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1.  Airway Smooth Muscle Hypercontractility in Asthma 
Journal of Allergy  2013;2013:185971.
In recent years, asthma has been defined primarily as an inflammatory disorder with emphasis on inflammation being the principle underlying pathophysiological characteristic driving airway obstruction and remodelling. Morphological abnormalities of asthmatic airway smooth muscle (ASM), the primary structure responsible for airway obstruction seen in asthma, have long been described, but surprisingly, until recently, relatively small number of studies investigated whether asthmatic ASM was also fundamentally different in its functional properties. Evidence from recent studies done on single ASM cells and on ASM-impregnated gel cultures have shown that asthmatic ASM is intrinsically hypercontractile. Several elements of the ASM contraction apparatus in asthmatics and in animal models of asthma have been found to be different from nonasthmatics. These differences include some regulatory contractile proteins and also some components of both the calcium-dependent and calcium-independent contraction signalling pathways. Furthermore, oxidative stress was also found to be heightened in asthmatic ASM and contributes to hypercontractility. Understanding the abnormalities and mechanisms driving asthmatic ASM hypercontractility provides a great potential for the development of new targeted drugs, other than the conventional current anti-inflammatory and bronchodilator therapies, to address the desperate unmet need especially in patients with severe and persistent asthma.
PMCID: PMC3613096  PMID: 23577039
2.  Chronic intermittent hypoxia increases rat sternohyoid muscle NADPH oxidase expression with attendant modest oxidative stress 
Chronic intermittent hypoxia (CIH) causes upper airway muscle dysfunction. We hypothesized that the superoxide generating NADPH oxidase (NOX) is upregulated in CIH-exposed muscle causing oxidative stress. Adult male Wistar rats were exposed to intermittent hypoxia (5% O2 at the nadir for 90 s followed by 210 s of normoxia), for 8 h per day for 14 days. The effect of CIH exposure on the expression of NOX subunits, total myosin and 4-hydroxynonenal (4-HNE) protein adducts in sternohyoid muscle was determined by western blotting and densitometry. Sternohyoid protein free thiol and carbonyl group contents were determined by 1D electrophoresis using specific fluorophore probes. Aconitase and glutathione reductase activities were measured as indices of oxidative stress. HIF-1α content and key oxidative and glycolytic enzyme activities were determined. Contractile properties of sternohyoid muscle were determined ex vivo in the absence and presence of apocynin (putative NOX inhibitor). We observed an increase in NOX 2 and p47 phox expression in CIH-exposed sternohyoid muscle with decreased aconitase and glutathione reductase activities. There was no evidence, however, of increased lipid peroxidation or protein oxidation in CIH-exposed muscle. CIH exposure did not affect sternohyoid HIF-1α content or aldolase, lactate dehydrogenase, or glyceraldehyde-3-phosphate dehydrogenase activities. Citrate synthase activity was also unaffected by CIH exposure. Apocynin significantly increased sternohyoid force and power. We conclude that CIH exposure upregulates NOX expression in rat sternohyoid muscle with concomitant modest oxidative stress but it does not result in a HIF-1α-dependent increase in glycolytic enzyme activity. Constitutive NOX activity decreases sternohyoid force and power. Our results implicate NOX-dependent reactive oxygen species in CIH-induced upper airway muscle dysfunction which likely relates to redox modulation of key regulatory proteins in excitation-contraction coupling.
PMCID: PMC4311627
apocynin; intermittent hypoxia; NADPH oxidase; oxidative stress; respiratory muscle; sternohyoid; sleep apnea; upper airway
3.  Altered CD38/Cyclic ADP-Ribose Signaling Contributes to the Asthmatic Phenotype 
Journal of Allergy  2012;2012:289468.
CD38 is a transmembrane glycoprotein expressed in airway smooth muscle cells. The enzymatic activity of CD38 generates cyclic ADP-ribose from β-NAD. Cyclic ADP-ribose mobilizes intracellular calcium during activation of airway smooth muscle cells by G-protein-coupled receptors through activation of ryanodine receptor channels in the sarcoplasmic reticulum. Inflammatory cytokines that are implicated in asthma upregulate CD38 expression and increase the calcium responses to contractile agonists in airway smooth muscle cells. The augmented intracellular calcium responses following cytokine exposure of airway smooth muscle cells are inhibited by an antagonist of cyclic ADP-ribose. Airway smooth muscle cells from CD38 knockout mice exhibit attenuated intracellular calcium responses to agonists, and these mice have reduced airway response to inhaled methacholine. CD38 also contributes to airway hyperresponsiveness as shown in mouse models of allergen or cytokine-induced inflammatory airway disease. In airway smooth muscle cells obtained from asthmatics, the cytokine-induced CD38 expression is significantly enhanced compared to expression in cells from nonasthmatics. This differential induction of CD38 expression in asthmatic airway smooth muscle cells stems from increased activation of MAP kinases and transcription through NF-κB, and altered post-transcriptional regulation through microRNAs. We propose that increased capacity for CD38 signaling in airway smooth muscle in asthma contributes to airway hyperresponsiveness.
PMCID: PMC3508580  PMID: 23213344
4.  Airway Smooth Muscle Sensitivity to Methacholine in Precision-Cut Lung Slices (PCLS) from Ovalbumin-induced Asthmatic Mice 
Asthma is a chronic inflammatory disease characterized by airway hyperresponsiveness (AHR) and reversible airway obstruction. Methacholine (MCh) is widely used in broncho-provocation test to evaluate airway resistance. For experimental investigation, ovalbumin-induced sensitization is frequently used in rodents (Ova-asthma). However, albeit the inflammatory histology and AHR in vivo, it remains unclear whether the MCh sensitivity of airway smooth muscle isolated from Ova-asthma is persistently changed. In this study, the contractions of airways in precision-cut lung slices (PCLS) from control, Ova-asthma, and IL-13 overexpressed transgenic mice (IL-13TG) were compared by analyzing the airway lumen space (AW). The airway resistance in vivo was measured using plethysmograph. AHR and increased inflammatory cells in BAL fluid were confirmed in Ova-asthma and IL-13TG mice. In the PCLS from all three groups, MCh concentration-dependent narrowing of airway lumen (ΔAW) was observed. In contrast to the AHR in vivo, the EC50 of MCh for ΔAW from Ova-asthma and IL-13TG were not different from control, indicating unchanged sensitivity to MCh. Although the AW recovery upon MCh-washout showed sluggish tendency in Ova-asthma, the change was also statistically insignificant. Membrane depolarization-induced ΔAW by 60 mM K+ (60K-contraction) was larger in IL-13TG than control, whereas 60K-contraction of Ova-asthma was unaffected. Furthermore, serotonin-induced ΔAW of Ova-asthma was smaller than control and IL-13TG. Taken together, the AHR in Ova-asthma and IL-13TG are not reflected in the contractility of isolated airways from PCLS. The AHR of the model animals seems to require intrinsic agonists or inflammatory microenvironment that is washable during tissue preparation.
PMCID: PMC4297764  PMID: 25605999
Airway; Asthma; Lung slice; Smooth muscle
5.  Glutathione Redox Control of Asthma: From Molecular Mechanisms to Therapeutic Opportunities 
Antioxidants & Redox Signaling  2012;17(2):375-408.
Asthma is a chronic inflammatory disorder of the airways associated with airway hyper-responsiveness and airflow limitation in response to specific triggers. Whereas inflammation is important for tissue regeneration and wound healing, the profound and sustained inflammatory response associated with asthma may result in airway remodeling that involves smooth muscle hypertrophy, epithelial goblet-cell hyperplasia, and permanent deposition of airway extracellular matrix proteins. Although the specific mechanisms responsible for asthma are still being unraveled, free radicals such as reactive oxygen species and reactive nitrogen species are important mediators of airway tissue damage that are increased in subjects with asthma. There is also a growing body of literature implicating disturbances in oxidation/reduction (redox) reactions and impaired antioxidant defenses as a risk factor for asthma development and asthma severity. Ultimately, these redox-related perturbations result in a vicious cycle of airway inflammation and injury that is not always amenable to current asthma therapy, particularly in cases of severe asthma. This review will discuss disruptions of redox signaling and control in asthma with a focus on the thiol, glutathione, and reduced (thiol) form (GSH). First, GSH synthesis, GSH distribution, and GSH function and homeostasis are discussed. We then review the literature related to GSH redox balance in health and asthma, with an emphasis on human studies. Finally, therapeutic opportunities to restore the GSH redox balance in subjects with asthma are discussed. Antioxid. Redox Signal. 17, 375–408.
I. Introduction and Conceptual Framework
II. Glutathione Synthesis
III. Distribution of Glutathione in the Body
IV. Glutathione Function and Homeostasis
A. Glutathione as a cysteine reservoir
B. Xenobiotic conjugation and detoxification
C. Free radical scavenging
D. Maintenance of thiol equilibrium
E. Protein S-glutathionylation
F. Regulation of cell surface proteins
G. Protection against nitrosative stress from RNS
V. Glutathione Redox Balance in Health
A. Intracellular glutathione redox status
B. Plasma glutathione redox status
C. Epithelial lining fluid glutathione redox status
VI. Glutathione Redox Balance in Asthma
A. Airway glutathione concentrations in asthma, measured invasively
B. Airway glutathione concentrations in asthma, measured noninvasively
C. Systemic glutathione concentrations in asthma
D. Glutathione redox balance in asthma: Effect of corticosteroids
VII. Other Glutathione-Related Proteins and Redox Systems in Asthma
A. Glutathione peroxidases
B. Glutathione reductases
C. Glutathione-S-transferases
D. Nitrosoglutathione
E. Thioredoxins
F. Glutaredoxins
G. Peroxiredoxins
VIII. Physiological and Biological Implications of Altered Glutathione Redox Balance in Asthma
IX. Altered Glutathione Redox Balance in Asthma: Therapeutic Opportunities
A. Glutathione and glutathione-ethyl esters
B. Cysteine precursors
C. Dietary interventions
1. Selenium
2. Whey protein
3. Sulfur amino acids
4. B vitamins
5. Glutamine and glycine
X. Clinical Implications and Future Directions
PMCID: PMC3353819  PMID: 22304503
6.  Myosin, Transgelin, and Myosin Light Chain Kinase 
Rationale: Airway smooth muscle (SM) of patients with asthma exhibits a greater velocity of shortening (Vmax) than that of normal subjects, and this is thought to contribute to airway hyperresponsiveness. A greater Vmax can result from increased myosin activation. This has been reported in sensitized human airway SM and in models of asthma. A faster Vmax can also result from the expression of specific contractile proteins that promote faster cross-bridge cycling. This possibility has never been addressed in asthma.
Objectives: We tested the hypothesis that the expression of genes coding for SM contractile proteins is altered in asthmatic airways and contributes to their increased Vmax.
Methods: We quantified the expression of several genes that code for SM contractile proteins in mild allergic asthmatic and control human airway endobronchial biopsies. The function of these contractile proteins was tested using the in vitro motility assay.
Measurements and Main Results: We observed an increased expression of the fast myosin heavy chain isoform, transgelin, and myosin light chain kinase in patients with asthma. Immunohistochemistry demonstrated the expression of these genes at the protein level. To address the functional significance of this overexpression, we purified tracheal myosin from the hyperresponsive Fisher rats, which also overexpress the fast myosin heavy chain isoform as compared with the normoresponsive Lewis rats, and found a faster rate of actin filament propulsion. Conversely, transgelin did not alter the rate of actin filament propulsion.
Conclusions: Selective overexpression of airway smooth muscle genes in asthmatic airways leads to increased Vmax, thus contributing to the airway hyperresponsiveness observed in asthma.
PMCID: PMC2633053  PMID: 19011151
asthma; airway hyperresponsiveness; gene expression; smooth muscle; myosin
7.  Role of Abl in airway hyperresponsiveness and airway remodeling 
Respiratory Research  2013;14(1):105.
Asthma is a chronic disease that is characterized by airway hyperresponsiveness and airway remodeling. The underlying mechanisms that mediate the pathological processes are not fully understood. Abl is a non-receptor protein tyrosine kinase that has a role in the regulation of smooth muscle contraction and smooth muscle cell proliferation in vitro. The role of Abl in airway hyperresponsiveness and airway remodeling in vivo is largely unknown.
To evaluate the role of Abl in asthma pathology, we assessed the expression of Abl in airway tissues from the ovalbumin sensitized and challenged mouse model, and human asthmatic airway smooth muscle cells. In addition, we generated conditional knockout mice in which Abl expression in smooth muscle was disrupted, and then evaluated the effects of Abl conditional knockout on airway resistance, smooth muscle mass, cell proliferation, IL-13 and CCL2 in the mouse model of asthma. Furthermore, we determined the effects of the Abl pharmacological inhibitors imatinib and GNF-5 on these processes in the animal model of asthma.
The expression of Abl was upregulated in airway tissues of the animal model of asthma and in airway smooth muscle cells of patients with severe asthma. Conditional knockout of Abl attenuated airway resistance, smooth muscle mass and staining of proliferating cell nuclear antigen in the airway of mice sensitized and challenged with ovalbumin. Interestingly, conditional knockout of Abl did not affect the levels of IL-13 and CCL2 in bronchoalveolar lavage fluid of animals treated with ovalbumin. However, treatment with imatinib and GNF-5 inhibited the ovalbumin-induced increase in IL-13 and CCL2 as well as airway resistance and smooth muscle growth in animals.
These results suggest that the altered expression of Abl in airway smooth muscle may play a critical role in the development of airway hyperresponsiveness and airway remodeling in asthma. Our findings support the concept that Abl may be a novel target for the development of new therapy to treat asthma.
PMCID: PMC3852349  PMID: 24112389
Airway hyperresponsiveness; Airway remodeling; Tyrosine kinase; Airway smooth muscle
8.  The laminin β1-competing peptide YIGSR induces a hypercontractile, hypoproliferative airway smooth muscle phenotype in an animal model of allergic asthma 
Respiratory Research  2010;11(1):170.
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.
PMCID: PMC3013082  PMID: 21129174
9.  Selective stimulation of IL-4 receptor on smooth muscle induces airway hyperresponsiveness in mice 
IL-4Rα expression on airway smooth muscle cells is sufficient for the development of airway hyperresponsiveness.
Production of the cytokines IL-4 and IL-13 is increased in both human asthma and mouse asthma models, and Stat6 activation by the common IL-4/IL-13R drives most mouse model pathophysiology, including airway hyperresponsiveness (AHR). However, the precise cellular mechanisms through which IL-4Rα induces AHR remain unclear. Overzealous bronchial smooth muscle constriction is thought to underlie AHR in human asthma, but the smooth muscle contribution to AHR has never been directly assessed. Furthermore, differences in mouse versus human airway anatomy and observations that selective IL-13 stimulation of Stat6 in airway epithelium induces murine AHR raise questions about the importance of direct IL-4R effects on smooth muscle in murine asthma models and the relevance of these models to human asthma. Using transgenic mice in which smooth muscle is the only cell type that expresses or fails to express IL-4Rα, we demonstrate that direct smooth muscle activation by IL-4, IL-13, or allergen is sufficient but not necessary to induce AHR. Five genes known to promote smooth muscle migration, proliferation, and contractility are activated by IL-13 in smooth muscle in vivo. These observations demonstrate that IL-4Rα promotes AHR through multiple mechanisms and provide a model for testing smooth muscle–directed asthma therapeutics.
PMCID: PMC3135339  PMID: 21464224
10.  The airway smooth muscle CCR3/CCL11 axis is inhibited by mast cells 
Allergy  2008;63(9):1148-1155.
Airway smooth muscle hyperplasia is a feature of asthma, and increases with disease severity. CCR3-mediated recruitment of airway smooth muscle progenitors towards the airway smooth muscle bundle has been proposed as one possible mechanism involved in airway smooth muscle hyperplasia. Mast cells are microlocalized to the airway smooth muscle bundle and whether mast cells influence CCR3-mediated migration is uncertain.
We examined the expression of CCR3 by primary cultures of airway smooth muscle cells from asthmatics and nonasthmatics. CCR3 function was examined using intracellular calcium measurements, chemotaxis, wound healing, cell proliferation and survival assays. We investigated the recovery and function of both recombinant and airway smooth muscle-derived CCL11 (eotaxin) after co-culture with β-tryptase and human lung mast cells.
Airway smooth muscle expressed CCR3. Airway smooth muscle CCR3 activation by CCL11 mediated intracellular calcium elevation, concentration-dependent migration and wound healing, but had no effect on proliferation or survival. Co-culture with β-tryptase or mast cells degraded recombinant and airway smooth muscle-derived CCL11, and β-tryptase inhibited CCL11-mediated airway smooth muscle migration.
CCL11 mediates airway smooth muscle migration. However co-culture with β-tryptase or mast cells degraded recombinant and airway smooth muscle-derived CCL11 and inhibited CCL11-mediated airway smooth muscle migration. Therefore these findings cast doubt on the importance of the CCL11/CCR3 axis in the development of airway smooth muscle hyperplasia in asthma.
PMCID: PMC3992370  PMID: 18699931
airway smooth muscle; asthma; CCL11; CCR3; mast cells
11.  Pathology of asthma 
Asthma is a serious health and socioeconomic issue all over the world, affecting more than 300 million individuals. The disease is considered as an inflammatory disease in the airway, leading to airway hyperresponsiveness, obstruction, mucus hyper-production and airway wall remodeling. The presence of airway inflammation in asthmatic patients has been found in the nineteenth century. As the information in patients with asthma increase, paradigm change in immunology and molecular biology have resulted in an extensive evaluation of inflammatory cells and mediators involved in the pathophysiology of asthma. Moreover, it is recognized that airway remodeling into detail, characterized by thickening of the airway wall, can be profound consequences on the mechanics of airway narrowing and contribute to the chronic progression of the disease. Epithelial to mesenchymal transition plays an important role in airway remodeling. These epithelial and mesenchymal cells cause persistence of the inflammatory infiltration and induce histological changes in the airway wall, increasing thickness of the basement membrane, collagen deposition and smooth muscle hypertrophy and hyperplasia. Resulting of airway inflammation, airway remodeling leads to the airway wall thickening and induces increased airway smooth muscle mass, which generate asthmatic symptoms. Asthma is classically recognized as the typical Th2 disease, with increased IgE levels and eosinophilic inflammation in the airway. Emerging Th2 cytokines modulates the airway inflammation, which induces airway remodeling. Biological agents, which have specific molecular targets for these Th2 cytokines, are available and clinical trials for asthma are ongoing. However, the relatively simple paradigm has been doubted because of the realization that strategies designed to suppress Th2 function are not effective enough for all patients in the clinical trials. In the future, it is required to understand more details for phenotypes of asthma.
PMCID: PMC3768124  PMID: 24032029
asthma; remodeling; epithelial to mesenchymal transition; Th2 cells; cytokines; Th17 cells; Th9 cell
12.  Inflammation signals airway smooth muscle cell proliferation in asthma pathogenesis 
Airway inflammation stimulates proliferation of airway smooth muscle cell, which contributes to the development of hyperplasia and hypertrophy of smooth muscle cell. The increase in airway smooth muscle cell mass is believed to be due to an up-regulation of inflammatory mediators in the airway. It is now well recognized that chronic inflammation as well as airway hyper-responsiveness and remodeling of airway during inflammation, are crucial to asthma. Airway hyper-responsiveness is caused by increased cell proliferation or by hypertrophy of airway smooth muscle cell depending on the nature of the inflammatory stimulation. Airway smooth muscle cell proliferation in asthma is regulated by the proinflammatory cytokines including IL-1β and TNF-α. These proinflammatory cytokines have been shown to influence human airway smooth muscle cell proliferation in vitro, which is due to cyclooxygenase-2 expression, production of prostaglandin E2, and increased cAMP levels.
This review highlights the role of different proinflammatory cytokines in regulating airway smooth muscle cell growth and also focuses on regulation of differential gene expression in airway smooth muscle cell by growth factors and cytokines, also to bestow unique insight into the effects of conventional asthma therapies on airway smooth muscle cell proliferation and development of new therapeutic strategies to control asthma.
PMCID: PMC3568740  PMID: 23388501
Airway inflammation; Airway smooth muscle cells; Asthma
13.  Airway TGFβ1 and oxidant stress in children with severe asthma: Association with airflow limitation 
Transforming growth factor beta-1 (TGFβ1) is thought to play a role in airway remodeling in asthma. TGFβ1 expression may be mediated by an excessive burden of reactive oxygen species and oxidant stress.
Given the profound airway oxidant stress we have previously observed in children with severe asthma, we sought to: 1) quantify TGFβ1 protein and mRNA gene expression in the airways of children with mild-to-moderate and severe atopic asthma; and to 2) determine the relationship of airway TGFβ1 concentrations to oxidant burden (i.e., lipid peroxidation), Th2-mediated eosinophilic inflammation, and airflow limitation.
Bronchoalveolar lavage fluid was collected from 68 atopic children with asthma (severe asthma, n = 28) and 12 atopic adult controls. Airway TGFβ1 expression and activation were assessed in relation to airway IL-13, 8-isoprostane, and malondialdehyde concentrations. The relationship of airway TGFβ1 expression to airflow limitation in children with asthma was also assessed.
Children with severe asthma had higher total airway concentrations of TGFβ1 that were associated with increased protein and mRNA expression of TGFβ1 in airway macrophages and an increase in the lipid peroxidation biomarkers 8-isoprostanes and malondialdehyde. TGFβ1 activation was also greater in children with severe asthma and was associated with higher airway 8-isoprostane, malondialdehyde and IL-13 concentrations. Total airway TGFβ1 concentrations were further associated with airflow limitation.
Children with severe asthma have increased airway TGFβ1 expression and activation associated with an increased airway oxidant burden. Oxidant stress may mediate the effects of TGFβ1 and promote airway remodeling in children with severe asthma.
PMCID: PMC3268912  PMID: 22206775
Airway remodeling; Asthma; Children; Lung function; Oxidant stress; Transforming growth factor beta-1
14.  Effects of nebulized ketamine on allergen-induced airway hyperresponsiveness and inflammation in actively sensitized Brown-Norway rats 
Since airway hyperresponsiveness (AHR) and allergic inflammatory changes are regarded as the primary manifestations of asthma, the main goals of asthma treatment are to decrease inflammation and maximize bronchodilation. These goals can be achieved with aerosol therapy. Intravenous administration of the anesthetic, ketamine, has been shown to trigger bronchial smooth muscle relaxation. Furthermore, increasing evidence suggests that the anti-inflammatory properties of ketamine may protect against lung injury. However, ketamine inhalation might yield the same or better results at higher airway and lower ketamine plasma concentrations for the treatment of asthma. Here, we studied the effect of ketamine inhalation on bronchial hyperresponsiveness and airway inflammation in a Brown-Norway rat model of ovalbumin(OVA)-induced allergic asthma. Animals were actively sensitized by subcutaneous injection of OVA and challenged by repeated intermittent (thrice weekly) exposure to aerosolized OVA for two weeks. Before challenge, the sensitizened rats received inhalation of aerosol of phosphate-buffered saline (PBS) or aerosol of ketamine or injection of ketamine respectivity. Airway reactivity to acetylcholine (Ach) was measured in vivo, and various inflammatory markers, including Th2 cytokines in bronchoalveolar lavage fluid (BALF), as well as induciable nitric oxide synthase (iNOS) and nitric oxide (NO) in lungs were examined. Our results revealed that delivery of aerosolized ketamine using an ultrasonic nebulizer markedly suppressed allergen-mediated airway hyperreactivity, airway inflammation and airway inflammatory cell infiltration into the BALF, and significantly decreased the levels of interleukin-4 (IL-4) in the BALF and expression of iNOS and the concentration of NO in the inflamed airways from OVA-treated rats. These findings collectively indicate that nebulized ketamine attenuated many of the central components of inflammatory changes and AHR in OVA-provoked experimental asthma, potentially providing a new therapeutic approach against asthma.
PMCID: PMC1876456  PMID: 17480224
15.  Mast Cells Promote Airway Smooth Muscle Cell Differentiation via Autocrine Up-Regulation of TGF-β11 
Asthma is a major cause of morbidity and mortality worldwide. It is characterized by airway dysfunction and inflammation. A key determinant of the asthma phenotype is infiltration of airway smooth muscle bundles by activated mast cells. We hypothesized that interactions between these cells promotes airway smooth muscle differentiation into a more contractile phenotype. In vitro coculture of human airway smooth muscle cells with β-tryptase, or mast cells with or without IgE/anti-IgE activation, increased airway smooth muscle-derived TGF-β1 secretion, α-smooth muscle actin expression and agonist-provoked contraction. This promotion to a more contractile phenotype was inhibited by both the serine protease inhibitor leupeptin and TGF-β1 neutralization, suggesting that the observed airway smooth muscle differentiation was driven by the autocrine release of TGF-β1 in response to activation by mast cell β-tryptase. Importantly, in vivo we found that in bronchial mucosal biopsies from asthmatics the intensity of α-smooth muscle actin expression was strongly related to the number of mast cells within or adjacent to an airway smooth muscle bundle. These findings suggest that mast cell localization in the airway smooth muscle bundle promotes airway smooth muscle cell differentiation into a more contractile phenotype, thus contributing to the disordered airway physiology that characterizes asthma.
PMCID: PMC3992381  PMID: 18802103
16.  Overexpression of Smad2 Drives House Dust Mite–mediated Airway Remodeling and Airway Hyperresponsiveness via Activin and IL-25 
Rationale: Airway hyperreactivity and remodeling are characteristic features of asthma. Interactions between the airway epithelium and environmental allergens are believed to be important in driving development of pathology, particularly because altered epithelial gene expression is common in individuals with asthma.
Objectives: To investigate the interactions between a modified airway epithelium and a common aeroallergen in vivo.
Methods: We used an adenoviral vector to generate mice overexpressing the transforming growth factor-β signaling molecule, Smad2, in the airway epithelium and exposed them to house dust mite (HDM) extract intranasally.
Measurements and Main Results: Smad2 overexpression resulted in enhanced airway hyperreactivity after allergen challenge concomitant with changes in airway remodeling. Subepithelial collagen deposition was increased and smooth muscle hyperplasia was evident resulting in thickening of the airway smooth muscle layer. However, there was no increase in airway inflammation in mice given the Smad2 vector compared with the control vector. Enhanced airway hyperreactivity and remodeling did not correlate with elevated levels of Th2 cytokines, such as IL-13 or IL-4. However, mice overexpressing Smad2 in the airway epithelium showed significantly enhanced levels of IL-25 and activin A after HDM exposure. Blocking activin A with a neutralizing antibody prevented the increase in lung IL-25 and inhibited subsequent collagen deposition and also the enhanced airway hyperreactivity observed in the Smad2 overexpressing HDM-exposed mice.
Conclusions: Epithelial overexpression of Smad2 can specifically alter airway hyperreactivity and remodeling in response to an aeroallergen. Moreover, we have identified novel roles for IL-25 and activin A in driving airway hyperreactivity and remodeling.
PMCID: PMC2913231  PMID: 20339149
asthma; lung; epithelium; smooth muscle; collagen
17.  NADPH Oxidases in Vascular Pathology 
Antioxidants & Redox Signaling  2014;20(17):2794-2814.
Significance: Reactive oxygen species (ROS) play a critical role in vascular disease. While there are many possible sources of ROS, nicotinamide adenine dinucleotide phosphate (NADPH) oxidases play a central role. They are a source of “kindling radicals,” which affect other enzymes, such as nitric oxide synthase endothelial nitric oxide synthase or xanthine oxidase. This is important, as risk factors for atherosclerosis (hypertension, diabetes, hypercholesterolemia, and smoking) regulate the expression and activity of NADPH oxidases in the vessel wall. Recent Advances: There are seven isoforms in mammals: Nox1, Nox2, Nox3, Nox4, Nox5, Duox1 and Duox2. Nox1, Nox2, Nox4, and Nox5 are expressed in endothelium, vascular smooth muscle cells, fibroblasts, or perivascular adipocytes. Other homologues have not been found or are expressed at very low levels; their roles have not been established. Nox1/Nox2 promote the development of endothelial dysfunction, hypertension, and inflammation. Nox4 may have a role in protecting the vasculature during stress; however, when its activity is increased, it may be detrimental. Calcium-dependent Nox5 has been implicated in oxidative damage in human atherosclerosis. Critical Issues: NADPH oxidase-derived ROS play a role in vascular pathology as well as in the maintenance of normal physiological vascular function. We also discuss recently elucidated mechanisms such as the role of NADPH oxidases in vascular protection, vascular inflammation, pulmonary hypertension, tumor angiogenesis, and central nervous system regulation of vascular function and hypertension. Future Directions: Understanding the role of individual oxidases and interactions between homologues in vascular disease is critical for efficient pharmacological regulation of vascular NADPH oxidases in both the laboratory and clinical practice. Antioxid. Redox Signal. 20, 2794–2814.
PMCID: PMC4026218  PMID: 24180474
18.  Breath Formate Is a Marker of Airway S-Nitrosothiol Depletion in Severe Asthma 
PLoS ONE  2010;5(7):e11919.
Children with severe asthma have poor symptom control and elevated markers of airway oxidative and nitrosative stress. Paradoxically, they have decreased airway levels of S-nitrosothiols (SNOs), a class of endogenous airway smooth muscle relaxants. This deficiency results from increased activity of an enzyme that both reduces SNOs to ammonia and oxidizes formaldehyde to formic acid, a volatile carboxylic acid that is more easily detected in exhaled breath condensate (EBC) than SNOs. We therefore hypothesize that depletion of airway SNOs is related to asthma pathology, and breath formate concentration may be a proxy measure of SNO catabolism.
Methods and Findings
We collected EBC samples from children and adolescents, including 38 with severe asthma, 46 with mild-to-moderate asthma and 16 healthy adolescent controls, and the concentration of ionic constituents was quantified using ion chromatography. The concentrations of EBC components with volatile conjugates were log-normally distributed. Formate was the principal ion that displayed a significant difference between asthma status classifications. The mean EBC formate concentration was 40% higher in samples collected from all asthmatics than from healthy controls (mean = 5.7 µM, mean±standard deviation = 3.1−10.3 µM vs. 4.0, 2.8−5.8 µM, p = 0.05). EBC formate was higher in severe asthmatics than in mild-to-moderate asthmatics (6.8, 3.7−12.3 µM vs. 4.9, 2.8−8.7 µM, p = 0.012). In addition, formate concentration was negatively correlated with methacholine PC20 (r = −0.39, p = 0.002, asthmatics only), and positively correlated with the NO-derived ion nitrite (r = 0.46, p<0.0001) as well as with total serum IgE (r = 0.28, p = 0.016, asthmatics only). Furthermore, formate was not significantly correlated with other volatile organic acids nor with inhaled corticosteroid dose.
We conclude that EBC formate concentration is significantly higher in the breath of children with asthma than in those without asthma. In addition, amongst asthmatics, formate is elevated in the breath of those with severe asthma compared to those with mild-to-moderate asthma. We suggest that this difference is related to asthma pathology and may be a product of increased catabolism of endogenous S-nitrosothiols.
PMCID: PMC2912922  PMID: 20689836
19.  Expression of IL-4 Receptor alpha on smooth muscle cells is not necessary for development of experimental allergic asthma 
Airflow in the lungs of patients with allergic asthma is impaired by excessive mucus production and airway smooth muscle contractions. Elevated levels of the cytokines IL-4 and IL-13 are associated with this pathology. In vitro studies have suggested that IL-4 receptor alpha (IL-4Rα) signalling on smooth muscle cells is critical for airway inflammation and airway hyperresponsiveness.
In order to define the contribution of IL-4 and IL-13 to the onset of asthmatic pathology the role of their key receptor IL-4Rα in smooth muscle cells was examined in vivo.
By using transgenic SMC-MHCcreIL-4Rα−/lox mice deficient for IL-4Rα in smooth muscle cells, in vivo effects of impaired IL-4Rα signalling in smooth muscle cells on the outcome of asthmatic disease were investigated for the first time. Allergic asthma was introduced in mice by repeated sensitisation with ovalbumin/aluminium hydroxide on days 0, 7 and 14 followed by intranasal allergen challenge on days 21–23. Mice were investigated for the presence of airway hyperresponsiveness, airway inflammation, allergen specific antibody production, Th2 type cytokine responses and lung pathology.
Airway hyperresponsiveness, airway inflammation, mucus production, Th2 cytokine production and specific antibody responses were unaffected in SMC-MHCcreIL-4Rα−/lox mice when compared to control animals.
The impairment of IL-4Rα on smooth muscle cells had no effect on major aetiological markers of allergic asthma. These findings suggest that IL-4Rα responsiveness in airway smooth muscle cells during the early phase of allergic asthma is not, as suggested, necessary for the outcome of the disease.
Clinical Implications
Therapies targeting the IL-4Rα might have no direct effect on smooth muscle cells in an allergic asthma response.
PMCID: PMC2917502  PMID: 20579713
Smooth muscle cell; Allergy; Asthma; Cytokine Receptors; IL-4; IL-13; gene-deficient mice
20.  Granulocyte–macrophage colony-stimulating factor expression in induced sputum and bronchial mucosa in asthma and COPD 
Thorax  2009;64(8):671-676.
Granulocyte–macrophage colony-stimulating factor (GM-CSF) has been implicated as an important mediator in the pathogenesis of asthma and chronic obstructive pulmonary disease (COPD). However, the expression of GM-CSF and its receptor in airway samples in asthma and COPD across disease severity needs to be further defined.
Sputum GM-CSF was measured in 18 control subjects, 45 subjects with asthma and 47 subjects with COPD. Enumeration of GM-CSF+ cells in the bronchial submucosa and airway smooth muscle bundle was performed in 29 control subjects, 36 subjects with asthma and 10 subjects with COPD.
The proportion of subjects with measurable GM-CSF in the sputum was raised in those with moderate (7/14) and severe (11/18) asthma, and in those with COPD GOLD (Global Initiative for Chronic Obstructive Lung Disease) stage II (7/16), III (8/17) and IV (7/14) compared with controls (1/18) and those with mild asthma (0/13); p = 0.001. The sputum GM-CSF concentration was correlated with the sputum eosinophilia in subjects with moderate to severe asthma (rs = 0.41; p = 0.018). The median (interquartile range) GM-CSF+ and GM-CSFR+ cells/mm2 of submucosa was increased in severe asthma (1.4 (3.0) and 2.1 (8.4)) compared with those with mild to moderate asthma (0 (2.5) and 1.1 (5)) and healthy controls (0 (0.5) and 0 (1.6)), (p = 0.004 and p = 0.02, respectively).
The findings support a potential role for GM-CSF in asthma and COPD and suggest that overexpression of GM-CSF in sputum and the bronchial mucosa is a particular feature of severe asthma.
PMCID: PMC2712140  PMID: 19213775
21.  Induction and regulation of matrix metalloproteinase-12in human airway smooth muscle cells 
Respiratory Research  2005;6(1):148.
The elastolytic enzyme matrix metalloproteinase (MMP)-12 has been implicated in the development of airway inflammation and remodeling. We investigated whether human airway smooth muscle cells could express and secrete MMP-12, thereby participating in the pathogenesis of airway inflammatory diseases.
Laser capture microdissection was used to collect smooth muscle cells from human bronchial biopsy sections. MMP-12 mRNA expression was analysed by quantitative real-time RT-PCR. MMP-12 protein expression and secretion from cultured primary airway smooth muscle cells was further analysed by Western blot. MMP-12 protein localization in bronchial tissue sections was detected by immunohistochemistry. MMP-12 activity was determined by zymography. The TransAM AP-1 family kit was used to measure c-Jun activation and nuclear binding. Analysis of variance was used to determine statistical significance.
We provide evidence that MMP-12 mRNA and protein are expressed by in-situ human airway smooth muscle cells obtained from bronchial biopsies of normal volunteers, and of patients with asthma, COPD and chronic cough. The pro-inflammatory cytokine, interleukin (IL)-1β, induced a >100-fold increase in MMP-12 gene expression and a >10-fold enhancement in MMP-12 activity of primary airway smooth muscle cell cultures. Selective inhibitors of extracellular signal-regulated kinase, c-Jun N-terminal kinase and phosphatidylinositol 3-kinase reduced the activity of IL-1β on MMP-12, indicating a role for these kinases in IL-1β-induced induction and release of MMP-12. IL-1β-induced MMP-12 activity and gene expression was down-regulated by the corticosteroid dexamethasone but up-regulated by the inflammatory cytokine tumour necrosis factor (TNF)-α through enhancing activator protein-1 activation by IL-1β. Transforming growth factor-β had no significant effect on MMP-12 induction.
Our findings indicate that human airway smooth muscle cells express and secrete MMP-12 that is up-regulated by IL-1β and TNF-α. Bronchial smooth muscle cells may be an important source of elastolytic activity, thereby participating in remodeling in airway diseases such as COPD and chronic asthma.
PMCID: PMC1363355  PMID: 16359550
22.  Inhibition of Aldose Reductase Prevents Experimental Allergic Airway Inflammation in Mice 
PLoS ONE  2009;4(8):e6535.
The bronchial asthma, a clinical complication of persistent inflammation of the airway and subsequent airway hyper-responsiveness, is a leading cause of morbidity and mortality in critically ill patients. Several studies have shown that oxidative stress plays a key role in initiation as well as amplification of inflammation in airways. However, still there are no good anti-oxidant strategies available for therapeutic intervention in asthma pathogenesis. Most recent studies suggest that polyol pathway enzyme, aldose reductase (AR), contributes to the pathogenesis of oxidative stress–induced inflammation by affecting the NF-κB-dependent expression of cytokines and chemokines and therefore inhibitors of AR could be anti-inflammatory. Since inhibitors of AR have already gone through phase-III clinical studies for diabetic complications and found to be safe, our hypothesis is that AR inhibitors could be novel therapeutic drugs for the prevention and treatment of asthma. Hence, we investigated the efficacy of AR inhibition in the prevention of allergic responses to a common natural airborne allergen, ragweed pollen that leads to airway inflammation and hyper-responsiveness in a murine model of asthma.
Methods and Findings
Primary Human Small Airway Epithelial Cells (SAEC) were used to investigate the in vitro effects of AR inhibition on ragweed pollen extract (RWE)-induced cytotoxic and inflammatory signals. Our results indicate that inhibition of AR prevents RWE -induced apoptotic cell death as measured by annexin-v staining, increase in the activation of NF-κB and expression of inflammatory markers such as inducible nitric oxide synthase (iNOS), cycloxygenase (COX)-2, Prostaglandin (PG) E2, IL-6 and IL-8. Further, BALB/c mice were sensitized with endotoxin-free RWE in the absence and presence of AR inhibitor and followed by evaluation of perivascular and peribronchial inflammation, mucin production, eosinophils infiltration and airway hyperresponsiveness. Our results indicate that inhibition of AR prevents airway inflammation and production of inflammatory cytokines, accumulation of eosinophils in airways and sub-epithelial regions, mucin production in the bronchoalveolar lavage fluid and airway hyperresponsiveness in mice.
These results suggest that airway inflammation due to allergic response to RWE, which subsequently activates oxidative stress-induced expression of inflammatory cytokines via NF-κB-dependent mechanism, could be prevented by AR inhibitors. Therefore, inhibition of AR could have clinical implications, especially for the treatment of airway inflammation, a major cause of asthma pathogenesis.
PMCID: PMC2717330  PMID: 19657391
23.  Airway Wall Expression of OX40/OX40L and Interleukin-4 in Asthma 
Chest  2010;137(4):797-804.
The costimulatory molecule OX40 and its ligand, OX40L, mediate key aspects of allergic airway inflammation in animal models of asthma, including eosinophilic airway inflammation, airway hyperresponsiveness, and T helper 2 polarization. We sought to examine OX40/OX40L and interleukin (IL)-4 expression in asthma across severities.
Bronchial biopsies were obtained from 27 subjects with asthma (mild Global Initiative for Asthma [GINA] 1 [n = 10], moderate GINA 2-3 [n = 7], and severe GINA 4-5 [n = 10]) and 13 healthy controls. The number of OX40+, OX40L+, IL-4+, and IL-4 receptor α (IL-4Rα)+ cells in the lamina propria and airway smooth muscle (ASM) bundle and the intensity of IL-4Rα+ expression by the ASM were assessed.
The number of OX40+, OX40L+, and IL-4+ cells in the lamina propria and OX40+ and IL-4+ cells in the ASM bundle was significantly increased in subjects with mild asthma, but not in those with moderate or severe asthma, compared with healthy controls. In the subjects with asthma, OX40/OX40L expression was positively correlated with the number of eosinophils and IL-4+ cells in the lamina propria. The number of IL-4Rα+ cells in the lamina propria was significantly increased in moderate-to-severe disease, but not in mild asthma, compared with controls. IL-4Rα expression by the ASM bundle was not different among groups.
OX40/OX40L expression is increased in the bronchial submucosa in mild asthma, but not in moderate-to-severe disease, and is related to the degree of tissue eosinophilia and IL-4 expression. Whether these costimulatory molecules have a role as targets for asthma requires further investigation.
PMCID: PMC2851558  PMID: 20139223
24.  Caveolin-1 is required for contractile phenotype expression by airway smooth muscle cells 
Airway smooth muscle cells exhibit phenotype plasticity that underpins their ability to contribute both to acute bronchospasm and to the features of airway remodelling in chronic asthma. A feature of mature, contractile smooth muscle cells is the presence of abundant caveolae, plasma membrane invaginations that develop from the association of lipid rafts with caveolin-1, but the functional role of caveolae and caveolin-1 in smooth muscle phenotype plasticity is unknown. Here, we report a key role for caveolin-1 in promoting phenotype maturation of differentiated airway smooth muscle induced by transforming growth factor (TGF)-β1. As assessed by Western analysis and laser scanning cytometry, caveolin-1 protein expression was selectively enriched in contractile phenotype airway myocytes. Treatment with TGF-β1 induced profound increases in the contractile phenotype markers sm-α-actin and calponin in cells that also accumulated abundant caveolin-1; however, siRNA or shRNAi inhibition of caveolin-1 expression largely prevented the induction of these contractile phenotype marker proteins by TGF-β1. The failure by TGF-β1 to adequately induce the expression of these smooth muscle specific proteins was accompanied by a strongly impaired induction of eukaryotic initiation factor-4E binding protein(4E-BP)1 phosphorylation with caveolin-1 knockdown, indicating that caveolin-1 expression promotes TGF-β1 signalling associated with myocyte maturation and hypertrophy. Furthermore, we observed increased expression of caveolin-1 within the airway smooth muscle bundle of guinea pigs repeatedly challenged with allergen, which was associated with increased contractile protein expression, thus providing in vivo evidence linking caveolin-1 expression with accumulation of contractile phenotype myocytes. Collectively, we identify a new function for caveolin-1 in controlling smooth muscle phenotype; this mechanism could contribute to allergic asthma.
PMCID: PMC3822954  PMID: 21199324
phenotype plasticity; asthma; airway remodelling; caveolae; TGF-β1
25.  Overexpression of endothelial nitric oxide synthase suppresses features of allergic asthma in mice 
Respiratory Research  2006;7(1):58.
Asthma is associated with airway hyperresponsiveness and enhanced T-cell number/activity on one hand and increased levels of exhaled nitric oxide (NO) with expression of inducible NO synthase (iNOS) on the other hand. These findings are in paradox, as NO also relaxes airway smooth muscle and has immunosuppressive properties. The exact role of the endothelial NOS (eNOS) isoform in asthma is still unknown. We hypothezised that a delicate regulation in the production of NO and its bioactive forms by eNOS might be the key to the pathogenesis of asthma.
The contribution of eNOS on the development of asthmatic features was examined. We used transgenic mice that overexpress eNOS and measured characteristic features of allergic asthma after sensitisation and challenge of these mice with the allergen ovalbumin.
eNOS overexpression resulted in both increased eNOS activity and NO production in the lungs. Isolated thoracic lymph nodes cells from eNOS overexpressing mice that have been sensitized and challenged with ovalbumin produced significantly less of the cytokines IFN-γ, IL-5 and IL-10. No difference in serum IgE levels could be found. Further, there was a 50% reduction in the number of lymphocytes and eosinophils in the lung lavage fluid of these animals. Finally, airway hyperresponsiveness to methacholine was abolished in eNOS overexpressing mice.
These findings demonstrate that eNOS overexpression attenuates both airway inflammation and airway hyperresponsiveness in a model of allergic asthma. We suggest that a delicate balance in the production of bioactive forms of NO derived from eNOS might be essential in the pathophysiology of asthma.
PMCID: PMC1456969  PMID: 16597326

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