Sarah R Slauson1, Donna M Peters2, Marie K Schwinn3, Paul L Kaufman4, B'Ann T Gabelt1, Curtis R Brandt4. 1. Department of Ophthalmology and Visual Sciences, University of Wisconsin, Madison, Wisconsin, United States. 2. Department of Ophthalmology and Visual Sciences, University of Wisconsin, Madison, Wisconsin, United States 2Department of Pathology and Laboratory Medicine, University of Wisconsin, Madison, Wisconsin, United States 3McPherson Eye Research Institute, Uni. 3. Department of Pathology and Laboratory Medicine, University of Wisconsin, Madison, Wisconsin, United States. 4. Department of Ophthalmology and Visual Sciences, University of Wisconsin, Madison, Wisconsin, United States 3McPherson Eye Research Institute, University of Wisconsin, Madison, Wisconsin, United States.
Abstract
PURPOSE: Purified Clostridium botulinum exoenzyme C3 transferase (C3) effects on the actin cytoskeleton in human trabecular meshwork cells (HTM) and on the outflow facility response in monkey organ-cultured anterior segments (MOCAS) were determined in the presence or absence of viral vectors. METHODS: Human adenovirus type 5 (AdV) and feline immunodeficiency virus (FIV) vectors were produced using kits. Cell soluble purified C3 (C3cs) was purchased commercially. Recombinant C3 (C3rec) cDNA was overexpressed in Escherichia coli and purified. The HTM cells were incubated with up to 10 μg/mL C3cs or with 5 μg of C3rec and/or viral vector (multiplicity of infection [MOI] = 25). Cells then were fixed and stained for actin. Outflow facility in MOCAS was measured at baseline, 4 hours, 24 hours, and 3 to 4 days following bolus injection of AdV (1.6 × 107 transducing units) and/or 2.5 μg C3rec. RESULTS: The HTM cells treated for 4 hours with C3cs (all doses) or for 24 hours with C3rec developed a rounded morphology and lost stress fibers. Cells transduced with vectors alone showed no changes at any time point. Cells exposed to C3rec and cotransduced with either viral vector showed significant disruption of the actin cytoskeleton within 4 hours after exposure, which persisted at 24 hours. In MOCAS, the AdV vector alone had no effect on outflow facility, but enhanced the response to C3rec at 4 hours. CONCLUSIONS: Coadministration of viral vectors enhances the ability of C3 transferase to disrupt actin stress fiber formation in HTM cells and increase outflow facility in MOCAS. Viral vectors potentially could be used to increase the bioavailability of proteins for cells that are difficult to transfect.
PURPOSE: Purified Clostridium botulinum exoenzyme C3 transferase (C3) effects on the actin cytoskeleton in human trabecular meshwork cells (HTM) and on the outflow facility response in monkey organ-cultured anterior segments (MOCAS) were determined in the presence or absence of viral vectors. METHODS:Human adenovirus type 5 (AdV) and feline immunodeficiency virus (FIV) vectors were produced using kits. Cell soluble purified C3 (C3cs) was purchased commercially. Recombinant C3 (C3rec) cDNA was overexpressed in Escherichia coli and purified. The HTM cells were incubated with up to 10 μg/mL C3cs or with 5 μg of C3rec and/or viral vector (multiplicity of infection [MOI] = 25). Cells then were fixed and stained for actin. Outflow facility in MOCAS was measured at baseline, 4 hours, 24 hours, and 3 to 4 days following bolus injection of AdV (1.6 × 107 transducing units) and/or 2.5 μg C3rec. RESULTS: The HTM cells treated for 4 hours with C3cs (all doses) or for 24 hours with C3rec developed a rounded morphology and lost stress fibers. Cells transduced with vectors alone showed no changes at any time point. Cells exposed to C3rec and cotransduced with either viral vector showed significant disruption of the actin cytoskeleton within 4 hours after exposure, which persisted at 24 hours. In MOCAS, the AdV vector alone had no effect on outflow facility, but enhanced the response to C3rec at 4 hours. CONCLUSIONS: Coadministration of viral vectors enhances the ability of C3 transferase to disrupt actin stress fiber formation in HTM cells and increase outflow facility in MOCAS. Viral vectors potentially could be used to increase the bioavailability of proteins for cells that are difficult to transfect.
Authors: William W MacDonald; Swarup S Swaminathan; Jae Young Heo; Alexandra Castillejos; Jessica Hsueh; Brian J Liu; Diane Jo; Annie Du; Hyunpil Lee; Min Hyung Kang; Douglas J Rhee Journal: Invest Ophthalmol Vis Sci Date: 2022-06-01 Impact factor: 4.925
Authors: Zeynep Aktas; Hongyu Rao; Sarah R Slauson; B'Ann T Gabelt; Inna V Larsen; Rachael T C Sheridan; Leonie Herrnberger; Ernst R Tamm; Paul L Kaufman; Curtis R Brandt Journal: Invest Ophthalmol Vis Sci Date: 2018-01-01 Impact factor: 4.799