Abstract 15775: Cyclosporin-a Mediated Inhibition of Nrf2 Nuclear Localization and Reduction in Superoxide Activity is Rescued by Tert-Butylhydroquinone in Human Coronary Artery Endothelial Cells
Background: Cyclosporin-A (CsA) is associated with the development of cardiac allograft vasculopathy (CAV) by as yet, unclear mechanisms. Endothelial dysfunction is a potential inciter of CAV. We have shown that CsA causes endothelial dysfunction and oxidant injury in rats. We also demonstrated in human cells that CsA impairs the important antioxidant Nrf2 pathway and inhibits nuclear localization of Nrf2. We hypothesized that CsA-mediated inhibition of Nrf2 nuclear translocation is associated with a reduction in superoxide dismutase (SOD) activity. We further hypothesized that this may be rescued by tert-Butylhydroquinone (tBHQ), an Nrf2 agonist.
Methods and Results: Human coronary artery endothelial cells (HCAEC, n=6-8) were incubated with CsA (10ug/ml), tBHQ (20uM), CsA and tBHQ, or DMSO (control) for 24hrs. Using Western Blotting, we measured Nrf2 whole cell expression and Nrf2 cellular localization (nuclear/cytosolic ratio: NCR). We also measured SOD activity. Compared to control, Nrf2 NCR was significantly decreased by CsA (0.25±0.04 vs 0.91±0.01, p<0.005). However, co-incubation with tBHQ rescued Nrf2 NCR compared to CsA alone (0.61±0.04 vs. 0.25±0.04, p<0.005). Although tBHQ alone did not significantly increase Nrf2 NCR at 24hrs compared to control (0.96±0.08 vs. 0.91±0.01, p=NS), it lead to whole cell upregulation of Nrf2 (0.03±0.01 vs. 0.12±0.02, p<0.001). SOD activity was significantly impaired by CsA compared to control (1.03±0.07 U/ml vs. 1.4±0.1 U/ml, p<0.05). Exposure to tBHQ, with or without concomitant exposure to CsA significantly enhanced SOD activity compared to both CsA alone and control (1.95±0.04 U/ml and 1.95±0.09 U/ml vs. 1.03±0.07 U/ml and 1.4±0.1 U/ml respectively, p<0.05).
Conclusions: Our study reveals the novel observation that tBHQ augments SOD activity in the presence of CsA and that this may be via activation of the Nrf2 pathway. CsA-mediated endothelial injury incurred after heart transplantation may be in part due to inhibition of Nrf2 nuclear translocation and reduction in SOD activity. tBHQ may be pharmacologically used in conjunction with CsA, to reduce cytotoxic effects of CsA via reduction in oxidant injury in these patients.
- © 2012 by American Heart Association, Inc.