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Division of Urology,3 Urology Research Laboratory, Los Angeles Biomedical Research Institute,4 Harbor-UCLA Medical Center, Torrance, California 90502
Department of Urology,5 David Geffen School of Medicine at UCLA, Los Angeles, California 90095
ABSTRACT
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.
aging,, apoptosis, cGMP, collagen, corporal veno-occlusive dysfunction, inducible nitric oxide synthase, nitric oxide, PDE5A inhibitors, penis, phosphodiesterases, ROS, smooth muscle, TGFB1
Since aging is one of the main risk factors for erectile dysfunction, most men during their lifetime will at some time be afflicted with this condition [1–3]. It is becoming increasingly apparent that the main culprit for erectile dysfunction in the majority of patients is corporal veno-occlusive dysfunction (CVOD) [4, 5]. Indeed, regardless of age or etiology, erectile dysfunction caused by CVOD ranges between 66% and 75%. Therefore, if the cause of CVOD itself can be prevented, then the possibility remains that erectile dysfunction could possibly become a preventable condition.
CVOD, which is also referred to as venous leakage, is characterized clinically in men as an inability to maintain an erection once it is achieved. Physiologically, CVOD is mainly due to the failure of the corporal smooth muscle (SM) mass to achieve sufficient relaxation, which is necessary for the process of passive veno-occlusion of the subtunical veins to occur. Therefore, any process that decreases the content or function of the corporal SM will predispose to the development of CVOD or venous leakage, specifically cavernosal nerve injury following radical prostatectomy and diabetes [6, 7]. A loss of smooth muscle cells (SMCs) and an increase in fibrosis have been detected in corporal tissue of erectile dysfunction patients [8–10].
Recently, CVOD as measured by dynamic infusion cavernosometry has been shown to occur in aged rats [11]. The corpora of these aged animals demonstrate a decrease in SMCs and an excessive deposit of collagen fibers that result in corporal fibrosis [12–15], and this also occurs in the media of the penile arteries [16]. It has been postulated that these histologic changes in the aged corpora are caused by increased oxidative stress and/or other profibrotic factors that stimulate SMC apoptosis and collagen deposition [16]. Cavernosal nerve damage in the rat models of erectile dysfunction after radical prostatectomy is also associated with corporal fibrosis and loss of SMCs [17–19], and the same occurs in rat models for types 1 and 2 diabetes [20, 21], so that these processes seem to uniformly underlie CVOD. However, in aging-related CVOD no functional and histologic determinations have been demonstrated concurrently in the same groups of animals.
In other models of fibrosis (e.g., in the penile tunica albuginea, where the fibrosis is characterized by an increase in collagen over the intracellular compartment), the fibrosis is associated with the production of profibrotic factors, such as transforming growth factor beta-1 (TGFB1), plasminogen activator inhibitor-1, and reactive oxygen species (ROS) during oxidative stress [22–29]. This is accompanied by the induction of the inducible nitric oxide synthase (NOS2A), which acts as an endogenous antifibrotic mechanism in response to the profibrotic processes both by producing nitric oxide, which quenches ROS in a reaction-originating peroxynitrite, and by a direct reduction of collagen synthesis by both nitric oxide and its downstream product, cGMP [24–27, 29–32]. The expression of NOS2A accompanying fibrosis and oxidative stress also has been seen in the above-mentioned rat models for aging of the arterial vessels, cavernosal nerve damage, and types 1 and 2 diabetes, as well as in chronic smoking [16, 17, 20, 21]. This agrees with studies in the NOS2A knockout mouse in which NOS2A depletion intensified experimental fibrosis of the kidney and liver, suggesting that the antifibrotic role of NOS2A is not restricted to the urogenital and vascular systems [33, 34].
Long-term overexpression of NOS2A and nitric oxide production via intratunical Nos2a cDNA gene transfer, or long-term oral administration of the phosphodiesterase 5 (PDE5A) inhibitors sildenafil and vardenafil, which elevate cGMP, or long-term treatment with the phosphodiesterase 4 inhibitor pentoxifylline, which increases cAMP, reduces penile fibrosis. This occurs in either the rat models of Peyronie disease and/or cavernosal nerve damage and, in the latter case, also prevents CVOD [17, 26, 32]. In addition, it is conceivable that long-term treatment with PDE5A inhibitors could upregulate NOS2A expression via cGMP modulation, and thus contribute to SMC protection [35–41]. Indeed, in the clinical setting there have been occasional reports in men that long-term continuous oral administration of pentoxifylline or sildenafil may ameliorate erectile dysfunction [42–44]. More recently, chronic treatment with tadalafil improved endothelial function and morning erections in patients with erectile dysfunction [45], agreeing with studies in rats in which long-term sildenafil both improved endothelium-dependent cavernosal relaxations and the erectile response to cavernosal nerve stimulation in young animals [46], and prolonged this response in old rats [47]. However, neither CVOD nor the underlying histologic changes in the SMC/collagen and NOS2A contents in cavernosal tissue were examined in these studies.
In the current work we address the issue of whether CVOD in the aged rat is associated in the corporal tissue with fibrosis, loss of SMCs, and oxidative stress, and whether these functional and histologic changes can be ameliorated by long-term PDE5A inhibition via, at least in part, stimulation of endogenous corporal NOS2A expression.
Twenty-mo-old (aged) male Fisher 344 rats (Harlan Sprague-Dawley, San Diego, CA) were treated under LA BioMed at Harbor-UCLA Institutional Animal Care Use Committee approved-protocol. The rats were either treated with continuous oral sildenafil from Pfizer Ltd. (0.3 mg/ml; Sandwich, UK) in the drinking water (aged-treated) or received regular drinking water (aged-untreated controls), and were killed 45 days later. As a washout process, treated animals were switched to regular drinking water 1 day prior to cavernosometry and killing. Five-mo-old (young-untreated) rats served as reference. The drinking volume was determined daily, and body weight was recorded weekly. The daily sildenafil dose given to these animals was approximately 20 mg/kg per day, which is equivalent to approximately 200 mg per day in men when corrected for differences in total body surface area [26].
Dynamic Infusion Cavernosometry
Cavernosometry was performed as previously described [11, 17, 20]. Briefly, basal intracavernosal pressure (ICP) was recorded, and papaverine at a dose of 5 mg/kg body weight was administered through a cannula into the corpus cavernosum. The ICP during tumescence was recorded as ICP after papaverine. Saline then was infused through another cannula placed into the corpus cavernosum, increasing the infusion rate by 0.05 ml/min every 10 sec until the ICP reached 100 mm Hg (infusion rate), and it was then adjusted to keep this pressure (maintenance rate). The drop rate was determined by recording the fall in ICP within the next 1 min after the infusion was stopped.
Histochemistry and Immunohistochemistry
After cavernosometry, animals were killed and the skin-denuded middle part of the penile shaft was fixed overnight in 10% formalin, washed, and stored in alcohol 70% at 4°C until processing for paraffin-embedded tissue cross-sections (5 µm). Adjacent sections at the middle region of the penis shaft were used [14, 17, 20, 24–29, 32] for: 1) Masson trichrome staining for collagen (blue) and SM (red); 2) picrosirius red under polarized microscopy for collagen III (green):I (red-orange) ratios; and 3) immunodetection with: a) monoclonal antibodies against 
-smooth muscle actin (ACTA2) as an SM marker (Sigma kit; Sigma Diagnostics, St. Louis, MO) and proliferating cell nuclear antigen (PCNA) as a marker of cell proliferation (Chemicon, Temecula, CA); and b) polyclonal antibody against NOS2A (Calbiochem, La Jolla, CA). The specificity of the antibodies was validated by Western blot.
Sections then were incubated with biotinylated anti-mouse IgG (ACTA2 and PCNA) or biotinylated anti-rabbit IgG (NOS2A), followed by ABC complex (Vector Laboratories, Temecula, CA) and 3,3' diaminobenzidine (PCNA and NOS2A; Sigma), or with the ACTA2 Sigma kit (ACTA2) and 3-amino-9-ethylcarbazole. TUNEL assay was performed as described [14–17, 20, 24–27, 29] using the Apoptag peroxidase detection assay (Chemicon) with TdT enzyme and anti-digoxigenin-conjugated peroxidase and 3,3' diaminobenzidine/H2O2. Sections were counterstained with hematoxylin. Negative controls in the immunohistochemical detections were generated by replacing the first antibody with IgG isotype. The negative control for TUNEL was generated by substituting buffer for the TdT enzyme. Testicular sections from old animals were used as a positive control only for the TUNEL assay.
Quantitative image analysis was performed by computerized densitometry using the ImagePro 5.1 program (Media Cybernetics, Silver Spring, MD) coupled with an Olympus BHS microscope equipped with an Olympus digital camera [14–17, 19, 20, 24–29, 32]. For Masson staining, 40x magnification pictures of the penis comprising half of the corpora cavernosa were analyzed for SM (stained in red) and collagen (stained in blue) areas, excluding the sinusoidal spaces, and were expressed as SM:collagen ratio. An identical approach was applied for collagen III:I ratios. For ACTA2 and NOS2A staining, only the corpora cavernosa tissue around lacunar spaces was analyzed in a computerized grid and expressed as percentage of positive versus total area of the corpora cavernosa. The intensity of immunostaining was determined as percentage of integrated optical density in the corpora cavernosa. For PCNA and TUNEL determinations, the number of positive cells at 400x magnification was counted, and results were expressed as a percentage of positive cells / total cells in the corpora cavernosa. If a staining procedure could not be applied simultaneously to all specimens, adjacent sections of a single positive control and of a given specimen within the series were run to standardize the optical intensity comparisons. Negative controls without primary or secondary antibodies were used to correct for antibody specificity and background intensity. In all cases, three fields at 40x magnification or 8 fields at 400x magnification were analyzed per tissue section, with at least four matched sections per animal and 6–11 animals per group.
Penile homogenates of frozen tissue (100 mg) were obtained in T-PER (Pierce, Rockford, IL) and protease inhibitors (3 µM leupeptin, 1 µM pepstatin A, 1 mM phenyl methyl sulfonyl fluoride), and were centrifuged at 10 000 x g for 5 min. Equal amounts of supernatant protein (30 µg) were run on 7.5% or 10% (ACTA2) polyacrylamide gels and submitted to Western blot immunodetection [20, 24–28, 32, 48–50] with: 1) monoclonal anti-mouse macrophage NOS2A IgG (1:500; BD Pharmingen, Transduction Laboratories, San Jose, CA,), detecting a 130-kDa band; 2) xanthine oxidoreductase (XDH) IgG (1:5000; AbCam, Cambridge, MA), detecting a 145-kDa band for xanthine dehydrogenase and a 70-kDa band for xanthine oxidase; 3) a polyclonal TGFB1 IgG (1:1000; Promega, Madison WI), detecting a 130-kDa monomer or a 26-kDa covalently linked dimer; 4) a monoclonal ACTA2 IgG (1:1000; Oncogene-Calbiochem, La Jolla, CA), detecting a 43-kDa band; 5) a monoclonal PTPN11 (Src homology region 2-containing protein tyrosine phosphatase) IgG (1:250; Santa Cruz Biotechnology, Santa Cruz, California), detecting a 70-kDa band; and 6) a monoclonal VAV IgG (1:500; Santa Cruz), detecting a 95-kDa band. Membranes were incubated with a secondary polyclonal horse anti-mouse IgG linked to horseradish peroxidase (1:2000; BD Transduction Laboratories), or anti-rabbit IgG linked to horseradish peroxidase (1:5000; Amersham, Piscataway, NJ), and bands were visualized with luminol (Pierce). A single positive control was run throughout all gels for each antibody to standardize for variations in exposures and staining intensities. Negative controls were performed omitting the primary antibody. Band intensities were determined by densitometry and were corrected by the respective intensities for a housekeeping protein, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), upon reprobing.
Collagen Estimation in Fresh Tissue
As previously described [20, 24, 49], the tissue was homogenized in saline and hydrolyzed with 2 N NaOH for 30 min at 120°C, followed by the estimation of hydroxyproline by a modification of the Neumann and Logan reaction using Chloramine T and Ehrlich reagent, using a hydroxyproline standard curve, and measuring at 550 nm. Values were expressed as milligrams of collagen per milligram of tissue.
Values were expressed as mean ± SEM. The normality distribution of the data was established using the Wilk-Shapiro test. Multiple comparisons were analyzed by a single factor analysis of variance (ANOVA), followed by post-hoc comparisons with the Student-Newman-Keuls test, according to the GraphPad Prism V 4.1 (San Diego, CA). Differences were considered significant at P < 0.05.
In order to determine the effects of sildenafil on CVOD in the aged rats, cavernosometry was performed after 45 days of treatment and a 24-h washout period. The latter reduces circulating and penile sildenafil concentrations to levels unlikely to affect the cavernosometry measurements, based on the pharmacokinetic 3- to 4-h half-life of sildenafil [43]. Figure 1 shows that, as expected, control aged rats not receiving sildenafil had a considerable decrease (38%) in the ICP induced by papaverine intracavernosal injection (top panel) and a concomitant 2.3-fold increase in the drop rate (bottom panel) of the ICP subsequent to saline infusion, compared with the young rats. This indicates the presence of venous leak or CVOD in the aged rats. Continuous sildenafil treatment for 45 days increased the papaverine-induced erection (top panel) and reduced the drop rate (bottom panel) to values not significantly different from the ones in the young untreated control rats. No priapism, lethargy, aggressiveness, or weight loss were noticed with sildenafil.
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This functional impairment of the corpora cavernosa associated with aging was accompanied by a considerable reduction (83%) of the ratio between the areas occupied by SMC and collagen fibers in the corpora of the aged untreated rats versus young untreated animals, as determined by Masson trichrome staining of cross-sections of penile shaft tissue (Fig. 2, top, middle, and bottom left panels). Treatment with sildenafil significantly improved the SMC:collagen ratio by 60% in comparison with the aged untreated controls, but in contrast to the correction of CVOD, the SM:collagen ratio still remained significantly lower than in the young untreated rats. At least part of this improvement was due to a significant 13% reduction in the amount of collagen fibers in the penile shaft, as measured by hydroxyproline in whole penile shaft tissue hydrolyzates (Fig. 2, bottom middle panel). Since determinations were restricted to the vicinity of the lacunar spaces lined by endothelium, where SMC were concentrated no attempts were made to study potential regional differences around the cross-sections (e.g., adjacent to the tunica albuginea vs. the lacunar spaces per se). However, with sildenafil treatment there was no change in the collagen III:I ratio as determined by picrosirius red and polarized light (Fig. 2, bottom right panel), which under these conditions do not measure total collagen content but a relative estimation of both types of collagen. No assays for hydroxyproline and collagens III and I were performed in the young rats.
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The other component in the reduction of the SMC:collagen ratio observed in the corporal tissue of the aged rat (Fig. 2) was the decrease in the actual content of SMC, as determined by immunostaining for ACTA2 (73%; Fig. 3, top, middle, and left panels). Sildenafil treatment of the aged rats increased this reduced SMC content by 60% compared with untreated aged animals, which is in line with the elevation of the SMC:collagen ratio observed in Figure 2. However, the increase estimated by Western blot of total penile shaft homogenates was much less, about 20% (Fig. 3, middle and right), probably reflecting a regional difference in SMC content or variations in the amount of ACTA2 expressed per SMC.
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This increase in ACTA2 content by sildenafil essentially was the result of a stimulation of cell replication, as estimated by PCNA expression. Figure 4 (top panel) shows an increase of more than 3-fold in PCNA immunostaining in the corpora of sildenafil-treated aged rats compared with untreated controls. This contrasts with the absence of any significant effect by sildenafil on the apoptotic index estimated by TUNEL immunostaining (middle panel), and as a result there was an increase in the ratio between PCNA and TUNEL, which can be considered as representative of the cell proliferation and death indexes, respectively (bottom panel). In other words, sildenafil induced a positive cell turnover in the corpora with a predominance of cell replication over death.
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We estimated NOS2A by immunohistochemistry in an attempt to elucidate the question of whether these effects of sildenafil are mediated directly by the elevation of cGMP levels or whether they are in part the result of an induction of NOS2A and the subsequent stimulation of the nitric oxide/cGMP pathway by cGMP stimulating NOS2A expression. Figure 5 (top, middle, and bottom left panels) shows that there was a moderate (about 34%) but not significant stimulation of NOS2A expression in the corpora of the aged sildenafil-treated rats compared with the respective aged controls. However, the Western blot analysis (bottom middle panel) of corporal homogenates showed a significant 86% stimulation of NOS2A induction by sildenafil.
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Despite this moderate effect of long-term sildenafil on NOS2A levels, this treatment did not vary the expression of TGFB1 and xanthine dehydrogenase levels, a key fibrotic factor and a marker of oxidative stress, respectively, as estimated by quantitative Western blot (data not shown). Similarly, the expression of PTPN11, a phosphatase that inhibits Rho kinase and hence counteracts vascoconstriction, and the expression of VAV, which activates Rho kinase and is a target of PTPN11, were not affected by sildenafil.
Our results demonstrate that CVOD in aged rats is associated with a significant reduction of the SMC:collagen ratio in the penile corpora cavernosa compared with young rats, which is in agreement with our previous data showing a similar association in a rat model of erectile dysfunction following bilateral cavernosal nerve resection [17]. Even more clinically relevant, our data show that long-term and continuous administration of a PDE5A inhibitor, in this case sildenafil, corrects aging-associated vasculogenic erectile dysfunction as measured by cavernosometry. Our study was intended as a proof of concept to demonstrate in the aging rat model that a PDE5A inhibitor corrects CVOD and ameliorates the underlying corporal fibrosis. The selected 45-day treatment was based on our previous similar work with PDE5 inhibitors in both Peyronie disease and corporal SM fibrosis subsequent to cavernosal nerve resection [17, 29]. The treatment duration was long enough to expect a measurable improvement of a chronic process and short enough to be experimentally convenient and shorter than in other studies [46, 47]. We speculate that longer treatments may be necessary to achieve a more considerable correction of the histologic alteration in the aged corpora cavernosa, but the chosen period validates our hypothesis.
The dose used in our current work (20 mg/kg per day) is one third lower than in other studies in rats that showed an improvement of endothelial function and penile erectile responses [46, 47]. At this dose the initial plasma concentration of sildenafil with a single oral administration is likely to be between 0.5 and 2 µg/ml, with a half-life between 30 and 60 min and a 98% clearance between 1.5 and 3.5 h [51]. This assured that our 24-h washout was completely effective to avoid pharmacologic acute effects of the drug during cavernosometry. When the 20 mg/kg per day dose is extrapolated to the human based on differences of surface area [26], the approximately 200 mg/day dose is still lower than the maximal dose tested without side effects for the treatment of pulmonary hypertension for 12 wk [52]. Our continuous oral regimen based on sildenafil dissolved in the drinking water is not feasible for men, although we have successfully given PDE5A inhibitors as bolus retrolingual administration to rats to prevent the development of a Peyronie-like fibrotic plaque and corporal SMC fibrosis after cavernosal nerve resection [29]. It is likely that our current administration regimen in the drinking water may be successfully replaced in the rat by two or three daily retrolingual treatments at lower doses. Further studies should determine which is the most adequate bolus dosage.
The improvement of the SMC:collagen ratio is likely due to the antifibrotic effects of cGMP [22, 30], and the fact that the release of another antifibrotic factor, nitric oxide, is also stimulated. This is evidenced by the moderate increase in NOS2A induction presumably exerted by the enhanced cGMP levels caused by the drug. This needs verification by measuring cGMP levels vis à vis the use of in vivo inhibitors of NOS2A activity, such as L-NIL or 1400W [16, 24, 37], or in vitro incubations of corporal SMCs with cGMP analogs [25, 39–41], but our results agree with studies showing that NOS2A expression was stimulated by sildenafil in the rat and mouse heart after ischemia reperfusion injury [36–38], and that cGMP analogs increased Nos2a mRNA and protein levels in cultures of mesangial cells, pulmonary artery SMCs, and human chondrocytes [17, 39–41]. The mechanism is not well characterized, but it may involve a crossover stimulation of PKA, biphasic Nos2A mRNA stabilization, and possibly transcriptional upregulation [39–41].
However, although sildenafil did significantly raise the SMC:collagen ratio in the corpora cavernosa of the aged rats, mainly by stimulation of cell proliferation, this value remained low in comparison to young rats. These observations are similar to what we found with the use of continuous long-term treatment with another PDE5A inhibitor, vardenafil, albeit in another model of erectile dysfunction, the bilateral cavernosal nerve resection model [17]. It would be interesting to determine the mechanism of the differential effect of sildenafil on SMC proliferation in two vascular beds, since in the arterial media cGMP and nitric oxide inhibit this process [53, 54]. This is in agreement with the fact that SMC turnover follows opposite directions with aging in the penile corpora cavernosa (with loss of SMCs leading to impaired compliance) versus the arterial media (often with SMC hyperplasia leading to wall thickening). In fact, it is known that cGMP acts as pro-proliferative or anti-proliferative agent according to the cell type [55].
In addition, the oxidative stress and TGFB1 levels were not affected by sildenafil, thus differing with the effects of vardenafil in the rat models of cavernosal nerve damage or Peyronie-like fibrotic plaque [17, 29]. This is not surprising, since cGMP is not a direct inhibitor of TGFB1 expression but does interfere with TGFB1 signaling both by blocking pSMAD 2 and 3 nuclear translocation or SMAD-induced gene expression and by the conversion of latent TGFB1 to its active form [56–58]. In fact, the inhibition of TGFB1 expression by nitric oxide is not mediated by cGMP [59]. In addition, in contrast to nitric oxide, cGMP is not a key modulator of oxidative stress, although it is possible that a sildenafil effect may be detected by markers of this process other than xanthine dehydrogenase. Sildenafil did not affect the collagen III:I ratio, as measured by the picrosirius red reaction, the alteration of which either as an increase [20, 29] or a decrease [17] is associated with tissue fibrosis in the penis. This finding may need confirmation by an alternative procedure, such as immunodetection in frozen sections. However, based on our current results we may assume that long-term sildenafil affects SM compliance by a mechanism additional to the elevation of the SMC:collagen ratio via the counteraction of oxidative stress or TGFB1 expression, or else by the alteration of collagen isoform composition, which we have postulated in those other models of penile fibrosis.
Taking together the normalization of CVOD by long-term pharmacologic interventions and the concurrent different degrees of amelioration of the corporal SMC:collagen ratio observed in the rat models, including in this work, of vasculogenic erectile dysfunction [17, 20], it may be concluded that correcting, at least partially, the relative SMC loss occurring with aging, diabetes, or cavernosal nerve damage should be a key therapeutic aim to prevent the erectile dysfunction associated with these conditions. Therefore, long-term and continuous treatment with sildenafil, and speculatively with the other available PDE5A inhibitors, may be pharmacologically effective for partially reversing the underlying alterations in the corpora that lead to erectile dysfunction, thus potentially curing this disorder, as opposed to the current discontinuous, on-demand, palliative administration of these compounds for eliciting an erection. We believe that by increasing SMC proliferation in the corpora cavernosa PDE5A inhibitors partially preserve or restore the number of SMCs and reduce collagen deposition, but whether this is the main factor in the beneficial long-term effects of PDE5A inhibitors may vary according to the degree of corporal oxidative stress, and thus of fibrosis.
Another factor that affects corporal SM compliance and that may be influenced by long-term PDE5A inhibitors, as opposed to acute effects on SM relaxation, is the expression of contractile proteins, such as ACTA2, smoothelin, and others, which are fundamental for the corporal SM relaxation/contradiction process that operates in penile tumescence and detumescence. This expression is associated with the functional phenotype of the SMC (e.g., contractile vs. synthetic) [53, 60]. Also, penile neuronal NOS and endothelial NOS levels, that directly elicit an erection by releasing nitric oxide upon sexual stimulation [16], may be elevated by sustained high cGMP levels and thus improve compliance. A third type of target that may be affected by PDE5 inhibitors is the Rho kinase system, by mechanisms alternative to PTPN11 induction or VAV downregulation. These mechanisms may be phosphorylation or direct inhibition of Rho kinase, or the availability of cGMP substrate for these processes [45, 61, 62]. Although the focus of the current work was placed on corporal SMCs and fibrosis, we cannot rule out that at least part of the amelioration of CVOD by long-term PDE5A inhibition could be due to the improvement of endothelial function via NOS3 activation [45–47, 63, 64], particularly at the arterial level, which would conceivably increase blood flow and thus compensate venous leak.
We speculate that the combination of improving the SMC:collagen ratio and putatively acting at other levels of corporal compliance is what eventually causes the beneficial effects of sildenafil on CVOD in the aged rat. If additional experimentation in animal models confirms the current results, determines whether lower doses are still efficacious, and further clarifies the mechanism of action, it may be warranted to evaluate clinically the daily administration of PDE5A inhibitors for long periods to prevent or correct aging-related CVOD. Long-term on-demand treatment with PDE5A inhibitors appears to be safe [65], and prolonged daily administration also may be well tolerated.
FOOTNOTES
1Supported by investigator-initiated research grants from Pfizer Corporation and National Institutes of Health (NIH) grant R01DK-53069 and, in certain aspects, NIHG12RR-03026. M.G.F. also is supported by grant N1 P20 MD000545 from the National Center on Minority Health and Health Disparities at the NIH. 
Correspondence: 2Nestor F. Gonzalez-Cadavid, LA BioMed at Harbor-UCLA Medical Center, Urology Research Laboratory, Bldg. F-6, 1124 West Carson St., Torrance, CA 90502. FAX: 310 222 1914; e-mail: ncadavid{at}ucla.edu
Received: 19 December 2006.
First decision: 8 January 2007.
Accepted: 26 January 2007.
REFERENCES
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  A. L. Burnett Molecular Pharmacotherapeutic Targeting of PDE5 for Preservation of Penile Health J Androl, January 1, 2008; 29(1): 3 - 14. [Abstract] [Full Text] [PDF]
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