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Monica Diaz-Coranguez, David Antonetti; Norrin Restores Barrier Properties After VEGF Stimulation.. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):55.
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© ARVO (1962-2015); The Authors (2016-present)
Accumulating evidence suggests that norrin and its receptors FZD4, LRP5 and TSPAN12, have a fundamental role in blood-retinal barrier (BRB) maintenance. Here, we examine the molecular mechanisms activated by norrin that may prevent VEGF-induced vascular permeability and promote proper BRB formation. Our overall hypothesis is that norrin induces the stabilization of the tight junction complex, thus promoting proper BRB formation.
The contribution of norrin signaling to endothelial permeability in vitro was tested by measures of transendothelial electrical resistence (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. mRNA was quantified by qPCR. Data was analyzed by one-way ANOVA with at least an n of 3.
BREC treated with norrin resulted in no change in TEER, while the addition of VEGF resulted in a decreased TEER ~30% after 24 hours. Pre-treatment with norrin for 24 hours, followed by the addition of VEGF, failed to prevent the VEGF induced loss of barrier. However, the addition of both VEGF and norrin together led to a decrease in barrier similar to VEGF alone for the first 24 hours but then the barrier recovered to control values by 72h. In BREC monolayers pre-treated with VEGF for 24 hours, followed by the addition of norrin, the TEER was largely restored to control values by 72h and norrin acted in a dose responsive manner. Consistent with these results, VEGF increased permeability (Po) 2-fold to dextran (P<0.0001). The addition of norrin and then VEGF failed to prevent the VEGF-mediated permeability, while the addition of VEGF for 24 hours and then norrin, or both simultaneously, restored barrier properties to 70kDa dextran (P<0.001 and P<0.05, respectively). The effect of norrin on barrier properties was not accounted for by inhibition of VEGFR Tyr phosphorylation or signal transduction through ERK phosphorylation, but β-catenin content increased after 48h treatment with norrin likely contributing to regulation of barrier properties. Importantly, VEGF increased the expression of norrin receptors, as measured by qRT-PCR.
Norrin induced barrier properties only after or co-incident with VEGF addition suggesting norrin signaling acts in coordination with VEGF to promote proper retinal vascular development and induction of the blood-retinal barrier.
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