Ji-Kan Ryu

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

INTRODUCTION It has been suggested that transforming growth factor-beta1 (TGF-beta1) 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-beta-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-beta1, phospho-Smad2 (P-Smad2), smooth muscle alpha-actin, and factor VIII (N = 12 per group). Penis specimens from a separate group of animals were used for TGF-beta1 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-beta1, 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-beta1 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.

Downregulation of angiogenic factors and their downstream target molecules affects the deterioration of erectile function in a rat model of hypercholesterolemia.

OBJECTIVES To evaluate how the expression of angiogenic factors and their downstream target molecules, which are potentially involved in penile homeostasis, is related to erectile dysfunction in a rat model of hypercholesterolemia. METHODS Fifty-six 2-month-old male Sprague-Dawley rats were included in this study. The control animals (n = 28) were fed a normal diet, and the experimental animals (n = 28) were fed a diet containing 4% cholesterol and 1% cholic acid for 3 months. Erectile function was evaluated by cavernous nerve electrical stimulation, and cavernous tissue was harvested for histologic examination (n = 12, respectively). Cavernous tissue specimens from the remaining rats were used for reverse transcriptase-polymerase chain reaction (RT-PCR), Western blot, or cyclic guanosine monophosphate (cGMP) measurement. RESULTS The ratio of maximal intracavernous pressure to mean arterial pressure was significantly lower in the hypercholesterolemic rats than in the controls (P <0.01). Analysis by RT-PCR and Western blot showed significantly lower gene expression of vascular endothelial growth factor (VEGF), angiopoietin-1, and angiopoietin-2 and significantly lower protein expression of VEGF, angiopoietin-1, angiopoietin-2, the ratio of phospho-Akt to Akt, and phospho-endothelial nitric oxide synthase (eNOS) to eNOS in hypercholesterolemic rats than in controls. Cavernous tissue cGMP concentrations and endothelial area were also significantly lower in hypercholesterolemic rats than in controls (P <0.01). CONCLUSIONS Downregulation of the expression of the angiogenic factors and their downstream signal molecules, and decreased endothelial content in the corpus cavernosum of hypercholesterolemic rats might play important roles in the deterioration of erectile function.

Functional and Morphologic Characterizations of the Diabetic Mouse Corpus Cavernosum: Comparison of a Multiple Low‐Dose and a Single High‐Dose Streptozotocin Protocols

INTRODUCTION With the advent of genetically modified mice, it seems particularly advantageous to develop a mouse model of diabetic erectile dysfunction. AIM To establish a mouse model of type I diabetes by implementation of either multiple low-dose streptozotocin (STZ) protocol or single high-dose STZ protocol and to evaluate morphologic alterations in the cavernous tissue and subsequent derangements in penile hemodynamics in vivo. METHODS Eight-week-old C57BL/6J mice were divided into three groups: a control group, a group administered the multiple low-dose STZ protocol (50 mg/kg x 5 days), and a group administered the single high-dose STZ protocol (200 mg/kg). MAIN OUTCOME MEASURES After 8 weeks, erectile function was measured by electrical stimulation of the cavernous nerve. The penis was then harvested and stained with hydroethidine (in situ analysis of superoxide anion), TUNEL, or antibodies to nitrotyrosine (marker of peroxynitrite formation), PECAM-1, smooth muscle alpha-actin, and phospho-eNOS. Penis specimens from a separate group of animals were used for phospho-eNOS and eNOS western blot or cGMP determination. RESULTS Erectile function was significantly less in diabetic groups than in control group. The generation of superoxide anion and nitrotyrosine and the number of apoptotic cells in both cavernous endothelial and smooth muscle cells were significantly higher in diabetic groups than in control group. Cavernous tissue phospho-eNOS and cGMP expression and the number of endothelial and smooth muscle cells were lower in diabetic groups than in control group. Both diabetic models resulted in similar structural and functional derangements in the corpus cavernosum; however, the mortality rate was higher in mice receiving single high-dose of STZ than in those receiving multiple low-doses. CONCLUSION The mouse model of type I diabetes is useful and technically feasible for the study of the pathophysiologic mechanisms involved in diabetic erectile dysfunction.

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

INTRODUCTION Transforming growth factor-beta1 (TGF-beta1) has been known to play a crucial role in the pathogenesis of Peyronies 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-beta, in an animal model of PD. METHODS PD was induced in rats through repeated injections of adenovirus expressing TGF-beta1 (days 0, 3, and 6; 1 x 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-beta signaling through the use of ALK5 inhibitors may represent an exciting new therapeutic strategy for the future treatment of PD.

A Mouse Model of Cavernous Nerve Injury-Induced Erectile Dysfunction: Functional and Morphological Characterization of the Corpus Cavernosum

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.

Intracavernous Delivery of Synthetic Angiopoietin-1 Protein as a Novel Therapeutic Strategy for Erectile Dysfunction in the Type II Diabetic db/db Mouse

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.

Intracavernous Delivery of Freshly Isolated Stromal Vascular Fraction Rescues Erectile Function by Enhancing Endothelial Regeneration in the Streptozotocin‐Induced Diabetic Mouse

INTRODUCTION Men with diabetic erectile dysfunction (ED) often have severe endothelial dysfunction and respond poorly to oral phosphodiesterase-5 inhibitors. AIM To examine whether and how freshly isolated stromal vascular fraction (SVF) promotes cavernous endothelial regeneration and restores erectile function in diabetic animals. METHODS Eight-week-old C57BL/6J mice were used. Diabetes was induced by intraperitoneal injection of streptozotocin. SVF was isolated from epididymal adipose tissues of green fluorescence protein transgenic mice. At 8 weeks after the induction of diabetes, the animals were divided into six groups: controls, diabetic mice, and diabetic mice treated with a single intracavernous injection of phosphate-buffered saline (PBS) or SVF (1 × 10(4) cells, 1 × 10(5) cells, or 2 × 10(5) cells/20 µL, respectively). MAIN OUTCOME MEASURES Two weeks later, erectile function was measured by cavernous nerve stimulation. The penis was stained with antibodies to CD31, CD34, phosphohistone H3, phospho-endothelial nitric oxide synthase (eNOS), and vascular endothelial growth factor-A (VEGF-A). We also performed Western blot for phospho-eNOS and eNOS, and determined cyclic guanosine monophosphate (cGMP) concentration in the corpus cavernosum tissue. RESULTS Significant improvement in erectile function was noted in diabetic mice treated with SVF at concentrations of 1 × 10(5) and 2 × 10(5) cells, which reached up to 82% of the control values. Local delivery of SVF significantly increased cavernous endothelial cell proliferation, eNOS phosphorylation, and cGMP expression compared with that in the untreated group and the PBS-treated diabetic group. Intracavernous injection of SVF increased cavernous VEGF-A expression and induced recruitment of CD34(+)CD31(-) progenitor cells. Some SVF underwent differentiation into cavernous endothelial cells. SVF-induced promotion of cavernous angiogenesis and erectile function was abolished in the presence of VEGF-Trap, a soluble VEGF-A neutralizing antibody. CONCLUSION The results support the concept of cavernous endothelial regeneration by use of SVF as a curative therapy for diabetic ED.

HS-173, a Novel PI3K Inhibitor, Attenuates the Activation of Hepatic Stellate Cells in Liver Fibrosis

Hepatic stellate cells (HSCs) are the primary source of matrix components in liver disease such as fibrosis. Phosphatidylinositol 3-kinase (PI3K) signaling in HSCs has been shown to induce fibrogenesis. In this study, we evaluated the anti-fibrotic activity of a novel imidazopyridine analogue (HS-173) in human HSCs as well as mouse liver fibrosis. HS-173 strongly suppressed the growth and proliferation of HSCs and induced the arrest at the G2/M phase and apoptosis in HSCs. Furthermore, it reduced the expression of extracellular matrix components such as collagen type I, which was confirmed by an in vivo study. We also observed that HS-173 blocked the PI3K/Akt signaling pathway in vitro and in vivo. Taken together, HS-173 suppressed fibrotic responses such as cell proliferation and collagen synthesis by blocking PI3K/Akt signaling. Therefore, we suggest that this compound may be an effective therapeutic agent for ameliorating liver fibrosis through the inhibition of PI3K signaling.

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.

Transforming Growth Factor (TGF)‐β Type I Receptor Kinase (ALK5) Inhibitor Alleviates Profibrotic TGF‐β1 Responses in Fibroblasts Derived from Peyronie's Plaque

INTRODUCTION Transforming growth factor-β1 (TGF-β1) has been identified as an important fibrogenic cytokine associated with Peyronies disease (PD). AIM The aim of this study was to study the differential expression of the TGF-β1 and Smad transcription factors in plaque tissue from PD patients and to determine the antifibrotic effect of SKI2162 (SK Chemicals, Seoul, South Korea), a novel small-molecule inhibitor of activin receptor-like kinase 5 (ALK5), a type I receptor of TGF-β, in primary fibroblasts derived from human PD plaque. METHODS Plaque tissue was isolated from five PD patients, and tunica albuginea tissue was obtained from four control patients. Plaque tissues from a patient with PD were used for primary fibroblast culture. Fibroblasts were pretreated with SKI2162 (10 µM) and then stimulated with TGF-β1 (10ng/mL). MAIN OUTCOME MEASURES The plaque or tunica albuginea tissue was stained with Massons trichrome or antibody to TGF-β1, phospho-Smad2 (P-Smad2), and P-Smad3. Protein was extracted from treated fibroblasts for Western blotting, and the membranes were probed with antibody to P-Smad2/Smad2, P-Smad3/Smad3, plasminogen activator inhibitor-1, fibronectin, collagen I, and collagen IV. We also determined the inhibitory effect of SKI2162 on TGF-β1-induced nuclear translocation of Smad2/3 in fibroblasts. RESULTS The plaque tissue from PD patients showed higher TGF-β1, P-Smad2, and P-Smad3 immunoreactivity than did the tunica albuginea tissue from control patients. SKI2162 not only blocked TGF-β1-induced phosphorylation and nuclear translocation of Smad2 and Smad3, but also inhibited the production of extracellular matrix markers in fibroblasts derived from human PD plaque. CONCLUSION In light of the pivotal role of TGF-β and Smads in the pathogenesis of PD, pharmacologic inhibition of ALK5 may represent a novel targeted approach to treating PD.