Hamza Yusuf Altun, Fan Yang, Giovanni Mann, Emrah Eroglu
Cellular redox status is tightly linked to ambient oxygen levels. Endothelial cells (EC) in vivo are exposed to O2 levels between 3-13 kPa, yet the majority in vitro studies maintain EC under atmospheric O2 levels (∼18 O2). Under 18 kPa O2, EC are exposed to hyperoxia, leading to persistent upregulation of NRF2 regulated antioxidant defences. Exposure of EC to physiological O2 levels for at least 5 days attenuates NRF2-regulated antioxidant gene expression in the absence of HIF-1a stabilisation.
This study aimed to determine whether O2 influences redox dynamics. EA.hy926 cells expressing the cytosolic Hyper7 biosensor were preadapted for 5 days under 5 kPa or 18 kPa in an O2 controlled Scitive dual workstation. Hyper7 ratio signals were measured in real-time in atmosphere-controlled plate reader, with cells treated acutely with exogenous H2O2 or auranofin (NRF2 inducer).
Our results showed reduced basal Hyper7 signals in cells adapted to 5 kPa compared to 18 kPa O2. Notably, cells adapted to 5 kPa O2 exhibited lower basal H2O2. Despite similar Hyper7 maximal responses to H2O2 (10mM), cells under 5 kPa O2 more effectively scavenged H2O2. EA.hy296 cells were then pretreated with PEG-SOD, PEG-CAT or vehicle under 18 or 5 kPa O2, followed by acute auranofin (1mM) challenge. Catalase significantly attenuated Hyper7 responses to auranofin under 18 and 5 kPa O2, and auranofin induced Hyper7 signals were markedly delayed under 5 kPa O2.
Our finding further underpin the importance of physiological O2 for cell culture in vitro, as ‘redox distress’ under standard atmospheric cell culture alters redox signaling, NRF2 gene transcription and thus influences experimental outcomes. As physiological O2 levels enhance EC antioxidant enzyme activity, this has implications for cell culture models and highlights the importance of considering oxygen levels in experimental design.