Abstract
Background: Oxidative stress and activation of cellular antioxidant defence pathways play critical roles in coronary ischaemia-reperfusion (IR) injury. Nuclear factor-E2-related factor 2 (Nrf2), a well-known antioxidant transcription factor, participates in maintaining cellular redox homeostasis. Notably, dysregulation of Zn homeostasis is associated with IR injury and linked with Nrf2 signalling pathway. Furthermore, the concept of cell culture under defined ambient oxygen levels that recapitulate pO2 experienced in vivo has recently acquired renewed research focus. The present study aims to characterise the redox state and metal profiles in coronary artery smooth muscle cells (HCASMCs) adapted long-term to hyperoxia (18kPa O2), physiological normoxia (5kPa O2) and hypoxia (1kPa O2). Compared to standard cell culture under 18kPa O2, which has mistakenly been defined as normoxia in the past, our study provides novel insights into the role of Nrf2 and Zn underlying hypoxia-reoxygenation (HR) in HCASMCs adapted to physiological normoxia (5kPa O2) and the crosstalk between Nrf2 signalling and Zn level.Methods: Redox signalling was examined in HCASMCs adapted long-term (5 days) to defined O2 levels (18, 5 and 1kPa). Intracellular redox status under different O2 levels was determined by assaying intracellular glutathione (GSH) levels and protein expression of antioxidant enzymes. Immunoblotting and qPCR were employed to determine protein and mRNA expression of select Nrf2 targets (e.g.HO-1 and GCLM) under basal different O2 levels and following treatment with the Nrf2 inducer sulforaphane (SFN, 2.5μM). Generation of reactive oxygen species (ROS) under basal conditions, following treatments and HR (18kPa/5kPa to 1kPa to 18kPa/5kPa), was measured using the chemiluminescence probe L-012. Metal profiles in HCASMCs under basal or HR were obtained using inductively coupled plasma mass spectrometry (ICP-MS). Crosstalk between Nrf2 and Zn was determined by measuring total Zn levels and protein expression of ZnT1 (main exporter of Zn on the membrane) and MT (main intracellular storage protein of Zn) under hyperoxia, normoxia and hypoxia and following pre-treatment (16h) with SFN, N,N,N′,N′-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN, a scavenger of free Zn2+, 1.25μM) or 2-mercaptopyridine N-oxide sodium salt (a Zn2+ ionophore, 0.5μM) with ZnCl2 (10μM).
Results: HCASMCs were adapted for 5 days to 18, 5 or 1kPa O2. Hypoxia inducible factor-1α (HIF-1α) stabilisation was only observed in cells under 1kPa O2. Lower basal intracellular antioxidant levels (e.g., GSH, CAT and SOD) and Nrf2-regulated target proteins (e.g.NQO1 and GCLM) were detected in cells adapted to 1kPa compared to 18 and 5kPa O2. Total intracellular Zn66 levels were 0.345±0.037ng/µg protein in HCASMCs under 18kPa and similar under 5 and 1kPa O2. Zn supplementation significantly increased total Zn content in HCASMC under 18 but not 5kPa O2. Zn supplementation increased Nrf2 nuclear accumulation in cells under 18 or 5kPa O2, whilst Nrf2 targeted HO-1 mRNA and protein expression in response to Zn supplementation was upregulated significantly in cells under 18kPa O2, which were attenuated in cells under 5kPa O2. Nrf2 siRNA silencing, overexpression or activation of Nrf2 with SFN did not significantly alter total Zn66 content.
When HCASMCs were loaded with probe L-012 and exposed to hypoxia (1h) and reoxygenation, superoxide generation (PEG-SOD inhibitable) was only detected on reoxygenation. Pre-treatment of cells with SFN or Zn attenuated reoxygenation-induced superoxide generation. Furthermore, total intracellular Zn66 levels were not significantly changed in cells under HR injury under 18 and 5kPa O2.
Conclusions: The present findings indicate that compared to physiological normoxia (5kPa O2),hyperoxia (18kPa O2) had negligible effects on the redox phenotype (e.g. GSH, antioxidant enzymes and Nrf2-regulated proteins) of HCASMCs, unlike umbilical vein and coronary artery endothelial cells. Notably, Zn supplementation activated the Nrf2 signalling pathway in HCASMCs, however activation of Nrf2 had negligible effects on total Zn66 levels. Abstract ROS generation, primarily superoxide, was detected in first 20min after reoxygenation in HCASMCs under 18kPa O2 and was significantly attenuated by pre-treatment with SFN or Zn, highlighting protective actions of Nrf2 activation and Zn supplementation in HR injury.
| Date of Award | 1 Oct 2023 |
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| Original language | English |
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| Supervisor | Giovanni Mann (Supervisor) & Luigi Gnudi (Supervisor) |