High glucose mediates prooxidant and antioxidant enzyme activities in coronary endothelial cells

Objective: Excess levels of free radicals such as nitric oxide (NO) and superoxide anion (O2-)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 (CMEC) exposed to hyperglycaemia. Methods: CMEC 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 eNOS, iNOS, NAD(P)H oxidase and antioxidant enzymes, namely, superoxide dismutase (SOD), catalase and glutahione peroxidase (GPx) were investigated by specific activity assays and Western analyses,respectively while the effects of hyperglycaemia on nitrite and O2 - 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/or 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 MPG (0.1-1 mol/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 prooxidant enzyme activity and diminished generation of 3 antioxidant glutathione. By increasing the antioxidant enzyme capacity CMEC may protect themselves against free radical-induced cell damage in diabetic conditions. The definitive version is available at http://www.blackwell-synergy.com

Internal mammary artery smooth muscle cells resist migration and possess high antioxidant capacity

Objective- This study investigated whether differences exist in atherogen-induced migratory behaviors and basal antioxidant enzyme capacity of vascular smooth muscle cells (VSMC) from human coronary (CA) and internal mammary (IMA) arteries. Methods- Migration experiments were performed using the Dunn chemotaxis chamber. The prooxidant [NAD(P)H oxidase] and antioxidant [NOS, superoxide dismutase, catalase and glutathione peroxidase] enzyme activities were determined by specific assays. Results- Chemotaxis experiments revealed that while both sets of VSMC migrated towards platelet-derived growth factor-BB (1-50 ng/ml) and angiotensin II (1-50 nM), neither oxidized-LDL (ox-LDL, 25-100 �g/ml) nor native LDL (100 �g/ml) affected chemotaxis in IMA VSMC. However, high dose ox-LDL produced significant chemotaxis in CA VSMC that was inhibited by pravastatin (100 nM), mevastatin (10 nM), losartan (10 nM), enalapril (1 �M), and MnTBAP (a free radical scavenger, 50��M). Microinjection experiments with isoprenoids i.e. geranylgeranylpyrophosphate (GGPP) and farnesylpyrophosphate (FPP) showed distinct involvement of small GTPases in atherogen-induced VSMC migration. Significant increases in antioxidant enzyme activities and nitrite production along with marked decreases in NAD(P)H oxidase activity and O2 .- levels were determined in IMA versus CA VSMC. Conclusions- Enhanced intrinsic antioxidant capacity may confer on IMA VSMC resistance to migration against atherogenic agents. Drugs that regulate ox-LDL or angiotensin II levels also exert antimigratory effects.