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Nrf2-regulated redox signalling in brain endothelial cells adapted to physiological O2 levels: consequences for ischaemia-reperfusion injury and protection by sulforaphane

Student thesis: Doctoral ThesisDoctor of Philosophy

Cerebral stroke remains the second most common cause of death and a major cause of disability worldwide. Ischaemic stroke accounts for 87% of stroke cases however treatments available for cerebral ischaemic stroke remains limited, with only about 25% of patients receiving pharmacological thrombolysis and 11% receiving endovascular thrombectomy. This is limited due to the narrow therapeutic window of intravenous thrombolysis and the need of specialised stroke centres and trained interventional radiologists in performing mechanical endovascular thrombectomy. Moreover, attempts to translate potential treatments into the clinic have so far been unsuccessful, largely due to failures in mimicking physiological in vivo conditions during pre-clinical studies. 
One aspect to consider to better mimic physiological in vivo conditions while conducting in vitro studies is the level of oxygen (O2) in the experimental setting. Most in vitro studies are conducted under room air conditions (18 kPa O2 at sea level); however cells in vivo experience O2 levels ranging from ~13 kPa to ~1 kPa, with brain endothelial cells experiencing O2 ranging ~3 – 7 kPa. Thus, the majority of cell culture studies unnecessarily expose cells to hyperoxia. The redox sensitive transcription factor NF-E2 related factor 2 (Nrf2) provides a first-line defence against oxidative stress, and recent studies with human endothelial cells have established that activation of Nrf2 target genes by the dietary isothiocyanate sulforaphane (SFN) is markedly attenuated in cells adapted to physiological normoxia (5 kPa O2) compared to 18 kPa O2
To better understand the consequence of physiological normoxia for reperfusion-induced oxidative stress and protection by SFN, mouse brain endothelial cell line (bEnd.3) was used as in vitro model of the blood-brain barrier (BBB). Using an O2-sensitive probe (MitoXpressINTRA), an intracellular O2 level of ~3.6 kPa was measured in bEnd.3 cells maintained under 5 kPa O2. bEnd.3 cells cultured under either 18 kPa or 5 kPa O2 were subsequently subjected to hypoxia (1 kPa O2, 1 h) and reoxygenation (either 18 or 5 kPa O2) to simulate cerebral ischaemic stroke. Reactive oxygen species production was measured using the chemiluminescent probe L-012 and mitochondrial-specific superoxide indicator MitoSOX™. In this study, reoxygenation-induced reactive oxygen species generation was negligible in bEnd.3 cells adapted initially to physiological normoxia (5 kPa O2). In contrast, hypoxia-reoxygenation induced a significant burst in reactive oxygen species in bEnd.3 cells adapted initially to 18 kPa O2, which was abrogated by polyethylene glycol superoxide dismutase (PEG-SOD) and SOD. Consequently, protection against reoxygenation induced reactive oxygen species generation afforded by pretreatment with SFN (2.5 µM, 20 h) was only observed in bEnd.3 cells cultured under 18 kPa O2. As adaptation of bEnd.3 cells to physiological normoxia (5 kPa O2) limited superoxide production associated with hypoxia-reoxygenation injury, this suggests that exaggerated responses from cultures exposed to hyperoxia (18 kPa O2) may potentially reflect the consequences of long-term adaptation to hyperoxia. 
Findings presented in this thesis highlight the importance of conducting vascular cell culture under the relevant physiological O2 levels encountered in vivo. The present study provides evidence that physiological normoxia has direct consequence for hypoxia-reoxygenation injury and protection afforded by activation of the redox transcription factor Nrf2. These findings may be of particular importance for the screening of potential therapies for pathology involving ischaemia-reperfusion injury. Future in vitro studies should consider a paradigm shift by conducting in vitro experiments under physiological O2 levels to enhance translation to the clinic.
Original languageEnglish
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Award date1 Jun 2020

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