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1.
Andrology ; 12(2): 447-458, 2024 Feb.
Article in English | MEDLINE | ID: mdl-37290397

ABSTRACT

BACKGROUND: The odds of erectile dysfunction are three times more prevalent in diabetes. Severe peripheral vascular and neural damage in diabetic patients responds poorly to phosphodiesterase-5 (PDE5) inhibitors. However, bone morphogenetic protein 2 is known to be involved in angiogenesis. OBJECTIVES: To assess the efficacy of bone morphogenetic protein 2 in stimulating angiogenesis and augmenting nerve regeneration in a mouse model of diabetic-induced erectile dysfunction. MATERIALS AND METHODS: The induction of diabetes mellitus was performed by streptozotocin (50 mg/kg daily) administered intraperitoneally for 5 successive days to male C57BL/6 mice that were 8 weeks old. Eight weeks post-inductions, animals were allocated to one of five groups: a control group, a streptozotocin-induced diabetic mouse group receiving two intracavernous 20 µL phosphate-buffered saline injections, or one of three bone morphogenetic protein 2 groups administered two injections of bone morphogenetic protein 2 protein (1, 5, or 10 µg) diluted in 20 µL of phosphate-buffered saline within a 3-day interval between the first and second injections. The erectile functions were assessed 2 weeks after phosphate-buffered saline or bone morphogenetic protein 2 protein injections by recording the intracavernous pressure through cavernous nerve electrical stimulation. Angiogenic activities and nerve regenerating effects of bone morphogenetic protein 2 were determined in penile tissues, aorta, vena cava, the main pelvic ganglions, the dorsal roots, and from the primary cultured mouse cavernous endothelial cells. Moreover, fibrosis-related factor protein expressions were evaluated by western blotting. RESULTS: Erectile function recovery to 81% of the control value in diabetic mice was found with intracavernous bone morphogenetic protein 2 injection (5 µg/20 µL). Pericytes and endothelial cells were extensively restored. It was confirmed that angiogenesis was promoted in the corpus cavernosum of diabetic mice treated with bone morphogenetic protein 2 through increased ex vivo sprouting of aortic rings, vena cava and penile tissues, and migration and tube formation of mouse cavernous endothelial cells. Bone morphogenetic protein 2 protein enhanced cell proliferation and reduced apoptosis in mouse cavernous endothelial cells and penile tissues, and promoted neurite outgrowth in major pelvic ganglia and dorsal root ganglia under high-glucose conditions. Furthermore, bone morphogenetic protein 2 suppressed fibrosis by reducing mouse cavernous endothelial cell fibronectin, collagen 1, and collagen 4 levels under high-glucose conditions. CONCLUSION: Bone morphogenetic protein 2 modulates neurovascular regeneration and inhibits fibrosis to revive the mouse erection function in diabetic conditions. Our findings propose that the bone morphogenetic protein 2 protein represents a novel and promising approach to treating diabetes-related erectile dysfunction.


Subject(s)
Diabetes Mellitus, Experimental , Erectile Dysfunction , Animals , Humans , Male , Mice , Bone Morphogenetic Protein 2/metabolism , Bone Morphogenetic Protein 2/pharmacology , Collagen/metabolism , Collagen/pharmacology , Diabetes Mellitus, Experimental/complications , Disease Models, Animal , Endothelial Cells/metabolism , Erectile Dysfunction/drug therapy , Erectile Dysfunction/etiology , Erectile Dysfunction/metabolism , Glucose/metabolism , Mice, Inbred C57BL , Penile Erection , Penis , Phosphates/metabolism , Phosphates/pharmacology , Streptozocin
2.
Exp Mol Med ; 54(5): 626-638, 2022 05.
Article in English | MEDLINE | ID: mdl-35562586

ABSTRACT

Diabetes mellitus (DM) is a chronic metabolic disorder characterized by inappropriate hyperglycemia, which causes endothelial dysfunction and peripheral neuropathy, ultimately leading to multiple complications. One prevalent complication is diabetic erectile dysfunction (ED), which is more severe and more resistant to treatment than nondiabetic ED. The serum glycoprotein leucine-rich ɑ-2-glycoprotein 1 (LRG1) is a modulator of TGF-ß-mediated angiogenesis and has been proposed as a biomarker for a variety of diseases, including DM. Here, we found that the adhesion GPCR latrophilin-2 (LPHN2) is a TGF-ß-independent receptor of LRG1. By interacting with LPHN2, LRG1 promotes both angiogenic and neurotrophic processes in mouse tissue explants under hyperglycemic conditions. Preclinical studies in a diabetic ED mouse model showed that LRG1 administration into the penile tissue, which exhibits significantly increased LPHN2 expression, fully restores erectile function by rescuing vascular and neurological abnormalities. Further investigations revealed that PI3K, AKT, and NF-κB p65 constitute the key intracellular signaling pathway of the LRG1/LPHN2 axis, providing important mechanistic insights into LRG1-mediated angiogenesis and nerve regeneration in DM. Our findings suggest that LRG1 can be a potential new therapeutic option for treating aberrant peripheral blood vessels and neuropathy associated with diabetic complications, such as diabetic ED.


Subject(s)
Diabetes Mellitus , Erectile Dysfunction , Animals , Erectile Dysfunction/etiology , Glycoproteins/metabolism , Humans , Male , Mice , Neovascularization, Pathologic , Receptors, Peptide , Receptors, Transforming Growth Factor beta , Transforming Growth Factor beta/metabolism
3.
Int J Mol Med ; 49(2)2022 Feb.
Article in English | MEDLINE | ID: mdl-34935051

ABSTRACT

Pericyte­derived extracellular vesicle­mimetic nanovesicles (PC­NVs) play an important role in the improvement of erectile function after cavernous nerve injury. However, the impact of PC­NVs on the peripheral nervous system (PNS), such as the sciatic nerve, is unclear. In this study, PC­NVs were isolated from mouse cavernous pericytes (MCPs). A sciatic nerve transection (SNT) model was established using 8­week­old C57BL/6J mice. The sciatic nerve was harvested 5 and 14 days for immunofluorescence and western blot studies. Function studies were evaluated by performing the rotarod test and walking track analysis. The results demonstrated that PC­NVs could stimulate endothelial cells, increase neuronal cell content, and increase macrophage and Schwann cell presence at the proximal stump rather than the distal stump in the SNT model, thereby improving angiogenesis and nerve regeneration in the early stage of sciatic nerve regeneration. In addition, PC­NVs also increased the expression of neurotrophic factors (brain­derived nerve growth factor, neurotrophin­3 and nerve growth factor) and the activity of the cell survival signaling pathway (PI3K/Akt signaling), and reduced the activity of the JNK signaling pathway. Additionally, after 8 weeks of local application of PC­NVs in SNT model mice, their motor and sensory functions were significantly improved, as assessed by performing the rotarod test and walking track analysis. In conclusion, the present study showed that the significant improvement of neurovascular regeneration in mice following treatment with PC­NVs may provide a favorable strategy for promoting motor and sensory regeneration and functional recovery of the PNS.


Subject(s)
Extracellular Vesicles/metabolism , Nanoparticles/chemistry , Nerve Regeneration/physiology , Pericytes/metabolism , Sciatic Nerve/physiopathology , Animals , Disease Models, Animal , Macrophages/pathology , Male , Mice, Inbred C57BL , Schwann Cells/pathology , Sciatic Nerve/pathology , Signal Transduction , Survival Analysis
4.
Sci Rep ; 11(1): 1114, 2021 01 13.
Article in English | MEDLINE | ID: mdl-33441910

ABSTRACT

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.


Subject(s)
Cell Cycle Proteins/metabolism , Cell Cycle Proteins/therapeutic use , Diabetes Mellitus, Experimental/complications , Erectile Dysfunction/drug therapy , Erectile Dysfunction/metabolism , Penile Erection , Penis/metabolism , Angiopoietin-1/antagonists & inhibitors , Angiopoietin-1/metabolism , Animals , Cell Cycle Proteins/administration & dosage , Diabetes Mellitus, Experimental/metabolism , Down-Regulation , Endothelial Cells/physiology , Male , Mice , Mice, Inbred C57BL , Neovascularization, Physiologic , Nitric Oxide Synthase Type III/metabolism , Penis/blood supply , Pericytes/physiology , Phosphorylation , Tight Junction Proteins/metabolism , Up-Regulation , Vascular Endothelial Growth Factor A/antagonists & inhibitors , Vascular Endothelial Growth Factor A/metabolism
5.
World J Mens Health ; 39(1): 131-138, 2021 Jan.
Article in English | MEDLINE | ID: mdl-31496149

ABSTRACT

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

6.
Investig Clin Urol ; 62(1): 90-99, 2021 01.
Article in English | MEDLINE | ID: mdl-33258323

ABSTRACT

PURPOSE: To investigate potential target genes associated with the diabetic condition in mouse cavernous endothelial cells (MCECs) for the treatment of diabetes-induced erectile dysfunction (ED). MATERIALS AND METHODS: Mouse cavernous tissue was embedded into Matrigel, and sprouted cells were subcultivated for other studies. To mimic diabetic conditions, MCECs were exposed to normal-glucose (NG, 5 mmoL) or high-glucose (HG, 30 mmoL) conditions for 72 hours. An RNA-sequencing assay was performed to evaluate gene expression profiling, and RT-PCR was used to validate the sequencing data. RESULTS: We isolated MCECs exposed to the two glucose conditions. MCECs showed well-organized tubes and dynamic migration in the NG condition, whereas tube formation and migration were significantly decreased in the HG condition. RNA-sequencing analysis showed that MCECs had different gene profiles in the NG and HG conditions. Among the significantly changed genes, which we classified into 14 major gene categories, we identified that aging-related (9.22%) and angiogenesis-related (9.06%) genes were changed the most. Thirteen genes from the two gene categories showed consistent changes on the RNA-sequencing assay, and these findings were validated by RT-PCR. CONCLUSIONS: Our gene expression profiling studies showed that Cyp1a1, Gclm, Igfbp5, Nqo1, Il6, Cxcl5, Olr1, Ctgf, Hbegf, Serpine1, Cyr61, Angptl4, and Loxl2 may play a critical role in diabetes-induced ED through aging and angiogenesis signaling. Additional research is necessary to help us understand the potential mechanisms by which these genes influence diabetes-induced ED.


Subject(s)
Aging/genetics , Diabetes Complications/complications , Endothelial Cells/physiology , Erectile Dysfunction/genetics , Gene Expression/drug effects , Animals , Cell Movement , Cells, Cultured , Endothelial Cells/drug effects , Endothelial Cells/pathology , Erectile Dysfunction/etiology , Gene Expression Profiling , Gene Ontology , Glucose/pharmacology , Male , Mice , Mice, Inbred C57BL , Neovascularization, Physiologic/genetics , Penis/blood supply , Primary Cell Culture , Sequence Analysis, RNA
7.
J Sex Med ; 18(1): 17-28, 2021 01.
Article in English | MEDLINE | ID: mdl-33243690

ABSTRACT

BACKGROUND: Radical prostatectomy for prostate cancer can not only induce cavernous nerve injury (CNI), but also causes cavernous hypoxia and cavernous structural changes, which lead to a poor response to phosphodiesterase 5 inhibitors. AIM: To investigate the therapeutic effect of oral administration of LM11A-31, a small molecule p75 neurotrophin receptor (p75NTR) ligand and proNGF antagonist, in a mouse model of bilateral CNI, which mimics nerve injury-induced erectile dysfunction after radical prostatectomy. METHODS: 8-week-old male C57BL/6 mice were divided into sham operation and CNI groups. Each group was divided into 2 subgroups: phosphate-buffered saline and LM11A-31 (50 mg/kg/day) being administered once daily starting 3 days before CNI via oral gavage. 2 weeks after CNI, we measured erectile function by electrical stimulation of the bilateral cavernous nerve. The penis was harvested for histologic examination and Western blot analysis. The major pelvic ganglia was harvested and cultured for assays of ex vivo neurite outgrowth. OUTCOMES: Intracavernous pressure, neurovascular regeneration in the penis, in vivo or ex vivo functional evaluation, and cell survival signaling were measured. RESULTS: Erectile function was decreased in the CNI group (44% of the sham operation group), while administration of LM11A-31 led to a significant improvement of erectile function (70% of the sham operation group) in association with increased neurovascular content, including cavernous endothelial cells, pericytes, and neuronal processes. Immunohistochemical and Western blot analyses showed significantly increased p75NTR expression in the dorsal nerve of CNI mice, which was attenuated by LM11A-31 treatment. Protein expression of active PI3K, AKT, and endothelial nitric oxide synthase was increased, and cell death and c-Jun N-terminal kinase signaling was significantly attenuated after LM11A-31 treatment. Furthermore, LM11A-31 promoted neurite sprouting in cultured major pelvic ganglia after lipopolysaccharide exposure. CLINICAL IMPLICATIONS: LM11A-31 may be used as a strategy to treat erectile dysfunction after radical prostatectomy or in men with neurovascular diseases. STRENGTHS & LIMITATIONS: Unlike biological therapeutics, such as proteins, gene therapies, or stem cells, the clinical application of LM11A-31 would likely be relatively less complex and low cost. Our study has some limitations. Future studies will assess the optimal dosing and duration of the compound. Given its plasma half-life of approximately 1 hour, it is possible that dosing more than once per day will provide added efficacy. CONCLUSION: Specific inhibition of the proNGF-p75NTR degenerative signaling via oral administration of LM11A-31 represents a novel therapeutic strategy for erectile dysfunction induced by nerve injury. Yin GN, Ock J, Limanjaya A, et al. Oral Administration of the p75 Neurotrophin Receptor Modulator, LM11A-31, Improves Erectile Function in a Mouse Model of Cavernous Nerve Injury. J Sex Med 2021;18:17-28.


Subject(s)
Erectile Dysfunction , Administration, Oral , Animals , Disease Models, Animal , Endothelial Cells , Erectile Dysfunction/drug therapy , Erectile Dysfunction/etiology , Humans , Isoleucine/analogs & derivatives , Male , Mice , Mice, Inbred C57BL , Morpholines , Penile Erection , Penis , Receptor, Nerve Growth Factor
8.
Andrology ; 9(1): 329-341, 2021 01.
Article in English | MEDLINE | ID: mdl-32696589

ABSTRACT

BACKGROUND: Radical prostatectomy induces some degree of cavernous nerve injury (CNI) and causes denervation-induced pathologic changes in cavernous vasculature, regardless of the advances in surgical techniques and robotic procedures. The precursor for nerve growth factor (proNGF) is known to be involved in neuronal cell apoptosis and microvascular dysfunction through its receptor p75NTR . OBJECTIVES: To determine the expression of proNGF/p75NTR and the efficacy of proNGF neutralizing antibody (anti-proNGF-Ab) in a mouse model of ED induced by CNI. MATERIALS AND METHODS: Age-matched 12-week-old C57BL/6 mice were distributed into three groups: sham group and bilateral CNI group treated with intracavernous injections of PBS (20 µL) or of anti-proNGF-Ab (20 µg in 20 µL of PBS) on days -3 and 0. Two weeks after treatment, erectile function was measured by electrical stimulation of cavernous nerve. Penis tissues from a separate group of animals were harvested for further analysis. We also determined the efficacy of anti-proNGF-Ab on neural preservation in major pelvic ganglion (MPG) ex vivo. RESULTS: We observed increased penile expression of proNGF and p75NTR after CNI. Intracavernous administration of anti-proNGF-Ab increased nNOS and neurofilament expression probably by enhancing the production of neurotrophic factors, such as neurotrophin-3, NGF, and brain-derived neurotrophic factor. Anti-proNGF-Ab preserved the integrity of cavernous sinusoids, such as pericytes, endothelial cells, and endothelial cell-to-cell junctions, possibly by controlling angiogenic factors (angiopoietin-1, angiopoietin-2, and vascular endothelial growth factor) and induced endogenous eNOS phosphorylation in CNI mice. And finally, treatment with anti-proNGF-Ab rescued erectile function in CNI mice. Anti-proNGF-Ab also enhanced neurite sprouting from MPG exposed to lipopolysaccharide. DISCUSSION AND CONCLUSION: The preservation of damaged cavernous neurovasculature through inhibition of the proNGF/p75NTR pathway may be a novel strategy to treat radical prostatectomy-induced erectile dysfunction.


Subject(s)
Antibodies, Neutralizing/therapeutic use , Erectile Dysfunction/drug therapy , Nerve Growth Factor/antagonists & inhibitors , Penis/drug effects , Peripheral Nerve Injuries/drug therapy , Protein Precursors/antagonists & inhibitors , Angiogenic Proteins/metabolism , Animals , Antibodies, Neutralizing/pharmacology , Disease Models, Animal , Drug Evaluation, Preclinical , Erectile Dysfunction/etiology , Male , Mice, Inbred C57BL , Nerve Growth Factor/metabolism , Penis/innervation , Penis/metabolism , Peripheral Nerve Injuries/metabolism , Prostatectomy/adverse effects , Protein Precursors/metabolism , Receptors, Nerve Growth Factor/metabolism
9.
J Sex Med ; 17(11): 2118-2128, 2020 11.
Article in English | MEDLINE | ID: mdl-32855091

ABSTRACT

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


Subject(s)
Erectile Dysfunction , Extracellular Vesicles , Animals , Disease Models, Animal , Endothelial Cells , Humans , Male , Mice , Mice, Inbred C57BL , Nerve Regeneration , Penile Erection , Penis , Pericytes , Regeneration
10.
Andrology ; 8(5): 1387-1397, 2020 09.
Article in English | MEDLINE | ID: mdl-32170840

ABSTRACT

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.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Diabetes Mellitus, Experimental/complications , Erectile Dysfunction/etiology , Erectile Dysfunction/metabolism , Neovascularization, Physiologic/physiology , Animals , Male , Mice , Mice, Inbred C57BL , Penile Erection/physiology
11.
World J Mens Health ; 38(4): 552-563, 2020 Oct.
Article in English | MEDLINE | ID: mdl-31496148

ABSTRACT

PURPOSE: To examine the therapeutic effect of Vactosertib, a small molecule inhibitor of transforming growth factor-ß (TGF-ß) type I receptor (activin receptor-like kinase-5, ALK5), in an experimental model of Peyronie's disease (PD) and determining anti-fibrotic mechanisms of Vactosertib in primary fibroblasts derived from human PD plaques. MATERIALS AND METHODS: Male rats were randomly divided into three groups (n=6 per group); control rats without treatment; PD rats receiving vehicle; and PD rats receiving Vactosertib (10 mg/kg). PD-like plaques were induced by administering 100 µL of each of human fibrin and thrombin solutions into the tunica albuginea on days 0 and 5. Vactosertib was given orally five times a week for 2 weeks. On day 30, we performed electrical stimulation of the cavernous nerve to measure erectile function, and the penis was obtained for histological examination. Fibroblasts isolated from human PD plaques were used to determine the anti-fibrotic effects of Vactosertib in vitro. RESULTS: Vactosertib induced significant regression of fibrotic plaques in PD rats in vivo through reduced infiltration of inflammatory cells and reduced expression of phospho-Smad2, which recovered erectile function. Vactosertib also abrogated TGF-ß1-induced enhancement of extracellular matrix protein production and hydroxyproline content in PD fibroblasts in vitro by hindering the TGF-ß1-induced Smad2/3 phosphorylation and nuclear translocation, and fibroblast-to-myofibroblast transdifferentiation. CONCLUSIONS: In view of the critical role of TGF-ß and the Smad pathway in the pathogenesis of PD, inhibition of this pathway with an ALK5 inhibitor may represent a novel, targeted therapy for PD.

12.
Sci Rep ; 9(1): 20072, 2019 12 27.
Article in English | MEDLINE | ID: mdl-31882614

ABSTRACT

Extracellular vesicles (EVs) have attracted particular interest in various fields of biology and medicine. However, one of the major hurdles in the clinical application of EV-based therapy is their low production yield. We recently developed cell-derived EV-mimetic nanovesicles (NVs) by extruding cells serially through filters with diminishing pore sizes (10, 5, and 1 µm). Here, we demonstrate in diabetic mice that embryonic stem cell (ESC)-derived EV-mimetic NVs (ESC-NVs) completely restore erectile function (~96% of control values) through enhanced penile angiogenesis and neural regeneration in vivo, whereas ESC partially restores erectile function (~77% of control values). ESC-NVs promoted tube formation in primary cultured mouse cavernous endothelial cells and pericytes under high-glucose condition in vitro; and accelerated microvascular and neurite sprouting from aortic ring and major pelvic ganglion under high-glucose condition ex vivo, respectively. ESC-NVs enhanced the expression of angiogenic and neurotrophic factors (hepatocyte growth factor, angiopoietin-1, nerve growth factor, and neurotrophin-3), and activated cell survival and proliferative factors (Akt and ERK). Therefore, it will be a better strategy to use ESC-NVs than ESCs in patients with erectile dysfunction refractory to pharmacotherapy, although it remains to be solved for future clinical application of ESC.


Subject(s)
Diabetes Mellitus, Experimental/therapy , Embryonic Stem Cells/cytology , Extracellular Vesicles , Nanostructures , Penile Erection , Penis/blood supply , Penis/innervation , Animals , Blood Vessels/growth & development , Male , Mice , Nerve Regeneration , Streptozocin
13.
Diabetes ; 67(6): 1149-1161, 2018 06.
Article in English | MEDLINE | ID: mdl-29559443

ABSTRACT

Penile erection requires well-coordinated interactions between vascular and nervous systems. Penile neurovascular dysfunction is a major cause of erectile dysfunction (ED) in patients with diabetes, which causes poor response to oral phosphodiesterase-5 inhibitors. Dickkopf2 (DKK2), a Wnt antagonist, is known to promote angiogenesis. Here, using DKK2-Tg mice or DKK2 protein administration, we demonstrate that the overexpression of DKK2 in diabetic mice enhances penile angiogenesis and neural regeneration and restores erectile function. Transcriptome analysis revealed that angiopoietin-1 and angiopoietin-2 are target genes for DKK2. Using an endothelial cell-pericyte coculture system and ex vivo neurite sprouting assay, we found that DKK2-mediated juxtacrine signaling in pericyte-endothelial cell interactions promotes angiogenesis and neural regeneration through an angiopoietin-1-Tie2 pathway, rescuing erectile function in diabetic mice. The dual angiogenic and neurotrophic effects of DKK2, especially as a therapeutic protein, will open new avenues to treating diabetic ED.


Subject(s)
Angiopoietin-1/agonists , Diabetes Mellitus, Type 1/metabolism , Endothelium, Vascular/metabolism , Intercellular Signaling Peptides and Proteins/metabolism , Penis/metabolism , Pericytes/metabolism , Receptor, TIE-2/agonists , Adult , Angiopoietin-1/genetics , Angiopoietin-1/metabolism , Animals , Cell Line, Tumor , Cells, Cultured , Coculture Techniques , Crosses, Genetic , Diabetes Mellitus, Type 1/complications , Diabetes Mellitus, Type 1/pathology , Diabetes Mellitus, Type 2/complications , Diabetes Mellitus, Type 2/metabolism , Diabetes Mellitus, Type 2/pathology , Diabetic Angiopathies/drug therapy , Diabetic Angiopathies/metabolism , Diabetic Angiopathies/pathology , Diabetic Nephropathies/drug therapy , Diabetic Nephropathies/metabolism , Diabetic Nephropathies/pathology , Endothelium, Vascular/drug effects , Endothelium, Vascular/innervation , Endothelium, Vascular/pathology , Erectile Dysfunction/complications , Erectile Dysfunction/drug therapy , Erectile Dysfunction/metabolism , Erectile Dysfunction/pathology , Humans , Intercellular Signaling Peptides and Proteins/chemistry , Intercellular Signaling Peptides and Proteins/genetics , Intercellular Signaling Peptides and Proteins/therapeutic use , Male , Mice, Inbred C57BL , Mice, Transgenic , Penis/blood supply , Penis/innervation , Penis/pathology , Pericytes/drug effects , Pericytes/pathology , Receptor, TIE-2/metabolism , Wnt Signaling Pathway , Young Adult
14.
Sci Rep ; 7(1): 17819, 2017 12 19.
Article in English | MEDLINE | ID: mdl-29259207

ABSTRACT

Penile erection is a neurovascular event and neurologic or vascular disturbances are major causes of erectile dysfunction (ED). Radical prostatectomy for prostate cancer not only induces cavernous nerve injury (CNI) but also results in cavernous angiopathy, which is responsible for poor responsiveness to oral phosphodiesterase-5 inhibitors. Dickkopf2 (DKK2) is known as a Wnt signaling antagonist and is reported to promote mature and stable blood vessel formation. Here, we demonstrated in CNI mice that overexpression of DKK2 by administering DKK2 protein or by using DKK2-Tg mice successfully restored erectile function: this recovery was accompanied by enhanced neural regeneration through the secretion of neurotrophic factors, and restoration of cavernous endothelial cell and pericyte content. DKK2 protein also promoted neurite outgrowth in an ex vivo major pelvic ganglion culture experiment and enhanced tube formation in primary cultured mouse cavernous endothelial cells and pericytes co-culture system in vitro. In light of critical role of neuropathy and angiopathy in the pathogenesis of radical prostatectomy-induced ED, reprogramming of damaged erectile tissue toward neurovascular repair by use of a DKK2 therapeutic protein may represent viable treatment option for this condition.


Subject(s)
Erectile Dysfunction/drug therapy , Intercellular Signaling Peptides and Proteins/metabolism , Intercellular Signaling Peptides and Proteins/pharmacology , Nerve Regeneration/drug effects , Penile Erection/drug effects , Penis/drug effects , Animals , Blood Vessels/drug effects , Blood Vessels/metabolism , Coculture Techniques/methods , Disease Models, Animal , Endothelial Cells/drug effects , Endothelial Cells/metabolism , Endothelium/drug effects , Endothelium/metabolism , Erectile Dysfunction/metabolism , Ganglion Cysts/drug therapy , Ganglion Cysts/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Nerve Growth Factors/metabolism , Neurites/drug effects , Neurites/metabolism , Penis/metabolism , Pericytes/drug effects , Pericytes/metabolism , Phosphodiesterase 5 Inhibitors/pharmacology , Prostatectomy/adverse effects , Trauma, Nervous System/drug therapy
15.
J Sex Med ; 13(11): 1618-1628, 2016 11.
Article in English | MEDLINE | ID: mdl-27770854

ABSTRACT

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


Subject(s)
Erectile Dysfunction/drug therapy , Hepatocyte Growth Factor/pharmacology , Penile Erection/drug effects , Animals , Cell Proliferation/physiology , Diabetes Mellitus, Experimental/physiopathology , Endothelial Cells/cytology , Endothelial Cells/physiology , Endothelium/metabolism , Erectile Dysfunction/physiopathology , Humans , Injections, Intralesional , Lipoproteins, LDL/metabolism , Male , Mice, Inbred C57BL , Nitric Oxide Synthase Type III/metabolism , Penis/blood supply , Phosphodiesterase 5 Inhibitors/pharmacology , Phosphorylation/physiology , Rats, Sprague-Dawley , Recombinant Proteins/pharmacology , Regeneration/physiology
16.
Int J Urol ; 23(7): 614-22, 2016 07.
Article in English | MEDLINE | ID: mdl-27109455

ABSTRACT

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


Subject(s)
Neovascularization, Physiologic , Penile Erection/physiology , Penis/metabolism , Animals , Erectile Dysfunction , Humans , Male , Matrix Metalloproteinase 2/metabolism , Mice , Mice, Inbred C57BL , Signal Transduction , Vascular Endothelial Growth Factor A/metabolism
17.
Sci Rep ; 5: 10891, 2015 Jun 05.
Article in English | MEDLINE | ID: mdl-26044953

ABSTRACT

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-ß 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.


Subject(s)
Erectile Dysfunction/metabolism , Erectile Dysfunction/physiopathology , Penile Erection/physiology , Pericytes/metabolism , Animals , Antibodies, Monoclonal/pharmacology , Biomarkers , Cell Separation , Coculture Techniques , Diabetes Mellitus, Experimental , Disease Models, Animal , Endothelial Cells/metabolism , Hepatocyte Growth Factor/pharmacology , Humans , Male , Mice , Penile Erection/drug effects , Penis/cytology , Pericytes/cytology , Pericytes/drug effects , Permeability , Receptor, Platelet-Derived Growth Factor beta/antagonists & inhibitors , Receptor, Platelet-Derived Growth Factor beta/metabolism
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