Circ_0047339 promotes the activation of fibroblasts and affects the development of urethral stricture by targeting the miR-4691-5p/TSP-1 axis.

Urethral stricture is related to scar tissue fibrosis, but its pathogenesis is still unclear. This study aims to explore the regulatory mechanism of circular RNA (circRNA) in the occurrence and development of urethral stricture. CircRNA microarray was employed to analyze circRNA expression profiles between human urethral scar tissue and normal urethral tissue. The results of circRNA microarray showed that there were 296 differentially expressed genes between urethral scar tissue and normal urethral tissue. The enrichment analysis of Kyoto encyclopedia of genes and genomes showed that these circRNAs were significantly correlated with ECM-receptor interaction. The first nine differentially expressed circRNA were selected to predict the circRNA-miRNA network. RT-qPCR results showed that circ_0047339 was upregulated considerably in urethral scar tissue. Urethral scar fibroblasts were isolated from human urethral scar tissue and cultured in vitro. After silencing circ_0047339, the proliferation of urethral scar cells decreased significantly, and the expressions of Collagen I (COL-1) and α-smooth muscle actin (α-SMA) also reduced. As a competing endogenous RNA, circ_0047339 could increase the expression of TSP-1 by competitively binding miR-4691-5p. In addition, miR-4691-5p mimic transfection could inhibit the proliferation of urethral scar fibroblasts and the presentation of thrombospondin-1 (TSP-1), α-SMA and COL-1, while circ_0047339 overexpression eliminated this inhibition. Our results showed that circ_0047339 might promote the growth and fibrosis of urethral scar fibroblasts through miR-4691-5p/TSP-1 axis, thus promoting the development of urethral stricture.

[Pirfenidone alleviates urethral stricture following urethral injury in rats by suppressing TGF-ß1 signaling and inflammatory response].

OBJECTIVE: To investigate the effect of pirfenidone for reducing urethral stricture following urethral injury in rats and explore the possible mechanism. METHODS: Thirty male SD rats were randomly assigned into negative control group, positive control group and pirfenidone group (n=10). In pirfenidone and positive control groups, the rats were subjected to incision of the posterior urethral cavernous body followed by daily intraperitoneal injection of pirfenidone (100 mg/kg) and an equivalent volume of solvent, respectively. The rats in the negative control group were given intraperitoneal injections of solvent without urethral injury. At two weeks after modeling, retrograde urethrography was performed for observing urethral stricture, and the injured urethral tissues were harvested for HE staining, Masson staining, immunohistochemical staining and Western blotting for detecting the protein expressions of α-SMA and TGF-ß1. The mRNA expressions of the inflammatory factors TNF-α, IL-6, and IL-1ß were detected using qRT-PCR. RESULTS: The body weight of the rats in pirfenidone group was significantly decreased compared with that in the other two groups (P < 0.05). Retrograde urethrography showed significant narrowing of the urethra in the positive control group but not in the pirfenidone group. HE staining of the injured urethral tissues showed obvious proliferation of urethral epithelial cells with narrow urethral cavity and increased inflammatory cells in positive control group. The pathological findings of the urethra were similar between pirfenidone group and the negative control group. Masson staining revealed obviously reduced collagen fibers and regular arrangement of the fibers in pirfenidone group as compared to the positive control group. Compared with those in the negative control group, the expressions of α-SMA and TGF-ß1 were significantly increased in the positive control group, and pirfenidone treatment significantly inhibited their expressions (P < 0.05 or 0.01). Pirfenidone also significantly inhibited the mRNA expressions of TNF-α, IL-6, and IL-1ß in the injured urethral tissue (P < 0.05 or 0.01). CONCLUSION: Pirfenidone can prevent urethral fibrosis and stricture after urethral injury possibly by inhibiting the TGF-ß1 pathway and inflammatory response.

Effect of uncultured adipose-derived stromal vascular fraction on preventing urethral stricture formation in rats.

Urethral stricture (US) remains a challenging disease without effective treatment options due to the high recurrence rate. This study aims to evaluate the preventive effect of uncultured adipose derived stromal vascular fraction (SVF) on urethral fibrosis in a rat model of US. Results demonstrated that US rats displayed hyperechogenic urethral wall with a narrowed lumen compared with sham rats, while SVF rats exhibited less extensive urethral changes. By histology, US rats showed obvious submucosal fibrosis in the urethral specimens, while SVF rats exhibited mild submucosal fibrosis with less extensive tissue changes. Furthermore, US rats showed increased gene and protein expression of collagen I (2.0 ± 0.2, 2.2 ± 0.2, all were normalized against GAPDH, including the following), collagen III (2.5 ± 0.3, 1.2 ± 0.1), and TGFß1R (2.8 ± 0.3, 1.9 ± 0.2), while SVF cells administration contributed to decreased gene and protein expression of collagen I (1.6 ± 0.2, 1.6 ± 0.2), collagen III (1.8 ± 0.4, 0.9 ± 0.1), and TGFß1R (1.8 ± 0.3, 1.3 ± 0.2), in parallel with the improvement of vascularization and increased expression of VEGF (1.7 ± 0.1) and bFGF (3.1 ± 0.3). Additionally, SVF served anti-inflammatory effect through regulation of inflammatory cytokines and cells, accompanied with conversion of the macrophage phenotype. Our findings suggested that uncultured SVF presented an inhibitory effect on stricture formation at an early stage of urethral fibrosis.

The Effects and Mechanisms of the Rapamycin-eluting Stent in Urethral Stricture Prevention in Rabbits.

Background: Rapamycin was shown to reduce transforming growth factor ß1 (TGF-ß1) expression, inhibit the Mammalian target of rapamycin function, and prevent TGF-ß1-induced pulmonary fibrosis. Rapamycin-eluting stents (RES) were successfully used to prevent coronary artery restenosis. Urethral stricture is one of the most challenging problems in urology. Thus, combining the pharmacological effects of rapamycin and the mechanical support of the stent on the urethra may prevent urethral stricture formation. However, the use of RES for urethral stricture treatment has not been studied. Aims: To observe the effects of RES in urethral stricture in a rabbit model. Study Design: Animal experimentation. Methods: Twenty adult male New Zealand rabbits were randomly divided into control, urethral stricture model, bare-metal stent, and RES groups. The rabbits in the control group underwent urethroscopy alone without electrocoagulation. The rabbit model of urethral stricture was established by electrocoagulation using a self-made electrocoagulation device under direct vision using ureteroscopy. After model establishment, the rabbits in the bare-metal stent and RES groups received stent placement by ureteroscopy. On day 30, retrograde urethrography was performed to assess urethral stricture formation, ureteroscopy to remove the stents, and histological examinations to assess the degree of fibrosis. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis were used to evaluate the expression levels of TGF-ß1, Smad3, and matrix metalloproteinase 1 (MMP1). Results: Urethral stricture formation was seen in the model group, whereas not in the bare-metal stent group. The bare-metal stents did not displace but were difficult to remove. In the RES group, RES was dislodged in itself at postoperative day 27 in one rabbit, whereas successfully removed by ureteroscopy in the remaining four rabbits, and urethral stricture formation was not seen on retrograde urethrography after stent removal. Histological examination revealed a large number of dense fibroblasts and blue-stained collagen fibers in the bare-metal stent group, whereas the number of fibroblasts and collagen fibers under the mucosa was reduced in the RES group. RT-qPCR and Western blot analyses showed that the messenger ribonucleic acid (mRNA) and protein expression of TGF-ß1and Smad3 was significantly decreased, and mRNA and protein expression of MMP1 was significantly increased in the RES group than that in the model ((P < 0.001) and bare-metal stent groups (P < 0.001). Conclusion: RES can effectively prevent electrocoagulation-induced urethral stricture in rabbits. The mechanism may be related to the effect of rapamycin on inhibiting TGF-ß1 and Smad3 expression and promoting MMP1 expression in urethral tissues.

Effects of a gamma-secretase inhibitor of notch signalling on transforming growth factor ß1-induced urethral fibrosis.

Urethral stricture (US) is a common disorder of the lower urinary tract in men caused by fibrosis. The recurrence rate of US is high; however, there are no effective therapies to prevent or treat urethral fibrosis. The pathogenesis of urethral fibrosis involves myofibroblast activation and excessive extracellular matrix (ECM) deposition. The molecular mechanisms underlying this pathological activation are not completely understood. It has been demonstrated that Notch signalling contributes to the development of fibrosis and inflammation. However, whether this contributes to urethral fibrosis remains unclear. In this study, activation of Notch signalling was observed in patients with US. Additionally, it was noted that activation of Notch signalling promoted ECM production and myofibroblast activation in human urethral scar fibroblasts (HUSFs) treated with transforming growth factor (TGF) ß1. However, the Notch inhibitor N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) suppressed activation of Notch signalling as well as proliferation and migration of the TGFß1-treated HUSFs. Additionally, DAPT ameliorated TGFß1-induced urethral fibrosis in Sprague Dawley rats by suppressing ECM production, myofibroblast activation and the TGFß signalling pathway. These findings demonstrate that Notch signalling may be a promising and potential target in the prevention or treatment of urethral fibrosis.

A Rho kinase inhibitor (Fasudil) suppresses TGF-ß mediated autophagy in urethra fibroblasts to attenuate traumatic urethral stricture (TUS) through re-activating Akt/mTOR pathway: An in vitro study.

AIMS: Transforming growth factor-ß (TGF-ß) mediated super-activation of urethra fibroblasts contributes to the progression of traumatic urethral stricture (TUS), and the Rho-associated kinase inhibitors, Fasudil, might be a novel therapeutic agent for TUS, but the underlying mechanisms had not been studied. MATERIALS AND METHODS: The primary urethral fibroblasts (PUFs) were isolated from rabbit urethral scar tissues and cultured in vitro, and the PUFs were subsequently treated with TGF-ß (10 µg/L) to simulate the realistic conditions of TUS pathogenesis. Next, the PUFs were exposed to Fasudil (50 µM) and autophagy inhibitor 3-methyladenine (3-MA) treatment. Genes expression was examined by Western Blot and immunofluorescence staining, and cellular functions were determined by MTT assay and Transwell assay. KEY FINDINGS: TGF-ß promoted cell proliferation, migration, autophagy, and secretion of extracellular matrix (ECM), including collagen I and collagen III, which were reversed by co-treating cells with both Fasudil and 3-MA. In addition, TGF-ß treatment decreased the expression levels of phosphorylated Akt (p-Akt) and mTOR (p-mTOR) to inactivate the Akt/mTOR pathway in the PUFs, which could be re-activated by Fasudil. Then, the fibroblasts were treated with the Pan-Akt inhibitor (GDC-0068), and we surprisingly found that GDC-0068 abrogated the inhibiting effects of Fasudil on cell autophagy and proliferation in the PUFs treated with TGF-ß. SIGNIFICANCE: Fasudil regulated Akt/mTOR pathway mediated autophagy to hamper TGF-ß-mediated super-activation in PUFs, which supported that Fasudil might be an ideal candidate therapeutic agent for TUS treatment for clinical utilization.

Evaluation of surgical procedures of mouse urethra by visualization and the formation of fistula.

Visualization of the surgically operated tissues is vital to improve surgical model animals including mouse. Urological surgeries for urethra include series of fine manipulations to treat the increasing number of birth defects such as hypospadias. Hence visualization of the urethral status is vital. Inappropriate urethral surgical procedure often leads to the incomplete wound healing and subsequent formation of urethro-cutaneous fistula or urethral stricture. Application of indocyanine green mediated visualization of the urethra was first performed in the current study. Indocyanine green revealed the bladder but not the urethral status in mouse. Antegrade injection of contrast agent into the bladder enabled to detect the urethral status in vivo. The visualization of the leakage of contrast agent from the operated region was shown as the state of urethral fistula in the current hypospadias mouse model and urethral stricture was also revealed. A second trial for contrast agent was performed after the initial operation and a tendency of accelerated urethral stricture was observed. Thus, assessment of post-surgical conditions of urogenital tissues can be improved by the current analyses on the urethral status.

Moderate and severe fetal pyelectasis: Correlation between prenatal aspects and postnatal outcome.

BACKGROUND: Renal pelvic dilatation (RPD) is a frequent finding in fetal ultrasound. The aim of the study is to correlate the prenatally detected moderate and severe pyelectasis with the postnatal outcome. METHODS: A retrospective analysis involving 90 cases of prenatally detected moderate and severe RPD referred to our prenatal diagnosis centre with 18 months of urological follow-up. Prenatal ultrasound was correlated with postnatal renal function, assessed by plasmatic creatinine and/or renal scintigraphy performed before surgery. RESULTS: Cases were divided between two groups according to postnatal management: group A including 35 newborns (38.9%) that needed surgical treatment and group B with 55 patients (61.1%) who were managed conservatively. The group A presented higher median RPD (18 mm, IQR 12-25 mm) compared to the group B (11 mm, IQR 10-14 mm). The most common anomaly detected within group A was pelvi-ureteric junction (PUI) obstruction (43%). Within group B 32 cases (58%) showed spontaneous resolution of hydronephrosis during postnatal follow up. In case of moderate pyelectasis the risk of postnatal surgery was 25% and raised to 60% for severe RPD. In our study, 29 newborns showed pathologic scintigraphies: 25 required surgery while 4 did not find indication for surgery due to ipsilateral renal function irreversible damage. 6 patients had high creatinine level (>0.6 mg/dl). 35 cases out of 90 (39%) developed monolateral irreversible renal function impairment. CONCLUSION: Moderate and severe RPD are often correlated with postnatal renal damage, therefore a close multidisciplinary follow-up is required. Prenatal scanning is highly predictive of postnatal outcome and can address properly the prenatal counseling.

Insights into the Pathophysiology of Urethral Stricture Disease due to Lichen Sclerosus: Comparison of Pathological Markers in Lichen Sclerosus Induced Strictures vs Nonlichen Sclerosus Induced Strictures.

PURPOSE: We evaluated the pathophysiology of lichen sclerosus and nonlichen sclerosus urethral stricture disease by comparing protein expression related to inflammation, cell cycle disruption, oxidative stress, hormone receptor status and infection. MATERIALS AND METHODS: Tissue samples were collected from the urethral strictures of 81 patients undergoing urethroplasty. Clinical and demographic data were obtained by chart review. After identifying areas pathognomonic for lichen sclerosus a tissue microarray was created with cores from each sample and immunohistochemistry was performed. RESULTS: Patients had similar baseline demographics and comorbidities. Of the 81 strictures 58 were and 23 were not due to lichen sclerosus. Lichen sclerosus strictures were significantly longer and showed higher levels of inflammation. The proportion of T cells which stained positive for CD8 was significantly higher in strictures due to lichen sclerosus (50% vs 13%, p = 0.004). CCL-4 was expressed significantly more in strictures due to lichen sclerosus (76% vs 42%, p = 0.01). Several other inflammatory markers were only found in strictures due to lichen sclerosus. Block-like p16, a surrogate for high risk human papillomavirus infection, and varicella zoster virus were found only in lichen sclerosus urethral stricture disease samples, although both were rare. Epstein-Barr virus RNA was found in significantly more lichen sclerosus samples (37% vs 10%, p = 0.024). CONCLUSIONS: To our knowledge this is the first study to evaluate protein expression in lichen sclerosus urethral stricture disease. These strictures demonstrate increased inflammation compared to nonlichen sclerosus urethral strictures. Markers of oxidative stress, cell cycle dysregulation and the androgen receptor do not appear to be uniquely associated with lichen sclerosus urethral stricture disease. Positive staining for several viruses in samples of lichen sclerosus urethral stricture disease suggests a possible infectious etiology.

Anti-fibrotic function of Cu-bearing stainless steel for reducing recurrence of urethral stricture after stent implantation.

Recurrent stenosis is the main reason inducing the failure of urethral stricture treatment. Our previous study has found that the 316L type Cu bearing stainless steel (316L-Cu SS) showed antimicrobial activity and anti-encrustation performance when it was used for relieving urethral obstructer. However, whether it can reduce the occurrence of fibrosis or not, we need further investigation to compare the cellular and molecular responses of human urethral scar fibroblast cells (USFCs) on 316L-Cu SS and medical grade 316L stainless (316L SS, as a control). [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4- sulfophenyl)- 2H-tetrazolium (MTS) and Transwell were used to assess the cellular responses, which confirmed that 316L-Cu SS could inhibit proliferation and migration of USFCs. Molecular expressions of fibrosis were evaluated by western blot, real-time quantitative polymerase chain reaction (qPCR), and Cu/Zn superoxide dismutase (CuZnSOD) measurement. The results indicated that up-regulating of CuZnSOD attenuated the transforming growth factor-ß1 expression and phosphorylation of Smad3 after exposure to 316L-Cu SS. Besides, the content of collagen type I (COL1) and collagen type III (COL3) secreting into the culture medium measured by enzyme-linked immunosorbent assay were in accord with the results of messenger ribonucleic acids. Both of them exhibited lower levels of COL1/COL3 exposure to 316L-Cu SS, demonstrating the inhibitory performance of 316L-Cu SS against fibrosis. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 2019-2028, 2018.

Protective effect of platelet-rich plasma on urethral injury model of male rats.

AIMS: Urethral stricture (US) formation is caused by fibrosis after excessive collagen formation following an injury or trauma to the urethra. In this study, we aimed to evaluate the effects of platelet-rich plasma (PRP) on a urethral injury (UI) model of male rats. METHODS: A UI model was used by applying a coagulation current to the urethras of male rats. There were four groups with six rats in each: control group, PRP applied to naive urethra, UI group, and UI with PRP application. PRP was applied to the urethra after a coagulation current-induced injury as soon as possible. On the 14th day, all rats were sacrificed and urethral tissues were investigated for collagen type I, collagen type III, platelet-derived growth factor-α, platelet-derived growth factor-ß, and transforming growth factor-ß using quantitative real-time polymerase chain reaction and Western blot analysis. The effect of urethral damage and healing was evaluated for collagen type I-to-collagen type III ratio. RESULTS: The collagen type I-to-collagen type III ratio was significantly higher in UI group (P < 0.05) than in the others, while UI with PRP application group had comparable results with the control group (P > 0.05). CONCLUSIONS: The results of this study show that PRP has a preventive effect on stricture formation in a UI model of rats, as shown by its effect on collagen synthesis. Further studies that eventually show the effects of PRP on human tissues are necessary and promising.

Impact of Altered WNT2B Expression on Bladder Wall Fibroblasts: Implications for Apoptosis Regulation in the Stroma of the Lower Urinary Tract.

BACKGROUND: Little is known about the role of WNT signalling in pathological processes involving the urinary tract stroma. Here the impact of WNT signalling on bladder wall fibroblasts (BWFs) was studied using integrated expression profiling. MATERIAL AND METHODS: WNT ligand and downstream WNT pathway component expression was profiled in human BWFs using qRT-PCR. Highly expressed WNT2B was knocked down using siRNA in BWFs. The expression of 730 mRNAs and 800 miRNAs was analyzed on the nCounter MAX platform in #WNT2B and control transfected BWFs. qRT-PCR was used for validation in vitro and in matched scar and healthy bladder wall tissue samples of 12 patients with vesico-urethral anastomotic stricture (VUAS). RESULTS: Thirteen genes and 9 miRNAs showed differential expression in #WNT2B cells. Among these were TNFSF10, a key apoptosis inductor, (0.22fold, p = 0.011) and miR-1246 (36.2fold, p = 0.031). miRNA target prediction indicated TNFSF10 to be regulated by miR-1246. qRT-PCR analysis confirmed differential expression of miR-1246 and TNFSF10 in #WNT2B BWFs. Furthermore, TNFSF10 was significantly underexpressed in VUAS tissue (p = 0.009). CONCLUSION: Perturbation of WNT signalling results in an altered expression of the apoptosis inductor TNFSF10. Similar changes are observed in VUAS. Further studies investigating the crosslink between WNT signalling and apoptosis regulation in the urinary tract stroma are warranted.

Low Testosterone Levels Result in Decreased Periurethral Vascularity via an Androgen Receptor-mediated Process: Pilot Study in Urethral Stricture Tissue.

OBJECTIVE: To compare expression of androgen receptor (AR) and angiopoietin 1 receptor TIE-2 and vessel density of urethral stricture tissue among eugonadal and hypogonadal men to identify a pathophysiological basis for our observations that low testosterone is associated with urethral atrophy. METHODS: Among 1200 men having urethroplasty at our institution, we retrospectively identified 11 patients with testosterone levels drawn within 2 years of surgery. Low testosterone was defined as <280 ng/dL and detected in 5 of 11 (45.5%) patients. Urethral tissue samples were analyzed using immunohistochemistry for AR, TIE-2 (a downstream target of activated AR linking it to angiogenesis), and CD31 expression. RESULTS: Mean testosterone was 179.4 ng/dL for patients classified as having low testosterone and 375.0 ng/dL for controls (P = .003). We found a significant decrease of AR expression (1.11%high power field [HPF] vs 1.62, P = .016), TIE-2 expression (1.84%HPF vs 3.08, P = .006), and vessel counts (44.47 vessels/HPF vs 98.33, P = .004) in men with low testosterone. Expression levels of AR and TIE-2 were directly correlated to testosterone levels (rho: 0.685, P = .029, and rho: 0.773, P = .005, respectively). We did not find a difference in age, radiation, or comorbidities among patients with normal or low testosterone levels, with the exception of higher body mass index in the latter. CONCLUSION: Men with low testosterone levels demonstrate decreased AR and TIE-2 expression and lower vessel counts in periurethral tissue samples of urethral strictures. Our results provide a rationale for a mechanistic relationship between low testosterone levels and decreased periurethral vascularity that may contribute to urethral atrophy in patients with urethral strictures.

The effect of rapamycin on TGFß1 and MMP1 expression in a rabbit model of urethral stricture.

PURPOSE: To investigate the effect of rapamycin on TGFß1 and MMP1 expression in a rabbit model of urethral stricture. METHODS: Twenty-four adult New Zealand male rabbits underwent an electrocoagulation of the bulbar urethra with a 13Fr pediatric resectoscope. Then rabbits were randomly divided into three groups: (1) normal control group: normal saline (NS), (2) the vehicle control group: dimethyl sulfoxide (DMSO), and (3) the treatment group: effective-dose rapamycin in DMSO (Ra), with 12, 6, and 6 rabbits in each group, respectively. Drugs were given by urethral irrigation daily for 4 weeks. Urethral tissue was harvested for histological and molecular analyses. TGFß1 and MMP1 expression levels were evaluated by real-time quantitative PCR and immunohistochemistry. RESULTS: Ten, six, and six rabbits were evaluated finally in Ra, DMSO, and NS group, respectively. Histological examination revealed the distribution of fibrosis and the degree of collagen deposition in the Ra group were smaller and slighter than the two control groups. Collagen content was significantly less in the Ra group than in the DMSO group (P < 0.001) and the NS group (P < 0.001). qRT-PCR analysis showed a higher expression of MMP1 mRNA in the Ra group than in the DMSO group (P < 0.001) and the NS group (P < 0.001). Immunohistochemistry showed the protein levels of MMP1 in the Ra group were significantly increased when compared with the DMSO group (P < 0.01) and the NS group (P < 0.01). On the other hand, no statistical difference could be found between every two groups in both mRNA and protein levels of TGFß1. CONCLUSIONS: Rapamycin enhances the expression of MMP1 in a rabbit model of urethral stricture, but has no direct effect on the expression of TGFß1.

Application of Wnt Pathway Inhibitor Delivering Scaffold for Inhibiting Fibrosis in Urethra Strictures: In Vitro and in Vivo Study.

OBJECTIVE: To evaluate the mechanical property and biocompatibility of the Wnt pathway inhibitor (ICG-001) delivering collagen/poly(L-lactide-co-caprolactone) (P(LLA-CL)) scaffold for urethroplasty, and also the feasibility of inhibiting the extracellular matrix (ECM) expression in vitro and in vivo. METHODS: ICG-001 (1 mg (2 mM)) was loaded into a (P(LLA-CL)) scaffold with the co-axial electrospinning technique. The characteristics of the mechanical property and drug release fashion of scaffolds were tested with a mechanical testing machine (Instron) and high-performance liquid chromatography (HPLC). Rabbit bladder epithelial cells and the dermal fibroblasts were isolated by enzymatic digestion method. (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay) and scanning electron microscopy (SEM) were used to evaluate the viability and proliferation of the cells on the scaffolds. Fibrolasts treated with TGF-ß1 and ICG-001 released medium from scaffolds were used to evaluate the anti-fibrosis effect through immunofluorescence, real time PCR and western blot. Urethrography and histology were used to evaluate the efficacy of urethral implantation. RESULTS: The scaffold delivering ICG-001 was fabricated, the fiber diameter and mechanical strength of scaffolds with inhibitor were comparable with the non-drug scaffold. The SEM and MTT assay showed no toxic effect of ICG-001 to the proliferation of epithelial cells on the collagen/P(LLA-CL) scaffold with ICG-001. After treatment with culture medium released from the drug-delivering scaffold, the expression of Collagen type 1, 3 and fibronectin of fibroblasts could be inhibited significantly at the mRNA and protein levels. In the results of urethrography, urethral strictures and fistulas were found in the rabbits treated with non-ICG-001 delivering scaffolds, but all the rabbits treated with ICG-001-delivering scaffolds showed wide caliber in urethras. Histology results showed less collagen but more smooth muscle and thicker epithelium in urethras repaired with ICG-001 delivering scaffolds. CONCLUSION: After loading with the Wnt signal pathway inhibitor ICG-001, the Collagen/P(LLA-CL) scaffold could facilitate a decrease in the ECM deposition of fibroblasts. The ICG-001 delivering Collagen/P(LLA-CL) nanofibrous scaffold seeded with epithelial cells has the potential to be a promising substitute material for urethroplasty. Longer follow-up study in larger animals is needed in the future.

Crosstalk between TGF-ß1 and CXCR3 signaling during urethral fibrosis.

Urethral fibrosis is an important pathological feature of urethral stricture. TGF-ß1 and CXC chemokine receptor 3 (CXCR3) signaling have been reported as the critical pathways involved in the pathology of fibrosis. Here, we collected the urine samples from the patients with recurring urethral stricture, recurring stricture treated by cystostomy, and age- and gender-matched healthy people. ELISA detection revealed that TGF-ß1 level was significantly up-regulated for the urethral stricture patients. By contrast, flow cytometry, real-time PCR detection, and immunofluoresecent staining showed that urethral stricture resulted in decreased expression of CXCR3. TGF-ß1 treatment could increase cell proliferation and migration ability of urethra fibroblasts, whereas IP-10/CXCR3 signaling showed the opposite effect. Further, we found a crosstalk between TGF-ß1 and CXCR3 signaling in the regulation of urethral fibrosis. Thus, pharmacological intervention of TGF-ß1 or CXCR3 signaling has a potential as the therapeutic target for the prevention of urethral fibrosis.

Metalloproteinase-1 usefulness in urethral stricture treatment.

BACKGROUND: Urethral stricture disease is an obstruction or thinning of the urethra that causes restriction of urinary flow from the bladder during micturition. The object of the present study was to evaluate the effectiveness of the proteolytic enzyme metalloproteinase-1 as treatment in urethral stricture disease. METHODS: An experimental study was carried out on rabbits in which urethral stricture was created endoscopically by cauterization. The rabbits were divided into three study groups. Metalloproteinase-1 was applied endoscopically in Group 1; phosphate-buffered saline solution was applied in Group 2; and Group 3 was the control group without stricture that received no treatment. The animals were euthanized after 25 days and histopathological slices of the strictured and control regions were stained with Masson's trichrome stain to quantify mean collagen concentration by means of densitometry. Student t test was used to compare the two different groups with parametric variables, and analysis of variance was used to compare more than two groups. RESULTS: Collagen concentration analysis in the three groups showed a statistically significant difference with P = 0.012 with two degrees of freedom. In the analysis among groups, there was a statistically significant difference that showed the metalloproteinase-1 group had lower collagen concentration than the phosphate-buffered saline group P = 0.009. Urethral opening area in the metalloproteinase-1 group was found to be larger than that in the phosphate-buffered saline group P = 0.048. CONCLUSIONS: Metalloproteinase-1 protein application in strictured urethral tissue is effective in reducing collagen concentration and maintaining or enlarging urethral opening.

Increased expression of connective tissue growth factor in patients with urethral stricture.

Urethral stricture (US) is a urologic disorder with a high morbidity and recurrence rate, and may arise due to external trauma, surgical procedures or urethritis. The pathogenesis of US is closely associated with scar formation, including excessive collagen-rich connective tissues. Previous studies have shown that connective tissue growth factor (CTGF) plays a key role in the fibrosis process of many tissues and organs, such as kidney and lung. We investigated the variation of CTGF expression in urethral tissues from 12 patients with US and normal urethral tissues. The stricture length range was 1 to 2.5 cm, and 4 patients underwent repeated urethral dilatation. The normal urethral tissues were obtained from 6 patients with heart disease, who were suffering from brain death. The expression of CTGF mRNA and protein was analyzed by real-time PCR, immunohistochemistry, and western blotting analyses. Results showed that the expression level of CTGF mRNA was significantly increased in US patients compared with the control. In addition, US patients who have longer history and more repeated surgical procedures have extremely high CTGF mRNA levels and its protein expression, while the age of patients, position of injury and stricture length were not found obvious relation with CTGF expression. Moreover, the degree of collagen deposition and muscle fiber proliferation in the submucosa is consistent with the increase in CTGF expression. In conclusion, our data suggest that the up-regulation of CTGF expression may be responsible for the fibrosis process of urethral tissues in US patients.

Synthesis and evaluation of long-acting D-penicillamine derivatives.

This study extends the use of two lathyrogens, ss-aminopropionitrile (BAPN) and D-penicillamine (DPA) from daily systemic or local-topical administration to long-time acting agents. This was achieved by converting the hydrophilic drugs into lipophilic derivatives. The synthesis of functional derivatives of DPA consisted in esterification with methyl-, hexyl-, or benzyl alcohols in the presence of thionylchloride. The esters formed were hydrochlorides, acidic and soluble in water. During neutralization in vitro or in vivo by tissue fluid, an oily substance is formed that elutes from a hydrogel polymer at a much slower rate than hydroplilic DPA itself. The degree of lipophilicity, measured as a partition coefficient between octanol/water, was highest for hexyl ester and lowest for methyl ester DPA. A single injection of either DPA hexyl ester HCl or 3-hexyl(amino) propionitrile into the full thickness skin incision wound in rats significantly lowered the breaking strength of the wound 12 days after injection, indicating the interference with collagen cross-linking. Both agents injected into the breast adenocarcinoma in Fisher rats significantly inhibited tumor growth without any signs of local or systemic toxicity. We conclude that these lipophilic lathyrogens with prolonged effectiveness are suitable in the treatment of pathologies, consisting of excessively cross-linked or deposited collagen (fibrotic adhesions, strictures, stenosis, and scar contractures) and in the treatment of single, solitary tumors, malignant and benign.

An experimental model for stricture studies in the anterior urethra of the male rabbit.

In this study, an animal model was developed for the examination of urethral strictures (US). Through a resectoscope, a resection was made in the urethras of 15 male rabbits. After 30 days, the rabbits were evaluated with urethrography, impedance planimetry and either histology or the determination of collagen content. Fifteen rabbits serving as controls were evaluated in the same way. Three rabbits in the resection group and one in the control group died before evaluation. Urethrography demonstrated a stricture in the remaining 12 animals in the resection group. The urethras of the control animals were all normal. Impedance planimetry confirmed that the luminal cross sectional area (CSA) of the strictures was significantly smaller than the CSA of the corresponding part of the urethra in the control group. No difference in CSA was found 1 cm proximal to the stricture. The strictures consisted of densely woven collagen which sent tongues into the adjacent normal parts of the urethra. No difference in collagen content was found between the two groups either at the stricture site or 1 cm proximally. The described method of producing US in the rabbit model was very consistent with all operated animals developing a stricture. The model might prove valuable in evaluating new methods for the treatment of US.