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1.  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.
PMCID: PMC3963906  PMID: 24663285
2.  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.
PMCID: PMC3937108  PMID: 24591822
chronic obstructive pulmonary disease; bacterial microbiome; lungs
3.  miR-638 regulates gene expression networks associated with emphysematous lung destruction 
Genome Medicine  2013;5(12):114.
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.
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.
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.
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.
PMCID: PMC3971345  PMID: 24380442
4.  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
6.  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.
PMCID: PMC3728203  PMID: 23936167
7.  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.
PMCID: PMC3359894  PMID: 22427533
COPD; bacteria; microbiome
9.  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.
PMCID: PMC3510026  PMID: 23209423
10.  Small-Airway Obstruction and Emphysema in Chronic Obstructive Pulmonary Disease 
The New England journal of medicine  2011;365(17):1567-1575.
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.
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.
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).
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.)
PMCID: PMC3238466  PMID: 22029978
11.  Ultrastructural changes in atherosclerotic plaques following the instillation of airborne particulate matter into the lungs of rabbits 
The Canadian Journal of Cardiology  2010;26(7):e258-e269.
Epidemiological studies have established that cardiovascular events account for the greatest number of air pollution-related deaths. However, the underlying structural changes are still unknown.
To investigate changes in the ultrastructure of atherosclerotic plaques in Watanabe heritable hyperlipidemic (WHHL) rabbits following the instillation of ambient particulate matter air pollution (particles smaller than 10 μm in diameter) into the lungs.
WHHL rabbits (n=8) exposed to 5 mg of ambient particles (Environmental Health Centre – 1993 [EHC-93]; suspended in saline and instilled in the airway) twice per week for four weeks were compared with control WHHL rabbits (n=8) treated with saline alone.
All abdominal aortic plaques were examined using light and electron microscopy, which showed the following: increased accumulation of macrophage-derived foam cells immediately below the endothelial plaque surface (P=0.04); increased contact between these foam cells and the dense subendothelial extracellular matrix (P<0.005) with reduction (P<0.0001) and fragmentation (P<0.0001) of this matrix; and emigration of macrophage-derived foam cells from the plaques in exposed rabbits. In addition, immunohistochemistry verified the presence of type IV collagen in the thickened extracellular matrix material subtending the endothelium.
The ultrastructure of atherosclerotic plaques in EHC-93-instilled rabbits differed from the ultrastructure observed in rabbits that did not receive EHC-93. These ultrastructural differences are consistent with greater endothelial instability in the plaques of atherosclerosis-prone rabbits.
PMCID: PMC2950725  PMID: 20847974
Air pollution (PM10); Atherosclerosis; Electron microscopy; Watanabe heritable hyperlipidemic rabbits
12.  Persistent Pneumocystis colonization leads to the development of chronic obstructive pulmonary disease (COPD) in a non-human primate model of AIDS 
The Journal of infectious diseases  2010;202(2):302-312.
HIV-infected patients are at increased risk for development of pulmonary complications, including chronic obstructive pulmonary disease (COPD). Inflammation associated with sub-clinical infection has been postulated to promote COPD. Persistence of Pneumocystis (Pc) is associated with HIV and COPD, although a causal relationship has not been established. We used a simian/human immunodeficiency virus (SHIV) model of HIV infection to study pulmonary effects of Pc colonization. SHIV-infected/Pc-colonized monkeys developed progressive obstructive pulmonary disease characterized by increased emphysematous tissue and bronchial-associated lymphoid tissue. Elevated Th2 cytokines and pro-inflammatory mediators in bronchoalveolar lavage fluid coincided with Pc colonization and pulmonary function decline. These results support the concept that an infectious agent contributes to development of HIV-associated lung disease and suggests that Pc colonization may be a risk factor for the development of HIV-associated COPD. Furthermore, this model allows examination of early host responses important to disease progression thus identifying potential therapeutic targets for COPD.
PMCID: PMC2946196  PMID: 20533880
Pneumocystis; COPD; SHIV; AIDS; HIV
13.  Differential Expression of Tissue Repair Genes in the Pathogenesis of Chronic Obstructive Pulmonary Disease 
Rationale: The airflow limitation that defines severity of chronic obstructive pulmonary disease (COPD) is caused by a combination of small airway obstruction and emphysematous lung destruction.
Objectives: To examine the hypothesis that small airway obstructive and emphysematous destructive lesions are produced by differential expression of genes associated with tissue repair.
Methods: The expression of 54 genes associated with repair of repetitively damaged tissue was measured in 136 paired samples of small bronchioles and surrounding lung tissue separated by laser capture microdissection. These samples were collected from 63 patients at different levels of disease severity who required surgery for either lung cancer or lung transplantation for very severe COPD. Gene expression was measured by quantitative polymerase chain reaction in these paired samples and compared with the FEV1 by linear regression analysis.
Measurements and Main Results: After corrections for false discovery rates, only 2 of 10 genes (serpin peptidase inhibitor/plasminogen activator inhibitor member 2 and matrix metalloproteinase [MMP] 10) increased, whereas 8 (MMP2, integrin-α1, vascular endothelial growth factor, a disintegrin and metallopeptidase domain 33, scatter factor/hepatocyte growth factor, tissue inhibitor of matrix metalloproteinase-2, fibronectin, and collagen 3α1) decreased in small airways in association with FEV1. In contrast, 8/12 genes (early growth response factor 1, MMP1, MMP9, MMP10, plasminogen activator urokinase, plasminogen activator urokinase receptor, tumor necrosis factor, and IL13) increased and 4/12 (MMP2, tissue inhibitor of matrix metalloproteinase-1, collagen 1α1, and transforming growth factor-β3) decreased in the surrounding lung tissue in association with progression of COPD.
Conclusions: The progression of COPD is associated with the differential expression of a cluster of genes that favor the degradation of the tissue surrounding the small conducting airways.
PMCID: PMC2894408  PMID: 20075389
emphysema; pulmonary disease, chronic obstructive; polymerase chain reaction; laser microdissection; nucleic acid amplification techniques
14.  Targeting Phosphoinositide-3-Kinase-δ with Theophylline Reverses Corticosteroid Insensitivity in Chronic Obstructive Pulmonary Disease 
Rationale: Patients with chronic obstructive pulmonary disease (COPD) show a poor response to corticosteroids. This has been linked to a reduction of histone deacetylase-2 as a result of oxidative stress and is reversed by theophylline.
Objectives: To determine the role of phosphoinositide-3-kinase-delta (PI3K-δ) on the development of corticosteroid insensitivity in COPD and under oxidative stress, and as a target for theophylline.
Methods: Corticosteroid sensitivity was determined as the 50% inhibitory concentration of dexamethasone on tumor necrosis factor-α–induced interleukin-8 release in peripheral blood mononuclear cells from patients with COPD (n = 17) and compared with that of nonsmoking (n = 8) and smoking (n = 7) control subjects. The effect of theophylline and a selective PI3K-δ inhibitor (IC87114) on restoration of corticosteroid sensitivity was confirmed in cigarette smoke–exposed mice.
Measurements and Main Results: Peripheral blood mononuclear cells of COPD (50% inhibitory concentration of dexamethasone: 156.8 ± 32.6 nM) were less corticosteroid sensitive than those of nonsmoking (41.2 ± 10.5 nM; P = 0.018) and smoking control subjects (47.5 ± 19.6 nM; P = 0.031). Corticosteroid insensitivity and reduced histone deacetylase-2 activity after oxidative stress were reversed by a non-selective PI3K inhibitor (LY294002) and low concentrations of theophylline. Theophylline was a potent selective inhibitor of oxidant-activated PI3K-δ, which was up-regulated in peripheral lung tissue of patients with COPD. Furthermore, cells with knock-down of PI3K-δ failed to develop corticosteroid insensitivity with oxidative stress. Both theophylline and IC87114, combined with dexamethasone, inhibited corticosteroid-insensitive lung inflammation in cigarette–smoke-exposed mice in vivo.
Conclusions: Inhibition of oxidative stress dependent PI3K-δ activation by a selective inhibitor or theophylline provides a novel approach to reversing corticosteroid insensitivity in COPD.
PMCID: PMC2970861  PMID: 20224070
Histone deacetylase 2; dexamethasone; cigarette smoke; oxidative stress; RNA interference
15.  Effects of CT Section Thickness and Reconstruction Kernel on Emphysema Quantification: Relationship to the Magnitude of the CT Emphysema Index 
Academic radiology  2010;17(2):146.
Rationale and Objectives
CT section thickness and reconstruction kernel each influence CT measurements of emphysema. This study was performed to assess whether their effects are related to the magnitude of the measurement.
Materials and Methods
Low-radiation-dose multidetector CT was performed in 21 subjects representing a wide range of emphysema severity. Images were reconstructed using 20 different combinations of section thickness and reconstruction kernel. Emphysema index values were determined as the percentage of lung pixels having attenuation lower than multiple thresholds ranging from −960 HU to −890 HU. The index values obtained from the different thickness-kernel combinations were compared by repeated measures ANOVA and Bland-Altman plots of mean vs. difference, and correlated with quantitative histology (mean linear intercept, Lm) in a subset of resected lung specimens.
The effects of section thickness and reconstruction kernel on the emphysema index were significant (p<0.001) and diminished as the index threshold was raised. The changes in index values due to changing the thickness-kernel combination were largest for subjects with intermediate index values (10–30%), and became progressively smaller for those with lower and higher index values. This pattern was consistent regardless of the thickness-kernel combinations compared and the HU threshold used. Correlations between the emphysema index values obtained with each thickness-kernel combination and Lm ranged from r=0.55–0.68 (p=0.007–0.03).
The effects of CT section thickness and kernel on emphysema index values varied systematically with the magnitude of the emphysema index. All reconstruction techniques provided significant correlations with quantitative histology.
PMCID: PMC2818169  PMID: 19931472
Emphysema; computed tomography; histology
16.  What Drives the Peripheral Lung–Remodeling Process in Chronic Obstructive Pulmonary Disease? 
The smaller airways (<2 mm in diameter) offer little resistance in normal lungs but become the major site of obstruction in chronic obstructive pulmonary disease (COPD). We examined bronchiolar remodeling in COPD by combining quantitative histology, micro-computed tomography (CT), and gene expression studies. Volumes of bronchiolar tissue, total collagen, collagen-1, and collagen-3 were measured in lung tissue from 52 patients with different levels of COPD severity. Micro-CT was used to measure the number and lumen area of terminal bronchioles in four lungs removed before lung transplantation and in four donor lungs that served as controls. Laser capture microdissection provided 136 paired samples of bronchiolar and surrounding lung tissue from 63 patients and the gene expression of a cluster of tissue repair genes was compared. This study shows that total bronchiolar tissue decreased with progression of COPD and was associated with a reduction in total collagen and relative increase in collagen-3 over collagen-1. The micro-CT studies showed a 10-fold reduction in terminal bronchiolar number and a 100-fold reduction in lumen area. Interestingly, most genes associated with tissue accumulation during repair decreased their expression in both airways and in the surrounding lung as FEV1 declined, but eight genes previously associated with COPD increased expression in the surrounding lung tissue. Our study shows that small airway remodeling is associated with narrowing and obliteration of the terminal bronchioles that begins before emphysematous destruction in COPD and in relation to differential expression of tissue repair genes in the airways and surrounding lung.
PMCID: PMC2797069  PMID: 20008873
bronchiolitis; small airway obstruction; emphysema; COPD
Respiratory medicine  2009;103(11):1672-1680.
Latent adenoviral infection may amplify cigarette smoke-induced lung inflammation and therefore play an important role in the development of chronic obstructive pulmonary disease (COPD). Adenoviruses can evade the human immune response via their 19-kDa protein (19K) which delays the expression of class I human leukocyte antigen (HLA) proteins. The 19K protein shows higher affinity to HLA-B7 and A2 compared with HLA-A1 and A3. The receptor for adenovirus (CXADR) and integrin β5 (ITGB5) are host factors which might affect adenovirus infection. Therefore, we investigated the contribution of HLA, CXADR, and ITGB5 genetic variants to the presence of the E1A gene and to level of lung function.
Study subjects were assayed for HLA-B7, A1, A2 and A3 by PCR-based assays using allele-specific primers. Polymorphisms of the CXADR and ITGB5 genes were genotyped by PCR-based restriction fragment length polymorphism assays. Detection of adenoviral E1A gene was performed by a real-time PCR TaqMan assay.
E1A positive individuals have a lower FEV1 compared with E1A negative individuals. However, there was no significant difference in E1A positivity rate between the high (HLA-B7 and A2) and low (HLA-A1 and A3) 19K affinity groups. There was also no significant difference in FEV1 level between each affinity group. There was no significant difference in E1A positivity rate or lung function among the CXADR and ITGB5 genotypes.
Genetic variants in HLA, CXADR and ITGB5 do not influence latent adenoviral infections and are not associated with COPD.
PMCID: PMC2757510  PMID: 19502044
18.  Quantification of lung surface area using computed tomography 
Respiratory Research  2010;11(1):153.
To refine the CT prediction of emphysema by comparing histology and CT for specific regions of lung. To incorporate both regional lung density measured by CT and cluster analysis of low attenuation areas for comparison with histological measurement of surface area per unit lung volume.
The histological surface area per unit lung volume was estimated for 140 samples taken from resected lung specimens of fourteen subjects. The region of the lung sampled for histology was located on the pre-operative CT scan; the regional CT median lung density and emphysematous lesion size were calculated using the X-ray attenuation values and a low attenuation cluster analysis. Linear mixed models were used to examine the relationships between histological surface area per unit lung volume and CT measures.
The median CT lung density, low attenuation cluster analysis, and the combination of both were important predictors of surface area per unit lung volume measured by histology (p < 0.0001). Akaike's information criterion showed the model incorporating both parameters provided the most accurate prediction of emphysema.
Combining CT measures of lung density and emphysematous lesion size provides a more accurate estimate of lung surface area per unit lung volume than either measure alone.
PMCID: PMC2976969  PMID: 21040527
19.  Effects of Diffusion Time on Short-Range Hyperpolarized 3He Diffusivity Measurements in Emphysema 
To characterize the effect of diffusion time on short-range hyperpolarized 3He MR diffusion measurements across a wide range of emphysema severity.
Materials and Methods
3He diffusion MR imaging was performed on 19 lungs or lobes resected from 18 subjects with varying degrees of emphysema using 3 diffusion times (1.6 msec, 5 msec, and 10 msec) at constant b value. Emphysema severity was quantified as the mean apparent diffusion coefficient (ADC) and as the percentage of pixels with ADC higher than multiple thresholds from 0.30–0.55 cm2/sec (ADC index). Quantitative histology (mean linear intercept) was obtained in 10 of the lung specimens from 10 of the subjects.
The mean ADCs with diffusion times of 1.6, 5.0, and 10.0 msec were 0.46, 0.40, and 0.37 cm2/sec, respectively (P <0.0001, ANOVA). There was no relationship between the ADC magnitude and the effect of diffusion time on ADC values. Mean linear intercept correlated with ADC (r=0.91–0.94, P<0.001) and ADC index (r=0.78–0.92, P<0.01) at all diffusion times.
Decreases in ADC with longer diffusion time were unrelated to emphysema severity. The strong correlations between the ADC at all diffusion times tested and quantitative histology demonstrate that the ADC is a robust measure of emphysema.
PMCID: PMC2844435  PMID: 19787725
Helium-3; diffusivity; emphysema
20.  Micro–Computed Tomography Measurements of Peripheral Lung Pathology in Chronic Obstructive Pulmonary Disease 
Background: The smaller airways, < 2 mm in diameter, offer little resistance in normal lungs, but become the major site of obstruction in chronic obstructive pulmonary disease (COPD).
Objective: To examine bronchiolar remodeling and alveolar destruction in COPD using micro–computed tomography (micro-CT).
Methods: Micro-CT was used to measure the number and cross-sectional lumen area of terminal bronchioles (TB) and alveolar mean linear intercept (Lm) in 4 lungs removed from patients with very severe (GOLD-4) COPD and 4 unused donor lungs that served as controls. These lungs were inflated with air to a transpulmonary pressure (PL) of 30 cm H2O and held at PL 10 cm H2O while they were frozen solid in liquid nitrogen vapor. A high resolution CT scan was performed on the frozen specimen prior to cutting it into 2-cm thick transverse slices. Representative core samples of lung tissue 2 cm long and 1 cm in diameter cut from each slice were fixed at −80°C in a 1% solution of gluteraldehyde in pure acetone, post-fixed in osmium, critically point dried, and examined by micro-CT.
Results: A 10-fold reduction in terminal bronchiolar number and a 100-fold reduction in their minimal cross-sectional lumen area were measured in both emphysematous and non-emphysematous regions of the COPD lungs.
Conclusions: The centrilobular emphysematous phenotype of COPD is associated with narrowing and obliteration of the terminal bronchioles that begins prior to the onset of emphysematous destruction.
PMCID: PMC3136953  PMID: 19741267
COPD, small airway obstruction; termination bronchioles, reduction of; emphysematous destruction
21.  Transpleural ventilation of explanted human lungs 
Thorax  2007;62(7):623-630.
The hypothesis that ventilation of emphysematous lungs would be enhanced by communication with the parenchyma through holes in the pleural surface was tested.
Fresh human lungs were obtained from patients with emphysema undergoing lung transplantation. Control human lungs were obtained from organ donors whose lungs, for technical reasons, were not considered suitable for implantation. Lungs were ventilated through the bronchial tree or transpleurally via a small hole communicating with the underlying parenchyma over which a flanged silicone tube had been cemented to the surface of the lung (spiracle). Measurements included flow‐volume‐time curves during passive deflation via each pathway; volume of trapped gas recovered from lungs via spiracles when no additional gas was obtainable passively from the airways; and magnetic resonance imaging assessment of spatial distribution of hyperpolarised helium (3He) administered through either the airways or spiracles.
In emphysematous lungs, passively expelled volumes at 20 s were 94% greater through spiracles than via the airways. Following passive deflation from the airways, an average of 1.07 litres of trapped gas volume was recoverable via spiracles. Regions were ventilated by spiracles that were less well ventilated via bronchi.
Because of the extensive collateral ventilation present in emphysematous lungs, direct communication with the lung parenchyma through non‐anatomical pathways has the potential to improve the mechanics of breathing and hence ventilation.
PMCID: PMC2117236  PMID: 17412776
22.  Decline in NRF2-regulated Antioxidants in Chronic Obstructive Pulmonary Disease Lungs Due to Loss of Its Positive Regulator, DJ-1 
Rationale: Oxidative stress is a key contributor in chronic obstructive pulmonary disease (COPD) pathogenesis caused by cigarette smoking. NRF2, a redox-sensitive transcription factor, dissociates from its inhibitor, KEAP1, to induce antioxidant expression that inhibits oxidative stress.
Objectives: To determine the link between severity of COPD, oxidative stress, and NRF2-dependent antioxidant levels in the peripheral lung tissue of patients with COPD.
Methods: We assessed the expression of NRF2, NRF2-dependent antioxidants, regulators of NRF2 activity, and oxidative damage in non-COPD (smokers and former smokers) and smoker COPD lungs (mild and advanced). Cigarette smoke–exposed human lung epithelial cells (Beas2B) and mice were used to understand the mechanisms.
Measurements and Main Results: When compared with non-COPD lungs, the COPD patient lungs showed (1) marked decline in NRF2-dependent antioxidants and glutathione levels, (2) increased oxidative stress markers, (3) significant decrease in NRF2 protein with no change in NRF2 mRNA levels, and (4) similar KEAP1 but significantly decreased DJ-1 levels (a protein that stabilizes NRF2 protein by impairing KEAP1-dependent proteasomal degradation of NRF2). Exposure of Bea2B cells to cigarette smoke caused oxidative modification and enhanced proteasomal degradation of DJ-1 protein. Disruption of DJ-1 in mouse lungs, mouse embryonic fibroblasts, and Beas2B cells lowered NRF2 protein stability and impaired antioxidant induction in response to cigarette smoke. Interestingly, targeting KEAP1 by siRNA or the small-molecule activator sulforaphane restored induction of NRF2-dependent antioxidants in DJ-1–disrupted cells in response to cigarette smoke.
Conclusions: NRF2-dependent antioxidants and DJ-1 expression was negatively associated with severity of COPD. Therapy directed toward enhancing NRF2-regulated antioxidants may be a novel strategy for attenuating the effects of oxidative stress in the pathogenesis of COPD.
PMCID: PMC2542433  PMID: 18556627
chronic obstructive pulmonary disease; NRF2; DJ-1; oxidative stress; antioxidants
24.  The Immunopathogenesis of Chronic Obstructive Pulmonary Disease 
Chronic obstructive pulmonary disease (COPD) progression is characterized by accumulation of inflammatory mucous exudates in the lumens of small airways, and thickening of their walls, which become infiltrated by innate and adaptive inflammatory immune cells. Infiltration of the airways by polymorphonuclear and mononuclear phagocytes and CD4 T cells increases with COPD stage, but the cumulative volume of the infiltrate does not change. By contrast, B cells and CD8 T cells increase in both the extent of their distribution and in accumulated volume, with organization into lymphoid follicles. This chronic lung inflammation is also associated with a tissue repair and remodeling process that determines the ultimate pathologic phenotype of COPD. Why these pathologic abnormalities progress in susceptible individuals, even after removal of the original noxious stimuli, remains mysterious. However, important clues are emerging from analysis of pathologic samples from patients with COPD and from recent discoveries in basic immunology. We consider the following relevant information: normal limitations on the innate immune system's ability to generate adaptive pulmonary immune responses and how they might be overcome by tobacco smoke exposure; the possible contribution of autoimmunity to COPD pathogenesis; and the potential roles of ongoing lymphocyte recruitment versus in situ proliferation, of persistently activated resident lung T cells, and of the newly described T helper 17 (Th17) phenotype. We propose that the severity and course of acute exacerbations of COPD reflects the success of the adaptive immune response in appropriately modulating the innate response to pathogen-related molecular patterns (“the Goldilocks hypothesis”).
PMCID: PMC2365762  PMID: 17878463
adaptive immunity; adhesion molecules; chemokines; cytokines; innate immunity
25.  Survival after Lung Volume Reduction in Chronic Obstructive Pulmonary Disease 
Rationale: COPD is associated with reduced life expectancy.
Objectives: To determine the association between small airway pathology and long-term survival after lung volume reduction in chronic obstructive pulmonary disease (COPD) and the effect of corticosteroids on this pathology.
Methods: Patients with severe (GOLD-3) and very severe (GOLD-4) COPD (n = 101) were studied after lung volume reduction surgery. Respiratory symptoms, quality of life, pulmonary function, exercise tolerance, chest radiology, and corticosteroid treatment status were assessed preoperatively. The severity of luminal occlusion, wall thickening, and the presence of small airways containing lymphoid follicles were determined in resected lung tissue. Kaplan-Meier survival analysis and Cox proportional hazards models were used to determine the relationship between survival and small airway pathology. The effect of corticosteroids on this pathology was assessed by comparing treated and untreated groups.
Measurements and Main Results: The quartile of subjects with the greatest luminal occlusion, adjusted for covariates, died earlier than subjects who had the least occlusion (hazard ratio, 3.28; 95% confidence interval, 1.55–6.92; P = 0.002). There was a trend toward a reduction in the number of airways containing lymphoid follicles (P = 0.051) in those receiving corticosteroids, with a statistically significant difference between the control and oral ± inhaled corticosteroid–treated groups (P = 0.019). However, corticosteroid treatment had no effect on airway wall thickening or luminal occlusion.
Conclusions: Occlusion of the small airways by inflammatory exudates containing mucus is associated with early death in patients with severe emphysema treated by lung volume reduction surgery. Corticosteroid treatment dampens the host immune response in these airways by reducing lymphoid follicles without changing wall thickening and luminal occlusion.
PMCID: PMC1976540  PMID: 17556723
premature death in COPD; airway remodeling; mucosal immune response; corticosteroids

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