Rapid Reactive Oxygen Species Production by Mitochondria in Endothelial Cells Exposed to Tumor Necrosis Factor- α Is Mediated by Ceramide

Abstract

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.

Pro-inflammatory cytokines increase reactive oxygen species through mitochondria and NADPH oxidase in cultured RPE cells

“…These results for TNF-α were consistent with previous studies in endothelial cells (Corda et al 2001). At the concentration of TNF-α (20 ng/ml) used for present study, there was no evidence of RPE cytotoxicity, as evaluated by trypan blue exclusion assays.…”

“…Although the increase in fluorescence induced by TNF-α required more than 30 min to reach maximal stimulation (Fig. 1), the time required to induce ROS in RPE cells was shorter than that reported for endothelial cells (Corda et al, 2001) and tumor cells (Goosens et al, 1995) where ROS production required at least 1 hr of exposure to TNF-α. The time required for IL-1β-and IFN-γ-induced intracellular and extracellular RPE ROS production was less than for TNF-α.…”

“…1D). As the intracellular accumulation of ROS in endothelial cells peaked at 2-3 hrs after TNF-α treatment (Corda et al, 2001), we tested whether longer treatment would be associated with more ROS accumulation in the RPE cells. By comparing ROS accumulation in the RPE cells stimulated by TNF-α at 0, 1, 2, 4, and 24 hr, we found that, unlike endothelial cells, there were no further increases in the intracellular ROS accumulation in RPE cells in response to TNF-α at 2, 4, or 24 hr, compared to the ROS accumulation at 1 hr.…”

“…Mitochondria have been identified as a major source of TNF-α-induced ROS production in other cell types (Schulze-Osthoff et al, 1993;Corda S et al, 2001). To determine whether mitochondria are the source of TNF-α-induced ROS generation in RPE cells, we used the mitochondrial complex inhibitors, rotenone, thenoyltrifluoroacetone (TTFA), and antimycin A, that block mitochondrial electron transport at various sites that may be associated with ROS production, while monitoring the formation of ROS in human RPE cells.…”

“…TNF-α increases mitochondrial ROS production in tumor cells, endothelial cells, and hepatocytes (Schulze-Osthoff, 1993;Corda et al, 2001). IL-1β stimulates ROS production in various cell types (Mendes et al, 2003;Brigelius-Flohe et al, 2004;Hwang et al, 2004;Kaur et al, 2004).…”