Development Of Outer Blood-Retinal Barrier And Regulation Of Tight Junction By Retinal Secretions

Objective:The retinal pigment epithelium(RPE) and outer layer of the neural retina collaborate to form a functional unit essential for vision. Interdigitating RPE microvilli communicate with photoreceptor outer segments across the subretinal space.There are inner and outer blood-retinal barriers to maitain the function of retina:the inner barrier consists of the tight junctions between the retinal microvascular endothelial cells and the pericytes,while the the tight junctions(TJ ) between RPE cells maitain the outer barrier,preventing passage of large molecules from choriocapillaris into the retina.Due to the existence of these barriers,the normal neural retina can keep itself dry and transparent.Dysfunction of the any barrier can lead to the leak of plasma components from vessels to retina and results in retinal edema or detachment.In retinal diseases,the malfunction of one partner of the retina leads to degeneration of the others.When transplanted,RPE redifferentiates best when transplanted before the retina degenerates or when cotransplanted with the neural retina,which indicates the neural retina can induce the differentiation of RPE.Most of the current studies about retinal diseases focus on the effect of the RPE on neural retina,however,less was known about the regulation of the neural retina on PRE.We studied both RPE development in vivo and the effect of neural retina on RPE,especially on the barrier function of RPE,in vitro by gene analysis technology and molecular biochemistry technology and hope to find the key factor of the regulation and provide theoretical basis for the treatment of retinal disease,especially the RPE transplantation.Methods:For in vivo study,chicken RPE was isolated from embryonic day 7(E7),E10,E14,and E18 embryos and total RNA extracted for probing the entire genome on Affymetrix microarray chips.For in vitro study,chick RPE from E7 and E14 were cultured on filters.Media conditioned by organ culture of E14 neural retinas was added to the apical medium chamber.RNA was isolated to probe the chick genome on Affymetrix microarrays,and expression was compared to native E14 RPE.Statistical parameters using ANOVA were adjusted to yield a theoretical false discovery rate of 5%.STEM software was used to cluster genes into statistically related patterns of expression.Gene ontology clustering,using Affymetrix software was used for functional clustering.The proteinlounge.com database was used as a source of known biological pathways. For the genes related to PRE barrier function,such as claudins and ZO family, expression was further analyzed by quantitative real-time PCR immunoblotting and immunocytochemistry to get the basic idea of the regulation during development and the compact of neural retina.SiRNA pilot experiment of claudin 20 was also done in order to study the function of each claudins.Results.Of the 37,694 probesets on the microarray,17,199 were absent. Of the 20,495 expressed probes,the expression of 8,889 was developmentally regulated.4,814 of these could be clustered into 12 patterns of expression that were statistically significant.The developmental patterns of 22 tight and adherens junction proteins were compared using hybridization to the microarray and quantitative PCR.Only two showed small variations from the patterns revealed by the microarray.The data indicate extensive remodeling of the extracellular matrix,cell surface receptors,cell-cell junctions,transcellular ion transport,and signal transduction pathways throughout development.Notably, the appearance of the mRNAs for claudin 20,ZO-3,and cadherins 13 and 20 very late in development suggest barrier properties continue to change after functional junctions are formed.In E7 and E14 cultrues,more than 86%of the genes expressed in vivo were expressed in basal culture conditions,including RPE-specific markers such as RPE65 and bestrophin.E14 retinal conditioned medium affected 15%of the transcriptome in E7 cultures,but only 1.9%in E14 cultures.Examination of 610 genes important for RPE function revealed that mRNAs for 17%were regulated by retinal conditioned medium alone in E7 cultures,compared to 6.2%for E14.For tight junctions,retinal conditioned medium had the most effect on members of the claudin family.Besides regulating mRNA levels,immunoblotting and immunocytochemistry suggested additional mechanisms whereby retinal secretions regulated protein expression and localization.Conclusions:The data in vivo reveal a far more dynamic view of the RPE and its interactions with its environment than would be expected from morphological examination.The remodeling of junctional complexes, extracellular matrix interactions and transcellular transport capabilities indicates a continuous remodeling of the blood-retinal barrier as the retina develops. These data provide a standard whereby culture models of RPE function and regulation may be judged.Gene expression in primary cultures of embryonic RPE resembled the native tissue,but differentiation and the levels of gene expression became more in vivo-like when elements of the retinal environment were introduced into the medium bathing the apical side of the cultures.Albeit insufficient,retinal secretions promoted differentiation of immature RPE and helped maintain the properties of more mature RPE.More detailed studies of claudin family proteins showed that,rcSF3 made the expression levels of claudins closer to the levels in vivo by regulating mRNA and protein expression respectively.