Bach1 differentially regulates distinct Nrf2-dependent genes in human venous and coronary artery endothelial cells adapted to physiological oxygen levels

Abstract

The effects of physiological oxygen tension on Nuclear Factor-E2-Related Factor 2 (Nrf2)-regulated redox signaling remain poorly understood. We report the first study of Nrf2-regulated signaling in human primary endothelial cells (EC) adapted long-term to physiological O2 (5%). Adaptation of EC to 5% O2 had minimal effects on cell ultrastructure, viability, basal redox status or HIF1-α expression. Affymetrix array profiling and subsequent qPCR/protein validation revealed that induction of select Nrf2 target genes, HO-1 and NQO1, was significantly attenuated in cells adapted to 5% O2, despite nuclear accumulation and DNA binding of Nrf2. Diminished HO-1 induction under 5% O2 was stimulus independent and reversible upon re-adaptation to air or silencing of the Nrf2 repressor Bach1, notably elevated under 5% O2. Induction of GSH-related genes xCT and GCLM were oxygen and Bach1-insensitive during long-term culture under 5% O2, providing the first evidence that genes related to GSH synthesis mediate protection afforded by Nrf2-Keap1 defense pathway in cells adapted to physiological O2 levels encountered in vivo.

A PP2A‐mediated feedback mechanism controls Ca 2+ ‐dependent NO synthesis under physiological oxygen

“…1 B ). When replotted against the corresponding mean intracellular O 2 level [as determined previously (4)], there was a leftward shift in the sigmoidal curve with a reduction in IC 50 from 4.5 to 2.3% O 2 . This shift is of particular physiologic significance, because blood oxygen (P o 2 ) level of 4.5% is normoxic for most cell types in vivo , yet 2.3% O 2 encroaches on hypoxia for endothelial cells.…”

“…Thus, HUVECs cultured in standard conditions in vitro are normally exposed to >4 times the O 2 level experienced in vivo . We have reported that culturing HUVECs in 5% ambient O 2 results in a cytosolic O 2 level of ∼3.5% (4), more accurately replicating levels measured in vivo and with important consequences for cellular antioxidant defense pathways.…”

Limitations of oxygen delivery to cells in culture: An underappreciated problem in basic and translational research

“…For example, one study simply cultured cells in ambient oxygen levels higher than the desired intracellular levels to successfully combat consumptive oxygen depletion in their system [58]. Another method that can be employed is to supplement the media with oxygen producing hydrogels.…”

Altered cellular redox homeostasis and redox responses under standard oxygen cell culture conditions versus physioxia

“…Significant changes in Nrf2-regulated redox signalling were observed after one day of physioxic culture and these changes were maintained through long-term (fiveday) culture. Chapple et al [75] also reported evidence of an O 2 gradient within in vitro cultures which could lead to the unintentional induction of hypoxia. The lack of effect on HIF1α expression (Fig.…”

“…Chapple et al [75] have reported that Nrf2 activity was attenuated in human primary endothelial cells adapted to physioxia (5% O 2 ) compared to cells cultured under air. Significant changes in Nrf2-regulated redox signalling were observed after one day of physioxic culture and these changes were maintained through long-term (fiveday) culture.…”