Anti-Senescent Effect of Photoreceptor Outer Segment Phagocytosis by Human Retinal Pigment Epithelial Cells In Vitro

Abstract

Purpose: Photoreceptor outer segment (POS) phagocytosis is crucial for the maintenance and stability of both the photoreceptor cells and the retinal pigment epithelium (RPE). Its possible association with oxidative stress attenuation has been previously discussed. Sirtuin 1 (SIRT1), a nicotinamide adenine dinucleotide-dependent deacetylase, has been pointed to play a protective role against cellular senescence in different tissues. Here we aim to investigate the physiological effect and the potential anti-aging role of POS phagocytosis by the RPE cells.

Methods: Human RPE cells (ARPE-19) were used. POS from the porcine eyes were added at the concentration of 3.0x10⁶/mL in culture medium for 3-9 hours. The mRNA levels for peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) and SIRT1 were measured using quantitative real-time PCR. SIRT1 protein expression was evaluated by ELISA. Mitochondrial biogenesis was evaluated by measuring mitochondrial DNA replication and western blotting for prohibitin, a mitochondrial marker protein. The mRNA levels for senescence markers were measured using quantitative real-time PCR.

Results: POS supplementation for 3 hours induced an elevation in the mRNA levels of PGC-1α (approximately 11 times increased, P= 0.0049, by ANOVA) and SIRT1 (approximately 2 times increased, P= 0.0043, by ANOVA). POS supplementation also increased the mitochondrial DNA replication (about 6 times increased, P<0.0001, by ANOVA) and prohibitin protein expression was significantly elevated 3 and 6 hours after adding the POS. Increased expression of SIRT1 protein was confirmed by ELISA 6h after POS supplementation. Furthermore, mRNA levels of senescence markers including apoliprotein J (Apo J), fibrotectin and connective tissue growth factor (CTGF) were decreased in the POS-supplemented ARPE-19 cells by approximately 30% (P=0.0157, by ANOVA), 50% (P=0.0072 by ANOVA) and 60% (P=0.0008 by ANOVA), respectively.

Conclusions: In RPE cells, POS phagocytosis may present a protective effect by inducing mitochondrial biogenesis and ameliorating senescence process. Increased expression of SIRT1 in ARPE19 cells after POS supplementation might have an association with anti-aging process.

Keywords: 688 retina • 701 retinal pigment epithelium • 648 photoreceptors