Redox and metal profiles in human coronary endothelial and smooth muscle cells under hyperoxia, physiological normoxia and hypoxia: Effects of NRF2 signaling on intracellular zinc

Zinc is an important component of cellular antioxidant defenses and dysregulation of zinc homeostasis is a risk factor for coronary heart disease and ischemia/reperfusion injury. Intracellular homeostasis of metals, such as zinc, iron and calcium are interrelated with cellular responses to oxidative stress. Most cells experience significantly lower oxygen levels in vivo (2–10 kPa O(2)) compared to standard in vitro cell culture (18kPa O(2)). We report the first evidence that total intracellular zinc content decreases significantly in human coronary artery endothelial cells (HCAEC), but not in human coronary artery smooth muscle cells (HCASMC), after lowering of O(2) levels from hyperoxia (18 kPa O(2)) to physiological normoxia (5 kPa O(2)) and hypoxia (1 kPa O(2)). This was paralleled by O(2)-dependent differences in redox phenotype based on measurements of glutathione, ATP and NRF2-targeted protein expression in HCAEC and HCASMC. NRF2-induced NQO1 expression was attenuated in both HCAEC and HCASMC under 5 kPa O(2) compared to 18 kPa O(2). Expression of the zinc efflux transporter ZnT1 increased in HCAEC under 5 kPa O(2), whilst expression of the zinc-binding protein metallothionine (MT) decreased as O(2) levels were lowered from 18 to 1 kPa O(2). Negligible changes in ZnT1 and MT expression were observed in HCASMC. Silencing NRF2 transcription reduced total intracellular zinc under 18 kPa O(2) in HCAEC with negligible changes in HCASMC, whilst NRF2 activation or overexpression increased zinc content in HCAEC, but not HCASMC, under 5 kPa O(2). This study has identified cell type specific changes in the redox phenotype and metal profile in human coronary artery cells under physiological O(2) levels. Our findings provide novel insights into the effect of NRF2 signaling on Zn content and may inform targeted therapies for cardiovascular diseases.