96 Cu,Zn­SOD­induced H2O2 Mediates Pulmonary Fibrosis via Down­regulation of ERK Chao He1, Shubha Murthy2, Alan J Ryan2, and A Brent Carter1,2 1The University of Iowa, 2 The University of Iowa Asbestosis is a prototypical pulmonary fibrosis and alveolar macrophages obtained from asbestosis patients are known to express high levels of H2O2. Pulmonary fibrosis is characterized by aberrant extracellular matrix deposition, and we have shown that macrophages-specific MMP-9 decreases collage deposition by fibroblasts. We have found that alveolar macrophages obtained from asbestosis patients have high expression and activity of Cu,Zn-SOD as well as decreased MMP-9 gene expression. The ERK MAP kinase is known to regulate MMP-9 expression. However, the link between Cu,Zn-SOD , H2O2 generation, and progression of pulmonary fibrosis is not known. We hypothesize that the Cu,Zn-SOD increases H2O2, which mediates pulmonary fibrosis by modulation of MMP-9 gene expression, via inhibition of ERK. Our data showed that over expression of Cu,Zn-SOD increased H2O2 generation, decreased MMP-9 promoter activity, MMP-9 mRNA, and ERK activation compared to cells over expressing an empty vector. MMP-9 promoter activity was recovered by over expression of constitutive active MEK1, an upstream kinase of ERK, in cells expressing Cu,Zn-SOD. These observations were recapitulated in vivo. Cu,Zn-SOD-/- mice had less H2O2-induced oxidative stress in the lung, increased MMP-9 mRNA in alveolar macrophages, and increased activation of ERK compared to wild-type (WT) mice. More importantly, Cu,Zn-SOD-/- mice did not develop pulmonary fibrosis. These data suggest that Cu,Zn-SOD mediates pulmonary fibrosis by increasing H2O2 production, leading to inhibition of ERK and decreased MMP-9 gene expression. Supported by NIH grants ES-015981 and ES-014871 to A.B.C. doi:

97

Pulmonary Oxygen Toxicity Protection by Adiponectin Sainath R Kotha1, Sean M Sliman1, Rishi B Patel1, Jason P Cruff1, Shariq I Sherwani1, Travis O Gurney1, Ulysses J Magalang1, and Narasimham L Parinandi1 1Ohio State University College of Medicine, Columbus Adiponectin (Ad), an adipokine exclusively secreted by the adipose tissue, has emerged as a paracrine metabolic regulator as well as a protectant against oxidative stress. Pharmacological approaches of protecting against clinical hyperoxic lung injury during oxygen therapy/treatment are limited. Earlier, we have reported that Ad inhibits the NADPH oxidase-catalyzed formation of superoxide from molecular oxygen in human neutrophils. Having this as the premise, we conducted studies to determine whether (i) exogenous adiponectin would protect against the hyperoxia-induced barrier dysfunction in the lung endothelial cells (ECs) in vitro and (ii) endogenously synthesized Ad would protect against hyperoxic lung injury in wild type (WT) and Ad-overexpressing transgenic (AdTg) mice in vivo. The results demonstrated that exogenous Ad protected against the hyperoxia-induced oxidative stress, loss of glutathione (GSH), cytoskeletal reorganization, barrier dysfunction, and leak in the lung ECs in vitro. Furthermore, the hyperoxia-induced lung injury, vascular leak, and lipid peroxidation were significantly attenuated in AdTg mice in vivo. Also, AdTg mice exhibited elevated levels of total thiols and GSH in the lungs as compared to WT mice. For the first time, our studies demonstrated that Ad protected against the hyperoxia-induced lung damage apparently through attenuation of oxygen toxicity and modulation of thiol-redox status. doi:

98 Sub­chronic Administration of Doxorubicin to Wistar Rats Results in Oxidative Stress and Unaltered Apoptotic Signaling in the Lung Nuno G. Machado1, Inês Baldeiras2, Gonçalo C. Pereira1, Susana P. Pereira1, and Paulo J. Oliveira1 1Center for Neurosciences and Cell Biology, Coimbra, Portugal, 2Laboratory of Neurochemistry, Department of Neurology, Coimbra University Hospital, Coimbra, Portugal Despite the wide range of published data on cardiac toxicity, there is still a shortage of evidence for chronic toxicity of the antineoplastic agent Doxorubicin (DOX) in the lung. The aim of the present work was to determine if DOX causes alterations in selected apoptotic proteins and oxidative stress in the lung, as it does in the heart. For that purpose, lungs from Wistar-Han rats sub-chronically treated with vehicle or DOX with seven weekly injections were collected and analyzed concerning several proteins involved in mitochondrial permeabilization and apoptotic pathways, including p53, Bax and Bcl-2 and different oxidative stress markers. After sub-chronic DOX treatment, no alterations in lung proteins involved in mitochondrial membrane permeabilization or caspase 3 and 9-like activities were found. Nevertheless, an increase of malondialdehyde levels and a decrease in the lung concentration of vitamin E after DOX toxicity was observed, despite no alterations in the reduced and oxidized forms of glutathione. The results obtained indicate for the first time that the lungs of DOX-treated rats appears to be susceptible to increased lipid peroxidation, which can explain some cases of DOX-induced lung toxicity. Financial support: NGM, GCP and SPP are supported by Ph.D. fellowships from the Foundation for Science and Technology, Portugal (SFRH/BD/66178/2009, SFRH/BD/36938/2007 and SFRH-BD/64247/2009 to SP, respectively). The work is supported by a FCT research grant (PTDC/SAU-OSM/104731/2008). doi:

99

Generation of Mice with Lung­specific Expression of Nuclear Heme oxygenase­1 Fumihiko Namba1, Ping La1, Amal P Fernando1, Guang Yang1, and Phyllis A Dennery1,2 1Children's Hospital of Philadelphia, 2Department of Pediatrics, University of Pennsylvania Heme oxygenase (HO)-1 can translocate to the nucleus under oxidative stress. This nuclear form lacks the C-terminus and enzymatic activity. Nevertheless, HO-1 knockout MEFs transfected with nuclear HO-1 demonstrated increased cell viability in hyperoxia compared with cytoplasmic HO-1. Newborn mice were injected with an adenovirus encoding C-terminally truncated HO-1 (TR) containing 3 nuclear localization sequences (NLS) into the lung. Controls were injected with empty vector (VEC) or with a full-length HO-1 (FL). In another model, constructs carrying FL or TR with 3 NLS driven by the human SP-C promoter were microinjected into fertilized mouse eggs. Transgenic mice were identified by PCR. The delivery of exogenous HO-1 into the lung resulted in overexpression of the protein for 48 h. The expression and the localization of HO-1 were confirmed by Western blot and immunohistochemistry. Poly-ADP ribose polymerase (PARP) and 8-oxoguanine glycosylase (Ogg1) levels were evaluated. PARP and Ogg-1 levels were lower in the animals injected with TR and TR HO-1 transgenic mice, suggesting that nuclear HO-1 may protect against cellular damage in vivo. These models will allow us to determine the physiologic significance of nuclear HO-1 in the neonatal mouse exposed to oxidative stress.

doi:

SFRBM/SFRRI 2010 S47

10.1016/j.freeradbiomed.2010.10.099

10.1016/j.freeradbiomed.2010.10.100

10.1016/j.freeradbiomed.2010.10.101

10.1016/j.freeradbiomed.2010.10.102