A 34-amino acid region of PEDF protects the RPE barrier from VEGF-induced breakdown

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