Homocysteine-Induced Endothelin-1 Release Is Dependent on Hyperglycaemia and Reactive Oxygen Species Production in Bovine Aortic Endothelial Cells

Background: Elevated plasma homocysteine (Hcy) is a risk factor for coronary disease. The objective of this study was to investigate whether Hcy either alone or in high glucose conditions induces endothelin-1 (ET-1) synthesis via the production of reactive oxygen species (ROS). Methods: Bovine aortic endothelial cells were grown in high (25 mmol/l) and low (5 mmol/l) glucose medium. Results: In high glucose, Hcy caused a time-dependent increase in ET-1 release, which was greatest with 50 µmol/l Hcy at 24 h (p < 0.01). This effect was not seen in low glucose conditions. In high glucose and 50 µmol/l Hcy, ET-1 mRNA levels were maximal after 1 h (p < 0.05). Tissue factor mRNA levels were raised at 4 h (p < 0.05) and functional activity was raised at 6 h (p < 0.01). Intracellular ROS production was increased by 50 µmol/l Hcy after 24 h (p < 0.05) but only in high glucose. To investigate the role of mitochondrial metabolism in ROS production, cells were incubated with thenoyltrifluoroacetone (inhibitor of complex II) or carbonyl cyanide m-chlorophenylhydrazone (uncoupler of oxidative phosphorylation). Both compounds abolished the Hcy-induced increase in ROS production and ET-1 release. There was an alteration in intracellular glutathione (GSH) levels with Hcy treatment with more oxidised GSH present. Conclusion: The combined metabolic burden of Hcy and high glucose stimulates ET-1 synthesis in bovine aortic endothelial cells via a mechanism dependent on the production of mitochondrial ROS, but may not be generalisable to all types of endothelial cells.