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Monica Diaz Coranguez, Chengmao Lin, Stefan Liebner, David A Antonetti; Signal interaction between Norrin and VEGF promotes blood-retinal barrier restoration. Invest. Ophthalmol. Vis. Sci. 2017;58(8):2519.
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Norrin signaling through FZD4, LRP5/6, and TSPAN12 receptors, is required for vascularization and formation of the blood-retinal barrier (BRB). Here, we examined how VEGF and Norrin signaling interact to alter BRB properties. Our overall hypothesis is that Norrin can reverse VEGF-induced permeability by inducing the stabilization of the tight junction (TJ).
Endothelial permeability in Vitro was tested by measures of transendothelial electrical resistance (TEER) and solute flux of 70kDa RITC-dextran in primary bovine retinal endothelial cells (BREC). The signal transduction crosstalk between Norrin and VEGF signaling pathways was assessed by Western blot and qRT-PCR. Norrin effect was corroborated in two animal models of diabetes: intravitreal injection of VEGF and 5-month STZ-diabetic rats. Data was analyzed with t-test, one- or two- way ANOVA with at least an n of 3.
VEGF decreased TEER ∼30-40% at 24h that was maintained for 72h, while Norrin addition simultaneous with or after VEGF addition restored TEER to control values at 72h. Similar results were found measuring the permeability of 70 kDa FITC-dextran across BREC monolayers. VEGF promoted Norrin signaling by the induction of TSPAN12 translocation to the cell membrane at 24h in a MEK/ERK dependent manner and increased mRNA content at 72h. Norrin induced βCatenin stabilization and its translocation to nuclear fractions. Chemical inhibitors and transfection of inhibitory or constitutively active forms of βCatenin revealed that this signaling is necessary but not sufficient to induce barrier properties after VEGF. Moreover, transfection of disheveled mutant that promotes planar cell polarity, resulted in a decrease of VEGF-induced permeability. VEGF and Norrin both increased the expression of the TJ protein Claudin-5 but only Norrin promotes its localization at the TJ. The effect of Norrin was further demonstrated in Vivo with intravitreal injection of Norrin, VEGF or both. VEGF increased permeability of Evans blue while Norrin/VEGF completely restored the barrier properties to control levels. Likewise, 5-month STZ-diabetic rats showed increased permeability to FITC-dextran that was completely restored after 24 hours of Norrin injection.
These results suggest that Norrin signaling acts in coordination with VEGF to promote proper induction of the BRB.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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