Viral Vector Effects on Exoenzyme C3 Transferase–Mediated Actin Disruption and on Outflow Facility

Slauson, Sarah R.; Peters, Donna M.; Schwinn, Marie K.; Kaufman, Paul L.; Gabelt, B’Ann T.; Brandt, Curtis R.

doi:10.1167/iovs.14-15909

Cited by 12 publications

(10 citation statements)

References 27 publications

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“…Consistent with the IOP lowering effect of Rho kinase inhibitors, lowering of IOP in rodent eyes has been achieved by inhibition of Rho GTPase activity, either via expression of recombinant C3 exoenzyme which inhibits RhoA through ADP-ribosylation, or expression of dominant negative RhoA in the anterior chamber of rodent eyes (Borras et al, 2015; Liu et al, 2005; Slauson et al, 2015). Similarly, reduction of RhoA activity in the AH outflow pathway using shRNA lowers IOP in rodents (Liu et al, 2012).…”

Section: Role Of Rho/rho Kinase Signaling In the Conventional Ah Omentioning

confidence: 98%

“…In addition to these studies of Rho kinase inhibition, inhibition of Rho GTPase activity in TM cells has been studied using dominant negative forms of RhoA (Borras et al, 2015; Vittitow et al, 2002), direct inhibitors of RhoA including C3 exoenzyme (Liu et al, 2005; Pattabiraman and Rao, 2010; Slauson et al, 2015), and inhibitors of protein isoprenylation, e.g. statins and geranylgeranyl transferase inhibitors-GGTIs (Pervan et al, 2015; Rao et al, 2008; Song et al, 2005; Von Zee et al, 2009).…”

Section: Role Of Rho/rho Kinase Signaling In the Conventional Ah Omentioning

confidence: 99%

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Role of the Rho GTPase/Rho kinase signaling pathway in pathogenesis and treatment of glaucoma: Bench to bedside research

Rao

Pattabiraman

Kopczynski

2017

Experimental Eye Research

142

166

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Glaucoma is a leading cause of irreversible blindness worldwide. Elevated intraocular pressure (IOP) is considered to be a predominant risk factor for primary open angle glaucoma, the most prevalent form of glaucoma. Although the etiological mechanisms responsible for increased IOP are not completely clear, impairment in aqueous humor (AH) drainage through the conventional or trabecular pathway is recognized to be a primary cause in glaucoma patients. Importantly, lowering of IOP has been demonstrated to reduce progression of vision loss and is a mainstay of treatment for all types of glaucoma. Currently however, there are limited therapeutic options available for lowering IOP especially as it relates to enhancement of AH outflow through the trabecular pathway. Towards addressing this challenge, bench and bedside research conducted over the course of the last decade and a half has identified the significance of inhibiting Rho kinase for lowering IOP. Rho kinase is a downstream effector of Rho GTPase signaling that regulates actomyosin dynamics in numerous cell types. Studies from several laboratories have demonstrated that inhibition of Rho kinase lowers IOP via relaxation of the trabecular meshwork which enhances AH outflow. By contrast, activation of Rho GTPase/Rho kinase signaling in the trabecular outflow pathway increases IOP by altering the contractile, cell adhesive and permeability barrier characteristics of the trabecular meshwork and Schlemm’s canal tissues, and by influencing extracellular matrix production and fibrotic activity. This article, written in honor of the late David Epstein, MD, summarizes findings from both basic and clinical studies that have been instrumental for recognition of the importance of the Rho/Rho kinase signaling pathway in regulation of AH outflow, and in the development of Rho kinase inhibitors as promising IOP- lowering agents for glaucoma treatment.

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Section: Role Of Rho/rho Kinase Signaling In the Conventional Ah Omentioning

confidence: 98%

Section: Role Of Rho/rho Kinase Signaling In the Conventional Ah Omentioning

confidence: 99%

Role of the Rho GTPase/Rho kinase signaling pathway in pathogenesis and treatment of glaucoma: Bench to bedside research

Rao

Pattabiraman

Kopczynski

2017

Experimental Eye Research

142

166

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“…Packaging of the FIV vector was carried out as we have described previously. 49 Briefly, the FIV-based vectors, pCDF1-MSC2-EF1-copGFP or pCDF1-CMV-mCherry--EF1-copGFP vector (1.74 mg; System Biosciences, Mountain View, CA, USA) were cotransfected into HEK293TN cells with pFIV-34-N (326 μg) and pVSVG (408 μg) in eight 500-cm 2 culture plates (no. 431110; Corning, Corning, NY, USA).…”

Section: Methodsmentioning

confidence: 99%

Proteasome Inhibition Increases the Efficiency of Lentiviral Vector-Mediated Transduction of Trabecular Meshwork

Aktaş

Rao

Slauson

et al. 2018

Invest. Ophthalmol. Vis. Sci.

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PurposeTo determine if proteasome inhibition using MG132 increased the efficiency of FIV vector–mediated transduction in human trabecular meshwork (TM)-1 cells and monkey organ-cultured anterior segments (MOCAS).MethodsTM-1 cells were pretreated for 1 hour with 0.5% dimethyl sulfoxide (DMSO; vehicle control) or 5 to 50 μM MG132 and transduced with FIV.GFP (green fluorescent protein)– or FIV.mCherry-expressing vector at a multiplicity of transduction (MOT) of 20. At 24 hours, cells were fixed and stained with antibodies for GFP, and positive cells were counted, manually or by fluorescence-activated cell sorting (FACS). Cells transduced with FIV.GFP particles alone were used as controls. The effect of 20 μM MG132 treatment on high- and low-dose (2 × 107 and 0.8 × 107 transducing units [TU], respectively) FIV.GFP transduction with or without MG132 was also evaluated in MOCAS using fluorescence microscopy. Vector genome equivalents in cells and tissues were quantified by quantitative (q)PCR on DNA.ResultsIn the MG132 treatment groups, there was a significant dose-dependent increase in the percentage of transduced cells at all concentrations tested. Vector genome equivalents were also increased in TM-1 cells treated with MG132. Increased FIV.GFP expression in the TM was also observed in MOCAS treated with 20 μM MG132 and the high dose of vector. Vector genome equivalents were also significantly increased in the MOCAS tissues. Increased transduction was not seen with the low dose of virus.ConclusionsProteasome inhibition increased the transduction efficiency of FIV particles in TM-1 cells and MOCAS and may be a useful adjunct for delivery of therapeutic genes to the TM by lentiviral vectors.

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“…However, an appreciable ADP-ribosylation of RhoA was detected already after few hours. In the majority of reports, the morphological changes are viewed as a clear indicator of a successful cellular uptake of C3 (Chardin et al 1989;Wiegers et al 1991;Just et al 1992;Miura et al 1993;Krska et al 2014;Slauson et al 2015). Since morphological changes were detectable not before 12-24 h (depending on the cell type), the majority of cell lines were classified as relatively insensitive to C3 (leading to the above-mentioned diverse delivery techniques of C3).…”

Section: Uptake Of C3 Exoenzyme Into Cellsmentioning

confidence: 99%

Cell Entry of C3 Exoenzyme from Clostridium botulinum

Rohrbeck

Just

2016

Current Topics in Microbiology and Immunology

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Clostridium botulinum C3 is the prototype of C3-like ADP-ribosyltransferases that selectively ADP-ribosylate the small GTP-binding proteins RhoA/B/C and inhibit their downstream signaling pathways. It is used as pharmacological tool to study cellular Rho functions. In addition, C3bot harbors a transferase-independent activity on neurons to promote axonal and dendritic growth and branching. Many bacterial protein toxins interact specifically with proteins and/or other membrane components at the surface of target cells. Binding enables access to the appropriate cellular compartment so that the knowledge of the receptor allows essential insight into the mechanism of these toxins. Unlike other bacterial protein toxins (such as the clostridial C1 and C2 toxins from C. botulinum), C3 exoenzyme is devoid of a binding and translocation domain, with which toxins usually initiate receptor-mediated endocytosis followed by access to the intact cell. To date, no specific mechanism for internalization of C3 exoenzyme has been identified. Recently, vimentin was identified as membranous C3-binding partner involved in binding and uptake of C3. Although vimentin is not detected in neurons, vimentin is re-expressed after damage in regenerating neurons. Reappearance of vimentin allows C3 to get access to lesioned neurons/axons to exhibit axonotrophic and dentritotrophic effects.

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Viral Vector Effects on Exoenzyme C3 Transferase–Mediated Actin Disruption and on Outflow Facility

Cited by 12 publications

References 27 publications

Role of the Rho GTPase/Rho kinase signaling pathway in pathogenesis and treatment of glaucoma: Bench to bedside research

Role of the Rho GTPase/Rho kinase signaling pathway in pathogenesis and treatment of glaucoma: Bench to bedside research

Proteasome Inhibition Increases the Efficiency of Lentiviral Vector-Mediated Transduction of Trabecular Meshwork

Cell Entry of C3 Exoenzyme from Clostridium botulinum

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