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1.  Increased Nicotinamide Adenine Dinucleotide Phosphate Oxidase 4 Expression Mediates Intrinsic Airway Smooth Muscle Hypercontractility in Asthma 
Rationale: Asthma is characterized by disordered airway physiology as a consequence of increased airway smooth muscle contractility. The underlying cause of this hypercontractility is poorly understood.
Objectives: We sought to investigate whether the burden of oxidative stress in airway smooth muscle in asthma is heightened and mediated by an intrinsic abnormality promoting hypercontractility.
Methods: We examined the oxidative stress burden of airway smooth muscle in bronchial biopsies and primary cells from subjects with asthma and healthy controls. We determined the expression of targets implicated in the control of oxidative stress in airway smooth muscle and their role in contractility.
Measurements and Main Results: We found that the oxidative stress burden in the airway smooth muscle in individuals with asthma is heightened and related to the degree of airflow obstruction and airway hyperresponsiveness. This was independent of the asthmatic environment as in vitro primary airway smooth muscle from individuals with asthma compared with healthy controls demonstrated increased oxidative stress–induced DNA damage together with an increased production of reactive oxygen species. Genome-wide microarray of primary airway smooth muscle identified increased messenger RNA expression in asthma of NADPH oxidase (NOX) subtype 4. This NOX4 overexpression in asthma was supported by quantitative polymerase chain reaction, confirmed at the protein level. Airway smooth muscle from individuals with asthma exhibited increased agonist-induced contraction. This was abrogated by NOX4 small interfering RNA knockdown and the pharmacological inhibitors diphenyleneiodonium and apocynin.
Conclusions: Our findings support a critical role for NOX4 overexpression in asthma in the promotion of oxidative stress and consequent airway smooth muscle hypercontractility. This implicates NOX4 as a potential novel target for asthma therapy.
doi:10.1164/rccm.201107-1281OC
PMCID: PMC3402550  PMID: 22108207
asthma; airway smooth muscle; airway hyperresponsiveness; NOX4; SOD2
2.  Functional KCa3.1 K+ channels are required for human fibrocyte migration 
Background
Fibrocytes are bone marrow–derived CD34+ collagen I–positive cells present in peripheral blood that develop α-smooth muscle actin expression and contractile activity in tissue culture. They are implicated in the pathogenesis of tissue remodeling and fibrosis in both patients with asthma and those with idiopathic pulmonary fibrosis. Targeting fibrocyte migration might therefore offer a new approach for the treatment of these diseases. Ion channels play key roles in cell function, but the ion-channel repertoire of human fibrocytes is unknown.
Objective
We sought to examine whether human fibrocytes express the KCa3.1 K+ channel and to determine its role in cell differentiation, survival, and migration.
Methods
Fibrocytes were cultured from the peripheral blood of healthy subjects and patients with asthma. Whole-cell patch-clamp electrophysiology was used for the measurement of ion currents, whereas mRNA and protein were examined to confirm channel expression. Fibrocyte migration and proliferation assays were performed in the presence of KCa3.1 ion-channel blockers.
Results
Human fibrocytes cultured from the peripheral blood of both healthy control subjects and asthmatic patients expressed robust KCa3.1 ion currents together with KCa3.1 mRNA and protein. Two specific and distinct KCa3.1 blockers (TRAM-34 and ICA-17043) markedly inhibited fibrocyte migration in transwell migration assays. Channel blockers had no effect on fibrocyte growth, apoptosis, or differentiation in cell culture.
Conclusions
The K+ channel KCa3.1 plays a key role in human fibrocyte migration. Currently available KCa3.1-channel blockers might therefore attenuate tissue fibrosis and remodeling in patients with diseases such as idiopathic pulmonary fibrosis and asthma through the inhibition of fibrocyte recruitment.
doi:10.1016/j.jaci.2011.07.047
PMCID: PMC3526791  PMID: 21872912
Pulmonary fibrosis; asthma; fibrocyte; cell migration; ion channel; KCa3.1; patch clamp electrophysiology; 1-EBIO, 1-Ethyl-2-benzimidazolinone; αSMA, α-Smooth muscle actin; ASM, Airway smooth muscle; IPF, Idiopathic pulmonary fibrosis; Kd, Concentration producing 50% block
3.  Expression of the T Helper 17-Associated Cytokines IL-17A and IL-17F in Asthma and COPD 
Chest  2010;138(5):1140-1147.
Background:
Asthma and COPD are characterized by airway dysfunction and inflammation. Neutrophilic airway inflammation is a common feature of COPD and is recognized in asthma, particularly in severe disease. The T helper (Th) 17 cytokines IL-17A and IL-17F have been implicated in the development of neutrophilic airway inflammation, but their expression in asthma and COPD is uncertain.
Methods:
We assessed IL-17A and IL-17F expression in the bronchial submucosa from 30 subjects with asthma, 10 ex-smokers with mild to moderate COPD, and 27 nonsmoking and 14 smoking control subjects. Sputum IL-17 concentration was measured in 165 subjects with asthma and 27 with COPD.
Results:
The median (interquartile range) IL-17A cells/mm2 submucosa was increased in mild to moderate asthma (2.1 [2.4]) compared with healthy control subjects (0.4 [2.8]) but not in severe asthma (P = .04). In COPD, IL-17A+ cells/mm2 submucosa were increased (0.5 [3.7]) compared with nonsmoking control subjects (0 [0]) but not compared with smoking control subjects (P = .046). IL-17F+ cells/mm2 submucosa were increased in severe asthma (2.7 [3.6]) and mild to moderate asthma (1.6 [1.0]) compared with healthy controls subjects (0.7 [1.4]) (P = .001) but was not increased in subjects with COPD. IL-17A and IL-17F were not associated with increased neutrophilic inflammation, but IL-17F was correlated with the submucosal eosinophil count (rs = 0.5, P = .005). The sputum IL-17 concentration in COPD was increased compared with asthma (2 [0-7] pg/mL vs 0 [0-2] pg/mL, P < .0001) and was correlated with post-bronchodilator FEV1% predicted (r = −0.5, P = .008) and FEV1/FVC (r = −0.4, P = .04).
Conclusions:
Our findings support a potential role for the Th17 cytokines IL-17A and IL-17F in asthma and COPD, but do not demonstrate a relationship with neutrophilic inflammation.
doi:10.1378/chest.09-3058
PMCID: PMC2972626  PMID: 20538817
4.  Airway Wall Expression of OX40/OX40L and Interleukin-4 in Asthma 
Chest  2010;137(4):797-804.
Background:
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.
Methods:
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.
Results:
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.
Conclusions:
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.
doi:10.1378/chest.09-1839
PMCID: PMC2851558  PMID: 20139223

Results 1-4 (4)