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Background

Airway smooth muscle (ASM) hyperplasia and mast cell localization within the ASM bundle are important features of asthma. The cause of this increased ASM mass is uncertain and whether it is a consequence of ASM–mast cell interactions is unknown.

Objective

We sought to investigate ASM proliferation and survival in asthma and the effects of co-culture with mast cells.

Methods

Primary ASM cultures were derived from 11 subjects with asthma and 12 non-asthmatic controls. ASM cells were cultured for up to 10 days in the presence or absence of serum either alone or in co-culture with the human mast cell line-1, unstimulated human lung mast cells (HLMC) or IgE/anti-IgE-activated HLMC. Proliferation was assessed by cell counts, CFSE assay and thymidine incorporation. Apoptosis and necrosis were analysed by Annexin V/propidium iodide staining using flow cytometry and by assessment of nuclear morphology using immunofluorescence. Mast cell activation was confirmed by the measurement of histamine release.

Results

Using a number of techniques, we found that ASM proliferation and survival was not significantly different between cells derived from subjects with or without asthma. Co-culture with mast cells did not affect the rate of proliferation or survival of ASM cells.

Conclusion

Our findings do not support a role for increased airway smooth proliferation and survival as the major mechanism driving ASM hyperplasia in asthma.

Cite this as: D. Kaur, F. Hollins, R. Saunders, L. Woodman, A. Sutcliffe, G. Cruse, P. Bradding and C. Brightling, Clinical & Experimental Allergy, 2010 (40) 279– 288.

Background

Airway smooth muscle (ASM) contraction underlies acute bronchospasm in asthma. ASM cells can switch between a synthetic-proliferative phenotype and a contractile phenotype. While the effects of extracellular matrix (ECM) components on modulation of ASM cells to a synthetic phenotype have been reported, the role of ECM components on maturation of ASM cells to a contractile phenotype in adult lung is unclear. As both changes in ECM components and accumulation of contractile ASM are features of airway wall remodelling in asthma, we examined the role of the ECM protein, laminin, in the maturation of contractile phenotype in human ASM cells.

Methods

Human ASM cells were made senescence-resistant by stable expression of human telomerase reverse transcriptase. Maturation to a contractile phenotype was induced by 7-day serum deprivation, as assessed by immunoblotting for desmin and calponin. The role of laminin on ASM maturation was investigated by comparing the effects of exogenous laminin coated on culture plates, and of soluble laminin peptide competitors. Endogenous expression of laminin chains during ASM maturation was also measured.

Results

Myocyte binding to endogenously expressed laminin was required for ASM phenotype maturation, as laminin competing peptides (YIGSR or GRGDSP) significantly reduced desmin and calponin protein accumulation that otherwise occurs with prolonged serum deprivation. Coating of plastic cell culture dishes with different purified laminin preparations was not sufficient to further promote accumulation of desmin or calponin during 7-day serum deprivation. Expression of α2, β1 and γ1 laminin chains by ASM cells was specifically up-regulated during myocyte maturation, suggesting a key role for laminin-2 in the development of the contractile phenotype.

Conclusion

While earlier reports suggest exogenously applied laminin slows the spontaneous modulation of ASM to a synthetic phenotype, we show for the first time that endogenously expressed laminin is required for ASM maturation to the contractile phenotype. As endogenously expressed laminin chains α2, β1 and γ1 are uniquely increased during myocyte maturation, these laminin chains may be key in this process. Thus, human ASM maturation appears to involve regulated endogenous expression of a select set of laminin chains that are essential for accumulation of contractile phenotype myocytes.

Asthma is a complex respiratory disease whose incidence has increased worldwide in the last decade. There is currently no cure for asthma. While bronchodilator and anti-inflammatory medications are effective medicines in some asthmatic patients, it is clear that an unmet therapeutic need persists for a subpopulation of individuals with severe asthma. This chronic lung disease is characterized by airflow limitation and lung inflammation and remodeling that includes increased airway smooth muscle (ASM) mass. In addition to its contractile properties, the ASM also contributes to the inflammatory process by producing active mediators, modifying the extracellular matrix composition, and interacting with inflammatory cells. These undesirable functions make interventions aimed at reducing ASM abundance an attractive strategy for novel asthma therapies. There are at least three mechanisms that could limit the accumulation of smooth muscle – decreased cell proliferation, augmented cell apoptosis, and reduced cell migration into the smooth muscle layer. Inhibitors of the mevalonate pathway or statins hold promise for asthma because they exhibit anti-inflammatory, anti-migratory, and anti-proliferative effects in pre-clinical and clinical studies, and they can target the SM. This review will discuss current knowledge of ASM biology and identify gaps in the field in order to stimulate future investigations of the cellular mechanisms controlling ASM overabundance in asthma. Targeting ASM has the potential to be an innovative venue of treatment for patients with asthma.

Background

Fibroproliferative airway remodelling, including increased airway smooth muscle (ASM) mass and contractility, contributes to airway hyperresponsiveness in asthma. In vitro studies have shown that maturation of ASM cells to a (hyper)contractile phenotype is dependent on laminin, which can be inhibited by the laminin-competing peptide Tyr-Ile-Gly-Ser-Arg (YIGSR). The role of laminins in ASM remodelling in chronic asthma in vivo, however, has not yet been established.

Methods

Using an established guinea pig model of allergic asthma, we investigated the effects of topical treatment of the airways with YIGSR on features of airway remodelling induced by repeated allergen challenge, including ASM hyperplasia and hypercontractility, inflammation and fibrosis. Human ASM cells were used to investigate the direct effects of YIGSR on ASM proliferation in vitro.

Results

Topical administration of YIGSR attenuated allergen-induced ASM hyperplasia and pulmonary expression of the proliferative marker proliferating cell nuclear antigen (PCNA). Treatment with YIGSR also increased both the expression of sm-MHC and ASM contractility in saline- and allergen-challenged animals; this suggests that treatment with the laminin-competing peptide YIGSR mimics rather than inhibits laminin function in vivo. In addition, treatment with YIGSR increased allergen-induced fibrosis and submucosal eosinophilia. Immobilized YIGSR concentration-dependently reduced PDGF-induced proliferation of cultured ASM to a similar extent as laminin-coated culture plates. Notably, the effects of both immobilized YIGSR and laminin were antagonized by soluble YIGSR.

Conclusion

These results indicate that the laminin-competing peptide YIGSR promotes a contractile, hypoproliferative ASM phenotype in vivo, an effect that appears to be linked to the microenvironment in which the cells are exposed to the peptide.

Airway smooth muscle (ASM) hyperplasia is a characteristic feature of the asthmatic airway but the underlying mechanisms that induce ASM hyperplasia remain unknown. Because transforming growth factor (TGF)-β is a potent regulator of ASM cell proliferation, we determined its expression and mitogenic signaling pathways in ASM cells. We obtained ASM cells by laser capture microdissection of bronchial biopsies and found that ASM cells from asthmatic patients expressed TGF-β1 mRNA and protein to a greater extent than non-asthmatic individuals using real-time RT-PCR and immunohistochemistry, respectively. TGF-β1 stimulated the growth of non-confluent and confluent ASM cells either in the presence or absence of serum in a time- and concentration-dependent manner. The mitogenic activity of TGF-β1 on ASM cells was inhibited by selective inhibitors of TGF-β receptor-I kinase (SD-208), of phosphatidylinositol 3-kinase (PI3K, LY294002), ERK (PD98059), JNK (SP600125) and NF-κB (AS602868). On the other hand, p38 MAPK inhibitor (SB203580) augmented TGF-β1-induced proliferation. To study role of the Smads, we transduced ASM cells with an adenovirus vector expressing Smad 4, Smad 7 or negative dominant Smad3 and found no involvement of these Smads in TGF-β1-induced proliferation. Dexamethasone caused a dose-dependent inhibition in TGF-β1-induced proliferation. Our findings suggest that TGF-β1 may act in an autocrine fashion to induce ASM hyperplasia, mediated by its receptor and several kinases including PI3K, ERK and JNK, while p38 MAPK is a negative regulator. NF-κB is also involved in the TGF-β1 mitogenic signaling but Smad pathway does not appear important.

Background

Asthma is a chronic inflammatory disorder of the lung airways that is associated with airway remodeling and hyperresponsiveness. Its is well documented that the smooth muscle mass in asthmatic airways is increased due to hypertrophy and hyperplasia of the ASM cells. Moreover, eosinophils have been proposed in different studies to play a major role in airway remodeling. Here, we hypothesized that eosinophils modulate the airways through enhancing ASM cell proliferation. The aim of this study is to examine the effect of eosinophils on ASM cell proliferation using eosinophils isolated from asthmatic and normal control subjects.

Methods

Eosinophils were isolated from peripheral blood of 6 mild asthmatics and 6 normal control subjects. ASM cells were incubated with eosinophils or eosinophil membranes and ASM proliferation was estimated using thymidine incorporation. The mRNA expression of extracellular matrix (ECM) in ASM cells was measured using quantitative real-time PCR. The effect of eosinophil-derived proliferative cytokines on ASM cells was determined using neutralizing antibodies. The role of eosinophil derived Cysteinyl Leukotrienes in enhancing ASM was also investigated.

Results

Co-culture with eosinophils significantly increased ASM cell proliferation. However, there was no significant difference in ASM proliferation following incubation with eosinophils from asthmatic versus normal control subjects. Co-culture with eosinophil membranes had no effect on ASM proliferation. Moreover, there was no significant change in the mRNA expression of ECM proteins in ASM cells following co-culture with eosinophils when compared with medium alone. Interestingly, blocking the activity of cysteinyl Leukotries using antagonists inhibited eosinophil-derived ASM proliferation.

Conclusions

Eosinophils enhances the proliferation of ASM cells. This role of eosinophil does not seem to depend on ASM derived ECM proteins nor on Eosinophil derived TGF-β or TNF-α. Eosinophil seems to induce ASM proliferation via the secretion of Cysteinyl Leukotrienes.

Breathing (especially deep breathing) antagonizes development and persistence of airflow obstruction during bronchoconstrictor stimulation. Force fluctuations imposed on contracted airway smooth muscle (ASM) in vitro result in its relengthening, a phenomenon called force fluctuation-induced relengthening (FFIR). Because breathing imposes similar force fluctuations on contracted ASM within intact lungs, FFIR represents a likely mechanism by which breathing antagonizes bronchoconstriction. While this bronchoprotective effect appears to be impaired in asthma, corticosteroid treatment can restore the ability of deep breaths to reverse artificially induced bronchoconstriction in asthmatic subjects. We previously demonstrated that FFIR is physiologically regulated through the p38 MAPK signaling pathway. While the beneficial effects of corticosteroids have been attributed to suppression of airway inflammation, we hypothesized that alternatively they might exert their action directly on ASM by augmenting FFIR as a result of inhibiting p38 MAPK signaling.

We tested this possibility in the present study by measuring relengthening in contracted canine tracheal smooth muscle (TSM) strips.

Our results indicate that dexamethasone treatment significantly augmented FFIR of contracted canine TSM. Canine tracheal ASM cells treated with dexamethasone demonstrated increased MAP kinase phosphatase (MKP)-1 expression and decreased p38 MAPK activity, as reflected in reduced phosphorylation of the p38 MAPK downstream target, HSP27.

These results suggest that corticosteroids may exert part of their therapeutic effect through direct action on ASM, by decreasing p38 MAPK activity and thus increasing FFIR.

Background

Chemokine receptors play an important role in cell migration and wound repair. In asthma, CCR3 and 7 are expressed by airway smooth muscle (ASM) and CCR7 has been implicated in the development of ASM hyperplasia. The expression profile of other chemokine receptors by ASM and their function needs to be further explored.

Objective

We sought to investigate ASM chemokine receptor expression and function in asthma.

Methods

ASM cells were derived from 17 subjects with asthma and 36 non-asthmatic controls. ASM chemokine receptor expression was assessed by flow cytometry and immunofluorescence. The function of chemokine receptors expressed by more than 10% of ASM cells was investigated by intracellular calcium measurements, chemotaxis, wound healing, proliferation and survival assays.

Results

In addition to CCR3 and 7, CXCR1, 3 and 4 were highly expressed by ASM. These CXC chemokine receptors were functional with an increase in intracellular calcium following ligand activation and promotion of wound healing [CXCL10 (100 ng/mL) 34 ± 2 cells/high-powered field (hpf) vs. control 29 ± 1; P=0.03; n=8]. Spontaneous wound healing was inhibited by CXCR3 neutralizing antibody (mean difference 7 ± 3 cells/hpf; P=0.03; n=3). CXC chemokine receptor activation did not modulate ASM chemotaxis, proliferation or survival. No differences in chemokine receptor expression or function were observed between ASM cells derived from asthmatic or non-asthmatic donors.

Conclusions

Our findings suggest that the chemokine receptors CXCR1, 3 and 4 modulate some aspects of ASM function but their importance in asthma is uncertain.

Background and Objective

Asthma is associated with airway narrowing in response to bronchoconstricting stimuli and increased airway smooth muscle (ASM) mass. In addition, some studies have suggested impaired β-agonist induced ASM relaxation in asthmatics, but the mechanism is not known.

Objective

To characterize the potential defect in β-agonist induced cAMP in ASM derived from asthmatic in comparison to non-asthmatic subjects and to investigate its mechanism.

Methods

We examined β2-adrenergic (β2AR) receptor expression and basal β-agonist and forskolin (direct activator of adenylyl cyclase) stimulated cAMP production in asthmatic cultured ASM (n = 15) and non-asthmatic ASM (n = 22). Based on these results, PDE activity, PDE4D expression and cell proliferation were determined.

Results

In the presence of IBMX, a pan PDE inhibitor, asthmatic ASM had ∼50% lower cAMP production in response to isoproterenol, albuterol, formoterol, and forskolin compared to non-asthmatic ASM. However when PDE4 was specifically inhibited, cAMP production by the agonists and forskolin was normalized in asthmatic ASM. We then measured the amount and activity of PDE4, and found ∼2-fold greater expression and activity in asthmatic ASM compared to non-asthmatic ASM. Furthermore, inhibition of PDE4 reduced asthmatic ASM proliferation but not that of non-asthmatic ASM.

Conclusion

Decreased β-agonist induced cAMP in ASM from asthmatics results from enhanced degradation due to increased PDE4D expression. Clinical manifestations of this dysregulation would be suboptimal β-agonist-mediated bronchodilation and possibly reduced control over increasing ASM mass. These phenotypes appear to be “hard-wired” into ASM from asthmatics, as they do not require an inflammatory environment in culture to be observed.

Severe asthma is associated with fixed airway obstruction attributable to inflammation, copious luminal mucus, and increased airway smooth muscle (ASM) mass. Paradoxically, studies demonstrated that the hypertrophic and hyperplastic ASM characteristic of severe asthma has reduced contractile capacity. We compared the G-protein–coupled receptor (GPCR)–induced Ca2+ mobilization and expression of GPCRs and signaling proteins related to procontractile signaling in ASM derived postmortem from subjects who died of nonrespiratory causes, with cells from subjects who died of asthma. Despite the increased or comparable expression of contraction-promoting GPCRs (bradykinin B2 or histamine H1 and protease-activated receptor 1, respectively) in asthmatic ASM cells relative to cells from healthy donors, asthmatic ASM cells exhibited reduced histamine-induced Ca2+ mobilization and comparable responses to bradykinin and thrombin, suggesting a postreceptor signaling defect. Accordingly, the expression of regulator of G-protein signaling–5 (RGS5), an inhibitor of ASM contraction, was increased in cultured, asthmatic ASM cells and in bronchial smooth muscle bundles of both human subjects with asthma and allergen-challenged mice, relative to those of healthy human subjects or naive mice. The overexpression of RGS5 impaired the release of Ca2+ to thrombin, histamine, and carbachol, and reduced the contraction of precision-cut lung slices to carbachol. These results suggest that increased RGS5 expression contributes to decreased myocyte shortening in severe and fatal asthma.

Backgrounds

Exaggerated bronchial constriction is the most significant and life threatening response of patients with asthma to inhaled stimuli. However, few studies have investigated the contractility of airway smooth muscle (ASM) from these patients. The purpose of this study was to establish a method to measure contraction of ASM cells by embedding them into a collagen gel, and to compare the contraction between subjects with and without asthma.

Methods

Gel contraction to histamine was examined in floating gels containing cultured ASM cells from subjects with and without asthma following overnight incubation while unattached (method 1) or attached (method 2) to casting plates. Smooth muscle myosin light chain kinase protein levels were also examined.

Results

Collagen gels containing ASM cells reduced in size when stimulated with histamine in a concentration‐dependent manner and reached a maximum at a mean (SE) of 15.7 (1.2) min. This gel contraction was decreased by inhibitors for phospholipase C (U73122), myosin light chain kinase (ML‐7) and Rho kinase (Y27632). When comparing the two patient groups, the maximal decreased area of gels containing ASM cells from patients with asthma was 19 (2)% (n = 8) using method 1 and 22 (3)% (n = 6) using method 2, both of which were greater than that of cells from patients without asthma: 13 (2)% (n = 9, p = 0.05) and 10 (4)% (n = 5, p = 0.024), respectively. Smooth muscle myosin light chain kinase levels were not different between the two groups.

Conclusion

The increased contraction of asthmatic ASM cells may be responsible for exaggerated bronchial constriction in asthma.

Airway smooth muscle (ASM) hyperplasia and angiogenesis are important features associated with airway remodeling. We investigated the effect of IL-4 and amphiregulin, an epidermal growth factor family member, on the proliferation of human ASM cells and on the release of vascular endothelial growth factor (VEGF) and monocyte chemotactic protein (MCP)-1 from human ASM cells. Human ASM cells were growth-arrested for 48 hr and incubated with platelet-derived growth factor (PDGF)-BB, interleukin (IL)-4, amphiregulin, and VEGF to evaluate cell proliferation. The cells were treated with PDGF, IL-4 and amphiregulin to evaluate the release of VEGF, MCP-1. IL-4 suppressed unstimulated and PDGF-stimulated ASM cell proliferation. Amphiregulin stimulated ASM cell proliferation in a dose-dependent manner. VEGF did not have any influence on ASM cell proliferation. IL-4 stimulated VEGF secretion by the ASM cells in a dose-dependent manner and showed added stimulatory effects when co-incubated with PDGF. Amphiregulin did not promote VEGF secretion. IL-4 and amphiregulin showed no stimulatory effects on MCP-1 secretion. The results of this study showed that IL-4 had bifunctional effects on airway remodeling, one was the suppression of the proliferation of the ASM cells and the other was the promotion of VEGF release by the ASM cells, and amphiregulin can promote human ASM cell proliferation.

Airway smooth muscle (ASM) plays a pivotal role in modulating bronchomotor tone but also orchestrates and perpetuates airway inflammation and remodeling. Despite substantial research, there remain important unanswered questions. In 2006, the National Heart, Lung, and Blood Institute sponsored a workshop to define new directions in ASM biology. Important questions concerning the key functions of ASM include the following: Does developmental dysregulation of ASM function promote airway disease, what key signaling pathways in ASM evoke airway hyperresponsiveness in vivo, do alterations in ASM mass affect excitation–contraction coupling, and can ASM modulate airway inflammation and remodeling in a physiologically relevant manner? This workshop identified critical issues in ASM biology to delineate areas for scientific investigation in the identification of new therapeutic and diagnostic approaches in asthma, chronic obstructive pulmonary disease, and cystic fibrosis.

Asthma is characterized by the association of airway hyperresponsiveness (AHR), inflammation, and remodelling. The aim of the present article is to review the pivotal role of airway smooth muscle (ASM) in the pathophysiology of asthma. ASM is the main effector of AHR. The mechanisms of AHR in asthma may involve a larger release of contractile mediators and/or a lower release of relaxant mediators, an improved ASM cell excitation/contraction coupling, and/or an alteration in the contraction/load coupling. Beyond its contractile function, ASM is also involved in bronchial inflammation and remodelling. Whereas ASM is a target of the inflammatory process, it can also display proinflammatory and immunomodulatory functions, through its synthetic properties and the expression of a wide range of cell surface molecules. ASM remodelling represents a key feature of asthmatic bronchial remodelling. ASM also plays a role in promoting complementary airway structural alterations, in particular by its synthetic function.

Excessive airway obstruction is the cause of symptoms and abnormal lung function in asthma.

As airway smooth muscle (ASM) is the effecter controlling airway calibre, it is suspected that dysfunction of ASM contributes to the pathophysiology of asthma. However, the precise role of ASM in the series of events leading to asthmatic symptoms is not clear. It is not certain whether, in asthma, there is a change in the intrinsic properties of ASM, a change in the structure and mechanical properties of the noncontractile components of the airway wall, or a change in the interdependence of the airway wall with the surrounding lung parenchyma. All these potential changes could result from acute or chronic airway inflammation and associated tissue repair and remodelling.

Anti-inflammatory therapy, however, does not “cure” asthma, and airway hyperresponsiveness can persist in asthmatics, even in the absence of airway inflammation. This is perhaps because the therapy does not directly address a fundamental abnormality of asthma, that of exaggerated airway narrowing due to excessive shortening of ASM.

In the present study, a central role for airway smooth muscle in the pathogenesis of airway hyperresponsiveness in asthma is explored.

Hyperplasia of airway smooth muscle (ASM) is a feature of the remodelled airway in asthmatics. We examined the antiproliferative effectiveness of the corticosteroid dexamethasone on expression of the key regulator of G1 cell cycle progression—cyclin D1—in ASM cells from nonasthmatics and asthmatics stimulated with the mitogen platelet-derived growth factor BB. While cyclin D1 mRNA and protein expression were repressed in cells from nonasthmatics in contrast, cyclin D1 expression in asthmatics was resistant to inhibition by dexamethasone. This was independent of a repressive effect on glucocorticoid receptor translocation. Our results corroborate evidence demonstrating that corticosteroids inhibit mitogen-induced proliferation only in ASM cells from subjects without asthma and suggest that there are corticosteroid-insensitive proliferative pathways in asthmatics.

Background

Mast cell microlocalisation within the airway smooth muscle (ASM) bundle is an important determinant of the asthmatic phenotype. We hypothesised that mast cells migrate towards ASM in response to ASM derived chemokines.

Methods

Primary ASM cultures from subjects with and without asthma were stimulated with interleukin (IL)‐1β, IL‐4, and IL‐13 alone and in combination. Mast cell chemotaxis towards these ASM supernatants was investigated, and the chemotaxins mediating migration by using specific blocking antibodies for stem cell factor (SCF) and the chemokine receptors CCR3, CXCR1, 3 and 4 as well as the Gi inhibitor pertussis toxin and the tyrosine kinase inhibitor genistein were defined. The concentrations of CCL11, CXCL8, CXCL10, TGF‐β, and SCF in the supernatants were measured and the effect of non‐asthmatic ASM supernatants on the mast cell chemotactic activity of asthmatic ASM was examined.

Results

Human lung mast cells and HMC‐1 cells migrated towards Th2 stimulated ASM from asthmatics but not non‐asthmatics. Mast cell migration was mediated through the combined activation of CCR3 and CXCR1. CCL11 and CXCL8 expression by ASM increased markedly after stimulation, but was similar in those with and without asthma. ASM supernatants from non‐asthmatics inhibited mast cell migration towards the asthmatic ASM supernatant.

Conclusion

Th2 stimulated ASM from asthmatics is chemotactic for mast cells. Non‐asthmatic ASM releases a mediator or mediators that inhibit mast cell migration towards stimulated asthmatic ASM. Specifically targeting mast cell migration into the ASM bundle may provide a novel treatment for asthma.

Histone deacetylase (HDAC) inhibitors may offer novel approaches in the treatment of asthma. We postulate that trichostatin A (TSA), a Class 1 and 2 inhibitor of HDAC, inhibits airway hyperresponsiveness in antigen-challenged mice. Mice were sensitized and challenged with Aspergillus fumigatus antigen (AF) and treated with TSA, dexamethasone, or vehicle. Lung resistance (RL) and dynamic compliance were measured, and bronchial alveolar lavage fluid (BALF) was analyzed for numbers of leukocytes and concentrations of cytokines. Human precision-cut lung slices (PCLS) were treated with TSA and their agonist-induced bronchoconstriction was measured, and TSA-treated human airway smooth muscle (ASM) cells were evaluated for the agonist-induced activation of Rho and intracellular release of Ca2+. The activity of HDAC in murine lungs was enhanced by antigen and abrogated by TSA. TSA also inhibited methacholine (Mch)-induced increases in RL and decreases in dynamic compliance in naive control mice and in AF-sensitized and -challenged mice. Total cell counts, concentrations of IL-4, and numbers of eosinophils in BALF were unchanged in mice treated with TSA or vehicle, whereas dexamethasone inhibited the numbers of eosinophils in BALF and concentrations of IL-4. TSA inhibited the carbachol-induced contraction of PCLS. Treatment with TSA inhibited the intracellular release of Ca2+ in ASM cells in response to histamine, without affecting the activation of Rho. The inhibition of HDAC abrogates airway hyperresponsiveness to Mch in both naive and antigen-challenged mice. TSA inhibits the agonist-induced contraction of PCLS and mobilization of Ca2+ in ASM cells. Thus, HDAC inhibitors demonstrate a mechanism of action distinct from that of anti-inflammatory agents such as steroids, and represent a promising therapeutic agent for airway disease.

In asthma, the increase in airway smooth muscle (ASM) can contribute to inflammation, airway wall remodeling and airway hyperresponsiveness (AHR). Targetting peroxisome proliferator-activated receptor γ (PPARγ), a receptor upregulated in ASM in asthmatic airways, may provide a novel approach to regulate these contributions. This review summarises experimental evidence that PPARγ ligands, such as rosiglitazone (RGZ) and pioglitazone (PGZ), inhibit proliferation and inflammatory cytokine production from ASM in vitro. In addition, inhaled administration of these ligands reduces inflammatory cell infiltration and airway remodelling in mouse models of allergen-induced airways disease. PPARγ ligands can also regulate ASM contractility, with acute treatment eliciting relaxation of mouse trachea in vitro through a PPARγ-independent mechanism. Chronic treatment can protect against the loss of bronchodilator sensitivity to β2-adrenoceptor agonists and inhibit the development of AHR associated with exposure to nicotine in utero or following allergen challenge. Of particular interest, a small clinical trial has shown that oral RGZ treatment improves lung function in smokers with asthma, a group that is generally unresponsive to conventional steroid treatment. These combined findings support further investigation of the potential for PPARγ agonists to target the noncontractile and contractile functions of ASM to improve outcomes for patients with poorly controlled asthma.

It remains unclear whether airway smooth muscle (ASM) mechanics is altered in asthma. While efforts have originally focussed on contractile force, some evidence points to an increased velocity of shortening. A greater rate of airway renarrowing after a deep inspiration has been reported in asthmatics compared to controls, which could result from a shortening velocity increase. In addition, we have recently shown in rats that increased shortening velocity correlates with increased muscle shortening, without increasing muscle force. Nonetheless, establishing whether or not asthmatic ASM shortens faster than that of normal subjects remains problematic. Endobronchial biopsies provide excellent tissue samples because the patients are well characterized, but the size of the samples allows only cell level experiments. Whole human lungs from transplant programs suffer primarily from poor patient characterization, leading to high variability. ASM from several animal models of asthma has shown increased shortening velocity, but it is unclear whether this is representative of human asthma. Several candidates have been suggested as responsible for increased shortening velocity in asthma, such as alterations in contractile protein expression or changes in the contractile apparatus structure. There is no doubt that more remains to be learned about the role of shortening velocity in asthma.

Airflow within the airways is determined directly by the lumenal area of that airway. In this paper, we consider several factors which can reduce airway lumenal area, including thickening and/or active constriction of the airway smooth muscle (ASM). The latter cell type can also contribute in part to inflammation, another feature of asthma, through its ability to take on a synthetic/secretory phenotype. The ASM therefore becomes a strategically important target in the treatment of asthma, given these key contributions to the pathophysiology of that disease. Pharmacological approaches have been developed to elicit relaxation of the ASM, but these are not always effective in all patients, nor do they address the long-term structural changes which impinge on the airway lumen. The recent discovery that thermal energy can be used to ablate smooth muscle has led to the development of a novel physical intervention—bronchial thermoplasty—in the treatment of asthma. Here, we review the evolution of this novel approach, consider some of the possible mechanisms that account for its salutary effects, and pose new questions which may lead to even better therapies for asthma.

Dysfunctional neural control of airway smooth muscle (ASM) is involved in inflammatory diseases, such as asthma. However, neurogenesis in the lung is poorly understood. This study uses mouse models to investigate developmental mechanisms of ASM innervation, a process that is highly coordinated with ASM formation during lung branching morphogenesis. We show that brain-derived neurotrophic factor (BDNF) is an essential ASM-derived signal for innervation. Although BDNF mRNA expression is temporally dissociated with ASM formation and innervation, BDNF protein is coordinately produced through post-transcriptional suppression by miR-206. Using a combination of chemical and genetic approaches to modulate sonic hedgehog (Shh) signaling, a pathway essential for lung branching and ASM formation, we show that Shh signaling blocks miR-206 expression, which in turn increases BDNF protein expression. Together, our work uncovers a functional cascade that involves Shh, miR-206 and BDNF to coordinate ASM formation and innervation.

CD4+ T helper (TH)1- and TH2-type cytokines reportedly play an important role in the pathobiology of asthma. Recent evidence suggests that proasthmatic changes in airway smooth muscle (ASM) responsiveness may be induced by the autocrine release of certain proinflammatory cytokines by the ASM itself. We examined whether TH1- and TH2-type cytokines are expressed by atopic asthmatic sensitized ASM and serve to autologously regulate the proasthmatic phenotype in the sensitized ASM. Expression of these cytokines and their receptors was examined in isolated rabbit and human ASM tissues and cultured cells passively sensitized with sera from atopic asthmatic patients or control subjects. Relative to controls, atopic sensitized ASM cells exhibited an early increased mRNA expression of the TH2-type cytokines, interleukin-5 (IL-5) and granulocyte–macrophage colony-stimulating factor (GM-CSF), and their receptors. This was later followed by enhanced mRNA expression of the TH1-type cytokines, IL-2, IL-12, and interferon-γ (IFN-γ), as well as their respective receptors. In experiments on isolated ASM tissue segments (a) exogenous administration of IL-2 and IFN-γ to atopic asthmatic serum–sensitized ASM ablated both their enhanced constrictor responsiveness to acetylcholine (ACh) and their attenuated relaxation responsiveness to β-adrenoceptor stimulation with isoproterenol, and (b) administration of IL-5 and GM-CSF to naive ASM induced significant increases in their contractility to ACh and impaired their relaxant responsiveness to isoproterenol. Collectively, these observations provide new evidence demonstrating that human ASM endogenously expresses both TH1- and TH2-type cytokines and their receptors, that these molecules are sequentially upregulated in the atopic asthmatic sensitized state, and that they act to downregulate and upregulate proasthmatic perturbations in ASM responsiveness, respectively.

An important interplay exists between specific viral respiratory infections and altered airway responsiveness in the development and exacerbations of asthma. However, the mechanistic basis of this interplay remains to be identified. This study addressed the hypothesis that rhinovirus (RV), the most common viral respiratory pathogen associated with acute asthma attacks, directly affects airway smooth muscle (ASM) to produce proasthmatic changes in receptor-coupled ASM responsiveness. Isolated rabbit and human ASM tissue and cultured ASM cells were inoculated with human RV (serotype 16) or adenovirus, each for 6 or 24 h. In contrast to adenovirus, which had no effect, inoculation of ASM tissue with RV induced heightened ASM tissue constrictor responsiveness to acetylcholine and attenuated the dose-dependent relaxation of ASM to beta-adrenoceptor stimulation with isoproterenol. These RV-induced changes in ASM responsiveness were largely prevented by pretreating the tissues with pertussis toxin or with a monoclonal blocking antibody to intercellular adhesion molecule-1 (ICAM-1), the principal endogenous receptor for most RVs. In extended studies, we found that the RV-induced changes in ASM responsiveness were associated with diminished cAMP accumulation in response to dose-dependent administration of isoproterenol, and this effect was accompanied by autologously upregulated expression of the Gi protein subtype, Gialpha3, in the ASM. Finally, in separate experiments, we found that the RV-induced effects on ASM responsiveness were also accompanied by autologously induced upregulated mRNA and cell surface protein expression of ICAM-1. Taken together, these findings provide new evidence that RV directly induces proasthmatic phenotypic changes in ASM responsiveness, that this effect is triggered by binding of RV to its ICAM-1 receptor in ASM, and that this binding is associated with the induced endogenously upregulated expression of ICAM-1 and enhanced expression and activation of Gi protein in the RV-infected ASM.

Bitter taste receptors (TAS2Rs) were shown to be expressed in human airway smooth muscle (ASM). They couple to specialized [Ca2+]i release, leading to membrane hyperpolarization, the relaxation of ASM, and marked bronchodilation. TAS2Rs are G-protein–coupled receptors, known to undergo rapid agonist-promoted desensitization that can limit therapeutic efficacy. Because TAS2Rs represent a new drug target for treating obstructive lung disease, we investigated their capacity for rapid desensitization, and assessed their potential mechanisms. The pretreatment of human ASM cells with the prototypic TAS2R agonist quinine resulted in a 31% ± 5.1% desensitization of the [Ca2+]i response from a subsequent exposure to quinine. No significant change in the endothelin-stimulated [Ca2+]i response was attributed to the short-term use of quinine, indicating a homologous form of desensitization. The TAS2R agonist saccharin also evoked desensitization, and cross-compound desensitization with quinine was evident. Desensitization of the [Ca2+]i response was attenuated by a dynamin inhibitor, suggesting that receptor internalization (a G-protein coupled receptor kinase [GRK]-mediated, β-arrestin–mediated process) plays an integral role in the desensitization of TAS2R. Desensitization was insensitive to antagonists of the second messenger kinases protein kinase A and protein kinase C. Using intact airways, short-term, agonist-promoted TAS2R desensitization of the relaxation response was also observed. Thus these receptors, which represent a potential novel target for direct bronchodilators, undergo a modest degree of agonist-promoted desensitization that may affect clinical efficacy. Collectively, the results of these mechanistic studies, along with the multiple serines and threonines in intracellular loop 3 and the cytoplasmic tail of TAS2Rs, suggest a GRK-mediated mode of desensitization.