Rapid reactive oxygen species production by mitochondria in endothelial cells exposed to tumor necrosis factor-alpha is mediated by ceramide

Am J Respir Cell Mol Biol. 2001 Jun;24(6):762-8. doi: 10.1165/ajrcmb.24.6.4228.


Tumor necrosis factor (TNF)-alpha increases mitochondrial reactive oxygen species (ROS) production in tumor cells and hepatocytes. However, whether TNF-alpha stimulates mitochondrial ROS production in endothelial cells (EC) has not yet been reported. We studied the effect of TNF-alpha on mitochondrial ROS generation in EC and the signaling pathways involved. Cultured human umbilical vein EC (HUVEC) were studied by fluorescence microscopy, using dichlorodihydrofluorescein diacetate (DCFH-DA) as a marker of ROS production and propidium iodide uptake for cell viability. TNF-alpha increased DCFH oxidation in HUVEC dose-dependently. To determine the source of ROS, the mitochondrial respiratory chain inhibitors rotenone + thenoyltrifluoroacetone (TTFA), which inhibit electron entry to ubiquinone, and antimycin A (AA), a blocker of ubisemiquinone, were used. Rotenone and TTFA inhibited (n = 7, P < 0.05), whereas AA increased (118% in 3 min; n = 4, P < 0.01) ROS generation in HUVEC. In contrast, ROS production was not abolished by the nicotinamide adenine dinucleotide phosphate-dependent oxidase inhibitor diphenylene iodonium, by the xanthine oxidase inhibitor allopurinol, nor by the nitric oxide and cyclooxygenase pathway inhibitors N(omega)-nitro-L-arginine and mefenamic acid. In addition, TNF-alpha-induced ROS production was inhibited by the acidic sphingomyelinase inhibitor desipramine (5 microM; -80%, n = 4, P < 0.01) and totally blocked by the ceramide-activated protein kinase (CAPK) inhibitor dimethylaminopurine (1 mM; n = 6, P < 0.05). Thus, TNF-alpha induces mitochondrial ROS production in HUVEC that primarily occurs at the ubisemiquinone site and is mediated by ceramide-dependent signaling pathways involving CAPK.

MeSH terms

  • Ceramides / metabolism*
  • Electron Transport
  • Endothelium, Vascular / drug effects
  • Endothelium, Vascular / metabolism*
  • Humans
  • Mitochondria / metabolism*
  • Models, Biological
  • Reactive Oxygen Species / metabolism*
  • Signal Transduction
  • Sphingomyelin Phosphodiesterase / metabolism
  • Tumor Necrosis Factor-alpha / pharmacology*


  • Ceramides
  • Reactive Oxygen Species
  • Tumor Necrosis Factor-alpha
  • Sphingomyelin Phosphodiesterase