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- Title
Hyperglycaemia-induced superoxide production decreases eNOS expression via AP-1 activation in aortic endothelial cells.
- Authors
Srinivasan, S.; Hatley, M. E.; Bolick, D. T.; Palmer, L. A.; Edelstein, D.; Brownlee, M.; Hedrick, C. C.
- Abstract
Aims/hypothesis. Hyperglycaemia is a primary cause of vascular complications in diabetes. A hallmark of these vascular complications is endothelial cell dysfunction, which is partly due to the reduced production of nitric oxide. The aim of this study was to investigate the regulation of endothelial nitric oxide synthase (eNOS) activity by acute and chronic elevated glucose. Methods. Human aortic endothelial cells were cultured in 5.5 mmol/l (NG) or 25 mmol/l glucose (HG) for 4 h, 1 day, 3 days or 7 days. Mouse aortic endothelial cells were freshly isolated from CS7BL/6J control and diabetic db/db mice. The expression and activity of eNOS were measured using quantitative PCR and nitrite measurements respectively. The binding of activator protein-I (AP-1) to DNA in nuclear extracts was determined using electrophoretic mobility-shift assays. Results. Acute exposure (4 h) of human aortic endothelial cells to 25 mmol/l glucose moderately increased eNOS activity and eNOS mRNA and protein expression. In contrast, chronic exposure to elevated glucose (25 mmol/l for 7 days) reduced total nitrite levels (46% reduction), levels of eNOS mRNA (46% reduction) and eNOS protein (65% reduction). In addition, AP- 1 DNA binding activity was increased in chronic HG- cultured human aortic endothelial cells, and this effect was reduced by the specific inhibition of reactive oxygen species production through the mitochondrial electron transport chain. Mutation of AP-I sites in the human eNOS promoter reversed the effects of HG. Compared with CS7BL/6J control mice, eNOS mRNA levels in diabetic db/db mouse aortic endothelial cells were reduced by 60%. This decrease was reversed by the overexpression of manganese su peroxide dismutase using an adenoviral construct. Conclusions/interpretation. In diabetes, the expression and activity of eNOS is regulated through glucose- mediated mitochondrial production of reactive oxygen species and activation of the oxidative stress transcription factor AP- 1.
- Subjects
HYPERGLYCEMIA; SUPEROXIDES; DIABETES complications; ENDOTHELIUM; NITRIC oxide; CELL culture; GENE expression; DNA
- Publication
Diabetologia, 2004, Vol 47, Issue 10, p1727
- ISSN
0012-186X
- Publication type
Article
- DOI
10.1007/s00125-004-1525-1