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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.

Background Severe peripheral angiopathy in patients with diabetes is a major contributing factor for low response rate to phosphodiesterase-5 inhibitors. Objectives To examine whether and how Dickkopf3 (DKK3), a secreted modulator of the Wnt pathway that known to be involved in endothelial cell repair and vascular progenitor cell migration, restores erectile function in diabetic mice. Methods Eight-week-old C57BL/6 mice received intraperitoneal injections of streptozotocin (50 mg/kg for 5 days). Eight weeks after the diabetes was induced, the efficacy of DKK3 was determined by three independent experiments: experiment 1 (DKK3 peptide [5 μg in 20 μL PBS]); experiment 2 (DKK3 plasmid DNA with electroporation [10, 40, or 100 μg in 20 μL PBS, respectively]); and experiment 3 (DKK3 adenovirus [1 × 107 , 1 × 108 , 1 × 109 virus particles per 20 μL, respectively]). Erectile function was measured by electrical stimulation of the cavernous nerve one week (for peptide) or two weeks (for genes) after treatment. The angiogenic activity of DKK3 was determined in diabetic penis in vivo and in primary cultured mouse cavernous endothelial cells (MCECs) in vitro. Results The cavernous expression of DKK3 protein was significantly lower in the diabetic mice than in controls. DKK3 peptide or adenovirus significantly improved erectile function in diabetic mice (70% of the control values). DKK3 adenovirus profoundly restored cavernous endothelial cell and pericyte contents and increased endothelial junction proteins in diabetic mice in vivo. DKK3 peptide induced upregulation of angiogenic factors (angiopoietin-1, vascular endothelial growth factor, and basic fibroblast growth factor) and accelerated tube formation in MCECs cultivated under the high-glucose condition in vitro. Conclusion DKK3 restored cavernous vascular integrity and improved erectile function in diabetic mice. Therapeutic cavernous angiogenesis by the use of DKK3 will be a promising therapeutic strategy to treat diabetic erectile dysfunction.

The mortality rate of ovarian cancer (OC) worldwide increases with age. OC is an often fatal cancer with a curative rate of only 20&ndash, 30%, as symptoms often appear after disease progression. Studies have reported that isolinderalactone (ILL), a furanosesquiterpene derivative extracted from the dried root of Lindera aggregata, can inhibit several cancer cell lines&rsquo, growth. However, the molecular mechanisms underlying ILL activities in human OC cells remain unexplored. This study investigated the antitumor activities of ILL in human OC cells by inducing mitochondrial superoxide (mtSO) and JAK-signal transducer and activator of transcription 3 (STAT3)-dependent cell death. ILL caused cell death in SKOV-3 and OVCAR-3 cells and increased the cell proportion in the subG1 phase. Additionally, ILL significantly induced mtSO production and reduced ROS production. Moreover, ILL downregulated mitochondrial membrane potential and the expression levels of anti-apoptotic Bcl-2 family proteins and superoxide dismutase (SOD)2. Results showed that ILL decreased phosphorylation of serine 727 and tyrosine 705 of STAT3 and expression of survivin, a STAT3-regulated gene. Furthermore, ILL-induced cell death was reversed by pretreatment of Mito-TEMPO, a mitochondria-specific antioxidant. These results suggest that ILL induces cell death by upregulation of mtSO, downregulation of mitochondrial SOD2, and inactivation of the STAT3-mediated pathway.

Glioblastoma multiforme (GBM) is a lethal and highly vascular type of brain tumor. We previously reported that isolinderalactone enhances GBM apoptosis in vitro and in vivo, but its role in tumor angiogenesis is unknown. Here, we investigated the anti-angiogenic activity of isolinderalactone and its mechanisms. In a human GBM xenograft mouse model, isolinderalactone significantly reduced tumor growth and vessels. Isolinderalactone decreased the expression of vascular endothelial growth factor (VEGF) mRNA, protein, and VEGF secretion in hypoxic U-87 GBM cells and also in xenograft GMB tissue. In addition, we demonstrated that isolinderalactone significantly inhibited the proliferation, migration, and capillary-like tube formation of human brain microvascular endothelial cells (HBMECs) in the presence of VEGF. We also found that isolinderalactone decreased sprout diameter and length in a 3D microfluidic chip, and strongly reduced VEGF-triggered angiogenesis in vivo Matrigel plug assay. Isolinderalactone downregulated hypoxia-inducible factor-1α (HIF-1α) and HIF-2α proteins, decreased luciferase activity driven by the VEGF promoter in U-87 cells under hypoxic conditions, and suppressed VEGF-driven phosphorylation of VEGFR2 in HBMECs. Taken together, our results suggest that isolinderalactone is a promising candidate for GBM treatment through tumor angiogenesis inhibition.

Hypoxic microenvironments exist in developing embryonic tissues and determine stem cell fate. We previously demonstrated that hypoxic priming plays roles in lineage commitment of embryonic stem cells. In the present study, we found that hypoxia-primed embryoid bodies (Hyp-EBs) efficiently differentiate into the myogenic lineage, resulting in the induction of the myogenic marker MyoD, which was not mediated by hypoxia-inducible factor 1α (HIF1α) or HIF2α, but rather by Sp1 induction and binding to the MyoD promoter. Knockdown of Sp1 in Hyp-EBs abrogated hypoxia-induced MyoD expression and myogenic differentiation. Importantly, in the cardiotoxin-muscle injury mice model, Hyp-EB transplantation facilitated muscle regeneration in vivo, whereas transplantation of Sp1-knockdown Hyp-EBs failed to do. Moreover, we compared microRNA (miRNA) expression profiles between EBs under normoxia versus hypoxia and found that hypoxia-mediated Sp1 induction was mediated by the suppression of miRNA-92a, which directly targeted the 3′ untranslated region (3′ UTR) of Sp1. Further, the inhibitory effect of miRNA-92a on Sp1 in luciferase assay was abolished by a point mutation in specific sequence in the Sp1 3′ UTR that is required for the binding of miRNA-92a. Collectively, these results suggest that hypoxic priming enhances EB commitment to the myogenic lineage through miR-92a/Sp1/MyoD regulatory axis, suggesting a new pathway that promotes myogenic-lineage differentiation.

Glioblastoma multiforme (GBM) is the most common malignant brain tumor, which remains incurable. Plant extracts are a potential source of potent anticancer medicines. In this study, we investigated the effect of isolinderalactone from Lindera aggregata on tumor growth using U-87 human glioblastoma cells. Treatment with isolinderalactone inhibited cell viability and promoted apoptotic cell death. In addition, intraperitoneal injection of isolinderalactone significantly inhibited tumor growth in a human GBM xenograft mouse model. To identify the proteins involved in the induction of apoptosis in isolinderalactone-treated cells, we performed a human apoptosis proteome array analysis and western blotting. Isolinderalactone suppressed the expression of B-cell lymphoma 2 (BCL-2), as well as of survivin and X-linked inhibitor of apoptosis protein (XIAP), known as apoptosis inhibitors, and increased the level of cleaved caspase-3. In addition, isolinderalactone treatment increased cleaved poly(ADP-ribose) polymerase (PARP) and DNA damage. In xenograft tumor tissues, we observed high immunofluorescence of cleaved caspase-3 and TUNEL in isolinderalactone-treated group. Taken together, isolinderalactone enhances U-87 GBM cell apoptosis in vitro and in vivo and retards tumor growth, suggesting that isolinderalactone may be a potential candidate for anti-glioblastoma drug development.

Induction of neovascularization is a promising strategy to treat erectile dysfunction (ED). Here we for the first time report the unexpected role of vasohibin-1, which is previously defined as an anti-angiogenic factor, in the restoration of erectile function by enhancing cavernous (penile) angiogenesis in an animal model of diabetic ED. The cavernous expression of vasohibin-1 was down-regulated in diabetic mice and in patients with diabetic ED; vasohibin-1 was mainly expressed in endothelial cells. Vasohibin-1 knockout mice revealed decrease in cavernous endothelial and pericyte content compared with wild type mice, which resulted in deterioration of erectile function. Local delivery of vasohibin-1 peptide into the corpus cavernosum of diabetic mice induced significant restoration of erectile function, which reached up to 85% of control values at the concentration of 4 µg/20 µL. Vasohibin-1 significantly increased cavernous endothelial cell content and induced eNOS phosphorylation (Ser1177). Vasohibin-1 decreased extravasation of oxidized-LDL by restoring pericytes and endothelial cell-cell junction proteins in the diabetic mice. By using proteome profiler array kit, we found that the induction of angiogenic factors, including angiopoietin-1, vascular endothelial growth factor, and basic fibroblasts growth factor, mainly in fibroblasts is a major molecular mechanism responsible for vasohibin-1-mediated cavernous angiogenesis and subsequent restoration of erectile function. Our findings suggest that vasohibin-1 is proangiogenic in diabetic penis and is a promising therapeutic target for ED from vascular causes.

Low oxygen or hypoxia can be observed in the central region of solid tumors. Hypoxia is a strong stimulus for new blood vessel formation or angiogenesis, which is essential for tumor growth and progression. Fibroblast growth factor 11 (FGF11) is an intracellular non-secretory FGF whose function has not yet been fully characterized. In the present study, we demonstrated that FGF11 expression is upregulated under hypoxic conditions in human umbilical vein endothelial cells (HUVECs). FGF11 overexpression stimulated capillary-like tube formation, yet did not affect cell migration. Notably, FGF11 markedly increased the levels of tight junction proteins including occludin, zonula occludens-1 (ZO-1) and claudin-5 in HUVECs. The FGF11 promoter contains hypoxia response elements (HREs), and hypoxia-inducible factor-1 (HIF-1) binds to HREs to activate hypoxia-related genes. We demonstrated that hypoxia or HIF-1 expression under normoxic conditions increased the luciferase activity driven by the FGF11 promoter. However, deletion of the HREs from the FGF11 promoter rendered reporter gene activity unresponsive to hypoxia or HIF-1. Taken together, we propose that FGF11 may be involved in the stabilization of capillary-like tube structures associated with angiogenesis and may act as a modulator of hypoxia-induced pathological processes such as tumorigenesis.

Artemisia capillaris, also called “InJin” in Korean, has been used as traditional oriental medicine in Korea because of its various pharmacological activities. These include hepatoprotective, analgesic, and antipyretic activities. The present study was designed to validate the beneficial effects of the aqueous extract of A. capillaris (AEAC) against acute gastric mucosal injury and investigate the underlying molecular mechanisms. The pharmacological efficacy of AEAC was evaluated using the gastric ulcer index and histological examination. AEAC decreased gastric mucosal lesions mediated by HCl/ethanol in vivo in a dose-dependent manner. Interestingly, the mucosal damage was almost prevented by pretreatment with 200 or 400 mg/kg AEAC. However, AEAC did not have acid-neutralizing activity in vitro and did not prevent histamine secretion in HMC-1 mast cells. In the gastric mucosa, AEAC also significantly inhibited lipid peroxide formation through superoxide dismutase (SOD) activation. Moreover, AEAC strongly reduced the generation of pro-inflammatory cytokines, such as interleukin-6 (IL-6) and interleukin-1β (IL-1β), through nuclear factor kappa B (NF-κB) downregulation. Taken together, our findings suggest that AEAC inhibits inflammation and maintains oxidant/antioxidant homeostasis, resulting in a gastro-protective effect against HCl/ethanol-induced gastric damage. Therefore, AEAC might be a promising drug or useful neutraceutical for treatment of gastritis and gastric ulcer.

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.

Ninjurin1 is involved in the pathogenesis of experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis, by mediating leukocyte extravasation, a process that depends on homotypic binding. However, the precise regulatory mechanisms of Ninjurin1 during inflammation are largely undefined. We therefore examined the pro-migratory function of Ninjurin1 and its regulatory mechanisms in macrophages. Interestingly, Ninjurin1-deficient bone marrow-derived macrophages exhibited reduced membrane protrusion formation and dynamics, resulting in the impairment of cell motility. Furthermore, exogenous Ninjurin1 was distributed at the membrane of filopodial structures in Raw264.7 macrophage cells. In Raw264.7 cells, RNA interference of Ninjurin1 reduced the number of filopodial projections, whereas overexpression of Ninjurin1 facilitated their formation and thus promoted cell motility. Ninjurin1-induced filopodial protrusion formation required the activation of Rac1. In Raw264.7 cells penetrating an MBEC4 endothelial cell monolayer, Ninjurin1 was localized to the membrane of protrusions and promoted their formation, suggesting that Ninjurin1-induced protrusive activity contributed to transendothelial migration. Taking these data together, we conclude that Ninjurin1 enhances macrophage motility and consequent extravasation of immune cells through the regulation of protrusive membrane dynamics. We expect these findings to provide insight into the understanding of immune responses mediated by Ninjurin1.

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.

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.

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.

INTRODUCTION AND OBJECTIVES: Induction of neovascularization is a promising strategy to treat diabetic erectile dysfunction (ED). Here we for the first time report the unexpected role of vasohibin-1, which is previously defined as an anti-angiogenic factor, in the restoration of erectile function by enhancing cavernous angiogenesis in an animal model of diabetic ED. METHODS: Diabetes was induced by intraperitoneal injection of streptozotocin into 8-week-old C57BL/6 male mice. At 8 weeks after the induction of diabetes, the animals were divided into 4 groups: controls, streptozotocin-induced diabetic mice receiving repeated intracavernous injections of PBS (days -3 and 0; 20 mL), or vasohibin-1 protein (days -3 and 0; 1 mg and 4 mg in 20 mL of PBS, respectively). Two weeks after treatment, we measured erectile function by electrical stimulation of the cavernous nerve. The penis was harvested for histologic and biochemical studies. RESULTS: The cavernous expression of vasohibin-1 was down-regulated in diabetic mice and in patients with diabetic ED; vasohibin-1 was mainly expressed in endothelial cells. Vasohibin-1 knockout mice revealed decrease in cavernous endothelial and pericyte content compared with wild type mice, which resulted in deterioration of erectile function. Local delivery of vasohibin-1 into the corpus cavernosum of diabetic mice induced significant restoration of erectile function, which reached up to 85% of control values at the concentration of 4 mg/20 mL. Vasohibin-1 significantly increased cavernous endothelial cell and induced eNOS phosphorylation (Ser1177). Vasohibin-1 decreased extravasation of oxidized-LDL by restoring pericyte content and endothelial cell-cell junction proteins in the diabetic mice. By using proteome profiler array kit, we found that the induction of angiogenic factors, including angiopoietin-1, vascular endothelial growth factor, and basic fibroblasts growth factor, mainly in fibroblasts is a major molecular mechanism responsible for vasohibin1-mediated cavernous angiogenesis and subsequent restoration of erectile function. CONCLUSIONS: Our findings suggest that vasohibin-1 is proangiogenic in diabetic penis and is a promising therapeutic target for ED from vascular causes.

We developed a novel method for harvesting endothelial cells from blood vessels of freshly obtained cancer and adjacent normal tissue of human breast, and compared the response of the cancer-derived endothelial cells (CECs) and normal tissue-derived endothelial cells (NECs) to ionizing radiation. In brief, when tissues were embedded in Matrigel and cultured in endothelial cell culture medium (ECM) containing growth factors, endothelial cells grew out of the tissues. The endothelial cells were harvested and cultured as monolayer cells in plates coated with gelatin, and the cells of 2nd-5th passages were used for experiments. Both CECs and NECs expressed almost the same levels of surface markers CD31, CD105 and TEM-8 (tumor endothelial marker-8), which are known to be expressed in angiogenic endothelial cells, i.e., mitotically active endothelial cells. Furthermore, both CECs and NECs were able to migrate into experimental wound in the monolayer culture, and also to form capillary-like tubes on Matrigel-coated plates. However, the radiation-induced suppressions of migration and capillary-like tube formations were greater for CECs than NECs from the same patients. In addition, in vitro clonogenic survival assays demonstrated that CECs were far more radiosensitive than NECs. In summary, we have developed a simple and efficient new method for isolating endothelial cells from cancer and normal tissue, and demonstrated for the first time that endothelial cells of human breast cancer are significantly more radiosensitive than their normal counterparts from the same patients.

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.

Neuregulin-1 (NRG1) signaling has multiple functions in neurons and glia. The data in this study show that NRG1 may also possess significant signaling and cytoprotective properties in human brain microvascular endothelial cells (BMECs). Neuregulin-1 mRNA and protein expression are present in these cells, and NRG1 receptors erbB2 and erbB3 are phosphorylated in response to NRG1. Neuregulin-1 triggers clear biologic responses in BMECs—elevated phospho-Akt levels, increased ring formation in a Matrigel assay, and decreased cell death after oxidative injury with H2O2. These data suggest that NRG1 signaling is functional and cytoprotective in BMECs.

The neurovascular unit is an emerging concept that emphasizes homeostatic interactions between endothelium and cerebral parenchyma. Here, we show that cerebral endothelium are not just inert tubes for delivering blood, but they also secrete trophic factors that can be directly neuroprotective. Conditioned media from cerebral endothelial cells broadly protects neurons against oxygen-glucose deprivation, oxidative damage, endoplasmic reticulum stress, hypoxia, and amyloid neurotoxicity. This phenomenon is largely mediated by endothelial-produced brain-derived neurotrophic factor (BDNF) because filtering endothelial-conditioned media with TrkB-Fc eliminates the neuroprotective effect. Endothelial production of BDNF is sustained by β-1 integrin and integrin-linked kinase (ILK) signaling. Noncytotoxic levels of oxidative stress disrupts ILK signaling and reduces endothelial levels of neuroprotective BDNF. These data suggest that cerebral endothelium provides a critical source of homeostatic support for neurons. Targeting these signals of matrix and trophic coupling between endothelium and neurons may provide new therapeutic opportunities for stroke and other CNS disorders.

Many diseases of the eye such as retinoblastoma, diabetic retinopathy, and retinopathy of prematurity are associated with blood–retinal barrier (BRB) dysfunction. Identifying the factors that contribute to BRB formation during human eye development and maintenance could provide insights into such diseases. Here we show that A-kinase anchor protein 12 (AKAP12) induces BRB formation by increasing angiopoietin-1 and decreasing vascular endothelial growth factor (VEGF) levels in astrocytes. We reveal that AKAP12 downregulates the level of hypoxia-inducible factor-1α (HIF-1α) protein by enhancing the interaction of HIF-1α with pVHL (von Hippel-Lindau tumor suppressor protein) and PHD2 (prolyl hydroxylase 2). Conditioned media from AKAP12-overexpressing astrocytes induced barriergenesis by upregulating the expression of tight junction proteins in human retina microvascular endothelial cells (HRMECs). Compared with the retina during BRB maturation, AKAP12 expression in retinoblastoma patient tissue was markedly reduced whereas that of VEGF was increased. These findings suggest that AKAP12 may induce BRB formation through antiangiogenesis and barriergenesis in the developing human eye and that defects in this mechanism can lead to a loss of tight junction proteins and contribute to the development of retinal pathologies such as retinoblastoma.

Pericytes are known to play critical roles in vascular development and homeostasis. However, the distribution of cavernous pericytes and their roles in penile erection is unclear. Herein we report that the pericytes are abundantly distributed in microvessels of the subtunical area and dorsal nerve bundle of mice, followed by dorsal vein and cavernous sinusoids. We further confirmed the presence of pericytes in human corpus cavernosum tissue and successfully isolated pericytes from mouse penis. Cavernous pericyte contents from diabetic mice and tube formation of cultured pericytes in high glucose condition were greatly reduced compared with those in normal conditions. Suppression of pericyte function with anti-PDGFR-β blocking antibody deteriorated erectile function and tube formation in vivo and in vitro diabetic condition. In contrast, enhanced pericyte function with HGF protein restored cavernous pericyte content in diabetic mice and significantly decreased cavernous permeability in diabetic mice and in pericytes-endothelial cell co-culture system, which induced significant recovery of erectile function. Overall, these findings showed the presence and distribution of pericytes in the penis of normal or pathologic condition and documented their role in the regulation of cavernous permeability and penile erection, which ultimately explore novel therapeutics of erectile dysfunction targeting pericyte function.

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.

The importance of epigenetic regulation for maintenance of embryonic stem cell (ESC) pluripotency or for initiation of differentiation is widely accepted. However, the molecular mechanisms are poorly understood. We recently reported that a hypoxic microenvironment induces ESC differentiation. In the present study, we found that hypoxia-responsive histone deacetylase 6 (HDAC6) performs an essential signaling function for myogenic differentiation of ESCs. HDAC6 was downregulated in hypoxic ESCs or during differentiation. A knock-down of HDAC6 in ESCs resulted in induction of myogenic markers, including Pax7. Suppression of HDAC6 increased acetylation of core histones H3 and H4, leading to enhanced binding of RNA polymerase II to the Pax7 promoter. Transplantation of HDAC6 knockdown cells facilitated muscle regeneration in vivo. Importantly, the downregulation of HDAC6 by hypoxia was not mediated by HIF1 alpha or HIF2 alpha, master transcription regulators under hypoxia, but by induction of microRNA-26a that directly targeted the 3'-untranslated region (3'-UTR) of HDAC6. A point mutation of the microRNA-26a-binding sequence in the HDAC6 3'-UTR diminished the luciferase reporter activity. Taken together, these results suggest that environmental cues of differentiation modulate the epigenetic machinery and guide stem cells to commit to a specific lineage.

The blood-neural barrier (BNB), including blood-brain barrier (BBB) and blood-retinal barrier (BRB), is an endothelial barrier constructed by an extensive network of endothelial cells, astrocytes and neurons to form functional "neurovascular units", which has an important role in maintaining a precisely regulated microenvironment for reliable neuronal activity. Although failure of the BNB may be a precipitating event or a consequence, the breakdown of BNB is closely related with the development and progression of CNS diseases. Therefore, BNB is most essential in the regulation of microenvironment of the CNS. The BNB is a selective diffusion barrier characterized by tight junctions between endothelial cells, lack of fenestrations, and specific BNB transporters. The BNB have been shown to be astrocyte dependent, for it is formed by the CNS capillary endothelial cells, surrounded by astrocytic end-foot processes. Given the anatomical associations with endothelial cells, it could be supposed that astrocytes play a role in the development, maintenance, and breakdown of the BNB. Therefore, astrocytes-endothelial cells interaction influences the BNB in both physiological and pathological conditions. If we better understand mutual interactions between astrocytes and endothelial cells, in the near future, we could provide a critical solution to the BNB problems and create new opportunities for future success of treating CNS diseases. Here, we focused astrocyte-endothelial cell interaction in the formation and function of the BNB.

In the developing brain, capillaries are differentiated and matured into the blood-brain barrier (BBB), which is composed of cerebral endothelial cells, astrocyte end-feet, and pericytes. Since the BBB regulates the homeostasis of central nervous system (CNS), the maintenance of the BBB is important for CNS function. The disruption of the BBB may result in many brain disorders including brain tumors. However, the molecular mechanism of BBB formation and maintenance is poorly understood. Here, we summarize recent advances in the role of oxygen tension and growth factors on BBB development and maintenance, and in BBB dysfunction related with brain tumors.

The maintenance of the blood–brain barrier (BBB) is important in the central nervous system because disruption of the BBB may contribute to many brain disorders, including Alzheimer's disease and stroke. However, the molecular mechanism of brain vessel development and BBB formation remains ill-defined. We found that the immunoreactivities of pimonidazol, hypoxic marker and vascular endothelial growth factor (VEGF) were detected in developing brain, and then disappeared after birth. However, the expression of occludin was gradually increased during BBB maturation. Moreover, we found src -suppressed C-kinase substrate (SSeCKS)-overexpressed in astrocytes during reoxygenation. Overexpressed SSeCKS strongly reduced the VEGF expression and angiogenesis. Furthermore, SSeCKS overexpression increased the expression of both angiopoietin-1 (Ang-1), an antipermeability factor, and tight junction proteins, consequently decreased [ 3 H]sucrose permeability. We therefore conclude that reoxygenation of astrocytes may act as a significant driving force for the BBB maturation, and SSeCKS is a novel regulator of BBB differentiation by modulating both brain angiogenesis and tight junction formation.

The blood-brain barrier (BBB) is essential for maintaining brain homeostasis and low permeability. BBB maintenance is important in the central nervous system (CNS) because disruption of the BBB may contribute to many brain disorders, including Alzheimer disease and ischemic stroke. The molecular mechanisms of BBB development remain ill-defined, however. Here we report that src-suppressed C-kinase substrate (SSeCKS) decreases the expression of vascular endothelial growth factor (VEGF) through AP-1 reduction and stimulates expression of angiopoietin-1 (Ang-1), an antipermeability factor in astrocytes. Conditioned media from SSeCKS-overexpressing astrocytes (SSeCKS-CM) blocked angiogenesis in vivo and in vitro. Moreover, SSeCKS-CM increased tight junction proteins in endothelial cells, consequently decreasing [3H]sucrose permeability. Furthermore, immunoreactivity to SSeCKS gradually increased during the BBB maturation period, and SSeCKS-expressing astrocytes closely interacted with zonula occludens (ZO)-1-expressing blood vessels in vivo. Collectively, our results suggest that SSeCKS regulates BBB differentiation by modulating both brain angiogenesis and tight junction formation.

Hepatocellular carcinoma (HCC), a prototype of hypervascular tumors, is one of the most common malignancies in the world, especially hyperendemic in the Far East where chronic hepatitis B virus (HBV) infection is highly prevalent. It is characterized by the clinical feature of a poor prognosis or a high mortality due to its already far advanced stages at diagnosis. It is so multifactorial that hepatocarcinogenesis cannot be explained by a single molecular mechanism. To date, a number of pathways have been known to contribute to the development, growth, angiogenesis, and even metastasis of HCC. Among the various factors, metastatic tumor antigens (MTAs) or metastasis-associated proteins have been vigorously investigated as an intriguing target in the field of hepatocarcinogenesis. According to recent studies including ours, MTAs are not only involved in the HCC development and growth (molecular carcinogenesis), but also closely associated with the post-operative recurrence and a poor prognosis or a worse response to post-operative anti-cancer therapy (clinical significance). Herein, we review MTAs in light of their essential structure, functions, and molecular mechanism in hepatocarcinogenesis. We will also focus in detail on the interaction between hepatitis B x protein (HBx) of HBV and MTA in order to clarify the HBV-associated HCC development. Finally, we will discuss the prognostic significance and clinical application of MTA in HCC. We believe that this review will help clinicians to understand the meaning and use of the detection of MTA in order to more effectively manage their HCC patients.

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.

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.

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.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)-β1 (10 ng/mL) to determine hydroxyproline levels, procollagen mRNA, apoptosis and protein expression of poly(ADP-ribose) polymerase 1 (PARP1) and cyclin D1.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.Specific inhibition of HDAC2 with RNA interference may represent a novel targeted therapy for PD.

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.

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.

Lately, the importance of the communication between different cell types and the understanding of the cell communication pathway has been emphasized, as it may provide a novel therapeutic strategy for the regeneration of damaged tissue. In the present study, we suggest that sonic hedgehog (Shh) is a mediator of cell communication between neurons and fibroblasts. Recombinant Shh (rShh) affected the expression of the angiogenic factors, angiopoietin (Ang)-1 and Ang-2, in fibroblasts, but not in neurons or neural progenitor cells (NPCs). The expression of the Shh downstream transcription factor, Gli1, was markedly increased in neurons and NPCs, indicating that neurons and NPCs responded to rShh. However, rShh did not affect Ang-1 and Ang-2 expression in neurons and NPCs. It should be noted that Shh was strongly expressed in neurons, but that Shh expression was undetectable in fibroblasts. We performed a co-culture assay using neurons and fibroblasts to investigate whether the expression of Ang-1 and Ang-2 is regulated by cell communication, without rShh treatment. Ang-1 expression in fibroblasts was markedly upregulated by co-culture with neurons, whereas Ang-2 expression was decreased by co-culture with neurons. Moreover, when an Shh-neutralizing antibody was added, this effect was diminished. Collectively, our data suggest that Shh-expressing neurons regulate angiopoietin expression in neighboring fibroblasts in a paracrine manner.

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.