Microvesicles-delivering Smad7 have advantages over microvesicles in suppressing fibroblast differentiation in a model of Peyronie's disease.

BACKGROUND: This study compared the differences of microvesicles (MVs) and microvesicles-delivering Smad7 (Smad7-MVs) on macrophage M1 polarization and fibroblast differentiation in a model of Peyronie's disease (PD). METHODS: Overexpression of Smad7 in rat BMSCs was obtained by pCMV5-Smad7 transfection. MVs were collected from rat BMSCs using ultracentrifugation. In cells, 100 µg/mL of MVs or Smad7-MVs were used to treat the 100 ng/mL of lipopolysaccharide (LPS)-induced RAW264.7 cells or 10 ng/mL of recombinant transforming growth factor-ß1 (TGF-ß1)-induced fibroblasts. The pro-inflammatory cytokines and markers of M1 macrophages were measured in RAW264.7 cells, and the migration and markers of fibroblast differentiation were measured in fibroblasts. In rats, 50 µg of MVs or Smad7-MVs were used to treat the TGF-ß1-induced animals. The pathology of tunica albuginea (TA), the markers of M1 macrophages and fibroblast differentiation in the TA were measured. RESULTS: The MVs or Smad7-MVs treatment suppressed the LPS-induced macrophage M1 polarization and TGF-ß1-induced fibroblast differentiation. Moreover, the Smad7-MVs treatment decreased the fibroblast differentiation compared with the MVs treatment. In the TGF-ß1-induced TA of rats, MVs or Smad7-MVs treatment ameliorated the TA fibrosis by suppressing the macrophage M1 polarization and fibroblast differentiation. There was no significance on the M1-polarized macrophages between the MVs treatment and the Smad7-MVs treatment. Meanwhile, the Smad7-MVs treatment had an edge in terms of suppressing the fibroblast differentiation in the TGF-ß1-induced PD model compared with the MVs treatment. CONCLUSIONS: This study demonstrated that Smad7-MVs treatment had advantages over MVs treatment in suppressing of fibroblast differentiation in a model of PD.

TGF-ß1 induces formation of TSG-6-enriched extracellular vesicles in fibroblasts which can prevent myofibroblast transformation by modulating Erk1/2 phosphorylation.

Extracellular vesicles have emerged as important mediators of cell-to-cell communication in the pathophysiology of fibrotic diseases. One such disease is Peyronie's disease (PD), a fibrotic disorder of the penis caused by uncontrolled transformation of resident fibroblasts to alpha-smooth muscle actin positive myofibroblasts. These cells produce large amounts of extracellular matrix, leading to formation of a plaque in the penile tunica albuginea (TA), causing pain, penile curvature, and erectile dysfunction. We have used primary fibroblasts derived from the TA of PD patients to explore the role of transforming growth factor beta 1 (TGF-ß1), a key signalling factor in this process. TGF-ß1 treatment elicited a range of responses from the myofibroblasts: (i) they secreted extracellular vesicles (EVs) that were more numerous and differed in size and shape from those secreted by fibroblasts, (ii) these EVs prevented TGF-ß1-induced transformation of fibroblasts in a manner that was dependent on vesicle uptake and (iii) they prevented phosphorylation of Erk1/2, a critical component in modulating fibrogenic phenotypic responses, but did not affect TGF-ß1-induced Smad-signalling. We posit that this effect could be linked to enrichment of TSG-6 in myofibroblast-derived EVs. The ability of myofibroblast-derived vesicles to prevent further myofibroblast transformation may establish them as part of an anti-fibrotic negative feedback loop, with potential to be exploited for future therapeutic approaches.

BMP4 and GREM1 are targets of SHH signaling and downstream regulators of collagen in the penis.

BACKGROUND: Cavernous nerve (CN) injury, caused by prostatectomy and diabetes, initiates a remodeling process (smooth muscle apoptosis and increased collagen) in the corpora cavernosa of the penis of patients and animal models that is an underlying cause of erectile dysfunction (ED), and the Sonic hedgehog (SHH) pathway plays an essential role in the response of the penis to denervation, as collagen increases with SHH inhibition and decreases with SHH treatment. AIM: We examined if part of the mechanism of how SHH prevents penile remodeling and increased collagen with CN injury involves bone morphogenetic protein 4 (BMP4) and gremlin1 (GREM1) and examined the relationship between SHH, BMP4, GREM1, and collagen in penis of ED patients and rat models of CN injury, SHH inhibition, and SHH, BMP4, and GREM1 treatment. METHODS: Corpora cavernosa of Peyronie's disease (control), prostatectomy, and diabetic ED patients were obtained (N = 30). Adult Sprague Dawley rats (n = 90) underwent (1) CN crush (1-7 days) or sham surgery; (2) CN injury and BMP4, GREM1, or mouse serum albumin (control) treatment via Affi-Gel beads or peptide amphiphile (PA) for 14 days; (3) 5E1 SHH inhibitor, IgG, or phosphate-buffered saline (control) treatment for 2 to 4 days; or (4) CN crush with mouse serum albumin or SHH for 9 days. OUTCOMES: Immunohistochemical and Western analysis for BMP4 and GREM1, and collagen analysis by hydroxyproline and trichrome stain were performed. RESULTS: BMP4 and GREM1 proteins were identified in corpora cavernosa smooth muscle of prostatectomy, diabetic, and Peyronie's patients, and in rat smooth muscle, sympathetic nerve fibers, perineurium, blood vessels, and urethra. Collagen decreased 25.4% in rats with CN injury and BMP4 treatment (P = .02) and increased 61.3% with CN injury and GREM1 treatment (P = .005). Trichrome stain showed increased collagen in rats treated with GREM1. Western analysis identified increased BMP4 and GREM1 in corpora cavernosa of prostatectomy and diabetic patients, and after CN injury (1-2 days) in our rat model. Localization of BMP4 and GREM1 changed with SHH inhibition. SHH treatment increased the monomer form of BMP4 and GREM1, altering their range of signaling. CLINICAL IMPLICATIONS: A better understanding of penile remodeling and how fibrosis occurs with loss of innervation is essential for development of novel ED therapies. STRENGTHS AND LIMITATIONS: The relationship between SHH, BMP4, GREM1, and collagen is complex in the penis. CONCLUSION: BMP4 and GREM1 are downstream targets of SHH that impact collagen and may be useful in collaboration with SHH to prevent penile remodeling and ED.

New in vivo model to assess macroscopic, histological, and molecular changes in Peyronie's disease.

BACKGROUND: Peyronie's Disease (PD) is a connective tissue disorder that affects the tunica albuginea (TA) of the penis causing curvature and erectile dysfunction. The pathophysiology is not well understood and, for this reason, treatment options are limited. OBJECTIVE: The aim of the present study is to analyze and compare whether single or multiple instillations of plasma in the TA of rats is capable of triggering macroscopic, histopathological, and molecular changes consistent with PD. MATERIAL/METHODS: Fifty male Wistar rats were divided into four groups: Group 1: a single instillation of plasma in the TA; Group 2: a single instillation of distilled water in the TA; Group 3: four instillations of plasma in the TA (1x per week); and Group 4: four instillations of distilled water in the TA (1× per week). Forty-five days after the last instillation a manual inspection of the corpus cavernosum, a penile erection test and a penectomy were performed to obtain material for histopathological and molecular analysis. RESULTS: It was observed that 31.25% of the rats that received repeated instillations of plasma presented penile curvature according to the erection test, while none of the rats from the control group or group with one instillation of plasma presented curvature. In the animals that received four instillations of plasma, the following differences were observed in relation to the control group: increase in fibrosis and the deposition of collagen I. The protein expression of heparanase (HPSE) and TGF-ß increased in the groups that received a single or four instillations of plasma, and the protein expression of heparanase-2 (HPSE-2), metalloproteinases (MMP-2, MMP-9) and metalloproteinase inhibitor (TIMP-2) showed an increase in the group that received four instillations of plasma. There was a significant increase in the gene expression of HPSE, MMP-9, and TGF-ß in the group that received four instillations of plasma. In the analysis of the glycosaminoglycans, an increase was observed in the secretion of galactosaminoglycans chondroitin sulfate and dermatan sulfate (CS/DS) in the group that received four instillations of plasma. DISCUSSION: Previous studies have demonstrated increased protein expression. of HPSE, MMP-9 and TGF-ß with instillation of blood in the TA; however, there was no increase in gene expression. In the present study, the increase in the expression of TGF-ß with plasma instillations, proved to be more reliable. The two models with plasma (one or four instillations) demonstrated significant histopathological and molecular changes when compared to the control group. However, only in the group with four plasma instillations there was a macroscopic change. The idea is that repeatedly extravasation of TGF-ß present in plasma of predisposed individuals acts as a trigger for the development and maintenance of changes in the extracellular matrix that perpetuate an anomalous inflammatory process present in PD. CONCLUSION: The present study shows that the repeated instillation of plasma is a low cost in vivo model for the study of PD.

Peyronie Disease as an Example of Nononcological, Inflammatory Uptake of 18F-Fluorocholine in Patient With Prostate Cancer.

F-fluorocholine PET/CT is commonly used for staging and assessing treatment response in prostate cancer patients. Growing clinical experience has shown that F-fluorocholine can actually accumulate in sites of inflammation. We report a rare case of a prostate cancer patient with incidentally detected Peyronie disease.

Verapamil and collagenase differentially affect collagen metabolism in experimental model of Peyronie's disease.

OBJECTIVES: Peyronie's disease (PD) is accompanied by remodelling of connective tissue into fibrotic plaque. Treatment of the inflammatory and fibrotic phases of the disease is not established. The aim of the study was to evaluate the effect of verapamil (VER) and bacterial collagenase (COLL) on collagen metabolism and cell migration in fibroblasts with experimental wound healing and inflammation as an in vitro model of PD. MATERIALS AND METHODS: In vitro model of PD was designed using experimental model of inflammation induced by Interleukin-1 (IL-1) in cultured fibroblasts and mechanical damage of the cells. Cell viability, cell proliferation, collagen biosynthesis, prolidase activity and cell migration were studied in both models of the cells treated with VER and COLL. RESULTS: VER decreased cell viability, DNA and collagen biosynthesis and increased prolidase activity in control fibroblast, while in "wounded" fibroblasts it significantly decreased all the processes. COLL did not affect cell viability and DNA biosynthesis, while inhibited collagen biosynthesis and prolidase activity in both control and "wounded" fibroblasts. In IL-1-treated fibroblasts VER inhibited all studied processes except prolidase activity, while COLL inhibited only collagen biosynthesis and prolidase activity. COLL accelerated cell migration, while VER attenuated the process in fibroblast model of wound healing, compared to control cells. CONCLUSION: VER and COLL attenuate collagen biosynthesis in both fibroblast models. The VER-dependent inhibition of collagen biosynthesis was accompanied by inhibition of DNA biosynthesis at high prolidase activity, while COLL affected this process through inhibition of prolidase activity at high rate of DNA biosynthesis. It shows that anti-fibrotic activity of VER/COLL and anti-inflammatory activity of VER may represent approach to establish standard treatment of PD.

Repeated Micro-Trauma of the Penile Tunica Albuginea: A New Animal Model of Peyronie's Disease.

OBJECTIVES: To compare effects of repairing injured tunica albuginea (PTA) of rat penis by single or repeated local injections of chlorhexidine ethanol (ChE) into the PTA and to establish a new animal model of Peyronie's disease (PD). MATERIALS AND METHODS: Forty-two rats were divided into 7 groups. Rats either served as the normal control group with 1-5 injections of 0.9% saline or they received a single injection, 2, 3, 4, or 5 injections of ChE (0.1% chlorhexidine gluconate plus 15% ethanol dissolved in saline); rats in the positive control group were injected with TGF-ß1. At 60 days after the last injection, the intracavernous pressure, degree of penile curvature, and histology were evaluated. RESULTS: Compared with the single injection of the ChE group, we found the following in the repeat ChE injections groups: an increase in the degree of penile curvature, fibrous plaques in the PTA and/or corpus cavernosum, broken elastic fibers, slightly decreased erectile function, and an increased expression of TGF-ß1 and αSMA. CONCLUSIONS: Repeated ChE injuries of PTA may lead to fibrosis. This represents an excellent model of PD that involves repeated injections of ChE into the local PTA as well as reveals the pathophysiologic mechanism of PD.

Alternative Splicing of IGF1 Gene as a Potential Factor in the Pathogenesis of Peyronie's Disease.

BACKGROUND/AIM: Peyronie's disease (PD) is a fibrotic entity for which the pathogenetic mechanism remains unclear and if resulting in severe deformity, its treatment is only surgical. In this study we investigated the possible role of insulin-like growth factor 1 (IGF1) expression in the pathogenesis of PD. MATERIALS AND METHODS: Tissue samples were obtained from plaques of 24 patients with PD. The expression of IGF1 isoforms was investigated using quantitative real-time polymerase chain reaction and immunofluorescence. RESULTS: All IGF1 isoform gene expression (Ea, Eb and Ec) were found significantly decreased in the affected tunica albuginea, compared to normal tunica albuginea, with Ec showing the greatest decrease. Staining of tissue sections with an antibody against IGF1Ec confirmed greater expression of IGF1Ec isoform in normal tunica albuginea. CONCLUSION: The expression of all IGF1 alternative spliced isoforms is decreased in patients with PD, suggestive of its possible participation in the pathophysiology of PD.

Increased aquaporin 1 expression in the tunica albuginea of Peyronie's disease patients: an in vivo pilot study.

Peyronie's disease (PD) is a localized disorder of the connective tissue of the tunica albuginea (TA) whose etiology has not been elucidated. Although several studies have implicated genetic susceptibility and/or mechanical trauma as triggering events for PD, the underlying molecular mechanisms remain largely unknown. Aquaporin 1 (AQP1) is a water channel protein potentially implicated in connective tissue resistance to mechanical stress, acting primarily by increasing tension within the collagen network. Although it represents a potentially attractive molecular target in PD, to date no studies had ever addressed whether AQP1 is detectable and/or differentially expressed in the TA of these patients. Herein the present study, through immunohistochemical and biochemical approaches, we were able to detect AQP1 expression in the TA of control and PD affected patients. We demonstrated that AQP1-like immunoreactivity and expression are significantly increased in plaques of PD patients Vs controls, implying that AQP1 overexpression might be the consequence of a localized maladaptive response of the connective tissue to repeated mechanical trauma. In summary, these data support the idea that AQP1 might represent a potentially useful biomarker of mechanical injury in the TA and a promising target for the treatment of PD.

Estradiol attenuates the TGF-ß1-induced conversion of primary TAFs into myofibroblasts and inhibits collagen production and myofibroblast contraction by modulating the Smad and Rho/ROCK signaling pathways.

The transformation of tunica albuginea-derived fibroblasts (TAFs) into myofibroblasts plays an important role in the pathological progress of Peyronie's disease (PD). However, no treatment which addresses this transformation is currently available. Estrogen has been shown to inhibit the progression of fibrosis in a number of fibrotic diseases. The aim of this study was to determine whether estrogen [17ß­estradiol (E2)] suppresses the diffentiation of primary rat TAFs into myofibroblasts in vitro. TAFs obtained from male Sprague­Dawley rats were stimulated with either transforming growth factor­ß1 (TGF­ß1) or E2. Western blot analysis and immunofluorescence staining were used to assess changes in the expression levels of α­smooth muscle actin (αSMA). The expression levels of additional proteins (GAPDH, p­Smad2, Smad2, Smad4, RhoA, Rac1, ROCK1 and ROCK2) were also measured by western blot analysis. We used collagen gel assays to assess cell contractility. Additionally, the concentration of hydroxyproline in the TAF cell culture medium was detected using commercially available kits. We found that E2 reduced αSMA expression which was induced by TGF­ß1. E2 also suppressed the TGF­ß1­induced increase in the concentration of hydroxyproline (a marker of collagen) in addition to suppressing the contraction of TAFs. The key processes affected by TGF­ß1 treatment included the phosphorylation of Smad2, ras homolog gene family, member A (RhoA) and Rho­associated, coiled-coil containing protein kinase 2 (ROCK2); this increase in phosphorylation was inhibited by treatment with E2. Collectively, these results demonstrate that by modulating the activation of the TGF­ß1­Smad and RhoA­ROCK2 signaling pathways, E2 inhibited the transformation of TAFs into myofibroblasts, decreased the expression of collagen and suppressed the contraction of myofibroblasts in response to TGF-ß1 stimulation.

Collagenase Clostridium Histolyticum: A Review in Peyronie's Disease.

Collagenase Clostridium Histolyticum (CCH) (Xiaflex(®), Xiapex(®)) intralesional injection is a mixture of class I (AUX-I) and class II (AUX-II) clostridial collagenases. It is indicated for the treatment of adult men with Peyronie's disease with a palpable plaque and curvature deformity of ≥ 30° at the start of therapy. This article reviews the efficacy and tolerability of CCH in this indication and briefly summarizes its pharmacology. CCH treatment significantly improved penile curvature deformity and reduced patient-reported bother associated with Peyronie's disease in the 52-week, double-blind, phase III IMPRESS I and II studies. Treatment benefit with CCH was also seen in 36-week, open-label studies, providing further support for its efficacy. CCH was generally well tolerated in patients with Peyronie's disease, with most treatment-related adverse events being of mild or moderate severity. Serious treatment-related adverse events (penile haematoma or corporal ruptures) were reported in <1% of CCH recipients in clinical studies. Although further studies assessing the long-term effects of CCH intralesional injection are needed, current evidence indicates that this is a minimally invasive, effective and generally well tolerated treatment option for patients with Peyronie's disease.

Pharmacologic therapy for Peyronie's disease: what should we prescribe?

INTRODUCTION: Peyronie's disease (PD) is a wound healing disorder of the penis with a myriad of proposed treatment options reported in the literature. Evaluating the available data and therapeutic management of PD can be challenging and confusing, even for the most experienced treating physician. This review provides a comprehensive overview of pharmacologic treatment options for PD, focusing on the best available evidence. AREAS COVERED: A comprehensive literature search for published articles evaluating oral, topical, and injectable pharmacologic agents for PD was completed. Prospective, controlled trials were given precedence for inclusion. EXPERT OPINION: Although a multitude of oral agents have been proposed and evaluated in PD patients, results vary widely and a reproducible objective benefit has not yet been strongly established for any single oral agent. Well-designed, large-scale, randomized controlled trials evaluating oral agents in PD patients are lacking. Consistent objective benefit from injectable agents has been supported for years by various non-controlled trials. Recently, injectable collagenase Clostridium histolyticum became the first pharmacologic agent to obtain FDA approval for use in PD patients, supported by data from a large-scale, Phase III randomized controlled trial. Further elucidation of the genetic and mechanistic pathways involved in the development and progression of PD will help define future therapeutic targets.

Tunica albuginea allograft: a new model of LaPeyronie's disease with penile curvature and subtunical ossification.

The pathophysiology of LaPeyronie's disease (PD) is considered to be multifactorial, involving genetic predisposition, trauma, inflammation and altered wound healing. However, these factors have not yet been validated using animal models. In this study, we have presented a new model obtained by tunica albuginea allograft. A total of 40, 16-week-old male rats were used. Of these, 8 rats served as controls and underwent a 10 × 2-mm-wide tunical excision with subsequent autografting, whereas the remaining 32 underwent the same excision with grafting of the defect with another rat's tunica. Morphological and functional testing was performed at 1, 3, 7 and 12 weeks after grafting. Intracavernous pressure, the degree of penile curvature and elastic fiber length were evaluated for comparison between the allograft and control groups. The tissues were obtained for histological examination. The penile curvature was significantly greater in the allografted rats as compared with the control rats. The erectile function was maintained in all rats, except in those assessed at 12 weeks. The elastin fiber length was decreased in the allografted tunica as compared to control. SMAD2 expression was detected in the inner part of the allograft, and both collagen-II- and osteocalcin-positive cells were also noted. Tunica albuginea (TA) allograft in rats is an excellent model of PD. The persistence of curvature beyond 12 weeks and the presence of ossification in the inner layer of the TA were similar to those observed in men with PD. Validation studies using this animal model would aid understanding of the PD pathophysiology for effective therapeutic interventions.

Inflammatory mechanisms and oxidative stress in Peyronie's disease: therapeutic "rationale" and related emerging treatment strategies.

Peyronie's disease (PD) is a connective tissue disorder characterized by a fibrous plaque involving the tunica albuginea of the penis. The inelastic fibrous plaque leads to a penile curvature. Several Authors have suggested an immunological genesis of this disease, others have linked PD with Dupuytren's contracture. Signs of this disease are curvature, penile pain, penile deformity, difficulty with coitus, shortening, hinging, narrowing and erectile dysfunction. The natural history of PD and the clinical course can develop from spontaneous resolution of symptoms to progressive penile deformity and impotence. Surgical treatment is indicated when patients fail the conservative medical treatment and however, only in case of disease stabilization with a condition of impossibility of penetration. The medical treatment is indicated in the development stage of PD for at least one year after diagnosis and whenever in case of penile pain. Current non-surgical therapy includes vitamin-E, verapamil, para-aminobenzoate, propoleum, colchicine, carnitine, tamoxifen, interferons, collagenase, hyaluronidase, cortisone, pentoxifylline, superoxide dismutase, iontophoresis, radiation, extracorporeal shock wave therapy (ESWT) and the penile extender. The etiology of this fibrotic disease is not widely known, although in recent years pathophysiological knowledge has evolved and new studies propose the penile trauma as cause of the disease. The penile trauma results in a delamination of the tunica albuginea with a consequent small hematoma, then the process evolves as an inflammation with accumulation of inflammatory cells and production of reactive oxygen species (ROS). In the course of the inflammation, Peyronie's disease occurs due to the activation of nuclear factor kappa-B, that induces the production of inducible nitric oxide synthase (iNOS), with an increase of nitric oxide, leading to increased production of peroxynitrite anion. All these processes result in the proliferation of fibroblasts and myo-fibroblasts and excessive production of collagen between the layers of the tunica albuginea (penile plaque). Referring to the current knowledge of inflammatory and oxidative mechanisms of PD, a possible therapeutic strategy is then analyzed.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its death receptor (DR5) in Peyronie's disease. A biomolecular study of apoptosis activation.

INTRODUCTION: Peyronie's disease (PD) is a connective tissue disorder of tunica albuginea (TA), a thick fibrous sheath surrounding the corpora cavernosa of the penis. Relatively, little is known about the disease itself. AIM: To investigate whether the apoptosis cascade in degenerated and macroscopically deformed TA from men with PD is activated through the extrinsic pathway, by assessing the immunoexpression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its death receptor, DR5. METHODS: TA plaques from 15 men with PD and from four unaffected men were processed for TRAIL and DR5 immunohistochemistry and Western blot analysis. MAIN OUTCOME MEASURES: A greater understanding of the pathophysiology of PD through a molecular approach, to gain insights that may lead to novel forms of treatment. RESULTS: Activation of the apoptosis mechanisms through the extrinsic pathway was demonstrated by TRAIL and DR5 overexpression in fibroblasts and myofibroblasts from affected TA. CONCLUSION: The finding that apoptosis activation in TA plaques occurs, at least in part, via the extrinsic pathway may help devise novel therapeutic options for these patients.

p53-Associated Parkin-like cytoplasmic protein (Parc) short-interfering RNA (siRNA) alters p53 location and biology of Peyronie's disease fibroblasts.

OBJECTIVE • To evaluate the impact of p53-associated Parkin-like cytoplasmic protein (Parc) short-interfering RNA (siRNA) on the location of p53 as well as the biology of Peyronie's disease (PD) plaque-derived fibroblasts after Parc knockdown. PATIENTS AND METHODS • Plaque tissue was excised from men with stable PD undergoing penile reconstructive surgery and used to produce cultured PD plaque-derived fibroblasts. • Immunofluorescence (IF) and reverse transcription-polymerase chain reaction (RT-PCR) were then used to define the location of p53 and Parc before and after siRNA. • Nuclear fractionation studies were used to assess the chronology of translocation of p53 from cytoplasm to nucleus on Parc knockdown. • The terminal transferase dUTP Nick end labelling (TUNEL) assay was used to assess the apoptotic indices of the PD fibroblasts after Parc knockdown. RESULTS • IF and PCR showed high cytoplasmic levels of p53 and Parc before siRNA. On IF, there was little or no p53 present within the nucleus before Parc knockdown. • After Parc siRNA, IF showed translocation of p53 to the fibroblast nucleus, while Parc levels dropped significantly, but what Parc remained was confined to the cytoplasm with none present in the nucleus. • Nuclear fractionation studies using RT-PCR confirmed this translocation phenomenon and showed the chronology of the event. All p53 had moved from the cytoplasm to the nucleus within 16 h of Parc siRNA. • On TUNEL assay, apoptotic indices increased dramatically after Parc siRNA. CONCLUSIONS • These data prove that Parc is a cytoplasmic anchor for p53 in PD plaque-derived fibroblasts and may be the primary cause of the stabilization and defunctionalization of p53 in these cells. • These findings support Parc as a novel target for PD pharmacotherapy, perhaps using human siRNA technologies once commercially available.

Transforming growth factor (TGF)-ß type I receptor kinase (ALK5) inhibitor alleviates profibrotic TGF-ß1 responses in fibroblasts derived from Peyronie's plaque.

INTRODUCTION: Transforming growth factor-ß1 (TGF-ß1) has been identified as an important fibrogenic cytokine associated with Peyronie's disease (PD). AIM: The aim of this study was to study the differential expression of the TGF-ß1 and Smad transcription factors in plaque tissue from PD patients and to determine the antifibrotic effect of SKI2162 (SK Chemicals, Seoul, South Korea), a novel small-molecule inhibitor of activin receptor-like kinase 5 (ALK5), a type I receptor of TGF-ß, in primary fibroblasts derived from human PD plaque. METHODS: Plaque tissue was isolated from five PD patients, and tunica albuginea tissue was obtained from four control patients. Plaque tissues from a patient with PD were used for primary fibroblast culture. Fibroblasts were pretreated with SKI2162 (10 µM) and then stimulated with TGF-ß1 (10ng/mL). MAIN OUTCOME MEASURES: The plaque or tunica albuginea tissue was stained with Masson's trichrome or antibody to TGF-ß1, phospho-Smad2 (P-Smad2), and P-Smad3. Protein was extracted from treated fibroblasts for Western blotting, and the membranes were probed with antibody to P-Smad2/Smad2, P-Smad3/Smad3, plasminogen activator inhibitor-1, fibronectin, collagen I, and collagen IV. We also determined the inhibitory effect of SKI2162 on TGF-ß1-induced nuclear translocation of Smad2/3 in fibroblasts. RESULTS: The plaque tissue from PD patients showed higher TGF-ß1, P-Smad2, and P-Smad3 immunoreactivity than did the tunica albuginea tissue from control patients. SKI2162 not only blocked TGF-ß1-induced phosphorylation and nuclear translocation of Smad2 and Smad3, but also inhibited the production of extracellular matrix markers in fibroblasts derived from human PD plaque. CONCLUSION: In light of the pivotal role of TGF-ß and Smads in the pathogenesis of PD, pharmacologic inhibition of ALK5 may represent a novel targeted approach to treating PD.

Effect of transdermal electromotive drug therapy on fibrogenic cytokine expression in Peyronie's disease.

OBJECTIVES: To assess the effect of transdermal electromotive drug therapy (EMDT) on transforming growth factor-beta (TGF-beta) and basic fibroblast growth factor (bFGF) expression and their receptors in plaques in patients with Peyronie's disease. METHODS: Tissue was obtained from 13 patients with stable Peyronie's disease who had undergone plaque excision because of penile curvature. Of the 13 patients, 7 underwent EMDT with dexamethasone, verapamil, and lidocaine as first-line therapy before plaque excision and 6 were therapy naive. TGF-beta and bFGF mRNA and protein expression and that of their receptors were measured using real-time polymerase chain reaction and Western blotting. RESULTS: The mean patient age was 52.83 years. The mean interval from the end of EMDT to plaque excision was 7.6 months, with stable disease for >or=5 months. The comparison of TGF-beta mRNA expression in the plaques showed no difference between the EMDT and therapy-naive patients (P = .17). Also, TGF-beta protein expression in the plaques was not significantly different between the EMDT and therapy-naive patients (P = .443). TGF-beta receptor 1 mRNA expression in the plaques was significantly different between the EMDT and therapy-naive patients (P = .023), but no difference was found for TGF-beta receptor 2 mRNA (P = .292). The expression of bFGF mRNA (P = .0005) and bFGF protein expression (P = .034) in the plaques was significantly lower after EMDT. bFGF receptor mRNA expression (P = .619) showed no significant differences. CONCLUSIONS: Patients with Peyronie's had significantly lower bFGF mRNA and bFGF protein expression in the plaques after EMDT. Also, overexpression of TGF-beta protein and the TGF-beta receptor was identified in the EMDT plaques compared with the therapy-naive plaques.

Testosterone deficiency and Peyronie's disease: pilot data suggesting a significant relationship.

INTRODUCTION: As testosterone (T) has been shown to influence wound healing, and serum T declines in the age group at risk for Peyronie's disease (PD), we explored the possibility that low serum T may be associated with PD. AIM: The purpose of this study was to evaluate the relationship between serum T concentrations and features of PD. METHODS: Medical records were reviewed for 121 consecutive patients with PD seen over a 2-year period. All patients were assessed for sociodemographic data, medical history, comorbid medical conditions, findings on physical examination, and severity of curvature. Laboratory testing included serum concentrations of total testosterone (TT) and free testosterone (FT). Testosterone deficiency (TD) was defined as TT values less than 300 ng/dL and/or FT less than 1.5 ng/dL. MAIN OUTCOME MEASURES: Prevalence of TD in men with PD and correlation of TT and FT with severity of curvature and plaque size. RESULTS: Mean patient age was 53.9 +/- 10.6 years (range 28-77). Penile curvature was 50.2 +/- 23.6 degrees (range 10-120). Mean TT was 411.6 +/- 203.6 ng/dL (range 69-877), and mean FT was 1.12 +/- 0.58 ng/dL (range 0.13-5.06). Low T was identified in 29.5% by TT alone and in 74.4% overall. Severity of curvature was greater for men with TD compared with men with normal T (54.3 vs. 37.1 degrees, P = 0.006). Men with low FT had greater penile curvature than men with normal FT (37.5 vs. 55.9 degrees, respectively, P = 0.003). Severity of penile curvature correlated significantly with FT (r = -0.314, P = 0.016) and estradiol/T (r = 0.476, P = 0.0001) but not TT (r = -0.199, P = 0.138). CONCLUSIONS: This pilot study suggests a possibly important relationship between low T and PD. Further prospective studies are needed to confirm this relationship.

Mechanisms of penile fibrosis.

INTRODUCTION: Penile fibrosis has been conceptually identified with the plaque that develops in the tunica albuginea in Peyronie's disease (PD), or with localized processes induced in the corpora cavernosa by ischemic or traumatic events. Recently, it has been proposed that a diffuse, progressive, and milder intracorporal fibrosis, which affects also the media of the penile arteries, is responsible for vasculogenic erectile dysfunction (ED) associated with aging, smoking, diabetes, hypertension, and post-radical prostatectomy. These processes differ in etiology, time course, target cells, and treatment, but have many features in common. AIM: To review the literature pertaining to fibrosis in the penis, related to PD and ED. METHODS: PubMed search for pertinent publications mainly during 2001-2008. RESULTS: This review focuses initially on PD and then deals with studies on ED in animal and cell culture models, discussing some of the pathophysiological similarities between tunical fibrosis in PD and corporal fibrosis in corporal veno-occlusive dysfunction (CVOD), and emerging therapeutic strategies. The role of profibrotic factors, the excessive deposit of collagen fibers and other extracellular matrix, the appearance of a synthetic cell phenotype in smooth muscle cells or the onset of a fibroblast-myofibroblast transition, and in the case of the corporal or penile arterial tissue the reduction of the smooth muscle cellular compartment, are discussed. This histopathology leads either to localized plaques or nodules in penile tissues, or to the diffuse fibrosis causing impairment of tissue compliance that underlies CVOD and arteriogenic ED. The antifibrotic role of the sustained stimulation of the nitric oxide/cyclic guanosine monophosphate pathway in the penis and its possible relevance to exogenous and endogenous stem cell differentiation is also briefly presented. CONCLUSIONS: Fibrotic processes in penile tissues share a similar cellular and molecular pathophysiology and common endogenous mechanisms of defense that have inspired novel pharmacological experimental approaches.