Nutrient deprivation is a stimulus for oxidative stress, and is an established method for induction of cell autophagy and apoptosis. The aims of the study were to identify conditions that evoke superoxide production in human cultured umbilical vein endothelial cells (HUVECs), determine the mechanism of action for this response, and examine whether the stimulus might facilitate the adhesion of human isolated neutrophils to the HUVECs. HUVECs were incubated in M199 media under conditions of serum-starvation (serum-free M199 media), low serum (media containing 2% fetal calf serum) and high serum (media containing 20% fetal calf serum). HUVECs were also incubated under pro-inflammatory conditions, in media supplemented with 50 ng/ml tumour necrosis factor-α (TNF-α) or neutrophils pre-activated with 10 nM phorbol 12-myristate 13-acetate (PMA). Superoxide production was increased 4-fold in serum-starved HUVECs compared to cells incubated in 20% media, and this was reduced by inhibitors of the mitochondrial electron transport chain and mitochondrial Ca2+ uniporter. Superoxide production was 23.6% higher in HUVECs incubated with TNF-α in 2% media compared to 2% media alone, but unchanged with TNF-α in 20% media. PMA-activated neutrophils adhered to morphologically aberrant HUVECs, which were mainly evident under the low serum condition. The findings show a role of mitochondrial enzymes in superoxide production in response to nutrient deprivation, and suggest that pro-inflammatory responses in HUVECs become manifest when HUVECs are in an already-compromised state.
Free Radical Biology and Medicine / Vol. 67, pp.408-415