Anton M Kolomeyer1, Ilene K Sugino, Marco A Zarbin. 1. Institute of Ophthalmology and Visual Science, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, New Jersey 07101, USA.
Abstract
PURPOSE: To characterize trophic factor secretion of cultured adult and fetal retinal pigment epithelial (RPE) cells and to assess the impact on porcine retinal survival in vitro. METHODS: Conditioned media (CM) were collected from cultured adult and fetal RPE cells and analyzed for trophic factor composition and concentration by multiplex ELISA. Trophic factor receptor occupancy was calculated to evaluate the potential biological effectiveness of the differences in trophic factor concentrations. Retina-preserving activity of the collected CM was evaluated using an in vitro model of degenerating porcine retina. Isobaric tag for relative and absolute quantification (iTRAQ) was used to identify additional proteins with a potential effect on porcine retinal survival. RESULTS: The overall trophic factor secretion profile of cultured fetal RPE cells remained stable over multiple passages and extended culture duration. Compared with CM from adult RPE cells, fetal RPE-CM had significantly higher concentrations of vascular endothelial growth factor-A (VEGF-A), brain-derived neurotrophic factor (BDNF), and pigment epithelium-derived factor (PEDF) and significantly lower concentrations of leukemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF), and nerve growth factor (NGF). Fetal RPE-CM was significantly better than adult RPE-CM at improving degenerating porcine retina survival. iTRAQ analysis identified insulin-like growth factor binding protein-3 (IGFBP-3), semaphorin-3B, transforming growth factor (TGF)-β, hepatoma-derived growth factor (HDGF), and gelsolin as factors potentially contributing to this activity. Co-culture of fetal RPE and porcine retina was significantly better than fetal RPE-CM at preserving porcine retinal survival. CONCLUSIONS: Adult and fetal RPE cells have significantly different trophic factor secretion profiles, which correlate with differences in their ability to support porcine retina survival. Combined with trophic factor receptor occupancy calculations, these data implicate VEGF-A and PEDF as key RPE-derived factors promoting preservation of retinal structure and function.
PURPOSE: To characterize trophic factor secretion of cultured adult and fetal retinal pigment epithelial (RPE) cells and to assess the impact on porcine retinal survival in vitro. METHODS: Conditioned media (CM) were collected from cultured adult and fetal RPE cells and analyzed for trophic factor composition and concentration by multiplex ELISA. Trophic factor receptor occupancy was calculated to evaluate the potential biological effectiveness of the differences in trophic factor concentrations. Retina-preserving activity of the collected CM was evaluated using an in vitro model of degenerating porcine retina. Isobaric tag for relative and absolute quantification (iTRAQ) was used to identify additional proteins with a potential effect on porcine retinal survival. RESULTS: The overall trophic factor secretion profile of cultured fetal RPE cells remained stable over multiple passages and extended culture duration. Compared with CM from adult RPE cells, fetal RPE-CM had significantly higher concentrations of vascular endothelial growth factor-A (VEGF-A), brain-derived neurotrophic factor (BDNF), and pigment epithelium-derived factor (PEDF) and significantly lower concentrations of leukemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF), and nerve growth factor (NGF). Fetal RPE-CM was significantly better than adult RPE-CM at improving degenerating porcine retina survival. iTRAQ analysis identified insulin-like growth factor binding protein-3 (IGFBP-3), semaphorin-3B, transforming growth factor (TGF)-β, hepatoma-derived growth factor (HDGF), and gelsolin as factors potentially contributing to this activity. Co-culture of fetal RPE and porcine retina was significantly better than fetal RPE-CM at preserving porcine retinal survival. CONCLUSIONS: Adult and fetal RPE cells have significantly different trophic factor secretion profiles, which correlate with differences in their ability to support porcine retina survival. Combined with trophic factor receptor occupancy calculations, these data implicate VEGF-A and PEDF as key RPE-derived factors promoting preservation of retinal structure and function.