Shaomin Peng

Integration of tight junctions and claudins with the barrier functions of the retinal pigment epithelium

The retinal pigment epithelium (RPE) forms the outer blood-retinal barrier by regulating the movement of solutes between the fenestrated capillaries of the choroid and the photoreceptor layer of the retina. Blood-tissue barriers use various mechanisms to accomplish their tasks including membrane pumps, transporters, and channels, transcytosis, metabolic alteration of solutes in transit, and passive but selective diffusion. The last category includes tight junctions, which regulate transepithelial diffusion through the spaces between neighboring cells of the monolayer. Tight junctions are extraordinarily complex structures that are dynamically regulated. Claudins are a family of tight junctional proteins that lend tissue specificity and selectivity to tight junctions. This review discusses how the claudins and tight junctions of the RPE differ from other epithelia and how its functions are modulated by the neural retina. Studies of RPE-retinal interactions during development lend insight into this modulation. Notably, the characteristics of RPE junctions, such as claudin composition, vary among species, which suggests the physiology of the outer retina may also vary. Comparative studies of barrier functions among species should deepen our understanding of how homeostasis is maintained in the outer retina. Stem cells provide a way to extend these studies of RPE-retinal interactions to human RPE.

Claudin-19 and the Barrier Properties of the Human Retinal Pigment Epithelium

PURPOSE The retinal pigment epithelium (RPE) separates photoreceptors from choroidal capillaries, but in age-related macular degeneration (AMD) capillaries breach the RPE barrier. Little is known about human RPE tight junctions or the effects of serum on the retinal side of the RPE. METHODS Cultured human fetal RPE (hfRPE) was assessed by the transepithelial electrical resistance (TER) and the transepithelial diffusion of methylated polyethylene glycol (mPEG). Claudins and occludin were monitored by quantitative RT-PCR, immunoblotting, and immunofluorescence. RESULTS Similar to freshly isolated hfRPE, claudin-19 mRNA was 25 times more abundant than claudin-3. Other detectable claudin mRNAs were found in even lesser amounts, as little as 3000 times less abundant than claudin-19. Claudin-1 and claudin-10b were detected only in subpopulations of cells, whereas others were undetectable. Knockdown of claudin-19 by small interfering RNA (siRNA) eliminated the TER. siRNAs for other claudins had minimal effects. Serum affected tight junctions only when presented to the retinal side of the RPE. The TER increased 2 times, and the conductance of K(+) relative to Na(+) decreased without affecting the permeability of mPEG. These effects correlated with increased steady-state levels of occludin. CONCLUSIONS Fetal human RPE is a claudin-19-dominant epithelium that has regional variations in claudin-expression. Apical serum decreases RPE permeability, which might be a defense mechanism that would retard the spread of edema due to AMD.

Claudin 5 is transiently expressed during the development of the retinal pigment epithelium.

During the development of chick retinal pigment epithelium (RPE), the permeability and selectivity of the epitheliums tight junctions are continuously modulated. Overall paracellular permeability decreases, but selectivity increases. Because the claudin family of transmembrane proteins appears to provide the structural basis for selectivity, we examined the expression of claudins as a function of development in chick RPE. Degenerate primers were used with the reverse transcriptase-polymerase chain reaction (RT-PCR) to obtain complete sequences of chick claudins 3 and 5. Northern blotting and semi-quantitative RT-PCR demonstrated that claudin 5 was expressed in RPE, but claudin 3 was expressed only in the choroid layer of the eye. Northern blotting, semiquantitative RT-PCR and immunoblotting demonstrated that the expression of claudin 5 was transient, with peak levels of expression between embryonic days 10 and 14. Primary cultures were used to demonstrate that factors secreted by the neural retina induced the expression of claudin 5 nearly 3-fold if RPE was isolated from embryonic day 7 embryos. There was little effect if RPE was isolated from embryonic day 14. The upregulation of claudin 5 correlates with permeability changes that occur during the intermediate stage of RPE development. Interestingly, claudin 5 must be replaced during the late stage of development when the number and complexity of tight junctional strands increases. This would imply more changes in selectivity.

The apical and basal environments of the retinal pigment epithelium regulate the maturation of tight junctions during development.

A culture model has been established to study the gradual development of tight junctions during the embryogenesis of the chick retinal pigment epithelium. This study asks how closely the culture model reflects normal development and how the composition, structure and function of embryonic tight junctions are affected by the apical and basal environments. The study focused on the expression of claudins, the fine-structure of tight junctional strands and the transepithelial electrical resistance. Between embryonic days 7 and 14, patches of junctional strands gradually expanded and coalesced to form a continuous junction, in vivo. Although there was a corresponding increase in claudin expression, different claudins appeared at different times. In culture, the apical and basal environments acted synergistically to promote a continuous network of tight junctions with higher electrical resistance. Independently, pituitary extract or the secretory products of either embryonic fibroblasts or the retina promoted the formation of tight junctions. In combination, three effects were identified. With basally placed fibroblast conditioned medium, apical retinal medium increased transepithelial electrical resistance by affecting structure alone. With basally placed pituitary extract, apical retinal conditioned medium increased transepithelial electrical resistance by affecting structure and by modulating claudin expression in a manner that was consistent with development in vivo. Although embryonic day 7 and 14 cultures in retinal medium exhibited similar structure, the transepithelial electrical resistance of the embryonic day 14 cultures was higher. This higher transepithelial electrical resistance correlated with differences in claudin expression and localization. Therefore, this experimental model can isolate the effects of retinal secretions on structure and claudin expression, and can help us to determine how claudins affect function when structure is held constant.

Minimal Effects of VEGF and Anti-VEGF Drugs on the Permeability or Selectivity of RPE Tight Junctions

PURPOSE Bevacizumab and ranibizumab are currently used to treat age-related macular degeneration by neutralizing vascular endothelial growth factor (VEGF). In this study, the potential side effects on the outer blood-retinal barrier were examined. METHODS Human fetal RPE (hfRPE) cells were used because they are highly differentiated in culture. The claudin composition of RPE tight junctions was determined by RT-PCR, immunoblot analysis, and immunofluorescence. ELISA assays monitored the secretion and trafficking of VEGF and a fluid-phase marker, methylpolyethylene glycol (mPEG). Tight junction functions were assessed by the conductance of K(+) and Na(+) (derived from the transepithelial electrical resistance, TER) and the flux of NaCl and mPEG. RESULTS Claudin-3, claudin-10, and claudin-19 were detected in RPE tight junctions. VEGF was secreted in equal amounts across the apical and basolateral membranes, but the apical membrane was more active in endocytosing and degrading VEGF. Exogenous VEGF and mPEG crossed the RPE monolayer by transcytosis, predominantly in the apical-to-basal direction. RPE tight junctions were selective for K(+), but did not discriminate between Na(+) and Cl(-). VEGF, bevacizumab, and ranibizumab had minimal effects on TER, permeation of mPEG, and selectivity for K(+), Na(+), and Cl(-). They had minimal effects on the expression and distribution of the claudins. CONCLUSIONS RPE has mechanisms for maintaining low concentrations of VEGF in the subretinal space that include endocytosis and degradation and fluid-phase transcytosis in the apical-to-basal direction. RPE tight junctions are selective for K(+) over Na(+) and Cl(-). Permeability and selectivity of the junctions are not affected by VEGF, bevacizumab, or ranibizumab.

Effects of Proinflammatory Cytokines on the Claudin-19 Rich Tight Junctions of Human Retinal Pigment Epithelium

PURPOSE Chronic, subclinical inflammation contributes to the pathogenesis of several ocular diseases, including age-related macular degeneration. Proinflammatory cytokines affect tight junctions in epithelia that lack claudin-19, but in the retinal pigment epithelium claudin-19 predominates. We examined the effects of cytokines on the tight junctions of human fetal RPE (hfRPE). METHODS hfRPE was incubated with interleukin 1-beta (IL-1β), interferon-gamma (IFNγ), or tumor necrosis factor-alpha (TNFα), alone or in combination. Permeability and selectivity of the tight junctions were assessed using nonionic tracers and electrophysiology. Claudins, occludin, and ZO-1 were examined using PCR, immunoblotting, and confocal immunofluorescence microscopy. RESULTS Only TNFα consistently reduced transepithelial electrical resistance (TER) >80%. A serum-free medium revealed two effects of TNFα: (1) decreased TER was observed only when TNFα was added to the apical side of the monolayer, and (2) expression of TNFα receptors and inhibitors of apoptosis were induced from either side of the monolayer. In untreated cultures, tight junctions were slightly cation selective, and this was affected minimally by TNFα. The results were unexplained by effects on claudin-2, claudin-3, claudin-19, occludin, and ZO-1, but changes in the morphology of the junctions and actin cytoskeleton may have a role. CONCLUSIONS Claudin-19-rich tight junctions have low permeability for ionic and nonionic solutes, and are slightly cation-selective. Claudin-19 is not a direct target of TNFα. TNFα may protect RPE from apoptosis, but makes the monolayer leaky when it is presented to the apical side of the monolayer. Unlike other epithelia, IFNγ failed to augment the effect of TNFα on tight junctions.

Engineering a Blood-Retinal Barrier With Human Embryonic Stem Cell-Derived Retinal Pigment Epithelium: Transcriptome and Functional Analysis

Retinal degenerations are a major cause of impaired vision in the elderly. Degenerations originate in either photoreceptors or the retinal pigment epithelium (RPE). RPE forms the outer blood‐retinal barrier and functions intimately with photoreceptors. Animal models and cultures of RPE are commonly used to screen potential pharmaceuticals or explore RPE replacement therapy, but human RPE differs from that of other species. Human RPE forms a barrier using tight junctions composed of a unique set of claudins, proteins that determine the permeability and selectivity of tight junctions. Human adult RPE fails to replicate these properties in vitro. To develop a culture model for drug development and tissue‐engineering human retina, RPE were derived from human embryonic stem cells (hESCs). Barrier properties of RPE derived from the H1 and H9 hESC lines were compared with a well‐regarded model of RPE function, human fetal RPE isolated from 16‐week‐gestation fetuses (hfRPE). A serum‐free medium (SFM‐1) that enhanced the redifferentiation of hfRPE in culture also furthered the maturation of hESC‐derived RPE. In SFM‐1, the composition, selectivity, and permeability of tight junctions were similar to those of hfRPE. Comparison of the transcriptomes by RNA sequencing and quantitative reverse transcription‐polymerase chain reaction revealed a high correlation between the hESCs and hfRPE, but there were notable differences in the expression of adhesion junction and membrane transport genes. These data indicated that hESC‐derived RPE is highly differentiated but may be less mature than RPE isolated from 16‐week fetuses. The study identified a panel of genes to monitor the maturation of RPE.

A Novel Rabbit Model for Studying RPE Transplantation

PURPOSE The goal of this project was to develop a model of retinal pigment epithelium (RPE) transplantation that permits extensive and reliable analysis of the transplants. METHODS Cultures of newborn rabbit RPE were evaluated by morphology, electrophysiology, and the expression of zonula occludens-1, cytokeratin, and the melanocyte marker S-100. Cells labeled with 5,6-carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) were transplanted into the subretinal space of rabbits with a 30-gauge needle without making a conjunctival flap or sclerotomy. The transplants were examined by fundus photography, confocal scanning laser ophthalmoscopy (cSLO), optical coherence tomography (OCT), and angiography. At 2 months, the retina was examined histochemically. RESULTS A 1-minute incubation at 37 degrees C with 20 muM CFDA-SE did not affect morphology or the expression of marker proteins. In coculture, the labeled cells integrated into monolayers that developed a normal transepithelial electrical resistance of 400 to 450 Omega . cm(-2). Dye was not transferred from labeled to nonlabeled RPE cells. Transplanted RPE was detectable for at least 2 months. Angiography demonstrated an intact blood-retinal barrier. The normal morphology of the retina and lack of debris in the subretinal space suggested that the transplanted RPE was functional. CONCLUSIONS Primary cultures of newborn rabbit RPE were highly differentiated, even when labeled with CFDA-SE. Labeled cells were observed long-term in vitro and in vivo. This model can be used to examine how culture and transplantation protocols affect the reformation of a functional RPE monolayer. The similar size of rabbit and human eyes will facilitate the translation of these protocols to the bedside.

TRP Channels Localize to Subdomains of the Apical Plasma Membrane in Human Fetal Retinal Pigment Epithelium

PURPOSE Calcium regulates many functions of the RPE. Its concentration in the subretinal space and RPE cytoplasm is closely regulated. Transient receptor potential (TRP) channels are a superfamily of ion channels that are moderately calcium-selective. This study investigates the subcellular localization and potential functions of TRP channels in a first-passage culture model of human fetal RPE (hfRPE). METHODS The RPE isolated from 15- to 16-week gestation fetuses were maintained in serum-free media. Cultures were treated with barium chloride (BaCl2) in the absence and presence of TRP channel inhibitors and monitored by the transepithelial electrical resistance (TER). The expression of TRP channels was determined using quantitative RT-PCR, immunoblotting, and immunofluorescence confocal microscopy. RESULTS Barium chloride substantially decreased TER and disrupted cell-cell contacts when added to the apical surface of RPE, but not when added to the basolateral surface. The effect could be partially blocked by the general TRP inhibitor, lanthanum chloride (LaCl3, ~75%), or an inhibitor of calpain (~25%). Family member-specific inhibitors, ML204 (TRPC4) and HC-067047 (TRPV4), had no effect on basal channel activity. Expression of TRPC4, TRPM1, TRPM3, TRPM7, and TRPV4 was detected by RT-PCR and immunoblotting. The TRPM3 localized to the base of the primary cilium, and TRPC4 and TRPM3 localized to apical tight junctions. The TRPV4 localized to apical microvilli in a small subset of cells. CONCLUSIONS The TRP channels localized to subdomains of the apical membrane, and BaCl2 was only able to dissociate tight junctions when presented to the apical membrane. The data suggest a potential role for TRP channels as sensors of [Ca(2+)] in the subretinal space.

Claudin-3 and claudin-19 partially restore native phenotype to ARPE-19 cells via effects on tight junctions and gene expression.

Mutations of claudin-19 cause severe ocular deficits that are not easily reconciled with its role in regulating the outer blood retinal barrier. ARPE-19 is a widely used culture model of the retinal pigment epithelium (RPE). ARPE-19 is unique among epithelial cell lines, because it expresses all tight junction proteins except claudin family members. ARPE-19 also loses aspects of the RPE phenotype with cell passage. This study asks whether exogenous expression of the main RPE claudins, claudin-3 and claudin-19, would restore RPE phenotype, and whether these claudins have distinct roles in RPE. An Ussing chamber was used to measure the transepithelial electrical resistance and transepithelial electrical potential. These measurements were used to estimate the permeability co-efficients of ions. The transepithelial diffusion of polyethylene glycols were used to examine the leak pathway of tight junctions. Wound-healing, quantitative RT-PCR and immunoblotting examined diverse aspects of the RPE phenotype. Over-expression of either claudin decreased the permeability of small ions and polyethylene glycol. Both claudins were slightly cation-specific, but claudin-3 was less permeable to large solutes. Claudin expression widely affected gene expression to partially restore RPE phenotype. Claudins redistributed filamentous actin from stress fibers to circumferential bands associated with tight junctions, and made wound-healing more epithelial-like. Both claudins increased the expression of genes related to RPE core functions and increased steady-state levels of phosphorylated-AKT. In conclusion, claudin-3 and claudin-19 formed general permeability barriers and affected cell morphology, proliferation, migration, AKT signaling, and gene expression. When claudins are exogenously expressed, ARPE-19 more closely model native RPE.