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Andreia Goncalves, Jason Keil, Sarah Sheskey, Chengmao Lin, David Antonetti; Contribution of occludin S490 phosphorylation on blood retinal barrier integrity and visual function.. Invest. Ophthalmol. Vis. Sci. 2018;59(9):3008. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
We have previously demonstrated both in vitro and in vivo that VEGF leads to phosphorylation of occludin at S490 promoting its ubiquitination and endocytosis of the junctional complex. We now aim to elucidate the role of this phospho-site in blood-retina barrier (BRB) permeability and visual function.
Mice with transgenic Wt human occludin (WtOCC+/+) or the nonphosphorylatable alanine mutant of occludin Ser490 (S490AOCC+/+) targeted to the ROSA26 site were crossed with mice expressing Tie2-iCre or with mice with endogenous occludin floxed (Occfl/fl)-PDGFb-iCre. Vascular endothelial restricted excision of endogenous occludin and expression of WtOCC+/+ or S490AOCC+/+ was achieved by tamoxifen injection at P3 to induce Cre.BRB permeability to texas red-70 kDa Dextran and FITC-BSA was assessed after 36h of intravitreal VEGF (200 ng/eye) or PBS. Retinal layer thickness was assessed in vivo using spectral domain optical coherence tomography (OCT). Diabetes was induced by streptozotocin injection in Tie2-iCre mice and visual function was evaluated over-time. Visual acuity and contrast sensitivity were determined by the changes in the optokinetic response using the Optometry system.
Conditional expression of S490AOCC+/+ under the Tie2-iCre promoter led to a decrease in BRB permeability induced by VEGF, when compared to Tie2-iCre mice. Suggesting that overexpressing the occludin mutant leads to a protective effect against VEGF-induced permeability. Endothelial conditional deletion of occludin appears to contribute to VEGF-induced edema but surprisingly, not to solute permeability. When endogenous occludin is conditionally deleted the effects of S490AOCC+/+ become more profound when compared to WtOCC+/+, as expression of the mutant reduces both the increase in VEGF-induced permeability as measured by solute flux and edema formation as determined by OCT measures of retinal thickness. Finally, conditional expression of the S490AOCC+/+ mutant prevented the reduction in visual acuity induced by diabetes at 4 months and blocked the loss of contrast sensitivity as well.
Our results show that occludin S490 phosphorylation contributes a major role in VEGF-induced retinal permeability and edema in vivo. Importantly, modulation of barrier properties can positively impact visual function in diabetes.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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