June 2013
Volume 54, Issue 15
Free
ARVO Annual Meeting Abstract  |   June 2013
A 34-amino acid region of PEDF protects the RPE barrier from VEGF-induced breakdown
Author Affiliations & Notes
  • Danielle Desjardins
    Ophthalmology, Medical University of South Carolina, Charleston, SC
  • Mohammad Dahrouj
    Ophthalmology, Medical University of South Carolina, Charleston, SC
  • Jason Kenealey
    Section of Protein Structure and Function, NEI, NIH, Bethesda, MD
  • S Patricia Becerra
    Section of Protein Structure and Function, NEI, NIH, Bethesda, MD
  • Yueying Liu
    Ophthalmology, Medical University of South Carolina, Charleston, SC
  • Kumar Sambamurti
    Neurosciences, Medical University of South Carolina, Charleston, SC
  • Craig Crosson
    Ophthalmology, Medical University of South Carolina, Charleston, SC
  • Zsolt Ablonczy
    Ophthalmology, Medical University of South Carolina, Charleston, SC
  • Footnotes
    Commercial Relationships Danielle Desjardins, None; Mohammad Dahrouj, None; Jason Kenealey, None; S Patricia Becerra, None; Yueying Liu, None; Kumar Sambamurti, None; Craig Crosson, Alimera Sciences (C), Lexicon Pharmaceuticals, Inc (R); Zsolt Ablonczy, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2013, Vol.54, 1187. doi:
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      Danielle Desjardins, Mohammad Dahrouj, Jason Kenealey, S Patricia Becerra, Yueying Liu, Kumar Sambamurti, Craig Crosson, Zsolt Ablonczy; A 34-amino acid region of PEDF protects the RPE barrier from VEGF-induced breakdown. Invest. Ophthalmol. Vis. Sci. 2013;54(15):1187.

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

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Abstract

Purpose: Pigment epithelium-derived factor (PEDF) inhibits the actions of vascular endothelial growth factor (VEGF)-induced RPE barrier breakdown in a γ-secretase-dependent manner. In addition to its actions on the RPE, two different structural regions of PEDF have been characterized in other systems as antiangiogenic (34-mer) and neurotrophic (44-mer). This study aims to identify if either of these domains protects the RPE barrier from VEGF-E induced dysfunction.

Methods: Barrier function was assessed by transepithelial resistance (TER) measurements on monolayer cultures of ARPE-19 cells. Wells were treated with VEGF-E (20ng/mL) and PEDF protein (0.01pg/mL - 10ng/mL), or PEDF peptides. The PEDF peptides are a 44-mer derived from positions 78-121 of human PEDF or an adjacent 34-mer derived from positions 44-77, both used at 10ng/mL- 0.01fg/mL. Inhibition of γ-secretase was performed by pretreatment with DAPT at 1µM for 1 hour. TER was measured over a 6-hour period. Concentration-dependence, inhibitory properties, and reversal of actions by DAPT were assessed and compared for PEDF and both its peptides based on TER measurements and immunoblotting.

Results: Treatment of ARPE-19 cells with VEGF-E (20ng/mL) induced a significant reduction in TER. This reduction was inhibited by co-treatment with PEDF or the 34-mer peptide, but not with the 44-mer peptide. Concentration dependence of the 34-mer peptide in the presence of VEGF-E established an EC50 of 2 fM. This was comparable to the full-length PEDF response (EC50 of 20 fM). Even at 100-fold higher concentrations, the 44-mer peptide did not alter the VEGF response to reduce the TER of ARPE-19 cells. Similar to full-length PEDF, the inhibitory response of the 34-mer peptide was reversed by pretreatment with DAPT at the peptide concentration of 265 pM.

Conclusions: The results show that the protective action of PEDF against VEGF-E-induced barrier breakdown resides within the 44-77 amino acid region of the PEDF polypeptide. Similar to full-length PEDF, the actions of this 34-amino acid peptide were also mediated by γ -secretase. These data provide evidence that the 34- amino acid peptide can modulate RPE barrier properties. Moreover, the data agree with previous observations in the endothelia showing that the 34-mer peptide antagonizes VEGF signaling, while the 44-mer peptide does not.

Keywords: 701 retinal pigment epithelium • 666 pump/barrier function • 543 growth factors/growth factor receptors  
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