Abstract 13883: Cyclosporin-A Inhibits Nuclear Localization of theTranscription Factor Nrf2 in Human Coronary Artery Endothelial Cells
Background: Cyclosporin-A (CsA) is known to cause endothelial dysfunction. However, the mechanisms behind CsA-induced damage have not been fully elucidated. We investigated the effect of CsA on the transcription factor Nrf2, which is the master regulator of native antioxidant defence systems. It has been suggested that increased levels of nitric oxide (NO) enhance Nrf2 activity via electrophile production. Thus we assessed the hypothesis that exposure to CsA impairs normal Nrf2 cellular localization in the presence of endothelial NO-enhancer drugs such as tetrahydrobiopterin (BH4) and exogenous endothelin-1 (ET-1).
Methods: Human coronary artery endothelial cells (n=4 to 6) were incubated with BH4 (100ng/ml), ET-1 (100nM) or 0.01% DMSO (control) in the presence or absence of CsA (10ug/ml) for 30min. Incubation with 20uM tert-Butylhydroquinone (tBHQ) for 30min was used as the positive control. Cells were then fixed and stained immunohistochemically. Confocal microscopy was used to obtain images and the data quantified. We analyzed Nrf2 cellular localization as measured by the nuclear/cytosolic ratio (NCR).
Results: Compared to control, treatment with tBHQ significantly increased Nrf2 NCR (0.61±0.04 vs. 3.95±0.47, p<0.001). Incubation with CsA did not significantly change the NCR compared to control (0.97±0.94 v.s 0.61±0.04, p=NS). The NCR significantly increased with either BH4 or ET-1 treatment compared to control (1.76±0.07 and 3.23±0.22 respectively vs 0.61±0.04, p<0.001 for both). However, co-incubation with CsA significantly attenuated nuclear localization of Nrf2 in cells treated with BH4 or ET-1 (0.91±0.07, and 0.84±0.22 respectively, p<0.001 for both).
Conclusions: Our study reveals the novel observation that CsA inhibits Nrf2 nuclear translocation in coronary artery endothelial cells. Furthermore, we found that CsA-induced Nrf2 nuclear localization inhibition is persistent despite increased levels of endothelial NO. This may suggest that CsA acts to desensitize the endothelial cells with respect to their ability to detect increased levels of cytosolic electrophiles. In conclusion, CsA-mediated endothelial damage incurred after heart transplantation may be in part due to inhibition of Nrf2 nuclear translocation.
- Transplantation/medical aspects
- Immunosuppressive therapy
- Oxidative stress
- Endothelial function
- Nitric oxide
- © 2010 by American Heart Association, Inc.