OP-08 - Recapitulating physiological normoxia in vitro to discriminate Nrf2 regulated gene transcription

Abstract

Cells have evolved endogenous defense mechanisms to combat oxidative/electrophilic stress, with Nrf2 playing a critical role in the induction of phase II and antioxidant defense enzymes. As molecular mechanisms regulating Nrf2 signaling have largely been studied in vitro in cells exposed to atmospheric O2, we investigated Nrf2 gene targets in human endothelial cells following long-term culture under standard (18%) or physiological (5%) O2 levels encountered in vivo (Chapple et al., FRBM 2016; 92:152-62). An O2-regulated Sci-tive workstation and CLARIOstar plate reader were used to obviate re-oxygenation during routine culture and to measure real-time changes in cellular O2 content using a phosphorescence, O2-sensitive nanoparticle probe (Luxcel Biosciences). Gene profiling established that Nrf2 nuclear translocation and induction of GSH-related genes were insensitive to alterations in ambient O2 levels. Induction of HO-1 and NQO1 by 4-hydroxynonenal, sulforaphane or NO was diminished in cells adapted to 5% O2 due to upregulation of the Nrf2 repressor Bach1. GSH-related genes may thus act as transducers of Nrf2 regulated cytoprotection under physiological normoxia. Our study of redox signaling in cells adapted to physiological normoxia provides novel insights for translation to cardiovascular and regenerative medicine.