to be due to the genetic regulation of angiogenic factorsand their receptors (Li &Wilson, 1997). Angiogenesis occurs asa result of replication of resident angioblasts in the airways andalso from recruitment of circulating CD34 positive mesenchy-mal stem cells (Kamihata et al., 2001; Seiler et al., 2001; Georgeet al., 2003; Herzog et al., 2003). Increased airway vascularityseen in asthma is dependent on VEGF and vascular endothelialgrowth factor receptor (VEGFR-1 and -2) expression (Hoshinoet al., 2001). The expression of the receptor is suggested to beparticularly important as the ligand is highly expressed inbleomycin-induced fibrotic lung injury without angiogenesis(Fehrenbach et al., 1999). VEGF promotes endothelial cellsurvival with Bcl-2 expression (Nor et al., 1999) and promotesMMP expression from vascular smooth muscle cells (Wang &Keiser, 1998). There is evidence of interaction between VEGFand TNF- and bFGF to generate tubular structures in the ECMnecessary for angiogenesis (Koolwijk et al., 1996). Multipleinflammatory mediators may cause vessel dilatation andmicrovascular leakage, thereby increasing tissue turgor andcontributing to airway wall changes (Orsida et al., 1999).

5. Current asthma treatmentsand their effects on airway remodelling

5.1. Glucocorticoids

Glucocorticoids are effective for the treatment of allergicinflammation in asthma and also result in reduced AHR which

Therapeutics 112 (2006) 474488is thought to be due to reduced inflammation (Djukanovic et al.,1992). However, long term treatment of asthmatics with inhaledcorticosteroids (ICS) does not completely eliminate AHR or

&resolve airway obstruction (van Essen-Zandvliet et al., 1994).This suggests that the corticosteroid agents may have a limitedimpact on the progression of airway remodelling changes.

However, there is some evidence that ICS may halt or evenreverse individual aspects of structural changes in the airways(Hoshino, 2004). One study on cultured sputum cells fromasthmatics showed that flunisolide treatment was able to inhibitTGF release and increase MMP-9 relative to TIMP1 (Profita etal., 2004). ICS therapy has been reported to reduce the RBMlayer thickness of asthmatic airways concomitant with areduction in fibroblast activity (Hoshino et al., 1998a, 1998b).However, other studies have failed to demonstrate a significanteffect of corticosteroid therapy on RBM layer thickness (Jefferyet al., 1992).

ICS have also been shown to be antimitogenic to smoothmuscle cells. However, Bonacci et al. (2003a, 2003b) haveshown this antimitogenic effect only occurs in the presence ofnormal BM (laminin) and not collagen (pathological BM)associated with airway remodelling. This suggests that theeffects of corticosteroids on fibrosis may be lessened in thesetting of established airway fibrosis. Corticosteroids inhibitlysyl oxidase activity leading to reduced collagen crosslinking(Benson & LuValle, 1981). Corticosteroids have been shown insome studies to provide improvement in epithelial shedding andcollagen deposition (Hoshino, 2004) but other studies haveshown little effect (Lundgren et al., 1988; Jeffery et al., 1992).Finally, it has been shown that corticosteroids given postnatallymay affect lung development in rabbits (Kovar et al., 2005).Corticosteroids affect tissue damping, hysteresis and resistanceof pressure curves in rabbit development, reducing lung size andperipheral airway wall diameter (Kovar et al., 2005).

Nevertheless, the observation that airway remodellingpersists despite the optimal use of corticosteroids (Ward &Walters, 2005) suggests these agents have a limited impact onstructural changes in the airway. This highlights the need fornovel therapies that can directly target the remodellingprocesses to prevent permanent changes in lung function.

5.2. agonists

Long-acting -agonists have shown some promise whenused with corticosteroids as combination therapy as the twodrugs appear to have synergistic or additive effects. For example,ASM cells express receptors for both corticosteroids and adrenergics and there is some evidence that combination therapymay inhibit cell proliferation and migration (Howarth et al.,2004). Long term studies are required to determine the efficacyof combination treatment in preventing the development ofairway remodelling changes (Currie et al., 2003). Combinationtherapy has also been shown to reduce airway vascularity in vivo(Johnson, 2002) and density of vessels in the lamina propria in a3-month study of asthmatics (Orsida et al., 2001).

5.3. Leukotriene modifiers

M.L.K. Tang et al. / PharmacologyIn an animal model of asthma a leukotriene receptorantagonist (LRA) prevented smooth muscle thickening aroundthe airways (Wang et al., 1993). LRA have also been shown toinhibit other aspects of airway remodelling in animal modelsand this may in part be due to suppression of cell infiltration intothe airway wall (Henderson et al., 2002).

Mast cell tryptase inhibitors may have potential in addres-sing not only bronchoconstriction but also airway remodelling,in particular fibrosis (Cairns, 2005).

6. Genetic susceptibility to airway remodelling

It is well known that asthma, like other allergic diseases, hasan inherited component.

Only a handful of asthma susceptibility genes have so farbeen identified but it is probable that many of these will affectairway remodelling. One example is an orphan G-coupledprotein receptor called GPRA which is differentially-expressedin epithelial cells and smooth muscle cells in asthmatics and inmice with OVA-induced AAD, compared to controls (Laitinenet al., 2004). The best known asthma gene is a disintegrin andmetalloprotease 33 (ADAM33), the product of which appears toplay a significant role in collagen homeostasis (Van Eerdeweghet al., 2002; Zou et al., 2004). Expression profiling of lungtissue from mice with experimental asthma has identified genesrelated to basic amino acid metabolism strongly upregulated(i.e. cationic amino acid transporter 2, arginase I and arginaseII). Arginase may be involved in the regulation of collagengeneration (Zimmermann et al., 2003).

7. Conclusion

Asthma is a common disease that frequently has its onset inchildhood and may persist lifelong. Airway remodelling is acharacteristic feature of asthma that has deleterious con-sequences on lung function, causing AHR and fixed airwayobstruction. The mechanisms regulating airway remodellingremain poorly understood. While airway inflammation isknown to play a role in the progression of remodellingchanges, other factors are also likely to be important in theregulation of this process. A primary defect of epithelial repairis thought to be a major factor leading to the development ofstructural change in the airways. In the setting of an epithelialrepair defect, repeated episodes of airway inflammation resultin the initiation of secondary repair processes that lead toepithelial metaplasia and subepithelial fibrosis. Subepithelialfibrosis is likely to represent one of the earliest changes ofremodelling and may act to drive the progression ofremodelling by promoting smooth muscle hyperplasia andinflammation, as well as providing a positive feedback signalfor fibrosis. ASM may also augment the remodelling processby producing ECM proteins and inflammatory mediators.Current asthma treatments are aimed at controlling airwayinflammation and therefore act indirectly to control airwayremodelling by reducing inflammation. This approach is onlypartially successful in retarding the development of structural

483Therapeutics 112 (2006) 474488changes in the airways. A more direct approach that targets thespecific components of remodelling may provide a moreeffective strategy to prevent or reverse structural changes and

&restore lung function. It is likely that successful therapies forairway remodelling will target more than one of thesecomponents. Improved understanding of the sequence ofevents that take place during the progression of remodellingand the factors regulating each of the components ofremodelling will facilitate the development of novel therapiesthat can directly target airway remodelling in asthma.

Acknowledgment

The authors wish to acknowledge Tiffany Bamford forproviding the stained sections in Fig. 1.

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