PURPOSE: To determine the effects of adenovirus-delivered exoenzyme C3 transferase (C3) gene expression on cultured human trabecular meshwork (HTM) cells and on outflow facility in organ cultured monkey anterior segments. METHODS: An adenoviral (Ad) vector expressing both C3 and green fluorescent protein (GFP) was used to transduce cultured HTM cells. Changes in cell morphology and the organization of actin, vinculin, and beta-catenin were assessed using immunofluorescence. Cultured monkey eye anterior segments were used to test the effects of AdC3GFP on outflow facility. RESULTS: Treatment of HTM cells with AdC3GFP resulted in dose-dependent morphological changes 3 or 4 days post-transduction. The AdC3GFP-transduced cells were either partially retracted, rounded, or very elongated compared to non-transduced cells. Compared to AdGFP-transduced cells, AdC3GFP-transduced cells demonstrated disrupted actin cytoskeleton, reduced vinculin-positive focal adhesions, and loss of beta-catenin staining. Cells transduced with AdGFP did not round up or retract. In organ culture studies, outflow facility was increased by 90+/-21% (n=15, p<0.001) in AdC3GFP-transduced eyes compared to baseline and corrected for AdGFP-transduced control eye washout on days 3-6 after transduction. CONCLUSIONS: C3 transduction is effective in disrupting actin filaments, cytoskeleton, and cellular adhesions in HTM cells and in increasing outflow facility in organ cultured monkey anterior segments, suggesting that expressing the C3 gene in the trabecular meshwork may be an effective approach for glaucoma therapy.
PURPOSE: To determine the effects of adenovirus-delivered exoenzyme C3 transferase (C3) gene expression on cultured human trabecular meshwork (HTM) cells and on outflow facility in organ cultured monkey anterior segments. METHODS: An adenoviral (Ad) vector expressing both C3 and green fluorescent protein (GFP) was used to transduce cultured HTM cells. Changes in cell morphology and the organization of actin, vinculin, and beta-catenin were assessed using immunofluorescence. Cultured monkey eye anterior segments were used to test the effects of AdC3GFP on outflow facility. RESULTS: Treatment of HTM cells with AdC3GFP resulted in dose-dependent morphological changes 3 or 4 days post-transduction. The AdC3GFP-transduced cells were either partially retracted, rounded, or very elongated compared to non-transduced cells. Compared to AdGFP-transduced cells, AdC3GFP-transduced cells demonstrated disrupted actin cytoskeleton, reduced vinculin-positive focal adhesions, and loss of beta-catenin staining. Cells transduced with AdGFP did not round up or retract. In organ culture studies, outflow facility was increased by 90+/-21% (n=15, p<0.001) in AdC3GFP-transduced eyes compared to baseline and corrected for AdGFP-transduced control eye washout on days 3-6 after transduction. CONCLUSIONS: C3 transduction is effective in disrupting actin filaments, cytoskeleton, and cellular adhesions in HTM cells and in increasing outflow facility in organ cultured monkey anterior segments, suggesting that expressing the C3 gene in the trabecular meshwork may be an effective approach for glaucoma therapy.
Authors: Michelle D Drewry; Pratap Challa; John G Kuchtey; Iris Navarro; Inas Helwa; Yanzhong Hu; Hongmei Mu; W Daniel Stamer; Rachel W Kuchtey; Yutao Liu Journal: Hum Mol Genet Date: 2018-04-01 Impact factor: 6.150
Authors: LaKisha K Buie; Carol A Rasmussen; Eric C Porterfield; Vinod S Ramgolam; Vivian W Choi; Silva Markovic-Plese; Richard J Samulski; Paul L Kaufman; Teresa Borrás Journal: Invest Ophthalmol Vis Sci Date: 2009-08-13 Impact factor: 4.799