Pericyte-derived heme-binding protein 1 promotes angiogenesis and improves erectile function in diabetic mice

윤국남 대한비뇨의학회 2022 Investigative and Clinical Urology Vol.63 No.4

Purpose: To comprehensively evaluate the effect on angiogenesis of heme-binding protein 1 (Hebp1) in the treatment of diabetes-induced erectile dysfunction. Materials and Methods: Mouse corpus cavernosum endothelial cells and pericytes were used for in vitro study. Four groups of mice were used: control nondiabetic mice and streptozotocin-induced diabetic mice receiving two intracavernous injections of phosphate-buffered saline, Hebp1 (1 µg), or Hebp1 (5 µg). The function of Hebp1 in diabetic conditions was evaluated by tube formation assay, aorta ring assay, migration assay, intracavernous pressure, immunofluorescence staining, and Western blot experiments. Results: We report that Hebp1 is more highly expressed in mouse corpus cavernosum pericytes and can effectively promote endothelial cell angiogenesis under high-glucose conditions. Following exogenous administration of Hebp1 protein, we found that elevated Hebp1 levels can improve the erectile function of diabetic mice, which is achieved by reducing reactive oxygen species levels and activating the PI3K/AKT/eNOS signaling pathway. Conclusions: Our findings demonstrate that Hebp1 can promote angiogenesis and improve erectile function under diabetic conditions.

Transcriptional profiling of mouse cavernous pericytes under high-glucose conditions: Implications for diabetic angiopathy

윤국남,Jitao Wu,Yuanshan Cui,Chunhua Li,Lei Shi,Zhen-Li Gao,서준규,류지간,Hai-Rong Jin 대한비뇨의학회 2021 Investigative and Clinical Urology Vol.62 No.1

Purpose: Penile erection requires integrative interactions between vascular endothelial cells, pericytes, smooth muscle cells, and autonomic nerves. Furthermore, the importance of the role played by pericytes in the pathogenesis of angiopathy has only recently been appreciated. However, global gene expression in pericytes in diabetes mellitus-induced erectile dysfunction (DMED) remains unclear. We aimed to identify potential target genes related to DMED in mouse cavernous pericytes (MCPs). Materials and Methods: Mouse cavernous tissue was allowed to settle under gravity in collagen I-coated dishes, and sprouted cells were subcultivated for experiments. To imitate diabetic conditions, MCPs were treated with normal-glucose (NG, 5 mM) or high-glucose (HG, 30 mM) media for 3 days. Microarray technology was used to evaluate gene expression profiles, and RT-PCR was used to validate sequencing data. Histological examinations and Western blot were used to validate final selected target genes related to DMED. Results: Decreased tube formation and increased apoptosis were detected in MCPs exposed to the HG condition. As shown by microarray analysis, the gene expression profiles of MCPs exposed to the NG or HG condition differed. A total of 2,523 genes with significantly altered expression were classified into 15 major gene categories. After further screening based on gene expression and RT-PCR and histologic results, we found that Hebp1 gene expression was significantly diminished under the HG condition and in DM mice. Conclusions: This gene profiling study provides new potential targets responsible for diabetes in MCPs. Validation studies suggest that Hebp1 may be a suitable biomarker for DMED.

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.

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.

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.

Regenerative therapies as a potential treatment of erectile dysfunction

정두용,류지간,윤국남 대한비뇨의학회 2023 Investigative and Clinical Urology Vol.64 No.4

Erectile dysfunction (ED) is the most common sexual dysfunction disease in adult males. ED can be caused by many factors, such as vascular disease, neuropathy, metabolic disturbances, psychosocial causes, and side effects of medications. Although current oral phosphodiesterase type 5 inhibitors can achieve a certain effect, they cause temporary dilatation of blood vessels with no curative treatment effects. Emerging targeted technologies, such as stem cell therapy, protein therapy, and low-intensity extracorporeal shock wave therapy (Li-ESWT), are being used to achieve more natural and long-lasting effects in treating ED. However, the development and application of these therapeutic methods are still in their infancy, and their pharmacological pathways and specific mechanisms have not been fully discovered. This article reviews the preclinical basic research progress of stem cells, proteins, and Li-ESWT therapy, as well as the current status of clinical application of Li-ESWT therapy.

Regulation of mast cell activation by extracellular vesicles in cow’s milk casein-induced allergic responses

Cho Young-Eun,Kim Hyun-Woo,민근영,Hwang Jin-Hyeon,김동하,김지인,윤국남,Lim Jae-Hwan,Kwun In-Sook,백문창,Kim Do-Kyun 대한독성 유전단백체 학회 2022 Molecular & cellular toxicology Vol.18 No.2

Background Food allergy is a hypersensitive immune reaction to food proteins including cow’s milk protein. Extracellular vesicles (EVs), such as exosomes, are newly discovered intercellular conveyors of functional molecular mechanisms and mediated intercellular interactions involving mast cells and are particularly relevant to allergy. Objective In this study, we investigated whether cow’s milk casein-induced allergy (CIA)-derived EVs can modulate mast cell activation. Results EVs in CIA mice and control mice were isolated using the ultracentrifugation method and the isolated EVs were quantified using BCA analysis. CIA responses were determined through changes in body temperature and systemic symptom score. The number of EVs was higher in CIA-derived EVs compared to normal EVs. EVs marker proteins such as CD63 and CD9 were elevated in CIA-derived EVs. The levels of EV-associated cytokines such as IL-6, IL-8, and TNF-α were significantly elevated in CIA-derived EVs. In addition, CIA-derived EVs significantly induced degranulation via Lyn kinase activation in mouse bone marrow-derived mast cells and human mast cells. Conclusion Our results demonstrate that CIA-derived EVs can induce a reaction similar to cow’s milk allergic reaction via mast cell activation. These results provide an insight into the pathology of cow’s milk allergy and a potential therapeutic approach through targeting EV release and/or uptake.