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.

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.

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.

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.

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.

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.

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.

Decreased expression of intelectin 1 in the human airway epithelium of smokers compared to nonsmokers.

Lectins are innate immune defense proteins that recognize bacterial cell wall components. Based on the knowledge that cigarette smoking is associated with an increased risk of infections, we hypothesized that cigarette smoking may modulate the expression of lectin genes in airway epithelium. Affymetrix microarrays were used to survey the expression of lectin genes in large airway epithelium from nine nonsmokers and 20 healthy smokers and in small airway epithelium from 13 nonsmokers and 20 healthy smokers. There were no changes (>2-fold change; p < 0.05) in lectin gene expression among healthy smokers compared with nonsmokers except for down-regulation of intelectin 1, a lectin that binds to galactofuranosyl residues in bacterial cell walls (large airway epithelium, p < 0.01; small airway epithelium, p < 0.01). This was confirmed by TaqMan RT-PCR in both large (p < 0.05) and small airway epithelium (p < 0.02). Immunohistochemistry assessment of airway biopsies demonstrated that intelectin 1 was expressed in secretory cells, while Western analysis confirmed the decreased expression of intelectin 1 in airway epithelium of healthy smokers compared with healthy nonsmokers (p < 0.02). Finally, compared with healthy nonsmokers, intelectin 1 expression was also decreased in small airway epithelium of smokers with lone emphysema and normal spirometry (n = 13, p < 0.01) and smokers with established chronic obstructive pulmonary disease (n = 14, p < 0.01). In the context that intelectin 1 plays a role in defense against bacteria, its down-regulation in response to cigarette smoking is another example of the immunomodulatory effects of smoking on the immune system and may contribute to the increase in susceptibility to infections observed in smokers.

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.

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.

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.

Overexpression of apoptotic cell removal receptor MERTK in alveolar macrophages of cigarette smokers.

Mononuclear phagocytes play an important role in the removal of apoptotic cells by expressing cell surface receptors that recognize and remove apoptotic cells. Based on the knowledge that cigarette smoking is associated with increased lung cell turnover, we hypothesized that alveolar macrophages (AMs) of normal cigarette smokers may exhibit enhanced expression of apoptotic cell removal receptor genes. AMs obtained by bronchoalveolar lavage of normal nonsmokers (n = 11) and phenotypic normal smokers (n = 13; 36 +/- 6 pack-years) were screened for mRNA expression of all known apoptotic cell removal receptors using Affymetrix HG-U133 Plus 2.0 microarray chips with TaqMan RT-PCR confirmation. Of the 14 known apoptotic receptors expressed, only MER tyrosine kinase (MERTK), a transmembrane tyrosine kinase receptor, was significantly up-regulated in smokers. MERTK expression was then assessed in AMs of smokers versus nonsmokers by TaqMan RT-PCR, immunocytochemistry, Western analysis, and flow analysis. Smoker AMs had up-regulation of MERTK mRNA levels (smoker vs. nonsmoker: 3.6-fold by microarray, P < 0.003; 9.5-fold by TaqMan RT-PCR, P < 0.02). Immunocytochemistry demonstrated a qualitative increase in MERTK protein expression on AMs of smokers. Increased protein expression of MERTK on AMs of smokers was confirmed by Western and flow analyses (P < 0.007 and P < 0.0002, respectively). MERTK, a cell surface receptor that recognizes apoptotic cells, is expressed on human AMs, and its expression is up-regulated in AMs of cigarette smokers. This up-regulation of MERTK may reflect an increased demand for removal of apoptotic cells in smokers, an observation with implications for the development of chronic obstructive pulmonary disease, a disorder associated with dysregulated apoptosis of lung parenchymal cells.

Variability in small airway epithelial gene expression among normal smokers.

BACKGROUND: Despite overwhelming data that cigarette smoking causes COPD, only a minority of long-term smokers are affected, strongly suggesting that genetic factors modify susceptibility to this disease. We hypothesized that individual variations exist in the response to cigarette smoking, with variability among smokers in expression levels of protective/susceptibility genes. METHODS: Affymetrix arrays and quantitative polymerase chain reaction were used to assess the variability of gene expression in the small airway epithelium obtained by fiberoptic bronchoscopy of 18 normal nonsmokers, 18 normal smokers, and 18 smokers with COPD. RESULTS: We identified 201 probe sets representing 152 smoking-responsive genes that were significantly up-regulated 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 probe sets and real-time polymerase chain reaction, and was observed in both normal smokers and smokers with COPD. Greater individual variability was found 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. CONCLUSIONS: We propose that genetic diversity is likely within this subset of genes, with highly variable individual-to-individual responses of the small airway epithelium to smoking, and that this subset of genes represents putative candidates for assessment of susceptibility/protection from disease in future gene-based epidemiologic studies of smokers' risk for COPD.

Modification of gene expression of the small airway epithelium in response to cigarette smoking.

The earliest morphologic evidence of changes in the airways associated with chronic cigarette smoking is in the small airways. To help understand how smoking modifies small airway structure and function, we developed a strategy using fiberoptic bronchoscopy and brushing to sample the human small airway (10th-12th order) bronchial epithelium to assess gene expression (Affymetrix HG-U133A and HG-133 Plus 2.0 array) in phenotypically normal smokers (n = 16, 25 +/- 7 pack-years) compared to matched nonsmokers (n = 17). Compared to samples from large (second to third order) bronchi, the small airway samples had a higher proportion of ciliated cells, but less basal, undifferentiated, and secretory cells, and contained Clara cells. Even though the smokers were phenotypically normal, microarray analysis of gene expression of the small airway epithelium of the smokers compared to the nonsmokers demonstrated up- and downregulation of genes in multiple categories relevant to the pathogenesis of chronic obstructive lung disease (COPD), including genes coding for cytokines/innate immunity, apoptosis, mucin, response to oxidants and xenobiotics, and general cellular processes. In the context that COPD starts in the small airways, these gene expression changes in the small airway epithelium in phenotypically normal smokers are candidates for the development of therapeutic strategies to prevent the onset of COPD.

Up-regulation of expression of the ubiquitin carboxyl-terminal hydrolase L1 gene in human airway epithelium of cigarette smokers.

Neuroendocrine differentiation is a common feature of lung cancer and increased numbers of neuroendocrine cells and their peptides have been described in chronic smokers. To understand the effects of cigarette smoking on the gene expression profile of neuroendocrine cells, microarray analysis with TaqMan confirmation was used to assess airway epithelial samples obtained by fiberoptic bronchoscopy from 81 individuals [normal nonsmokers, normal smokers, smokers with early chronic obstructive lung disease (COPD), and smokers with established COPD]. Of 11 genes considered to be neuroendocrine cell specific, only ubiquitin carboxyl-terminal hydrolase L1 (UCHL1), a member of the ubiquitin proteasome pathway, was consistently up-regulated in smokers compared with nonsmokers. Up-regulation of UCHL1 at the protein level was observed with immunohistochemical analysis of bronchial biopsies of smokers compared with nonsmokers. UCHL1 expression was evident only in neuroendocrine cells of the airway epithelium in nonsmokers; however, UCHL1 was also expressed in ciliated epithelial cells in smokers. This observation may add further weight to recent observations that ciliated cells are capable of transdifferentiating to other airway epithelial cells. In the context that UCHL1 is involved in the degradation of unwanted, misfolded, or damaged proteins within the cell and is overexpressed in >50% of lung cancers, its overexpression in chronic smokers may represent an early event in the complex transformation from normal epithelium to overt malignancy.

Responses of the human airway epithelium transcriptome to in vivo injury.

To identify genes participating in human airway epithelial repair, we used bronchoscopy and brushing to denude the airway epithelium of healthy individuals, sequentially sampled the same region 7 and 14 days later, and assessed gene expression by Affymetrix microarrays with TaqMan RT-PCR confirmation. Histologically, the injured area was completely covered by a partially redifferentiated epithelial layer after 7 days; by 14 days the airway epithelium was very similar to the uninjured state. At day 7 compared with resting epithelium, there were substantial differences in gene expression pattern, with a distinctive airway epithelial "repair transcriptome" of actively proliferating cells in the process of redifferentiation. The repair transcriptome at 7 days was dominated by cell cycle, signal transduction, metabolism and transport, and transcription genes. Interestingly, the majority of differentially expressed cell cycle genes belonged to the G2 and M phases, suggesting that the proliferating cells were relatively synchronized 1 wk following injury. At 14 days postinjury, the expression profile was similar to that of resting airway epithelium. These observations provide a baseline of the functional gene categories participating in the process of normal human airway epithelial repair that can be used in future studies of injury and repair in airway epithelial diseases.

Gene expression profiling of human alveolar macrophages of phenotypically normal smokers and nonsmokers reveals a previously unrecognized subset of genes modulated by cigarette smoking.

Cigarette smoking is the leading cause of the respiratory diseases collectively known as chronic obstructive pulmonary disease (COPD). While the pathogenesis of COPD is complex, there is abundant evidence that alveolar macrophages (AM) play an important role. Based on the concept that COPD is a slow-progressing disorder likely involving multiple mediators released by AM activated by cigarette smoke, the present study focuses on the identification of previously unrecognized genes that may be linked to early events in the molecular pathogenesis of COPD, as opposed to factors associated with the presence of disease. To accomplish this, microarray analysis using Affymetrix microarrays was used to carry out an unbiased survey of the differences in gene expression profiles in the AM of phenotypically normal, approximately 20 pack-year smokers compared to healthy nonsmokers. Although smoking did not alter the global gene expression pattern of AM, 75 genes were modulated by smoking, with 40 genes up-regulated and 35 down-regulated in the AM of smokers compared to nonsmokers. Most of these genes belong to the functional categories of immune/inflammatory response, cell adhesion and extracellular matrix, proteolysis and antiproteolysis, lysosomal function, antioxidant-related function, signal transduction, and regulation of transcription. Of these 75 genes, 69 have not been previously recognized to be up- or down-regulated in AM in association with smoking or COPD, including genes coding for proteins belonging to all of the above categories, and others belonging to various functional categories or of unknown function. These observations suggest that gene expression responses of AM associated with the stress of cigarette smoking are more complex than previously thought, and offer a variety of new insights into the complex pathogenesis of smoking-induced lung diseases.

Monoallelic up-regulation of the imprinted H19 gene in airway epithelium of phenotypically normal cigarette smokers.

H19, a paternally imprinted gene, is postulated to have regulatory functions in normal development and oncogenesis. Loss of imprinting (LOI) of H19 is observed in human malignancies, including lung cancer. Microarray assessment of gene expression patterns in airway epithelium of healthy 20 pack-year smokers versus nonsmokers revealed that smokers have dramatically elevated H19 RNA levels without alteration of expression of other imprinted genes. Interestingly, the up-regulation of H19 was not attributable to LOI, i.e., expression of H19 in smokers was monoallelic. These observations suggest that cigarette smoking initially induces up-regulation of the active H19 allele and that there is likely progression to LOI as the burden of smoking increases and as the epithelium undergoes transition from normal to neoplastic. Overexpression and eventual LOI of H19 may represent early markers in the progression of airway epithelium toward lung cancer.