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Y. Hu, B. T. Gabelt, D. M. Peters, P. L. Kaufman; Effects of Noladin Ether (NE) on the Actin Cytoskeleton, Focal Adhesions and Cell-Cell Junctions in Human Trabecular Meshwork (HTM) Cells and Outflow Facility (OF) in Monkey Organ-Cultured Anterior Segments (MOCAS). Invest. Ophthalmol. Vis. Sci. 2008;49(13):677.
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To determine whether NE alters the actin cytoskeleton, focal adhesions and cell-cell junctions in HTM cells and OF in MOCAS.
Proliferating HTM cells were treated with 0, 12.5, 25, 50 or 100 µM NE for 24 h or with 100 µM for 1, 3, 6 or 24 h. 7-day differentiated HTM cells were also treated with 100 µM NE for 20 h. Cells were fixed and labeled with phalloidin, anti-paxillin, anti-vinculin or anti-β catenin antibody. The changes in the actin cytoskeleton, focal adhesions and cell-cell junctions were determined using fluorescence microscopy. For OF measurements, 3 pairs of rhesus or 3 pairs of baboon monkey anterior segments were exchanged with DMEM media containing 1 (n=1), 50 (n=4), 100 (n=1) or 200 (n=3) µM NE. 3 of 6 pairs of anterior segments were treated with both 50 and 200 µM NE. The contralateral anterior segments were exchanged with DMSO vehicle. Anterior segments were continuously infused with NE or vehicle at 2.5 µl/min overnight. OF was measured by two-level constant pressure perfusion at baseline prior to treatment and after overnight treatment.
100 µM NE caused proliferating HTM cells to round up and spread less after 3 h. The actin cytoskeleton showed shorter stress fibers and focal adhesions were decreased. However, in 7-day differentiated HTM cells, 100 µM NE did not cause any changes in the actin cytoskeleton, focal adhesions or cell-cell junctions. OF prior to treatment was 0.47±0.04 for all eyes combined (n=12). OF was not significantly changed by overnight treatment with various concentrations of NE.
Although NE can alter the actin cytoskeleton and focal adhesions in proliferating HTM cells, it had no effect on differentiated HTM cells in culture. NE also did not affect OF in MOCAS. This indicates that proliferating TM cells may not be the best model in all situations to study therapeutics for glaucoma in vitro.
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