Pericyte-Derived Extracellular Vesicle-Mimetic Nanovesicles Restore Erectile Function by Enhancing Neurovascular Regeneration in a Mouse Model of Cavernous Nerve Injury.

BACKGROUND: Extracellular vesicle (EV)-mimetic nanovesicles (NVs) from embryonic stem cells have been observed to stimulate neurovascular regeneration in the streptozotocin-induced diabetic mouse. Pericytes play important roles in maintaining penile erection, yet no previous studies have explored the effects of pericyte-derived NVs (PC-NVs) in neurovascular regeneration in the context of erectile dysfunction. AIM: To investigate the potential effect of PC-NVs in neurovascular regeneration. METHODS: PC-NVs were isolated from mouse cavernous pericytes, and neurovascular regeneration was evaluated in an in vitro study. Twelve-week-old C57BL/6J mice were used to prepare cavernous nerve injury model. Erectile function evaluation, histologic examination of the penis, and Western blots were assessed 2 weeks after model creation and PC-NVs treatment. OUTCOMES: The main outcomes of this study are PC-NVs characterization, intracavernous pressure, neurovascular regeneration in the penis, and in vitro functional evaluation. RESULTS: The PC-NVs were extracted and characterized by cryotransmission electron microscopy and EV-positive (Alix, TSG101, CD81) and EV-negative (GM130) markers. In the in vivo studies, PC-NVs successfully improved erectile function in cavernous nerve injury mice (∼82% of control values). Immunofluorescence staining showed significant increases in pericytes, endothelial cell, and neuronal contents. In the in vitro studies, PC-NVs significantly increased mouse cavernous endothelial cells tube formation, Schwann cell migration, and dorsal root ganglion and major pelvic ganglion neurite sprouting. Finally, Western blot analysis revealed that PC-NVs upregulated cell survival signaling (Akt and eNOS) and induced the expression of neurotrophic factors (brain-derived neurotrophic factor, neurotrophin-3, and nerve growth factor). CLINICAL IMPLICATIONS: PC-NVs may be used as a strategy to treat erectile dysfunction after radical prostatectomy or in men with neurovascular diseases. STRENGTHS & LIMITATIONS: We evaluated the effect of PC-NVs in vitro and in a mouse nerve injury model, cavernous nerve injury. Additional studies are necessary to determine the detailed mechanisms of neurovascular improvement. Further study is needed to test whether PC-NVs are also effective when given weeks or months after nerve injury. CONCLUSION: PC-NVs significantly improved erectile function by enhancing neurovascular regeneration. Local treatment with PC-NVs may represent a promising therapeutic strategy for the treatment of neurovascular diseases. Yin GN, Park S-H, Ock J, et al. Pericyte-Derived Extracellular Vesicle-Mimetic Nanovesicles Restore Erectile Function by Enhancing Neurovascular Regeneration in a Mouse Model of Cavernous Nerve Injury. J Sex Med 2020;17:2118-2128.

Inhibition of proNGF and p75NTR Pathway Restores Erectile Function Through Dual Angiogenic and Neurotrophic Effects in the Diabetic Mouse.

INTRODUCTION: Penile neurovascular dysfunction is a major cause of erectile dysfunction (ED) in diabetic patients, which causes poor response to oral phosphodiesterase-5 inhibitors. Nerve growth factor precursor (proNGF) and its p75 neurotrophin receptor (p75NTR) have been known to be involved in microvascular complications and neurodegeneration. AIM: To examine the role of proNGF and its receptor p75NTR signaling pathway in diabetic ED, and to determine the effectiveness of proNGF neutralizing antibody (proNGF-Ab) in restoring erectile function in streptozotocin (STZ)-induced diabetic mice. METHODS: Diabetes mellitus was induced by intraperitoneal injection of STZ (50 mg/kg) into 8-week-old C57BL/6 male mice for 5 consecutive days. At 8 weeks after the induction of diabetes mellitus, the animals were distributed into 3 groups: controls and STZ-induced diabetic mice receiving 2 intracavernous injections of either saline (days -3 and 0; 20 µL) or proNGF-Ab (days -3 and 0; 20 µg in 20 µL of saline). We also examined the effect of proNGF-Ab or p75NTR small interfering RNA in primary cultured mouse cavernous endothelial cells, pericytes, and major pelvic ganglion. MAIN OUTCOME MEASURES: Erectile function was measured by electrical stimulation of the cavernous nerve at 2 weeks after treatment, and the penis was then harvested for histologic and biochemical studies. RESULTS: The cavernous expression of proNGF and p75NTR was upregulated under diabetic conditions. Intracavernous injection of proNGF-Ab successfully restored erectile function in diabetic mice, which reach 93-96% of control values. ProNGF-Ab significantly restored cavernous endothelial cell, pericyte, and neuronal cell content, and increased endothelial cell-to-cell junction proteins in the diabetic mice. Under the high-glucose condition, proNGF-Ab or p75NTR small interfering RNA promoted tube formation in mouse cavernous endothelial cells and pericytes, decreased apoptosis of endothelial cells and pericytes, and enhanced neurite sprouting from major pelvic ganglion. CLINICAL IMPLICATIONS: The ProNGF/p75NTR pathway will be a new therapeutic target for diabetic ED. STRENGTH & LIMITATIONS: This is the first study demonstrating the efficacy of the inhibition of proNGF/p75NTR pathway in diabetic ED. Further studies are needed to test whether a different dosing of proNGF-Ab would induce more durable erectile function recovery. CONCLUSION: Our findings suggest that inhibition of the proNGF/p75NTR signaling pathway is a promising therapeutic strategy for diabetic ED. Nguyen NM, Song K-M, Choi M-J, et al. Inhibition of proNGF and p75NTR Pathway Restores Erectile Function Through Dual Angiogenic and Neurotrophic Effects in the Diabetic Mouse. J Sex Med 2019;16:351-364.

Inhibition of Ninjurin 1 restores erectile function through dual angiogenic and neurotrophic effects in the diabetic mouse.

Penile erection is a neurovascular phenomenon, and erectile dysfunction (ED) is caused mainly by vascular risk factors or diseases, neurologic abnormalities, and hormonal disturbances. Men with diabetic ED often have severe endothelial dysfunction and peripheral nerve damage, which result in poor response to oral phosphodiesterase-5 inhibitors. Nerve injury-induced protein 1 (Ninjurin 1, Ninj1) is known to be involved in neuroinflammatory processes and to be related to vascular regression during the embryonic period. Here, we demonstrate in streptozotocin-induced diabetic mice that inhibition of the Ninj1 pathway by administering Ninj1-neutralizing antibody (Ninj1-Ab) or by using Ninj1-knockout mice successfully restored erectile function through enhanced penile angiogenesis and neural regeneration. Angiopoietin-1 (Ang1) expression was down-regulated and angiopoietin-2 expression was up-regulated in the diabetic penis compared with that in controls, and these changes were reversed by treatment with Ninj1-Ab. Ninj1 blockade-mediated penile angiogenesis and neural regeneration as well as recovery of erectile function were abolished by inhibition of Ang1-Tie2 (tyrosine kinase with Ig and epidermal growth factor homology domain-2) signaling with soluble Tie2 antibody or Ang1 siRNA. The present results suggest that inhibition of the Ninj1 pathway will be a novel therapeutic strategy for treating ED.

Sac-1004, a Pseudo-Sugar Derivative of Cholesterol, Restores Erectile Function through Reconstruction of Nonleaky and Functional Cavernous Angiogenesis in the Streptozotocin Induced Diabetic Mouse.

PURPOSE: We examined whether and how Sac-1004, a vascular leakage blocker, would restore erectile function in an animal model of diabetic erectile dysfunction. MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Effectiveness of Intracavernous Delivery of Recombinant Human Hepatocyte Growth Factor on Erectile Function in the Streptozotocin-Induced Diabetic Mouse.

INTRODUCTION: Diabetic erectile dysfunction is a disease mostly of vascular origin and men with diabetic erectile dysfunction respond poorly to oral phosphodiesterase-5 inhibitors. Hepatocyte growth factor (HGF) is a pleiotropic factor that plays an essential role in the regulation of cell proliferation, survival, and angiogenesis. AIM: To determine the effectiveness of recombinant human (rh)-HGF in restoring erectile function in diabetic mice. METHODS: Four groups of mice were used: control non-diabetic mice and streptozotocin-induced diabetic mice receiving two successive intracavernous injections of phosphate buffered saline (days -3 and 0), a single intracavernous injection of rh-HGF (day 0), or two successive intracavernous injections of rh-HGF (days -3 and 0). We also examined the effect of rh-HGF in primary cultured mouse cavernous endothelial cells and in major pelvic ganglion culture in vitro, which was incubated under a normal-glucose (5 mmol/L) or a high-glucose (30 mmol/L) condition. MAIN OUTCOME MEASURES: Two weeks after treatment, we measured erectile function by electrical stimulation of the cavernous nerve and the penis was harvested for histologic studies. RESULTS: Repeated intracavernous injections of rh-HGF protein induced significant restoration of erectile function in diabetic mice (89-100% of control values), whereas a single intracavernous injection of rh-HGF protein elicited modest improvement. Rh-HGF significantly induced phosphorylation of its receptor c-Met, increased the content of endothelial cells and smooth muscle cells, and decreased the generation of reactive oxygen species (superoxide anion and peroxynitrite) and extravasation of oxidized low-density lipoprotein in diabetic mice. Under the high-glucose condition, rh-HGF protein also promoted tube formation in mouse cavernous endothelial cells and enhanced neurite sprouting in major pelvic ganglion culture in vitro. CONCLUSION: The dual angiogenic and neurotrophic effects of HGF could open a new avenue through which diabetic erectile dysfunction can be treated.

Penile erection induces angiogenic, survival, and antifibrotic signals: molecular events associated with penile erection induced by cavernous nerve stimulation in mice.

OBJECTIVES: To determine the molecular events related to penile erection in the corpus cavernosum tissue of mice after electrical stimulation of the cavernous nerve. METHODS: Twelve-week-old male C57BL/6 mice were used in this study. Electrical stimulation of the cavernous nerve was carried out to induce penile erection. Corpus cavernosum tissues were then harvested to determine the effect of nerve-induced penile erection on signaling pathway involved in angiogenesis (vascular endothelial growth factor, hepatocyte growth factor, angiopoietin-1, matrix metalloproteinase 2, and matrix metalloproteinase 9), cell survival and proliferation (phosphatidylinositol 3-kinase, phospho-Akt/Akt, and phospho-ERK/ERK), and tissue fibrosis (phospho-Smad2/Smad2, phospho-Smad3/Smad3, and plasminogen activator inhibitor-1). RESULTS: Cavernous nerve stimulation enhanced the expression of factors involved in angiogenesis (vascular endothelial growth factor, hepatocyte growth factor, angiopoietin-1, matrix metalloproteinase 2, and metalloproteinase 9), and activated intracellular signaling mediators related to cell survival and proliferation (phosphatidylinositol 3-kinase, phospho-Akt/Akt, and phospho-ERK/ERK), while suppressing the pathways involved in tissue fibrosis (phospho-Smad2/Smad2, phospho-Smad3/Smad3, and plasminogen activator inhibitor-1). CONCLUSIONS: Penile erection in mice is accompanied by the activation of a cascade of signaling pathways involved in angiogenesis, cell survival and proliferation, and antifibrosis. The present results might provide a theoretical and molecular basis for understanding the importance of penile rehabilitation and subsequent restoration of nocturnal or sexually-mediated penile erections.

Xenogenic transplantation of human breast adipose-derived stromal vascular fraction enhances recovery of erectile function in diabetic mice.

The adipose tissue-derived stromal vascular fraction (SVF) is an ideal source of stem and stromal cells. The aim of this study was to examine whether and how xenogenic transplantation of human breast SVF restores erectile function in diabetic mice. Human SVF was isolated from five patients (age, 20-45 yr) undergoing reduction mammoplasty. Eight-week-old C57BL/6J mice were used, and diabetes was induced by intraperitoneal injection of streptozotocin. At 8 wk after induction of diabetes, the animals were randomly distributed into controls and diabetic mice treated with a single intracavernous injection of PBS, human SVF at different concentrations, or human SVF lysate. Two weeks later, erectile function was measured by cavernous nerve stimulation, and the penis was then harvested for biochemical examinations. Erectile function was significantly improved in diabetic mice treated with human SVF (2 × 10(5), 5 × 10(5), and 1 × 10(6) cells/20 µl) and SVF lysate. Human SVF treatment in diabetic mice significantly increased cavernous endothelial and smooth muscle cell contents, induced eNOS phosphorylation, and restored penile nNOS-positive nerve fibers. Human SVF lysate induced secretion of angiogenic factors and expression of their receptors. Human SVF did not increase serum levels of proinflammatory cytokines. A limitation of this study was that the exact composition of the human SVF was not examined. In summary, xenogenic transplantation of human SVF did not induce systemic inflammation and successfully improved erectile function in diabetic mice through enhanced penile angiogenesis and neural regeneration.

Silencing histone deacetylase 2 using small hairpin RNA induces regression of fibrotic plaque in a rat model of Peyronie's disease.

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 × 10(8) pfu/0.1 mL phosphate-buffered saline [PBS]); and PD rats receiving a single injection of Ad-HDAC2 shRNA (day 15; 1 × 10(8) 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)-ß1 (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-ß1-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.

Intracavernous delivery of clonal mesenchymal stem cells restores erectile function in a mouse model of cavernous nerve injury.

INTRODUCTION: Recently, much attention has focused on stem cell therapy; bone marrow-derived stem cells (BMSCs) are one of the most studied mesenchymal stem cells used in the field of erectile dysfunction (ED). However, a major limitation for the clinical application of stem cell therapy is the heterogeneous nature of the isolated cells, which may cause different treatment outcomes. AIM: We investigated the effectiveness of mouse clonal BMSCs obtained from a single colony by using subfractionation culturing method (SCM) for erectile function in a mouse model of cavernous nerve injury (CNI). METHODS: Twelve-week-old C57BL/6J mice were divided into four groups: sham operation group, bilateral CNI group receiving a single intracavernous (IC) injection of phosphate-buffered saline (20 µL) or clonal BMSCs (3 × 10(5) cells/20 µL), and receiving a single intraperitoneal (IP) injection of clonal BMSCs (3 × 10(5) cells/20 µL). MAIN OUTCOME MEASURES: The clonal BMSC line was analyzed for cell-surface epitopes by using fluorescence-activated cell sorting and for differentiation potential. Two weeks after CNI and treatment, erectile function was measured by electrically stimulating the cavernous nerve. The penis was harvested for histologic examinations and Western blot analysis. RESULTS: Clonal BMSCs expressed cell surface markers for mesenchymal stem cells and were capable of differentiating into several lineages, including adipogenic, osteogenic, and chondrogenic cells. Both IC and IP injections of clonal BMSCs significantly restored cavernous endothelial and smooth muscle content, and penile nNOS and neurofilament content in CNI mice. IC injection of clonal BMSCs induced significant recovery of erectile function, which reached 90-100% of the sham control values, whereas IP injection of clonal BMSCs partially restored erectile function. CONCLUSION: We established a homogeneous population of mouse clonal BMSCs using SCM; clonal BMSCs successfully restored erectile function in CNI mice. The homogeneous nature of clonal mesenchymal stem cells may allow their clinical applications.

Intracavernous delivery of stromal vascular fraction restores erectile function through production of angiogenic factors in a mouse model of cavernous nerve injury.

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.

Effectiveness of intracavernous delivery of adenovirus encoding Smad7 gene on erectile function in a mouse model of cavernous nerve injury.

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-ß1 (TGF-ß1) 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-ß signaling pathway by use of Smad7 may represent a promising therapeutic strategy for ED induced by radical prostatectomy.

Erectile dysfunction precedes other systemic vascular diseases due to incompetent cavernous endothelial cell-cell junctions.

PURPOSE: 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. MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Effect of intracavernous administration of angiopoietin-4 on erectile function in the streptozotocin-induced diabetic mouse.

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.

Expression of the apelin-APJ pathway and effects on erectile function in a mouse model of vasculogenic erectile dysfunction.

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.

Nerve injury-induced protein 1 (Ninjurin-1) is a novel therapeutic target for cavernous nerve injury-induced erectile dysfunction in mice.

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 injury-induced protein 1, Ninjurin-1 (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 (Ninj1-Ab) restores erectile function in mice with CNI. METHODS: Twelve-week-old 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, low-dose Ninj1-Ab (1.0 µg/20 µL), or high-dose Ninj1-Ab (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 Ninj1-Ab 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 angiopoietin-1 and downregulating angiopoietin-2. High-dose Ninj1-Ab induced profound restoration of erectile function in the CNI mice (91% of sham control values), whereas low-dose Ninj1-Ab 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.

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.

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-ß1 (TGF-ß1) 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.

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.

Vasohibin-1 rescues erectile function through up-regulation of angiogenic factors in the diabetic mice.

Neovascularization of the erectile tissue emerges as a beneficial curative approach to treat erectile dysfunction (ED). Here we for the first time report the unexpected role of vasohibin-1 (VASH1), mainly known as an anti-angiogenic factor, in restoring erectile function in diabetic mice. A diabetic patient has lower cavernous VASH1 expression than in the potent man. VASH1 was mainly expressed in endothelial cells. There were significant decreases in cavernous endothelial cell and pericyte contents in VASH1 knockout mice compared with those in wild-type mice, which resulted in impairments in erectile function. Intracavernous injection of VASH1 protein successfully restored erectile function in the diabetic mice (~ 90% of control values). VASH1 protein reinstated endothelial cells, pericytes, and endothelial cell-cell junction proteins and induced phosphorylation of eNOS (Ser1177) in the diabetic mice. The induction of angiogenic factors, such as angiopoietin-1 and vascular endothelial growth factor, is responsible for cavernous angiogenesis and the restoration of erectile function mediated by VASH1. Altogether, these findings suggest that VASH1 is proangiogenic in diabetic penis and is a new potential target for diabetic ED.

Three-Dimensional Reconstruction of Neurovascular Network in Whole Mount Preparations and Thick-Cut Transverse Sections of Mouse Urinary Bladder.

PURPOSE: Proper functional and structural integrity of nervous and vascular system in urinary bladder plays an important role in normal bladder function and the disruption of these structures is known to be related to lower urinary tract symptoms. Here, we present an immunohistochemical staining method that delineates neurovascular structures in the mouse urinary bladder by using immunohistochemical staining with three-dimensional reconstruction. MATERIALS AND METHODS: The urinary bladder was harvested from 8-week-old C57BL/6 male mouse. Lamina propria and detrusor muscle layer were dissected for whole mount staining, and thick-cut (60-µm) sections were prepared for full-thickness bladder staining. Immunofluorescent staining of bladder tissue was performed with antibodies against CD31 (an endothelial cell marker), smooth muscle α-actin (a smooth muscle cell marker), NG2 (a pericyte marker), and ßIII-tubulin (a neuronal marker). We reconstructed three-dimensional images of bladder neurovascular system from stacks of two-dimensional images. RESULTS: Three-dimensional images obtained from thick-cut sections clearly provided good anatomic information about neurovascular structures in the three layers of bladder, such as urothelium, lamina propria, and detrusor muscle layer. Whole mount images of lamina propria and detrusor muscle layer also clearly delineated spatial relationship between nervous and vascular systems. The microvessel density was higher in the lamina propria than in the detrusor muscle layer. Nerve fibers were evenly innervated into the lamina propria and detrusor muscle. CONCLUSIONS: This study provides comprehensive insight into three-dimensional neurovascular structures of mouse urinary bladder. Our technique may constitute a standard tool to evaluate pathologic changes in a variety of urinary bladder diseases.