Modeling the diverse features of asthma

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Drug Discovery Today: Disease Models

DRUG DISCOVERY

TODAY

DISEASE

MODELS

Editors-in-Chief Jan Tornell – AstraZeneca, Sweden Denis Noble – University of Oxford, UK

Respiratory diseases

Modeling the diverse features of asthma Barry J. Moynihan, James G. Martin* Meakins-Christie Laboratories, 3626 St. Urbain Street, Montreal, Que., Canada H2X 2P2

Asthma is characterized by complexity resulting from the interactions among a variety of biomechanical, immunological and biochemical processes that lead to airway narrowing. Multiple models are therefore required to address particular aspects of its molecular pathophysiology. Exploration of the disease and its treatment requires experimental methodologies that include a range of in vitro and in vivo approaches. This article will contextualize recent advances in our under-

Section Editor: Alastair Stewart – University of Melbourne, Australia The vexed issue of animal models of asthma has been debated for as long as these have been in use, and despite refinements in models, the intensity of the debate surrounding the value of such models does not appear to have diminished. James Martin has dedicated at least two decades to the development of a better understanding of asthma using a series of investigations in the Brown Norway rat model of allergic inflammation. Here, he and his colleague Barry Moynihan review recent developments in the complexity of animal models that have resulted from an increasing appreciation of the complexity and diversity of human asthma.

standing of asthma through the development and exploration of various models. The relative strengths and weaknesses of the various models and their utility for target identification will be reviewed.

Introduction Asthma is a common disease whose incidence (5–10%) is still increasing. Pathogenetic factors include allergen exposure, viral infections, pollution and obesity. Asthma is often the result of an inappropriate T cell response to foreign allergenic proteins, causing airway inflammation, airway hyperresponsiveness (AHR; excessive irritability) and undue mucus production. Airway inflammation is accompanied by epithelial cell activation, enhanced airway smooth muscle cell contractility (Fig. 1) and changes in airway architecture. Therapy for asthma has the unique challenge of targeting the airways with topical drugs. The two current areas of focus for treatment are anti-inflammatory agents (steroids, anti-cytokines, anti-IgE, anti-adhesion molecules) and bronchodilators *Corresponding author: (J.G. Martin) [email protected] 1740-6757/$ ß 2004 Elsevier Ltd. All rights reserved.

DOI: 10.1016/j.ddmod.2004.11.020

(selective b-2 agonists, anti-cholinergics, leukotriene modifiers, phosphodiesterase inhibitors) that interfere with smooth muscle contraction. Some agents such as corticosteroids can have multiple sites of action. Experimental models of the inflammatory process spanning the spectrum of potential experimental approaches are available. This article will review some of the strategies in use for the exploration of asthma.

Cellular and tissue culture models of asthma Cultured explanted lung tissues are an alternative in vitro tool to the classical organ baths. Explant models retain intact tissue architecture. Airway responses to allergen or bronchoconstrictive agonists such as methacholine can be measured by videomicroscopy. Intrapulmonary airways harvested from hyperresponsive rats and mice retain exaggerated rates of shortening in response to methacholine or serotonin [1]. Explant models can be used to study cellular and biochemical events following allergen challenge. For example, early and late bronchoconstriction are evoked by allergen challenge and, similar to in vivo models, are cysteinyl-leukotriene www.drugdiscoverytoday.com

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Figure 1. Molecular mechanisms of airway hyperresponsiveness. Airway smooth muscle cells are the primary effectors of bronchoconstriction. The schema outlined above indicates some of the mechanisms characterised that facilitate the development of AHR. Abbreviations: PLC, phospholipase C; IP3, inositol-triphosphate; RhoA, RAS homologue gene family member A; ROK, Rho-activated kinase; MLCP, myosin light chain phosphatase; MLCK, myosin light chain kinase; MAPK, mitogen activated protein kinase; SR, sarcoplasmic reticulum; AHR, airway hyperresponsiveness.

(Cys-LT) driven [2]. Thin slices of lung can be used to study the role of resident cells in pathobiology as well as certain aspects of intracellular signaling such as calcium transients [3]. Although not yet exploited for this purpose, explants are amenable also to the study of the responses of the lung tissues to cyclical stresses. An obvious drawback is that the lung responses are evaluated in isolation from the circulation and the bone marrow, sources of inflammatory cells recruited in asthma. Human nasal explants are a useful surrogate for lower airway tissues. In situ eosinophil maturation in response to interleukin-5 (IL-5) and lipopolysaccharide modulation of Th2 cytokine synthesis has been studied using explants of nasal biopsies [4,5].

Single cell culture models Single cell culture models have utility in exploring the properties of isolated epithelium, fibroblasts and smooth muscle. Epithelial cells can be polarized using an air–liquid interface. Ciliogenesis, normal ion transport in cultured epithelial cells and experimental infection with respiratory syncytial virus (RSV) require polarization [6–8]. Monolayers are suitable for the study of injury and repair. Airway smooth muscle (ASM) cells in culture are subject to phenotypic variation, losing contractile proteins and assuming a secretory phenotype. Restoration of the contractile phenotype can be achieved 312

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using prolonged serum starvation [9] or by culturing on laminin and type IV collagen substrata, which inhibit ASM growth in contrast to fibronectin and type III collagen that enhance it [10]. Growing ASM cells on a homologous cell substrate induces a differentiated phenotype and is particularly suitable for the assay of contractile responses [11]. Investigators have used the above conditions to study the influence of cytokines on resident airway cells. In vitro studies have shown that cytokines modulate the contractility of ASM via both upregulation of CysLT1 receptors (IL-13) [12] and altering calcium signaling in ASM (IL-13, tumour necrosis factor-a (TNF-a) [13]. IL-4 and IL-13 increase fibroblast proliferation, and can also induce a myofibroblast phenotype; this effect is attenuated by interferon-g (IFN-g) [14]. Transforming growth factor-b (TGF-b1) also increases myofibroblast differentiation of human lung fibroblasts, as evidenced by a-smooth muscle actin staining. Cells in the airways are subjected to periodic mechanical stress in vivo. Efforts to integrate variations in mechanical strain with matrix and cell culture techniques have been made. Cultured ASM cells have been studied under conditions of strain by subjecting integrin-bound ferromagnetic beads on their surface to torque through magnetic fields (magnetic twisting cytometry) [15] or by cyclic mechanical strain to flexible membranes on which the cells are layered

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[16]. Mechanical stress causes cytoskeletal rearrangement in ASM with increased contractility, stiffness and proliferation, demonstrating the plasticity of the cytoskeleton in ASM [17]. To simulate the effects of respiration, cells are subjected to cyclical mechanical stress on flexible membranes (e.g. FlexcellTM (Flexcell International: http://www.flexcellint.com/)). The plates also allow different matrices to be used. Mechanical strain increases the contractility of ASM, influences the synthetic functions of ASM and increases IL-8 production by HASM in an AP-1 dependent manner via the MAPK pathway [18]. Mechanical stress also interacts with the matrix in influencing ASM growth. Collagen enhances growth more than laminin in cells subjected to cyclic mechanical strain but strain reduces growth on both substrata [19]. The precise alterations in strain in asthmatic airways require elucidation. Tissue engineering approaches have been used to produce bilayers of epithelium and fibroblasts and exposed to an air– liquid interface. The cells in culture retain characteristics relevant to asthma. Epithelium reconstituted from asthmatic subjects demonstrates increased fragility and promotes IL-5 production by T cells [20]. Recently, Choe et al. engineered a similar model of the airways with a ciliated epithelium on fibrocytes to study the effects of strain on epithelial structure. Activated eosinophils increased epithelial thickness synergistically with mechanical strain [21]. Epithelial stress releases fibrogenic growth factors [22]. This approach has substantial promise for the exploration of mechanisms of airway remodeling.

Animal models of asthma Animal models of asthma showing airway hyperresponsiveness, bronchoconstriction, airway inflammation and remodeling are available. Allergen driven models predominate but models of innate hyperresponsiveness as well as viral and exercise-triggered asthma are also available. Cats and horses suffer naturally from a form of allergic asthma but most animals have an asthma-like phenotype induced by allergen sensitization and challenge. The early research in asthma was performed on dogs and cats because of the ease with which airway mechanics could be studied. Improved techniques for measurements in small animals are available and smaller animals are easier to manage and more economical. Larger animals, even primates with their closer genetic links to humans, are outbred and show variability in responses. Both mice and rats are available as highly inbred strains. Mice have the added advantage of easy genetic manipulation.

Innate hyperresponsiveness The guinea pig, selected rat and mouse strains are hyperresponsive to challenge with contractile agonists. The Basenji greyhound cross is another well-characterized model of AHR but is not readily accessible. Innate responsiveness of the airways is related to ASM mass and nitric oxide production in

Drug Discovery Today: Disease Models | Respiratory diseases

the airways but other factors are involved. Altered phosphoinositol metabolism causing increased calcium mobilization by contractile agonists accounts for hyperresponsiveness in Fischer344 rats compared to the normo-responsive Lewis strain. Mechanisms related to protein-kinase C (PKC) and phosphoinositol specific 50 phosphatase contribute to the observed differences in growth and contractile responses of ASM [23]. A short sequence insert in the myosin heavy chain that makes it a faster contracting isoform affects the rate of bronchoconstriction of airways on exposure to methacholine and can be a mechanism of AHR in mice [24]. A/J mice display a relative AHR. The differential sensitivity of A/J mice compared to the C57/Bl6 strain has been exploited to identify susceptibility loci for AHR.

Allergen induced asthma AHR following allergen challenge is the usual surrogate for asthma used in drug testing. Various protocols for sensitization and subsequent aerosolized allergen challenge are used to induce AHR. Nasal instillation of allergen in lightly anesthetized mice results in the aspiration of allergen into the airways and is a satisfactory alternative to aerosol. Adjuvants (aluminium hydroxide, Bordetella pertussis) are often used to potentiate the sensitizing effect of parenteral allergen. These models replicate other features of asthma, including eosinophil-rich inflammation. Studies on chronic models are comparatively few. Airway remodeling, a consequence of ongoing inflammation and repair, occurs following repeated allergen exposures in several species. The airways show an increase in ASM mass, subepithelial fibrosis, a chronic inflammatory cell infiltrate and epithelial dysfunction. Rats and mice are the principal models used to explore immunological mechanisms. Precise pulmonary mechanics measurements can be made in both species. However, the limitations of non-invasive techniques, popular for the assessment of respiratory responses to airway challenge in mice, need to be noted. The Brown Norway (BN) rat is the preferred rat strain because it develops a Th2 biased inflammatory response and high IgE levels following allergen exposure. It has leukotriene-driven early and late responses to challenge, consistent with human asthma. Adoptive transfer techniques are well described for the study of T cells in this model; antigen primed CD4+ T cells cause late responses, AHR and eosinophilic inflammation, independently of IgE [25]. Mice are used for trait analysis and knockout and transgenic models have identified central roles of several cytokines in airway inflammation (Tables 1 and 2). The architecture of murine airways differs from larger mammals, being only a few cell layers thick and devoid of a bronchial circulation. The alterations in lung function induced by allergen are very dependent on airway closure. Epithelial function is central www.drugdiscoverytoday.com

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Table 1. Knockout models used to study asthma Knockout models Adenosine deaminase Adenosine(3) receptor Aquaporin 5 (AQP5) B cell b-AdrenoReceptor b-Arrestin-2 C3a receptor Calcitonin gene-related peptide (CGRP) CD23 CD28 CD4 CD40 Clara cell secretory protein (CCSP) Class IA phosphoinositide-3 kinase (PI-3kinase) C2GlcNAcT c-Rel Eosinophil peroxidase (EPO) EP receptor E-selectin FceRIa Gq-deficient GRK3 GRK5 IgE Inducible costimulator (ICOS) Inducible T cell kinase (ITK) Intercellular adhesion molecule-1 (ICAM-1) Invariant chain (Ii) IP receptor Ja18 JunB-deficient CD4+ T cells L-Histidine

decarboxylase LPS binding protein (LBP) mAChR1, 2, and 3 Major basic protein-1 (mMBP-1) Mast cell MMP-2 MMP-9 nNOS NOD NOS1, 2, and 3 OX40 ligand p50 subunit of nuclear factor k B p75-neurotrophin-receptor PDE4D Plasminogen activator inhibitor (PAI)-1 Poly(ADP-ribose) polymerase-1 (PARP-1) Proteinase-activated receptor 2 (PAR2) P-selectin STAT6 T cell T-bet TCRb TCRd Cytokine-related knockouts IFN-g receptor IL-10 IL-12

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Table 1 (Continued ) Knockout models IL-12p40 knockout DC IL-12Rb1 IL-12Rb2 IL-13 IL-13R a 2 IL-18 IL-1R1 IL-4 IL-4R a IL-5 IL-5R Interferon-g TNF-a TNF-a receptor knockout (TNFR) Chemokine-related knockouts CCR3 CCR4 CCR5 CCR6 CCR8 CXCR2 Eotaxin IP-10

to murine asthma. Murine models of asthma have exploited the distribution of Clara cells (CC) throughout the airways. The 10 kDa protein (CC10) promoter allows selective overexpression of effector molecules of interest. A confounding variable with most transgenic models is that effects begin in utero and it can be difficult to separate developmental changes from pathology. This can be addressed using inducible tetracycline-based CC10 models. This is well illustrated by the studies of Elias and co-workers [26] who have identified the importance of IL-13 in such a model, upregulating vascular endothelial growth factor (VEGF) and TGF-b, in addition forming a positive feedback system with adenosine. Studies to date in other animal models of asthma have shown similar responses to the above-mentioned murine and rodent models. The allergic sheep model has been extensively characterized pharmacologically. These animals have early and late responses and AHR after airway allergen challenge. Although these animals are easily instrumented and studied they are not readily available. The rhesus monkey can develop an asthma-like phenotype on sensitization with dust mite allergen [27] and recent reports indicate remodeling can occur with post-natal exposure to allergen. The consistency in findings among human, sub-human primates and small animal models is reassuring. The horse has a clinical disease termed heaves that is similar to human allergic asthma. Although characterized by neutrophilic inflammation, the T cell cytokine expression profile is Th2 in type and the airways show extensive remodeling similar to the human condition [28].

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Table 2. Transgenic models used to study asthma Antigen-specific IgE transgenic mice b-2AdrenoReceptor CCR5 CD23 DO11.10 (ova specific TCR) Dominant-negative TGF-b type II receptor Endothelin-1 Eotaxin hCysLT(1)R HLA-class II HLA-DQ/hCD4+ HLA-DQ6/hCD4+ HLA-DQ8 Human ALX (Lipoxin A4 receptor) Human haptoglobin IL-11

IL-13 IL-2Rb/IL-4Ra chimera IL-4 CC10 IL-5 IL-6 IL-9 CC10 IP-10 Nerve growth factor (epithelium) PAF receptor (PAFR) Proteinase-activated receptor 2 (PAR2) RANTES SMAD7 SOCS-3 T cell receptor-transgenic CD4(+) T cell-specific dnRas Thymidine kinase

Non-allergic models of asthma There is potential to over-emphasize the role of acquired immunity in asthma through allergen-based models. Other stimuli are important. Dry air hyperpnea causes a neurokinin and prostanoid dependent bronchoconstriction in several species. This challenge mimics the conditions that trigger exercise induced asthma. The guinea pig is perhaps the best responder to this challenge modality. The responses of the dog to dry gas challenge can be measured using a bronchoscope to deliver the dry gas and to record the peripheral airway and lung responses to challenge [29]. Ozone and chlorine exposures of various species have been explored as causes of airway inflammation and AHR and are useful models of irritant-induced asthma. Mouse models of viral infection have been developed to address both acute infection and chronic airways disease. RSV instillation into the airways predominantly causes epithelial infection and a transient viremia. Viral clearance is linked to IFN-production and rapid natural killer T cell response. RSV induces both Th1 and Th2 responses and seems to predispose to allergen induced asthma in the post-infection period. AHR is augmented by exposure to allergen synchronously with RSV [30]. In vitro models of RSV infection with human rhinovirus (HRV16) have proved difficult to develop but have been successfully developed with the minor subtype HRV1B, a factor dependent on intracellular adhesion molecule 1 (ICAM-1). Paramyxovirus murine models show AHR that persist for up to a year, independent of the acute inflammation but dependent on ICAM-1 [31].

In silico models Gene chips in asthma Conventional genotyping of populations has identified genes of interest, such as interleukin-4 receptor a (IL-4Ra), in atopy and asthma. Replication of findings in different populations

has met with variable success; this implies that genetic risk factor stratification will differ, perhaps substantially, between populations. Single gene analysis cannot also address wholegenome changes in asthma. Despite these caveats, genomewide screening has identified several genes of interest in asthma along with several single nucleotide polymorphisms (SNPs) that can be of importance (e.g. in IL-13, IL-4R-a, ADAM-33 and CTLA4). In contrast to genotyping-based approaches, gene chips or microarrays are available to assess whole genome changes in asthma by simultaneously quantifying the expression levels of thousands of genes. The analysis of gene chips is of greatest utility from a hypothesis-generating standpoint by identifying hundreds of differentially expressed genes. Genes of interest (arbitrarily defined usually as genes with at least a two-fold change in expression) can be selected for further study using complementary techniques such as quantitative polymerase chain reaction (PCR). Analysing the pattern of altered genes using cluster analysis can identify trends in the genomic expression response. The interpretation of large data sets generated by most gene chip experiments can be problematic; typically, hundreds of genes are altered by experimental conditions. One approach that has been used successfully is to profile two models of asthma and identify genes similarly regulated in both. The smaller data set is more probable to contain genes of importance. For example, Zimmermann et al. [32] analysed two different murine models of allergic airways disease with gene chips and identified genes involved in arginine metabolism as important mediators of AHR. The two allergic airways models generated distinct transcript profiles. Importantly, the authors linked arginine metabolism with IL-13 and performed supportive experiments with transgenic mice overexpressing IL-4 and signal transducer and activator of transcription 6 (STAT6). This emphasises the need for complementary conventional molecular approaches to follow up on in silico data. Genes of interest can be investigated using conventional genotyping techniques. Karp et al. [33] have examined innate AHR in murine strains to identify asthma susceptibility genes using a combination of microarray technology, quantitative trait locus analysis and SNP genotyping. The authors generated crossbreeds of susceptible and resistant mice and matched chip data to AHR. This approach identified complement factor 5 (C5) as a gene of interest; C5 regulates IL-12 production, a cytokine involved in T cell differentiation. The response of individual cell types to cytokines can also be addressed using gene chips. For example, the response to IL-13 of human bronchial epithelial cells, human bronchial smooth muscle cells and human lung fibroblasts showed no common gene co-regulation among the three cell types and minimal overlap between groups [34]. Gene chip analysis of whole lung or bronchial biopsies can, therefore, suffer from data loss because of differential expression among cell types. www.drugdiscoverytoday.com

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These data also imply that any change in the cellular composition of the airways can introduce bias into microarray studies of asthma; a potential solution could be the use of laser capture microscopy. Microarrays can be performed on human endobronchial biopsies for analysis of genes involved in asthma pathogenesis or response to treatment. In one such study, a comparison of gene expression profiles in asthmatics and controls using bronchial biopsy specimens was made. Seventy-nine genes differentially expressed in asthmatic bronchi were identified, and microarrays were repeated in the asthmatic patients after inhaled corticosteroids, with onethird of the genes responding [35]. This type of comparison could allow meaningful interpretations of responses to drug therapy in asthma and could allow individualization of therapy in the future. A further advantage of this approach is that unannotated genes, or expressed sequence tags, can be assessed simultaneously with known genes. Although not widely available as yet, it is possible to perform genotyping using microarrays to identify single nucleotide polymorphisms in genes of interest. In silico approaches to studying asthma are useful in addressing aspects of asthma that are difficult to assess using standard techniques, yet can also complement in vivo and in vitro approaches. Careful consideration needs to be given to study design and findings need to be confirmed using complementary techniques.

Applications of mathematical modeling to asthma The effects of airway remodeling in asthma have been addressed using mathematical techniques and useful predictions have been made. Mucosal folding, subepithelial fibrosis and thickening of the airway wall are all predicted to influence airway narrowing. Inflammation reduces the load impeding ASM contraction [36]. Increased force generation by hyperplastic smooth muscle and faster shortening velocity

can enhance airway narrowing. Mathematical models suggest that increased smooth muscle mass is the single most important factor in AHR. Although most structural changes in the asthmatic airway potentially contribute to enhanced airway narrowing, some of the changes, such as collagen deposition, can limit airway narrowing. Thickening of the airway wall could require more force to effect the same airway narrowing [37]. Anatomically accurate computational models derived from high-resolution CT scans of mouse lungs might augment the predictive value of mathematical modeling. Species differences will of course be important factors in applying findings in animal models to humans. An integral aspect of asthma therapeutics is drug delivery to the airways. Inhaled therapy offers the opportunity to minimize side effects but poses the challenge of effective delivery. Airway and airflow modeling has been applied to both murine and human subjects in an effort to improve drug delivery of medications under normal and disease conditions. Modeling of the effects of ventilation and airway narrowing on aerosol delivery in human airways using single photon emission computerized tomography (SPECT) and MRI holds great promise as a tool to improve drug delivery in asthma. Particle size and ventilatory parameters affect aerosol deposition, with large airways receiving relatively larger doses than peripheral airways because of substantial differences in crosssectional surface area. Modeling also indicates that delivery of aerosols is reduced during asthma exacerbations; inhaled therapy can be more effective in preventing, rather than treating, exacerbations. Finer aerosols will be more successful in reaching the peripheral airways [38]. Combining anatomical data and computational modeling techniques with lung mechanics measurements, Bates and co-workers [39] have identified airway closure as an important factor in murine models of allergic airways disease, linking closure to mucosal thickening and altered surface tension in the

Table 3. Comparison summary table In vitro models, lung explants

In vivo models, Brown Norway rat

In silico models, gene chip comparison of two murine models of allergen-induced AHRa

Pros

Allow pharmacological and immunological studies on intrapulmonary airways in small animals Preservation of lung architecture

Naturally atopic Inbred strain Reproduces most of the characteristics of human asthma

Reduces the number of potential genes of interest Hypothesis generating Provides genomic phenotype of asthma

Cons

Isolation from bone marrow and circulation Heterogeneous airway responses

Less readily manipulated than murine models Variability in characteristics as a function of source

Less sensitive to absolute changes in copy number Cost Uncertain number of false positives

Best use of model

Structure–function correlations on isolated intra-parenchymal airways in small animals

Allergen induced airway responses, inflammation and remodelling

Identifying novel asthma-related genes

Reference

[1]

[40]

[32]

a

Airway hyperresponsiveness.

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air–liquid interface. High-resolution CT scans of murine lungs will undoubtedly provide useful information in modeling AHR.

Conclusions Asthma is a complex disease that requires adequate models for study of its different components (Outstanding issues). These have been successfully developed for the in vivo study of the bronchorelaxant and bronchoprotective effects of various drugs. A substantial range of animal models of allergic sensitization is available for the study of airway responses to challenge with allergen. Large animals such as the allergic sheep have been very informative in the past but are less convenient for studies requiring terminal experiments or expensive reagents. Anti-inflammatory mechanisms of drug actions, including immunobiologicals are currently best studied in murine and rodent models. T cells and cytokine related airway responses to allergen are well characterized in both mice and rats. The murine models are heavily dependent on changes in epithelial function. The vast majority of studies have been performed in acute models of ‘asthma’. Features of asthma such as airway remodeling require repeated airway insults. Few well-characterized models are as yet available. However, the BN rat undergoing repeated allergen challenge shows hyperplastic muscle growth and alterations of the matrix proteins and the sensitivity of some of these processes to leukotrienes and endothelin have been demonstrated. There is a paucity of studies addressing the phenotypic changes of structural cells such as smooth muscle following remodeling. Such information is essential for the prediction of the functional consequences of the observed remodeling. Murine models are emerging for the study of allergen driven remodeling. The horse with heaves can be useful for the study of drug therapy for remodeling in a ‘real life’ setting of episodic and uncontrolled allergen exposures. Models of non-allergic triggers of asthma such as dry gas challenge, oxidant exposed animals and viral infection are also available. In summary, all of the major characteristics of human asthma have been well modeled in animals. In vivo models are complemented by in vitro and in silico studies, their relative features being summarized in Table 3. Cultured cells allow the dissection of pharmacological responses and signaling pathways activated in airway cells of interest, an area of paramount importance in drug development. Cells cultured from the airways of asthmatics retain certain characteristics of relevance, such as faster growth rates for smooth muscle and decreased collagen degradation by fibroblasts. Gene chips, by contrast, provide a map of the genomic expression response to allergen, bronchoactive agents and pharmaceuticals. Dissecting the disease causing mechanisms from beneficial adaptive responses will require a concerted investigation of in silico and in vivo models.

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Outstanding issues  Molecular mechanisms of airway dysfunction in asthma models.  Development and characterization of chronic models.  Elucidation of the growth mechanisms of airway smooth muscle in vivo.  Clarification of the phenotypic changes of airway structural cells in asthma.  Establishing the links between immune responses, inflammatory cells and AHR in asthma.  Potential reversibility of established airway remodelling.  Clarification of the reliability and pertinence of gene chip findings for asthma.

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