Pigment epithelium-derived factor and osteopontin protect human retinal pigment epithelial cells from oxidative stress in 2D and 3D models

Retinal pigment epithelial (RPE) cells are particularly vulnerable to oxidative stress. Neighboring choroidal melanocytes (CMs) secrete trophic factors, such as pigment epithelium-derived factor (PEDF) and osteopontin (OPN), which may mitigate oxidative damage in RPE cells. Herein, we investigated the ability of PEDF and OPN to protect RPE cells from extended NaIO₃-induced oxidative stress damage. Primary human RPE cells were seeded on plastic (2D culture) or on a 3D tissue-engineered choroidal stroma (TECS) containing (or not) CMs. PEDF or OPN was added before, during, and after 5 days of NaIO₃ exposure. Analyses included assessment of PEDF and OPN expression, intracellular reactive oxygen species (ROS), lipid peroxidation, mitochondrial superoxide, cell death, antioxidant capacity (TAC) and p-Nrf2 nuclear translocation. Adding exogenous PEDF to the growth media stimulated de novo PEDF secretion by RPE cells, whereas OPN concentration detected in the growth media corresponded to the amount added. PEDF and OPN levels were consistently lower in the growth media of 3D TECS compared to the 2D cultures. Oxidative stress significantly increased intracellular ROS, lipid peroxidation, and RPE cell death in both 2D and 3D cultures, which were reduced by PEDF or OPN supplementation. Oxidative stress and PEDF or OPN supplementation did not alter the TAC of RPE cells. PEDF or OPN supplementation promoted p-Nrf2 nuclear translocation in RPE cells on plastic, even without oxidative stress, whereas this response was not observed in 3D TECS cultures. Adding CMs to the 3D TECS did not influence p-Nrf2 nuclear translocation in RPE cells. Overall, PEDF and OPN supplementation significantly protected human RPE cells against NaIO₃-induced oxidative stress damage. In 3D TECS, CMs secreted insufficient levels of PEDF and OPN for RPE cells to manage oxidative stress-induced damage. These findings demonstrate that exogenous supplementation with PEDF and OPN proteins may be beneficial for oxidative-stress related eye diseases.