June 2020
Volume 61, Issue 7
Free
ARVO Annual Meeting Abstract  |   June 2020
Super resolution microscopy and mutational analysis reveal novel role of occludin in VEGF-driven neovascularization
Author Affiliations & Notes
  • David A Antonetti
    Kellogg Eye Center Univ of Michigan, Ann Arbor, Michigan, United States
  • Xuwen Liu
    Kellogg Eye Center Univ of Michigan, Ann Arbor, Michigan, United States
  • Footnotes
    Commercial Relationships   David Antonetti, None; Xuwen Liu, None
  • Footnotes
    Support  NIH EY012021; NIH HL055374
Investigative Ophthalmology & Visual Science June 2020, Vol.61, 2371. doi:
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      David A Antonetti, Xuwen Liu; Super resolution microscopy and mutational analysis reveal novel role of occludin in VEGF-driven neovascularization. Invest. Ophthalmol. Vis. Sci. 2020;61(7):2371.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Vascular endothelial growth factor (VEGF) induction of endothelial permeability requires occludin phosphorylation on Ser490 and alanine point mutants preventing this phosphorylation reduce or prevent VEGF-driven permeability both in cell culture and in transgenic mouse models. However, previous studies also demonstrated that the same Ser490 phosphorylation of occludin is required for VEGF-driven endothelial cell proliferation and that occludin could be found in the centrosomes of dividing cells. The current research explored the hypothesis that VEGF-driven phosphorylation of occludin and centrosomal localization regulates cell proliferation.

Methods : Transgenic mice were created with occludin containing Ser 490 mutated to Ala (S490A) following a floxed stop sequence at the ROSA26 locus. Cell proliferation and neovascularization were evaluated in S490A mice in the animal models of middle cerebral arterial occlusion (MCAO), branch retinal vein occlusion (BRVO) and ex vivo retinal explant culture. Occludin mutations were generated and its centrosomal localization was tested by transfecting U2OS cells that have well-characterized centrosomes. VEGF-induced proliferation was examined after nucleofection of occludin mutants in bovine retinal endothelial cells (BREC) using Click-iT EdU cell proliferation assay in 3D culture. Occludin localization was examined by immunofluorescence confocal super-resolution microscopy.

Results : Cell proliferation and neovascularization were significantly inhibited in S490A mice. Localization to centrosomes requires both the C-terminal coiled-coil domain with Ser490 phosphorylation and the fourth transmembrane domain of occludin. A truncation mutant lacking all external loops, that mimics a naturally occurring splice variant, localized to centrosomes but an S490A point mutant inhibited VEGF-driven proliferation. BODIPY staining demonstrated that phospho-occludin co-localizes with lipid markers at centrosomes during mitosis. Super-resolution microscopy revealed occludin could be observed in multiple vesicles at the start of mitosis and occludin vesicles fuse to a single vesicle by metaphase with concurrent peak phosphorylation.

Conclusions : These studies demonstrate novel mechanistic insight into retinal neovascularization revealing a required role for occludin phosphorylation and regulation of mitosis through centrosomal localized vesicles.

This is a 2020 ARVO Annual Meeting abstract.

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