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ACTIVATION OF CAVERNOUS TRANSFORMING GROWTH FACTOR-β1 AND SMAD SIGNALING PATHWAY AFFECTS ERECTILE DYSFUNCTION IN MEN WITH SPINAL CORD INJURY
THE JOURNAL OF UROLOGY®
Vol. 181, No. 4, Supplement, Monday, April 27, 2009
dephosphorylation by activating SM myosin light chain phosphatase, thus desensitizing the SM myocyte to Ca2+ and favoring relaxation. Our studies showed that in corpus cavernosum (CC) smooth muscle (CCSM) of Type 1 diabetic (T1D) patients with erectile dysfunction (ED) telokin expression is significantly down regulated. Further objectives were to determine 1) if telokin and the calcium sensitization related Rho-kinase (ROK) pathway are coordinately regulated and 2) if in vivo overexpression of telokin in rats with T1D-induced ED could restore erectile function (EF) . METHODS: 1) Human samples were obtained from T1D and nondiabetic patients during penile implant surgery and immediately frozen 2) Telokin was amplified from human CC cDNA and cloned into the pVAX1 vector. The construct pVAX1/telokin or a pVAX1 control were then transfected into rat A7r5 and A10 SM cell lines and cells harvested 48 hours later 3) For in vivo studies, 2 month streptozotocin-induced diabetic male Sprague-Dawley rats with ED were injected in the CC with 100 μg pVAX1/ telokin (n=5) or a pVAX1 control (n=3) and EF assessed via cavernous nerve stimulation-induced increase in intracavernous pressure (ICP) one week post-injection. Total RNA and protein extracts were prepared and mRNA and protein expression levels of molecular targets were quantitated by Real Time PCR and Western Blot, respectively. RESULTS: 1) Telokin expression was decreased ~40% in CC of T1D compared to nondiabetic men with ED. 2) Telokin both in vitro and in vivo down regulated expression of the ROKA and ROKB isoforms at mRNA and protein levels by 30-50%. Telokin overexpression in vivo restored ICP levels in STZ-induced diabetic rats from levels ~50% decreased to near control levels across all stimulations. CONCLUSIONS: We demonstrate for the first time that telokin is downregulated in T1D men with ED compared to nondiabetic men and that telokin gene therapy can nearly completely restore EF in T1D diabetic rats. Further, we identify a novel coordinated regulation between calcium sensitization and desensitization pathways in CCSM. Taken together, our results suggest telokin modulation as a novel strategy to treat ED. Source of Funding: NIH R01 DK077116
671 POTENTIAL ROLE FOR NESTIN, A NEURAL STEM CELL MARKER, IN THE PATHOPHYSIOLOGY OF INJURY AND REPAIR OF THE CAVERNOUS NERVE Trinity J Bivalacqua*, Travis D Strong, Edward M Schaeffer, Christian P Pavlovich, Hunter C Champion, Arthur L Burnett, Baltimore, MD INTRODUCTION AND OBJECTIVE: One of the major complications after radical prostatectomy is the development of neurogenic erectile dysfunction (ED) secondary to injury of the neurovascular bundle. The purpose of the present study was to: 1) Use genomic expression profiling (Microarray Analysis) to identify candidate’s genes which are involved in injury and repair of the cavernous nerve after injury, and 2) evaluate the change in expression of the intermediate filament protein Nestin which is a known neural stem cell maker. METHODS: Two groups of rats were utilized: 1) sham-operated, and 2) BCNI (crush of cavernous nerve bilaterally). The major pelvic ganglion (MPG) was isolated and removed 48 hrs and 14 days after BCNI. Once the MPG was isolated, Haematoxylin and Eosin stain, protein and RNA were isolated. Agilent 44K whole rat genome array was performed to evaluate genomic changes in the MPG after nerve injury and compared to MPG of sham at 48 hrs and 14 days. Nestin protein (Western Blot), immunolocalization (Confocal microscopy), and RNA (quantitative PCR) expression was determined 48 hrs after injury and compared to sham MPG. RESULTS: Microarray analysis identified >2,000 genes which were differentially induced and repressed at 48 hrs and 14 days postBCNI. In the present study, our focus was in evaluating genes involved in neurotrophism. The neural stem cell marker, Nestin was increased 4 fold at 48 hrs after BCNI. There was no change at 14 days. Immunolocalization demonstrated increased expression in the ganglion cells of the MPG and in the injured cavernous nerve at 48hrs. We found significantly increased (P<0.05) protein and RNA expression in the MPG at 48 hrs after BCNI when compared to sham-operated control MPG.
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CONCLUSIONS: Nestin is widely used as a predominant marker for stem/progenitor cells. In the present study we show early (48 hrs) increased expression of Nestin in the MPG and cavernous nerve after nerve injury. There was no change in expression at 14 days after BCNI. These data suggest that Nestin may be involved in early injury repair and differentiation of the cavernous nerves after nerve injury. Source of Funding: AUA Foundation
672 EFFECTS OF NH02D ON EJACULATION LATENCY AND SEXUAL BEHAVIOR IN RATS Lesley Marson*, Chapel Hill, NC; Neal M Farber, Newton, MA INTRODUCTION AND OBJECTIVE: Modafinil (diphenylmethyl sulphinyl-2-acetamide), first identified as a novel wake-promoting agent for the treatment of narcolepsy, is a racemate of two stereoisomers with different pk properties. During clinical testing of NH02D (the shorteracting d-modafinil isomer) for an unrelated indication, an unexpected significant delay in ejaculatory time during sexual activity was reported. The aim of the current study was to investigate the effect of NH02D in a rat model of male sexual behavior. METHODS: The efficacy of NH02D was tested in groups [total N=25] of sexually experienced male Sprague Dawley rats exposed to sexually receptive females. The behavioral trials lasted 50 min, during which the timing and number of mounts, intromissions, ejaculations, and post ejaculatory refractory period (time from ejaculation to the next intromission) were recorded. All animals were sexually experienced (Sura etal 2001). In two series of experiments, rats were treated with NH02D (dose range tested 10-300 mg/kg, p.o. and i.p.) or vehicle (0.25% methylcellulose in saline) administered 15-20 min before the behavioral test using a crossover design. RESULTS: Oral administration of NH02D produced a dose dependant increase in the latency to ejaculation (LEJN, time from the 1st intromission to ejaculation) [p <0.05] and an increase in the number of intromissions and attempted mounts by the male. This response was most pronounced in the 1st ejaculatory series with 30-100mg/kg doses, with a tendency to reduce ejaculation latency in subsequent ejaculatory series. When animals with baseline ejaculation latencies of less than 9 min were analyzed, the effect of NH02D to delay ejaculation was more pronounced [LEJN for vehicle 5.1±0.5 min; for 30mg/kg dose 11.2±1.2 min, (p<0.001, compared to vehicle) and 100mg/kg 9.0±1.4 min (p<0.05, compared to vehicle), mean± SE]. IP administration did not produce any significant changes compared to vehicle, presumably because of lack of absorption due to a solubility problem. CONCLUSIONS: These results demonstrate that acute oral administration of NH02D can delay latency to ejaculation in rats without reducing the mounting frequency or number of intromissions. The greatest delay in ejaculation was observed in animals with shorter baseline ejaculatory latencies. No adverse reactions from NH02D treatment were observed. Source of Funding: NeuroHealing Pharmaceuticals
673 ACTIVATION OF CAVERNOUS TRANSFORMING GROWTH FACTOR-B1 AND SMAD SIGNALING PATHWAY AFFECTS ERECTILE DYSFUNCTION IN MEN WITH SPINAL CORD INJURY Tae-Young Shin*, Shuguang Piao, Munkhbayar Tumurbaatar, Lu Wei Zhang, Sun Hwa Shin, Buyankhuu Tuvshintur, Sang-Min Yoon, Jee-Young Han, Woo Jean Kim, Ji-Kan Ryu, Jun-Kyu Suh, Incheon, Republic of Korea INTRODUCTION AND OBJECTIVE: Transforming growth factor-B1 (TGF-B1)-Smad2/3 pathway is known to inhibit proliferation of smooth muscle and endothelial cells, and to induce apoptosis of these cells. However, it is as yet unknown whether the altered TGF-B1-Smad2/3 pathway is involved in the pathogenesis of spinal cord injury (SCI)-induced erectile dysfunction (ED). In this study, we investigated whether and how
THE JOURNAL OF UROLOGY®
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Vol. 181, No. 4, Supplement, Monday, April 27, 2009
TGF-B1-Smad pathway is involved in the pathogenesis of ED from SCI. METHODS: After obtaining informed consent and approval from patients and IRB, we enrolled five patients with psychogenic ED (mean age, 36.8 ± 9.6 yr) and 18 patients with neurogenic ED from SCI (mean age, 41 ± 8.8 yr; mean duration of injury, 12.1 ± 6.3 mo; range, 3-21 mo) for this study. Cavernous tissues were obtained by percutaneous biopsy and stained with Masson trichrome, or antibody to TGF-B1 and phosphoSmad2. Quantitative analysis of collagen in cavernous tissue was done with an image analyzer and semi-quantitative analysis was performed to evaluate the intensity of TGF-B1 and phospho-Smad2 staining (0, absent; 1, weak; 2, moderate; 3, strong). We also performed TUNEL assay and evaluated apoptotic index. RESULTS: Erectile function domain score of IIEF was lower in SCI-induced ED patients than in psychogenic ED patients (*P < 0.01). The expression of TGF-B1 and phospho-Smad2 protein was significantly increased in SCI-induced ED patients compared with that in psychogenic ED patients (*P < 0.01). TUNEL assay revealed a higher apoptotic index in SCI-induced ED patients than in psychogenic ED patients (*P < 0.01). Compared with that in psychogenic ED patients, higher TGF-B1 and phospho-Smad2 expression, and numerous apoptotic cells were noted mainly in smooth muscle cells, endothelial cells, and fibroblasts of SCIinduced ED patients. Cavernous collagen content was also higher in SCI-induced ED patients than in psychogenic ED patients (†P < 0.05). CONCLUSIONS: Up-regulation of cavernous TGF-B1 and Smad signaling pathways might play important roles in SCI-induced cavernous fibrosis and deterioration of erectile function, which validates early pharmacological intervention to protect erectile tissue from irreversible damage. Semiquantitative analysis of immunoreactivity and apoptotic index (mean ± SD). TGF-B1 Phospho-Smad2 Apoptotic index (%) Collagen content (%)
Psychogenic ED 0.98±0.43 1.23±0.36 17.7±10.6 16.3±5.8
SCI-induced ED 2.18±0.51* 2.54±0.59* 58.7±23.4* 23.1±14.3†
Source of Funding: This study was supported by the Korea Science and Engineering Foundation (KOSEF) through the National Research Lab. Program funded by the Ministry of Science and Technology (No. R0A-2007-000-20018-0, JunKyu Suh).
674 VCSA1-INDUCED EXPERIMENTAL PRIAPISM IN RATS IS ACCOMPANIED BY ACTIVATION OF ARGININE CATABOLISM AND POLYAMINE SYNTHESIS. Nirmala D Kanika*, Moses Tar, Yuehong Tong, Dwaraka S Kuppam, Arnold Melman, Kelvin P Davies, Bronx, NY INTRODUCTION AND OBJECTIVE: The molecular mechanisms involved in priapism are not well characterized. Potentially a better understanding of the underlying molecular mechanism will identify therapeutic targets for treatment. We have previously reported that the overexpression of genes encoding opiorphins (Vcsa1, ProL1 and hSMR3A/B) in the corpora of retired breeder rats will result in a priapic-like condition. To gain insight into the molecular mechanism by which opiorphin expression might induce a priapic-like condition in the rat, we determined molecular changes that occur following intracorporal gene transfer Vcsa1. METHODS: Gene transfer studies were carried out by intracorporal injection of retired breeder Sprague-Dawley rats with 1000 μg pVAX-Vcsa1 plasmid. Control animals were treated with plasmids which do not result in the priapic-like condition (pVAX, pVAX-hSlo). Global gene changes in priapism were analyzed by microarray analysis, quantitative RT-PCR and Western blot analysis. The erectile function of corporal tissues were determined histologically, visually and by measuring intracorporal/ blood pressure (ICP/BP). RESULTS: Intracorporal gene transfer of pVAX-Vcsa1 resulted in a priapic-like condition Microarray analysis of gene expression indicated a significant up-regulation of the ornithine decarboxylase (ODC) gene in these animals compared to controls. We confirmed not only the up-
regulation of the ODC gene but also other genes involved in polyamine synthetic pathway, such as arginase, polyamine oxidase, spermidine synthase, spermidine acetyltransferase (SAT) and S-adenosylmethionine decarboxylase. Western blot analysis also showed the upregulation of ODC and SAT at the protein level. The polyamine spermidine levels were up-regulated in pVAX-Vcsa1 treated animals compared to controls. We hypothesized that the up-regulation of polyamine synthesis in corpora tissue may contribute to the priapic-like condition. This hypothesis was supported by the observation that the ODC inhibitor (1,3-Diaminopropane) when added to the drinking water of animals treated with pVAX-Vcsa1 can prevent the priapic-like condition. CONCLUSIONS: Our results suggest that the up-regulation of ODC, a key enzyme in the polyamine synthetic pathway, may play a role in the development of Vcsa1-induced experimental priapism. It indicates that ODC inhibitors, or other interventions targeting the polyamine synthetic pathway, maybe useful in preventing priapic-like pathologies. Source of Funding: NIH/NIDDK
675 CALORIC RESTRICTION INCREASES PENILE AND INTERNAL ILIAC ARTER ENDOTHELIAL NITRIC OXIDE SYNTHASE AND NEURONAL AND ENDOTHELIAL NITRIC OXIDE SYNTHASE ACTIVITIES IN RAT CAVERNOUSAL TISSUES Emin Ozbek*, Yusuf O Ilbey, Abdulmuttalip Simsek, Adnan Somay, Istanbul, Turkey INTRODUCTION AND OBJECTIVE: Moderate diatery or caloric restriction (CR) in epidemiologic and animal studies suggest a positive link between cardiovascular system and long-term health outcomes. Because of positive corelation between healthy cardiovascular system and sexual life we aimed to evaluate the effect of CR on endothelial and neuronal nitric oxide synthase (e NOS, n NOS) activities in cavernousal tissues and e NOS activity in the internal iliac artery in young and aged rats. METHODS: Young (3 mo, n=8) and aged (24 mo, n=8) male Sprague-Dawley rats were subjected to 40% CR and were allowed free access to water for 3 mounths. Control rats (n=8) fed ad libitum had free access to food and water at all times. On day 90, all rats were sacrified and internal iliac arteries and penises were removed and parafinized. e NOS, n NOS expression in tissues evaluated with immunohistochemistry using spesific antibodies. Results were evaluated semiquantitatively. RESULTS: e NOS, n NOS expression in cavernousal tissue in CR rats were more strong than control group in both young and old rats. e NOS expression was also higher in the internal iliac arteries of CR rats than control in young and old rats. CONCLUSIONS: As a result of our study we can say that there is a positive link between CR and neurotransmitter of erection in cavernousal tissues and internal iliac arteries. CR not only improve cardiovascular function and decrease occurence of age-associated diseases, but also has beneficial effect on sexual life in young and old animals and possible humans. Source of Funding: None
676 PENILE MORPHOLOGY, SONIC HEDGEHOG SIGNALING AND TESTOSTERONE Christopher W. Bond, Carol A Podlasek*, Chicago, IL INTRODUCTION AND OBJECTIVE: 30-87% of patients treated by radical prostatectomy experience erectile dysfunction (ED) and current treatments are only partially effective in this patient population. Abundant apoptosis observed in penile smooth muscle is a major contributing factor to ED development. A critical regulator of penile smooth muscle and apoptosis is Sonic hedgehog (SHH). SHH signaling is decreased in ED models and SHH treatment of cavernous nerve (CN) injured rats can prevent smooth muscle apoptosis. How SHH signaling is regulated in the penis remains unknown. A close association between androgen deficiency and ED has been suggested in the literature however few studies have examined the
Vol. 181, No. 4, Supplement, Monday, April 27, 2009
dephosphorylation by activating SM myosin light chain phosphatase, thus desensitizing the SM myocyte to Ca2+ and favoring relaxation. Our studies showed that in corpus cavernosum (CC) smooth muscle (CCSM) of Type 1 diabetic (T1D) patients with erectile dysfunction (ED) telokin expression is significantly down regulated. Further objectives were to determine 1) if telokin and the calcium sensitization related Rho-kinase (ROK) pathway are coordinately regulated and 2) if in vivo overexpression of telokin in rats with T1D-induced ED could restore erectile function (EF) . METHODS: 1) Human samples were obtained from T1D and nondiabetic patients during penile implant surgery and immediately frozen 2) Telokin was amplified from human CC cDNA and cloned into the pVAX1 vector. The construct pVAX1/telokin or a pVAX1 control were then transfected into rat A7r5 and A10 SM cell lines and cells harvested 48 hours later 3) For in vivo studies, 2 month streptozotocin-induced diabetic male Sprague-Dawley rats with ED were injected in the CC with 100 μg pVAX1/ telokin (n=5) or a pVAX1 control (n=3) and EF assessed via cavernous nerve stimulation-induced increase in intracavernous pressure (ICP) one week post-injection. Total RNA and protein extracts were prepared and mRNA and protein expression levels of molecular targets were quantitated by Real Time PCR and Western Blot, respectively. RESULTS: 1) Telokin expression was decreased ~40% in CC of T1D compared to nondiabetic men with ED. 2) Telokin both in vitro and in vivo down regulated expression of the ROKA and ROKB isoforms at mRNA and protein levels by 30-50%. Telokin overexpression in vivo restored ICP levels in STZ-induced diabetic rats from levels ~50% decreased to near control levels across all stimulations. CONCLUSIONS: We demonstrate for the first time that telokin is downregulated in T1D men with ED compared to nondiabetic men and that telokin gene therapy can nearly completely restore EF in T1D diabetic rats. Further, we identify a novel coordinated regulation between calcium sensitization and desensitization pathways in CCSM. Taken together, our results suggest telokin modulation as a novel strategy to treat ED. Source of Funding: NIH R01 DK077116
671 POTENTIAL ROLE FOR NESTIN, A NEURAL STEM CELL MARKER, IN THE PATHOPHYSIOLOGY OF INJURY AND REPAIR OF THE CAVERNOUS NERVE Trinity J Bivalacqua*, Travis D Strong, Edward M Schaeffer, Christian P Pavlovich, Hunter C Champion, Arthur L Burnett, Baltimore, MD INTRODUCTION AND OBJECTIVE: One of the major complications after radical prostatectomy is the development of neurogenic erectile dysfunction (ED) secondary to injury of the neurovascular bundle. The purpose of the present study was to: 1) Use genomic expression profiling (Microarray Analysis) to identify candidate’s genes which are involved in injury and repair of the cavernous nerve after injury, and 2) evaluate the change in expression of the intermediate filament protein Nestin which is a known neural stem cell maker. METHODS: Two groups of rats were utilized: 1) sham-operated, and 2) BCNI (crush of cavernous nerve bilaterally). The major pelvic ganglion (MPG) was isolated and removed 48 hrs and 14 days after BCNI. Once the MPG was isolated, Haematoxylin and Eosin stain, protein and RNA were isolated. Agilent 44K whole rat genome array was performed to evaluate genomic changes in the MPG after nerve injury and compared to MPG of sham at 48 hrs and 14 days. Nestin protein (Western Blot), immunolocalization (Confocal microscopy), and RNA (quantitative PCR) expression was determined 48 hrs after injury and compared to sham MPG. RESULTS: Microarray analysis identified >2,000 genes which were differentially induced and repressed at 48 hrs and 14 days postBCNI. In the present study, our focus was in evaluating genes involved in neurotrophism. The neural stem cell marker, Nestin was increased 4 fold at 48 hrs after BCNI. There was no change at 14 days. Immunolocalization demonstrated increased expression in the ganglion cells of the MPG and in the injured cavernous nerve at 48hrs. We found significantly increased (P<0.05) protein and RNA expression in the MPG at 48 hrs after BCNI when compared to sham-operated control MPG.
241
CONCLUSIONS: Nestin is widely used as a predominant marker for stem/progenitor cells. In the present study we show early (48 hrs) increased expression of Nestin in the MPG and cavernous nerve after nerve injury. There was no change in expression at 14 days after BCNI. These data suggest that Nestin may be involved in early injury repair and differentiation of the cavernous nerves after nerve injury. Source of Funding: AUA Foundation
672 EFFECTS OF NH02D ON EJACULATION LATENCY AND SEXUAL BEHAVIOR IN RATS Lesley Marson*, Chapel Hill, NC; Neal M Farber, Newton, MA INTRODUCTION AND OBJECTIVE: Modafinil (diphenylmethyl sulphinyl-2-acetamide), first identified as a novel wake-promoting agent for the treatment of narcolepsy, is a racemate of two stereoisomers with different pk properties. During clinical testing of NH02D (the shorteracting d-modafinil isomer) for an unrelated indication, an unexpected significant delay in ejaculatory time during sexual activity was reported. The aim of the current study was to investigate the effect of NH02D in a rat model of male sexual behavior. METHODS: The efficacy of NH02D was tested in groups [total N=25] of sexually experienced male Sprague Dawley rats exposed to sexually receptive females. The behavioral trials lasted 50 min, during which the timing and number of mounts, intromissions, ejaculations, and post ejaculatory refractory period (time from ejaculation to the next intromission) were recorded. All animals were sexually experienced (Sura etal 2001). In two series of experiments, rats were treated with NH02D (dose range tested 10-300 mg/kg, p.o. and i.p.) or vehicle (0.25% methylcellulose in saline) administered 15-20 min before the behavioral test using a crossover design. RESULTS: Oral administration of NH02D produced a dose dependant increase in the latency to ejaculation (LEJN, time from the 1st intromission to ejaculation) [p <0.05] and an increase in the number of intromissions and attempted mounts by the male. This response was most pronounced in the 1st ejaculatory series with 30-100mg/kg doses, with a tendency to reduce ejaculation latency in subsequent ejaculatory series. When animals with baseline ejaculation latencies of less than 9 min were analyzed, the effect of NH02D to delay ejaculation was more pronounced [LEJN for vehicle 5.1±0.5 min; for 30mg/kg dose 11.2±1.2 min, (p<0.001, compared to vehicle) and 100mg/kg 9.0±1.4 min (p<0.05, compared to vehicle), mean± SE]. IP administration did not produce any significant changes compared to vehicle, presumably because of lack of absorption due to a solubility problem. CONCLUSIONS: These results demonstrate that acute oral administration of NH02D can delay latency to ejaculation in rats without reducing the mounting frequency or number of intromissions. The greatest delay in ejaculation was observed in animals with shorter baseline ejaculatory latencies. No adverse reactions from NH02D treatment were observed. Source of Funding: NeuroHealing Pharmaceuticals
673 ACTIVATION OF CAVERNOUS TRANSFORMING GROWTH FACTOR-B1 AND SMAD SIGNALING PATHWAY AFFECTS ERECTILE DYSFUNCTION IN MEN WITH SPINAL CORD INJURY Tae-Young Shin*, Shuguang Piao, Munkhbayar Tumurbaatar, Lu Wei Zhang, Sun Hwa Shin, Buyankhuu Tuvshintur, Sang-Min Yoon, Jee-Young Han, Woo Jean Kim, Ji-Kan Ryu, Jun-Kyu Suh, Incheon, Republic of Korea INTRODUCTION AND OBJECTIVE: Transforming growth factor-B1 (TGF-B1)-Smad2/3 pathway is known to inhibit proliferation of smooth muscle and endothelial cells, and to induce apoptosis of these cells. However, it is as yet unknown whether the altered TGF-B1-Smad2/3 pathway is involved in the pathogenesis of spinal cord injury (SCI)-induced erectile dysfunction (ED). In this study, we investigated whether and how
THE JOURNAL OF UROLOGY®
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Vol. 181, No. 4, Supplement, Monday, April 27, 2009
TGF-B1-Smad pathway is involved in the pathogenesis of ED from SCI. METHODS: After obtaining informed consent and approval from patients and IRB, we enrolled five patients with psychogenic ED (mean age, 36.8 ± 9.6 yr) and 18 patients with neurogenic ED from SCI (mean age, 41 ± 8.8 yr; mean duration of injury, 12.1 ± 6.3 mo; range, 3-21 mo) for this study. Cavernous tissues were obtained by percutaneous biopsy and stained with Masson trichrome, or antibody to TGF-B1 and phosphoSmad2. Quantitative analysis of collagen in cavernous tissue was done with an image analyzer and semi-quantitative analysis was performed to evaluate the intensity of TGF-B1 and phospho-Smad2 staining (0, absent; 1, weak; 2, moderate; 3, strong). We also performed TUNEL assay and evaluated apoptotic index. RESULTS: Erectile function domain score of IIEF was lower in SCI-induced ED patients than in psychogenic ED patients (*P < 0.01). The expression of TGF-B1 and phospho-Smad2 protein was significantly increased in SCI-induced ED patients compared with that in psychogenic ED patients (*P < 0.01). TUNEL assay revealed a higher apoptotic index in SCI-induced ED patients than in psychogenic ED patients (*P < 0.01). Compared with that in psychogenic ED patients, higher TGF-B1 and phospho-Smad2 expression, and numerous apoptotic cells were noted mainly in smooth muscle cells, endothelial cells, and fibroblasts of SCIinduced ED patients. Cavernous collagen content was also higher in SCI-induced ED patients than in psychogenic ED patients (†P < 0.05). CONCLUSIONS: Up-regulation of cavernous TGF-B1 and Smad signaling pathways might play important roles in SCI-induced cavernous fibrosis and deterioration of erectile function, which validates early pharmacological intervention to protect erectile tissue from irreversible damage. Semiquantitative analysis of immunoreactivity and apoptotic index (mean ± SD). TGF-B1 Phospho-Smad2 Apoptotic index (%) Collagen content (%)
Psychogenic ED 0.98±0.43 1.23±0.36 17.7±10.6 16.3±5.8
SCI-induced ED 2.18±0.51* 2.54±0.59* 58.7±23.4* 23.1±14.3†
Source of Funding: This study was supported by the Korea Science and Engineering Foundation (KOSEF) through the National Research Lab. Program funded by the Ministry of Science and Technology (No. R0A-2007-000-20018-0, JunKyu Suh).
674 VCSA1-INDUCED EXPERIMENTAL PRIAPISM IN RATS IS ACCOMPANIED BY ACTIVATION OF ARGININE CATABOLISM AND POLYAMINE SYNTHESIS. Nirmala D Kanika*, Moses Tar, Yuehong Tong, Dwaraka S Kuppam, Arnold Melman, Kelvin P Davies, Bronx, NY INTRODUCTION AND OBJECTIVE: The molecular mechanisms involved in priapism are not well characterized. Potentially a better understanding of the underlying molecular mechanism will identify therapeutic targets for treatment. We have previously reported that the overexpression of genes encoding opiorphins (Vcsa1, ProL1 and hSMR3A/B) in the corpora of retired breeder rats will result in a priapic-like condition. To gain insight into the molecular mechanism by which opiorphin expression might induce a priapic-like condition in the rat, we determined molecular changes that occur following intracorporal gene transfer Vcsa1. METHODS: Gene transfer studies were carried out by intracorporal injection of retired breeder Sprague-Dawley rats with 1000 μg pVAX-Vcsa1 plasmid. Control animals were treated with plasmids which do not result in the priapic-like condition (pVAX, pVAX-hSlo). Global gene changes in priapism were analyzed by microarray analysis, quantitative RT-PCR and Western blot analysis. The erectile function of corporal tissues were determined histologically, visually and by measuring intracorporal/ blood pressure (ICP/BP). RESULTS: Intracorporal gene transfer of pVAX-Vcsa1 resulted in a priapic-like condition Microarray analysis of gene expression indicated a significant up-regulation of the ornithine decarboxylase (ODC) gene in these animals compared to controls. We confirmed not only the up-
regulation of the ODC gene but also other genes involved in polyamine synthetic pathway, such as arginase, polyamine oxidase, spermidine synthase, spermidine acetyltransferase (SAT) and S-adenosylmethionine decarboxylase. Western blot analysis also showed the upregulation of ODC and SAT at the protein level. The polyamine spermidine levels were up-regulated in pVAX-Vcsa1 treated animals compared to controls. We hypothesized that the up-regulation of polyamine synthesis in corpora tissue may contribute to the priapic-like condition. This hypothesis was supported by the observation that the ODC inhibitor (1,3-Diaminopropane) when added to the drinking water of animals treated with pVAX-Vcsa1 can prevent the priapic-like condition. CONCLUSIONS: Our results suggest that the up-regulation of ODC, a key enzyme in the polyamine synthetic pathway, may play a role in the development of Vcsa1-induced experimental priapism. It indicates that ODC inhibitors, or other interventions targeting the polyamine synthetic pathway, maybe useful in preventing priapic-like pathologies. Source of Funding: NIH/NIDDK
675 CALORIC RESTRICTION INCREASES PENILE AND INTERNAL ILIAC ARTER ENDOTHELIAL NITRIC OXIDE SYNTHASE AND NEURONAL AND ENDOTHELIAL NITRIC OXIDE SYNTHASE ACTIVITIES IN RAT CAVERNOUSAL TISSUES Emin Ozbek*, Yusuf O Ilbey, Abdulmuttalip Simsek, Adnan Somay, Istanbul, Turkey INTRODUCTION AND OBJECTIVE: Moderate diatery or caloric restriction (CR) in epidemiologic and animal studies suggest a positive link between cardiovascular system and long-term health outcomes. Because of positive corelation between healthy cardiovascular system and sexual life we aimed to evaluate the effect of CR on endothelial and neuronal nitric oxide synthase (e NOS, n NOS) activities in cavernousal tissues and e NOS activity in the internal iliac artery in young and aged rats. METHODS: Young (3 mo, n=8) and aged (24 mo, n=8) male Sprague-Dawley rats were subjected to 40% CR and were allowed free access to water for 3 mounths. Control rats (n=8) fed ad libitum had free access to food and water at all times. On day 90, all rats were sacrified and internal iliac arteries and penises were removed and parafinized. e NOS, n NOS expression in tissues evaluated with immunohistochemistry using spesific antibodies. Results were evaluated semiquantitatively. RESULTS: e NOS, n NOS expression in cavernousal tissue in CR rats were more strong than control group in both young and old rats. e NOS expression was also higher in the internal iliac arteries of CR rats than control in young and old rats. CONCLUSIONS: As a result of our study we can say that there is a positive link between CR and neurotransmitter of erection in cavernousal tissues and internal iliac arteries. CR not only improve cardiovascular function and decrease occurence of age-associated diseases, but also has beneficial effect on sexual life in young and old animals and possible humans. Source of Funding: None
676 PENILE MORPHOLOGY, SONIC HEDGEHOG SIGNALING AND TESTOSTERONE Christopher W. Bond, Carol A Podlasek*, Chicago, IL INTRODUCTION AND OBJECTIVE: 30-87% of patients treated by radical prostatectomy experience erectile dysfunction (ED) and current treatments are only partially effective in this patient population. Abundant apoptosis observed in penile smooth muscle is a major contributing factor to ED development. A critical regulator of penile smooth muscle and apoptosis is Sonic hedgehog (SHH). SHH signaling is decreased in ED models and SHH treatment of cavernous nerve (CN) injured rats can prevent smooth muscle apoptosis. How SHH signaling is regulated in the penis remains unknown. A close association between androgen deficiency and ED has been suggested in the literature however few studies have examined the