High glucose mediates pro-oxidant and antioxidant enzyme activities in coronary endothelial cells

Aim: Excess levels of free radicals such as nitric oxide (NO) and superoxide anion (O-2(-)) are associated with the pathogenesis of endothelial cell dysfunction in diabetes mellitus. This study was designed to investigate the underlying causes of oxidative stress in coronary microvascular endothelial cells (CMECs) exposed to hyperglycaemia. Methods: CMECs were cultured under normal (5.5 mmol/l) or high glucose (22 mmol/l) concentrations for 7 days. The activity and expression (protein level) of endothelial NO synthase (eNOS), inducible NOS (iNOS), NAD(p)H oxidase and antioxidant enzymes, namely, superoxide dismutase (SOD), catalase and glutathione peroxidase (GPx) were investigated by specific activity assays and Western analyses, respectively, while the effects of hyperglycaemia on nitrite and O-2(-) generation were investigated by Griess reaction and cytochrome C reduction assay, respectively. Results: Hyperglycaemia did not alter eNOS or iNOS protein expressions and overall nitrite generation, an index of NO production. However, it significantly reduced the levels of intracellular antioxidant glutathione by 50% (p < 0.05) and increased the protein expressions and activities of p22-phox, a membrane-bound component of pro-oxidant NAD(p)H oxidase and antioxidant enzymes (p < 0.05). Free radical scavengers, namely, Tiron and mercaptopropionylglycine (MPG) (0.1-1 mumol/l) reduced hyperglycaemia-induced antioxidant enzyme activity and increased glutathione and nitrite generation to the levels observed in CMEC cultured in normoglycaemic medium (p < 0.01). The differences in enzyme activity and expressions were independent of the increased osmolarity generated by high glucose levels as investigated by using equimolar concentrations of mannitol in parallel experiments. Conclusions: These results suggest that hyperglycaemia-induced oxidative stress may arise in CMEC as a result of enhanced pro-oxidant enzyme activity and diminished generation of antioxidant glutathione. By increasing the antioxidant enzyme capacity, CMEC may protect themselves against free radical-induced cell damage in diabetic conditions.