Inhibition of Ninjurin 1 restores erectile function through dual angiogenic and neurotrophic effects in the diabetic mouse.

Penile erection is a neurovascular phenomenon, and erectile dysfunction (ED) is caused mainly by vascular risk factors or diseases, neurologic abnormalities, and hormonal disturbances. Men with diabetic ED often have severe endothelial dysfunction and peripheral nerve damage, which result in poor response to oral phosphodiesterase-5 inhibitors. Nerve injury-induced protein 1 (Ninjurin 1, Ninj1) is known to be involved in neuroinflammatory processes and to be related to vascular regression during the embryonic period. Here, we demonstrate in streptozotocin-induced diabetic mice that inhibition of the Ninj1 pathway by administering Ninj1-neutralizing antibody (Ninj1-Ab) or by using Ninj1-knockout mice successfully restored erectile function through enhanced penile angiogenesis and neural regeneration. Angiopoietin-1 (Ang1) expression was down-regulated and angiopoietin-2 expression was up-regulated in the diabetic penis compared with that in controls, and these changes were reversed by treatment with Ninj1-Ab. Ninj1 blockade-mediated penile angiogenesis and neural regeneration as well as recovery of erectile function were abolished by inhibition of Ang1-Tie2 (tyrosine kinase with Ig and epidermal growth factor homology domain-2) signaling with soluble Tie2 antibody or Ang1 siRNA. The present results suggest that inhibition of the Ninj1 pathway will be a novel therapeutic strategy for treating ED.

Xenogenic transplantation of human breast adipose-derived stromal vascular fraction enhances recovery of erectile function in diabetic mice.

The adipose tissue-derived stromal vascular fraction (SVF) is an ideal source of stem and stromal cells. The aim of this study was to examine whether and how xenogenic transplantation of human breast SVF restores erectile function in diabetic mice. Human SVF was isolated from five patients (age, 20-45 yr) undergoing reduction mammoplasty. Eight-week-old C57BL/6J mice were used, and diabetes was induced by intraperitoneal injection of streptozotocin. At 8 wk after induction of diabetes, the animals were randomly distributed into controls and diabetic mice treated with a single intracavernous injection of PBS, human SVF at different concentrations, or human SVF lysate. Two weeks later, erectile function was measured by cavernous nerve stimulation, and the penis was then harvested for biochemical examinations. Erectile function was significantly improved in diabetic mice treated with human SVF (2 × 10(5), 5 × 10(5), and 1 × 10(6) cells/20 µl) and SVF lysate. Human SVF treatment in diabetic mice significantly increased cavernous endothelial and smooth muscle cell contents, induced eNOS phosphorylation, and restored penile nNOS-positive nerve fibers. Human SVF lysate induced secretion of angiogenic factors and expression of their receptors. Human SVF did not increase serum levels of proinflammatory cytokines. A limitation of this study was that the exact composition of the human SVF was not examined. In summary, xenogenic transplantation of human SVF did not induce systemic inflammation and successfully improved erectile function in diabetic mice through enhanced penile angiogenesis and neural regeneration.

Silencing histone deacetylase 2 using small hairpin RNA induces regression of fibrotic plaque in a rat model of Peyronie's disease.

OBJECTIVES: To examine the therapeutic effect of adenovirus encoding histone deacetylase 2 (HDAC2) small hairpin RNA (Ad-HDAC2 shRNA) in a rat model of Peyronie's disease (PD) and to determine the mechanisms by which HDAC2 knockdown ameliorates fibrotic responses in primary fibroblasts derived from human PD plaque. MATERIALS AND METHODS: Rats were distributed into four groups (n = 6 per group): age-matched controls without treatment; rats in which PD has been induced (PD rats) without treatment; PD rats receiving a single injection of control adenovirus encoding scrambled small hairpin RNA (Ad-shRNA) (day 15; 1 × 10(8) pfu/0.1 mL phosphate-buffered saline [PBS]); and PD rats receiving a single injection of Ad-HDAC2 shRNA (day 15; 1 × 10(8) pfu/0.1 mL PBS) into the lesion. PD-like plaque was induced by repeated intratunical injections of 100 µL each of human fibrin and thrombin solutions on days 0 and 5. On day 30, the penis was harvested for histological examination. Fibroblasts isolated from human PD plaque were pretreated with HDAC2 small interfering (si)RNA (100 pmoL) and then stimulated with transforming growth factor (TGF)-ß1 (10 ng/mL) to determine hydroxyproline levels, procollagen mRNA, apoptosis and protein expression of poly(ADP-ribose) polymerase 1 (PARP1) and cyclin D1. RESULTS: We observed that Ad-HDAC2 shRNA decreased inflammatory cell infiltration, reduced transnuclear expression of phospho-Smad3 and regressed fibrotic plaque of the tunica albuginea in PD rats in vivo. siRNA-mediated silencing of HDAC2 significantly decreased the TGF-ß1-induced transdifferentiation of fibroblasts into myofibroblasts and collagen production, and induced apoptosis by downregulating the expression of PARP1, and decreased the expression of cyclin D1 (a positive cell-cycle regulator) in primary cultured fibroblasts derived from human PD plaque in vitro. CONCLUSION: Specific inhibition of HDAC2 with RNA interference may represent a novel targeted therapy for PD.

Intracavernous delivery of stromal vascular fraction restores erectile function through production of angiogenic factors in a mouse model of cavernous nerve injury.

INTRODUCTION: Erectile dysfunction (ED) is a major complication of radical prostatectomy. Men with radical prostatectomy-induced ED respond less positively to oral phosphodiesterase-5 inhibitors. AIM: The study aims to examine whether and how stromal vascular fraction (SVF) restores erectile function in mice with cavernous nerve injury (CNI). METHODS: Twelve-week-old male C57BL/6J mice were used and the animals were distributed into five groups: sham operation group and CNI group receiving a single intracavernous injection of phosphate-buffered saline (PBS) or SVF (1 × 10(4) , 1 × 10(5) , or 3 × 10(5) cells/20 µL, respectively). SVF was isolated from epididymal adipose tissues of green fluorescence protein transgenic mice. MAIN OUTCOME MEASURES: Two weeks after injection, erectile function was measured by cavernous nerve stimulation. The penis was stained with antibodies to platelet/endothelial cell adhesion molecule-1, phosphohistone H3, and phosphorylated endothelial nitric oxide synthase (phospho-eNOS). We also performed Western blot for angiopoietin-1 (Ang-1), vascular endothelial growth factor-A, hepatocyte growth factor, phospho-eNOS, and eNOS in the corpus cavernosum tissue. RESULTS: Local delivery of SVF restored erectile function in a dose-dependent manner in CNI mice. The highest erectile response was noted at a dose of 3 × 10(5) cells, for which the response was comparable with that in the sham operation group. Local delivery of SVF significantly increased the expression of angiogenic factor proteins and induced cavernous endothelial cell proliferation and eNOS phosphorylation compared with that in the PBS-treated CNI group. SVF-induced promotion of cavernous angiogenesis and erectile function was diminished in the presence of soluble antibody to Tie2, a receptor tyrosine kinase of Ang-1. CONCLUSION: Secretion of angiogenic factors from SVF is an important mechanism by which SVF induces cavernous endothelial regeneration and restores erectile function. These findings suggest that cavernous endothelial regeneration by using SVF may represent a promising treatment strategy for radical prostatectomy-induced ED.

Erectile dysfunction precedes other systemic vascular diseases due to incompetent cavernous endothelial cell-cell junctions.

PURPOSE: Erectile dysfunction is often a harbinger of cardiovascular disease. We sought to gain mechanistic insight at the cellular and molecular levels into why erectile dysfunction precedes the clinical consequences of cardiovascular disease. MATERIALS AND METHODS: Diabetes was induced by intraperitoneal streptozotocin injection in 8-week-old C57BL/6J mice. At 8 weeks after diabetes induction, we determined the expression of endothelial cell-cell junction proteins and vascular endothelial permeability in the penis, heart and hind limb by systemic injection of various vascular space markers (350 Da to 2,000 kDa) or by immunohistochemical staining with antibody to oxidized low density lipoprotein. We also investigated the effect of recombinant Ang1 protein on cavernous endothelial permeability. RESULTS: Alterations in the integrity of the endothelial cell-cell junction, including a decrease in endothelial cell-cell junction proteins and an increase in vascular permeability to fluorescent tracers or oxidized low density lipoprotein, were prominent in the cavernous tissue of diabetic mice. In contrast, no significant changes in endothelial cell-cell junction proteins or vascular permeability were noted in heart or hind limb tissue according to the diabetic condition. Intracavernous injection of Ang1 protein, an anti-permeability factor, significantly decreased cavernous endothelial permeability to oxidized low density lipoprotein by restoring endothelial cell-cell junction proteins in diabetic mice. CONCLUSIONS: The incompetent cavernous endothelial cell-cell junction in the diabetic condition provides an important clue to why erectile dysfunction is highly prevalent and often precedes other systemic vascular diseases.

Effect of intracavernous administration of angiopoietin-4 on erectile function in the streptozotocin-induced diabetic mouse.

INTRODUCTION: Erectile dysfunction (ED) is a highly prevalent complication of diabetes, and the severity of endothelial dysfunction is one of the most important factors in reduced responsiveness to oral phosphodiesterase type 5 inhibitors. AIM: To study the effects of human angiopoietin-4 (Ang-4) protein on erectile function in diabetic mice. METHODS: Diabetes was induced by intraperitoneal injection of streptozotocin into 8-week-old C57BL/6J male mice. At 8 weeks after the induction of diabetes, the animals were divided into four groups: control nondiabetic mice and diabetic mice receiving two successive intracavernous injections of phosphate buffered saline (days -3 and 0), a single intracavernous injection of Ang-4 protein (day 0), or two successive intracavernous injections of Ang-4 protein (days -3 and 0). MAIN OUTCOME MEASURES: One week after treatment, we measured erectile function by electrical stimulation of the cavernous nerve. The penis was harvested and stained with hydroethidine or antibodies to Ang-4, platelet/endothelial cell adhesion molecule-1, and phosphorylated endothelial nitric oxide synthase (eNOS). We also determined the differential expression of Ang-4 in cavernous tissue in the control and diabetic mice. The effect of Ang-4 protein on the phosphorylation of Tie-2, Akt, and eNOS was determined in human umbilical vein endothelial cells (HUVECs) by Western blot. RESULTS: The cavernous expression of Ang-4 was downregulated in diabetic mice; Ang-4 was mainly expressed in endothelial cells. Local delivery of Ang-4 protein significantly increased cavernous endothelial content, induced eNOS phosphorylation, and decreased the generation of superoxide anion and apoptosis in diabetic mice. Ang-4 protein strongly increased the phosphorylation of Tie-2, Akt, and eNOS in HUVECs. Repeated intracavernous injections of Ang-4 induced significant restoration of erectile function in diabetic mice (87% of control values), whereas a single intracavernous injection of Ang-4 protein elicited modest improvement. CONCLUSIONS: Cavernous endothelial regeneration by use of Ang-4 protein may have potential for the treatment of vascular disease-induced ED, such as diabetic ED.

Expression of the apelin-APJ pathway and effects on erectile function in a mouse model of vasculogenic erectile dysfunction.

INTRODUCTION: Much attention has recently been focused on therapeutic angiogenesis as a treatment for erectile dysfunction (ED). The apelin and apelin receptor (APJ) system is known to cause endothelium-dependent vasodilatation and to be involved in angiogenesis. AIM: To examine the differential expression of apelin and APJ in animal models of vasculogenic ED and to determine whether and how enhancement of apelin-APJ signaling restores erectile function in hypercholesterolemic mice. METHODS: Acute cavernous ischemia was induced in C57BL/6J mice by bilateral occlusion of internal iliac arteries, and chronic vasculogenic ED was induced by feeding a high-cholesterol diet or by intraperitoneal injection of streptozotocin. MAIN OUTCOME MEASURES: Messenger RNA (mRNA) levels of apelin and APJ were determined in cavernous tissue of each vasculogenic ED model by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR). We evaluated erectile function by electrical stimulation of the cavernous nerve in hypercholesterolemic mice 1, 3, 7, and 14 days after a single intracavernous injection of apelin protein (5 µg/20 µL). The penis was harvested for histologic examinations and Western blot analysis. RESULTS: The cavernous mRNA expression of apelin and APJ was up-regulated in acute ischemia model and down-regulated in chronic vasculogenic ED models. A significant restoration of erectile function was noted 1 day after injection of apelin protein into the penis of hypercholesterolemic mice; however, erectile function returned to baseline values thereafter. The beneficial effects of apelin on erectile function resulted mainly from an activation of endothelial nitric oxide synthase and increase in nitric oxide bioavailability through reduction in reactive oxygen species-mediated endothelial apoptosis rather than through direct endothelial cell proliferation. CONCLUSION: These findings suggest that apelin-APJ signaling is a potential therapeutic target in the treatment of vasculogenic ED. Further studies are needed to develop a potent agonist for APJ and to determine the role of repeated dosing of apelin on long-term recovery of erectile function.

Nerve injury-induced protein 1 (Ninjurin-1) is a novel therapeutic target for cavernous nerve injury-induced erectile dysfunction in mice.

INTRODUCTION: Radical prostatectomy for prostate cancer can not only induce cavernous nerve injury (CNI) but also result in structural changes in the cavernous tissues. Nerve injury-induced protein 1, Ninjurin-1 (Ninj1), is known to be involved in neuroinflammatory processes and to be related to vascular regression during the embryonic period. AIM: The study aims to determine whether and how Ninj1 neutralizing antibody (Ninj1-Ab) restores erectile function in mice with CNI. METHODS: Twelve-week-old C57BL/6J mice were used and distributed into four groups: sham operation group and CNI groups receiving a single intracavernous injection of immunoglobulin G (IgG) control antibody, low-dose Ninj1-Ab (1.0 µg/20 µL), or high-dose Ninj1-Ab (2.5 µg/20 µL). MAIN OUTCOME MEASURES: One week after bilateral cavernous nerve crush, erectile function was measured by electrical stimulation of the cavernous nerve. The penis was harvested for histologic examinations and Western blot analysis. RESULTS: The cavernous expression of Ninj1 protein was upregulated up to 7 days after CNI and returned to baseline levels thereafter. Local delivery of Ninj1-Ab significantly increased penile neuronal nitric oxide synthase and neurofilament contents, induced cavernous endothelial proliferation and phosphorylation of Akt and endothelial nitric oxide synthase, and decreased endothelial cell apoptosis in the CNI mice by upregulating angiopoietin-1 and downregulating angiopoietin-2. High-dose Ninj1-Ab induced profound restoration of erectile function in the CNI mice (91% of sham control values), whereas low-dose Ninj1-Ab elicited partial improvement. CONCLUSION: The dual neurotrophic and angiogenic effects of Ninj1 blockade may provide a good opportunity for treating erectile dysfunction resulting from radical prostatectomy.

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.

Isolinderalactone suppresses human glioblastoma growth and angiogenic activity in 3D microfluidic chip and in vivo mouse models.

Glioblastoma multiforme (GBM) is a lethal and highly vascular type of brain tumor. We previously reported that isolinderalactone enhances GBM apoptosis in vitro and in vivo, but its role in tumor angiogenesis is unknown. Here, we investigated the anti-angiogenic activity of isolinderalactone and its mechanisms. In a human GBM xenograft mouse model, isolinderalactone significantly reduced tumor growth and vessels. Isolinderalactone decreased the expression of vascular endothelial growth factor (VEGF) mRNA, protein, and VEGF secretion in hypoxic U-87 GBM cells and also in xenograft GMB tissue. In addition, we demonstrated that isolinderalactone significantly inhibited the proliferation, migration, and capillary-like tube formation of human brain microvascular endothelial cells (HBMECs) in the presence of VEGF. We also found that isolinderalactone decreased sprout diameter and length in a 3D microfluidic chip, and strongly reduced VEGF-triggered angiogenesis in vivo Matrigel plug assay. Isolinderalactone downregulated hypoxia-inducible factor-1α (HIF-1α) and HIF-2α proteins, decreased luciferase activity driven by the VEGF promoter in U-87 cells under hypoxic conditions, and suppressed VEGF-driven phosphorylation of VEGFR2 in HBMECs. Taken together, our results suggest that isolinderalactone is a promising candidate for GBM treatment through tumor angiogenesis inhibition.

Topical cell-free conditioned media harvested from adipose tissue-derived stem cells promote recovery from corneal epithelial defects caused by chemical burns.

Corneal chemical burns can lead to blindness following serious complications. As most of these complications are caused by failure of reepithelization during the acute phase, treatment at this stage is critical. Although there have been some studies on corneal injury recovery using adipose tissue-derived stem cells (ADSCs), none has reported the effect of topical cell-free conditioned culture media (CM) derived from ADSCs on corneal epithelial regeneration. Here, the best conditions for CM were selected and used for in vitro and in vivo experiments. Corneal burn in rats was induced using 100% alcohol. The chosen CM was administered to corneal burn rats (CM-treated [CT] group) four times a day for three days and this group was compared with the normal control and corneal burn (CB) groups. Biomicroscopic fluorescence images and the actual physical corneas were taken over time and used for analysis. mRNA levels of hepatocyte growth factor and epidermal growth factor (EGF) were significantly increased, whereas those of vascular endothelial growth factor, interleukin (IL)-1ß, IL-6, IL-10, and matrix metalloproteinase-9 were significantly decreased in the CT group compared with those in the CB group. The numbers of proliferating cell nuclear antigen- and zonular occludens-1-positive cells in the CT group were significantly higher than those in the CB group. The macrophage-infiltrating corneas in the CT group expressed significantly more of the M2 marker arginase than corneas in the CB group. Optimal CM (× 0.5 concentration) treatment significantly accelerated the migration of corneal epithelial cells and induced upregulation of the expression of IL-6, EGF, and C-X-C chemokine receptor type 4 mRNAs. Overall, in this study, topical administration of cell-free CM promoted regeneration of the corneal epithelium after induction of chemical burns.

Volatile pollutants emitted from selected liquid household products.

BACKGROUND, AIM, AND SCOPE: To identify household products that may be potential sources of indoor air pollution, the chemical composition emitted from the products should be surveyed. Although this kind of survey has been conducted by certain research groups in Western Europe and the USA, there is still limited information in scientific literature. Moreover, chemical components and their proportions of household products are suspected to be different with different manufacturers. Consequently, the current study evaluated the emission composition for 42 liquid household products sold in Korea, focusing on five product classes (deodorizers, household cleaners, color removers, pesticides, and polishes). MATERIALS AND METHODS: The present study included two phase experiments. First, the chemical components and their proportions in household products were determined using a gas chromatograph and mass spectrometer system. For the 19 target compounds screened by the first phase of the experiment and other selection criteria, the second phase was done to identify their proportions in the purged-gas phase. RESULTS: The number of chemicals in the household products surveyed ranged from 9 to 113. Eight (product class of pesticides) to 17 (product class of cleaning products) compounds were detected in the purged-gas phase of each product class. Several compounds were identified in more than one product class. Six chemicals (acetone, ethanol, limonene, perchloroethylene (PCE), phenol, and 1-propanol) were identified in all five product classes. There were 13 analytes occurring with a frequency of more than 10% in the household products: limonene (76.2%), ethanol (71.4%), PCE (66.7%), phenol (40.5%), 1-propanol (35.7%), decane (33%), acetone (28.6%), toluene (19.0%), 2-butoxy ethanol (16.7%), o-xylene (16.7%), chlorobenzene (14.3%), ethylbenzene (11.9%), and hexane (11.9%). All of the 42 household products analyzed were found to contain one or more of the 19 compounds. DISCUSSION: The chemical composition varied broadly along with the product classes or product categories, and it was different from that reported in other studies abroad, although certain target chemicals were identified in both studies. This finding supports an assertion that chemical components emitted from household products may be different in different products and with different manufacturers. The chlorinated pollutants identified in the present study have not been reported to be components of cleaning products in papers published since the early 1990s. Limonene was identified as having the highest occurrence in the household products in the present study, although it was not detected in any of 67 household products sold in the U.S. CONCLUSIONS: The emission composition of selected household products was successfully examined by purge-and-trap analysis. Along with other exposure information such as use pattern of household products and the indoor climate, this composition data can be used to estimate personal exposure levels of building occupants. This exposure data can be employed to link environmental exposure to health risk. It is noteworthy that many liquid household products sold in Korea emitted several toxic aromatic and chlorinated organic compounds. Moreover, the current finding suggests that product types and manufacturers should be considered, when evaluating building occupants' exposure to chemical components emitted from household products. RECOMMENDATIONS AND PERSPECTIVES: The current findings can provide valuable information for the semiquantitative estimation of the population inhalation exposure to these compounds in indoor environments and for the selection of safer household products. However, although the chemical composition is known, the emissions of household products might include compounds formed during the use of the product or compounds not identified as ingredients by this study. Accordingly, further studies are required, and testing must be done to determine the actual composition being emitted. Similar to eco-labeling of shampoos, shower gels, and foam baths proposed by a previous study, eco-labeling of other household products is suggested.

Characterization of emissions composition for selected household products available in Korea.

The present study investigated the emission composition for 59 household products currently sold in Korea, using a headspace analysis. The chemical composition and concentrations of total volatile organic compounds (VOCs) broadly varied along with products, even within the same product category. Up to 1-17 organic compounds were detected in the headspace gas phase of any one of the products. The chemical composition of certain household products determined in the current study was different from that of other studies from other countries. Between 4 and 37 compounds were detected in the headspace gas phase of each product class. Several compounds were identified in more than one product class. Of the 59 household products analyzed, 58 emitted one or more of the 72 compounds at chromatographic peak areas above 10(4). There were 11 analytes which occurred with a frequency of more than 10%: limonene (44.2%), ethanol (30.5%), acetone (18.6%), alpha-pinene (18.6%), o,m,p-xylenes (18.6%), decane (17.0%), toluene (17.0%), beta-myrcene (11.9%), ammonia (10.2%), ethylbenzene (10.2%), and hexane (10.2%).

Multi-route trihalomethane exposure in households using municipal tap water treated with chlorine or ozone-chlorine.

In Korea, data for multi-route trihalomethane (THM) exposure in households using municipal tap water treated with ozone-chlorine or chlorine are unavailable or very limited. Accordingly, the present study was designed to obtain those data by measurements of the THM concentrations in the tap water and indoor and outdoor air in the two types of households, along with an estimation of THM exposure from water ingestion, showering, and the inhalation of indoor air. Chloroform was the most abundant THM in all three media, yet no bromoform was detected in any sample. Similar to previous findings, the winter chloroform concentration in tap water treated with chlorine (22.1 microg/l, median) was significantly higher than that in the tap water treated with ozone-chlorine (16.8 microg/l, median). However, the summer water chloroform concentrations and summer and winter water concentrations of the other two THMs (bromodichloromethane and dibromochloromethane) exhibited no significant difference between the chlorine and ozone-chlorine-treated water. It was suggested that the effects of the water parameters including biochemical oxygen demand of raw water entering water treatment plants should be considered when evaluating the advantage of ozone-chlorine disinfection for THM formation over chlorine disinfection. The indoor air THM concentration trend was also consistent with the water concentration trend. The indoor to outdoor air concentration ratios were comparable with previous studies. The THM exposure estimates from water ingestion, showering, and the inhalation of apartment indoor air when not in the shower suggested that, for residents living in the surveyed households, their exposure to THMs in the home was mostly associated with their household water uses. The THM exposure estimates from tap water ingestion were similar to those from showering.

Effect of SMAD7 gene overexpression on TGF-ß1-induced profibrotic responses in fibroblasts derived from Peyronie's plaque.

Transforming growth factor-ß1 (TGF-ß1) has been identified as one of the most important fibrogenic cytokines associated with Peyronie's disease (PD). The mothers against decapentaplegic homolog 7 (SMAD7) is an inhibitory Smad protein that blocks TGF-ß signaling pathway. The aim of this study was to examine the anti-fibrotic effect of the SMAD7 gene in primary fibroblasts derived from human PD plaques. PD fibroblasts were pretreated with the SMAD7 gene and then stimulated with TGF-ß1. Treated fibroblasts were used for Western blotting, fluorescent immunocytochemistry, hydroxyproline determination, and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assays. Overexpression of the SMAD7 gene inhibited TGF-ß1-induced phosphorylation and nuclear translocation of SMAD2 and SMAD3, transdifferentiation of fibroblasts into myofibroblasts, and quashed TGF-ß1-induced production of extracellular matrix protein and hydroxyproline. Overexpression of the SMAD7 gene decreased the expression of cyclin D1 (a positive cell cycle regulator) and induced the expression of poly (ADP-ribose) polymerase 1, which is known to terminate Smad-mediated transcription, in PD fibroblasts. These findings suggest that the blocking of the TGF-ß pathway by use of SMAD7 may be a promising therapeutic strategy for the treatment of PD.

Inhibition of histone deacetylase 2 mitigates profibrotic TGF-ß1 responses in fibroblasts derived from Peyronie's plaque.

Epigenetic modifications, such as histone acetylation/deacetylation, have been shown to play a role in the pathogenesis of fibrotic disease. Peyronie's disease (PD) is a localized fibrotic process of the tunica albuginea, which leads to penile deformity. This study was undertaken to determine the anti-fibrotic effect of small interfering RNA (siRNA)-mediated silencing of histone deacetylase 2 (HDAC2) in primary fibroblasts derived from human PD plaque. PD fibroblasts were pre-treated with HDAC2 siRNA and then stimulated with transforming growth factor-ß1 (TGF-ß1). Protein was extracted from treated fibroblasts for Western blotting and the membranes were probed with antibody to phospho-Smad2/Smad2, phospho-Smad3/Smad3, smooth muscle α-actin and extracellular matrix proteins, including plasminogen activator inhibitor-1, fibronectin, collagen I and collagen IV. We also performed immunocytochemistry to detect the expression of extracellular matrix proteins and to examine the effect of HDAC2 siRNA on the TGF-ß1-induced nuclear translocation of Smad2/3 in fibroblasts. Knockdown of HDAC2 in PD fibroblasts abrogated TGF-ß1-induced extracellular matrix production by blocking TGF-ß1-induced phosphorylation and nuclear translocation of Smad2 and Smad3, and by inhibiting TGF-ß1-induced transdifferentiation of fibroblasts into myofibroblasts. Decoding the individual function of the HDAC isoforms by use of siRNA technology, preferably siRNA for HDAC2, may lead to the development of specific and safe epigenetic therapies for PD.