Correction: Diabetes-Induced Superoxide Anion and Breakdown of the Blood-Retinal Barrier: Role of the VEGF/uPAR Pathway

<p>Correction: Diabetes-Induced Superoxide Anion and Breakdown of the Blood-Retinal Barrier: Role of the VEGF/uPAR Pathway</p

Blockade of diabetes-induced increase in retinal vascular permeability and MMP9 activity in uPAR-/- mice.

<p>Diabetic and non-diabetic uPAR-/- mice and their congenic controls (uPAR+/+, uPAR+/-) were injected intravenously with Alexa-Fluor 488-BSA (10 mg/kg) and permeability was quantified as described in the methods section. Permeability was significantly increased in the diabetic uPAR+/+ or uPAR+/- mice compared with the non-diabetic controls (A). Permeability was not increased in the uPAR-/- mice. n=4-6. C, control; D, diabetic; *, <i>P</i><0.01 <i>versus</i> non-diabetic control; #, <i>P</i><0.01 <i>versus</i> uPAR+/+ and uPAR+/- diabetic. Gelatin zymograms prepared using vitreous samples collected from the same mice showed prominent increases in MMP9 activity in the uPAR+/+ and uPAR+/- diabetic mice. MMP9 activity was absent in retinas from uPAR-/- diabetic mice and non-diabetic controls (B). Retinal sections from diabetic and control mice were immunolabeled with anti-phospho-GSK3β antibody (green) and Texas red-isolectin B4 (red) (C). This analysis showed that phospho-GSK3β co-localizes with the lectin-positive vessels in the diabetic retina (arrows) and is present in microglial-like cells (arrowheads) in both control and diabetic retinas.</p

High glucose-induced permeability increase.

<p>Endothelial monolayers grown in ECIS microarray slides were treated with conditioned media from endothelial cells treated with high glucose (HG CM) or VEGF (VEGF CM) and tested for effects on TER (transcellular electrical resistance). Serum free medium was used as control. TER was markedly reduced by HG CM or VEGF CM (A). HG CM-induced decreases in TER were prominently reduced by pretreatment of the cultures with anti-uPA or anti-uPAR antibodies (B). Gelatin zymograms prepared using medium conditioned by cells treated with high glucose for 1 day (HG1) or 3 days (HG3) or with VEGF or uPA showed prominent increases in MMP9 activity as compared with medium from normal glucose controls (NG) (C). +Control = MMP9.</p

PEG SOD prevents HG glucose induced VEGF expression.

<p>Quantitative RT-PCR and Western blotting analyses were performed with RNA and protein isolated from endothelial cells cultured for 3 days in serum-free medium containing 5.5 mM glucose (NG) or 25 mM glucose (HG) with or without superoxide dismutase (SOD, 80 U/ml) or VEGFR inhibitor (VEGFRI, 2 µM). HG treatment increased VEGF mRNA levels as compared with NG treated cells and this effect was blocked by SOD (A). Western blot analysis confirmed increased levels of VEGF in conditioned media from HG treated endothelial cells as compared with the NG control and HG + SOD cultures (B).</p

PEG SOD prevents HG induced uPAR expression.

<p>Quantitative RT-PCR analysis was performed with RNA isolated from endothelial cells cultured for 1 to 5 days in serum-free medium containing 5.5 mM glucose (NG), 25 mM glucose (HG) with or without superoxide dismutase (SOD, 80 U/ml) or VEGFR inhibitor (VEGFRI, 2 µM). HG treatment caused a time-dependent increase in uPAR (A) that was blocked by SOD or VEGFRI (B).</p

High glucose-induced nuclear translocation of

<p>β-catenin. Endothelial cells were grown in serum-free medium with normal glucose (NG, 5.5mM) or high glucose (HG, 25 mM) for 3 days, fixed and processed for immunocytochemistry and confocal microscopy or used for cell fractionation. Confocal imaging of NG cultures using anti β-catenin antibody (green) and propidium iodide nuclear staining (red) shows membrane bound β-catenin (arrows) (A). Following high glucose treatment β-catenin was redistributed into the cytosol (arrowheads) and nucleus (as shown in z-series optical slices above and at left) (B). Western blotting showed increased cytoplasmic and nuclear β-catenin following HG treatment (C). Densitomitric analysis showed significant increases in cytosolic and nuclear β-catenin levels following HG or VEGF treatment (D) (* = P < 0.05 vs Control). Gray bars represent cytosolic β-catenin. Black bars represent nuclear β-catenin.</p