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Purpose Penile erection requires integrative interactions between vascular endothelial cells, pericytes, smooth muscle cells, and autonomic nerves. Furthermore, the importance of the role played by pericytes in the pathogenesis of angiopathy has only recently been appreciated. However, global gene expression in pericytes in diabetes mellitus-induced erectile dysfunction (DMED) remains unclear. We aimed to identify potential target genes related to DMED in mouse cavernous pericytes (MCPs). Materials and Methods Mouse cavernous tissue was allowed to settle under gravity in collagen I-coated dishes, and sprouted cells were subcultivated for experiments. To imitate diabetic conditions, MCPs were treated with normal-glucose (NG, 5 mM) or high-glucose (HG, 30 mM) media for 3 days. Microarray technology was used to evaluate gene expression profiles, and RT-PCR was used to validate sequencing data. Histological examinations and Western blot were used to validate final selected target genes related to DMED. Results Decreased tube formation and increased apoptosis were detected in MCPs exposed to the HG condition. As shown by microarray analysis, the gene expression profiles of MCPs exposed to the NG or HG condition differed. A total of 2,523 genes with significantly altered expression were classified into 15 major gene categories. After further screening based on gene expression and RT-PCR and histologic results, we found that Hebp1 gene expression was significantly diminished under the HG condition and in DM mice. Conclusions This gene profiling study provides new potential targets responsible for diabetes in MCPs. Validation studies suggest that Hebp1 may be a suitable biomarker for DMED., Graphical Abstract

Purpose To establish a simple and nonenzymatic technique to isolate endothelial cells (ECs) and pericytes from human corpus cavernosum tissue and to evaluate the angiogenic ability of the human cavernous EC or pericytes for the study of high glucose-induced angiopathy. Materials and methods For primary human cavernous EC culture, cavernous tissues were implanted into Matrigel in dishes. For primary human cavernous pericyte culture, cavernous tissues were settled by gravity into dishes. We performed immunocytochemistry and Western blot to determine phenotype and morphologic changes from passage 1 to 5. The primary cultured cells were exposed to a normal-glucose (5 mmol/L) or a high-glucose (30 mmol/L) condition, and then tube formation assay was done. Results We successfully isolated high-purity EC and pericytes from human corpus cavernosum tissue. Primary cultured EC showed highly positive staining for von Willebrand factor, and pericyte revealed positive staining for NG2 and platelet-derived growth factor receptor-β. Primary cultured EC and pericytes maintained their cellular characteristics up to passage 2 or 3. However, we observed significant changes in their typical phenotype from the passage 4 and morphological characteristics from the passage 3. Human cavernous EC or pericytes formed well-organized capillary-like structures in normal-glucose condition, whereas severely impaired tube formation was detected in high-glucose condition. Conclusions This study provides a simple and nonenzymatic method for primary culture of human cavernous EC and pericytes. Our study will aid us to understand the pathophysiology of diabetic erectile dysfunction, and also be a valuable tool for determining the efficacy of candidate therapeutic targets.

Men with diabetic erectile dysfunction (ED) respond poorly to the currently available oral phosphodiesterase-5 inhibitors. Therefore, functional therapies for diabetic ED are needed. Stromal vascular fraction (SVF) and the adenovirus-mediated cartilage oligomeric matrix angiopoietin-1 (Ad-COMP-Ang1) gene are known to play critical roles in penile erection. We previously reported that SVF and Ad-COMP-Ang1 have only a short-term effect in restoring erectile function. Further improvements to ED therapy are needed for long-lasting effects. In the present study, we aimed to test if the combination of SVF and Ad-COMP-Ang1 could extend the erection effect in diabetic ED. We found that the combination therapy showed a long-term effect in restoring erectile function through enhanced penile endothelial and neural cell regeneration. Combination therapy with SVF and Ad-COMP-Ang1 notably restored cavernous endothelial cell numbers, pericyte numbers, endothelial cell-cell junctions, decreased cavernous endothelial cell permeability, and promoted neural regeneration for at least 4 weeks in diabetic mice. In summary, this is an initial description of the long-term effect of combination therapy with SVF and Ad-COMP-Ang1 in restoring erectile function through a dual effect on endothelial and neural cell regeneration. Such combination therapy may have therapeutic potential for the treatment of diabetic ED.

Penile erection requires well-coordinated interactions between vascular and nervous systems. Penile neurovascular dysfunction is a major cause of erectile dysfunction (ED) in patients with diabetes, which causes poor response to oral phosphodiesterase-5 inhibitors. Dickkopf2 (DKK2), a Wnt antagonist, is known to promote angiogenesis. Here, using DKK2-Tg mice or DKK2 protein administration, we demonstrate that the overexpression of DKK2 in diabetic mice enhances penile angiogenesis and neural regeneration and restores erectile function. Transcriptome analysis revealed that angiopoietin-1 and angiopoietin-2 are target genes for DKK2. Using an endothelial cell-pericyte coculture system and ex vivo neurite sprouting assay, we found that DKK2-mediated juxtacrine signaling in pericyte-endothelial cell interactions promotes angiogenesis and neural regeneration through an angiopoietin-1-Tie2 pathway, rescuing erectile function in diabetic mice. The dual angiogenic and neurotrophic effects of DKK2, especially as a therapeutic protein, will open new avenues to treating diabetic ED.

Background The efficacy of exercise training in patients with lung cancer after lung resection has not been well established yet. Therefore, we performed a meta-analysis to investigate the efficiency of exercise training in patients with lung cancer after lung resection. Methods Several databases were searched for eligible randomised controlled trials (RCTs). The primary outcome was quality of life, and the secondary outcomes included 6-min walk distance (6MWD), forced expiratory volume in 1 s (FEV1) and postoperative complications (POCs). Weighted mean differences (WMDs) and relative risks (RRs) with 95% confidence intervals (CIs) were calculated by random-effects model. Results Six RCTs involving 438 patients were enrolled in this meta-analysis. The pooled WMDs of the scores were 2.41 (95% CI = −5.20 to 10.02; P = 0.54) and −0.46 (95% CI = −20.52 to 19.61; P = 0.96) for the physical and mental components of the 36-item short-form scale, respectively. The pooled WMDs were 23.50 m (95% CI = −22.04 to 69.03; P = 0.31) for 6MWD and 0.03 L (95% CI = −0.19 to 0.26; P = 0.76) for FEV1. Finally, the pooled RRs were 0.79 (95% CI = 0.41 to 1.53; P = 0.49) for POCs. Conclusions Insufficient evidence is available to support the efficacy of exercise training in patients with lung cancer after lung resection. Further studies must confirm our findings and investigate the long-term effects of exercise training on patients with lung cancer following lung resection. Electronic supplementary material The online version of this article (doi:10.1186/s12957-017-1233-1) contains supplementary material, which is available to authorized users.

Diabetic erectile dysfunction (ED) has multiple causative factors, such as endothelial and smooth muscle dysfunction and cavernous fibrosis. Wnt signalling is essential for normal embryonic development and for tissue homeostasis in adults. Aberrant activation of Wnt family members has been implicated in tissue fibrosis and in angiogenesis. In this study, we investigated the differential expression of Wnts in the penises of mice with streptozotocin-induced diabetic ED. We also examined the effect of transforming growth factor-β1 (TGF-β1) on the expression of Wnts in primary cultured fibroblasts isolated from human tunica albuginea. Among the mouse and human Wnts tested, 16 mouse Wnts and 14 human Wnts were detected in the corpus cavernosum tissue of normal mice and in fibroblasts derived from human tunica albuginea respectively. We observed up-regulation of Wnt10b (known to be involved in tissue fibrosis) and down-regulation of Wnt16 (known to be involved in vasculogenesis and hematopoiesis), both in the diabetic condition in vivo and with treatment of fibroblasts with TGF-β1 in vitro. Wnt10b was mainly expressed in fibroblasts and Wnt16 was colocalized with smooth muscle cells in the corpus cavernosum tissue. Cavernous TGF-β1 protein expression and the degree of cavernous fibrosis determined by the ratio of collagen to smooth muscle content were significantly higher in diabetic mice than in controls. Cavernous endothelial content was significantly decreased by the diabetic condition. Overexpression of Wnt16 with plasmid vector accelerated tube formation in primary cultured mouse cavernous endothelial cells. However, down-regulation of Wnt10b with small interfering RNA did not decrease the production of extracellular matrix protein in human fibroblasts. This is the first report demonstrating the differential expression of Wnts in diabetic mouse penis. Aberrant Wnt expression might contribute to the pathogenesis of ED.

Introduction Radical prostatectomy for prostate cancer can not only induce cavernous nerve injury (CNI) but also result in structural changes in the cavernous tissues. Nerve injuryinduced protein 1, Ninjurin1 (Ninj1), is known to be involved in neuroinflammatory processes and to be related to vascular regression during the embryonic period. Aim The study aims to determine whether and how Ninj1 neutralizing antibody (Ninj1Ab) restores erectile function in mice with CNI. Methods Twelveweekold C57BL/6J mice were used and distributed into four groups: sham operation group and CNI groups receiving a single intracavernous injection of immunoglobulin G (IgG) control antibody, lowdose Ninj1Ab (1.0 μg/20 μL), or highdose Ninj1Ab (2.5 μg/20 μL). Main Outcome Measures One week after bilateral cavernous nerve crush, erectile function was measured by electrical stimulation of the cavernous nerve. The penis was harvested for histologic examinations and Western blot analysis. Results The cavernous expression of Ninj1 protein was upregulated up to 7 days after CNI and returned to baseline levels thereafter. Local delivery of Ninj1Ab significantly increased penile neuronal nitric oxide synthase and neurofilament contents, induced cavernous endothelial proliferation and phosphorylation of Akt and endothelial nitric oxide synthase, and decreased endothelial cell apoptosis in the CNI mice by upregulating angiopoietin1 and downregulating angiopoietin2. Highdose Ninj1Ab induced profound restoration of erectile function in the CNI mice (91% of sham control values), whereas lowdose Ninj1Ab elicited partial improvement. Conclusion The dual neurotrophic and angiogenic effects of Ninj1 blockade may provide a good opportunity for treating erectile dysfunction resulting from radical prostatectomy.

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. Ryu J‐K, Tumurbaatar M, Jin H‐R, Kim WJ, Kwon M‐H, Piao S, Choi MJ, Yin GN, Song K‐M, Kang Y‐J, Koh YJ, Koh GY, and Suh J‐K. Intracavernous delivery of freshly isolated stromal vascular fraction rescues erectile function by enhancing endothelial regeneration in the streptozotocin‐induced diabetic mouse. J Sex Med 2012;9:3051–3065.

Lung cancer is the most common cancer worldwide, and morbidity and mortality associated with lung cancer has been increasing annually in recent decades. MicroRNAs (miRNAs), which are short non‑coding RNA sequences that are involved in the regulation of gene expression, have been previously demonstrated to be key regulators in cancer. The present study aimed to clarify the role of miRNA (miR)‑1284 in lung cancer. A549 lung carcinoma cells were transfected with miR‑1284 mimic or miR‑1284 inhibitor using Lipofectamine 2000. Subsequently, cell viability, growth and apoptosis of A459 cells in the miR‑1284 mimic, miR‑1284 inhibitor and control groups were assayed by MTT assay, bromodeoxyuridine assay and flow cytometry, respectively. Furthermore, the protein expression levels of p27, p21, Bax, pro‑caspase‑3, activated caspase‑3 and Myc were detected by western blot analysis to investigate the molecular mechanisms underlying the effect of miR‑1284 on A549 cells. The cell viability and growth of A549 cells were significantly decreased in the miR‑1284 mimic group compared with the control group, whereas the percentage of apoptotic cells was significantly increased. By contrast, miR‑1284 inhibitor transfection significantly increased the cell viability and growth compared with control, and decreased apoptosis. Furthermore, expression of p27 was increased in miR‑1284 mimic‑transfected A549 cells compared with the control group, whereas p21 was unaffected by miR‑1284 overexpression or inhibition. The expression of Myc was decreased by miR‑1284 mimic transfection compared with the control group. For the other apoptosis‑associated proteins that were investigated (Bax, pro‑caspase‑3 and active caspase‑3), the expression levels in the miR‑1284 mimic transfected cells were higher than in the other two groups (control and miR‑1284 inhibitor). In conclusion, the results suggest that miR‑1284 affects cell proliferation and apoptosis of lung cancer cells, indicating that miR‑1284 may have a key role in lung tumorigenesis.

Introduction A proper cavernous endothelial cell culture system would be advantageous for the study of the pathophysiologic mechanisms involved in endothelial dysfunction and erectile dysfunction (ED). Aim To establish a nonenzymatic technique, which we termed the “Matrigel‐based sprouting endothelial cell culture system,” for the isolation of mouse cavernous endothelial cells (MCECs) and an in vitro model that mimics in vivo situation for diabetes‐induced ED. Methods For primary MCEC culture, mouse cavernous tissue was implanted into Matrigel and sprouting cells from the tissue were subcultivated. To establish an in vitro model for diabetes‐induced ED, the primary cultured MCECs were exposed to a normal‐glucose (5 mmoL) or a high‐glucose (30 mmoL) condition for 48 hours. Main Outcome Measures The purity of isolated cells was determined by fluorescence‐activated cell sorting analysis. MCECs incubated under the normal‐ or the high‐glucose condition were used for Western blot, cyclic guanosine monophosphate (cGMP) quantification, and in vitro angiogenesis assay. Results We could consistently isolate high‐purity MCECs (about 97%) with the Matrigel‐based sprouting endothelial cell culture system. MCECs were subcultured up to the fifth passage and no significant changes were noted in endothelial cell morphology or purity. The phosphorylation of Akt and eNOS and the cGMP concentration were significantly lower in MCECs exposed to high glucose than in those exposed to normal glucose. MCECs exposed to the normal‐glucose condition formed well‐organized capillary‐like structures, whereas derangements in tube formation were noted in MCECs exposed to high glucose. The protein expression of transforming growth factor‐β1 (TGF‐β1) and phospho‐Smad2 was significantly increased by exposure to high glucose. Conclusion The Matrigel‐based sprouting endothelial cell culture system is a simple, technically feasible, and reproducible technique for isolating pure cavernous endothelial cells in mice. An in vitro model for diabetic ED will be a valuable tool for evaluating the angiogenic potential of novel endogenous or synthetic modulators. Yin GN, Ryu J‐K, Kwon M‐H, Shin SH, Jin HR, Song K‐M, Choi MJ, Kang D‐Y, Kim WJ, and Suh J‐K. Matrigel‐based sprouting endothelial cell culture system from mouse corpus cavernosum is potentially useful for the study of endothelial and erectile dysfunction related to high‐glucose exposure. J Sex Med 2012;9:1777–1789.

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. Jin H-R, Kim WJ, Song JS, Piao S, Tumurbaatar M, Shin SH, Choi MJ, Tuvshintur B, Song K-M, Kwon M-H, Yin GN, Koh GY, Ryu J-K, and Suh J-K. Intracavernous delivery of synthetic angiopoietin-1 protein as a novel therapeutic strategy for erectile dysfunction in the type II diabetic db/db mouse.

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-β1 (TGF-β1), 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-β1 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. Jin H-R, Chung YG, Kim WJ, Zhang LW, Piao S, Tuvshintur B, Yin GN, Shin SH, Tumurbaatar M, Han J-Y, Ryu J-K, and Suh J-K. A mouse model of cavernous nerve injury-induced erectile dysfunction: Functional and morphological characterization of the corpus cavernosum.

Introduction Transforming growth factor-β1 (TGF-β1) 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-β, in an animal model of PD. Methods PD was induced in rats through repeated injections of adenovirus expressing TGF-β1 (days 0, 3, and 6; 1 × 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-β signaling through the use of ALK5 inhibitors may represent an exciting new therapeutic strategy for the future treatment of PD. Ryu J-K, Piao S, Shin H-Y, Choi MJ, Zhang LW, Jin H-R, Kim WJ, Han J-Y, Hong SS, Park SH, Lee S-J, Kim I-H, Lee CR, Kim D-K, Mamura M, Kim S-J, and Suh J-K. IN-1130, a novel transforming growth factor-β 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. J Sex Med 2009;6:1284–1296.

Introduction It has been suggested that transforming growth factor‐β1 (TGF‐β1) 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‐β‐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‐β1, phospho‐Smad2 (P‐Smad2), smooth muscle α‐actin, and factor VIII (N = 12 per group). Penis specimens from a separate group of animals were used for TGF‐β1 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‐β1, 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‐β1 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. Zhang LW, Piao S, Choi MJ, Shin H‐Y, Jin H‐R, Kim WJ, Song SU, Han J‐Y, Park SH, Mamura M, Kim S‐J, Ryu J‐K, and Suh J‐K. Role of increased penile expression of transforming growth factor‐β1 and activation of the Smad signaling pathway in erectile dysfunction in streptozotocin‐induced diabetic rats. J Sex Med 2008;5:2318–2329.

Despite the advent of oral phosphodiesterase-5 inhibitors, curative treatment for erectile dysfunction (ED) remains unavailable. Recently, the link between ED and cardiovascular disease was unveiled and the main etiology of ED was found to be vasculogenic. Therefore, neovascularization is a promising strategy for curing ED. Angiopoietin-1 (Ang1) is an angiogenic growth factor that promotes the generation of stable and functional vasculature. Here, we demonstrate that local delivery of the soluble, stable and potent Ang1 variant, COMP-Ang1 gene or protein, into the penises of hypercholesterolemic mice increases cavernous angiogenesis, eNOS phosphorylation and cGMP expression, resulting in full recovery of erectile function and cavernous blood flow up to 8 weeks after treatment. COMP-Ang1-induced promotion of cavernous angiogenesis and erectile function was abolished in Nos3-/- mice and in the presence of the NOS inhibitor, L-NAME. COMP-Ang1 also restored the integrity of endothelial cell-cell junction by down-regulating the expression of histone deacetylase 2 in the penis of hypercholesterolemic mice and in primary cultured mouse cavernous endothelial cells. These findings constitute a new paradigm toward curative treatment of both cavernous angiopathy and ED.

Introduction Erectile dysfunction (ED) is a major complication of radical prostatectomy. Men with radical prostatectomy‐induced ED respond less positively to oral phosphodiesterase‐5 inhibitors. Aim The study aims to examine whether and how stromal vascular fraction (SVF) restores erectile function in mice with cavernous nerve injury (CNI). Methods Twelve‐week‐old male C57BL/6J mice were used and the animals were distributed into five groups: sham operation group and CNI group receiving a single intracavernous injection of phosphate‐buffered saline (PBS) or SVF (1 × 10 4 , 1 × 10 5 , or 3 × 10 5 cells/20 μL, respectively). SVF was isolated from epididymal adipose tissues of green fluorescence protein transgenic mice. Main Outcome Measures Two weeks after injection, erectile function was measured by cavernous nerve stimulation. The penis was stained with antibodies to platelet/endothelial cell adhesion molecule‐1, phosphohistone H3, and phosphorylated endothelial nitric oxide synthase (phospho‐eNOS). We also performed Western blot for angiopoietin‐1 (Ang‐1), vascular endothelial growth factor‐A, hepatocyte growth factor, phospho‐eNOS, and eNOS in the corpus cavernosum tissue. Results Local delivery of SVF restored erectile function in a dose‐dependent manner in CNI mice. The highest erectile response was noted at a dose of 3 × 10 5 cells, for which the response was comparable with that in the sham operation group. Local delivery of SVF significantly increased the expression of angiogenic factor proteins and induced cavernous endothelial cell proliferation and eNOS phosphorylation compared with that in the PBS‐treated CNI group. SVF‐induced promotion of cavernous angiogenesis and erectile function was diminished in the presence of soluble antibody to Tie2, a receptor tyrosine kinase of Ang‐1. Conclusion Secretion of angiogenic factors from SVF is an important mechanism by which SVF induces cavernous endothelial regeneration and restores erectile function. These findings suggest that cavernous endothelial regeneration by using SVF may represent a promising treatment strategy for radical prostatectomy‐induced ED. Song K‐M, Jin H‐R, Park J‐M, Choi MJ, Kwon M‐H, Kwon K‐D, Batbold D, Yin GN, Kim WJ, Koh GY, Ryu J‐K, and Suh J‐K. Intracavernous delivery of stromal vascular fraction restores erectile function through production of angiogenic factors in a mouse model of cavernous nerve injury. J Sex Med 2014;11:1962–1973.

Introduction Men with erectile dysfunction (ED) respond poorly to oral phosphodiesterase-5 inhibitors following radical prostatectomy. Recent studies have reported that up-regulation of transforming growth factor-β1 (TGF-β1) and activation of the Smad signaling pathway play important roles in cavernous fibrosis and in the deterioration of erectile function in a mouse model of cavernous nerve injury (CNI) and in patients with spinal cord injury. The mothers against decapentaplegic homolog 7 (Smad7) is known to inhibit the phosphorylation of Smad2 and Smad3. Aim To investigate the effectiveness of adenoviruses encoding Smad7 gene (Ad-Smad7) on erectile function in a mouse model of CNI. Methods Twelve-week-old C57BL/6J mice were used and distributed into 7 groups: sham operation group, untreated CNI group, and CNI groups receiving a single intracavernous injection of adenovirus encoding LacZ (1 × 10 8 virus particles [vp]/20 μL) or adenovirus encoding Smad7 (Ad-Smad7; 1 × 10 7 , 1 × 10 8 , 2 × 10 8 , or 1 × 10 9 vp/20 μL). Main Outcome Measures Two weeks after bilateral cavernous nerve crushing and treatment, erectile function was measured by electrical stimulation of the cavernous nerve. The penis was harvested for histologic examinations and Western blot analysis. Results The highest erectile response was noted in CNI mice treated with Ad-Smad7 at a dose of 1 × 10 8 vp, which reached up to 82–85% of sham control values. Local delivery of Ad-Smad7 significantly decreased endothelial cell apoptosis and the production of extracellular matrix proteins, including plasminogen activator inhibitor-1, fibronectin, collagen I, and collagen IV, and induced endothelial nitric oxide synthase phosphorylation in the corpus cavernosum tissue of CNI mice. Conclusion The adenovirus-mediated gene transfer of Smad7 successfully restored erectile function by enhancing endothelial cell function and through antifibrotic effects. These findings suggest that inhibition of the TGF-β signaling pathway by use of Smad7 may represent a promising therapeutic strategy for ED induced by radical prostatectomy. Song KM, Chung J-S, Choi MJ, Jin H-R, Yin GN, Kwon M-H, Park J-M, Kim WJ, Lee S-J, Kim S-J, Ryu J-K, and Suh J-K. Effectiveness of intracavernous delivery of adenovirus encoding Smad7 gene on erectile function in a mouse model of cavernous nerve injury. J Sex Med 2014;11:51–63.

Introduction Erectile dysfunction (ED) is a highly prevalent complication of diabetes, and the severity of endothelial dysfunction is one of the most important factors in reduced responsiveness to oral phosphodiesterase type 5 inhibitors. Aim To study the effects of human angiopoietin‐4 (Ang‐4) protein on erectile function in diabetic mice. Methods Diabetes was induced by intraperitoneal injection of streptozotocin into 8‐week‐old C57BL/6J male mice. At 8 weeks after the induction of diabetes, the animals were divided into four groups: control nondiabetic mice and diabetic mice receiving two successive intracavernous injections of phosphate buffered saline (days −3 and 0), a single intracavernous injection of Ang‐4 protein (day 0), or two successive intracavernous injections of Ang‐4 protein (days −3 and 0). Main Outcome Measures One week after treatment, we measured erectile function by electrical stimulation of the cavernous nerve. The penis was harvested and stained with hydroethidine or antibodies to Ang‐4, platelet/endothelial cell adhesion molecule‐1, and phosphorylated endothelial nitric oxide synthase (eNOS). We also determined the differential expression of Ang‐4 in cavernous tissue in the control and diabetic mice. The effect of Ang‐4 protein on the phosphorylation of Tie‐2, Akt, and eNOS was determined in human umbilical vein endothelial cells (HUVECs) by Western blot. Results The cavernous expression of Ang‐4 was downregulated in diabetic mice; Ang‐4 was mainly expressed in endothelial cells. Local delivery of Ang‐4 protein significantly increased cavernous endothelial content, induced eNOS phosphorylation, and decreased the generation of superoxide anion and apoptosis in diabetic mice. Ang‐4 protein strongly increased the phosphorylation of Tie‐2, Akt, and eNOS in HUVECs. Repeated intracavernous injections of Ang‐4 induced significant restoration of erectile function in diabetic mice (87% of control values), whereas a single intracavernous injection of Ang‐4 protein elicited modest improvement. Conclusions Cavernous endothelial regeneration by use of Ang‐4 protein may have potential for the treatment of vascular disease‐induced ED, such as diabetic ED. Kwon M‐H, Ryu J‐K, Kim WJ, Jin H‐R, Song K‐M, Kwon K‐D, Batbold D, Yin GN, Koh GY, and Suh J‐K. Effect of intracavernous administration of angiopoietin‐4 on erectile function in the streptozotocin‐induced diabetic mouse. J Sex Med 2013;10:2912–2927.

Introduction Much attention has recently been focused on therapeutic angiogenesis as a treatment for erectile dysfunction (ED). The apelin and apelin receptor (APJ) system is known to cause endothelium‐dependent vasodilatation and to be involved in angiogenesis. Aim To examine the differential expression of apelin and APJ in animal models of vasculogenic ED and to determine whether and how enhancement of apelin–APJ signaling restores erectile function in hypercholesterolemic mice. Methods Acute cavernous ischemia was induced in C57BL/6J mice by bilateral occlusion of internal iliac arteries, and chronic vasculogenic ED was induced by feeding a high‐cholesterol diet or by intraperitoneal injection of streptozotocin. Main Outcome Measures Messenger RNA (mRNA) levels of apelin and APJ were determined in cavernous tissue of each vasculogenic ED model by semiquantitative reverse transcriptase‐polymerase chain reaction (RT‐PCR). We evaluated erectile function by electrical stimulation of the cavernous nerve in hypercholesterolemic mice 1, 3, 7, and 14 days after a single intracavernous injection of apelin protein (5 μg/20 μL). The penis was harvested for histologic examinations and Western blot analysis. Results The cavernous mRNA expression of apelin and APJ was up‐regulated in acute ischemia model and down‐regulated in chronic vasculogenic ED models. A significant restoration of erectile function was noted 1 day after injection of apelin protein into the penis of hypercholesterolemic mice; however, erectile function returned to baseline values thereafter. The beneficial effects of apelin on erectile function resulted mainly from an activation of endothelial nitric oxide synthase and increase in nitric oxide bioavailability through reduction in reactive oxygen species‐mediated endothelial apoptosis rather than through direct endothelial cell proliferation. Conclusion These findings suggest that apelin–APJ signaling is a potential therapeutic target in the treatment of vasculogenic ED. Further studies are needed to develop a potent agonist for APJ and to determine the role of repeated dosing of apelin on long‐term recovery of erectile function. Kwon M‐H, Tuvshintur B, Kim WJ, Jin H‐R, Yin GN, Song K‐M, Choi MJ, Kwon K‐D, Batbold D, Ryu J‐K, and Suh J‐K. Expression of the apelin–APJ pathway and effects on erectile function in a mouse model of vasculogenic erectile dysfunction. J Sex Med 2013;10:2928–2941.

INTRODUCTION AND OBJECTIVES: Patients with diabetic erectile dysfunction (ED) often have severe endothelial dysfunction, which results in poor response to oral phosphodiesterase-5 inhibitors. Nerve injury-induced protein 1, Ninjurin-1 (Ninj1) has been known to be up-regulated after peripheral nerve injury and to be related to vascular regression during embryonic period. In the present study, we determined whether and how Ninjurin 1 neutralizing antibody (Ninj1-Ab) restores erectile function in diabetic animals. METHODS: Five groups of mice were used: control nondiabetic mice, streptozotocin-induced diabetic mice, and diabetic mice treated with a single intracavernous injection of IgG control antibody or Ninj1-Ab (1.0 g or 2.5 g/20 l, respectively). Two weeks later, erectile function was measured by electrical stimulation of cavernous nerve. The penis was then harvested and stained with hydroethidine or antibodies to PECAM-1, phosphohistone H3, and nitrotyrosine. We performed western blot analysis of angiopoietin-1 (Ang1), angiopoietin-2 (Ang2), Akt, phospho-Akt, eNOS, phospho-eNOS, p47phox, and iNOS. RESULTS: Local delivery of Ninj1-Ab significantly increased cavernous endothelial content through enhanced endothelial cell proliferation, and induced phosphorylation of Akt and eNOS compared with that in the untreated or IgG-treated diabetic group. Endothelial protective effects, such as decreased expression of p47phox and inducible NOS, and decrease in the generation of reactive oxygen species including superoxide anion and peroxinitrite, and decrease in the number of apoptotic cells in the cavernous endothelium, was noted in the diabetic mice treated with Ninj1-Ab. The Ang1 expression was down-regulated and Ang2 expression was up-regulated in the diabetic penis compared with that in controls, which was reversed by treatment with Ninj1-Ab. High-dose of Ninj1-Ab (2.5 g/20 l) induced profound restoration of erectile function in the diabetic mice (up to 90% of the control values), whereas low-dose of Ninj1-Ab (1 g/20 l) elicited partial improvement. CONCLUSIONS: Cavernous endothelial regeneration by using Ninj1-Ab is a novel therapeutic strategy for the treatment of vasculogenic ED.

Erectile dysfunction is often a harbinger of cardiovascular disease. We sought to gain mechanistic insight at the cellular and molecular levels into why erectile dysfunction precedes the clinical consequences of cardiovascular disease.Diabetes was induced by intraperitoneal streptozotocin injection in 8-week-old C57BL/6J mice. At 8 weeks after diabetes induction, we determined the expression of endothelial cell-cell junction proteins and vascular endothelial permeability in the penis, heart and hind limb by systemic injection of various vascular space markers (350 Da to 2,000 kDa) or by immunohistochemical staining with antibody to oxidized low density lipoprotein. We also investigated the effect of recombinant Ang1 protein on cavernous endothelial permeability.Alterations in the integrity of the endothelial cell-cell junction, including a decrease in endothelial cell-cell junction proteins and an increase in vascular permeability to fluorescent tracers or oxidized low density lipoprotein, were prominent in the cavernous tissue of diabetic mice. In contrast, no significant changes in endothelial cell-cell junction proteins or vascular permeability were noted in heart or hind limb tissue according to the diabetic condition. Intracavernous injection of Ang1 protein, an anti-permeability factor, significantly decreased cavernous endothelial permeability to oxidized low density lipoprotein by restoring endothelial cell-cell junction proteins in diabetic mice.The incompetent cavernous endothelial cell-cell junction in the diabetic condition provides an important clue to why erectile dysfunction is highly prevalent and often precedes other systemic vascular diseases.

Summary A prerequisite for the successful clinical application of gene therapy in erectile dysfunction (ED) is the availability of safe and efficient gene delivery systems. The aim of this study was to examine the effectiveness of guanidinylated bioreducible polymer (GBP) polyplexes for gene delivery systems, which take advantage of the biodegradability of reducible disulfide bonds and the cell-penetrating ability of guanidine groups. For in vitro transfection experiments, we used mouse cavernous endothelial cells and A7r5 rat vascular smooth muscle cells. For in vivo experiments, we used a mouse model of hypercholesterolaemic ED in which 2-month-old male C57BL/6 mice were fed a diet containing 4% cholesterol and 1% cholic acid for 3 months. Animals or cells were treated with pCMV-Luc, poly(ethyleneimine) (PEI)25k/pCMV-Luc polyplex (weight ratio: 1) and GBP/pCMV-Luc polyplexes (weight ratio: 20, 40, 60 and 80). Gene expression was evaluated by luciferase assay, and the gene expression area was evaluated by immunohistochemistry. GBP had greater transfection efficiency as the weight ratio increased. GBP had sevenfold higher gene delivery efficiency in A7r5 cells at a weight ratio of 80 than did PEI25k. Moreover, the gene expression was more profoundly induced by GBP/pCMV-Luc than by pCMV-Luc in both the corpus cavernosum tissue of hypercholesterolaemic mice and in mouse cavernous endothelial cells, although the expression levels induced by the GBP gene delivery system were lower than those induced by the PEI25k gene delivery system. GBP revealed no considerable cytotoxicity to A7r5 cells and mouse cavernous endothelial cells (relative cell viability: 95 and 88% respectively), whereas PEI25k resulted in high cytotoxicity. Interestingly, immunofluorescent double staining revealed that luciferase expression induced by the GBP polyplex mainly overlapped with cavernous endothelial cells, but rarely with smooth muscle cells. The GBP-based non-viral gene expression system may be useful for the development of gene therapy in vasculogenic ED.

Cavernous hypoxia is an important factor in the pathogenesis of vasculogenic erectile dysfunction (ED). Therefore, the hypoxia-inducible gene expression system can be exploited as gene therapy for vasculogenic ED. This study was undertaken to examine the effectiveness of a hypoxia-inducible gene expression system, namely, the RTP801 promoter or the erythropoietin enhancer, in a mouse model of hypercholesterolemic ED in vivo and in primary cultured mouse cavernous endothelial cells in vitro. Two-month-old male C57BL/6 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. Mouse cavernous endothelial cells were isolated and cultured under normoxic or hypoxic conditions. After treatment of animals or endothelial cells with pSV-Luc, pRTP801-Luc, or pEpo-SV-Luc vector, gene expression was evaluated by luciferase assay, and the gene expression area was evaluated by immunohistochemistry. Plasmids pRTP801-Luc and pEpo-SV-Luc induced gene expression significantly in the hypercholesterolemic mice and in cavernous endothelial cells under hypoxia, and the highest gene expression was noted in the group treated with pEpo-SV-Luc. Gene expression was higher for more than 7 days in the hypercholesterolemic mice injected with pEpo-SV-Luc than in mice injected with pSV-Luc. As shown by immunohistochemistry, the gene expression area was also greater in the pEpo-SV-Luc group than in the pSV-Luc group, but the difference was not as great as that in luciferase activity. The hypoxia-specific gene expression system could be a valuable tool for facilitating gene delivery into ischemic corpus cavernosum tissue resulting from vascular causes.

OBJECTIVE Patients with diabetic erectile dysfunction often have severe endothelial dysfunction and respond poorly to oral phosphodiesterase-5 inhibitors. We examined the effectiveness of the potent angiopoietin-1 (Ang1) variant, cartilage oligomeric matrix protein (COMP)-Ang1, in promoting cavernous endothelial regeneration and restoring erectile function in diabetic animals. RESEARCH DESIGN AND METHODS Four groups of mice were used: controls; streptozotocin (STZ)-induced diabetic mice; STZ-induced diabetic mice treated with repeated intracavernous injections of PBS; and STZ-induced diabetic mice treated with COMP-Ang1 protein (days −3 and 0). Two and 4 weeks after treatment, we measured erectile function by electrical stimulation of the cavernous nerve. The penis was harvested for histologic examinations, Western blot analysis, and cGMP quantification. We also performed a vascular permeability test. RESULTS Local delivery of the COMP-Ang1 protein significantly increased cavernous endothelial proliferation, endothelial nitric oxide (NO) synthase (NOS) phosphorylation, and cGMP expression compared with that in the untreated or PBS-treated STZ-induced diabetic group. The changes in the group that received COMP-Ang1 restored erectile function up to 4 weeks after treatment. Endothelial protective effects, such as marked decreases in the expression of p47phox and inducible NOS, in the generation of superoxide anion and nitrotyrosine, and in the number of apoptotic cells in the corpus cavernosum tissue, were noted in COMP-Ang1–treated STZ-induced diabetic mice. An intracavernous injection of COMP-Ang1 completely restored endothelial cell-cell junction proteins and decreased cavernous endothelial permeability. COMP-Ang1–induced promotion of cavernous angiogenesis and erectile function was abolished by the NOS inhibitor, N-nitro-L-arginine methyl ester, but not by the NADPH oxidase inhibitor, apocynin. CONCLUSIONS These findings support the concept of cavernous endothelial regeneration by use of the recombinant Ang1 protein as a curative therapy for diabetic erectile dysfunction.

Introduction Transforming growth factor-β1 (TGF-β1) 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-β1 and the Smad transcription factor, the key molecule for the initiation of TGF-β-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-β1 and phospho-Smad2. Main Outcome Measures Semi-quantitative analysis of TGF-β1 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-β1 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-β1 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-β1 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. Shin T-Y, Ryu J-K, Jin H-R, Piao S, Tumurbaatar M, Yin GN, Shin SH, Kwon M-H, Song K-M, Fang Z-H, Han J-Y, Kim WJ, and Suh J-K. Increased cavernous expression of transforming growth factor-β1 and activation of the smad signaling pathway affects erectile dysfunction in men with spinal cord injury.

Introduction Transforming growth factor-β1 (TGF-β1) 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-β1 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-β, 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-β1 (10 ng/mL). Main Outcome Measures The plaque or tunica albuginea tissue was stained with Masson's trichrome or antibody to TGF-β1, 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-β1-induced nuclear translocation of Smad2/3 in fibroblasts. Results The plaque tissue from PD patients showed higher TGF-β1, P-Smad2, and P-Smad3 immunoreactivity than did the tunica albuginea tissue from control patients. SKI2162 not only blocked TGF-β1-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-β and Smads in the pathogenesis of PD, pharmacologic inhibition of ALK5 may represent a novel targeted approach to treating PD. Piao S, Choi MJ, Tumurbaatar M, Kim WJ, Jin H-R, Shin SH, Tuvshintur B, Yin GN, Song JS, Kwon M-H, Lee S-J, Han J-Y, Kim S-J, Ryu J-K, and Suh J-K. Transforming growth factor (TGF)-β type I receptor kinase (ALK5) inhibitor alleviates profibrotic TGF-β1 responses in fibroblasts derived from Peyronie's plaque.

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 × 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 α‐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. Jin H‐R, Kim WJ, Song JS, Choi MJ, Piao S, Shin SH, Tumurbaatar M, Tuvshintur B, Nam M‐S, Ryu J‐K, and Suh, J‐K. Functional and morphologic characterizations of the diabetic mouse corpus cavernosum: comparison of a multiple low‐dose and a single high‐dose streptozotocin protocols.

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. Ryu J-K, Zhang LW, Jin H-R, Piao S, Choi MJ, Tuvshintur B, Tumurbaatar M, Shin SH, Han J-Y, Kim WJ, and Suh J-K. Derangements in endothelial cell-to-cell junctions involved in the pathogenesis of hypercholesterolemia-induced erectile dysfunction. J Sex Med 2009;6:1893–1907.

Purpose: To examine the effectiveness of small-molecule inhibitor of transforming growth factor-β (TGF-β) type I receptor, an activin receptor-like kinase 5 (ALK5), on erectile dysfunction (ED) in a rat model of cavernous fibrosis, in which fibrosis was induced by intracavernous injection of adenovirus expressing TGF-β1 (Ad-TGF-β1). Materials and Methods: Four-month-old Sprague-Dawley rats were divided into four groups (n=10 per group): age-matched controls without treatment, age-matched controls receiving intracavernous injection of LacZ adenovirus, and cavernous fibrosis rats receiving an intracavernous injection of saline or ALK5 inhibitor (5 mg/kg). ALK5 inhibitor or saline was administered on day 5 after injection of Ad-TGF-β1. On day 30, erectile function was assessed by electrical stimulation of the cavernous nerve and the penis was then harvested for histologic studies (n=6 per group) and for the measurement of the hydroxyproline level (n=4 per group). Results: Ad-TGF-β1-induced cavernous fibrosis rats treated with saline showed a significant decrease in cavernous smooth muscle and endothelial content, and an increase in collagen deposition, which resulted in profound deterioration of all erectile function parameters, such as the ratios of maximal intracavernous pressure (ICP), total ICP, and slope to mean arterial pressure. ALK5 inhibitor significantly restored erectile function in a rat model of cavernous fibrosis by increasing cavernous smooth muscle and endothelial content, and by blocking cavernous fibrosis. Conclusions: The results suggest that inhibition of the TGF-β pathway is a promising therapeutic strategy for the treatment of ED related to cavernous fibrosis from various cause