Whereas the role of bronchial smooth muscle remains controversial in healthy subjects its role is well established in asthmatics. Bronchial smooth muscle contraction induces airway narrowing. The smooth muscle also contributes to bronchial inflammation by secreting a range of inflammatory mediators, recruiting and activating inflammatory cells, such as mast cells or T-lymphocytes. In addition, bronchial smooth muscle mass is significantly increased in asthma. Such an increase has been related to a deposition of extracellular matrix proteins, and an increase in both cell size and number. However, the mechanisms of this smooth muscle remodelling are complex and not completely understood. The article will review recent data regarding the pathophysiology of bronchial smooth muscle remodelling in asthma.
Asthma is a chronic inflammatory disease, characterised by the association of bronchial hyperresponsiveness, inflammation and remodelling 1–3. Current medications are effective in treating acute airway narrowing and decreasing inflammation but are relatively less effective in preventing chronic structural changes 4. Bronchial remodelling is described as an increased thickening of the bronchial wall due to various structural alterations including: abnormal epithelium; sub-epithelial membrane thickening; alteration in extracellular matrix (ECM) deposition; neoangiogenesis; mucus gland hypertrophy; and an increased bronchial smooth muscle (BSM) mass (fig. 1). The latter appears to be the most important feature of bronchial remodelling since increased BSM mass is associated with a decrease in lung function in severe asthma 5, 6. However, major anti-asthmatic treatments, such as corticosteroids, remain totally ineffective in decreasing BSM mass 4. As a result, innovative treatments such as bronchial thermoplasty 7, 8 aim to target BSM.
Representative optic microscopic images from bronchial sections stained with Haematoxylin, Eosin and safranin stain were obtained from a) a control subject or b) an asthmatic subject. E: epithelium; G: mucous gland; SM: smooth muscles. Scale bars = 50 μm.
The physiological role of BSM remains controversial. BSM is known to contribute to the normal branching of the respiratory tree during lung embryogenesis 9, 10. In healthy subjects, BSM may play a role in co-ordinating the distribution of ventilation within the airways 11, 12, in mucus propulsion 13 or in helping exhalation 14. However, these potential roles have not been experimentally validated. Mitzner 15 suggested that BSM is vestigial and has no physiological function, stating that BSM is “the appendix of the lung”. Paradoxically, the pathophysiological role of BSM in asthma is well established. BSM is the main effector of bronchial contraction in response to various stimuli, including inflammatory mediators. Moreover, BSM has also been considered as an inflammatory cell per se 16. It can contribute to an auto-activation loop involving mast cells and implicating the production of cytokines 17. Upon stimulation, BSM cells produce a wide range of cytokines and chemokines including CXCL10 (IP-10) and CX3CL1 (Fraktalkine), which participate in this auto-activation loop 18, 19. As a result, mast cells are attracted by BSM and preferentially infiltrate the BSM layer of both fatal and nonfatal asthmatics 20, 21. As part of this auto-activation loop, mast cells can adhere to BSM cells 2, 22, 23, promoting both survival and proliferation of mast cells 24. Mast cell activation and degranulation can be allergen dependent or independent 25–28, and can be responsible for an important extracellular deposition of inflammatory products that may facilitate the increase in BSM mass, as well as bronchial hyperresponsiveness 16, 29, 30. T-lymphocytes may also participate in BSM remodelling. Lazaar et al. 31 demonstrated that the adhesion of T-lymphocytes to BSM cells induced BSM cell DNA synthesis. More recently, this increased BSM proliferation was related to a direct contact between activated CD4 + T-cells and BSM cells using cells from a rat experimental model of asthma 32.
Bronchial chronic asthmatic inflammation causes tissue injuries leading to repetitive repair processes. Remodelling was initially thought to be the consequence of an incomplete repair process in asthma 33. However, the early onset of this process 34, 35 sometimes before eosinophilic inflammation 36 suggests that bronchial inflammation and remodelling may occur simultaneously in asthma. BSM remodelling is characterised by an increased deposition of ECM proteins in and around the BSM bundles, an increased BSM cell size or hypertrophy, and an increased BSM cell number or hyperplasia (fig. 2). The aim of our article is to review recent data regarding these specific aspects of the pathophysiology of BSM remodelling in asthma.
Mechanisms of asthmatic bronchial smooth muscle (BSM) remodelling. The three main characteristics of BSM remodelling in asthma are presented. BSM cell hyperplasia can be related to an increased cell proliferation, a decreased cell apoptosis or the recruitment of mesenchymal cells. EMT: epithelial mesenchymal transition; ECM: extracelluar matrix.
There is a growing body of evidence indicating that the BSM ECM is altered in asthma 29, 37–39. Indeed, ECM is increased around each individual BSM cell within the muscle bundles 37, by large bland amount of protein deposits 29. Such an increased ECM contains a higher amount of collagen 38 and both fibronectin and elastic fibres, although the latter has only been found within the BSM from fatal asthma 39. Several of these characteristics have been described in both large and small airways 39. Cultured human nonasthmatic BSM cells produce a wide range of matrix proteins, including fibronectin, perlecan, elastin, laminin, thrombospondin, chondroitin sulfate, collagen I, III, IV and V, versican and decorin 40. Interestingly, asthmatic BSM cells produce an altered profile of ECM proteins in vitro, characterised by more collagen I and perlecan, but less laminin-α1, collagen IV 41 and hyaluronan 42. Such an altered ECM production by BSM cells could contribute to the altered ECM composition of the whole asthmatic bronchial wall. Indeed, asthmatic bronchial ECM is characterised by an increased amount of collagen I, collagen III and fibronectin 43–45 and a decreased amount of collagen IV 46. However, bronchial ECM also presents higher amount of hyaluronan, versican, and laminin 43, 47, which may be produced by cells different from BSM, such as epithelial cells and/or an imbalance between matrix production and degradation.
The increased ECM deposition may also be due to decreased matrix metalloproteinases (MMP) or increased tissue inhibitors of matrix metalloproteinases (TIMP). However, in biopsies from fatal asthmatics, both MMP-9 and MMP-12 were increased within the BSM, whereas no change was observed in the expression of MMP-1, MMP-2, TIMP-1 and TIMP-2 39. However, these findings seem to be restricted to fatal asthma cases since no significant difference has been demonstrated in the BSM from nonfatal asthmatics 39. MMP-9 degrades collagen IV, a major component of the airway sub-epithelial basement membrane 48, and MMP-12 is implicated in elastin, collagen IV, fibronectin and laminin digestion 49, 50. In vitro, BSM cells from nonasthmatics have been shown to express only a small amount of MMP-9 but also MMP-2, MMP-3, membrane type-1-MMP 51. Nevertheless, the overall BSM MMP activity remains low due to an excess expression of TIMP-1 and TIMP-2 51. Whether MMP-9 production and activity can be upregulated under inflammatory conditions remains unknown. In contrast, MMP-12, which is also expressed by BSM cells, is upregulated by interleukin (IL)-1β or tumour necrosis factor (TNF)-α 52, although such upregulation was not observed in a single report on asthmatic BSM cells in vitro 52. Nevertheless, an increased expression of both MMP-9 and MMP-3 has been found in the bronchoalveolar lavage (BAL) fluid from asthmatics 53 and could be related to other cell types. For example, eosinophils and neutrophils are also known to be a major source of MMP-9 48, 54. In addition, levels of TIMP-1 are higher in untreated asthmatics than in treated subjects 55 although the role of BSM cells in down regulating MMPs by upregulation of TIMPs in asthma remains to be established.
The increased and abnormal asthmatic ECM could interact with growth factors. In particular, transforming growth factor (TGF)-β is stored within the ECM as an inactive form combined with the latency-associated peptide 17. Amongst various enzymes capable of activating TGF-β, MMP-9 releases the active form of TGF-β 56. TGF-β is increased within asthmatic airways 57, 58 and more specifically in the BSM layer 17. TGF-β induces fibronectin and collagen I deposition from BSM cells through connective tissue growth factor (CTGF)-dependent and -independent pathways 59. Interestingly, CTEF is increased in asthmatic BSM cells 60. In addition, TGF-β, which is secreted by BSM cells after mast cell degranulation, induces mast cell chemotaxis and thus participates in an auto-activation loop 17.
Finally, ECM proteins may also modulate BSM phenotype, as well as its functions including contraction, migration and proliferation 61. On the one hand, fibronectin reduces both the contractility and expression of α-actin, calponin and myosin heavy chain in bovine BSM strips 62. On the other hand, laminin increases the contractility of bovine BSM strips 62, and induces the maturation of human BSM cells into a contractile phenotype 63. Conversely, fibronectin enhances BSM cell proliferation in response to platelet-derived growth factor (PDGF) or thrombin, whereas laminin decreases BSM cell proliferation 64. Thus, asthmatic BSM cells that produce an altered ECM influence their own environment, and may, as a consequence, contribute to modulate their own function.
Whether BSM hypertrophy is present in asthma remains controversial 29, 65–67. For some authors, there is evidence that BSM hypertrophy contributes to airway remodelling in asthma. Ebina et al. 67 have examined the airways of fatal asthma, and described two asthmatic subtypes. In particular, the second subtype includes an increased BSM cell size throughout the bronchial tree. More recently, Benayoun et al. 65 studied bronchial biopsies and found that patients with asthma had larger BSM cell diameter compared to control subjects. Furthermore, severe asthmatics presented the highest BSM cell size 65. Interestingly, it has also been shown that asthmatic BSM hypertrophy was associated with an increased expression of myosin light chain kinase (MLCK), whereas that of both α-smooth muscle actin (SMA) and myosin was unchanged 65. In addition, using an ultrastructural approach, Begueret et al. 29 also showed an increased BSM cell size in atopic asthmatics. Conversely, using a three-dimensional approach Woodruff et al. 66 did not find any evidence of an increase in the BSM cell size in patients with mild-to-moderate asthma. Thus, BSM cell hypertrophy may be related to asthma severity.
The cellular mechanisms of such BSM cell hypertrophy have been addressed using nonasthmatic BSM cells only. In vitro, primary cultured BSM cells obtained from nonasthmatic donors and even from animals or immortalised human BSM cell lines have been examined 68–70. On the one hand, BSM cell hypertrophy has been reproduced in vitro using serum deprivation 69 or cell stimulation with TGF-β, endothelin or cardiotrophin-1 70–72. On the other hand, a BSM cell line has been obtained using a temperature-sensitive simian virus-40 large T-antigen, which binds to and inactivates p53 68. In such a cell line there is an increase in both cell size and amount of α-SMA and MLCK in a post-transcriptional manner 68. BSM hypertrophy involved complex transduction pathways (fig. 3), recently reviewed by Bentley and Hershenson 73. As a summary, two distinct pathways could activate BSM cell hypertrophy. The first pathway involves the mammalian target of rapamycin (i.e. mTOR). mTOR induces the phosphorylation of 4E-binding protein (4E-BP), which releases the transcription factor eIF4E leading to BSM cell hypertrophy 74. In addition, mTOR also phosphorylates p70S6-kinase, which activates S6 kinase 75. Such a pathway is necessary and sufficient for BSM cell hypertrophy. In addition, when TGF-β is used to induce BSM cell hypertrophy in vitro the phosphorylation of 4E-BP appears to be more phosphatidylinositol 3-kinase (PI3)-kinase-dependent than mTOR-dependent, whereas that of p70S6-kinase only requires mTOR activation 70. The possible upstream inhibition of mTOR by tuberous sclerosis complex-2 has not been demonstrated in BSM cells but has been confirmed in other cell types, including HEK293 76. The second pathway involves the inhibition of glycogen synthase kinase (GSK)-3β, for instance by Akt. GSK-3β usually inhibits the translation initiation by eIF2B in many cell types 77, 78. Inhibition of GSK3-β induces BSM cell hypertrophy through an eIF2B-dependent manner 79. Furthermore, in a recent in vivo study using ovalbumin-sensitised mice, Bentley et al. 80 have demonstrated that GSK3-β is phosphorylated and thus inactivated within the hypertrophic BSM cells. Whether these transduction pathways are actually implicated in human asthmatic BSM cell hypertrophy remains to be established and further studies are needed to explore the involvement of such pathways in asthmatic BSM cells.
Mechanisms of bronchial smooth muscle (BSM) cell hypertrophy. Signal transduction mechanisms of BSM cell hypertrophy involve both mammalian target of rapamycin (mTOR) and glycogen synthase kinase (GSK)-3β. Upstream and down-stream transduction cascades are presented. →: activation; –––|: inhibition; ······: indicates that the transduction pathway has not yet been demonstrated in BSM cells.