Nrf2-regulated redox signaling in brain endothelial cells adapted to physiological oxygen levels: Consequences for sulforaphane mediated protection against hypoxia-reoxygenation
Ischemic stroke is associated with a surge in reactive oxygen species generation during reperfusion. The narrow therapeutic window for the delivery of intravenous thrombolysis and endovascular thrombectomy limits therapeutic options for patients. Thus, understanding the mechanisms regulating neurova...
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| Format: | Article |
| Language: | English |
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Elsevier
2020-10-01
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| Series: | Redox Biology |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2213231720309137 |
| _version_ | 1818921933609107456 |
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| author | Gabriela Warpsinski Matthew J. Smith Salil Srivastava Thomas P. Keeley Richard C.M. Siow Paul A. Fraser Giovanni E. Mann |
| author_facet | Gabriela Warpsinski Matthew J. Smith Salil Srivastava Thomas P. Keeley Richard C.M. Siow Paul A. Fraser Giovanni E. Mann |
| author_sort | Gabriela Warpsinski |
| collection | DOAJ |
| description | Ischemic stroke is associated with a surge in reactive oxygen species generation during reperfusion. The narrow therapeutic window for the delivery of intravenous thrombolysis and endovascular thrombectomy limits therapeutic options for patients. Thus, understanding the mechanisms regulating neurovascular redox defenses are key for improved clinical translation. Our previous studies in a rodent model of ischemic stroke established that activation of Nrf2 defense enzymes by pretreatment with sulforaphane (SFN) affords protection against neurovascular and neurological deficits. We here further investigate SFN mediated protection in mouse brain microvascular endothelial cells (bEnd.3) adapted long-term (5 days) to hyperoxic (18 kPa) and normoxic (5 kPa) O2 levels. Using an O2-sensitive phosphorescent nanoparticle probe, we measured an intracellular O2 level of 3.4 ± 0.1 kPa in bEnd 3 cells cultured under 5 kPa O2. Induction of HO-1 and GCLM by SFN (2.5 μM) was significantly attenuated in cells adapted to 5 kPa O2, despite nuclear accumulation of Nrf2. To simulate ischemic stroke, bEnd.3 cells were adapted to 18 or 5 kPa O2 and subjected to hypoxia (1 kPa O2, 1 h) and reoxygenation. In cells adapted to 18 kPa O2, reoxygenation induced free radical generation was abrogated by PEG-SOD and significantly attenuated by pretreatment with SFN (2.5 μM). Silencing Nrf2 transcription abrogated HO-1 and NQO1 induction and led to a significant increase in reoxygenation induced free radical generation. Notably, reoxygenation induced oxidative stress, assayed using the luminescence probe L-012 and fluorescence probes MitoSOX™ Red and FeRhoNox™-1, was diminished in cells cultured under 5 kPa O2, indicating an altered redox phenotype in brain microvascular cells adapted to physiological normoxia. As redox and other intracellular signaling pathways are critically affected by O2, the development of antioxidant therapies targeting the Keap1-Nrf2 defense pathway in treatment of ischemia-reperfusion injury in stroke, coronary and renal disease will require in vitro studies conducted under well-defined O2 levels. |
| first_indexed | 2024-12-20T01:45:31Z |
| format | Article |
| id | doaj.art-047eb14f816f4bad9543c59bb2faf295 |
| institution | Directory Open Access Journal |
| issn | 2213-2317 |
| language | English |
| last_indexed | 2024-12-20T01:45:31Z |
| publishDate | 2020-10-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Redox Biology |
| spelling | doaj.art-047eb14f816f4bad9543c59bb2faf2952022-12-21T19:57:46ZengElsevierRedox Biology2213-23172020-10-0137101708Nrf2-regulated redox signaling in brain endothelial cells adapted to physiological oxygen levels: Consequences for sulforaphane mediated protection against hypoxia-reoxygenationGabriela Warpsinski0Matthew J. Smith1Salil Srivastava2Thomas P. Keeley3Richard C.M. Siow4Paul A. Fraser5Giovanni E. Mann6King's British Heart Foundation Centre for Research Excellence, School of Cardiovascular Medicine & Sciences, Faculty of Life Sciences & Medicine, King's College London, 150 Stamford Street, London, SE1 9NH, UKKing's British Heart Foundation Centre for Research Excellence, School of Cardiovascular Medicine & Sciences, Faculty of Life Sciences & Medicine, King's College London, 150 Stamford Street, London, SE1 9NH, UKKing's British Heart Foundation Centre for Research Excellence, School of Cardiovascular Medicine & Sciences, Faculty of Life Sciences & Medicine, King's College London, 150 Stamford Street, London, SE1 9NH, UKKing's British Heart Foundation Centre for Research Excellence, School of Cardiovascular Medicine & Sciences, Faculty of Life Sciences & Medicine, King's College London, 150 Stamford Street, London, SE1 9NH, UKKing's British Heart Foundation Centre for Research Excellence, School of Cardiovascular Medicine & Sciences, Faculty of Life Sciences & Medicine, King's College London, 150 Stamford Street, London, SE1 9NH, UKKing's British Heart Foundation Centre for Research Excellence, School of Cardiovascular Medicine & Sciences, Faculty of Life Sciences & Medicine, King's College London, 150 Stamford Street, London, SE1 9NH, UKCorresponding author.; King's British Heart Foundation Centre for Research Excellence, School of Cardiovascular Medicine & Sciences, Faculty of Life Sciences & Medicine, King's College London, 150 Stamford Street, London, SE1 9NH, UKIschemic stroke is associated with a surge in reactive oxygen species generation during reperfusion. The narrow therapeutic window for the delivery of intravenous thrombolysis and endovascular thrombectomy limits therapeutic options for patients. Thus, understanding the mechanisms regulating neurovascular redox defenses are key for improved clinical translation. Our previous studies in a rodent model of ischemic stroke established that activation of Nrf2 defense enzymes by pretreatment with sulforaphane (SFN) affords protection against neurovascular and neurological deficits. We here further investigate SFN mediated protection in mouse brain microvascular endothelial cells (bEnd.3) adapted long-term (5 days) to hyperoxic (18 kPa) and normoxic (5 kPa) O2 levels. Using an O2-sensitive phosphorescent nanoparticle probe, we measured an intracellular O2 level of 3.4 ± 0.1 kPa in bEnd 3 cells cultured under 5 kPa O2. Induction of HO-1 and GCLM by SFN (2.5 μM) was significantly attenuated in cells adapted to 5 kPa O2, despite nuclear accumulation of Nrf2. To simulate ischemic stroke, bEnd.3 cells were adapted to 18 or 5 kPa O2 and subjected to hypoxia (1 kPa O2, 1 h) and reoxygenation. In cells adapted to 18 kPa O2, reoxygenation induced free radical generation was abrogated by PEG-SOD and significantly attenuated by pretreatment with SFN (2.5 μM). Silencing Nrf2 transcription abrogated HO-1 and NQO1 induction and led to a significant increase in reoxygenation induced free radical generation. Notably, reoxygenation induced oxidative stress, assayed using the luminescence probe L-012 and fluorescence probes MitoSOX™ Red and FeRhoNox™-1, was diminished in cells cultured under 5 kPa O2, indicating an altered redox phenotype in brain microvascular cells adapted to physiological normoxia. As redox and other intracellular signaling pathways are critically affected by O2, the development of antioxidant therapies targeting the Keap1-Nrf2 defense pathway in treatment of ischemia-reperfusion injury in stroke, coronary and renal disease will require in vitro studies conducted under well-defined O2 levels.http://www.sciencedirect.com/science/article/pii/S2213231720309137Brain endothelial cellsKeap1-Nrf2Heme oxygenase-1Glutamate cysteine ligaseIschemia-reoxygenationRedox signaling |
| spellingShingle | Gabriela Warpsinski Matthew J. Smith Salil Srivastava Thomas P. Keeley Richard C.M. Siow Paul A. Fraser Giovanni E. Mann Nrf2-regulated redox signaling in brain endothelial cells adapted to physiological oxygen levels: Consequences for sulforaphane mediated protection against hypoxia-reoxygenation Redox Biology Brain endothelial cells Keap1-Nrf2 Heme oxygenase-1 Glutamate cysteine ligase Ischemia-reoxygenation Redox signaling |
| title | Nrf2-regulated redox signaling in brain endothelial cells adapted to physiological oxygen levels: Consequences for sulforaphane mediated protection against hypoxia-reoxygenation |
| title_full | Nrf2-regulated redox signaling in brain endothelial cells adapted to physiological oxygen levels: Consequences for sulforaphane mediated protection against hypoxia-reoxygenation |
| title_fullStr | Nrf2-regulated redox signaling in brain endothelial cells adapted to physiological oxygen levels: Consequences for sulforaphane mediated protection against hypoxia-reoxygenation |
| title_full_unstemmed | Nrf2-regulated redox signaling in brain endothelial cells adapted to physiological oxygen levels: Consequences for sulforaphane mediated protection against hypoxia-reoxygenation |
| title_short | Nrf2-regulated redox signaling in brain endothelial cells adapted to physiological oxygen levels: Consequences for sulforaphane mediated protection against hypoxia-reoxygenation |
| title_sort | nrf2 regulated redox signaling in brain endothelial cells adapted to physiological oxygen levels consequences for sulforaphane mediated protection against hypoxia reoxygenation |
| topic | Brain endothelial cells Keap1-Nrf2 Heme oxygenase-1 Glutamate cysteine ligase Ischemia-reoxygenation Redox signaling |
| url | http://www.sciencedirect.com/science/article/pii/S2213231720309137 |
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