Photobiomodulation as a Potential Therapy for Erectile Function: A Preclinical Study in a Cavernous Nerve Injury Model.

PURPOSE: To identify the optimal photobiomodulation (PBM) parameters using molecular, histological, and erectile function analysis in cavernous nerve injury. MATERIALS AND METHODS: A cavernous nerve injury was induced in 8-week-old C57BL/6J male mice that were subsequently divided randomly into age-matched control groups. Erectile function tests, penile histology, and Western blotting were performed 2 weeks after surgery and PBM treatment. RESULTS: The PBM treatment was administered for five consecutive days with a light-emitted diode (LED) device that delivers 660 nm±3% RED light, and near infra-red 830 nm±2% promptly administered following nerve-crushing surgery and achieved a notable restoration of erectile function approximately 90% of the control values. Subsequent in-vitro and ex-vivo analyses revealed the regeneration of neurovascular connections in both the dorsal root ganglion and major pelvic ganglion, characterized by the sprouting of neurites. Furthermore, the expression levels of neurotrophic, survival, and angiogenic factors exhibited a substantial increase across all groups subjected to PBM treatment. CONCLUSIONS: The utilization of PBM employing LED with 660 nm, 830 nm, and combination of both these wavelengths, exhibited significant efficacy to restore erectile function in a murine model of cavernous nerve injury. Thus, the PBM emerges as a potent therapeutic modality with notable advantages such as efficacy, noninvasiveness, and non-pharmacological interventions for erectile dysfunction caused by nerve injury.

FLRT2 prevents endothelial cell senescence and vascular aging by regulating the ITGB4/mTORC2/p53 signaling pathway.

The roles of fibronectin leucine-rich transmembrane protein 2 (FLRT2) in physiological and pathological processes are not well known. Here, we identify a potentially novel function of FLRT2 in preventing endothelial cell senescence and vascular aging. We found that FLRT2 expression was lower in cultured senescent endothelial cells as well as in aged rat and human vascular tissues. FLRT2 mediated endothelial cell senescence via the mTOR complex 2, AKT, and p53 signaling pathway in human endothelial cells. We uncovered that FLRT2 directly associated with integrin subunit beta 4 (ITGB4) and thereby promoted ITGB4 phosphorylation, while inhibition of ITGB4 substantially mitigated the induction of senescence triggered by FLRT2 depletion. Importantly, FLRT2 silencing in mice promoted vascular aging, and overexpression of FLRT2 rescued a premature vascular aging phenotype. Therefore, we propose that FLRT2 could be targeted therapeutically to prevent senescence-associated vascular aging.

Heme-binding protein 1 delivered via pericyte-derived extracellular vesicles improves neurovascular regeneration in a mouse model of cavernous nerve injury.

As a peripheral nerve injury disease, cavernous nerve injury (CNI) caused by prostate cancer surgery and other pelvic surgery causes organic damage to cavernous blood vessels and nerves, thereby significantly attenuating the response to phosphodiesterase-5 inhibitors. Here, we investigated the role of heme-binding protein 1 (Hebp1) in erectile function using a mouse model of bilateral CNI, which is known to promote angiogenesis and improve erection in diabetic mice. We found a potent neurovascular regenerative effect of Hebp1 in CNI mice, demonstrating that exogenously delivered Hebp1 improved erectile function by promoting the survival of cavernous endothelial-mural cells and neurons. We further found that endogenous Hebp1 delivered by mouse cavernous pericyte (MCP)-derived extracellular vesicles promoted neurovascular regeneration in CNI mice. Moreover, Hebp1 achieved these effects by reducing vascular permeability through regulation of claudin family proteins. Our findings provide new insights into Hebp1 as a neurovascular regeneration factor and demonstrate its potential therapeutic application to various peripheral nerve injuries.

Vactosertib, TGF-ß receptor I inhibitor, augments the sensitization of the anti-cancer activity of gemcitabine in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) exhibits a pronounced extracellular matrix (ECM)-rich response, which is produced by an excessive amount of transforming growth factor ß (TGF-ß), resulting in tumor progression and metastasis. In addition, TGF-ß signaling contributes to rapidly acquired resistance and incomplete response to gemcitabine. Recently, selective inhibitors of the TGF-ß signaling pathway have shown promise in PDAC treatment, particularly as an option for augmenting responses to chemotherapy. Here, we investigated the synergistic anticancer effects of a small-molecule TGF-ß receptor I kinase inhibitor (vactosertib/EW-7197) in the presence of gemcitabine, and its mechanism of action in pancreatic cancer. Vactosertib sensitized pancreatic cancer cells to gemcitabine by synergistically inhibiting their viability. Importantly, the combination of vactosertib and gemcitabine significantly attenuated the expression of major ECM components, including collagens, fibronectin, and α-SMA, in pancreatic cancer compared with gemcitabine alone. This resulted in potent induction of mitochondrial-mediated apoptosis, gemcitabine-mediated cytotoxicity, and inhibition of tumor ECM by vactosertib. Additionally, the combination decreased metastasis through inhibition of migration and invasion, and exhibited synergistic anti-cancer activity by inhibiting the TGF-ß/Smad2 pathway in pancreatic cancer cells. Furthermore, co-treatment significantly suppressed tumor growth in orthotopic models. Therefore, our findings demonstrate that vactosertib synergistically increased the antitumor activity of gemcitabine via inhibition of ECM component production by inhibiting the TGF-ß/Smad2 signaling pathway. This suggests that the combination of vactosertib and gemcitabine may be a potential treatment option for patients with pancreatic cancer.

A novel DDR1 inhibitor enhances the anticancer activity of gemcitabine in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is an extracellular matrix (ECM)-rich carcinoma, which promotes chemoresistance by inhibiting drug diffusion into the tumor. Discoidin domain receptor 1 (DDR1) increases tumor progression and drug resistance by binding to collagen, a major component of tumor ECM. Therefore, DDR1 inhibition may be helpful in cancer therapeutics by increasing drug delivery efficiency and improving drug sensitivity. In this study, we developed a novel DDR1 inhibitor, KI-301690 and investigated whether it could improve the anticancer activity of gemcitabine, a cytotoxic agent widely used for the treatment of pancreatic cancer. KI-301690 synergized with gemcitabine to suppress the growth of pancreatic cancer cells. Importantly, its combination significantly attenuated the expression of major tumor ECM components including collagen, fibronectin, and vimentin compared to gemcitabine alone. Additionally, this combination effectively decreased mitochondrial membrane potential (MMP), thereby inducing apoptosis. Further, the combination synergistically inhibited cell migration and invasion. The enhanced anticancer efficacy of the co-treatment could be explained by the inhibition of DDR1/PYK2/FAK signaling, which significantly reduced tumor growth in a pancreatic xenograft model. Our results demonstrate that KI-301690 can inhibit aberrant ECM expression by DDR1/PYK2/FAK signaling pathway blockade and attenuation of ECM-induced chemoresistance observed in desmoplastic pancreatic tumors, resulting in enhanced antitumor effect through effective induction of gemcitabine apoptosis.

Functional and Immunofluorescence Evaluations of Vascular and Neural Integrities in Urinary Bladder of Streptozotocin-Induced Diabetic Mice.

PURPOSE: To assess functional and structural changes in vascular and neural structures associated with diabetic bladder dysfunction (DBD) in the bladders of streptozotocin (STZ)-induced diabetic mice. METHODS: Eight-week-old C57BL/6 mice were injected with STZ at 50 mg/kg daily for 5 consecutive days. Catheters were inserted 12 weeks later, and 5 days after catheter placement bladder functions were assessed by conscious cystometry. Neurovascular and extracellular matrix marker changes in harvested urinary bladders were investigated by immunofluorescent staining. Body weights and fasting and postprandial blood glucose levels were measured 12 weeks after STZ injection. RESULTS: STZ-induced diabetic mice had significantly lower body weights and significantly higher blood glucose levels. Assessment of bladder function in STZ-induced diabetic mice revealed a nearly 3-fold increase in bladder capacity and intercontractile interval compared to controls. However, basal pressure, maximal bladder pressure, and threshold pressure were not significantly different. Morphological and structural analysis showed that STZ-induced diabetic mice had significantly reduced microvascular density in lamina propria (33% of the nondiabetic control values), and severely decreased nerve contents in the detrusor region (42% of the nondiabetic control values). CONCLUSION: STZ-induced diabetic mice exhibit functional and structural derangements in urinary bladder. The present study provides a foundation and describes a useful means of evaluating the efficacies of therapeutic targets and exploring the detailed mechanism of DBD.

Heat Shock Protein 70 in Penile Neurovascular Regeneration Requires Cystathionine Gamma-Lyase.

PURPOSE: Diabetes mellitus, one of the major causes of erectile dysfunction, leads to a poor response to phosphodiesterase-5 inhibitors. Heat shock protein 70 (Hsp70), a ubiquitous molecular chaperone, is known to play a role in cell survival and neuroprotection. Here, we aimed to assess whether and how Hsp70 improves erectile function in diabetic mice. MATERIALS AND METHODS: Eight-week-old male C57BL/6 mice and Hsp70-Tg mice were used in this study. We injected Hsp70 protein into the penis of streptozotocin (STZ)-induced diabetic mice. Detailed mechanisms were evaluated in WT or Hsp70-Tg mice under normal and diabetic conditions. Primary MCECs, and MPG and DRG tissues were cultivated under normal-glucose and high-glucose conditions. RESULTS: Using Hsp70-Tg mice or Hsp70 protein administration, we demonstrate that elevated levels of Hsp70 restores erectile function in diabetic mice. We found that cystathionine gamma-lyase (Cse) is a novel target of Hsp70 in this process, showing that Hsp70-Cse acts through the SDF1/HO-1/PI3K/Akt/eNOS/NF-κB p65 pathway to exert its neurovascular regeneration-promoting effects. Coimmunoprecipitation and pull-down assays using mouse cavernous endothelial cells treated with Hsp70 demonstrated physical interactions between Hsp70 and Cse with a dissociation constant of 1.8 nmol/L. CONCLUSIONS: Our findings provide novel and solid evidence that Hsp70 acts through a Cse-dependent mechanism to mediate neurovascular regeneration and restoration of erectile function under diabetic conditions.

Pericyte-derived extracellular vesicle-mimetic nanovesicles ameliorate erectile dysfunction via lipocalin 2 in diabetic mice.

Diabetes mellitus is one of the main causes of erectile dysfunction (ED). Men with diabetic ED do not respond well to oral phosphodiesterase-5 inhibitors owing to neurovascular dysfunction. Pericyte-derived extracellular vesicle-mimetic nanovesicles (PC-NVs) are known to promote nerve regeneration in a mouse model of cavernous nerve injury. Here, we report that administration of PC-NVs effectively promoted penile angiogenesis and neural regeneration under diabetic conditions, thereby improving erectile function. Specifically, PC-NVs induced endothelial proliferation and migration and reduced cell apoptosis under diabetic conditions. In addition, PC-NVs induced neural regeneration in STZ-induced diabetic mice in dorsal root ganglion and major pelvic ganglion explants in vivo and ex vivo under high-glucose conditions. We found that lipocalin 2 (Lcn2) is a new target of PC-NVs in this process, demonstrating that PC-NVs exert their angiogenic and nerve-regeneration effects by activating MAP kinase and PI3K/Akt and suppressing P53 signaling pathway in an Lcn2-dependent manner. Our findings provide new conclusive evidence that PC-NVs can promote neurovascular regeneration and recovery of erectile function under diabetic conditions via an Lcn2-dependent mechanism. Thus, local administration of PC-NVs may be a promising treatment strategy for the treatment of diabetic ED.

Latrophilin-2 is a novel receptor of LRG1 that rescues vascular and neurological abnormalities and restores diabetic erectile function.

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.

Pericyte­derived extracellular vesicles­mimetic nanovesicles improves peripheral nerve regeneration in mouse models of sciatic nerve transection.

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.

RNA-sequencing profiling analysis of pericyte-derived extracellular vesicle-mimetic nanovesicles-regulated genes in primary cultured fibroblasts from normal and Peyronie's disease penile tunica albuginea.

BACKGROUND: Peyronie's disease (PD) is a severe fibrotic disease of the tunica albuginea that causes penis curvature and leads to penile pain, deformity, and erectile dysfunction. The role of pericytes in the pathogenesis of fibrosis has recently been determined. Extracellular vesicle (EV)-mimetic nanovesicles (NVs) have attracted attention regarding intercellular communication between cells in the field of fibrosis. However, the global gene expression of pericyte-derived EV-mimetic NVs (PC-NVs) in regulating fibrosis remains unknown. Here, we used RNA-sequencing technology to investigate the potential target genes regulated by PC-NVs in primary fibroblasts derived from human PD plaque. METHODS: Human primary fibroblasts derived from normal and PD patients was cultured and treated with cavernosum pericytes isolated extracellular vesicle (EV)-mimetic nanovesicles (NVs). A global gene expression RNA-sequencing assay was performed on normal fibroblasts, PD fibroblasts, and PD fibroblasts treated with PC-NVs. Reverse transcription polymerase chain reaction (RT-PCR) was used for sequencing data validation. RESULTS: A total of 4135 genes showed significantly differential expression in the normal fibroblasts, PD fibroblasts, and PD fibroblasts treated with PC-NVs. However, only 91 contra-regulated genes were detected among the three libraries. Furthermore, 20 contra-regulated genes were selected and 11 showed consistent changes in the RNA-sequencing assay, which were validated by RT-PCR. CONCLUSION: The gene expression profiling results suggested that these validated genes may be good targets for understanding potential mechanisms and conducting molecular studies into PD.

Vasohibin-1 rescues erectile function through up-regulation of angiogenic factors in the diabetic mice.

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.

Oral Administration of the p75 Neurotrophin Receptor Modulator, LM11A-31, Improves Erectile Function in a Mouse Model of Cavernous Nerve Injury.

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.

Endothelial cells under therapy-induced senescence secrete CXCL11, which increases aggressiveness of breast cancer cells.

The effects of senescence associated secretory phenotype (SASP) from therapy-induced senescent endothelial cells on tumor microenvironment (TME) remains to be clarified. Here, we investigated effects of ionizing radiation (IR)- and doxorubicin-induced senescent HUVEC on TME. MDA-MB-231 cancer cells treated with conditioned medium (CM) from senescent HUVEC or co-cultured with senescent HUVEC significantly increased cancer cell proliferation, migration, and invasion. We found that CXCL11 plays a principal role in the senescent CM-induced aggressive activities of MDA-MB-231 cells. When we treated HUVEC with a neutralizing anti-CXCL11 antibody or CXCL11 SiRNA, or treated MDA-MB-231 cells with CXCR3 SiRNA, we observed synergistic diminution of the ability of the HUVEC SASP to alter the migration and spheroid invasion of cancer cells. ERK activation was involved in the HUVEC SASP-induced aggressive activity of MDA-MB-231 cells. Finally, we observed the in vivo effect of CXCL11 from the senescent HUVEC in tumor-bearing mice. Together, our results demonstrate that SASP from endothelial cells experiencing therapy-induced senescence promotes the aggressive behavior of cancer cells, and that CXCL11 can potentially be targeted to prevent the adverse effects of therapy-induced senescent endothelial cells on the tumor microenvironment.

Intracavernous delivery of Dickkopf3 gene or peptide rescues erectile function through enhanced cavernous angiogenesis in the diabetic mouse.

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.

Labial paraffinoma in menopause women.

Age-related changes in menopause women not only affect the physical appearance of the female external genitalia but also disrupt sexual functioning, for which many menopause women may require treatment to resolve these aesthetic problems or sexual dysfunction. Unfortunately, it is not infrequent that women seek non-medical solutions, including local injection of an unapproved agent into external genitalia, and it is extremely rare for it to be reported in literature. This case study reports on the experiences of treating two menopause women who had labial granuloma induced by local injection of paraffin.

Roles of Exosome-Like Vesicles Released from Inflammatory C2C12 Myotubes: Regulation of Myocyte Differentiation and Myokine Expression.

BACKGROUND/AIMS: The complicated differentiation processes of cells in skeletal muscle against inflammation that induce muscle atrophy are not fully elucidated. Given that skeletal muscle is a secretory organ, we evaluated the effects of inflammation on myogenic signals and myokine expression, and the roles of inflammatory exosomes released by myotubes in myogenic differentiation. METHODS: Inflammation was induced by treatment of fully differentiated C2C12 myotubes with a cytokine mixture of TNF-α and INF-γ. Exosome-like vesicles (ELVs) were isolated from conditioned media of control or inflamed myotubes and incubated with myoblasts. The expression of molecular switches that contribute to myogenic differentiation, including several kinases, their downstream targets, and myokines, were evaluated using immunoblot analysis in inflamed myotubes and in myoblasts treated with ELVs. RESULTS: Inflammation activated molecular mechanisms contributing to muscle atrophy, including AMPK, p-38 MAPK and JNK, while inhibiting Akt-mediated myogenic signals. In addition, inflammation induced myostatin expression with suppression of a myostatin-counteracting myokine, decorin. Well-characterized ELVs released from inflamed myotubes induced myoblast inflammation and inhibited myogenic mechanisms while stimulating atrophic signals. CONCLUSION: Inflammation of skeletal muscle induces muscle atrophy via multiple mechanisms, including the regulation of myokines and kinases. Inflammatory ELVs are likely to contribute to inflammation-induced muscle atrophy.

Combination of stromal vascular fraction and Ad-COMP-Ang1 gene therapy improves long-term therapeutic efficacy for diabetes-induced erectile dysfunction.

Men with diabetic erectile dysfunction (ED) respond poorly to the currently available oral phosphodiesterase-5 inhibitors. Therefore, functional therapies for diabetic ED are needed. Stromal vascular fraction (SVF) and the adenovirus-mediated cartilage oligomeric matrix angiopoietin-1 (Ad-COMP-Ang1) gene are known to play critical roles in penile erection. We previously reported that SVF and Ad-COMP-Ang1 have only a short-term effect in restoring erectile function. Further improvements to ED therapy are needed for long-lasting effects. In the present study, we aimed to test if the combination of SVF and Ad-COMP-Ang1 could extend the erection effect in diabetic ED. We found that the combination therapy showed a long-term effect in restoring erectile function through enhanced penile endothelial and neural cell regeneration. Combination therapy with SVF and Ad-COMP-Ang1 notably restored cavernous endothelial cell numbers, pericyte numbers, endothelial cell-cell junctions, decreased cavernous endothelial cell permeability, and promoted neural regeneration for at least 4 weeks in diabetic mice. In summary, this is an initial description of the long-term effect of combination therapy with SVF and Ad-COMP-Ang1 in restoring erectile function through a dual effect on endothelial and neural cell regeneration. Such combination therapy may have therapeutic potential for the treatment of diabetic ED.

Pericyte-Derived Dickkopf2 Regenerates Damaged Penile Neurovasculature Through an Angiopoietin-1-Tie2 Pathway.

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.

Dickkopf2 rescues erectile function by enhancing penile neurovascular regeneration in a mouse model of cavernous nerve injury.

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.