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Objectives To examine the therapeutic effect of adenovirus encoding histone deacetylase 2 (HDAC2) small hairpin RNA (Ad-HDAC2 shRNA) in a rat model of Peyronie's disease (PD) and to determine the mechanisms by which HDAC2 knockdown ameliorates fibrotic responses in primary fibroblasts derived from human PD plaque. Materials and Methods Rats were distributed into four groups (n = 6 per group): age-matched controls without treatment; rats in which PD has been induced (PD rats) without treatment; PD rats receiving a single injection of control adenovirus encoding scrambled small hairpin RNA (Ad-shRNA) (day 15; 1 × 108 pfu/0.1 mL phosphate-buffered saline [PBS]); and PD rats receiving a single injection of Ad-HDAC2 shRNA (day 15; 1 × 108 pfu/0.1 mL PBS) into the lesion. PD-like plaque was induced by repeated intratunical injections of 100 μL each of human fibrin and thrombin solutions on days 0 and 5. On day 30, the penis was harvested for histological examination. Fibroblasts isolated from human PD plaque were pretreated with HDAC2 small interfering (si)RNA (100 pmoL) and then stimulated with transforming growth factor (TGF)-β1 (10 ng/mL) to determine hydroxyproline levels, procollagen mRNA, apoptosis and protein expression of poly(ADP-ribose) polymerase 1 (PARP1) and cyclin D1. Results We observed that Ad-HDAC2 shRNA decreased inflammatory cell infiltration, reduced transnuclear expression of phospho-Smad3 and regressed fibrotic plaque of the tunica albuginea in PD rats in vivo. siRNA-mediated silencing of HDAC2 significantly decreased the TGF-β1-induced transdifferentiation of fibroblasts into myofibroblasts and collagen production, and induced apoptosis by downregulating the expression of PARP1, and decreased the expression of cyclin D1 (a positive cell-cycle regulator) in primary cultured fibroblasts derived from human PD plaque in vitro. Conclusion Specific inhibition of HDAC2 with RNA interference may represent a novel targeted therapy for PD.

Epigenetic modifications, such as histone acetylation/deacetylation, have been shown to play a role in the pathogenesis of fibrotic disease. Peyronie's disease (PD) is a localized fibrotic process of the tunica albuginea, which leads to penile deformity. This study was undertaken to determine the anti-fibrotic effect of small interfering RNA (siRNA)-mediated silencing of histone deacetylase 2 (HDAC2) in primary fibroblasts derived from human PD plaque. PD fibroblasts were pre-treated with HDAC2 siRNA and then stimulated with transforming growth factor-β1 (TGF-β1). Protein was extracted from treated fibroblasts for Western blotting and the membranes were probed with antibody to phospho-Smad2/Smad2, phospho-Smad3/Smad3, smooth muscle α-actin and extracellular matrix proteins, including plasminogen activator inhibitor-1, fibronectin, collagen I and collagen IV. We also performed immunocytochemistry to detect the expression of extracellular matrix proteins and to examine the effect of HDAC2 siRNA on the TGF-β1-induced nuclear translocation of Smad2/3 in fibroblasts. Knockdown of HDAC2 in PD fibroblasts abrogated TGF-β1-induced extracellular matrix production by blocking TGF-β1-induced phosphorylation and nuclear translocation of Smad2 and Smad3, and by inhibiting TGF-β1-induced transdifferentiation of fibroblasts into myofibroblasts. Decoding the individual function of the HDAC isoforms by use of siRNA technology, preferably siRNA for HDAC2, may lead to the development of specific and safe epigenetic therapies for PD.

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.

We examined whether and how Sac-1004, a vascular leakage blocker, would restore erectile function in an animal model of diabetic erectile dysfunction.Eight-week-old C57BL/6J mice were used. Diabetes was induced by intraperitoneal injection of streptozotocin. Eight weeks after diabetes induction the animals were divided into 6 groups, including controls, diabetic mice that received repeat intracavernous injections of phosphate buffered saline (20 μl) on days -3 and 0, and diabetic mice that received repeat intracavernous injections of Sac-1004 on days -3 and 0 (1, 2, 5 and 10 μg, respectively, in 20 μl phosphate buffered saline). One week after injection erectile function was measured by cavernous nerve stimulation. The penis was then harvested for histological examinations and Western blot analysis.Local delivery of Sac-1004 in the corpus cavernosum restored erectile function in diabetic mice. The highest erectile response was noted at a dose of 5 μg with a response comparable to that in the control group. Sac-1004 significantly increased cavernous endothelial and smooth muscle contents, and induced endothelial nitric oxide synthase phosphorylation (Ser1177). Sac-1004 decreased extravasation of oxidized low density lipoprotein by restoring endothelial cell-cell junction proteins and pericyte content. Sac-1004 also promoted tube formation in primary cultured mouse cavernous endothelial cells in vitro. Sac-1004 mediated cavernous angiogenesis and erectile function recovery was abolished by inhibiting angiopoietin-1-Tie2 signaling with soluble Tie2 antibody.With the effects of angiogenesis and antipermeability Sac-1004 reestablishes structural and functional cavernous sinusoids. This is highly promising for future treatment of erectile dysfunction from vascular causes.

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 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.