Project description:Chronic obstructive pulmonary disease (COPD) is one of the most prevalent lung diseases, and involves persistent airflow limitation and incorporates both emphysema and chronic bronchitis. Cigarette smoking has been identified as the main risk factor for disease development and progression. In a basic model of COPD, the disease is initiated when the physiologic response mechanisms to cigarette smoke exposure are overwhelmed; for example, because of long-term exposure effects or other aging-related changes. In this parallel-group case-controlled clinical study we asked to what extent the different transitions in a chronic-exposure-to-disease model are reflected in the proteome and cellular transcriptome of induced sputum samples from the lung. For this, we selected 60 age- and gender-matched individuals for each of four study groups: current healthy smokers, current-smoker COPD patients, former smokers, and never smokers (a total of 240 individuals). Induced sputum was collected, the cell-free supernatant was analyzed by quantitative proteomics (isobaric-tag based), and the cellular mRNA fraction was analyzed by microarray-based expression profiling. The sputum proteome of current smokers (healthy or COPD patients) clearly reflected the common physiological responses to smoke exposure, including alterations in mucin/trefoil proteins (e.g., MUC5AC and TFF1/3up-regulation), peptidase regulators (e.g., TIMP1 up-regulation), and a prominent xenobiotic/oxidative stress response (e.g., NQO1 and ALDH3A1 up-regulation). The latter response also was observed in the sputum transcriptome, which additionally demonstrated an immune-related polarization change (toward a M2 signature). The (long-term) former smoker group showed nearly complete reversal of the observable biological effects. Thirteen differentially abundant proteins between the COPD and healthy smoker groups were identified. These abundant proteins included previously reported COPD-associated proteins (e.g., TIMP1 (up-regulation) and APOA1 (down-regulation)) and novel proteins such as C6orf58 and BPIFB1 (LPLUNC1) (both up-regulated in the COPD group compared with the healthy smokers). In summary, our study demonstrates that sputum proteomics/transcriptomics can capture the complex and reversible physiological response to cigarette smoke exposure, which appears to be only slightly modulated in early-stage COPD patients. The study has been registered on ClinicalTrials.gov with identifier NCT01780298.

Project description:Background: Chronic obstructive pulmonary disease (COPD) is a heterogeneous disease of the lungs that is currently the fourth leading cause of death worldwide. Genetic factors account for only a small amount of COPD risk, but epigenetic mechanisms including DNA methylation, have the potential to mediate the interactions between an individual?s genetics and environmental exposure. DNA methylation is highly cell type specific and individual cell type studies of DNA methylation in COPD are sparse. Fibroblasts are present within the airway and parenchyma of the lung and contribute to the aberrant deposition of extracellular matrix in COPD. No assessment or comparison of genome-wide DNA methylation profiles in airway and parenchymal fibroblasts from individuals with and without COPD has been undertaken. These data provide valuable insight into the molecular mechanisms contributing to COPD and the differing pathologies of small airways disease and emphysema in COPD. Methods: Genome-wide DNA methylation was evaluated at over 485,000 CpG sites using the Illumina Infinium HumanMethylation450 BeadChip array in airway (non-COPD n=8, COPD n=7) and parenchymal fibroblasts (non-COPD n=18, COPD n=28) isolated from individuals with and without COPD. Targeted gene expression was assessed by qPCR in matched RNA samples. Results: Differentially methylated DNA regions were identified between cells isolated from individuals with and without COPD in both airway and parenchymal fibroblasts. Only in parenchymal fibroblasts was differential DNA methylation associated with differential gene expression. A second analysis of differential DNA methylation variability identified 359 individual differentially variable CpG sites in parenchymal fibroblasts. No differentially variable CpG sites were identified in airway fibroblasts. Five differentially variable methylated CpG sites, associated with three genes were subsequently assessed for gene expression differences. Two genes (OAT and GRIK2) displayed significantly increased gene expression in cells isolated from individuals with COPD. Conclusions: Differential and variable DNA methylation was associated with COPD status in parenchymal fibroblasts but not airway fibroblasts. Aberrant DNA methylation was associated with altered gene expression imparting biological function to DNA methylation changes. Changes in DNA methylation are therefore implicated in the molecular mechanisms underlying COPD pathogenesis and may represent novel therapeutic targets.

Project description:Rationale: DNA methylation is an epigenetic modification that is highly disrupted in response to cigarette smoke and involved in a wide spectrum of malignant and non-malignant diseases, but surprisingly not previously assessed in small airways of patients with chronic obstructive pulmonary disease (COPD). Small airways are the primary sites of airflow obstruction in COPD. We sought to determine whether DNA methylation patterns are disrupted in small airway epithelia of COPD patients, and evaluate whether changes in gene expression are associated with these disruptions. Methods: Genome-wide methylation and gene expression analysis were performed on small airway epithelial DNA and RNA obtained from the same patient during bronchoscopy, using Illumina's Infinium HM27 and Affymetrix's Genechip Human Gene 1.0 ST arrays. To control for known effects of cigarette smoking on DNA methylation, methylation and gene expression profiles were compared between former smokers (FS) with and without COPD matched for age, pack years and years of smoking cessation. Results: Our results indicate that aberrant DNA methylation is i) a genome-wide phenomenon in small airways of patients with COPD and ii) associated with altered expression of genes and pathways important to COPD, such as the Nrf2 oxidative response pathway. Conclusions: DNA methylation is likely an important mechanism contributing to modulation of genes important to COPD pathology. Since these methylation events may underlie disease-specific gene-expression changes, their characterization is a critical first step towards the development of epigenetic markers and an opportunity for developing novel epigenetic therapeutic interventions for COPD. Bisulphite converted DNA from small airway (airways less than <2 mm in diameter) from 38 former smokers: 15 subjects with COPD (post bronchodilator FEV1/FVC ratio <70% and FEV1 predicted M-bM-^IM-$ 80%) and 21 with normal lung function, were hybridized to the Illumina Infinium 27k Human Methylation Beadchip.

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HLF COPD DNA Methylomics

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