Persistence of circulating endothelial microparticles in COPD despite smoking cessation.

INTRODUCTION: Increasing evidence links COPD pathogenesis with pulmonary capillary apoptosis. We previously demonstrated that plasma levels of circulating microparticles released from endothelial cells (EMPs) due to apoptosis are elevated in smokers with normal spirometry but low diffusion capacity, that is, with early evidence of lung destruction. We hypothesised that pulmonary capillary apoptosis persists with the development of COPD and assessed its reversibility in healthy smokers and COPD smokers following smoking cessation. METHODS: Pulmonary function and high-resolution CT (HRCT) were assessed in 28 non-smokers, 61 healthy smokers and 49 COPD smokers; 17 healthy smokers and 18 COPD smokers quit smoking for 12 months following the baseline visit. Total EMP (CD42b-CD31+), pulmonary capillary EMP (CD42b-CD31+ACE+) and apoptotic EMP (CD42b-CD62E+/CD42b-CD31+) levels were quantified by flow cytometry. RESULTS: Compared with non-smokers, healthy smokers and COPD smokers had elevated levels of circulating EMPs due to active pulmonary capillary endothelial apoptosis. Levels remained elevated over 12 months in healthy smokers and COPD smokers who continued smoking, but returned to non-smoker levels in healthy smokers who quit. In contrast, levels remained significantly abnormal in COPD smokers who quit. CONCLUSIONS: Pulmonary capillary apoptosis is reversible in healthy smokers who quit, but continues to play a role in COPD pathogenesis in smokers who progressed to airflow obstruction despite smoking cessation. TRIAL REGISTRATION NUMBER: NCT00974064; NCT01776398.

Risk of COPD with obstruction in active smokers with normal spirometry and reduced diffusion capacity.

Smokers are assessed for chronic obstructive pulmonary disease (COPD) using spirometry, with COPD defined by the Global Initiative for Chronic Obstructive Lung Disease (GOLD) as airflow limitation that is not fully reversible with bronchodilators. There is a subset of smokers with normal spirometry (by GOLD criteria), who have a low diffusing capacity of the lung for carbon monoxide (DLCO), a parameter linked to emphysema and small airway disease. The natural history of these "normal spirometry/low DLCO" smokers is unknown.From a cohort of 1570 smokers in the New York City metropolitian area, all of whom had normal spirometry, two groups were randomly selected for lung function follow-up: smokers with normal spirometry/normal DLCO (n=59) and smokers with normal spirometry/low DLCO (n=46). All had normal history, physical examination, complete blood count, urinalysis, HIV status, α1-antitrypsin level, chest radiography, forced expiratory volume in 1 s (FEV1), forced vital capacity (FVC), FEV1/FVC ratio and total lung capacity. Throughout the study, all continued to be active smokers.In the normal spirometry/normal DLCO group assessed over 45±20 months, 3% developed GOLD-defined COPD. In contrast, in the normal spirometry/low DLCO group, followed over 41±31 months, 22% developed GOLD-defined COPD.Despite appearing "normal" according to GOLD, smokers with normal spirometry but low DLCO are at significant risk of developing COPD with obstruction to airflow.

Persistence of smoking-induced dysregulation of miRNA expression in the small airway epithelium despite smoking cessation.

Even after quitting smoking, the risk of the development of chronic obstructive pulmonary disease (COPD) and lung cancer remains significantly higher compared to healthy nonsmokers. Based on the knowledge that COPD and most lung cancers start in the small airway epithelium (SAE), we hypothesized that smoking modulates miRNA expression in the SAE linked to the pathogenesis of smoking-induced airway disease, and that some of these changes persist after smoking cessation. SAE was collected from 10th to 12th order bronchi using fiberoptic bronchoscopy. Affymetrix miRNA 2.0 arrays were used to assess miRNA expression in the SAE from 9 healthy nonsmokers and 10 healthy smokers, before and after they quit smoking for 3 months. Smoking status was determined by urine nicotine and cotinine measurement. There were significant differences in the expression of 34 miRNAs between healthy smokers and healthy nonsmokers (p<0.01, fold-change >1.5), with functions associated with lung development, airway epithelium differentiation, inflammation and cancer. After quitting smoking for 3 months, 12 out of the 34 miRNAs did not return to normal levels, with Wnt/ß-catenin signaling pathway being the top identified enriched pathway of the target genes of the persistent dysregulated miRNAs. In the context that many of these persistent smoking-dependent miRNAs are associated with differentiation, inflammatory diseases or lung cancer, it is likely that persistent smoking-related changes in SAE miRNAs play a role in the subsequent development of these disorders.

Smoking accelerates aging of the small airway epithelium.

BACKGROUND: Aging involves multiple biologically complex processes characterized by a decline in cellular homeostasis over time leading to a loss and impairment of physiological integrity and function. Specific cellular hallmarks of aging include abnormal gene expression patterns, shortened telomeres and associated biological dysfunction. Like all organs, the lung demonstrates both physiological and structural changes with age that result in a progressive decrease in lung function in healthy individuals. Cigarette smoking accelerates lung function decline over time, suggesting smoking accelerates aging of the lung. Based on this data, we hypothesized that cigarette smoking accelerates the aging of the small airway epithelium, the cells that take the initial brunt of inhaled toxins from the cigarette smoke and one of the primary sites of pathology associated with cigarette smoking. METHODS: Using the sensitive molecular parameters of aging-related gene expression and telomere length, the aging process of the small airway epithelium was assessed in age matched healthy nonsmokers and healthy smokers with no physical manifestation of lung disease or abnormalities in lung function. RESULTS: Analysis of a 73 gene aging signature demonstrated that smoking significantly dysregulates 18 aging-related genes in the small airway epithelium. In an independent cohort of male subjects, smoking significantly reduced telomere length in the small airway epithelium of smokers by 14% compared to nonsmokers. CONCLUSION: These data provide biologic evidence that smoking accelerates aging of the small airway epithelium.

Intraflagellar transport gene expression associated with short cilia in smoking and COPD.

Smoking and COPD are associated with decreased mucociliary clearance, and healthy smokers have shorter cilia in the large airway than nonsmokers. We hypothesized that changes in cilia length are consistent throughout the airway, and we further hypothesized that smokers with COPD have shorter cilia than healthy smokers. Because intraflagellar transport (IFT) is the process by which cilia of normal length are produced and maintained, and alterations in IFT lead to short cilia in model organisms, we also hypothesized that smoking induces changes in the expression of IFT-related genes in the airway epithelium of smokers and smokers with COPD. To assess these hypotheses, airway epithelium was obtained via bronchoscopic brushing. Cilia length was assessed by measuring 100 cilia (10 cilia on each of 10 cells) per subject and Affymetrix microarrays were used to evaluate IFT gene expression in nonsmokers and healthy smokers in 2 independent data sets from large and small airway as well as in COPD smokers in a data set from the small airway. In the large and small airway epithelium, cilia were significantly shorter in healthy smokers than nonsmokers, and significantly shorter in COPD smokers than in both healthy smokers and nonsmokers. The gene expression data confirmed that a set of 8 IFT genes were down-regulated in smokers in both data sets; however, no differences were seen in COPD smokers compared to healthy smokers. These results support the concept that loss of cilia length contributes to defective mucociliary clearance in COPD, and that smoking-induced changes in expression of IFT genes may be one mechanism of abnormally short cilia in smokers. Strategies to normalize cilia length may be an important avenue for novel COPD therapies.

High correlation of the response of upper and lower lobe small airway epithelium to smoking.

The distribution of lung disease induced by inhaled cigarette smoke is complex, depending on many factors. With the knowledge that the small airway epithelium (SAE) is the earliest site of smoking-induced lung disease, and that the SAE gene expression is likely sensitive to inhaled cigarette smoke, we compared upper vs. lower lobe gene expression in the SAE within 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 upper and lower lobe SAE in order to compare upper vs lower lobe genome-wide and smoking-responsive gene expression by microarray. Cluster and principal component analysis demonstrated that, for each individual, the expression of the known SAE smoking-responsive genes were highly correlated in upper and lower lobe pairs, although, as expected, there were differences in the smoking-induced changes in gene expression from individual to individual. These observations support the concept that the heterogeneity observed among smokers in the anatomic distribution of smoking-induced disease are not secondary to the topographic differences in the effects of cigarette smoke on the airway epithelium.

Circulating endothelial microparticles as a measure of early lung destruction in cigarette smokers.

RATIONALE: There is increasing evidence that emphysema is associated with primary loss of pulmonary capillary endothelium. Plasma levels of endothelial microparticles (EMPs), small vesicles released from activated or apoptotic endothelial cells, are elevated in vascular-related disorders. OBJECTIVES: To evaluate whether plasma EMP levels are elevated in smokers with early lung destruction as assessed by normal spirometry but reduced diffusing capacity of the lung for carbon monoxide (Dl(co)). METHODS: Lung health was assessed by pulmonary function tests (PFTs: spirometry, total lung capacity, Dl(co)) and chest X-ray; smoking status was assessed by urine nicotine and cotinine. EMP levels (CD42b(-)CD31(+) microparticles) were quantified as activated or apoptotic. The initial cohort (n = 92) included healthy nonsmokers (normal PFTs), healthy smokers (normal PFTs), and smokers with early evidence of lung destruction (normal spirometry, low Dl(co)). Two prospective cohorts were then tested: a group similar to the initial cohort and an HIV1(+) cohort. MEASUREMENTS AND MAIN RESULTS: Healthy smokers had mildly increased levels of EMPs. Strikingly, 95% of smokers with normal spirometry, low Dl(co) had increased EMPs, with reduced CD62(+)/CD31(+) ratios (P < 10(-4)) and elevated CD42b(-)CD31(+) annexin V(+) EMPs (P < 10(-4)), suggesting derivation from endothelial apoptosis. Most elevated EMPs were angiotensin-converting enzyme positive, suggesting derivation from pulmonary capillaries. Both prospective cohorts confirmed the initial cohort data. CONCLUSIONS: Plasma EMPs with apoptotic characteristics are elevated in smokers with normal spirometry but reduced Dl(co), consistent with the concept that emphysema is associated, in part, with capillary endothelium apoptosis, suggesting that the early development of emphysema might be monitored with plasma EMP levels.

Cigarette smoking reprograms apical junctional complex molecular architecture in the human airway epithelium in vivo.

The apical junctional complex (AJC), composed of tight and adherens junctions, maintains epithelial barrier function. Since cigarette smoking and chronic obstructive pulmonary disease (COPD), the major smoking-induced disease, are associated with increased lung epithelial permeability, we hypothesized that smoking alters the transcriptional program regulating airway epithelial AJC integrity. Transcriptome analysis revealed global down-regulation of physiological AJC gene expression in the airway epithelium of healthy smokers (n = 59) compared to nonsmokers (n = 53) in association with changes in canonical epithelial differentiation pathways such as PTEN signaling accompanied by induction of cancer-related AJC components. The overall expression of AJC-related genes was further decreased in COPD smokers (n = 23). Exposure of airway epithelial cells to cigarette smoke extract in vitro resulted in down-regulation of several AJC genes paralleled by decreased transepithelial resistance. Thus, cigarette smoking induces transcriptional reprogramming of airway epithelial AJC architecture from its physiological pattern necessary for barrier function toward a disease-associated molecular phenotype.

The air-liquid interface and use of primary cell cultures are important to recapitulate the transcriptional profile of in vivo airway epithelia.

Organotypic cultures of primary human airway epithelial cells have been used to investigate the morphology, ion and fluid transport, innate immunity, transcytosis, infection, inflammation, signaling, cilia, and repair functions of this complex tissue. However, we do not know how closely these cultures resemble the airway surface epithelium in vivo. In this study, we examined the genome-wide expression profile of tracheal and bronchial human airway epithelia in vivo and compared it with the expression profile of primary cultures of human airway epithelia grown at the air-liquid interface. For comparison, we also investigated the expression profile of Calu-3 cells grown at the air-liquid interface and primary cultures of human airway epithelia submerged in nutrient media. We found that the transcriptional profile of differentiated primary cultures grown at the air-liquid interface most closely resembles that of in vivo airway epithelia, suggesting that the use of primary cultures and the presence of an air-liquid interface are important to recapitulate airway epithelia biology. We describe a high level of similarity between cells of tracheal and bronchial origin within and between different human donors, which suggests a very robust expression profile that is specific to airway cells.

Dramatic improvement in pulmonary hypertension with rapamycin.

This is a report of a 61-year-old woman with improved pulmonary arterial hypertension following treatment with rapamycin for an islet cell tumor of the pancreas with liver metastases.

Threshold of biologic responses of the small airway epithelium to low levels of tobacco smoke.

RATIONALE: Epidemiologic data demonstrate that individuals exposed to low levels of tobacco smoke have decrements in lung function and higher risk for lung disease compared with unexposed individuals. Although this risk is small, low-level tobacco smoke exposure is so widespread, it is a significant public health concern. OBJECTIVES: To identify biologic correlates of this risk we hypothesized that, compared with unexposed individuals, individuals exposed to low levels of tobacco smoke have biologic changes in the small airway epithelium, the site of the first abnormalities associated with smoking. METHODS: Small airway epithelium was obtained by bronchoscopy from 121 individuals; microarrays were used to assess genome-wide gene expression; urine nicotine and cotinine were used to categorize subjects as "nonsmokers," "active smokers," and "low exposure." Gene expression data were used to determine the threshold and induction half maximal level (ID50) of urine nicotine and cotinine at which the small airway epithelium showed abnormal responses. MEASUREMENTS AND MAIN RESULTS: There was no threshold of urine nicotine without a small airway epithelial response, and only slightly above detectable urine cotinine threshold with a small airway epithelium response. The ID50 for nicotine was 25 ng/ml and for cotinine it was 104 ng/ml. CONCLUSIONS: The small airway epithelium detects and responds to low levels of tobacco smoke with transcriptome modifications. This provides biologic correlates of epidemiologic studies linking low-level tobacco smoke exposure to lung health risk, identifies the genes most sensitive to tobacco smoke, and defines thresholds at which the lung epithelium responds to low levels of tobacco smoke.

Proinflammatory phenotype and increased caveolin-1 in alveolar macrophages with silenced CFTR mRNA.

The inflammatory milieu in the respiratory tract in cystic fibrosis (CF) has been linked to the defective expression of the cystic transmembrane regulator (CFTR) in epithelial cells. Alveolar macrophages (AM), important contibutors to inflammatory responses in the lung, also express CFTR. The present study analyzes the phenotype of human AM with silenced CFTR. Expression of CFTR mRNA and the immature form of the CFTR protein decreased 100-fold and 5.2-fold, respectively, in AM transfected with a CFTR specific siRNA (CFTR-siRNA) compared to controls. Reduction of CFTR expression in AM resulted in increased secretion of IL-8, increased phosphorylation of NF-kappaB, a positive regulator of IL-8 expression, and decreased expression of IkappaB-alpha, the inhibitory protein of NF-kappaB activation. AM with silenced CFTR expression also showed increased apoptosis. We hypothesized that caveolin-1 (Cav1), a membrane protein that is co-localized with CFTR in lipid rafts and that is related to inflammation and apoptosis in macrophages, may be affected by decreased CFTR expression. Messenger RNA and protein levels of Cav1 were increased in AM with silenced CFTR. Expression and transcriptional activity of sterol regulatory element binding protein (SREBP), a negative transcriptional regulator of Cav1, was decreased in AM with silenced CFTR, but total and free cholesterol mass did not change. These findings indicate that silencing of CFTR in human AM results in an inflammatory phenotype and apoptosis, which is associated to SREBP-mediated regulation of Cav1.

Smoking is associated with shortened airway cilia.

BACKGROUND: Whereas cilia damage and reduced cilia beat frequency have been implicated as causative of reduced mucociliary clearance in smokers, theoretically mucociliary clearance could also be affected by cilia length. Based on models of mucociliary clearance predicting that cilia length must exceed the 6-7 microm airway surface fluid depth to generate force in the mucus layer, we hypothesized that cilia height may be decreased in airway epithelium of normal smokers compared to nonsmokers. METHODOLOGY/PRINCIPAL FINDINGS: Cilia length in normal nonsmokers and smokers was evaluated in aldehyde-fixed, paraffin-embedded endobronchial biopsies, and air-dried and hydrated samples were brushed from human airway epithelium via fiberoptic bronchoscopy. In 28 endobronchial biopsies, healthy smoker cilia length was reduced by 15% compared to nonsmokers (p<0.05). In 39 air-dried samples of airway epithelial cells, smoker cilia length was reduced by 13% compared to nonsmokers (p<0.0001). Analysis of the length of individual, detached cilia in 27 samples showed that smoker cilia length was reduced by 9% compared to nonsmokers (p<0.05). Finally, in 16 fully hydrated, unfixed samples, smoker cilia length was reduced 7% compared to nonsmokers (p<0.05). Using genome-wide analysis of airway epithelial gene expression we identified 6 cilia-related genes whose expression levels were significantly reduced in healthy smokers compared to healthy nonsmokers. CONCLUSIONS/SIGNIFICANCE: Models predict that a reduction in cilia length would reduce mucociliary clearance, suggesting that smoking-associated shorter airway epithelial cilia play a significant role in the pathogenesis of smoking-induced lung disease.

Disparate oxidant gene expression of airway epithelium compared to alveolar macrophages in smokers.

BACKGROUND: The small airway epithelium and alveolar macrophages are exposed to oxidants in cigarette smoke leading to epithelial dysfunction and macrophage activation. In this context, we asked: what is the transcriptome of oxidant-related genes in small airway epithelium and alveolar macrophages, and does their response differ substantially to inhaled cigarette smoke? METHODS: Using microarray analysis, with TaqMan RT-PCR confirmation, we assessed oxidant-related gene expression in small airway epithelium and alveolar macrophages from the same healthy nonsmoker and smoker individuals. RESULTS: Of 155 genes surveyed, 87 (56%) were expressed in both cell populations in nonsmokers, with higher expression in alveolar macrophages (43%) compared to airway epithelium (24%). In smokers, there were 15 genes (10%) up-regulated and 7 genes (5%) down-regulated in airway epithelium, but only 3 (2%) up-regulated and 2 (1%) down-regulated in alveolar macrophages. Pathway analysis of airway epithelium showed oxidant pathways dominated, but in alveolar macrophages immune pathways dominated. CONCLUSION: Thus, the response of different cell-types with an identical genome exposed to the same stress of smoking is different; responses of alveolar macrophages are more subdued than those of airway epithelium. These findings are consistent with the observation that, while the small airway epithelium is vulnerable, alveolar macrophages are not "diseased" in response to smoking. TRIAL REGISTRATION: ClinicalTrials.gov ID: NCT00224185 and NCT00224198.

Quality control in microarray assessment of gene expression in human airway epithelium.

BACKGROUND: Microarray technology provides a powerful tool for defining gene expression profiles of airway epithelium that lend insight into the pathogenesis of human airway disorders. The focus of this study was to establish rigorous quality control parameters to ensure that microarray assessment of the airway epithelium is not confounded by experimental artifact. Samples (total n = 223) of trachea, large and small airway epithelium were collected by fiberoptic bronchoscopy of 144 individuals and hybridized to Affymetrix microarrays. The pre- and post-chip quality control (QC) criteria established, included: (1) RNA quality, assessed by RNA Integrity Number (RIN) > or = 7.0; (2) cRNA transcript integrity, assessed by signal intensity ratio of GAPDH 3' to 5' probe sets < or = 3.0; and (3) the multi-chip normalization scaling factor < or = 10.0. RESULTS: Of the 223 samples, all three criteria were assessed in 191; of these 184 (96.3%) passed all three criteria. For the remaining 32 samples, the RIN was not available, and only the other two criteria were used; of these 29 (90.6%) passed these two criteria. Correlation coefficients for pairwise comparisons of expression levels for 100 maintenance genes in which at least one array failed the QC criteria (average Pearson r = 0.90 +/- 0.04) were significantly lower (p < 0.0001) than correlation coefficients for pairwise comparisons between arrays that passed the QC criteria (average Pearson r = 0.97 +/- 0.01). Inter-array variability was significantly decreased (p < 0.0001) among samples passing the QC criteria compared with samples failing the QC criteria. CONCLUSION: Based on the aberrant maintenance gene data generated from samples failing the established QC criteria, we propose that the QC criteria outlined in this study can accurately distinguish high quality from low quality data, and can be used to delete poor quality microarray samples before proceeding to higher-order biological analyses and interpretation.

Smoking-dependent reprogramming of alveolar macrophage polarization: implication for pathogenesis of chronic obstructive pulmonary disease.

When exposed to a specific microenvironment, macrophages acquire either M1- or M2-polarized phenotypes associated with inflammation and tissue remodeling, respectively. Alveolar macrophages (AM) directly interact with environmental stimuli such as cigarette smoke, the major risk factor for chronic obstructive pulmonary disease (COPD), a disease characterized by lung inflammation and remodeling. Transcriptional profiling of AM obtained by bronchoalveolar lavage of 24 healthy nonsmokers, 34 healthy smokers, and 12 COPD smokers was performed to test the hypothesis whether smoking alters AM polarization, resulting in a disease-relevant activation phenotype. The analysis revealed that AM of healthy smokers exhibited a unique polarization pattern characterized by substantial suppression of M1-related inflammatory/immune genes and induction of genes associated with various M2-polarization programs relevant to tissue remodeling and immunoregulation. Such reciprocal changes progressed with the development of COPD, with M1-related gene expression being most dramatically down-regulated (p < 0.0001 vs healthy nonsmokers, p < 0.002 vs healthy smokers). Results were confirmed with TaqMan real-time PCR and flow cytometry. Among progressively down-regulated M1-related genes were those encoding type I chemokines CXCL9, CXCL10, CXCL11, and CCL5. Progressive activation of M2-related program was characterized by induction of tissue remodeling and immunoregulatory genes such as matrix metalloproteinase (MMP)2, MMP7, and adenosine A3 receptor (ADORA3). Principal component analysis revealed that differential expression of polarization-related genes has substantial contribution to global AM phenotypes associated with smoking and COPD. In summary, the data provide transcriptome-based evidence that AM likely contribute to COPD pathogenesis in a noninflammatory manner due to their smoking-induced reprogramming toward M1-deactivated, partially M2-polarized macrophages.

Cigarette smoking induces overexpression of a fat-depleting gene AZGP1 in the human.

BACKGROUND: Smokers weigh less and have less body fat than nonsmokers. Increased body fat and weight gain are observed following smoking cessation. To assess a possible molecular mechanism underlying the inverse association between smoking and body weight, we hypothesized that smoking may induce the expression of a fat-depleting gene in the airway epithelium, the cell population that takes the brunt of the stress of cigarette smoke. METHODS: To assess whether smoking up-regulates expression in the airway epithelium of genes associated with weight loss, microarray analysis was used to evaluate genes associated with fat depletion in large airway epithelial samples obtained by fiberoptic bronchoscopy from healthy smokers and healthy nonsmokers. As a candidate gene we further evaluated the expression of alpha(2)-zinc-glycoprotein 1 (AZGP1), a soluble protein that stimulates lipolysis, induces a reduction in body fat in mice, is associated with the cachexia related to cancer, and is known to be expressed in secretory cells of lung epithelium. AZGP1 protein expression was assessed by Western analysis and localization in the large airway epithelium by immunohistochemistry. RESULTS: Both microarray and TaqMan analysis demonstrated that AZGP1 messenger RNA levels were higher in the large airway epithelium of healthy smokers compared to healthy nonsmokers (p < 0.05, all comparisons). Western analysis of airway biopsy specimens from smokers compared with those from nonsmokers demonstrated up-regulation of AZGP1 at the protein level, and immunohistochemical analysis demonstrated up-regulation of AZGP1 in secretory as well as neuroendocrine cells of smokers. CONCLUSIONS: In the context that AZGP1 is involved in lipolysis and fat loss, its overexpression in the airway epithelium of chronic smokers may represent one mechanism for the weight difference in smokers vs nonsmokers.

Down-regulation of the notch pathway in human airway epithelium in association with smoking and chronic obstructive pulmonary disease.

RATIONALE: The airway epithelium of smokers is subject to a variety of mechanisms of injury with consequent modulation of epithelial regeneration and disordered differentiation. Several signaling pathways, including the Notch pathway, control epithelial differentiation in lung morphogenesis, but little is known about the role of these pathways in adults. OBJECTIVES: We tested the hypotheses that Notch-related genes are expressed in the normal nonsmoker small airway epithelium of human adults, and that Notch-related gene expression is down-regulated in healthy smokers and smokers with chronic obstructive pulmonary disease (COPD). METHODS: We used microarray technology to evaluate the expression of 55 Notch-related genes in the small airway epithelium of nonsmokers. We used TaqMan quantitative polymerase chain reaction (PCR) to confirm the expression of key genes and we used immunohistochemistry to assess the expression of Notch-related proteins in the airway epithelium. Changes in expression of Notch genes in healthy smokers and smokers with COPD compared with nonsmokers were evaluated by PCR. MEASUREMENTS AND MAIN RESULTS: Microarray analysis demonstrated that 45 of 55 Notch-related genes are expressed in the small airway epithelium of adults. TaqMan PCR confirmed the expression of key genes with highest expression of the ligand DLL1, the receptor NOTCH2, and the downstream effector HES1. Immunohistochemistry demonstrated the expression of Jag1, Notch2, Hes1, and Hes5 in airway epithelium. Several Notch ligands, receptors, and downstream effector genes were down-regulated in smokers, with more genes down-regulated in smokers with COPD than in healthy smokers. CONCLUSIONS: These observations are consistent with the hypothesis that the Notch pathway likely plays a role in the human adult airway epithelium, with down-regulation of Notch pathway gene expression in association with smoking and COPD.

Emphysema mediated by lung overexpression of ADAM10.

Cigarette smoking is the major risk factor for emphysema, a disorder of the lung parenchyma characterized by destruction of the alveolar walls. Current concepts of the pathogenesis of emphysema hold that the destruction of the lung parenchyma results, in part, from a local imbalance of proteases and antiproteases. Based on the knowledge that human alveolar macrophages express ADAM 10, a protease capable of destroying basement membrane collagen but not previously implicated in emphysema, we used adenovirus-mediated lung expression of ADAM 10 in a mouse model to assess whether an increased burden of ADAM 10 was capable of inducing emphysema. To assess this, the human ADAM 10 cDNA under control of a constitutive promoter was inserted into an adenovirus gene transfer vector (AdhADAMlO), and the vector (10(11) particle units) administered to the respiratory tract of wild type C57BI/6 mice. Lung levels of ADAM 10 mRNA and protein were upregulated following AdhADAMlO administration. After 8 weeks, quantitative morphometry of the lung parenchyma demonstrated that AdhADAMlO administration induced emphysema (mean linear intercept of 60.6 + 1.3 microm compared with 55.6 + 0.8 in mice treated with a control vector, p < 0.003). These results suggest a role of ADAM 10 in the pathogenesis of emphysema, adding to the list of proteases expressed in the lung that are capable of contributing to the development of lung destruction.