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IMPAIRED BLADDER FUNCTION IN A NEW MUTANT MOUSE MODEL WITH INCREASED LEVELS OF SUPEROXIDE
THE JOURNAL OF UROLOGY®
Vol. 181, No. 4, Supplement, Sunday, April 26, 2009
Bladder & Urethra: Anatomy, Physiology and Pharmacology (II) Moderated Poster 14 Sunday, April 26, 2009
1:00 pm - 3:00 pm
404 R11: A NOVEL CELL PERMEABLE PEPTIDE ALLOWING BLADDER-SPECIFIC TARGETING Jian Zhou*, Crystal Gore, Jer-Tsong Hsieh, Philippe E. Zimmern, Dallas, TX INTRODUCTION AND OBJECTIVE: Cell permeable peptides (CPPs) are short cationic peptides that can cross the plasma membrane efficiently and result in a rapid uptake by mammalian cells in vitro and in vivo (ref.1). We have shown that, in uro-genital organs, a polyarginine-R11 is the most efficient CPP over several other synthetic CPPs (TAT, PENE, KALA and K11) (ref 2). However, the glycosaminoglycan layer may represent an impassable barrier for R-11. With drug delivery in mind, we tested the uptake efficiency of R11 administered intra-vesically. METHODS: An in vivo model (athymic nude mouse) approved by Institution Animal Care and Usage Committee was employed. Synthetic R11 conjugated with fluorescein isothiocyanate (FITC) was tested at the concentrations of 1 nM and 5 nM. Controls received FITC without CPP conjugation. All animals were instilled with R11 for 30 minutes and sacrificed 24 hours later to harvest their bladder for the measurement of CPP uptake and tissue localization using OCT frozen section. Bladder uptake specificity was determined by averaging relative FITC intensity with each tissue weight; these data were then compared with other major organs. RESULTS: As shown in the histogram, although the uptake of R11 varied among animals, a 2 to 6 fold higher amount of R11 was detected in the bladder 24 hours after intra-vesical instillation at 1nM or 5 nM concentrations over the detection rate in each animal control group (n=4) or other organs. Tissue sections further confirmed the localization of R11 in the lamina propria of the bladder wall. In contrast to R11, other tested CPP’s exhibited poor bladder specificity (data not shown). CONCLUSIONS: Because of its high affinity for bladder, both systemically in prior studies (ref.2) and after intra-vesical instillation as reported herein, R11 can be further tested in this animal model as a delivery vector for therapeutic agents in bladder diseases. Ref 1: Science 1999, 285:1569; Trends in Biotech 2003, 21:498; Nat Med 2004,10:305. Ref 2: Cancer Res. 2006, 66:8954.
Source of Funding: DOD (W81XWH-08-1-0305)
405 IMPAIRED BLADDER FUNCTION IN A NEW MUTANT MOUSE MODEL WITH INCREASED LEVELS OF SUPEROXIDE Claudius Fullhase*, Roberto Soler, Baisong Lu, Winston-salem, NC; Christian Gratzke, Munich, Germany; Collin Bishop, Karl-Erik Andersson, Winston-salem, NC INTRODUCTION AND OBJECTIVE: Urethral smooth muscle relaxation is mediated by parasympathetic postganglionic released nitric oxide (NO). Increased outflow resistance due to deficient urethral
145
relaxation has been shown in male nNOS-deficient mice. In streptozotocininduced diabetic female rats a decreased NO responsiveness was associated with decreased urethral smooth muscle relaxation. Using transgenic insertional mutagenesis strategy, we generated a mouse mutation in the inner mitochondrial membrane peptidase 2-like (Immp2l) gene. This mutation leads to high superoxide ion levels with consequent decrease of NO levels and increase in reactive oxygen species. Recently erectile dysfunction, objectified i.n corpus cavernosum organ bath experiments, has been described for male Immp2l-mutants. We studied bladder function in vivo and in vitro in this model. METHODS: Homozygote mutation for the Immp2l gene was demonstrated by PCR and Western blot. 4-6 months old male mutants (n=5) and healthy controls (wildtypes; n=5) underwent bladder catheterization. Three days later urodynamic evaluation was performed in conscious animals, with continuous bladder filling. Non-operated mutants (n=5) and wildtypes (n=5) were sacrificed and bladders removed. Strips of smooth muscle were dissected and mounted in an oxygenated organ bath. Strips were exposed to pharmacological and electrical stimulation. RESULTS: Urodynamically, in vivo, the mutants showed significant lower micturition volumes (0.08±0.07 vs. 0.2±0.1ml, p<0.05) and higher residual volumes (0.17±0.03 vs. 0.04±0.03ml, p<0.001). Mutants had pronounced difficulties in initiating micturition, and massive straining was seen before voiding. This could be objectified in measuring the time between the first raise in bladder pressure till the maximal pressure during micturition (77.9±29.3 vs. 24.1±15.1sec, p=0.007). However, in vitro, no statistically significant differences could be shown in the response to carbachol or to electrical field stimulation. CONCLUSIONS: These results suggest that Immp2l mutant mice exhibit bladder dysfunction mainly characterized by emptying abnormalities, with a preserved detrusor function. This may be explained by a defect urethral relaxation secondary to a reduction in available NO. Since these are young animals, further evaluation of old animals will bring valuable information about chronic exposition to high superoxide and low NO levels. This makes these animals an interesting model to study diseases like diabetes and aging. Source of Funding: None
406 EVIDENCE THAT SONIC HEDGEHOG INDUCES BLADDER SMOOTH MUSCLE DEVELOPMENT Mei Cao*, Gerald R Cunha, Laurence S Baskin, San Francisco, CA INTRODUCTION AND OBJECTIVE: Background: We have proposed that the bladder smooth muscle (SM) inducing factor in urothelium is Sonic Hedgehog (Shh). This hypothesis is supported by the observation that Shh localizes to urothelium and a Shh secreting cell line induces SM development in bladder mesenchyme. The goal of this study is to confirm the hypothesis that Shh directly induces bladder SM differentiation. METHODS: Timed pregnant FVB mice were euthanized, and fetuses obtained on gestational day 12. In 12 day embryos (E) the bladder mesenchyme was separated from the urothelium with chemical and forceps dissection. Shh protein was diluted with DMEM High Glucose 50% F-12 50% plus insulin organ culture medium at 48nm and 480nm. After 72hrs in culture medium the explants were analyzed for SM differentiation, urothelial contamination and proliferation. Three different experimental protocols were performed: 1) E12 intact bladder + organ culture medium without Shh (positive control); 2) E12 bladder mesenchyme + organ culture medium without Shh (negative control); and 3) E12 bladder mesenchyme + organ culture medium with 480nm Shh. Each experiment was repeated in triplicate. RESULTS: Gross examination (Figure top row) showed that the specimens (group 3) treated with 480nm Shh were closer in size to the intact E12 bladders (group 1) than the bladder mesenchyme grown alone (group 2). Immunochemistry revealed that SM alpha-actin localized to the bladder mesenchyme in groups 1, and 3 (Figure 2 bottom row) with no expression in the bladder mesenchyme alone. Terminal markers of SM differentiation, SM myosin heavy chain, calponin, caldesmon and SM
Vol. 181, No. 4, Supplement, Sunday, April 26, 2009
Bladder & Urethra: Anatomy, Physiology and Pharmacology (II) Moderated Poster 14 Sunday, April 26, 2009
1:00 pm - 3:00 pm
404 R11: A NOVEL CELL PERMEABLE PEPTIDE ALLOWING BLADDER-SPECIFIC TARGETING Jian Zhou*, Crystal Gore, Jer-Tsong Hsieh, Philippe E. Zimmern, Dallas, TX INTRODUCTION AND OBJECTIVE: Cell permeable peptides (CPPs) are short cationic peptides that can cross the plasma membrane efficiently and result in a rapid uptake by mammalian cells in vitro and in vivo (ref.1). We have shown that, in uro-genital organs, a polyarginine-R11 is the most efficient CPP over several other synthetic CPPs (TAT, PENE, KALA and K11) (ref 2). However, the glycosaminoglycan layer may represent an impassable barrier for R-11. With drug delivery in mind, we tested the uptake efficiency of R11 administered intra-vesically. METHODS: An in vivo model (athymic nude mouse) approved by Institution Animal Care and Usage Committee was employed. Synthetic R11 conjugated with fluorescein isothiocyanate (FITC) was tested at the concentrations of 1 nM and 5 nM. Controls received FITC without CPP conjugation. All animals were instilled with R11 for 30 minutes and sacrificed 24 hours later to harvest their bladder for the measurement of CPP uptake and tissue localization using OCT frozen section. Bladder uptake specificity was determined by averaging relative FITC intensity with each tissue weight; these data were then compared with other major organs. RESULTS: As shown in the histogram, although the uptake of R11 varied among animals, a 2 to 6 fold higher amount of R11 was detected in the bladder 24 hours after intra-vesical instillation at 1nM or 5 nM concentrations over the detection rate in each animal control group (n=4) or other organs. Tissue sections further confirmed the localization of R11 in the lamina propria of the bladder wall. In contrast to R11, other tested CPP’s exhibited poor bladder specificity (data not shown). CONCLUSIONS: Because of its high affinity for bladder, both systemically in prior studies (ref.2) and after intra-vesical instillation as reported herein, R11 can be further tested in this animal model as a delivery vector for therapeutic agents in bladder diseases. Ref 1: Science 1999, 285:1569; Trends in Biotech 2003, 21:498; Nat Med 2004,10:305. Ref 2: Cancer Res. 2006, 66:8954.
Source of Funding: DOD (W81XWH-08-1-0305)
405 IMPAIRED BLADDER FUNCTION IN A NEW MUTANT MOUSE MODEL WITH INCREASED LEVELS OF SUPEROXIDE Claudius Fullhase*, Roberto Soler, Baisong Lu, Winston-salem, NC; Christian Gratzke, Munich, Germany; Collin Bishop, Karl-Erik Andersson, Winston-salem, NC INTRODUCTION AND OBJECTIVE: Urethral smooth muscle relaxation is mediated by parasympathetic postganglionic released nitric oxide (NO). Increased outflow resistance due to deficient urethral
145
relaxation has been shown in male nNOS-deficient mice. In streptozotocininduced diabetic female rats a decreased NO responsiveness was associated with decreased urethral smooth muscle relaxation. Using transgenic insertional mutagenesis strategy, we generated a mouse mutation in the inner mitochondrial membrane peptidase 2-like (Immp2l) gene. This mutation leads to high superoxide ion levels with consequent decrease of NO levels and increase in reactive oxygen species. Recently erectile dysfunction, objectified i.n corpus cavernosum organ bath experiments, has been described for male Immp2l-mutants. We studied bladder function in vivo and in vitro in this model. METHODS: Homozygote mutation for the Immp2l gene was demonstrated by PCR and Western blot. 4-6 months old male mutants (n=5) and healthy controls (wildtypes; n=5) underwent bladder catheterization. Three days later urodynamic evaluation was performed in conscious animals, with continuous bladder filling. Non-operated mutants (n=5) and wildtypes (n=5) were sacrificed and bladders removed. Strips of smooth muscle were dissected and mounted in an oxygenated organ bath. Strips were exposed to pharmacological and electrical stimulation. RESULTS: Urodynamically, in vivo, the mutants showed significant lower micturition volumes (0.08±0.07 vs. 0.2±0.1ml, p<0.05) and higher residual volumes (0.17±0.03 vs. 0.04±0.03ml, p<0.001). Mutants had pronounced difficulties in initiating micturition, and massive straining was seen before voiding. This could be objectified in measuring the time between the first raise in bladder pressure till the maximal pressure during micturition (77.9±29.3 vs. 24.1±15.1sec, p=0.007). However, in vitro, no statistically significant differences could be shown in the response to carbachol or to electrical field stimulation. CONCLUSIONS: These results suggest that Immp2l mutant mice exhibit bladder dysfunction mainly characterized by emptying abnormalities, with a preserved detrusor function. This may be explained by a defect urethral relaxation secondary to a reduction in available NO. Since these are young animals, further evaluation of old animals will bring valuable information about chronic exposition to high superoxide and low NO levels. This makes these animals an interesting model to study diseases like diabetes and aging. Source of Funding: None
406 EVIDENCE THAT SONIC HEDGEHOG INDUCES BLADDER SMOOTH MUSCLE DEVELOPMENT Mei Cao*, Gerald R Cunha, Laurence S Baskin, San Francisco, CA INTRODUCTION AND OBJECTIVE: Background: We have proposed that the bladder smooth muscle (SM) inducing factor in urothelium is Sonic Hedgehog (Shh). This hypothesis is supported by the observation that Shh localizes to urothelium and a Shh secreting cell line induces SM development in bladder mesenchyme. The goal of this study is to confirm the hypothesis that Shh directly induces bladder SM differentiation. METHODS: Timed pregnant FVB mice were euthanized, and fetuses obtained on gestational day 12. In 12 day embryos (E) the bladder mesenchyme was separated from the urothelium with chemical and forceps dissection. Shh protein was diluted with DMEM High Glucose 50% F-12 50% plus insulin organ culture medium at 48nm and 480nm. After 72hrs in culture medium the explants were analyzed for SM differentiation, urothelial contamination and proliferation. Three different experimental protocols were performed: 1) E12 intact bladder + organ culture medium without Shh (positive control); 2) E12 bladder mesenchyme + organ culture medium without Shh (negative control); and 3) E12 bladder mesenchyme + organ culture medium with 480nm Shh. Each experiment was repeated in triplicate. RESULTS: Gross examination (Figure top row) showed that the specimens (group 3) treated with 480nm Shh were closer in size to the intact E12 bladders (group 1) than the bladder mesenchyme grown alone (group 2). Immunochemistry revealed that SM alpha-actin localized to the bladder mesenchyme in groups 1, and 3 (Figure 2 bottom row) with no expression in the bladder mesenchyme alone. Terminal markers of SM differentiation, SM myosin heavy chain, calponin, caldesmon and SM