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Elisabeth Obert, Gautam Ghatnekar, Robert Gourdie, Baerbel Rohrer; Mechanistic Insight into the Effects of the αCT1 Peptide on RPE Cell Integrity in Models of AMD. Invest. Ophthalmol. Vis. Sci. 2017;58(8):3383. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
A critical target tissue in age-related macular degeneration (AMD) is the retinal pigment epithelium (RPE), which forms the outer blood-retina barrier (BRB). RPE-barrier dysfunction might result from attenuation/disruption of intercellular tight junctions (TJs). Zonula occludens-1 (ZO-1) is a major structural protein of TJs. A connexin43 (Cx43)-based peptide mimetic, αCT1 was developed to competitively block interactions at the PDZ2 domain of ZO-1, thereby inhibiting ligands that selectively bind to this domain. We hypothesized that targeting ZO-1 signaling using αCT1 would maintain BRB integrity and reduce RPE pathophysiology by stabilizing gap- and/or tight-junctions and reducing cytokine activity.
Transepithelial resistance (TER) experiments on ARPE-19 (express Cx43) and MDCK (express negligible levels of Cx43) monolayers were employed to study the integrity of TJs. In mice, choroidal neovascularization (CNV) was induced using laser-photocoagulation; this wet AMD-like model is known to lead to VEGF-dependent loss of cell junctions as well as increased cytokine production. Animals were injected with anti-VEGF antibody in addition to αCT1 eye drops to measure a potential synergistic effect on CNV development. A cytokine array assay determined the relative quantity of cytokines and chemokines in αCT1-treated animals compared to vehicles three and six days after CNV induction.
Barrier dysfunction was induced by VEGF or calcium chelation (EGTA) in ARPE-19 and MDCK cells, respectively. αCT1 ameliorated reduction in TER in both experimental paradigms. In mice, the VEGF and αCT1 co-treatment study revealed no synergistic effects on reducing CNV, but were equally effective on their own. Furthermore, decreased cytokine activity was measured in αCT1-treated animals after induction of laser-photocoagulation compared to non-treated animals.
In vitro experiments using RPE and MDCK monolayers indicated that αCT1 stabilizes TJs, independent of its effects on Cx43. In vivo experiments in mouse also suggest that αCT1 stabilizes epithelial monolayers by inhibiting the VEGF-dependent breakdown of TJs, but in addition, reduces an inflammatory immune response. Targeting TJ stability may serve as a promising new treatment paradigm in retinal diseases in which the RPE barrier is affected, either as monotherapy, or in combination with existing therapeutics.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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