IMPACT OF RHOGDI GENE TRANSFECTION OF BLADDER SMOOTH MUSCLE CONTRACTILITY IN A VALIDATED EX-VIVO MURINE MODEL
Title
IMPACT OF RHOGDI GENE TRANSFECTION OF BLADDER SMOOTH MUSCLE CONTRACTILITY IN A VALIDATED EX-VIVO MURINE MODEL
Creator
Joice G; Bell J M; La Favor J; Yoshida T; Torga G; Harris K; Liu X; Kiedrowski M; Penn M; Bivalacqua T
Publisher
Journal of Sexual Medicine
Date
2019
2019-04
Description
19th Annual Fall Scientific Meeting of SMSNA
Introduction Plasmid-based gene therapy is an intriguing option for treating malignant and bladder pathologies. The RhoA pathway is involved in bladder smooth muscle regulation, cancer invasion and metastasis. Rho GDP-dissociation inhibitor (RhoGDI) is an inhibitor of the RhoA pathway. We validated ex-vivo bladder gene transfer to facilitate assessment of gene targets for treating bladder pathology. Methods Basic Local Alignment Search Tool was used to identify human RhoGDI coding sequences and to compare between rats and humans, which were cloned into a eCMV-based expression vector. NBTII rat bladder cancer cell lines were transfected using FuGENE (Promega, USA) and human protein expression and interaction with endogenous RhoA were tested using flow cytometry, immunofluorescence, and RNA expression analysis. Bladders were harvested from female Lewis Rats (∼250g) and sectioned and cultured for 72-hours following transfection with RhoGDI and FuGENE. Transfected bladder tissues were analyzed as described above. Non-transfected cultured bladder segments were analyzed using myography for viability and intact smooth muscle physiology in response to 120 mM KCl and 30uM carbachol. Results Human and rodent RhoGDI protein homology is 96%. Human RhoGDI was successfully detected exclusively in transfected NBTII cells with a top efficiency of 26%. qPCR analysis demonstrated rodent RhoA and RhoGDI levels were not impacted but ROCK1 and ROCK2 mRNA were significantly decreased by 23.6% (p=0.034) and 40.0% (p=0.015), respectively following human RhoGDI transfection. Human RhoGDI was detected in transfected ex-vivo cultured bladder segments in both FuGENE and microinjection experiments. Similar to NBTII cells, qPCR analysis demonstrated rodent RhoA and RhoGDI levels were not impacted but ROCK1 and ROCK2 mRNA were significantly decreased by 15.0% (p=0.035) and 22.4% (p=0.010) after FuGENE transfection and 20.5% (p=0.024) and 21.4% (p=0.015) after microinjections. Ex-vivo cultured bladder strips successfully contracted to KCl (mean 0.88+/-0.48 mN/mg tissue) and carbachol (1.82+/-0.97 mN/mg tissue) stimulation. After microinjection, RhoGDI caused a significant reduction in KCl mediated constriction although this was not observed in FuGENE experiments. Conclusion Ex-vivo bladder culture, transfection, and physiological assessment are feasible and may provide a high-throughput method to test novel gene transfer technologies before in-vivo testing. RhoGDI plasmid microinjection transfection appears to decrease contractility of ex-vivo bladder smooth muscle.
Introduction Plasmid-based gene therapy is an intriguing option for treating malignant and bladder pathologies. The RhoA pathway is involved in bladder smooth muscle regulation, cancer invasion and metastasis. Rho GDP-dissociation inhibitor (RhoGDI) is an inhibitor of the RhoA pathway. We validated ex-vivo bladder gene transfer to facilitate assessment of gene targets for treating bladder pathology. Methods Basic Local Alignment Search Tool was used to identify human RhoGDI coding sequences and to compare between rats and humans, which were cloned into a eCMV-based expression vector. NBTII rat bladder cancer cell lines were transfected using FuGENE (Promega, USA) and human protein expression and interaction with endogenous RhoA were tested using flow cytometry, immunofluorescence, and RNA expression analysis. Bladders were harvested from female Lewis Rats (∼250g) and sectioned and cultured for 72-hours following transfection with RhoGDI and FuGENE. Transfected bladder tissues were analyzed as described above. Non-transfected cultured bladder segments were analyzed using myography for viability and intact smooth muscle physiology in response to 120 mM KCl and 30uM carbachol. Results Human and rodent RhoGDI protein homology is 96%. Human RhoGDI was successfully detected exclusively in transfected NBTII cells with a top efficiency of 26%. qPCR analysis demonstrated rodent RhoA and RhoGDI levels were not impacted but ROCK1 and ROCK2 mRNA were significantly decreased by 23.6% (p=0.034) and 40.0% (p=0.015), respectively following human RhoGDI transfection. Human RhoGDI was detected in transfected ex-vivo cultured bladder segments in both FuGENE and microinjection experiments. Similar to NBTII cells, qPCR analysis demonstrated rodent RhoA and RhoGDI levels were not impacted but ROCK1 and ROCK2 mRNA were significantly decreased by 15.0% (p=0.035) and 22.4% (p=0.010) after FuGENE transfection and 20.5% (p=0.024) and 21.4% (p=0.015) after microinjections. Ex-vivo cultured bladder strips successfully contracted to KCl (mean 0.88+/-0.48 mN/mg tissue) and carbachol (1.82+/-0.97 mN/mg tissue) stimulation. After microinjection, RhoGDI caused a significant reduction in KCl mediated constriction although this was not observed in FuGENE experiments. Conclusion Ex-vivo bladder culture, transfection, and physiological assessment are feasible and may provide a high-throughput method to test novel gene transfer technologies before in-vivo testing. RhoGDI plasmid microinjection transfection appears to decrease contractility of ex-vivo bladder smooth muscle.
Subject
Urology & Nephrology
Identifier
Rights
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URL Address
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Pages
S92–S92
Issue
4
Volume
16
ISSN
1743-6095
Citation
Joice G; Bell J M; La Favor J; Yoshida T; Torga G; Harris K; Liu X; Kiedrowski M; Penn M; Bivalacqua T, “IMPACT OF RHOGDI GENE TRANSFECTION OF BLADDER SMOOTH MUSCLE CONTRACTILITY IN A VALIDATED EX-VIVO MURINE MODEL,” NEOMED Bibliography Database, accessed April 19, 2024, https://neomed.omeka.net/items/show/6437.