Vascular protection afforded by zinc supplementation in human coronary artery smooth muscle cells mediated by NRF2 signaling under hypoxia/reoxygenation

Abstract

Zinc (Zn) has antioxidant, anti-inflammatory and anti-proliferative actions, with Zn dysregulation associated with coronary ischemia/reperfusion injury and smooth muscle cell dysfunction. As the majority of studies concerning Zn have been conducted under non-physiological hyperoxic conditions, we compare the effects of Zn chelation or supplementation on total intracellular Zn content, antioxidant NRF2 targeted gene transcription and hypoxia/reoxygenation-induced reactive oxygen species generation in human coronary artery smooth muscle cells (HCASMC) pre-adapted to hyperoxia (18 kPa O₂) or normoxia (5 kPa O₂). Expression of the smooth muscle marker SM22-α was unaffected by lowering pericellular O₂, whereas calponin-1 was significantly upregulated in cells under 5 kPa O₂, indicating a more physiological contractile phenotype under 5 kPa O₂. Inductively coupled plasma mass spectrometry established that Zn supplementation (10 μM ZnCl₂ + 0.5 μM pyrithione) significantly increased total Zn content in HCASMC under 18 but not 5 kPa O₂. Zn supplementation increased metallothionein mRNA expression and NRF2 nuclear accumulation in cells under 18 or 5 kPa O₂. Notably, NRF2 regulated HO-1 and NQO1 mRNA expression in response to Zn supplementation was only upregulated in cells under 18 but not 5 kPa. Furthermore, whilst hypoxia increased intracellular glutathione (GSH) in cells pre-adapted to 18 but not 5 kPa O₂, reoxygenation had negligible effects on GSH or total Zn content. Reoxygenation-induced superoxide generation in cells under 18 kPa O₂ was abrogated by PEG-superoxide dismutase but not by PEG-catalase, and Zn supplementation, but not Zn chelation, attenuated reoxygenation-induced superoxide generation in cells under 18 but not 5kPaO₂, consistent with a lower redox stress under physiological normoxia. Our findings highlight that culture of HCASMC under physiological normoxia recapitulates an in vivo contractile phenotype and that effects of Zn on NRF2 signaling are altered by oxygen tension.

Keywords: Coronary artery smooth muscle cells; Hyperoxia; Hypoxia; Hypoxia-reoxygenation; Metallomics; Metallothionein; Metals; NRF2; Oxygen; Physiological normoxia; Redox status; Zinc.