A Simple and Nonenzymatic Method to Isolate Human Corpus Cavernosum Endothelial Cells and Pericytes for the Study of Erectile Dysfunction

A Method to Isolate Pericytes From the Mouse Urinary Bladder for the Study of Diabetic Bladder Dysfunction / 대한배뇨장애요실금학회지

Purpose@#Pericytes surround the endothelial cells in microvessels and play a distinct role in controlling vascular permeability and maturation. The loss of pericyte function is known to be associated with diabetic retinopathy and erectile dysfunction. This study aimed to establish a technique for the isolation of pericytes from the mouse urinary bladder and an in vitro model that mimics in vivo diabetic bladder dysfunction. @*Methods@#To avoid contamination with epithelial cells, the urothelial layer was meticulously removed from the underlying submucosa and detrusor muscle layer. The tissues were cut into multiple pieces, and the fragmented tissues were settled by gravity into collagen I-coated culture plates. The cells were cultured under normal-glucose (5 mmol/L) or high-glucose (30 mmol/L) conditions, and tube formation, cell proliferation, and TUNEL assays were performed. We also performed hydroethidine staining to measure superoxide anion production. @*Results@#We successfully isolated high-purity pericytes from the mouse urinary bladder. The cells were positively stained for platelet-derived growth factor receptor-β and NG2 and negatively stained for smooth muscle cell markers (desmin and myosin) and an endothelial cell marker (CD31). The number of tubes formed and the number of proliferating cells were significantly lower when the pericytes were exposed to high-glucose conditions compared with normal-glucose conditions. In addition, there were significant increases in superoxide anion production and the number of apoptotic cells when the pericytes were cultured under high-glucose conditions. @*Conclusions@#To the best of our knowledge, this is the first study to isolate and culture pericytes from the mouse urinary bladder. Our model would be a useful tool for screening the efficacy of therapeutic candidates targeting pericyte function in diabetic bladder dysfunction and exploring the functional role of specific targets at the cellular level.

A Method to Isolate Pericytes From the Mouse Urinary Bladder for the Study of Diabetic Bladder Dysfunction / 대한배뇨장애요실금학회지

Purpose@#Pericytes surround the endothelial cells in microvessels and play a distinct role in controlling vascular permeability and maturation. The loss of pericyte function is known to be associated with diabetic retinopathy and erectile dysfunction. This study aimed to establish a technique for the isolation of pericytes from the mouse urinary bladder and an in vitro model that mimics in vivo diabetic bladder dysfunction. @*Methods@#To avoid contamination with epithelial cells, the urothelial layer was meticulously removed from the underlying submucosa and detrusor muscle layer. The tissues were cut into multiple pieces, and the fragmented tissues were settled by gravity into collagen I-coated culture plates. The cells were cultured under normal-glucose (5 mmol/L) or high-glucose (30 mmol/L) conditions, and tube formation, cell proliferation, and TUNEL assays were performed. We also performed hydroethidine staining to measure superoxide anion production. @*Results@#We successfully isolated high-purity pericytes from the mouse urinary bladder. The cells were positively stained for platelet-derived growth factor receptor-β and NG2 and negatively stained for smooth muscle cell markers (desmin and myosin) and an endothelial cell marker (CD31). The number of tubes formed and the number of proliferating cells were significantly lower when the pericytes were exposed to high-glucose conditions compared with normal-glucose conditions. In addition, there were significant increases in superoxide anion production and the number of apoptotic cells when the pericytes were cultured under high-glucose conditions. @*Conclusions@#To the best of our knowledge, this is the first study to isolate and culture pericytes from the mouse urinary bladder. Our model would be a useful tool for screening the efficacy of therapeutic candidates targeting pericyte function in diabetic bladder dysfunction and exploring the functional role of specific targets at the cellular level.

Combination of stromal vascular fraction and Ad-COMP-Ang1 gene therapy improves long-term therapeutic efficacy for diabetes-induced erectile dysfunction / 亚洲男科学杂志(英文版)

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.

Silencing Histone Deacetylase 7 Alleviates Transforming Growth Factor-β1-Induced Profibrotic Responses in Fibroblasts Derived from Peyronie's Plaque

PURPOSE: Epigenetic modifications, such as histone acetylation/deacetylation and DNA methylation, play a crucial role in the pathogenesis of inflammatory disorders and fibrotic diseases. The aim of this study was to study the differential gene expression of histone deacetylases (HDACs) in fibroblasts isolated from plaque tissue of Peyronie's disease (PD) or normal tunica albuginea (TA) and to examine the anti-fibrotic effect of small interfering RNA (siRNA)-mediated silencing of HDAC7 in fibroblasts derived from human PD plaque. MATERIALS AND METHODS: For differential gene expression study, we performed reverse-transcriptase polymerase chain reaction for HDAC isoforms (1–11) in fibroblasts isolated from PD plaque or normal TA. Fibroblasts isolated from PD plaque were pretreated with HDAC7 siRNA (100 pmol) and then stimulated with transforming growth factor-β1 (TGF-β1, 10 ng/mL). Protein was extracted from treated fibroblasts for Western blotting. We also performed immunocytochemistry to detect the expression of extracellular matrix proteins and to examine the effect of HDAC2 siRNA on the TGF-β1-induced nuclear translocation of Smad2/3 and myofibroblastic differentiation. RESULTS: The mRNA expression of HDAC2, 3, 4, 5, 7, 8, 10, and 11 was higher in fibroblasts isolated from PD plaque than in fibroblasts isolated from normal TA tissue. Knockdown of HDAC7 in PD fibroblasts inhibited TGF-β1-induced nuclear shuttle of Smad2 and Smad3, transdifferentiation of fibroblasts into myofibroblasts, and abrogated TGF-β1-induced production of extracellular matrix protein. CONCLUSIONS: These findings suggest that specific inhibition of HDAC7 with RNA interference may represent a promising epigenetic therapy for PD.

Differential Expression of Nerve Injury-Induced Protein 1 (Ninjurin 1) in In Vivo and In Vitro Models for Diabetic Erectile Dysfunction

PURPOSE: Endothelial dysfunction and peripheral neuropathy are important mechanisms responsible for diabetes-induced erectile dysfunction (ED). Nerve injury-induced protein 1 (Ninjurin 1) is known to be related to neuroinflammatory processes and is also reported to induce vascular regression during the developmental period. In the present study, we determined the differential expression of Ninjurin 1 in penile tissue of streptozotocin (STZ)-induced diabetic mice with ED. MATERIALS AND METHODS: Diabetes was induced in 8-week-old C57BL/6J mice by intraperitoneal injections of STZ (50 mg/kg for 5 days). Eight weeks later, erectile function was measured by electrical stimulation of the cavernous nerve (n=6 per group). The penis was then harvested for immunohistochemical analysis and Western blot analysis for Ninjurin 1 (n=4 per group). We also determined Ninjurin 1 expression in primary cultured mouse cavernous endothelial cells (MCECs) incubated under the following conditions: normal glucose condition (5 mM), high-glucose condition (30 mM), and high-glucose condition (30 mM)+insulin (1 nM). RESULTS: The expression of Ninjurin 1 protein was significantly higher in both cavernous endothelial cells and the dorsal nerve bundle of diabetic mice than in those of controls. In the in vitro study in MCECs, Ninjurin 1 expression was also significantly increased by the high-glucose condition and was returned to baseline levels by treatment with insulin. CONCLUSIONS: Regarding the role of Ninjurin 1 in neuropathy and vascular regression, it would be interesting to examine the effects of inhibition of Ninjurin 1 on erectile function in animal models of ED with a vascular or neurogenic cause.