- Email: [email protected]
The role of mitochondrial reactive oxygen species in the response of the pulmonary vasculature to hypoxia and right heart remodeling
A. Stepanova et al. / Free Radical Biology and Medicine 120 (2018) S45–S166
Methods: To study 2-ClHDA-mediated protein modification in cardiomyocytes, click-chemistry with proteomic approaches was used. Therefore, an alkyne containing bioortholog of 2-ClHDA, 2-chlorohexadec-15yn-1-al (2-ClHDyA), was synthesized. Protein lysates of 2-ClHDyA-modified cardiomyocytes were clicked with tetramethylrhodamine (TAMRA) azide and separated by 2-DE. TAMRA-positive spots were trypsinized and identified by LC-MS/MS. Results: During these analyses 51 2-ClHDyA-modified proteins were identified. Those proteins were mainly attributed to cellular processes involved in stress response, the cytoskeleton, and enzymes responsible for energy metabolism. These results suggest that cellular damage following myocardial infarction could be caused by impaired protein function due to modification by 2-ClHDA.
E-mail address: [email protected]
S101
P-188
Enterococcus faecalis FK-23 may improve the bactericidal activity of human neutrophil via enhancing ROS production and phagocytosis Hiroshi Ichikawa 1, Yutaro Yamada 1, Shuhei Saiki 1, Keiko Kobayasi 2, Yukiko Minamiyama 2 1 Graduate School of Life and Medical Sciences, Doshisha University, Kyoto, Japan 2 Graduate School of Life and Environmental Sciences, Kyoto Prefectural University, Kyoto, Japan
http://dx.doi.org/10.1016/j.freeradbiomed.2018.04.333
P-187
Nox 5 overexpression induces the activation of the unfolded protein response in human aortic endothelial cells Álvaro Pejenaute Martinez de Lizarrondo, Adriana Cortés Jiménez, Eduardo Ansorena Artieda, Silvia Cenoz Zubillaga, Carlos de Miguel Vázquez, Guillermo Zalba Goñi Biochemistry and Genetics Department, University of Navarra IdiSNA, Pamplona, Spain
Oxidative stress is a critical mechanism that underlies the pathophysiology of cardiovascular diseases. At the vascular level, an enhanced NADPH oxidase-mediated superoxide production associates with endothelial dysfunction, metalloprotease secretion, cellular senescence, and more recently activation of the unfolded protein response (UPR). Human aortic endothelial cells (HAEC) were infected with Nox 5 or GFP adenovirus, RNA was extracted 12, 18 and 24 hours after infection and used to determine mRNA expression by a Gene Chip Human Transcriptome Array 2.0 (Affymetrix). Nox 5 overexpression after adenoviral infection in HAEC resulted in a significant enhancement of extracellular superoxide and hydrogen peroxide production and led to a significant increase in cellular apoptosis by caspases 3 and 7. The ontological analysis of the array showed an upregulation of UPR. Infection of HAEC with Nox 5 adenovirus activated the UPR pathway, increasing Ire1α and CHOP mRNA expression, GRP78 protein levels and reducing calnexin protein expression. Additionally, Nox 5 overexpression significantly reduced Nox 4 and NOS3 mRNA expression. Nox 5 overexpression promotes a situation of oxidative stress in HAEC, which alters correct protein folding and activates the UPR pathway.
Purpose: Many studies have shown the importance of keeping oral health to prevent against various diseases. Neutrophils (PMNs) play a very important role to keep oral health by its bactericidal activity such as reactive oxygen species (ROS) production and phagocytosis. We investigated the effects of lysed and heat-treated Enterococcus faecalis FK-23 (LFK) on ROS production and phagocytosis of human PMNs and its mechanism. Method: I. PMNs (1 106 cells/ml) isolated from peripheral blood were stimulated with water-soluble LFK (5 mg/ml) and/or phorbol 12-myristate 13-acetate (PMA). Then, ROS production was measured using MCLA-dependent chemiluminescence. II. PMNs (1 106 cells/ml) were incubated for 1 h at 37 °C with/without LFK. Then, carboxylate-modified fluorescent beads were added to PMNs suspension and incubated for 30 min at 37 °C. After that, phagocytosis was measured by flow cytometry. Results: LFK transiently increased ROS production from PMNs without change in total amounts. This effect was decreased by some inhibitors of mitochondrial complex. Thus, LFK might increase the reactivity of PMNs by enhancing mitochondrial functional ability or phagocytosis ability. These results suggest LFK may increase bactericidal activity of human PMNs to improve oral health.
E-mail address: [email protected] http://dx.doi.org/10.1016/j.freeradbiomed.2018.04.335
P-189
The role of mitochondrial reactive oxygen species in the response of the pulmonary vasculature to hypoxia and right heart remodeling Susan Scheibe 1, Oleg Pak 1, Azadeh Esfandiary 1, Akylbek Sydykov 1, Michael Murphy 2, Werner Seeger 1, Norbert Weissmann 1, Natascha Sommer 1
E-mail address: [email protected] 1
University of Giessen, Germany MRC Mitochondrial Biology Unit, Cambridge, UK
Acknowledgements MINECO SAF2016–7915
2
http://dx.doi.org/10.1016/j.freeradbiomed.2018.04.334
Increased release of mitochondrial superoxide has been suggested to mediate acute hypoxic pulmonary vasoconstriction (HPV) as well as chronic
S102
A. Stepanova et al. / Free Radical Biology and Medicine 120 (2018) S45–S166
hypoxia-induced pulmonary hypertension (PH) and right heart remodeling. Thus, we investigated the superoxide release during HPV, chronic hypoxiainduced PH and after pulmonary arterial banding (PAB), as well as the effect of the mitochondria-targeted antioxidant MitoQ on these processes. Superoxide levels were increased in PASMC during acute hypoxia and decreased after 5 days of hypoxia. In parallel MitoQ, but not its inactive carrier substance, TPPþ , significantly inhibited acute HPV and the rise in superoxide concentration induced by acute hypoxia. However, MitoQ application did not affect the hypoxia-induced proliferation of PASMC or the development of chronic hypoxia-induced PH. In contrast, MitoQ application attenuated right ventricular remodeling after chronic hypoxic exposure as well as after PAB with regard to development of right heart hypertrophy and dilatation. Increased superoxide concentration mediates acute HPV, while decreased superoxide levels were detected in chronic hypoxia-induced PH. MitoQ may be beneficial under conditions of exaggerated acute HPV and to prevent the development of right heart remodeling.
E-mail address: [email protected] http://dx.doi.org/10.1016/j.freeradbiomed.2018.04.336
P-190
Reactions of the Trp-radical – dimerisation versus addition of superoxide Luke Carroll 1,2,3,4,5, David Pattison 1,2,3,4,5, Justin Davies 1,2,3,4,5, Robert Anderson 1,2,3,4,5, Camilo Lopez-Alarcon 1,2,3,4,5, Michael Davies 1,2,3,4,5 1
Heart Research Institute, Sydney, Australia; University of Sydney, Australia 2 University of Copenhagen, Denmark 3 Australian Nuclear Science and Technology Organisation, Australia 4 University of Auckland, Auckland, New Zealand 5 Pontificia Universidad Catolica de Chile, Chile
Trp is a preferential target for radical reactions due to its low radical reduction potential, and these reactions result in the formation of indolyl radicals (Trp). Trp can then undergo reactions, including dimerisation and addition reactions with superoxide (O2-). The addition of O2- to Trp is very rapid, with rate k 1–2 109 M-1s-1, and rates correlated with positive peptide charge. The primary product of the reaction are peroxides, and decay products including N-formylkynurenine and kynurenine detected. The dimerization reaction of Trp has rates of k 2–6 108 M -1s-1. Dimerisation of Trp yield novel dimer products of Trp, with four isomers detected. Oxidation of peptides and proteins by peroxynitrous acid or H2O2 in the presence of bovine superoxide dismutase and carbonate ions, yield Trp dimers that can be isolated through alkaline hydrolysis. These data explore the fates of Trp, and based on the rate constants, the addition of O2- should be the preferred pathway. Under conditions where O2- is limited, Trp dimerization would be favored. The peroxidation and dimerization of Trp could play a major role protein degradation, playing a role in cellular dysfunction and disease.
E-mail address: [email protected] http://dx.doi.org/10.1016/j.freeradbiomed.2018.04.337
P-191
Suppressive effects on lung fibrosis and the polarization and populations of macrophages by liposomal clodronate combined with pirfenidone Michihito Toda, Shinjiro Mizuguchi, Yukiko Minamiyama, Shigekazu Takemura, Hiroko Oka-Yamamoto, Takanori Aota, Hikaru Miyamoto, Noritoshi Nishiyama Department of Thoracic Surgery, Osaka City University Medical School, Osaka, Japan
Purpose: Effective treatment of idiopathic pulmonary fibrosis (IPF) is still difficult. Macrophages (Mφ) are closely related to IPF. M1 phenotype Mφ exhibit proinflammatory properties, M2 phenotype Mφ promote fibrogenesis. We found that pirfenidone (PFD), a representative medication of IPF, suppresses lung fibrosis through M2 polarization. Furthermore, liposomal clodronate (LC) is reported to suppress lung fibrosis by depleting Mφ. This study aimed to investigate the effects of combined LC and PFD on lung fibrosis and M2 Mφ polarization and populations. Method: Rats were randomly divided into five groups: sham, bleomycin (BLM), PFD, LC, and PFDþ LC. Following intratracheal instillation of BLM, rats were orally administered PFD daily. Rats were administered LC intravenously at 7 days and sacrificed at 14 days. Fibrosis and Mφ polarization and populations were analyzed in lung. Results: Fibrosis and M2 marker levels were increased in BLM group, whereas PFD, LC, and PFDþLC groups showed significantly suppressed fibrosis and M2 markers. However, there were no synergistic effects of PFDþLC. Conclusion: PFD or LC regulated lung fibrosis and M2 markers, but the suppressive effect on lung fibrosis and M2 Mφ polarization and populations was not be enhanced by the combination of PFD and LC.
E-mail address: [email protected] http://dx.doi.org/10.1016/j.freeradbiomed.2018.04.338
P-192
Exposure of tropoelastin to peroxynitrous acid gives high yields of nitrated tyrosine residues, di-tyrosine cross-links and altered protein structure and function Georg Degendorfer 1,2,3,4,5,6,7, Lasse G. Lorentzen 1,2,3,4,5,6,7, Christine Y. Chuang 1,2,3,4,5,6,7, Michele Mariotti 1,2,3,4,5,6,7, Astrid Hammer 1,2,3,4,5,6,7, Gerald Hoefler 1,2,3,4,5,6,7, Per Hägglund 1,2,3,4,5,6,7, Ernst Malle 1,2,3,4,5,6,7, Steven Wise 1,2,3,4,5,6,7, Michael J. Davies 1,2,3,4,5,6,7 1
The Heart Research Institute, Sydney, Australia Dept. of Biomedical Sciences, Panum Institute, University of Copenhagen, Copenhagen, Denmark 3 Department of Biotechnology and Biomedicine, Technical University of Denmark, Kongens Lyngby, Denmark 4 Institute of Cell Biology, Histology and Embryology, Medical University of Graz, Graz, Austria 5 Institute of Pathology, Medical University of Graz, Graz, Austria 6 Institute of Molecular Biology and Biochemistry, Medical University of Graz, Graz, Austria 7 Faculty of Medicine, University of Sydney, Sydney, Australia 2
Methods: To study 2-ClHDA-mediated protein modification in cardiomyocytes, click-chemistry with proteomic approaches was used. Therefore, an alkyne containing bioortholog of 2-ClHDA, 2-chlorohexadec-15yn-1-al (2-ClHDyA), was synthesized. Protein lysates of 2-ClHDyA-modified cardiomyocytes were clicked with tetramethylrhodamine (TAMRA) azide and separated by 2-DE. TAMRA-positive spots were trypsinized and identified by LC-MS/MS. Results: During these analyses 51 2-ClHDyA-modified proteins were identified. Those proteins were mainly attributed to cellular processes involved in stress response, the cytoskeleton, and enzymes responsible for energy metabolism. These results suggest that cellular damage following myocardial infarction could be caused by impaired protein function due to modification by 2-ClHDA.
E-mail address: [email protected]
S101
P-188
Enterococcus faecalis FK-23 may improve the bactericidal activity of human neutrophil via enhancing ROS production and phagocytosis Hiroshi Ichikawa 1, Yutaro Yamada 1, Shuhei Saiki 1, Keiko Kobayasi 2, Yukiko Minamiyama 2 1 Graduate School of Life and Medical Sciences, Doshisha University, Kyoto, Japan 2 Graduate School of Life and Environmental Sciences, Kyoto Prefectural University, Kyoto, Japan
http://dx.doi.org/10.1016/j.freeradbiomed.2018.04.333
P-187
Nox 5 overexpression induces the activation of the unfolded protein response in human aortic endothelial cells Álvaro Pejenaute Martinez de Lizarrondo, Adriana Cortés Jiménez, Eduardo Ansorena Artieda, Silvia Cenoz Zubillaga, Carlos de Miguel Vázquez, Guillermo Zalba Goñi Biochemistry and Genetics Department, University of Navarra IdiSNA, Pamplona, Spain
Oxidative stress is a critical mechanism that underlies the pathophysiology of cardiovascular diseases. At the vascular level, an enhanced NADPH oxidase-mediated superoxide production associates with endothelial dysfunction, metalloprotease secretion, cellular senescence, and more recently activation of the unfolded protein response (UPR). Human aortic endothelial cells (HAEC) were infected with Nox 5 or GFP adenovirus, RNA was extracted 12, 18 and 24 hours after infection and used to determine mRNA expression by a Gene Chip Human Transcriptome Array 2.0 (Affymetrix). Nox 5 overexpression after adenoviral infection in HAEC resulted in a significant enhancement of extracellular superoxide and hydrogen peroxide production and led to a significant increase in cellular apoptosis by caspases 3 and 7. The ontological analysis of the array showed an upregulation of UPR. Infection of HAEC with Nox 5 adenovirus activated the UPR pathway, increasing Ire1α and CHOP mRNA expression, GRP78 protein levels and reducing calnexin protein expression. Additionally, Nox 5 overexpression significantly reduced Nox 4 and NOS3 mRNA expression. Nox 5 overexpression promotes a situation of oxidative stress in HAEC, which alters correct protein folding and activates the UPR pathway.
Purpose: Many studies have shown the importance of keeping oral health to prevent against various diseases. Neutrophils (PMNs) play a very important role to keep oral health by its bactericidal activity such as reactive oxygen species (ROS) production and phagocytosis. We investigated the effects of lysed and heat-treated Enterococcus faecalis FK-23 (LFK) on ROS production and phagocytosis of human PMNs and its mechanism. Method: I. PMNs (1 106 cells/ml) isolated from peripheral blood were stimulated with water-soluble LFK (5 mg/ml) and/or phorbol 12-myristate 13-acetate (PMA). Then, ROS production was measured using MCLA-dependent chemiluminescence. II. PMNs (1 106 cells/ml) were incubated for 1 h at 37 °C with/without LFK. Then, carboxylate-modified fluorescent beads were added to PMNs suspension and incubated for 30 min at 37 °C. After that, phagocytosis was measured by flow cytometry. Results: LFK transiently increased ROS production from PMNs without change in total amounts. This effect was decreased by some inhibitors of mitochondrial complex. Thus, LFK might increase the reactivity of PMNs by enhancing mitochondrial functional ability or phagocytosis ability. These results suggest LFK may increase bactericidal activity of human PMNs to improve oral health.
E-mail address: [email protected] http://dx.doi.org/10.1016/j.freeradbiomed.2018.04.335
P-189
The role of mitochondrial reactive oxygen species in the response of the pulmonary vasculature to hypoxia and right heart remodeling Susan Scheibe 1, Oleg Pak 1, Azadeh Esfandiary 1, Akylbek Sydykov 1, Michael Murphy 2, Werner Seeger 1, Norbert Weissmann 1, Natascha Sommer 1
E-mail address: [email protected] 1
University of Giessen, Germany MRC Mitochondrial Biology Unit, Cambridge, UK
Acknowledgements MINECO SAF2016–7915
2
http://dx.doi.org/10.1016/j.freeradbiomed.2018.04.334
Increased release of mitochondrial superoxide has been suggested to mediate acute hypoxic pulmonary vasoconstriction (HPV) as well as chronic
S102
A. Stepanova et al. / Free Radical Biology and Medicine 120 (2018) S45–S166
hypoxia-induced pulmonary hypertension (PH) and right heart remodeling. Thus, we investigated the superoxide release during HPV, chronic hypoxiainduced PH and after pulmonary arterial banding (PAB), as well as the effect of the mitochondria-targeted antioxidant MitoQ on these processes. Superoxide levels were increased in PASMC during acute hypoxia and decreased after 5 days of hypoxia. In parallel MitoQ, but not its inactive carrier substance, TPPþ , significantly inhibited acute HPV and the rise in superoxide concentration induced by acute hypoxia. However, MitoQ application did not affect the hypoxia-induced proliferation of PASMC or the development of chronic hypoxia-induced PH. In contrast, MitoQ application attenuated right ventricular remodeling after chronic hypoxic exposure as well as after PAB with regard to development of right heart hypertrophy and dilatation. Increased superoxide concentration mediates acute HPV, while decreased superoxide levels were detected in chronic hypoxia-induced PH. MitoQ may be beneficial under conditions of exaggerated acute HPV and to prevent the development of right heart remodeling.
E-mail address: [email protected] http://dx.doi.org/10.1016/j.freeradbiomed.2018.04.336
P-190
Reactions of the Trp-radical – dimerisation versus addition of superoxide Luke Carroll 1,2,3,4,5, David Pattison 1,2,3,4,5, Justin Davies 1,2,3,4,5, Robert Anderson 1,2,3,4,5, Camilo Lopez-Alarcon 1,2,3,4,5, Michael Davies 1,2,3,4,5 1
Heart Research Institute, Sydney, Australia; University of Sydney, Australia 2 University of Copenhagen, Denmark 3 Australian Nuclear Science and Technology Organisation, Australia 4 University of Auckland, Auckland, New Zealand 5 Pontificia Universidad Catolica de Chile, Chile
Trp is a preferential target for radical reactions due to its low radical reduction potential, and these reactions result in the formation of indolyl radicals (Trp). Trp can then undergo reactions, including dimerisation and addition reactions with superoxide (O2-). The addition of O2- to Trp is very rapid, with rate k 1–2 109 M-1s-1, and rates correlated with positive peptide charge. The primary product of the reaction are peroxides, and decay products including N-formylkynurenine and kynurenine detected. The dimerization reaction of Trp has rates of k 2–6 108 M -1s-1. Dimerisation of Trp yield novel dimer products of Trp, with four isomers detected. Oxidation of peptides and proteins by peroxynitrous acid or H2O2 in the presence of bovine superoxide dismutase and carbonate ions, yield Trp dimers that can be isolated through alkaline hydrolysis. These data explore the fates of Trp, and based on the rate constants, the addition of O2- should be the preferred pathway. Under conditions where O2- is limited, Trp dimerization would be favored. The peroxidation and dimerization of Trp could play a major role protein degradation, playing a role in cellular dysfunction and disease.
E-mail address: [email protected] http://dx.doi.org/10.1016/j.freeradbiomed.2018.04.337
P-191
Suppressive effects on lung fibrosis and the polarization and populations of macrophages by liposomal clodronate combined with pirfenidone Michihito Toda, Shinjiro Mizuguchi, Yukiko Minamiyama, Shigekazu Takemura, Hiroko Oka-Yamamoto, Takanori Aota, Hikaru Miyamoto, Noritoshi Nishiyama Department of Thoracic Surgery, Osaka City University Medical School, Osaka, Japan
Purpose: Effective treatment of idiopathic pulmonary fibrosis (IPF) is still difficult. Macrophages (Mφ) are closely related to IPF. M1 phenotype Mφ exhibit proinflammatory properties, M2 phenotype Mφ promote fibrogenesis. We found that pirfenidone (PFD), a representative medication of IPF, suppresses lung fibrosis through M2 polarization. Furthermore, liposomal clodronate (LC) is reported to suppress lung fibrosis by depleting Mφ. This study aimed to investigate the effects of combined LC and PFD on lung fibrosis and M2 Mφ polarization and populations. Method: Rats were randomly divided into five groups: sham, bleomycin (BLM), PFD, LC, and PFDþ LC. Following intratracheal instillation of BLM, rats were orally administered PFD daily. Rats were administered LC intravenously at 7 days and sacrificed at 14 days. Fibrosis and Mφ polarization and populations were analyzed in lung. Results: Fibrosis and M2 marker levels were increased in BLM group, whereas PFD, LC, and PFDþLC groups showed significantly suppressed fibrosis and M2 markers. However, there were no synergistic effects of PFDþLC. Conclusion: PFD or LC regulated lung fibrosis and M2 markers, but the suppressive effect on lung fibrosis and M2 Mφ polarization and populations was not be enhanced by the combination of PFD and LC.
E-mail address: [email protected] http://dx.doi.org/10.1016/j.freeradbiomed.2018.04.338
P-192
Exposure of tropoelastin to peroxynitrous acid gives high yields of nitrated tyrosine residues, di-tyrosine cross-links and altered protein structure and function Georg Degendorfer 1,2,3,4,5,6,7, Lasse G. Lorentzen 1,2,3,4,5,6,7, Christine Y. Chuang 1,2,3,4,5,6,7, Michele Mariotti 1,2,3,4,5,6,7, Astrid Hammer 1,2,3,4,5,6,7, Gerald Hoefler 1,2,3,4,5,6,7, Per Hägglund 1,2,3,4,5,6,7, Ernst Malle 1,2,3,4,5,6,7, Steven Wise 1,2,3,4,5,6,7, Michael J. Davies 1,2,3,4,5,6,7 1
The Heart Research Institute, Sydney, Australia Dept. of Biomedical Sciences, Panum Institute, University of Copenhagen, Copenhagen, Denmark 3 Department of Biotechnology and Biomedicine, Technical University of Denmark, Kongens Lyngby, Denmark 4 Institute of Cell Biology, Histology and Embryology, Medical University of Graz, Graz, Austria 5 Institute of Pathology, Medical University of Graz, Graz, Austria 6 Institute of Molecular Biology and Biochemistry, Medical University of Graz, Graz, Austria 7 Faculty of Medicine, University of Sydney, Sydney, Australia 2