NF-κB and antioxidant genes in mouse lung. Six to eight weeks aged FoxO3 knock-out (KO) mice and wild type (WT) littermates were exposed to CS for 3 days to 4 months. FoxO3 KO mice showed increased susceptibility to inflammatory response by CS exposure as evidenced by increased inflammatory cell influx and pro-inflammatory cytokines release, such as MCP-1 and KC, and downregulation of MnSOD and catalase genes as compared to CS-exposed WT mice. RelA/p65 DNA binding activity was significantly increased in FoxO3 KO mice as compared to WT mice exposed to CS. We further showed that CS induced the translocation of FoxO3 into the nucleus where FoxO3 interacted with NF-κB and disrupted its DNA-binding ability leading to inhibition of its activity associated with downregulation of antioxidant genes in WT mice. Interestingly, genetic ablation of FoxO3 led to augmented pulmonary emphysema by CS exposure as compared to WT. Thus, FoxO3 plays a pivotal role in regulation of inflammatory response and antioxidant genes, and deficiency of FoxO3 results in development of emphysema/COPD. Funding: Supported by the NIH-NHLBI Grants- 1R01HL085613, 1R01HL097751, and 1R01HL092842 (IR). doi: 107 PTEN is a Key Mediator of Vascular Remodeling in Pulmonary Hypertension Yazhini Ravi1,2, Selvendiran Karuppaiyah1, Sarath Meduru1, Mahmood Khan1, Lucas Citro1, Alex Dayton1, Chittoor B Sai-Sudhakar2, and Periannan Kuppusamy1 1Dept of Internal Medicine: DHLRI:The Ohio State University, 2Division of Cardiac Surgery: DHLRI: The Ohio State University Introduction: Pulmonary hypertension (PH) induces vascular remodeling and impairs cardiac function. In addition, PH, which is secondary to CHF, is a major hurdle in the management of cardiac failure. PTEN, a phosphatase-and-tensin homolog deleted on chromosome 10 and implicated in multiple advanced cancers, has been shown to have a causative role in vascular restenosis. The objective of the present study was to test our hypothesis that PTEN may be a key mediator of vascular remodeling in PH. Methods: PH was induced in rats (male Sprague Dawley; N=6 per group) by three different methods: (i) Monocrotaline administration (60 mg/kg sc) - pharmacological;(ii) Hypoxic exposure (10% O2) – physiological; (iii) Ligation of left-anterior-descending (LAD) coronary artery - clinical. High-frequency ultrasound (ECHO) and micro-MRI were used to monitor cardiac function during the development of PH. Western-blot analysis was used to measure PTEN and other key downstream proteins involved in PH.  Results: All three methods resulted in the development of PH in 2-4 weeks as verified by RV systolic pressure monitoring. Noninvasive serial imaging studies showed a significant deterioration of cardiac function, particularly LV-EF (37% vs 75% in control). Phosphorylated PTEN was markedly decreased to 45.1% in lung, 59.5% in LV, 37.2% in RV when compared to respective tissues in control. In contrast, pAkt levels were significantly upregulated in the tissues of the PH group (341% lung, 157% LV, 128% in RV).  Conclusion: The study showed that PH was associated with reduction of PTEN levels in the cardiac and pulmonary tissues. The study suggests that PTEN may be a potential therapeutic target for the management of PH.  doi:

108 Maternal Inflammation and Neonatal Hyperoxia Results in Adult Pulmonary Fibrosis Lynette K Rogers1,2, Trent E Tipple1,2, Kathryn M Heyob1, and Markus Velten1 1The Research Institute at Nationwide Children's Hospital, 2The Ohio State University Rationale: Maternal inflammation is implicated as a causative factor in preterm birth. Preterm infants are often exposed to hyperoxia as life-sustaining therapy. While younger and more immature infants are surviving, the long term effects of the combination of extreme prematurity and hyperoxia exposure have not been extensively investigated. We tested the hypothesis that systemic maternal inflammation and transient neonatal hyperoxia exposure induces alterations in pulmonary structure and function that persist into adulthood. Methods: Timed pregnant C3H/HeN mice were injected on E16 with LPS (80 µg/kg) or saline i.p. Newborns were placed in room air (RA) or 85% O2 for 14 days followed by RA exposure until 8 weeks. Lung structure was assessed by morphometric analyses and pulmonary function was assessed by FlexiVent (SCIREQ). Results: The combination of systemic maternal inflammation and postnatal hyperoxia exposure induced persistent alterations in pulmonary structure (alveoli/HPF: saline/RA 135±14; LPS/hyperoxia 55±6) and function (tissue resistance: saline/RA 4.89±0.24; LPS/hyperoxia 5.51±0.25 cmH2O/ml; static compliance: saline/RA 0.071±0.004; LPS/hyperoxia 0.058±0.003 ml/cmH2O). Furthermore, these changes were associated with a fibrotic phenotype as indicated by increases in collagen I (saline/RA 0.7±0.1; LPS/hyperoxia 3.8±1.3 arbitrary density) and collagen III (saline/RA 0.8±0.2; LPS/hyperoxia 15.9±7.3 arbitrary density). Histological assessments indicated increases in elastin deposition and redistribution of α smooth muscle actin in lung tissue sections. Conclusion: Our data indicate that systemic maternal inflammation and neonatal hyperoxia exposure induces persistent alterations in lung structure and function which is characterized by fibrosis. This data could provide insights into the long-term pulmonary deficits observed in adults with a history of preterm birth. doi: 109 A Non­invasive Tool for Examining the Intracellular Redox Environment of Mycobacterium tuberculosis Anup Srivastava1, Ashwani Kumar1, David K. Crossman1, James S. Remington2, and Adrie J.C. Steyn1 1University of Alabama at Birmingham, 2University of Oregon Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is a global health problem causing ~2 million deaths annually, and has latently infected approximately one third of the world’s population. Several studies suggest that the ability of Mtb to undergo long-term persistence hinges on its ability to resist host-generated oxido-reductive stress during infection. A clear understanding of the molecular mechanisms required to maintain redox homeostasis in Mtb is limited due to the lack of tools capable of measuring the mycobacterial intracellular redox potential. In this study, we have creatively developed a novel, non-invasive tool based on redox sensitive green fluorescent protein (roGFP) to quantify the intracellular redox environment of mycobacteria in real-time. Using roGFP, we have demonstrated that the intracellular redox potential of Mycobacterium smegmatis (Msm) is -266 ± 4 mV and that of Mtb is -236 mV. We have also examined the influence of host-generated redox agents •NO, O2

•–, and H2O2, and the frontline anti-TB drugs isoniazid, rifampicin, ethionamide, and ethambutol on the intracellular redox

SFRBM/SFRRI 2010S50

10.1016/j.freeradbiomed.2010.10.109

10.1016/j.freeradbiomed.2010.10.110

10.1016/j.freeradbiomed.2010.10.111

environment of Msm. Lastly, we provide genetic evidence that mycobacterial redox homeostasis is predominantly maintained by the pseudodipeptide, mycothiol (MSH). We anticipate that roGFP will contribute towards understanding virulence mechanisms utilized by Mtb to cause pathogenesis and persist, which may lead to the development of better strategies for the control of TB. doi:

110 Glutathione Reductase Deficiency Alters Lung Development in Newborn Mice Joseph Luchsinger1, Lynette K Rogers1,2, Morgan L Locy1, Cynthia L Hill1, Markus Velten1, Leif D Nelin1,2, and Trent E Tipple1,2 1Nationwide Children's Hospital, 2The Ohio State University College of Medicine Adult glutathione reductase (GR)-knockout (KO) mice are more resistant to hyperoxic lung injury through mechanisms that likely involve compensation by the thioredoxin (Trx) system. Exposure of neonatal wild-type mice to 85% O2 results in a phenotype similar to that seen in infants with bronchopulmonary dysplasia (BPD) with lung growth arrest manifested by fewer and larger alveoli. However, the roles of the GSH and Trx systems in this pathologic phenotype are not well defined. The present studies tested the hypothesis that neonatal GR-KO mice exposed to 85% O2 would not exhibit the BPD phenotype following hyperoxia exposure. Within 24 h of birth, wild-type C3H/HeN and GR-KO mice were exposed to 85% O2 or were kept in room air (RA). Morphometric analyses were performed on lung sections from 14 d mice. Homogenates prepared from the lungs of 7 d mice were analyzed for Trx1 and Trx reductase-1 (TrxR1) contents by western blotting. Data (in arbitrary units) were analyzed by 2-way ANOVA with differences noted at p<0.05. Lung morphometric analyses of RA-raised animals indicated that 14 d GR-KO mice had statistically fewer and smaller alveoli than wild-type C3H/HeN mice. GR-KO mice exposed to 85% O2 exhibited much less severe growth arrest than did hyperoxia-exposed wild-type controls. Lung Trx1 protein levels were greater in GR-knockout mice than in wild-type mice (31.0±2.8 vs 7.0±2.6) raised in RA but no differences in TrxR1 protein levels between strains were detected (6.3±0.4 vs 5.3±0.7). In contrast to wild-type animals, exposure of GR-KO mice to hyperoxia increased both Trx1 (49.5±5.5) and TrxR1 (12.0±0.7) lung protein levels. Our data indicate that GR is necessary for normal lung development. Interestingly, GR deficient mice exhibit less severe hyperoxia-induced lung phenotypic alterations. Increases in Trx1 and TrxR1 protein levels in the lungs of GR-KO mice suggest compensation for GR deficiency by the Trx system. We speculate that the Trx system may protect against the effects of hyperoxia on lung development in prematurely born human infants. doi: 111 Addition of Açai (Euterpe oleracea) to Cigarettes has a Protective Effect against Emphysema in Mice Samuel Santos Valenca1, Karla Pereira Pires2, Thiago Santos Ferreira1,2, Alan Aguiar Lopes2, Renata Tiscoski Nesi1, Angela Castro Resende2, Pergentino Cunha Sousa3, Luís Cristóvão Porto2, and Roberto Soares Moura2 1UFRJ, 2UERJ, 3UFPa Chronic inhalation of cigarette smoke (CS) induces emphysema by the damage contributed by oxidative stress during inhalation of CS. Ingestion of açai fruits (Euterpe oleracea) in animals has both antioxidant and anti-inflammatory effects. This study compared lung damage in mice induced by chronic (60-day) inhalation of regular CS and smoke from cigarettes containing 100 mg of

hydroalcoholic extract of açai stone extract (CS+A). Sham smoke-exposed mice served as the control group. Mice were sacrificed on day 60, bronchoalveolar lavage was performed, and lungs were removed for histological and biochemical analyses. Histopathological investigation showed enlargement of alveolar space in CS mice compared to CS+A and control mice. The increase in leukocytes in the CS group was higher than the increase observed in the CS+A group (p<0.001). Oxidative stress, as evaluated by antioxidant enzyme activities, myeloperoxidase, glutathione, and 4-hydroxynonenal, was reduced in mice exposed to CS+A versus CS (at least p<0.05). Macrophage and neutrophil elastase levels were reduced in mice exposed to CS+A versus CS (p<0.05). Thus, the presence of açai extract in cigarettes had a protective effect against emphysema in mice, probably by reducing oxidative and inflammatory reactions. These results raise the possibility that addition of açai extract to normal cigarettes could reduce their harmful effects. Supported by: CNPq, CAPES, FAPERJ and UERJ. doi: 112 Hypoxia Inducible Factor (HIF­1) Protects Against Oxidant Induced Airway Epithelial Barrier Dysfunction Through the Modulation of the Anti­oxidant Enzymes Peroxiredoxin and Sestrin­2 Nels C Olson1,2, Nicholas H Heintz1,2, Karen M Lounsbury1,3, and Albert van der Vliet1,2 1College of Medicine, The University of Vermont, Departments of, 2Pathology and, 3Pharmacology The respiratory epithelium creates an important barrier to minimize permeability to inhaled pollutants and organisms, and disruption of epithelial barrier function is a common feature of inflammatory airway diseases such as asthma. One feature of epithelial activation during airway inflammation is the activation of the heterodimeric transcription factor hypoxia inducible factor 1 (HIF-1), a moderator of inflammation and innate immunity as well as epithelial integrity. Based on previous findings suggesting the importance of HIF-1 in maintenance of intestinal epithelial integrity during colitis, we explored the importance of HIF-1 activation on airway epithelial barrier function in an in vitro model of oxidant-induced epithelial injury, using cultured monolayers of immortalized human bronchial epithelial cells (16HBE14o-) or primary mouse tracheal epithelial (MTE) cells seeded on Transwell inserts. Exposure to 16HBE14o- or MTE cells to H2O2 resulted in significant loss of transepithelial electrical resistance (TER) and increased permeability to FITC-dextran (4 kDa), which was associated with reduced levels of the tight junction protein occludin and hyperoxidation of the antioxidant enzyme peroxiredoxin (Prx-SO2H). Prior exposure of these cells to hypoxia (2% O2) or overnight incubation with the HIF activators CoCl2 or Dimethyloxalylglycine (DMOG) delayed or prevented H2O2-mediated disruption of epithelial barrier function, as well as loss of occludin and Prx hyperoxidation. To demonstrate a role for HIF-1α in these protective effects, we performed similar experiments in the presence of the pharmacological HIF-1 inhibitor YC-1 or in 16HBE14o- cells stably transfected with HIF-1α shRNA, which demonstrated the importance of HIF-1 activation in these protective effects. Activation of HIF-1 by either CoCl2 or DMOG also resulted in induction of sestrin-2, an enzyme known to reduce Prx-SO2H. Taken together, our results suggest that loss of epithelial barrier integrity by oxidative stress may be associated with Prx hyperoxidation as a stress signal, and that activation of HIF-1 may serve a protective role against oxidant-induced epithelial injury during airway inflammation, in part by inducing anti-oxidant enzymes such as sestrin-2. doi:

SFRBM/SFRRI 2010 S51

10.1016/j.freeradbiomed.2010.10.112

10.1016/j.freeradbiomed.2010.10.113

10.1016/j.freeradbiomed.2010.10.114

10.1016/j.freeradbiomed.2010.10.115