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Hydrogen sulfide inhibits bronchial epithelial–mesenchymal transition
Abstracts/Nitric Oxide 47 (2015) S14–S60
Platelets are key drivers of acute cardiovascular events such as myocardial infarction. Hydrogen sulphide (H2S) is a gasotransmitter with emerging roles in cardiovascular biology although there is currently no consensus on the regulation of platelets by H2S. We aimed to define the role of H2S in regulating platelets and investigate the enzymatic source of H2S in platelets. We determined the effect of the slow-releasing H2S compound GYY4137 upon human platelet aggregation in vitro and in a mouse model of radiolabelled platelet thromboembolism. We also assessed expression and catalytic activity of H2S-generating enzymes in human platelets. The slow releasing H2S donor GYY4137 inhibited thrombinand collagen-induced human platelet aggregation in a concentrationdependent manner and to a significantly larger extent than the decayed control compound. Inhibition occurred at equivalent H2S concentrations in the 40–400 nM range. In vivo, GYY4137 (50 mg/ kg) significantly (P < 0.05, unpaired t-test) reduced collageninduced radiolabelled platelet aggregation from 22.3 ± 3.5% to 15.5 ± 1.5. Western blotting showed that human platelet expresses the H2S generating enzyme cystathione-β-synthase (CBS) but not cystathione-γ-lysase (CSE). In addition, CBS but not CSE was catalytically active in platelets under basal conditions. Experiments with the H2S selective probe WSP-1 showed that platelets generated H2S and that H2S production was inhibited by the CBS antagonist aminooxyacetate. H2S, when applied to platelets in a manner that mimics slow, physiological release from enzymes, inhibits platelet aggregation through mechanisms that remain to be identified. Platelets generate H2S endogenously from CBS although the functional relevance of endogenous H2S remains to be evaluated. H2S may therefore be an important regulator of platelet activation and thrombotic events. Further research is warranted to fully evaluate the role of H2S in the negative regulation of platelets. http://dx.doi.org/10.1016/j.niox.2015.02.033
PP4 A persulfide analog of the nitrosothiol SNAP: From a molecular tool to a biologically active compound Erwan Galardon a, Vida Terzic a, Dominique Padovani a, Isabelle Artaud a, Burkhard Kloesch b a UMR 8601 CNRS, Université Paris Descartes, Sorbonne Paris Cité, Paris, France b Ludwig Boltzmann Institut für Rheumatologie und Balneologie, Vienna, Austria The proposal of the post-translational modification "S-sulfhydration" [1] as a major pathway for H2S-induced signaling in biology has recently shed a new light on persulfides, a class of compounds known by chemists for decades. However, the study of these species under biologically relevant conditions is hampered by their instability, which requires their generation in situ immediately prior to use. Current methods to prepare persulfides in aqueous solution suffer from several drawbacks, in particular they require or liberate hydrogen sulfide, resulting in difficulties to decipher the persulfide reactivity from that of H2S [2]. Using the following pH-driven H2S-free strategy, we were able to access [3] a water-soluble persulfide analog of the nitrosothiol SNAP, a widely used nitric oxide donor. The characterization and the reactivity of P toward relevant substrates will be discussed. Besides being an interesting molecular tool, P also shows promising biological activities, due to its ability to release H2S under reductive conditions. Results on the anti-inflammatory effects of P in different systems will be presented.
S15
References [1] A.K. Mustafa, et al., Sci. Signal 2 (2009) ra72. [2] N.E. Francoleon, et al., Arch. Biochem. Biophys. 516 (2011) 146. [3] (a) I. Artaud, et al., Chembiochem 15 (2014) 2361–2364; (b) N. Goodman, Nat. Chem. Biol. 10 (2014) 876. http://dx.doi.org/10.1016/j.niox.2015.02.034
PP5 Hydrogen sulfide inhibits bronchial epithelial–mesenchymal transition Chengcheng Liao a, Yahong Chen a, Fan Lin a, Yongfen Qi b a Pulmonary and Critical Care Medicine, Peking University Third Hospital, Beijing 100191, China b Key Laboratory of Molecular Cardiovascular Science, Ministry of Education, Beijing 100191, China Objective: To investigate the effects of hydrogen sulfide on TGFβ1-induced epithelial–mesenchymal transition in human bronchial epithelial cell. Methods: Human bronchial epithelial cell line 16HBE was used in the in vitro experiments. Incubation with GYY4137, a H2S slowly release donor, prior to TGF-β1 exposure to induce EMT was employed and EMT markers we measured by western blotting. Changes in cell morphology were assessed under phase-contrast light microscopy. Results: The cell assumed an elongated shape, and many cells lost contact with each other and displayed spindle-shaped, fibroblastlike morphological features after treatment with TGF-β1. In contrast, on application of GYY4137, cells maintained a classic cobblestone epithelial morphology and were tightly attached to each other. TGF-β1 treatment significantly increased the expression of mesenchymal marker α-SMA and decreased the expression of epithelial marker E-cadherin. Compared with the TGF-β1 group, GYY4137 could reverse these markers by reducing the expression of α-SMA by 45.1% and increasing the expression of E-cadherin by 57.6% (P < 0.05). Conclusions: Hydrogen sulfide inhibits TGF-β1-induced cell morphological changes and EMT in 16HBE. Grant: National Natural Science Foundation of China (Nos. 81170012 and 81370141). http://dx.doi.org/10.1016/j.niox.2015.02.035
PP6 Reduction of endogenous hydrogen sulfide generation increases cytokine expression in kidney ischemia–reperfusion injury Karmin O a,b,c, Pengqi Wang a,b, Cara K. Isaak c,d, Yaw L. Siow a,c,d a St. Boniface Hospital Research Centre, Winnipeg, Canada b Department of Animal Science, University of Manitoba, Canada c Department of Physiology and Pathophysiology, University of Manitoba, Canada d Agriculture and Agri Food Canada, Canada Endogenous hydrogen sulfide is a gasotransmitter that has been implicated as a mediator in regulating inflammatory response. Cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE) are two major enzymes responsible for hydrogen sulfide production. The kidney has a high expression of CBS and CSE which contributes significantly to the regulation of hydrogen sulfide homeostasis. Inflammation plays a critical role in kidney
Platelets are key drivers of acute cardiovascular events such as myocardial infarction. Hydrogen sulphide (H2S) is a gasotransmitter with emerging roles in cardiovascular biology although there is currently no consensus on the regulation of platelets by H2S. We aimed to define the role of H2S in regulating platelets and investigate the enzymatic source of H2S in platelets. We determined the effect of the slow-releasing H2S compound GYY4137 upon human platelet aggregation in vitro and in a mouse model of radiolabelled platelet thromboembolism. We also assessed expression and catalytic activity of H2S-generating enzymes in human platelets. The slow releasing H2S donor GYY4137 inhibited thrombinand collagen-induced human platelet aggregation in a concentrationdependent manner and to a significantly larger extent than the decayed control compound. Inhibition occurred at equivalent H2S concentrations in the 40–400 nM range. In vivo, GYY4137 (50 mg/ kg) significantly (P < 0.05, unpaired t-test) reduced collageninduced radiolabelled platelet aggregation from 22.3 ± 3.5% to 15.5 ± 1.5. Western blotting showed that human platelet expresses the H2S generating enzyme cystathione-β-synthase (CBS) but not cystathione-γ-lysase (CSE). In addition, CBS but not CSE was catalytically active in platelets under basal conditions. Experiments with the H2S selective probe WSP-1 showed that platelets generated H2S and that H2S production was inhibited by the CBS antagonist aminooxyacetate. H2S, when applied to platelets in a manner that mimics slow, physiological release from enzymes, inhibits platelet aggregation through mechanisms that remain to be identified. Platelets generate H2S endogenously from CBS although the functional relevance of endogenous H2S remains to be evaluated. H2S may therefore be an important regulator of platelet activation and thrombotic events. Further research is warranted to fully evaluate the role of H2S in the negative regulation of platelets. http://dx.doi.org/10.1016/j.niox.2015.02.033
PP4 A persulfide analog of the nitrosothiol SNAP: From a molecular tool to a biologically active compound Erwan Galardon a, Vida Terzic a, Dominique Padovani a, Isabelle Artaud a, Burkhard Kloesch b a UMR 8601 CNRS, Université Paris Descartes, Sorbonne Paris Cité, Paris, France b Ludwig Boltzmann Institut für Rheumatologie und Balneologie, Vienna, Austria The proposal of the post-translational modification "S-sulfhydration" [1] as a major pathway for H2S-induced signaling in biology has recently shed a new light on persulfides, a class of compounds known by chemists for decades. However, the study of these species under biologically relevant conditions is hampered by their instability, which requires their generation in situ immediately prior to use. Current methods to prepare persulfides in aqueous solution suffer from several drawbacks, in particular they require or liberate hydrogen sulfide, resulting in difficulties to decipher the persulfide reactivity from that of H2S [2]. Using the following pH-driven H2S-free strategy, we were able to access [3] a water-soluble persulfide analog of the nitrosothiol SNAP, a widely used nitric oxide donor. The characterization and the reactivity of P toward relevant substrates will be discussed. Besides being an interesting molecular tool, P also shows promising biological activities, due to its ability to release H2S under reductive conditions. Results on the anti-inflammatory effects of P in different systems will be presented.
S15
References [1] A.K. Mustafa, et al., Sci. Signal 2 (2009) ra72. [2] N.E. Francoleon, et al., Arch. Biochem. Biophys. 516 (2011) 146. [3] (a) I. Artaud, et al., Chembiochem 15 (2014) 2361–2364; (b) N. Goodman, Nat. Chem. Biol. 10 (2014) 876. http://dx.doi.org/10.1016/j.niox.2015.02.034
PP5 Hydrogen sulfide inhibits bronchial epithelial–mesenchymal transition Chengcheng Liao a, Yahong Chen a, Fan Lin a, Yongfen Qi b a Pulmonary and Critical Care Medicine, Peking University Third Hospital, Beijing 100191, China b Key Laboratory of Molecular Cardiovascular Science, Ministry of Education, Beijing 100191, China Objective: To investigate the effects of hydrogen sulfide on TGFβ1-induced epithelial–mesenchymal transition in human bronchial epithelial cell. Methods: Human bronchial epithelial cell line 16HBE was used in the in vitro experiments. Incubation with GYY4137, a H2S slowly release donor, prior to TGF-β1 exposure to induce EMT was employed and EMT markers we measured by western blotting. Changes in cell morphology were assessed under phase-contrast light microscopy. Results: The cell assumed an elongated shape, and many cells lost contact with each other and displayed spindle-shaped, fibroblastlike morphological features after treatment with TGF-β1. In contrast, on application of GYY4137, cells maintained a classic cobblestone epithelial morphology and were tightly attached to each other. TGF-β1 treatment significantly increased the expression of mesenchymal marker α-SMA and decreased the expression of epithelial marker E-cadherin. Compared with the TGF-β1 group, GYY4137 could reverse these markers by reducing the expression of α-SMA by 45.1% and increasing the expression of E-cadherin by 57.6% (P < 0.05). Conclusions: Hydrogen sulfide inhibits TGF-β1-induced cell morphological changes and EMT in 16HBE. Grant: National Natural Science Foundation of China (Nos. 81170012 and 81370141). http://dx.doi.org/10.1016/j.niox.2015.02.035
PP6 Reduction of endogenous hydrogen sulfide generation increases cytokine expression in kidney ischemia–reperfusion injury Karmin O a,b,c, Pengqi Wang a,b, Cara K. Isaak c,d, Yaw L. Siow a,c,d a St. Boniface Hospital Research Centre, Winnipeg, Canada b Department of Animal Science, University of Manitoba, Canada c Department of Physiology and Pathophysiology, University of Manitoba, Canada d Agriculture and Agri Food Canada, Canada Endogenous hydrogen sulfide is a gasotransmitter that has been implicated as a mediator in regulating inflammatory response. Cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE) are two major enzymes responsible for hydrogen sulfide production. The kidney has a high expression of CBS and CSE which contributes significantly to the regulation of hydrogen sulfide homeostasis. Inflammation plays a critical role in kidney