In vitro Transfection of Plasmid DNA Encoding Pigment Epithelum–Derived Factor (PEDF) Into Retinal Cells Protects Against Hydrogen Peroxide–Induced Cell Death

Abstract

Abstract: : Purpose: To examine the ability of introducing human pigment epithelium derived factor (hPEDF) gene into cultured rat retinal cells using non–viral constructs to protect against hydrogen peroxide (H₂O₂)–induced cell death. Methods: Rat Müller cell, retinal pigment epithelium (RPE) cell and primary retinal neuronal cultures were transfected with pBLAST49–hPEDF, pSV40–hPEDF–EGFP, and pSV40–EGFP. The level of hPEDF in the culture medium was determined using the commercially available hPEDF ELISA kit (CYT 420, Chemicon). The in vitro protection by transfection of hPEDF against H₂O₂–induced cell death was measured by cell viability assay and Flow Cytometry analysis. Results:The basal level of PEDF protein in the culture medium was 16 ng/ml from primary neuronal culture, 58 ng/ml from Müller cell culture and 69 ng/ml from RPE cell culture. The medium from the cultures transfected with pBLAST49–hPEDF showed that PEDF protein levels were 228 ng/ml for neurons, 362 ng/ml for Müller cells and 432 ng/ml for RPE cells. Transfection with pBLASY49–hPEDF significantly protected cultured neurons, Müller and RPE cells against H₂O₂–induced cell death, whereas pSV40–hPEDF–EGFP did not. The observation that pSV40–hPEDF–EGFP transfection did not protect any type of retinal cells tested in this study suggests that PEDF may lose its protective function when fused to EGFP. Conclusions: We demonstrated that the secreted PEDF from those cells transfected with a plasmid encoding PEDF cDNA can protect against H₂O₂–mediated cell death. These will provide a solid base to further develop the approach employing plasmid DNA encoding PEDF as a therapeutic treatment to delay or prevent retinal degeneration in vivo.

Keywords: neuroprotection • growth factors/growth factor receptors • oxidation/oxidative or free radical damage