April 2014
Volume 55, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2014
ProNGF causes retinal endothelial cell permeability via activation of p75NTR/RhoA pathway
Author Affiliations & Notes
  • Barbara A Mysona
    UGA Clinical and Experimental Therapeutics, University of Georgia, Augusta, GA
    Culver Vision Discovery Institute, Georgia Regents University, Augusta, GA
  • Dorothy Rodenbeck
    UGA Clinical and Experimental Therapeutics, University of Georgia, Augusta, GA
    Culver Vision Discovery Institute, Georgia Regents University, Augusta, GA
  • Ahmed Y Shanab
    UGA Clinical and Experimental Therapeutics, University of Georgia, Augusta, GA
    Culver Vision Discovery Institute, Georgia Regents University, Augusta, GA
  • Suraporn Matragoon
    UGA Clinical and Experimental Therapeutics, University of Georgia, Augusta, GA
    Culver Vision Discovery Institute, Georgia Regents University, Augusta, GA
  • Azza B El-Remessy
    UGA Clinical and Experimental Therapeutics, University of Georgia, Augusta, GA
    Culver Vision Discovery Institute, Georgia Regents University, Augusta, GA
  • Footnotes
    Commercial Relationships Barbara Mysona, None; Dorothy Rodenbeck, None; Ahmed Shanab, None; Suraporn Matragoon, None; Azza El-Remessy, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science April 2014, Vol.55, 5827. doi:https://doi.org/
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      Barbara A Mysona, Dorothy Rodenbeck, Ahmed Y Shanab, Suraporn Matragoon, Azza B El-Remessy; ProNGF causes retinal endothelial cell permeability via activation of p75NTR/RhoA pathway. Invest. Ophthalmol. Vis. Sci. 2014;55(13):5827. doi: https://doi.org/.

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

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Abstract

Purpose: Our aim is to understand the molecular mechanisms contributing to breakdown of the inner blood-retinal barrier (BRB), an event which leads to macular edema, a major cause of diabetic blindness. Previously, our lab has shown that p75NTR deletion or inhibition blocks diabetes and proNGF-induced inflammation and BRB breakdown. Here, we test the hypothesis that proNGF acts directly on endothelial cells to stimulate p75NTR mediated activation of RhoA leading to barrier dysfunction.

Methods: Human retinal endothelial (HRE) cell permeability was assayed by measuring extravasation of FITC-dextran (70 kD) through HRE monolayers grown on collagen/fibronectin coated transwells. Cells were treated in the presence or absence of human cleavage-resistant mutant proNGF, (hm-proNGF, 1 to 50 ng/ml) with or without pretreatment with p75NTR inhibitors compound A (Uri Saragovi, McGill University) or LM11A-31 (Dr. Frank Longo, Stanford University) and Rho-kinase inhibitor Y-27632, 10 μM. Cells were also grown in high (25 mM) compared to normal (5 mM) glucose in the presence or absence of hm-proNGF (10 ng/ml). Barrier function was measured by monitoring accumulation of fluorescence in the abluminal compartment. Lysates from confluent HRE cells were analyzed by Western blot for RhoA activation, VEGF, and TNF-α.

Results: Treatment with hm-proNGF yielded a maximal 2-fold increase in HRE cell permeability at 10 ng/ml (n=6, P < 0.05) that was blocked by p75NTR and Rho-kinase inhibition (n=6-8, P < 0.05). Analysis of HRE lysates revealed that cells treated with hm-proNGF exhibited a 50% increase in active RhoA that was blocked by inhibition of p75NTR. In HRE cells, treatment with proNGF did not stimulate increased expression of VEGF or TNF-α.

Conclusions: Our data provide evidence that proNGF/p75NTR can directly mediate RhoA activation and increased HRE cell permeability independent of VEGF or TNF-α. The p75NTR/Rho kinase pathway may be a potential therapeutic target for treating macular edema, a major cause of diabetic blindness.

Keywords: 499 diabetic retinopathy • 505 edema • 688 retina  
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