Project description:Nuclear factor erythroid 2-related factor 2 (NFE2L2, Nrf2) is an oxidant responsive transcription factor known to induce phase 2 detoxifying and antioxidant genes to protect cells from oxidative stress. Cigarette smoke, with its large oxidant content, is a major stressor to the small airway epithelium, the cells of which are vulnerable to oxidant damage and consequent malignant transformation. In this study, we assessed the role of cigarette smoke in activation of Nrf2 in the human small airway epithelium in vivo. Fiberoptic bronchoscopy was used to sample a pure population of small airway epithelium in 38 healthy nonsmokers and 45 healthy smokers, and gene expression was assessed using Affymetrix HG-U133 Plus 2.0 microarrays. Compared to that of healthy nonsmokers, Nrf2 protein was significantly activated in the small airway epithelium of healthy normal smokers and localized in the nucleus (p<0.05). Of the human homologs of 201 known murine Nrf2-mediated genes, 13 highly smoking-responsive genes were identified (p<10-4, all comparisons smokers to nonsmokers). Using a “Nrf2-index” to quantify the extent of expression in the small airway epithelium of these 13 known Nrf2 genes, variability in the level of expression was observed among the 45 healthy smokers, but the variability was coordinately modulated among the 13 genes, an observation confirmed by TaqMan quantitative PCR. This variability in the coordinate level of expression of the 13 Nrf2-mediated genes was independent of the smoking history. Based on these observations, the “Nrf2 index” was used to evaluate whether other genes modulated by smoking in the small airway epithelium were also coordinately up- or down- modulated among the 45 healthy smokers. Two genes, pirin (PIR) and UDP glucuronosyltransferase 1 family polypeptide A4 (UGT1A4), not previously known to be modulated by Nrf2 were identified as being coordinately modulated among the 45 smokers. Both genes contain several functional antioxidant response elements in the promoter region. Using an electrophoretic mobility shift assay, these antioxidant response elements in the promoters of PIR and UGT1A4 responded in vitro to activated Nrf2. These observations are consistent with the concept that Nrf2 plays an important role in regulating cellular defenses against smoking in the highly vulnerable small airway epithelium cell population, and that there is variability among the population in the relative Nrf2 responsiveness to a similar oxidant burden. Affymetrix arrays were used to assess gene expression data in small airway epithelium obtained by fiberoptic bronchoscopy of 38 healthy non-smokers and 45 healthy smokers
Project description:Nuclear factor erythroid 2-related factor 2 (NFE2L2, Nrf2) is an oxidant responsive transcription factor known to induce phase 2 detoxifying and antioxidant genes to protect cells from oxidative stress. Cigarette smoke, with its large oxidant content, is a major stressor to the small airway epithelium, the cells of which are vulnerable to oxidant damage and consequent malignant transformation. In this study, we assessed the role of cigarette smoke in activation of Nrf2 in the human small airway epithelium in vivo. Fiberoptic bronchoscopy was used to sample a pure population of small airway epithelium in 38 healthy nonsmokers and 45 healthy smokers, and gene expression was assessed using Affymetrix HG-U133 Plus 2.0 microarrays. Compared to that of healthy nonsmokers, Nrf2 protein was significantly activated in the small airway epithelium of healthy normal smokers and localized in the nucleus (p<0.05). Of the human homologs of 201 known murine Nrf2-mediated genes, 13 highly smoking-responsive genes were identified (p<10-4, all comparisons smokers to nonsmokers). Using a “Nrf2-index” to quantify the extent of expression in the small airway epithelium of these 13 known Nrf2 genes, variability in the level of expression was observed among the 45 healthy smokers, but the variability was coordinately modulated among the 13 genes, an observation confirmed by TaqMan quantitative PCR. This variability in the coordinate level of expression of the 13 Nrf2-mediated genes was independent of the smoking history. Based on these observations, the “Nrf2 index” was used to evaluate whether other genes modulated by smoking in the small airway epithelium were also coordinately up- or down- modulated among the 45 healthy smokers. Two genes, pirin (PIR) and UDP glucuronosyltransferase 1 family polypeptide A4 (UGT1A4), not previously known to be modulated by Nrf2 were identified as being coordinately modulated among the 45 smokers. Both genes contain several functional antioxidant response elements in the promoter region. Using an electrophoretic mobility shift assay, these antioxidant response elements in the promoters of PIR and UGT1A4 responded in vitro to activated Nrf2. These observations are consistent with the concept that Nrf2 plays an important role in regulating cellular defenses against smoking in the highly vulnerable small airway epithelium cell population, and that there is variability among the population in the relative Nrf2 responsiveness to a similar oxidant burden.
Project description:Disparate Oxidant-related Gene Expression of Human Small Airway Epithelium Compared to Autologous Alveolar Macrophages in Response to the In Vivo Oxidant Stress of Cigarette Smoking The oxidant burden of cigarette smoking induces lung cell dysfunction, and play a significant role in the pathogenesis of lung disease. Two cell populations directly exposed to the oxidants in cigarette smoke are the small airway epithelium and alveolar macrophages. Of these, the epithelium appears to be more vulnerable to smoking, becoming disordered in differentiation, repair and function, while alveolar macrophages become activated, without becoming diseased. In this context, we asked: for the same individuals, what is the baseline trancriptome of oxidant-related genes in small airway epithelium compared to alveolar macrophages and do the responses of the transcriptome of these 2 cell populations differ substantially to inhaled cigarette smoke? To address these questions we used microarray gene expression and TaqMan analysis to assess the gene expression profile of known oxidant-related genes in paired samples recovered by bronchoscopy from small airway epithelium and alveolar macrophages from the same healthy nonsmokers and normal smokers. Of the 155 oxidant-related genes surveyed, 122 (77%) were expressed in both cell populations in nonsmokers. However, of the genes expressed by both cell populations, oxidant related gene expression levels were higher in alveolar macrophages (67 genes, 43%) than small airway epithelium (37 genes, 24%). There were more oxidant-related genes uniquely expressed in the small airway epithelium (17%), than in alveolar macrophages (5%). In healthy smokers, the majority of oxidant-related genes were expressed in both cell populations, but there were marked differences in the numbers of oxidant-related genes that smoking up- or down-regulated. While smoking up-regulated 15 genes (10%) and down-regulated 7 genes (5%) in the small airway epithelium, smoking had far less effect on alveolar macrophages [only 4 (3%) genes up-regulated, and only 1 (0.6%) down-regulated]. Only a small number of smoking responsive oxidant-related genes overlapped between the two cell types (2 up-regulated, and no down-regulated genes). Consistent with this observation, pathway analysis of smoking-responsive genes in the small airway epithelium showed oxidant-related pathways dominated, but in alveolar macrophages immune-response pathways dominated. Thus, the responses of the oxidant-related transcriptome of cells with an identical genome and exposed to the same oxidant stress of cigarette smoking are very different, with responses of oxidant-related genes of alveolar macrophages far more subdued than that of small airway epithelium, consistent with the clinical observation that, while the small airway epithelium is vulnerable, alveolar macrophages are not "diseased" in response to the oxidant stress of cigarette smoking. Gene expression profiles of known oxidant-related genes in paired samples recovered by bronchoscopy from small airway epithelium and alveolar macrophages from the same healthy nonsmokers and normal smokers.
Project description:Prior microarray studies of smokers at high risk for lung cancer have demonstrated that heterogeneity in bronchial airway epithelial cell gene expression response to smoking can serve as an early diagnostic biomarker for lung cancer. This study examines the relationship between gene expression variation and genetic variation in a central molecular pathway (NRF2-mediated antioxidant response) associated with smoking exposure and lung cancer. We assessed global gene expression in histologically normal airway epithelial cells obtained at bronchoscopy from smokers who developed lung cancer (SC, n=20), smokers without lung cancer (SNC, n=24), and never smokers (NS, n=8). Functional enrichment showed that the NRF2-mediated antioxidant response pathway differed significantly among these groups. Keywords: Global mRNA expression profiling 21 total arrays (20 unique patients) run on total RNA obtained from Bronchial Epithelium of Smokers with Lung Cancer 30 total arrays (24 unique patients) run on total RNA obtained from Bronchial Epithelium of Smokers without Lung Cancer 9 total arrays (8 unique patients) run on total RNA obtained from Bronchial Epithelium of Never Smokers
Project description:Despite overwhelming data that cigarette smoking causes chronic obstructive pulmonary disease (COPD), only a minority of chronic smokers are affected, strongly suggesting that genetic factors modify susceptibility to this disease. We hypothesized that there are individual variations in the response to cigarette smoking, with variability among smokers in expression levels of protective / susceptibility genes. Affymetrix arrays and TaqMan PCR were used to assess the variability of gene expression in the small airway epithelium obtained by fiberoptic bronchoscopy of 18 normal non-smokers, 18 normal smokers and 18 smokers with COPD. We identified 201 probesets representing 150 smoking-responsive genes that were significantly up- or down-regulated, and assessed the coefficient of variation in expression levels among the study population. Variation was a reproducible property of each gene as assessed by different microarray probesets and realtime PCR and was observed in both normal smokers and smokers with COPD. There was greater individual variability in smokers with COPD than in normal smokers. The majority of these highly variable smoking responsive genes were in the functional categories of signal transduction, xenobiotic degradation, metabolism, transport, oxidant-related and transcription. A similar pattern of the same highly variable genes was observed in an independent data set.We propose that there is likely genetic diversity within this subset of genes with highly variable individual to individual responses of the small airway epithelium to smoking, and this subset of genes represent putative candidates for assessment of susceptibility/protection from disease for future gene-based epidemiological studies for the risk of smokers for COPD. Experiment Overall Design: Affymetrix arrays and TaqMan PCR were used to assess the variability of gene expression in the small airway epithelium obtained by fiberoptic bronchoscopy of 18 normal non-smokers, 18 normal smokers and 18 smokers with COPD.
Project description:Disparate Oxidant-related Gene Expression of Human Small Airway Epithelium Compared to Autologous Alveolar Macrophages in Response to the In Vivo Oxidant Stress of Cigarette Smoking The oxidant burden of cigarette smoking induces lung cell dysfunction, and play a significant role in the pathogenesis of lung disease. Two cell populations directly exposed to the oxidants in cigarette smoke are the small airway epithelium and alveolar macrophages. Of these, the epithelium appears to be more vulnerable to smoking, becoming disordered in differentiation, repair and function, while alveolar macrophages become activated, without becoming diseased. In this context, we asked: for the same individuals, what is the baseline trancriptome of oxidant-related genes in small airway epithelium compared to alveolar macrophages and do the responses of the transcriptome of these 2 cell populations differ substantially to inhaled cigarette smoke? To address these questions we used microarray gene expression and TaqMan analysis to assess the gene expression profile of known oxidant-related genes in paired samples recovered by bronchoscopy from small airway epithelium and alveolar macrophages from the same healthy nonsmokers and normal smokers. Of the 155 oxidant-related genes surveyed, 122 (77%) were expressed in both cell populations in nonsmokers. However, of the genes expressed by both cell populations, oxidant related gene expression levels were higher in alveolar macrophages (67 genes, 43%) than small airway epithelium (37 genes, 24%). There were more oxidant-related genes uniquely expressed in the small airway epithelium (17%), than in alveolar macrophages (5%). In healthy smokers, the majority of oxidant-related genes were expressed in both cell populations, but there were marked differences in the numbers of oxidant-related genes that smoking up- or down-regulated. While smoking up-regulated 15 genes (10%) and down-regulated 7 genes (5%) in the small airway epithelium, smoking had far less effect on alveolar macrophages [only 4 (3%) genes up-regulated, and only 1 (0.6%) down-regulated]. Only a small number of smoking responsive oxidant-related genes overlapped between the two cell types (2 up-regulated, and no down-regulated genes). Consistent with this observation, pathway analysis of smoking-responsive genes in the small airway epithelium showed oxidant-related pathways dominated, but in alveolar macrophages immune-response pathways dominated. Thus, the responses of the oxidant-related transcriptome of cells with an identical genome and exposed to the same oxidant stress of cigarette smoking are very different, with responses of oxidant-related genes of alveolar macrophages far more subdued than that of small airway epithelium, consistent with the clinical observation that, while the small airway epithelium is vulnerable, alveolar macrophages are not "diseased" in response to the oxidant stress of cigarette smoking.
Project description:The initial site of smoking-induced lung disease is the small airway epithelium, which is difficult and time consuming to sample by fiberoptic bronchoscopy. We developed a rapid, office-based procedure to obtain trachea epithelium without conscious sedation from healthy nonsmokers (n=26) and healthy smokers (n=19, 27 ± 15 pack-yr). Gene expression differences [fold-change >1.5, p< 0.01, Benjamini-Hochberg correction] were assessed with Affymetrix microarrays. 1,057 probe sets were differentially expressed in healthy smokers vs nonsmokers, representing >500 genes. Trachea gene expression was compared to an independent group of small airway epithelial samples (n=23 healthy nonsmokers, n=19 healthy smokers, 25 ± 12 pack-yr). The trachea epithelium is more sensitive to smoking, responding with 3-fold more differentially-expressed genes than small airway epithelium. The trachea transcriptome paralleled the small airway epithelium, with 156 of 167 (93%) genes that are significantly up- and down-regulated by smoking in the small airway epithelium showing similar direction and magnitude of response to smoking in the trachea. Trachea epithelium can be obtained without conscious sedation, representing a less invasive surrogate “canary” for smoking-induced changes in the small airway epithelium. This should prove useful in epidemiologic studies correlating gene expression with clinical outcome in assessing smoking-induced lung disease.
Project description:The initial site of smoking-induced lung disease is the small airway epithelium, which is difficult and time consuming to sample by fiberoptic bronchoscopy. We developed a rapid, office-based procedure to obtain trachea epithelium without conscious sedation from healthy nonsmokers (n=26) and healthy smokers (n=19, 27 ± 15 pack-yr). Gene expression differences [fold-change >1.5, p< 0.01, Benjamini-Hochberg correction] were assessed with Affymetrix microarrays. 1,057 probe sets were differentially expressed in healthy smokers vs nonsmokers, representing >500 genes. Trachea gene expression was compared to an independent group of small airway epithelial samples (n=23 healthy nonsmokers, n=19 healthy smokers, 25 ± 12 pack-yr). The trachea epithelium is more sensitive to smoking, responding with 3-fold more differentially-expressed genes than small airway epithelium. The trachea transcriptome paralleled the small airway epithelium, with 156 of 167 (93%) genes that are significantly up- and down-regulated by smoking in the small airway epithelium showing similar direction and magnitude of response to smoking in the trachea. Trachea epithelium can be obtained without conscious sedation, representing a less invasive surrogate “canary” for smoking-induced changes in the small airway epithelium. This should prove useful in epidemiologic studies correlating gene expression with clinical outcome in assessing smoking-induced lung disease. Experiment Overall Design: Tracheal gene expression: matched group of small airway epithelial samples (n=23 healthy non-smokers, n= 19 healthy smokers)
Project description:Although smoking-induced lung disease tends to be more common in the upper lobe, it is not known if this results from the skewed distribution of inhaled cigarette smoke or increased susceptibility of the upper lobes to these disorders. The distribution of inhaled cigarette smoke within the lung is complex, depending on lung pressure-volume relationships, gravity, individual smoking habits and the properties of the individual components of cigarette smoke. With the knowledge that the small airway epithelium is the earliest site of smoking-induced lung disease, and that the small airway epithelium is acutely sensitive to inhaled cigarette smoke with significant changes in the up- and down-regulation of hundreds of genes, we compared upper vs lower lobe gene expression in the small airway epithelium of the same cigarette smokers to determine if the gene expression patterns were similar or different. Active smokers (n=11) with early evidence of smoking-induced lung disease (normal spirometry but low diffusing capacity) underwent bronchoscopy and brushing of the small airway epithelium to compare upper vs lower lobe genome-wide gene expression assessed by microarray. Interestingly, cluster and principal component analysis demonstrated that, for each individual, the expression of the known small airway epithelium smoking-responsive genes were remarkably similar as upper vs lower lobe pairs, although, as expected, there were differences in the smoking-induced changes in gene expression from individual to individual. Thus, while there may be topographic differences in the distribution of cigarette smoke, sufficient smoke reaches the upper vs lower lobe small airway epithelium so that, within each smoker, the upper vs lower lobe gene expression are similar. These observations support the concept that the topographic differences in the occurrence of the smoking-induced lung diseases are likely secondary to topographic differences in the susceptibility of the upper vs lower lobes to cigarette smoke, not the topographic differences in distribution of inhaled cigarette smoke.
Project description:Despite overwhelming data that cigarette smoking causes chronic obstructive pulmonary disease (COPD), only a minority of chronic smokers are affected, strongly suggesting that genetic factors modify susceptibility to this disease. We hypothesized that there are individual variations in the response to cigarette smoking, with variability among smokers in expression levels of protective / susceptibility genes. Affymetrix arrays and TaqMan PCR were used to assess the variability of gene expression in the small airway epithelium obtained by fiberoptic bronchoscopy of 18 normal non-smokers, 18 normal smokers and 18 smokers with COPD. We identified 201 probesets representing 150 smoking-responsive genes that were significantly up- or down-regulated, and assessed the coefficient of variation in expression levels among the study population. Variation was a reproducible property of each gene as assessed by different microarray probesets and realtime PCR and was observed in both normal smokers and smokers with COPD. There was greater individual variability in smokers with COPD than in normal smokers. The majority of these highly variable smoking responsive genes were in the functional categories of signal transduction, xenobiotic degradation, metabolism, transport, oxidant-related and transcription. A similar pattern of the same highly variable genes was observed in an independent data set.We propose that there is likely genetic diversity within this subset of genes with highly variable individual to individual responses of the small airway epithelium to smoking, and this subset of genes represent putative candidates for assessment of susceptibility/protection from disease for future gene-based epidemiological studies for the risk of smokers for COPD. Keywords: disease state analysis