Jin-Mi Park

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

Significance Curative treatment modalities for erectile dysfunction (ED) are not available. Penile erection is a neurovascular phenomenon, and ED is caused mainly by vascular and neurologic disturbances. Here we demonstrate that inhibition of nerve injury-induced protein 1 promotes penile angiogenesis and neural regeneration through angiopoietin-1–Tie2 signaling and rescues erectile function in diabetic mice. Our preclinical work shed light on the application of therapeutic angiogenesis and neural regeneration for the treatment of human ED. 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

To examine the therapeutic effect of adenovirus encoding histone deacetylase 2 (HDAC2) small hairpin RNA (Ad‐HDAC2 shRNA) in a rat model of Peyronies disease (PD) and to determine the mechanisms by which HDAC2 knockdown ameliorates fibrotic responses in primary fibroblasts derived from human PD plaque.

Inhibition of histone deacetylase 2 mitigates profibrotic TGF-β1 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. Peyronies 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.

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.

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.

Aberrant expression of Wnt family contributes to the pathogenesis of diabetes‐induced erectile dysfunction

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.

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-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.

Effect of SMAD7 gene overexpression on TGF-β1-induced profibrotic responses in fibroblasts derived from Peyronie's plaque.

Transforming growth factor-β1 (TGF-β1) has been identified as one of the most important fibrogenic cytokines associated with Peyronie′s disease (PD). The mothers against decapentaplegic homolog 7 (SMAD7) is an inhibitory Smad protein that blocks TGF-β signaling pathway. The aim of this study was to examine the anti-fibrotic effect of the SMAD7 gene in primary fibroblasts derived from human PD plaques. PD fibroblasts were pretreated with the SMAD7 gene and then stimulated with TGF-β1. Treated fibroblasts were used for Western blotting, fluorescent immunocytochemistry, hydroxyproline determination, and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assays. Overexpression of the SMAD7 gene inhibited TGF-β1-induced phosphorylation and nuclear translocation of SMAD2 and SMAD3, transdifferentiation of fibroblasts into myofibroblasts, and quashed TGF-β1-induced production of extracellular matrix protein and hydroxyproline. Overexpression of the SMAD7 gene decreased the expression of cyclin D1 (a positive cell cycle regulator) and induced the expression of poly (ADP-ribose) polymerase 1, which is known to terminate Smad-mediated transcription, in PD fibroblasts. These findings suggest that the blocking of the TGF-β pathway by use of SMAD7 may be a promising therapeutic strategy for the treatment of PD.