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Nrf2 Polymorphisms and Association With Susceptibility To Oxidative Lung Injury in Mice
Future work will evaluate whether the enhanced PH in mice lacking SMC SOD3 is mediated by alterations in NO metabolism or inflammation.
Pulmonary Diseases 261 Role of Endoplasmic Reticulum Stress and Oxidative Stress in Influenza Virus Induced Lung Fibrosis Yvonne Janssen-Heininger1, and Vikas Anathy1 1 University of Vermont Several environmental insults are known to cause lung fibrosis in humans. Fibrosis is an irreversible, progressive and ultimately fatal disease process. Several respiratory viruses including influenza virus, SARS-corona virus and respiratory syncytial virus can cause acute respiratory distress syndrome (ARDS) and lung fibrosis. the respiratory viral infections are known to cause apoptosis of lung epithelial cells which may lead to lung remodeling and ultimately fatal fibrosis. So far, there are no in vivo comprehensive studies linking the mechanisms of respiratory virus-triggered epithelial apoptosis in lung fibroproliferative diseases. Our recent study demonstrates that influenza virus infection in mice enhances endoplasmic reticulum (ER) stress, oxidative stress (in the form of protein S-glutathionylation) in lung epithelial cells. This enhancement in ER stress leads to Fas or caspase-8 independent apoptosis of lung epithelial cells and ultimately resulting in lung fibrosis in mice. Our study, demonstrate a novel mechanistic link between respiratory virus induced ER stress, oxidative stress and activation of death receptor independent epithelial cell apoptosis during lung fibrosis.
doi:10.1016/j.freeradbiomed.2011.10.344
262 Conditional Knock Down of Extracellular Superoxide Dismutase in Smooth Muscle Cells Augments Chronic Hypoxic Pulmonary Hypertension. Rahul Birari1, Leah Villegas1, Julia Locke1, David Harrison2, and Eva Nozik-Grayck1 1 2 university of Colorado Denver, Vanderbilt University Excess superoxide has been implicated in chronic hypoxiainduced pulmonary hypertension (PH). We have previously shown that lung over-expression of the antioxidant extracellular superoxide dismutase (SOD3) attenuates chronic hypoxia pulmonary vascular remodeling and PH. SOD3 is highly expressed in arteries, thus we hypothesize that loss of SOD3 specifically from pulmonary artery smooth muscle cells (PA SMC) will augment hypoxia-induced PH and remodeling within the medial wall. To test our hypothesis, the SOD3 gene was deleted loxp/loxp in adult mice using the Cre-Lox technology. Briefly SOD3 × cre/esr Tg mice (SMC SOD3 KO) were used and the Crerecombinase was activated by the intraperitoneal injection of tamoxifen (0.4mg/20 gm of body weight) for 5 consecutive days. loxp/loxp SOD3 (SOD3 control) mice were used for control experiments. Mice were exposed to hypobaric hypoxia (395 torr) for 3 or 21 days and evaluated for PH and medial wall remodeling. Conditional knockout of SMC SOD3 depleted aortic SOD3 by 95% and total lung SOD3 protein by 50% (P<0.05). SMC SOD3 KO increased PH and right ventricular (RV) hypertrophy, measured by RV systolic pressures and RV/LV+septum weights compared to SOD3 controls both at baseline and following 21 days of chronic hypoxia (P<0.05). Loss of SMC SOD3 did not enhance cell proliferation in small PA at 3 days nor did it augment PA medial wall thickening or the number of muscularized small PA at 21 days. We conclude that SMC SOD3 promotes lower pulmonary vascular tone at baseline and attenuates chronic hypoxic PH, and the increased PH in mice lacking SMC SOD3 was not due to PA medial wall remodeling.
S110
doi:10.1016/j.freeradbiomed.2011.10.345
263 Nrf2 Polymorphisms and Association With Susceptibility To Oxidative Lung Injury in Mice Hye-Youn Cho1, Anne E Jedlicka2, Wesley Gladwell1, Jacqui Marzec1, and Steven R Kleeberger1 1 National Institute of Environmental Health Sciences, National 2 Institutes of Health, Johns Hopkins University Bloomberg School of Public Health Nrf2 is a master transcription factor of cytoprotective genes through binding on antioxidant response element (ARE). Hyperoxia exposure to laboratory rodents induces pulmonary injury which resembles acute lung injury (ALI) sub-phenotypes. Genome-wide linkage analysis in mice revealed Nrf2 as a susceptibility gene of hyperoxia-induced ALI, and its protective roles have been determined. the current study was designed to discover sequence variation in Nrf2 and to determine the association with hyperoxia susceptibility in mice. Single nucleotide polymorphisms (SNPs) in Nrf2 genome including 5 kb upstream promoter (chr2:75547698-75510174bp) were compiled for 73 inbred strains from publicly available data base. Bialleric matrix mapping of the SNPs categorized the mice into 3 distinct haplotypes: C3H/HeJ (C3)-like 27 strains (class I), C57BL/6J (B6)-like 23 strains (class II), and P/J-like 16 strains (class III). Representative 16 strains (class I: C3, A/J, DBA/2J, BALB/cJ, CBA/J, LP/J; class II: B6, C57BL10/J, C57L/J, AKR/J, NZW/LACJ, SJL/J, SWR/J; class III: P/J, SM/J, SEA/GnJ) were re-sequenced for validation and further elucidation of SNPs. a total of 230 SNPs included 29 in 5’ upstream, 18 in exons 1, 2, 3, and 5, 163 in introns, and 20 in 3’ downstream regions. Sixteen strains were exposed to hyperoxia (>95% O2, 72 h), and lung injury was compared by bronchoalveolar lavage analysis. Functional roles of SNP haplotypes were determined by comparison of lung expression and activity of Nrf2 and downstream effectors. Hyperoxia-induced body weight loss and lung injury was relatively greater in class II and III strains than in class I. Promoter SNPs including T-103C which adds additional Sp1 binding site in class II suppressed the promoter activity and Nrf2 induction after hyperoxia relative to class I. Nuclear proteins from class III mice bearing two non-synonymous SNPs located in (1862A>T, His543Gln) and adjacent to (1417T>C, Thr395Ile) Neh1 domain of exon 5 showed diminished ARE binding activity after hyperoxia. Results indicate that genetic variations of Nrf2 associated with its functions confer significant influence on hyperoxia-induced ALI susceptibility in mice.
doi:10.1016/j.freeradbiomed.2011.10.346
264 Alveolar Epithelial Type I Cells Attenuate Cigarette Smoke Extract-Induced Production of Hydrogen Peroxide by Microvascular Endothelial Cells Charles a Downs1, David W. Montgomery2,3, and Carrie J Merkle2,3 1 2 Emory University, Southern Arizona VA HealthCare System, 3 The University of Arizona Cigarette smoke causes oxidative stress in the lung. the response of alveolar epithelial type I (AT I) cells and
SFRBM 2011
Pulmonary Diseases 261 Role of Endoplasmic Reticulum Stress and Oxidative Stress in Influenza Virus Induced Lung Fibrosis Yvonne Janssen-Heininger1, and Vikas Anathy1 1 University of Vermont Several environmental insults are known to cause lung fibrosis in humans. Fibrosis is an irreversible, progressive and ultimately fatal disease process. Several respiratory viruses including influenza virus, SARS-corona virus and respiratory syncytial virus can cause acute respiratory distress syndrome (ARDS) and lung fibrosis. the respiratory viral infections are known to cause apoptosis of lung epithelial cells which may lead to lung remodeling and ultimately fatal fibrosis. So far, there are no in vivo comprehensive studies linking the mechanisms of respiratory virus-triggered epithelial apoptosis in lung fibroproliferative diseases. Our recent study demonstrates that influenza virus infection in mice enhances endoplasmic reticulum (ER) stress, oxidative stress (in the form of protein S-glutathionylation) in lung epithelial cells. This enhancement in ER stress leads to Fas or caspase-8 independent apoptosis of lung epithelial cells and ultimately resulting in lung fibrosis in mice. Our study, demonstrate a novel mechanistic link between respiratory virus induced ER stress, oxidative stress and activation of death receptor independent epithelial cell apoptosis during lung fibrosis.
doi:10.1016/j.freeradbiomed.2011.10.344
262 Conditional Knock Down of Extracellular Superoxide Dismutase in Smooth Muscle Cells Augments Chronic Hypoxic Pulmonary Hypertension. Rahul Birari1, Leah Villegas1, Julia Locke1, David Harrison2, and Eva Nozik-Grayck1 1 2 university of Colorado Denver, Vanderbilt University Excess superoxide has been implicated in chronic hypoxiainduced pulmonary hypertension (PH). We have previously shown that lung over-expression of the antioxidant extracellular superoxide dismutase (SOD3) attenuates chronic hypoxia pulmonary vascular remodeling and PH. SOD3 is highly expressed in arteries, thus we hypothesize that loss of SOD3 specifically from pulmonary artery smooth muscle cells (PA SMC) will augment hypoxia-induced PH and remodeling within the medial wall. To test our hypothesis, the SOD3 gene was deleted loxp/loxp in adult mice using the Cre-Lox technology. Briefly SOD3 × cre/esr Tg mice (SMC SOD3 KO) were used and the Crerecombinase was activated by the intraperitoneal injection of tamoxifen (0.4mg/20 gm of body weight) for 5 consecutive days. loxp/loxp SOD3 (SOD3 control) mice were used for control experiments. Mice were exposed to hypobaric hypoxia (395 torr) for 3 or 21 days and evaluated for PH and medial wall remodeling. Conditional knockout of SMC SOD3 depleted aortic SOD3 by 95% and total lung SOD3 protein by 50% (P<0.05). SMC SOD3 KO increased PH and right ventricular (RV) hypertrophy, measured by RV systolic pressures and RV/LV+septum weights compared to SOD3 controls both at baseline and following 21 days of chronic hypoxia (P<0.05). Loss of SMC SOD3 did not enhance cell proliferation in small PA at 3 days nor did it augment PA medial wall thickening or the number of muscularized small PA at 21 days. We conclude that SMC SOD3 promotes lower pulmonary vascular tone at baseline and attenuates chronic hypoxic PH, and the increased PH in mice lacking SMC SOD3 was not due to PA medial wall remodeling.
S110
doi:10.1016/j.freeradbiomed.2011.10.345
263 Nrf2 Polymorphisms and Association With Susceptibility To Oxidative Lung Injury in Mice Hye-Youn Cho1, Anne E Jedlicka2, Wesley Gladwell1, Jacqui Marzec1, and Steven R Kleeberger1 1 National Institute of Environmental Health Sciences, National 2 Institutes of Health, Johns Hopkins University Bloomberg School of Public Health Nrf2 is a master transcription factor of cytoprotective genes through binding on antioxidant response element (ARE). Hyperoxia exposure to laboratory rodents induces pulmonary injury which resembles acute lung injury (ALI) sub-phenotypes. Genome-wide linkage analysis in mice revealed Nrf2 as a susceptibility gene of hyperoxia-induced ALI, and its protective roles have been determined. the current study was designed to discover sequence variation in Nrf2 and to determine the association with hyperoxia susceptibility in mice. Single nucleotide polymorphisms (SNPs) in Nrf2 genome including 5 kb upstream promoter (chr2:75547698-75510174bp) were compiled for 73 inbred strains from publicly available data base. Bialleric matrix mapping of the SNPs categorized the mice into 3 distinct haplotypes: C3H/HeJ (C3)-like 27 strains (class I), C57BL/6J (B6)-like 23 strains (class II), and P/J-like 16 strains (class III). Representative 16 strains (class I: C3, A/J, DBA/2J, BALB/cJ, CBA/J, LP/J; class II: B6, C57BL10/J, C57L/J, AKR/J, NZW/LACJ, SJL/J, SWR/J; class III: P/J, SM/J, SEA/GnJ) were re-sequenced for validation and further elucidation of SNPs. a total of 230 SNPs included 29 in 5’ upstream, 18 in exons 1, 2, 3, and 5, 163 in introns, and 20 in 3’ downstream regions. Sixteen strains were exposed to hyperoxia (>95% O2, 72 h), and lung injury was compared by bronchoalveolar lavage analysis. Functional roles of SNP haplotypes were determined by comparison of lung expression and activity of Nrf2 and downstream effectors. Hyperoxia-induced body weight loss and lung injury was relatively greater in class II and III strains than in class I. Promoter SNPs including T-103C which adds additional Sp1 binding site in class II suppressed the promoter activity and Nrf2 induction after hyperoxia relative to class I. Nuclear proteins from class III mice bearing two non-synonymous SNPs located in (1862A>T, His543Gln) and adjacent to (1417T>C, Thr395Ile) Neh1 domain of exon 5 showed diminished ARE binding activity after hyperoxia. Results indicate that genetic variations of Nrf2 associated with its functions confer significant influence on hyperoxia-induced ALI susceptibility in mice.
doi:10.1016/j.freeradbiomed.2011.10.346
264 Alveolar Epithelial Type I Cells Attenuate Cigarette Smoke Extract-Induced Production of Hydrogen Peroxide by Microvascular Endothelial Cells Charles a Downs1, David W. Montgomery2,3, and Carrie J Merkle2,3 1 2 Emory University, Southern Arizona VA HealthCare System, 3 The University of Arizona Cigarette smoke causes oxidative stress in the lung. the response of alveolar epithelial type I (AT I) cells and
SFRBM 2011