Antioxidant Mechanism of Xiaojin Pill () for Treatment of Peyronie's Disease in Rats Based on Matrix Metalloproteinases.

OBJECTIVE: To evaluate the effects of Xiaojin Pill () in the treatment of Peyronie's disease (PD) in a rat model. METHODS: Twenty-four male Sprague-Dawley rats were randomly divided into four groups with 6 in each: sham operation, PD model, vehicle control and Xiaojin Pill groups. The rats in the sham operation group received penile tunica albsginea (TA) injection with 50 µL vehicle, while the rats in the other 3 groups received 50 µL penile TA injection of 50 µg transforming growth factor (TGF)-ß1. Forty-two days after the injection, rats in the vehicle control and Xiaojin Pill groups received 0.5 mL water and Xiaojin Pill solution (107 mg/kg of body weight), respectively by gavage for 28 days, while those in the sham operation and PD model groups did not receive any intervention. After intervention, the expressions of matrix metalloproteinase 2/9 (MMP2/9), nitric oxidesynthase (NOS), superoxide dismutase (SOD) and malondialdehyde (MDA) were measured. RESULTS: Rats in the PD model and vehicle control groups presented obvious fibrosis in corpus cavernosum (CC) and demonstrated a significantly increased expressions of MMP2 and MMP9 in the CC compared with the sham operation group (all P<0.01). In contrast, the expressions of MMP2 and MMP9 in the Xiaojin Pill group were significantly down-regulated (both P<0.01). In addition, the levels of NOS and MDA in CC were significantly increased while the activity of SOD was decreased in the PD model and vehicle control groups compared with the sham operation group (all P<0.01). After Xiaojin Pill treatment, the levels of MDA, NOS and SOD appeared to be corrected (all P<0.01). CONCLUSIONS: Xiaojin Pill could reduce fibrosis in the CC by decreasing the expressions of MMPs, NOS and MDA, and by increasing the activity of SOD. Therefore, Xiaojin Pill might be a therapeutic option for PD.

Antifibrotic Synergy Between Phosphodiesterase Type 5 Inhibitors and Selective Oestrogen Receptor Modulators in Peyronie's Disease Models.

BACKGROUND: Peyronie's disease (PD) is a fibrotic disorder of the penile tunica albuginea, characterised by the formation of a localised fibrous plaque that can lead to deformity and erectile dysfunction. Nonsurgical therapeutic options for PD are limited in efficacy and safety. Myofibroblasts are key cells in the pathogenesis of PD, and inhibition of myofibroblast transformation has been suggested as a therapeutic option. OBJECTIVE: To identify potential drugs using a novel phenotypic assay and then to test them using in vitro and in vivo models of PD. DESIGN, SETTING, AND PARTICIPANTS: We have developed and validated a phenotypic screening assay that measures myofibroblast transformation, by which we tested 21 compounds that were suggested to be efficacious in treating PD. The successful hits from this assay were further tested using in vitro and in vivo models of PD. RESULTS AND LIMITATIONS: The new assay was able to detect transforming growth factor-ß1-induced myofibroblast transformation. Using this assay, phosphodiesterase type 5 inhibitors (PDE5i) and selective oestrogen receptor modulators (SERMs) were identified to significantly inhibit myofibroblast transformation. A PDE5i (vardenafil) and an SERM (tamoxifen) inhibited myofibroblast transformation, collagen gel contraction, and extracellular matrix production in a synergistic fashion. In a rat model of PD, the antifibrotic effect of the combination of vardenafil and tamoxifen was greater than that of each drug alone. This study is limited by not providing a molecular mechanism for the proposed synergy. CONCLUSIONS: This is the first demonstration of a synergistic activity between a PDE5i and an SERM discovered through a phenotypic screening approach. Future clinical trials using a combination of these drugs should be considered during the active phase of PD, given the early evidence of benefit in both in vitro and in vivo models. PATIENT SUMMARY: This report suggests that the combination of a phosphodiesterase type 5 inhibitor and a selective oestrogen receptor modulator may be efficacious in treating Peyronie's disease in its active phase.

The role of intrinsic pathway in apoptosis activation and progression in Peyronie's disease.

Peyronie's disease (PD) is characterized with formation of fibrous plaques which result in penile deformity, pain, and erectile dysfunction. The aim of this study was to investigate the activation of the intrinsic apoptotic pathway in plaques from PD patients. Tunica albuginea from either PD or control patients was assessed for the expression of bax, bcl-2 and caspases 9 and 3 using immunohistochemistry and by measurement of apoptotic cells using TUNEL assay. Bax overexpression was observed in metaplastic bone tissue, in fibroblasts, and in myofibroblast of plaques from PD patients. Little or no bcl-2 immunostaining was detected in samples from either patients or controls. Caspase 3 immunostaining was very strong in fibrous tissue, in metaplasic bone osteocytes, and in primary ossification center osteoblasts. Moderate caspase 9 immunostaining was seen in fibrous cells plaques and in osteocytes and osteoblasts of primary ossification centers from PD patients. Control samples were negative for caspase 9 immunostaining. In PD patients the TUNEL immunoassay showed intense immunostaining of fibroblasts and myofibroblasts, the absence of apoptotic cells in metaplasic bone tissue and on the border between fibrous and metaplastic bone tissue. Apoptosis occurs in stabilized PD plaques and is partly induced by the intrinsic pathway.

Treatment of Peyronie's disease with PDE5 inhibitors: an antifibrotic strategy.

Peyronie's disease (PD) is a localized fibrotic condition of the tunica albuginea that is associated with risk factors for corpora cavernosa fibrosis (such as advanced age and diabetes) and Dupuytren contracture, another localized fibrotic process. Most of the current pharmacological treatments for PD are not based on antifibrotic approaches that have shown promising results in animal models and clinical efficacy in other fibrotic conditions, which may explain why they are generally unsuccessful. Evidence gathered in human specimens and animal models of PD have elucidated aspects of its etiology and histopathology, showing that overexpression of transforming growth factor beta1, plasminogen activator inhibitor 1, reactive oxygen species and other profibrotic factors, which are, in most cases, assumed to be induced by trauma to the tunica albuginea, leads to myofibroblast accumulation and excessive deposition of collagen. At the same time, a steady overexpression of inducible nitric oxide synthase, leading to increased nitric oxide and cGMP levels, seems to act as an endogenous antifibrotic mechanism. This process has also been reported in corporal and cardiovascular fibrosis, and has led to the demonstration that long-term continuous administration of phosphodiesterase type 5 inhibitors counteracts the development of a PD-like fibrotic plaque in a rat model, and later extended to the prevention of corporal fibrosis in animal models of erectile dysfunction.

Differential calcium independent regulation of matrix metalloproteinases and tissue inhibitors of matrix metalloproteinases by interleukin-1beta and transforming growth factor-beta in Peyronie's plaque fibroblasts.

PURPOSE: Peyronie's disease is a fibrotic disorder of the tunica albuginea characterized by the localized formation of an inelastic plaque. We characterized matrix metalloproteinases and TIMPs (tissue inhibitors of matrix metalloproteinase) in Peyronie's disease tissue. MATERIALS AND METHODS: Matrix metalloproteinases and TIMPs were investigated in Peyronie's disease plaque tunica removed from patients with stable Peyronie's disease. Immunological methods were used to characterize the matrix metalloproteinases and TIMPs produced by cell cultures stimulated with transforming growth factor-beta or interleukin-1beta (PreproTech, Rocky Hill, New Jersey). Enzyme activity was quantified with a fluorescent substrate and correlated with mRNA levels using real-time polymerase chain reaction. RESULTS: Interleukin-1beta significantly induced (p <0.01) matrix metalloproteinase-1, 3, 10 and 13 protein production, endogenous matrix metalloproteinase-13 activity (12-fold) and matrix metalloproteinase-13 mRNA expression (11.2-fold) through a Ca(2+) independent mechanism in cultured fibroblasts. Transforming growth factor-beta stimulation failed to induce any detectable matrix metalloproteinase protein production or activity and conditioned culture medium even had the capacity to inhibit (p <0.01) the activity of purified recombinant human matrix metalloproteinase-13. Intact Peyronie's disease plaques were highly enriched with TIMP-1 to 4 compared to donor matched perilesional tunica. CONCLUSIONS: These data show that, while interleukin-1beta strongly induces matrix metalloproteinase expression, transforming growth factor-beta strongly induces TIMP expression without any effect on matrix metalloproteinases and may represent an important downstream biochemical mechanism that leads to the progression of Peyronie's disease. The localized accumulation of TIMPs together with decreased matrix metalloproteinase activity in the Peyronie's disease lesion may be the biochemical consequence of the transforming growth factor-beta over expression that has been reported in many fibrotic disorders, including Peyronie's disease.

Liposomal recombinant human superoxide dismutase for the treatment of Peyronie's disease: a randomized placebo-controlled double-blind prospective clinical study.

OBJECTIVE: To demonstrate the efficacy and safety of a topical gel containing liposomally encapsulated recombinant human Superoxide Dismutase (lrhSOD) in the treatment of painful Peyronie's Disease. The theoretical background is that lrhSOD, by scavenging of free oxygen radicals, might interrupt inflammatory cascades and thereby limit further disease progression. METHODS: In a placebo-controlled randomized clinical trial, 39 patients with Peyronie's Disease and significant pain symptoms were treated with lrhSOD or placebo for a 4 week period. At this time, statistical evaluation of pain resolution was performed as primary study endpoint. Patients then were continued in a cross-over study design to ensure a total of 8 weeks of lrhSOD therapy for all study participants. Pain, plaque and curvature assessment was performed at study entry and every 4 weeks until week 12. RESULTS: LrhSOD treatment resulted in a statistically significant reduction of pain (p=0.017) compared to placebo already after 4 weeks. At week 12 pain was significantly reduced in 89% of patients who all had received 8 weeks of lrhSOD therapy at that time. Response to other disease parameters was assessed at week 12: plaque size was reduced in 47% of patients, as was plaque consistence in 38%. Penile curvature was improved at 5-30 degrees in 23% of patients. The expected spontaneous disease progression rate of up to 40%, as reported by several investigators, was significantly reduced to <10% under lrhSOD therapy, and patients satisfaction was high, also consequent to the lack of therapy-related side effects observed in the present study. CONCLUSION: LrhSOD is an easily administrable, safe and effective local therapeutic for the painful phase of Peyronie's Disease.

L-arginine and phosphodiesterase (PDE) inhibitors counteract fibrosis in the Peyronie's fibrotic plaque and related fibroblast cultures.

Inducible nitric oxide synthase (iNOS) is expressed in both the fibrotic plaque of Peyronie's disease (PD) in the human, and in the PD-like plaque elicited by injection of TGFbeta1 into the penile tunica albuginea (TA) of the rat. Long-term inhibition of iNOS activity, presumably by blocking nitric oxide (NO)- and cGMP-mediated effects triggered by iNOS expression, exacerbates tissue fibrosis through an increase in: (a) collagen synthesis, (b) levels of reactive oxygen species (ROS), and (c) the differentiation of fibroblasts into myofibroblasts. We have now investigated whether: (a) phosphodiesterase (PDE) isoforms, that regulate the interplay of cGMP and cAMP pathways, are expressed in both the human and rat TA; and (b) L-arginine, that stimulates NOS activity and hence NO synthesis, and PDE inhibitors, that increase the levels of cGMP and/or cAMP, can inhibit collagen synthesis and induce fibroblast/myofibroblast apoptosis, thus acting as antifibrotic agents. We have found by immunohistochemistry, RT/PCR, and Western blot that PDE5A-3 and PDE4A, B, and D variants are indeed expressed in human and rat normal TA and PD plaque tissue, as well as in their respective fibroblast cultures. As expected, in the PD fibroblast cultures, pentoxifylline (non-specific cAMP-PDE inhibitor) increased cAMP levels without affecting cGMP levels, whereas sildenafil (PDE5A inhibitor) raised cGMP levels. Both agents and L-arginine reduced the expression of collagen I (but not collagen III) and the myofibroblast marker, alpha-smooth muscle actin, as determined by immunocytochemistry and quantitative image analysis. These effects were mimicked by incubation with 8-Br-cGMP, which in addition increased apoptosis, as measured by TUNEL. When L-arginine (2.25 g/kg/day), pentoxifylline (10 mg/kg/day), or sildenafil (10 mg/kg/day) was given individually in the drinking water for 45 days to rats with a PD-like plaque induced by TGF beta1, each treatment resulted in a 80-95% reduction in both plaque size and in the collagen/fibroblast ratio, as determined by Masson trichrome staining. Both sildenafil and pentoxiphylline stimulated fibroblast apoptosis within the TA. Our results support the hypothesis that the increase in NO and/or cGMP/cAMP levels by long-term administration of nitrergic agents or inhibitors of PDE, may be effective in reversing the fibrosis of PD, and more speculatively, other fibrotic conditions.

Evaluation of nitric oxide synthase and arginase in the induction of a Peyronie's-like condition in the rat.

Peyronie's disease is an idiopathic, localized connective tissue disorder of the penis, involving the tunica albuginea of the corpus cavernosum and adjacent areolar space. Current proposals as to the origin of Peyronie's disease suggest that fibrosis and collagen changes of the tunica are the result of an inflammatory process following vascular trauma. Our laboratory and other investigators have recently proposed an animal model for the study of Peyronie's disease. When transforming growth factor-beta1 (TGF-beta1) was injected into the rat tunica albuginea, tissue fibrosis was observed at 6 weeks. Therefore, our aim was to assess arginase II, endothelial and inducible nitric oxide synthase isoforms, and nitrotyrosine levels--all factors involved in inflammatory reactions--in the cavernosal tissue of saline-injected and TGF-beta1-injected rats after 6 weeks in order to evaluate the roles these enzymes may play in the induction of a Peyronie's-like condition in the rat. To examine the expression of endothelial nitric oxide synthase (eNOS), iNOS, and arginase II protein, and mRNA in the corpus cavernosum, immunoblot analysis, and reverse transcriptase-polymerase chain reaction were performed. We also determined immunohistochemically the expression of nitrotyrosine, a marker of peroxynitrite formation, in the rat penis. After 6 weeks, iNOS protein and gene expression was up-regulated and eNOS protein and gene expression was down-regulated in the corpora cavernosa of the TGF-beta1-injected penises. Furthermore, arginase II protein expression as well as immunohistochemical localization of nitrotyrosine was significantly higher in the TGF-beta1-injected corpora cavernosa. These results suggest that iNOS is the key control element for peroxynitrite formation, arginase II expression, and eNOS down-regulation in the induction of a Peyronie's-like condition in the rat.