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Mei Chin Lee, Boon Tin Chua, Shuang Ru Goh, Yaan Fun Chong, Tin Aung, Walter Hunziker, Eranga Nishanthie Vithana; Cellular barrier integrity in multicellular non-pigmented ciliary epithelial spheroids with modulated levels of Pleckstrin homology-like domain family member 7 (PLEKHA7) gene.. Invest. Ophthalmol. Vis. Sci. 2016;57(12):6002.
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© ARVO (1962-2015); The Authors (2016-present)
1. To understand the functional role of primary angle closure glaucoma (PACG) susceptibility gene, PLEKHA7, for the maintenance of the blood aqueous barrier (BAB).2. To establish a cellular model of multicellular non-pigmented ciliary epithelial cells (NPCE) spheroids, for functional characterization of PLEKHA7 molecular mechanisms and expression and recruitment of tight junction (TJ) and adherens junction (AJ) proteins.
Three-dimensional (3-D) multicellular NPCE spheroids (cultivated in suspension culture plates) and conventional two-dimensional (2-D) cultures (cultivated in fibronectin collagen coated plates) with modulated PLEKHA7 expression levels were established, via overexpression constructs or with PLEKHA7-specific shRNA constructs, and assessed thus. To ascertain the importance of PLEKHA7 for recruitment of TJ marker protein, Occludin; and AJ marker protein, b-catenin, confocal immunofluorescent analyses were performed. Barrier function was also assessed by indirect transepithelial electrical resistance (TEER) measurements on an xCELLigence real-time cell analysis platform.
3-D multicellular NPCE spheroids and 2-D conventional cultures with up-regulated levels of PLEKHA7 showed, an increase in the level of Occludin and b-catenin distribution at the cell-cell junctions and higher impedance readings, respectively. NPCE speheroids with depleted PLEKHA7 levels had lower Occludin and b-catenin protein recruitment to the cell-cell junctions whilst PLEKHA7 depleted NPCE cells in 2-D cultures gave a lower TEER impedance readings compared to the controls.
The 3-D confocal imaging data taken together with the 2-D impedance data indicates PLEKHA7 expression levels to be pivotal for the recruitment of apical junctional complexes, which are essential for maintenance of BAB at the NPCE cell layer. Our data also suggests the potential of these more physiologically relevant systems: The 3-D spheroids model of NPCE and conventional 2-D cultures, for the analysis of regulatory molecules may impact the barrier integrity studies of the BAB.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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