at SciVerse ScienceDirect

Pulmonary Pharmacology & Therapeutics 26 (2013) 95e104

Contents lists available

Pulmonary Pharmacology & Therapeutics

journal homepage: www.elsevier .com/locate/ypupt

Functional phenotype of airway myocytes from asthmatic airways

David B. Wright a, Thomas Trian b, Sana Siddiqui c,d, Chris D. Pascoe e, Oluwaseun O. Ojo f, Jill R. Johnson g,Bart G.J. Dekkers h, Shyamala Dakshinamurti i, Rushita Bagchi j, Janette K. Burgess k, Varsha Kanabar l,*aMedical Research Council and Asthma UK Centre in Allergic Mechanisms of Asthma, King’s College London, London, United KingdombCentre de Recherche Cardio-thoracique de Bordeaux, INSERM, U1045, Equipe: Remodelage bronchique, Université Bordeaux2, Bordeaux Cedex, FrancecMeakins-Christie Laboratories, Department of Medicine, McGill University, Montréal, Québec, CanadadReceptor Pharmacology Unit, National Institute on Aging, National Institutes of Health, Baltimore, MD, USAeUniversity of British Columbia, Department of Medicine, CanadafDepartment of Respiratory Physiology, University of Manitoba, Biology of Breathing Group, Manitoba Institute of Child Health, Winnipeg, Manitoba, Canadag Leukocyte Biology Section, National Heart and Lung Institute, Imperial College London, London, United KingdomhDepartment of Molecular Pharmacology, Groningen Research Institute for Asthma and COPD, University of Groningen, Groningen, The Netherlandsi Section of Neonatology, WS012 Women’s Hospital, Winnipeg, University of Manitoba, Manitoba, CanadajDepartment of Physiology, University of Manitoba, Institute of Cardiovascular Sciences, St. Boniface Hospital Research Centre, Winnipeg, Manitoba, CanadakCell Biology Group, Woolcock Institute of Medical Research and Discipline of Pharmacology, The University of Sydney, Australial Sackler Institute of Pulmonary Pharmacology, 150 Stamford Street, Pharmaceutical Science Division, King’s College London, London SE1 9NH, United Kingdom

a r t i c l e i n f o

Article history:Received 5 May 2012Received in revised form8 August 2012Accepted 8 August 2012

Keywords:Airway smooth muscleVascular smooth muscleAsthmaExtracellular matrixAnimal models

Abbreviations: AHR, airway hyperresponsiveness;C/EBP a, CCAAT/enhancer binding protein a; CTGF, conECM, extracellular matrix; FGF, fibroblast growth facepidermal growth factor; MLCK, myosin light chainchain; mtTFA, mitochondrial transcription factor A; NPDE, phosphodiesterase; PGC1 a, peroxisome prolifertivator-1a; PDGF, platelet derived growth factor; smactin; TNF, tumour necrosis factor; TGF, transformingendothelial growth factor; VSM, vascular smooth mu* Corresponding author. Tel.: þ44 7848 4819.

E-mail address: varsha.kanabar@kcl.ac.uk (V. Kana

1094-5539/$ e see front matter � 2012 Elsevier Ltd.http://dx.doi.org/10.1016/j.pupt.2012.08.003

a b s t r a c t

In asthma, the airway smooth muscle (ASM) cell plays a central role in disease pathogenesis throughcellular changes which may impact on its microenvironment and alter ASM response and function. Theanswer to the long debated question of what makes a ‘healthy’ ASM cell become ‘asthmatic’ still remainsspeculative. What is known of an ‘asthmatic’ ASM cell, is its ability to contribute to the hallmarks ofasthma such as bronchoconstriction (contractile phenotype), inflammation (synthetic phenotype) andASM hyperplasia (proliferative phenotype).

The phenotype of healthy or diseased ASM cells or tissue for the most part is determined by expressionof key phenotypic markers. ASM is commonly accepted to have different phenotypes: the contractile(differentiated) state versus the synthetic (dedifferentiated) state (with the capacity to synthesizemediators, proliferate and migrate). There is now accumulating evidence that the synthetic functions ofASM in culture derived from asthmatic and non-asthmatic donors differ. Some of these differencesinclude an altered profile and increased production of extracellular matrix proteins, pro-inflammatorymediators and adhesion receptors, collectively suggesting that ASM cells from asthmatic subjects havethe capacity to alter their environment, actively participate in repair processes and functionally respondto changes in their microenvironment.

� 2012 Elsevier Ltd. All rights reserved.

ASM, airway smooth muscle;nective tissue growth factor;tor; HB-EGF, heparin bindingkinase; MHC, myosin heavyRF, nuclear respiratory factor;ator-activated receptor coac--a-actin, smooth muscle-a-

growth factor; VEGF, vascularscle.

bar).

All rights reserved.

1. Introduction

The state of a healthy or diseased ASM cell or tissue is dictatedby expression of key phenotypic markers. Airway smooth muscle(ASM) is commonly accepted to have different phenotypes: thecontractile (differentiated) state and the synthetic (dediffer-entiated) state (which has the capacity to synthesize mediators,proliferate and migrate). Increased ASM mass is a hallmark featureof airway remodelling in the asthmatic airway; other featuresinclude thickening of the basement membrane, increased deposi-tion of and alterations in the composition of the extracellularmatrix (ECM), metaplasia of the epithelial cells and an increase inthe number of blood vessels (angiogenesis). In addition, accumu-lating evidence indicates that the synthetic functions of ASM cellsderived from asthmatic and non-asthmatic donors differ.

D.B. Wright et al. / Pulmonary Pharmacology & Therapeutics 26 (2013) 95e10496

2. What are the functional differences between ASM cellsderived from non-asthmatic and asthmatic subjects?

The answer to whether ASM derived from asthmatic donors ismechanically or phenotypically different from that of non-asthmatic derived ASM is still largely unknown. Several studies todate have shown that in asthma there is an increase in ASM forceproduction, degree of shortening and sensitivity to agonists [1e4],and increased proliferative markers [5]. However, an equal numberof studies have shown no mechanical differences between ASMderived from asthmatic and non-asthmatic donors [6e9].

2.1. ASM contractile responses in asthma

Airway hyperresponsiveness (AHR) is characterized by anexaggerated narrowing of the airways to a variety of chemical,physical and pharmacological stimuli [10,11]. Two components ofAHR have been observed, namely variable and persistent AHR.Variable AHR is suggested to be due to increased sensitivity of theASM to contractile stimuli and is associated with inflammatoryresponses, whereas persistent or baseline AHR is found in themajority of chronic asthmatics and is considered to relate to airwayremodelling [10,12e16]. Persistent AHR in asthma may beexplained by increased ASMmass, but also by increased contractileproperties of the ASM [17].

Studies on contractile and relaxant responses have shownintrinsic differences between ASM obtained from asthmatic andnon-asthmatic donors. The contractile force of tracheal smoothmuscle strips from donors with asthma was increased compared tonon-asthmatic controls [7,18], however, contraction of bronchialsmooth muscle from asthmatic and non-asthmatic donors was notdifferent [4,19]. In another study investigating asthmatic and non-asthmatic trachealis muscle function, it was shown that there wasno difference between the contractile forces generated by asthmatictissue [18]. In this study, force generated by the muscle wasnormalized to the cross sectional area to give the stress (force perunit area) generated by the muscle. This is an appropriate normali-zation because there is more ASM in the airways of asthmatics soone would expect it to produce more force overall but not neces-sarily more force per contractile unit. In animal models of allergicAHR, increased contractile force of the tracheal smooth muscle hasbeen observed after repeated allergen challenge [20e22]. In a guineapig model, repeated allergen challenge increased responsiveness tothe contractile agonist methacholine in perfused tracheae ex vivo[22]. In addition to increased contractile force, increased cell short-ening velocity (Vmax) and maximal shortening capacity have beenobserved in ASM cells isolated from asthmatics [18,23]. Moreover,Vmax has been shown to be increased in various other models of AHR[17]. When cultured in collagen gels, ASM derived from asthmaticsincreased gel compaction compared to non-asthmatic derived ASMcells following histamine exposure [24], suggestive of increased ASMcontraction. In addition to changes in contractile responsiveness,ASM derived from asthmatic donors is less responsive to pharma-cological relaxants [4,7], which may contribute to AHR. In support ofthis, the relaxant capabilities of hyperresponsive tissue were shownto be decreased in animal models of AHR [25,26]. However, it shouldbe noted that in whole tissue preparations from asthmatic and non-asthmatic subjects, there was no observable difference with regardsto shortening velocity or maximal shortening [18]. These seeminglycontradictory results highlight a need for more research intowhether there is a difference between asthmatic and non-asthmaticASM tissue preparations (muscle strips and airway tubes).

Smooth muscle contraction is regulated by the intracellularcontractile apparatus, which is comprised of thin (ae and g�actin;the regulatory proteins tropomyosin, caldesmon and calponin and

SM22) and thick filaments (myosin). For a review on the ASMcontractile apparatus and contraction, the reader is referred to [27]and [17]. Studies on the expression of components of the contractilemachinery in endobronchial biopsies from asthmatic donorsshowed increased mRNA expression of transgelin (SM-22), myosinlight chain kinase (MLCK), and total smooth muscle myosin heavychain (sm-MHC) compared to non-asthmatic donors [28,29].Moreover, Benayoun et al. showed that in asthmatic donors, MLCKexpressionwas found to be increased with disease severity and wasmost prominent in the ASM bundles of severe asthmatics [30]. Incontrast to these findings, Woodruff et al. showed no change inMLCK mRNA levels in biopsies from asthmatic and non-asthmaticsubjects [31]. Increased expression of the sm-MHC isoforms SM-2, SM-A and SM-B were also detected in asthmatic ASM bundles,whereas no differences were observedwith SM-1, tropomyosin b ora-actin. Conversely, expression of tropomyosin a and g-actin wasdecreased in these patients [28]. In culture, ASM cells derived fromasthmatics expressed MLCK mRNA more abundantly than non-asthmatic derived ASM cells, whereas no difference in expressionof SM-A was observed and expression of SM-B was not detectable[23]. In contrast to in vitro findings, enhanced expression of the SM-B isoform of sm-MHC within the ASM bundles in vivo may be ofmore interest in relation to AHR, as this isoform contains an addi-tional 7-amino acid insert which increases ATPase activity andincreases shortening capacity compared to the SM-A isoform [17].Since MLCK phosphorylates myosin light chains, one of the keyevents in the generation of force by smooth muscle [27], increasedexpression of the SM-B isoform may enhance contraction andcontribute to AHR in asthma. In support of a contribution of SM-Band MLCK to AHR in vivo, expression of SM-B and MLCK havebeen shown to be increased in hyperresponsive Fisher ratscompared to normoresponsive Lewis rats [26,32]. In animal modelsof allergic AHR, increased pulmonary expression of sm-MHC hasbeen observed after repeated allergen challenge, which was asso-ciated with increased contractile force produced by the ASM[20,21]. An imbalance between MLCK expression and myosin lightchain phosphatase (MLCP) may lead to enhanced myosin lightchain phosphorylation and consequently enhanced ASM contrac-tion. Studies on the RhoA/Rho-kinase pathway have indicatedincreased expression of RhoA after allergen challenge, leading todecreased MLCP activity and increased contraction [33,34]. Inaddition, increased expression of the contractile proteins sm-MHC,sm-a-actin and desmin have been shown to correlate with meth-acholine responsiveness in subjects with asthma [28,35]. Collec-tively, these findings seem to indicate that increased expression ofcontractile proteins in ASM from asthmatic donors may enhancecontractile responses, however more studies on human asthmatictissue are needed to add evidence to this argument.

2.2. ASM cytoskeleton e is the cellular architecture of ASM differentin asthma or in allergic animal models?

Chin et al. reported that in response to electrical field stimula-tion (EFS), tracheal smooth muscle from asthmatic donors did notproduce more force per unit area nor did it shorten more exten-sively or faster in comparison to non-asthmatic tissue [18]. Oneproperty of asthmatic-derived ASM that differs from non-asthmatic-derived ASM unequivocally is the response to mechan-ical perturbation. It has beenwell documented that there is a lack ofa bronchodilating effect following a deep inspiration in asthmatics[36e38]. It has been suggested that this loss is due to increasedtone in the ASM of asthmatics [39], possibly indicating that it is theenvironment surrounding the ASM bundles that differs in asthma.However, studies have shown that asthmatic-derived muscle itselfmay have an altered response to oscillations. Chin et al. showed

D.B. Wright et al. / Pulmonary Pharmacology & Therapeutics 26 (2013) 95e104 97

that asthmatic-derived trachealis muscle oscillated at a supra-maximal length of 30% reference length (i.e. at a greater length thanexpected in vivo), showed a smaller drop in EFS-induced force thanthe non-asthmatic-derived muscle and it also recovered beyondthe normalized force when compared to the non-asthmatic musclewhich did not recover to pre-oscillation levels [18]. There are manypossible explanations for this unique finding, including increasedstability of the contractile apparatus or attached cross-bridges,increased stability of the myosin thick filaments that preventdissociation upon oscillations [18] and increased stability of theadhesion junctions at the cell membrane. The last point warrantsfurther investigation as Huang et al. have shown that inhibitingproteins at these junctions leads to a decrease in the force trans-mitted by the muscle [40]. Increased stability at the adhesionjunctions, and therefore increased actin tethering at these sites,would enhance the resistance of the muscle to oscillations. It islikely that a combination of inherent differences in the ASM(genetically determined) and differences in the environmentaround the ASM are causative in the lack of bronchoprotectionreceived by asthmatics following a deep inspiration.

Another recent phenomenon identified by Bossé et al. known asforce adaptation [41], has been suggested to play a pivotal role inAHR in asthma. Force adaptation is defined as an increase in forcegenerating capacity over time in the continued presence ofa spasmogen (inflammatory or otherwise). It has recently beenshown to occur under conditions that more closely mimic thein vivo strains that ASM is subjected to during normal breathingpatterns [42]. While the mechanism of force adaptation remainsunknown, it is possible that similar changes to the muscle that giveit the oscillatory resistance properties may also allow it to undergoforce adaptation. In particular, filamentogenesis of actin andmyosin or rearrangement of the contractile apparatus may beimportant [43,44]. It is unlikely that the spasmogens are inducingchanges in gene expression as force adaptation occurs withinminutes and is quickly and easily reversible. This phenomenoncould help explain why asthmatic-derived ASM dissected from thelung does not produce more force than its non-asthmatic coun-terpart. If spasmogens are inducing the increase inmuscle force andcytoskeletal rearrangement, then removal of the muscle from thatenvironment should abolish the increase in force.

As described above, increased expression of specific myosinisoforms in asthma has been suggested to account for functionaldifferences observed in ASM from asthmatics. In addition, recentfindings have shown that allergic animal models of AHR withovalbumin [45] or house dust mite antigen (Dermatophagoidespteronyssinus) challenge [46] have increased ASM mass withincreased sm-a-actin expression which correlates directly withairway resistance [45]. Encouraging data suggests that features ofairway remodelling such as increased ASM mass and goblet cellhyperplasia, subepithelial fibrosis and collagen deposition aredecreased by an IkB kinase (IKK) b inhibitor preventing nuclearfactor (NF)-kB activation [46]. Overexpression of the fast SM-Bmyosin isoform in asthmatics leads to an increase in the velocityof shortening and correlates to a decrease in the PC20 to inhaledmethacholine [28]. Interestingly, loss of the SM-B isoform in VSMcauses a decrease in smooth muscle shortening velocity as well asforce production [47,48]. It will be important, in the future, todetermine the role of the different myosin isoforms in contractionof ASM in asthma.

It has been proposed that altered ASM mass could account forAHR in asthma [49]. It was predicted that with increasing airwaywall thickening, the amount of luminal narrowing, as a conse-quence of ASM shortening would be enhanced [50]. Increased ASMmass in asthma could contribute to an increase in force develop-ment and thus airway narrowing [51], as well as an enhanced

biosynthetic capability. In a recent in vivo study, the relationshipbetween the site of ASM mass and AHR was examined. There wasa lack of association between these two parameters with AHR beinglargely present in the peripheral lung whereas ASM growth wasfound throughout the airway tree [52]. In vivo, airway narrowingfollowing an early allergic response was initially reported in thelarge airways and subsequently in the periphery of the lung [53]. Itis therefore important to understand the intrinsic properties ofASM, including the phenotypic changes as well as regulators of thecontractile apparatus in the context of AHR. Preliminary in vivowork has reported that, in the presence of AHR, there is nodownregulation of gene expression of contractile proteinsincluding sm-MHC and total MLCK despite evidence of smoothmuscle proliferation [54]. It has also been proposed that an increasein the shortening velocity of ASM could contribute to AHR. A cross-bridge model showed that an increase in the total amount ofshortening with increased isotonic velocity could be explained bychanges in either the cycling rate of phosphorylated cross-bridgesor the rate of myosin light chain phosphorylation. Thus, if asthmainvolves increased ASM velocity, this could be important in theassociated AHR [55].

Future work should be focused on determining the role of theinflammatory and remodelled environment in the asthmaticairway on the expression of ASM cytoskeletal genes to furtherunderstand the regulation of the contractility of ASM. Determiningthe mechanism behind force adaptation may help us gain insightinto the role of inflammation in the exacerbation of asthmasymptoms. More research elucidating the mechanical properties ofASM is required, particularly studies that mimic in vivo conditions.

3. ASM remodelling

Persistent asthma is associated with structural changes to theairway wall, termed remodelling, which increases with diseaseseverity and includes alterations in the extracellular matrix (ECM)protein profile, thickening of the basement membrane andincreases in the ASM bulk. The degree of airway remodelling in anasthmatic airway has a direct effect on the airway obstruction andclosure which contributes to hyperresponsiveness of the airway.The nature of airway remodelling has been most commonlydescribed in adult central airway samples obtained by bronchos-copy, or from postmortem studies of asthmatics and in animalmodels [56]. The lack of functional and histological studies inpaediatric asthma has largely been due to a limitation of tissue sizeand availability. That the increased ASM mass contributes to thepathophysiology of adult asthma has been demonstrated in part bythe utility of bronchial thermoplasty, resulting in alleviation of thesymptoms of severe [57], but not moderate asthmatics [58].Paediatric data on thermoplasty are unavailable to date [59].

Morphometric studies incorporating paediatric and adult asth-matics together correlate airway thickness with clinical severityrather than age of onset or duration of symptoms, suggesting thatASM remodelling is an early event which may in fact determineprogressive asthma severity, independent of the additive effects ofchronic inflammation [60]. Already in paediatric asthma airwayremodelling is present and characterized by ASM hypertrophy and/or hyperplasia [61] and reticular basement membrane thickening[62]. However, basement membrane composition does not differbetween asthmatics and non-asthmatic controls [63]. Neither thedegree of inflammation nor basement membrane thickening inbronchial biopsies from children with refractory asthma correlatewith their degree of airflow limitation or responsiveness to therapy[64]. Bronchial biopsies from children with poorly controlledasthma demonstrate a greater smooth muscle area situated closerto the basement membrane, and increased expression of MLCK,

D.B. Wright et al. / Pulmonary Pharmacology & Therapeutics 26 (2013) 95e10498

when compared with children without chronic airway obstruction.These parameters correlate with asthma severity, but not withasthma duration [65]. Treatment-resistant asthma in atopic chil-dren features a marked increase in ASM mass in the presence ofvariable degrees of eosinophilia and absence of Th2 mediatoractivity, indicating that the mechanisms driving paediatric ASMgrowth may differ from the inflammatory pathways typical of adultasthma [66]. Surveying other aetiologies of paediatric airflowobstruction, for example infants with bronchopulmonary dysplasia,has demonstrated substantial airway thickening contributing toairway resistance in the absence of epithelial damage or otherevidence of airway inflammation [67]. ASM mass is also increasedin childrenwith cystic fibrosis and bronchiectasis, regardless of theduration of inflammation [68]. Collectively, these findings suggestthat severe airflow obstruction may propagate ASM growth inchildren independent of the chronicity of the associatedinflammation.

ASM cells isolated from asthmatics proliferate faster in culturecompared to those from non-asthmatics [69]. In vitro hyperplasia isassociatedwith a loss of contractile proteins, while in vivo, but thereare only a few studies addressing this issue [28,31,70,71]. Hyper-plastic growth of ASM has been observed after repeated allergenchallenges in animal models [20,21,72]. Some studies suggestdownregulation of contractile protein gene expression [70] andprotein content of contractile proteins following allergen chal-lenges in experimental models [70,71]. In repeatedly challengedguinea pigs, contractile protein expression and contractilityincrease [20,21]. Recent preliminary in vivo work using lasercapture microdissection to capture ASM cells [54] suggests thata decrease in gene expression of contractile protein does not occurdespite the ASM proliferation induced by repeated ovalbuminchallenge. Additionally, in vivo and in vitro studies using humanASM have failed to show such a downregulation of contractileproteins or even suggest an increase in contractile proteinexpression [23,28,31]. One human study that used laser capturemicrodissection to isolate ASM did not find an increase in MLCKgene expression between asthmatic and non-asthmatic biopsies[31], in contrast to findings of an upregulation of MLCK mRNA inmild asthmatic bronchial biopsies compared to non-asthmaticcontrols [28].

Reports indicate that in severe asthmatics there are proliferatingcells in the ASM bundles in vivo and that they express an epidermalgrowth factor receptor ligand, heparin-binding epidermal growthfactor (HB-EGF) [5]. These datawere confirmed in proliferating cellsin vitro (see Table 1 for mediators which induce ASM cell prolifer-ation in vitro). There is also a report of a proliferating compartment

Table 1Proliferative factors expressed by inflammatory cells in the asthmatic bronchial wall. Ma detailed review of these mediators, the reader is referred to [137].

Factors secreted byinflammatory cells

Source

PDGF Monocyte, macrophFGF2 Epithelial and endoTNFa T-lymphocyte, monTGFb T-lymphocyte, eosiHistamine (not mitogenic alone

but enhances growth factor function)Mast cell, basophil

Acetylcholine (not mitogenic alonebut enhances growth factor function)

Macrophages, mastmesenchymal cells

Endothelin-1 monocyte-macrophThromboxane A2 Mast cell, monocytLeukotrienes Monocyte, macrophTryptase Mast cellElastase NeutrophilROS Monocyte-macroph

of subepithelial sm-a-actinþ cells, non-organized airway contrac-tile elements in asthmatics, suggestive of a phenotypic gradientfrom undifferentiated cells to smooth muscle-like cells [73]. Insupport of phenotypic differences in asthmatic ASM, there is alsoliterature reporting that multiple VSM phenotypes exist in vivo.

4. How can the phenotype and function of vascular smoothmuscle in health and disease inform the airways?

Phenotypically, ASM and VSM express a number of smoothmuscle markers, including sm-a-actin, sm-MHC, desmin, telokin,meta-vinculin, calponin, caldesmon, leiomodin, smoothelin-A and-B and SM22 [74,75]. However, some cytoskeletal proteins thatdifferentiate these two types of smooth muscle cells have beenidentified, including different isoforms of smooth muscle myosinand smoothelin with ASM-specific expression (SM-2 and SM-B)[76,77] and VSM-specific expression (smoothelin B) [78]. More-over, the transcription factors GATA-5 and Foxf1 are preferentiallyexpressed in bronchial ASM cells, but not in VSM cells [79,80].

Angiogenesis in asthma has been recognised for many years andhas been confirmed in a large number of studies. These changesinclude an up to three-fold increase in the degree of vascularity ofthe large airways [81,82], and have been detected even in the earlystages of the disease [83]. This increase in vascularity is thought tohave mostly deleterious consequences, including lung oedema,increased inflammatory cell trafficking, airway lumen narrowingand loss of distensibility [84]. In the large airways, changes in thebronchial microcirculation are thought to be driven by increasedexpression of vascular endothelial growth factor (VEGF) byremodelled ASM cells, induced in part by TGFb [85e87].

Similar to the phenotypic changes to ASM in asthma, phenotypicand structural changes to VSM have been found to occur in thisdisease. Studies in human asthmatics and in animal models ofallergic AHR have described VSM thickening [88,89], likely drivenby increased proliferation of VSM cells under chronic inflammatoryconditions [90,91]. Furthermore, increased collagen depositionaround the pulmonary vessels in a mouse model of chronic housedust mite-driven allergic AHR [92] suggests phenotypic changes tothese cells, characterised by increased ECM deposition by VSM cellsin asthma. Moreover, these changes have also been described insmaller vessels not associated with the bronchi [93]. However, theimpact of these VSM changes on lung function has not yet beendescribed. Furthermore, it is not clear as to whether the changes toVSM in asthma are a direct consequence of chronic airwayinflammation, or if these changes occur as a secondary effectdownstream of changes to ASM.

any other mediators have either pro- or anti-mitogenic effects on ASM cells. For

Ref.

age, eosinophils, mesenchymal cells [114e116]thelial cells [117e119]ocyte, macrophage, mast cells [120e122]nophils, macrophages, epithelial cells [118,123e126]

[127]

cells, lymphocytes, granulocytes,, epithelial cells

[128,129]

age [130,131]e, macrophage [132]age, eosinophils, mast cells, fibrocytes [133]

[134][135]

age, neutrophil, eosinophils, mast cells [136]

D.B. Wright et al. / Pulmonary Pharmacology & Therapeutics 26 (2013) 95e104 99

5. Do asthmatic ASM cells exist? or are these healthy cellsbehaving differently due to an asthmatic environment?

Increased ASM mass may be attributed to the following events:an increase in ASM cells (hyperplasia) [31,68,69,72,94e96], anincrease in ASM cell size (hypertrophy) [30,68,94] mediated bycytokines such as TGFb [97], migration of ASM cells or subepithelialmyofibroblasts (intermediary between fibroblasts and myocytes)[30], infiltration of circulating fibrocytes [98] and local mesen-chymal stem cells [99] as well as epithelial-to-mesenchymal tran-sition [100]. The balance between decreased apoptosis andincreased ASM hyperplasia may also be important in regulating theASM mass [101]. Altered calcium homeostasis may also contributeto excessive ASM proliferation in severe asthma via increasedmitochondrial biogenesis [102]. Alterations in the ECM surroundingthe ASM bundles may also contribute to the increased ASM bulk[103,104]. The increased muscle bulk observed in asthmaticsubjects correlates with the severity of disease and is one feature ofairway remodelling insensitive to all currently available asthmatherapeutics [15,105,106]. The major question to address is whetherASM derived from asthmatics is intrinsically and functionallydifferent in comparison to its otherwise healthy counterpart and iftherapies can be discovered to reverse these differences? Gluco-corticosteroid treatment is efficient in reducing proliferation ofASM derived from non-asthmatics but is completely ineffective inasthma derived ASM cells [106], particularly in the presence oftype-I collagen [107,108]. Roth et al. reported that asthmatic-derived ASM cells do not express CCAAT/enhancer bindingprotein a (C/EBP a) which is necessary for the translocation of thecorticosteroid/receptor complex to the nucleus, an essential steprequired for the glucocorticosteroid cascade to be effective [106].Furthermore, ASM cells derived from asthmatic donors proliferateat a higher rate, when treated with FBS, in comparison to thatderived from non-asthmatic subjects [69,102], again suggestive ofan intrinsic difference between asthmatic-derived and non-asthmatic-derived ASM cells. Additionally, this functional differ-ence is retained in ASM cultured for several weeks (around fivepassages) in the absence of an endogenous asthmatic environment(beyond that which the cells release themselves) [69,102,106].

In the last decade, a growing list of factors and/or modificationshas been identified which support the hypothesis that ‘asthmatic’ASM cells are intrinsically and functionally different from ‘non-

Table 2Mediators known to be differentially expressed by asthmatic ASM.

Mediator Function in ASM

Mediators increased in asthmatic ASMCollagen type-I Increased in basal and growth factor-induced

of a proliferative, hypocontractile ASM phensynthesis, migration and survival.

Fibronectin Increased in basal and growth factor-inducedSwitching to a proliferative, hypocontractileIncreased ASM cytokine synthesis, migrationEnhanced synthetic function of asthmatic AS

Perlecan Function in ASM unknown. Inhibition of VSMCTGF Fibronectin and collagen I expression by ASMEotaxin Increased in basal and IL-13 induced ASM. InIP-10 Increased in cytokine stimulated ASM and wHB-EGF Upregulated in severe asthmatic tissues. May

potential biomarker of remodelling.Mediators decreased in asthmatic ASMLaminin a1 Inhibition of growth factor-induced proliferaCollagen IV Function in ASM unknown.Hyaluronan Increased ASM proliferation.Chondroitin sulphate Inhibition of growth factor-induced proliferaPGE2 Inhibition of ASM proliferation. Inhibition of

fibronectin and CTGF expression.

asthmatic’ ASM cells (see Table 2). For example, it has been reportedthat ASM cells in culture derived from asthmatics, compared to non-asthmatics, express increased amounts of phosphodiesterase (PDE) 4,nuclear respiratory factor-1 (NRF-1), peroxisome proliferator-activated receptor coactivator-1a (PGC-1a), mitochondrial transcrip-tion factor A (mtTFA), collagen type-I,fibronectin, perlecan, eotaxin-1(CCL11), VEGF and connective tissue growth factor (CTGF) andinversely express less SERCA2B, C/EBPa, PGE2, laminin a1, collagentype-IV, chondroitin sulphate and hyaluronan [85,102,106,109e113].However, it is also clear that the pathological environment thatsurrounds the ASMcells in vivo is also implicated in thismodification.Inflammation characteristic of asthma acts on the ASM cells and caninduce proliferation, migration or secretion of cytokines, chemokinesand growth factors. As an example, see the non-exhaustive list ofinflammatory factors increased in the asthmatic bronchial wall andwhich are found to induce ASM proliferation (see Table 2).

ASM cells themselves produce a wide range of cytokines andchemokines that can potentially attract and sustain direct interac-tions between the ASM and infiltrating inflammatory cells. One ofthe best described is the interaction between ASM and mast cells.ASM cells from asthmatic subjects produce more CXCL10 (IP-10)[148] and CX3CL1 (fraktalkine) [155] following proinflammatorycytokine treatment compared to ASM from healthy donors. Boththese chemokines induce mast cell migration towards the ASMlayer in asthma. As a result, mast cells can adhere to ASM cells,thereby promoting both the survival and proliferation of mast cells.Mast cell activation and degranulation in the vicinity of the ASMcells leads to extracellular deposition of inflammatory productsthat may contribute to the mechanisms underlying the increase inASMmass and AHR [156,157]. T lymphocytes can also interact withASM cells, with T lymphocyte engagement of cell surface receptorson ASM cells inducing DNA synthesis in the ASM cells [158]. Morerecently, the interaction between ASM cells and CD4þ T cells hasbeen reported to increase ASM proliferation in vivo [101]. In addi-tion to targeting the mast cell and T-cell interactions with ASM,platelets have also been demonstrated to influence syntheticfunctions of ASM [150,159e161], and warrant further investiga-tions. Features of airway remodelling such as inflammation,increased muscle mass and collagen deposition are suppressed inplatelet-depleted mouse models of allergic asthma [162,163], sug-gesting that factors external to ASM can influence the progressionof asthma disease.

Ref.

ASM cell proliferation. Inductionotype. Increased ASM cytokine

[20,109,138e143]

ASM cell proliferation.ASM phenotype.and survival.M.

[20,112,138,139,141,143]

proliferation [109,144]cells [145,146]

duction of ASM migration [112,147]hich induces mast cell migration [148]serve as a [5]

tion and ASM phenotype switching [20,109,138,139][109][149,150]

tion [109,151]TGFb-induced collagen I, [113,152e154]

Fig. 1. Influence of ASM derived ECM on ASM phenotype and function. Alterations in the ECM microenvironment dictate ASM function such that non-asthmatic derived ECMreverses the enhanced proliferative and secretory capacity of ASM derived from asthmatic subjects or vice versa.

D.B. Wright et al. / Pulmonary Pharmacology & Therapeutics 26 (2013) 95e104100

It appears that ASM derived from asthmatic subjects hasa functional phenotype that differentiates them from its non-asthmatic counterpart, at least in ASM cells in culture. However,there is also the possibility that the environment present in anasthmatic bronchus is different to that of ‘asthmatic’ ASM cells inculture. Therefore, differential functional observations betweenhealthy and asthmatic ASM require characterization in vivo. Thecomposition of ECM surrounding the ASM bundles of healthy andasthmatic subjects are continuously being explored with some

Fig. 2. Phenotype switching. The question if ASM from healthy and asthmatic subjects arecells reside drives the cellular phenotype and dictates switching between the two phenoty

histological studies suggesting no differences and others demon-strating some variation [16,116,164]. What is clear is that an alteredECM environment, of which collagen type-I, fibronectin andlaminin-111 and -211 have been predominantly explored, dramat-ically influences ASM function [116,164]. Furthermore, ASM cellsderived from asthmatics secrete an altered profile of ECMcompared to cells derived from healthy donors, which caninduce an otherwise functionally healthy ASM cell into behavingmore like a highly proliferative and secretory asthmatic phenotype

intrinsically different remains to be answered. Whether the environment in which thepes is currently unknown.

D.B. Wright et al. / Pulmonary Pharmacology & Therapeutics 26 (2013) 95e104 101

[109,112,165] (see Figs. 1 and 2). A complete profile of ECMcomponents surrounding ASM bundles in asthma of varyingseverity is warranted and transcriptome-based studies may bea useful tool [166] to inform key targets for further exploration in2D and 3D culture-based studies and to identify signalling path-ways and pharmacologically relevant targets.

Accumulating evidence indicates that ASM cells derived fromasthmatic donors have specific phenotypic differences that distin-guish them from ASM cells derived from non-asthmatic donors.However, it is also clear that the environment inwhich the ASM cellis located within the airway of an asthmatic is different to that inthe airway of a non-asthmatic person. (see review by Bossé et al.[167]). This combination of alterations at many levels within theairways of an asthmatic increase the difficulty of establishingaccurate model systems for elucidating and understanding thecontribution of the ASM cells in driving the pathophysiology ofasthma.

6. Overview

The ASM cell plays a central role in the mechanisms underlyingasthma. How these cells are altered in the asthmatic patient,compared to the non-asthmatic, is a question that challenges thefield. A catalogue of differences is emerging between these two celltypes from changes in force production, degree of shortening,increased contractile properties and alterations in the profile ofsynthetic products released from the ASM cells. We have learnta great deal about the consequences of airway remodelling forasthma symptoms, but we are no closer to establishing the initialtrigger or order of events leading to disease, particularly in child-hood asthma.

Current asthma treatments are ineffective in reversing airwayremodelling in moderate to severe asthmatics. Addressing the keydifferences in the molecular and biochemical mechanisms whichdefine an ASM cell as asthmatic and differentiate it from an ASMcell in a healthy donor could provide novel therapeutic strategies toswitch asthmatic ASM cells back to their relatively quiescent stateas found in their non-asthmatic counterparts.

Nonetheless, studies have established that the ASM cellpossesses an immunomodulatory phenotype that contracts,proliferates andmodulates its environment, and at the same time isaltered by the milieu within the remodelled airway. Understandingthe contribution to and the response of the ASM to the diseasepathobiology in asthma will provide significant knowledge for thefield.

Acknowledgements

Sana Siddiqui is the recipient of the Alexander McFee Student-ship, Faculty of Medicine, McGill University, Montréal, QC. JanetteBurgess is supported by a National Health & Medical ResearchCouncil, Australia Career Development Fellowship #1032695. Jill R.Johnson is supported by an Imperial College London JuniorResearch Fellowship. Rushita Bagchi is the recipient of an MHRC/SBRC Coordinated Graduate Studentship awarded by the ManitobaHealth Research Council, Canada.

References

[1] Thomson RJ, Bramley AM, Schellenberg RR. Airway muscle stereology:implications for increased shortening in asthma. Am J Respir Crit Care Med1996;154:749e57.

[2] Bramley AM, Thomson RJ, Roberts CR, Schellenberg RR. Hypothesis: exces-sive bronchoconstriction in asthma is due to decreased airway elastance. EurRespir J 1994;7:337e41.

[3] Schellenberg RR, Foster A. In vitro responses of human asthmatic airway andpulmonary vascular smooth muscle. Int Arch Allergy Appl Immunol 1984;75:237e41.

[4] Bai TR. Abnormalities in airway smooth muscle in fatal asthma. Am RevRespir Dis 1990;141:552e7.

[5] Hassan M, Jo T, Risse PA, Tolloczko B, Lemiere C, Olivenstein R, et al. Airwaysmooth muscle remodeling is a dynamic process in severe long-standingasthma. J Allergy Clin Immunol 2010;125:1037e45. e3.

[6] Whicker SD, Armour CL, Black JL. Responsiveness of bronchial smoothmuscle from asthmatic patients to relaxant and contractile agonists. PulmPharmacol 1988;1:25e31.

[7] Bai TR. Abnormalities in airway smooth muscle in fatal asthma. A compar-ison between trachea and bronchus. Am Rev Respir Dis 1991;143:441e3.

[8] Cerrina J, Le Roy Ladurie M, Labat C, Raffestin B, Bayol A, Brink C. Comparisonof human bronchial muscle responses to histamine in vivo with histamineand isoproterenol agonists in vitro. Am Rev Respir Dis 1986;134:57e61.

[9] Björck T, Gustafsson LE, Dahlén SE. Isolated bronchi from asthmatics arehyperresponsive to adenosine, which apparently acts indirectly by liberationof leukotrienes and histamine. Am Rev Respir Dis 1992;145:1087e91.

[10] Meurs H, Gosens R, Zaagsma J. Airway hyperresponsiveness in asthma:lessons from in vitro model systems and animal models. Eur Respir J 2008;32:487e502.

[11] Postma DS, Kerstjens HA. Characteristics of airway hyperresponsiveness inasthma and chronic obstructive pulmonary disease. Am J Respir Crit CareMed 1998;158:S187e92.

[12] Cockcroft DW, Davis BE. Mechanisms of airway hyperresponsiveness.J Allergy Clin Immunol 2006;118:551e9. quiz 60e1.

[13] Bourdin A, Neveu D, Vachier I, Paganin F, Godard P, Chanez P. Specificity ofbasement membrane thickening in severe asthma. J Allergy Clin Immunol2007;119:1367e74.

[14] Chetta A, Foresi A, Del Donno M, Bertorelli G, Pesci A, Olivieri D. Airwaysremodeling is a distinctive feature of asthma and is related to severity ofdisease. Chest 1997;111:852e7.

[15] Pepe C, Foley S, Shannon J, Lemiere C, Olivenstein R, Ernst P, et al. Differencesin airway remodeling between subjects with severe and moderate asthma.J Allergy Clin Immunol 2005;116:544e9.

[16] Yick CY, Ferreira DS, Annoni R, von der Thusen JH, Kunst PW, Bel EH, et al.Extracellular matrix in airway smooth muscle is associated with dynamics ofairway function in asthma. Allergy 2012;67:552e9.

[17] Leguillette R, Lauzon AM. Molecular mechanics of smooth muscle contractileproteins in airway hyperresponsiveness and asthma. Proc Am Thorac Soc2008;5:40e6.

[18] Chin LY, Bosse Y, Pascoe C, Hackett TL, Seow CY, Pare PD. Mechanicalproperties of asthmatic airway smooth muscle. Eur Respir J 2012;40:45e54.

[19] Thomson NC. In vivo versus in vitro human airway responsiveness todifferent pharmacologic stimuli. Am Rev Respir Dis 1987;136:S58e62.

[20] Dekkers BG, Bos IS, Gosens R, Halayko AJ, Zaagsma J, Meurs H. The integrin-blocking peptide RGDS inhibits airway smooth muscle remodeling ina guinea pig model of allergic asthma. Am J Respir Crit Care Med 2010;181:556e65.

[21] Gosens R, Bos IS, Zaagsma J, Meurs H. Protective effects of tiotropiumbromide in the progression of airway smooth muscle remodeling. Am JRespir Crit Care Med 2005;171:1096e102.

[22] Maarsingh H, Dekkers BG, Zuidhof AB, Bos IS, Menzen MH, Klein T, et al.Increased arginase activity contributes to airway remodelling in chronicallergic asthma. Eur Respir J 2011;38:318e28.

[23] Ma X, Cheng Z, Kong H, Wang Y, Unruh H, Stephens NL, et al. Changes inbiophysical and biochemical properties of single bronchial smooth musclecells from asthmatic subjects. Am J Physiol Lung Cell Mol Physiol 2002;283:L1181e9.

[24] Matsumoto H, Moir LM, Oliver BG, Burgess JK, Roth M, Black JL, et al.Comparison of gel contraction mediated by airway smooth muscle cells frompatients with and without asthma. Thorax 2007;62:848e54.

[25] Stephens NL, Fust A, Jiang H, Li W, Ma X. Isotonic relaxation of control andsensitized airway smooth muscle. Can J Physiol Pharmacol 2005;83:941e51.

[26] Blanc FX, Coirault C, Oliviero P, Lecarpentier Y. Relaxation of tracheal smoothmuscle is impaired in innate airway hyperresponsiveness. Eur Respir J 2009;34:417e24.

[27] Pfitzer G. Invited review: regulation of myosin phosphorylation in smoothmuscle. J Appl Physiol 2001;91:497e503.

[28] Leguillette R, Laviolette M, Bergeron C, Zitouni N, Kogut P, Solway J, et al.Myosin, transgelin, and myosin light chain kinase: expression and functionin asthma. Am J Respir Crit Care Med 2009;179:194e204.

[29] Sun Q, Chen G, Streb JW, Long X, Yang Y, Stoeckert Jr CJ, et al. Defining themammalian CArGome. Genome Res 2006;16:197e207.

[30] Benayoun L, Druilhe A, Dombret MC, Aubier M, Pretolani M. Airway struc-tural alterations selectively associated with severe asthma. Am J Respir CritCare Med 2003;167:1360e8.

[31] Woodruff PG, Dolganov GM, Ferrando RE, Donnelly S, Hays SR, Solberg OD,et al. Hyperplasia of smooth muscle in mild to moderate asthma withoutchanges in cell size or gene expression. Am J Respir Crit Care Med 2004;169:1001e6.

[32] Gil FR, Zitouni NB, Azoulay E, Maghni K, Lauzon AM. Smooth muscle myosinisoform expression and LC20 phosphorylation in innate rat airway hyper-responsiveness. Am J Physiol Lung Cell Mol Physiol 2006;291:L932e40.

D.B. Wright et al. / Pulmonary Pharmacology & Therapeutics 26 (2013) 95e104102

[33] Chiba Y, Sakai H, Suenaga H, Kamata K, Misawa M. Enhanced Ca2þ sensiti-zation of the bronchial smooth muscle contraction in antigen-inducedairway hyperresponsive rats. Res Commun Mol Pathol Pharmacol 1999;106:77e85.

[34] Schaafsma D, Gosens R, Bos IS, Meurs H, Zaagsma J, Nelemans SA. Allergicsensitization enhances the contribution of Rho-kinase to airway smoothmuscle contraction. Br J Pharmacol 2004;143:477e84.

[35] Slats AM, Janssen K, van SA, van der Plas DT, Schot R, van den Aardweg JG,et al. Expression of smooth muscle and extracellular matrix proteins inrelation to airway function in asthma. J Allergy Clin Immunol 2008;121:1196e202.

[36] Scichilone N, Permutt S, Bellia V, Togias A. Inhaled corticosteroids and thebeneficial effect of deep inspiration in asthma. Am J Respir Crit Care Med2005;172:693e9.

[37] Scichilone N, Pyrgos G, Kapsali T, Anderlind C, Brown R, Permutt S, et al.Airways hyperresponsiveness and the effects of lung inflation. Int ArchAllergy Immunol 2001;124:262e6.

[38] Skloot G, Togias A. Bronchodilation and bronchoprotection by deep inspi-ration and their relationship to bronchial hyperresponsiveness. Clin RevAllergy Immunol 2003;24:55e72.

[39] Pyrgos G, Scichilone N, Togias A, Brown RH. Bronchodilation response todeep inspirations in asthma is dependent on airway distensibility and airtrapping. J Appl Physiol 2011;110:472e9.

[40] Huang Y, Zhang W, Gunst SJ. Activation of vinculin induced by cholinergicstimulation regulates contraction of tracheal smooth muscle tissue. J BiolChem 2011;286:3630e44.

[41] Bosse Y, Chin LYM, Pare PD, Seow CY. Adaptation of airway smooth muscle tobasal tone: relevance to airway hyperresponsiveness. Am J Respir Cell MolBiol 2009;40:13e8.

[42] Pascoe C, Jiao Y, Seow CY, Pare PD, Bosse Y. Force oscillations simulatingbreathing maneuvers do not prevent force adaptation. Am J Respir Cell MolBiol 2012;47:44e9.

[43] Seow CY. Myosin filament assembly in an ever-changing myofilament latticeof smooth muscle. Am J Physiol Cell Physiol 2005;289:C1363e8.

[44] Zhang W, Gunst SJ. Interactions of airway smooth muscle cells with theirtissue matrix: implications for contraction. Proc Am Thorac Soc 2008;5:32e9.

[45] Plant PJ, North ML, Ward A, Ward M, Khanna N, Correa J, et al. Hyper-trophic airway smooth muscle mass correlates with increased airwayresponsiveness in a murine model of asthma. Am J Respir Crit Care Med2012;46:532e40.

[46] Ogawa H, Azuma M, Muto S, Nishioka Y, Honjo A, Tezuka T, et al. IkappaBkinase beta inhibitor IMD-0354 suppresses airway remodelling in a derma-tophagoides pteronyssinus-sensitized mouse model of chronic asthma. ClinExp Allergy 2011;41:104e15.

[47] Babu GJ, Loukianov E, Loukianova T, Pyne GJ, Huke S, Osol G, et al. Loss of SM-B myosin affects muscle shortening velocity and maximal force develop-ment. Nat Cell Biol 2001;3:1025.

[48] Babu GJ, Pyne GJ, Zhou Y, Okwuchukuasanya C, Brayden JE, Osol G, et al.Isoform switching from SM-B to SM-A myosin results in decreasedcontractility and altered expression of thin filament regulatory proteins. AmJ Physiol Cell Physiol 2004;287:C723e9.

[49] Martin JG, Duguet A, Eidelman DH. The contribution of airway smoothmuscle to airway narrowing and airway hyperresponsiveness in disease. EurRespir J 2000;16:349e54.

[50] Moreno RH, Hogg JC, Pare PD. Mechanics of airway narrowing. Am RevRespir Dis 1986;133:1171e80.

[51] Lambert RK, Wiggs BR, Kuwano K, Hogg JC, Pare PD. Functional-significanceof increased airway smooth-muscle in asthma and Copd. J Appl Physiol 1993;74:2771e81.

[52] Siddiqui S, Jo T, Tamaoka M, Shalaby KH, Ghezzo H, Bernabeu M, et al. Sites ofallergic airway smooth muscle remodeling and hyperresponsiveness are notassociated in the rat. J Appl Physiol 2010;109:1170e8.

[53] Siddiqui S, Tsuchiya K, Risse PA, Bullimore SR, Benedetti A, Martin JG. Site ofallergic airway narrowing and the influence of exogenous surfactant in theBrown Norway rat. PLoS One 2012;7:e29381.

[54] Siddiqui S, Tsuchiya K, Jo T, Risse PA, MacSharry J, Marchica CL, et al. Theeffects of inhibition of the epidermal growth factor receptor on airwaysmooth muscle phenotype and airway hyperresponsiveness in the ratinduced by repeated allergen challenges. Am J Respir Crit Care Med 2011:A2460.

[55] Bullimore SR, Siddiqui S, Donovan GM, Martin JG, Sneyd J, Bates JHT, et al.Could an increase in airway smooth muscle shortening velocity cause airwayhyperresponsiveness? Am J Physiol-Lung C 2011;300:L121e31.

[56] Hyde DM, Hamid Q, Irvin CG. Anatomy, pathology, and physiology of thetracheobronchial tree: emphasis on the distal airways. J Allergy ClinImmunol 2009;124:S72e7.

[57] Castro M, Rubin A, Laviolette M, Hanania NA, Armstrong B, Cox G. Persistenceof effectiveness of bronchial thermoplasty in patients with severe asthma.Ann Allergy Asthma Immunol 2011;107:65e70.

[58] Wu Q, Xing Y, Zhou X, Wang D. Meta-analysis of the efficacy and safety ofbronchial thermoplasty in patients with moderate-to-severe persistentasthma. J Int Med Res 2011;39:10e22.

[59] Bush A, Saglani S. Management of severe asthma in children. Lancet 2010;376:814e25.

[60] James AL, Bai TR, Mauad T, Abramson MJ, Dolhnikoff M, McKay KO, et al.Airway smooth muscle thickness in asthma is related to severity but notduration of asthma. Eur Respir J 2009;34:1040e5.

[61] Bentley JK, Hershenson MB. Airway smooth muscle growth in asthma:proliferation, hypertrophy, and migration. Proc Am Thorac Soc 2008;5:89e96.

[62] Kim ES, Kim SH, Kim KW, Park JW, Kim YS, Sohn MH, et al. Basementmembrane thickening and clinical features of children with asthma. Allergy2007;62:635e40.

[63] Saglani S, Molyneux C, Gong H, Rogers A, Malmstrom K, Pelkonen A, et al.Ultrastructure of the reticular basement membrane in asthmatic adults,children and infants. Eur Respir J 2006;28:505e12.

[64] Jenkins HA, Cool C, Szefler SJ, Covar R, Brugman S, Gelfand EW, et al.Histopathology of severe childhood asthma: a case series. Chest 2003;124:32e41.

[65] Tillie-Leblond I, de Blic J, Jaubert F, Wallaert B, Scheinmann P, Gosset P.Airway remodeling is correlated with obstruction in children with severeasthma. Allergy 2008;63:533e41.

[66] Bossley CJ, Fleming L, Gupta A, Regamey N, Frith J, Oates T, et al. Pediatricsevere asthma is characterized by eosinophilia and remodeling without T(H)2 cytokines. J Allergy Clin Immunol 2012;129:974e82. e13.

[67] Tiddens HA, Hofhuis W, Casotti V, Hop WC, Hulsmann AR, de Jongste JC.Airway dimensions in bronchopulmonary dysplasia: implications for airflowobstruction. Pediatr Pulmonol 2008;43:1206e13.

[68] Regamey N, Ochs M, Hilliard TN, Muhlfeld C, Cornish N, Fleming L, et al.Increased airway smooth muscle mass in children with asthma, cysticfibrosis, and non-cystic fibrosis bronchiectasis. Am J Respir Crit Care Med2008;177:837e43.

[69] Johnson PR, Roth M, Tamm M, Hughes M, Ge Q, King G, et al. Airway smoothmuscle cell proliferation is increased in asthma. Am J Respir Crit Care Med2001;164:474e7.

[70] Labonte I, Hassan M, Risse PA, Tsuchiya K, Laviolette M, Lauzon AM, et al. Theeffects of repeated allergen challenge on airway smooth muscle structuraland molecular remodeling in a rat model of allergic asthma. Am J Physiol-Lung C 2009;297:L698e705.

[71] Moir LM, Leung SY, Eynott PR, McVicker CG, Ward JP, Chung KF, et al.Repeated allergen inhalation induces phenotypic modulation of smoothmuscle in bronchioles of sensitized rats. Am J Physiol Lung Cell Mol Physiol2003;284:L148e59.

[72] Tamaoka M, Hassan M, McGovern T, Ramos-Barbon D, Jo T, Yoshizawa Y,et al. The epidermal growth factor receptor mediates allergic airwayremodelling in the rat. Eur Respir J 2008;32:1213e23.

[73] Ramos-Barbon D, Fraga-Iriso R, Brienza NS, Montero-Martinez C, Verea-Hernando H, Olivenstein R, et al. T cells localize with proliferating smoothmuscle alpha-actin(þ) cell compartments in asthma. Am J Resp Crit Care2010;182:317e24.

[74] Low RB, White SL. Lung smooth muscle differentiation. Int J Biochem Cell Biol1998;30:869e83.

[75] Jostarndt-Fogen K, Djonov V, Draeger A. Expression of smooth musclemarkers in the developing murine lung: potential contractile properties andlineal descent. Histochem Cell Biol 1998;110:273e84.

[76] Woodcock-Mitchell J, White S, Stirewalt W, Periasamy M, Mitchell J, Low RB.Myosin isoform expression in developing and remodeling rat lung. Am JRespir Cell Mol Biol 1993;8:617e25.

[77] Low RB, Mitchell J, Woodcock-Mitchell J, Rovner AS, White SL. Smooth-muscle myosin heavy-chain SM-B isoform expression in developing andadult rat lung. Am J Respir Cell Mol Biol 1999;20:651e7.

[78] Rensen SS, Thijssen VL, De Vries CJ, Doevendans PA, Detera-Wadleigh SD,Van Eys GJ. Expression of the smoothelin gene is mediated by alternativepromoters. Cardiovasc Res 2002;55:850e63.

[79] Morrisey EE, Ip HS, Tang Z, Lu MM, Parmacek MS. GATA-5: a transcriptionalactivator expressed in a novel temporally and spatially-restricted patternduring embryonic development. Dev Biol 1997;183:21e36.

[80] Kalinichenko VV, Lim L, Shin B, Costa RH. Differential expression of forkheadbox transcription factors following butylated hydroxytoluene lung injury.Am J Physiol Lung Cell Mol Physiol 2001;280:L695e704.

[81] Chetta A, Zanini A, Torre O, Olivieri D. Vascular remodelling and angiogenesisin asthma: morphological aspects and pharmacological modulation. InflammAllergy Drug Targets 2007;6:41e5.

[82] Hashimoto M, Tanaka H, Abe S. Quantitative analysis of bronchial wallvascularity in the medium and small airways of patients with asthma andCOPD. Chest 2005;127:965e72.

[83] Barbato A, Turato G, Baraldo S, Bazzan E, Calabrese F, Panizzolo C, et al.Epithelial damage and angiogenesis in the airways of children with asthma.Am J Respir Crit Care Med 2006;174:975e81.

[84] Zanini A, Chetta A, Imperatori AS, Spanevello A, Olivieri D. The role of thebronchial microvasculature in the airway remodelling in asthma and COPD.Respir Res 2010;11:132.

[85] Simcock DE, Kanabar V, Clarke GW, Mahn K, Karner C, O’Connor BJ, et al.Induction of angiogenesis by airway smooth muscle from patients withasthma. Am J Respir Crit Care Med 2008;178:460e8.

[86] Fehrenbach H, Kasper M, Haase M, Schuh D, Muller M. Differential immu-nolocalization of VEGF in rat and human adult lung, and in experimental ratlung fibrosis: light, fluorescence, and electron microscopy. Anat Rec 1999;254:61e73.

D.B. Wright et al. / Pulmonary Pharmacology & Therapeutics 26 (2013) 95e104 103

[87] Shin JH, Shim JW, Kim DS, Shim JY. TGF-beta effects on airway smoothmuscle cell proliferation, VEGF release and signal transduction pathways.Respirology 2009;14:347e53.

[88] Rydell-Tormanen K, Uller L, Erjefalt JS. Allergic airway inflammation initiateslong-term vascular remodeling of the pulmonary circulation. Int Arch AllergyImmunol 2009;149:251e8.

[89] Green FH, Butt JC, James AL, Carroll NG. Abnormalities of the bronchialarteries in asthma. Chest 2006;130:1025e33.

[90] Tormanen KR, Uller L, Persson CG, Erjefalt JS. Allergen exposure of mouseairways evokes remodeling of both bronchi and large pulmonary vessels. AmJ Respir Crit Care Med 2005;171:19e25.

[91] Daley E, Emson C, Guignabert C, de Waal Malefyt R, Louten J, Kurup VP, et al.Pulmonary arterial remodeling induced by a Th2 immune response. J ExpMed 2008;205:361e72.

[92] Rydell-Tormanen K, Johnson JR, Fattouh R, Jordana M, Erjefalt JS. Induction ofvascular remodeling in the lung by chronic house dust mite exposure. Am JRespir Cell Mol Biol 2008;39:61e7.

[93] Rydell-Tormanen K, Uller L, Erjefalt JS. Remodeling of extra-bronchial lungvasculature following allergic airway inflammation. Respir Res 2008;9:18.

[94] Ebina M, Takahashi T, Chiba T, Motomiya M. Cellular hypertrophy andhyperplasia of airway smooth muscles underlying bronchial asthma. A 3-Dmorphometric study. Am Rev Respir Dis 1993;148:720e6.

[95] Heard BE, Hossain S. Hyperplasia of bronchial muscle in asthma. J Pathol1973;110:319e31.

[96] Panettieri JA, Murray RK, Eszterhas AJ, Bilgen G, Martin JG. Repeated allergeninhalations induce DNA synthesis in airway smooth muscle and epithelialcells in vivo. Am J Physiol-Lung C 1998;274:L417e24.

[97] Goldsmith AM, Bentley JK, Zhou LM, Jia Y, Bitar KN, Fingar DC, et al. Trans-forming growth factor-beta induces airway smooth muscle hypertrophy. AmJ Respir Cell Mol Biol 2006;34:247e54.

[98] Schmidt M, Sun G, Stacey MA, Mori L, Mattoli S. Identification of circulatingfibrocytes as precursors of bronchial myofibroblasts in asthma. J Immunol2003;171:380e9.

[99] Summer R, Fitzsimmons K, Dwyer D, Murphy J, Fine A. Isolation of an adultmouse lung mesenchymal progenitor cell population. Am J Respir Cell MolBiol 2007;37:152e9.

[100] Yang YW, Haitchi HM, Cakebread J, Sammut D, Harvey A, Powell RM, et al.Epigenetic mechanisms silence a disintegrin and metalloprotease 33expression in bronchial epithelial cells. J Allergy Clin Immun 2008;121:1393e9.

[101] Ramos-Barbon D, Presley JF, Hamid QA, Fixman ED, Martin JG. Antigen-specific CD4þ T cells drive airway smooth muscle remodeling in experi-mental asthma. J Clin Investig 2005;115:1580e9.

[102] Trian T, Benard G, Begueret H, Rossignol R, Girodet PO, Ghosh D, et al.Bronchial smooth muscle remodeling involves calcium-dependent enhancedmitochondrial biogenesis in asthma. J Exp Med 2007;204:3173e81.

[103] Araujo BB, Dolhnikoff M, Silva LF, Elliot J, Lindeman JH, Ferreira DS, et al.Extracellular matrix components and regulators in the airway smoothmuscle in asthma. Eur Respir J 2008;32:61e9.

[104] Dekkers BG, Maarsingh H, Meurs H, Gosens R. Airway structural componentsdrive airway smooth muscle remodeling in asthma. Proc Am Thorac Soc2009;6:683e92.

[105] Kaminska M, Foley S, Maghni K, Storness-Bliss C, Coxson H, Ghezzo H, et al.Airway remodeling in subjects with severe asthma with or without chronicpersistent airflow obstruction. J Allergy Clin Immunol 2009;124:45e51.e1e4.

[106] Roth M, Johnson PR, Borger P, Bihl MP, Rudiger JJ, King GG, et al. Dysfunc-tional interaction of C/EBPalpha and the glucocorticoid receptor in asthmaticbronchial smooth-muscle cells. N Engl J Med 2004;351:560e74.

[107] Dekkers BG, Pehlic A, Mariani R, Bos IS, Meurs H, Zaagsma J. Glucocorticos-teroids and beta2-adrenoceptor agonists synergize to inhibit airway smoothmuscle remodelling. J Pharmacol Exp Ther 2012.

[108] Bonacci JV, Schuliga M, Harris T, Stewart AG. Collagen impairs glucocorticoidactions in airway smooth muscle through integrin signalling. Br J Pharmacol2006;149:365e73.

[109] Johnson PR, Burgess JK, Underwood PA, Au W, Poniris MH, Tamm M, et al.Extracellular matrix proteins modulate asthmatic airway smooth muscle cellproliferation via an autocrine mechanism. J Allergy Clin Immunol 2004;113:690e6.

[110] Trian T, Burgess JK, Niimi K, Moir LM, Ge Q, Berger P, et al. Beta2-agonistinduced cAMP is decreased in asthmatic airway smooth muscle due toincreased PDE4D. PLoS One 2011;6:e20000.

[111] Mahn K, Hirst SJ, Ying S, Holt MR, Lavender P, Ojo OO, et al. Diminishedsarco/endoplasmic reticulum Ca2þ ATPase (SERCA) expression contributesto airway remodelling in bronchial asthma. Proc Natl Acad Sci U S A 2009;106:10775e80.

[112] Chan V, Burgess JK, Ratoff JC, O’Connor BJ, Greenough A, Lee TH, et al.Extracellular matrix regulates enhanced eotaxin expression in asthmaticairway smooth muscle cells. Am J Respir Crit Care Med 2006;174:379e85.

[113] Chambers LS, Black JL, Ge Q, Carlin SM, Au WW, Poniris M, et al. PAR-2activation, PGE2, and COX-2 in human asthmatic and nonasthmatic airwaysmooth muscle cells. Am J Physiol Lung Cell Mol Physiol 2003;285:L619e27.

[114] Hirst SJ, Barnes PJ, Twort CH. PDGF isoform-induced proliferation andreceptor expression in human cultured airway smooth muscle cells. Am JPhysiol 1996;270:L415e28.

[115] Ohno I, Nitta Y, Yamauchi K, Hoshi H, Honma M, Woolley K, et al. Eosinophilsas a potential source of platelet-derived growth factor B-chain (PDGF-B) innasal polyposis and bronchial asthma. Am J Respir Cell Mol Biol 1995;13:639e47.

[116] Burgess JK, Ceresa C, Johnson SR, Kanabar V, Moir LM, Nguyen TT, et al.Tissue and matrix influences on airway smooth muscle function. PulmPharmacol Ther 2009;22:379e87.

[117] Hawker KM, Johnson PR, Hughes JM, Black JL. Interleukin-4 inhibits mitogen-induced proliferation of human airway smooth muscle cells in culture. Am JPhysiol 1998;275:L469e77.

[118] Boxall C, Holgate ST, Davies DE. The contribution of transforming growthfactor-beta and epidermal growth factor signalling to airway remodelling inchronic asthma. Eur Respir J 2006;27:208e29.

[119] Shute JK, Solic N, Shimizu J, McConnell W, Redington AE, Howarth PH.Epithelial expression and release of FGF-2 from heparan sulphate bindingsites in bronchial tissue in asthma. Thorax 2004;59:557e62.

[120] Stewart AG, Tomlinson PR, Fernandes DJ, Wilson JW, Harris T. Tumornecrosis factor alpha modulates mitogenic responses of human culturedairway smooth muscle. Am J Respir Cell Mol Biol 1995;12:110e9.

[121] Babu SK, Puddicombe SM, Arshad HH, Wilson SJ, Ward J, Gozzard N, et al.Tumor necrosis factor alpha (TNF-alpha) autoregulates its expression andinduces adhesion molecule expression in asthma. Clin Immunol 2011;140:18e25.

[122] Corren J. Cytokine inhibition in severe asthma: current knowledge andfuture directions. Curr Opin Pulm Med 2011;17:29e33.

[123] Xie S, Sukkar MB, Issa R, Khorasani NM, Chung KF. Mechanisms of inductionof airway smooth muscle hyperplasia by transforming growth factor-beta.Am J Physiol Lung Cell Mol Physiol 2007;293:L245e53.

[124] Cohen MD, Ciocca V, Panettieri Jr RA. TGF-beta 1 modulates human airwaysmooth-muscle cell proliferation induced by mitogens. Am J Respir Cell MolBiol 1997;16:85e90.

[125] Minshall EM, Leung DY, Martin RJ, Song YL, Cameron L, Ernst P, et al.Eosinophil-associated TGF-beta1 mRNA expression and airways fibrosis inbronchial asthma. Am J Respir Cell Mol Biol 1997;17:326e33.

[126] Magnan A, Frachon I, Rain B, Peuchmaur M, Monti G, Lenot B, et al. Trans-forming growth factor beta in normal human lung: preferential location inbronchial epithelial cells. Thorax 1994;49:789e92.

[127] Maruno K, Absood A, Said SI. VIP inhibits basal and histamine-stimulatedproliferation of human airway smooth muscle cells. Am J Physiol 1995;268:L1047e51.

[128] Kistemaker LE, Oenema TA, Meurs H, Gosens R. Regulation of airwayinflammation and remodeling by muscarinic receptors: perspectives onanticholinergic therapy in asthma and COPD. Life Sci 2012.

[129] Gosens R, Dueck G, Rector E, Nunes RO, Gerthoffer WT, Unruh H, et al.Cooperative regulation of GSK-3 by muscarinic and PDGF receptors isassociated with airway myocyte proliferation. Am J Physiol Lung Cell MolPhysiol 2007;293:L1348e58.

[130] Tomlinson PR, Wilson JW, Stewart AG. Inhibition by salbutamol of theproliferation of human airway smooth muscle cells grown in culture. Br JPharmacol 1994;111:641e7.

[131] Giaid A, Polak JM, Gaitonde V, Hamid QA, Moscoso G, Legon S, et al. Distri-bution of endothelin-like immunoreactivity and mRNA in the developingand adult human lung. Am J Respir Cell Mol Biol 1991;4:50e8.

[132] Capra V, Habib A, Accomazzo MR, Ravasi S, Citro S, Levy-Toledano S, et al.Thromboxane prostanoid receptor in human airway smooth muscle cells:a relevant role in proliferation. Eur J Pharmacol 2003;474:149e59.

[133] Ravasi S, Citro S, Viviani B, Capra V, Rovati GE. CysLT1 receptor-induced humanairway smooth muscle cells proliferation requires ROS generation, EGFreceptor transactivation and ERK1/2 phosphorylation. Respir Res 2006;7:42.

[134] Berger P, Perng DW, Thabrew H, Compton SJ, Cairns JA, McEuen AR, et al.Tryptase and agonists of PAR-2 induce the proliferation of human airwaysmooth muscle cells. J Appl Physiol 2001;91:1372e9.

[135] Huang CD, Chen HH, Wang CH, Chou CL, Lin SM, Lin HC, et al. Humanneutrophil-derived elastase induces airway smooth muscle cell proliferation.Life Sci 2004;74:2479e92.

[136] Pandya HC, Snetkov VA, Twort CH, Ward JP, Hirst SJ. Oxygen regulatesmitogen-stimulated proliferation of fetal human airway smooth muscle cells.Am J Physiol Lung Cell Mol Physiol 2002;283:L1220e30.

[137] Gosens R, Roscioni SS, Dekkers BG, Pera T, Schmidt M, Schaafsma D, et al.Pharmacology of airway smooth muscle proliferation. Eur J Pharmacol 2008;585:385e97.

[138] Hirst SJ, Twort CH, Lee TH. Differential effects of extracellular matrix proteinson human airway smooth muscle cell proliferation and phenotype. Am J ResCell Mol Biol 2000;23:335e44.

[139] Dekkers BG, Schaafsma D, Nelemans SA, Zaagsma J, Meurs H. Extracellularmatrix proteins differentially regulate airway smooth muscle phenotype andfunction. Am J Physiol Lung Cell Mol Physiol 2007;292:L1405e13.

[140] Dekkers BG, Bos IS, Zaagsma J, Meurs H. Functional consequences of humanairway smoothmuscle phenotype plasticity. Br J Pharmacol 2012;166:359e67.

[141] Bonacci JV, Harris T, Stewart AG. Impact of extracellular matrix and strain onproliferation of bovine airway smooth muscle. Clin Exp Pharmacol Physiol2003;30:324e8.

[142] Freyer AM, Johnson SR, Hall IP. Effects of growth factors and extracellularmatrix on survival of human airway smooth muscle cells. Am J Respir CellMol Biol 2001;25:569e76.

D.B. Wright et al. / Pulmonary Pharmacology & Therapeutics 26 (2013) 95e104104

[143] Parameswaran K, Radford K, Zuo J, Janssen LJ, O’Byrne PM, Cox PG. Extra-cellular matrix regulates human airway smooth muscle cell migration. EurRespir J 2004;24:545e51.

[144] Garl PJ, Wenzlau JM, Walker HA, Whitelock JM, Costell M, Weiser-Evans MC.Perlecan-induced suppression of smooth muscle cell proliferation is medi-ated through increased activity of the tumor suppressor PTEN. Circ Res 2004;94:175e83.

[145] Burgess JK, Johnson PR, Ge Q, Au WW, Poniris MH, McParland BE, et al.Expression of connective tissue growth factor in asthmatic airway smoothmuscle cells. Am J Respir Crit Care Med 2003;167:71e7.

[146] Johnson PR, Burgess JK, Ge Q, Poniris M, Boustany S, Twigg SM, et al.Connective tissue growth factor induces extracellular matrix in asthmaticairway smooth muscle. Am J Respir Crit Care Med 2006;173:32e41.

[147] Joubert P, Lajoie-Kadoch S, Labonte I, Gounni AS, Maghni K, Wellemans V,et al. CCR3 expression and function in asthmatic airway smooth muscle cells.J Immunol 2005;175:2702e8.

[148] Brightling CE, Ammit AJ, Kaur D, Black JL, Wardlaw AJ, Hughes JM, et al. TheCXCL10/CXCR3 axis mediates human lung mast cell migration to asthmaticairway smooth muscle. Am J Respir Crit Care Med 2005;171:1103e8.

[149] Klagas I, Goulet S, Karakiulakis G, Zhong J, Baraket M, Black JL, et al.Decreased hyaluronan in airway smooth muscle cells from patients withasthma and COPD. Eur Respir J 2009;34:616e28.

[150] Svensson Holm AC, Bengtsson T, Grenegard M, Lindstrom EG. Hyaluronicacid influence on platelet-induced airway smooth muscle cell proliferation.Exp Cell Res 2012;318:632e40.

[151] Kanabar V, Hirst SJ, O’Connor BJ, Page CP. Some structural determinants ofthe antiproliferative effect of heparin-like molecules on human airwaysmooth muscle. Br J Pharmacol 2005;146:370e7.

[152] Burgess JK, Ge Q, Poniris MH, Boustany S, Twigg SM, Black JL, et al.Connective tissue growth factor and vascular endothelial growth factor fromairway smooth muscle interact with the extracellular matrix. Am J PhysiolLung Cell Mol Physiol 2006;290:L153e61.

[153] Roscioni SS, Dekkers BG, Prins AG, Menzen MH, Meurs H, Schmidt M, et al.cAMP inhibits modulation of airway smooth muscle phenotype via theexchange protein activated by cAMP (Epac) and protein kinase A. Br JPharmacol 2011;162:193e209.

[154] Yan H, Deshpande DA, Misior AM, Miles MC, Saxena H, Riemer EC, et al. Anti-mitogenic effects of {beta}-agonists and PGE2 on airway smooth muscle arePKA dependent. FASEB J 2011;25:389e97.

[155] El-Shazly A, Berger P, Girodet PO, Ousova O, Fayon M, Vernejoux JM, et al.Fraktalkine produced by airway smooth muscle cells contributes to mast cellrecruitment in asthma. J Immunol 2006;176:1860e8.

[156] Begueret H, Berger P, Vernejoux JM, Dubuisson L, Marthan R, Tunon-de-Lara JM. Inflammation of bronchial smooth muscle in allergic asthma. Thorax2007;62:8e15.

[157] Berger P, Compton SJ, Molimard M, Walls AF, N’Guyen C, Marthan R, et al.Mast cell tryptase as a mediator of hyperresponsiveness in human isolatedbronchi. Clin Exp Allergy 1999;29:804e12.

[158] Lazaar AL, Albelda SM, Pilewski JM, Brennan B, Pure E, Panettieri Jr RA.T lymphocytes adhere to airway smooth muscle cells via integrins and CD44and induce smooth muscle cell DNA synthesis. J Exp Med 1994;180:807e16.

[159] Svensson Holm AC, Bengtsson T, Grenegard M, Lindstrom EG. Plateletmembranes induce airway smooth muscle cell proliferation. Platelets 2011;22:43e53.

[160] Svensson Holm AC, Bengtsson T, Grenegard M, Lindstrom EG. Plateletsstimulate airway smooth muscle cell proliferation through mechanismsinvolving 5-lipoxygenase and reactive oxygen species. Platelets 2008;19:528e36.

[161] Kanabar V, Upton N, Page CP, Pitchford SC. Platelet and airway smoothmuscle interactions in asthma. Am J Respir Crit Care Med 2011;183:A4056.

[162] Pitchford SC, Momi S, Baglioni S, Casali L, Giannini S, Rossi R, et al. Allergeninduces the migration of platelets to lung tissue in allergic asthma. Am JRespir Crit Care Med 2008;177:604e12.

[163] Pitchford SC, Riffo-Vasquez Y, Sousa A, Momi S, Gresele P, Spina D, et al.Platelets are necessary for airway wall remodeling in a murine model ofchronic allergic inflammation. Blood 2004;103:639e47.

[164] Fernandes DJ, Bonacci JV, Stewart AG. Extracellular matrix, integrins, andmesenchymal cell function in the airways. Curr Drug Targets 2006;7:567e77.

[165] Moir LM, Burgess JK, Black JL. Transforming growth factor beta 1 increasesfibronectin deposition through integrin receptor alpha 5 beta 1 on humanairway smooth muscle. J Allergy Clin Immunol 2008;121:1034e9. e4.

[166] Manabe R, Tsutsui K, Yamada T, Kimura M, Nakano I, Shimono C, et al.Transcriptome-based systematic identification of extracellular matrixproteins. Proc Natl Acad Sci U S A 2008;105:12849e54.

[167] Bossé Y, Paré PD, Boss Y. Airway smooth muscle in asthma symptoms:culprit but maybe innocent. In: Irusen Elvis Malcolm, editor. Lung diseases eselected state of the art reviews. InTech, ISBN 978-953-51-0180-2; 2012.