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The NADPH oxidase DUOX1 Mediates Airway Epithelial Regeneration Following Naphthalene-Induced Injury
lesions, lipid and protein damage, and inflammatory responses. Recent studies in rodents and non-human primates have shown that single- and multi-day exposures to ozone induce mitochondrial damage. However, what transpires during the initial phases of exposure remains poorly defined. the goal of this study was to determine the temporal and spatial distribution of early ozone-induced airway injury and the role of mitochondrial damage and dysfunction on injury initiation and progression. Methods: Male Sprague-Dawley rats (250-300g) were exposed to filtered air or ozone (1 ppm) for 1, 2, 3, 4, or 8 hours. Immediately after exposure cessation, (i) lungs were evaluated for airway cell injury via differential permeability using nuclear fluorochromes and whole mount airway imaging and high resolution histopathology, (ii) airways dissected, snap frozen, and used for mitochondrial DNA (mtDNA) damage by quantitative PCR and for superoxide dismutase and aconitase activity assays, or (iii) airways dissected, mitochondria isolated and respirometry studies conducted. Results: Injury was observed in distal airways as early as 1 hour, and in the proximal airways at 3 hours. Mitochondrial DNA (mtDNA) lesions correlated temporally and spatially with the distribution of cell injury. Conclusions: Ozone mediated airway injury is site- and timespecific. MtDNA damage coincides with plasma membrane permeability which may indicate mitochondrial involvement in early injury processes induced by ozone inhalation. Supported by P01 ES-11617 and DRTC BARB Core P60 DK079626.
doi:10.1016/j.freeradbiomed.2011.10.362
280 The NADPH oxidase DUOX1 Mediates Airway Epithelial Regeneration Following NaphthaleneInduced Injury Stefan Gorissen1, Milena Hristova1, Lynn M. Sipsey1, Page C. Spiess1, and Albert van der Vliet1 1 University of Vermont The respiratory epithelium plays an important role in mediating innate or adaptive immune responses to airborne pathogens and/or pollutants, and alterations in its structural integrity are important features of chronic lung diseases such as asthma, emphysema, or lung cancer. Defining the mechanisms that regulate the repair of bronchiolar epithelium is critical for understanding the pathogenesis of both acute and chronic lung diseases associated with airway remodeling. the dual oxidases DUOX1 and DUOX2 are expressed in airway epithelia and produce reactive oxygen species (ROS) as a mechanism of airway mucosal host defense and epithelial responses to infection and injury. We previously showed that DUOX1, the major DUOX isoform, participates in in vitro wound responses in human airway epithelial cells. in the present study, we observed a similar function of DUOX1 in primary murine tracheal epithelial cells. DUOX1-dependent cell migration and wound healing were mediated by oxidant-dependent activation of epidermal growth factor receptor (EGFR) and the transcription factor STAT3, previously shown to be critical in cell migration and wound responses. To explore the significance of DUOX1 for epithelial regeneration upon injury in vivo, we induced airway injury in adult mice by administration of naphthalene, which transiently depletes nonciliated respiratory epithelial cells (Clara cells). Epithelial injury was maximal 2 days after naphthalene administration, with dramatic loss of non-ciliated cells and activation of STAT3. Silencing of DUOX1 using oropharyngeal administration of DUOX1-targeted siRNA delayed airway re-epithelialization, with significantly reduced levels of non-ciliated epithelial cell types at 7 days after naphthalene injury, which was normalized after 14 days. Collectively, these studies demonstrate an important function for epithelial DUOX1 in lung epithelial regeneration by
S116
promoting cell migration as a critical event in initial repair pathways. Supported by NIH R01 HL085646
doi:10.1016/j.freeradbiomed.2011.10.363
281 Superoxide Dismutase Mimetic Attenuates HypoxiaInduced Pulmonary Vascular Remodeling, ECM Hyaluronan Expression, and NALP3-Mediated Inflammation Leah Redondo Villegas1, Carlie Field1, Dylan Kluck1, Micheal Yeager1, Karim El Kasmi1, Rebecca Oberley-Deegan2, Russell Bowler2, Rashmin Savani3, and Eva Nozik-Grayck1 1 2 3 University of Colorado, National Jewish Health, University of Texas Southwestern Medical Center We hypothesize that activation of the NALP3 inflammasome is one pathway that is important in ROS-mediated lung injury. There is an apparent need to fully understand the role of oxidative stress in chronic hypoxic PH in order to develop effective and efficient therapeutic interventions. This study sought to investigate the protective role of a pharmacological catalytic antioxidant, an SOD mimetic (AEOL 10113), in hypoxia-induced vascular remodeling, ECM modulation, and NALP3-mediated inflammation. Mice (C57/BL6) were exposed to hypobaric hypoxic conditions while sub-cutaneous injections of AEOL 10113 (5 to 10 mg/kg) or PBS were given 3x weekly for up to 35 days. To assess the development of PH, right ventricular systolic pressure (RVSP) was measured in a closed chest by direct RV puncture. the RV and LV of hearts were dissected and weighed. Fixed and embedded lung sections were immunostained with anti-Ki-67, anti-α-smooth muscle actin, and hyaluronan binding protein. RNA and protein were isolated from flash frozen right lungs to quantify hyaluronan synthases, hyaluronidases, NALP3, IL-1β and IL-18 expression by semi-quantitative RT-PCR and Western blot. Hypoxia-induced vascular remodeling, ECM modulation, and inflammation were evident by increased vascular cell proliferation, development of vessel muscularization, increased hyaluronan expression and activation of NALP3 pathway. Ki-67 nuclear staining detected significant increase of proliferating cells in response to 3-day hypoxic exposure. After 21-day hypoxic exposure there was significant increase in α-smooth muscle staining indicative of newly developed muscularized small vessels, and activation of the NALP3 inflammasome. Treatment with SOD mimetic, AEOL 10113, attenuated the development of these pathophysiological characteristics in the lung, demonstrating 50% decrease in Ki-67 nuclear staining, 30% decrease in α-smooth muscle staining, 50% decreases in NALP3, IL-1β, and IL-18 expression, compared to PBS treated groups. Although there were no changes in hyaluronan sythase and hyaluronidase gene expression, there was increased hyaluronan content upon hypoxic exposure and decreased content in AEOL 10113 treated groups. in addition, AEOL 10113 treated groups demonstrated overall protection against increased RVSP and RV hypertrophy, an indirect measure of pulmonary artery pressure and development of pulmonary hypertension.
doi:10.1016/j.freeradbiomed.2011.10.364
SFRBM 2011
doi:10.1016/j.freeradbiomed.2011.10.362
280 The NADPH oxidase DUOX1 Mediates Airway Epithelial Regeneration Following NaphthaleneInduced Injury Stefan Gorissen1, Milena Hristova1, Lynn M. Sipsey1, Page C. Spiess1, and Albert van der Vliet1 1 University of Vermont The respiratory epithelium plays an important role in mediating innate or adaptive immune responses to airborne pathogens and/or pollutants, and alterations in its structural integrity are important features of chronic lung diseases such as asthma, emphysema, or lung cancer. Defining the mechanisms that regulate the repair of bronchiolar epithelium is critical for understanding the pathogenesis of both acute and chronic lung diseases associated with airway remodeling. the dual oxidases DUOX1 and DUOX2 are expressed in airway epithelia and produce reactive oxygen species (ROS) as a mechanism of airway mucosal host defense and epithelial responses to infection and injury. We previously showed that DUOX1, the major DUOX isoform, participates in in vitro wound responses in human airway epithelial cells. in the present study, we observed a similar function of DUOX1 in primary murine tracheal epithelial cells. DUOX1-dependent cell migration and wound healing were mediated by oxidant-dependent activation of epidermal growth factor receptor (EGFR) and the transcription factor STAT3, previously shown to be critical in cell migration and wound responses. To explore the significance of DUOX1 for epithelial regeneration upon injury in vivo, we induced airway injury in adult mice by administration of naphthalene, which transiently depletes nonciliated respiratory epithelial cells (Clara cells). Epithelial injury was maximal 2 days after naphthalene administration, with dramatic loss of non-ciliated cells and activation of STAT3. Silencing of DUOX1 using oropharyngeal administration of DUOX1-targeted siRNA delayed airway re-epithelialization, with significantly reduced levels of non-ciliated epithelial cell types at 7 days after naphthalene injury, which was normalized after 14 days. Collectively, these studies demonstrate an important function for epithelial DUOX1 in lung epithelial regeneration by
S116
promoting cell migration as a critical event in initial repair pathways. Supported by NIH R01 HL085646
doi:10.1016/j.freeradbiomed.2011.10.363
281 Superoxide Dismutase Mimetic Attenuates HypoxiaInduced Pulmonary Vascular Remodeling, ECM Hyaluronan Expression, and NALP3-Mediated Inflammation Leah Redondo Villegas1, Carlie Field1, Dylan Kluck1, Micheal Yeager1, Karim El Kasmi1, Rebecca Oberley-Deegan2, Russell Bowler2, Rashmin Savani3, and Eva Nozik-Grayck1 1 2 3 University of Colorado, National Jewish Health, University of Texas Southwestern Medical Center We hypothesize that activation of the NALP3 inflammasome is one pathway that is important in ROS-mediated lung injury. There is an apparent need to fully understand the role of oxidative stress in chronic hypoxic PH in order to develop effective and efficient therapeutic interventions. This study sought to investigate the protective role of a pharmacological catalytic antioxidant, an SOD mimetic (AEOL 10113), in hypoxia-induced vascular remodeling, ECM modulation, and NALP3-mediated inflammation. Mice (C57/BL6) were exposed to hypobaric hypoxic conditions while sub-cutaneous injections of AEOL 10113 (5 to 10 mg/kg) or PBS were given 3x weekly for up to 35 days. To assess the development of PH, right ventricular systolic pressure (RVSP) was measured in a closed chest by direct RV puncture. the RV and LV of hearts were dissected and weighed. Fixed and embedded lung sections were immunostained with anti-Ki-67, anti-α-smooth muscle actin, and hyaluronan binding protein. RNA and protein were isolated from flash frozen right lungs to quantify hyaluronan synthases, hyaluronidases, NALP3, IL-1β and IL-18 expression by semi-quantitative RT-PCR and Western blot. Hypoxia-induced vascular remodeling, ECM modulation, and inflammation were evident by increased vascular cell proliferation, development of vessel muscularization, increased hyaluronan expression and activation of NALP3 pathway. Ki-67 nuclear staining detected significant increase of proliferating cells in response to 3-day hypoxic exposure. After 21-day hypoxic exposure there was significant increase in α-smooth muscle staining indicative of newly developed muscularized small vessels, and activation of the NALP3 inflammasome. Treatment with SOD mimetic, AEOL 10113, attenuated the development of these pathophysiological characteristics in the lung, demonstrating 50% decrease in Ki-67 nuclear staining, 30% decrease in α-smooth muscle staining, 50% decreases in NALP3, IL-1β, and IL-18 expression, compared to PBS treated groups. Although there were no changes in hyaluronan sythase and hyaluronidase gene expression, there was increased hyaluronan content upon hypoxic exposure and decreased content in AEOL 10113 treated groups. in addition, AEOL 10113 treated groups demonstrated overall protection against increased RVSP and RV hypertrophy, an indirect measure of pulmonary artery pressure and development of pulmonary hypertension.
doi:10.1016/j.freeradbiomed.2011.10.364
SFRBM 2011