The two phases of the clinical validation of preclinical translational mechanistic research on PDE5 inhibitors since Viagra's advent. A personal perspective.

The FDA approval of Viagra (sildenafil) for the on demand treatment of erectile dysfunction (ED) through relaxation of the corporal and cavernosal vascular smooth muscle that results in an increase in blood flow to the corporal tissues stemmed from 2 decades of research, mainly at academic centers. This culminated in the finding of the nitric oxide/cGMP pathway as the mediator of penile erection, followed by some years of basic studies and clinical validation at Pfizer. Further on, new translational laboratory and animal research from our group initiated a second phase when we proposed an alternative therapeutic schedule and mechanism of action for PDE5 inhibitors (PDE5i) in both corporal veno-occlusive dysfunction (CVOD) and Peyronie's disease (PD), specifically, continuous long-term administration (CLTA) to achieve sustained levels of cGMP within the penis. Due to the extended half-life of the long-acting PDE5i, tadalafil, this new alternative encompasses preferentially daily administration, although shorter half-life PDE5i, like sildenafil and vardenafil work too, depending on the duration, dose, and frequency of their administration This novel use was initially supported by showing the antifibrotic/antioxidant effects of nitric oxide and cGMP, produced by the induction of iNOS, as a mechanism of defense against collagen deposition in the localized fibrotic plaque of PD in an avascular tissue, the tunica albuginea. Our studies on iNOS and the progressive diffuse fibrosis occurring in the smooth muscle in CVOD, led to proposing the CLTA of PDE5i for maintaining sustained cGMP levels both in PD and in CVOD in order to halt or regress the penile fibrosis. In CVOD, we showed that PDE5i protect the corporal smooth muscle and reduce myofibroblast activation and number, counteracting the underlying corporal tissue pathology that causes CVOD, and potentially ameliorating long-term CVOD or even curing it. This review is focused on this novel PDE5i anti-fibrotic therapeutic concept.

Myostatin, a profibrotic factor and the main inhibitor of striated muscle mass, is present in the penile and vascular smooth muscle.

Myostatin is present in striated myofibers but, except for myometrial cells, has not been reported within smooth muscle cells (SMC). We investigated in the rat whether myostatin is present in SMC within the penis and the vascular wall and, if so, whether it is transcriptionally expressed and associated with the loss of corporal SMC occurring in certain forms of erectile dysfunction (ED). Myostatin protein was detected by immunohistochemistry/fluorescence and western blots in the perineal striated muscles, and also in the SMC of the penile corpora, arteries and veins, and aorta. Myostatin was found in corporal SMC cultures, and its transcriptional expression (and its receptor) was shown there by DNA microarrays. Myostatin protein was measured by western blots in the penile shaft of rats subjected to bilateral cavernosal nerve resection (BCNR), that were left untreated, or treated (45 days) with muscle-derived stem cells (MDSC), or concurrent daily low-dose sildenafil. Myostatin was not increased by BCNR (compared with sham operated animals), but over expressed after treatment with MDSC. This was reduced by concurrent sildenafil. The presence of myostatin in corporal and vascular SMC, and its overexpression in the corpora by MDSC therapy, may have relevance for the stem cell treatment of corporal fibrosis and ED.

Oral Bisphenol A (BPA) given to rats at moderate doses is associated with erectile dysfunction, cavernosal lipofibrosis and alterations of global gene transcription.

Bisphenol A (BPA), a suspected reproductive biohazard and endocrine disruptor, released from plastics is associated with ED in occupationally exposed workers. However, in rats, despite the induction of hypogonadism, apoptosis of the penile corporal smooth muscle (SM), fat infiltration into the cavernosal tissue and changes in global gene expression with the intraperitoneal administration of high dose BPA, ED was not observed. We investigated whether BPA administered orally rather than intraperitoneally to rats for longer periods and lower doses will lead to ED. Main outcome measures are ED, histological, and biochemical markers in rat penile tissues. In all, 2.5-month-old rats were given drinking water daily without and with BPA at 1 and 0.1 mg kg(-1) per day. Two months later, erectile function was determined by cavernosometry and electrical field stimulation (EFS) and serum levels of testosterone (T), estradiol (E2) and BPA were measured. Penile tissue sections were assayed by Masson (SM/collagen), Oil Red O (fat), terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) (apoptosis), immunohistochemistry for Oct4 (stem cells), and α-SM actin/calponin (SM and myofibroblasts), applying quantitative image analysis. Other markers were assayed by western blotting. DNA microarrays/microRNA (miR) assays defined transcription profiles. Orally administered BPA did not affect body weight, but (1) decreased serum T and E2; (2) reduced the EFS response and increased the drop rate; (3) increased within the corporal tissue the presence of fat, myofibroblasts and apoptosis; (4) lowered the contents of SM and stem cells, but not nerve terminals; and (5) caused alterations in the transcriptional profiles for both mRNA and miRs within the penile shaft. Long-term exposure of rats to oral BPA caused a moderate corporal veno-occlusive dysfunction (CVOD), possibly due to alterations within the corporal tissue that pose gene transcriptional changes related to inflammation, fibrosis and epithelial/mesenchymal transition (EMT).

Early onset erectile dysfunction is usually not associated with abnormal cavernosal arterial Inflow.

Endothelial dysfunction, a marker for atherosclerosis and hence arterial disease, has recently been proffered as the main offender within the vascular system to predict not only the future onset of erectile dysfunction (ED) but also as the main cause of the ED. To glean more insight into whether arterial disease is indeed operative during the early onset of ED, we reviewed the duplex ultrasound scans of 23 men with ED who were younger than 50 years of age. Depending on the criteria used for abnormal arterial responses, it was determined in this cohort of young men that there was only a 4-13% incidence of abnormal arterial responses. These observations suggest that the penile arterial system does not appear to be primarily involved in the etiology of the majority cases of ED that occur in young men.

Discovery of NO in the penis.

In 1989, the recognition by our group at UCLA that nitric oxide was indeed the chemical mediator of penile erection turned out to have major influences on our society and on some of those who participated in this research. Not only did it ultimately lead to the development of drugs like sildenafil, vardenafil and tadalafil, it launched new businesses, it gave insight into the way the vascular system functioned, it changed the way many physicians, particularly urologists, practiced sexual medicine and by bringing the topic of erectile dysfunction out of the closet, talk of sexual issues became mainstream. For one of us, a Nobel Prize lay in the future. This review, which comes one decade following the introduction of Viagra into the marketplace, chronicles the actual events involved in the discovery.

Rationale for phosphodiesterase 5 inhibitor use post-radical prostatectomy: experimental and clinical review.

Erectile dysfunction (ED) is a common complication after radical prostatectomy and results from trauma sustained by the cavernosal nerves. This is a major concern for patients and often affects treatment decisions. The likely mechanism for post-prostatectomy ED is through corporal veno-occlusive dysfunction. There is an increasing amount of evidence to suggest that phosphodiesterase 5 inhibitors (PDE5 inhibitors), when given on a continuous long-term basis, can help to prevent and reverse ED after surgery. In this review article we will examine the pathophysiology of post-prostatectomy ED and discuss the experimental and available clinical evidence for administering PDE5 inhibitors after prostatectomy.

Long-term continuous sildenafil treatment ameliorates corporal veno-occlusive dysfunction (CVOD) induced by cavernosal nerve resection in rats.

It was recently reported in the rat that vardenafil given in a continuous long-term manner was successful in preventing smooth muscle fibrosis in the penile corpora cavernosa and corporal veno-occlusive dysfunction (CVOD) that occur following bilateral cavernosal nerve resection (BCNR), a model for human erectile dysfunction after radical prostatectomy. To expand on this finding and to determine whether this effect was common to other PDE5 inhibitors, and occurred in part by stimulation of the spontaneous induction of inducible nitric oxide synthase (iNOS, also known as NOS2), male Fischer 344 rats (N=10/group) were subjected to either BCNR or unilateral cavernosal nerve resection (UCNR) and treated with sildenafil (20 mg kg(-1) day(-1)) in the drinking water daily for 45 days. Additional BCNR groups received L-NIL (6.7 mg kg(-1) day(-1)) as inhibitor of iNOS activity, with or without concurrent sildenafil administration. It was determined that sildenafil, like vardenafil, (1) prevented the 30% decrease in the smooth muscle cell/collagen ratio, and the 3-4-fold increase in apoptosis and reduction in cell proliferation, and partially counteracted the increase in collagen, seen with both UCNR and BCNR; and (2) normalized the CVOD, measured by dynamic infusion cavernosometry, induced by both BCNR and UCNR. The long-term inhibition of iNOS activity exacerbated corporal fibrosis and CVOD in the BCNR rats, but sildenafil functional effects were not affected by L-NIL. These data suggest that the salutary effects of continuous long-term PDE5 inhibitors on erectile function post-cavernosal nerve resection involve their ability to prevent the alterations in corporal histology induced by cavernosal nerve damage, in a process apparently independent from endogenous iNOS induction.

Long-term continuous treatment with sildenafil ameliorates aging-related erectile dysfunction and the underlying corporal fibrosis in the rat.

Aging-related erectile dysfunction is characterized by a loss of smooth muscle cells (SMCs) and fibrosis in the corpora cavernosa, and functionally by corporal veno-occlusive dysfunction (CVOD). Phosphodiesterase 5 (PDE5A) inhibitors, in part via upregulating inducible nitric oxide synthase (NOS2A), have antifibrotic properties in penile tissues. We aimed to determine whether in the aged rat the chronic long-term treatment with sildenafil ameliorates corporal SMC loss and fibrosis, stimulates NOS2A induction, and corrects the associated CVOD. Aged male rats (20 mo old) received sildenafil in their drinking water (20 mg/kg per day) or plain water for 45 days, and untreated young rats (5 mo old) served as controls (n = 8 per group). CVOD was assessed by dynamic infusion cavernosometry (DIC). Collagen:SMC (Masson trichrome) and collagen III:I (picrosirius red) ratios, SMC content (alpha-smooth muscle actin [ACTA2]), cell proliferation (proliferating nuclear antigen [PCNA]), apoptotic death (TUNEL), and NOS2A induction were measured by histochemistry and immunohistochemistry followed by quantitative image analysis. Collagen content was determined by hydroxyproline assay, and transforming growth factor beta-1 (TGFB1); xanthine oxidoreductase (XDH); ACTA2; NOS2A; and the Rho kinase inhibitor protein tyrosine phosphatase, nonreceptor type 11 (PTPN11), and activator, VAV, were measured by quantitative Western blot. In the aged rats treated with sildenafil, the erectile response by DIC was normalized, and the corporal SMC:collagen ratio and SMC number were increased. In addition, sildenafil reduced the corporal collagen content without affecting the collagen III:I ratio, increased the PCNA:apoptosis ratio, and stimulated NOS2A induction, although there was no effect on XDH, TGFB1, PTPN11, or VAV levels. These data show that long-term PDE5A treatment corrected CVOD in the aged rat and partially reversed the aging-related fibrosis and loss of SMC in the corpora cavernosa without affecting TGFB1 or PTPN11 levels, which are markers of oxidative stress. It may be speculated that similar effects may be achieved with this paradigm in men.

Tadalafil dosed once a day in men with erectile dysfunction: a randomized, double-blind, placebo-controlled study in the US.

The efficacy and safety of tadalafil, dosed once a day for the treatment of erectile dysfunction, was assessed in a randomized, double-blind, placebo-controlled, parallel-design study at 15 US centers. Following a 4-week treatment-free run-in period, patients (>or=18 years of age) were randomly assigned to 24 weeks treatment with tadalafil 2.5 mg, tadalafil 5 mg or placebo. Primary efficacy endpoints were change at 24 weeks in International Index of Erectile Function Erectile Function (EF) Domain score and mean per-patient percentage 'yes' responses to Sexual Encounter Profile diary questions 2 and 3. Tadalafil significantly improved erectile function compared with placebo for all three co-primary efficacy endpoints. Few patients discontinued because of adverse events (2.1%, placebo; 6.3%, tadalafil 2.5 mg; 4.1%, tadalafil 5 mg). Common treatment-emergent adverse events (>or=5%) were nasopharyngitis, influenza, viral gastroenteritis and back pain. Tadalafil 2.5 mg and 5 mg, dosed once a day for 24 weeks, was well tolerated and significantly improved erectile function.

The evolving role of testosterone in the treatment of erectile dysfunction.

Hypogonadism may play a significant role in the pathophysiology of erectile dysfunction (ED). A threshold level of testosterone may be necessary for normal erectile function. Testosterone replacement therapy is indicated in hypogonadal patients and is beneficial in patients with ED and hypogonadism. Monotherapy with testosterone for ED is of limited effectiveness and may be most promising in young patients with hypogonadism and without vascular risk factors for ED. A number of laboratory and human studies have shown the combination of testosterone and other ED treatments, such as phosphodiesterase type 5 (PDE5) inhibitors, to be beneficial in patients with ED and hypogonadism, who fail PDE5 inhibitor therapy alone. There is increasing evidence that combination therapy is effective in treating the symptoms of ED in patients for whom treatment failed with testosterone or PDE5 inhibitors alone. Testosterone replacement therapy has potentially evolved from a monotherapy for ED in cases of low testosterone, to a combination therapy with PDE5 inhibitors. Screening for hypogonadism may be useful in men with ED who fail prior PDE5 inhibitors, especially in populations at risk for hypogonadism such as type 2 diabetes and the metabolic syndrome.

The pleiotropic effects of inducible nitric oxide synthase (iNOS) on the physiology and pathology of penile erection.

The contribution of the neuronal and endothelial isoforms of nitric oxide synthase (nNOS and eNOS, respectively) in the synthesis of nitric oxide as a mediator of penile erection, at the levels of both the penile corpora cavernosa and the hypothalamic regions that control the erectile response, are well established. More recently, the role of the third NOS isoform, the inducible NOS (iNOS), has also started to be elucidated. iNOS does not appear to intervene directly in physiological penile erection or in its central control, but its transcriptional induction is postulated to be a key factor in two opposite related pathological processes, namely neurotoxicity in critical related regions of the hypothalamus during senescence, and as a defense mechanism against the aging or injury-associated fibrosis in the penile corpora cavernosa, the media of the penile arteries, and the tunica albuginea. By counteracting fibrosis that impairs cavernosal smooth muscle compliance, iNOS would protect the erectile tissue. However, further studies are needed to conclusively evaluate these putative roles in the two organs involved in reproductive function. In addition, whether iNOS induction during aging is a major cause in the net loss of trabecular smooth muscle in the corpora cavernosa through apoptosis, remains to be elucidated. The overall evaluation of these conflicting effects is important in order to decide whether pharmacological iNOS induction, or alternatively NO donors or L-arginine, may constitute a valid approach to prevent or treat penile fibrosis and vasculogenic erectile dysfunction.

Corporal veno-occlusive dysfunction in aging rats: evaluation by cavernosometry and cavernosography.

OBJECTIVES: To determine whether the rat develops corporal veno-occlusive dysfunction (CVOD) during the aging process. METHODS: We divided male Fisher 344 rats into four groups with 5 to 8 rats in each group. Group 1 rats were 5 months old and not orchiectomized (young intact); group 2 were 5 months old and had undergone castration (control CVOD); group 3 were 24 months old and not orchiectomized (aged intact); and group 4 rats were 24 months old, nonorchiectomized rats who had undergone deep dorsal vein (DDV) ligation (aged ligated). Serum testosterone was measured, and dynamic infusion cavernosometry and cavernosography (by dual-energy x-ray absorptiometry scan) were performed. RESULTS: Compared with the young intact animals, the aged intact rats had a 73% reduction in testosterone, a 30% reduction in intracavernosal pressure after papaverine injection, and an 84% and a 44% increase in the maintenance and drop rate, respectively, during dynamic infusion cavernosometry. The extent of CVOD as measured by cavernosometry in the aged rats was similar to that in the young castrated rats, but was more pronounced when measured by dynamic infusion cavernosometry. Cavernosography confirmed that the aged rats had venous leakage primarily by way of the DDV. By applying DDV ligation, distal penile engorgement occurred, showing that a small venous leakage occurred primarily by way of the cavernosal veins. CONCLUSIONS: These data have demonstrated that CVOD occurs in the aged rat, mainly through the DDV. This supports the concept that the aged rat is an appropriate animal model to study erectile dysfunction associated with CVOD.

Molecular pathophysiology and gene therapy of aging-related erectile dysfunction.

Erectile dysfunction (ED) is a major public health problem that seriously affects the quality of life of patients and their partners. ED is mainly associated with vascular disease, diabetes, smoking, and radical prostatectomy, and its prevalence increases significantly with aging. Vasculogenic ED, specifically corporal veno-occlusive dysfunction (CVOD), is caused by the impairment of the relaxation of the smooth muscle in the penile corpora cavernosa and occurs in 2/3 of cases, whereas the less common neurogenic ED is due to a defective nitrergic neurotransmission triggered by the sexual stimulus, either at the central hypothalamic and spinal levels or at the penile nerves. Based on animal and cell studies, neurogenic ED is assumed to be caused mainly by: (a) an insufficient synthesis of nitric oxide (NO) due to a decrease in the levels of the penile neuronal nitric oxide synthase (PnNOS) or the impairment of its regulation by protein effectors (NMDA receptor, protein inhibitor of nNOS: PIN), occurring in the neuronal bodies or nerve terminals, or (b) a loss of the cells themselves by apoptosis caused by the induction of inducible NOS (iNOS) and the production of peroxynitrite. In contrast vasculogenic ED, although may involve endothelial damage and down-regulation of endothelial NOS (eNOS), appears to be mainly caused by the relative loss of smooth muscle cells and replacement by collagen fibers (fibrosis) that impairs tissue compliance. In this case, iNOS induction may not be deleterious, but a defense mechanism preventing excessive collagen deposition. Gene therapy to the penile corpora cavernosa of cDNAs expressing PnNOS or eNOS, or counteracting PIN, has been effective in ameliorating ED in the aging rat model that exhibits both neurogenic ED and CVOD. cDNA constructs for other genes involved in the control of penile erection have also been successfully tested. Gene transfer into the penis may soon translate to the clinic as a therapy aimed to cure the underlying conditions in ED, including fibrosis, as opposed to the facilitation of erection on demand offered by the current oral therapies.

Comparison of gene expression profiles between Peyronie's disease and Dupuytren's contracture.

OBJECTIVES: To compare the gene expression alterations in human Peyronie's disease (PD) and Dupuytren's disease (DD) to determine whether they share a common pathophysiology. Multiple mRNA expression profiles of human PD have previously shown that genes that regulate fibroblast replication, myofibroblast differentiation, collagen metabolism, tissue repair, and ossification are involved. DD, a palmar fascia fibrosis, may be associated with PD. METHODS: Total RNA samples from PD plaques, normal tunica albuginea, Dupuytren's nodules, and normal palmar fascia (nine samples per group) were subjected to differential gene expression profile analysis (Clontech Atlas DNA microarray) comparing PD with tunica albuginea and DD with normal palmar fascia. Changes of more than 2.0 in PD and DD compared with tunica albuginea and normal palmar fascia, respectively, were recorded. Reverse transcriptase-polymerase chain reactions were performed for some genes whose expression was altered in PD. RESULTS: Some of the gene families upregulated in both PD and DD were (a) collagen degradation: matrix metalloproteinase (MMP), with MMP2 and MMP9, and thymosins (MMP activators), with TMbeta10 and TMbeta4; (b) ossification: osteoblast-specific factors (OSFs) OSF-1 and OSF-2 (DD only); and (c) myofibroblast differentiation: RhoGDP dissociation inhibitor 1. The genes upregulated in PD only were decorin (an inhibitor of transforming growth factor-beta1 and a part of fibroblast replication/collagen synthesis) and early growth response protein. Reverse transcriptase-polymerase chain reaction confirmed these changes. CONCLUSIONS: These data demonstrate that the pattern of alterations in the expression of certain gene families in PD and DD is similar, suggesting that they share a common pathophysiology and may be amenable to the same therapeutic regimens.

The efficacy and safety of tadalafil: an update.

OBJECTIVE: To provide an update on the efficacy and safety of tadalafil, a phosphodiesterase-5 inhibitor, in the treatment of erectile dysfunction (ED). PATIENTS AND METHODS: In all, 2102 men (mean age 56 years) with mild-to-severe ED of various causes were randomized to placebo or tadalafil, taken as needed with no food restrictions, at fixed 'on-demand' doses of 10 or 20 mg in 11 randomized, double-blind, placebo-controlled trials lasting 12 weeks. The three co-primary outcomes were changes from baseline in the erectile function domain of the International Index of Erectile Function (IIEF) and the proportion of 'yes' responses to questions 2 and 3 of the Sexual Encounter Profile (SEP). Additional efficacy instruments included a Global Assessment Question (GAQ). RESULTS: Compared with placebo, tadalafil gave significantly better outcomes. Patients receiving either dose of tadalafil had a significant mean improvement of 6.5 and 8.6, respectively, in the IIEF erectile function domain score from baseline (P < 0.001 vs placebo). At both doses the mean success rate for intercourse attempts (SEP-Q3) was 58% and 68%, respectively, compared with 31% in the placebo group (P < 0.001), and 71% and 84% reported improved erections at the endpoint (GAQ), vs 33% on placebo (P < 0.001). Tadalafil was effective up to 36 h after dosing and was effective regardless of disease severity and causes, and in patients of all ages. The most frequent adverse events were headache, dyspepsia, back pain and myalgia. CONCLUSION: Tadalafil was an effective and well-tolerated treatment for ED.

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

OBJECTIVES: 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). MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Protein inhibitor of nitric oxide synthase (NOS) and the N-methyl-D-aspartate receptor are expressed in the rat and mouse penile nerves and colocalize with penile neuronal NOS.

Nitrergic neurotransmission triggering penile erection is mediated by nitric oxide (NO) synthesized in the cavernosal nerves of the penis by penile neuronal NO synthase (PnNOS). In the central nervous system, nNOS is activated by the N-methyl-D-aspartate receptor (NMDAR) and, presumably, is inhibited by the protein inhibitor of NOS (PIN). The PnNOS and NMDAR are expressed in the penis, and PnNOS has been localized in penile nerves. Both proteins colocalize with PIN in the hypothalamus and the spinal cord involved in the control of erection. The present study aimed to elucidate the relationship between PnNOS, PIN, and NMDAR in the penis. It was found that in the rat, PIN was expressed in the pelvic ganglion and the cavernosal nerve, and penile PIN cDNA was cloned, sequenced, and expressed. Immunohistochemistry localized PIN to the cavernosal and dorsal nerve of the penis, whereas NMDAR was not detected in the latter. Dual-fluorescence labeling showed that PnNOS colocalized with PIN in both nerves but with NMDAR only in the cavernosal nerve. Aging did not affect the mRNA levels of PnNOS, nNOS, NMDAR, and PIN. Both PIN and NMDAR were detected in penile nerves of the wild-type and nNOS(-/-) mouse. The PIN protein did not inhibit or bind NOS in penile extracts, and in vivo, PIN cDNA reduced the erectile response to electrical field stimulation. In conclusion, PIN and NMDAR colocalize with PnNOS in penile nerves, but the functional significance of these protein interactions for penile erection remains to be elucidated.

Gene expression in Peyronie's disease.

Currently, surgical intervention is the only efficacious treatment for Peyronie's disease (PD), a fibromatosis of the tunica albuginea of the penis. Therapies based on the molecular pathways for this disease could provide alternatives to surgical treatment but only recently has the pathophysiology of the Peyronie's disease plaque been investigated at the molecular level. In this review, we examine the current knowledge of gene expression in the PD plaque and the relationship of PD with other fibrotic conditions such as Dupytren's disease. TGFbeta1, along with other growth factors, pro-fibrotic genes, and collagen, are expressed in fibroblasts and myofibroblasts. Myofibroblasts are normally involved in wound contracture and largely eliminated via apoptosis during the late stages of wound remodeling. In the PD plaque, however, these cells persist and may play an important role in the PD plaque fibrosis. The expression levels of TGFbeta1 and pro- and anti-fibrotic gene products, along with the nitric oxide/reactive oxygen species (NO/ROS) ratio in the tunica albuginea, appear to be essential for the formation and progression of the PD plaque and effect the expression of multiple genes. This can be assessed with the recently developed DNA-based chip arrays and results with the PD plaque have been encouraging. OSF-1 (osteoblast recruitment), MCP-1 (macrophage recruitment), procollagenase IV (collagenase degradation), and other fibrotic genes have been identified as being possible candidate regulatory genes. Finally, possible therapeutic avenues for gene-based therapy in the treatment of PD are discussed that may eventually reduce the need for surgical intervention.

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.

Improvement in emotional well-being and relationships of users of sildenafil.

PURPOSE: We estimated the association of sildenafil use with erectile function, relationship with sexual partner, functional status and emotional well-being in men with erectile dysfunction. MATERIALS AND METHODS: Letters were mailed to eligible patients at a university hospital urology and internal medicine clinic, and university affiliated community primary care clinics by the primary care provider or urologist inviting them to participate in the study. Of the eligible sample 124 men (53%) completed and returned a survey, including 85 who reported current sildenafil use. Change scores in these patients were calculated using the International Index of Erectile Function, marital interaction scale from the Cancer Rehabilitation Evaluation System Short Form, 5-item emotional well-being scale of the RAND 36-Item Health Survey and 12-Item Short Form Health Survey. RESULTS: Sildenafil users reported an 88% increase in erectile function scores, 60% increase in overall sexual satisfaction and 36% increase in intercourse satisfaction related to the use of sildenafil (p <0.001). Of the respondents 38% indicated that using sildenafil had definitely improved quality of life. Likewise 29% of respondents indicated that using sildenafil had definitely improved the relationship with their partner. With sildenafil there was a statistically significant improvement in the scores of erectile and sexual function (p <0.001), sexual partner relationship (p = 0.007) and emotional well-being (p <0.001). In a multivariate model improved erectile function and sexual partner relationship were each significantly associated with improved emotional well-being (R2 = 0.20, p <0.001). CONCLUSIONS: Sildenafil users reported significant improvements in erectile and sexual function that were associated with positive changes in emotional well-being and the sexual partner relationships with their sexual partner.