Phenotype modulation of airway smooth muscle in asthma

Pulmonary Pharmacology & Therapeutics 26 (2013) 42e49

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Phenotype modulation of airway smooth muscle in asthma David B. Wright a, Thomas Trian b, Sana Siddiqui c, d, Chris D. Pascoe e, Jill R. Johnson f, Bart G.J. Dekkers g, Shyamala Dakshinamurti h, Rushita Bagchi i, Janette K. Burgess j, Varsha Kanabar k, Oluwaseun O. Ojo l, * a

Medical Research Council and Asthma UK Centre in Allergic Mechanisms of Asthma, King’s College London, London, United Kingdom Centre de Recherche Cardio-thoracique de Bordeaux, INSERM, U1045, Equipe: Remodelage bronchique, Université Bordeaux2, Bordeaux Cedex, France c Meakins-Christie Laboratories, Department of Medicine, McGill University, Montréal, Québec, Canada d Receptor Pharmacology Unit, National Institute on Aging, National Institutes of Health, Baltimore, MD, United States of America e University of British Columbia, Department of Medicine, Canada f Leukocyte Biology Section, National Heart and Lung Institute, Imperial College London, London, United Kingdom g Department of Molecular Pharmacology, Groningen Research Institute for Asthma and COPD, University of Groningen, Groningen, The Netherlands h Section of Neonatology, WS012 Women’s Hospital, Winnipeg, University of Manitoba, Manitoba, Canada i Department of Physiology, University of Manitoba, Institute of Cardiovascular Sciences, St. Boniface Hospital Research Centre, Winnipeg, Manitoba, Canada j Cell Biology Group Woolcock Institute of Medical Research, The University of Sydney and Discipline of Pharmacology, The University of Sydney, Australia k Sackler Institute of Pulmonary Pharmacology, King’s College London, London, United Kingdom l Department of Respiratory Physiology, University of Manitoba, Biology of Breathing Group, Manitoba Institute of Child Health, Winnipeg, Manitoba, Canada b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 18 July 2012 Received in revised form 11 August 2012 Accepted 13 August 2012

The biological responses of airway smooth muscle (ASM) are diverse, in part due to ASM phenotype plasticity. ASM phenotype plasticity refers to the ability of ASM cells to change the degree of a variety of functions, including contractility, proliferation, migration and secretion of inflammatory mediators. This plasticity occurs due to intrinsic or acquired abnormalities in ASM cells, and these abnormalities or predisposition of the ASM cell may alter the ASM response and in some cases recapitulate disease hallmarks of asthma. These phenotypic changes are ultimately determined by multiple stimuli and occur due to alterations in the intricate balance or reversible state that maintains ASM cells in either a contractile or synthetic state, through processes termed maturation or modulation, respectively. To elucidate the role of ASM phenotype in disease states, numerous in vitro studies have suggested a phenotypic switch in ASM primary cell cultures as an explanation for the plethora of responses mediated by ASM cells. Moreover, there is overwhelming evidence suggesting that the immunomodulatory response of ASM is due to the acquisition of a synthetic phenotype; however, whether this degree of plasticity is present in vivo as opposed to cell culture-based models remains speculative. Nonetheless, this review will give an overall scope of ASM phenotypic markers, triggers of ASM phenotype modulation and novel therapeutic approaches to control ASM phenotype plasticity. Ó 2012 Elsevier Ltd. All rights reserved.

Keywords: Airway smooth muscle Asthma Phenotypic plasticity Contractile response Synthetic response

Airway smooth muscle (ASM) responses play an important role in asthma, which is evident in the key hallmarks of asthma; for example, ASM contraction gives rise to bronchoconstriction, ASM proliferation contributes towards increased ASM mass during airway remodelling and the secretion of pro-inflammatory mediators by ASM perpetuates airway inflammation. Thus, ASM

phenotype plasticity can dictate or drive toward key hallmarks of asthma pathogenesis. This review will attempt to provide an indepth overview of our current understanding of phenotype modulation in ASM, with specific focus on the key markers of ASM phenotypes including triggers that may be responsible for driving or switching towards a certain phenotype. Finally, we will postulate avenues for the development of novel therapies that aim to prevent or revert ASM modulation towards an asthma phenotype.

* Corresponding author. Department of Respiratory Physiology, University of Manitoba, Biology of Breathing Group, Manitoba Institute of Child Health, Room 641, John Buhler Research Centre, 715 McDermott Avenue, Winnipeg, Manitoba R3E 3P4, Canada. Tel.: þ1 204 789 3778. E-mail address: [email protected] (O.O. Ojo).

1. ASM phenotype switching

1094-5539/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.pupt.2012.08.005

The behaviour of ASM cells in culture varies greatly depending on both intrinsic factors and exposure to extrinsic factors. These

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phenotypic characteristics are highly dynamic and cells may undergo “modulation” from a contractile phenotype to a more synthetic, proliferative one, which may be reversed by “maturation”, returning the phenotype back to a state that expresses an abundance of contractile markers [1,2]. This process of switching between phenotypes is also referred to as phenotypic plasticity. Studies by Halayko et al. showed that ASM phenotype plasticity occurs in ASM cells by demonstrating that ASM cells in culture grown to sub-confluency in the presence of serum developed a proliferative ASM phenotype characterised by decreased expression of contractile proteins including smooth muscle-myosin heavy chain (sm-MHC), calponin, smooth muscle a actin (sm-aactin), desmin, myosin light chain kinase (MLCK) and caldesmon [3]. In addition, other groups have shown that ASM cells can switch from one phenotype to another in response to many different stimuli including extracellular matrix (ECM) proteins, platelet derived growth factor (PDGF) and transforming growth factor (TGF) b [4,5]. In support of ASM phenotypic plasticity towards a synthetic phenotype, ASM cells derived from non-asthmatic donors that are relatively less proliferative (to a mitogen stimulus) compared to ASM derived from asthmatic donors [6] can be altered towards a more proliferative phenotype [7]. Studies by Mahn et al. show that diminished sarcoendoplasmic reticulum calcium ATPase in ASM cells derived from healthy donors can recapitulate a synthetic phenotype that mediates increased ASM proliferation and increased eotaxin-1 secretion [8]. In this study, diminishing expression of sarcoendoplasmic reticulum calcium ATPase (a calcium transporter) by approximately 70% using siRNA followed by exposure to IL-13 and PDGF-BB was sufficient to make these ASM cells recapitulate a more secretory and proliferative phenotype respectively. Similar studies have also shown that ECM proteins, in particular collagen type I and fibronectin, can alter nonasthmatic derived ASM cells towards a proliferative phenotype [4]. Several factors affect the state of modulation and maturation, including serum concentration and confluency during cell culture [9,10]. To differentiate between these ASM phenotypes, it is important to know what unique markers are present in each state. However, it is unclear whether there is any distinction between populations of ASM which have the capacity to either switch between distinct functional phenotypes (contractile, proliferative, secretory, migratory) or have the ability to perform a combination of functions whilst in any given phenotype. Interestingly, Sukkar et al. showed that an overlapping population of ASM cells does exist [11]. They observed a population of ASM cells both releasing cytokines and undergoing proliferation, including a separate population they identified as nonproliferating ASM cells that secreted cytokines. This observation of ASM responses clearly depicts the importance of ASM phenotypic plasticity in asthma [11]. 2. ASM contractile phenotype and markers Smooth muscle cells in the contractile phenotype are characterised by a high density of contractile proteins and retain their ability to contract in response to spasmogens. In ASM (see Fig. 1) these contractile phenotype markers include sm-a-actin, smooth muscle g-actin, smooth muscle myosin heavy chain (sm-MHC), calponin, h-caldesmon [12], transgelin (SM22), smoothelin and metavinculin [3], including endogenous laminin [13] and lamininbinding integrin a7 [14] which are important for ASM maturation. Caveolin-1 is another potential marker that is markedly increased in TGFb induced ASM maturation [15]. Additionally, Sharma et al. have identified dystrophineglycoprotein complex as a marker of ASM phenotype maturation [16]. Ultra-structural markers such as high cytoplasmic volume fraction of contractile

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Fig. 1. Proteins expressed by ASM in the contractile or synthetic state.

apparatus and few biosynthetic intracellular organelles that result in mitotic quiescence are also present in the contractile phenotype. 3. ASM synthetic phenotype and markers Modulation towards a synthetic phenotype is characterised by an increase in synthetic organelles for protein and lipid synthesis, such as the Golgi apparatus and numerous mitochondria. These cells have an increased proliferative capacity, become mitotically active and exhibit a diminished abundance of contractile apparatus-associated proteins with a concomitant attenuation of responsiveness to contractile agonists [17e20]. In a population of ASM cells in culture, 20e60% of the cells had a secretory phenotype whilst 50% of the total population of cells demonstrated proliferative capacity [11], suggesting that cytokine production and proliferation are not functions of distinct cell populations but overlap. Halayko et al. showed that non-muscle MHC, l-caldesmon, vimentin, a/beprotein kinase C and CD44 homing cellular adhesion molecule all increase by one- to six-fold as cells become proliferative, thus making them putative markers for the synthetic phenotype [3]. Neither the contractile nor the synthetic states appear to be mutually exclusive; therefore, intermediary and extreme phenotypes of each state are likely to exist. Interestingly, prolonged serum starvation in canine ASM cells has revealed a third putative ASM phenotype that is hyper-contractile [10,21], however, this has not been replicated in human ASM cells to date. The hyper-contractile markers include a lack of smooth muscle myosin-B (SM-B; an MHC isoform linked to cycling velocity) and a 30-fold increase in MLCK expression, possibly to mitigate this loss. Another classical contractile marker of this “hyper-contractile” phenotype is the muscarinic M3 receptor [10,22]. In human ASM cells prolonged serum starvation increases M3 receptor cell surface expression in non-asthmatic but not asthmatic derived cells [23]. 4. Ancillary changes during phenotype switching The plasticity of human ASM cells from a contractile to proliferative phenotype is associated with changes in ion channel expression that may be functionally significant [24]. Specifically, this transition is associated with the expression of voltage-gated sodium and inwardly rectifying potassium channels, and a significant change in the relative proportions of three types of large conductance potassium channels [24]. On the other hand, the transition of cultured arterial myocytes from a contractile to

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a proliferative phenotype is associated with modulations in calcium handling regulators: sarcoplasmic reticular calcium stores, increased store-operated calcium entry (SOCE), receptor-operated calcium entry (ROCE), as well as an increased expression of transient receptor potential channel type C (TRPC), Orai and stromal interacting molecule 1 (STIM1) proteins [25]. 5. In vitro ASM phenotype modulation-cell culture artefact or recapitulation of in vivo conditions? In vitro cell based models provide a relatively controlled environment that allows scientists to assess long-term control of cellular responses (reviewed in [26]). In vitro studies indicate that ASM expresses contractile markers immediately after isolation from ASM bundles, but this rapidly changes to recapitulate a noncontractile phenotype (acquiring synthetic/proliferative properties) when exposed to serum-rich medium [3]. For example, if canine ASM cells are serum-deprived for prolonged periods (up to 19 days), they can acquire “hypercontractile” properties by re-expressing contractile protein markers such as sm-a-actin [10]. Serum deprivation of human ASM cells for an extended period of time (14 days) does not alter the size or complexity of either nonasthmatic or asthmatic cells [23]. The expression of contractile protein markers has not been examined in serum-deprived human ASM cells to date, however these cells have a reduced synthetic capacity. When addressing the question whether phenotypic modulation is an artefact of culture conditions or whether it does indeed occur in vivo, there are a number of points to consider. Firstly, the in vitro cell culture model is a relatively isolated system, while in vivo, there are an overwhelming number of simultaneous networks of signalling pathways occurring in parallel which may impact the ASM state. Many scientists are now recognising the limitations of the 2-D single cell type culture system and advances are being made to increase the physiological relevance of these models [27]. An important point to consider is that the phenotype of smooth muscle is highly influenced by the surrounding milieu. Thus, factors including, but not limited to, the density at which the cells are plated [28], the ECM on which the cells are grown [4,5,7,29e34] and the presence of a homologous cell substrate [35], may differentially affect the ASM phenotype. Another factor that influences cell behaviour and phenotype is mechanical strain or stretch [36,37]. For cell culture conditions, cell stretching experiments require the use of uniform uniaxial or equibiaxial stretch plates to replicate the non-uniform stretch distributions in vivo. Depending on the modality of the stretching technique, the ASM phenotype can be skewed towards a pro-contractile phenotype [36] or synthetic phenotype that releases CXCL8 (interleukin (IL)-8) [38], among other factors. 6. Regulation of phenotype switching Smooth muscle phenotype switching is a dynamic process influenced by both profound and subtle changes in the microenvironment of the cell. Many factors increase ASM cell proliferation in vitro, including peptide growth factors, Gq/i protein coupled receptor agonists, inflammatory mediators and ECM proteins such as collagen type I and fibronectin [4,5,19,20,29,30,39,40]. In asthma, many of these factors are increased in the vicinity of the ASM cell, by structural cells of the airways, including the ASM itself [7,17,19,33,41e44] and inflammatory cells. Exposure of nonasthmatic ASM cells and intact tracheal tissue strips (human and bovine) to mitogens, such as PDGF or the ECM proteins collagen type-I and fibronectin, results in the induction of a hypocontractile ASM phenotype, characterised by decreased contractile

protein expression and decreased contractile function [4,5,20,29]. Conversely, ASM proliferation is inhibited by many factors and mediators, including glucocorticosteroids, Gs coupled receptor agonists, nitric oxide, insulin, prostaglandins and ECM proteins, like chondroitin sulphate, decorin and laminins [4,5,32,34,39,45e 50]. Furthermore, variability in the responses of non-asthmatic and asthmatic derived ASM cells to the aforementioned factors suggests that further research is needed to explore the full repertoire of their functions [33,50,51]. Prolonged serum deprivation and/or contact inhibition, in the presence of insulin or TGFb, induces a hypercontractile phenotype, characterised by decreased proliferative responses, increased contractile function and increased expression of the contractile proteins sm-a-actin, sm-MHC, sm-MLCK and calponin [47,52e54]. The effects of many of the factors mentioned above on ASM contractile protein expression and contractile function, however, remain to be determined. 7. Pathways associated with phenotype switching The differential effects of the multiple factors on ASM phenotype are associated with the activation of specific signalling pathways, with a major role for the extracellular regulated kinase (ERK) and phosphoinositol 3-kinase (PI3-K) pathways. Activation of these pathways leads to changes in the transcription and translation of genes required for contractile protein expression and cell cycle progression. 7.1. Serum response factor The transcription factor serum response factor (SRF) is ubiquitously expressed and appears to play a critical role in phenotype switching. It also regulates transcription of smooth muscle genes including cardiac and skeletal muscle-specific genes [55]. Activation and nuclear translocation of SRF and transcription of smooth muscle-specific genes are tightly controlled by the Rho kinase pathway [54,56,57] and after translocation, SRF activates transcription by binding to CC(A/T)6GG (CArG) box elements [58] in the nucleus, including cofactors which direct SRF to smooth musclespecific genes such as SM22 and sm-MHC in ASM cells [59]. In airway smooth muscle, myocardin expression is increased by Wnt2 signalling and plays an important role in airway development and ASM programming. Goss et al. report an increased expression in SM22, PDGF receptor and sm-a-actin in lung explants treated with recombinant Wnt2 [60] and perhaps downstream myocardin may be important for the differentiation of immature ASM to mature ASM cells including the expression of these key ASM markers. Moreover, the binding of myocardin to SRF may be displaced by other cofactors such as phospho-Elk-1, which is phosphorylated by ERK1/2 in response to mitogens, leading to reduced SRF-dependent transcription of contractile proteins and enhanced transcription of proliferative genes [59,61]. As of yet, no ASM specific transcription factors have been discovered, but one possibility is that such transcription factors do exist and mediate their effects in a similar way to known tissue-specific transcription factors such as SRF or perhaps a cell type specific complex of transcription factors may determine ASM specific genes. Activation of the PI3-K/Akt signalling axis has been implicated as a major player in gene translation, leading not only to ASM proliferation [18,39,62], but also to increased contractile protein expression and contractility [47,54,63,64]. Growth factor-induced ASM phenotype switching is inhibited by Gs coupled receptor agonists, including b2-agonists and prostaglandin (PG) E2 [45,46,65]. These downstream effectors of cyclic adenosine monophosphate (cAMP), protein kinase A (PKA) and exchange

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protein activated by cAMP (Epac), have been implicated in the inhibition of ASM proliferation and phenotype switching in human and bovine ASM cells and tissue [45,46,66,67]. Inhibition of ASM proliferation by PKA was associated with inhibition of both ERK and PI3-K signalling activities [45,46], whereas Epac inhibited ASM proliferation by inhibiting solely ERK signalling [45]. 7.2. Micro RNAs Transcription of genes involved in smooth muscle phenotype switching is fine-tuned by micro ribonucleic acids (miRNAs). Although several miRNAs are expressed by ASM [68,69], studies on the role of miRNAs in ASM phenotype switching are currently limited (see review on microRNA and epigenetics: regulation of smooth muscle phenotype and function within this volume). miRNA25 was found to affect ASM phenotype, as inhibition of miRNA-25 increased Kruppel-like factor-4 (KLF4) expression, resulting in decreased expression of the sm-MHC gene and protein [68]. In VSM, several other miRNAs control phenotype switching [70]; miRNA-143 and miRNA-145 repress expression of pro-mitogenic targets such as Elk1 and KLF4, increase contractile protein expression and decrease proliferation. In addition, the expression of miRNA-143 and miRNA-145 is increased by cytokines that increase smooth muscle maturation, such as TGFb, via a mechanism involving SRF cofactors such as myocardin [71], whereas mitogens decrease expression of miRNA-145 [72]. Conversely, miRNA-146a and miRNA-221 have been associated with PDGF-induced proliferation of VSM cells, acting by reducing the expression of cyclin dependent kinase inhibitors and myocardin [73,74]. Expression of miRNA-143, miRNA-145 and miRNA-146a has also been observed in ASM [68,69], suggesting that these miRNAs may also contribute to ASM phenotype switching. 7.3. Integrins ECM components affect smooth muscle phenotype switching through interactions with integrin receptors. Of the integrins expressed by the ASM [75,76], the a5b1 integrin has been found to be important in phenotype switching, as inhibition of integrin signalling with the integrin blocking peptide Arg-Gly-Asp-Ser (RGDS) inhibits both basal and mitogen-induced ASM proliferation and phenotype switching on collagen type I and fibronectin matrices [5,30]. Moreover, a5b1 integrin contributes to growth factor-induced proliferation [30,76e78]. Integrin activation results in the formation of focal adhesion complexes and activation of focal adhesion kinase (FAK) and integrin-linked kinase (ILK) [79], which may link to ERK and PI3-K signalling pathways and contribute to ASM phenotype switching [80,81]. In addition to these pathways, interactions between integrins and ECM proteins promote cytoskeletal reorganisation, which is required for ASM cells to respond to mechanical forces [82]. 8. What is the initial trigger that induces an otherwise dormant/healthy ASM into an “asthmatic” cell and how does this affect cell phenotype markers? ASM cells have been described as “the appendix of the lung” [83], with their role in the post natal airway only becoming evident in disease. The predisposition or drive towards modulation or maturation in ASM cells derived from asthmatic donors may be determined by specific triggers. Identification of the initial trigger which switches an ASM cell to the asthmatic state lies in our understanding of asthma pathophysiology, including the uni- or multi-factorial signalling pathways that drive towards key asthmatic hallmarks.

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8.1. Inflammatory mediators There appears to be an important role for inflammatory mediators in the reported changes or altered ASM responses that describe ASM phenotype plasticity. Sathish et al. reported that treatment of non-asthmatic derived ASM cells with inflammatory cytokines interleukin-13 (IL-13) and tumour necrosis factor-alpha (TNFa) resulted in diminished SERCA-2 expression and agonist induced calcium store release [84]. Mahn et al. showed that SERCA2 levels in ASM cells correlated inversely with asthma severity (mild, moderate, severe asthma) and a reduction in SERCA-2 levels in non-asthmatic ASM recapitulated an asthmatic phenotype. These induced phenotypic asthmatic responses are associated with a proliferative or synthetic profile that comprises increased cytokine release (eotaxin-1) and increased motility (lamellipodia and filopodia formation) [8]. Likewise, others have shown that ASM exposure to inflammatory cytokines alters calcium transients in vitro and leads to ASM hyperresponsiveness through an augmented ASM constrictive force and diminished relaxation response in vivo [85,86]. Interestingly, the onset of respiratory syncytial virus (RSV) infection in childhood asthma and the ensuing inflammatory response has also been correlated with an asthmatic phenotype in this cohort of patients [87,88]. In addition to these functional changes, inflammatory triggers also alter phenotypic markers associated with ASM plasticity. For example, IL-4, IL-13 and TNFa have been shown to induce a more synthetic ASM phenotype characterised by cytokine and chemokine secretion, expression of adhesion molecules [89,90] and altered expression of CCAAT enhanced binding protein-a (C/EBPa) [91]. The latter is a negative regulator of gene expression that can silence/ attenuate inflammatory and/or proliferative responses. For example, a report by Zhang et al. showed that C/EBPa attenuates p40 transcription and IL-12p40 cytokine release [92]. Additionally, studies in asthmatic derived ASM cells have revealed diminished expression of C/EBPa [91]. Lin et al. demonstrated that IL-4 inhibits C/EBPa [93] and this may further preserve and sustain the inflammatory and proliferative response of ASM derived from asthmatic donors. Furthermore, Miglino et al. reported that house dust mite extract exposure in asthmatic bronchial smooth muscle cells down regulates C/EBPa, increases IL-6 secretion and ASM hyperplasia in vitro [94]. 8.2. Extracellular matrix proteins A second trigger or co-trigger in the ASM microenvironment that modulates phenotype plasticity and the induction of an asthmatic ASM cell phenotype is the ECM. Fibronectin and collagen are two ECM proteins that have been extensively studied for their role in phenotype plasticity. Exposure of non-asthmatic derived ASM cells to fibronectin or collagen in the presence of a mitogen induces ASM modulation and recapitulation of a proliferative phenotype compared to mitogen exposure alone. Similar conditions were also associated with low expression of contractile markers such as MHC, calponin, sm-a-actin [4,11]. ECM proteins such as laminin-111 and laminin-211 delay ASM modulation and proliferation but induce ASM maturation and recapitulation of a pro-contractile phenotype with elevated expression of contractile proteins, including desmin, calponin, sm-a-actin and sm-MHC [13,34,47]. In addition, the laminin competing peptide Tyr-Ile-Gly-Ser-Arg (YIGSR), inhibits ASM proliferation in the presence of mitogens and induces a hypercontractile phenotype in a guinea pig allergic model of asthma [34], whereas fibronectin and collagen type-I support proliferation following exposure to mitogens [4,5]. In the ASM bundle of asthmatics, increased expression of collagen type I and fibronectin has been reported [41,95], which probably contributes to ASM phenotype switching [30].

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These triggers suggest that (1) inflammation induces a proliferative/synthetic phenotype, possibly through altered calcium signalling, (2) ECM proteins induce a differential ASM phenotype by modulation (fibronectin, collagen type-I) or maturation (laminin111, laminin-211) to recapitulate an asthmatic ASM cell. 9. Based upon the above collective findings, is it possible to therapeutically control or switch cells back into a dormant/ quiescent cell? With the collective possibilities mentioned thus far, key questions still remain as to how we can therapeutically target ASM cells and switch their “asthmatic” phenotype back to that of a “non-asthmatic” ASM cell. Several clinical trials (including pre-clinical studies) that aimed to completely revert an asthmatic phenotype with antiinflammatory antibodies have shown limited efficacy [96e98]. However, with the onset of gene therapy, it may be possible to directly target correlative markers of asthma, such as SERCA-2, that are altered by inflammatory cytokines. Targeting the diminished SERCA2b isoform in asthma may provide a future approach for therapy. Promisingly, over-expression of SERCA-2a in cardiac myocytes, where diminished SERCA-2 expression contributes to heart failure, led to a significant improvement in cardiac function in several models [99,100]. Furthermore, the effects of ECM proteins, in particular collagen type I, fibronectin or laminin on ASM proliferation also indicate an avenue to control phenotype switching; however, given our lack of understanding of the role of the individual ECM proteins, the apparent redundancy within the roles of these proteins suggests that a more feasible approach perhaps is to target the receptors of these ECM proteins, for example integrins or a-dystroglycan. 10. Overview e where to go next? Asthma is a heterogeneous disorder clinically manifested as varying levels of asthma severity with altered cellular infiltrates and reduced responses to current therapies. In order to treat asthma effectively, we need to identify the key functional

differences between these clinically relevant asthma phenotypes by examining one of the major effector cells: ASM in healthy and asthmatic subjects. Despite what we have learnt about the consequences of phenotype plasticity for asthma symptoms, we are no closer to establishing the initial trigger or order of events leading to disease phenotypes. Key findings thus far implicate C/EBPa as a major player in the phenotype modulation of ASM cells, particularly the proliferative and secretory phenotypes. A reduction in the anti-proliferative inhibitory protein C/EBPa in asthmatic ASM cells compared to non-asthmatic ASM cells was reported by Roth and colleagues, indicative of perhaps an intrinsic or acquired abnormality in ASM cells that resonates with asthmatic ASM responses. Similar to other proliferation studies [51,91], a reduction in C/EBPa has been strongly linked with a proliferative phenotype. Conversely, a secretory phenotype was found by John et al. to be associated with an increase in C/EBPa binding to the CXCL8 promoter in ASM cells [101]. Despite the contrasting opinions with regards to C/EBPa levels in ASM phenotype change/response, identification of the precise early events that alter C/EBPa expression and thus subsequent phenotype change may take us closer to a uniform approach to tackle phenotype changes in ASM. Other major findings on phenotype modulation include changes in mitochondrial biogenesis as reported by Trian et al. and altered calcium homeostasis as reflected in studies by Mahn and colleagues. Both studies allude to changes in calcium homeostasis; the former specifically focuses on calcium dependent changes in mitochondrial transcription activity [102] and the latter links diminished SERCA-2 expression in healthy ASM cells to the recapitulation of a synthetic asthmatic phenotype [8]. Again, identifying the precise early modulators involved in this altered calcium homeostasis may bring us closer to understanding the intricacy and precise control of phenotype switching in ASM cells. By improving our cell culture models to better depict an asthmatic environment, we are starting to identify the pathways that are deregulated in asthma. However, there is still a need to optimise in vitro culture conditions/models in order to investigate in vivo

Fig. 2. The intricacies of airway smooth muscle phenotype switching. In vitro, factors influencing ASM phenotype include: serum, cell density, cellecell contacts (e.g. T-Cells), ECM, homologous substrates, mechanical stretch and micro RNAs. The onset and regulation of phenotype switching in asthma remains to be determined.

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contractile ASM under controlled circumstances. By establishing such models, we will be able to investigate the major regulators of ASM phenotype in vitro (Fig. 2), while ensuring that these ASM cells continue to be representative of in vivo cells. From these in vitro studies, we can examine the ultrastructural, transcriptional and protein translational mechanisms involved, the architecture of the ASM cytoskeleton, and the regulation of phenotype expression and how these processes adapt to triggers from a pathological environment. Acknowledgements Sana Siddiqui is the recipient of the Alexander McFee Studentship, Faculty of Medicine, McGill University, Montréal, QC. Jill R. Johnson is supported by a Research Fellowship from Imperial College London. Janette K. Burgess is supported by a National Health & Medical Research Council, Australia Career Development Fellowship #1032695. Rushita Bagchi is the recipient of an MHRC/ SBRC Coordinated Graduate Studentship awarded by the Manitoba Health Research Council, Canada. Abbreviations

AHR airway hyperresponsiveness ASM airway smooth muscle ECM extracellular matrix sm-a-actin smooth muscle-a-actin MLCK myosin light chain kinase MHC myosin heavy chain PDGF platelet derived growth factor VSM vascular smooth muscle SERCA sarcoendoplasmic reticulum calcium ATPase ERK extracellular regulated kinase PI3K phosphoinositol 3-kinase C/EBPa CCAAT enhanced binding protein-a miRNA micro ribonucleic acid IL interleukin tumour necrosis factor a TNFa TGFb transforming growth factor b SM-B smooth muscle myosin-B SOCE store-operated calcium entry ROCE receptor-operated calcium entry TRPC transient receptor potential channel type C STIM1 stromal interacting molecule 1 SRF serum response factor KLF4 Kruppel-like factor-4 PKA protein kinase A PG prostaglandin cAMP cyclic adenosine monophosphate EPAC exchange protein activated by cAMP FAK focal adhesion kinase ILK integrin-linked kinase RSV respiratory syncytial virus YIGSR Tyr-Ile-Gly-Ser-Arg RGDS Arg-Gly-Asp-Ser References [1] Halayko AJ, Rector E, Stephens NL. Characterization of molecular determinants of smooth muscle cell heterogeneity. Can J Physiol Pharmacol 1997; 75(7):917e29. [2] Halayko AJ, Tran T, Ji SY, Yamasaki A, Gosens R. Airway smooth muscle phenotype and function: interactions with current asthma therapies. Curr Drug Targets 2006;7(5):525e40.

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[3] Halayko AJ, Salari H, Ma X, Stephens NL. Markers of airway smooth muscle cell phenotype. Am J Physiol 1996;270(6 Pt 1):L1040e51. [4] Hirst SJ, Twort CH, Lee TH. Differential effects of extracellular matrix proteins on human airway smooth muscle cell proliferation and phenotype. Am J Respir Cell Mol Biol 2000;23(3):335e44. [5] Dekkers BG, Schaafsma D, Nelemans SA, Zaagsma J, Meurs H. Extracellular matrix proteins differentially regulate airway smooth muscle phenotype and function. Am J Physiol Lung Cell Mol Physiol 2007;292(6):L1405e13. [6] Johnson PR, Roth M, Tamm M, Hughes M, Ge Q, King G, et al. Airway smooth muscle cell proliferation is increased in asthma. Am J Respir Crit Care Med 2001;164(3):474e7. [7] Johnson PR, Burgess JK, Underwood PA, Au W, Poniris MH, Tamm M, et al. Extracellular matrix proteins modulate asthmatic airway smooth muscle cell proliferation via an autocrine mechanism. J Allergy Clin Immunol 2004; 113(4):690e6. [8] Mahn K, Hirst SJ, Ying S, Holt MR, Lavender P, Ojo OO, et al. Diminished sarco/endoplasmic reticulum Ca2þ ATPase (SERCA) expression contributes to airway remodelling in bronchial asthma. Proc Natl Acad Sci U S A 2009; 106(26):10775e80. [9] Chamley-Campbell J, Campbell GR, Ross R. The smooth muscle cell in culture. Physiol Rev 1979;59(1):1e61. [10] Halayko AJ, Camoretti-Mercado B, Forsythe SM, Vieira JE, Mitchell RW, Wylam ME, et al. Divergent differentiation paths in airway smooth muscle culture: induction of functionally contractile myocytes. Am J Physiol 1999; 276(1 Pt 1):L197e206. [11] Sukkar MB, Stanley AJ, Blake AE, Hodgkin PD, Johnson PR, Armour CL, et al. ‘Proliferative’ and ‘synthetic’ airway smooth muscle cells are overlapping populations. Immunol Cell Biol 2004;82(5):471e8. [12] Ueki N, Sobue K, Kanda K, Hada T, Higashino K. Expression of high and low molecular weight caldesmons during phenotypic modulation of smooth muscle cells. Proc Natl Acad Sci U S A 1987;84(24):9049e53. [13] Tran T, McNeill KD, Gerthoffer WT, Unruh H, Halayko AJ. Endogenous laminin is required for human airway smooth muscle cell maturation. Respir Res 2006;7:117. [14] Tran T, Ens-Blackie K, Rector ES, Stelmack GL, McNeill KD, Tarone G, et al. Laminin-binding integrin alpha7 is required for contractile phenotype expression by human airway myocytes. Am J Respir Cell Mol Biol 2007; 37(6):668e80. [15] Gosens R, Stelmack GL, Bos ST, Dueck G, Mutawe MM, Schaafsma D, et al. Caveolin-1 is required for contractile phenotype expression by airway smooth muscle cells. J Cell Mol Med 2011;15(11):2430e42. [16] Sharma P, Tran T, Stelmack GL, McNeill K, Gosens R, Mutawe MM, et al. Expression of the dystrophin-glycoprotein complex is a marker for human airway smooth muscle phenotype maturation. Am J Physiol Lung Cell Mol Physiol 2008;294(1):L57e68. [17] Halayko AJ, Tran T, Gosens R. Phenotype and functional plasticity of airway smooth muscle: role of caveolae and caveolins. Proc Am Thorac Soc 2008; 5(1):80e8. [18] Hirota JA, Nguyen TT, Schaafsma D, Sharma P, Tran T. Airway smooth muscle in asthma: phenotype plasticity and function. Pulm Pharmacol Ther 2009; 22(5):370e8. [19] Hirst SJ, Martin JG, Bonacci JV, Chan V, Fixman ED, Hamid QA, et al. Proliferative aspects of airway smooth muscle. J Allergy Clin Immunol 2004;114(2 Suppl.):S2e17. [20] Gosens R, Meurs H, Bromhaar MM, McKay S, Nelemans SA, Zaagsma J. Functional characterization of serum- and growth factor-induced phenotypic changes in intact bovine tracheal smooth muscle. Br J Pharmacol 2002; 137(4):459e66. [21] Hirst SJ, Walker TR, Chilvers ER. Phenotypic diversity and molecular mechanisms of airway smooth muscle proliferation in asthma. Eur Respir J 2000; 16(1):159e77. [22] Mitchell RW, Halayko AJ, Kahraman S, Solway J, Wylam ME. Selective restoration of calcium coupling to muscarinic M(3) receptors in contractile cultured airway myocytes. Am J Physiol Lung Cell Mol Physiol 2000;278(5): L1091e100. [23] Hughes JM, Qawasmeh A, Blake AE, Ge Q, Armour CL. Serum starvation induces phenotypic and functional changes in human airway smooth muscle cells. Am J Respir Crit Care Med 2003;167:A32. [24] Snetkov VA, Hirst SJ, Ward JPT. Ion channels in freshly isolated and cultured human bronchial smooth muscle cells. Exp Physiol 1996;81(5):791e804. [25] Berra-Romani R, Mazzocco-Spezzia A, Pulina MV, Golovina VA. Ca2þ handling is altered when arterial myocytes progress from a contractile to a proliferative phenotype in culture. Am J Physiol Cell Physiol 2008;295(3):C779e90. [26] Hall IP, Kotlikoff M. Use of cultured airway myocytes for study of airway smooth-muscle. Am J Physiol Lung Cell Mol Physiol 1995;268(1):L1e11. [27] Ceresa CC, Knox AJ, Johnson SR. Use of a three-dimensional cell culture model to study airway smooth muscle-mast cell interactions in airway remodeling. Am J Physiol Lung Cell Mol Physiol 2009;296(6):L1059e66. [28] Chamley-Campbell JH, Campbell GR. What controls smooth muscle phenotype? Atherosclerosis 1981;40(3e4):347e57. [29] Dekkers BG, Bos IS, Zaagsma J, Meurs H. Functional consequences of human airway smooth muscle phenotype plasticity. Br J Pharmacol 2012;166(1): 359e67. [30] Dekkers BG, Bos IS, Gosens R, Halayko AJ, Zaagsma J, Meurs H. The integrinblocking peptide RGDS inhibits airway smooth muscle remodeling in

48

[31] [32]

[33]

[34]

[35]

[36]

[37]

[38]

[39]

[40]

[41]

[42]

[43]

[44]

[45]

[46]

[47]

[48]

[49]

[50]

[51]

[52]

[53]

[54]

[55] [56]

D.B. Wright et al. / Pulmonary Pharmacology & Therapeutics 26 (2013) 42e49 a guinea pig model of allergic asthma. Am J Respir Crit Care Med 2010; 181(6):556e65. Black JL, Burgess JK, Johnson PR. Airway smooth muscleeits relationship to the extracellular matrix. Respir Physiol Neurobiol 2003;137(2e3):339e46. D’Antoni ML, Torregiani C, Ferraro P, Michoud MC, Mazer B, Martin JG, et al. Effects of decorin and biglycan on human airway smooth muscle cell proliferation and apoptosis. Am J Physiol Lung Cell Mol Physiol 2008;294(4):L764e71. Chan V, Burgess JK, Ratoff JC, O’Connor BJ, Greenough A, Lee TH, et al. Extracellular matrix regulates enhanced eotaxin expression in asthmatic airway smooth muscle cells. Am J Respir Crit Care Med 2006;174(4):379e85. Dekkers BG, Bos IS, Halayko AJ, Zaagsma J, Meurs H. The laminin beta1competing peptide YIGSR induces a hypercontractile, hypoproliferative airway smooth muscle phenotype in an animal model of allergic asthma. Respir Res 2010;11:170. Tao F, Chaudry S, Tolloczko B, Martin JG, Kelly SM. Modulation of smooth muscle phenotype in vitro by homologous cell substrate. Am J Physiol Cell Physiol 2003;284(6):C1531e41. Smith PG, Moreno R, Ikebe M. Strain increases airway smooth muscle contractile and cytoskeletal proteins in vitro. Am J Physiol 1997;272(1 Pt 1): L20e7. Pasternyk SM, D’Antoni ML, Venkatesan N, Siddiqui S, Martin JG, Ludwig MS. Differential effects of extracellular matrix and mechanical strain on airway smooth muscle cells from ovalbumin- vs saline-challenged brown Norway rats. Respir Physiol Neurobiol 2012;181(1):36e43. Kumar A, Knox AJ, Boriek AM. CCAAT/enhancer-binding protein and activator protein-1 transcription factors regulate the expression of interleukin-8 through the mitogen-activated protein kinase pathways in response to mechanical stretch of human airway smooth muscle cells. J Biol Chem 2003; 278(21):18868e76. 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(2e3):385e97. Hirst SJ, Barnes PJ, Twort CH. PDGF isoform-induced proliferation and receptor expression in human cultured airway smooth muscle cells. Am J Physiol 1996;270(3 Pt 1):L415e28. Araujo BB, Dolhnikoff M, Silva LF, Elliot J, Lindeman JH, Ferreira DS, et al. Extracellular matrix components and regulators in the airway smooth muscle in asthma. Eur Respir J 2008;32(1):61e9. Bai TR, Cooper J, Koelmeyer T, Pare PD, Weir TD. The effect of age and duration of disease on airway structure in fatal asthma. Am J Respir Crit Care Med 2000;162(2 Pt 1):663e9. Dekkers BG, Maarsingh H, Meurs H, Gosens R. Airway structural components drive airway smooth muscle remodeling in asthma. Proc Am Thorac Soc 2009;6(8):683e92. Howarth PH, Knox AJ, Amrani Y, Tliba O, Panettieri Jr RA, Johnson M. Synthetic responses in airway smooth muscle. J Allergy Clin Immunol 2004; 114(2 Suppl.):S32e50. Roscioni SS, Dekkers BG, Prins AG, Menzen MH, Meurs H, Schmidt M, et al. cAMP inhibits modulation of airway smooth muscle phenotype via the exchange protein activated by cAMP (Epac) and protein kinase A. Br J Pharmacol 2011;162(1):193e209. Yan H, Deshpande DA, Misior AM, Miles MC, Saxena H, Riemer EC, et al. Antimitogenic effects of {beta}-agonists and PGE2 on airway smooth muscle are PKA dependent. FASEB J 2011;25(1):389e97. Dekkers BG, Schaafsma D, Tran T, Zaagsma J, Meurs H. Insulin-induced laminin expression promotes a hypercontractile airway smooth muscle phenotype. Am J Respir Cell Mol Biol 2009;41(4):494e504. Kanabar V, Hirst SJ, O’Connor BJ, Page CP. Some structural determinants of the antiproliferative effect of heparin-like molecules on human airway smooth muscle. Br J Pharmacol 2005;146(3):370e7. Dekkers BG, Pehlic A, Meurs H, Zaagsma J. Glucocorticosteroids and á2adrenoceptor agonists synergistically prevent the induction of a proliferative, hypocontractile airway smooth muscle phenotype. Am J Respir Crit Care Med 2010;181:A5308. Burgess JK, Ge Q, Boustany S, Black JL, Johnson PR. Increased sensitivity of asthmatic airway smooth muscle cells to prostaglandin E2 might be mediated by increased numbers of E-prostanoid receptors. J Allergy Clin Immunol 2004;113(5):876e81. Roth M, Johnson PR, Borger P, Bihl MP, Rudiger JJ, King GG, et al. Dysfunctional interaction of C/EBPalpha and the glucocorticoid receptor in asthmatic bronchial smooth-muscle cells. N Engl J Med 2004;351(6):560e74. Ma X, Wang Y, Stephens NL. Serum deprivation induces a unique hypercontractile phenotype of cultured smooth muscle cells. Am J Physiol 1998; 274(5 Pt 1):C1206e14. Gosens R, Nelemans SA, Hiemstra M, Grootte Bromhaar MM, Meurs H, Zaagsma J. Insulin induces a hypercontractile airway smooth muscle phenotype. Eur J Pharmacol 2003;481(1):125e31. Schaafsma D, McNeill KD, Stelmack GL, Gosens R, Baarsma HA, Dekkers BG, et al. Insulin increases the expression of contractile phenotypic markers in airway smooth muscle. Am J Physiol Cell Physiol 2007;293(1):C429e39. Sun Q, Chen G, Streb JW, Long X, Yang Y, Stoeckert Jr CJ, et al. Defining the mammalian CArGome. Genome Res 2006;16(2):197e207. Liu HW, Halayko AJ, Fernandes DJ, Harmon GS, McCauley JA, Kocieniewski P, et al. The RhoA/Rho kinase pathway regulates nuclear localization of serum response factor. Am J Respir Cell Mol Biol 2003;29(1):39e47.

[57] Camoretti-Mercado B, Liu HW, Halayko AJ, Forsythe SM, Kyle JW, Li B, et al. Physiological control of smooth muscle-specific gene expression through regulated nuclear translocation of serum response factor. J Biol Chem 2000; 275(39):30387e93. [58] Solway J, Forsythe SM, Halayko AJ, Vieira JE, Hershenson MB, CamorettiMercado B. Transcriptional regulation of smooth muscle contractile apparatus expression. Am J Respir Crit Care Med 1998;158(5 Pt 3):S100e8. [59] Wang Z, Wang DZ, Hockemeyer D, McAnally J, Nordheim A, Olson EN. Myocardin and ternary complex factors compete for SRF to control smooth muscle gene expression. Nature 2004;428(6979):185e9. [60] Goss AM, Tian Y, Cheng L, Yang J, Zhou D, Cohen ED, et al. Wnt2 signaling is necessary and sufficient to activate the airway smooth muscle program in the lung by regulating myocardin/Mrtf-B and Fgf10 expression. Dev Biol 2011;356(2):541e52. [61] Taurin S, Sandbo N, Yau DM, Sethakorn N, Kach J, Dulin NO. Phosphorylation of myocardin by extracellular signal-regulated kinase. J Biol Chem 2009; 284(49):33789e94. [62] Gosens R, Dueck G, Rector E, Nunes RO, Gerthoffer WT, Unruh H, et al. Cooperative regulation of GSK-3 by muscarinic and PDGF receptors is associated with airway myocyte proliferation. Am J Physiol Lung Cell Mol Physiol 2007;293(5):L1348e58. [63] Halayko AJ, Kartha S, Stelmack GL, McConville J, Tam J, Camoretti-Mercado B, et al. Phophatidylinositol-3 kinase/mammalian target of rapamycin/p70S6K regulates contractile protein accumulation in airway myocyte differentiation. Am J Respir Cell Mol Biol 2004;31(3):266e75. [64] Ge Q, Moir LM, Trian T, Niimi K, Poniris M, Shepherd PR, et al. The phosphoinositide 30 -kinase p110delta modulates contractile protein production and IL-6 release in human airway smooth muscle. J Cell Physiol 2012;227(8): 3044e52. [65] Tomlinson PR, Wilson JW, Stewart AG. Inhibition by salbutamol of the proliferation of human airway smooth muscle cells grown in culture. Br J Pharmacol 1994;111(2):641e7. [66] Roscioni SS, Prins AG, Elzinga CR, Menzen MH, Dekkers BG, Halayko AJ, et al. Protein kinase A and the exchange protein directly activated by cAMP (Epac) modulate phenotype plasticity in human airway smooth muscle. Br J Pharmacol 2011;164(3):958e69. [67] Kassel KM, Wyatt TA, Panettieri Jr RA, Toews ML. Inhibition of human airway smooth muscle cell proliferation by beta 2-adrenergic receptors and cAMP is PKA independent: evidence for EPAC involvement. Am J Physiol Lung Cell Mol Physiol 2008;294(1):L131e8. [68] Kuhn AR, Schlauch K, Lao R, Halayko AJ, Gerthoffer WT, Singer CA. MicroRNA expression in human airway smooth muscle cells: role of miR-25 in regulation of airway smooth muscle phenotype. Am J Respir Cell Mol Biol 2010; 42(4):506e13. [69] Larner-Svensson HM, Williams AE, Tsitsiou E, Perry MM, Jiang X, Chung KF, et al. Pharmacological studies of the mechanism and function of interleukin1beta-induced miRNA-146a expression in primary human airway smooth muscle. Respir Res 2010;11:68. [70] Davis-Dusenbery BN, Wu C, Hata A. Micromanaging vascular smooth muscle cell differentiation and phenotypic modulation. Arterioscler Thromb Vasc Biol 2011;31(11):2370e7. [71] Davis-Dusenbery BN, Chan MC, Reno KE, Weisman AS, Layne MD, Lagna G, et al. Down-regulation of Kruppel-like factor-4 (KLF4) by microRNA-143/145 is critical for modulation of vascular smooth muscle cell phenotype by transforming growth factor-beta and bone morphogenetic protein 4. J Biol Chem 2011;286(32):28097e110. [72] Cheng Y, Liu X, Yang J, Lin Y, Xu DZ, Lu Q, et al. MicroRNA-145, a novel smooth muscle cell phenotypic marker and modulator, controls vascular neointimal lesion formation. Circ Res 2009;105(2):158e66. [73] Davis BN, Hilyard AC, Nguyen PH, Lagna G, Hata A. Induction of microRNA221 by platelet-derived growth factor signaling is critical for modulation of vascular smooth muscle phenotype. J Biol Chem 2009;284(6):3728e38. [74] Liu X, Cheng Y, Zhang S, Lin Y, Yang J, Zhang C. A necessary role of miR-221 and miR-222 in vascular smooth muscle cell proliferation and neointimal hyperplasia. Circ Res 2009;104(4):476e87. [75] Freyer AM, Johnson SR, Hall IP. Effects of growth factors and extracellular matrix on survival of human airway smooth muscle cells. Am J Respir Cell Mol Biol 2001;25(5):569e76. [76] Nguyen TT, Ward JP, Hirst SJ. Beta1-integrins mediate enhancement of airway smooth muscle proliferation by collagen and fibronectin. Am J Respir Crit Care Med 2005;171(3):217e23. [77] Moir LM, Burgess JK, Black JL. Transforming growth factor beta 1 increases fibronectin deposition through integrin receptor alpha 5 beta 1 on human airway smooth muscle. J Allergy Clin Immunol 2008;121(4):1034e9. e4. [78] Hedin UL, Daum G, Clowes AW. Disruption of integrin alpha 5 beta 1 signaling does not impair PDGF-BB-mediated stimulation of the extracellular signal-regulated kinase pathway in smooth muscle cells. J Cell Physiol 1997; 172(1):109e16. [79] Liu S, Calderwood DA, Ginsberg MH. Integrin cytoplasmic domain-binding proteins. J Cell Sci 2000;113(Pt 20):3563e71. [80] Wu Y, Huang Y, Herring BP, Gunst SJ. Integrin-linked kinase regulates smooth muscle differentiation marker gene expression in airway tissue. Am J Physiol Lung Cell Mol Physiol 2008;295(6):L988e97. [81] Dekkers BG, van der Schuyt RD, Spanjer AIR, Kuik JW, Zaagsma J, Meurs H. Activation of focal adhesion kinase regulates collagen I-induced airway

D.B. Wright et al. / Pulmonary Pharmacology & Therapeutics 26 (2013) 42e49

[82] [83] [84]

[85]

[86]

[87] [88]

[89]

[90]

[91]

[92]

smooth muscle phenotype switching. Am J Respir Crit Care Med 2011;181: A3657. Parsons JT, Horwitz AR, Schwartz MA. Cell adhesion: integrating cytoskeletal dynamics and cellular tension. Nat Rev Mol Cell Biol 2010;11(9):633e43. Mitzner W. Airway smooth muscle: the appendix of the lung. Am J Respir Crit Care Med 2004;169(7):787e90. Sathish V, Thompson MA, Bailey JP, Pabelick CM, Prakash YS, Sieck GC. Effect of proinflammatory cytokines on regulation of sarcoplasmic reticulum Ca2þ reuptake in human airway smooth muscle. Am J Physiol Lung Cell Mol Physiol 2009;297(1):L26e34. Hakonarson H, Herrick DJ, Serrano PG, Grunstein MM. Autocrine role of interleukin 1beta in altered responsiveness of atopic asthmatic sensitized airway smooth muscle. J Clin Invest 1997;99(1):117e24. Hakonarson H, Herrick DJ, Grunstein MM. Mechanism of impaired betaadrenoceptor responsiveness in atopic sensitized airway smooth muscle. Am J Physiol 1995;269(5 Pt 1):L645e52. Kato M, Kimura H. Respiratory syncytial virus induces inflammation in bronchial asthma: role of eosinophils. Allergol Int 2004;53(4):301e7. Matsuse H, Behera AK, Kumar M, Rabb H, Lockey RF, Mohapatra SS. Recurrent respiratory syncytial virus infections in allergen-sensitized mice lead to persistent airway inflammation and hyperresponsiveness. J Immunol 2000; 164(12):6583e92. Amrani Y, Chen H, Panettieri Jr RA. Activation of tumor necrosis factor receptor 1 in airway smooth muscle: a potential pathway that modulates bronchial hyper-responsiveness in asthma? Respir Res 2000;1(1):49e53. Ammit AJ, Hoffman RK, Amrani Y, Lazaar AL, Hay DW, Torphy TJ, et al. Tumor necrosis factor-alpha-induced secretion of RANTES and interleukin-6 from human airway smooth-muscle cells. Modulation by cyclic adenosine monophosphate. Am J Respir Cell Mol Biol 2000;23(6):794e802. Borger P, Miglino N, Baraket M, Black JL, Tamm M, Roth M. Impaired translation of CCAAT/enhancer binding protein alpha mRNA in bronchial smooth muscle cells of asthmatic patients. J Allergy Clin Immunol 2009; 123(3):639e45. Zhang Y, Ma X. Triptolide inhibits IL-12/IL-23 expression in APCs via CCAAT/enhancer-binding protein alpha. J Immunol 2010;184(7): 3866e77.

49

[93] Lin SJ, Shu PY, Chang C, Ng AK, Hu CP. IL-4 suppresses the expression and the replication of hepatitis B virus in the hepatocellular carcinoma cell line Hep3B. J Immunol 2003;171(9):4708e16. [94] Miglino N, Roth M, Tamm M, Borger P. House dust mite extract downregulates C/EBPalpha in asthmatic bronchial smooth muscle cells. Eur Respir J: Off J Eur Soc Clin Respir Physiol 2011;38(1):50e8. [95] Thomson RJ, Schellenberg RR. Increased amount of airway smooth muscle does not account for excessive bronchoconstriction in asthma. Can Respir J 1998;5(1):61e2. [96] Leckie MJ, ten Brinke A, Khan J, Diamant Z, O’Connor BJ, Walls CM, et al. Effects of an interleukin-5 blocking monoclonal antibody on eosinophils, airway hyper-responsiveness, and the late asthmatic response. Lancet 2000; 356(9248):2144e8. [97] Kips JC, O’Connor BJ, Langley SJ, Woodcock A, Kerstjens HA, Postma DS, et al. Effect of SCH55700, a humanized anti-human interleukin-5 antibody, in severe persistent asthma: a pilot study. Am J Respir Crit Care Med 2003; 167(12):1655e9. [98] Bryan SA, O’Connor BJ, Matti S, Leckie MJ, Kanabar V, Khan J, et al. Effects of recombinant human interleukin-12 on eosinophils, airway hyperresponsiveness, and the late asthmatic response. Lancet 2000;356(9248): 2149e53. [99] Miyamoto MI, del Monte F, Schmidt U, DiSalvo TS, Kang ZB, Matsui T, et al. Adenoviral gene transfer of SERCA2a improves left-ventricular function in aortic-banded rats in transition to heart failure. Proc Natl Acad Sci U S A 2000;97(2):793e8. [100] Schmidt U, del Monte F, Miyamoto MI, Matsui T, Gwathmey JK, Rosenzweig A, et al. Restoration of diastolic function in senescent rat hearts through adenoviral gene transfer of sarcoplasmic reticulum Ca(2þ)-ATPase. Circulation 2000;101(7):790e6. [101] John AE, Zhu YM, Brightling CE, Pang L, Knox AJ. Human airway smooth muscle cells from asthmatic individuals have CXCL8 hypersecretion due to increased NF-kappa B p65, C/EBP beta, and RNA polymerase II binding to the CXCL8 promoter. J Immunol 2009;183(7):4682e92. [102] Trian T, Benard G, Begueret H, Rossignol R, Girodet PO, Ghosh D, et al. Bronchial smooth muscle remodeling involves calcium-dependent enhanced mitochondrial biogenesis in asthma. J Exp Med 2007;204(13):3173e81.