A genome-wide analysis of the response to inhaled ß2-agonists in chronic obstructive pulmonary disease.

Short-acting ß2-agonist bronchodilators are the most common medications used in treating chronic obstructive pulmonary disease (COPD). Genetic variants determining bronchodilator responsiveness (BDR) in COPD have not been identified. We performed a genome-wide association study (GWAS) of BDR in 5789 current or former smokers with COPD in one African-American and four white populations. BDR was defined as the quantitative spirometric response to inhaled ß2-agonists. We combined results in a meta-analysis. In the meta-analysis, single-nucleotide polymorphisms (SNPs) in the genes KCNK1 (P=2.02 × 10(-7)) and KCNJ2 (P=1.79 × 10(-7)) were the top associations with BDR. Among African Americans, SNPs in CDH13 were significantly associated with BDR (P=5.1 × 10(-9)). A nominal association with CDH13 was identified in a gene-based analysis in all subjects. We identified suggestive association with BDR among COPD subjects for variants near two potassium channel genes (KCNK1 and KCNJ2). SNPs in CDH13 were significantly associated with BDR in African Americans.The Pharmacogenomics Journal advance online publication, 27 October 2015; doi:10.1038/tpj.2015.65.

NiO and Co3O4 nanoparticles induce lung DTH-like responses and alveolar lipoproteinosis.

Lung exposure to metal oxide nanoparticles (NPs) comprising soluble metal haptens may produce T-helper cell type 1 (Th1)- and Th17-associated delayed-type hypersensitivity (DTH) responses and pulmonary alveolar proteinosis (PAP). In order to study this, haptenic metal oxide NPs (NiO, Co(3)O(4), Cr(2)O(3) and CuO) were instilled into the lungs of female Wistar rats, and the immunoinflammatory responses were assessed at 24 h and 4 weeks post-instillation. Primary culture of alveolar macrophages from Wistar rats was used to evaluate the effect of the NPs on the ability to clear surfactant. NiO NPs induced chronic interstitial inflammation and pro-inflammatory Th1 and Th17 immune responses characterised by increases in the cytokines monocyte chemotactic protein (MCP)-1/CCL2, interleukin (IL)-12 p40, interferon-γ and IL-17A, whilst similar pathological responses induced by Co(3)O(4) NPs were associated with increases in MCP-1/CCL2 and IL-12 p40. However, neither Cr(2)O(3) nor CuO NPs elicited immunoinflammatory reactions. PAP was induced by both NiO and Co(3)O(4) NPs during the chronic phase. PAP was associated with over-production of surfactant by proliferation of type II cells and impaired clearance of surfactant by macrophages. These findings have implications for the risk management of occupational NP exposure and provide evidence that haptenic metal oxide NPs can induce chronic progressive lung immune responses via a DTH-like mechanism.

Computed tomographic emphysema distribution: relationship to clinical features in a cohort of smokers.

Computed tomography (CT) scanning allows precise assessment of both the extent and distribution of emphysema. There has been little work on the relationship between the distribution of emphysema and clinical features of the disease. The current study investigated the association between clinical features and distribution of emphysema. A total of 129 patients with smoking-related chronic obstructive pulmonary disease underwent CT assessment of the extent and distribution of their emphysema (core/rind and upper/lower zone predominance). Emphysema was found predominantly in the upper/core zone and this distribution was related to the extent of disease. Core predominance was associated with lower forced expiratory volume in one second (FEV(1)), FEV(1)/forced vital capacity ratio and body mass index (BMI); and with higher BODE (BMI, airflow obstruction, dyspnoea and exercise capacity) index and Medical Research Council dyspnoea score. Upper-zone predominance was associated with female sex and an increased total St George's Respiratory Questionnaire score. Using multiple linear regression age, sex and whole lung emphysema severity were independently associated with core/rind distribution, while sex and whole lung emphysema severity were independently related to upper/lower distribution. Distribution of emphysema related best to clinical features when divided into core/rind predominance. However, the effects were not independent of the extent of emphysema. Increased age and female sex were related to disease distribution independent of emphysema severity. These findings may be related to differences in development of emphysema.

Increased arterial stiffness in patients with chronic obstructive pulmonary disease: a mechanism for increased cardiovascular risk.

RATIONALE: Chronic obstructive pulmonary disease (COPD) is associated with a 2-3-fold increase in the risk of ischaemic heart disease, stroke and sudden death. The mechanisms responsible for this association are not clear and appear to be independent of smoking history. OBJECTIVE: We test the hypothesis that patients with COPD have increased arterial stiffness and blood pressure in comparison with age and smoking matched controls. METHODS: In a prospective case control study, we recruited 102 patients with COPD and 103 healthy controls matched for age and smoking status. Patients were assessed by clinical history and spirometry, with arterial stiffness and blood pressure determined using radial artery applanation tonometry and sphygmomanometry. RESULTS: Patients with COPD had increased arterial stiffness compared with matched controls, with elevated augmentation pressure (17 (1) vs 14 (1) mm Hg; p = 0.005) and a reduced time to wave reflection (131 (1) vs 137 (2) ms; p = 0.004). These differences were associated with increases in both diastolic (82 (1) vs 78 (1) mm Hg; p = 0.005) and systolic blood pressure (147 (2) vs 132 (2) mm Hg; p<0.001). Serum C reactive protein concentrations were threefold higher in patients (6.1 (0.9) vs 2.3 (0.4) mg/l; p = 0.001). Data are presented as mean (SEM). CONCLUSIONS: Patients with COPD have increased arterial stiffness and blood pressure in comparison with controls matched for age and smoking status. We speculate that increased systemic inflammation and vascular dysfunction could potentially explain the excess cardiovascular morbidity and mortality associated with COPD.

Nanoparticle-driven DNA damage mimics irradiation-related carcinogenesis pathways.

The epidemiological association between cancer and exposure to ambient air pollution particles (particles with a 50% cut-off aerodynamic diameter of 10 microm (PM(10))) has been related to the ability of PM(10) and its constituent nanoparticles (NPs) to cause reactive oxidative species (ROS)-driven DNA damage. However, there are no data on the molecular response to these genotoxic effects. In order to assess whether PM(10), NP and ROS-driven DNA damage induce carcinogenesis pathways, A549 cells were treated with tert-butyl-hyperperoxide (Tbh), urban dust (UD), carbon black (CB), nanoparticulate CB (NPCB), benzo(a)pyrene (BaP) and NPCB coated with BaP for

Evaluation of COPD Longitudinally to Identify Predictive Surrogate End-points (ECLIPSE).

Chronic obstructive pulmonary disease (COPD) is a heterogeneous disease and not well understood. The forced expiratory volume in one second is used for the diagnosis and staging of COPD, but there is wide acceptance that it is a crude measure and insensitive to change over shorter periods of time. Evaluation of COPD Longitudinally to Identify Predictive Surrogate End-points (ECLIPSE) is a 3-yr longitudinal study with four specific aims: 1) definition of clinically relevant COPD subtypes; 2) identification of parameters that predict disease progression in these subtypes; 3) examination of biomarkers that correlate with COPD subtypes and may predict disease progression; and 4) identification of novel genetic factors and/or biomarkers that both correlate with clinically relevant COPD subtypes and predict disease progression. ECLIPSE plans to recruit 2,180 COPD subjects in Global Initiative for Chronic Obstructive Lung Disease categories II-IV and 343 smoking and 223 nonsmoking control subjects. Study procedures are to be performed at baseline, 3 months, 6 months and every 6 months thereafter. Assessments include pulmonary function measurements (spirometry, impulse oscillometry and plethysmography), chest computed tomography, biomarker measurement (in blood, sputum, urine and exhaled breath condensate), health outcomes, body impedance, resting oxygen saturation and 6-min walking distance. Evaluation of COPD Longitudinally to Identify Predictive Surrogate End-points is the largest study attempting to better describe the subtypes of chronic obstructive pulmonary disease, as well as defining predictive markers of its progression.

Genetic variants of microsomal epoxide hydrolase and glutamate-cysteine ligase in COPD.

The genetic factors that contribute to the development of chronic obstructive pulmonary disease (COPD) are poorly understood. Many candidate genes have been proposed, including enzymes that protect the lung against oxidative stress, such as microsomal epoxide hydrolase (EPHX1) and glutamate-cysteine ligase (GCL). To date, most reported findings have been for EPHX1, particularly in relation to functional variants associated with fast and slow metabolism of epoxide intermediates. The present study aimed to identify any association of variation in these genes with COPD susceptibility or severity. In total, 1,017 white COPD patients and 912 nondiseased age and sex matched smoking controls were genotyped for six single nucleotide polymorphisms (SNPs) in EPHX1 (including the fast and slow variants and associated haplotypes), and eight SNPs in the two genes encoding GCL. GCL is a rate-limiting enzyme in the synthesis of glutathione, a major contributor to anti-oxidant protection in the lung. No association of variation was found in EPHX1 or GCL with susceptibility to COPD or disease severity. This is the largest reported study to date and is well powered to detect associations that have been previously suggested. The current data indicate that these genetic variants are unlikely to be related to susceptibility or disease severity in white chronic obstructive pulmonary disease patients.

Outcomes for COPD pharmacological trials: from lung function to biomarkers.

The American Thoracic Society/European Respiratory Society jointly created a Task Force on "Outcomes for COPD pharmacological trials: from lung function to biomarkers" to inform the chronic obstructive pulmonary disease research community about the possible use and limitations of current outcomes and markers when evaluating the impact of a pharmacological therapy. Based on their review of the published literature, the following document has been prepared with individual sections that address specific outcomes and markers, and a final section that summarises their recommendations.

Interacting effects of particulate pollution and cold temperature on cardiorespiratory mortality in Scotland.

OBJECTIVES: To determine whether the effect of black smoke on cardiorespiratory mortality is modified by cold temperatures. METHODS: Poisson regression models were used to investigate the relationship between lagged black smoke concentration and daily mortality, and whether the effect of black smoke on mortality was modified by cold temperature for three Scottish cities from January 1981 to December 2001. MAIN RESULTS: For all-cause respiratory and non-cardiorespiratory mortality, there was a significant association between mortality and lagged black smoke concentration. Generally the maximum black smoke effect occurred at lag 0, although these estimates were not statistically significant. A 10 mugm(-3) increase in the daily mean black smoke concentration on any given day was associated with a 1.68% (95% CI 0.72 to 2.65) increase in all-cause mortality and a 0.43% (95% CI -0.97 to 1.86), 5.36% (95% CI 2.93 to 7.84) and 2.13% (95% CI 0.82 to 3.47) increase in cardiovascular, respiratory and non-cardiorespiratory mortality, respectively, over the ensuing 30-day period. The effect of black smoke on mortality did not vary significantly between seasons (cool and warm periods). For all-cause, cardiovascular and non-cardiorespiratory mortality the inclusion of interaction terms did not improve the models, although for all-cause and non-cardiorespiratory mortality there was a suggestion for interaction between temperature and recent black smoke exposure. CONCLUSIONS: The results of this study suggested a greater effect of black smoke on mortality at low temperatures. Since extremes of cold and particulate pollution may coexist, for example during temperature inversion, these results may have important public health implications.

Nanoparticle carbon black driven DNA damage induces growth arrest and AP-1 and NFkappaB DNA binding in lung epithelial A549 cell line.

To assess whether nanoparticle (NP) driven DNA damage induces the expression of proinflammatory transcription factors such as NFB and AP-1 A549, lung epithelial cells were treated with Carbon Black (CB), nanoparticulate CB (NPCB), NPCB coated with BaP (BaP-NPCB) for various times ranging from 30 min to 24 h. DNA strand break was determined by the comet assay and cell cycle status was analyzed using flow cytometry. Nuclear extracts were used for WB analysis of P approximately Ser15-p53. EMSA was used to detect DNA binding. Tested NP caused single strand breaks and significantly altered cell cycle kinetics. NF-kappaB and AP-1 DNA binding were increased at early time points (2.3 and 2.6 fold at 1 hour, respectively). Effects were also found on Ser15-p53 phosphorylation. N-acetylcysteine blocked NP driven effects. In conclusion, NPCB and BaP-NPCB induce DNA damage, activating p53, proteins related to DNA repair and proinflammatory transcription factors.

Cytoplasm-nuclear trafficking of CREB and CREB phosphorylation at Ser133 during therapy of chronic obstructive pulmonary disease.

cAMP responsive element binding protein (CREB) plays an important role in transcriptional machinery. CREB signaling is altered in patients with asthma. However, the role of CREB in chronic obstructive pulmonary disease (COPD) is less clear. In the present study we assessed changes in subcellular CREB distribution and activation (CREB-P) in 35 stable COPD patients treated with formoterol (F), formoterol+budesonide (F/ICS), and formoterol+budesonide+theophylline (F/ICS/Th) b.i.d. for 4 weeks, using SDS-PAGE/WB in cytosol and nuclear extracts of induced sputum cells. The expression of CREB was increased after F/ICS in both cytosolic and nuclear fractions by about 40% and 24%, respectively (P<0.001, P<0.01), while CREB-P increased after F/ICS by about 50% (P<0.01) in both compartments. These changes were not affected by theophylline. In F/ICS-treated patients, relative accumulation of CREB in cytosol was observed. These findings indicate, that poor response to ICS therapy may be related to increased CREB-associated signaling.

Is oxidative stress central to the pathogenesis of chronic obstructive pulmonary disease?

There is now considerable evidence for an increased oxidant burden in patients with chronic obstructive pulmonary disease (COPD). Oxidative stress is a critical feature in the pathogenesis of COPD, since it results in inactivation of antiproteinases, airspace epithelial injury, MUCUS HYPERSECRETION, increased influx of neutrophils into the lungs, transcription factor activation and gene expression of pro-inflammatory mediators. Antioxidants should therefore not only protect against the direct injurious effects of oxidants, but also may fundamentally alter the inflammatory events which have a central role in the pathogenesis of COPD.

Soluble transition metals in welding fumes cause inflammation via activation of NF-kappaB and AP-1.

We previously reported that the molecular pro-inflammatory effects of welding fumes in vitro were caused by soluble transition metals via an oxidative stress-mediated mechanism. Herein, we tested the hypothesis that transition metals in welding fume drive the in vivo inflammatory response caused by welding fume. Rats were instilled with either whole, soluble extract or washed welding fume particulates or soluble extracts pre-treated with a transition metal chelator. Markers of pulmonary inflammation were measured in the bronchoalveolar lavage fluid (BALF) and nuclear translocation of transcription factor was assessed in BAL cells by electrophoretic mobility shift assay. Instillation of either whole or soluble fractions of welding fume caused a significant influx of inflammatory cells and other markers of inflammation in the BALF 24 h later. MIP-2 protein in BALF and nuclear translocation of NF-kappaB and AP-1 were significantly greater following instillation of whole and soluble fractions than in saline-instilled lungs. Chelation of the soluble fraction, to remove transition metals, abolished the ability to cause inflammation, MIP-2 increase or transcription factor translocation to the nucleus. Instillation of washed particulates alone caused no significant change in any end-point compared to saline. This study demonstrates that soluble transition metals present in welding fumes cause inflammation via activation of the redox-sensitive transcription factors NF-kappaB and AP-1 and confirms the validity of utilising in vitro models to assess inflammatory responses to such particles.

The procoagulant potential of environmental particles (PM10).

BACKGROUND AND AIMS: Epidemiology studies have shown that cardiovascular (CV) disease is primarily responsible for the mortality associated with increased pulmonary environmental particle (PM10) exposure. The mechanisms involved in PM10 mediated CV effects are unknown although changes in plasma viscosity and in the homoeostasis of blood coagulation have been implicated. It was hypothesised that PM10 exposure would result in an inflammatory response and enhance the activation of the extrinsic coagulation mechanisms in pulmonary and vascular cells in culture. METHODS: Primary human monocyte derived macrophages and human umbilical cord vein endothelial, human alveolar type II epithelial (A549), and human bronchial epithelial (16HBE) cells were tested for their inflammatory and procoagulant response to PM10 exposure. IL-8, tissue factor (TF), and tissue plasminogen activator (tPA) gene expression and protein release, and coagulation enhancing ability of culture media were determined 6 and 24 hours following exposure. RESULTS: The culture media from macrophages and 16HBE bronchial epithelial cells, but not A549 cells, exposed to PM10 had an enhanced ability to cause clotting. Furthermore, H2O2 also increased the clotting activity. Apoptosis was significantly increased in macrophages exposed to PM10 and LPS as shown by annexin V binding. TF gene expression was enhanced in macrophages exposed to PM10, and HUVEC tissue factor and tPA gene and protein expression were inhibited. CONCLUSIONS: These data indicate that PM10 has the ability to alter macrophage, epithelial, and endothelial cell function to favour blood coagulation via activation of the extrinsic pathway and inhibition of fibrinolysis pathways.

Anti-inflammatory effects of atorvastatin treatment in chronic obstructive pulmonary disease. A controlled pilot study.

UNLABELLED: Observational studies have suggested that statins may have beneficial effects on outcomes in chronic obstructive pulmonary disease (COPD) patients. These effects may be mediated through an anti-inflammatory effect of statins. The purpose of this pilot-study was to determine whether statins have an anti-inflammatory effect on the lungs of COPD patients. We conducted randomized, controlled, parallel group pilot-study to compare the effects of atorvastatin (n=12) or placebo (n=6) on lung inflammation in patients with mild to moderate COPD. The primary endpoint was change in CD45+ cells expression measured by immunohistochemistry and changes in expression of genes measured using microarrays in lung biopsy (TBB) samples before and after 12 weeks of treatment with atorvastatin 40 mg/day. All subjects had spirometry, lung volumes, diffusing capacity of the lungs for carbon monoxide (DLCO), St George's Respiratory Questionnaire (SGRQ), 6 minute walk distance (6 MWD), serum lipids, hs-CRP, induced sputum (IS), bronchoscopy and TBB carried out at baseline and after treatment. TBB specimens were processed for histology, immunohistochemistry and genome-wide association studies (GWAS) profiling. Seventeen subjects completed the study. There was a significant improvement in SGRQ with mean SGRQ decreased by 12 points after treatment with atorvastatin (P=0.012). Atorvastatin treatment produced a significant 34% reduction in sputum neutrophil count, and a 57% reduction in CD45+ cells in lung biopsies (expressed as integrated optical density -IOD; median IOD 62.51% before, 27.01% after atorvastatin treatment, P=0.008). In patients' lung tissue atorvastatin treatment produced downregulation of key genes involved in inflammatory processes, immune response, and leukocyte activation. These data demonstrate the pulmonary anti-inflammatory effects of atorvastatin in COPD patients with the potential for beneficial clinical effects. TRIAL REGISTRATION: ClinicalTrials.gov: NCT01748279.

Regulation of redox glutathione levels and gene transcription in lung inflammation: therapeutic approaches.

Glutathione (L-gamma-glutamyl-L-cysteinylglycine, GSH), is a vital intra- and extracellular protective antioxidant. Glutathione is synthesized from its constituent amino acids by the sequential action of gamma-glutamylcysteine synthetase (gamma-GCS) and GSH synthetase. The rate-limiting enzyme in GSH synthesis is gamma-GCS. Gamma-GCS expression is modulated by oxidants, phenolic antioxidants, and inflammatory and anti-inflammatory agents in various mammalian cells. The intracellular GSH redox homeostasis is strictly regulated to govern cell metabolism and protect cells against oxidative stress. Growing evidence has suggested that cellular oxidative processes have a fundamental role in inflammation through the activation of stress kinases (JNK, MAPK, p38) and redox-sensitive transcription factors such as NF-kappaB and AP-1, which differentially regulate the genes for proinflammatory mediators and protective antioxidant genes such as gamma-GCS, Mn-SOD, and heme oxygenase-1. The critical balance between the induction of proinflammatory mediators and antioxidant genes and the regulation of the levels of GSH in response to oxidative stress at the site of inflammation is not known. Knowledge of the mechanisms of redox GSH regulation and gene transcription in inflammation could lead to the development of novel therapies based on the pharmacological manipulation of the production of this important antioxidant in inflammation and injury. This FORUM article features the role of GSH levels in the regulation of transcription factors, whose activation and DNA binding leads to proinflammatory and antioxidant gene transcription. The potential role of thiol antioxidants as a therapeutic approach in inflammatory lung diseases is also discussed.

Role of oxidants/antioxidants in smoking-induced lung diseases.

An imbalance between oxidants and antioxidants has been considered in the pathogenesis of smoking-induced lung diseases, such as chronic obstructive pulmonary disease (COPD), particularly emphysema. Recent evidence indicates that increased neutrophil sequestration and activation occurs in the pulmonary microvasculature in smokers and in patients with COPD, with the potential to release reactive oxygen species (ROS). ROS generated by airspace phagocytes or inhaled directly from the environment also increase the oxidant burden and may contribute to the epithelial damage. Although much research has focused on the protease/antiprotease theory of the pathogenesis of emphysema, less attention has been paid to the role of ROS in this condition. The injurious effects of the increased oxidant burden in smokers and in patients with COPD are opposed by the lung antioxidant defences. Hence, determining the mechanisms regulating the antioxidant responses is critical to our understanding of the role of oxidants in the pathogenesis of smoking-induced lung disease and to devising future strategies for antioxidant therapy. In this article we have reviewed the evidence for the presence of an oxidant/antioxidant imbalance in smoking-induced lung disease and its relevance to therapy in these conditions.

Characterisation of gamma-glutamylcysteine synthetase-heavy subunit promoter: a critical role for AP-1.

The 5'-flanking region of human gamma-glutamylcysteine synthetase-heavy subunit (gamma-GCS-HS) was characterised by creating a series of chloramphenicol acetyl transferase (CAT) reporter deletion constructs. Analysis of various deleted CAT constructs revealed that a putative AP-1 consensus sequence is required to direct the constitutive and oxidant-mediated promoter activity. Gel mobility shift and mutation analysis of the sequence (-269 to -263 bp), showed binding of AP-1 is involved in the oxidant-mediated regulation of gamma-GCS-HS promoter activity.

Induction of gamma-glutamylcysteine synthetase by cigarette smoke is associated with AP-1 in human alveolar epithelial cells.

Increased levels of glutathione (GSH) occur in the epithelial lining fluid (ELF) of chronic cigarette smokers. Therefore we investigated the effect of cigarette smoke condensate solution (CSC) on GSH synthesis and the regulation of gamma-glutamylcysteine synthetase (gammaGCS) in human type II alveolar epithelial cells (A549). CSC exposure increased GSH levels, gammaGCS activity and gammaGCS heavy subunit (HS) mRNA, as well as increasing DNA binding of the activator protein-1 (AP-1) and the human antioxidant response element (hARE). Transfection of deletion constructs of the gammaGCS-HS promoter in a chloramphenicol acetyl transferase (CAT) reporter system revealed that an hARE, present within promoter, is not required for the CSC mediated induction. We conclude that CSC induction of gammaGCS-HS expression is associated with AP-1/AP-1-like responsive elements.

Regulation of iNOS expression and glutathione levels in rat liver by oxygen tension.

Molecular oxygen (O(2)) regulates the expression of a variety of genes. We hypothesized that O(2) tension may regulate iNOS expression in rat liver through the production of reactive oxygen species (ROS) and the reduction of intracellular glutathione (GSH) levels. To investigate this hypothesis, we determined the effects of hyperoxia upon iNOS induction (both at the protein and mRNA level) and the intracellular concentration of GSH in an isolated in vitro perfused rat liver preparation. To study the potential involvement of ROS in the intracellular signaling pathway linking changes in oxygen tension to gene expression, we repeated these determinations in the presence of the thiol antioxidant N-acetyl-L-cysteine (NAC). We found that 95% O(2) tension caused a significant induction of the iNOS protein and mRNA levels paralleled by a significant fall in intracellular GSH concentration. The addition of NAC (1 mM) to the perfusate during hyperoxia blocked the induction of iNOS and restored GSH levels. These results indicate that molecular O(2) regulates the expression of iNOS in rat liver at the transcriptional level, most likely through the production of ROS and the reduction of intracellular GSH levels.