Immunofluorescence reaction of pVEGF-R2 and pSHP1 in retinal sections.

<p>There was marked increase in the pVEGF immunoreactivity in retinal sections from diabetic mice in comparison to the control. This was prevented by baicalein treatment (A). Note that pVEGF-R2 expression was localized mainly in Muller cells in the control (long arrows), however the reaction was also increased in retinal vessels during diabetes (short arrows) * P<0.05 vs control and # P<0.05 vs baicalein treated group (n = 5). pSHP immunolocalization (B) revealed marked decrease in diabetes which was restored by baicalein treatment. Note that pSHP is expressed in different retinal layers and also in relation to retinal vessels (arrows) in normal retina. In diabetes the decrease in pSHP occurred mainly in inner retina including retinal vessels. * P<0.05 vs control and # P<0.05 vs baicalein treated group (n = 5).</p

Effect of HETE on ROS generation and expression NOX2 in retinal endothelial cells.

<p>DHE staining (A) and DCF assay (B) showed marked increase in ROS generation in REC by 12-HETE. Note significant reduction of 12-HETE-induced ROS generation by apocynin and SOD. Western blotting analysis of NOX2 expression (C) in REC showed upregulation by 12- and 15-HETE compared to the control. *P<0.05 vs Control, # P<0.05 vs 12-HETE (n = 6).</p

Effect of 12/15-LOX inhibition on the levels of inflammatory mediators in retina of diabetic mice.

<p>Analysis of IL-6 and adhesion molecules expression in mouse retina using multiplex system showed significant increase in the levels of ICAM-1 (A), VCAM-1 (B) and IL-6 (C) in diabetic group compared to the control. These increases were prevented in baicalein treated group. *P<0.05 vs Control, # P<0.05 vs Diabetic (n = 7).</p

Effect of VEGF-R2 inhibition on 12-HETE-induced REC hyperpermeability.

<p>Retinal endothelial cells were treated with 0.1 µM 12-HETE in the presence or absence of the VEGF-R2 inhibitor, ZM323881 hydrochloride (10 nM) for 12 hrs before adding the FITC-dextran to the upper chamber of the transwell. Four hrs later the fluorescence intensity in the lower chamber was measured by the plate reader and corrected to the intensity in the upper one. The permeability effect of 12-HETE was significantly prevented by the ZM323881 hydrochloride (12-HETE+I). * P<0.05 vs control and # P<0.05 vs 12-HETE (n = 5).</p

Effect of HETE on ZO-1 expression in retinal endothelial cells.

<p>Immunofluorescence of ZO-1 (green) in REC treated by 12- or 15-HETE. There was marked decrease in ZO-1 expression by 12- and 15-HETE. Effect of 12- and15-HETE on ZO-1 expression in the REC was prevented by apocynin, DPI and NAC. DAPI (blue color) is a nuclear marker *P<0.05 vs control, # P<0.05 vs 12- or 15-HETE (n = 6).</p

Proposed cascade of events following activation of 12/15-LOX by hyperglycemia.

<p>Lipid metabolites of 12/15-LOX (12- and 15-HETE) activate vascular NADPH oxidase leading to overproduction of reactive oxygen species (ROS). Generation of ROS suppresses the activity of protein tyrosine phosphatases with subsequent activation of VEGF-R2 signal pathway and disruption of retinal endothelial cell barrier.</p

Effect of baicalein on ROS generation and NOX2 expression in diabetic retina.

<p>DHE staining (A) of retinal section showed marked increase in ROS generation in diabetic mice which was prevented by baicalein. Immunofluorescence (B) and Western blot analysis of NOX2 (C) in mouse retina demonstrated marked upregulation by diabetes and this increase was significantly reduced by baicalein. *P<0.05 vs C, # P<0.05 vs D (n = 6).</p

Effect of HETE on REC barrier function.

<p>Measurement of transcellular electrical resistance (TER)in REC (A) showed significant decreases in the TER by VEGF, 12- and 15-HETE, compared to the control (* P<0.05 vs control, n = 3). Assessment of REC permeability by FITC-dextran flux method (B). Retinal endothelial cells were incubated with VEGF (100 ng/ml) 12-HETE or 15-HETE (0.1 µM) for 12 hrs before adding FITC-Dextran to the top chamber of the transwell. Four hours later fluorescence intensity in the medium of the lower chamber was measured by a plate reader and normalized to the fluorescence intensity in the upper chamber. *There was significant increase in the FITC-dextran leakage by VEGF, 12- and 15-HETE in comparison to the control. Effect of 12- and 15-HETE on FITC-dextran leakage was blocked by the NADPH oxidase inhibitors (apocynin and DPI), and the antioxidant N-acetyl-L- cystein (NAC). *P<0.05 vs control, # P<0.05 vs 12-HETE, ∧ P<0.05 vs 15-HETE (n = 7).</p

12-HETE induces phosphorylation of VEGF-R2 and dephosphorylation of protein tyrosine phosphatase (PTP) SHP1 in REC.

<p>Western blotting analysis (A) demonstrated significant increase in the level of pVEGF-R2 after 5 and 60 minutes from the beginning of treatment with 12-HETE. *P<0.05 vs Control (C) and other time points. There was simultaneous decrease in the level of pSHP1. Immunofluorescence of pSHP1 (green) in REC also showed marked decrease in the pSHP1 immunoreactivity by 12-HETE compared to the control. * P<0.05 vs control (n = 3).</p