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Rationale: Nuclear factor erythroid 2–related factor 2 (Nrf2), an important regulator of lung antioxidant defenses, declines in chronic obstructive pulmonary disease (COPD). However, Nrf2 also regulates the proteasome system that degrades damaged and misfolded proteins. Because accumulation of misfolded proteins in the endoplasmic reticulum (ER) causes ER stress and ER stress-induced apoptosis, Nrf2 may potentially prevent ER stress-mediated apoptosis in COPD.

Objectives: To determine whether Nrf2-regulated proteasome function affects ER stress-mediated apoptosis in COPD.

Methods: We assessed the expression of Nrf2, Nrf2-dependent proteasomal subunits, proteasomal activity, markers of ER stress, and apoptosis in emphysematous lungs of mice exposed to cigarette smoke (CS) as well as peripheral lung tissues from normal control subjects and patients with COPD.

Measurements and Main Results: Compared with wild-type mice, emphysematous lungs of CS-exposed Nrf2-deficient mice exhibited markedly lower proteasomal activity and elevated markers of ER stress and apoptosis. Furthermore, compared with normal control subjects, lungs of patients with mild and advanced COPD showed a marked decrease in the expression of Nrf2-regulated proteasomal subunits and total proteasomal activity. However, they were associated with greater levels of ER stress and apoptosis markers. In vitro studies have demonstrated that enhancing proteasomal activity in Beas2B cells either by sulforaphane, an activator of Nrf2, or overexpression of Nrf2-regulated proteasomal subunit PSMB6, significantly inhibited cigarette smoke condensate (CSC)-induced ER stress and cell death.

Conclusions: Impaired Nrf2 signaling causes significant decline in proteasomal activity and heightens ER stress response in lungs of patients with COPD and CS-exposed mice. Accordingly, pharmacological approaches that augment Nrf2 activity may protect against COPD progression by both up-regulating antioxidant defenses and relieving ER stress.

Exposure to cigarette smoke (CS) is the primary factor associated with the development of chronic obstructive pulmonary disease (COPD). CS increases the level of oxidants in the lungs, resulting in a depletion of antioxidants, which promotes oxidative stress and the destruction of alveolar tissue. In response to CS, pulmonary epithelial cells counteract increased levels of oxidants by activating Nrf2-dependent pathways to augment the expression of detoxification and antioxidant enzymes, thereby protecting the lung from injury. We hypothesize that increasing the pathways activated by Nrf2 will afford protection against CS-induced lung damage. To this end we have developed a novel mouse model in which the cytosolic inhibitor of Nrf2, Keap1, is genetically deleted in Clara cells, which predominate in the upper airways in mice. Deletion of Keap1 in Clara cells resulted in increased expression of Nrf2-dependent genes, such as Nqo1 and Gclm, as determined by microarray analysis and quantitative PCR. Deletion of Keap1 in airway epithelium decreased Keap1 protein levels and significantly increased the total level of glutathione in the lungs. Increased Nrf2 activation protected Clara cells against oxidative stress ex vivo and attenuated oxidative stress and CS-induced inflammation in vivo. Expression of KEAP1 was also decreased in human epithelial cells through siRNA transfection, which increased the expression of Nrf2-dependent genes and attenuated oxidative stress. In conclusion, activating Nrf2 pathways in tissue-specific Keap1 knockout mice represents an important genetic approach against oxidant-induced lung damage.

Oxidative stress results in protein oxidation and is involved in the pathogenesis of lung diseases such as chronic obstructive pulmonary disorder (COPD). Sulfiredoxin-1 (Srx1) catalyzes reduction of cysteine sulfinic acid to sulfenic acid in oxidized proteins and protects them from inactivation. This study examined the mechanism of transcriptional regulation of Srx1 and its possible protective role during oxidative stress associated with COPD. Nrf2, a transcription factor known to influence susceptibility to pulmonary diseases, upregulates Srx1 expression during oxidative stress caused by cigarette smoke exposure in the lungs of mice. Disruption of Nrf2 signaling by genetic knockout in mice or RNAi in cells downregulated the expression of Srx1. In silico analysis of the 5′-promoter flanking region of Srx1 identified multiple antioxidant response elements that are highly conserved. Reporter and chromatin-immunoprecipation assays demonstrated that ARE1 at −228 is critical for the Nrf2-mediated response. Attenuation of Srx1 expression with RNAi potentiated the toxicity of hydrogen peroxide (H2O2), whereas overexpression of Srx1 protected against H2O2 mediated cell death in vitro. Immunoblot analysis revealed dramatic decreases in Srx1 expression in lungs from patients with COPD relative to non-emphysematous lungs together with a decline in Nrf2 protein. Thus, Srx1, a key Nrf2-regulated gene, contributes to protection against oxidative injury in the lung.

Although inflammation and protease/antiprotease imbalance have been postulated to be critical in cigarette smoke–induced (CS-induced) emphysema, oxidative stress has been suspected to play an important role in chronic obstructive pulmonary diseases. Susceptibility of the lung to oxidative injury, such as that originating from inhalation of CS, depends largely on its upregulation of antioxidant systems. Nuclear factor, erythroid-derived 2, like 2 (Nrf2) is a redox-sensitive basic leucine zipper protein transcription factor that is involved in the regulation of many detoxification and antioxidant genes. Disruption of the Nrf2 gene in mice led to earlier-onset and more extensive CS-induced emphysema than was found in wild-type littermates. Emphysema in Nrf2-deficient mice exposed to CS for 6 months was associated with more pronounced bronchoalveolar inflammation; with enhanced alveolar expression of 8-oxo-7,8-dihydro-2′-deoxyguanosine, a marker of oxidative stress; and with an increased number of apoptotic alveolar septal cells — predominantly endothelial and type II epithelial cells — as compared with wild-type mice. Microarray analysis identified the expression of nearly 50 Nrf2-dependent antioxidant and cytoprotective genes in the lung that may work in concert to counteract CS-induced oxidative stress and inflammation. The responsiveness of the Nrf2 pathway may act as a major determinant of susceptibility to tobacco smoke–induced emphysema by upregulating antioxidant defenses and decreasing lung inflammation and alveolar cell apoptosis.

Chronic obstructive pulmonary disease (COPD) is a global health problem, and current therapy for COPD is poorly effective and the mainstays of pharmacotherapy are bronchodilators. A better understanding of the pathobiology of COPD is critical for the development of novel therapies. In the present review, we have discussed the roles of oxidative/aldehyde stress, inflammation/immunity, and chromatin remodeling in the pathogenesis of COPD. Imbalance of oxidant/antioxidant balance caused by cigarette smoke and other pollutants/biomass fuels plays an important role in the pathogenesis of COPD by regulating redox-sensitive transcription factors (e.g. NF-κB), autophagy and unfolded protein response leading to chronic lung inflammatory response. Cigarette smoke also activates canonical/alternative NF-κB pathways and their upstream kinases leading to sustained inflammatory response in lungs. Recently, epigenetic regulation has been shown to be critical for the development of COPD because the expression/activity of enzymes that regulate these epigenetic modifications have been reported to be abnormal in airways of COPD patients. Hence, the significant advances made in understanding the pathophysiology of COPD as described herein will identify novel therapeutic targets for intervening COPD.

Background

Oxidative stress plays a potential role in the pathogenesis and progression of chronic obstructive pulmonary disease (COPD). Glutathione S-transferases (GSTs) detoxify toxic compounds in tobacco smoke via glutathione-dependent mechanisms. Little is known about the regulation and expression of GSTs in COPD lung and their presence in airway secretions.

Methods

GST alpha, pi and mu were investigated by immunohistochemistry in 72 lung tissue specimens and by Western analysis in total lung homogenates and induced sputum supernatants from non-smokers, smokers and patients with variable stages of COPD severity.

Results

GST alpha was expressed mainly in the airway epithelium. The percentage of GST alpha positive epithelial cells was lower in the central airways of patients with very severe (Stage IV) COPD compared to mild/moderate COPD (p = 0.02). GST alpha by Western analysis was higher in the total lung homogenates in mild/moderate COPD compared to cases of very severe disease (p < 0.001). GST pi was present in airway and alveolar epithelium as well as in alveolar macrophages. GST mu was expressed mainly in the epithelium. Both GST alpha and pi were detectable in sputum supernatants especially in patients with COPD.

Conclusion

This study indicates the presence of GST alpha and pi especially in the epithelium and sputum supernatants in mild/moderate COPD and low expression of GST alpha in the epithelium in cases of very severe COPD. The presence of GSTs in the airway secretions points to their potential protective role both as intracellular and extracellular mediators in human lung.

Research highlights

► Nrf2 anti-oxidant function is impaired when HDAC activity is inhibited. ► HDAC inhibition decreases Nrf2 protein stability. ► HDAC2 is involved in reduced Nrf2 stability and both correlate in COPD samples. ► HDAC inhibition increases Nrf2 acetylation.

Nuclear factor erythroid 2-related factor 2 (Nrf2) plays a crucial role in cellular defence against oxidative stress by inducing the expression of multiple anti-oxidant genes. However, where high levels of oxidative stress are observed, such as chronic obstructive pulmonary disease (COPD), Nrf2 activity is reduced, although the molecular mechanism for this defect is uncertain. Here, we show that down-regulation of histone deacetylase (HDAC) 2 causes Nrf2 instability, resulting in reduced anti-oxidant gene expression and increase sensitivity to oxidative stress. Although Nrf2 protein was clearly stabilized after hydrogen peroxide (H2O2) stimulation in a bronchial epithelial cell line (BEAS2B), Nrf2 stability was decreased and Nrf2 acetylation increased in the presence of an HDAC inhibitor, trichostatin A (TSA). TSA also reduced Nrf2-regulated heme-oxygenase-1 (HO-1) expression in these cells, and this was confirmed in acute cigarette-smoke exposed mice in vivo. HDAC2 knock-down by RNA interference resulted in reduced H2O2-induced Nrf2 protein stability and activity in BEAS2B cells, whereas HDAC1 knockdown had no effect. Furthermore, monocyte-derived macrophages obtained from healthy volunteers (non-smokers and smokers) and COPD patients showed a significant correlation between HDAC2 expression and Nrf2 expression (r = 0.92, p < 0.0001). Thus, reduced HDAC2 activity in COPD may account for increased Nrf2 acetylation, reduced Nrf2 stability and impaired anti oxidant defences.

BACKGROUND—It has been suggested that oxidative stress is an important factor in the pathogenesis of chronic obstructive pulmonary disease (COPD). We have shown that an oxidant/antioxidant imbalance occurs in the distal air spaces of smokers and in patients with COPD which is reflected systemically in the plasma. A study was undertaken to determine whether plasma antioxidant status correlated with lung function as assessed by forced expiratory volume in one second (FEV1) and forced vital capacity (FVC) in smokers and patients with COPD.
METHODS—Plasma antioxidant capacity, assessed by the Trolox equivalent antioxidant capacity (TEAC) as an index of overall systemic oxidative stress, and protein thiol levels were measured in 95patients with stable COPD, in 82 healthy smokers, and in 37 healthy non-smokers.
RESULTS—Mean (SE) plasma TEAC levels were significantly decreased in patients with COPD (0.81 (0.03) mmol/l, p<0.001) and in healthy smokers (0.87 (0.04) mmol/l, p<0.001) compared with healthy non-smokers (1.31 (0.11) mmol/l). The mean differences in plasma antioxidant capacity (mM) were (0.81, 95% confidence interval (CI) 0.22 to 1.48), (0.87, 95% CI 0.2 to 1.46), and (1.31, 95% CI 1.09 to 1.58) in patients with COPD, healthy smokers, and healthy non-smokers, respectively. This reduction was associated with a 29% (95% CI 18 to 38) and a 30% (95% CI 19 to 40) decrease in plasma protein thiol levels in COPD patients and smokers, respectively. Current smoking was not the main contributor to the reduction in antioxidant capacity in patients with COPD as those patients who were still smokers had similar TEAC levels (mean (SE) 0.78 (0.05); n = 25) to those who had stopped smoking (0.84 (0.02); n = 70). No significant correlations were found between spirometric data measured as FEV1 % predicted or FEV1/FVC % predicted and the plasma levels of TEAC in patients with COPD, healthy smokers, or healthy non-smokers. Similarly, there was no significant correlation between FEV1 %predicted or FEV1/FVC % predicted and the levels of plasma protein thiols in the three groups.
CONCLUSIONS—These data confirm decreased antioxidant capacity in smokers and patients with COPD, indicating the presence of systemic oxidative stress. However, no relationship was found between protein thiols or TEAC levels and measurements of airflow limitation in either smokers or in patients with COPD.



Background

Oxidative Stress contributes to the pathogenesis of many diseases. The NRF2/KEAP1 axis is a key transcriptional regulator of the anti-oxidant response in cells. Nrf2 knockout mice have implicated this pathway in regulating inflammatory airway diseases such as asthma and COPD. To better understand the role the NRF2 pathway has on respiratory disease we have taken a novel approach to define NRF2 dependent gene expression in a relevant lung system.

Methods

Normal human lung fibroblasts were transfected with siRNA specific for NRF2 or KEAP1. Gene expression changes were measured at 30 and 48 hours using a custom Affymetrix Gene array. Changes in Eotaxin-1 gene expression and protein secretion were further measured under various inflammatory conditions with siRNAs and pharmacological tools.

Results

An anti-correlated gene set (inversely regulated by NRF2 and KEAP1 RNAi) that reflects specific NRF2 regulated genes was identified. Gene annotations show that NRF2-mediated oxidative stress response is the most significantly regulated pathway, followed by heme metabolism, metabolism of xenobiotics by Cytochrome P450 and O-glycan biosynthesis. Unexpectedly the key eosinophil chemokine Eotaxin-1/CCL11 was found to be up-regulated when NRF2 was inhibited and down-regulated when KEAP1 was inhibited. This transcriptional regulation leads to modulation of Eotaxin-1 secretion from human lung fibroblasts under basal and inflammatory conditions, and is specific to Eotaxin-1 as NRF2 or KEAP1 knockdown had no effect on the secretion of a set of other chemokines and cytokines. Furthermore, the known NRF2 small molecule activators CDDO and Sulphoraphane can also dose dependently inhibit Eotaxin-1 release from human lung fibroblasts.

Conclusions

These data uncover a previously unknown role for NRF2 in regulating Eotaxin-1 expression and further the mechanistic understanding of this pathway in modulating inflammatory lung disease.

Rationale: Abnormal inflammation and accelerated decline in lung function occur in patients with chronic obstructive pulmonary disease (COPD). Human sirtuin (SIRT1), an antiaging and antiinflammatory protein, is a metabolic NAD+-dependent protein/histone deacetylase that regulates proinflammatory mediators by deacetylating histone and nonhistone proteins.

Objectives: To determine the expression of SIRT1 in lungs of smokers and patients with COPD, and to elucidate the regulation of SIRT1 in response to cigarette smoke in macrophages, and its impact on nuclear factor (NF)-κB regulation.

Methods: SIRT1 and NF-κB levels were assessed in lung samples of nonsmokers, smokers, and patients with COPD. Human monocyte–macrophage cells (MonoMac6) were treated with cigarette smoke extract (CSE) to determine the mechanism of CSE-mediated regulation of SIRT1 and its involvement in RelA/p65 regulation and IL-8 release.

Measurements and Main Results: Peripheral lungs of smokers and patients with COPD showed decreased levels of nuclear SIRT1, as compared with nonsmokers, associated with its post-translational modifications (formation of nitrotyrosine and aldehyde carbonyl adducts). Treatment of MonoMac6 cells with CSE showed decreased levels of SIRT1 associated with increased acetylation of RelA/p65 NF-κB. Mutation or knockdown of SIRT1 resulted in increased acetylation of nuclear RelA/p65 and IL-8 release, whereas overexpression of SIRT1 decreased IL-8 release in response to CSE treatment in MonoMac6 cells.

Conclusions: SIRT1 levels were reduced in macrophages and lungs of smokers and patients with COPD due to its post-translational modifications by cigarette smoke–derived reactive components, leading to increased acetylation of RelA/p65. Thus, SIRT1 plays a pivotal role in regulation of NF-κB–dependent proinflammatory mediators in lungs of smokers and patients with COPD.

Nrf2 is the key transcription factor regulating the antioxidant response. Nrf2 signaling is repressed by Keap1 at basal condition and induced by oxidative stress. Keap1 is recently identified as a Cullin 3-dependent substrate adaptor protein. A two-sites binding “hinge & latch” model vividly depicts how Keap1 can efficiently present Nrf2 as substrate for ubiquitination. Oxidative perturbation can impede Keap1-mediated Nrf2 ubiquitination but fail to disrupt Nrf2/Keap1 binding. Nrf2 per se is a redox-sensitive transcripon factor. A new Nrf2-mediated redox signaling model is proposed based on these new discoveries. Free floating Nrf2 protein functions as a redox-sensitive probe. Keap1 instead functions as a gate keeper to control the availability of Nrf2 probes and thus regulates the overall sensitivity of the redox signaling.

Lung cancer (LC) and chronic obstructive pulmonary disease (COPD) commonly coexist in smokers, and the presence of COPD increases the risk of developing LC. Cigarette smoke causes oxidative stress and an inflammatory response in lung cells, which in turn may be involved in COPD and lung cancer development. The aim of this study was to identify differential proteomic profiles related to oxidative stress response that were potentially involved in these two pathological entities. Protein content was assessed in the bronchoalveolar lavage (BAL) of 60 patients classified in four groups: COPD, COPD and LC, LC, and control (neither COPD nor LC). Proteins were separated into spots by two dimensional polyacrylamide gel electrophoresis (2D-PAGE) and examined by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF/TOF). A total of 16 oxidative stress regulatory proteins were differentially expressed in BAL samples from LC and/or COPD patients as compared with the control group. A distinct proteomic reactive oxygen species (ROS) protein signature emerged that characterized lung cancer and COPD. In conclusion, our findings highlight the role of the oxidative stress response proteins in the pathogenic pathways of both diseases, and provide new candidate biomarkers and predictive tools for LC and COPD diagnosis.

The transcription factor Nrf2 regulates cellular redox homeostasis. Under basal conditions, Keap1 recruits Nrf2 into the Cul3-containing E3 ubiquitin ligase complex for ubiquitin conjugation and subsequent proteasomal degradation. Oxidative stress triggers activation of Nrf2 through inhibition of E3 ubiquitin ligase activity, resulting in increased levels of Nrf2 and transcriptional activation of Nrf2-dependent genes. In this study, we identify Keap1 as a key postinduction repressor of Nrf2 and demonstrate that a nuclear export sequence (NES) in Keap1 is required for termination of Nrf2-antioxidant response element (ARE) signaling by escorting nuclear export of Nrf2. We provide evidence that ubiquitination of Nrf2 is carried out in the cytosol. Furthermore, we show that Keap1 nuclear translocation is independent of Nrf2 and the Nrf2-Keap1 complex does not bind the ARE. Collectively, our results suggest the following mechanism of postinduction repression: upon recovery of cellular redox homeostasis, Keap1 translocates into the nucleus to dissociate Nrf2 from the ARE. The Nrf2-Keap1 complex is then transported out of the nucleus by the NES in Keap1. Once in the cytoplasm, the Keap1-Nrf2 complex associates with the E3 ubiquitin ligase, resulting in degradation of Nrf2 and termination of the Nrf2 signaling pathway. Hence, postinduction repression of the Nrf2-mediated antioxidant response is controlled by the nuclear export function of Keap1 in alliance with the cytoplasmic ubiquitination and degradation machinery.

Background

One typical feature in chronic obstructive pulmonary disease (COPD) is the disturbance of the oxidant/antioxidant balance. Glutaredoxins (Grx) are thiol disulfide oxido-reductases with antioxidant capacity and catalytic functions closely associated with glutathione, the major small molecular weight antioxidant of human lung. However, the role of Grxs in smoking related diseases is unclear.

Methods

Immunohistochemical and Western blot analyses were conducted with lung specimens (n = 45 and n = 32, respectively) and induced sputum (n = 50) of healthy non-smokers and smokers without COPD and at different stages of COPD.

Results

Grx1 was expressed mainly in alveolar macrophages. The percentage of Grx1 positive macrophages was significantly lower in GOLD stage IV COPD than in healthy smokers (p = 0.021) and the level of Grx1 in total lung homogenate decreased both in stage I–II (p = 0.045) and stage IV COPD (p = 0.022). The percentage of Grx1 positive macrophages correlated with the lung function parameters (FEV1, r = 0.45, p = 0.008; FEV1%, r = 0.46, p = 0.007, FEV/FVC%, r = 0.55, p = 0.001). Grx1 could also be detected in sputum supernatants, the levels being increased in the supernatants from acute exacerbations of COPD compared to non-smokers (p = 0.013) and smokers (p = 0.051).

Conclusion

The present cross-sectional study showed that Grx1 was expressed mainly in alveolar macrophages, the levels being decreased in COPD patients. In addition, the results also demonstrated the presence of Grx1 in extracellular fluids including sputum supernatants. Overall, the present study suggests that Grx1 is a potential redox modulatory protein regulating the intracellular as well as extracellular homeostasis of glutathionylated proteins and GSH in human lung.

Influenza virus is a common respiratory tract viral infection. Although influenza can be fatal in patients with chronic pulmonary diseases such as chronic obstructive pulmonary disease, its pathogenesis is not fully understood. The Nrf2-mediated antioxidant system is essential to protect the lungs from oxidative injury and inflammation. In the present study, we investigated the role of Nrf2 in protection against influenza virus-induced pulmonary inflammation after cigarette smoke exposure with both in vitro and in vivo approaches. For in vitro analyses, peritoneal macrophages isolated from wild-type and Nrf2-deficient mice were treated with poly(I:C) and/or cigarette smoke extract. For in vivo analysis, these mice were infected with influenza A virus with or without exposure to cigarette smoke. In Nrf2-deficient macrophages, NF-κB activation and the induction of its target inflammatory genes were enhanced after costimulation with cigarette smoke extract and poly(I:C) compared with wild-type macrophages. The induction of antioxidant genes was observed for the lungs of wild-type mice but not those of Nrf2-deficient mice after cigarette smoke exposure. Cigarette smoke-exposed Nrf2-deficient mice showed higher rates of mortality than did wild-type mice after influenza virus infection, with enhanced peribronchial inflammation, lung permeability damage, and mucus hypersecretion. Lung oxidant levels and NF-κB-mediated inflammatory gene expression in the lungs were also enhanced in Nrf2-deficient mice. Our data indicate that the antioxidant pathway controlled by Nrf2 is pivotal for protection against the development of influenza virus-induced pulmonary inflammation and injury under oxidative conditions.

Redox imbalance has been implicated in the pathogenesis of many acute and chronic lung diseases. The b-Zip transcription factor Nrf2 acts via an antioxidant/electrophilic response element to regulate antioxidants and maintain cellular redox homeostasis. Our previous studies have shown that Nrf2-deficient mice (Nrf2−/−) show reduced pulmonary expression of several antioxidant enzymes, which renders them highly susceptible to hyperoxia-induced lung injury. To better understand the physiologic significance of Nrf2-induced redox signaling, we have used primary cells isolated from the lungs of Nrf2+/+ and Nrf2−/− mice. Our studies were focused on type II cells because these cells are constantly exposed to the oxidant environment and play key roles in host defense, injury, and repair processes. Using this system, we now report that an Nrf2 deficiency leads to defects in type II cell proliferation and greatly enhances the cells' sensitivity to oxidant-induced cell death. These defects were closely associated with high levels of reactive oxygen species (ROS) and redox imbalance in Nrf2−/− cells. Glutathione (GSH) supplementation rescued these phenotypic defects associated with the Nrf2 deficiency. Intriguingly, although the antioxidant N-acetyl-cysteine drastically squelched ROS levels, it was unable to counteract growth arrest in Nrf2−/− cells. Moreover, despite their elevated levels of ROS, Nrf2−/− type II cells were viable and, like their wild-type counterparts, exhibited normal differentiation characteristics. Our data suggest that dysfunctional Nrf2-regulated GSH-induced signaling is associated with deregulation of type II cell proliferation, which contributes to abnormal injury and repair and leads to respiratory impairment.

Purpose:

Cigarette smoking and advanced age are well known as risk factors for chronic obstructive pulmonary disease (COPD), and nutritional abnormalities are important in patients with COPD. However, little is known about the nutritional status in non-COPD aging men with smoking history. We therefore investigated whether reduced lung function is associated with lower blood markers of nutritional status in those men.

Subjects and methods:

This association was examined in a cross-sectional study of 65 Japanese male current or former smokers aged 50 to 80 years: 48 without COPD (non-COPD group), divided into tertiles according to forced expiratory volume in one second as percent of forced vital capacity (FEV1/FVC), and 17 with COPD (COPD group).

Results:

After adjustment for potential confounders, lower FEV1/FVC was significantly associated with lower red blood cell count (RBCc), hemoglobin, and total protein (TP); not with total energy intake. The difference in adjusted RBCc and TP among the non-COPD group tertiles was greater than that between the bottom tertile in the non-COPD group and the COPD group.

Conclusion:

In non-COPD aging men with smoking history, trends toward reduced nutritional status and anemia may independently emerge in blood components along with decreased lung function even before COPD onset.

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 Fcγ 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.

Cigarette smoke causes significant oxidant stress which is further enhanced by recruitment and activation of inflammatory cells to the lung. Polymorphisms in some detoxification enzymes are thought to increase the risk of developing chronic obstructive pulmonary disease (COPD), but the ultimate role of genetic variability in antioxidant and/or detoxification enzymes in COPD remains obscure. Some antioxidant enzymes are inducted, but the extent of induction is insufficient to protect the lung/alveolar epithelium against cigarette smoke. Exogenous antioxidants such as vitamins do not seem to protect against cigarette smoke related lung injury. Glutathione related synthetic drugs such as N-acetylcysteine have shown some benefits, but they may have pro-oxidant side effects. Synthetic compounds with superoxide dismutase and catalase activities have shown promising results in animal models against a variety of oxidant exposures including cigarette smoke in the lung. These results are in agreement with studies highlighting the importance of alveolar antioxidant protection mechanisms in oxidant stress and their inducibility. These new drugs need to be tested in cigarette smoking related lung injury/inflammation since inflammation/oxidant stress can continue after discontinuation of smoking.

Chronic obstructive pulmonary disease (COPD) is associated with high incidence of morbidity and mortality. Oxidative stress is intimately associated with the progression and exacerbation of COPD and therefore targeting oxidative stress with antioxidants or boosting the endogenous levels of antioxidants is likely to have beneficial outcome in the treatment of COPD. Among the various antioxidants tried so far, thiol antioxidants and mucolytic agents, such as glutathione, N-acetyl-L-cysteine, N-acystelyn, erdosteine, fudosteine, and carbocysteine; Nrf2 activators, and dietary polyphenols (curcumin, resveratrol, green tea, and catechins/quercetin) have been reported to increase intracellular thiol status alongwith induction of GSH biosynthesis. Such an elevation in the thiol status in turn leads to detoxification of free radicals and oxidants as well as inhibition of ongoing inflammatory responses. In addition, specific spin traps, such as a-phenyl-N-tert-butyl nitrone, a catalytic antioxidant (ECSOD mimetic), porphyrins (AEOL 10150 and AEOL 10113), and a SOD mimetic M40419 have also been reported to inhibit cigarette smoke-induced inflammatory responses in vivo in the lung. Since a variety of oxidants, free radicals and aldehydes are implicated in the pathogenesis of COPD; it is possible that therapeutic administration of multiple antioxidants and mucolytics will be effective in management of COPD. However, a successful outcome will critically depend upon the choice of antioxidant therapy for a particular clinical phenotype of COPD, whose pathophysiology should be first properly understood. This article will review the various approaches adopted to enhance lung antioxidant levels, antioxidant therapeutic advances and recent past clinical trials of antioxidant compounds in COPD.

Background

Patients with bronchitis type of chronic obstructive pulmonary disease (COPD) have raised vascular endothelial growth factor (VEGF) levels in induced sputum. This has been associated with the pathogenesis of COPD through apoptotic and oxidative stress mechanisms. Since, chronic airway inflammation is an important pathological feature of COPD mainly initiated by cigarette smoking, aim of this study was to assess smoking as a potential cause of raised airway VEGF levels in bronchitis type COPD and to test the association between VEGF levels in induced sputum and airway inflammation in these patients.

Methods

14 current smokers with bronchitis type COPD, 17 asymptomatic current smokers with normal spirometry and 16 non-smokers were included in the study. VEGF, IL-8, and TNF-α levels in induced sputum were measured and the correlations between these markers, as well as between VEGF levels and pulmonary function were assessed.

Results

The median concentrations of VEGF, IL-8, and TNF-α were significantly higher in induced sputum of COPD patients (1,070 pg/ml, 5.6 ng/ml and 50 pg/ml, respectively) compared to nonsmokers (260 pg/ml, 0.73 ng/ml, and 15.4 pg/ml, respectively, p < 0.05) and asymptomatic smokers (421 pg/ml, 1.27 ng/ml, p < 0.05, and 18.6 pg/ml, p > 0.05, respectively). Significant correlations were found between VEGF levels and pack years (r = 0.56, p = 0.046), IL-8 (r = 0.64, p = 0.026) and TNF-α (r = 0.62, p = 0.031) levels both in asymptomatic and COPD smokers (r = 0.66, p = 0.027, r = 0.67, p = 0.023, and r = 0.82, p = 0.002, respectively). No correlation was found between VEGF levels in sputum and pulmonary function parameters.

Conclusion

VEGF levels are raised in the airways of both asymptomatic and COPD smokers. The close correlation observed between VEGF levels in the airways and markers of airway inflammation in healthy smokers and in smokers with bronchitis type of COPD is suggestive of VEGF as a marker reflecting the inflammatory process that occurs in smoking subjects without alveolar destruction.

Background

Oxidative stress induced by smoking is considered to be important in the pathogenesis of Chronic Obstructive Pulmonary Disease (COPD). Heme oxygenase-1 (HMOX1) is an essential enzyme in heme catabolism that is induced by oxidative stress and may play a protective role as an antioxidant in the lung. We determined whether HMOX1 polymorphisms were associated with lung function in COPD patients and whether the variants had functional effects.

Methods

We genotyped five single nucleotide polymorphisms (SNPs) in the HMOX1 gene in Caucasians who had the fastest (n = 278) and the slowest (n = 304) decline of FEV1 % predicted, selected from smokers in the NHLBI Lung Health Study. These SNPs were also studied in Caucasians with the lowest (n = 535) or the highest (n = 533) baseline lung function. Reporter genes were constructed containing three HMOX1 promoter polymorphisms and the effect of these polymorphisms on H2O2 and hemin-stimulated gene expression was determined. The effect of the HMOX1 rs2071749 SNP on gene expression in alveolar macrophages was investigated.

Results

We found a nominal association (p = 0.015) between one intronic HMOX1 SNP (rs2071749) and lung function decline but this did not survive correction for multiple comparisons. This SNP was in perfect linkage disequilibrium with rs3761439, located in the promoter of HMOX1. We tested rs3761439 and two other putatively functional polymorphisms (rs2071746 and the (GT)n polymorphism) in reporter gene assays but no significant effects on gene expression were found. There was also no effect of rs2071749 on HMOX1 gene expression in alveolar macrophages.

Conclusions

We found no association of the five HMOX1 tag SNPs with lung function decline and no evidence that the three promoter polymorphisms affected the regulation of the HMOX1 gene.

Oxidative stress-mediated destruction of normal parenchymal cells during hepatic inflammatory responses contributes to the pathogenesis of immune-mediated hepatitis and is implicated in the progression of acute inflammatory liver injury to chronic inflammatory liver disease. The transcription factor NF-E2-related factor 2 (Nrf2) regulates the expression of a battery of antioxidative enzymes and Nrf2 signaling can be activated by small-molecule drugs that disrupt Keap1-mediated repression of Nrf2 signaling. Therefore, genetic and pharmacologic approaches were used to activate Nrf2 signaling to assess protection against inflammatory liver injury. Profound increases in ind of cell death were observed in both Nrf2 wild-type (Nrf2-WT) mice and Nrf2-disrupted (Nrf2-KO) mice 24-hr following intravenous injection of concanavalin A (12.5 mg/kg, ConA), a model for T cell-mediated acute inflammatory liver injury. However, hepatocyte-specific conditional Keap1 null (Alb-Cre:Keap1flox/−, cKeap1-KO) mice with constitutively enhanced expression of Nrf2-regulated antioxidative genes as well as Nrf2-WT mice but not Nrf2-KO mice pretreated with three daily doses of a triterpenoid that potently activates Nrf2 (30 µmole/kg, CDDO-Im) were highly resistant to ConA-mediated inflammatory liver injury. CDDO-Im pretreatment of both Nrf2-WT and Nrf2-KO mice resulted in equivalent suppression of serum pro-inflammatory soluble proteins suggesting that the hepatoprotection afforded by CDDO-Im pretreatment of Nrf2-WT mice but not Nrf2-KO mice was not due to suppression of systemic pro-inflammatory signaling, but instead was due to activation of Nrf2 signaling in the liver. Enhanced hepatic expression of Nrf2-regulated antioxidative genes inhibited inflammation-mediated oxidative stress, thereby preventing hepatocyte necrosis. Attenuation of hepatocyte death in cKeap1-KO mice and CDDO-Im pretreated Nrf2-WT mice resulted in decreased late-phase pro-inflammatory gene expression in the liver thereby diminishing the sustained influx of inflammatory cells initially stimulated by the ConA challenge. Taken together, these results clearly illustrate that targeted cytoprotection of hepatocytes through Nrf2 signaling during inflammation prevents the amplification of inflammatory responses in the liver.

Disruption of NF-E2–related factor (Nrf2), a redox-sensitive basic leucine zipper transcription factor, causes early-onset and more severe emphysema due to chronic cigarette smoke. Nrf2 determines the susceptibility of lungs to cigarette smoke–induced emphysema in mice through the transcriptional induction of numerous antioxidant genes. The lungs of Nrf2−/− mice have higher oxidative stress as evident from the increased levels of lipid peroxidation (4-hydroxy-2-nonenal) and oxidative DNA damage (7,8-dihydro-8-Oxo-2′deoxyguanosine) in response to cigarette smoke. Glutathione peroxidases (GPX) are the primary antioxidant enzymes that scavenge hydrogen peroxide and organic hydroperoxides. Among the five GPX isoforms, expression of GPX2 was significantly induced at both mRNA and protein levels in the lungs of Nrf2+/+ mice, in response to cigarette smoke. Activation of Nrf2 by specific knock down of the cytosolic inhibitor of Nrf2, Keap1, by small inhibitory RNA (siRNA) upregulated the expression of GPx2, whereas Nrf2 siRNA down-regulated the expression of GPX2 in lung epithelial cells. An ARE sequence located in the 5′ promoter–flanking region of exon 1 that is highly conserved between mouse, rat, and human was identified. Mutation of this ARE core sequence completely abolished the activity of promoter–reporter gene construct. The binding of Nrf2 to the GPX2 antioxidant response element was confirmed by chromatin immunoprecipation, electrophoretic mobility shift assays, and site-directed mutagenesis. This study shows that GPX2 is the major oxidative stress–inducible cellular GPX isoform in the lungs, and that its basal as well as inducible expression is dependent on Nrf2.

The transcription factor Nrf2 has emerged as a master regulator of cellular redox homeostasis. As an adaptive response to oxidative stress, Nrf2 activates the transcription of a battery of genes encoding antioxidants, detoxification enzymes, and xenobiotic transporters by binding the cis-antioxidant response element in the promoter regions of genes. The magnitude and duration of inducible Nrf2 signaling is delicately controlled at multiple levels by Keap1, which targets Nrf2 for redox-sensitive ubiquitin-mediated degradation in the cytoplasm and exports Nrf2 from the nucleus. However, it is not clear how Keap1 gains access to the nucleus. In this study, we show that Keap1 is constantly shuttling between the nucleus and the cytoplasm under physiological conditions. The nuclear import of Keap1 requires its C-terminal Kelch domain and is independent of Nrf1 and Nrf2. We have determined that importin α7, also known as karyopherin α6 (KPNA6), directly interacts with the Kelch domain of Keap1. Overexpression of KPNA6 facilitates Keap1 nuclear import and attenuates Nrf2 signaling, whereas knockdown of KPNA6 slows down Keap1 nuclear import and enhances the Nrf2-mediated adaptive response induced by oxidative stress. Furthermore, KPNA6 accelerates the clearance of Nrf2 protein from the nucleus during the postinduction phase, therefore promoting restoration of the Nrf2 protein to basal levels. These findings demonstrate that KPNA6-mediated Keap1 nuclear import plays an essential role in modulating the Nrf2-dependent antioxidant response and maintaining cellular redox homeostasis.