PEDF and Erythropoietin Share Protective Properties: on Retinal Neuronal Precursor Cell Lines

Abstract

Abstract: : Purpose: Pigment epithelium–derived factor (PEDF) and erythropoietin (Epo) protect photoreceptors against light–induced damage. Epo is a survival and neurite–outgrowth factor for retinal ganglion cells (RGCs). Photoreceptors and RGCs contain receptors for both factors. Immortalized retinal neuronal cell lines are useful tools for the study of retina survival. The purpose of this work was to develop survival assays and compare the effects of the two cytokines on immortalized retinal precursor cell lines. Methods: R–28 cells are an immortalized rat retinal precursor cell line, RGC–5 cells are immortalized rat retinal ganglion cells, and HER–10 cells are immortalized human embryonic retinoblasts. Cell viability was assessed with MTS, MTT or Cell–Titer Glo Viability kits. Cell death was induced by serum deprivation or cell membrane depolarization. Cell–surface receptor binding assays were performed with radiolabeled ligand in solution, or by cell adhesion to PEDF–coated wells. Binding to soluble Epo receptor (sEpoR) was tested by size–exclusion ultrafiltration or Surface Plasmon Resonance. Hematopoietic assays were performed in primary adult erythroid progenitor cells, and glycophorin A–positive and benzidine positive cells were determined as indication of erythroid differentiation. Results: PEDF and Epo increased the viability of serum–deprived R28 and RGC–5 cells in a dose–dependent fashion, Epo being 1000–fold more potent than PEDF. PEDF also increased the viability of depolarized HER–10 cells. ¹²⁵I–PEDF specifically bound to R28 and HER–10 cells. Viable RGC–5 cells adhered to PEDF–coated wells and an excess of anti–PEDF prevented cell adhesion. Although PEDF bound and formed complexes with sEpoR after incubation for 16 h, it did not bind to sEpoR in real–time under identical conditions for optimal Epo–sEpoR binding. Unlike Epo, PEDF did not promote erythroid differentiation and showed only marginal survival activity. Conclusions: Epo and PEDF protected retinal precursor cells from natural and induced cell death, the effects of Epo being more potent than those of PEDF. The retinal precursor cells had PEDF binding sites consistent with cell–surface receptors specific for PEDF. Like Epo, PEDF is a survival factor for RGCs. However, PEDF did not share hematopoietic properties or affinity for EpoR with Epo. These results demonstrate that Epo and PEDF share protective activities, and suggest that they might have distinct mechanisms of action in the retina.

Keywords: apoptosis/cell death • retinal culture • neuroprotection