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1.  Findings on Thoracic Computed Tomography Scans and Respiratory Outcomes in Persons with and without Chronic Obstructive Pulmonary Disease: A Population-Based Cohort Study 
PLoS ONE  2016;11(11):e0166745.
Background
Thoracic computed tomography (CT) scans are widely performed in clinical practice, often leading to detection of airway or parenchymal abnormalities in asymptomatic or minimally symptomatic individuals. However, clinical relevance of CT abnormalities is uncertain in the general population.
Methods
We evaluated data from 1361 participants aged ≥40 years from a Canadian prospective cohort comprising 408 healthy never-smokers, 502 healthy ever-smokers, and 451 individuals with spirometric evidence of chronic obstructive pulmonary disease (COPD) who had thoracic CT scans. CT images of subjects were visually scored for respiratory bronchiolitis(RB), emphysema(E), bronchial-wall thickening(BWT), expiratory air-trapping(AT), and bronchiectasis(B). Multivariable logistic regression models were used to assess associations of CT features with respiratory symptoms, dyspnea, health status as determined by COPD assessment test, and risk of clinically significant exacerbations during 12 months follow-up.
Results
About 11% of life-time never-smokers demonstrated emphysema on CT scans. Prevalence increased to 30% among smokers with normal lung function and 36%, 50%, and 57% among individuals with mild, moderate or severe/very severe COPD, respectively. Presence of emphysema on CT was associated with chronic cough (OR,2.11; 95%CI,1.4–3.18); chronic phlegm production (OR,1.87; 95% CI,1.27–2.76); wheeze (OR,1.61; 95% CI,1.05–2.48); dyspnoea (OR,2.90; 95% CI,1.41–5.98); CAT score≥10(OR,2.17; 95%CI,1.42–3.30) and risk of ≥2 exacerbations over 12 months (OR,2.17; 95% CI, 1.42–3.0).
Conclusions
Burden of thoracic CT abnormalities is high among Canadians ≥40 years of age, including never-smokers and smokers with normal lung function. Detection of emphysema on CT scans is associated with pulmonary symptoms and increased risk of exacerbations, independent of smoking or lung function.
doi:10.1371/journal.pone.0166745
PMCID: PMC5115801  PMID: 27861566
2.  CT-Definable Subtypes of Chronic Obstructive Pulmonary Disease: A Statement of the Fleischner Society 
Radiology  2015;277(1):192-205.
Integration of visual characterization of emphysema and airway abnormalities with physiologic and quantitative CT assessment permits categorization of chronic obstructive pulmonary disease into distinct structurally and functionally defined subtypes.
The purpose of this statement is to describe and define the phenotypic abnormalities that can be identified on visual and quantitative evaluation of computed tomographic (CT) images in subjects with chronic obstructive pulmonary disease (COPD), with the goal of contributing to a personalized approach to the treatment of patients with COPD. Quantitative CT is useful for identifying and sequentially evaluating the extent of emphysematous lung destruction, changes in airway walls, and expiratory air trapping. However, visual assessment of CT scans remains important to describe patterns of altered lung structure in COPD. The classification system proposed and illustrated in this article provides a structured approach to visual and quantitative assessment of COPD. Emphysema is classified as centrilobular (subclassified as trace, mild, moderate, confluent, and advanced destructive emphysema), panlobular, and paraseptal (subclassified as mild or substantial). Additional important visual features include airway wall thickening, inflammatory small airways disease, tracheal abnormalities, interstitial lung abnormalities, pulmonary arterial enlargement, and bronchiectasis.
© RSNA, 2015
doi:10.1148/radiol.2015141579
PMCID: PMC4613878  PMID: 25961632
3.  Molecular mechanisms underlying variations in lung function: a systems genetics analysis 
The Lancet. Respiratory medicine  2015;3(10):782-795.
Summary
Background
Lung function measures reflect the physiological state of the lung, and are essential to the diagnosis of chronic obstructive pulmonary disease (COPD). The SpiroMeta-CHARGE consortium undertook the largest genome-wide association study (GWAS) so far (n=48 201) for forced expiratory volume in 1 s (FEV1) and the ratio of FEV1 to forced vital capacity (FEV1/FVC) in the general population. The lung expression quantitative trait loci (eQTLs) study mapped the genetic architecture of gene expression in lung tissue from 1111 individuals. We used a systems genetics approach to identify single nucleotide polymorphisms (SNPs) associated with lung function that act as eQTLs and change the level of expression of their target genes in lung tissue; termed eSNPs.
Methods
The SpiroMeta-CHARGE GWAS results were integrated with lung eQTLs to map eSNPs and the genes and pathways underlying the associations in lung tissue. For comparison, a similar analysis was done in peripheral blood. The lung mRNA expression levels of the eSNP-regulated genes were tested for associations with lung function measures in 727 individuals. Additional analyses identified the pleiotropic effects of eSNPs from the published GWAS catalogue, and mapped enrichment in regulatory regions from the ENCODE project. Finally, the Connectivity Map database was used to identify potential therapeutics in silico that could reverse the COPD lung tissue gene signature.
Findings
SNPs associated with lung function measures were more likely to be eQTLs and vice versa. The integration mapped the specific genes underlying the GWAS signals in lung tissue. The eSNP-regulated genes were enriched for developmental and inflammatory pathways; by comparison, SNPs associated with lung function that were eQTLs in blood, but not in lung, were only involved in inflammatory pathways. Lung function eSNPs were enriched for regulatory elements and were over-represented among genes showing differential expression during fetal lung development. An mRNA gene expression signature for COPD was identified in lung tissue and compared with the Connectivity Map. This in-silico drug repurposing approach suggested several compounds that reverse the COPD gene expression signature, including a nicotine receptor antagonist. These findings represent novel therapeutic pathways for COPD.
Interpretation
The system genetics approach identified lung tissue genes driving the variation in lung function and susceptibility to COPD. The identification of these genes and the pathways in which they are enriched is essential to understand the pathophysiology of airway obstruction and to identify novel therapeutic targets and biomarkers for COPD, including drugs that reverse the COPD gene signature in silico.
Funding
The research reported in this article was not specifically funded by any agency. See Acknowledgments for a full list of funders of the lung eQTL study and the Spiro-Meta CHARGE GWAS.
doi:10.1016/S2213-2600(15)00380-X
PMCID: PMC5021067  PMID: 26404118
4.  Host Response to the Lung Microbiome in Chronic Obstructive Pulmonary Disease 
Rationale: The relatively sparse but diverse microbiome in human lungs may become less diverse in chronic obstructive pulmonary disease (COPD). This article examines the relationship of this microbiome to emphysematous tissue destruction, number of terminal bronchioles, infiltrating inflammatory cells, and host gene expression.
Methods: Culture-independent pyrosequencing microbiome analysis was used to examine the V3–V5 regions of bacterial 16S ribosomal DNA in 40 samples of lung from 5 patients with COPD (Global Initiative for Chronic Obstructive Lung Disease [GOLD] stage 4) and 28 samples from 4 donors (controls). A second protocol based on the V1–V3 regions was used to verify the bacterial microbiome results. Within lung tissue samples the microbiome was compared with results of micro–computed tomography, infiltrating inflammatory cells measured by quantitative histology, and host gene expression.
Measurements and Main Results: Ten operational taxonomic units (OTUs) was found sufficient to discriminate between control and GOLD stage 4 lung tissue, which included known pathogens such as Haemophilus influenzae. We also observed a decline in microbial diversity that was associated with emphysematous destruction, remodeling of the bronchiolar and alveolar tissue, and the infiltration of the tissue by CD4+ T cells. Specific OTUs were also associated with neutrophils, eosinophils, and B-cell infiltration (P < 0.05). The expression profiles of 859 genes and 235 genes were associated with either enrichment or reductions of Firmicutes and Proteobacteria, respectively, at a false discovery rate cutoff of less than 0.1.
Conclusions: These results support the hypothesis that there is a host immune response to microorganisms within the lung microbiome that appears to contribute to the pathogenesis of COPD.
doi:10.1164/rccm.201502-0223OC
PMCID: PMC4595667  PMID: 25945594
COPD; microbiome; inflammation; bacteria
5.  Susceptibility loci for lung cancer are associated with mRNA levels of nearby genes in the lung 
Carcinogenesis  2014;35(12):2653-2659.
Summary
This study extends the functional meaning of lung cancer susceptibility loci to gene expression in the lung and suggests that the risk alleles on 15q25 and 6p21 mediate their effect by regulating the expression levels of CHRNA5 and APOM, respectively.
Recent studies identified three genetic loci reproducibly associated with lung cancer in populations of European ancestry, namely 15q25, 5p15 and 6p21. The goals of this study are first to confirm whether these loci are associated with lung cancer in a French Canadian population and second to identify disease-associated single nucleotide polymorphisms (SNPs) influencing messenger RNA (mRNA) expression levels of genes in the lung, that is expression quantitative trait loci (eQTLs). SNPs were genotyped in 420 patients undergoing lung cancer surgery and compared with 3151 controls of European ancestry. Genome-wide gene expression levels in non-tumor lung tissues of the same 420 patients were also measured to identify eQTLs. Significant eQTLs were then followed-up in two replication sets (n = 339 and 363). SNPs found in the three susceptibility loci were associated with lung cancer in the French Canadian population. Strong eQTLs were found on chromosome 15q25 with the expression levels of CHRNA5 (P = 2.23 × 10− 22 with rs12907966). The CHRNA5-rs12907966 eQTL was convincingly validated in the two replication sets (P = 3.46 × 10− 16 and 2.01 × 10− 15). On 6p21, a trend was observed for rs3131379 to be associated with the expression of APOM (P = 3.58 × 10− 4) and validated in the replication sets (P = 1.11 × 10− 8 and 6.84 × 10− 4). On 5p15, no significant eQTLs were found. This study confirmed that chromosomes 15q25, 5p15 and 6p21 harbored susceptibility loci for lung cancer in French Canadians. Most importantly, this study suggests that the risk alleles at 15q25 and 6p21 may mediate their effect by regulating the mRNA expression levels of CHRNA5 and APOM in the lung.
doi:10.1093/carcin/bgu184
PMCID: PMC4247514  PMID: 25187487
6.  Three Dimensional Imaging of Paraffin Embedded Human Lung Tissue Samples by Micro-Computed Tomography 
PLoS ONE  2015;10(6):e0126230.
Background
Understanding the three-dimensional (3-D) micro-architecture of lung tissue can provide insights into the pathology of lung disease. Micro computed tomography (µCT) has previously been used to elucidate lung 3D histology and morphometry in fixed samples that have been stained with contrast agents or air inflated and dried. However, non-destructive microstructural 3D imaging of formalin-fixed paraffin embedded (FFPE) tissues would facilitate retrospective analysis of extensive tissue archives of lung FFPE lung samples with linked clinical data.
Methods
FFPE human lung tissue samples (n = 4) were scanned using a Nikon metrology µCT scanner. Semi-automatic techniques were used to segment the 3D structure of airways and blood vessels. Airspace size (mean linear intercept, Lm) was measured on µCT images and on matched histological sections from the same FFPE samples imaged by light microscopy to validate µCT imaging.
Results
The µCT imaging protocol provided contrast between tissue and paraffin in FFPE samples (15mm x 7mm). Resolution (voxel size 6.7 µm) in the reconstructed images was sufficient for semi-automatic image segmentation of airways and blood vessels as well as quantitative airspace analysis. The scans were also used to scout for regions of interest, enabling time-efficient preparation of conventional histological sections. The Lm measurements from µCT images were not significantly different to those from matched histological sections.
Conclusion
We demonstrated how non-destructive imaging of routinely prepared FFPE samples by laboratory µCT can be used to visualize and assess the 3D morphology of the lung including by morphometric analysis.
doi:10.1371/journal.pone.0126230
PMCID: PMC4452358  PMID: 26030902
7.  Loss of GD1-positive Lactobacillus correlates with inflammation in human lungs with COPD 
BMJ Open  2015;5(2):e006677.
Objectives
The present study assesses the relationship between contents of GD1 (glycerol dehydratase)-positive Lactobacillus, presence of Lactobacillus and the inflammatory response measured in host lung tissue in mild to moderate chronic obstructive pulmonary disease (COPD). We hypothesise that there will be a loss of GD1 producing Lactobacillus with increasing severity of COPD and that GD1 has anti-inflammatory properties.
Setting
Secondary care, 1 participating centre in Vancouver, British Columbia, Canada.
Participants
74 individuals who donated non-cancerous portions of their lungs or lobes removed as treatment for lung cancer (normal lung function controls (n=28), persons with mild (GOLD 1) (n=21) and moderate (GOLD 2) COPD (n=25)).
Outcome measures
Primary outcome measure was GD1 positivity within each group and whether or not this impacted quantitative histological measures of lung inflammation. Secondary outcome measures included Lactobacillus presence and quantification, and quantitative histological measurements of inflammation and remodelling in early COPD.
Results
Total bacterial count (p>0.05) and prevalence of Lactobacillus (p>0.05) did not differ between groups. However, the GD1 gene was detected more frequently in the controls (14%) than in either mild (5%) or moderate (0%) COPD (p<0.05) samples. Macrophage and neutrophil volume fractions (0.012±0.005 (mean±SD) vs 0.026±0.017 and 0.005±0.002 vs 0.015±0.014, respectively) in peripheral lung tissue were reduced in samples positive for the GD1 gene (p<0.0035).
Conclusions
A reduction in GD1 positivity is associated with an increased tissue immune inflammatory response in early stage COPD. There is potential for Lactobacillus to be used as a possible therapeutic, however, validation of these results need to be completed before an anti-inflammatory role of Lactobacillus in COPD can be confirmed.
doi:10.1136/bmjopen-2014-006677
PMCID: PMC4322209  PMID: 25652802
8.  Administration IL-17 soluble receptor C suppresses TH17 cells, oxidative stress and hypertension in response to placental ischemia during pregnancy 
Hypertension  2013;62(6):1068-1073.
Pre-eclampsia (PE), new onset hypertension with proteinuria during pregnancy, is associated with chronic inflammation and placental oxidative stress (ROS). Chronic IL-17 increases blood pressure (MAP), autoantibodies (AT1-AA) and ROS during pregnancy. The objective of this study was to determine if TH17 suppression via IL-17RC (recombinant receptor C) decreases pathophysiology associated with placental ischemia (RUPP). On gestation day 14, mini-osmotic pumps infusing 100 pg/day of IL-17RC were implanted into pregnant rats undergoing RUPP (Reduced Uterine Perfusion Pressure), gestation day18 carotid catheters were inserted, day 19 MAP was recorded, TH17 cells, oxidative stress and AT1-AA were measured and analyzed via one-way ANOVA. MAP increased from 101 ±2 mmHg in normal pregnant, NP (n=19), to 120 ±1 mmHg in RUPP (n=17),but decreased to 110±2 mmHg in RUPP+IL-17RC rats (n=22). Pup weight decreased from 2.28 ± 0.2 g in NP to 1.96 ± 0.3 g in RUPP rats, but was significantly increased to 2.01 ± 0.1 in RUPP+IL-17RC rats. TH17 cells were 1.77% in RUPP but decreased to 0.65% in RUPP+IL-17RC rats. Urinary isoprostanes normalized in RUPP +IL-17RC rats (52 pg/μg) compared to 89 pg/μg in RUPP controls. Placental ROS was 652 RLU in RUPP, but decreased to 337 RLU in RUPP+IL-17RC rats. AT1-AA was 17.27 ± 0.7 bpm in RUPP but decreased to 5.00 ± 0.5 bpm in RUPP+IL-17RC rats. With this study, we show that infusion of IL-17RC blunts TH17s, oxidative stress, AT1-AA, and hypertension in the RUPP model of PE indicating that TH17 cells may play an important role in disease pathophysiology.
doi:10.1161/HYPERTENSIONAHA.113.01514
PMCID: PMC3899693  PMID: 24060899
hypertension; pregnancy; inflammation; oxidative stress
9.  Genes related to emphysema are enriched for ubiquitination pathways 
BMC Pulmonary Medicine  2014;14:187.
Background
Increased small airway resistance and decreased lung elasticity contribute to the airflow limitation in chronic obstructive pulmonary disease (COPD). The lesion that corresponds to loss of lung elasticity is emphysema; the small airway obstruction is due to inflammatory narrowing and obliteration. Despite their convergence in altered physiology, different mechanisms contribute to these processes. The relationships between gene expression and these specific phenotypes may be more revealing than comparison with lung function.
Methods
We measured the ratio of alveolar surface area to lung volume (SA/V) in lung tissue from 43 smokers. Two samples from 21 subjects, in which SA/V differed by >49 cm2/mL were profiled to select genes whose expression correlated with SA/V. Significant genes were tested for replication in the 22 remaining subjects.
Results
The level of expression of 181 transcripts was related to SA/V ( p < 0.05). When these genes were tested in the 22 remaining subjects as a replication, thirty of the 181 genes remained significantly associated with SA/V (P < 0.05) and the direction of association was the same in 164/181. Pathway and network analysis revealed enrichment of genes involved in protein ubiquitination, and western blotting showed altered expression of genes involved in protein ubiquitination in obstructed individuals.
Conclusion
This study implicates modified protein ubiquitination and degradation as a potentially important pathway in the pathogenesis of emphysema.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2466-14-187) contains supplementary material, which is available to authorized users.
doi:10.1186/1471-2466-14-187
PMCID: PMC4280711  PMID: 25432663
Pulmonary emphysema; Surface area to lung volume ratio; Gene expression; Transcriptional analysis; mRNA; Cigarette smoking; Protein ubquitination
10.  Changes in the Bacterial Microbiota in Gut, Blood, and Lungs following Acute LPS Instillation into Mice Lungs 
PLoS ONE  2014;9(10):e111228.
Introduction
Previous reports have shown that the gastrointestinal (GI) bacterial microbiota can have profound effects on the lungs, which has been described as the “gut-lung axis”. However, whether a “lung-gut” axis exists wherein acute lung inflammation perturbs the gut and blood microbiota is unknown.
Methods
Adult C57/Bl6 mice were exposed to one dose of LPS or PBS instillation (n = 3 for each group) directly into lungs. Bacterial microbiota of the bronchoalveolar lavage fluid, blood, and cecum were determined using 454 pyrotag sequencing and quantitative polymerase chain reaction (qPCR) at 4 through 168 hours post-instillation. We then investigated the effects of oral neomycin and streptomycin (n = 8) on the microbiota at 4 and 24 hours post LPS instillation versus control treatment (n = 5 at baseline and 4 hours, n = 7 at 24 hours).
Results
At 24 hours post LPS instillation, the total bacterial count was significantly increased in the cecum (P<0.05); whereas the total bacterial count in blood was increased at 4, 48, and 72 hours (P<0.05). Antibiotic treatment reduced the total bacteria in blood but not in the cecum. The increase in total bacteria in the blood correlated with Phyllobacteriaceae OTU 40 and was significantly reduced in the blood for both antibiotic groups (P<0.05).
Conclusion
LPS instillation in lungs leads to acute changes in the bacterial microbiota in the blood and cecum, which can be modulated with antibiotics.
doi:10.1371/journal.pone.0111228
PMCID: PMC4205020  PMID: 25333938
11.  A Comparison between Droplet Digital and Quantitative PCR in the Analysis of Bacterial 16S Load in Lung Tissue Samples from Control and COPD GOLD 2 
PLoS ONE  2014;9(10):e110351.
Background
Low biomass in the bacterial lung tissue microbiome utilizes quantitative PCR (qPCR) 16S bacterial assays at their limit of detection. New technology like droplet digital PCR (ddPCR) could allow for higher sensitivity and accuracy of quantification. These attributes are needed if specific bacteria within the bacterial lung tissue microbiome are to be evaluated as potential contributors to diseases such as chronic obstructive pulmonary disease (COPD). We hypothesize that ddPCR is better at quantifying the total bacterial load in lung tissue versus qPCR.
Methods
Control (n = 16) and COPD GOLD 2 (n = 16) tissue samples were obtained from patients who underwent lung resection surgery, were cut on a cryotome, and sections were assigned for use in quantitative histology or for DNA extraction. qPCR and ddPCR were performed on these samples using primers spanning the V2 region on the 16S rRNA gene along with negative controls. Total 16S counts were compared between the two methods. Both methods were assessed for correlations with quantitative histology measurements of the tissue.
Results
There was no difference in the average total 16S counts (P>0.05) between the two methods. However, the negative controls contained significantly lower counts in the ddPCR (0.55 ± 0.28 16S/uL) than in the qPCR assay (1.00 ± 0.70 16S copies) (P <0.05). The coefficient of variation was significantly lower for the ddPCR assay (0.18 ± 0.14) versus the qPCR assay (0.62 ± 0.29) (P<0.05).
Conclusion
Overall the ddPCR 16S assay performed better by reducing the background noise in 16S of the negative controls compared with 16S qPCR assay.
doi:10.1371/journal.pone.0110351
PMCID: PMC4199711  PMID: 25329701
12.  Gas exchange and pulmonary hypertension following acute pulmonary thromboembolism: has the emperor got some new clothes yet? 
Pulmonary Circulation  2014;4(2):220-236.
Patients present with a wide range of hypoxemia after acute pulmonary thromboembolism (APTE). Recent studies using fluorescent microspheres demonstrated that the scattering of regional blood flows after APTE, created by the embolic obstruction unique in each patient, significantly worsened regional ventilation/perfusion (V/Q) heterogeneity and explained the variability in gas exchange. Furthermore, earlier investigators suggested the roles of released vasoactive mediators in affecting pulmonary hypertension after APTE, but their quantification remained challenging. The latest study reported that mechanical obstruction by clots accounted for most of the increase in pulmonary vascular resistance, but that endothelin-mediated vasoconstriction also persisted at significant level during the early phase.
doi:10.1086/675985
PMCID: PMC4070768  PMID: 25006441
fluorescent microspheres; pulmonary vasoconstriction; regional blood flow; ventilation-perfusion mismatch
13.  A Dynamic Bronchial Airway Gene Expression Signature of Chronic Obstructive Pulmonary Disease and Lung Function Impairment 
Rationale: Molecular phenotyping of chronic obstructive pulmonary disease (COPD) has been impeded in part by the difficulty in obtaining lung tissue samples from individuals with impaired lung function.
Objectives: We sought to determine whether COPD-associated processes are reflected in gene expression profiles of bronchial airway epithelial cells obtained by bronchoscopy.
Methods: Gene expression profiling of bronchial brushings obtained from 238 current and former smokers with and without COPD was performed using Affymetrix Human Gene 1.0 ST Arrays.
Measurements and Main Results: We identified 98 genes whose expression levels were associated with COPD status, FEV1% predicted, and FEV1/FVC. In silico analysis identified activating transcription factor 4 (ATF4) as a potential transcriptional regulator of genes with COPD-associated airway expression, and ATF4 overexpression in airway epithelial cells in vitro recapitulates COPD-associated gene expression changes. Genes with COPD-associated expression in the bronchial airway epithelium had similarly altered expression profiles in prior studies performed on small-airway epithelium and lung parenchyma, suggesting that transcriptomic alterations in the bronchial airway epithelium reflect molecular events found at more distal sites of disease activity. Many of the airway COPD-associated gene expression changes revert toward baseline after therapy with the inhaled corticosteroid fluticasone in independent cohorts.
Conclusions: Our findings demonstrate a molecular field of injury throughout the bronchial airway of active and former smokers with COPD that may be driven in part by ATF4 and is modifiable with therapy. Bronchial airway epithelium may ultimately serve as a relatively accessible tissue in which to measure biomarkers of disease activity for guiding clinical management of COPD.
doi:10.1164/rccm.201208-1449OC
PMCID: PMC3707363  PMID: 23471465
chronic obstructive pulmonary disease; gene expression profiling; biologic markers
14.  Impact of Cigarette Smoke on the Human and Mouse Lungs: A Gene-Expression Comparison Study 
PLoS ONE  2014;9(3):e92498.
Cigarette smoke is well known for its adverse effects on human health, especially on the lungs. Basic research is essential to identify the mechanisms involved in the development of cigarette smoke-related diseases, but translation of new findings from pre-clinical models to the clinic remains difficult. In the present study, we aimed at comparing the gene expression signature between the lungs of human smokers and mice exposed to cigarette smoke to identify the similarities and differences. Using human and mouse whole-genome gene expression arrays, changes in gene expression, signaling pathways and biological functions were assessed. We found that genes significantly modulated by cigarette smoke in humans were enriched for genes modulated by cigarette smoke in mice, suggesting a similar response of both species. Sixteen smoking-induced genes were in common between humans and mice including six newly reported to be modulated by cigarette smoke. In addition, we identified a new conserved pulmonary response to cigarette smoke in the induction of phospholipid metabolism/degradation pathways. Finally, the majority of biological functions modulated by cigarette smoke in humans were also affected in mice. Altogether, the present study provides information on similarities and differences in lung gene expression response to cigarette smoke that exist between human and mouse. Our results foster the idea that animal models should be used to study the involvement of pathways rather than single genes in human diseases.
doi:10.1371/journal.pone.0092498
PMCID: PMC3963906  PMID: 24663285
15.  Bacterial microbiome of lungs in COPD 
Chronic obstructive pulmonary disease (COPD) is currently the third leading cause of death in the world. Although smoking is the main risk factor for this disease, only a minority of smokers develop COPD. Why this happens is largely unknown. Recent discoveries by the human microbiome project have shed new light on the importance and richness of the bacterial microbiota at different body sites in human beings. The microbiota plays a particularly important role in the development and functional integrity of the immune system. Shifts or perturbations in the microbiota can lead to disease. COPD is in part mediated by dysregulated immune responses to cigarette smoke and other environmental insults. Although traditionally the lung has been viewed as a sterile organ, by using highly sensitive genomic techniques, recent reports have identified diverse bacterial communities in the human lung that may change in COPD. This review summarizes the current knowledge concerning the lung microbiota in COPD and its potential implications for pathogenesis of the disease.
doi:10.2147/COPD.S38932
PMCID: PMC3937108  PMID: 24591822
chronic obstructive pulmonary disease; bacterial microbiome; lungs
16.  miR-638 regulates gene expression networks associated with emphysematous lung destruction 
Genome Medicine  2013;5(12):114.
Background
Chronic obstructive pulmonary disease (COPD) is a heterogeneous disease characterized by varying degrees of emphysematous lung destruction and small airway disease, each with distinct effects on clinical outcomes. There is little known about how microRNAs contribute specifically to the emphysema phenotype. We examined how genome-wide microRNA expression is altered with regional emphysema severity and how these microRNAs regulate disease-associated gene expression networks.
Methods
We profiled microRNAs in different regions of the lung with varying degrees of emphysema from 6 smokers with COPD and 2 controls (8 regions × 8 lungs = 64 samples). Regional emphysema severity was quantified by mean linear intercept. Whole genome microRNA and gene expression data were integrated in the same samples to build co-expression networks. Candidate microRNAs were perturbed in human lung fibroblasts in order to validate these networks.
Results
The expression levels of 63 microRNAs (P < 0.05) were altered with regional emphysema. A subset, including miR-638, miR-30c, and miR-181d, had expression levels that were associated with those of their predicted mRNA targets. Genes correlated with these microRNAs were enriched in pathways associated with emphysema pathophysiology (for example, oxidative stress and accelerated aging). Inhibition of miR-638 expression in lung fibroblasts led to modulation of these same emphysema-related pathways. Gene targets of miR-638 in these pathways were amongst those negatively correlated with miR-638 expression in emphysema.
Conclusions
Our findings demonstrate that microRNAs are altered with regional emphysema severity and modulate disease-associated gene expression networks. Furthermore, miR-638 may regulate gene expression pathways related to the oxidative stress response and aging in emphysematous lung tissue and lung fibroblasts.
doi:10.1186/gm519
PMCID: PMC3971345  PMID: 24380442
17.  A brief review of chronic obstructive pulmonary disease 
A recent study, based on a combination of multidetector computed tomography scanning of an intact specimen with microcomputed tomography and histological analysis of lung tissue samples, reported that the number of terminal bronchioles were reduced from approximately 44,500/lung pair in control (donor) lungs to approximately 4800/lung pair in lungs donated by individuals with very severe (Global initiative for chronic Obstructive Lung Disease stage 4) chronic obstructive pulmonary disease (COPD) treated by lung transplantation. The present short review discusses the hypothesis that a rapid rate of terminal bronchiolar destruction causes the rapid decline in lung function leading to advanced COPD. With respect to why the terminal bronchioles are targeted for destruction, the postulated mechanisms of this destruction and the possibility that new treatments are able to either prevent or reverse the underlying cause of airway obstruction in COPD are addressed.
PMCID: PMC3603763  PMID: 23248802
Airway obstruction; Chronic airway inflammation; Emphysematous tissue destruction
19.  Refining Susceptibility Loci of Chronic Obstructive Pulmonary Disease with Lung eqtls 
PLoS ONE  2013;8(7):e70220.
Chronic obstructive pulmonary disease (COPD) is the fourth leading cause of mortality worldwide. Recent genome-wide association studies (GWAS) have identified robust susceptibility loci associated with COPD. However, the mechanisms mediating the risk conferred by these loci remain to be found. The goal of this study was to identify causal genes/variants within susceptibility loci associated with COPD. In the discovery cohort, genome-wide gene expression profiles of 500 non-tumor lung specimens were obtained from patients undergoing lung surgery. Blood-DNA from the same patients were genotyped for 1,2 million SNPs. Following genotyping and gene expression quality control filters, 409 samples were analyzed. Lung expression quantitative trait loci (eQTLs) were identified and overlaid onto three COPD susceptibility loci derived from GWAS; 4q31 (HHIP), 4q22 (FAM13A), and 19q13 (RAB4B, EGLN2, MIA, CYP2A6). Significant eQTLs were replicated in two independent datasets (n = 363 and 339). SNPs previously associated with COPD and lung function on 4q31 (rs1828591, rs13118928) were associated with the mRNA expression of HHIP. An association between mRNA expression level of FAM13A and SNP rs2045517 was detected at 4q22, but did not reach statistical significance. At 19q13, significant eQTLs were detected with EGLN2. In summary, this study supports HHIP, FAM13A, and EGLN2 as the most likely causal COPD genes on 4q31, 4q22, and 19q13, respectively. Strong lung eQTL SNPs identified in this study will need to be tested for association with COPD in case-control studies. Further functional studies will also be needed to understand the role of genes regulated by disease-related variants in COPD.
doi:10.1371/journal.pone.0070220
PMCID: PMC3728203  PMID: 23936167
20.  The Lung Tissue Microbiome in Chronic Obstructive Pulmonary Disease 
Rationale: Based on surface brushings and bronchoalveolar lavage fluid, Hilty and coworkers demonstrated microbiomes in the human lung characteristic of asthma and chronic obstructive pulmonary disease (COPD), which have now been confirmed by others.
Objectives: To extend these findings to human lung tissue samples.
Methods: DNA from lung tissue samples was obtained from nonsmokers (n = 8); smokers without COPD (n = 8); patients with very severe COPD (Global Initiative for COPD [GOLD] 4) (n = 8); and patients with cystic fibrosis (CF) (n = 8). The latter served as a positive control, with sterile water as a negative control. All bacterial community analyses were based on polymerase chain reaction amplifying 16S rRNA gene fragments. Total bacterial populations were measured by quantitative polymerase chain reaction and bacterial community composition was assessed by terminal restriction fragment length polymorphism analysis and pyrotag sequencing.
Measurement and Main Results: Total bacterial populations within lung tissue were small (20–1,252 bacterial cells per 1,000 human cells) but greater in all four sample groups versus the negative control group (P < 0.001). Terminal restriction fragment length polymorphism analysis and sequencing distinguished three distinct bacterial community compositions: one common to the nonsmoker and smoker groups, a second to the GOLD 4 group, and the third to the CF-positive control group. Pyrotag sequencing identified greater than 1,400 unique bacterial sequences and showed an increase in the Firmicutes phylum in GOLD 4 patients versus all other groups (P < 0.003) attributable to an increase in the Lactobacillus genus (P < 0.0007).
Conclusions: There is a detectable bacterial community within human lung tissue that changes in patients with very severe COPD.
doi:10.1164/rccm.201111-2075OC
PMCID: PMC3359894  PMID: 22427533
COPD; bacteria; microbiome
22.  Lung eQTLs to Help Reveal the Molecular Underpinnings of Asthma 
PLoS Genetics  2012;8(11):e1003029.
Genome-wide association studies (GWAS) have identified loci reproducibly associated with pulmonary diseases; however, the molecular mechanism underlying these associations are largely unknown. The objectives of this study were to discover genetic variants affecting gene expression in human lung tissue, to refine susceptibility loci for asthma identified in GWAS studies, and to use the genetics of gene expression and network analyses to find key molecular drivers of asthma. We performed a genome-wide search for expression quantitative trait loci (eQTL) in 1,111 human lung samples. The lung eQTL dataset was then used to inform asthma genetic studies reported in the literature. The top ranked lung eQTLs were integrated with the GWAS on asthma reported by the GABRIEL consortium to generate a Bayesian gene expression network for discovery of novel molecular pathways underpinning asthma. We detected 17,178 cis- and 593 trans- lung eQTLs, which can be used to explore the functional consequences of loci associated with lung diseases and traits. Some strong eQTLs are also asthma susceptibility loci. For example, rs3859192 on chr17q21 is robustly associated with the mRNA levels of GSDMA (P = 3.55×10−151). The genetic-gene expression network identified the SOCS3 pathway as one of the key drivers of asthma. The eQTLs and gene networks identified in this study are powerful tools for elucidating the causal mechanisms underlying pulmonary disease. This data resource offers much-needed support to pinpoint the causal genes and characterize the molecular function of gene variants associated with lung diseases.
Author Summary
Recent genome-wide association studies (GWAS) have identified genetic variants associated with lung diseases. The challenge now is to find the causal genes in GWAS–nominated chromosomal regions and to characterize the molecular function of disease-associated genetic variants. In this paper, we describe an international effort to systematically capture the genetic architecture of gene expression regulation in human lung. By studying lung specimens from 1,111 individuals of European ancestry, we found a large number of genetic variants affecting gene expression in the lung, or lung expression quantitative trait loci (eQTL). These lung eQTLs will serve as an important resource to aid in the understanding of the molecular underpinnings of lung biology and its disruption in disease. To demonstrate the utility of this lung eQTL dataset, we integrated our data with previous genetic studies on asthma. Through integrative techniques, we identified causal variants and genes in GWAS–nominated loci and found key molecular drivers for asthma. We feel that sharing our lung eQTLs dataset with the scientific community will leverage the impact of previous large-scale GWAS on lung diseases and function by providing much needed functional information to understand the molecular changes introduced by the susceptibility genetic variants.
doi:10.1371/journal.pgen.1003029
PMCID: PMC3510026  PMID: 23209423
23.  A gene expression signature of emphysema-related lung destruction and its reversal by the tripeptide GHK 
Genome Medicine  2012;4(8):67.
Background
Chronic obstructive pulmonary disease (COPD) is a heterogeneous disease consisting of emphysema, small airway obstruction, and/or chronic bronchitis that results in significant loss of lung function over time.
Methods
In order to gain insights into the molecular pathways underlying progression of emphysema and explore computational strategies for identifying COPD therapeutics, we profiled gene expression in lung tissue samples obtained from regions within the same lung with varying amounts of emphysematous destruction from smokers with COPD (8 regions × 8 lungs = 64 samples). Regional emphysema severity was quantified in each tissue sample using the mean linear intercept (Lm) between alveolar walls from micro-CT scans.
Results
We identified 127 genes whose expression levels were significantly associated with regional emphysema severity while controlling for gene expression differences between individuals. Genes increasing in expression with increasing emphysematous destruction included those involved in inflammation, such as the B-cell receptor signaling pathway, while genes decreasing in expression were enriched in tissue repair processes, including the transforming growth factor beta (TGFβ) pathway, actin organization, and integrin signaling. We found concordant differential expression of these emphysema severity-associated genes in four cross-sectional studies of COPD. Using the Connectivity Map, we identified GHK as a compound that can reverse the gene-expression signature associated with emphysematous destruction and induce expression patterns consistent with TGFβ pathway activation. Treatment of human fibroblasts with GHK recapitulated TGFβ-induced gene-expression patterns, led to the organization of the actin cytoskeleton, and elevated the expression of integrin β1. Furthermore, addition of GHK or TGFβ restored collagen I contraction and remodeling by fibroblasts derived from COPD lungs compared to fibroblasts from former smokers without COPD.
Conclusions
These results demonstrate that gene-expression changes associated with regional emphysema severity within an individual's lung can provide insights into emphysema pathogenesis and identify novel therapeutic opportunities for this deadly disease. They also suggest the need for additional studies to examine the mechanisms by which TGFβ and GHK each reverse the gene-expression signature of emphysematous destruction and the effects of this reversal on disease progression.
doi:10.1186/gm367
PMCID: PMC4064320  PMID: 22937864
24.  Small-Airway Obstruction and Emphysema in Chronic Obstructive Pulmonary Disease 
The New England journal of medicine  2011;365(17):1567-1575.
BACKGROUND
The major sites of obstruction in chronic obstructive pulmonary disease (COPD) are small airways (<2 mm in diameter). We wanted to determine whether there was a relationship between small-airway obstruction and emphysematous destruction in COPD.
METHODS
We used multidetector computed tomography (CT) to compare the number of airways measuring 2.0 to 2.5 mm in 78 patients who had various stages of COPD, as judged by scoring on the Global Initiative for Chronic Obstructive Lung Disease (GOLD) scale, in isolated lungs removed from patients with COPD who underwent lung transplantation, and in donor (control) lungs. MicroCT was used to measure the extent of emphysema (mean linear intercept), the number of terminal bronchioles per milliliter of lung volume, and the minimum diameters and cross-sectional areas of terminal bronchioles.
RESULTS
On multidetector CT, in samples from patients with COPD, as compared with control samples, the number of airways measuring 2.0 to 2.5 mm in diameter was reduced in patients with GOLD stage 1 disease (P = 0.001), GOLD stage 2 disease (P = 0.02), and GOLD stage 3 or 4 disease (P<0.001). MicroCT of isolated samples of lungs removed from patients with GOLD stage 4 disease showed a reduction of 81 to 99.7% in the total cross-sectional area of terminal bronchioles and a reduction of 72 to 89% in the number of terminal bronchioles (P<0.001). A comparison of the number of terminal bronchioles and dimensions at different levels of emphysematous destruction (i.e., an increasing value for the mean linear intercept) showed that the narrowing and loss of terminal bronchioles preceded emphysematous destruction in COPD (P<0.001).
CONCLUSIONS
These results show that narrowing and disappearance of small conducting airways before the onset of emphysematous destruction can explain the increased peripheral airway resistance reported in COPD. (Funded by the National Heart, Lung, and Blood Institute and others.)
doi:10.1056/NEJMoa1106955
PMCID: PMC3238466  PMID: 22029978
25.  Reduced Denitration Activity in Peripheral Lung of Chronic Obstructive Pulmonary Disease 
Tanaffos  2012;11(4):23-29.
Background
Accumulation of nitrated protein is seen in peripheral lung and cells from patients with chronic obstructive pulmonary disease (COPD). Nitrated protein causes abnormal protein function, but the nitration was believed to be an irreversible process. However, there are accumulating evidences that this process is reversible by an active denitration pathway. The aim of this study is to detect denitration activity in protein extracts from peripheral lung tissue of COPD and to compare with those in healthy subjects.
Materials and Methods
Peripheral lung tissue from 4 healthy, 4 smokers without COPD, 4 GOLD stage 1 and 4 GOLD stage 2 were used for denitration assay. Denitration activity was determined as reduction of nitro-tyrosine level of nitrated histone protein after incubation with protein extracts from peripheral lung, which was determined by western blotting. In addition, RNA is extracted from peripheral lung of 8 healthy, 7 smoking control, 8 stage 1 and 2 COPD and 10 stage 3 and 4 COPD and nitrate reductase mRNA expression was determined by real time RT-PCR.
Results
Peripheral lung protein extracts from healthy subjects reduced nitro-tyrosine level of nitrated histone. Thus, we were able to show denitration activity in peripheral lungs. The denitration activity was slightly reduced in smoking controls, and significantly reduced in COPD patients. We also showed that the expression of the human homologue of nitrate reductase (chytochrome β2 reductase), a potential candidate of denitrase, was significanty reduced in COPD lung.
Conclusion
This study suggests that accumulation of nitrated protein in lung tissue of COPD may, at least in part, be induced by a reduction in denitration activity or nitrate reductase.
PMCID: PMC4153218  PMID: 25191434
COPD; Nitrotyrosine; Denitration; Nitrate reductase; Nitrative stress

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