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X. Liu, M.S. Filla, C.R. Brandt, D.P. Peters, P.L. Kaufman; EXOENZYME C3 TRANSFERASE GENE TRANSFER TO CULTURED HUMAN TRABECULAR MESHWORK CELLS AND CILIARY MUSCLE CELLS––TOWARDS GLAUCOMA GENE THERAPY . Invest. Ophthalmol. Vis. Sci. 2004;45(13):4563.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To determine the effects of adenovirus–mediated exoenzyme C3 transferase (C3) gene expression on cultured human trabecular meshwork (HTM) and ciliary muscle (HCM) cells. Methods: An adenovirus vector was constructed which expressed C3 under the control of a human promoter. In vitro studies were performed using HTM and HCM cells infected with C3–expressing adenovirus and the changes in morphology, actin, vinculin and ß–catenin were detected. Cells treated with medium (untreated cells) and virus vector only were used as controls. Results: Treatment of both HTM and HCM cells with C3–expressing adenovirus resulted in dose–dependent morphological changes 4 days post–infection. C3–treated cells were either partially retracted, rounded up completely or very elongated and attenuated in appearance compared to untreated cells. Compared to virus–treated control cells, which demonstrated prominent stress fibers, C3–treated cells demonstrated either a disrupted or absent actin cytoskeleton. C3–treated cells of both types demonstrated reduced numbers of vinculin–positive focal adhesions compared to controls. In C3–treated HTM cells, vinculin staining at cell–cell junctions was also partially reduced, and there was a near complete loss of ß–catenin staining even in cells that still exhibited an intact actin cytoskeleton, suggesting that cell–cell junctions may be more sensitive to C3 transferase than actin and cell–matrix contacts. Cells treated with the negative control construct did not round up or retract; however some cells appeared somewhat elongated and irregular compared to untreated cells. Conclusions: Transduced C3 is effective in disrupting actin and cellular adhesions in HTM and HCM cells, resembling the effects of the IOP–reducing reagents H–7 and Lat–A on the cells, indicating that over–expressing the C3 gene may provide an effective approach for glaucoma treatment.
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