Hyperglycaemia-induced superoxide production decreases eNOS expression via AP-1 activation in aortic endothelial cells

S. Srinivasan, M. E. Hatley, D. T. Bolick, L. A. Palmer, D. Edelstein, M. Brownlee, C. C. Hedrick

Research output: Contribution to journalArticle

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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 C57BL/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-1 (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-1 sites in the human eNOS promoter reversed the effects of HG. Compared with C57BL/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 superoxide 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.

Original languageEnglish (US)
Pages (from-to)1727-1734
Number of pages8
JournalDiabetologia
Volume47
Issue number10
DOIs
StatePublished - Oct 2004

Fingerprint

Nitric Oxide Synthase Type III
Transcription Factor AP-1
Superoxides
Hyperglycemia
Endothelial Cells
Glucose
Nitrites
Messenger RNA
Blood Vessels
Reactive Oxygen Species
DNA
Electrophoretic Mobility Shift Assay
Diabetes Complications
Electron Transport
Superoxide Dismutase
Carrier Proteins
Nitric Oxide
Proteins
Oxidative Stress
Transcription Factors

Keywords

  • AP-1
  • Diabetes
  • Endothelial
  • eNOS
  • Superoxide

ASJC Scopus subject areas

  • Internal Medicine
  • Endocrinology, Diabetes and Metabolism

Cite this

Srinivasan, S., Hatley, M. E., Bolick, D. T., Palmer, L. A., Edelstein, D., Brownlee, M., & Hedrick, C. C. (2004). Hyperglycaemia-induced superoxide production decreases eNOS expression via AP-1 activation in aortic endothelial cells. Diabetologia, 47(10), 1727-1734. https://doi.org/10.1007/s00125-004-1525-1

Hyperglycaemia-induced superoxide production decreases eNOS expression via AP-1 activation in aortic endothelial cells. / Srinivasan, S.; Hatley, M. E.; Bolick, D. T.; Palmer, L. A.; Edelstein, D.; Brownlee, M.; Hedrick, C. C.

In: Diabetologia, Vol. 47, No. 10, 10.2004, p. 1727-1734.

Research output: Contribution to journalArticle

Srinivasan, S, Hatley, ME, Bolick, DT, Palmer, LA, Edelstein, D, Brownlee, M & Hedrick, CC 2004, 'Hyperglycaemia-induced superoxide production decreases eNOS expression via AP-1 activation in aortic endothelial cells', Diabetologia, vol. 47, no. 10, pp. 1727-1734. https://doi.org/10.1007/s00125-004-1525-1
Srinivasan, S. ; Hatley, M. E. ; Bolick, D. T. ; Palmer, L. A. ; Edelstein, D. ; Brownlee, M. ; Hedrick, C. C. / Hyperglycaemia-induced superoxide production decreases eNOS expression via AP-1 activation in aortic endothelial cells. In: Diabetologia. 2004 ; Vol. 47, No. 10. pp. 1727-1734.
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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 C57BL/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-1 (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-1 sites in the human eNOS promoter reversed the effects of HG. Compared with C57BL/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 superoxide 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.",
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AU - Hatley, M. E.

AU - Bolick, D. T.

AU - Palmer, L. A.

AU - Edelstein, D.

AU - Brownlee, M.

AU - Hedrick, C. C.

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N2 - 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 C57BL/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-1 (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-1 sites in the human eNOS promoter reversed the effects of HG. Compared with C57BL/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 superoxide 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.

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