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.

中文翻译：

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在血管内皮生长因子诱导的通透性后，Norrin恢复了血视网膜屏障的特性。

在包括糖尿病性视网膜病在内的几种致盲眼疾病中，血管内皮生长因子（VEGF）会导致血视网膜屏障（BRB）功能障碍。通过卷曲的类受体4（FZD4）/ LDL受体相关蛋白5-6（LRP5-6）/四跨膜蛋白12（TSPAN12）受体复合物通过分泌的蛋白诺林进行信号传导是发育血管形成和BRB形成所必需的。在这里，我们测试了以下假设：在糖尿病大鼠或玻璃体内注射细胞因子的动物中，诺林可在VEGF诱导的血管通透性后恢复BRB特性。玻璃体腔内注射Norrin和VEGF可完全消除VEGF诱导的BRB对Evans Blue-Albumin的通透性。同样，5个月的糖尿病大鼠表现出FITC-白蛋白的通透性增加，单次Norrin注射即可恢复BRB特性。这些结果在体外得到证实，其中在血管内皮生长因子的共同刺激下或在暴露于血管内皮生长因子后刺激诺兰蛋白恢复了屏障性能，这由原代内皮细胞培养物中的电阻或70kDa RITC-葡聚糖渗透性表示。有趣的是，VEGF通过以MAPK / ERK激酶（MEK）/ ERK依赖性的方式增加细胞膜上的FZD4共受体TSPAN12来促进Norrin信号传导。BRB恢复需要通过β-catenin进行Norrin信号传导，但糖原合酶激酶3α/β（GSK-3α/β）的抑制不能恢复BRB的特性。此外，norrin和VEGF或单独使用VEGF会增加紧密连接蛋白claudin-5的水平，但norrin和VEGF都需要在紧密连接处富集claudin-5。这些结果表明，VEGF同时诱导血管通透性并促进对诺里灵的响应性。反过来，Norrin以β-catenin依赖的方式恢复紧密连接的复杂组织和BRB属性。