BACKGROUND: Cultured retinal pigment epithelial (RPE) cells exposed to vitreous lose their cuboidal epithelial phenotype and transform to a fibroblast-like morphology. This study investigates whether the loss of RPE phenotype will affect the ability of RPE to phagocytize photoreceptor outer segments (ROS). METHODS: First-passage porcine RPE cells were grown to confluence and exposed to an equal mixture of freshly homogenized vitreous and DMEM (10% FBS) for 48 h to allow transformation to occur. The phagocytic ability of normal epithelial and vitreous-transformed RPE cultures was evaluated by determining the number of ROS bound and ingested. The expression and distribution of alphavbeta5 integrin in control and vitreous-treated RPE were examined by immunoprecipitation coupled with immunoblotting and immunohistochemistry and the contribution of alphavbeta5 integrin to ROS phagocytosis was evaluated using a functional blocking antibody. RESULTS: Bound ROS on vitreous-transformed RPE decreased to 33% of control and ingested ROS decreased to 47%. The expression of alphavbeta5 integrin was decreased significantly following vitreous transformation as compared with controls and the immunolocalization of alphavbeta5 on control RPE was restricted to apical patches while on vitreous-treated RPE, labeling was diminished and randomly dispersed. Five times less blocking antibody was required to attain maximal phagocytic inhibition in these cultures, but some phagocytic activity remained in both normal and transformed cultures even at saturating concentrations. CONCLUSION: The decrease in ROS phagocytosis observed in transformed RPE correlated with the down- regulation of alphavbeta5 integrin expression and its random distribution on the RPE.
BACKGROUND: Cultured retinal pigment epithelial (RPE) cells exposed to vitreous lose their cuboidal epithelial phenotype and transform to a fibroblast-like morphology. This study investigates whether the loss of RPE phenotype will affect the ability of RPE to phagocytize photoreceptor outer segments (ROS). METHODS: First-passage porcine RPE cells were grown to confluence and exposed to an equal mixture of freshly homogenized vitreous and DMEM (10% FBS) for 48 h to allow transformation to occur. The phagocytic ability of normal epithelial and vitreous-transformed RPE cultures was evaluated by determining the number of ROS bound and ingested. The expression and distribution of alphavbeta5 integrin in control and vitreous-treated RPE were examined by immunoprecipitation coupled with immunoblotting and immunohistochemistry and the contribution of alphavbeta5 integrin to ROS phagocytosis was evaluated using a functional blocking antibody. RESULTS: Bound ROS on vitreous-transformed RPE decreased to 33% of control and ingested ROS decreased to 47%. The expression of alphavbeta5 integrin was decreased significantly following vitreous transformation as compared with controls and the immunolocalization of alphavbeta5 on control RPE was restricted to apical patches while on vitreous-treated RPE, labeling was diminished and randomly dispersed. Five times less blocking antibody was required to attain maximal phagocytic inhibition in these cultures, but some phagocytic activity remained in both normal and transformed cultures even at saturating concentrations. CONCLUSION: The decrease in ROS phagocytosis observed in transformed RPE correlated with the down- regulation of alphavbeta5 integrin expression and its random distribution on the RPE.
Authors: P M D'Cruz; D Yasumura; J Weir; M T Matthes; H Abderrahim; M M LaVail; D Vollrath Journal: Hum Mol Genet Date: 2000-03-01 Impact factor: 6.150