March 2012
Volume 53, Issue 14
Free
ARVO Annual Meeting Abstract  |   March 2012
Molecular Mechanisms of Blood-Retinal Barrier Breakdown in Ischemia-Reperfusion Injury
Author Affiliations & Notes
  • Arivalagan Muthusamy
    Department of Ophthalmology & Visual Sciences, University of Michigan Kellogg Eye Center, Ann Arbor, Michigan
  • Cheng-Mao Lin
    Department of Ophthalmology & Visual Sciences, University of Michigan Kellogg Eye Center, Ann Arbor, Michigan
  • Heather Lindner
    Department of Ophthalmology & Visual Sciences, University of Michigan Kellogg Eye Center, Ann Arbor, Michigan
  • Sumathi Shanmugam
    Department of Ophthalmology & Visual Sciences, University of Michigan Kellogg Eye Center, Ann Arbor, Michigan
  • Steven F. Abcouwer
    Department of Ophthalmology & Visual Sciences, University of Michigan Kellogg Eye Center, Ann Arbor, Michigan
  • David A. Antonetti
    Department of Ophthalmology & Visual Sciences, University of Michigan Kellogg Eye Center, Ann Arbor, Michigan
  • Footnotes
    Commercial Relationships  Arivalagan Muthusamy, None; Cheng-Mao Lin, None; Heather Lindner, None; Sumathi Shanmugam, None; Steven F. Abcouwer, None; David A. Antonetti, None
  • Footnotes
    Support  JDRF
Investigative Ophthalmology & Visual Science March 2012, Vol.53, 5327. doi:
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      Arivalagan Muthusamy, Cheng-Mao Lin, Heather Lindner, Sumathi Shanmugam, Steven F. Abcouwer, David A. Antonetti; Molecular Mechanisms of Blood-Retinal Barrier Breakdown in Ischemia-Reperfusion Injury. Invest. Ophthalmol. Vis. Sci. 2012;53(14):5327.

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

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Abstract

Purpose: : Formation and maintenance of the blood-retinal barrier (BRB) requires assembly of multiple tight junction (TJ) proteins including the MARVEL family (occludin, tricellulin, and marvel D3 proteins) and the claudin family. Recently, we demonstrated that ischemia-reperfusion (IR) injury induces retinal vascular permeability that occurs within 4 h after reperfusion and persists for at least 48 h. Previous studies demonstrated that VEGF causes vascular permeability through rapid phosphorylation of the TJ protein occludin on serine 490 (Ser-490), which leads to its ubiquitination and translocation away from TJ complexes. Therefore, the present study was designed to test the hypotheses that retinal IR injury causes vascular permeability through rapid alterations in tight junction protein expression, modification and complex formation.

Methods: : Rats were subjected to ischemia for 45 minutes followed by reperfusion up to 4 h. Blood-retinal barrier breakdown after IR was confirmed by leakage of intravenously injected Evans blue dye. Changes in retinal TJ protein content and occludin phosphorylation were assessed by Western blotting. Occludin ubiquitination was determined by immunoprecipitation (IP) followed by Western blotting. To observe the effect of IR on TJ organization, the localization of TJ proteins was examined by immunohistochemistry (IHC) of retinal flat mounts.

Results: : Retinal vascular permeability was significantly increased 4, 24 and 48 h after reperfusion. IR caused rapid occludin Ser-490 phosphorylation occurring between 15 and 60 min after reperfusion, while poly-ubiquitination of occludin was evident after 15 min. IHC indicated that IR diminished TJ protein content at endothelial cell junctions; however, there were no changes in total protein contents of occludin, claudins (1, 5, 10, and 16) and Zonula occluden-1 (ZO-1).

Conclusions: : These data are consistent with retinal IR injury causing vascular permeability through rapid phosphorylation and ubiquitination of occludin leading to its translocation and subsequent disassembly of the tight junction complexes. This model suggests that occludin phosphorylation may represent a valid target for therapeutic prevention of BRB breakdown and subsequent edema.

Keywords: ischemia • retina • diabetic retinopathy 
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