Human Peyronie's Disease Plaque Research Articles

The transcriptional signatures of cells from the human Peyronie's disease plaque and the ability of these cells to generate a plaque in a rat model suggest potential therapeutic targets.

The success of medical therapies for Peyronie's disease (PD) has not been optimal, possibly because many of them went directly to clinical application without sufficient preclinical scientific research. Previous studies revealed cellular and molecular pathways involved in the formation of the PD plaque and in particular the role of the myofibroblast. The current work aimed to determine under normal and fibrotic conditions what differentiates PD cells from tunica albuginea (TA) and corpora cavernosa (CC) cells by defining their global transcriptional signatures and testing in vivo whether PD cells can generate a PD-like plaque. Human TA, PD, and CC cells were grown with transforming growth factor beta 1 (TGFβ1; TA+, PD+, CC+) or without it (TA-, PD-, CC-) and assayed by (i) immunofluorescence, Western blot and RT-PCR for myofibroblast, smooth muscle cell and stem cell markers; (ii) collagen content; and (iii) DNA microarray analysis. The ability of PD+ cells to induce a PD-like plaque in an immuno-suppressed rat model was assessed by Masson trichrome and Picrosirius Red stainings. Fibroproliferative features of PD cells and identification of related key genes as novel targets to reduce plaque size. Upon TGFβ1stimulation, collagen levels were increased by myofibroblasts in the PD+ but not in the CC+ cells. The transcriptional signature of the PD- cells identified fibroproliferative, myogenic (myofibroblasts), inflammatory, and collagen turnover genes that differentiate them from TA- or CC- cells and respond to TGFβ1 with a PD+ fibrotic phenotype, by upregulation of IGF-1, ACTG2, MYF5, ACTC1, PSTN, COL III, MMP3, and others. The PD+ cells injected into the TA of the rat induce a PD-like plaque. This suggests a novel combination therapy to eliminate a PD plaque by targeting the identified genes to (i) improve collagenase action by stimulating endogenous metalloproteinases specific to key collagen types and (ii) counteract fibromatosis by inhibiting myofibroblast generation, proliferation, and/or apoptosis.

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

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. 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)-β1 (10 ng/mL) to determine hydroxyproline levels, procollagen mRNA, apoptosis and protein expression of poly(ADP-ribose) polymerase 1 (PARP1) and cyclin D1. 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-β1-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. Specific inhibition of HDAC2 with RNA interference may represent a novel targeted therapy for PD.

Inhibition of histone deacetylase 2 mitigates profibrotic TGF-β1 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-β1 (TGF-β1). 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-β1-induced nuclear translocation of Smad2/3 in fibroblasts. Knockdown of HDAC2 in PD fibroblasts abrogated TGF-β1-induced extracellular matrix production by blocking TGF-β1-induced phosphorylation and nuclear translocation of Smad2 and Smad3, and by inhibiting TGF-β1-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.

Profibrotic Role of Myostatin in Peyronie's Disease

The primary histologic finding in many urologic disorders, including Peyronie's disease (PD), is fibrosis, mainly mediated by the transforming growth factor beta1 (TGFbeta1). To determine whether another member of the TGFbeta family, myostatin, (i) is expressed in the human PD plaque and normal tunica albuginea (TA), their cell cultures, and the TGFbeta1-induced PD lesion in the rat model; (ii) is responsible for myofibroblast generation, collagen deposition, and plaque formation; and (iii) mediates the profibrotic effects of TGFbeta1 in PD. Human TA and PD tissue sections, and cell cultures from both tissues incubated with myostatin and TGFbeta1 were subjected to immunocytochemistry for myostatin and alpha-smooth muscle actin (ASMA). The cells were assayed by western blot, Real time-Polymerase chain reaction (RT-PCR), and ribonuclease protection. Myostatin cDNA and shRNA were injected, with or without TGFbeta1, in the rat penile TA, and plaque size was estimated by Masson. Myostatin expression in the human TA, the PD plaque, and their cell cultures, and myostatin effects on the PD-like plaque in the rat. A threefold overexpression of myostatin was found in the PD plaque as compared with the TA. In PD cells, myostatin expression was mainly in the myofibroblasts, and in the TA cells, it increased upon passage paralleling myofibroblast differentiation and was up-regulated by TGFbeta1. Myostatin or its cDNA construct increased the myofibroblast number and collagen in TA cells. Myostatin was detected in the TGFbeta1-induced PD-like plaque of the rat partly in the myofibroblasts, and in the TA. Myostatin cDNA injected in the TA induced a plaque and intensified the TGFbeta1 lesion, which was not reduced by myostatin shRNA. Myostatin is overexpressed in the PD plaque, partly because of myofibroblast generation. Although myostatin induces a plaque in the rat TA, it does not appear to mediate the one triggered by TGFbeta1, thus suggesting that both proteins act concurrently and that therapy should target their common downstream effectors.

Fibrin as an inducer of fibrosis in the tunica albuginea of the rat: a new animal model of Peyronie's disease.

To investigate the role of fibrin in inducing fibrosis in the tunica albuginea (TA) of the rat penis, to develop a new animal model for Peyronie's disease (PD). The TA of rats (five per group per period) were injected with either saline, fibrin, transforming growth factor-beta1 (TGF-beta1) or TGF-beta1 plus fibrin; the rats were killed at 1, 3, and 6 weeks after injection. Images were analysed quantitatively from tissue sections stained for collagen (Masson trichrome), fibrin (Verhoeff's stain) and elastin (Hart's stain), and immunostained for TGF-beta1, inducible nitric oxide synthase (iNOS), heme oxygenase 1 (HO1), alpha-smooth muscle actin (ASMA), apoptosis (TUNEL) and plasminogen activator inhibitor (PAI). Collagen fibre organization was characterized by electron microscopy. Human PD plaque tissue and normal human TA were assayed for fibrin by immunohistochemistry in nine samples. At 1 week after injection of fibrin into the rat TA, only oedema was present; at 3 weeks, the oedema developed into a characteristic fibrotic PD-like plaque. The injection of TGF-beta1 into the TA also induced oedema in the TA at 1 and 3 weeks but there was very little evidence of a recognisable plaque at either time. Injection with TGF-beta1 plus fibrin resulted in oedema at 1 week but at 3 weeks there was a smaller plaque than with fibrin only. At 6 weeks the induced plaques in the fibrin-only and fibrin + TGF-beta1 groups persisted, and were comparable with those elicited at this time by TGF-beta1 alone. The control animals showed no pathology at any of the sample times. At 3 weeks the PD plaque induced by injection with fibrin alone had not only greater expression of TGF-beta1 than the TA of the animals receiving TGF-beta1 alone, but also greater levels of other markers of fibrosis, e.g. HO1 (reactive oxygen species), ASMA (presence of myofibroblasts), apoptosis, and PAI (inhibitor of fibrinolysis). iNOS, a known antifibrotic agent, was also increased. In human PD plaque tissue, fibrin was detected by immunohistochemistry in all nine specimens. These results suggest that fibrin, when introduced into the TA of the rat penis, acts as a potential profibrotic protein, possibly via the local release of TGF-beta1, and induces a plaque not only histologically similar to that induced by TGF-beta1 but to that of the human condition. Because fibrin can extravasate from the blood into the human TA after an injury to the TA, and because fibrin persists in the plaque tissue, we hypothesise that fibrin may play a key role in the pathogenesis of human PD.

Antifibrotic Role of Inducible Nitric Oxide Synthase

Long-term treatment in rats with l-NAME, an isoform-non-specific inhibitor of nitric oxide synthase (NOS), leads to fibrosis of the heart and kidney, suggesting that nitric oxide (NO) may play a role in preventing tissue fibrosis. In this process, a likely target of NO is the quenching of reactive oxygen species (ROS) through peroxynitrite formation, and one possible source for this NO is inducible NOS (iNOS). Using Peyronie's disease (PD) tissue from both human specimens and from a rat model of PD as the source of fibrotic tissue, we investigated if NO derived from iNOS could act as such an antifibrogenic defense mechanism by determining whether: (a) tunical ROS and iNOS are increased in PD; and (b) the long-term inhibition of iNOS activity decreases the NO/ROS balance in the tunica albuginea thereby promoting collagen deposition. It was determined that in the human PD plaque, iNOS mRNA and protein, ROS, collagen, and the peroxynitrite marker, nitrotyrosine, were all increased in comparison to the normal tunica. In the rat model of PD, the fibrotic plaque also showed significant increases in iNOS mRNA and protein, nitrotyrosine, ROS as measured by heme oxygenase-1, and collagen when compared with the normal control tunica. When a selective inhibitor of iNOS, L-NIL, was given to rats with the PD-like plaque, this resulted in a decrease in nitrotyrosine levels but intensified ROS levels and collagen deposition. These data demonstrate that: (a) iNOS induction occurs in both the human and rat PD fibrotic plaque; and (b) that the NO derived from iNOS appears to counteract ROS formation and collagen deposition. Because the inhibition of iNOS activity leads to a decrease in the NO/ROS ratio, thereby favoring the development of fibrosis, it is proposed that iNOS induction in this tissue may be a protective mechanism against fibrosis and abnormal wound healing.