Abstract
Vascular endothelial growth factor (VEGF) contributes to blood-retinal barrier (BRB) dysfunction in several blinding eye diseases, including diabetic retinopathy. Signaling via the secreted protein norrin through the frizzled class receptor 4 (FZD4)/LDL receptor-related protein 5-6 (LRP5-6)/tetraspanin 12 (TSPAN12) receptor complex is required for developmental vascularization and BRB formation. Here, we tested the hypothesis that norrin restores BRB properties after VEGF-induced vascular permeability in diabetic rats or in animals intravitreally injected with cytokines. Intravitreal co-injection of norrin with VEGF completely ablated VEGF-induced BRB permeability to Evans Blue-albumin. Likewise, 5-month diabetic rats exhibited increased permeability of FITC-albumin, and a single norrin injection restored BRB properties. These results were corroborated in vitro, where co-stimulation of norrin with VEGF or stimulation of norrin after VEGF exposure restored barrier properties, indicated by electrical resistance or 70-kDa RITC-dextran permeability in primary endothelial cell culture. Interestingly, VEGF promoted norrin signaling by increasing the FZD4 co-receptor TSPAN12 at cell membranes in an MAPK/ERK kinase (MEK)/ERK-dependent manner. Norrin signaling through β-catenin was required for BRB restoration, but glycogen synthase kinase 3 α/β (GSK-3α/β) inhibition did not restore BRB properties. Moreover, levels of the tight junction protein claudin-5 were increased with norrin and VEGF or with VEGF alone, but both norrin and VEGF were required for enriched claudin-5 localization at the tight junction. These results suggest that VEGF simultaneously induces vascular permeability and promotes responsiveness to norrin. Norrin, in turn, restores tight junction complex organization and BRB properties in a β-catenin-dependent manner.
Highlights
Vascular endothelial growth factor (VEGF) contributes to blood-retinal barrier (BRB) dysfunction in several blinding eye diseases, including diabetic retinopathy
The hypovascular phenotype observed in both Norrie and familial exudative vitreopathy (FEVR) diseases has been recapitulated in knockout mice models of norrin or the frizzled class receptor 4 (FZD4) receptor complex (1, 5, 8, 28 –34), in which retinal vascular growth, mural cell recruitment, endothelial differentiation, and barrier properties are affected as a consequence of a low Sox7, Sox17, and Sox18 gene expression [35] or due to an increased expression of the Wnt signaling inhibitor APCDD1 (APC down-regulated 1 protein) [36]
We have found that permeability in response to norrin injection was not different from control values, whereas, as expected, retinas injected with VEGF showed a significant increase in the accumulation of Evans Blue dye
Summary
Vascular endothelial growth factor (VEGF) contributes to blood-retinal barrier (BRB) dysfunction in several blinding eye diseases, including diabetic retinopathy. The hypovascular phenotype observed in both Norrie and FEVR diseases has been recapitulated in knockout mice models of norrin or the FZD4 receptor complex (1, 5, 8, 28 –34), in which retinal vascular growth, mural cell recruitment, endothelial differentiation, and barrier properties are affected as a consequence of a low Sox, Sox, and Sox gene expression [35] or due to an increased expression of the Wnt signaling inhibitor APCDD1 (APC down-regulated 1 protein) [36] These retinas show high retinal vascular permeability that correlates with reduced border immunostaining of the tight junction protein claudin-5 and increased expression of the transcytosis marker PLVAP, a phenotype that can be reversed by the expression of stabilized -catenin [5]. These data indicate a requirement of norrin signaling through -catenin in deep capillary angiogenesis, BRB formation and maintenance
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