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V. S. Rao, S. Peng, R. A. Adelman, L. J. Rizzolo; Effects of Ectopic Serum on the Barrier Properties of the Retinal Pigment Epithelium. Invest. Ophthalmol. Vis. Sci. 2010;51(13):2790.
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In wet AMD, leaky capillaries invade the subretinal space, exposing the apical (subretinal) side of the retinal pigment epithelium (RPE) to serum. Subretinal serum may alter the permeability and selectivity of the RPE tight junctions that are essential for the outer blood-retinal barrier.
Human fetal RPE (hfRPE) was cultured on filters in a serum-containing growth medium until quiescent monolayers were established. Cultures were maintained in this medium or switched to a serum-free medium formulated for hfRPE (Gamm et al., IOVS 49:788, 2008). After the cells adapted, serum was added to the serum-free medium in the apical, basal, or both media chambers. Transepithelial electrical resistance (TER) was used to estimate the relative conductance of Na+and K+. Effects on claudins, which determine tight junction properties, were measured at the mRNA, protein, and localization levels using real-time RT-PCR, immunoblotting, and immunofluorescence, respectively. The expression of claudin mRNA was compared to native hfRPE.
TER of cells maintained in growth medium was ~1000 Ω-cm2 throughout the study, but the TER of cells in serum-free medium decreased to ~200 Ω-cm2. This decrease was reversible. With apical, but not basal, serum the TER increased 2-3 fold. Apical serum and base media altered ion selectivity of the tight junctions. The ratio of the conductance for Na+/K+ increased to 0.89 in serum-free medium from 0.39 in growth medium. Apical serum further increased the Na+/K+ ratio to 1.32. The expression of the most plentiful claudin mRNA, claudin 19, was unaffected but changes in the minor claudins did accompany changes in selectivity. Claudins 2, 10, and 15 increased with the shift to serum-free medium. Apical, but not basal, serum increased claudin 3 and 10 mRNA without affecting other claudins. The mRNA levels observed in native RPE were closest to those of serum-free medium. Immunoblotting and immunofluorescence revealed corresponding changes in protein expression.
hfRPE maintained in serum-free medium appear to have the most in vivo-like properties with respect to claudin expression and TER (Quinn and Miller, IOVS, 33:3513, 1992). Both apical serum and base medium affected the permeability and selectivity of the tight junctions. Apical serum appears to make RPE tight junctions less permeable, perhaps helping to retard the lateral spread of CNV.
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