RPE secreted proteins and antibody influence photoreceptor cell survival and maturation

Proteins in media conditioned by retinal pigment epithelial cells (RPE-CM) and an antibody against these proteins (RPE-SP) were tested for their respective effects on rat retinal development in vitro and in vivo. Proteins of RPE-CM were separated in denaturing gels and evaluated by Western blot analysis. Retinal explants from postnatal day 2 (P2) rats were cultured in RPE-CM only or CM diluted with the RPE-SP antibody and, after 7 days, the explants were dissociated into single cells that were immunostained for opsin. RPE-CM or antibody was also injected into the vitreous of postnatal day 7 (P7) Long-Evans rats and analyzed 7 and 21 days later. Electrophoretic analysis of RPE-CM predominantly showed 60-70 kDa proteins; when these proteins were probed with RPE-SP antibody by Western blot, immunoreactive proteins were restricted to this narrow molecular weight range. In P2 retinal explant cultures supplemented with RPE-CM, long ganglion cell-like neurites were detected in 3 days. This activity was nullified in explant cultures grown in RPE-CM titrated with antibody, and these explants appeared to degenerate within 5 days. Over 80% of dissociated retinal cells from explants 7 days after treatment with RPE-CM expressed opsin, compared to only 20% of cells from explants grown in defined medium or serum. Retinas of P14 rats injected intravitreally with RPE-CM at P7 had increased numbers of ectopic photoreceptor cells within the inner nuclear layer when compared to retinas of sham-injected eyes. In contrast, retinas of eyes injected intravitreally with RPE-SP antibody exhibited shorter outer (OS) and inner (IS) segments and thinner outer nuclear (ONL) and outer plexiform (OPL) layers than retinas of sham-injected eyes. In conclusion, proteins in RPE-CM appeared to accelerate and maximize the development of rat photoreceptor cells in vitro, while intravitreal injections of its antibody caused an apparent retardation of outer segment maturation. These results suggest that a protein(s) secreted by RPE plays a key role in normal retinal development, particularly in photoreceptor cell survival and outer segment maturation.