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C. J. Dougherty, M. R. Biswal, G. W. Smith, H. M. Prentice, C. K. Dorey, K. A. Webster, J. C. Blanks; Hypoxia-Enhanced and Endothelial-Specific Expression of a Mitochondria-Targeted Catalase Decreases Damage to Microvascular Endothelial Cells Due to Hypoxia and Elevated Glucose. Invest. Ophthalmol. Vis. Sci. 2008;49(13):4905. doi: https://doi.org/.
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To the test the hypothesis that an endothelial cell-specific and hypoxia-enhanced expression of a mitochondria-targeted catalase will reduce or prevent apoptosis and associated permeability in endothelial cells cultured in normoxia, hypoxia and/or elevated glucose (30mM).
An endothelial-specific and hypoxia-responsive promoter - (consisting of nine tandem hypoxia-response elements (HREs) and a truncated Tie2 promoter (Tie2)) - was employed to drive luciferase transgene expression in a pGL3 luciferase plasmid. Dual luciferase assays (DLA) were used to evaluate cell specificity and response to hypoxia. The HRE-Tie2 promoter was incorporated into in a self-complimentary AAV encoding a mitochondria-targeted catalase (mCatalase). The effects of hypoxia and/or elevated glucose on apoptosis, and permeability were assayed in control RF/6A retinal choroidal microvascular endothelial monolayers and in those that were transfected with the AAV.HRE.Tie2.mCatalase.
The HRE-Tie2 promoter was selectively activated in endothelial cells in comparison to cultured HT22 hippocampal neurons. Hypoxia alone induced a 21-fold increase in expression of luciferase in the endothelial cells. In normal glucose, hypoxia did not induce apoptosis. However, in elevated glucose, hypoxia induced a dramatic increase in apoptosis that was accompanied by increased permeability of control RF/6A endothelial monolayers. Under these same conditions, RF/6A cells transfected with the HRE-Tie2-mCatalase vector exhibited a 58% decrease in apoptosis, and a 46% decrease in endothelial cell permeability.
The results indicate damage to microvascular endothelial cells by elevated glucose is enhanced by hypoxia. This damage was significantly reduced by an AAV-vector delivering mitochondria-targeted catalase. These data indicate that mitochondrial catalase serves to protect microvascular endothelial cells from the damaging consequences of a hypoxic, hyperglycemic environment in diabetic retinopathy.
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