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S. L. Rudrabhatla, J. Cai, A. Baharani, D. Nguyen, M. E. Boulton; RPE Junctional Complex Proteins are Regulated via a Signaling Pathway Involving PEDF, VEGFR-1 and -Secretase. Invest. Ophthalmol. Vis. Sci. 2008;49(13):481.
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It has been previously demonstrated that VEGFR-1 is a critical regulator of VEGF-induced RPE permeability. The aim of this study was to determine if PEDF and γ-secretase, both of which have both been linked to VEGFR-1 regulation in other cell types, modulate the expression and integrity of junctional proteins in RPE cells.
ARPE-19 cells maintained at confluence for two weeks were treated with VEGFA, PEDF, PLGF and VEGFC (100ng/ml), or a combination thereof, in the presence and absence a γ-secretase inhibitor for up to 24hrs. Transepithelial resistance (TER) measurements were performed to determine changes in permeability. γ-secretase activity was determined by ELISA. Immuno-precipitation and Western blot were employed to determine the association between VEGFR-1 and components of the γ-secretase complex (i.e. presenilin and nicastrin). Immunocytochemistry was used to identify changes in ZO-1, occludin, cadherin and β-catenin localization and expression. PCR and Western blot were used to quantitate changes in expression of junctional proteins.
VEGFA and VEGFC, but none of the other angiogenic factors tested, caused a 2 fold reduction in TER and this could be blocked by PEDF. The inhibitory effect of PEDF on VEGFA-induced permeability was largely reversed in the presence of a γ-secretase inhibitor or neutralization of VEGFR-1. Addition of PEDF increased γ-secretase activity by 1.5 fold and this increase was abolished in the presence of a γ-secretase inhibitor. Immunoprecipitation demonstrated that both presenilin and nicastrin were associated with VEGFR-1 and that the degree of association was dependent on the VEGF:PEDF ratio. Immunocytochemistry demonstrated relocation of junctional complexes as permeability increased and a decrease in the expression of junctional proteins.
VEGF-induced RPE permeability is regulated by a complex association between γ-secretase, VEGFR-1 and PEDF. Modulation of this signaling pathway may be important in the prevention of retinal edema.
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