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.
Peyronie’s disease (PD) is a connective tissue disorder where formation of fibrous plaques in tunica albuginea (TA) and erectile tissue can result in penile deformity, pain, and erectile dysfunction [
The mechanisms underpinning PD are unclear [
Apoptosis, or programmed cell death, is a fundamental mechanism with a key role throughout development [
Apoptosis works through two main, alternative pathways: death receptor-mediated (or extrinsic) and mitochondria-dependent (or intrinsic). The former pathway is initiated by ligation of specific death receptors by their ligands. The main death receptors—Fas and tumour necrosis factor- (TNF-) related apoptosis inducing ligand (TRAIL) receptors DR4 and DR5—induce cell death following ligation with Fas ligand (FasL) or TRAIL, respectively, followed by recruitment of procaspase 8. This process gives rise to caspase 8 activation. The latter induces apoptosis by directly activating caspase 3 or by cleaving bid (BH3 interacting domain death agonist), resulting in mitochondrial dysfunction and subsequent release of cytochrome C and activation of caspases 9 and 3. Caspase 3 promotes the typical apoptosis features, including DNA fragmentation and cell death in several tissues. The mitochondrial pathway is partly influenced by bcl family members bound to the mitochondrial membrane, including bax and bcl-2, which are, respectively, pro- or antiapoptotic regulatory proteins. The antiapoptotic proteins bcl-2 and bcl-XL inhibit cytochrome c release, whereas bcl-2—associated X protein(bax), bcl-2 homologous antagonist/killer (bak), and bid, all proapoptotic proteins, promote its release from mitochondria. Cytochrome C and deoxyadenosine triphosphate (dATP) bind to apoptotic protease activating factor (APAF-1) to form a multimeric complex that recruits and activates procaspase 9, an apoptosis-mediating executioner protease that in turn activates caspase 3, resulting in cell apoptosis.
The intrinsic mitochondrial pathway is activated by a range of exogenous and endogenous stimuli including DNA damage, ischemia, and oxidative stress (Figure
We have previously described apoptotic cell death in TA plaques from patients with PD and demonstrated its activation via the extrinsic pathway by assessing an overexpression of tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) and its death receptor, DR5, in fibroblast and myofibroblast cells [
Wedge-shaped biopsy specimens (approximately 3 × 5 mm) were collected from 15 patients (mean age
The study protocol was approved by the ethics committee of the Clinic of Urology, Clinical Center Nis, Nis, Serbia. The informed consent of each patient was obtained before tissue collection.
During preoperative examination all patients reported having spontaneous erections but being prevented from regular sexual intercourse by a penile curvature of above 45°. Degree of penile curvature and rigidity were evaluated with Doppler ultrasonography after intracavernous injection of 10–20
Control samples were specimens from 4 patients (mean age 23 ± 3 years; range 21–27) with congenital penile curvature who underwent Nesbit’s corrective procedure [
For the immunohistochemical studies, PD plaques were fixed overnight in 10% neutral buffered formalin (Bio-Optica, Italy). After fixation and overnight washing in water they were dehydrated in graded ethanol and paraffin-embedded. They were then cut into 5
The following primary antibodies were used: rabbit polyclonal antihuman bax (1 : 100, Dako, Denmark), mouse monoclonal antibody antihuman bcl-2 (Dako, Denmark), mouse monoclonal antihuman caspase 9 (1 : 100, Santa Cruz Biotechnology, USA), and rabbit monoclonal antihuman caspase 3 (1 : 50, Abcam, UK). The primary antibodies were applied directly onto sections and slides were incubated overnight (4°C) in a humid chamber. The sections were then washed in PBS and treated with a biotinylated antibody and detected using peroxidase-labeled streptavidin, both incubated for 10 min at room temperature (LSAB+System-HRP, Dako Italy).
The immunoreaction was assessed using a Zeiss Axioplan light microscope (Oberkochen, Germany) after incubating sections in 0.1% 3,3′-diaminobenzidine and 0.02% hydrogen peroxide solution (DAB substrate kit, Vector Laboratories, Burlingame, CA, USA) for 4 min. Sections were lightly counterstained with Mayer’s hematoxylin and then mounted on GVA mount (Zymed Laboratories, San Francisco, CA, USA).
Staining for bax, bcl-2, caspase 9, and caspase 3 was classified as negative/positive. Immunohistochemical staining was brown chromogen detected on the edge of the hematoxylin-stained cell nucleus or distributed in the cytoplasm or the membrane. Intensity of staining (IS) was graded in a semiquantitative manner using a 5-point scale: 0 = no detectable staining, 1 = weak staining, 2 = moderate staining, 3 = strong staining, and 4 = very strong staining. The proportion of immunopositive cells (extent score = ES) was evaluated at 20x magnification blindly by two anatomists and a histologist. ES was scored as a proportion of the final number of 100 cells into five classes: <5% (0), 5–30% (+), 31–50% (++), 51–75% (+++), and >75% (++++). Counting was performed at 20x magnification.
Positive and negative controls were run to test the specific reaction of the primary antibodies used. Positive controls were basal cell carcinoma specimens. For the negative controls, randomly selected sections from PD patients were treated with normal rabbit serum instead of the specific antibodies.
In situ detection of apoptosis at the single cell level was performed by terminal deoxynucleotidyl transferase- (TdT-) mediated dUTP-biotin nick end labeling, TUNEL (In Situ Cell Death Detection Kit, POD, Roche) as previously described [
To quantify immunohistochemical staining, 10 sections/sample were analyzed in stepwise fashion as a series of consecutive fields with a 40x magnification; the stained area was expressed as pixels/field. Randomly selected fields from each section were analyzed and the percent area staining for bax, bcl-2, caspase 9, and caspase 3 was calculated using AxioVision rel. 4.8.2 image analysis software and an AxioVision 4 Module AutoMeasure (Zeiss, Göttingen, Germany) to quantify the level of immunolabeling in each field. Values from all consecutive images of each biopsy were averaged. Digital pictures were taken using an Axiocam camera (Zeiss, Göttingen).
Statistical analysis was performed using SPSS software (rel. 16.0, Chicago, IL, USA). Comparisons between mean values were tested with Student’s unpaired
On hematoxylin and eosin-stained specimens the collagen fiber arrangement was mostly disrupted. Fibers were affected to different degrees in all PD patients, the damage involving fragmentation, tears, and splitting. Elastic fibers were often fragmented; clumps of collagen bundles and surrounding elastic fibers were also noted. In contrast, control TA samples exhibited preserved collagen bundles displaying longitudinal fiber orientation in the outer layer and circular fibers in the inner layer (not shown).
PD plaques showed areas of osseous metaplasia with heterotopic ossification (Figure
Bax immunostaining in plaque from a patient with Peyronie’s disease (a, b, c) and in control tissue (d); magnification is 20x for all. Scale bar = 100
Immunohistochemical examination of sections from PD plaques showed bax overexpression (IS: 4; ES: ++++) in some osteocytes in metaplastic bone (Figure
Little or no bcl-2 immunostaining was detected in samples from PD patients (IS: 1; ES: 0) or from controls (IS: 2; ES: +) (Figures
Bcl-2 immunostaining in plaque from a patient with Peyronie’s disease (a) and in control tissue (b); magnification is 20x for all. Scale bar = 100
Caspase 3 immunostaining was very strong (IS: 4; ES: ++++) in fibroblasts lying on the border between fibrous and metaplastic bone tissue; it was also very strong in fibrous tissue (Figure
Caspase 3 immunostaining in plaque from a patient with Peyronie’s disease ((a) and (b)) and in control tissue (c); magnification is 20x for all. Scale bar = 100
Moderate immunostaining for caspase 9 was seen in fibrous cells from PD plaques (Figure
Caspase 9 immunostaining in plaque from a patient with Peyronie’s disease (a and b) and in control tissue (c); magnification is 20x for all. Scale bar = 100
The TUNEL immunoassay showed intense staining of fibroblasts and myofibroblasts from PD plaques (Figure
TUNEL staining in plaque from a patient with Peyronie’s disease ((a) and (b)) and in control tissue (c); magnification is 20x for all. Scale bar = 100
Quantitative analysis of bax (a), bcl-2 (b), caspase 3 (c), and caspase 9 (d) immunostaining in PD samples. In both panels, asterisks indicate significant differences versus the control group (
Quantitative analysis of TUNEL-positive cell staining in PD samples. Different cell positivity can be seen in areas with osseous metaplasia and in fibrous tissue areas. Asterisks indicate significant differences versus the control group (
Even though PD has been described in the 18th century, its pathogenic mechanism and molecular basis are poorly understood and its therapeutic approach is still empirical [
A previous study by our group documented the involvement of the extrinsic pathway in the activation of programmed cell death in PD. The present study examines the possible activation of the intrinsic apoptotic pathway through the immunohistochemical demonstration of staining for a number of key molecules: bcl-2, bax, anti- and proapoptotic oncoproteins, respectively, involved in apoptosis regulation, modulating cell survival or death [
TA tissue from PD patients also exhibited upregulation of caspase 3, the executioner caspase of the apoptotic cascade. The apoptotic cascade has been subdivided into three sets of stages.
The caspase 3 immunopositivity found in the plaques from the PD patients, both in fibrous and in metaplastic bone tissue, seems to be in contrast with the TUNEL test results, which suggest that apoptotic cells are found in fibrous tissue but are absent at the level of the osseous metaplasia. However, the discrepancy can be accounted for by the fact that the anticaspase 3 antibody used in these experiments recognizes both the cleaved and the noncleaved form of the protein. This suggests that whereas the apoptotic process takes place in fibrous tissue as a defense mechanism, at the level of the osseous metaplasia, where a degenerative process is already under way, the apoptosis mechanism is already activated but stops and does not continue with caspase 3 activation (cleavage). Apoptosis can therefore be regarded as an injury-limiting mode of cell disposal [
A study by Lucattelli et al. [
A further consideration emerging from the analysis of our data is that the moderate expression of caspase 9 and overexpression of caspase 3 indicate a weak role for the intrinsic pathway in programmed cell death, a role that is played principally by the extrinsic pathway as demonstrated by our previous findings. This is reasonable, since this pathway is basically activated by death receptor binding with ligands such as TRAIL, whereas the intrinsic pathway is activated by exogenous stimuli such as inflammation-induced oxidative stress (ROS) [
Recent studies have shown that phosphoinositide 3-kinase (PI3K)/Akt signaling regulates fibrotic responses including collagen synthesis and cell proliferation [
Previous studies have advanced the hypothesis of an abnormal wound healing process in PD, possibly through a failure of fibroblast apoptosis [
In this study we tested the involvement of the intrinsic apoptosis pathway and of executioner caspases in PD and completed the experimental work with the TUNEL assay. Novel therapeutic strategies, for instance, based on TRAIL agonists, could help PD patients through enhancement of myofibroblast death in the attempt to induce plaque stabilization and prevent disease progression.
Apoptotic cell death occurs in stabilized PD plaques and is partly induced by the intrinsic mitochondrial pathway. The present findings can have clinical implications and may help devise improved treatment strategies. A therapeutic approach aimed at enhancing apoptosis-inducing molecules would at least help delay the progression of PD. Identification of target molecules for gene construct or biological or chemical reagent delivery to target sites could contribute to inducing PD plaque stabilization.
The authors declare that there is no conflict of interests regarding the publication of this paper.