Dual oxidase-1 is required for airway epithelial cell migration and bronchiolar reepithelialization after injury.

The respiratory epithelium plays a critical role in innate defenses against airborne pathogens and pollutants, and alterations in epithelial homeostasis and repair mechanisms are thought to contribute to chronic lung diseases associated with airway remodeling. Previous studies implicated the nicotinamide adenine dinucleotide phosphate-reduced oxidase dual oxidase-1 (DUOX1) in redox signaling pathways involved in in vitro epithelial wound responses to infection and injury. However, the importance of epithelial DUOX1 in in vivo epithelial repair pathways has not been established. Using small interfering (si)RNA silencing of DUOX1 expression, we show the critical importance of DUOX1 in wound responses in murine tracheal epithelial (MTE) cells in vitro, as well as its contribution to epithelial regeneration in vivo in a murine model of epithelial injury induced by naphthalene, a selective toxicant of nonciliated respiratory epithelial cells (club cells [Clara]). Whereas naphthalene-induced club-cell injury is normally followed by epithelial regeneration after 7 and 14 days, such airway reepithelialization was significantly delayed after the silencing of airway DUOX1 by oropharyngeal administration of DUOX1-targeted siRNA. Wound closure in MTE cells was related to DUOX1-dependent activation of the epidermal growth factor receptor (EGFR) and the transcription factor signal transducer and activator of transcription-3 (STAT3), known mediators of epithelial cell migration and wound responses. Moreover, in vivo DUOX1 silencing significantly suppressed naphthalene-induced activation of STAT3 and EGFR during early stages of epithelial repair. In conclusion, these experiments demonstrate for the first time an important function for epithelial DUOX1 in lung epithelial regeneration in vivo, by promoting EGFR-STAT3 signaling and cell migration as critical events in initial repair.

Anti-malarial drug artesunate ameliorates oxidative lung damage in experimental allergic asthma.

Oxidative stress is a critical pathophysiological factor in the development of allergic airway inflammation, resulting in oxidative damage to lipids, proteins, and DNA. Our recent report revealed potent anti-inflammatory effects of the antimalarial drug artesunate in experimental allergic asthma. The present study investigated potential antioxidative effects of artesunate in a murine model of allergic asthma in comparison with dexamethasone, a potent corticosteroid. Mice were sensitized and challenged with ovalbumin and developed airway inflammation and oxidative lung damage. Artesunate markedly suppressed ovalbumin-induced increases in total cell, eosinophil, and neutrophil counts. In contrast, dexamethasone failed to inhibit neutrophil recruitment. Levels of the oxidative damage markers 8-isoprostane, 8-hydroxy-2-deoxyguanosine, and 3-nitrotyrosine were potently repressed by artesunate. However, dexamethasone showed weaker inhibitory effects on 3-nitrotyrosine production. Ovalbumin-induced increases in the expression of the pro-oxidants iNOS and NADPH oxidase (NOX1, 2, 3, and 4) were significantly abated by artesunate. Gene expression of regulatory subunits of NOX, p22phox and p67phox, was also reduced by artesunate. The expression and activities of the antioxidants superoxide dismutase and catalase were substantially reversed with artesunate in ovalbumin-challenged mice. Artesunate significantly enhanced nuclear levels of nuclear factor erythroid-2-related factor 2 (Nrf2) in lung tissues from ovalbumin-challenged mice and in TNF-α-stimulated human bronchial epithelial cells. Our findings implicate a potential therapeutic value for artesunate in the treatment of asthma via the amelioration of oxidative damage in allergic airways, and it may act by suppressing pro-oxidants and restoring the activities and expression of antioxidants via activation of Nrf2. Artesunate may be a potential novel anti-asthma drug capable of controlling both inflammation and oxidative damage in chronic severe asthma.

Nitric oxide inhibits highly selective sodium channels and the Na+/K+-ATPase in H441 cells.

Nitric oxide (NO) is an important regulator of Na(+) reabsorption by pulmonary epithelial cells and therefore of alveolar fluid clearance. The mechanisms by which NO affects epithelial ion transport are poorly understood and vary from model to model. In this study, the effects of NO on sodium reabsorption by H441 cell monolayers were studied in an Ussing chamber. Two NO donors, (Z)-1-[N-(3-aminopropyl)-N-(n-propyl)amino]diazen-1-ium-1,2-diolate and diethylammonium (Z)-1-(N,N-diethylamino)diazen-1-ium-1,2-diolate, rapidly, reversibly, and dose-dependently reduced amiloride-sensitive, short-circuit currents across H441 cell monolayers. This effect was neutralized by the NO scavenger hemoglobin and was not observed with inactive NO donors. The effects of NO were not blocked by 8-bromoguanosine-3',5'-cyclic monophosphate or by soluble guanylate cyclase inhibitors (methylene blue and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one) and were therefore independent of soluble guanylate cyclase signaling. NO targeted apical, highly selective, amiloride-sensitive Na(+) channels in basolaterally permeabilized H441 cell monolayers. NO had no effect on the activity of the human epithelial sodium channel heterologously expressed in Xenopus oocytes. NO decreased Na(+)/K(+)-ATPase activity in apically permeabilized H441 cell monolayers. The inhibition of Na(+)/K(+)-ATPase activity by NO was reversed by mercury and was mimicked by N-ethylmaleimide, which are agents that reverse and mimic, respectively, the reaction of NO with thiol groups. Consistent with these data, S-NO groups were detected on the Na(+)/K(+)-ATPase α subunit in response to NO-donor application, using a biotin-switch approach coupled to a Western blot. These data demonstrate that, in the H441 cell model, NO impairs Na(+) reabsorption by interfering with the activity of highly selective Na(+) channels and the Na(+)/K(+)-ATPase.

Production and activation of matrix metalloproteinase 7 (matrilysin 1) in the lungs of patients with idiopathic pulmonary fibrosis.

CONTEXT: Idiopathic pulmonary fibrosis (IPF) is characterized by diffuse interstitial inflammation and fibroblast proliferation with accelerated remodeling of extracellular matrix, which result in irreversible destruction of the lung's architecture. OBJECTIVE: To elucidate the production levels, tissue localization, and activation of matrix metalloproteinase 7 (MMP-7) in the lungs of patients with IPF. DESIGN: Bronchoalveolar lavage analysis was performed in 17 IPF patients and 6 healthy volunteers. Levels of MMP-7 in blood were assayed in 23 IPF patients and 20 controls. Histologic and immunohistochemical analyses were performed on paraffin sections of the lung tissues from patients with IPF, interstitial pneumonia associated with rheumatoid arthritis, or nonspecific interstitial pneumonia. RESULTS: The proMMP-7 levels in bronchoalveolar lavage fluids from IPF patients were significantly higher than those from healthy controls, although there was no difference in the serum levels between the 2 groups. By immunohistochemistry, proMMP-7 was localized mainly to the hyperplastic alveolar and metaplastic bronchiolar epithelial cells in the lung tissues from IPF patients. Active MMP-7 was immunolocalized on alveolar macrophages and hyperplastic epithelial cells, which were also immunostained with antibody against CD151, a molecule associated with activation of proMMP-7. Immunoblot analysis indicated the overproduction of proMMP-7 together with a small amount of active MMP-7 in bronchoalveolar lavage fluids from IPF patients. The MMP-7 activity was detected in a cross-linked carboxymethylated transferrin film assay. CONCLUSIONS: proMMP-7 is excessively produced by hyperplastic alveolar and metaplastic bronchiolar epithelial cells and activated locally in the lungs of IPF patients, suggesting that MMP-7 may contribute to the pathology of IPF.

Mechanisms underlying epithelium-dependent relaxation in rat bronchioles: analogy to EDHF-type relaxation in rat pulmonary arteries.

This study investigated the mechanisms underlying epithelium-derived hyperpolarizing factor (EpDHF)-type relaxation in rat bronchioles. Immunohistochemistry was performed, and rat bronchioles and pulmonary arteries were mounted in microvascular myographs for functional studies. An opener of small (SK(Ca)) and intermediate (IK(Ca))-conductance calcium-activated potassium channels, NS309 (6,7-dichloro-1H-indole-2,3-dione 3-oxime) was used to induce EpDHF-type relaxation. IK(Ca) and SK(Ca)3 positive immunoreactions were observed mainly in the epithelium and endothelium of bronchioles and arteries, respectively. In 5-hydroxytryptamine (1 microM)-contracted bronchioles (828 +/- 20 microm, n = 84) and U46619 (0.03 microM)-contracted arteries (720 +/- 24 microm, n = 68), NS309 (0.001-10 microM) induced concentration-dependent relaxations that were reduced by epithelium/endothelium removal and by blocking IK(Ca) channels with charybdotoxin and in bronchioles also by blocking SK(Ca) channels with apamin. Inhibition of cyclooxygenase, nitric oxide synthase, and cytochrome 2C isoenzymes, or blockade of large (BK(Ca))-conductance calcium-activated potassium channels with iberiotoxin, failed to reduce NS309 relaxation. In contrast to the pulmonary arteries, relaxations to a beta(2)-adrenoceptor agonist, salbutamol, were reduced in bronchioles by removing the epithelium or blocking IK(Ca) and/or SK(Ca) channels. Extracellular K(+) (2-20 mM) induced relaxation in both bronchioles and arteries. An inhibitor of Na(+)-K(+)-ATPase, ouabain, abolished relaxations to NS309, salbutamol, and K(+). These results suggest that IK(Ca) and SK(Ca)3 channels are located in the epithelium of bronchioles and endothelium of pulmonary arteries. Analog to the endothelium-derived hyperpolarizing factor (EDHF)-type relaxation in pulmonary arteries, these channels may be involved in EpDHF-type relaxation of bronchioles caused by epithelial K(+) efflux followed by activation of Na(+)-K(+)-ATPase in the underlying smooth muscle layer.

Aging enhances susceptibility to cigarette smoke-induced inflammation through bronchiolar chemokines.

Cigarette smoking and aging are major risk factors for chronic obstructive pulmonary disease. An unsolved question is whether elderly lungs are particularly vulnerable to cigarette smoke (CS) exposure. In this study, we used a mouse model to test the hypothesis that aging increases the susceptibility to CS-induced pulmonary inflammation. We subjected 9-week-old and 69-week-old C57BL/6J mice to CS (whole-body exposure, 90 min/d), and evaluated neutrophil infiltration in the lungs, the levels of keratinocyte-derived chemokine (KC) and macrophage inflammatory protein (MIP)-2 in bronchoalveolar lavage fluid, and mRNA expression in bronchiolar epithelium retrieved by laser capture microdissection. The 69-week-old mice showed a greater number of neutrophils and higher levels of bronchiolar KC and MIP-2 expression than 9-week-old mice after 9 days of CS exposure. Furthermore, single CS exposure induced the rapid up-regulation of KC and MIP-2 in bronchiolar epithelium in both 9-week-old and 69-week-old mice, and the much higher levels in 69-week-old mice were associated with greater nuclear translocation of NF-kappaB. In contrast, no age-related differences were observed in the bronchiolar expression of NF-E2-related factor 2-regulated antioxidant and detoxification genes, heme oxygenase-1, reduced nicotinamide adenine dinucleotide phosphate quinone reductase 1, and glutamate-cysteine ligase, modifier unit, or antioxidant activity in bronchoalveolar lavage fluid, regardless of CS exposure. In summary, aging increases susceptibility to CS-induced inflammation in a mouse model, and robust mRNA up-regulation and nuclear translocation of NF-kappaB in bronchiolar epithelium may be involved.

Atypical protein kinase C{iota} is required for bronchioalveolar stem cell expansion and lung tumorigenesis.

Protein kinase Ciota (PKCiota) is an oncogene required for maintenance of the transformed phenotype of non-small cell lung cancer cells. However, the role of PKCiota in lung tumor development has not been investigated. To address this question, we established a mouse model in which oncogenic Kras(G12D) is activated by Cre-mediated recombination in the lung with or without simultaneous genetic loss of the mouse PKCiota gene, Prkci. Genetic loss of Prkci dramatically inhibits Kras-initiated hyperplasia and subsequent lung tumor formation in vivo. This effect correlates with a defect in the ability of Prkci-deficient bronchioalveolar stem cells to undergo Kras-mediated expansion and morphologic transformation in vitro and in vivo. Furthermore, the small molecule PKCiota inhibitor aurothiomalate inhibits Kras-mediated bronchioalveolar stem cell expansion and lung tumor growth in vivo. Thus, Prkci is required for oncogene-induced expansion and transformation of tumor-initiating lung stem cells. Furthermore, aurothiomalate is an effective antitumor agent that targets the tumor-initiating stem cell niche in vivo. These data have important implications for PKCiota as a therapeutic target and for the clinical use of aurothiomalate for lung cancer treatment.

Mouse specific lung tumors from CYP2F2-mediated cytotoxic metabolism: an endpoint/toxic response where data from multiple chemicals converge to support a mode of action.

It is proposed that metabolism of several structurally-related chemicals by CYP2F isoforms of the cytochromes P450 family results in a cytotoxicity-driven mode of action in organs high in CYP2F; namely, CYP2F2 in nasal and lung tissue in mice and CYP2F4 in nasal tissues in rats. Importantly, the CYP2F1 isozyme expressed in humans appears to have a low capacity to metabolize these compounds. In mice, the resultant cytotoxicity and subsequent regenerative hyperplasia is hypothesized drive an increase in lung tumors that are mostly benign and are not life shortening. Although a complete picture of the mode of action has not been developed in any one model compound, data from the individual compounds can be combined to synthesize and reinforce confidence in the CYP2F toxicity hypothesis. For coumarin, naphthalene, and styrene, inhibition of toxicity with inhibition of CYP2F2 has been demonstrated. Rat CYP2F4 appears to be equally active in metabolizing these chemicals; however, CYP2F4 occurs to a much lower extent in rat Clara cells and levels of metabolites produced are not sufficient to cause lung cytotoxicity. Human lungs contain far fewer of Clara cells than rats or mice, and human lung microsomes failed to, or only marginally, metabolize these compounds. In addition, the human lung differs markedly from the mouse lung in the morphology of its Clara cells, which make humans much less sensitive than mice to toxicity due to reactive metabolites. The absence of a role for CYP2E1-generated metabolites (primarily alkyl oxidation vs. ring-oxidation) in mouse pulmonary effects was demonstrated by the lack of protection from styrene toxicity by CYP2E1 inhibitor, or reduction of toxicity in CYP2E1-knockout mice, and lack of lung toxicity of the primary metabolite of ethylbenzene. The chemicals used as examples of this mode of action generally are negative in standard genotoxicity assays. Apart from increased SCE, no consistent pattern in genotoxicity results was found among these chemicals. Thus, while lung tumors from bronchiolar cell cytotoxicity are theoretically possible in humans, it is unlikely that metabolism by CYP2F1 would produce levels of cytotoxic metabolites in human lungs sufficient to result in lung cytotoxic responses and thus tumors. Therefore, it is unlikely several chemicals that cause mouse lung tumors via CYP2F2 metabolism will cause lung tumors in humans.

Matrilysin-1 mediates bronchiolization of alveoli, a potential premalignant change in lung cancer.

Matrilysin-1 (also called matrix metalloproteinase-7) is expressed in injured lung and in cancer but not in normal epithelia. Bronchiolization of the alveoli (BOA), a potential precursor of lung cancer, is a histologically distinct type of metaplasia that is composed of cells resembling airway epithelium in the alveolar compartment. We demonstrate that there is increased expression of matrilysin-1 in human lesions and BOA in the CC10-human achaete-scute homolog-1 transgenic mouse model. Forced expression of the matrilysin-1 gene in immortalized human normal airway epithelial BEAS-2B and HPLD1 cells, which do not normally express matrilysin-1, promoted cellular migration, suggesting a functional link for BOA formation via bronchiolar cell migration. In addition, matrilysin-1 stimulated proliferation and inhibited Fas-induced apoptosis, while a knockdown by RNA interference decreased cell growth, migration, and increased sensitivity to apoptosis. Western blotting demonstrated increased levels of phospho-p38 and phospho-Erk1/2 kinases after matrilysin-1 expression. Gene expression analysis uncovered several genes that were related to cell growth, migration/movement, and death, which could potentially facilitate bronchiolization. In vivo, the formation of BOA lesions was reduced when CC10-human achaete-scute homolog-1 mice were crossed with matrilysin-1 null mice and was correlated with reduced matrilysin-1 expression in BOA. We conclude that matrilysin-1 may play an important role in the bronchiolization of alveoli by promoting proliferation, migration, and attenuation of apoptosis involving multiple genes in the MAP kinase pathway.

Early and late changes of MMP-2 and MMP-9 in bleomycin-induced pulmonary fibrosis.

PURPOSE: Matrix metalloproteinases (MMPs) have been implicated in the pathogenesis of pulmonary fibrosis. To understand the role of MMP-2 and MMP-9 in pulmonary fibrosis, we evaluated the sequential dynamic change and different cellular sources of the 2 MMPs along the time course and their differential expression in the bronchoalveolar lavage (BAL) fluid and in the lung parenchyma of the bleomycin-induced pulmonary fibrosis models in rats. MATERIALS AND METHODS: The level of MMPs in BAL fluid of 54 bleomycin-treated rats was assessed by zymography from 1 to 28 days after intratracheal bleomycin instillation. The level of MMPs in lung parenchyma was evaluated by immunohistochemistry. RESULTS: MMP-2 and MMP-9 were markedly increased in both the BAL fluid and in the lung parenchyma of the bleomycin-treated rats, especially in the early phase with the peak on the 4th day. The levels of both MMPs in the BAL fluid correlated generally well to those in lung parenchyma, although the level of MMP-9 in BAL fluid was higher than MMP-2. In the lung parenchyma, the 2 MMPs, in early stage, were predominantly expressed in the inflammatory cells. In late stage, type II pneumocytes and alveolar epithelial cells at the periphery of the fibrotic foci retained MMP expression, which was more prominent in the cells showing features of cellular injury and/or repair. CONCLUSION: In bleomycin-induced pulmonary fibrosis, MMP-2 and MMP-9 may play important roles, especially in the early phase. In the late stage, the MMP-2 and MMP-9 may play a role in the process of repair.

Early and Late Changes of MMP-2 and MMP-9 in Bleomycin-Induced Pulmonary Fibrosis

PURPOSE: Matrix metalloproteinases (MMPs) have been implicated in the pathogenesis of pulmonary fibrosis. To understand the role of MMP-2 and MMP-9 in pulmonary fibrosis, we evaluated the sequential dynamic change and different cellular sources of the 2 MMPs along the time course and their differential expression in the bronchoalveolar lavage (BAL) fluid and in the lung parenchyma of the bleomycin-induced pulmonary fibrosis models in rats. MATERIALS AND METHODS: The level of MMPs in BAL fluid of 54 bleomycin-treated rats was assessed by zymography from 1 to 28 days after intratracheal bleomycin instillation. The level of MMPs in lung parenchyma was evaluated by immunohistochemistry. RESULTS: MMP-2 and MMP-9 were markedly increased in both the BAL fluid and in the lung parenchyma of the bleomycin-treated rats, especially in the early phase with the peak on the 4th day. The levels of both MMPs in the BAL fluid correlated generally well to those in lung parenchyma, although the level of MMP-9 in BAL fluid was higher than MMP-2. In the lung parenchyma, the 2 MMPs, in early stage, were predominantly expressed in the inflammatory cells. In late stage, type II pneumocytes and alveolar epithelial cells at the periphery of the fibrotic foci retained MMP expression, which was more prominent in the cells showing features of cellular injury and/or repair. CONCLUSION: In bleomycin-induced pulmonary fibrosis, MMP-2 and MMP-9 may play important roles, especially in the early phase. In the late stage, the MMP-2 and MMP-9 may play a role in the process of repair.