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.
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.
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.
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.
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.
asthma; bronchial smooth muscle; signal transduction; G-protein–coupled receptors
Inhaled β2-adrenoreceptor agonists are widely used in asthma and chronic obstructive pulmonary disease (COPD) for bronchoconstriction relief. β2-adrenoreceptor agonists relax airway smooth muscle cells via cyclic adenosine monophosphate (cAMP) mediated pathways. However, prolonged stimulation induces functional desensitization of the β2-adrenoreceptors (β2-AR), potentially leading to reduced clinical efficacy with chronic or prolonged administration. ASM-024, a small synthetic molecule in clinical stage development, has shown activity at the level of nicotinic receptors and possibly at the muscarinic level and presents anti-inflammatory and bronchodilator properties. Aerosolized ASM-024 reduces airway resistance in mice and promotes in-vitro relaxation of tracheal and bronchial preparations from animal and human tissues. ASM-024 increased in vitro relaxation response to maximally effective concentration of short—acting beta-2 agonists in dog and human bronchi. Although the precise mechanisms by which ASM-024 promotes airway smooth muscle (ASM) relaxation remain unclear, we hypothesized that ASM-024 will attenuate and/or abrogate agonist-induced contraction and remain effective despite β2-AR tachyphylaxis. β2-AR tachyphylaxis was induced with salbutamol, salmeterol and formoterol on guinea pig tracheas. The addition of ASM-024 relaxed concentration-dependently intact or β2-AR desensitized tracheal rings precontracted with methacholine. ASM-024 did not induce any elevation of intracellular cAMP in isolated smooth muscle cells; moreover, blockade of the cAMP pathway with an adenylate cyclase inhibitor had no significant effect on ASM-024-induced guinea pig trachea relaxation. Collectively, these findings show that ASM-024 elicits relaxation of β2-AR desensitized tracheal preparations and suggest that ASM-024 mediates smooth muscle relaxation through a different target and signaling pathway than β2-adrenergic receptor agonists. These findings suggest ASM-024 could potentially provide clinical benefit when used adjunctively with inhaled β2-adrenoreceptor agonists in those patients exhibiting a reduced response to their chronic use.
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.
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.
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.
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.
Mast cell localization within the airway smooth muscle (ASM)-bundle plays an important role in the development of airway hyper-responsiveness (AHR). Genomewide association studies implicate the ‘alarmin’ IL-33 in asthma, but its role in mast cell–ASM interactions is unknown.
We examined the expression and functional role of IL-33 in bronchial biopsies of patients with and without asthma, ex vivo ASM, mast cells, cocultured cells and in a mouse model system.
IL-33 protein expression was assessed in human bronchial tissue from 9 healthy controls, and 18 mild-to-moderate and 12 severe asthmatic patients by immunohistochemistry. IL-33 and ST2 mRNA and protein expression in human-derived ASM, epithelial and mast cells were assessed by qPCR, immunofluorescence and/or flow cytometry and ELISA. Functional assays were used to assess calcium signalling, wound repair, proliferation, apoptosis and contraction. AHR and inflammation were assessed in a mouse model.
Bronchial epithelium and ASM expressed IL-33 with the latter in asthma correlating with AHR. ASM and mast cells expressed intracellular IL-33 and ST2. IL-33 stimulated mast cell IL-13 and histamine secretion independent of FcεR1 cross-linking and directly promoted ASM wound repair. Coculture of mast cells with ASM activated by IL-33 increased agonist-induced ASM contraction, and in vivo IL-33 induced AHR in a mouse cytokine installation model; both effects were IL-13 dependent.
IL-33 directly promotes mast cell activation and ASM wound repair but indirectly promotes ASM contraction via upregulation of mast cell-derived IL-13. This suggests that IL-33 may present an important target to modulate mast cell–ASM crosstalk in asthma.
ASM; IL-33; mast cells
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.
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.
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.
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.
BACKGROUND AND PURPOSE
Platelet-derived growth factor (PDGF) modulates the airway smooth muscle (ASM) ‘contractile’ phenotype to a more ‘proliferative’ phenotype, resulting in increased proliferation and reduced contractility. Such phenotypic modulation may contribute to airway remodelling in asthma. We have previously shown that the cAMP effector molecules, protein kinase A (PKA) and the exchange protein directly activated by cAMP (Epac) inhibited PDGF-induced phenotypic modulation in bovine ASM. Here, we investigated these mechanisms in human ASM strips and cells.
ASM strips were incubated with PDGF in the absence or presence of the activators of Epac (8-pCPT-2′-O-Me-cAMP) or of PKA (6-Bnz-cAMP) for 4 days. Strips were mounted for isometric contraction experiments or analysed for the expression of contractile markers. Cell proliferation was measured and proliferative markers were analysed under similar conditions.
Activation of Epac and PKA prevented PDGF-induced ASM strip hypocontractility, and restored the expression of smooth muscle actin, myosin and calponin, which had been markedly diminished by PDGF. Epac and PKA activation inhibited the PDGF-induced ASM cell proliferation and G1/S phase transition and the expression and phosphorylation of cell cycle regulators.
CONCLUSIONS AND IMPLICATIONS
Epac and PKA maintain a normally contractile ASM phenotype in a mitogenic environment, suggesting that specific activators of Epac and PKA may be beneficial in the treatment of airway remodelling in asthma.
airway smooth muscle; Epac; PKA; airway remodelling; airway smooth muscle contraction; proliferation; PDGF; cAMP; asthma; contractile proteins; phenotypic plasticity; β2-agonists
BACKGROUND AND PURPOSE
Previous studies have linked a reduction in pH in airway, caused by either environmental factors, microaspiration of gastric acid or inflammation, with airway smooth muscle (ASM) contraction and increased airway resistance. Neural mechanisms have been shown to mediate airway contraction in response to reductions in airway pH to < 6.5; whether reduced extracellular pH (pHo) has direct effects on ASM is unknown.
Intracellular signalling events stimulated by reduced pHo in human cultured ASM cells were examined by immunoblotting, phosphoinositide hydrolysis and calcium mobilization assays. ASM cell contractile state was examined using magnetic twisting cytometry. The expression of putative proton-sensing GPCRs in ASM was assessed by real-time PCR. The role of ovarian cancer G protein-coupled receptor 1 (OGR1 or GPR68) in acid-induced ASM signalling and contraction was assessed in cultures subjected to siRNA-mediated OGR1 knockdown.
ASM cells responded to incremental reductions in pHo (from pH 8.0 to pH 6.8) by activating multiple signalling pathways, involving p42/p44, PKB, PKA and calcium mobilization. Coincidently, ASM cells contracted in response to decreased pHo with similar ‘dose’-dependence. Real-time PCR suggested OGR1 was the only proton-sensing GPCR expressed in ASM cells. Both acid-induced signalling (with the exception of PKB activation) and contraction were significantly attenuated by knockdown of OGR1.
CONCLUSIONS AND IMPLICATIONS
These studies reveal OGR1 to be a physiologically relevant GPCR in ASM cells, capable of pleiotropic signalling and mediating contraction in response to small reductions in extracellular pH. Accordingly, ASM OGR1 may contribute to asthma pathology and represent a therapeutic target in obstructive lung diseases.
airway smooth muscle; asthma; bronchoconstriction; calcium; cAMP-dependent protein kinase; contraction; GPR68; G protein-coupled receptor; inflammation; OGR1
Background: Airway smooth muscle (ASM) from asthmatics is in a hyperproliferative state and releases more cytokines than healthy individuals.
Results: Inhibition of BET bromodomains reduces ASM proliferation and cytokine release by reducing Brd4 binding to the promoter regions of IL-6 and CXCL8.
Conclusion: BET bromodomain mimics inhibit aberrant ASM proliferation and inflammation in asthmatic patients.
Significance: These compounds may reduce airway remodeling in asthma.
Airway smooth muscle (ASM) mass is increased in asthma, and ASM cells from patients with asthma are hyperproliferative and release more IL-6 and CXCL8. The BET (bromo- and extra-terminal) family of proteins (Brd2, Brd3, and Brd4) govern the assembly of histone acetylation-dependent chromatin complexes. We have examined whether they modulate proliferation and cytokine expression in asthmatic ASM cells by studying the effect of BET bromodomain mimics JQ1/SGCBD01 and I-BET762. ASM cells from healthy individuals and nonsevere and severe asthmatics were pretreated with JQ1/SGCBD01 and I-BET762 prior to stimulation with FCS and TGF-β. Proliferation was measured by BrdU incorporation. IL-6 and CXCL8 release was measured by ELISA, and mRNA expression was measured by quantitative RT-PCR. ChIP using a specific anti-Brd4 antibody and PCR primers directed against the transcriptional start site of IL-6 and CXCL8 gene promoters was performed. Neither JQ1/SGCBD01 nor I-BET762 had any effect on ASM cell viability. JQ1/SGCBD01 and I-BET762 inhibited FCS+TGF-β-induced ASM cell proliferation and IL-6 and CXCL8 release in healthy individuals (≥ 30 nm) and in nonsevere and severe asthma patients (≥100 nm), with the latter requiring higher concentrations of these mimics. JQ1/SGCBD01 reduced Brd4 binding to IL8 and IL6 promoters induced by FCS+TGF-β. Mimics of BET bromodomains inhibit aberrant ASM cell proliferation and inflammation with lesser efficiency in those from asthmatic patients. They may be effective in reducing airway remodeling in asthma.
Asthma; Bromodomain-containing Protein 4 (BRD4); Cell Proliferation; Interleukin 6 (IL-6); Myc (c-Myc); Airway Smooth Muscle; CXCL8; I-BET762; JQ1/SGCBD01
BACKGROUND AND PURPOSE
Airway smooth muscle (ASM) phenotype plasticity, characterized by reversible switching between contractile and proliferative phenotypes, is considered to contribute to increased ASM mass and airway hyper-responsiveness in asthma. Further, increased expression of collagen I has been observed within the ASM bundle of asthmatics. Previously, we showed that exposure of intact bovine tracheal smooth muscle (BTSM) to collagen I induces a switch from a contractile to a hypocontractile, proliferative phenotype. However, the functional relevance of this finding for intact human ASM has not been established.
We investigated the effects of exposure of human tracheal smooth muscle (HTSM) strips to monomeric collagen I and PDGF on contractile responses to methacholine and KCl. Expression of contractile proteins sm-α-actin and sm-MHC was assessed by Western blot analysis. The proliferation of HTSM cells was assessed by cell counting, measuring mitochondrial activity (Alamarblue conversion) and [3H]-thymidine incorporation. Proliferation of intact tissue slices was assessed by [3H]-thymidine incorporation.
Culturing HTSM strips in the presence of collagen I or PDGF for 4 days reduced maximal contractile responses to methacholine or KCl and the expression of contractile proteins. Conversely, collagen I and PDGF increased proliferation of HTSM cells and proliferative responses in tissue slices. PDGF additively increased the proliferation of HTSM cells cultured on collagen I; this additive effect was not observed on contractility, contractile protein expression or proliferation of intact tissue.
CONCLUSION AND IMPLICATIONS
These findings indicate that collagen I and PDGF induce a functionally hypocontractile, proliferative phenotype of human ASM, which may contribute to airway remodelling in asthma.
collagen type I; platelet-derived growth factor; airway smooth muscle contractility; airway smooth muscle proliferation; phenotype modulation
Caveolin-1 is a fundamental signalling scaffold protein involved in contraction; however, the role of caveolin-1 in airway responsiveness remains unclear. We evaluated the relationship between caveolin-1 expression in airway smooth muscle (ASM) and antigen-induced airway responsiveness and obstruction in a guinea pig asthma model.
Airway obstruction in sensitised guinea pigs, induced by antigenic (ovalbumin) challenges administered every 10 days, was measured. Antigen-induced responsiveness to histamine and the expression of caveolin-1 and cavin 1, 2 and 3 were evaluated at the third ovalbumin challenge. The control group received saline solution instead of ovalbumin.
After the first challenge, antigen exposure induced a transient airway obstruction and airway hyperresponsiveness, high levels of IL-4 and IL-5 in lung and airway globet cells proliferation at the third antigenic challenge. Caveolin-1 mRNA levels in total lung decreased in the experimental group compared with controls. Flow cytometric analysis of ASM from the experimental group showed a high number of cells expressing caveolin-1 compared with controls. This increase was confirmed by western blot. Airway obstruction and hyperresponsiveness correlated with the degree of increased caveolin-1 expression in ASM cells (P < 0.05; r = 0.69 and −0.52, respectively). The expression of cavins 1, 2 and 3 in ASM also increased in the experimental group compared to controls. Immunohistochemical findings reveal that differences in ASM caveolin-1 were not evident between groups. Nevertheless, a marked decrease in caveolin-1 and caspase 3 was observed in the pulmonary vascular smooth muscle of asthma model compared with controls. Histological analysis did not reveal differences in smooth muscles mass or subepithelial fibrosis levels in airways between groups. However, an enlargement of smooth muscle mass was observed in the pulmonary microvessels of experimental animals. This enlargement did not induce changes in pulmonary or systemic arterial pressures.
Our data suggest that caveolin-1 expression in ASM has a crucial role in the development of antigen-induced airway obstruction and hyperresponsiveness in a guinea pig asthma model. In addition, the asthma model in guinea pigs appears to induce a contractile smooth muscle phenotype in the airways and a proliferative smooth muscle phenotype in pulmonary vessels.
Airway hyperresponsiveness; Airway obstruction; Airway smooth muscle; Asthma; caspase 3; Caveolin-1; Cavin; Pulmonary arterial smooth muscle
Leukotriene B4 (LTB4) increases in induced sputum and exhaled breath condensate in people with asthma. Furthermore, the TH2-type immune response and airway hyperresponsiveness induced by ovalbumin sensitization is markedly suppressed in LTB4 receptor (BLT) 1 null mice. These studies suggest that LTB4 may contribute to asthma pathophysiology. However, the direct effects of LTB4 on human airway smooth muscle (ASM) have not been studied.
We sought to determine the expression of LTB4 receptors on human ASM and its functional role in mediating responses of human ASM cells, and the effect of LTB4 on these cells.
Immunohistochemistry, RT-PCR, Western blotting, and flow cytometry were used to determine the expression of LTB4 receptors. To determine the effect of LTB4 on human ASM cells, cell proliferation was assessed by counting cells, and chemokinesis was assessed by gold particle phagokinesis assay.
We confirmed expression of both BLT1 and BLT2 in human ASM cells in bronchial tissue and in cell culture. LTB4 markedly induced cyclin D1 expression, proliferation, and chemokinesis of human ASM cells. LTB4 also induced phosphorylation of both p42/p44 mitogen-activated protein kinase (MAPK) and downstream PI3 kinase effector, Akt1. However, we observed no induction of c-Jun N-terminal kinase or p38 MAPK. Notably, LTB4-induced migration and proliferation of ASM cells were inhibited by the BLT1 specific antagonist, U75302, and by inhibitors of p42/p44 MAPK phosphorylation (U1026), and PI3 kinase (LY294002).
These observations are the first to suggest a role for a LTB4-BLT1 signaling axis in ASM responses that may contribute to the pathogenesis of airway remodeling in asthma.
Asthma; airway remodeling; airway smooth muscle cells; LTB4; BLT
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.
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.
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.
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.
mast cells; chemokine receptors; chemokines; airway smooth muscle; asthma
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.
Asthma is a chronic respiratory disease characterized by reversible airway obstruction with persistent airway inflammation and airway remodeling. Features of airway remodeling include increased airway smooth muscle (ASM) mass. A disintegrin and metalloproteinase (ADAM)–33 has been identified as playing a role in the pathophysiology of asthma. ADAM-33 is expressed in ASM cells and is suggested to play a role in the function of these cells. However, the regulation of ADAM-33 is not fully understood. Vascular endothelial growth factor (VEGF) has been implicated in inflammatory and airway blood vessel remodeling in asthmatics. Although VEGF was initially thought of as an endothelial-specific growth factor, recent reports have found that VEGF can promote proliferation of other cell types, including ASM cells. To investigate the precise mechanism of VEGF's effect on ASM cell proliferation, we tested the expression of ADAM-33, phospho-extracellularsignal-regulated kinase 1/2 (ERK1/2), and phospho-Akt in VEGF-stimulated ASM cells. We found that VEGF up-regulates ADAM-33 mRNA and protein levels in a dose- and time-dependent manner as well as phosphorylation of ERK1/2 and Akt. We also found that VEGF-induced ASM cell proliferation is inhibited by both ADAM-33 knockdown and a selective VEGF receptor 2 (VEGFR2) inhibitor (SU1498). Furthermore, VEGF-induced ADAM-33 expression and ASM cell proliferation were suppressed by inhibiting ERK1/2 activity, but not by inhibiting Akt activity. Collectively, our findings suggest that VEGF enhances ADAM-33 expression and ASM cell proliferation by activating the VEGFR2/ERK1/2 signaling pathway, which might be involved in the pathogenesis of airway remodeling. Further elucidation of the mechanisms underlying these observations might help develop therapeutic strategies for airway diseases associated with smooth muscle hyperplasia such as asthma.
Akt; airway remodeling; ADAM-33; ERK1/2; VEGF
Airway remodelling describes the histopathological changes leading to fixed airway obstruction in patients with asthma and includes extra-cellular matrix (ECM) deposition. Matrix metalloproteinase-1 (MMP-1) is present in remodelled airways but its relationship with ECM proteins and the resulting functional consequences are unknown. We used airway smooth muscle cells (ASM) and bronchial biopsies from control donors and patients with asthma to examine the regulation of MMP-1 by ECM in ASM cells and the effect of MMP-1 on ASM contraction. Collagen-I and tenascin-C induced MMP-1 protein expression, which for tenascin-C, was greater in asthma derived ASM cells. Tenascin-C induced MMP-1 expression was dependent on ERK1/2, JNK and p38 MAPK activation and attenuated by function blocking antibodies against the β1 and β3 integrin subunits. Tenascin-C and MMP-1 were not expressed in normal airways but co-localised in the ASM bundles and reticular basement membrane of patients with asthma. Further, ECM from asthma derived ASM cells stimulated MMP-1 expression to a greater degree than ECM from normal ASM. Bradykinin induced contraction of ASM cells seeded in 3D collagen gels was reduced by the MMP inhibitor ilomastat and by siRNA knockdown of MMP-1. In summary, the induction of MMP-1 in ASM cells by tenascin-C occurs in part via integrin mediated MAPK signalling. MMP-1 and tenascin-C are co-localised in the smooth muscle bundles of patients with asthma where this interaction may contribute to enhanced airway contraction. Our findings suggest that ECM changes in airway remodelling via MMP-1 could contribute to an environment promoting greater airway narrowing in response to broncho-constrictor stimuli and worsening asthma symptoms.
T-helper type 2 (Th2) cytokines have been implicated in the pathogenesis of the pulmonary inflammatory response and altered bronchial responsiveness in allergic asthma. To elucidate the mechanism of Th2-dependent mediation of altered airway responsiveness in the atopic asthmatic state, the expression and actions of specific cytokines were examined in isolated rabbit and human airway smooth muscle (ASM) tissues and cultured cells passively sensitized with sera from atopic asthmatic patients or nonatopic/nonasthmatic (control) subjects. Relative to control tissues, the atopic asthmatic sensitized ASM exhibited significantly enhanced maximal isometric contractility to acetylcholine and attenuated relaxation responses to isoproterenol. These proasthmatic changes in agonist responsiveness were ablated by pretreating the atopic sensitized tissues with either an IL-5 receptor blocking antibody (IL-5ra) or the human recombinant IL-1 receptor antagonist (IL-1ra), whereas an IL-4 neutralizing antibody had no effect. Moreover, relative to controls, atopic asthmatic sensitized ASM cells demonstrated an initial, early (after 3 hours of incubation) increased mRNA expression and protein release of IL-5. This was followed (after 6 hours of incubation) by an enhanced mRNA expression and release of IL-1β protein, an effect that was inhibited in sensitized cells pretreated with IL-5ra. Extended studies demonstrated that naive ASM exposed to exogenously administered IL-5 exhibited an induced upregulated mRNA expression and protein release of IL-1β associated with proasthmatic-like changes in ASM constrictor and relaxant responsiveness, and that these effects were ablated in tissues pretreated with IL-1ra. Taken together, these observations provide new evidence that (a) the Th2 cytokine IL-5 and the pleiotropic proinflammatory cytokine IL-1β are endogenously released by atopic asthmatic sensitized ASM and mechanistically interact to mediate the proasthmatic perturbations in ASM responsiveness; and (b) the nature of this interaction is given by an initial endogenous release of IL-5, which then acts to induce the autologous release of IL-1β by the sensitized ASM itself, resulting in its autocrine manifestation of the proasthmatic phenotype.
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.
Laser capture microdissection; TGF-β1 expression; airway smooth muscle cells; asthma; corticosteroids
Background and purpose:
Airway remodelling in asthma is manifested, in part, as increased airway smooth muscle (ASM) mass, reflecting myocyte proliferation. We hypothesized that calcitriol, a secosteroidal vitamin D receptor (VDR) modulator, would inhibit growth factor-induced myocyte proliferation.
Human ASM cell cultures were derived from bronchial samples taken during surgery. ASM cells were treated with platelet-derived growth factor (PDGF) (10 ng·mL−1) for 24 h in the presence of calcitriol, dexamethasone or a checkpoint kinase 1 (Chk1) inhibitor (SB218078). The effects of calcitriol on PDGF-mediated cell proliferation were assessed by thymidine incorporation assay, propidium iodide-based cell cycle analysis, caspase-3 assay and immunoblotting for specific cell cycle modulators.
Calcitriol, but not dexamethasone, inhibited PDGF-induced ASM DNA synthesis concentration dependently (IC50= 520 ± 52 nM). These effects were associated with VDR-mediated expression of cytochrome CYP24A1 with no effects on ASM apoptosis. Calcitriol substantially inhibited (P < 0.01) PDGF-stimulated cell growth in ASM derived from both normal (59 ± 8%) and asthmatic subjects (57 ± 9%). Calcitriol inhibited PDGF-induced phosphorylation of retinoblastoma protein (Rb) and Chk1, with no effects on PDGF-mediated activation of extracellular signal-regulated kinases 1/2, PI3-kinase and S6 kinase, or expression of p21Waf/Cip-1, p27Kip1, cyclin D and E2F-1. Consistent with these observations, SB218078 also inhibited (IC50= 450 ± 100 pM) PDGF-induced cell cycle progression.
Conclusions and implications:
Calcitriol decreased PDGF-induced ASM cell growth by inhibiting Rb and Chk1 phosphorylation.
This Research Paper is the subject of a Commentary in this issue by Clifford and Knox (pp. 1426–1428). To view this article visit http://www3.interscience.wiley.com/journal/121548564/issueyear?year=2009
asthma; airway remodelling; cell cycle; hyperplasia; chronic obstructive pulmonary disease
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.
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.
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.
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.
Here we explored the impact of hydrogen sulfide (H2S) on biophysical properties of the primary human airway smooth muscle (ASM)–the end effector of acute airway narrowing in asthma. Using Magnetic Twisting Cytometry (MTC), we measured dynamic changes in the stiffness of isolated ASM, at the single-cell level, in response to varying doses of GYY4137 (1–10 mM). GYY4137 slowly released appreciable levels of H2S in the range of 10–275 µM, and H2S released was long lived. In isolated human ASM cells, GYY4137 acutely decreased stiffness (i.e. an indicator of the single-cell relaxation) in a dose-dependent fashion, and stiffness decreases were sustained in culture for 24h. Human ASM cells showed protein expressions of cystathionine-γ-lyase (CSE; a H2S synthesizing enzyme) and ATP-sensitive potassium (KATP) channels. The KATP channel opener pinacidil effectively relaxed isolated ASM cells. In addition, pinacidil-induced ASM relaxation was completely inhibited by the treatment of cells with the KATP channel blocker glibenclamide. Glibenclamide also markedly attenuated GYY4137-mediated relaxation of isolated human ASM cells. Taken together, our findings demonstrate that H2S causes the relaxation of human ASM and implicate as well the role for sarcolemmal KATP channels. Finally, given that ASM cells express intrinsic enzymatic machinery of generating H2S, we suggest thereby this class of gasotransmitter can be further exploited for potential therapy against obstructive lung disease.
asthma; airway smooth muscle; single cell contraction; H2S; ATP-sensitive potassium channels
Increased proliferation of airway smooth muscle (ASM) cells leading to hyperplasia and increased ASM mass is one of the most characteristic features of airway remodelling in asthma. A bioactive lipid, sphingosine-1-phosphate (S1P), has been suggested to affect airway remodelling by stimulation of human ASM cell proliferation.
To investigate the effect of S1P on signalling and regulation of gene expression in ASM cells from healthy and asthmatic individuals.
Airway smooth muscle cells grown from bronchial biopsies of healthy and asthmatic individuals were exposed to S1P. Gene expression was analysed using microarray, real-time PCR and Western blotting. Receptor signalling and function were determined by mRNA knockdown and intracellular calcium mobilization experiments.
S1P potently regulated the expression of more than 80 genes in human ASM cells, including several genes known to be involved in the regulation of cell proliferation and airway remodelling (HBEGF, TGFB3, TXNIP, PLAUR, SERPINE1, RGS4). S1P acting through S1P2 and S1P3 receptors activated intracellular calcium mobilization and extracellular signal-regulated and Rho-associated kinases to regulate gene expression. S1P-induced responses were not inhibited by corticosteroids and did not differ significantly between ASM cells from healthy and asthmatic individuals.
S1P induces a steroid-resistant, pro-remodelling pathway in ASM cells. Targeting S1P or its receptors could be a novel treatment strategy for inhibiting airway remodelling in asthma.
airway smooth muscle; asthma; remodelling; sphingosine-1-phosphate
Endobronchial ultrasonography (EBUS) revealed differences in the thickness of the layer representing subepithelial tissues (L2) between human asthmatics and controls, but whether this measurement correlates with airway smooth muscle (ASM) remodeling in asthma is unknown. In this study, we sought to determine the ability of EBUS to predict histological ASM remodeling in normal and equine asthmatic airways. We studied 109 isolated bronchi from the lungs of 13 horses. They underwent EBUS examination using a 30 MHz radial probe before being processed for histology. ASM remodeling parameters were evaluated in EBUS images (L2 thickness, L2 area, L2 area/internal perimeter [Pi] and L2 area/Pi2) and histological cuts (ASM area/Pi2), and compared. EBUS was then performed ex vivo on the lungs of 4 horses with heaves, an asthma-like condition of horses, and 7 controls to determine whether central bronchial remodeling could be detected with this technique. An optimized approach was developed based on data variability within airways, subjects, and groups, and then validated in 7 horses (3 controls, 4 with heaves) that underwent EBUS in vivo. L2 area was significantly associated to ASM area in isolated lungs (p<0.0001), in the absence of significant bias related to the airway size. Bronchial size significantly affected EBUS ASM-related parameters, except for L2 area/Pi2. L2 area/Pi2 was increased in the airways of asthmatic horses compared to controls, both ex vivo and in vivo (p<0.05). Bronchial histology confirmed our findings (AASM/Pi2 was increased in asthmatic horses compared to controls, p<0.05). In both horses with heaves and controls, L2 was composed of ASM for the outer 75% of its thickness and by ECM for the remaining inner 25%. In conclusion, EBUS reliably allows assessment of asthma-associated ASM remodeling of central airways in a non-invasive way.
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.
Airway inflammation is a key aspect of diseases such as asthma. Several inflammatory cytokines (e.g., TNFα and IL-13) increase cytosolic Ca2+ ([Ca2+]cyt) responses to agonist stimulation and Ca2+ sensitivity of force generation, thereby enhancing airway smooth muscle (ASM) contractility (hyper-reactive state). Inflammation also induces ASM proliferation and remodeling (synthetic state). In normal ASM, the transient elevation of [Ca2+]cyt induced by agonists leads to a transient increase in mitochondrial Ca2+ ([Ca2+]mito) that may be important in matching ATP production with ATP consumption. In human ASM (hASM) exposed to TNFα and IL-13, the transient increase in [Ca2+]mito is blunted despite enhanced [Ca2+]cyt responses. We also found that TNFα and IL-13 induce reactive oxidant species (ROS) formation and endoplasmic/sarcoplasmic reticulum (ER/SR) stress (unfolded protein response) in hASM. ER/SR stress in hASM is associated with disruption of mitochondrial coupling with the ER/SR membrane, which relates to reduced mitofusin 2 (Mfn2) expression. Thus, in hASM it appears that TNFα and IL-13 result in ROS formation leading to ER/SR stress, reduced Mfn2 expression, disruption of mitochondrion–ER/SR coupling, decreased mitochondrial Ca2+ buffering, mitochondrial fragmentation, and increased cell proliferation.
inflammation; ER/SR stress; airway; asthma; unfolded protein response