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Cigarette Smoke-Stimulated Epithelial-Mesenchymal Transition Through Src Activation
microvascular endothelial cells (MVECs) to cigarette smoke extract (CSE) have not been fully elucidated. the purpose here was to begin examining the effects of CSE on the redox state of AT I and MVECs cultured separately and as co-cultures. Primary AT I and MVECs were obtained from the lungs of male Fischer 344 rats. Cells were studied as single cultures and in co-culture with AT I and MVECs seeded on opposites sides of Transwell Fluroblok™ filters with 1 micron pores. the concentration of CSE was standardized spectrophotometrically using the smoke from one research grade (1R5F) cigarette dissolved in 10 mL of RPMI 1640 with 10% FBS. Cells were exposed to CSE for 24 hours and intracellular ROS, extracellular ROS, and total antioxidant capacity measured in AT I and MVECs from single and cocultures. ROS production in AT I cells did not change even when exposed to 100% CSE (1-100%). AT I cells grown as a single culture did not produce significant H2O2 while MVECs demonstrated a 2 to 2.5 fold increase. Interestingly, when AT I and MVECs were grown as co-cultures, H2O2 levels were reduced 1 to 4 fold compared to MVECs cultured alone. the total antioxidant capacity of single cultures of AT I cells was greater (~8 fold) than MVECs which may explain the reduction of H2O2 levels. Gene expression profiling specific to oxidant and antioxidant pathways revealed 9 genes that were significantly different in MVECs grown in co-culture compared to single cultures. the results of these studies may provide insight into mechanisms of CSE-induced injury and possible therapeutic strategies to reduce oxidant burden and lung injury secondary to smoking and related environmental exposures.
doi:10.1016/j.freeradbiomed.2011.10.347
265 Cigarette Smoke-Stimulated Epithelial-Mesenchymal Transition Through Src Activation Hongqiao Zhang1, Honglei Liu1, Zea Borok1, Kelvin J.A. Davies1, Fulvio Ursini2, and Henry J Forman1,3 1 2 University of Southern California, Università di Padova, Italy 3 University of California, Merced Epithelial-mesenchymal transition (EMT) is implicated in the pathogenesis of lung fibrosis and cancer metastasis, two conditions associated with cigarette smoke (CS). CS has been reported to promote the EMT process. CS is the major cause of lung cancer and nearly half of lung cancer patients are active smokers. Nonetheless, the mechanism whereby CS induces EMT remains largely unknown. in this study we investigated the induction of EMT by CS extract (CSE) and explored the underlying mechanisms in the human non-small cell lung carcinoma (H358) cell line. We demonstrate that CSE exposure decreases E-cadherin and increases N-cadherin and vimentin, markers of EMT. Pretreatment with N-acetyl cysteine (NAC), a potent antioxidant and precursor of glutathione, abrogated changes in these EMT markers. Furthermore, CSE activated Src kinase (shown as increased phosphorylation of Src at Tyr418) and the Src kinase inhibitor, PP2, inhibited CS-stimulated EMT changes, suggesting that Src is critical in CSE-stimulated EMT induction.
doi:10.1016/j.freeradbiomed.2011.10.348
266 Mechanisms by Which the EC-SOD R213G SNP Reduces the Risk for Lung Disease David A. Goldstrohm1, Rebecca E. Oberley-Deegan1, Michael R. Weaver1, Zuzana Valnickova2, Jan J. Enghild2, Robert J. Mason1, Tim D. Oury3, Angela K. Tollefson1, Steen V. Petersen2, and Russell P. Bowler1 1 2 National Jewish Health, Center for Insoluble Protein Structures 3 University of and Interdisciplinary Nanoscience Center, Pittsburgh School of Medicine Chronic Obstructive Pulmonary Disease (COPD) is the fourth leading cause of death and second leading cause of hospital days in the United States. Large human genetic studies have reproducibly identified a nonsynonymous single nucleotide polymorphism (SNP) in SOD3 (the gene encoding the antioxidant extracellular superoxide dismutase; EC-SOD) associated with a decreased susceptibility to and exacerbations of COPD. Because the R213G SNP in SOD3 only occurs in humans, we have limited knowledge of how this SNP might protect lung function. To elucidate these mechanisms, we recently created a R213G SNP knockin mouse. Similar to humans, these mice have a significant 3-fold increase in EC-SOD protein levels in their plasma. the R213G mice demonstrated a significant 1.5-fold increase in ECSOD protein levels in lung epithelial lining fluid and significant 3.8fold and 4.2-fold decreases in EC-SOD levels in lung tissue and aorta, respectively. Because COPD exacerbations are associated with bacterial infections, we tested whether the R213G mice were less susceptible to LPS-induced lung injury. Following LPS exposure, the R213G mice had a significant 3.3-fold decrease in the recruitment of neutrophils from LPS-induced inflammation. the R213G mice also had significant 2.1-fold and 1.5-fold decreases in proinflammatory TNF-α cytokines and protein nitration, respectively. R213G alveolar macrophages also contained diminished levels of EC-SOD. These results suggest the R213G polymorphism attenuates the risk of COPD by shifting the distribution of EC-SOD activity from lung tissue towards the extracellular spaces, thus blunting redox sensitive proinflammatory signaling pathways. This is the first R213G knockin mouse in existence for a SNP that has been associated with COPD and permits us to perform experiments that would be risky or unethical in humans (e.g. toxin exposure and organ resection). Understanding how the R213G SNP alters the distribution of EC-SOD may lead to better-targeted antioxidant therapies for lung disease. the R213G mouse model may also lead to a better understanding of cardiovascular disease since the R213G SNP is a major risk factor for stroke and myocardial infarction.
doi:10.1016/j.freeradbiomed.2011.10.349
267 Cu,Zn-SOD Induces Pulmonary Fibrosis via Alternatively Activation of Macrophages Chao He1, Alan J Ryan2, Shubha Murthy2, and a Brent Carter1,2 1 2 Free Radical and Radiation Biology Program, Department of Internal Medicine, the University of Iowa Asbestos-induced pulmonary fibrosis is associated with increasing H2O2 production by alveolar macrophages. We have shown that mitochondrial Cu,Zn-SOD regulates H2O2 levels, and -/Cu,Zn-SOD mice do not develop pulmonary fibrosis. Alternatively activated macrophages (AAM, M2) are known to have an important role in fibrosis. We hypothesize that Cu,ZnSOD mediates pulmonary fibrosis via increasing M2-phenotype. Our data show that over expression of Cu,Zn-SOD increased chemokine (CCL-18) expression. To further support this notion, Tg macrophages obtained from WT and Cu,Zn-SOD mice showed Tg that macrophages from Cu,Zn-SOD mice have increased
SFRBM 2011
S111
doi:10.1016/j.freeradbiomed.2011.10.347
265 Cigarette Smoke-Stimulated Epithelial-Mesenchymal Transition Through Src Activation Hongqiao Zhang1, Honglei Liu1, Zea Borok1, Kelvin J.A. Davies1, Fulvio Ursini2, and Henry J Forman1,3 1 2 University of Southern California, Università di Padova, Italy 3 University of California, Merced Epithelial-mesenchymal transition (EMT) is implicated in the pathogenesis of lung fibrosis and cancer metastasis, two conditions associated with cigarette smoke (CS). CS has been reported to promote the EMT process. CS is the major cause of lung cancer and nearly half of lung cancer patients are active smokers. Nonetheless, the mechanism whereby CS induces EMT remains largely unknown. in this study we investigated the induction of EMT by CS extract (CSE) and explored the underlying mechanisms in the human non-small cell lung carcinoma (H358) cell line. We demonstrate that CSE exposure decreases E-cadherin and increases N-cadherin and vimentin, markers of EMT. Pretreatment with N-acetyl cysteine (NAC), a potent antioxidant and precursor of glutathione, abrogated changes in these EMT markers. Furthermore, CSE activated Src kinase (shown as increased phosphorylation of Src at Tyr418) and the Src kinase inhibitor, PP2, inhibited CS-stimulated EMT changes, suggesting that Src is critical in CSE-stimulated EMT induction.
doi:10.1016/j.freeradbiomed.2011.10.348
266 Mechanisms by Which the EC-SOD R213G SNP Reduces the Risk for Lung Disease David A. Goldstrohm1, Rebecca E. Oberley-Deegan1, Michael R. Weaver1, Zuzana Valnickova2, Jan J. Enghild2, Robert J. Mason1, Tim D. Oury3, Angela K. Tollefson1, Steen V. Petersen2, and Russell P. Bowler1 1 2 National Jewish Health, Center for Insoluble Protein Structures 3 University of and Interdisciplinary Nanoscience Center, Pittsburgh School of Medicine Chronic Obstructive Pulmonary Disease (COPD) is the fourth leading cause of death and second leading cause of hospital days in the United States. Large human genetic studies have reproducibly identified a nonsynonymous single nucleotide polymorphism (SNP) in SOD3 (the gene encoding the antioxidant extracellular superoxide dismutase; EC-SOD) associated with a decreased susceptibility to and exacerbations of COPD. Because the R213G SNP in SOD3 only occurs in humans, we have limited knowledge of how this SNP might protect lung function. To elucidate these mechanisms, we recently created a R213G SNP knockin mouse. Similar to humans, these mice have a significant 3-fold increase in EC-SOD protein levels in their plasma. the R213G mice demonstrated a significant 1.5-fold increase in ECSOD protein levels in lung epithelial lining fluid and significant 3.8fold and 4.2-fold decreases in EC-SOD levels in lung tissue and aorta, respectively. Because COPD exacerbations are associated with bacterial infections, we tested whether the R213G mice were less susceptible to LPS-induced lung injury. Following LPS exposure, the R213G mice had a significant 3.3-fold decrease in the recruitment of neutrophils from LPS-induced inflammation. the R213G mice also had significant 2.1-fold and 1.5-fold decreases in proinflammatory TNF-α cytokines and protein nitration, respectively. R213G alveolar macrophages also contained diminished levels of EC-SOD. These results suggest the R213G polymorphism attenuates the risk of COPD by shifting the distribution of EC-SOD activity from lung tissue towards the extracellular spaces, thus blunting redox sensitive proinflammatory signaling pathways. This is the first R213G knockin mouse in existence for a SNP that has been associated with COPD and permits us to perform experiments that would be risky or unethical in humans (e.g. toxin exposure and organ resection). Understanding how the R213G SNP alters the distribution of EC-SOD may lead to better-targeted antioxidant therapies for lung disease. the R213G mouse model may also lead to a better understanding of cardiovascular disease since the R213G SNP is a major risk factor for stroke and myocardial infarction.
doi:10.1016/j.freeradbiomed.2011.10.349
267 Cu,Zn-SOD Induces Pulmonary Fibrosis via Alternatively Activation of Macrophages Chao He1, Alan J Ryan2, Shubha Murthy2, and a Brent Carter1,2 1 2 Free Radical and Radiation Biology Program, Department of Internal Medicine, the University of Iowa Asbestos-induced pulmonary fibrosis is associated with increasing H2O2 production by alveolar macrophages. We have shown that mitochondrial Cu,Zn-SOD regulates H2O2 levels, and -/Cu,Zn-SOD mice do not develop pulmonary fibrosis. Alternatively activated macrophages (AAM, M2) are known to have an important role in fibrosis. We hypothesize that Cu,ZnSOD mediates pulmonary fibrosis via increasing M2-phenotype. Our data show that over expression of Cu,Zn-SOD increased chemokine (CCL-18) expression. To further support this notion, Tg macrophages obtained from WT and Cu,Zn-SOD mice showed Tg that macrophages from Cu,Zn-SOD mice have increased
SFRBM 2011
S111