Russell P. Bowler

The Role of Oxidative Stress in Chronic Obstructive Pulmonary Disease

Tobacco smoke is the number one risk factor for chronic obstructive pulmonary disease (COPD) and contains a high concentration of oxidants. The lung has a high concentration of antioxidants and antioxidant enzymes; however, COPD patients show evidence of increased oxidative stress suggesting that endogenous antioxidants may be insufficient to prevent oxidative damage from cigarette smoke. The consequences of increased oxidative stress in the lung include increased transcription of inflammatory genes, increased protease activity, and increased mucus secretion. Oxidative stress is often associated with impaired skeletal muscle function and may be one of the causes of glucocorticoid resistance. While current pharmacologic approaches to the treatment of chronic obstructive pulmonary disease do not commonly include antioxidants, preclinical studies involving animal models suggest that antioxidant superoxide dismutase mimetics offer a potential new therapeutic approach to the prevention and treatment of chronic obstructive pulmonary disease.

Cigarette Smoke Impairs Clearance of Apoptotic Cells through Oxidant-dependent Activation of RhoA

RATIONALE Cigarette smoke (CS) is the primary cause of chronic obstructive pulmonary disease (COPD), an effect that is, in part, due to intense oxidant stress. Clearance of apoptotic cells (efferocytosis) is a critical regulator of lung homeostasis, which is defective in smokers and in patients with COPD, suggesting a role in disease pathogenesis. OBJECTIVES We hypothesized that CS would impair efferocytosis through oxidant-dependent activation of RhoA, a known inhibitor of this process. METHODS We investigated the effect of CS on efferocytosis in vivo and ex vivo, using acute, subacute, and long-term mouse exposure models. MEASUREMENTS AND MAIN RESULTS Acute and subacute CS exposure suppressed efferocytosis by alveolar macrophages in a dose-dependent, reversible, and cell type-independent manner, whereas more intense CS exposure had an irreversible effect. In contrast, CS did not alter ingestion through the Fc gamma receptor. The inhibitory effect of CS on apoptotic cell clearance depended on oxidants, because the effect was blunted in oxidant-resistant ICR mice, and was prevented by either genetic or pharmacologic antioxidant strategies in vivo and ex vivo. CS inhibited efferocytosis through oxidant-dependent activation of the RhoA-Rho kinase pathway because (1) CS activated RhoA, (2) antioxidants prevented RhoA activation by CS, and (3) inhibitors of the RhoA-Rho kinase pathway reversed the suppressive effect of CS on apoptotic cell clearance in vivo and ex vivo. CONCLUSIONS These findings advance the hypothesis that impaired efferocytosis may contribute to the pathogenesis of COPD and suggest the therapeutic potential of drugs targeting the RhoA-Rho kinase pathway.

Function and regulation of SPLUNC1 protein in mycoplasma infection and allergic inflammation

Respiratory infections, including Mycoplasma pneumoniae (Mp), contribute to asthma pathobiology. To date, the mechanisms underlying the increased susceptibility of asthmatics to airway Mp infection remain unclear. Short palate, lung, and nasal epithelium clone 1 (SPLUNC1) protein is a recently described large airway epithelial cell-derived molecule that was predicted to exert host defense activities. However, SPLUNC1 function and regulation in an infectious or allergic milieu are still unknown. We determined host defense and anti-inflammatory functions of SPLUNC1 protein in Mp infection and the regulation of SPLUNC1 by Mp and allergic inflammation (e.g., IL-13). SPLUNC1 function was examined in Mp or human airway epithelial cell cultures by using SPLUNC1 recombinant protein, overexpression and RNA interference. Human and mouse bronchial epithelial SPLUNC1 was examined using immunostaining, Western blotting, ELISA, laser capture microdissection, and real-time PCR. Mouse models of Mp infection and allergic inflammation and air-liquid interface cultures of normal human primary bronchial epithelial cells were used to study SPLUNC1 regulation by Mp and IL-13. We found that: 1) SPLUNC1 protein decreased Mp levels and inhibited epithelial IL-8 production induced by Mp-derived lipoproteins; 2) normal human and mouse large airway epithelial cells expressed high levels of SPLUNC1; and 3) although Mp infection increased SPLUNC1, IL-13 significantly decreased SPLUNC1 expression and Mp clearance. Our results suggest that SPLUNC1 serves as a novel host defense protein against Mp and that an allergic setting markedly reduces SPLUNC1 expression, which may in part contribute to the persistent nature of bacterial infections in allergic airways.

Effects of metalloporphyrin catalytic antioxidants in experimental brain ischemia

Reactive oxygen species play a role in the response of brain to ischemia. The effects of metalloporphyrin catalytic antioxidants (AEOL 10113 and AEOL 10150) were examined after murine middle cerebral artery occlusion (MCAO). Ninety minutes after reperfusion from 90 min MCAO in the rat, AEOL 10113, AEOL 10150, or vehicle were given intracerebroventricularly. AEOL 10113 and AEOL 10150 similarly reduced infarct size (35%) and neurologic deficit. AEOL 10113 caused behavioral side effects at twice the neuroprotective dose while AEOL 10150 required a 15-fold increase from the neuroprotective dose to cause behavioral changes. AEOL 10150, given 6 h after 90 min MCAO, reduced total infarct size by 43% without temperature effects. Brain AEOL 10150 elimination t(1/2) was 10 h. In the mouse, intravenous AEOL 10150 infusion post-MCAO reduced both infarct size (25%) and neurologic deficit. Brain AEOL 10150 uptake, greater in the ischemic hemisphere, was dose- and time-dependent. AEOL 10150 had direct effects on proteomic events and ameliorated changes caused by ischemia. In primary mixed neuronal/glial cultures exposed to 2 h of O(2)/glucose deprivation, AEOL 10150 reduced lactate dehydrogenase release dose-dependently and selectively preserved aconitase activity in concentrations consistent with neuroprotection in vivo. AEOL 10150 is an effective neuroprotective compound offering a wide therapeutic window with a large margin of safety against adverse behavioral side effects.

A combined pulmonary -radiology workshop for visual evaluation of COPD: study design, chest CT findings and concordance with quantitative evaluation

Abstract The purposes of this study were: to describe chest CT findings in normal non-smoking controls and cigarette smokers with and without COPD; to compare the prevalence of CT abnormalities with severity of COPD; and to evaluate concordance between visual and quantitative chest CT (QCT) scoring. Methods: Volumetric inspiratory and expiratory CT scans of 294 subjects, including normal non-smokers, smokers without COPD, and smokers with GOLD Stage I-IV COPD, were scored at a multi-reader workshop using a standardized worksheet. There were 58 observers (33 pulmonologists, 25 radiologists); each scan was scored by 9–11 observers. Interobserver agreement was calculated using kappa statistic. Median score of visual observations was compared with QCT measurements. Results: Interobserver agreement was moderate for the presence or absence of emphysema and for the presence of panlobular emphysema; fair for the presence of centrilobular, paraseptal, and bullous emphysema subtypes and for the presence of bronchial wall thickening; and poor for gas trapping, centrilobular nodularity, mosaic attenuation, and bronchial dilation. Agreement was similar for radiologists and pulmonologists. The prevalence on CT readings of most abnormalities (e.g. emphysema, bronchial wall thickening, mosaic attenuation, expiratory gas trapping) increased significantly with greater COPD severity, while the prevalence of centrilobular nodularity decreased. Concordances between visual scoring and quantitative scoring of emphysema, gas trapping and airway wall thickening were 75%, 87% and 65%, respectively. Conclusions: Despite substantial inter-observer variation, visual assessment of chest CT scans in cigarette smokers provides information regarding lung disease severity; visual scoring may be complementary to quantitative evaluation.

Superoxide Dismutase 3 Polymorphism Associated with Reduced Lung Function in Two Large Populations

RATIONALE Superoxide dismutase (SOD) 3 inhibits oxidative fragmentation of lung matrix components collagen I, hyaluronan, and heparan sulfate. Inherited change in SOD3 expression or function could affect lung matrix homeostasis and influence pulmonary function. OBJECTIVES To identify novel SOD3 polymorphisms that are associated with lung function or chronic obstructive pulmonary disease (COPD). METHODS Resequencing of 182 individuals identified two novel polymorphisms, E1 (rs8192287) and I1 (rs8192288), in a conserved region of the SOD3 gene of potential relationship to lung function. We next genotyped 9,093 individuals from the Copenhagen City Heart Study for the polymorphisms and recorded spirometry, and admissions and deaths due to COPD during 26-year follow-up. Finally, we validated our findings in a cross-sectional analysis of 35,635 individuals from the Copenhagen General Population Study. MEASUREMENTS AND MAIN RESULTS Genotyping the Copenhagen City Heart Study identified 35 E1/I1 homozygotes, 1,050 heterozygotes, and 8,008 noncarriers (Hardy-Weinberg equilibrium: P = 0.93). Using quadruple lung function measurements, we found that E1/I1 homozygotes had 7% lower FVC % predicted (P = 0.006) and 4% lower FEV(1) % predicted (P = 0.12) compared with noncarriers. In the Copenhagen General Population Study, E1/I1 homozygotes also had lower FVC % predicted than noncarriers (P = 0.03), confirming an association between E1/I1 genotype and reduced lung function. E1/I1 homozygotes had adjusted hazard ratios for COPD hospitalization and COPD mortality of 2.5 (95% confidence interval, 1.0-5.9) and 3.7 (95% confidence interval, 0.9-15), respectively; the results were independent of influence from the R213G allele of the SOD3 gene. CONCLUSIONS We identified two novel polymorphisms in a conserved region of the SOD3 gene and show that individuals that are homozygous for these polymorphisms have reduced FVC % predicted in two large, population-based studies.

Genome-wide association study of smoking behaviours in patients with COPD

Background Cigarette smoking is a major risk factor for chronic obstructive pulmonary disease (COPD) and COPD severity. Previous genome-wide association studies (GWAS) have identified numerous single nucleotide polymorphisms (SNPs) associated with the number of cigarettes smoked per day (CPD) and a dopamine beta-hydroxylase (DBH) locus associated with smoking cessation in multiple populations. Objective To identify SNPs associated with lifetime average and current CPD, age at smoking initiation, and smoking cessation in patients with COPD. Methods GWAS were conducted in four independent cohorts encompassing 3441 ever-smoking patients with COPD (Global Initiative for Obstructive Lung Disease stage II or higher). Untyped SNPs were imputed using the HapMap (phase II) panel. Results from all cohorts were meta-analysed. Results Several SNPs near the HLA region on chromosome 6p21 and in an intergenic region on chromosome 2q21 showed associations with age at smoking initiation, both with the lowest p=2×10−7. No SNPs were associated with lifetime average CPD, current CPD or smoking cessation with p<10−6. Nominally significant associations with candidate SNPs within cholinergic receptors, nicotinic, alpha 3/5 (CHRNA3/CHRNA5; eg, p=0.00011 for SNP rs1051730) and cytochrome P450, family 2, subfamily A, polypeptide 6 (CYP2A6; eg, p=2.78×10−5 for a non-synonymous SNP rs1801272) regions were observed for lifetime average CPD, however only CYP2A6 showed evidence of significant association with current CPD. A candidate SNP (rs3025343) in DBH was significantly (p=0.015) associated with smoking cessation. Conclusion The authors identified two candidate regions associated with age at smoking initiation in patients with COPD. Associations of CHRNA3/CHRNA5 and CYP2A6 loci with CPD and DBH with smoking cessation are also likely of importance in the smoking behaviours of patients with COPD.

Phenotypes of Chronic Obstructive Pulmonary Disease

The current clinical classification of smoking-related lung disease fails to take into account the heterogeneity of chronic obstructive pulmonary disease (COPD). With an increased understanding of pathophysiologic variation, COPD now clearly represents a spectrum of overlapping diseases with important extrapulmonary consequences. A “phenotype” describes the outward physical manifestations of a particular disease, and compromises anything that is part of the observable structure, function or behavior of an individual. Such phenotypic distinctions in COPD include: frequent exacerbator, pulmonary cachectic, rapid decliner, airways hyperresponsiveness, impaired exercise tolerance, and emphysema versus airways disease. These variable manifestations, each with unique prognostic, clinical and physiologic ramifications, represent distinct phenotypes within COPD. While all of these phenotypes have smoking as a common risk factor, the other risk factors that determine these phenotypes remain poorly understood. An individual smoker has variable expression of each phenotype and there is mounting evidence that COPD phenotypes have different clinical outcomes. These phenotypes can be broadly classified into one of three groups: clinical, physiologic and radiographic. This review presents the evidence for the spectrum of COPD phenotypes with a focused discussion on the pathophysiologic, epidemiologic and clinical significance of each subtype.

Computed Tomographic Measures of Pulmonary Vascular Morphology in Smokers and Their Clinical Implications

RATIONALE Angiographic investigation suggests that pulmonary vascular remodeling in smokers is characterized by distal pruning of the blood vessels. OBJECTIVES Using volumetric computed tomography scans of the chest we sought to quantitatively evaluate this process and assess its clinical associations. METHODS Pulmonary vessels were automatically identified, segmented, and measured. Total blood vessel volume (TBV) and the aggregate vessel volume for vessels less than 5 mm(2) (BV5) were calculated for all lobes. The lobe-specific BV5 measures were normalized to the TBV of that lobe and the nonvascular tissue volume (BV5/T(issue)V) to calculate lobe-specific BV5/TBV and BV5/T(issue)V ratios. Densitometric measures of emphysema were obtained using a Hounsfield unit threshold of -950 (%LAA-950). Measures of chronic obstructive pulmonary disease severity included single breath measures of diffusing capacity of carbon monoxide, oxygen saturation, the 6-minute-walk distance, St Georges Respiratory Questionnaire total score (SGRQ), and the body mass index, airflow obstruction, dyspnea, and exercise capacity (BODE) index. MEASUREMENTS AND MAIN RESULTS The %LAA-950 was inversely related to all calculated vascular ratios. In multivariate models including age, sex, and %LAA-950, lobe-specific measurements of BV5/TBV were directly related to resting oxygen saturation and inversely associated with both the SGRQ and BODE scores. In similar multivariate adjustment lobe-specific BV5/T(issue)V ratios were inversely related to resting oxygen saturation, diffusing capacity of carbon monoxide, 6-minute-walk distance, and directly related to the SGRQ and BODE. CONCLUSIONS Smoking-related chronic obstructive pulmonary disease is characterized by distal pruning of the small blood vessels (<5 mm(2)) and loss of tissue in excess of the vasculature. The magnitude of these changes predicts the clinical severity of disease.

Furin Proteolytically Processes the Heparin-binding Region of Extracellular Superoxide Dismutase

Extracellular superoxide dismutase (EC-SOD) is an antioxidant enzyme that attenuates brain and lung injury from oxidative stress. A polybasic region in the carboxyl terminus distinguishes EC-SOD from other superoxide dismutases and determines EC-SODs tissue half-life and affinity for heparin. There are two types of EC-SOD that differ based on the presence or absence of this heparin-binding region. It has recently been shown that proteolytic removal of the heparin-binding region is an intracellular event (Enghild, J. J., Thogersen, I. B., Oury, T. D., Valnickova, Z., Hojrup, P., and Crapo, J. D. (1999) J. Biol. Chem. 274, 14818–14822). By using mammalian cell lines, we have now determined that removal of the heparin-binding region occurs after passage through the Golgi network but before being secreted into the extracellular space. Specific protease inhibitors and overexpression of intracellular proteases implicate furin as a processing protease. In vitro experiments using furin and purified EC-SOD suggest that furin proteolytically cleaves EC-SOD in the middle of the polybasic region and then requires an additional carboxypeptidase to remove the remaining lysines and arginines. A mutation in Arg213renders EC-SOD resistant to furin processing. These results indicate that furin-dependent processing of EC-SOD is important for determining the tissue distribution and half-life of EC-SOD.