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M.A. Behzadian, A.B. El-Remessy, T. Franklin, R.B. Caldwell; Mechanism of Glucose Induced Urokinase Receptor (uPAR) Expression in Retinal Endothelial Cells, Possible Role of the ROS and Peroxynitrite Formation . Invest. Ophthalmol. Vis. Sci. 2003;44(13):3905.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Using bovine retinal microvascular endothelial (BRE) cells, we have shown that VEGF increases paracellular permeability by a mechanism involving ß-catenin nuclear translocation and induction of uPAR gene expression. In the streptozotocin diabetic rat, we have also shown that vascular hyperpermeability in retina is accompanied by upregulation of VEGF and uPAR in a process that involves superoxide anion and peroxynitrite formation. Increased reactive oxygen species (ROS) have been implicated in the induction of vascular complications in diabetic patients. However, the mechanism of hyperglycemia induced vascular hyperpermeability is not clear. The aim of this study is to define the intracellular signaling pathway by which these oxidative species increase uPAR gene expression. Our general hypothesis is that high glucose-induced VEGF expression causes transcriptional activation of beta-catenin and uPAR expression through phosphorylation / deactivation of glycogen synthase kinase (GSK-3). Methods: BRE cells were incubated in serum free medium with 5mM or 25 mM glucose and the designated reagents for various times. RNA was extracted and real-time quantitative RT-PCR was carried out to quantify the VEGF and uPAR gene expression. Western blot analysis was employed to demonstrate GSK-3 phosphorylation, it's co-precipitation with phosphorylated Akt-1 or PI-3 kinase and to evaluate the VEGF contents of the conditioned media. Nuclear-translocation of ß-catenin was analyzed by confocal microscopy. Results: High glucose increased VEGF and uPAR expression. These effects were blocked by the superoxide scavenger SOD (80 U/ml) or the NOS inhibitor LNAME, (5 mM). Exogenous peroxynitrite also increased the VEGF expression. Moreover, high glucose increased nuclear translocation of ß-catenin. This effect was accompanied by phosphorylation of GSK-3 and its co-precipitation with phosphorylated PI-3 kinase and with Akt. Conclusion: Our data suggest that glucose-induced superoxide and peroxynitrite formation is responsible for increasing the expression of VEGF and uPAR, hence, breakdown of the blood/retinal barrier in diabetes. The mechanism involves inactivation of GSK-3 by PI-3 kinase/Akt pathways, leading to the nuclear translocation of ß-catenin.
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