expression of AAM markers, including surface mannose receptors, metabolic factor (arginase-1), and pro-fibrotic cytokine (TGF-β) compared to WT mice. This was also associated with reduced expression of pro-inflammatory (M1) cytokines (IL-1β and TNF-α) and iNOS in Cu,Zn-SOD Tg mice. Stimulation with the Th2 cytokine, IL-4, Cu,Zn-SOD Tg macrophages have increased TGF-β production compared to WT macrophages. These observations suggest that Cu,Zn-SOD mediates alternatively activation of macrophages, which is linked to fibrosis. To demonstrate the role of Cu,Zn-SOD in AAM and provide biological relevance, we measured TNF-α and IL-1β level in bronchoalveolar lavage fluid and IL-13 level in lung homogenates of Cu,Zn-SOD -/- mice following asbestos exposure. Cu,Zn-SOD -/- mice have significantly less IL-13 and increased TNF-α and IL-1β compared to WT mice. Moreover, Cu,Zn-SOD Tg mice have advanced stage of pulmonary fibrosis compared to WT mice after asbestos exposure. These data strongly suggest that Cu,Zn-SOD mediates pulmonary fibrosis by alternatively activation of macrophages. (Supported by NIH grants ES-015981 and ES-014871 to A.B.C.) doi:10.1016/j.freeradbiomed.2011.10.350 268 Knockdown of Thioredoxin-1 Attenuates Hypoxia- induced Pulmonary Artery Smooth Muscle Cell Proliferation Cynthia Hill 1 , Viktoria Nelin 1 , Yi Gin 1 , Lynette Rogers 1 , Leif Nelin 1 , and Trent Tipple 1 1 The Research Institute at Nationwide Children's Hospital Pulmonary hypertension (PH) is a disease which manifests itself in the lungs of both adults and children. Vascular proliferation and remodeling are the hallmarks of PH and occur mainly in the pulmonary arterial smooth muscle cells (PASMC). the cause of PASMC proliferation and vascular remodeling in PH is poorly understood. Hypoxia underlies many forms of PH and results in alterations in the redox balance within the PASMC. Therefore, the aim of this study was to examine the role of the thioredoxin-1 (Trx1) in hypoxia-induced proliferation in PASMC. Protein was isolated from human PASMC which were exposed to either hypoxia (1% O 2 , 5% CO 2 , balance N 2 ) or normoxia (21% O 2 , 5% CO 2 and balance N 2 ). Trx1 levels were determined by western blotting. Proliferation studies were done by seeding 6 well plates with 10,000 PASMC per well, incubating in either normoxia or hypoxia for 5 days and counting viable cells using trypan blue exclusion. We found that Trx1 protein levels in hypoxia were significantly greater after 48 and 72 hours of exposure than in PASMC grown in normoxia. We also found that PASMC proliferate more in hypoxia than in normoxia. To determine the role of Trx1 in hypoxia-induced PASMC proliferation we knocked down Trx1 protein in PASMC using Trx1-specific siRNA. Treatment with the Trx1 siRNA completely prevented hypoxia- induced proliferation in PASMC. Hypoxia response element (HRE) luciferase reporter studies were performed using scramble and Trx1 siRNA. We detected a 3.5-fold increase in HRE- luciferase activity in PASMCs exposed to hypoxia that was prevented by Trx1 siRNA. These findings demonstrate that Trx1 protein is necessary for the hypoxia-induced proliferation of PASMC likely through hypoxia-inducible factor-mediated pathways. We speculate that Trx1 may represent a novel therapeutic target for the vascular remodeling that underlies PH. doi:10.1016/j.freeradbiomed.2011.10.351 269 Retinoids Attenuate Hyperoxia-Induced Alveolar Simplification and Abnormal Lung Function in the Newborn Mouse Lung Masheika L. James 1 , A. Catherine Ross 2 , and Namasivayam Ambalavanan 1 1 University of Alabama at Birmingham, 2 Pennsylvania State Univeristy We have recently shown that a 10:1 molar combination of VA and RA (VA+RA) synergistically increases lung retinol content in newborn rodents. We hypothesized that the increase in lung retinol would translate into functional benefits in the attenuation of alveolar simplification and abnormal lung function in newborn mice exposed to hyperoxia. Newborn C57BL/6 mice were exposed to 85% O 2 (hyperoxia) or air (normoxia) for 7 or 14 days and given vehicle or VA+RA every other day. Lung retinol content was measured by HPLC, lung function was assessed by flexiVent, and lung development was evaluated by radial alveolar counts, mean linear intercept, and secondary septal crest density. Additionally, mediators of alveolar development and hyperoxia induced inhibition of alveolar development were evaluated by qPCR and by ELISA in lung homogenates. We observed that VA+RA attenuated the hyperoxia-induced alveolar simplification and improved lung function, with higher compliance and lower resistance, associated with increased lung retinyl ester stores. We also found that VA+RA attenuated the hyperoxia-induced increases in protein oxidation accompanied by no changes in MDA-adduct or nitrotyrosine levels. IFN-Ȗ and MIP-2α mRNA and protein were observed to increase with hyperoxia and this increase was attenuated by VA+RA. Supplementation and inhibition experiments revealed that MIP-2α and IFN-Ȗ were sufficient but not necessary for hyperoxia-induced inhibition of alveolar development. in addition, VA+RA increased FGF-10 mRNA expression in hyperoxia-exposed mouse pups. Further investigation is required to define more precisely mechanisms by which retinoids benefit lung development during hyperoxia exposure. doi:10.1016/j.freeradbiomed.2011.10.352 270 Redox Potential of Glutathione Affects ENaC Activity Lisa Kreiner 1 , and My Helms 1 1 Emory University, Atlanta Lung cells produce high levels of reactive oxygen species (ROS) due to their exposure to free oxygen in inspired air. High concentrations of ROS can act as signaling molecules and have been shown to modulate the activity of epithelial Na + channels (ENaC) which regulate lung fluid clearance. Glutathione is an efficient antioxidant present in high quantities in the lung. the relative concentrations of oxidized and reduced glutathione determine the redox state of cells, which has been shown to be less negative in diseases such as alcoholism, type 2 diabetes and some cancers. in this study, we propose that glutathione in its reduced form may scavenge free radicals and alter ENaC activity, ultimately affecting lung fluid clearance. To test this, we performed cell-attached single channel recordings from alveolar type 1 or type 2 cells isolated from mouse lungs or from rat lung slices. Oxidized and reduced forms of glutathione (0.4 mM) were added to the extracellular solution in ratios that corresponded to specific redox potentials. We found that at less negative redox potentials (-25 mV) a significant reduction in NP o of ENaC was observed (from 0.41± 0.06 (control) to 0.20 ± 0.06 (oxidized), P<0.05). When the redox potential was then made more negative by the addition of reduced glutathione (-300 mV) an increased NP o of ENaC was observed (from 0.20± 0.06 (oxidized) to 0.35 ± SFRBM 2011 S112