Breathlessness is a predominant symptom of chronic obstructive pulmonary disease (COPD), making it a valuable outcome in addition to lung function to assess treatment benefit. The phosphodiesterase-4 inhibitor roflumilast has been shown to provide small but significant improvements in dyspnea, as measured by the transition dyspnea index (TDI), in two 1-year studies in patients with severe to very severe COPD.
Patients and methods
To provide a more comprehensive assessment of the impact of roflumilast on dyspnea, post hoc analyses of four 1-year roflumilast studies (M2-111, M2-112, M2-124, and M2-125) in patients with moderate to very severe COPD were conducted.
In this pooled analysis (N=5,595), roflumilast significantly improved TDI focal scores versus placebo at week 52 (treatment difference, 0.327; P<0.0001). Roflumilast was associated with significantly greater TDI responders and significantly fewer TDI deteriorators (≥1-unit increase or decrease from baseline, respectively) versus placebo at week 52 (P<0.01, both); these significant differences were apparent by week 8 and maintained until study end (P<0.05, all). At study end, the postbronchodilator forced expiratory volume in 1 second improvement in TDI responders was significantly greater with roflumilast versus placebo (P<0.05). Similar to the overall population, improvements in TDI focal scores at week 52 were small but consistently significant over placebo in patients with chronic bronchitis, regardless of exacerbation history, concomitant treatment with short-acting muscarinic antagonists or long-acting β2-agonists, or pretreatment with inhaled corticosteroids.
This analysis shows that patients treated with roflumilast to reduce exacerbation risk may also experience small but significant improvements in dyspnea, with accompanying improvements in lung function.
phosphodiesterase-4 inhibitor; breathlessness; lung function; subgroup analyses
Chronic obstructive pulmonary disease (COPD) is a major cause of
morbidity and mortality, yet research suggests this disease is greatly
underdiagnosed. This literature review sought to summarize the most common
and significant variables associated with case-finding or missed cases of
COPD to inform more effective and efficient detection of high-risk COPD
patients in primary care.
PubMed and EMBASE were searched for articles describing case-finding
and epidemiologic research to detect or characterize new cases of COPD.
International studies in primary and non-primary care settings, published in
English from 2002–2014, were eligible for inclusion. Studies related
to risk factors for development of COPD were excluded.
Of the 33 studies identified and reviewed, 21 were case-finding or
screening and 12 were epidemiological, including cross-sectional,
longitudinal, and retrospective designs. A range of variables were
identified within and across studies. Variables common to both screening and
epidemiological studies included age, smoking status, and respiratory
symptoms. Seven significant predictors from epidemiologic studies did not
appear in screening tools. No studies targeted discovery of higher risk
patients such as those with reduced lung function or risks for
Variables used to identify new cases of COPD or differentiate COPD
cases and non-cases are wide- ranging, (from sociodemographic to
self-reported health or health history variables), providing insight into
important factors for case identification. Further research is underway to
develop and test the best, smallest variable set that can be used as a
screening tool to identify people with undiagnosed, high-risk COPD in
chronic obstructive pulmonary disease; primary care; screening; literature review
Rationale: Chronic neutrophilic inflammation is a hallmark in the
pathogenesis of chronic obstructive pulmonary disease (COPD) and persists after
cigarette smoking has stopped. Mechanisms involved in this ongoing inflammatory
response have not been delineated.
Objectives: We investigated changes to the leukotriene A4
hydrolase (LTA4H)–proline-glycine-proline (PGP) pathway and chronic
inflammation in the development of COPD.
Methods: A/J mice were exposed to air or cigarette smoke for 22 weeks
followed by bronchoalveolar lavage and lung and cardiac tissue analysis. Two human
cohorts were used to analyze changes to the LTA4H–PGP pathway in
never smokers, control smokers, COPD smokers, and COPD former smokers. PGP/AcPGP and
LTA4H aminopeptidase activity were detected by mass spectroscopy,
LTA4H amounts were detected by ELISA, and acrolein was detected by
Measurements and Main Results: Mice exposed to cigarette smoke developed
emphysema with increased PGP, neutrophilic inflammation, and selective inhibition of
LTA4H aminopeptidase, which ordinarily degrades PGP. We recapitulated
these findings in smokers with and without COPD. PGP and AcPGP are closely associated
with cigarette smoke use. Once chronic inflammation is established, changes to
LTA4H aminopeptidase remain, even in the absence of ongoing cigarette
use. Acrolein modifies LTA4H and inhibits aminopeptidase activity to the
same extent as cigarette smoke.
Conclusions: These results demonstrate a novel pathway of aberrant
regulation of PGP/AcPGP, suggesting this inflammatory pathway may be intimately
involved in disease progression in the absence of ongoing cigarette smoke exposure.
We highlight a mechanism by which acrolein potentiates neutrophilic inflammation
through selective inhibition of LTA4H aminopeptidase activity.
Clinical trial registered with www.clinicaltrials.gov (NCT
COPD; inflammation; PGP; leukotriene A4 hydrolase; acrolein
Objective and design
Reduced expression of histone deacetylase 2 (HDAC2) in alveolar macrophages and epithelial cells may account for reduced response of chronic obstructive pulmonary disease (COPD) patients to glucocorticoids. HDAC2 expression and its role in mediating glucocorticoid effects on fibroblast functions, however, has not been fully studied. This study was designed to investigate whether HDAC2 mediates glucocorticoid effects on release of inflammatory cytokines and matrix metalloproteinases (MMPs) from human lung fibroblasts.
Human lung fibroblasts (HFL-1 cells) were stimulated with interleukin (IL)-1 β plus tumor necrosis factor (TNF)-α in the presence or absence of the glucocorticoid budesonide. Cytokines (IL-6 and IL-8) were quantified by enzyme linked immunosorbent assay (ELISA) and MMPs (MMP-1 and MMP-3) by immunoblotting in culture medium. The role of HDAC2 was investigated using a pharmacologic inhibitor as well as a small interfering ribonucleic acid (siRNA) targeting HDAC2.
We have demonstrated that budesonide concentration-dependently (10−10–10−7 M) inhibited IL-6, IL-8, MMP-1, and MMP-3 release by HFL-1 cells in response to IL-1β plus TNF-α. While an HDAC inhibitor significantly blocked the inhibitory effect of budesonide on human bronchial epithelial cells (HBECs) and monocytes (THP-1 cells), it did not block the inhibitory effect of budesonide on release of cytokines and MMPs from HFL-1 cells. Similarly, an HDAC2-siRNA blocked budesonide inhibition of cytokine release in HBECs, but it did not block the inhibitory effect of budesonide on HFL-1 cytokine and MMP release. Furthermore, budesonide significantly blocked release of cytokines and MMPs to a similar degree in normal and COPD lung fibroblasts as well as in HFL-1 cells exposed or not exposed to cigarette smoke extract.
These findings suggest that, in contrast to airway epithelial cells and monocytes/macrophages, HDAC2 is not required for budesonide to inhibit MMP and cytokine release by lung fibroblasts and this inhibitory pathway appears to be intact in cultured fibroblasts from COPD patients. These results also suggest that budesonide has the potential to modulate fibroblast-mediated tissue remodeling following airway inflammation in COPD, which is mediated via an HDAC2 independent pathway.
budesonide; fibroblasts; HDAC2
Many cases of chronic obstructive pulmonary disease (COPD) are diagnosed only after significant loss of lung function or during exacerbations.
This study is part of a multi-method approach to develop a new screening instrument for identifying undiagnosed, clinically significant COPD in primary care.
Subjects with varied histories of COPD diagnosis, risk factors and history of exacerbations were recruited through five US clinics (four pulmonary, one primary care). Phase I: Eight focus groups and six telephone interviews were conducted to elicit descriptions of risk factors for COPD, recent or historical acute respiratory events, and symptoms to inform the development of candidate items for the new questionnaire. Phase II: A new cohort of subjects participated in cognitive interviews to assess and modify candidate items. Two peak expiratory flow (PEF) devices (electronic, manual) were assessed for use in screening.
Of 77 subjects, 50 participated in Phase I and 27 in Phase II. Six themes informed item development: exposure (smoking, second-hand smoke); health history (family history of lung problems, recurrent chest infections); recent history of respiratory events (clinic visits, hospitalisations); symptoms (respiratory, non-respiratory); impact (activity limitations); and attribution (age, obesity). PEF devices were rated easy to use; electronic values were significantly higher than manual (P<0.0001). Revisions were made to the draft items on the basis of cognitive interviews.
Forty-eight candidate items are ready for quantitative testing to select the best, smallest set of questions that, together with PEF, can efficiently identify patients in need of diagnostic evaluation for clinically significant COPD.
The balance between production and degradation of extracellular matrix is crucial in maintaining normal tissue structure. This study was designed to investigate the effect of budesonide on fibroblast-mediated tissue repair and remodeling.
Using human fetal lung fibroblasts in a three-dimensional collagen gel culture system, we investigated the effect of budesonide (1-1000 nM) on collagen gel contraction and degradation in the presence or absence of Inflammatory cytokines (interleukin-1β and tumor necrosis factor α; 5 ng/mL each) and, in order to activate latent proteases, serine protease trypsin 0.25 μg/mL. The effects of budesonide on metalloproteinase production and activation were also investigated.
Inflammatory cytokines significantly inhibited collagen gel contraction mediated by lung fibroblasts. Budesonide counteracted the effect of cytokines in a concentration-dependent manner (to 50%, P< 0.01). Budesonide 100 nM almost completely inhibited the release and mRNA expression of metalloproteinase-1, metalloproteinase-3, and metalloproteinase-9 induced by the cytokines (P< 0.05). Exposure to the cytokines plus trypsin increased collagen degradation and conversion of the metalloproteinases to lower molecular weight forms corresponding to their active forms. Budesonide blocked both enhanced collagen degradation (P< 0.01) and suppressed trypsin-mediated conversion of cytokine-induced metalloproteinase-9 and metalloproteinase-3 to lower molecular weight forms. Similar effects were observed with dexamethasone 1 μM, suggesting a class effect.
These findings demonstrate that budesonide directly modulates contraction of collagen gels and can decrease collagen degradation under Inflammatory conditions. The mechanism of this effect is through suppressing gene expression, release, and activation of metalloproteinases. By modulating the release and activity of metalloproteinases, inhaled budesonide may be able to modify airway tissue repair and remodeling.
metalloproteinase; budesonide; tissue remodeling
Fibroblasts are the major mesenchymal cells present within the interstitium of the lung and are a major source of vascular endothelial growth factor (VEGF), which modulates the maintenance of pulmonary microvasculature. Prostaglandin E2 (PGE2) acts on a set of E-prostanoid (EP) receptors that activate multiple signal transduction pathways leading to downstream responses. We investigated the modulation by PGE2 of VEGF release by human lung fibroblasts. Human lung fibroblasts were cultured until reaching 90% confluence in tissue culture plates, after which the culture media were changed to serum-free Dulbecco's modified Eagle's medium, with or without PGE2, and with specific agonists or antagonists for each EP receptor. After 2 days, culture media were assayed for VEGF by ELISA. The results demonstrated that PGE2 and the EP2 agonist ONO-AE1-259-01 significantly stimulated the release of VEGF in a concentration-dependent manner. Agonists for other EP receptors did not stimulate the release of VEGF. The stimulatory effect of PGE2 was blocked by the EP2 antagonist AH6809, but was not blocked by antagonists for other EP receptors. The protein kinase–A (PKA) inhibitor KT-5720 also blocked the stimulatory effect of PGE2. The increased release of VEGF induced by PGE2 was accompanied by a transient increase in the concentration of VEGF mRNA. These findings demonstrate that PGE2 can modulate the release of VEGF by human lung fibroblasts through its actions in the EP2 receptor/PKA pathway. This activity may contribute to the maintenance of pulmonary microvasculature in the alveolar wall.
prostaglandin E2; vascular endothelial growth factor; human lung fibroblasts; tissue repair
Rationale: Muscle wasting in chronic obstructive pulmonary disease (COPD) is associated with a poor prognosis and is not readily assessed by measures of body mass index (BMI). BMI does not discriminate between relative proportions of adipose tissue and lean muscle and may be insensitive to early pathologic changes in body composition. Computed tomography (CT)–based assessments of the pectoralis muscles may provide insight into the clinical significance of skeletal muscles in smokers.
Objectives: We hypothesized that objective assessment of the pectoralis muscle area on chest CT scans provides information that is clinically relevant and independent of BMI.
Methods: Data from the ECLIPSE (Evaluation of COPD Longitudinally to Identify Predictive Surrogate Endpoints) Study (n = 73) were used to assess the relationship between pectoralis muscle area and fat-free mass. We then used data in a subset (n = 966) of a larger cohort, the COPDGene (COPD Genetic Epidemiology) Study, to explore the relationship between pectoralis muscle area and COPD-related traits.
Measurements and Main Results: We first investigated the correlation between pectoralis muscle area and fat-free mass, using data from a subset of participants in the ECLIPSE Study. We then further investigated pectoralis muscle area in COPDGene Study participants and found that higher pectoralis muscle area values were associated with greater height, male sex, and younger age. On subsequent clinical correlation, compared with BMI, pectoralis muscle area was more significantly associated with COPD-related traits, including spirometric measures, dyspnea, and 6-minute-walk distance (6MWD). For example, on average, each 10-cm2 increase in pectoralis muscle area was associated with a 0.8-unit decrease in the BODE (Body mass index, Obstruction, Dyspnea, Exercise) index (95% confidence interval, –1.0 to –0.6; P < 0.001). Furthermore, statistically significant associations between pectoralis muscle area and COPD-related traits remained even after adjustment for BMI.
Conclusions: CT-derived pectoralis muscle area provides relevant indices of COPD morbidity that may be more predictive of important COPD-related traits than BMI. However, the relationship with clinically relevant outcomes such as hospitalization and death requires additional investigation. Pectoralis muscle area is a convenient measure that can be collected in the clinical setting in addition to BMI.
COPD; wasting; pectoral muscle area; imaging
Chronic obstructive pulmonary disease (COPD) is characterized by the frequent association of disease outside the lung. The objective of this study was to determine the presence of biomechanical gait abnormalities in COPD patients compared to healthy controls while well rested and without rest.
Patients with COPD (N = 17) and aged-matched, healthy controls (N = 21) walked at their self-selected pace down a 10-meter walkway while biomechanical gait variables were collected. A one-minute rest was given between each of the five collected trials to prevent tiredness (REST condition). Patients with COPD then walked at a self-selected pace on a treadmill until the onset of self-reported breathlessness or leg tiredness. Subjects immediately underwent gait analysis with no rest between each of the five collected trials (NO REST condition). Statistical models with and without covariates age, gender, and smoking history were used.
After adjusting for covariates, COPD patients demonstrated more ankle power absorption in mid-stance (P = 0.006) than controls during both conditions. Both groups during NO REST demonstrated increased gait speed (P = 0.04), stride length (P = 0.03), and peak hip flexion (P = 0.04) with decreased plantarflexion moment (P = 0.04) and increased knee power absorption (P = 0.04) as compared to REST. A significant interaction revealed that peak ankle dorsiflexion moment was maintained from REST to NO REST for COPD but increased for controls (P < 0.01). Stratifying by disease severity did not alter these findings, except that step width decreased in NO REST as compared to REST (P = 0.01). Standardized effect sizes of significant effects varied from 0.5 to 0.98.
Patients with COPD appear to demonstrate biomechanical gait changes at the ankle as compared to healthy controls. This was seen not only in increased peak ankle power absorption during no rest but was also demonstrated by a lack of increase in peak ankle dorsiflexion moment from the REST to the NO REST condition as compared to the healthy controls. Furthermore, a wider step width has been associated with fall risk and this could account for the increased incidence of falls in patients with COPD.
Pulmonary disease; Locomotion; Joint kinematics; Joint kinetics; Biomechanics
In 2010 the COPD Foundation established the COPD Biomarkers Qualification Consortium (CBQC) as a partnership between the Foundation, the Food and Drug Administration (FDA), and the pharmaceutical industry to pool publicly-funded and industry data to develop innovative tools to facilitate the development and approval of new therapies for COPD. We present data from the initial project seeking regulatory qualification of fibrinogen as a biomarker for the stratification of COPD patients into clinical trials.
This analysis pooled data from 4 publicly-funded studies and 1 industry study into a common database resulting in 6376 individuals with spirometric evidence of COPD. We used a threshold of 350 mg/dL to determine high vs. low fibrinogen, and determined the subsequent risk of hospitalizations from exacerbations and death using Cox proportional hazards models.
High fibrinogen levels at baseline were present in 2853 (44.7%) of individuals with COPD. High fibrinogen was associated with an increased risk of hospitalized COPD exacerbations within 12 months (hazard ratio [HR]: 1.64; 95% confidence interval [CI]: 1.39–1.93) among participants in the Atherosclerosis Risk in Communities Study (ARIC), the Cardiovascular Health Study (CHS), and the Evaluation of COPD Longitudinally to Identify Predictive Surrogate Endpoints (ECLIPSE) study. High fibrinogen was associated with an increased risk of death within 36 months (HR: 1.94; 95% CI: 1.62–2.31) among all participants.
Fibrinogen levels ≥ 350 mg/dL identify COPD individuals at an increased risk of exacerbations and death and could be a useful biomarker for enriching clinical trials in the COPD population.
fibrinogen; COPD; biomarker
Subpopulations and Intermediate Outcomes in COPD Study (SPIROMICS) is a multi-center longitudinal, observational study to identify novel phenotypes and biomarkers of chronic obstructive pulmonary disease (COPD). In a subset of 300 subjects enrolled at six clinical centers, we are performing flow cytometric analyses of leukocytes from induced sputum, bronchoalveolar lavage (BAL) and peripheral blood. To minimize several sources of variability, we use a “just-in-time” design that permits immediate staining without pre-fixation of samples, followed by centralized analysis on a single instrument.
The Immunophenotyping Core prepares 12-color antibody panels, which are shipped to the six Clinical Centers shortly before study visits. Sputum induction occurs at least two weeks before a bronchoscopy visit, at which time peripheral blood and bronchoalveolar lavage are collected. Immunostaining is performed at each clinical site on the day that the samples are collected. Samples are fixed and express shipped to the Immunophenotyping Core for data acquisition on a single modified LSR II flow cytometer. Results are analyzed using FACS Diva and FloJo software and cross-checked by Core scientists who are blinded to subject data.
Thus far, a total of 152 sputum samples and 117 samples of blood and BAL have been returned to the Immunophenotyping Core. Initial quality checks indicate useable data from 126 sputum samples (83%), 106 blood samples (91%) and 91 BAL samples (78%). In all three sample types, we are able to identify and characterize the activation state or subset of multiple leukocyte cell populations (including CD4+ and CD8+ T cells, B cells, monocytes, macrophages, neutrophils and eosinophils), thereby demonstrating the validity of the antibody panel.
Our study design, which relies on bi-directional communication between clinical centers and the Core according to a pre-specified protocol, appears to reduce several sources of variability often seen in flow cytometric studies involving multiple clinical sites. Because leukocytes contribute to lung pathology in COPD, these analyses will help achieve SPIROMICS aims of identifying subgroups of patients with specific COPD phenotypes. Future analyses will correlate cell-surface markers on a given cell type with smoking history, spirometry, airway measurements, and other parameters.
This study was registered with ClinicalTrials.gov as NCT01969344.
Human; COPD; Flow cytometry; Sputum; Bronchoalveolar lavage; Immunophenotyping
Fibroblast heterogeneity is recognized and fibroblasts from diseased tissues, including asthma, have functional phenotypes that differ from normal. However, progenitor progeny relationships and the factors that control fibroblast differentiation are poorly defined.
To determine if interleukin-4 could alter the functional phenotype of fibroblasts during their differentiation from stem/progenitor cells.
Using a three-dimensional collagen gel system, we embryoid bodies derived from human embryonic stem cells and recovered spindle-shaped cells consistent with fibroblasts that had differentiated in the presence or absence of interleukin-4.
Interleukin-4 induced fibroblast-like cells were more active in contracting collagen gels, in migration and in producing fibronectin than control (without interleukin-4) cells. Interleukin-4 induced cells demonstrated less expression of miR-155 that modulated contraction, migration and fibronectin production. These differences persisted in culture without further addition of interleukin-4, suggesting the differentiated phenotype might be a permanent alteration.
The current study demonstrates that interleukin-4 induces differentiation of stem/precursor cells into fibroblast-like cells that demonstrate a more “fibrogenic” phenotype, which is due to reduced expression of miR-155. These findings provide a novel mechanism for the persistent abnormalities in IL-4 related diseases and a novel target to regulate tissue remodeling by fibroblasts.
embryonic stem cells; fibroblasts; IL-4; fibrosis; asthma; microRNA; miR-155; chemotaxis; collagen gel contraction; TGF-β
Exacerbations of chronic obstructive pulmonary disease (COPD), characterized by acute deterioration in symptoms, may be due to bacterial or viral infections, environmental exposures, or unknown factors. Exacerbation frequency may be a stable trait in COPD patients, which could imply genetic susceptibility. Observing the genes, networks, and pathways that are up- and down-regulated in COPD patients with differing susceptibility to exacerbations will help to elucidate the molecular signature and pathogenesis of COPD exacerbations.
Gene expression array and plasma biomarker data were obtained using whole-blood samples from subjects enrolled in the Treatment of Emphysema With a Gamma-Selective Retinoid Agonist (TESRA) study. Linear regression, weighted gene co-expression network analysis (WGCNA), and pathway analysis were used to identify signatures and network sub-modules associated with the number of exacerbations within the previous year; other COPD-related phenotypes were also investigated.
Individual genes were not found to be significantly associated with the number of exacerbations. However using network methods, a statistically significant gene module was identified, along with other modules showing moderate association. A diverse signature was observed across these modules using pathway analysis, marked by differences in B cell and NK cell activity, as well as cellular markers of viral infection. Within two modules, gene set enrichment analysis recapitulated the molecular signatures of two gene expression experiments; one involving sputum from asthma exacerbations and another involving viral lung infections. The plasma biomarker myeloperoxidase (MPO) was associated with the number of recent exacerbations.
A distinct signature of COPD exacerbations may be observed in peripheral blood months following the acute illness. While not predictive in this cross-sectional analysis, these results will be useful in uncovering the molecular pathogenesis of COPD exacerbations.
Electronic supplementary material
The online version of this article (doi:10.1186/s12920-014-0072-y) contains supplementary material, which is available to authorized users.
Network analysis; Chronic obstructive pulmonary disease; Gene expression profiling; Biomarker
Comorbidities are common in COPD, but quantifying their burden is difficult. Currently there is a COPD-specific comorbidity index to predict mortality and another to predict general quality of life. We sought to develop and validate a COPD-specific comorbidity score that reflects comorbidity burden on patient-centered outcomes.
Materials and Methods
Using the COPDGene study (GOLD II-IV COPD), we developed comorbidity scores to describe patient-centered outcomes employing three techniques: 1) simple count, 2) weighted score, and 3) weighted score based upon statistical selection procedure. We tested associations, area under the Curve (AUC) and calibration statistics to validate scores internally with outcomes of respiratory disease-specific quality of life (St. George's Respiratory Questionnaire, SGRQ), six minute walk distance (6MWD), modified Medical Research Council (mMRC) dyspnea score and exacerbation risk, ultimately choosing one score for external validation in SPIROMICS.
Associations between comorbidities and all outcomes were comparable across the three scores. All scores added predictive ability to models including age, gender, race, current smoking status, pack-years smoked and FEV1 (p<0.001 for all comparisons). Area under the curve (AUC) was similar between all three scores across outcomes: SGRQ (range 0·7624–0·7676), MMRC (0·7590–0·7644), 6MWD (0·7531–0·7560) and exacerbation risk (0·6831–0·6919). Because of similar performance, the comorbidity count was used for external validation. In the SPIROMICS cohort, the comorbidity count performed well to predict SGRQ (AUC 0·7891), MMRC (AUC 0·7611), 6MWD (AUC 0·7086), and exacerbation risk (AUC 0·7341).
Quantifying comorbidity provides a more thorough understanding of the risk for patient-centered outcomes in COPD. A comorbidity count performs well to quantify comorbidity in a diverse population with COPD.
There is growing evidence that many diseases develop, progress, and respond to therapy differently in men and women. This variability may manifest as a result of sex-specific structures in gene regulatory networks that influence how those networks operate. However, there are few methods to identify and characterize differences in network structure, slowing progress in understanding mechanisms driving sexual dimorphism.
Here we apply an integrative network inference method, PANDA (Passing Attributes between Networks for Data Assimilation), to model sex-specific networks in blood and sputum samples from subjects with Chronic Obstructive Pulmonary Disease (COPD). We used a jack-knifing approach to build an ensemble of likely networks for each sex. By adapting statistical methods to compare these network ensembles, we were able to identify strong differential-targeting patterns associated with functionally-related sets of genes, including those involved in mitochondrial function and energy metabolism. Network analysis also identified several potential sex- and disease-specific transcriptional regulators of these pathways.
Network analysis yielded insight into potential mechanisms driving sexual dimorphism in COPD that were not evident from gene expression analysis alone. We believe our ensemble approach to network analysis provides a principled way to capture sex-specific regulatory relationships and could be applied to identify differences in gene regulatory patterns in a wide variety of diseases and contexts.
Electronic supplementary material
The online version of this article (doi:10.1186/s12918-014-0118-y) contains supplementary material, which is available to authorized users.
Network modeling; Gene regulation; Regulatory networks; Sexual-dimorphism; Chronic Obstructive Lung Disease
Rationale: Persistent inflammation plays a major role in chronic obstructive pulmonary disease (COPD) pathogenesis, but its mechanisms are incompletely defined. Overproduction of the inflammatory mediator prostaglandin (PG) E2 by COPD fibroblasts contributes to reduced repair function.
Objectives: The present study determined if fibroblasts from subjects with COPD overproduce PGE2 after stimulation with the inflammatory cytokines IL-1β and tumor necrosis factor-α, and further defined the mechanism for overproduction.
Methods: Fibroblasts were isolated from parenchymal tissue obtained from smokers with and without COPD undergoing lung surgery. PGE2, cyclooxygenases (COX), and miR-146a in these cells were evaluated by in vitro studies.
Measurements and Main Results: After stimulation with inflammatory cytokines, COPD fibroblasts produced 2.7-fold more PGE2 compared with controls with similar smoking history. The increase in PGE2 depended on induction of COX-2, which increased to a greater degree in fibroblasts from subjects with COPD. Cytokines also induced microRNA miR-146a expression in both fibroblasts, but significantly less in COPD fibroblasts. miR-146a caused degradation of COX-2 mRNA; reduced expression prolonged COX-2 mRNA half-life in fibroblasts from subjects with COPD. Cytokine-stimulated PGE2 production and miR-146a expression in cultured fibroblasts correlated with clinical severity assessed by expiratory airflow and diffusion capacity.
Conclusions: miR-146a seems to play a pathogenetic role in the abnormal inflammatory response in COPD. Increased half-life of inflammatory mRNAs is a mechanism of abnormal inflammation in this disease.
chronic obstructive pulmonary disease; miR-146a; prostaglandin E2; cyclooxygenase-2; fibroblasts
Chronic obstructive pulmonary disease (COPD) has been classically divided into blue bloaters and pink puffers. The utility of these clinical subtypes is unclear. However, the broader distinction between airway-predominant and emphysema-predominant COPD may be clinically relevant. The objective was to define clinical features of emphysema-predominant and non-emphysematous COPD patients.
Current and former smokers from the Genetic Epidemiology of COPD Study (COPDGene) had chest computed tomography (CT) scans with quantitative image analysis. Emphysema-predominant COPD was defined by low attenuation area at -950 Hounsfield Units (LAA-950) ≥10%. Non-emphysematous COPD was defined by airflow obstruction with minimal to no emphysema (LAA-950 < 5%).
Out of 4197 COPD subjects, 1687 were classified as emphysema-predominant and 1817 as non-emphysematous; 693 had LAA-950 between 5–10% and were not categorized. Subjects with emphysema-predominant COPD were older (65.6 vs 60.6 years, p < 0.0001) with more severe COPD based on airflow obstruction (FEV1 44.5 vs 68.4%, p < 0.0001), greater exercise limitation (6-minute walk distance 1138 vs 1331 ft, p < 0.0001) and reduced quality of life (St. George’s Respiratory Questionnaire score 43 vs 31, p < 0.0001). Self-reported diabetes was more frequent in non-emphysematous COPD (OR 2.13, p < 0.001), which was also confirmed using a strict definition of diabetes based on medication use. The association between diabetes and non-emphysematous COPD was replicated in the ECLIPSE study.
Non-emphysematous COPD, defined by airflow obstruction with a paucity of emphysema on chest CT scan, is associated with an increased risk of diabetes. COPD patients without emphysema may warrant closer monitoring for diabetes, hypertension, and hyperlipidemia and vice versa.
Clinicaltrials.gov identifiers: COPDGene NCT00608764, ECLIPSE NCT00292552.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2466-14-164) contains supplementary material, which is available to authorized users.
Airway disease; CT scan; Diabetes mellitus; Emphysema; Spirometry
Combining two long-acting bronchodilators with complementary mechanisms of action may provide treatment benefits to patients with chronic obstructive pulmonary disease (COPD) that are greater than those derived from either treatment alone. The efficacy and safety of a fixed-dose combination (FDC) of aclidinium bromide, a long-acting muscarinic antagonist, and formoterol fumarate, a long-acting β2-agonist, in patients with moderate to severe COPD are presented.
In this 24-week double-blind study, 1692 patients with stable COPD were equally randomized to twice-daily treatment with FDC aclidinium 400 μg/formoterol 12 μg (ACL400/FOR12 FDC), FDC aclidinium 400 μg/formoterol 6 μg (ACL400/FOR6 FDC), aclidinium 400 μg, formoterol 12 μg, or placebo administered by a multidose dry powder inhaler (Genuair®/Pressair®)*. Coprimary endpoints were change from baseline to week 24 in 1-hour morning postdose FEV1 (FDCs versus aclidinium) and change from baseline to week 24 in morning predose (trough) FEV1 (FDCs versus formoterol). Secondary endpoints were change from baseline in St. George’s Respiratory Questionnaire (SGRQ) total score and improvement in Transition Dyspnea Index (TDI) focal score at week 24. Safety and tolerability were also assessed.
At study end, improvements from baseline in 1-hour postdose FEV1 were significantly greater in patients treated with ACL400/FOR12 FDC or ACL400/FOR6 FDC compared with aclidinium (108 mL and 87 mL, respectively; p < 0.0001). Improvements in trough FEV1 were significantly greater in patients treated with ACL400/FOR12 FDC versus formoterol (45 mL; p = 0.0102), a numerical improvement of 26 mL in trough FEV1 over formoterol was observed with ACL400/FOR6 FDC. Significant improvements in both SGRQ total and TDI focal scores were observed in the ACL400/FOR12 FDC group at study end (p < 0.0001), with differences over placebo exceeding the minimal clinically important difference of ≥4 points and ≥1 unit, respectively. All treatments were well tolerated, with safety profiles of the FDCs similar to those of the monotherapies.
Treatment with twice-daily aclidinium 400 μg/formoterol 12 μg FDC provided rapid and sustained bronchodilation that was greater than either monotherapy; clinically significant improvements in dyspnea and health status were evident compared with placebo. Aclidinium/formoterol FDC may be an effective and well tolerated new treatment option for patients with COPD.
*Registered trademarks of Almirall S.A., Barcelona, Spain; for use within the US as Pressair® and Genuair® within all other licensed territories.
Electronic supplementary material
The online version of this article (doi:10.1186/s12931-014-0123-0) contains supplementary material, which is available to authorized users.
Aclidinium; Formoterol; COPD; Lung function; Dyspnea; Health status
NicVAX®, a nicotine vaccine (3’AmNic-rEPA), has been clinically evaluated to determine if higher antibody concentrations are associated with higher smoking abstinence rates and if doses and frequency of administration are associated with increased antibody response. This randomized, double-blinded, placebo-controlled multicenter clinical trial (N=301 smokers) tested 200 and 400 µg doses administered 4 or 5 times over 6 months compared to placebo. 3’AmNic-rEPA recipients with the highest serum anti-nicotine antibody response (top 30% by AUC) were significantly more likely to attain 8 weeks continuous abstinence from weeks 19 through 26 than the placebo recipients (24.6% vs. 12.0%, p=0.024, OR=2.69, 95% CI, 1.14–6.37). The 5 injection 400 µg dose regimen had the greatest antibody response and had significantly higher abstinence rates than placebo. This study demonstrates proof-of-concept that 3’AmNic-rEPA elicits antibodies to nicotine and is associated with higher continuous abstinence rates, justifying its further development as a treatment for nicotine dependence.
3’AmNic-rEPA; NicVAX; nicotine immunotherapeutic; nicotine vaccine; cigarette; smoking cessation; antibody; P. aeruginosa r-Exoprotein A; aminomethyl nicotine; cotinine; CO
Concepts relating to the natural history of chronic obstructive pulmonary disease (COPD) arise most importantly from the classic study of Fletcher and colleagues (The Natural History of Chronic Bronchitis and Emphysema, Oxford University Press, New York, 1976). This study, which evaluated working English men over 8 years, was used to construct a proposed life-long natural history. Although this is a classic study that has greatly advanced understanding of COPD, it has a number of limitations. Its duration is relatively short compared with the duration of COPD, so it is more cross-sectional than longitudinal. It was unable to distinguish among varied “natural histories.” It assessed primarily the FEV1, and the natural history of other features of COPD is largely undescribed. With advances in understanding the clinical features of COPD and with the development of evaluating new tools to assess patients with COPD, longitudinal studies evaluating COPD in novel ways and for longer durations are needed.
lung function; severity; symptoms; biomarkers
Migration of fibroblasts plays an essential role in tissue repair after injury. Sphingosine 1-phosphate (S1P) is a multifunctional mediator released by many cells that can be released in inflammation and after injury. This study evaluated the effect of S1P on fibroblast chemotaxis toward fibronectin. S1P alone did not affect fibroblast migration, but S1P enhanced fibronectin-directed chemotaxis in a concentration-dependent manner. The effect of S1P was not mimicked by dihydro (dh) S1P or the S1P1 receptor agonist SEW2871. S1P augmentation of fibroblast chemotaxis, however, was completely blocked by JTE-013, an S1P2 antagonist, but not by suramin, an S1P3 antagonist. Suppression of the S1P2 receptor by small interfering (si)RNA also completely blocked S1P augmentation of fibroblast chemotaxis to fibronectin. S1P stimulated Rho activation and focal adhesion kinase (FAK) phosphorylation, and these were also significantly inhibited by the S1P2 receptor antagonist (JTE-013) or by S1P2 siRNA. Further, the potentiation of S1P signaling was blocked by the Rho-kinase inhibitor Y-27632 in a concentration-dependent manner. Inhibition of FAK with siRNA reduced basal chemotaxis toward fibronectin slightly but significantly, and almost completely blocked S1P augmented chemotaxis. These results suggest that S1P-augmented fibroblast chemotaxis toward fibronectin depends on the S1P2 receptor and requires Rho and Rho-kinase, and FAK phosphorylation. By augmenting fibroblast recruitment, S1P has the potential to modulate tissue repair after injury. The pathways by which S1P mediates this effect, therefore, represent a potential therapeutic target to affect tissue repair and remodeling.
sphingosine 1-phosphate; fibroblasts; migration; fibronectin
Rationale: Fibroblasts are believed to be the major cells responsible for the production and maintenance of extracellular matrix. Alterations in fibroblast functional capacity, therefore, could play a role in the pathogenesis of pulmonary emphysema, which is characterized by inadequate maintenance of tissue structure.
Objectives: To evaluate the hypothesis that deficient fibroblast repair characterizes cells obtained from individuals with chronic obstructive pulmonary disease (COPD) compared with control subjects.
Methods: Fibroblasts were cultured from lung tissue obtained from individuals undergoing thoracotomy and were characterized in vitro. Measurements and Main Results: Fibroblasts from individuals with COPD, defined by reduced FEV1, manifested reduced chemotaxis toward fibronectin and reduced contraction of three-dimensional collagen gels, two bioassays associated with fibroblast repair function. At least two mechanisms appear to account for these differences. Prostaglandin E (PGE), a known inhibitor of fibroblast repair functions, was produced in increased amount by fibroblasts from subjects with COPD, which also expressed increased amounts of the receptors EP2 and EP4, both of which signal through cyclic AMP. Incubation of fibroblasts with indomethacin or with the PKA inhibitor KT-5720 partially restored COPD subject fibroblast function. In addition, fibroblasts from subjects with COPD produced more transforming growth factor (TGF)-β1, but manifested reduced response to TGF-β1. The functional alterations in fibroblasts correlated with both lung function assessed by FEV1 and, for the data available, with severity of emphysema assessed by DlCO.
Conclusions: Fibroblasts from individuals with COPD have reduced capability to sustain tissue repair, which suggests that this may be one mechanism that contributes to the development of emphysema.
fibroblasts; prostaglandin E; transforming growth factor-β; chemotaxis; contraction
Fibroblast-mediated collagen gel contraction has been used as an in vitro model of tissue remodeling. Thrombin is one of the mediators present in the milieu of airway inflammation and may be involved in airway tissue remodeling. We have previously reported that thrombin stimulates fibroblast-mediated collagen gel contraction partially through the PAR1/PKCε signaling pathway (Fang et al, ERJ, 2004; 24: 918-924). Here we further report that the delta-isoform of PKC (PKCδ) is also activated by thrombin and involved in the thrombin-mediated augmentation of collagen gel contraction. Thrombin (10nM) significantly increased PKCδ activity (over 5-fold increase after 15-30 min stimulation) and stimulated phosphorylation of PKCδ. Rottlerin, a PKCδ inhibitor, completely inhibited activation of PKCδ and partially blocked collagen gel contraction stimulated by thrombin. Similarly, PKCδ -specific siRNA significantly inhibited PKCδ activation without affecting PKCε expression and activation. Furthermore, suppression of PKCδ by siRNA resulted in partial blockade of thrombin-augmented collagen gel contraction. These results suggest that thrombin contributes to the tissue remodeling in inflammatory airways and lung diseases at least partially through both PKCδ and PKCε signaling.
Asthmatic airway remodeling is characterized by goblet cell hyperplasia, angiogenesis, smooth muscle hypertrophy, and subepithelial fibrosis. This study evaluated whether acquired changes in fibroblast phenotype could contribute to this remodeling. Airway and parenchymal fibroblasts from control or chronically ovalbumin (OVA)-sensitized and challenged “asthmatic” mice were assessed for several functions related to repair and remodeling ± exogenous transforming growth factor (TGF)-β. All OVA-challenged mouse fibroblasts demonstrated augmented gel contraction (P < 0.05) and chemotaxis (P < 0.05); increased TGF-β1 (P < 0.05), fibronectin (P < 0.05), and vascular endothelial growth factor (P < 0.05) release; and expressed more α-smooth muscle actin (P < 0.05). TGF-β1 stimulated both control and asthmatic fibroblasts, which retained all differences from control fibroblasts for all features(P < 0.05, all comparisons). Parenchymal fibroblasts proliferated more rapidly (P < 0.05), while airway fibroblasts proliferated similarly compared with control fibroblasts (P = 0.25). Thus, in this animal model, OVA-challenged mouse fibroblasts acquire a distinct phenotype that differs from control fibroblasts. The augmented profibrotic activity and mediator release of asthmatic fibroblasts could contribute to airway remodeling in asthma.
remodeling; fibroblast; phenotype; mouse model
Hedgehog Interacting Protein (HHIP) was implicated in chronic obstructive pulmonary disease (COPD) by genome-wide association studies (GWAS). However, it remains unclear how HHIP contributes to COPD pathogenesis. To identify genes regulated by HHIP, we performed gene expression microarray analysis in a human bronchial epithelial cell line (Beas-2B) stably infected with HHIP shRNAs. HHIP silencing led to differential expression of 296 genes; enrichment for variants nominally associated with COPD was found. Eighteen of the differentially expressed genes were validated by real-time PCR in Beas-2B cells. Seven of 11 validated genes tested in human COPD and control lung tissues demonstrated significant gene expression differences. Functional annotation indicated enrichment for extracellular matrix and cell growth genes. Network modeling demonstrated that the extracellular matrix and cell proliferation genes influenced by HHIP tended to be interconnected. Thus, we identified potential HHIP targets in human bronchial epithelial cells that may contribute to COPD pathogenesis.
Hedgehog interacting protein (HHIP); Gene expression profiling; COPD (Chronic obstructive pulmonary disease); extracellular matrix (ECM); network modeling