This proposal investigates how the neural retina regulates the development of polarity and barrier function in the retinal pigment epithelium (RPE). It tests the hypothesis that during development, retinal-derived factors, induce the maturation of tight junctions. This maturation is required for the RPE to form the outer blood-retinal barrier. Reestablishing this barrier is the goal of therapies aimed at repairing RPE damaged by surgical intervention or disease. Barrier function requires the RPE to develop two related, but distinct properties. First, it must form tight junctions, a barrier to diffusion between the choroid and the subretinal space. Second, it must polarize the distribution of proteins that regulate transport across the barrier. In most epithelia, these properties are induced by interactions at the basal and lateral membranes. In RPE, this induction is modulated by interaction with the neural retina at the apical membrane. Novel interactions between the neural retina and the RPE were revealed by examining the gradual development of polarity during embryogenesis. Furthermore, these studies revealed a previously undescribed maturation of the tight junctions. The current proposal will reconstitute this developmental process in culture. RPE will be isolated from chick embryos before the outer blood-retinal barrier forms. These immature cells will be cultured with medium that was conditioned by retinas isolated from older embryos, when the barrier is forming. The retinal-derived factors will be characterized according to functional properties: the ability to increase barrier function, and the ability to induce the changes in junction structure and composition that occur in vivo. Accordingly, the study will provide the first molecular and morphological description of tight junctions as they are converted from a leaky to a tight form in vivo. The maturation of tight junctions will be monitored with electron microscopic and immunofluorescence techniques to examine changes in morphology, and with immunofluorescence and immunobiochemical techniques to examine the assembly of the tight junction proteins 7H6, occludin, cingulin and ZO-l. The functional assays will be use to characterize and purify the retinal factors on an analytical scale. These studies address fundamental issues of epithelial cell biology, and examine an aspect of RPE/retinal interactions that have been previously ignored. This information is required to understand the function of RPE in normal tissue and in proliferative disease. These studies may help identify factors that promote the regeneration of a functional monolayer from endogenous or transplanted RPE.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
5R01EY008694-05
Application #
2019755
Study Section
Visual Sciences C Study Section (VISC)
Project Start
1993-07-01
Project End
1998-11-30
Budget Start
1996-12-01
Budget End
1997-11-30
Support Year
5
Fiscal Year
1997
Total Cost
Indirect Cost
Name
Yale University
Department
Surgery
Type
Schools of Medicine
DUNS #
082359691
City
New Haven
State
CT
Country
United States
Zip Code
06520
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Cong, Lidan; Sun, Dawei; Zhang, Zhongyu et al. (2008) A novel rabbit model for studying RPE transplantation. Invest Ophthalmol Vis Sci 49:4115-25
Rizzolo, Lawrence J (2007) Development and role of tight junctions in the retinal pigment epithelium. Int Rev Cytol 258:195-234
Rizzolo, Lawrence J; Chen, Xiang; Weitzman, Matthew et al. (2007) Analysis of the RPE transcriptome reveals dynamic changes during the development of the outer blood-retinal barrier. Mol Vis 13:1259-73
Luo, Yan; Fukuhara, Masayuki; Weitzman, Matthew et al. (2006) Expression of JAM-A, AF-6, PAR-3 and PAR-6 during the assembly and remodeling of RPE tight junctions. Brain Res 1110:55-63
Luo, Yan; Zhuo, Yehong; Fukuhara, Masayuki et al. (2006) Effects of culture conditions on heterogeneity and the apical junctional complex of the ARPE-19 cell line. Invest Ophthalmol Vis Sci 47:3644-55
Rahner, Christoph; Fukuhara, Masayuki; Peng, Shaomin et al. (2004) The apical and basal environments of the retinal pigment epithelium regulate the maturation of tight junctions during development. J Cell Sci 117:3307-18
Peng, Shaomin; Rahner, Christoph; Rizzolo, Lawrence J (2003) Apical and basal regulation of the permeability of the retinal pigment epithelium. Invest Ophthalmol Vis Sci 44:808-17
Kojima, S; Rahner, C; Peng, S et al. (2002) Claudin 5 is transiently expressed during the development of the retinal pigment epithelium. J Membr Biol 186:81-8
Bumsted, K M; Rizzolo, L J; Barnstable, C J (2001) Defects in the MITF(mi/mi) apical surface are associated with a failure of outer segment elongation. Exp Eye Res 73:383-92

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