MicroRNA-148a-3p in pericyte-derived extracellular vesicles improves erectile function in diabetic mice by promoting cavernous neurovascular regeneration.

BACKGROUND: To investigate the regulatory role of microRNA (miR)-148a-3p in mouse corpus cavernous pericyte (MCPs)-derived extracellular vesicles (EVs) in the treatment of diabetes-induced erectile dysfunction (ED). METHODS: Mouse corpus cavernous tissue was used for MCP primary culture and EV isolation. Small-RNA sequencing analysis was performed to assess the type and content of miRs in MCPs-EVs. Four groups of mice were used: control nondiabetic mice and streptozotocin-induced diabetic mice receiving two intracavernous injections (days - 3 and 0) of phosphate buffered saline, MCPs-EVs transfected with reagent control, or MCPs-EVs transfected with a miR-148a-3p inhibitor. miR-148a-3p function in MCPs-EVs was evaluated by tube-formation assay, migration assay, TUNEL assay, intracavernous pressure, immunofluorescence staining, and Western blotting. RESULTS: We extracted EVs from MCPs, and small-RNA sequencing analysis showed miR-148a-3p enrichment in MCPs-EVs. Exogenous MCPs-EV administration effectively promoted mouse cavernous endothelial cell (MCECs) tube formation, migration, and proliferation, and reduced MCECs apoptosis under high-glucose conditions. These effects were significantly attenuated in miR-148a-3p-depleted MCPs-EVs, which were extracted after inhibiting miR-148a-3p expression in MCPs. Repetitive intracavernous injections of MCPs-EVs improved erectile function by inducing cavernous neurovascular regeneration in diabetic mice. Using online bioinformatics databases and luciferase report assays, we predicted that pyruvate dehydrogenase kinase-4 (PDK4) is a potential target gene of miR-148a-3p. CONCLUSIONS: Our findings provide new and reliable evidence that miR-148a-3p in MCPs-EVs significantly enhances cavernous neurovascular regeneration by inhibiting PDK4 expression in diabetic mice.

RNA-sequencing profiling analysis of pericyte-derived extracellular vesicle-mimetic nanovesicles-regulated genes in primary cultured fibroblasts from normal and Peyronie's disease penile tunica albuginea.

BACKGROUND: Peyronie's disease (PD) is a severe fibrotic disease of the tunica albuginea that causes penis curvature and leads to penile pain, deformity, and erectile dysfunction. The role of pericytes in the pathogenesis of fibrosis has recently been determined. Extracellular vesicle (EV)-mimetic nanovesicles (NVs) have attracted attention regarding intercellular communication between cells in the field of fibrosis. However, the global gene expression of pericyte-derived EV-mimetic NVs (PC-NVs) in regulating fibrosis remains unknown. Here, we used RNA-sequencing technology to investigate the potential target genes regulated by PC-NVs in primary fibroblasts derived from human PD plaque. METHODS: Human primary fibroblasts derived from normal and PD patients was cultured and treated with cavernosum pericytes isolated extracellular vesicle (EV)-mimetic nanovesicles (NVs). A global gene expression RNA-sequencing assay was performed on normal fibroblasts, PD fibroblasts, and PD fibroblasts treated with PC-NVs. Reverse transcription polymerase chain reaction (RT-PCR) was used for sequencing data validation. RESULTS: A total of 4135 genes showed significantly differential expression in the normal fibroblasts, PD fibroblasts, and PD fibroblasts treated with PC-NVs. However, only 91 contra-regulated genes were detected among the three libraries. Furthermore, 20 contra-regulated genes were selected and 11 showed consistent changes in the RNA-sequencing assay, which were validated by RT-PCR. CONCLUSION: The gene expression profiling results suggested that these validated genes may be good targets for understanding potential mechanisms and conducting molecular studies into PD.

[Diagnosis and treatment of 12 cases of sexual activity-related hematuria: Experience and literature review].

OBJECTIVE: To summarize and analyze the experience in the diagnosis and treatment of sexual activity-related hematuria. METHODS: A retrospective analysis was conducted on 12 cases of sexual activity-related hematuria treated in Changhai Hospital from October 2015 to April 2019. The patients ranged in age between 31 and 59 years, with a disease course of 2 weeks to 25 years, 6 complaining of urethral bleeding at penile erection and another 6 hematuria immediately after ejaculation, including 2 accompanied by hemospermia. All the patients underwent urethroscopy and cauterization of the lesioned urethral mucosa with the electric excision ring or holmium laser. In addition, one of the patients received seminal tract endoscopic exploration and seminal vesicle irrigation, and another one seminal tract endoscopy and transurethral resection of the prostate. RESULTS: All the patients were diagnosed with posterior urethral varicosity, one accompanied with bulbar and posterior urethral varicosity, one with seminal vesiculitis, and still another with BPH. The patients were followed up for 3－45 (mean 23.5) months, during which the symptoms of sexual activity-related hematuria disappeared in 11 cases, with smooth urination and no recurrence, and post-ejaculation hematuria developed in one case at 2 and 10 months postoperatively but never again thereafter. No complications, such as epididymitis, urethral stricture and ED, were observed in any of the patients. CONCLUSIONS: Urethral varicosity should be first considered in patients with painless hematuria immediately after penile erection or sexual activity though other conditions such as seminal vesicle bleeding can also be taken into account. Urethroscopy combined with seminal tract endoscopy is effective in the diagnosis and treatment of sexual activity-related hematuria.

[Surgical treatment of azoospermia caused by iatrogenic injury to bilateral vas deferens].

OBJECTIVE: To investigate the treatment of azoospermia induced by iatrogenic injury to the bilateral vas deferens. METHODS: We retrospectively analyzed 11 cases of azoospermia caused by iatrogenic injury to bilateral vas deferens. The patients were aged 20－33 years, all diagnosed with azoospermia preoperatively and none with a history of pelvic operation. Seven of them had received bilateral inguinal hernia repair and the other 4 undergone bilateral orchidopexy in the childhood. RESULTS: Intraoperative exploration of the bilateral inguinal region was performed in all the patients. Bilateral vas deference atresia was found in the inguinal canal in 6 cases, which was treated by microscopic vasovasostomy following removal of the atresic segment. Vas deferens residual was observed in or near the deep inguinal ring in the other 5 cases, with the distal vas deferens inaccessible, which was treated by bilateral vasovasostomy in 3 cases and unilateral vasovasostomy in 2 (for longer defect segment than could be anastomosed) following combined laparoscopic exploration of the abdominal cavity. The patients were followed up for 3－12 months postoperatively, during which sperm were detected in 7 cases, with sperm concentration ranging from 0.4×106/ml to 35×106/ml and grade a+b sperm from 15% to 46%. CONCLUSIONS: For the diagnosis of azoospermia, especially in patients with no history of pelvic operation, special attention should be paid to iatrogenic injury to the vas deferens. For the treatment of the disease, non-tension vasovasostomy is essential and, when necessary, the vas deferens can be reconstructed by changing its anatomical path and shortening its length.

[Transurethral seminal vesiculoscopy for refractory or recurrent hemospermia: clinical analysis of 162 cases].

OBJECTIVE: To investigate the feasibility and effect of transurethral seminal vesiculoscopy in the diagnosis and treatment of refractory or recurrent hemospermia. METHODS: We retrospectively analyzed 162 cases of refractory or recurrent hemospermia examined and treated by transurethral seminal vesiculoscopy. The patients ranged in age from 19 to 76 years and had a hemospermia history of 3 months to 11 years, admitted due to poor therapeutic results or recurrence after 4 weeks of antibiotic medication. All the patients underwent serum PSA examination, transrectal ultrasonography, seminal vesicle ultrasonography and pelvis CT or MRI before surgery. RESULTS: Wine- or magenta-colored colloid and inflammation were found in one or both sides of the seminal vesicle in all the cases. Pathological biopsy revealed chronic inflammatory mucosa of the seminal vesicle in all the patients, and even calculi in the ejaculatory duct or seminal vesicle in 15 cases. Postoperative follow-up averaged 21.7 (12 -29) months. Hemospermia disappeared or was alleviated in 150 (92.64%) of the cases after 1-15 ejaculations, in which 7 experienced recurrence 3 months later. Four cases failed to respond, and 1 developed acute bilateral epididymitis after surgery. No such complications as retrograde ejaculation, urinary incontinence or rectal injury were observed postoperatively. CONCLUSION: Transurethral seminal vesiculoscopy is a safe, effective and feasible new method for the treatment of refractory or recrudescent hemospermia.

RNA sequencing profiles reveals progressively reduced spermatogenesis with progression in adult cryptorchidism.

Introduction: The fertility of cryptorchidism patients who didn't perform corrective surgery will decrease with age. Herein, we elucidate the histological alterations and underlying molecular mechanism in patients with an increase in the disease duration from 20 to 40 years. Methods: Testicular tissues were obtained from three patients with cryptorchidism, ranging in age from 22 to 44 years. Three benign paracancerous testicular samples of matched ages were used as controls. The normal and undescended testicular tissues were stained with hematoxylin and eosin (HE) and immunofluorescence and all six testicular samples were subjected to RNA sequencing. RNA sequencing data were subjected to gene set enrichment analysis (GSEA), Kyoto Encyclopedia of Genes and Genomes (KEGG), protein-protein interaction (PPI) network analysis, and Gene Ontology (GO) searches. Real-time reverse transcriptase polymerase chain reaction was used to confirm the DEGs. Results: The seminiferous tubules' basement membrane thickens with age in healthy testes. As the period of cryptorchidism in the cryptorchid testis extended, the seminiferous tubules significantly atrophy, the number of spermatogenic cells declines, and the amount of interstitial fibrous tissue increases in comparison to normal tissues. The number of germ cells per cross-section of seminiferous tubules was significantly lower in cryptorchidism than in normal testicular tissues, according to immunofluorescence staining, but the number of Sertoli cells remained stable. RNA sequencing analysis identified 1150 differentially expressed genes (DEGs) between cryptorchidism and normal testicular tissues (fold change >2 and p<0.05), of which 61 genes were noticeably upregulated and 1089 were significantly downregulated. These genes were predominantly linked to sperm development and differentiation, and fertilization, according to GO analysis. Meiosis pathways were significantly downregulated in cryptorchidism, according to KEGG pathway analysis and GSEA (P<0.001). PPI analysis was used to identify the top seven downregulated hub genes (PLCZ1, AKAP4, IZUMO1, SPAG6, CAPZA3, and ROPN1L), which were then further verified by qPCR. Discussion: By describing the histological changes and differential gene expression patterns in adult cryptorchid patients of different age groups, we discovered the progression mechanisms of undescended testes in adults with aging and identified seven significantly downregulated hub genes (PLCZ1, AKAP4, IZUMO1, SPAG6, CAPZA3, and ROPN1L) in cryptorchid testis compared to normal testicular tissues. These genes played a role in the process of spermgenesis and are directly linked to the steady decline in fertility caused by cryptorchidism. Our study provided a better understanding of the molecular mechanisms underlying the loss of spermatogenesis in adult cryptorchidism, and give support for the development of adult cryptorchidism treatments.

Frenulum Protection Technique in Disposable Circumcision Suture Device for Adult Males.

PURPOSE: To evaluate the efficacy of frenulum protection technique of the disposable circumcision suture device (DCSD) in adult males. MATERIALS AND METHODS: Atotal of 53 adult males were diagnosed with redundant prepuce and underwent circumcision with DCSD using frenulum protection technique. The main preoperative and postoperative measure of the length of penile frenulum was evaluated. Other data such as edema rate, intraoperative blood loss, operation time, postoperative pain, staple falling off time, incision infection rate, and evaluation of satisfaction rate with penis appearance were documented in the study. RESULTS: There was no significant difference in preoperative and postoperative frenulum length for each patient. The mean length of the penile frenulum before and after surgery was 2.25 ± 0.36 cm and 2.23 ± 0.39 cm, respectively (p = .31). The rate of frenulum length preservation was 100%. All the patients had no excessive resection of the frenulum and no serious complication happened after surgery. The satisfaction rate of postoperative penis appearance from patients' evaluation was 98.1% (52/53). CONCLUSION: The frenulum protection technique was simple and operable, which could help the operator to accurately identify the most distal position of the frenulum and retain a sufficient length of frenulum during DCSD circumcision.

Intracavernous delivery of freshly isolated stromal vascular fraction rescues erectile function by enhancing endothelial regeneration in the streptozotocin-induced diabetic mouse.

INTRODUCTION: Men with diabetic erectile dysfunction (ED) often have severe endothelial dysfunction and respond poorly to oral phosphodiesterase-5 inhibitors. AIM: To examine whether and how freshly isolated stromal vascular fraction (SVF) promotes cavernous endothelial regeneration and restores erectile function in diabetic animals. METHODS: Eight-week-old C57BL/6J mice were used. Diabetes was induced by intraperitoneal injection of streptozotocin. SVF was isolated from epididymal adipose tissues of green fluorescence protein transgenic mice. At 8 weeks after the induction of diabetes, the animals were divided into six groups: controls, diabetic mice, and diabetic mice treated with a single intracavernous injection of phosphate-buffered saline (PBS) or SVF (1 × 10(4) cells, 1 × 10(5) cells, or 2 × 10(5) cells/20 µL, respectively). MAIN OUTCOME MEASURES: Two weeks later, erectile function was measured by cavernous nerve stimulation. The penis was stained with antibodies to CD31, CD34, phosphohistone H3, phospho-endothelial nitric oxide synthase (eNOS), and vascular endothelial growth factor-A (VEGF-A). We also performed Western blot for phospho-eNOS and eNOS, and determined cyclic guanosine monophosphate (cGMP) concentration in the corpus cavernosum tissue. RESULTS: Significant improvement in erectile function was noted in diabetic mice treated with SVF at concentrations of 1 × 10(5) and 2 × 10(5) cells, which reached up to 82% of the control values. Local delivery of SVF significantly increased cavernous endothelial cell proliferation, eNOS phosphorylation, and cGMP expression compared with that in the untreated group and the PBS-treated diabetic group. Intracavernous injection of SVF increased cavernous VEGF-A expression and induced recruitment of CD34(+)CD31(-) progenitor cells. Some SVF underwent differentiation into cavernous endothelial cells. SVF-induced promotion of cavernous angiogenesis and erectile function was abolished in the presence of VEGF-Trap, a soluble VEGF-A neutralizing antibody. CONCLUSION: The results support the concept of cavernous endothelial regeneration by use of SVF as a curative therapy for diabetic ED.

Increased cavernous expression of transforming growth factor-ß1 and activation of the Smad signaling pathway affects erectile dysfunction in men with spinal cord injury.

INTRODUCTION: Transforming growth factor-ß1 (TGF-ß1) is implicated in bladder fibrosis after spinal cord injury (SCI) and in the fibrosis in the corpus cavernosum tissue after cavernous nerve injury. AIM: We investigated the differential expression of TGF-ß1 and the Smad transcription factor, the key molecule for the initiation of TGF-ß-mediated fibrosis, in cavernous tissue from SCI patients. METHODS: After obtaining informed consent and approval from the patients and our institutional review board, we enrolled 5 patients with psychogenic erectile dysfunction (ED) (mean age 36.8 years; range 20-50 years) and 10 patients with neurogenic ED from SCI (mean age 38.8 years; range 18-50 years). Cavernous tissues were obtained by percutaneous biopsy and stained with Masson trichrome, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL), or antibodies to TGF-ß1 and phospho-Smad2. MAIN OUTCOME MEASURES: Semi-quantitative analysis of TGF-ß1 and phospho-Smad2 was performed, and the numbers of apoptotic cells were counted. We also quantified the cavernous collagen area with the use of an image analyzer system. RESULTS: The expression of TGF-ß1 and phospho-Smad2 protein was significantly higher in the SCI group than in the psychogenic group. The TUNEL assay revealed a higher apoptotic index in the SCI group than in the psychogenic group. Higher TGF-ß1 and phospho-Smad2 expression and more apoptotic cells were noted mainly in endothelial cells, smooth muscle cells, and fibroblasts of the SCI group. Double labeling of cavernous tissue with TUNEL and antibody to phospho-Smad2 revealed that most TUNEL-positive cells showed immunoreactivity to phospho-Smad2 staining. Cavernous collagen content was significantly greater in the SCI group than in the psychogenic group. CONCLUSION: Upregulation of TGF-ß1 and activation of the Smad signaling pathway may play important roles in SCI-induced cavernous fibrosis and deterioration of erectile function, which warrants early pharmacological intervention to protect erectile tissue from irreversible damage.

[Efficacy and safety of long-term small-dose tadalafil in the treatment of erectile dysfunction].

OBJECTIVE: Erectile dysfunction (ED) is now recognized as a comorbid condition, especially in men with cardiovascular disease or diabetes mellitus. This randomized controlled trial was to examine the effect of long-term small-dose tadalafil in the treatment of ED. METHODS: A total of 98 men older than 18 years with at least a 6-month ED history were enlisted and divided into two groups to receive once-daily treatment with tadalafil at 5 mg (n = 60) and 20 mg (n = 38), respectively, for 12 months. The effects of medication were analyzed and compared using IIEF, Global Assessment Questionnaire (GAQ) and Sexual Encounter Profile (SEP), and so were the safety and tolerance of the two doses. RESULTS: There were no statistically significant differences in the therapeutical results between the 5 mg and 20 mg groups (P < 0.05). The IIEF-5 score was raised by 8.1 points in the former and 7.9 points in the latter; the YES answers to SEP2 in the two groups were 51.3% and 49.2% before the treatment and 82.6% and 84.9% after it. No serious adverse events were observed, except some common ones, such as rubeosis (11.9% vs 8.7%) and headache (5.3% vs 4.9%) in the 5 mg and 20 mg groups. CONCLUSION: Oral tadalafil at 5 mg once daily is efficacious with good tolerance in the treatment of ED, and it can be an alternative to on-demand medication for some men to eliminate the inconvenience of planned intercourse within a limited timeframe.

A mouse model of cavernous nerve injury-induced erectile dysfunction: functional and morphological characterization of the corpus cavernosum.

INTRODUCTION: With the advent of genetically engineered mice, it seems important to develop a mouse model of cavernous nerve injury (CNI). AIM: To establish a mouse model of CNI induced either by nerve crushing or by neurectomy and to evaluate time-dependent derangements in penile hemodynamics in vivo and subsequent histologic alterations in the cavernous tissue. METHODS: Twelve-week-old C57BL/6J mice were divided into 4 groups (N=36 per group): control, sham operation, bilateral cavernous nerve crush, and bilateral cavernous neurectomy group. MAIN OUTCOME MEASURES: Three days and 1, 2, 4, 8, and 12 weeks after CNI, erectile function was measured by electrical stimulation of the cavernous nerve. The penis was then harvested and TUNEL was performed. Immunohistochemical analysis was performed assaying for caspase-3, transforming growth factor-ß1 (TGF-ß1), phospho-Smad2, PECAM-1, factor VIII, and smooth muscle α-actin. The numbers of apoptotic cells and phospho-Smad2-immunopositive cells in endothelial cells or smooth muscle cells were counted. RESULTS: Erectile function was significantly less in the cavernous nerve crushing and neurectomy groups than in the control or sham group. This difference was observed at the earliest time point assayed (day 3) and persisted up to 4 weeks after nerve crushing and to 12 weeks after neurectomy. The apoptotic index peaked at 1 or 2 weeks after CNI and decreased thereafter. Cavernous TGF-ß1 and phospho-Smad expression was also increased after CNI. The numbers of apoptotic cells and phospho-Smad2-immunopositive cells in cavernous endothelial cells and smooth muscle cells were significantly greater in the cavernous nerve crush and cavernous neurectomy groups than in the control or sham group. Conclusion. The mouse is a useful model for studying pathophysiologic mechanisms involved in erectile dysfunction after CNI. Early intervention to prevent apoptosis in smooth muscle cells and endothelial cells or to inhibit cavernous tissue fibrosis is required to restore erectile function.

Transforming growth factor (TGF)-ß type I receptor kinase (ALK5) inhibitor alleviates profibrotic TGF-ß1 responses in fibroblasts derived from Peyronie's plaque.

INTRODUCTION: Transforming growth factor-ß1 (TGF-ß1) has been identified as an important fibrogenic cytokine associated with Peyronie's disease (PD). AIM: The aim of this study was to study the differential expression of the TGF-ß1 and Smad transcription factors in plaque tissue from PD patients and to determine the antifibrotic effect of SKI2162 (SK Chemicals, Seoul, South Korea), a novel small-molecule inhibitor of activin receptor-like kinase 5 (ALK5), a type I receptor of TGF-ß, in primary fibroblasts derived from human PD plaque. METHODS: Plaque tissue was isolated from five PD patients, and tunica albuginea tissue was obtained from four control patients. Plaque tissues from a patient with PD were used for primary fibroblast culture. Fibroblasts were pretreated with SKI2162 (10 µM) and then stimulated with TGF-ß1 (10ng/mL). MAIN OUTCOME MEASURES: The plaque or tunica albuginea tissue was stained with Masson's trichrome or antibody to TGF-ß1, phospho-Smad2 (P-Smad2), and P-Smad3. Protein was extracted from treated fibroblasts for Western blotting, and the membranes were probed with antibody to P-Smad2/Smad2, P-Smad3/Smad3, plasminogen activator inhibitor-1, fibronectin, collagen I, and collagen IV. We also determined the inhibitory effect of SKI2162 on TGF-ß1-induced nuclear translocation of Smad2/3 in fibroblasts. RESULTS: The plaque tissue from PD patients showed higher TGF-ß1, P-Smad2, and P-Smad3 immunoreactivity than did the tunica albuginea tissue from control patients. SKI2162 not only blocked TGF-ß1-induced phosphorylation and nuclear translocation of Smad2 and Smad3, but also inhibited the production of extracellular matrix markers in fibroblasts derived from human PD plaque. CONCLUSION: In light of the pivotal role of TGF-ß and Smads in the pathogenesis of PD, pharmacologic inhibition of ALK5 may represent a novel targeted approach to treating PD.

Intracavernous delivery of synthetic angiopoietin-1 protein as a novel therapeutic strategy for erectile dysfunction in the type II diabetic db/db mouse.

INTRODUCTION: Patients with erectile dysfunction (ED) associated with type II diabetes often have impaired endothelial function and tend to respond poorly to oral phosphodiesterase type 5 inhibitors. Therefore, neovascularization is a promising strategy for curing diabetic ED. AIM: To determine the effectiveness of a soluble, stable, and potent angiopoietin-1 (Ang1) variant, cartilage oligomeric matrix protein (COMP)-Ang1, in promoting cavernous angiogenesis and erectile function in a mouse model of type II diabetic ED. Methods. Sixteen-week-old male db/db mice (in which obesity and type II diabetes are caused by a mutation in the leptin receptor) and control C57BL/6J mice were used and divided into four groups (N=14 per group): age-matched controls; db/db mice receiving two successive intracavernous injections of phosphate-buffered saline (PBS) (days -3 and 0; 20 µL); db/db mice receiving a single intracavernous injection of COMP-Ang1 protein (day 0; 5.8 µg/20 µL); and db/db mice receiving two successive intracavernous injections of COMP-Ang1 protein (days -3 and 0; 5.8 µg/20 µL). MAIN OUTCOME MEASURES: Two weeks later, erectile function was measured by electrical stimulation of the cavernous nerve. The penis was then harvested and stained with antibodies to platelet/endothelial cell adhesion molecule-1 (PECAM-1) (endothelial cell marker), phosphohistone H3 (PH3, a nuclear protein indicative of cell proliferation), phospho-endothelial nitric oxide synthase (eNOS), and eNOS. Penis specimens from a separate group of animals were used for cyclic guanosine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP) quantification. RESULTS: Local delivery of COMP-Ang1 protein significantly increased eNOS phosphorylation and cGMP and cAMP expression compared with that in the group treated with PBS. Repeated intracavernous injections of COMP-Ang1 protein completely restored erectile function and cavernous endothelial content through enhanced cavernous neoangiogenesis as evaluated by PECAM-1 and PH3 immunohistochemistry and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling assay, whereas a single injection of COMP-Ang1 protein elicited partial improvement. CONCLUSION: Cavernous neovascularization using recombinant Ang1 protein is a novel therapeutic strategy for the treatment of ED resulting from type II diabetes.

Derangements in endothelial cell-to-cell junctions involved in the pathogenesis of hypercholesterolemia-induced erectile dysfunction.

INTRODUCTION: Endothelial cell-to-cell junctions are crucial for vascular formation, networking, and remodeling of blood vessels as well as for inducing and integrating intracellular signals. AIM: We investigated the differential expression and distribution of endothelial cell-to-cell junction proteins in the penis of mice with hypercholesterolemia-induced erectile dysfunction. METHODS: Two-month-old C57BL/6J mice were fed a diet containing 4% cholesterol and 1% cholic acid, and age-matched control animals were fed a normal diet, for 3 months. We performed dual priming oligonucleotide (DPO)-based multiplex polymerase chain reaction (PCR) (Seegene, Seoul, Korea) to screen the differential gene expression of 21 endothelial cell-to-cell junctions. MAIN OUTCOME MEASURES: At 5 months, erectile function was measured by electrical stimulation of the cavernous nerve, and the penis was harvested and stained with antibody to claudin-5, vascular endothelial (VE)-cadherin, and platelet/endothelial cell adhesion molecule (PECAM)-1 (N = 8 per group). Cavernous specimens from a separate group of animals were used for claudin-5, VE-cadherin, and PECAM-1 reverse transcriptase-PCR and Western blot analysis. RESULTS: Erectile function was significantly lower in hypercholesterolemic mice than in controls. DPO-based multiplex PCR revealed a profound decrease in the gene expression of endothelium-specific cell-to-cell junction proteins, including claudin-5, VE-cadherin, and PECAM-1, in hypercholesterolemic mice compared with that in controls. The expression of claudin-5, VE-cadherin, and PECAM-1 protein evaluated by Western blot or immunohistochemistry was significantly lower in hypercholesterolemic mice than in controls. These endothelial cell-to-cell junction proteins were more sparsely distributed in the endothelium of cavernous sinusoids than in the endothelium of cavernous artery and dorsal blood vessels. CONCLUSION: Down-regulation of the endothelial cell-to-cell junctions and decreased endothelial content in the corpus cavernosum might play a major role in the deterioration of erectile function in hypercholesterolemic mice.

Functional and morphologic characterizations of the diabetic mouse corpus cavernosum: comparison of a multiple low-dose and a single high-dose streptozotocin protocols.

INTRODUCTION: With the advent of genetically modified mice, it seems particularly advantageous to develop a mouse model of diabetic erectile dysfunction. AIM: To establish a mouse model of type I diabetes by implementation of either multiple low-dose streptozotocin (STZ) protocol or single high-dose STZ protocol and to evaluate morphologic alterations in the cavernous tissue and subsequent derangements in penile hemodynamics in vivo. METHODS: Eight-week-old C57BL/6J mice were divided into three groups: a control group, a group administered the multiple low-dose STZ protocol (50 mg/kg x 5 days), and a group administered the single high-dose STZ protocol (200 mg/kg). MAIN OUTCOME MEASURES: After 8 weeks, erectile function was measured by electrical stimulation of the cavernous nerve. The penis was then harvested and stained with hydroethidine (in situ analysis of superoxide anion), TUNEL, or antibodies to nitrotyrosine (marker of peroxynitrite formation), PECAM-1, smooth muscle alpha-actin, and phospho-eNOS. Penis specimens from a separate group of animals were used for phospho-eNOS and eNOS western blot or cGMP determination. RESULTS: Erectile function was significantly less in diabetic groups than in control group. The generation of superoxide anion and nitrotyrosine and the number of apoptotic cells in both cavernous endothelial and smooth muscle cells were significantly higher in diabetic groups than in control group. Cavernous tissue phospho-eNOS and cGMP expression and the number of endothelial and smooth muscle cells were lower in diabetic groups than in control group. Both diabetic models resulted in similar structural and functional derangements in the corpus cavernosum; however, the mortality rate was higher in mice receiving single high-dose of STZ than in those receiving multiple low-doses. CONCLUSION: The mouse model of type I diabetes is useful and technically feasible for the study of the pathophysiologic mechanisms involved in diabetic erectile dysfunction.

IN-1130, a novel transforming growth factor-beta type I receptor kinase (activin receptor-like kinase 5) inhibitor, promotes regression of fibrotic plaque and corrects penile curvature in a rat model of Peyronie's disease.

INTRODUCTION: Transforming growth factor-beta1 (TGF-beta1) has been known to play a crucial role in the pathogenesis of Peyronie's disease (PD). AIM: The aim of this paper was to investigate the therapeutic effect of IN-1130, a novel small molecule inhibitor of activin receptor-like kinase (ALK)5, a type I receptor of TGF-beta, in an animal model of PD. METHODS: PD was induced in rats through repeated injections of adenovirus expressing TGF-beta1 (days 0, 3, and 6; 1 x 10(10) particles/0.1 mL, respectively) into the tunica albuginea. The rats were divided into five groups (N = 10 per group): group 1, age-matched controls without treatment; group 2, age-matched controls receiving repeated injections of IN-1130 (days 30 and 37; 5 mg/kg in 0.1 mL saline, respectively); group 3, PD rats without treatment; group 4, PD rats receiving repeated injections of saline (days 30 and 37; 0.1 mL, respectively); group 5, PD rats receiving repeated injections of IN-1130 (days 30 and 37; 5 mg/kg in 0.1 mL saline, respectively) into the lesion. MAIN OUTCOME MEASURES: Penile curvature was evaluated by use of an artificial erection test at day 45, and the penis was then harvested for histologic examination. Collagen in the plaque was quantitatively assessed by hydroxyproline determination. RESULTS: IN-1130 induced significant regression of fibrotic plaque through reduced infiltration of inflammatory cells, reduced transnuclear expression of phospho-Smad2/phospho-Smad3, reduced hydroxyproline content, and reduced cartilage content and restoration of elastin fibers in the fibrotic plaque of PD rats, which was accompanied by the correction of penile curvature. CONCLUSION: Antagonizing TGF-beta signaling through the use of ALK5 inhibitors may represent an exciting new therapeutic strategy for the future treatment of PD.

Repeated intratunical injection of adenovirus expressing transforming growth factor-beta1 in a rat induces penile curvature with tunical fibrotic plaque: a useful model for the study of Peyronie's disease.

This study was undertaken to establish a Peyronie's disease model with penile curvature by using recombinant transforming growth factor-beta1 (TGF-beta1) protein or adenovirus (ad-TGF-beta1). Four-month-old male Sprague-Dawley rats were divided into seven groups (n = 18 per group): G1 received a single injection of saline into the tunica albuginea (0.1 mL); G2, repeated injections of ad-LacZ (days 0, 3, and 6; 1 x 10(10) particles/0.1 mL respectively); G3, a single injection of recombinant TGF-beta1 protein (700 ng/0.1 mL); G4, repeated injections of recombinant TGF-beta1 protein (days 0, 3 and 6; 700 ng/0.1 mL respectively); G5, a single injection of low-dose ad-TGF-beta1 (1 x 10(10) particles/0.1 mL); G 6, a single injection of high-dose ad-TGF-beta1 (3 x 10(10) particles/0.1 mL); and G7, repeated injections of low-dose ad-TGF-beta1 (days 0, 3, and 6; 1 x 10(10) particles/0.1 mL respectively). Penile curvature was evaluated 30, 45 and 60 days after treatment, and the penis was then harvested for histological examination. Repeated injection of low-dose ad-TGF-beta1 not only induced fibrous scar in the tunica, which lasted up to 60 days after injection, but also resulted in significant penile curvature by artificial erection test 45 days after treatment. A peculiar histological finding in this group was trapping of inflammatory cells in the tunica, subsequent fibrosis, and formation of cartilage and calcification as well as loss of elastin fibres. This model involving repeated injection of ad-TGF-beta1 may contribute to further investigation of the pathogenesis of Peyronie's disease and the development of new therapeutics targeting this pathway.

Efficient gene expression system using the RTP801 promoter in the corpus cavernosum of high-cholesterol diet-induced erectile dysfunction rats for gene therapy.

INTRODUCTION: The application of gene therapy for a nonlife-threatening disease, such as erectile dysfunction (ED), requires a higher safety level and more efficacious systems for gene transfer. AIM: To establish a novel technique for gene expression in a rat model of hypercholesterolemic ED that uses the RTP801 promoter, a hypoxia-inducible promoter. METHODS: Two-month-old male Sprague-Dawley rats were fed a diet containing 4% cholesterol and 1% cholic acid, and age-matched control animals were fed a normal diet, for 3 months. MAIN OUTCOME MEASURES: Cavernous expression of hypoxia-inducible factor (HIF)-1alpha was evaluated by Western blot. After intracavernous injection of pSV-Luc or pRTP801-Luc, gene expression was evaluated by luciferase assay, and the gene expression area was evaluated by immunohistochemistry. RESULTS: HIF-1alpha was up-regulated in the corpus cavernosum of hypercholesterolemic rats. Although pSV-Luc did not induce gene expression in either the control or the cholesterol group, pRTP801-Luc significantly induced gene expression in the cholesterol group and resulted in higher luciferase activity than did pSV-Luc up to 14 days after injection. Immunohistochemistry showed that the gene expression area was also greater in the pRTP801-Luc group than in the pSV-Luc group, but the difference was not as great as that in luciferase activity. This suggests that pRTP801-Luc exerts its effect mainly by inducing promoter activity under hypoxia, not by increasing the number of transfected cells. CONCLUSION: The RTP801 promoter-driven gene expression system increased gene expression in the corpus cavernosum tissue of rats with cholesterol-induced ED. This may be a useful system for the development of gene therapy in vasculogenic ED.

Role of increased penile expression of transforming growth factor-beta1 and activation of the Smad signaling pathway in erectile dysfunction in streptozotocin-induced diabetic rats.

INTRODUCTION: It has been suggested that transforming growth factor-beta1 (TGF-beta1) plays an important role in the pathogenesis of diabetes-induced erectile dysfunction. AIM: To investigate the expression and activity of Smad transcriptional factors, the key molecules for the initiation of TGF-beta-mediated fibrosis, in the penis of streptozotocin (STZ)-induced diabetic rats. METHODS: Fifty-two 8-week-old Sprague-Dawley rats were used and divided into control and diabetic groups. Diabetes was induced by an intravenous injection of STZ. MAIN OUTCOME MEASURES: Eight weeks later, erectile function was measured by electrical stimulation of the cavernous nerve (N = 12 per group). The penis was harvested and stained with Masson trichrome or antibody to TGF-beta1, phospho-Smad2 (P-Smad2), smooth muscle alpha-actin, and factor VIII (N = 12 per group). Penis specimens from a separate group of animals were used for TGF-beta1 enzyme-linked immunosorbent assay (ELISA), P-Smad2/Smad2, phospho-Smad3 (P-Smad3)/Smad3, fibronectin, collagen I, and collagen IV western blot, or hydroxyproline determination. RESULTS: Erectile function was significantly reduced in diabetic rats compared with that in controls. The expression of TGF-beta1, P-Smad2, and P-Smad3 protein evaluated by ELISA or western blot was higher in diabetic rats than in controls. Compared with that in control rats, P-Smad2 expression was higher mainly in smooth muscle cells and fibroblasts of diabetic rats, whereas no significant differences were noted in endothelial cells or in the dorsal nerve bundle. Cavernous smooth muscle and endothelial cell contents were lower in diabetic rats than in controls. Cavernous fibronectin, collagen IV, and hydroxyproline content was significantly higher in diabetic rats than in controls. CONCLUSION: Upregulation of TGF-beta1 and activation of the Smad signaling pathway in the penis of diabetic rats might play important roles in diabetes-induced structural changes and deterioration of erectile function.