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T. W. Prow, K. Uno, I. Bhutto, C. Merges, R. Grebe, G. Lutty; The Antioxidant Response Element as a Novel Biosensor for Diabetes. Invest. Ophthalmol. Vis. Sci. 2007;48(13):3640.
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© ARVO (1962-2015); The Authors (2016-present)
This study was designed to test the hypothesis that the antioxidant response element (ARE) could be used to biologically detect oxidative stress in diabetes or hyperglycemia in living tissue and potentially be used as a conditional promoter for therapeutic gene expression to treat oxidative stress in diabetes. The first steps in this study were to characterize the activity of the ARE system in vitro during hyperglycemia and then evaluate levels of ARE-mediated expression in diabetic human eyes. Nrf2, the major transcription factor in this system, is known to localize to the nucleus to activate antioxidant gene expression during oxidative stress.
Human retinal endothelial (HREC) and HL60 (a surrogate for circulating monocytes) cells were exposed to normoglycemic and hyperglycemic culture conditions (5, 10, and 30 mM glucose) for 3 days. Then, these cells were evaluated for nuclear translocation of the Nrf2 transcription factor through immunocytochemistry and Western blot analysis. HREC were also transfected with an ARE-driven EGFP reporter plasmid and evaluated for ARE activity through EGFP expression. Finally, adult human eye tissues from individuals with diabetes of varying severity were evaluated for nuclear localization of the Nrf2 transcription factor and HO-1 expression (an ARE driven antioxidant gene) by immunohistochemistry.
Both HREC and HL60 cells had increased Nrf2 nuclear localization with increased glucose concentration by Western blot analysis. However, the HL60 cells had a stronger response than the HREC. ARE mediated EGFP expression also increased with glucose concentration in HREC. The human immunohistochemical staining in diabetic human retina revealed a clear correlation between HO1 levels and the severity of diabetes. HO-1 expression increased significantly with severity of diabetes throughout inner retina including the retinal vasculature and perivascular cells. Nuclear localization of Nrf2 was also evident in many retinal cell types in severe diabetic patients.
These data support the hypothesis that the ARE system could be used to drive synthetic antioxidant enzymes to treat oxidative stress in diabetes. Additionally, these data suggest that ARE functionality may play a role in diabetes progression.
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