Airway smooth muscle dynamics: a common pathway of airway obstruction in asthma

Difference in response to deep inspiration implicates smooth muscle involvement

Fish et al. 16 were the first to show that a difference in response to deep inspiration distinguishes asthmatic from nonasthmatic subjects. They compared the effect of deep inspiration on G_aw and forced expiratory volume in one second (FEV₁) before and after inhalation of methacholine in asthmatic and nonasthmatic allergic subjects (i.e. rhinitis sufferers). Subjects with allergic rhinitis responded to deep inspirations in a similar way to healthy subjects 15, 16. That is, prior to bronchoprovocation, deep inspiration induced no change in the G_aw, but, after methacholine-induced bronchoconstriction, deep inspiration caused a marked increase in G_aw. Conversely, the G_aw of asthmatic subjects decreased transiently after a deep inspiration in the baseline state, and deep inspiration after methacholine-induced bronchoconstriction caused no significant improvement in G_aw. Subsequent studies of induced bronchial obstruction have shown that timing of the measurements after a deep inspiration is important. Pellegrino et al. 19 showed that with methacholine-induced obstruction in asthmatics, a deep inspiration caused consistent, but transient, bronchodilation when lung resistance was measured on a breath-by-breath basis. The rate of restitution of pre-deep inspiration R_aw was much more rapid in asthmatics than in nonasthmatics. Jensen et al. 21 confirmed that after airway provocation at the peak of a deep inspiration, healthy subjects could still reduce their R_aw (i.e. dilate their airways) to levels similar to that achievable at baseline. In striking contrast, at baseline, asthmatics dilated their airways less with a deep inspiration compared with healthy subjects, and their dilating ability was subsequently diminished after provocation. Moreover, the same study suggests that airways re-constrict faster in asthmatics than in healthy subjects. Jackson et al. 35 showed consistent findings and found an exponential return to pre-deep inspiration levels, a finding compatible with a first order process; whereas, Thorpe et al. 36 reported a time course following a power law, which is inconsistent with a first order process. Nevertheless, these findings are all consistent with the greater velocity of contraction of asthmatic smooth muscle, as has been shown by Ma et al. 37 in isolated human asthmatic ASM cells and by Mitchell et al. 38 in passively sensitised human bronchial smooth muscle. In the studies by Pellegrino et al. 19 and Jackson et al. 35, the relative magnitude of the reversal was less in asthmatic subjects. Thus the relative magnitude of the reversal, rather than the reversal itself, appears to differentiate the asthmatic response to induced obstruction. The constrictor response to a deep inspiration with spontaneous obstruction, however, clearly distinguishes the asthmatic subjects.

Possible roles of ASM in mediating effects of deep inspiration

What makes asthmatic airways uniquely different in their response to deep inspiration? At least three possible answers are presented in the literature. The first is provided by Burns et al. 17, Pichurko et al. 39, Lim and co-workers 40, 41 and Pliss et al. 42, which is based on the concept of relative hysteresis of airway versus lung and first developed by Froeb and Mead 43. They found, as did Fish et al. 16, that in asthmatics, not only did deep inspiration fail to reverse spontaneous obstruction, it sometimes resulted in worsening of airway narrowing. Furthermore, Lim and co-workers 40, 41 showed that the degree of bronchoconstriction induced by deep inspiration was related to the severity of spontaneous obstruction at the time of testing. That is, asthmatic subjects with severe spontaneous airway obstruction responded to deep inspiration with a further decrement in maximal expiratory flow (on maximal as opposed to partial expiratory flow–volume curves). The deep inspiration-induced bronchoconstriction waned during the course of intensive anti-inflammatory treatment, and when the same subjects were subsequently challenged to reduce their maximal expiratory flow to a level comparable to that during the spontaneous exacerbation of their disease, deep inspiration became an effective bronchodilating manoeuvre. These effects are illustrated in figure 1⇓. Using the concept of relative parenchymal airway hysteresis, Lim and co-workers 40, 41 postulated that during a spontaneous asthma attack, peripheral parenchymal hysteresis was much greater than that of the airways. An increase in lung pressure–volume hysteresis is characterised by greatly increased elastic recoil pressures during inflation and much lower recoil pressures during deflation at any given volume. As the forces acting on the airways are proportional to the recoil pressures, diminished radial forces acting on airways during deflation will lead to smaller airways than before a deep inhalation, with a concomitant increase in resistance. Although bronchial provocation does not explicitly alter parenchymal tissue properties 44, it is likely that ASM participates in changes in both parenchymal and airway hysteresis. One important factor determining the magnitude of airway hysteresis is ASM tone and the response of that tone to the stretch induced by deep inspiration; this will be discussed later in the current study. Therefore, the analysis of Burns et al. 17, Pichurko et al. 39, Lim and co-workers 40, 41 and Pliss et al. 42 highlights the importance of ASM in mediating the deep inspiration-induced changes in airway calibre.

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Fig. 1—

Maximal (M)/partial (P) ratio measured during spontaneous asthma (•), following treatment (○) and following acutely induced bronchoconstriction (□; induced by either dry air or histamine). To calculate the M/P ratio, the maximal mid-expiratory flow on a P and complete M expiratory flow volume curve are compared. An M/P ratio of 1 indicates that the deep inspiration accompanying the complete flow–volume manoeuvre causes neither an increase nor a decrease in mid-expiratory flow. An M/P ratio of <1 means that the deep inspiration decreased mid-expiratory flow; while an M/P ratio of >1 indicates that the deep inspiration increased mid-expiratory flow. During spontaneous asthma, a deep inspiration causes further airway narrowing. This paradoxical bronchoconstriction declines with effective treatment and, when comparable airway narrowing is induced by inhalation of histamine or dry air, a bronchodilating effect of deep inspiration is apparent. FEV₁: forced expiratory volume in one second; % pred: % predicted. Modified from 41 with permission.

The second possible answer takes a complementary view to that of the first [17, 39, 40–42] and is based on a distinctly different mechanism 22, 23, 45–51. Both Gunst and co-workers 22, 23 and Fredberg et al. 52 focused not so much on the parenchyma and changes of parenchymal hysteresis, but rather called attention to the changes in ASM itself. They subjected isolated bronchial segments and isolated ASM tissues in vitro to tidal oscillations and observed that the oscillatory perturbations caused a marked reduction in the contractile responsiveness of the tissues. These observations suggested that volume or load oscillations imposed on the airways during normal breathing might be an important physiological mechanism for reducing airway responsiveness in vivo. Gunst 22 initially suggested that the reduction in airway muscle stiffness and contractility caused by tidal volume oscillations might result from a slow rate of cross-bridge cycling relative to the oscillation rate of the muscle. However, Gunst and co-workers 53–55 subsequently hypothesised that the mechanical stretch and oscillation of ASM might trigger changes in the organisation of the cytoskeletal and contractile filaments that decreased the stiffness and contractility of the muscle.

Fredberg et al. 52 noted that the increase of muscle hysteresis referred to by Lim and co-workers 40, 41 and Sasaki and Hoppin 56 and upon which their argument hinges, happens if, and only if, the muscle is activated and subject to large ongoing tidal stretches; the opposite response occurs to smaller tidal stretches. That is, the hysteresis decreases and does so rather dramatically. Moreover, this progressive decrease of muscle hysteresis with activation has been linked directly, quantitatively, and at the molecular level to progressive decreases of shortening velocity, downregulation of cross-bridge cycling rates, decreases of myosin adenosine triphosphatase activity and the conversion of rapidly cycling cross-bridges to slowly cycling latch-bridges, all of which are now understood to be characteristics of the different phases of smooth muscle activation 57–59. In the process of activation and tension generation, the muscle can become so stiff that in response to a deep inspiration, it stretches little; it can then become frozen, and virtually stuck in the high-stiffness, low-hysteresis latch state. Such behaviour is consistent with observations from studies in isolated nonasthmatic ASM or in situ in human asthmatics 21, 27, 58, 60, 61. Such an outcome becomes all the more likely when the ASM mass is increased, when the muscle becomes uncoupled from the lung parenchyma, when expansion of the chest wall is restricted, or when large lung inflations are prohibited during a bronchial challenge 18, 62, 63, all of which are factors that reduce the stretch experienced by the smooth muscle and circumstances relevant to AHR 49, 64. That being the case, this muscle-based molecular mechanism explains not only how the airways can become refractory to the effects of a deep inspiration, but also how it can exhibit a bronchoconstrictor response.

The third possible answer stems from studies in which the protective effect of deep inspirations taken before experimentally induced bronchoconstriction is examined 18, 27, 60, 65–68. Malmberg et al. 65 were the first to notice that if a deep inspiration was taken shortly before (<6 min) acutely induced bronchoconstriction, the extent of airway narrowing during the induced constriction was reduced. Subsequently, Scichilone et al. 68 found that deep inspiration exhibited its bronchoprotective effect only in healthy subjects. Using a somewhat different protocol, King et al. 60 found that deep inspiration also had a limited bronchoprotective effect in mild asthmatics. This cannot be easily explained by the mechanical perturbation of activated smooth muscle described immediately above. However, length oscillation applied to resting ASM was observed to diminish the muscle's ability to generate force in the subsequent contractions 69. It was postulated by King et al. 26 that this post-oscillation refractoriness of resting ASM was responsible for the bronchoprotective effect of deep inspiration taken before bronchoprovocation seen in nonasthmatics, and that in asthmatics, this refractoriness might be absent. Crimi et al. 70, however, found that this effect was only evident when lung function parameters preceded by a full inflation were used, such as FEV₁ or forced vital capacity. Using parameters not preceded by full inflation, (e.g. isovolume partial forced expiratory flow, residual volume after partial expiration, or specific G_aw), multiple deep inspirations taken prior to methacholine inhalation were found to be associated with increased rather than depressed response, especially in asthmatics. Whether the response to deep inspiration taken before bronchoconstriction is associated with ASM is still a matter of debate.

Morphological evidence of ASM involvement in asthma

There is general agreement that the total amount of smooth muscle is increased in asthma 71–73, and increased muscle mass, even without a change in contractile phenotype, has the potential to enhance airway responsiveness, thus contributing to asthma symptoms 74. The increased area of the layer of smooth muscle involves small and large airways and may be related to the clinical severity 75 and duration 76 of asthma. Modelling studies predict amplified responsiveness consequent to increased smooth muscle mass. It is important to recognise that this prediction relies on the assumption that the maximal tension that the muscle can generate increases in proportion to its mass. Thickening of the muscle layer alone without such an assumption will only mildly enhance reactivity 74, 77. Measurements of the area of ASM have usually been made on transverse sections of airways obtained from autopsies. Results from these measurements will depend on the number and size of smooth muscle cells, as well as the extracellular matrix (ECM) between cells 78. There have been three studies showing more cells (hyperplasia), two in cases of fatal asthma 72, 79 and one in a case of asthma of mild-to-moderate severity 80. Although Ebina et al. 72 also reported hypertrophy of ASM cells, their measurements included ECM and therefore may have over-estimated cell size.

Benayoun et al. 81 excluded the ECM elements from their measurements and reported hypertrophy of ASM based on cell thickness in patients with severe persistent asthma, based upon thickness of the smooth muscle cells. However, the effects of cell shortening on cell thickness may have confounded these measurements. Bai et al. 76 showed both increased smooth muscle and ECM within the smooth muscle layer in cases of asthma. This study was undertaken on “thick” (relative to submicron-thin sheets of ECM) sections and may have overestimated the true volume fraction of the ECM due to an overlap effect. To date, there are few published quantitative studies regarding the type of ECM within the smooth muscle bundle in asthmatic airways. Recently, Pini et al. 82 have shown that the area fractions of the proteoglycans lumican and biglycan, but not decorin or versican, are increased within the smooth muscle layer in cases of moderate asthma, compared with both nonasthmatic and severe asthma cases. Changes in the matrix components of ASM may alter the mechanical properties of the muscle.

There are no published studies showing increased proliferation of smooth muscle in situ in asthma. Data presented in abstract form 83–85 have not shown differences between cases of asthma and nonasthmatic controls using either proliferating cell nuclear antigen or cyclin D1 to label proliferating smooth muscle cells. Conversely, increased expression of proliferation markers has been observed in the epithelium of patients with asthma 86, 87. The increased expression of proliferation markers in ASM in animal models of asthma 88 suggests that, in humans, proliferation may not be an important mechanism for increased ASM in asthma. Alternate explanations may be that current techniques do not detect subtle increases in the rate of smooth muscle proliferation in the chronically inflamed airway, or that increased proliferation occurs in “bursts”, in relation to airway development, early at the “onset” of asthma or during episodes of acute inflammation. Since different signals may be involved in smooth muscle hypertrophy, hyperplasia or increased deposition of ECM, sorting out the contribution of these elements to the increased thickness of the smooth muscle layer in asthma is an important research goal.

Apoptosis and increased ASM mass

Reduced apoptosis could in part explain increased ASM mass in asthmatics and account for limited evidence of increased proliferation of ASM cells in situ 89. Peptide growth factors, which are elevated in asthmatic airways, play pivotal roles in modulating cell survival, proliferation and migration in the lung 90. For example, fibroblast growth factor, which is elevated in premature and neonatal lungs exposed to hyperoxia, suppresses apoptosis required for normal alveologenesis, thus underpinning the development of chronic neonatal lung injury 91, 92. To date, only a few studies have assessed the impact of asthma-associated biomolecules on ASM apoptosis.

Human ASM cell apoptosis is potentiated by TNF-α, which concomitantly upregulates myocyte-expressed Fas, a transmembrane component of the TNF-α receptor 93. Binding of inflammatory cell-derived Fas-Ligand (FasL) greatly induces human ASM apoptosis 93. However, the pro-apoptotic role of TNF-α on ASM in vitro is concentration dependent 94, and appears to be modulated by other mediators, since pro-mitogenic effects of TNF-α have also been reported 94, 95. Notably, cardiotrophin-1, a member of the gp130/IL-6 cytokine family that is synthesised by ASM cells, inhibits, via p42/p44 mitogen-activated protein kinase (MAPK), human ASM cell apoptosis induced by serum deprivation or FasL/TNF-α exposure 96. A subsequent study revealed that endothelin-1, which is elevated in asthmatic airways 97, may also promote increased ASM mass via its effects on cell growth and apoptosis 98. Indeed, endothelin-1 markedly reduced the apoptosis of human ASM cells induced by serum withdrawal, and, in combination with cardiotrophin-1, further promoted ASM hypertrophy and accumulation of contractile apparatus-associated proteins 96, 98. Freyer et al. 99 reported that ECM components, such as fibronectin, laminin-1 and collagens I and IV, provide strong survival signals to cultured human ASM cells, an effect that is mediated, in part, via integrin α5β1. Interestingly, proteinases derived from inflammatory cells, such as neutrophils, degrade ECM proteins, including fibronectin, to promote human ASM apoptosis 100. Collectively, these observations suggest that inflammation and changes in ECM associated with airway remodelling in asthma could directly modulate apoptotic responses of ASM cells.

The few studies of apoptosis of ASM in animal models of airway remodelling support the notion that the rate of apoptosis could be a contributor to the regulation of ASM mass. Using an adoptive transfer model of CD4+ T-cell-driven remodelling, Ramos-Barbon et al. 101 observed a reduction in ASM apoptosis at the same time that there was an increase in proliferation of ASM. The airway remodelling was triggered by three consecutive allergen exposures. Interestingly, a change in matrix proteins was also induced by allergen challenge in this model. In horses with heaves, a form of equine asthma, increase in ASM proliferation was also confirmed by proliferating cell nuclear antigen immunoreactivity. However, there was an increase in rates of apoptosis 88. In the steady state, it seems more likely that an increase in apoptosis will occur to adjust tissue mass. Therapeutic approaches to alter rates of apoptosis might well be useful to diminishing airway remodelling. Peroxisome proliferator-activated receptor (PPAR)-γ ligands have also been shown to reduce ASM proliferation in response to thrombin, basic fibroblast growth factor and foetal bovine serum 102, 103. The PPAR-γ ligand ciglitazone induces ASM cell apoptosis 102 at concentrations exceeding that required to inhibit proliferation 103. Future studies are needed in this important area to assess the therapeutic potential of pro-apoptotic agents in reducing or reversing ASM tissue thickening.

ASM growth responses

The controversies over the relative importance of hyperplastic and hypertrophic growth in asthma appear to have had little influence on the extent to which smooth muscle proliferation or hypertrophy have been explored in culture as potential mechanisms for the increase in smooth muscle volume. Proliferation has been extensively explored in smooth muscle cultures from post mortem tissue, resections, lung transplants and biopsies. There are a number of recent studies that identify the diversity of growth stimuli that range from growth factors (fibroblast growth factor (FGF)-β), spasmogens (cysteinyl leukotrienes), cytokines (TNF-α), and the ECM components (monomeric type I collagen) through to physical influences, such as stretch (supra-tidal breathing volumes) and stasis, when smooth muscle is cultured on flexible supports that can be subjected to pressure cycles that simulate the impact of different breathing patterns on the forces acting on smooth muscle 104–106. Furthermore, signal transduction pathways have been extensively examined, leading to identification of roles for extracellular signal-related kinase 1 and 2, phosphoinositide-3 kinase and p38MAPK in the initial signalling followed by activation of cyclin-dependent kinases and diminished levels of cyclin-dependent kinase inhibitors, including p27^kip1 andp21^cip1, which culminate in retinoblastoma phosphorylation to allow cells to continue through the final stages of G1 and onto S-phase.

The range of inhibitors of proliferation that have been identified include a number of endogenous regulators including 2-methoxyestradiol 107, cortisol, adrenaline, natriuretic peptides and interferon-β 108. There has been an even greater level of interest in the impact of existing and potential anti-asthma agents. Both short- and long-acting β₂-adrenoceptor agonists reduce proliferation, as do synthetic glucocorticoids, including budesonide and fluticasone propionate, which are used clinically in combination with the long-acting β₂-agonists (LABA). Combinations of LABA and glucocorticoids show some synergy in regulating smooth muscle proliferation 109. However, there are differences amongst mitogens in their susceptibility to regulation, with thrombin responses appearing to be more readily regulated than those to stronger mitogens, such as FGF-β. Although stimulus-dependency of inhibition by glucocorticoids is also observed for cytokine production, this response is more readily regulated when thrombin, rather than IL-1α, is the stimulus 110.

The ECM may also modulate drug-responsiveness of remodelling responses, such as proliferation and migration 111, 112, as cells cultured on monomeric collagen show steroid-resistance whereas cells cultured directly on plastic or on laminin are highly steroid-responsive 113. Interest in migration of smooth muscle is increasing in light of the possibility that smooth muscle proliferation does not appear to take place in the muscle bundle, but rather in the subepithelial tissue, where the source of the proliferating cells may be stem cells recruited from the bone marrow 114, fibroblasts that have undergone differentiation 115 or smooth muscle cells that have migrated off the potentially antiproliferative ECM that is rich in proteoglycans and laminins 89. Migration is required for each of these alternative mechanisms for smooth muscle hyperplasia. Migration of ASM can be induced by platelet-derived growth factor 116, 117, enhanced by cys-leukotrienes 118 modulated by β₂-adrenoceptor agonists and glucocorticoids 119, with the latter agents losing activity when cells are seeded onto a matrix containing monomeric type-1 collagen 120, 121.

Hypertrophic growth of cultured ASM has been largely ignored, possibly because it is more difficult to establish. Work in human ASM identified the peptide cardiotrophin as an hypertrophic (and anti-apoptotic) influence 96, and in guinea pig ASM culture, IL-1 has reported hypertrophic actions. The most widely investigated hypertrophic factor for vascular smooth muscle, transforming growth factor-β, has no detectable effect on the size of cultured human ASM (T. Harris and A. G. Stewart, Dept of Pharmacology, University of Melbourne, Parkville, Australia; unpublished communication).

Removal of smooth muscle from asthmatic airways

Recently, another line of evidence supporting a role of ASM in asthma has emerged from studies in which ASM mass of asthmatic subjects was reduced partially and permanently through a procedure called bronchial thermoplasty 122. Rationale for the procedure is perhaps based on the fact that there is no clearly demonstrated physiological benefit of ASM contraction, and that only negative consequences accompany enhanced ASM tone 28, 123. It should be pointed out that there is evidence to suggest various roles of ASM contraction, from controlling the ventilation/perfusion ratio 124 to stiffening of airways 12, 125, 126. A review by Seow and Fredberg 28 gives more details. Although a complete ablation of ASM is not feasible with current technology, a recent report by Cox et al. 127, with comments by Bel 128, has shown that partial removal of ASM in asthmatic lungs alleviates asthma symptoms without introducing severe or persistent undesirable side-effects. By reducing the mass of smooth muscle in the walls of conducting airways (intralobar bronchi with diameters >3 mm), Cox et al. 127 showed in a group of mild-to-moderate asthmatics, that the treatment was effective in reducing AHR and asthma exacerbation, and that the reduction persisted throughout the 2 yrs following the procedure, during which the subjects were monitored. The direct effect of bronchial thermoplasty on airway calibre during methacholine challenge was assessed by Brown et al. 129, 130 and Cox et al. 127, who found that the decrease in ASM resulting from thermoplasty led to an increased airway calibre at any dose of inhaled methacholine, compared with the untreated airways. One cautionary note here is that airway calibre data relies on imaging that can detect only a limited number of airways per subject, with these being primarily larger airways. Based on imaging and oscillatory mechanical data 131, it is difficult to imagine that asthmatics do not experience substantial constriction in airways smaller in size than those treated with thermoplasty. Nevertheless, while the thermoplasty studies do not provide much insight into the pathogenesis of the disease, their results argue strongly that ASM is involved in the exacerbation of asthma. If the disease could be cured by eliminating the smooth muscle, there might be a shift in emphasis from understanding the pathogenic role of muscle in the disease to this novel therapy. However, the risk and cost associated with the procedure will probably warrant a continued search for a better therapy, of lower invasiveness and expense, based on a thorough understanding of the disease mechanism.

Coupling between the airway and the lung parenchyma in situ

First, the term “coupling” must be clarified. As lung volume increases, two forces act to potentially dilate the airways and hence stretch the ASM. One relates to the bulk modulus and is associated with the pressure difference acting directly across the airway wall, transmitted from the pleural space through the parenchyma and relative to the intraluminal pressure for each airway. This is sometimes referred to as airway–parenchymal interdependence. The other force relates to the shear modulus of the lung parenchyma and occurs because of local tethering, or pulling, of the surrounding parenchyma as it expands with lung volume 132, 133. The forces of interdependence have a potent inhibitory effect on airway narrowing and can prevent airway closure at high transpulmonary pressures 9, 12. In the context of lung inflation, the former force is likely to be more important than the latter 133, 134; however, shear forces have been thought to be important in the context of airway narrowing produced by bronchial provocation, where they may contribute to forces opposing airway narrowing and limit the capacity of the airways to close off or to reach high levels of resistance 8, 9, 12, 135, 136. Hence, what is of primary concern in the context of the present review is the coupling between the length of ASM and increases in the recoil pressure of the lung during a deep inspiration, rather than the coupling of the length of ASM and actual change in lung volume itself. This is rather important because, if there is a deficiency in the ability to stretch ASM with a deep breath, one needs to distinguish whether it arises simply from a reduced ability to generate an ∼2.9 kPa transmural pressure at total lung capacity (TLC), or whether it arises because of a real stiffening in the local airway wall, perhaps due to alterations in ASM properties or other wall constituents. A recent study by Fredberg and Kamm 137 gives a thorough description of force transmission in the lung.

Two recent in situ approaches show the best promise for assessing potential coupling. One method involves direct imaging of the airways. High resolution computed tomography (HRCT) has been used to show that airways dilate during lung inflation from functional residual capacity (FRC) to TLC 138–140. The studies by Brown et al. 138, 139 suggest that the degree of dilation is similar in milder asthmatics and healthy subjects, even after provocation. However, in the studies by Brown et al. 138, 139, only relatively few large airways were imaged in each subject and changes in airway dimension (during bronchial provocation and during a deep inspiration) occurring in the huge number of small airways were not accessible with the HRCT. Also, closure of these small airways will probably contribute to changes in lung volume (“air trapping”) and could affect coupling between the length of ASM and increases in recoil pressure. Subsequent imaging studies, reviewed by Brown and Mitzner 140, by the same group suggest that the constriction of airways is highly heterogeneous, that they may be capable of closing, and that airways in more severe asthmatics are likely to dilate less during deep inspiration than in those with healthy lungs. Of course, the imaging approach is inherently static, that is, each image is acquired during zero-flow conditions at a fixed lung volume. Thus, one cannot distil the dynamic force–length behaviour of the ASM in situ as it responds to deep inspiration. Another less explicit approach, but one that might permit inference of dynamic force–length behaviour, is to track R_aw (or conductance) during a deep inspiration using a forced oscillation method. The R_aw obtained from this method is used as a surrogate for airway diameter 21. An advantage of this approach is that it explicitly captures the functional impact of the deep inspiration on the net change in airway diameters averaged over the entire airway tree. Also, one can track the dynamics of airway reconstriction 21, 63, 141, which may be sensitive to altered ASM function in situ. A disadvantage is that one cannot distinguish airway dilation from either airway or alveolar recruitment. Generally, this approach shows that at TLC, despite achieving a normal recoil pressure, asthmatics cannot reach the same degree of reduction in R_aw at baseline as normal subjects 21. This problem is amplified by bronchoprovocation with inhaled methacholine (or similar bronchoconstrictors). Even if baseline R_aw increases in healthy subjects to that of symptomatic asthmatics, the asthmatics have a lesser decrease in R_aw with a deep inspiration 63. The implication is that the tracheobronchial tree is inherently stiffer in asthma 125. There is also evidence from animal models that allergen exposure increases airway stiffness 142.

An inherent limitation of these approaches is that one cannot determine whether the reduced ability to dilate airways, and hence to stretch ASM with a deep inspiration, lies at the level of ASM. For example, the reduced response to deep inspiration could be due to a change in airway distensibility due to remodelling in those portions of the wall distinct from the ASM, such as the adventitia, the lamina propria or the reticular layer under the airway epithelium, where deposition of connective tissue proteins can alter mechanical properties of the whole airway. Mauad et al. 143 have shown that the content of elastic fibres is decreased in the adventitial wall of the small airways in patients who died of asthma. This could contribute to the increased airway wall stiffness associated with asthma. A role for local connective tissue elements in the airway wall in the dynamics of bronchoconstriction has been shown in nondiseased isolated airways 144, 145. Airway remodelling is a well-documented phenomenon associated with asthma development. Thickening of some airway wall components seen in asthmatic airways may contribute to excessive airway narrowing 146; thickening of other components may have a protective effect against excessive narrowing 147–151. Many of these predictions were based on computer simulations of geometrical changes in airway components and, therefore, were limited by the assumptions associated with the simulation. As highlighted by Pare 152, “geometry is not everything”; analysis of the effect of airway remodelling has to be based not only on altered geometry, but also on the mechanical properties of the altered airway components. For example, deposition of connective tissue in the adventitia could attenuate the cyclically varying strain from the surrounding parenchyma to smooth muscle if the material deposited in this layer was stiff. Conversely, if the deposited material was highly compliant, the parenchymal strain could not be effectively transmitted to the smooth muscle either. It appears, therefore, that there should exist an optimal coupling stiffness that allows maximal transmission of undulating strain from parenchyma to smooth muscle in order for the airways to receive the maximal benefit of the bronchodilating effect of tidal breathing and deep inspirations.

In situ HRCT imaging of canine airways of sizes ranging 1.8–19.1 mm by Brown and Mitzner 153 showed that the diameters of fully relaxed airways increased with transpulmonary pressures up to 0.5–0.7 kPa, where they reached a plateau, suggesting that in these relatively large airways, the diameter does not relate linearly to the cube root of lung volume, at least not at volumes near TLC. This is in contrast to studies 154–156, which concluded that relaxed airways expanded isotropically with the lung. The discrepancy appears to be in the sizes of airways examined. Klingele and Staub 154 based their conclusion on the measurements of diameters of terminal bronchioles. Hahn et al. 156 showed that in airways <2 mm in diameter, isotropic expansion with lung volume was observed throughout the entire range of lung volumes in both relaxed and activated airways. The larger airways deviated from the isotropic behaviour when smooth muscle tone was introduced; they expanded significantly less with lung volume in the high volume range 156. The effect of smooth muscle tone was also observed by Brown and Mitzner 153. They showed that with a moderate tone, the airways were less distensible in the low-pressure range, but their diameters continued to increase with increasing transpulmonary pressure at high pressures.

In summary, there is accumulating in situ evidence suggesting that airways (especially small airways) in the lung are sufficiently compliant to be stretched during a deep inspiration, although the extent of stretch is still debatable. In other words, ASM in healthy lungs is likely to be stretched periodically during tidal breathing and deep inspirations, and responds to that stretch by relaxation, even after pharmacological stimulation. Modulation of the mechanical properties of ASM by oscillatory strains, either in the resting or activated state, is discussed elsewhere in the present study. In asthma, it appears that ASM is either refractory to the modulating effect of oscillatory strain, or that the oscillatory strain is not transmitted to the ASM.

AHR and lung volume

Although ASM contraction may not be the only factor that contributes to AHR, it is the final common pathway leading to excessive airway narrowing. Other components, such as inflammatory changes and cellular infiltrates, and structural changes, such as increased or decreased elastin and collagen, as well as proteoglycans, can modify the ASM responses and thus contribute to AHR. These latter changes are commonly referred to as airway remodelling, which has been defined as changes in the composition, quantity and organisation of the cellular and molecular constituents of the airway wall. The topic of airway remodelling is discussed in more detail elsewhere in the present study. Certain aspects of airway remodelling may affect the relationship between AHR and lung volume, while other types may not. For example, airway wall oedema may thicken the wall, but may have little effect on the airway luminal diameter. Brown et al. 157 have shown in a sheep model that airway oedema has a minimal effect on R_aw in general, and on the relationship between lung volume and extent of airway constriction, specifically. They found that a 50% increase in airway wall oedema caused a 13% decrease in airway luminal area; and at high lung volume, luminal area was not affected by the oedema. These observations are consistent with another study in which the effects of lung volume on oedematous airways were measured using HRCT 158. In that study, airway narrowing caused by wall oedema at low lung volume was completely reversed by lung expansion. These findings in sheep are analogous to the changes in airway dimensions observed using computed tomography scanning in patients with asthma. Nakano et al. 159 showed that although patients with asthma had increased thickness of the airway wall, their lumen area was not significantly reduced, compared with nonasthmatic control cases.

In contrast, Brown et al. 138, 139 found that the airway wall thickness was increased and the lumen diameter was decreased in patients with mild asthma. In that same study, it was shown that the airway dilatation caused by lung inflation was comparable in asthmatics and healthy individuals. That is, both at baseline and after methacholine challenge, deep inspiration dilated airways to a similar extent. However, after the lung inflation manoeuvre, when the lung volumes returned to FRC, there was a difference in response. The airways of the healthy individuals remained somewhat dilated, while the airways of the subjects with mild asthma constricted further. This visual evidence of differential hysteresis confirms the early studies of Fish et al. 16, and suggests that the difference between mild asthmatics and nonasthmatics is not in the transmission of the strain to the airway. It remains to be determined whether a comparable dilatation occurs following deep inspiration in moderate or severe asthmatics in whom airway remodelling may cause baseline airway narrowing and/or an increase in airway wall thickness.

Response of activated ASM to oscillatory stress or strain

As discussed briefly above, it has been known for a long time that length or force oscillations at physiologically relevant frequencies and amplitudes applied to activated ASM result in a reduction in the ability of the muscle to contract 22, 23, 45, 56, 58, 160–164. Evidence that tidal volume oscillations had similar effects on airway calibre and R_aw in experimental animals in vivo led Warner et al. 24, Tepper et al. 25 and Shen et al. 160 to suggest that the oscillation of the airways that occurs during tidal breathing might be a physiologically important mechanism for decreasing airway responsiveness.

A theoretical explanation for this muscle behaviour was provided by Fredberg et al. 58 based on predictions by Mijailovich et al. 59 of a four-state cross-bridge model of smooth muscle contraction. Fredberg et al. 58 and Mijailovich et al. 59 were able to describe the relationship between oscillation amplitude and the extent of shortening in the muscle, and these studies 58, 59 suggest that if tidal breathing or deep inspiration causes mechanical perturbation to contracted ASM in situ, bronchodilation could result. The relatively small amplitude of length oscillation (comparable to that associated with tidal breathing) required to produce a substantial decrease in active force in ASM prompted Gump et al. 163 to suggest that tidal breathing is a potent bronchodilator.

Structural rearrangement of cytoskeleton and contractile apparatus during active contraction may also be responsible for the bronchodilatory effect. Fredberg et al. 58 observed that after large-amplitude oscillations, a muscle temporarily lost some of its ability to shorten, and this loss of ability was not related to perturbed actomyosin interaction, but was likely to be related to plastic rearrangement of cell structure. It has been theorised that actin filament length may be a factor determining the extent of muscle relaxation (lengthening) due to oscillatory stress 165. Abnormally long actin filaments may increase the range of sliding of contractile filaments without diminishing the overlap between myosin and actin filaments, and thus render the muscle more resistant to the relaxing effect of oscillatory stress. This was proposed as one of the possible defects of asthmatic ASM 165. Computer simulation of this scenario 166, 167 also demonstrated that longer actin filaments were associated with greater resistance of the simulated muscle to relax in the presence of oscillatory strain.

Response of resting ASM to mechanical perturbation

It was mentioned briefly previously that length oscillation applied to ASM in the relaxed state has been found to have an inhibitory effect on the muscle's ability to generate force in the subsequent contractions 69. The same study also revealed that the inhibitory effect was linearly correlated to the amplitude and duration of oscillation, but was relatively independent of oscillation frequency. This finding suggests that if deep inspirations are taken just before a bronchial provocation (e.g. with methacholine inhalation), the induced bronchoconstriction may be attenuated due to the inability of the muscle to generate maximal force. This interpretation, of course, is subject to the caveats mentioned above regarding mechanical coupling between changes in lung volume and ASM length. This interesting response of resting muscle to mechanical strain is transient and appears to be part of the process of the muscle's adaptation to length change. Kuo et al. 168 have shown that the length-oscillation-induced decrease in force is accompanied by a similar amount of reduction in myosin filament density in the muscle. Transmission of force, therefore, can occur throughout the whole lung structure right down to the molecules 137. Length adaptation appears to be responsible for many aspects of the unique behaviour of ASM and may be an integral part of normal airway function. A failure of length adaptation, due to either changes within the smooth muscle or external strain coupling to the smooth muscle, could explain some abnormalities seen in asthma.

Length adaptation in ASM

As in striated muscle, a sufficiently large change in length (from one that corresponds to optimal overlap of contractile filaments) in ASM can reduce force production 47, 169, 170. What is different in the smooth muscle response is that the force decrease is often transient. Different extents of force recovery have been observed after a length change if the muscle is allowed to relax and is re-activated repeatedly at the new length 47, 170–172. The dynamic process of force recovery is termed length adaptation 173. Adaptation of ASM to a new length leads to a shift in the length–force relationship along the length axis, as illustrated in figure 2⇓, and consequently alters the muscle's ability to regulate airway diameter. Although the mechanism for length adaptation is still largely unknown, the ability of smooth muscle to adapt to length change is likely to stem from structural malleability in the muscle's cytoskeleton and contractile apparatus. This novel concept deviates from the static model of fixed myofilament lattice that describes striated muscle. The molecular events associated with structural reorganisation in smooth muscle probably involve rearrangement of actin and myosin filaments, attachment of actin filaments to dense bodies and plaques, and polymerisation and depolymerisation of these filaments. The reorganisation is thought to serve the purpose of facilitating the cell in maintaining optimal contractile filament overlap and orientation at different cell lengths 174.

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Fig. 2—

Shift in the length–force relationship of airway smooth muscle due to length adaptation. The solid curve (———) represents the length–force relationship of a muscle adapted at an arbitrarily chosen reference length (L_ref). Upon shortening by an amount (X), the actively developed force decreases from A to B. After adaptation at the new length (L_ref - X), usually through repeated activation and relaxation, the maximal active force for the muscle at the new length recovers to the level before the length change (C), and the muscle now possesses a new length–force relationship (- - - - -).

Gunst and co-workers 53, 169 suggest that actin filaments may not attach firmly to dense plaques in the relaxed state; they do so only when the muscle is activated. Thus, it was proposed that the reorganisation of the attachment of actin filaments to membrane adhesion sites might be accompanied by changes in the organisation of the actin filament lattice resulting in reorientation of the contractile apparatus 53, 169. This would allow ASM in the relaxed state to change length without altering contractile-filament overlap and enable the muscle to optimise the organisation of the contractile filaments to maximise the contractility of the muscle when length changes are imposed on it by the external environment. The details on how the attachment of thin filaments to adhesion proteins is regulated, especially in partially activated states, remain to be established. However, there is evidence that contractile stimulation of ASM activates a complex of cytoskeletal proteins that associate with transmembrane integrins at sites of cell adhesion 175, 176, and that the activation of these proteins is sensitive to mechanical strain 177, 178. The cytoskeletal protein complex that forms at cell adhesion sites may transduce mechanical signals sensed by transmembrane integrin proteins, and initiate signalling pathways that regulate cytoskeletal events, such as the polymerisation of actin, the anchoring of the actin cytoskeleton to adhesion proteins, and the strain-sensitive activation of contractile proteins 55, 179–181.

The “fluidity” of the cell structure is not limited to the anchorage sites where actin filaments attach to dense plaques. In fact, the whole cell structure appears to be in a constant state of restructuring and adaptation to external stress and strain 182–184. Depression of force and stiffness due to length change can be explained by a disruption or weakening of structures along the force-generating and -transmitting pathway. The actomyosin cross-bridge is only one of many such structures. Other structures include myosin filaments, actin filaments, cytoskeletal cross-linker molecules, cytosolic dense bodies, membrane adhesion plaques and cell–cell connections 55, 185. There is ample evidence for each of these candidates to be somehow involved in the response to external perturbation 168, 174, 186–191.

The overall interaction of these subcellular components can be described as weakly interacting discrete elements in a “crowded”, out-of-equilibrium micro-environment. In other words, the cell behaves like soft glass 182, 183. According to the theory of soft glass, as applied to smooth muscle, the ability to deform, to flow and to remodel is governed by nonthermal agitation (motion) energy of the cytoskeletal elements relative to the height of bond energies that constrain their motion 182, 183. A mechanical strain can act as an energy source that helps individual elements to jump out of their local energy well, so the cytoskeleton essentially “heats up” and “melts”. Over time, however, the cytoskeletal elements evolve into configurations that are more and more stable and stiffer 183. Slowly evolving dynamics of this kind are called “ageing”, in the terminology of material sciences. External forces can reverse the ageing process, and the system can be “rejuvenated” by shear stress, as found by Bursac et al. 183 in ASM. The theory explains many aspects of time-dependent behaviour observed in ASM 29.

By examining the details of length-dependent properties of ASM, including adapted and nonadapted isometric force, shortening velocity, power output, rate of adenosine tiphosphate utilisation and change in myosin filament density (or filament mass), Kuo et al. 188, Qi et al. 192 and Herrera and co-workers 192–195 have provided evidence that length adaptation in ASM involves changes in the number of contractile units in series and in parallel. They also showed that externally applied strain on ASM in the relaxed state led directly to partial disassembly of the myosin filaments 168, thus providing an explanation for the strain-induced inhibition of muscle force, which could underlie the phenomenon of deep-inspiration-induced bronchoprotection, as discussed in the previous section.

The mechanisms described are not mutually exclusive, as suggested by simulations of dynamic networks mimicking intracellular structure of smooth muscle 166.

Changes in morphology and cell attachment in culture

It is now well established that mature ASM cells with contractile phenotype undergo spontaneous, reversible phenotype switching when placed in cell culture 221, 226. This has promoted understanding of the functional properties of ASM cells that contribute to airway remodelling, including myocyte proliferation, secretion of pro-inflammatory bio-molecules, and synthesis of ECM components 232–234. Nonetheless, key ultrastructural changes are evident when smooth muscle cells are plated as a monolayer in culture dishes, and this underpins a need for careful assessment of the effects of external mechanical strain on myocyte function. In tissues, the contractile apparatus of smooth muscle cells is dotted with a more-or-less regular pattern of dense bodies to which thin filaments are anchored 185, 235. Furthermore, dense plaques, arranged in discrete linear plasma membrane domains between caveolae-rich regions, anchor the contractile apparatus to the cell periphery and serve as junctions between adjacent cells, thereby creating a contractile filament syncytium that is sensitive to externally applied mechanical strain 185, 236. In contrast, cultured myocytes characteristically possess multiple, longitudinally oriented cables of actin and myosin called “stress fibres”. In specialised, long-term, serum-deficient culture conditions, the protein content of contractile cables in cultured cells can be induced to a higher level resembling the contractile apparatus of smooth muscle cells in intact tissue, and dense bodies are visible within contractile cables 237, 238. Moreover, as seen in tissue, some ASM cells in these cultures exhibit discrete longitudinal caveolae-rich plasma membrane domains, and these appear to be in register with underlying contractile cables 238.

In cultured ASM cells, stress fibres are anchored at either end to the extracellular substratum via focal adhesion complexes 179. These junctions, like dense plaques in tissue-situated myocytes, transmit strain due to deformation of the substratum, and are composed of integrins and associated proteins, such as talin, α-actinin and vinculin. However, a notable difference between myocytes in tissue and in culture is that there is little or no connection between the cytoskeleton of adjacent cells in the latter environment. Since this difference is likely to have direct effects on the response of myocytes to external mechanical strain, it needs to be considered in analysis of data obtained using in vitro systems. It seems likely that by placing myocytes in monolayer culture on inflexible plastic plates, structural constraints are imposed so as to preclude cytoskeletal organisation mimicking the in situ situation. A potential solution may lie in recent advances in developing cultures in which cells are cast in three-dimensional matrices of biopolymers 239–241. Though a systematic appraisal of the ultrastructure and phenotype of myocytes in such cultures has not yet been reported, future directions using this approach should provide valuable insight, and may significantly extend the utility of in vitro systems to study the molecular signalling, cytoskeletal remodelling and functional consequences of oscillatory mechanical strain on airway myocytes.

Changes in mechanical properties after chronic strain

Application of chronic mechanical strain in cultured ASM cells leads to several changes in cell structure and function. These changes depend on strain magnitude and orientation, as discussed in sections below. In several studies, Smith and co-workers 230, 242–244 have demonstrated that a predominantly uniaxial (stretch in one direction) strain of ∼10% applied for several days in culture increases proliferation, force generation, expression of contractile proteins, calcium sensitivity and cytoskeletal stiffness with contractile activation. Perhaps the most dramatic changes they have been shown are a doubling in shortening capacity and shortening velocity 245. Furthermore, while the ability to relax and decrease cell stiffness following acute stretches in cultured cells (whether they have been exposed to strain or not) is unaltered and the response is comparable to that of intact tissues 52, 246, the ability to recover following such stretches is significantly altered by previous exposure to chronic strain. In single cells lifted from culture dishes and then subjected to large acute stretches of 12% (simulating stretches caused by sighs) followed by smaller stretches of 2% (simulating tidal breathing), cells that had been grown under conditions of chronic cyclic strain for several days recovered their stiffness rapidly within tens of seconds, compared with cells that were not grown under chronic cyclic strain, which recovered after >100 s 246. This more rapid recovery following an acute stretch may be important as it is reminiscent of the more rapid re-narrowing (and thus lack of bronchodilatory effect) observed following a deep inspiration in asthma 20, 35, 138, 139. Together, these changes suggest that chronic strain may play an important role in promoting the contractile function of ASM, and possibly in the pathogenesis of asthma.

Cell alignment and dependence on strain profile and underlying matrix

The changes described for ASM cells cultured in the presence of oscillatory mechanical strain appear to depend on the magnitude, nature and orientation of the strain that is applied. In the studies described in the preceding paragraph, cyclical negative pressure applied below the circular flexible membranes strained the cultured cells. This method results in uneven strain across the membranes with biaxial strain (i.e. the stretch is the same in every direction in a two-dimensional plane) of ∼10% in the central region, changing gradually to uniaxial stretch of 10% in the radial direction near the membrane edge 246, 247. After 2–4 days, the cells in the region of predominantly uniaxial strain re-orient, forming a ring of cells aligned circumferentially, while cells in the central region do not show any particular preferred direction. Thus, over most of the dish, stretch is primarily across the “waists” of the cells, with very little stretch occurring along the cell's long axis 242, 243. This is illustrated in figure 3⇓.

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Fig. 3—

a) Airway smooth muscle cells being stretched in circular dishes by oscillating negative pressure below the flexible membrane. b) The strain field is anisotropic with nearly constant strain in the radial strain (–––––––) but gradually decreasing stretch in the circumferential strain (-------), which results in nearly uniaxial strain near the edge where the membrane is held. c, d, e) Cells are shown in Hoffmann contrast microscopy after 7 days of continuous cyclic stretching in culture. Cells do not align in the biaxial strain region, where strain is the same in every direction in the plane (arrows, as shown in c), but near the edge become almost completely aligned transverse to the applied strain direction (e). c, d, e) Microscopy shows the strain profile near radial positions 0, 0.5. and 1.0, respectively. As indicated by the normalised radial position in (b). Reproduced from 246 with permission.

It has been observed for many cell types that cells align in response to anisotropic or uniaxial strain, including fibroblasts 248 and vascular endothelial cells 249, 250. Interestingly, the cellular response to strain also depends on the orientation of the applied strain relative to the cells. For example, in response to uniaxial strain, fibroblasts that are forced to align in grooves with the applied strain increase their expression of α-actin more than cells aligned perpendicular to the strain direction, although α-actin is increased in both cases. Interestingly, fibroblasts forced to align in grooves perpendicular to the applied strain increase secretory behaviour compared with those aligned parallel to the strain 248. In contrast, in response to biaxial strain, no orientation behaviour is observed in fibroblast or ASM cells 229, 248.

It is unknown why almost all cells re-orient in response to mostly uniaxial or anisotropic strain; however, there is recent evidence that a difference in strain profile leads to quite different changes in cell structure and function in ASM cells. Most studies of mechanically strained ASM cells including the results described above were primarily from cells in the outer area of the membranes on which they were grown, and thus the results represent the response to a primarily uniaxial strain. In contrast to these results, Wang et al. 229 found that when subjected to uniform biaxial strain, there was no cell alignment, no increase in proliferation, a ∼50% decrease in smooth muscle protein 22 and smooth muscle myosin heavy chain promoter activity, and decreased filamentous to globular-actin ratio, compared with cells not exposed to any strain. These observations are consistent with decreased Rho activation, which is in contrast to the increased Rho-A activation observed in response to predominantly uniaxial strain 231 and would appear to represent anticontractile effects of strain rather than pro-contractile effects of strain as occurring with primarily uniaxial strain. Furthermore, increases in amount of myosin light chain kinase after 11 days of strain seem to occur primarily in uniaxially strained cells 243, 246. Interestingly, proliferation is reduced in cells grown on laminin and subjected to lower strains of 4% compared with cells grown on collagen with either 0 or 4% strain. Conversely, proliferation is enhanced in cells grown on collagen subjected to 10% strain, which indicates that the response to strain is sensitive to both strain magnitude and ECM 242, 251. While these results point to the importance to cell function of strain profile and magnitude, how relevant are the strain conditions experienced by cells in culture to strain experienced in vivo, and do strain conditions change in disease?

Strain in culture and strain in vivo

In vivo, the strain produced by lung expansion is three-dimensional, and thus applied to all cell surfaces, in contrast to most in vitro strain systems. In vivo, the airways are constantly being stretched circumferentially and longitudinally. The magnitude of the strain depends on airway size as discussed previously, but typically is ∼2–4% during tidal breathing, ∼12% during sighs, with a maximal of ∼25% during inflation to TLC 52, 154–156. Thus in healthy lungs, smooth muscle strain more closely matches the biaxial strain that does not promote contractile changes in cultured cells. However, the strain profile must be altered in the presence of elevated ASM tone and with airway narrowing, such as that occurs in asthma. As ASM is predominantly oriented circumferentially about the airways with an average pitch close to 13° 252, increased tone would increase circumferential stiffness relative to the stiffness along the length of the airway. Thus, the stretch proffered by breathing would tend to be more axial than circumferential, leading to development of strain anisotropy in vivo. Indeed, while this has not yet been examined directly, if true, more stretch would be conferred across the waists of the ASM cells, matching conditions leading to pro-contractile changes in ASM in vitro.

Thus, increased tone may lead to enhanced contractile function by enhancing strain anisotropy in vivo. Interestingly, elevated tone appears to be required to elicit stretch-induced changes in cultured ASM cells. For example, cytoskeletal remodelling and stiffening in response to mechanical stress are inhibited during β-agonist-induced reduction of tone in culture 253. While it has yet to be demonstrated that reduced tone would inhibit the pro-contractile changes previously demonstrated in uniaxially strained cells, this suggests that in addition to promoting shortened ASM as discussed previously 34, tone may play a harmful role in the pathogenesis of asthma by also leading to strain anisotropy and potentiation of structural remodelling of the ASM cells. However, while changes in cell structure and function with strain are well documented in vivo, the acquisition of a more contractile phenotype due to mechanical strain in vivo has not been established.

Parenchyma-airway interdependence

As mentioned previously, there appears to be a lack of parenchymal-airway interdependence in the pig lung, at least as far as mid-sized airways are concerned 289. If this is also found in the human lung, it will drastically change current interpretation of many previous studies, and indeed, it will change the view of how the lung works. Unanswered questions remain from the studies of Noble et al. 289, an obvious one being “does human lung behave in the same way as pig lung?”. More exact determination of the interdependence of airway size on parenchymal mechanics also needs to be derived for the small airways at the periphery of the lung. The static conditions under which the interdependence was assessed by Noble et al. 289 do not represent the real environfment of the lung 297. The viscoelastic lung parenchyma under dynamic conditions interacts with the airways very differently 298, 299, and this should be kept in mind if the experiments of Noble et al. 289 are revisited.

Stretch-induced release of transmitters in the parenchyma and airways

The influence of mechanical stretch on ASM contractility has so far been interpreted as strictly a “physical” phenomenon: 1) perturbation of actomyosin cross-bridge interaction 58; 2) disruption of myosin filament assembly 168; or 3) changes in cytoskeletal organisation 55. It is entirely possible that a mechanical signal can be transduced into other signals via some kind of stretch receptor coupled to chemical signalling pathways 231, 300. It has been speculated 68 that stretch-induced bronchodilation acts through inhibition of cholinergic tone or activation of the nonadrenergic noncholinergic system and results in the release of nitric oxide 140. Stretch is also known to increase prostaglandin production, and certain ion channels may be directly influenced. Surfactant release after a deep inspiration could also affect airway impedance, especially that of the small airways 301. It is crucial to establish the relative importance of the various contributing factors involved in stretch-induced smooth muscle response and their relevance to asthma.

Discrepancies in findings from airway segments and ASM

So far, there is no clear explanation as to why periodic stretches applied, by pressure oscillation, to ASM embedded in intact airways increase muscle contractility, and why stretches applied to isolated ASM bundles have an opposite effect 295, 296. This question needs to be answered before any interpretation of stretch-induced alteration in ASM properties in terms of lung function can be taken seriously. Perhaps more studies need to be performed using airway segments from different animal species and from different generations of the airway tree, so that the generality of this observation can be confirmed. It will be informative to perform similar experiments using the whole lung, which is one-step closer to the in vivo lung function test, compared with the isolated airway experiment. In such experiments, R_aw could be measured in a lung lobe before and after a period of volume oscillation. Airway diameter should be monitored throughout the experiment, during volume oscillation and during broncho-challenge under static conditions. Experimentally, there are at least two ways by which to oscillate lung volume. One is through changes in airway pressure (i.e. in a system where a closed airway tree in the lung is connected to a pressure source); the other is by changing the volume of pleural cavity or an experimental device serving the same function. The latter is more physiologically relevant. Different modes of volume oscillation may produce different experimental outcomes. If this is found to be the case, then the reason for the disparity may provide insights into the mechanism responsible for the altered ASM properties.

Differences between normal and asthmatic ASM

Excessive shortening of ASM leading to excessive narrowing of the airways could occur under at least four scenarios. Each scenario is associated with a different mechanism. One scenario is that the local airway environment allows the muscle to adapt at abnormally short lengths and, when the muscle is subsequently activated, it shortens further than it would do normally. Although it is not clear whether it happens in vivo, this behaviour has been reproduced in vitro in healthy ASM 34, 195. Hence, excessive shortening of airways does not have to be associated with altered intrinsic muscle properties 33. Therefore, any experiment designed to illustrate the relationship between airway narrowing and ASM contractility needs to establish what the normal in situ length is for the muscle cells. Without that reference, comparison of the amounts of shortening in two muscle preparations would be meaningless, because the amount of shortening is partially determined by the initial length of the muscle 195. For the same reason, comparison of shortening velocities needs to be carried out at appropriate lengths of reference.

The second scenario involves changes in the mechanical properties or the amount of the ECM surrounding ASM. Bramley et al. 302 have shown that treatment of human bronchial smooth muscle with collagenase augments the muscle's ability to shorten. Assuming that the collagenase has no direct effect on muscle contractility, the results indicate that collagen fibres (and probably other components of the ECM) constrain muscle shortening, perhaps through restricting lateral expansion of the muscle cells, as elegantly demonstrated by Meiss 303 in an in vitro model. Changes in the ECM composition could also affect ASM phenotype by increasing its proliferative state and altering its secretory properties 234.

The third scenario is that the smooth muscle mass within the airway wall has increased, due to hypertrophy and/or hyperplasia, without a change in the intrinsic contractile properties of individual muscle cells. This could lead to excessive airway narrowing simply because of the increased constricting force associated with the greater muscle mass, if other factors influencing airway narrowing remain the same. This emphasises the importance of proper normalisation of muscle properties by appropriate parameters, such as muscle cross-section and length, and local airway geometry and mechanical properties. More quantitative information is needed about smooth muscle cell number and size, and the amount and type of ECM between the muscle cells in cases of asthma of varying duration and severity.

The fourth scenario is that the intrinsic contractile properties of asthmatic ASM are indeed different from normal. A change in intrinsic properties does not automatically mean that there will be a change in the degree of airway narrowing. Other changes in the airway property may negate or enhance the influence of smooth muscle. If an increase in contractility of ASM is a contributing factor in the excessive airway narrowing in asthma, then further questions should be asked. Can overexpression of MLCK 37, 304 be responsible for the exaggerated airway narrowing 305? Can a change in myosin or actin filament length and integrity alter the response of ASM to length fluctuation associated with lung volume change 164? Can a change in the composition of myosin heavy chain isoforms in asthmatics 306 lead to excessive airway narrowing and through what mechanism? Studies on a rodent model of innate AHR suggest that the abnormalities in the pathways of biochemical signalling and the isoform mix of myosin molecules may underlie the hyperresponsiveness. Tracheal smooth muscle from the hyperresponsive Fisher rat generates more force 307, 308 and shortens faster 309 compared with the Lewis rat (control). Experiments on airway explants from Fisher rats also demonstrate a more rapid and excessive airway narrowing in response to agonist stimulation, compared with explants from the Lewis rats 310, 311. In cultured ASM cells from Fisher rats, the observed excessive calcium response can be attributed to higher levels of IP₃, which in turn could be caused by a slower rate of IP₃ degradation in the cells from the Fisher rats 312. Increased activation of the contractile pathway is also shown by the increased phosphorylation of the regulatory myosin light chain 308. The Fisher ASM cells are also characterised by a greater complement of the fast myosin isoform (SM-B) 308. Preliminary results from bronchial biopsies of asthmatic subjects show that the fast myosin isoform is also expressed to a greater extent than that found in normal subjects 306. Examination of the differences in biochemical pathways and myosin isoforms in airway tissues from animal models of hyperresponsiveness may point to mechanisms pertinent to human asthma.

Effects of steroids on smooth muscle length adaptation

Inhaled steroids are the most effective drugs in reducing AHR in asthma 313. The working hypothesis is that this is due to a reduction in airway inflammation 314, which indirectly changes the stimulation and the micro-environment of ASM, leading to reduced airway narrowing at a given dose of inhaled agonist. However, smooth muscle itself responds to steroids by changing its expression of a host of genes that are involved in cell adhesion, proliferation, ECM formation, cell signalling, cytokine production and signal-transduction molecules regulating smooth muscle responsiveness 315–318. It can be postulated that this also influences the structure of the cytoskeleton and the arrangement of contractile proteins, and thus affects the muscle's ability to adapt to length or force perturbation 319. Hence, it needs to be investigated whether the benefits of steroids in asthma are based on changing the dynamics in structure and function of ASM rather than suppressing the surrounding airway inflammation alone. As suggested by the recent reports regarding the lack of antiproliferative activity of steroids on smooth muscle cells from asthmatics 320 and the impairment of glucocorticoid action by type I monomeric collagen (presumably present in the micro-environment where ASM cells reside in asthmatic airways) 251, 321, the efficacy of steroids in modulating smooth muscle gene transcription may be diminished in asthmatics. This indicates a need to look for alternative mechanisms for steroid action that are beneficial to (at least some) asthmatics. Given the interaction of β-agonists with steroids 109, as well as the relaxing effect of mechanical stretch in smooth muscle 163, examination is required as to whether there is a three-party interaction functioning in normal ASM and whether this interaction is impaired in ASM obtained from patients with asthma.

Possible abnormal relaxation of ASM in asthma

The role of impaired relaxation needs to be dealt with in consideration of airway hyperreactivity, since it could contribute to bronchospasm. Several investigators have reported alterations in relaxation in ASM from animal models of asthma 322–325. Unfortunately, most of them dealt with isometric relaxation (relaxation of tension in muscle maintained at a constant length after cessation of stimulation), which provided information relating to stiffening of the airway wall and not to the relevant parameter of airway lumen narrowing 322–324. In vivo measurements of FEV₁ and specific G_aw are better in this regard, but suffer from the drawback that multiple confounding variables are operating simultaneously. Jiang and Stephens 325 have studied isotonic relaxation (lengthening of muscle under a constant load after active shortening) and have shown that its half-relaxation time is prolonged in sensitised ASM. The magnitude of the residual failure to relax could account for the magnitude of bronchospasm seen in the sensitised ASM. No such experiment has been carried out using human ASM. The bottom line, therefore, is that in studies of ASM dynamics in asthma, relaxation must be included and that for it to be relevant the studies should be in isotonic mode.