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1.  Cluster Analysis and Clinical Asthma Phenotypes 
Heterogeneity in asthma expression is multidimensional, including variability in clinical, physiologic, and pathologic parameters. Classification requires consideration of these disparate domains in a unified model.
To explore the application of a multivariate mathematical technique, k-means cluster analysis, for identifying distinct phenotypic groups.
We performed k-means cluster analysis in three independent asthma populations. Clusters of a population managed in primary care (n = 184) with predominantly mild to moderate disease, were compared with a refractory asthma population managed in secondary care (n = 187). We then compared differences in asthma outcomes (exacerbation frequency and change in corticosteroid dose at 12 mo) between clusters in a third population of 68 subjects with predominantly refractory asthma, clustered at entry into a randomized trial comparing a strategy of minimizing eosinophilic inflammation (inflammation-guided strategy) with standard care.
Measurements and Main Results
Two clusters (early-onset atopic and obese, noneosinophilic) were common to both asthma populations. Two clusters characterized by marked discordance between symptom expression and eosinophilic airway inflammation (early-onset symptom predominant and late-onset inflammation predominant) were specific to refractory asthma. Inflammation-guided management was superior for both discordant subgroups leading to a reduction in exacerbation frequency in the inflammation-predominant cluster (3.53 [SD, 1.18] vs. 0.38 [SD, 0.13] exacerbation/patient/yr, P = 0.002) and a dose reduction of inhaled corticosteroid in the symptom-predominant cluster (mean difference, 1,829 μg beclomethasone equivalent/d [95% confidence interval, 307–3,349 μg]; P = 0.02).
Cluster analysis offers a novel multidimensional approach for identifying asthma phenotypes that exhibit differences in clinical response to treatment algorithms.
PMCID: PMC3992366  PMID: 18480428
taxonomy; corticosteroid response; multivariate classification
2.  Stress in the ER (Endoplasmic Reticulum) 
PMCID: PMC2720126  PMID: 18832553
3.  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.
PMCID: PMC2654975  PMID: 19106307
gene expression; microarray analysis; delta-like ligand; basic helix-loop-helix transcription factors; Notch receptors
4.  The Prognostic Value of Cardiopulmonary Exercise Testing in Idiopathic Pulmonary Fibrosis 
Rationale: Idiopathic pulmonary fibrosis (IPF) is characterized by progressive dyspnea, impaired gas exchange, and ultimate mortality.
Objectives: To test the hypothesis that maximal oxygen uptake during cardiopulmonary exercise testing at baseline and with short-term longitudinal measures would predict mortality in patients with idiopathic pulmonary fibrosis.
Methods: Data from 117 patients with IPF and longitudinal cardiopulmonary exercise tests were examined retrospectively. Survival was calculated from the date of the first cardiopulmonary exercise test.
Measurements and Main Results: Patients with baseline maximal oxygen uptake less than 8.3 ml/kg/min had an increased risk of death (n = 8; hazard ratio, 3.24; 95% confidence interval, 1.10–9.56; P = 0.03) after adjusting for age, gender, smoking status, baseline forced vital capacity, and baseline diffusion capacity for carbon monoxide. We were unable to define a unit change in maximal oxygen uptake that predicted survival in our cohort.
Conclusions: We conclude that a threshold maximal oxygen uptake of 8.3 ml/kg/min during cardiopulmonary exercise testing at baseline adds prognostic information for patients with IPF.
PMCID: PMC2648909  PMID: 19074597
idiopathic pulmonary fibrosis; exercise test; mortality
5.  Variants in TGFB1, Dust Mite Exposure, and Disease Severity in Children with Asthma 
Rationale: Polymorphisms in the gene for transforming growth factor-β1 (TGFB1) have been associated with asthma, but not with airway responsiveness or disease exacerbations in subjects with asthma.
Objectives: To test for association between single nucleotide polymorphisms (SNPs) in TGFB1 and markers of asthma severity in childhood.
Methods: We tested for the association between nine SNPs in TGFB1 and indicators of asthma severity (lung function, airway responsiveness, and disease exacerbations) in two cohorts: 416 Costa Rican parent-child trios and 465 families of non-Hispanic white children in the Childhood Asthma Management Program (CAMP). We also tested for the interaction between these polymorphisms and exposure to dust mite allergen on asthma severity.
Measurements and Main Results: The A allele of promoter SNP rs2241712 was associated with increased airway responsiveness in Costa Rica (P = 0.0006) and CAMP (P = 0.005), and the C allele of an SNP in the promoter region (rs1800469) was associated with increased airway responsiveness in both cohorts (P ≤ 0.01). Dust mite exposure modified the effect of the C allele of exonic SNP rs1800471 on airway responsiveness (P = 0.03 for interactions in both cohorts). The T allele of a coding SNP (rs1982073) was associated with a reduced risk of asthma exacerbations in Costa Rica (P = 0.009) and CAMP (P = 0.005). Dust mite exposure also significantly modified the effect of the A allele of the promoter SNP rs2241712 on asthma exacerbations in both cohorts.
Conclusions: SNPs in TGFB1 are associated with airway responsiveness and disease exacerbations in children with asthma. Moreover, dust mite exposure may modify the effect of TGFB1 SNPs on airway responsiveness and asthma exacerbations.
PMCID: PMC2648908  PMID: 19096005
airway responsiveness; asthma; dust mite allergen; single nucleotide polymorphisms; transforming growth factor-β1
6.  Expiratory Pharyngeal Narrowing during Central Hypocapnic Hypopnea 
Rationale: Ventilatory motor output is an important determinant of upper airway patency during sleep.
Objectives: We hypothesized that central hypocapnic hypopnea would lead to increased expiratory upper airway resistance and pharyngeal narrowing during non-REM sleep.
Methods: Noninvasive positive pressure ventilation was used to induce hypocapnic hypopnea in 20 healthy subjects. Expiratory pressure was set at the lowest pressure (2 cm H2O), and inspiratory pressure was increased gradually during each 3-minute noninvasive positive pressure ventilation trial by increments of 2 cm H2O. Analysis 1 (n = 9) included measured retropalatal cross-sectional area (CSA) using nasopharyngoscope to compare CSA at five points of the respiratory cycle between control (eupneic) and hypopneic breaths. The pharyngeal pressure (Pph) was measured using a catheter positioned at the palatal rim. Analysis 2 (n = 11) included measured supraglottic pressure and airflow to compare inspiratory and expiratory upper airway resistance (RUA) at peak flow between eupneic and hypopneic breaths.
Measurements and Main Results: Expiratory CSA during hypopneic breaths was decreased relative to eupnea (CSA at beginning of expiration [BI]: 101.5 ± 6.3 vs. 121.6 ± 8.9%; P < 0.05); Pph-BI was lower than that generated during eupnea (1.5 ± 0.3 vs. 3.3 ± 0.9 cm H2O; P < 0.05). Body mass index was an independent predictor of retropalatal narrowing during hypopnea. Hypopnea-RUA increased during expiration relative to eupnea (14.0 ± 5.7 vs. 10.6 ± 2.5 cm H2O/L/s; P = 0.01), with no change in inspiratory resistance.
Conclusions: Expiratory pharyngeal narrowing occurs during central hypocapnic hypopnea. Reduced ventilatory drive leads to increased expiratory, but not inspiratory, upper airway resistance. Central hypopneas are obstructive events because they cause pharyngeal narrowing.
PMCID: PMC2643080  PMID: 19201929
ventilatory motor output; expiratory; upper airway resistance
7.  Post-Transcriptional Regulation of Urokinase-type Plasminogen Activator Receptor Expression in Lipopolysaccharide-induced Acute Lung Injury 
Rationale: Urokinase-type plasminogen activator (uPA) receptor (uPAR) is required for the recruitment of neutrophils in response to infection. uPA induces its own expression in lung epithelial cells, which involves its interaction with cell surface uPAR. Regulation of uPAR expression is therefore crucial for uPA-mediated signaling in infectious acute lung injury (ALI).
Objectives: To determine the role of uPA in uPAR expression during ALI caused by sepsis.
Methods: We used Western blot, Northern blot, Northwestern assay, and immunohistochemistry. Phosphate-buffered saline– and lipopolysaccharide (LPS)-treated wild-type and uPA−/− mice were used.
Measurements and Main Results: Biological activities of uPA, including proteolysis, cell adhesion, migration, proliferation, and differentiation, are dependent on its association with uPAR. Bacterial endotoxin (LPS) is a major cause of pulmonary dysfunction and infection-associated mortality. The present study shows that LPS induces uPAR expression both in vitro and in vivo, and that the mechanism involves post-transcriptional stabilization of uPAR mRNA by reciprocal interaction of phosphoglycerate kinase (PGK) and heterogeneous nuclear ribonucleoprotein C (hnRNPC) with uPAR mRNA coding region and 3′ untranslated region determinants, respectively. The process involves tyrosine phosphorylation of PGK and hnRNPC. uPA−/− mice failed to induce uPAR expression after LPS treatment. In these mice, LPS treatment failed to alter the binding of PGK and hnRNPC protein with uPAR mRNA due to lack of tyrosine phosphorylation.
Conclusions: Our study shows that induction of LPS-mediated uPAR expression is mediated through tyrosine phosphorylation of PGK and hnRNPC. This involves expression of uPA as an obligate intermediary.
PMCID: PMC2643078  PMID: 19029002
LPS; urokinase-type plasminogen activator; urokinase-type plasminogen activator receptor; tyrosine phosphorylation
8.  Cigarette Smoke Induces Cellular Senescence via Werner's Syndrome Protein Down-regulation 
Rationale: Werner's syndrome is a genetic disorder that causes premature aging due to loss-of-function mutations in a gene encoding a member of the RecQ helicase family. Both Werner's syndrome and cigarette smoking accelerate aging. No studies have examined the effect of cigarette smoke on Werner's syndrome protein.
Objectives: To investigate the role of Werner's syndrome protein in cigarette smoke–induced cellular senescence.
Methods: Cellular senescence and amounts of Werner's syndrome protein were measured in fibroblasts isolated from patients with emphysema and compared with age-matched nonsmokers. The in vitro effects of cigarette smoke on amounts of Werner's syndrome protein, function, and senescence were also evaluated in primary human lung fibroblasts and epithelial cells.
Measurements and Main Results: Cultured lung fibroblasts isolated from patients with emphysema exhibited a senescent phenotype accompanied by a decrease in Werner's syndrome protein. Cigarette smoke extract decreased Werner's syndrome protein in cultured fibroblasts and epithelial cells. Werner's syndrome protein–deficient fibroblasts were more susceptible to cigarette smoke–induced cellular senescence and cell migration impairment. In contrast, exogenous overexpression of Werner's syndrome protein attenuated the cigarette smoke effects.
Conclusions: Cigarette smoke induces cellular senescence and cell migration impairment via Werner's syndrome protein down-regulation. Rescue of Werner's syndrome protein down-regulation may represent a potential therapeutic target for smoking-related diseases.
PMCID: PMC2643077  PMID: 19011155
aging; smoking; emphysema; oxidative stress; cell migration
9.  Attenuated P2X7 Pore Function as a Risk Factor for Virus-induced Loss of Asthma Control 
Rationale: Upper respiratory tract infection is a guideline accepted risk domain for the loss of asthma control. The ionotrophic nucleotide receptor P2X7 regulates compartmentalized acute inflammation and the immune response to airway pathogens.
Objectives: We hypothesized that variability in P2X7 function contributes to neutrophilic airway inflammation during a cold and thereby is linked to acute asthma.
Methods: Research volunteers with asthma were enrolled at the onset of a naturally occurring cold and monitored through convalescence, assessing symptoms, lung function, and airway inflammation. P2X7 pore activity in whole blood samples was measured using a genomically validated flow cytometric assay.
Measurements and Main Results: Thirty-five participants with mild to moderate allergic asthma were enrolled and 31 completed all visits. P2X7 pore function correlated with the change in nasal lavage neutrophil counts during the cold (Rs = 0.514, P = 0.004) and was inversely related to the change in asthma symptoms (Rs = −0.486, P = 0.009). The change in peak expiratory flow recordings, precold use of inhaled corticosteroids, and P2X7 pore function were multivariate predictors of asthma symptoms (P = 0.001, < 0.001 and = 0.003 respectively). Attenuated P2X7 activity was associated with the risk of losing asthma control (crude odds ratio, 11.0; 95% confidence interval, 1.1–106.4) even after adjustment for inhaled corticosteroids and rhinovirus (odds ratio, 15.0).
Conclusions: A whole blood P2X7 pore assay robustly identifies participants with loss-of-function genotypes. Using this assay as an epidemiologic tool, attenuated P2X7 pore activity may be a novel biomarker of virus-induced loss of asthma control.
PMCID: PMC2643076  PMID: 19201928
asthma; virus; neutrophils; P2X7
10.  Alterations in Glucose Disposal in Sleep-disordered Breathing 
Rationale: It is well established that sleep-disordered breathing (SDB) is independently associated with insulin resistance, glucose intolerance, and type 2 diabetes mellitus. However, data on whether SDB alters in vivo kinetics of glucose and insulin are lacking.
Objectives: The primary goal of this study was to use the frequently sampled intravenous glucose tolerance test (FSIVGTT) in subjects with and without SDB to model the in vivo kinetics of glucose and insulin. Minimal model analysis of the FSIVGTT data was used to derive parameters of insulin sensitivity, glucose effectiveness (a measure of the ability of glucose to mediate its own disposal), and pancreatic β-cell function.
Results: A total of 118 nondiabetic subjects underwent polysomnography, the FSIVGTT, and body composition measurements including determination of percent body fat. Compared with normal subjects (apnea-hypopnea index < 5 events/h), those with mild, moderate, and severe SDB displayed a 26.7, 36.5 and 43.7% reduction in insulin sensitivity, respectively, independent of age, sex, race, and percent body fat. The disposition index, an integrated measure of pancreatic β-cell function, was also reduced in patients with moderate to severe SDB. The decrease in insulin sensitivity and the disposition index were correlated with the average degree of oxyhemoglobin desaturation. In contrast, glucose effectiveness was negatively correlated with the frequency of respiratory event–related arousals.
Conclusions: The results of this study suggest that, independent of adiposity, SDB is associated with impairments in insulin sensitivity, glucose effectiveness, and pancreatic β-cell function. Collectively, these defects may increase the risk of glucose intolerance and type 2 diabetes mellitus in SDB.
PMCID: PMC2633056  PMID: 19011148
sleep-disordered breathing; sleep apnea; glucose metabolism; insulin resistance; beta-cell function
11.  Obstructive Sleep Apnea, Insulin Resistance, and Steatohepatitis in Severe Obesity 
Rationale: Obstructive sleep apnea is associated with insulin resistance and liver injury. It is unknown whether apnea contributes to insulin resistance and steatohepatitis in severe obesity.
Objectives: To examine whether sleep apnea and nocturnal hypoxemia predict the severity of insulin resistance, systemic inflammation, and steatohepatitis in severely obese individuals presenting for bariatric surgery.
Methods: We performed sleep studies and measured fasting blood glucose, serum insulin, C-reactive protein, and liver enzymes in 90 consecutive severely obese individuals, 75 women and 15 men, without concomitant diabetes mellitus or preexistent diagnosis of sleep apnea or liver disease. Liver biopsies (n = 20) were obtained during bariatric surgery.
Measurements and Main Results: Obstructive sleep apnea with a respiratory disturbance index greater than 5 events/hour was diagnosed in 81.1% of patients. The median respiratory disturbance index was 15 ± 29 events/hour and the median oxygen desaturation during apneic events was 4.6 ± 1.8%. All patients exhibited high serum levels of C-reactive protein, regardless of the severity of apnea, whereas liver enzymes were normal. Oxygen desaturation greater than 4.6% was associated with a 1.5-fold increase in insulin resistance, according to the homeostasis model assessment index. Histopathology data suggested that significant nocturnal desaturation might predispose to hepatic inflammation, hepatocyte ballooning, and liver fibrosis. Fasting blood glucose levels and steatosis scores were not affected by nocturnal hypoxia. There was no relationship between the respiratory disturbance index and insulin resistance or liver histopathology.
Conclusions: Hypoxic stress of sleep apnea may be implicated in the development of insulin resistance and steatohepatitis in severe obesity.
PMCID: PMC2633055  PMID: 18990675
hypoxemia; fatty liver disease; metabolic syndrome; sleep-disordered breathing; liver injury
12.  Insulin-like Growth Factor-I Receptor Blockade Improves Outcome in Mouse Model of Lung Injury 
Rationale: The insulin-like growth factor-I (IGF-I) pathway is an important determinant of survival and proliferation in many cells. However, little is known about the role of the IGF-I pathway in lung injury. We previously showed elevated levels of IGF-I in bronchoalveolar lavage fluid from patients with acute respiratory distress syndrome. Furthermore, immunodepletion of IGF from acute respiratory distress syndrome bronchoalveolar lavage increased fibroblast apoptosis.
Objectives: We examined the effect of blockade of type 1 IGF tyrosine kinase receptor (IGF-IR) in a murine model of bleomycin-induced lung injury and fibrosis.
Methods: Mice were treated with a monoclonal antibody against the IGF-I receptor (A12) or vehicle after intratracheal bleomycin instillation.
Measurements and Main Results: Mice treated with A12 antibody had significantly improved survival after bleomycin injury compared with control mice. Both groups of mice had a similar degree of fibrosis on days 7 and 14, but by Day 28 the A12-treated group had significantly less fibrosis. Delayed treatment with A12 also resulted in decreased fibrosis. A12-treated mice had significantly decreased apoptotic cells on Day 28 compared with control mice. We confirmed that A12 treatment induced mouse lung fibroblast apoptosis in vitro. In addition, IGF-I increased lung fibroblast migration. The primary pathway activated by IGF-I in lung fibroblasts was the insulin receptor substrate-2/phosphatidylinositol 3-kinase/Akt axis.
Conclusions: IGF-I regulated survival and migration of fibrogenic cells in the lung. Blockade of the IGF pathway increased fibroblast apoptosis and subsequent resolution of pulmonary fibrosis. Thus, IGF-IR may be a potential target for treatment of lung injury and fibrosis.
PMCID: PMC2633054  PMID: 19011156
insulin-like growth factor; lung injury; lung fibrosis
13.  Myosin, Transgelin, and Myosin Light Chain Kinase 
Rationale: Airway smooth muscle (SM) of patients with asthma exhibits a greater velocity of shortening (Vmax) than that of normal subjects, and this is thought to contribute to airway hyperresponsiveness. A greater Vmax can result from increased myosin activation. This has been reported in sensitized human airway SM and in models of asthma. A faster Vmax can also result from the expression of specific contractile proteins that promote faster cross-bridge cycling. This possibility has never been addressed in asthma.
Objectives: We tested the hypothesis that the expression of genes coding for SM contractile proteins is altered in asthmatic airways and contributes to their increased Vmax.
Methods: We quantified the expression of several genes that code for SM contractile proteins in mild allergic asthmatic and control human airway endobronchial biopsies. The function of these contractile proteins was tested using the in vitro motility assay.
Measurements and Main Results: We observed an increased expression of the fast myosin heavy chain isoform, transgelin, and myosin light chain kinase in patients with asthma. Immunohistochemistry demonstrated the expression of these genes at the protein level. To address the functional significance of this overexpression, we purified tracheal myosin from the hyperresponsive Fisher rats, which also overexpress the fast myosin heavy chain isoform as compared with the normoresponsive Lewis rats, and found a faster rate of actin filament propulsion. Conversely, transgelin did not alter the rate of actin filament propulsion.
Conclusions: Selective overexpression of airway smooth muscle genes in asthmatic airways leads to increased Vmax, thus contributing to the airway hyperresponsiveness observed in asthma.
PMCID: PMC2633053  PMID: 19011151
asthma; airway hyperresponsiveness; gene expression; smooth muscle; myosin
14.  Altered Upper Airway and Soft Tissue Structures in the New Zealand Obese Mouse 
Rationale: The effect of obesity on upper airway soft tissue structure and size was examined in the New Zealand Obese (NZO) mouse and in a control lean mouse, the New Zealand White (NZW).
Objectives: We hypothesized that the NZO mouse has increased volume of neck fat and upper airway soft tissues and decreased pharyngeal airway caliber.
Methods: Pharyngeal airway size, volume of the upper airway soft tissue structures, and distribution of fat in the neck and body were measured using magnetic resonance imaging (MRI). Dynamic MRI was used to examine the differences in upper airway caliber between inspiration and expiration in NZO versus NZW mice.
Measurements and Main Results: The data support the hypothesis that, in obese NZO versus lean NZW mice, airway caliber was significantly smaller (P < 0.03), with greater parapharyngeal fat pad volumes (P < 0.0001) and a greater volume of other upper airway soft tissue structures (tongue, P = 0.003; lateral pharyngeal walls, P = 0.01; soft palate, P = 0.02). Dynamic MRI showed that the airway of the obese NZO mouse dilated during inspiration, whereas in the lean NZW mouse, the upper airway was reduced in size during inspiration.
Conclusions: In addition to the increased volume of pharyngeal soft tissue structures, direct fat deposits within the tongue may contribute to airway compromise in obesity. Pharyngeal airway dilation during inspiration in NZO mice compared with narrowing in NZW mice suggests that airway compromise in obese mice may lead to muscle activation to defend upper airway patency during inspiration.
PMCID: PMC2633061  PMID: 18996996
obstructive sleep apnea; magnetic resonance imaging; body fat distribution; tongue; upper airway
15.  Antiviral Activity of Nrf2 in a Murine Model of Respiratory Syncytial Virus Disease 
Rationale: Respiratory syncytial virus (RSV) is the most frequent cause of significant lower respiratory illness in infants and young children, but its pathogenesis is not fully understood. The transcription factor Nrf2 protects lungs from oxidative injury and inflammation via antioxidant response element (ARE)-mediated gene induction.
Objectives: The current study was designed to determine the role of Nrf2-mediated cytoprotective mechanisms in murine airway RSV disease.
Methods: Nrf2-deficient (Nrf2−/−) and wild-type (Nrf2+/+) mice were intranasally instilled with RSV or vehicle. In a separate study, Nrf2+/+ and Nrf2−/− mice were treated orally with sulforaphane (an Nrf2-ARE inducer) or phosphate-buffered saline before RSV infection.
Measurements and Main Results: RSV-induced bronchopulmonary inflammation, epithelial injury, and mucus cell metaplasia as well as nasal epithelial injury were significantly greater in Nrf2−/− mice than in Nrf2+/+ mice. Compared with Nrf2+/+ mice, significantly attenuated viral clearance and IFN-γ, body weight loss, heightened protein/lipid oxidation, and AP-1/NF-κB activity along with suppressed antioxidant induction was found in Nrf2−/− mice in response to RSV. Sulforaphane pretreatment significantly limited lung RSV replication and virus-induced inflammation in Nrf2+/+ but not in Nrf2−/− mice.
Conclusions: The results of this study support an association of oxidant stress with RSV pathogenesis and a key role for the Nrf2-ARE pathway in host defense against RSV.
PMCID: PMC2633060  PMID: 18931336
airway; oxidative stress; antioxidant response element; inflammation; sulforaphane
16.  Growth of the Lung Parenchyma Early in Life 
Rationale: Early in life, lung growth can occur by alveolarization, an increase in the number of alveoli, as well as expansion. We hypothesized that if lung growth early in life occurred primarily by alveolarization, then the ratio of pulmonary diffusion capacity of carbon monoxide (DlCO) to alveolar volume (VA) would remain constant; however, if lung growth occurred primarily by alveolar expansion, then DlCO/VA would decline with increasing age, as observed in older children and adolescents.
Objectives: To evaluate the relationship between alveolar volume and pulmonary diffusion capacity early in life.
Methods: In 50 sleeping infants and toddlers, with equal number of males and females between the ages of 3 and 23 months, we measured DlCO and VA using single breath-hold maneuvers at elevated lung volumes.
Measurements and Main Results: DlCO and VA increased with increasing age and body length. Males had higher DlCO and VA when adjusted for age, but not when adjusted for length. DlCO increased with VA; there was no gender difference when DlCO was adjusted for VA. The ratio of DlCO/VA remained constant with age and body length.
Conclusions: Our results suggest that surface area for diffusion increases proportionally with alveolar volume in the first 2 years of life. Larger DlCO and VA for males than females when adjusted for age, but not when adjusted for length, is primarily related to greater body length in boys. The constant ratio for DlCO/VA in infants and toddlers is consistent with lung growth in this age occurring primarily by the addition of alveoli rather than the expansion of alveoli.
PMCID: PMC2633059  PMID: 18996997
pulmonary diffusion capacity; alveolar volume; lung development
17.  Endothelin-1 Impairs Alveolar Epithelial Function via Endothelial ETB Receptor 
Rationale: Endothelin-1 (ET-1) is increased in patients with high-altitude pulmonary edema and acute respiratory distress syndrome, and these patients have decreased alveolar fluid reabsorption (AFR).
Objectives: To determine whether ET-1 impairs AFR via activation of endothelial cells and nitric oxide (NO) generation.
Methods: Isolated perfused rat lung, transgenic rats deficient in ETB receptors, coincubation of lung human microvascular endothelial cells (HMVEC-L) with rat alveolar epithelial type II cells or A549 cells, ouabain-sensitive 86Rb+ uptake.
Measurements and Main Results: The ET-1–induced decrease in AFR was prevented by blocking the endothelin receptor ETB, but not ETA. Endothelial–epithelial cell interaction is required, as direct exposure of alveolar epithelial cells (AECs) to ET-1 did not affect Na,K-ATPase function or protein abundance at the plasma membrane, whereas coincubation of HMVEC-L and AECs with ET-1 decreased Na,K-ATPase activity and protein abundance at the plasma membrane. Exposing transgenic rats deficient in ETB receptors in the pulmonary vasculature (ET-B−/−) to ET-1 did not decrease AFR or Na,K-ATPase protein abundance at the plasma membrane of AECs. Exposing HMVEC-L to ET-1 led to increased NO, and the ET-1–induced down-regulation of Na,K-ATPase was prevented by the NO synthase inhibitor l-NAME, but not by a guanylate cyclase inhibitor.
Conclusions: We provide the first evidence that ET-1, via an endothelial–epithelial interaction, leads to decreased AFR by a mechanism involving activation of endothelial ETB receptors and NO generation leading to alveolar epithelial Na,K-ATPase down-regulation in a cGMP-independent manner.
PMCID: PMC2633058  PMID: 18948426
endothelium; lung injury; sodium-potassium-exchanging ATPase; acute respiratory distress syndrome
18.  Extracellular Superoxide Dismutase Haplotypes Are Associated with Acute Lung Injury and Mortality 
Rationale: Extracellular superoxide dismutase (EC-SOD) is a potent antioxidant that plays an important role in controlling oxidant-mediated stress and inflammation. High levels of EC-SOD are found in the lung. Acute lung injury (ALI) frequently occurs in patients with infection, and levels of EC-SOD have been shown to modulate severity of lung injury in transgenic animal models of endotoxemia-induced ALI. An R213G single nucleotide polymorphism (SNP) has been shown to alter levels of EC-SOD and patient outcomes in chronic obstructive pulmonary disease (COPD) and ischemic heart disease.
Objectives: To determine genetic variation in the promoter and EC-SOD gene and to examine whether EC-SOD haplotype blocks are associated with clinical outcomes.
Methods: We sequenced the EC-SOD promoter and gene to determine genetic variation and linkage disequilibrium (LD) patterns in a European American population. Two separate patient populations with infection-associated ALI were also evaluated to determine whether EC-SOD haplotypes were associated with clinical outcomes.
Measurements and Main Results: Sequencing resulted in the identification of 28 SNPs with relatively strong LD and 1 block consisting of 4691-5321-5360-5955-5982. This specific block was shown to be protective in two separate patient populations with infection associated ALI. In particular, patients with a GCCT haplotype had a reduced risk of time on the ventilator and mortality.
Conclusions: These results indicate that a GCCT haplotype may reduce inflammation in the lung, thereby decreasing the severity of lung injury and ultimately protecting patients from mortality associated with infection-induced ALI.
PMCID: PMC2633057  PMID: 18948423
EC-SOD; haplotypes; acute lung injury; single nucleotide polymorphism
19.  High Dietary Inorganic Phosphate Increases Lung Tumorigenesis and Alters Akt Signaling 
Rationale: Phosphate (Pi) is an essential nutrient to living organisms. Recent surveys indicate that the intake of Pi has increased steadily. Our previous studies have indicated that elevated Pi activates the Akt signaling pathway. An increased knowledge of the response of lung cancer tissue to high dietary Pi may provide an important link between diet and lung tumorigenesis.
Objectives: The current study was performed to elucidate the potential effects of high dietary Pi on lung cancer development.
Methods: Experiments were performed on 5-week-old male K-rasLA1 lung cancer model mice and 6-week-old male urethane-induced lung cancer model mice. Mice were fed a diet containing 0.5% Pi (normal Pi) and 1.0% Pi (high Pi) for 4 weeks. At the end of the experiment, all mice were killed. Lung cancer development was evaluated by diverse methods.
Measurement and Main Results: A diet high in Pi increased lung tumor progression and growth compared with normal diet. High dietary Pi increased the sodium-dependent inorganic phosphate transporter-2b protein levels in the lungs. High dietary consumption of Pi stimulated pulmonary Akt activity while suppressing the protein levels of tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 as well as Akt binding partner carboxyl-terminal modulator protein, resulting in facilitated cap-dependent protein translation. In addition, high dietary Pi significantly stimulated cell proliferation in the lungs of K-rasLA1 mice.
Conclusions: Our results showed that high dietary Pi promoted tumorigenesis and altered Akt signaling, thus suggesting that careful regulation of dietary Pi may be critical for lung cancer prevention as well as treatment.
PMCID: PMC2615662  PMID: 18849498
inorganic phosphate; lung tumorigenesis; Akt signaling
20.  Surrogate Decision-Makers' Perspectives on Discussing Prognosis in the Face of Uncertainty 
Rationale: Many physicians are reluctant to discuss a patient's prognosis when there is significant prognostic uncertainty.
Objectives: We sought to understand surrogate decision makers' views regarding whether physicians should discuss prognosis in the face of uncertainty.
Methods: We conducted semi-structured interviews with 179 surrogates for 142 incapacitated patients at high risk of death in four intensive care units at an academic medical center. The interviews explored surrogates' attitudes about whether physicians should discuss prognosis when they cannot be certain their prognostic estimates are correct. We used constant comparative methods to analyze the transcripts. Validation methods included triangulation by multidisciplinary analysis and member checking.
Measurements and Main Results: Eighty-seven percent (155/179) of surrogates wanted physicians to discuss an uncertain prognosis. We identified five main reasons for this, including surrogates' belief that prognostic uncertainty is unavoidable, that physicians are their only source for prognostic information, and that discussing prognostic uncertainty leaves room for realistic hope, increases surrogates' trust in the physician, and signals a need to prepare for possible bereavement. Twelve percent (22/179) of surrogates felt that discussions about an uncertain prognosis should be avoided. The main explanation was that it is not worth the potential emotional distress if the prognostications are incorrect. Surrogates suggested that physicians should explicitly discuss uncertainty when prognosticating.
Conclusions: The majority of surrogates of patients that are critically ill want physicians to disclose their prognostic estimates even if they cannot be certain they are correct. This stems from surrogates' belief that prognostic uncertainty is simultaneously unavoidable and acceptable.
PMCID: PMC2615661  PMID: 18931332
surrogate decision making; ethics; critical care; prognostication
21.  Integrating the MicroRNome into the Study of Lung Disease 
Over the last 15 years, investigators have identified small noncoding RNAs as regulators of gene expression. One type of noncoding RNAs are termed microRNAs (miRNAs). miRNAs are evolutionary conserved, approximately 22-nucleotide single-stranded RNAs that target genes by inducing mRNA degradation or by inhibiting translation. miRNAs are implicated in many critical cellular processes, including apoptosis, proliferation, and differentiation. Furthermore, it is estimated that miRNAs may be responsible for regulating the expression of nearly one-third of the human genome. Despite the identification of greater than 500 mature miRNAs, very little is known about their biological functions and functional targets. In the last 5 years, researchers have increasingly focused on the functional relevance and role that miRNAs play in the pathogenesis of human disease. miRNAs are known to be important in solid organ and hematological malignancies, heart disease, as potential modulators of the immune response, and organ development. It is anticipated that miRNA analysis will emerge as an important complement to proteomic and genomic studies to further our understanding of disease pathogenesis. Despite the application of genomics and proteomics to the study of human lung disease, few studies have examined miRNA expression. This perspective is not meant to be an exhaustive review of miRNA biology but will provide an overview of both miRNA biogenesis and our current understanding of the role of miRNAs in lung disease as well as a perspective on the importance of integrating this analysis as a tool for identifying and understanding the biological pathways in lung-disease pathogenesis.
PMCID: PMC2615660  PMID: 18787215
microRNA; epigenetics; genomics
22.  Rhinovirus Disrupts the Barrier Function of Polarized Airway Epithelial Cells 
Rationale: Secondary bacterial infection following rhinovirus (RV) infection has been recognized in chronic obstructive pulmonary disease.
Objectives: We sought to understand mechanisms by which RV infection facilitates secondary bacterial infection.
Methods: Primary human airway epithelial cells grown at air–liquid interface and human bronchial epithelial (16HBE14o-) cells grown as polarized monolayers were infected apically with RV. Transmigration of bacteria (nontypeable Haemophilus influenzae and others) was assessed by colony counting and transmission electron microscopy. Transepithelial resistance (RT) was measured by using a voltmeter. The distribution of zona occludins (ZO)-1 was determined by immunohistochemistry and immunoblotting.
Measurements and Main Results: Epithelial cells infected with RV showed 2-log more bound bacteria than sham-infected cultures, and bacteria were recovered from the basolateral media of RV- but not sham-infected cells. Infection of polarized airway epithelial cell cultures with RV for 24 hours caused a significant decrease in RT without causing cell death or apoptosis. Ultraviolet-treated RV did not decrease RT, suggesting a requirement for viral replication. Reduced RT was associated with increased paracellular permeability, as determined by flux of fluorescein isothiocyanate (FITC)-inulin. Neutralizing antibodies to tumor necrosis factor (TNF)-α, IFN-γ and IL-1β reversed corresponding cytokine-induced reductions in RT but not that induced by RV, indicating that the RV effect is independent of these proinflammatory cytokines. Confocal microscopy and immunoblotting revealed the loss of ZO-1 from tight junction complexes in RV-infected cells. Intranasal inoculation of mice with RV1B also caused the loss of ZO-1 from the bronchial epithelium tight junctions in vivo.
Conclusions: RV facilitates binding, translocation, and persistence of bacteria by disrupting airway epithelial barrier function.
PMCID: PMC2599868  PMID: 18787220
COPD; exacerbation; Haemophilus influenzae; tight junction; ZO-1
23.  Race-Ethnic Differences in Factors Associated with Inhaled Steroid Adherence among Adults with Asthma 
Rationale: Adherence to inhaled corticosteroid (ICS) medication is known to be low overall, but tends to be lower among African-American patients when compared with white patients.
Objectives: To understand the factors that contribute to ICS adherence among African-American and white adults with asthma.
Methods: Eligible individuals had a prior diagnosis of asthma, one or more ICS prescriptions, and were members of a large health maintenance organization in southeast Michigan. Individuals were sent a survey that included questions about internal factors (e.g., patient beliefs, knowledge, and motivation) and external factors (e.g., socioeconomic status, barriers to care, social support, and stressors) potentially related to ICS adherence. Adherence was calculated using electronic prescription and fill data. Stepwise regression was used to identify factors associated with adherence before and after stratifying by race-ethnicity.
Measurements and Main Results: Surveys were returned by 1,006 (56.3%) of 1,787 eligible patients. Adjusting for internal factors, but not external factors, diminished the relationship between race-ethnicity and ICS adherence. Among African-American patients, readiness to take ICS medication was the only internal or external factor significantly associated with ICS adherence; it explained 5.6% of the variance in adherence. Among white patients, perceived ICS necessity, ICS knowledge, doctors being perceived as the source of asthma control, and readiness to take medication were the internal factors associated with ICS adherence; these accounted for 19.8% of the variance in adherence.
Conclusions: Factors associated with ICS adherence appear to differ between African-American and white patients, suggesting that group-specific approaches are needed to improve adherence.
PMCID: PMC2599867  PMID: 18849496
medication adherence; inhaled corticosteroids; asthma; race-ethnicity; patient compliance
24.  Effects of Acute Administration of Corticosteroids during Mechanical Ventilation on Rat Diaphragm 
Rationale: Mechanical ventilation is known to induce ventilator-induced diaphragm dysfunction. Patients submitted to mechanical ventilation often receive massive doses of corticosteroids that may cause further deterioration of diaphragm function.
Objectives: To examine whether the combination of 24 hours of controlled mechanical ventilation with corticosteroid administration would exacerbate ventilator-induced diaphragm dysfunction.
Methods: Rats were randomly assigned to a group submitted to 24 hours of controlled mechanical ventilation receiving an intramuscular injection of saline or 80 mg/kg methylprednisolone, a group submitted to 24 hours of spontaneous breathing receiving saline, or methylprednisolone and a control group.
Measurements and Main Results: The diaphragm force–frequency curve was shifted downward in the mechanical ventilation group, but this deleterious effect was prevented when corticosteroids were administered. Diaphragm cross-sectional area of type I fibers was similarly decreased in both mechanical ventilation groups while atrophy of type IIx/b fibers was attenuated after corticosteroid administration. The mechanical ventilation-induced reduction in diaphragm MyoD and myogenin protein expression was attenuated after corticosteroids. Plasma cytokine levels were unchanged while diaphragm lipid hydroperoxides were similarly increased in both mechanical ventilation groups. Diaphragmatic calpain activity was significantly increased in the mechanical ventilation group, but calpain activation was abated with corticosteroid administration. Inverse correlations were found between calpain activity and diaphragm force.
Conclusions: A single high dose of methylprednisolone combined with controlled mechanical ventilation protected diaphragm function from the deleterious effects of controlled mechanical ventilation. Inhibition of the calpain system is most likely the mechanism by which corticosteroids induce this protective effect.
PMCID: PMC3266049  PMID: 18849500
respiratory muscles; steroids; proteolysis; calpain
25.  Smoking-related Genomic Signatures in Non–Small Cell Lung Cancer 
Rationale: Tobacco smoking is responsible for 85% of all lung cancers. To further our understanding of the molecular pathogenesis of lung cancer, we determined whether smoking history leads to the emergence of specific genomic alterations found in non–small cell lung cancer (NSCLC).
Objectives: To identify gene copy number alterations in NSCLCs associated with smoking history or DNA repair capacity.
Methods: Seventy-five NSCLCs were selected for this study from patients with current, none, or past smoking history, including pack year information. Tissue sections were microdissected, and DNA was extracted, purified, and labeled by random priming before hybridization onto bacterial artificial chromosome (BAC) arrays. Normalized ratios were correlated with smoking history and DNA repair capacity was measured by an in vitro lymphocyte assay in the same patients.
Measurements and Main Results: We identified smoking-related genomic signatures in NSCLCs that could be predicted with an overall 74% accuracy. Lung tumors arising from current-smokers had the greatest number of copy number alterations. The genomic regions most significantly associated with smoking were located within 60 regions and were functionally associated with genes controlling the M phase of the cell cycle, the segregation of chromosomes, and the methylation of DNA. Verification of the data is provided from data in the public domain and by quantitative real-time polymerase chain reaction. The associations between genomic abnormalities and DNA repair capacity did not reach statistical significance.
Conclusions: These findings indicate that smoking history leaves a specific genomic signature in the DNA of lung tumors and suggest that these alterations may reflect new molecular pathways to cancer development.
PMCID: PMC2720147  PMID: 18776155
array comparative genomic hybridization; tobacco; profile; microarray

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