Airway hyper-responsiveness, a key feature of asthma, is the enhanced contraction of the airway smooth muscle layer in response to inhaled stimuli, which leads to variable airway obstruction. This phenomenon is responsible for the acute exacerbations associated with asthma. Recurrent exacerbations are a feature of severe asthma. Similarly, the structural changes of airway remodelling are also commonly associated with cases of severe asthma (1
) and deteriorating lung function over time (2
). It has recently been shown that broncho-constriction can induce features of airway remodelling in mild asthmatics, including collagen deposition and goblet cell hyperplasia (4
), however, the mechanism responsible for this is unknown.
Transforming growth factor-β (TGF-β) has been implicated in the pathogenesis of airway remodelling in asthma (5
). TGF-β promotes airway remodelling due, in part, to its effects on ASM cell proliferation, epithelial cell apoptosis and its potent pro-fibrotic actions, including increasing synthesis of collagen and fibronectin (6
). It promotes extracellular matrix deposition, ASM proliferation and mucous production in an animal model of allergic asthma without affecting existing airway inflammation (9
). Over-expression of Smad2, a TGF-β signalling protein, causes thickening of the ASM layer and deposition of collagen following allergen challenge (10
). Moreover, the importance of TGF-β signalling in asthma pathogenesis is supported by a genome-wide association studying demonstrating a link between a single nucleotide polymorphism in the SMAD3 gene and asthma (11
TGF-β is secreted from cells in non-covalent association with its pro-peptide, the latency associated peptide (LAP), which renders it inactive. Activation of latent TGF-β (L-TGF-β) is the rate limiting step in its bioavailability (12
) and mechanisms of TGF-β activation are fundamental to disease. Several mechanisms of activation have been described in vitro
including proteolytic activation by plasmin, matrix metalloproteases (MMP’s) and tryptase, physical activation by extremes of heat and oxidation, and activation by thrombospondin-1 (13
). Several studies have described increased TGF-β activity in asthma (18
). Activation of TGF-β in asthma may occur by several mechanisms. Epithelial cells may activate TGF-β in response to damage to the epithelial layer. Mast cells, which are present in large numbers in the asthmatic bronchial mucosa, can activate TGF-β proteolytically through the release of proteases from their granules (15
). In vivo
integrins appear to play the major role in TGF-β activation, at least in development (24
), and recently fibroblast specific deletion of the αvβ8 integrin has been shown to reduce airway remodelling by reducing TGF-β induced CCL2 and CCL20 dependent dendritic cell migration (26
). However, whether smooth muscle cell TGF-β activation can directly contribute to airway remodelling is unknown.
Integrins are heterodimeric cell surface molecules involved in cell-cell interactions and cell-matrix interactions. Six of the 24 currently described integrins recognise and bind arginine-glycine-aspartate (RGD) motifs in the LAP of both TGF-β1 and TGF-β3. Four of these have been reported to activate TGF-β in vitro including αVβ6 (27
), αVβ8 (28
), αVβ3 (29
) and αVβ5 (30
). Integrin meditaed TGFβ activation has been best characterised for the αvβ6 and the αvβ8 integrins. Activation of TGF-β by the αvβ8 integrin involves MMP14 and proteolytic cleavage of the latent TGF-β molecule, whereas αVβ3, αVβ5 and αvβ6 integrins activate TGF-β by a mechanism requiring an intact cytoskeleton and cell contraction (27
). Activation of TGF-β by αVβ6 integrins is spatially restricted to epithelial cells, whereas αVβ5, and to a lesser extent αVβ3, are present on mesenchymal cells and are able to activate mesenchymal TGF-β (30
). This raises the possibility that cellular contraction during broncoconstriction may promote TGF-β activation via cell surface integrins.
The aims of this study were to investigate whether contraction agonists could promote TGF-β activation in human ASM (HASM) cells, and determine whether this process was dysregulated in asthma. We found that LPA induced TGF-β activation by HASM cells via an integrin αVβ5-mediated mechanism that involved reorganisation of the cytoskeleton. Furthermore, methacholine also induced TGF-β activation by HASM cells. HASM cells isolated from asthmatic patients activated more TGF-β in response to both contraction agonists than cells from non-asthmatic individuals. Furthermore, a polymorphism in the cytoplasmic domain of the integrin β5 (itgb5) gene resulted in a β5 subunit that was unable to activate TGF-β. Using the ovalbumin (OVA) model of airway remodelling we demonstrated co-association of αVβ5 integrins and phospho-Smad2 staining in the airway smooth muscle (ASM) layer of remodelled airways, as well as global increases in TGF-β activation. Finally, using two distinct murine models of asthma, we show that both inhibition, and genetic loss, of the αVβ5 integrin results in significantly less ASM surrounding the airways despite enhanced inflammation. This suggests that the αvβ5 integrin may play an important role mediating airway remodelling, as well as supporting the notion that inflammation and remodelling can be dissociated in asthma. These data suggest a potential novel mechanism through which contraction of the ASM layer during asthma attacks could promote airway remodelling in patients with poorly controlled disease.