Optimizing in vivo gene transfer into mouse corpus cavernosum by use of surface electroporation

송강문,최민지,권미혜,Kalyan Ghatak,박수환,류동수,류지간,서준규 대한비뇨의학회 2015 Investigative and Clinical Urology Vol.56 No.3

Purpose: Electroporation is known to enhance the efficiency of gene transfer through a transient increase in cell membrane permeability. The aim of this study was to determine the optimal conditions for in vivo electroporation-mediated gene delivery into mouse corpus cavernosum. Materials and Methods: Diabetes was induced in C57BL/6 mice by intraperitoneal injections of streptozotocin. After intracavernous injection of pCMV-Luc (100 μg/40 μL), different electroporation settings (5–50 V, 8–16 pulses with a duration of 40–100 ms) were applied to the penis to establish the optimal conditions for electroporation. Gene expression was evaluated by luciferase assay. We also assessed the undesired consequences of electroporation by visual inspection and hematoxylin-eosin staining of penile tissue. Results: Electroporation profoundly induced gene expression in the corpus cavernosum tissue of normal mice in a voltage-dependent manner. We observed electrical burn scars in the penis of normal mice who received electroporation with eight 40-ms pulses at a voltage of 50 V and sixteen 40-ms pulses, eight 100-ms pulses, and sixteen 100-ms pulses at a voltage of 30 V. No detectable burn scars were noted in normal mice stimulated with eight 40-ms pulses at a voltage of 30 V. Electroporation also significantly induced gene expression in diabetic mice stimulated with 40-ms pulse at a voltage of 30 V without injury to the penis. Conclusions: We have established the optimal electroporation conditions for maximizing gene transfer into the corpus cavernosum of mice while avoiding damage to the erectile tissue. The electroporation-mediated gene delivery technique will be a valuable tool for gene therapy in the field of erectile dysfunction.

마그네슘 합금의 압출 가공기술 개발

村井 勉(T. Murai),松岡信一(S. Matsuoka),向山準(H. Mukaiyama),中川文昭(F. Nakagawa),?本進(S. Miyamoto) 한국소성·가공학회 2006 소성가공 : 한국소성가공학회지 Vol.15 No.4

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

강동혁,윤국남,최민지,송강문,Kalyan Ghatak,Nguyen Nhat Minh,권미혜,성도환,류지간,서준규 대한남성과학회 2018 The World Journal of Men's Health Vol.36 No.2

Purpose: Epigenetic modifications, such as histone acetylation/deacetylation and DNA methylation, play a crucial role in thepathogenesis of inflammatory disorders and fibrotic diseases. The aim of this study was to study the differential gene expressionof histone deacetylases (HDACs) in fibroblasts isolated from plaque tissue of Peyronie’s disease (PD) or normal tunicaalbuginea (TA) and to examine the anti-fibrotic effect of small interfering RNA (siRNA)-mediated silencing of HDAC7 in fibroblastsderived from human PD plaque. Materials and Methods: For differential gene expression study, we performed reverse-transcriptase polymerase chain reactionfor HDAC isoforms (1–11) in fibroblasts isolated from PD plaque or normal TA. Fibroblasts isolated from PD plaque were pretreatedwith HDAC7 siRNA (100 pmol) and then stimulated with transforming growth factor-β1 (TGF-β1, 10 ng/mL). Proteinwas extracted from treated fibroblasts for Western blotting. We also performed immunocytochemistry to detect the expressionof extracellular matrix proteins and to examine the effect of HDAC2 siRNA on the TGF-β1-induced nuclear translocation ofSmad2/3 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 infibroblasts isolated from normal TA tissue. Knockdown of HDAC7 in PD fibroblasts inhibited TGF-β1-induced nuclear shuttleof Smad2 and Smad3, transdifferentiation of fibroblasts into myofibroblasts, and abrogated TGF-β1-induced production ofextracellular matrix protein. Conclusions: These findings suggest that specific inhibition of HDAC7 with RNA interference may represent a promising epigenetictherapy for PD.

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

윤국남,옥지연,Min-Ji Choi,송강문,Kalyan Ghatak,Nguyen Nhat Minh,Mi Hye Kwon,Do-Hwan Seong,Hai-Rong Jin,Ji-Kan Ryu,Jun Kyu Suh 대한남성과학회 2020 The World Journal of Men's Health Vol.38 No.1

ChinaPurpose: To establish a simple and nonenzymatic technique to isolate endothelial cells (ECs) and pericytes from human corpus cavernosum tissue and to evaluate the angiogenic ability of the human cavernous EC or pericytes for the study of high glucose-induced angiopathy.Materials and Methods: For primary human cavernous EC culture, cavernous tissues were implanted into Matrigel in dishes. For primary human cavernous pericyte culture, cavernous tissues were settled by gravity into dishes. We performed immunocytochemistry and Western blot to determine phenotype and morphologic changes from passage 1 to 5. The primary cultured cells were exposed to a normal-glucose (5 mmol/L) or a high-glucose (30 mmol/L) condition, and then tube formation assay was done.Results: We successfully isolated high-purity EC and pericytes from human corpus cavernosum tissue. Primary cultured EC showed highly positive staining for von Willebrand factor, and pericyte revealed positive staining for NG2 and platelet-derived growth factor receptor-β. Primary cultured EC and pericytes maintained their cellular characteristics up to passage 2 or 3. However, we observed significant changes in their typical phenotype from the passage 4 and morphological characteristics from the passage 3. Human cavernous EC or pericytes formed well-organized capillary-like structures in normal-glucose condition, whereas severely impaired tube formation was detected in high-glucose condition.Conclusions: This study provides a simple and nonenzymatic method for primary culture of human cavernous EC and pericytes. Our study will aid us to understand the pathophysiology of diabetic erectile dysfunction, and also be a valuable tool for determining the efficacy of candidate therapeutic targets.

Gene expression profiling of mouse cavernous endothelial cells for diagnostic targets in diabetes-induced erectile dysfunction

윤국남,옥지연,최민지,Anita Limanjaya,Kalyan Ghatak,송강문,권미혜,서준규,류지간 대한비뇨의학회 2021 Investigative and Clinical Urology Vol.62 No.1

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.

Transforming Growth Factor-β Type I Receptor Inhibitor Induces Functional and Morphologic Recovery in a Rat Model of Erectile Dysfunction and Cavernous Fibrosis

류지간,Jun Kyu Suh,Seung Min Oh,Hai Rong Jin,송강문,Mi Hye Kwon,Do Kyung Kim 대한남성과학회 2012 The World Journal of Men's Health Vol.30 No.1

Purpose: To examine the effectiveness of small-molecule inhibitor of transforming growth factor-β (TGF-β) type I receptor, an activin receptor-like kinase 5 (ALK5), on erectile dysfunction (ED) in a rat model of cavernous fibrosis, in which fibrosis was induced by intracavernous injection of adenovirus expressing TGF-β1 (Ad-TGF-β1). Materials and Methods: Four-month-old Sprague-Dawley rats were divided into four groups (n=10 per group): age-matched controls without treatment, age-matched controls receiving intracavernous injection of LacZ adenovirus, and cavernous fibrosis rats receiving an intracavernous injection of saline or ALK5 inhibitor (5 mg/kg). ALK5 inhibitor or saline was administered on day 5 after injection of Ad-TGF-β1. On day 30, erectile function was assessed by electrical stimulation of the cavernous nerve and the penis was then harvested for histologic studies (n=6 per group) and for the measurement of the hydroxyproline level (n=4 per group). Results: Ad-TGF-β1-induced cavernous fibrosis rats treated with saline showed a significant decrease in cavernous smooth muscle and endothelial content, and an increase in collagen deposition, which resulted in profound deterioration of all erectile function parameters, such as the ratios of maximal intracavernous pressure (ICP), total ICP, and slope to mean arterial pressure. ALK5 inhibitor significantly restored erectile function in a rat model of cavernous fibrosis by increasing cavernous smooth muscle and endothelial content, and by blocking cavernous fibrosis. Conclusions: The results suggest that inhibition of the TGF-β pathway is a promising therapeutic strategy for the treatment of ED related to cavernous fibrosis from various causes.