Formation of peroxynitrite in vascular endothelial cells exposed to cyclosporine A

Abstract

ABSTRACT Cyclosporine A (CsA) therapy is associated with side effects related to oxidative stress. We characterized the reactive species produced in bovine aortic endothelial cells (BAEC) exposed to CsA. Electron spin resonance demonstrated that the cell‐permeable probe dihydroethidium is a specific tool to detect superoxide anion (O 2 • − ) in BAEC. Treatment of BAEC with CsA produced a dose‐dependent increase in the intracellular O 2 − , a process inhibited by the superoxide dismutase mimetic MnTMPyP. We used the nitric oxide (NO•)‐sensitive dye diaminofluorescein/diacetate (DAF‐2/DA) to detect NO• by flow cytometry in BAEC and in Sf9 cells infected with a recombinant baculovirus expressing eNOS. In BAEC, CsA induced a L‐NAME‐sensitive increase of the intracellular oxidation of DAF‐2/DA and increased the chemiluminescence‐based detection of NO 2 − . In BAEC treated with CsA for 2 h, an increase in peroxynitrite formation, measured with dihydrorhodamine 123, was detected and inhibited by N ‐acetyl‐cysteine (NAC) and L‐NAME. Furthermore, immunocytochemistry studies showed that CsA treatment of BAEC increased nitrotyrosine formation, an effect abrogated by NAC and MnTMPyP. Cytotoxicity after 24 and 48 h of CsA studied with propidium iodide exclusion was found to be higher in L‐NAME‐treated BAEC. Peroxynitrite formation induced by CsA in endothelial cells provides a new mechanism to explain endothelial injury observed in patients treated with this drug.