PEDF78-121 Limits Oxidative Stress in RPE Cells Through Akt and Erk Signaling and Reduces Stress Promoted Expression of VEGFR1

Abstract

Introduction: : PEDF treatment can reduce both retinal cell death and neovascularization. The neurotrophic activity of the protein is localized to an N terminal fragment corresponding to amino acid residues 78-121. In this study we examined the effects of this peptide on oxidative stress in RPE cells.

Methods: : PEDF_78-121 peptide was cloned into pET32a expressed in E.coli, and peptide purified using Ni-NTA chromatography. The effects of purified PEDF_78-121 peptide were examined in ARPE19 cultures challenged with 640µM H₂O₂. Cells were treated for 2h with H₂O₂ followed by treatment with various concentration of PEDF_78-121 for 24h. The protective effects of PEDF_78-121 on H₂O₂ toxicity were assessed using LDH assay and PI staining. Modulations in VEGF, VEGF receptors, and various signaling pathways were also determined.

Results: : PEDF_78-121 protects RPE cells from hydrogen peroxide toxicity at concentrations as low as 25 ng/ml. Approximately 30% decrease in cell death is seen in cultures after treatment with the peptide. This protective effect is mediated through the Akt and Erk signaling pathways. H₂0₂ decreased the phosphorylation of both signal molecules after 2h, a condition immediately reversed by PEDF_78-121. Specificity of PEDF_78-121 actions was shown with the pharmacological inhibitors Akt and MEK1/2 which blocked Akt and Erk1/2 activation, respectively by PEDF_78-121. Inhibition of Akt and Erk signaling abrogated the protective effects of PEDF_78-121 on RPE cells. We also show that oxidative stress increases the expression of VEGFR1 in RPE cells and that PEDF_78-121 reduced this expression by 3.4 fold, an effect that was abolished when Akt and Erk signaling pathways were blocked.

Conclusions: : Oxidative stress can modulate Akt and Erk signaling and induce expression of VEGR1 in RPE cells. PEDF_78-121 is an effective pharmacological tool that reduces oxidative stress induced death in RPE cells and may limit the actions of these cells in angiogenic events in the retina.

Keywords: cell survival • neuropeptides • retinal pigment epithelium