June 2013
Volume 54, Issue 15
Free
ARVO Annual Meeting Abstract  |   June 2013
Early Ischemia-reperfusion Injury Induces Retinal Vascular Permeability in a VEGF Receptor 2 Dependent Manner Followed by Occludin Phosphorylation and Ubiquitination
Author Affiliations & Notes
  • Arivalagan Muthusamy
    Ophthalmology & Vis Sciences, Univ of Michigan Kellogg Eye Ctr, Ann Arbor, MI
  • Cheng-mao Lin
    Ophthalmology & Vis Sciences, Univ of Michigan Kellogg Eye Ctr, Ann Arbor, MI
  • Heather Lindner
    Ophthalmology & Vis Sciences, Univ of Michigan Kellogg Eye Ctr, Ann Arbor, MI
  • Sumathi Shanmugam
    Ophthalmology & Vis Sciences, Univ of Michigan Kellogg Eye Ctr, Ann Arbor, MI
  • Steven Abcouwer
    Ophthalmology & Vis Sciences, Univ of Michigan Kellogg Eye Ctr, Ann Arbor, MI
  • David Antonetti
    Ophthalmology & Vis Sciences, Univ of Michigan Kellogg Eye Ctr, Ann Arbor, MI
  • Footnotes
    Commercial Relationships Arivalagan Muthusamy, None; Cheng-mao Lin, None; Heather Lindner, None; Sumathi Shanmugam, None; Steven Abcouwer, None; David Antonetti, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2013, Vol.54, 2474. doi:
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      Arivalagan Muthusamy, Cheng-mao Lin, Heather Lindner, Sumathi Shanmugam, Steven Abcouwer, David Antonetti, ; Early Ischemia-reperfusion Injury Induces Retinal Vascular Permeability in a VEGF Receptor 2 Dependent Manner Followed by Occludin Phosphorylation and Ubiquitination. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2474.

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

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Abstract

Purpose: Ischemia-reperfusion (IR) injury rapidly induces retinal vascular permeability by an unknown mechanism. Multiple tight junction (TJ) proteins including the MARVEL family (occludin, tricellulin, and marvel D3 proteins) and the claudin family are required for the formation and maintenance of the blood-retinal barrier (BRB). Vascular endothelial growth factor- (VEGF)-induced permeability of retinal endothelial cell cultures requires phosphorylation of occludin on serine 490 (Ser-490), which leads to its ubiquitination and endocytosis. The present study tests the hypotheses that retinal IR injury stimulates vascular permeability through VEGF receptor 2 (VEGFR2) activation and 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. BRB permeability was quantified by retinal accumulation of Evans blue dye measured 2 h after its intravitreal injection. Changes in retinal TJ protein content, occludin phosphorylation and ubiquitination were assessed by Western blotting. To observe TJ organization, localization of TJ proteins was examined by immunohistochemistry (IHC) of flat-mounted retinas.

Results: IR caused a rapid significant increase in permeability assessed during 0.25-2.25 h and 4-6 h following reperfusion. IR had no effects on total protein content of any TJ protein examined, but rapidly diminished TJ protein localization at endothelial cell junctions within 15 min of reperfusion. IR caused phosphorylation of VEGFR2 on tyrosine 1175 (Tyr-1175) at 15 min after reperfusion, coinciding with occludin phosphorylation at Ser-490 and poly-ubiquitination. Intravitreal injection of a tyrosine kinase inhibitor specifically targeting VEGFR2 efficiently blocks both IR-induced VEGFR2 Tyr-1175 phosphorylation and occludin Ser-490 phosphorylation.

Conclusions: These data are consistent with retinal IR injury causing vascular permeability through VEGFR2 activation and phosphorylation and ubiquitination of occludin leading to rapid disassembly of tight junction complexes. This model suggests that occludin phosphorylation may represent a valid target for therapeutic prevention of VEGF-induced BRB breakdown and subsequent edema.

Keywords: 499 diabetic retinopathy • 572 ischemia • 748 vascular endothelial growth factor  
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