Macrophages in trigeminal ganglion contribute to ectopic mechanical hypersensitivity following inferior alveolar nerve injury in rats.

BACKGROUND: Accidental mandibular nerve injury may occur during tooth extraction or implant procedures, causing ectopic orofacial pain. The exact mechanisms underlying ectopic orofacial pain following mandibular nerve injury is still unknown. Here, we investigated the role of macrophages and tumor necrosis factor alpha (TNFα) in the trigeminal ganglion (TG) in ectopic orofacial pain following inferior alveolar nerve transection (IANX). METHODS: IANX was performed and the mechanical head-withdrawal threshold (MHWT) in the whisker pad skin ipsilateral to IANX was measured for 15 days. Expression of Iba1 in the TG was examined on day 3 after IANX, and the MHWT in the whisker pad skin ipsilateral to IANX was measured following successive intra-ganglion administration of the macrophage depletion agent liposomal clodronate Clophosome-A (LCCA). TNFα expression in the TG and the MHWT in the whisker pad skin ipsilateral to IANX following successive intra-ganglion administration of the TNFα blocker etanercept were measured on day 3 after IANX, and tumor necrosis factor receptor-1 (TNFR1) immunoreactive (IR) cells in the TG were analyzed immunohistochemically on day 3. RESULTS: The MHWT in the whisker pad skin was significantly decreased for 15 days, and the number of Iba1-IR cells was significantly increased in the TG on day 3 after IANX. Successive intra-ganglion administration of the macrophage depletion agent LCCA significantly reduced the increased number of Iba1-IR cells in the TG and reversed the IANX-induced decrease in MHWT in the whisker pad skin. TNFα expression was increased in the TG on day 3 after IANX and was reduced following successive intra-ganglion administration of the TNFα inhibitor etanercept. The decreased MHWT was also recovered by etanercept administration, and TNFR1-IR cells in the TG were increased on day 3 following IANX. CONCLUSIONS: These findings suggest that signaling cascades resulting from the production of TNFα by infiltrated macrophages in the TG contributes to the development of ectopic mechanical allodynia in whisker pad skin following IANX.

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.

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.

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.

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.

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.

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.

Inhibition of histone deacetylase 2 mitigates profibrotic TGF-ß1 responses in fibroblasts derived from Peyronie's plaque.

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-ß1 (TGF-ß1). 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-ß1-induced nuclear translocation of Smad2/3 in fibroblasts. Knockdown of HDAC2 in PD fibroblasts abrogated TGF-ß1-induced extracellular matrix production by blocking TGF-ß1-induced phosphorylation and nuclear translocation of Smad2 and Smad3, and by inhibiting TGF-ß1-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.

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.

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.