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

Ghatak Kalyan,Yin Guo Nan,Hong Soon-Sun,Kang Ju-Hee,Suh Jun-Kyu,Ryu Ji-Kan 대한남성과학회 2022 The World Journal of Men's Health Vol.40 No.4

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 normalglucose 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.

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.

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.

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

Yin, Guo Nan,Jin, Hai-Rong,Choi, Min-Ji,Limanjaya, Anita,Ghatak, Kalyan,Minh, Nguyen Nhat,Ock, Jiyeon,Kwon, Mi-Hye,Song, Kang-Moon,Park, Heon Joo,Kim, Ho Min,Kwon, Young-Guen,Ryu, Ji-Kan,Suh, Jun-Kyu American Diabetes Association 2018 Diabetes Vol.67 No.6

<P>Penile erection requires well-coordinated interactions between vascular and nervous systems. Penile neurovascular dysfunction is amajor 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 co-culture 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.</P>

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

Kang, Dong Hyuk,Yin, Guo Nan,Choi, Min-Ji,Song, Kang-Moon,Ghatak, Kalyan,Minh, Nguyen Nhat,Kwon, Mi-Hye,Seong, Do-Hwan,Ryu, Ji-Kan,Suh, Jun-Kyu Korean Society for Sexual Medicine and Andrology 2018 The World Journal of Men's Health Vol.36 No.2

<P><B>Purpose</B></P><P>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.</P><P><B>Materials and Methods</B></P><P>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.</P><P><B>Results</B></P><P>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.</P><P><B>Conclusions</B></P><P>These findings suggest that specific inhibition of HDAC7 with RNA interference may represent a promising epigenetic therapy for PD.</P>

Optimizing <i>in vivo</i> gene transfer into mouse corpus cavernosum by use of surface electroporation

Song, Kang-Moon,Choi, Min Ji,Kwon, Mi-Hye,Ghatak, Kalyan,Park, Soo-Hwan,Ryu, Dong-Soo,Ryu, Ji-Kan,Suh, Jun-Kyu 대한비뇨기과학회 2015 Investigative and Clinical Urology Vol.56 No.3

<P><B>Purpose</B></P><P>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 <I>in vivo</I> electroporation-mediated gene delivery into mouse corpus cavernosum.</P><P><B>Materials and Methods</B></P><P>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.</P><P><B>Results</B></P><P>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.</P><P><B>Conclusions</B></P><P>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.</P>

Vactosertib, a Novel, Orally Bioavailable Activin Receptor-Like Kinase 5 Inhibitor, Promotes Regression of Fibrotic Plaques in a Rat Model of Peyronie’s Disease

Kang-Moon Song,Doo Yong Chung,Min Ji Choi,Kalyan Ghatak,Nguyen Nhat Minh,Anita Limanjaya,Mi-Hye Kwon,옥지연,Guo Nan Yin,Dae-Kee Kim,Ji-Kan Ryu,Jun-Kyu Suh 대한남성과학회 2020 The World Journal of Men's Health Vol.38 No.4

Purpose: To examine the therapeutic effect of Vactosertib, a small molecule inhibitor of transforming growth factor-β (TGF-β) 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- β1-induced enhancement of extracellular matrix protein production and hydroxyproline content in PD fibroblasts in vitro by hindering the TGF-β1-induced Smad2/3 phosphorylation and nuclear translocation, and fibroblast-to-myofibroblast transdifferentiation. Conclusions: In view of the critical role of TGF-β 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.

Three-Dimensional Reconstruction of Neurovascular Network in Whole Mount Preparations and Thick-Cut Transverse Sections of Mouse Urinary Bladder

Nguyen Nhat Minh,Song Kang-Moon,Choi Min-Ji,Ghatak Kalyan,Limanjaya Anita,Kwon Mi-Hye,Chung Doo Yong,Ock Jiyeon,Yin Guo Nan,Park Chang-Shin,Suh Jun-Kyu,Ryu Ji-Kan 대한남성과학회 2021 The World Journal of Men's Health Vol.39 No.1

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 βIII-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.