Sandra Filippi

Testosterone Partially Ameliorates Metabolic Profile and Erectile Responsiveness to PDE5 Inhibitors in an Animal Model of Male Metabolic Syndrome

INTRODUCTION Metabolic syndrome (MetS) is a clustering of cardio-metabolic risk factors (hyperglycemia, hypertension, dyslipidemia, visceral fat accumulation) that is also associated with hypogonadism and erectile dysfunction (ED). AIM To clarify the relationships among MetS, hypogonadism, and ED, we developed an animal model of MetS. METHODS Male rabbits fed a high-fat diet (HFD), with or without testosterone (T) supplementation, were compared with control rabbits (fed a standard chow) and with rabbits made hypogonadal by a single injection of a long-acting GnRH-analog, triptorelin. MAIN OUTCOME MEASURES Evaluation of metabolic disturbances (plasma glucose, cholesterol, triglycerides, testosterone, LH, FSH level, glucose tolerance, mean arterial pressure, visceral fat accumulation), and corpora cavernosa (CC) relaxant capacity (in vitro contractility study) in HFD animals as compared with control, GnRH analog-treated rabbits, and T-supplemented HFD rabbits. RESULTS HFD rabbits showed all the features of MetS. HFD induced hypogonadotropic hypogonadism is characterized by a reduction of plasma T, FSH, LH levels, testis and seminal vesicles weight, and testicular steroidogenic enzymes. Such a phenotype is similar to that induced by triptorelin administration. A reduced GnRH immunopositivity in hypothalamus suggests a central origin of HFD-related hypogonadism. HFD also induced penile alterations, as demonstrated by a reduction of acetylcholine-and electrical field stimulation-induced CC relaxation, hyper-responsiveness to the NO donor, SNP, and unresponsiveness to PDE5 inhibitors. Similar penile alterations were observed in triptorelin treated rabbit. In HFD, as well as in triptorelin treated rabbits, PDE5 and eNOS mRNA expression quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) were significantly decreased. T administration prevented almost all penile alterations observed in HFD rabbits. T treatment dramatically reduced HFD-induced visceral obesity, partially ameliorating also the metabolic profile. CONCLUSION We have developed an animal model of MetS associated with hypogonadotropic hypogonadism and penile alterations including unresponsiveness to PDE5 inhibitors. T supplementation was able to partially revert HFD-induced phenotype.

Testosterone protects from metabolic syndrome-associated prostate inflammation: an experimental study in rabbit.

Metabolic syndrome (MetS) and benign prostatic hyperplasia (BPH)/lower urinary tract symptoms (LUTS) are often associated. One of their common denominators is hypogonadism. However, testosterone supplementation is limited by concerns for potential prostatic side effects. The objective was to determine whether MetS-associated prostate alterations are prevented by testosterone supplementation. We used a previously described animal model of MetS, obtained by feeding male rabbits a high-fat diet (HFD) for 12 weeks. Subsets of HFD rabbits were treated with testosterone or with the farnesoid X receptor agonist INT-747. Rabbits fed a standard diet were used as controls. HFD-animals develop hypogonadism and all the MetS features: hyperglycemia, glucose intolerance, dyslipidemia, hypertension, and visceral obesity. In addition, HFD-animals show a prostate inflammation. Immunohistochemical analysis demonstrated that HFD-induced prostate fibrosis, hypoxia, and inflammation. The mRNA expression of several proinflammatory (IL8, IL6, IL1β, and TNFα), T lymphocyte (CD4, CD8, Tbet, Gata3, and ROR γt), macrophage (TLR2, TLR4, and STAMP2), neutrophil (lactoferrin), inflammation (COX2 and RAGE), and fibrosis/myofibroblast activation (TGFβ, SM22α, αSMA, RhoA, and ROCK1/ROCK2) markers was significantly increased in HFD prostate. Testosterone, as well as INT-747, treatment prevented some MetS features, although only testosterone normalized all the HFD-induced prostate alterations. Interestingly, the ratio between testosterone and estradiol plasma level retains a significant, negative, association with all the fibrosis and the majority of inflammatory markers analyzed. These data highlight that testosterone protects rabbit prostate from MetS-induced prostatic hypoxia, fibrosis, and inflammation, which can play a role toward the development/progression of BPH/LUTS.

Phosphodiesterase Type 5 Expression in Human and Rat Lower Urinary Tract Tissues and the Effect of Tadalafil on Prostate Gland Oxygenation in Spontaneously Hypertensive Rats

INTRODUCTION In humans, prostate phosphodiesterase type 5 inhibitors (PDE5) expression was prominently localized in the endothelial and smooth muscle cells of the vascular bed, suggesting a possible action of PDE5 inhibitors (PDE5i) on prostate blood flow. AIM To investigate PDE5 expression in human and rat lower urinary tract (LUT) tissues, including vasculature, and determine the effects of PDE5 inhibition with tadalafil on prostatic blood perfusion. MAIN OUTCOME MEASURES Human vesicular-deferential arteries (which originate from the inferior vesical artery, the main arterial source of blood supply to the bladder and prostate) were analyzed for PDE5 expression and activity. The effects of tadalafil on prostate oxygenation were studied in spontaneously hypertensive rats (SHR), characterized by ischemia/hypoxia of the genitourinary tract. METHODS PDE5 expression was evaluated by quantitative reverse transcription-polymerase chain reaction and immunohistochemistry. SHR were treated with tadalafil (2 mg/kg/day) for 1, 7, or 28 days and compared with untreated SHR and the unaffected counterpart Wistar-Kyoto (WKY) rats. Prostate oxygenation was detected by Hypoxyprobe-1 and hypoxia markers (hypoxia-inducible factor-1α[HIF-1α] and endothelin-1 type B [ETB]) immunostaining. RESULTS Human vesicular-deferential artery expressed high levels of PDE5, similar to corpora cavernosa, immunolocalized in the endothelial and smooth muscle layer. In these arteries, tadalafil inhibited cyclic guanosine monophosphate breakdown (half maximal inhibitory concentration (IC(50) ) in the low nanomolar range, as in corpora cavernosa) and increased the relaxant response to sodium nitroprusside. SHR prostate resulted markedly hypoxic (hypoxyprobe immunopositivity) and positive for HIF-1α and ETB, while tadalafil treatment restored oxygenation to WKY level at each time point. The mRNA expression of the HIF-1α target gene, BCL2/adenovirus E1B 19 kDa interacting protein 3, was significantly increased in SHR prostate and partially restored to WKY level by tadalafil. CONCLUSION Human vesicular-deferential artery is characterized by a high expression and activity of PDE5, which was inhibited by tadalafil in vitro. In SHR, tadalafil increases prostate tissue oxygenation, thus suggesting a possible mechanism through which PDE5i exert beneficial effects on LUT symptoms.

Characterization of Phosphodiesterase Type 5 Expression and Functional Activity in the Human Male Lower Urinary Tract

INTRODUCTION Phosphodiesterase type 5 (PDE5) inhibitors ameliorate low urinary tract (LUT) symptoms in men with ED and symptomatic benign prostatic hyperplasia (BPH). PDE5 is highly expressed in rat and human bladder, where it regulates cyclic guanosine monophosphate (cGMP) degradation, muscle tone, and proliferation. AIM To investigate PDE5 tissue distribution and activity in human LUT tissues (urethra, prostate, and bladder). MAIN OUTCOME MEASURES PDE5 expression and activity were analyzed and compared within the same BPH patient in LUT tissues and in smooth muscle cells (SMCs) cultured from urethra, prostate, and bladder. METHODS In LUT tissues, PDE5 was localized by immunohistochemistry and mRNA expression by quantitative real-time polymerase chain reaction. Proliferation assay was used as readout of PDE5 activity, evaluated as ability of vardenafil to increase the antiproliferative effect of different nitric oxide (NO)/cGMP pathway activators [the PDE5-resistant cGMP analog Sp-8-Br-PET-cGMPS, the NO donor sodium nitroprusside (SNP), and the soluble guanylate cyclase (sGC) stimulator BAY 41-8543]. RESULTS In all the LUT tissues, PDE5 was immunolocalized in blood vessels and in muscular fibres, but not in epithelium. PDE5 mRNA expression was higher in urethra and bladder than in prostate SMC. The antiproliferative effect of Sp-8-Br-PET-cGMPS was similar in all LUT SMC. In prostatic SMC, SNP and BAY 41-8543 show a dose-dependent antiproliferative effect that resulted marginally enhanced by vardenafil. Conversely, in urethra and bladder SMC the antiproliferative effect of SNP and BAY 41-8543 was lower than in prostatic SMC, but it was significantly enhanced by vardenafil. In urethral and bladder cells vardenafil half-maximal response inhibiting concentration was in the subnanomolar range, whereas in prostate cells it resulted significantly higher. CONCLUSIONS The highest expression and biological activity of PDE5 was found in bladder. However, a consistent PDE5 expression and activity was also found in prostatic urethra. In contrast, the prostate gland showed the lowest PDE5 abundance and cultures derived from this tissue were less sensitive to vardenafil.

Which patients with sexual dysfunction are suitable for testosterone replacement therapy

According to all the consensus and statements of the major societies, hypogonadism should be considered a medical problem, termed late onset hypogonadism (LOH) or testosterone deficiency syndrome (TDS), only when symptoms are present. One of the most common symptoms of LOH/TDS is sexual dysfunction (SD). The main purpose of this review is to discuss the role of testosterone (T) in men’s sexual function, including epidemiology, pathophysiology, diagnostic procedures, and treatment efficacy in patients affected by erectile dysfunction (ED). The prevalence of hypogonadism in men with ED ranges from 1.7% to 35%. In ED patients, hypogonadism is often associated with reduced sexual desire and nocturnal penile erections, while association with sex-induced erection is less evident. This is because T regulates not only cyclic guanosine monophosphate (cGMP) formation, through nitric oxide synthase (NOS) stimulation, but also its catabolism, through phosphodiesterase-5 (PDE5) activity. The androgen-dependent PDE5 expression could explain the reduced effectiveness of PDE5 inhibitors (PDE5i) in the treatment of erectile dysfunction in hypogonadal patients. Accordingly, T substitution in these subjects restores responsiveness to PDE5i. Recognising hypogonadism in patients with ED is essential in order to appropriately treat the disease. However, suspecting LOH/TDS in SD patients is not an easy task. Recently published structured inventories, such as ANDROTEST, might help physicians to recognize hypogonadism and to further pursue its appropriate diagnosis and treatment.

Atorvastatin Ameliorates Sildenafil-Induced Penile Erections in Experimental Diabetes by Inhibiting Diabetes-Induced RhoA/Rho-Kinase Signaling Hyperactivation

INTRODUCTION One of the proposed mechanisms responsible for diabetes-related erectile dysfunction (ED) is overactivity of RhoA/ROCK signaling, as seen in experimental models of chemical diabetes. AIM Because statins may interfere with RhoA/Rho-kinase (ROCK) signaling through the reduction of geranyl-geranyl pyrophosphate (GGPP), required for RhoA activation, we investigated whether atorvastatin ameliorated diabetes-related ED. METHODS Streptozotocin-induced (8 weeks) diabetic rats and alloxan-induced (8 weeks) diabetic rabbits received atorvastatin (5 mg/kg daily) for the last 2 weeks. In vitro contractility studies were conducted in the rabbit model. In the rat model, sildenafil effect on electrical stimulation (ES)-induced erection was investigated. Atorvastatin action was also analyzed using human fetal penile smooth muscle cells (hfPSMCs) exposed to low (5 mM), high (22 mM), and very high (40 mM) glucose. MAIN OUTCOME MEASURES Atorvastatin effect on hyperglycemia-induced RhoA/ROCK signaling was evaluated using the ROCK inhibitor Y-27632 in both animal models and by analyzing functional effects downstream to RhoA activation in hfPSMCs. RESULTS In both diabetic models, atorvastatin did not affect glycemia, lipid plasma levels, and the hypogonadal state. In diabetic rats, atorvastatin ameliorated the erectile response to the ES of the cavernous nerve and normalized sildenafil effect on erectile function, strongly decreased by diabetes. In penile tissue from diabetic animals, atorvastatin completely restored the diabetes-induced hypersensitivity to Y-27632 and prevented RhoA membrane translocation/activation. In hfPSMCs, high glucose significantly increased not only membrane RhoA expression, but also ROCK activity (increased phosphorylation of the ROCK substrate myosin phosphatase target subunit 1) and several RhoA-dependent functions such as proliferation, migration, and smooth muscle-related gene expression. Atorvastatin restored all the high-glucose-induced effects, an action specifically reverted by GGPP. CONCLUSION Atorvastatin improves diabetes-related ED and restores sildenafil responsiveness, most probably by inhibiting RhoA/ROCK signaling, which underlies several high-glucose-induced derangements in penile smooth muscle cell commitment.

Antiinflammatory effect of androgen receptor activation in human benign prostatic hyperplasia cells

Progression of benign prostatic hyperplasia (BPH) involves chronic inflammation and immune dysregulation. Preclinical studies have demonstrated that prostate inflammation and tissue remodeling are exacerbated by hypogonadism and prevented by testosterone supplementation. We now investigated whether, in humans, hypogonadism was associated with more severe BPH inflammation and the in vitro effect of the selective androgen receptor agonist dihydrotestosterone (DHT) on cultures of stromal cells derived from BPH patients (hBPH). Histological analysis of inflammatory infiltrates in prostatectomy specimens from a cohort of BPH patients and correlation with serum testosterone level was performed. Even after adjusting for confounding factors, hypogonadism was associated with a fivefold increased risk of intraprostatic inflammation, which was also more severe than that observed in eugonadal BPH patients. Triggering hBPH cells by inflammatory stimuli (tumor necrosis factor α, lipopolysaccharide, or CD4(+)T cells) induced abundant secretion of inflammatory/growth factors (interleukin 6 (IL6), IL8, and basic fibroblast growth factor (bFGF)). Co-culture of CD4(+)T cells with hBPH cells induced secretion of Th1 inducer (IL12), Th1-recruiting chemokine (interferon γ inducible protein 10, IP10), and Th2 (IL9)- and Th17 (IL17)-specific cytokines. Pretreatment with DHT inhibited NF-κB activation and suppressed secretion of several inflammatory/growth factors, with the most pronounced effects on IL8, IL6, and bFGF. Reduced inflammatory cytokine production by T-cells, an increase in IL10, and a significant reduction of T cells proliferation suggested that DHT exerted a broad anti inflammatory effect on testosterone cells [corrected]. In conclusion, our data demonstrate that DHT exerts an immune regulatory role on human prostatic stromal cells, inhibiting their potential to actively induce and/or sustain autoimmune and inflammatory responses.

Acute vardenafil administration improves bladder oxygenation in spontaneously hypertensive rats.

INTRODUCTION In human bladder, phosphodiesterase type 5 (PDE5) is present not only in the muscular wall but also in the vascular beds, suggesting a role for PDE5 inhibitors in favoring bladder blood flow and tissue oxygenation. AIM To investigate whether acute administration of vardenafil could affect bladder oxygenation in spontaneously hypertensive rats (SHR), an animal model of naturally occurring overactive bladder. MAIN OUTCOME MEASURES The effect of vardenafil on hypoxia-induced alterations was studied in vivo in SHR by acute dosing (10 mg/kg, 90 minutes before sacrifice) and in vitro in human bladder smooth muscle cells (hBCs). METHODS Bladder oxygenation was detected using the hypoxyprobe immunostaining. The expression of some hypoxia markers (vascular endothelial growth factor [VEGF] and endothelin-1 type B [ETB] receptor) was also evaluated by immunohistochemistry and Western blot. Gene expression in hBC was quantified by real-time reverse transcription-polymerase chain reaction. RESULTS Rat bladder PDE5 immunopositivity was detected in the muscular wall and in the endothelial and smooth muscle cells of blood vessels. In SHR bladder, a significant increase of hypoxic cells, VEGF, and ETB expression was observed when compared with their normotensive counterpart Wistar Kyoto rats (WKY). Vardenafil treatment dramatically decreased hypoxyprobe staining, as well as VEGF and ETB expression in SHR bladder up to WKY level. Accordingly, in SHR bladder, vardenafil administration significantly blunted relaxation induced by the selective ETB agonist IRL-1620. In hBCs, experimental hypoxia significantly induced gene expression of hypoxia markers (carbonic anhydrase IX and VEGF), which was not changed by simultaneous treatment with vardenafil. Conversely, the hypoxia-related induction of smooth muscle-specific genes (alphaSMA, SM22alpha, and desmin) was significantly reduced by vardenafil. CONCLUSIONS SHR showed bladder hypoxia which was significantly reduced by acute vardenafil treatment. Thus, besides relaxing muscular wall, PDE5 inhibition may positively affect urinary vesicle blood perfusion.

Vardenafil modulates bladder contractility through cGMP-mediated inhibition of RhoA/Rho kinase signaling pathway in spontaneously hypertensive rats.

INTRODUCTION Phosphodiesterase type 5 inhibitors (PDE5i), the most widely used drugs for erectile dysfunction, could also improve lower urinary tract symptoms, essentially due to overactive bladder (OAB), a condition hypothesized to be a result of an increased RhoA/Rho-kinase (ROCK) signaling. Phosphorylation/inactivation of RhoA by cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG) activity has been described in vascular smooth muscle. AIM The aim of this paper was to investigate whether vardenafil-induced cGMP accumulation reduces RhoA/ROCK signaling in bladder. METHODS Spontaneously hypertensive rats (SHRs), a strain genetically prone to develop OAB, were treated with vardenafil (10 mg/kg/day) for 2 weeks. Wistar-Kyoto rats (WKY) were used as control. In vitro experiments were performed in human bladder smooth muscle cells (hBCs). MAIN OUTCOME MEASURES Urodynamic parameters were registered in vivo in anesthetized WKY and SHRs. RhoA/ROCK activity in bladder was evaluated by molecular and functional studies in tissues and cells. RESULTS The intercontraction interval and bladder capacity, and were decreased in SHRs and restored by vardenafil. The in vitro relaxant effect of the ROCK inhibitor Y-27632 was higher in bladder strips from SHR than from WKY and reduced by vardenafil. Nomega-nitro-L-arginine-methyl-ester (a NO-synthase inhibitor, 40 mg/kg/day during the last week of the 2-week treatment with vardenafil) partially antagonized vardenafil effect on Y-27632 responsiveness. Vardenafil prevented RhoA membrane translocation/activation, decreased ROCK activity, and increased cGMP levels in vivo (rat) and in vitro (hBCs). Exposing hBCs to vardenafil increased Ser(188) RhoA phosphorylation, to the same extent as the PDE5-insensitive PKG agonist Sp-8-Br-PET-cGMP. Moreover, vardenafil inhibited several RhoA-dependent functions in hBCs, including smooth muscle gene transcription and endothelin-1-induced migration. These effects were reverted by the PKG inhibitor KT 5823, further suggesting a cGMP/PKG-dependency. In hBCs, vardenafil was active in the low nanomolar range. CONCLUSIONS This is the first study demonstrating that the effect of vardenafil on OAB could be partially determined by a cGMP-dependent RhoA/ROCK signaling inhibition.

Farnesoid X Receptor Activation Improves Erectile Function in Animal Models of Metabolic Syndrome and Diabetes

INTRODUCTION The farnesoid X receptor (FXR) is critically involved in the regulation of the hepato-biliary system. Recent data suggest a role for FXR in modulating other metabolic pathways and vascular function. AIM To investigate whether long-term administration of the selective FXR agonist INT-747 ameliorates erectile function, we tested it in two animal models of metabolic derangements: a rabbit model of high-fat diet (HFD)-induced metabolic syndrome (MetS) and a rat model of streptozotocin (STZ)-induced type 1 diabetes. METHODS HFD rabbit or STZ rats with or without chronic INT-747 dosing (10 mg/kg/day for 12 weeks). INT-747 addition to rabbit penile smooth muscle cells (rpSMCs). MAIN OUTCOME MEASURE Effects of INT-747 on metabolic features and erectile function in animal models and clarification of mechanism of action in isolated cells. RESULTS INT-747 dosing normalized visceral adiposity and glucose intolerance in HFD rabbits. INT-747 increased penile FXR expression and partially restored endothelial nitric oxide synthase and dimethylarginine dimethylaminohydrolase 1 expression as well as impaired nitric oxide (NO)-dependent relaxation (improved responsiveness to acetylcholine and electrical field stimulation). INT-747 was also effective in regulating NO downstream events, as shown by increased sodium nitroprusside-induced relaxation. Because phosphodiesterase type 5 and protein kinase G (PKG) were unaltered by INT-747, we analyzed the calcium-sensitizing RhoA/ROCK pathway. HFD increased, and INT-747 normalized, RhoA membrane translocation/activation. RhoA/ROCK signaling inhibition by INT-747 was confirmed in rpSMCs by confocal microscopy, MYPT1-phosphorylation, cytoskeleton remodeling, cell migration, and smooth muscle-related genes expression. In STZ rats, FXR penile expression was not altered but was significantly upregulated by INT-747 dosing. In this model, INT-747 improved penile erection induced by electrical stimulation of cavernous nerve and hypersensitivity to intracavernous injection of a ROCK-inhibitor, Y-27632, without improving hyperglycemia. CONCLUSION In HFD rabbits, INT-747 dosing improved glucose sensitivity and MetS-associated erectile dysfunction, via upregulation of NO transmission and inhibition of RhoA/ROCK pathway. In STZ rats, INT-747 restored in vivo penile erection and sensitivity to ROCK inhibition, independently of effects on glycemia.