Smoking-mediated up-regulation of GAD67 expression in the human airway epithelium

<p>Abstract</p> <p>Background</p> <p>The production of gamma-amino butyric acid (GABA) is dependent on glutamate decarboxylases (GAD65 and GAD67), the enzymes that catalyze the decarboxylation of glutamate to GABA. Based on studies suggesting a role of the airway epithelial GABAergic system in asthma-related mucus overproduction, we hypothesized that cigarette smoking, another disorder associated with increased mucus production, may modulate GABAergic system-related gene expression levels in the airway epithelium.</p> <p>Methods</p> <p>We assessed expression of the GABAergic system in human airway epithelium obtained using bronchoscopy to sample the epithelium and microarrays to evaluate gene expression. RT-PCR was used to confirm gene expression of GABAergic system gene in large and small airway epithelium from heathy nonsmokers and healthy smokers. The differences in the GABAergic system gene was further confirmed by TaqMan, immunohistochemistry and Western analysis.</p> <p>Results</p> <p>The data demonstrate there is a complete GABAergic system expressed in the large and small human airway epithelium, including glutamate decarboxylase, GABA receptors, transporters and catabolism enzymes. Interestingly, of the entire GABAergic system, smoking modified only the expression of GAD67, with marked up-regulation of GAD67 gene expression in both large (4.1-fold increase, p < 0.01) and small airway epithelium of healthy smokers (6.3-fold increase, p < 0.01). At the protein level, Western analysis confirmed the increased expression of GAD67 in airway epithelium of healthy smokers compared to healthy nonsmokers (p < 0.05). There was a significant positive correlation between GAD67 and MUC5AC gene expression in both large and small airway epithelium (p < 0.01), implying a link between GAD67 and mucin overproduction in association with smoking.</p> <p>Conclusions</p> <p>In the context that GAD67 is the rate limiting enzyme in GABA synthesis, the correlation of GAD67 gene expression with MUC5AC expressions suggests that the up-regulation of airway epithelium expression of GAD67 may contribute to the increase in mucus production observed in association with cigarette smoking.</p> <p>Trial registration</p> <p>NCT00224198; NCT00224185</p

RNA-Seq quantification of the human small airway epithelium transcriptome

<p>Abstract</p> <p>Background</p> <p>The small airway epithelium (SAE), the cell population that covers the human airway surface from the 6^thgeneration of airway branching to the alveoli, is the major site of lung disease caused by smoking. The focus of this study is to provide quantitative assessment of the SAE transcriptome in the resting state and in response to chronic cigarette smoking using massive parallel mRNA sequencing (RNA-Seq).</p> <p>Results</p> <p>The data demonstrate that 48% of SAE expressed genes are ubiquitous, shared with many tissues, with 52% enriched in this cell population. The most highly expressed gene, SCGB1A1, is characteristic of Clara cells, the cell type unique to the human SAE. Among other genes expressed by the SAE are those related to Clara cell differentiation, secretory mucosal defense, and mucociliary differentiation. The high sensitivity of RNA-Seq permitted quantification of gene expression related to infrequent cell populations such as neuroendocrine cells and epithelial stem/progenitor cells. Quantification of the absolute smoking-induced changes in SAE gene expression revealed that, compared to ubiquitous genes, more SAE-enriched genes responded to smoking with up-regulation, and those with the highest basal expression levels showed most dramatic changes. Smoking had no effect on SAE gene splicing, but was associated with a shift in molecular pattern from Clara cell-associated towards the mucus-secreting cell differentiation pathway with multiple features of cancer-associated molecular phenotype.</p> <p>Conclusions</p> <p>These observations provide insights into the unique biology of human SAE by providing quantit-ative assessment of the global transcriptome under physiological conditions and in response to the stress of chronic cigarette smoking.</p

Biologic Phenotyping of the Human Small Airway Epithelial Response to Cigarette Smoking

BACKGROUND: The first changes associated with smoking are in the small airway epithelium (SAE). Given that smoking alters SAE gene expression, but only a fraction of smokers develop chronic obstructive pulmonary disease (COPD), we hypothesized that assessment of SAE genome-wide gene expression would permit biologic phenotyping of the smoking response, and that a subset of healthy smokers would have a "COPD-like" SAE transcriptome. METHODOLOGY/PRINCIPAL FINDINGS: SAE (10th-12th generation) was obtained via bronchoscopy of healthy nonsmokers, healthy smokers and COPD smokers and microarray analysis was used to identify differentially expressed genes. Individual responsiveness to smoking was quantified with an index representing the % of smoking-responsive genes abnormally expressed (I(SAE)), with healthy smokers grouped into "high" and "low" responders based on the proportion of smoking-responsive genes up- or down-regulated in each smoker. Smokers demonstrated significant variability in SAE transcriptome with I(SAE) ranging from 2.9 to 51.5%. While the SAE transcriptome of "low" responder healthy smokers differed from both "high" responders and smokers with COPD, the transcriptome of the "high" responder healthy smokers was indistinguishable from COPD smokers. CONCLUSION/SIGNIFICANCE: The SAE transcriptome can be used to classify clinically healthy smokers into subgroups with lesser and greater responses to cigarette smoking, even though these subgroups are indistinguishable by clinical criteria. This identifies a group of smokers with a "COPD-like" SAE transcriptome

Down-Regulation of the Canonical Wnt β-Catenin Pathway in the Airway Epithelium of Healthy Smokers and Smokers with COPD

Background: The Wnt pathway mediates differentiation of epithelial tissues; depending on the tissue types, Wnt can either drive or inhibit the differentiation process. We hypothesized that key genes in the Wnt pathway are suppressed in the human airway epithelium under the stress of cigarette smoking, a stress associated with dysregulation of the epithelial differentiated state. Methodology/Principal Findings: Microarrays were used to assess the expression of Wnt-related genes in the small airway epithelium (SAE) obtained via bronchoscopy and brushing of healthy nonsmokers, healthy smokers, and smokers with COPD. Thirty-three of 56 known Wnt-related genes were expressed in the SAE. Wnt pathway downstream mediators b-catenin and the transcription factor 7-like 1 were down-regulated in healthy smokers and smokers with COPD, as were many Wnt target genes. Among the extracellular regulators that suppress the Wnt pathway, secreted frizzled-related protein 2 (SFRP2), was up-regulated 4.3-fold in healthy smokers and 4.9-fold in COPD smokers, an observation confirmed by TaqMan Real-time PCR, Western analysis and immunohistochemistry. Finally, cigarette smoke extract mediated up-regulation of SFRP2 and down-regulation of Wnt target genes in airway epithelial cells in vitro. Conclusions/Significance: Smoking down-regulates the Wnt pathway in the human airway epithelium. In the context that Wnt pathway plays an important role in differentiation of epithelial tissues, the down-regulation of Wnt pathway ma

Effets de la fumée de nargilé sur la santé du poumon

Waterpipe, an instrument for smoking fruit-flavored tobacco, is used by millions of people worldwide. There is limited data on the health effects of waterpipe smoking, and no regulations to its use. We hypothesized that even young, light-use waterpipe smokers have abnormalities relevant to lung health. Based on the knowledge that the first abnormalities associated with cigarette smoking are in lung cells long before there are clinical abnormalities, we compared young, light-use waterpipe smokers to nonsmokers, using a variety of lung-related parameters, including: blood carboxyhemoglobin (CO) levels; cough and sputum scores; lung function; metabolites present in lower respiratory tract epithelial lining fluid (ELF); cell differentials and transcriptome of small airway epithelium (SAE); cellular composition of ELF; transcriptome of alveolar macrophages (AM); and levels of total and apoptotic endothelial microparticles (EMPs). Light-use waterpipe smokers displayed abnormalities in all parameters assessed. Compared to nonsmokers, waterpipe smokers had more cough and sputum, higher CO levels, reduced diffusing capacity for carbon monoxide (DLCO), abnormal ELF metabolome profile, increased proportions of SAE secretory and intermediate cells, reduced proportions of SAE ciliated and basal cells, markedly abnormal SAE and AM transcriptomes, and elevated levels of total and apoptotic EMPs.DLCO, a lung function parameter linked to emphysema and small airway disease, was affected by light-use waterpipe smoking. The relevance of this comes from our studies that demonstrated, in a separate cohort of cigarette smokers with normal spirometry, that reduced DLCO predicted a high risk for developing chronic obstructive pulmonary disease (COPD), a leading cause of death worldwide. We assessed the risk for developing COPD, a clinical disorder characterized by a mixture of small airway disease and parenchymal destruction (emphysema), with a serial lung function in cigarette smokers with normal spirometry and no emphysema as assessed by HRCT, by comparing smokers with reduced DLCO vs normal DLCO. Despite having normal spirometry, cigarette smokers with reduced DLCO were at significantly higher risk for developing COPD within <4 years compared to those with normal DLCO i.e., the DLCO can be used to identify smokers at high risk for developing COPD, and could be a unique parameter in future studies to assess waterpipe smokers over time.Plasma levels of total and apoptotic EMPs, indicative of pulmonary capillary endothelial apoptosis, were elevated in light-use waterpipe smokers. The possible importance of this observation was highlighted by a parallel study, where we assessed the stability and reversibility of EMP levels in nonsmokers, healthy cigarette smokers and COPD cigarette smokers at 4 time points over a period of 1 year. The levels of total and apoptotic EMPs remained high with continuous smoking in healthy and COPD cigarette smokers. A subset of the healthy cigarette smokers and COPD cigarette smokers agreed to quit smoking. Following smoking cessation for 1 year, total and apoptotic EMP levels returned to normal nonsmoker levels in healthy cigarette smokers but remained abnormally high in COPD cigarette smokers. High levels of circulating and apoptotic EMPs are indicative of persistent and irreversible destruction of pulmonary capillaries and may be another unique parameter to assess waterpipe smokers over time.In summary, young, light-use waterpipe smokers have a number of lung clinical and biologic abnormalities compared to nonsmokers, including reduced DLCO, found to predict high risk for developing COPD in cigarette smokers, and elevated plasma levels of total and apoptotic EMPs, a marker of alveolar destruction, shown to be persistent and irreversible in COPD cigarette smokers despite smoking cessation. Together, these studies suggest that even light-use waterpipe smokers may be at risk for developing lung disease.La Chicha qui sert à fumer du tabac parfumé est utilisé par des millions de personnes. Il y a peu de données sur les effets du chicha sur la santé, peu de régulation et les utilisateurs pensent que la chicha n’est ni addictif ni nocif. Nous avons émis l’hypothèse que la consommation même occasionnelle de la chicha chez le sujet jeune a des conséquences sur la biologie pulmonaire. Nous avons ainsi comparé 21 sujets jeunes fumeur occasionnel de Chicha à un groupe de 19 non fumeur apparié pour le sexe et l’ethnicité. Les premières anomalies chez le fumeur de cigarette étant présent au niveau des cellules pulmonaires nous avons évalué plusieurs paramètres : (1) taux plasmatique de carboxyhemoglobine (CO), (2) Score de toux et d’expectoration; (3) fonction pulmonaire; (4) Métabolites présent dans les fluides des voies respiratoires basses (ELF); (5différences cellulaires et de transcriptome des petites voies aériennes (6) composition cellulaire des lavages broncho-alvéolaires (7) le transcriptome et (9) niveau des microparticules endothéliales circulantes. Le groupe d’étude montrait des anomalies dans tous les paramètres étudiés. Comparé au groupe contrôle les fumeurs avaient plus de toux et d’expectoration, un niveau de CO plus élevé, une diminution de la capacité de diffusion du CO , des anomalies du profil métabolique des fluides alvéolaires, une augmentation des cellules sécrétoires et intermédiaires et une diminution des cellules ciliées et basales, des anomalies du transcriptome des cellules pulmonaires et de macrophages alvéolaires et une augmentation des microparticules endothéliales.LA capacité de diffusion du monoxyde de carbone qui est un paramètre lié à l’emphysème et aux pathologies des petites voies pulmonaires était affectée par l’utilisation de la chicha. Nos précédentes études avaient montré que chez les sujets fumeurs de cigarette la réduction de la capacité de diffusion malgré une spirométrie normale était associée à un risque de développer un BPCO. Nous avons ainsi évalué le risque de développer une BPCO chez le sujet fumeur avec spirométrie normale par des scanners haute résolution comparant des groupes avec capacité de diffusion diminuée (46) et normale (59). La réduction de la capacité de diffusion était associée à un risque élevé de développer une BPCO dans les 4 ans.Par ailleurs les niveaux plasmatiques des microparticules endothéliales totales et apoptotiques était élevé dans le groupe d’étude. Dans une étude chez les sujets fumeurs de cigarette, nous avons comparé des non fumeurs (28) à des fumeurs sains (61) et des fumeurs BPCO (49) sur un an. Nous avons montré que le niveau de microparticules endothéliales apoptotiques étaient élevé en continu chez les sujets fumeurs sains et avec BPCO. Un sous groupe des fumeurs sains (17) et BPCO (18) a accepté d’arrêter de fumer. 12 mois après l’arrêt de la cigarette le niveau des microparticules endothéliales totale et apoptotique était retourné à la normale pour les fumeurs sains mais restait élevé chez les fumeurs BPCO. Ainsi le niveau élevé de ces microparticules indiquait des lésions persistantes et irréversibles des capillaires pulmonaires et pourrait servir à évaluer les fumeurs de chicha au long cours.Au total, l’utilisation occasionnelle de chicha chez le sujet jeune a des conséquence clinique et biologique pulmonaire en relation avec une diminution de la capacité de diffusion. Certaines anomalies mises en évidence dans notre étude (diminution de la capacité de diffusion, Microparticules endothéliales) pourraient prédire la survenue de maladies pulmonaires chroniques obstructives

Genes associated with MUC5AC expression in small airway epithelium of human smokers and non-smokers

Abstract Background Mucus hypersecretion contributes to the morbidity and mortality of smoking-related lung diseases, especially chronic obstructive pulmonary disease (COPD), which starts in the small airways. Despite progress in animal studies, the genes and their expression pattern involved in mucus production and secretion in human airway epithelium are not well understood. We hypothesized that comparison of the transcriptomes of the small airway epithelium of individuals that express high vs low levels of MUC5AC, the major macromolecular component of airway mucus, could be used as a probe to identify the genes related to human small airway mucus production/secretion. Methods Flexible bronchoscopy and brushing were used to obtain small airway epithelium (10th to 12th order bronchi) from healthy nonsmokers (n=60) and healthy smokers (n=72). Affymetrix HG-U133 plus 2.0 microarrays were used to assess gene expression. Massive parallel sequencing (RNA-Seq) was used to verify gene expression of small airway epithelium from 5 nonsmokers and 6 smokers. Results MUC5AC expression varied 31-fold among the healthy nonsmokers. Genome-wide comparison between healthy nonsmokers (n = 60) grouped as “high MUC5AC expressors” vs “low MUC5AC expressors” identified 528 genes significantly up-regulated and 15 genes significantly down-regulated in the high vs low expressors. This strategy identified both mucus production and secretion related genes under control of a network composed of multiple transcription factors. Based on the literature, genes in the up-regulated list were used to identify a 73 “MUC5AC-associated core gene” list with 9 categories: mucus component; mucus-producing cell differentiation-related transcription factor; mucus-producing cell differentiation-related pathway or mediator; post-translational modification of mucin; vesicle transport; endoplasmic reticulum stress-related; secretory granule-associated; mucus secretion-related regulator and mucus hypersecretory-related ion channel. As a validation cohort, we assessed the MUC5AC-associated core gene list in the small airway epithelium of an independent set of healthy smokers (n = 72). There was up-regulation of MUC5AC in the small airway epithelium of smokers (2.3-fold, p -8) associated with a coordinated up-regulation of MUC5AC-associated core gene expression pattern in the small airway epithelium of smokers (p  Conclusion The identification of the genes associated with increased airway mucin production in humans should be useful in understanding the pathogenesis of airway mucus hypersecretion and identifying therapeutic targets. Author summary Mucus hypersecretion contributes to the morbidity and mortality of smoking-related lung diseases, especially chronic obstructive pulmonary disease (COPD), which starts in the small airways. Little is known about the gene networks associated with the synthesis and secretion of mucins in the human small airway epithelium. Taking advantage of the knowledge that MUC5AC is a major mucin secreted by the small airway epithelium, the expression of MUC5AC in small airway epithelium is highly regulated at the transcriptional level and our observation that healthy nonsmokers have variable numbers of MUC5AC+ secretory cells in the human small airway epithelium, we compared genome-wide gene expression of the small airway epithelium of high vs low MUC5AC expressors from 60 nonsmokers to identify the genes associated with MUC5AC expression. This novel strategy enabled identification of a 73 “MUC5AC-associated core gene” list with 9 categories, which control a series of processes from mucin biosynthesis to mucus secretion. The coordinated gene expression pattern of MUC5AC-associated core genes were corroborated in an independent cohort of 72 healthy smokers. Deep sequencing of small airway epithelium RNA confirmed these observations. This finding will be useful in identifying therapeutic targets to treat small airway mucus hypersecretion.</p