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Tadalafil Effect on Metabolic Syndrome-Associated Bladder Alterations: An Experimental Study in a Rabbit Model Linda Vignozzi, MD, PhD,* Sandra Filippi, PhD,† Paolo Comeglio, PhD,* Ilaria Cellai, PhD,* Annamaria Morelli, PhD,‡ Elena Maneschi, PhD,* Erica Sarchielli, PhD,‡ Mauro Gacci, MD,§ Marco Carini, MD,§ Gabriella Barbara Vannelli, MD,‡ and Mario Maggi, MD* *Sexual Medicine and Andrology Unit, Department of Experimental and Clinical Biomedical Sciences, University of Florence, Florence, Italy; †Interdepartmental Laboratory of Functional and Cellular Pharmacology of Reproduction, Department of Experimental and Clinical Biomedical Sciences and Department of NEUROFARBA, University of Florence, Florence, Italy; ‡Section of Anatomy and Histology, Department of Experimental and Clinical Medicine, University of Florence, Florence, Italy; §Urology Unit, Department of Experimental and Clinical Medicine, University of Florence, Florence, Italy DOI: 10.1111/jsm.12478
ABSTRACT
Introduction. Metabolic syndrome (MetS) and lower urinary tract symptoms (LUTS) are often associated. Bladder detrusor hyper-contractility—a major LUTS determinant—is characterized by increased Ras homolog gene family, member A/Rho-associated protein kinase (RhoA/ROCK) signaling, which is often upregulated in MetS. Aim. This study investigated the effects of tadalafil dosing on RhoA/ROCK signaling in bladder, in a rabbit model of high-fat diet (HFD)-induced MetS. Methods. Adult male rabbits feeding a HFD for 12 weeks. A subset of HFD animals was treated with tadalafil (2 mg/kg/day, 1 week: the last of the 12 weeks) and compared with HFD and control (feeding a regular diet) rabbits. Main Outcome Measures. In vitro contractility studies to evaluate the relaxant effect of the selective ROCK inhibitor, Y-27632, in carbachol precontracted bladder strips. Evaluation of RhoA activation by its membrane translocation. Immunohistochemistry for ROCK expression has been performed to evaluate ROCK expression in bladder from the different experimental groups. mRNA expression of inflammation, pro-fibrotic markers by quantitative RT-PCR has been performed to evaluate the effect of tadalafil on MetS-induced inflammation and fibrosis within the bladder. The in vitro effect of tadalafil on RhoA/ROCK signaling in bladder smooth muscle cells was evaluated by using chemotaxis assay. Results. Bladder strips from HFD rabbits showed hyper-responsiveness to Y-27632, indicating RhoA/ROCK overactivity in HFD bladder compared with matched controls. Accordingly, the fraction of activated (translocated to the membrane) RhoA as well as ROCK expression are increased in HFD bladder. Tadalafil dosing normalized HFD-induced bladder hypersensitivity to Y-27632, by reducing RhoA membrane translocation and ROCK overexpression. Tadalafil dosing reduced mRNA expression of inflammatory, pro-fibrotic, and hypoxia markers. A direct inhibitory effect of tadalafil on RhoA/ROCK signaling in bladder smooth muscle cell was demonstrated by using chemotaxis assay. Pre-treatment with tadalafil inhibited both basal and PDGF-induced migration of bladder smooth muscle cells. Conclusions. Tadalafil dosing reduced RhoA/ROCK signaling and smooth muscle overactivity in an animal model of MetS-associated bladder alterations. Our findings suggest a novel mechanism of action of tadalafil in alleviating LUTS in MetS patients. Vignozzi L, Filippi S, Comeglio P, Cellai I, Morelli A, Maneschi E, Sarchielli E, Gacci M, Carini M, Vannelli GB, and Maggi M. Tadalafil effect on metabolic syndrome-associated bladder alterations: An experimental study in a rabbit model. J Sex Med 2014;11:1159–1172. Key Words. LUTS; Metabolic Syndrome; Tadalafil; RhoA/ROCK Signaling; OAB; Rabbit Bladder; Prostatic Enlargement
Funding: The study was supported by PRIN (Programmi di ricerca di Rilevante InteresseNazionale) funds by the Italian Minister of University, Research and Instruction (prot number:2009WLNXNT_002), and by FIRB (Programma Futuro in Ricerca) funds by the Italian Minister of University, Research and Instruction (prot number: RBFR10VJ56_002).
© 2014 International Society for Sexual Medicine
J Sex Med 2014;11:1159–1172
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T
he historical, simplistic, causal relationship among prostatic enlargement and lower urinary tract symptoms (LUTS) has been challenged nowadays by the recognition of an incomplete overlap between voiding symptoms and urodynamic markers of prostatic conditions [1]. Indeed, many men with benign prostatic hyperplasia (BPH) do not develop LUTS, and LUTS may occur independently of BPH [2]. Additionally, epidemiological evidence indicates that these bothersome urinary symptoms are not gender-specific, having a high prevalence also in women [3]. Hence, although an enlarged prostate can contribute to the onset of LUTS, other extra-prostatic factors are supposed to be equally important in LUTS determinism [2]. Bladder dysfunction— characterized by detrusor overactivity and/or overactive bladder (OAB)—is considered one of the main extra-prostatic LUTS determinants in both genders [4]. The pathogenesis of LUTS, including OAB, is complex and largely unknown. Increasing evidence links a number of age-related disorders, including metabolic and cardiovascular disorders, with the pathogenesis of LUTS [5]. In particular, it is epidemiologically well-established that metabolic syndrome (MetS) is not only frequently associated with, but also poses an increased risk for, the development and progression of LUTS in men. Several MetS features, including abdominal obesity [6], hypertension [7–9], hyperglycemia and type 2 diabetes mellitus (T2DM) [8,10], low high-density lipoprotein cholesterol and increased triglycerides plasma level [10–12], have all been related to LUTS. Therefore, recent investigations were aimed at understanding the potential pathogenic mechanisms underlying the association between MetS and LUTS. Animal models have provided a great deal of information. In a genetic mouse model of T2DM/obesity, both bladder dysfunction and prostatitis were described [13]. However, monogenic alterations related to metabolic derangements have rarely associated to human LUTS [14]. Bladder dysfunctions, including overactivity [15] or fibrosis [16], were often observed in hyperlipidemic rats and were associated with pelvic ischemia and smooth muscle atrophy. In male rats fed a fructose enriched diet, unstable bladder contractions and increased concentrations of M2,3muscarinic receptors, suggestive of bladder overactivity [17], were reported. Similar results J Sex Med 2014;11:1159–1172
Vignozzi et al. were observed in rats with streptozotocininduced diabetes [18,19]. We recently developed a non-genomic animal model of MetS, feeding adult male rabbits a highfat diet (HFD) for 12 weeks [20–25]. This animal model essentially recapitulates the human phenotype, showing, along with the classical features of MetS (hyperglycemia, impaired glucose tolerance, dyslipidemia, hypertension, increased visceral obesity, and hypogonadism), also severe alterations at the low urinary tract (LUT) level. In MetS rabbits, we characterized a prostatitis-like syndrome [22] and peculiar bladder alterations, including fibrosis, hypoxia, and inflammation [23], confirming that MetS could affect the whole of the LUT. More important, bladder from MetS animals showed increased signaling of Ras homolog gene family, member A (RhoA)/Rhoassociated protein kinase (ROCK) [23], a pathway deeply involved in detrusor hyper-contractility and bladder dysfunction, as often observed in human LUTS [4]. We previously tested the effect of chronic (2 mg/kg/day, for 12 weeks; preventive) or acute (2 mg/kg/day, for the last of the 12 weeks; curative) tadalafil dosing in HFD animals, focusing on prostate alterations [25]. Interestingly, we found that not only chronic (i.e., preventive), but also acute (i.e., curative), tadalafil dosing in HFD animals was effective in reducing MetS-associated prostatic abnormalities, including fibrosis and inflammation [25]. Aims
This study was aimed at investigating the effect of acute (curative) tadalafil dosing on MetSassociated increased signaling of RhoA/ROCK in bladder. Materials and Methods
MetS Rabbit Model The HFD-induced rabbit model of MetS has been obtained as previously described [20]. Male New Zealand White rabbits (Charles River, Calco, Lecco, Italy), weighing about 3 kg, were individually caged, under standard conditions, in a temperature- and humidity-controlled room, on a 12-hour light/dark cycle. Water and food were unrestricted throughout the study. After 1 week of standard rabbit diet, animals were randomly numbered and assigned to different groups: control (N = 43) or treatment group
Tadalafil Inhibits Bladder RhoA/ROCK Activity (N = 51). The control group continued to receive a standard diet (regular diet, RD), while the treatment group fed a HFD constituted by 0.5% cholesterol and 4% peanut oil (HFD rabbits) for 12 weeks, as previously described [20]. A subset of HFD rabbits was treated with tadalafil (2 mg/ kg/day, administered ad libitum in water drank in the entire day, 1 week; N = 14). Tadalafil powder, provided by Eli Lilly and Company (Indianapolis, IN, USA), was dissolved in dimethyl sulfoxide (10 mg/mL), then first diluted in ethanol (1:100) and finally in drinking water (28 mL). Mean arterial blood pressure was measured by using a polyethylene catheter inserted into a femoral artery at week 12, after pentobarbital (45 mg/kg) sedation as previously described [20]. After 12 weeks of treatment, rabbits were sacrificed with a lethal dose of pentobarbital, and specimens of bladder, seminal vesicles, prostates, and testis were harvested and processed for subsequent analyses. Biochemical and hormonal serum analyses were performed as previously described [20]. Animal handling will be complied with the Institutional Animal Care and Use Committee of the University of Florence, Florence, Italy, in accordance to the Italian Ministerial Law #116/92.
Measurement of Cholesterol,Triglycerides, Glycemia, Testosterone, and 17β Estradiol in Rabbits Blood samples for glucose, total cholesterol, triglycerides, testosterone (T), and 17β estradiol analyses were obtained from the animals via marginal ear vein at week 0 (baseline) and at week 12 in all groups, early in the morning after an overnight fasting. Plasma cholesterol, triglycerides, and glucose levels were measured by using an Automated System (ADVIA 2004 Siemens Chemistry System; Siemens Science Medical Solution Diagnostic, Tarrytown, NY, USA). Plasma T and 17β estradiol levels were measured by using an automated chemiluminescence system (Immunolite 2000 Siemens, Siemens Healthcare Diagnostics, Deerfield, IL, USA), after appropriate extraction. For extraction, samples were mixed with four volumes of diethyl ether for 15 minutes, centrifuged for 5 minutes at 2,000 rpm, and the aqueous phase frozen in dry ice. The organic phase was recovered, evaporated to dryness under a nitrogen stream, and reconstituted in the assay buffer. Oral Glucose Tolerance Test Oral glucose tolerance test (OGTT) was performed in accordance with the published method
1161 [20]. After an overnight fasting, a 50% glucose solution was orally administered to the animals, at a dose of 1.5 g/kg. Blood samples were collected via the marginal ear vein before and 15, 30, and 120 minutes after glucose loading. Samples were stored on ice and centrifuged at 3.000 rpm for 20 minutes at 4°C to obtain plasma. Plasma glucose was measured as described above. The incremental area under the curve (iAUC), was calculated by using the GRAPHPAD PRISM software version 4.0 for Windows (San Diego, CA, USA).
In Vitro Contractility Studies After harvesting bladder samples were immediately placed and maintained in cold Krebs solution and “in vitro” contractility experiments were performed in organ chambers, as previously described [26]. The maximal tonic tension elicited by high potassium salt solution (KCl, 80 mM) was taken as 100%, and the contractile effect induced by different concentrations of carbachol was referred to this value. The degree of contractile response induced by 10 μm carbachol was taken as 100% and the relaxant effect induced by Y-27632 was referred to this value. Drug cumulative concentrations were added to the bath, at 7-minute intervals, in order to obtain concentration-relaxant effect curves. Preparation of Membrane/Cytosolic Fractions and Western Blot Analysis for RhoA Activation Assay For protein extraction from bladder samples, the frozen tissues were ground in liquid nitrogen. Membrane and cytosolic fractions were prepared using the ProteoExtract subcellular proteome extraction kit (Calbiochem, Darmstadt, Germany), according to the manufacturer’s instructions. Protein extracts were quantified with the bicinchoninic acid (BCA) reagent (Pierce, Rockford, IL, USA). Western blot analysis with antiRhoA antibody (1:500; Santa Cruz Biotechnology, Dallas, TX, USA) was performed as previously described [23,26]. Densitometry analysis of band intensity was performed using Photoshop 5.5 software (Adobe Systems Inc. Italia srl, Agrate Brianza, Italy). RNA Extraction and Quantitative RT-PCR RNA isolation from tissue was performed as described previously [22,23]. Specific primers for all the target genes were previously described [20,22–25]. The expression of the 18S ribosomal RNA subunit was quantified with a pre-developed assay (Applied Biosystems, Foster City, CA, USA). J Sex Med 2014;11:1159–1172
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Immunohistochemistry Immunohistochemical studies were carried out as previously described [21]. Briefly, rabbit bladder sections (fixed in Bouin’s solution and embedded in paraffin) were incubated for 1 hour in 2% fetal bovine serum (FBS) in phosphate buffered saline (PBS, Sigma-Aldrich, Milan, Italy), to block nonspecific antibody binding. Sections were then incubated overnight at 4°C with the ROCK1 (1:1,000, Santa Cruz Biotechnology) primary antibody and then with the corresponding specific immunoglobulin peroxidase conjugated for 30 minutes (dilution 1:1,000). Demonstration of peroxidase activity and controls for antiserum specificity were performed as previously described [21]. Rabbit Bladder Smooth Muscle Cell Cultures Rabbit bladder smooth muscle cells (rbBSMCs) were isolated and characterized as previously described [23]. Briefly, bladder tissue samples were digested with 2 mg/mL bacterial collagenase type IV (Worthington Biochemical Corporation, Lakewood, NJ, USA) for 1 hour at 37°C, then fragments were extensively washed in PBS and cultured in Dulbecco’s modified Eagle medium (DMEM)/ Ham’s F-12 medium 1:1; Sigma-Aldrich) supplemented with 10% FBS) (Sigma-Aldrich), antibiotics (penicillin, 100 IU/mL; streptomycin 100 mg/mL; Sigma-Aldrich), and 1 mg/mL amphotericin B (Sigma-Aldrich) in a fully humidified atmosphere of 95% air/5% CO2. rbBSMCs began to emerge within 2 days and were used within the seventh passage. Characterization of cells showed positive staining for desmin and α-smooth muscle actin (α-SMA) and negative staining for cytokeratin (not shown). The percent of stained cells over the total number in 10 fields for each slide was >95% for both desmin and α-SMA. To test the capacity of tadalafil to reduce the mRNA expression of RhoA-dependent genes in rbBSMC cells primed with lipopolysaccharide (LPS, 100 ng/mL for 5 hours) with or without pretreatment with tadalafil (100 nM, 24 hours). Rabbit Smooth Muscle Cell Migration rbBSMC migration was studied in a P48 multiwell Boyden’s chamber (Nuclepore Inc., Pleasanton, CA, USA) as previously described [23]. Briefly, rbBSMCs were treated or not with tadalafil (100 nM, for 24 hours). Cells in serum free culture medium, in the absence of any stimulus, were taken as control for the rbBSMC basal migration. Platelet-derived growth factor (PDGF; 10 ng/mL, J Sex Med 2014;11:1159–1172
Vignozzi et al. for 5 hours) was added to the lower wells, alone or adding C3 exoenzyme (1 μg/mL, for 5 hours), a known selective inhibitor of RhoA, in the upper well. In the second set of experiments, rbBSMC migration was evaluated for 5 hours in the absence or in the presence of PDGF (10 ng/mL) in multiwell Boyden chamber. The effect of pretreatment with tadalafil (100 nM for 24 hours), with or without co-treatment with the protein kinase G (PKG) inhibitor 11H-2,7b,11a-trizadibenzo-(a,g)cycloocta-(c,d,e)-trinden-1-one (KT5823) (10 nM) or the membrane-permeant and phosphodiesterase (PDE)-resistant PKG agonist, Sp-8-Br-PET cyclic guanosine monophosphate (cGMP) (8Br-cGMP, 10 microM), was also evaluated. Methanol-fixed cells were stained with Diff-Quick (DADE Behring AG, Düdingen, Switzerland), and cell migration was measured by microscopic evaluation of the number of cells that moved across the filter. The number of migrated cells over the total number in 10 fields for each well was counted. Each experimental point was replicated at least five times, in three independent experiments.
Statistical Analysis Results are expressed as mean ± standard error of the mean (SEM) for n experiments as specified. The statistical analysis was performed with a oneway anova test followed by the Tukey–Kramer post hoc analysis in order to evaluate differences between the groups, and P < 0.05 was considered significant. When data were non-normally distributed, statistical differences were calculated with Kruskal–Wallis test and Mann–Whitney U-test was used for comparisons between groups. Correlations were assessed using Spearman’s method and statistical analysis was performed with the Statistical Package for the Social Sciences (SPSS, Inc., Chicago, IL, USA) for Windows 20.0. Stepwise multiple linear regressions were applied for multivariate analysis, whenever appropriate. Halfmaximal excitatory (EC50) or inhibitory (IC50) concentration values were calculated using the computer program ALLFIT [27], and reported as −logEC50 (pEC50) or −logIC50 (pIC50). IC50s potency ratio was calculated as the ratio between IC50 value in the examined experimental groups, and reported with the relative confidence interval. Main Outcome Measures In vitro contractility studies to evaluate the relaxant effect of the selective ROCK inhibitor,
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Tadalafil Inhibits Bladder RhoA/ROCK Activity Y-27632, in carbachol precontracted bladder strips. Evaluation of RhoA activation by its membrane translocation. Immunohistochemistry for ROCK expression. mRNA expression of inflammation, pro-fibrotic markers by quantitative RT-PCR. The in vitro effect of tadalafil on RhoA/ ROCK signaling in bladder smooth muscle cells was evaluated by using chemotaxis assay. Results
Metabolic, Hormonal, and Biochemical Parameters Metabolic, cardiovascular, and biochemical effects of feeding rabbits with a HFD for 12 weeks are reported in Table 1. As previously demonstrated [20–25], HFD induced features of a MetS-like syndrome (increased visceral fat, increased blood pressure, high cholesterol and triglyceride plasma levels, hyperglycemia and glucose intolerance, as demonstrated by the area under the curve of OGTT). Overt hypogonadism was also present, as demonstrated by combined low testosterone and decreased seminal vesicles, testis and prostate weights. A parallel increase of 17β-estradiol plasma
Table 1
level was observed in HFD rabbits. Confirming previous data [25], tadalafil treatment (2 mg/kg/ day, during the last out of 12 weeks, induced a significant reduction of visceral adipose tissue accumulation and triglyceride levels, without affecting testosterone or 17β estradiol plasma levels (Table 1).
Effect of Tadalafil on Carbachol-Induced Bladder Contractility To investigate the effect of HFD on bladder contractility, we first studied in vitro contractile response to increasing concentration of a muscarinic receptor agonist, carbachol (0.001–10 μm), in control and HFD rabbit bladder strips. As shown in Figure 1A, carbachol induced a higher dose dependent increase in tension in HFD than in control. Mathematical modeling of sigmoidal relationships (program ALLFIT, [27]) indicates that neither EC50s nor slopes (Hill coefficient) were different (shared –logEC50 = 5.89 ± 0.16, shared slope = 0.73 ± 0.17). In contrast, Emaxs were different: control Emax = 155.8 ± 15.2 and HFD Emax = 220.3 ± 20.1 (P = 0.017).
Metabolic and hormonal parameters in experimental rabbit
Total body weight (g) Baseline Week 12 Blood glucose (g/L) Baseline Week 12 OGTT (iAUC) Week 12 Cholesterol (mg/dL) Baseline Week 12 Triglycerides (mg/dL) Baseline Week 12 MAP (mmHg) Week 12 VAT weight (g) Week 12 Testosterone (nmol/L) Week 12 17β-estradiol (pmol/L) Week 12 Seminal vesicles weight (mg) Week 12 Testis weight (g) Week 12 Prostate weight (g) Week 12
Control (N = 43)
HFD (N = 37)
HFD + tadalafil (N = 14)
3,277.2 ± 36.7 3,923.9 ± 36.5***
3,295 ± 41.4 3,770.5 ± 32.4***
3,498 ± 45.7 3,791.2 ± 72.9**
1.1 ± 0.04 1.21 ± 0.04 149.6 ± 5 36.7 ± 1.97 41.6 ± 2.8 78.5 ± 4.3 92.2 ± 4.2
1.19 ± 0.03 1.96 ± 0.07***°°°
0.99 ± 0.06 1.85 ± 0.06***°°°
224.7 ± 7.1°°°
221.9 ± 13.3°°°
43.6 ± 2 1,451.1 ± 63.6***°°°
35.8 ± 2 1,675.8 ± 168.4***°°°
85.9 ± 4.1 301.7 ± 24.7***°°°
89.5 ± 2.1
133 ± 3.4°°°
37.5 ± 1.5
43.6 ± 1.5°
60.4 ± 3.9 171.6 ± 27.7**°°°∧ 125.6 ± 4.1°°° 32.9 ± 2∧∧
6.9 ± 0.93
1.5 ± 0.31°°°
1.27 ± 0.34°°°
168.7 ± 12.7
307.6 ± 38.4°°°
477 ± 153°°°
715.7 ± 34.7
517.7 ± 24.3°°°
446.9 ± 31.9°°
3.4 ± 0.1 0.6 ± 0.05
3 ± 0.05°°°
2.84 ± 0.08°°°
0.35 ± 0.04°°° ∧
0.46 ± 0.04 ∧∧
∧∧∧
*P < 0.05, **P < 0.01, ***P < 0.001 vs. baseline; °P < 0.05, °°P < 0.01, °°°P < 0.001 vs. control week 12; P < 0.05, P < 0.01, P < 0.001 vs. HFD week 12. Results are reported as mean ± SEM. iAUC = incremental area under the curve of glucose blood level during oral glucose tolerance test (OGTT). MAP = mean arterial pressure; VAT = visceral adipose tissue
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A
B
C
D
Figure 1 Effect of tadalafil on bladder contractility. (A) Effect of increasing concentrations of carbachol in bladder strips from control (closed circles) and HFD (closed squares) rabbits. Contractile response to carbachol is expressed in % (ordinate) of contraction obtained in bladder strips by KCl 80 mM; abscissa: concentrations of carbachol (0.001–10 μM). (B) Effect of carbachol (10 μM) in bladder strips from control (black bar), HFD (white bar) and tadalafil-treated HFD (gray bar) rabbits. Maximal response to KCl (80 mM), before the addition of carbachol, was taken as 100%. The effect of carbachol was evaluated as percentage of this response. Results were obtained from seven independent experiments and reported as mean ± SEM (C) Relaxation elicited by increasing concentrations of Y-27632 (abscissa) in bladder strips precontracted with 10 μM carbachol from control (N = 13 independent experiments; 9 animals), HFD (N = 10 independent experiments; 6 animals), HFD + tadalafil (N = 7 independent experiments; 7 animals) groups. Relaxant response to Y-27632 is expressed in % (ordinate) of contraction obtained with 10 μM carbachol. Each point represents mean ± SEM of n samples for each experimental group. (D) Relationship between the visceral fat weight (abscissa) and the response to Y-27632 (expressed as area under the curve [AUC] ordinate). This relationship are derived from univariate Spearman’s regression analysis and the relative coefficient of correlation (r), level of significance (P) and number of samples (N) are reported within the panel. Control, black circles; HFD, white squares; HFD + tadalafil, black triangles. *P < 0.05, **P < 0.01 vs. control.
We then tested the effect of tadalafil treatment on bladder tension induced by a fixed dose of carbachol (10 μm). This dose of carbachol (10 μm) was selected, based on the present (see Figure 1A) and a previous study [26], as that one eliciting maximal increase in bladder tension. Carbachol (10 μm) induced a significantly higher response in HFD bladder strips than in control ones (P < 0.05). Tadalafil treatment completely normalized carbachol responsiveness (see Figure 1B). J Sex Med 2014;11:1159–1172
Effect of Tadalafil on RhoA/ROCK Pathway in MetS Bladder In bladder strips precontracted with carbachol, we next evaluated the relaxant response to the selective ROCK inhibitor Y-27632 (Figure 1C). The relative IC50 values (−logIC50) and potency ratios were then calculated, using the program ALLFIT [27]. The amplitude of the response to Y-27632 was normalized to maximal carbachol responsiveness (taken as 100%), because it was significantly different among groups (see before). In control
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rabbits, Y-27632 dose-dependently relaxed carbachol-precontracted bladder strips (−logIC50 = 5.25 +/− 0.1) (Figure 1C). HFD induced a highly significant (P < 0.0001 vs. control), leftward shift of the Y-27632 sigmoidal relationship (−logIC50 = 6.7 +/− 0.08; IC50s potency ratio = 28.654 [10.97– 46.34]). This potentiating effect of HFD on responsiveness to Y-27632 was completely normalized (P = 0.2 vs. control) by tadalafil dosing (−logIC50 = 5.11 +/− 0.1; IC50s potency ratio = 0.7 [0.243–1.224]; Figure 1C). When all the experimental groups were considered, at univariate Spearman’s regression analysis, the responsiveness to Y-27632 (expressed as area under the curve) resulted positively related to the presence of MetS (r = 0.450, P < 0.05, not shown). Among MetS factors, visceral fat accumulation (r = 0.552, P < 0.01; see Figure 1D) and blood pressure (r = 0.453, P < 0.05, not shown) were both positively associated with responsiveness to Y-27632.
Effect of Tadalafil on Bladder Expression of RhoA/ROCK System To further investigate the HFD-induced RhoA/ ROCK pathway activation, we analyzed RhoA intracellular distribution (membrane vs. cytosol), in bladder extracts. As shown in Figure 2, membrane fraction of RhoA (which is indicative of its activation) was significantly increased in HFD rabbits, as compared with control (P < 0.05). Tadalafil dosing completely counteracted this effect, thereby causing cytosolic accumulation (P < 0.01 vs. HFD) (Figure 2). Accordingly, mRNA expression of several HFD-induced [23] and RhoA-dependent myofibroblast markers, (such as αSMA; desmin; transgelin, SM22α; ROCK1, [28] was significantly reduced in the bladder of tadalafil-treated rabbits, when compared with HFD group (Figure 3). Tadalafil treatment reduced also RhoA and ROCK2 mRNA expression; however, without reaching statistical significance (Figure 3). Consistent with a positive effect of tadalafil dosing on HFD-induced bladder alterations, also the mRNAs expression of other myofibroblast transdifferentiation/fibrosis (transforming growth factor β, TGFβ1; fibronectin 1, FN1; metallopeptidase inhibitor 2, TIMP2; matrix metallopeptidase 9, MMP9), inflammatory (cytochrome C oxidase subunit II, COX2, interleukin1β, IL1β; toll-like receptor type 2, TLR2; toll-like receptor type 4, TLR4; six-transmembrane protein of prostate 2, STAMP2; lactoferrin, LACT), and hypoxia (carbonic anhydrase IX, CAIX) markers
Figure 2 Effect of tadalafil on RhoA/ROCK pathway in MetS bladder. Intracellular distribution of RhoA evaluated by Western blot analysis with anti-RhoA primary antibody on membrane and cytosolic fractions obtained from bladder tissue of the indicated experimental groups. The bottom panel shows representative immunoblots with anti-RhoA and anti-β-actin, used as loading control. The top panel shows a graphical representation of densitometry data obtained from membrane/cytosol RhoA, calculated as % of control group and expressed as mean ± SEM from three independent experiments. *P < 0.05 vs. control; P < 0.01 vs. HFD.
were significantly reduced by tadalafil treatment (Figure 3). A significant reduction of estrogen receptor α was also observed (Figure 3). Interestingly, the expression of all these genes was positively associated with PDE type 5 (PDE5) mRNA expression in bladder (see Table 2). In addition, PDE5 mRNA expression in bladder from HFD tended to be increased, without, however, reaching statistical significance (data not shown). No significant changes for PDE5 expression were observed in tadalafil-treated HFD group (data not shown). The increased RhoA activation, observed in HFD bladder, was associated with an increased expression of its downstream effector ROCK1, as evaluated by immunohistochemistry. In control rabbit, ROCK1 protein was prominently immunolocalized in the muscular compartment, whereas urothelial layer was negative (Figure 4A). J Sex Med 2014;11:1159–1172
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Figure 3 Effect of tadalafil treatment on mRNA expression of genes related to inflammation, fibrosis, and steroid receptors in rabbit bladder. Bars represents tadalafil-induced variations of bladder genes, compared with HFD. Data were obtained by quantitative RT-PCR, then were expressed as percentage of HFD (HFD, N = 37, HFD + tadalafil, N = 14). *P < 0.05, **P < 0.01 vs. HFD.
HFD induced an increase in ROCK1 immunopositivity (at computer-assisted quantitative analysis P < 0.001 vs. control, Figure 4D), which was more evident in the smooth muscle bundles and blood vessels of sub-urothelium (Figure 4B). These changes were normalized by tadalafil dosing (Figure 4C, D).
In Vitro Effect of Tadalafil in Bladder Smooth Muscle Cells In order to further investigate the effect of tadalafil on RhoA/ROCK signaling, according to functional and immunohistochemical results (see before), we initiated studies on smooth muscle cells from isolated rabbit bladder (rbBSMC), evaluating two RhoA-mediated cellular functions: cell migration (Figure 5A, B) and mRNA expression of RhoA-dependent smooth muscle genes (Figure 5C). Using the Boyden’s chamber technique, we analyzed rbBSMC migration, as a read-out for RhoA/ROCK activation status (Figure 5A–B). Cell migration was studied in basal J Sex Med 2014;11:1159–1172
conditions and after the addition of PDGF, a wellknown stimulator of RhoA-mediated migration. As expected, PDGF (10 ng/mL, for 5 hours) induced a sustained increase in migration of rbBSMC across filters (188.61 ± 7.1% P < 0.0001 vs. control cells). Pretreatment with tadalafil (100 nM, 24 hours) abrogated PDGF-induced rbBSMC chemotaxis (P < 0.0001 vs. PDGF), to an extent similar to that observed with the RhoA selective inhibitor C3 exoenzyme (1 μg/mL; P < 0.0001 vs. PDGF). Interestingly, tadalafil was also able to reduce rbBSMC basal migration (P = 0.0001 vs. control cells; Figure 5). We next studied the molecular mechanism through which tadalafil blunted PDGF-induced migration. Exposing rbBSMC cells to a PDE-insensitive PKG agonist, Sp-8-Br-PET-cGMP, PDGFinduced migration was significantly reduced, to an extent similar to that observed with tadalafil treatment (Figure 5B). The effects of tadalafil was completely reverted by the PKG inhibitor KT 5823 (10 nM) (Figure 5B).
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Tadalafil Inhibits Bladder RhoA/ROCK Activity Table 2 Association between mRNA expression of PDE5 and genes of fibrosis, inflammation, steroid receptors and NO pathway in rabbit bladder
Fibrosis RhoA ROCK1 ROCK2 αSMA SM22α Desmin Vimentin TGFβ1 FN1 TIMP1 TIMP2 MMP2 MMP9 Inflammation IL1β TLR4 RAGE STAMP2 Steroid receptors AR PR GPR30 ERα NO pathway eNOS PDE11
r
P value
N
0.464 0.487 0.585 0.241 0.475 0.373 0.329 0.510 0.367 0.353 0.528 0.277 0.535
Tadalafil Effect on Metabolic Syndrome-Associated Bladder Alterations: An Experimental Study in a Rabbit Model Linda Vignozzi, MD, PhD,* Sandra Filippi, PhD,† Paolo Comeglio, PhD,* Ilaria Cellai, PhD,* Annamaria Morelli, PhD,‡ Elena Maneschi, PhD,* Erica Sarchielli, PhD,‡ Mauro Gacci, MD,§ Marco Carini, MD,§ Gabriella Barbara Vannelli, MD,‡ and Mario Maggi, MD* *Sexual Medicine and Andrology Unit, Department of Experimental and Clinical Biomedical Sciences, University of Florence, Florence, Italy; †Interdepartmental Laboratory of Functional and Cellular Pharmacology of Reproduction, Department of Experimental and Clinical Biomedical Sciences and Department of NEUROFARBA, University of Florence, Florence, Italy; ‡Section of Anatomy and Histology, Department of Experimental and Clinical Medicine, University of Florence, Florence, Italy; §Urology Unit, Department of Experimental and Clinical Medicine, University of Florence, Florence, Italy DOI: 10.1111/jsm.12478
ABSTRACT
Introduction. Metabolic syndrome (MetS) and lower urinary tract symptoms (LUTS) are often associated. Bladder detrusor hyper-contractility—a major LUTS determinant—is characterized by increased Ras homolog gene family, member A/Rho-associated protein kinase (RhoA/ROCK) signaling, which is often upregulated in MetS. Aim. This study investigated the effects of tadalafil dosing on RhoA/ROCK signaling in bladder, in a rabbit model of high-fat diet (HFD)-induced MetS. Methods. Adult male rabbits feeding a HFD for 12 weeks. A subset of HFD animals was treated with tadalafil (2 mg/kg/day, 1 week: the last of the 12 weeks) and compared with HFD and control (feeding a regular diet) rabbits. Main Outcome Measures. In vitro contractility studies to evaluate the relaxant effect of the selective ROCK inhibitor, Y-27632, in carbachol precontracted bladder strips. Evaluation of RhoA activation by its membrane translocation. Immunohistochemistry for ROCK expression has been performed to evaluate ROCK expression in bladder from the different experimental groups. mRNA expression of inflammation, pro-fibrotic markers by quantitative RT-PCR has been performed to evaluate the effect of tadalafil on MetS-induced inflammation and fibrosis within the bladder. The in vitro effect of tadalafil on RhoA/ROCK signaling in bladder smooth muscle cells was evaluated by using chemotaxis assay. Results. Bladder strips from HFD rabbits showed hyper-responsiveness to Y-27632, indicating RhoA/ROCK overactivity in HFD bladder compared with matched controls. Accordingly, the fraction of activated (translocated to the membrane) RhoA as well as ROCK expression are increased in HFD bladder. Tadalafil dosing normalized HFD-induced bladder hypersensitivity to Y-27632, by reducing RhoA membrane translocation and ROCK overexpression. Tadalafil dosing reduced mRNA expression of inflammatory, pro-fibrotic, and hypoxia markers. A direct inhibitory effect of tadalafil on RhoA/ROCK signaling in bladder smooth muscle cell was demonstrated by using chemotaxis assay. Pre-treatment with tadalafil inhibited both basal and PDGF-induced migration of bladder smooth muscle cells. Conclusions. Tadalafil dosing reduced RhoA/ROCK signaling and smooth muscle overactivity in an animal model of MetS-associated bladder alterations. Our findings suggest a novel mechanism of action of tadalafil in alleviating LUTS in MetS patients. Vignozzi L, Filippi S, Comeglio P, Cellai I, Morelli A, Maneschi E, Sarchielli E, Gacci M, Carini M, Vannelli GB, and Maggi M. Tadalafil effect on metabolic syndrome-associated bladder alterations: An experimental study in a rabbit model. J Sex Med 2014;11:1159–1172. Key Words. LUTS; Metabolic Syndrome; Tadalafil; RhoA/ROCK Signaling; OAB; Rabbit Bladder; Prostatic Enlargement
Funding: The study was supported by PRIN (Programmi di ricerca di Rilevante InteresseNazionale) funds by the Italian Minister of University, Research and Instruction (prot number:2009WLNXNT_002), and by FIRB (Programma Futuro in Ricerca) funds by the Italian Minister of University, Research and Instruction (prot number: RBFR10VJ56_002).
© 2014 International Society for Sexual Medicine
J Sex Med 2014;11:1159–1172
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T
he historical, simplistic, causal relationship among prostatic enlargement and lower urinary tract symptoms (LUTS) has been challenged nowadays by the recognition of an incomplete overlap between voiding symptoms and urodynamic markers of prostatic conditions [1]. Indeed, many men with benign prostatic hyperplasia (BPH) do not develop LUTS, and LUTS may occur independently of BPH [2]. Additionally, epidemiological evidence indicates that these bothersome urinary symptoms are not gender-specific, having a high prevalence also in women [3]. Hence, although an enlarged prostate can contribute to the onset of LUTS, other extra-prostatic factors are supposed to be equally important in LUTS determinism [2]. Bladder dysfunction— characterized by detrusor overactivity and/or overactive bladder (OAB)—is considered one of the main extra-prostatic LUTS determinants in both genders [4]. The pathogenesis of LUTS, including OAB, is complex and largely unknown. Increasing evidence links a number of age-related disorders, including metabolic and cardiovascular disorders, with the pathogenesis of LUTS [5]. In particular, it is epidemiologically well-established that metabolic syndrome (MetS) is not only frequently associated with, but also poses an increased risk for, the development and progression of LUTS in men. Several MetS features, including abdominal obesity [6], hypertension [7–9], hyperglycemia and type 2 diabetes mellitus (T2DM) [8,10], low high-density lipoprotein cholesterol and increased triglycerides plasma level [10–12], have all been related to LUTS. Therefore, recent investigations were aimed at understanding the potential pathogenic mechanisms underlying the association between MetS and LUTS. Animal models have provided a great deal of information. In a genetic mouse model of T2DM/obesity, both bladder dysfunction and prostatitis were described [13]. However, monogenic alterations related to metabolic derangements have rarely associated to human LUTS [14]. Bladder dysfunctions, including overactivity [15] or fibrosis [16], were often observed in hyperlipidemic rats and were associated with pelvic ischemia and smooth muscle atrophy. In male rats fed a fructose enriched diet, unstable bladder contractions and increased concentrations of M2,3muscarinic receptors, suggestive of bladder overactivity [17], were reported. Similar results J Sex Med 2014;11:1159–1172
Vignozzi et al. were observed in rats with streptozotocininduced diabetes [18,19]. We recently developed a non-genomic animal model of MetS, feeding adult male rabbits a highfat diet (HFD) for 12 weeks [20–25]. This animal model essentially recapitulates the human phenotype, showing, along with the classical features of MetS (hyperglycemia, impaired glucose tolerance, dyslipidemia, hypertension, increased visceral obesity, and hypogonadism), also severe alterations at the low urinary tract (LUT) level. In MetS rabbits, we characterized a prostatitis-like syndrome [22] and peculiar bladder alterations, including fibrosis, hypoxia, and inflammation [23], confirming that MetS could affect the whole of the LUT. More important, bladder from MetS animals showed increased signaling of Ras homolog gene family, member A (RhoA)/Rhoassociated protein kinase (ROCK) [23], a pathway deeply involved in detrusor hyper-contractility and bladder dysfunction, as often observed in human LUTS [4]. We previously tested the effect of chronic (2 mg/kg/day, for 12 weeks; preventive) or acute (2 mg/kg/day, for the last of the 12 weeks; curative) tadalafil dosing in HFD animals, focusing on prostate alterations [25]. Interestingly, we found that not only chronic (i.e., preventive), but also acute (i.e., curative), tadalafil dosing in HFD animals was effective in reducing MetS-associated prostatic abnormalities, including fibrosis and inflammation [25]. Aims
This study was aimed at investigating the effect of acute (curative) tadalafil dosing on MetSassociated increased signaling of RhoA/ROCK in bladder. Materials and Methods
MetS Rabbit Model The HFD-induced rabbit model of MetS has been obtained as previously described [20]. Male New Zealand White rabbits (Charles River, Calco, Lecco, Italy), weighing about 3 kg, were individually caged, under standard conditions, in a temperature- and humidity-controlled room, on a 12-hour light/dark cycle. Water and food were unrestricted throughout the study. After 1 week of standard rabbit diet, animals were randomly numbered and assigned to different groups: control (N = 43) or treatment group
Tadalafil Inhibits Bladder RhoA/ROCK Activity (N = 51). The control group continued to receive a standard diet (regular diet, RD), while the treatment group fed a HFD constituted by 0.5% cholesterol and 4% peanut oil (HFD rabbits) for 12 weeks, as previously described [20]. A subset of HFD rabbits was treated with tadalafil (2 mg/ kg/day, administered ad libitum in water drank in the entire day, 1 week; N = 14). Tadalafil powder, provided by Eli Lilly and Company (Indianapolis, IN, USA), was dissolved in dimethyl sulfoxide (10 mg/mL), then first diluted in ethanol (1:100) and finally in drinking water (28 mL). Mean arterial blood pressure was measured by using a polyethylene catheter inserted into a femoral artery at week 12, after pentobarbital (45 mg/kg) sedation as previously described [20]. After 12 weeks of treatment, rabbits were sacrificed with a lethal dose of pentobarbital, and specimens of bladder, seminal vesicles, prostates, and testis were harvested and processed for subsequent analyses. Biochemical and hormonal serum analyses were performed as previously described [20]. Animal handling will be complied with the Institutional Animal Care and Use Committee of the University of Florence, Florence, Italy, in accordance to the Italian Ministerial Law #116/92.
Measurement of Cholesterol,Triglycerides, Glycemia, Testosterone, and 17β Estradiol in Rabbits Blood samples for glucose, total cholesterol, triglycerides, testosterone (T), and 17β estradiol analyses were obtained from the animals via marginal ear vein at week 0 (baseline) and at week 12 in all groups, early in the morning after an overnight fasting. Plasma cholesterol, triglycerides, and glucose levels were measured by using an Automated System (ADVIA 2004 Siemens Chemistry System; Siemens Science Medical Solution Diagnostic, Tarrytown, NY, USA). Plasma T and 17β estradiol levels were measured by using an automated chemiluminescence system (Immunolite 2000 Siemens, Siemens Healthcare Diagnostics, Deerfield, IL, USA), after appropriate extraction. For extraction, samples were mixed with four volumes of diethyl ether for 15 minutes, centrifuged for 5 minutes at 2,000 rpm, and the aqueous phase frozen in dry ice. The organic phase was recovered, evaporated to dryness under a nitrogen stream, and reconstituted in the assay buffer. Oral Glucose Tolerance Test Oral glucose tolerance test (OGTT) was performed in accordance with the published method
1161 [20]. After an overnight fasting, a 50% glucose solution was orally administered to the animals, at a dose of 1.5 g/kg. Blood samples were collected via the marginal ear vein before and 15, 30, and 120 minutes after glucose loading. Samples were stored on ice and centrifuged at 3.000 rpm for 20 minutes at 4°C to obtain plasma. Plasma glucose was measured as described above. The incremental area under the curve (iAUC), was calculated by using the GRAPHPAD PRISM software version 4.0 for Windows (San Diego, CA, USA).
In Vitro Contractility Studies After harvesting bladder samples were immediately placed and maintained in cold Krebs solution and “in vitro” contractility experiments were performed in organ chambers, as previously described [26]. The maximal tonic tension elicited by high potassium salt solution (KCl, 80 mM) was taken as 100%, and the contractile effect induced by different concentrations of carbachol was referred to this value. The degree of contractile response induced by 10 μm carbachol was taken as 100% and the relaxant effect induced by Y-27632 was referred to this value. Drug cumulative concentrations were added to the bath, at 7-minute intervals, in order to obtain concentration-relaxant effect curves. Preparation of Membrane/Cytosolic Fractions and Western Blot Analysis for RhoA Activation Assay For protein extraction from bladder samples, the frozen tissues were ground in liquid nitrogen. Membrane and cytosolic fractions were prepared using the ProteoExtract subcellular proteome extraction kit (Calbiochem, Darmstadt, Germany), according to the manufacturer’s instructions. Protein extracts were quantified with the bicinchoninic acid (BCA) reagent (Pierce, Rockford, IL, USA). Western blot analysis with antiRhoA antibody (1:500; Santa Cruz Biotechnology, Dallas, TX, USA) was performed as previously described [23,26]. Densitometry analysis of band intensity was performed using Photoshop 5.5 software (Adobe Systems Inc. Italia srl, Agrate Brianza, Italy). RNA Extraction and Quantitative RT-PCR RNA isolation from tissue was performed as described previously [22,23]. Specific primers for all the target genes were previously described [20,22–25]. The expression of the 18S ribosomal RNA subunit was quantified with a pre-developed assay (Applied Biosystems, Foster City, CA, USA). J Sex Med 2014;11:1159–1172
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Immunohistochemistry Immunohistochemical studies were carried out as previously described [21]. Briefly, rabbit bladder sections (fixed in Bouin’s solution and embedded in paraffin) were incubated for 1 hour in 2% fetal bovine serum (FBS) in phosphate buffered saline (PBS, Sigma-Aldrich, Milan, Italy), to block nonspecific antibody binding. Sections were then incubated overnight at 4°C with the ROCK1 (1:1,000, Santa Cruz Biotechnology) primary antibody and then with the corresponding specific immunoglobulin peroxidase conjugated for 30 minutes (dilution 1:1,000). Demonstration of peroxidase activity and controls for antiserum specificity were performed as previously described [21]. Rabbit Bladder Smooth Muscle Cell Cultures Rabbit bladder smooth muscle cells (rbBSMCs) were isolated and characterized as previously described [23]. Briefly, bladder tissue samples were digested with 2 mg/mL bacterial collagenase type IV (Worthington Biochemical Corporation, Lakewood, NJ, USA) for 1 hour at 37°C, then fragments were extensively washed in PBS and cultured in Dulbecco’s modified Eagle medium (DMEM)/ Ham’s F-12 medium 1:1; Sigma-Aldrich) supplemented with 10% FBS) (Sigma-Aldrich), antibiotics (penicillin, 100 IU/mL; streptomycin 100 mg/mL; Sigma-Aldrich), and 1 mg/mL amphotericin B (Sigma-Aldrich) in a fully humidified atmosphere of 95% air/5% CO2. rbBSMCs began to emerge within 2 days and were used within the seventh passage. Characterization of cells showed positive staining for desmin and α-smooth muscle actin (α-SMA) and negative staining for cytokeratin (not shown). The percent of stained cells over the total number in 10 fields for each slide was >95% for both desmin and α-SMA. To test the capacity of tadalafil to reduce the mRNA expression of RhoA-dependent genes in rbBSMC cells primed with lipopolysaccharide (LPS, 100 ng/mL for 5 hours) with or without pretreatment with tadalafil (100 nM, 24 hours). Rabbit Smooth Muscle Cell Migration rbBSMC migration was studied in a P48 multiwell Boyden’s chamber (Nuclepore Inc., Pleasanton, CA, USA) as previously described [23]. Briefly, rbBSMCs were treated or not with tadalafil (100 nM, for 24 hours). Cells in serum free culture medium, in the absence of any stimulus, were taken as control for the rbBSMC basal migration. Platelet-derived growth factor (PDGF; 10 ng/mL, J Sex Med 2014;11:1159–1172
Vignozzi et al. for 5 hours) was added to the lower wells, alone or adding C3 exoenzyme (1 μg/mL, for 5 hours), a known selective inhibitor of RhoA, in the upper well. In the second set of experiments, rbBSMC migration was evaluated for 5 hours in the absence or in the presence of PDGF (10 ng/mL) in multiwell Boyden chamber. The effect of pretreatment with tadalafil (100 nM for 24 hours), with or without co-treatment with the protein kinase G (PKG) inhibitor 11H-2,7b,11a-trizadibenzo-(a,g)cycloocta-(c,d,e)-trinden-1-one (KT5823) (10 nM) or the membrane-permeant and phosphodiesterase (PDE)-resistant PKG agonist, Sp-8-Br-PET cyclic guanosine monophosphate (cGMP) (8Br-cGMP, 10 microM), was also evaluated. Methanol-fixed cells were stained with Diff-Quick (DADE Behring AG, Düdingen, Switzerland), and cell migration was measured by microscopic evaluation of the number of cells that moved across the filter. The number of migrated cells over the total number in 10 fields for each well was counted. Each experimental point was replicated at least five times, in three independent experiments.
Statistical Analysis Results are expressed as mean ± standard error of the mean (SEM) for n experiments as specified. The statistical analysis was performed with a oneway anova test followed by the Tukey–Kramer post hoc analysis in order to evaluate differences between the groups, and P < 0.05 was considered significant. When data were non-normally distributed, statistical differences were calculated with Kruskal–Wallis test and Mann–Whitney U-test was used for comparisons between groups. Correlations were assessed using Spearman’s method and statistical analysis was performed with the Statistical Package for the Social Sciences (SPSS, Inc., Chicago, IL, USA) for Windows 20.0. Stepwise multiple linear regressions were applied for multivariate analysis, whenever appropriate. Halfmaximal excitatory (EC50) or inhibitory (IC50) concentration values were calculated using the computer program ALLFIT [27], and reported as −logEC50 (pEC50) or −logIC50 (pIC50). IC50s potency ratio was calculated as the ratio between IC50 value in the examined experimental groups, and reported with the relative confidence interval. Main Outcome Measures In vitro contractility studies to evaluate the relaxant effect of the selective ROCK inhibitor,
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Tadalafil Inhibits Bladder RhoA/ROCK Activity Y-27632, in carbachol precontracted bladder strips. Evaluation of RhoA activation by its membrane translocation. Immunohistochemistry for ROCK expression. mRNA expression of inflammation, pro-fibrotic markers by quantitative RT-PCR. The in vitro effect of tadalafil on RhoA/ ROCK signaling in bladder smooth muscle cells was evaluated by using chemotaxis assay. Results
Metabolic, Hormonal, and Biochemical Parameters Metabolic, cardiovascular, and biochemical effects of feeding rabbits with a HFD for 12 weeks are reported in Table 1. As previously demonstrated [20–25], HFD induced features of a MetS-like syndrome (increased visceral fat, increased blood pressure, high cholesterol and triglyceride plasma levels, hyperglycemia and glucose intolerance, as demonstrated by the area under the curve of OGTT). Overt hypogonadism was also present, as demonstrated by combined low testosterone and decreased seminal vesicles, testis and prostate weights. A parallel increase of 17β-estradiol plasma
Table 1
level was observed in HFD rabbits. Confirming previous data [25], tadalafil treatment (2 mg/kg/ day, during the last out of 12 weeks, induced a significant reduction of visceral adipose tissue accumulation and triglyceride levels, without affecting testosterone or 17β estradiol plasma levels (Table 1).
Effect of Tadalafil on Carbachol-Induced Bladder Contractility To investigate the effect of HFD on bladder contractility, we first studied in vitro contractile response to increasing concentration of a muscarinic receptor agonist, carbachol (0.001–10 μm), in control and HFD rabbit bladder strips. As shown in Figure 1A, carbachol induced a higher dose dependent increase in tension in HFD than in control. Mathematical modeling of sigmoidal relationships (program ALLFIT, [27]) indicates that neither EC50s nor slopes (Hill coefficient) were different (shared –logEC50 = 5.89 ± 0.16, shared slope = 0.73 ± 0.17). In contrast, Emaxs were different: control Emax = 155.8 ± 15.2 and HFD Emax = 220.3 ± 20.1 (P = 0.017).
Metabolic and hormonal parameters in experimental rabbit
Total body weight (g) Baseline Week 12 Blood glucose (g/L) Baseline Week 12 OGTT (iAUC) Week 12 Cholesterol (mg/dL) Baseline Week 12 Triglycerides (mg/dL) Baseline Week 12 MAP (mmHg) Week 12 VAT weight (g) Week 12 Testosterone (nmol/L) Week 12 17β-estradiol (pmol/L) Week 12 Seminal vesicles weight (mg) Week 12 Testis weight (g) Week 12 Prostate weight (g) Week 12
Control (N = 43)
HFD (N = 37)
HFD + tadalafil (N = 14)
3,277.2 ± 36.7 3,923.9 ± 36.5***
3,295 ± 41.4 3,770.5 ± 32.4***
3,498 ± 45.7 3,791.2 ± 72.9**
1.1 ± 0.04 1.21 ± 0.04 149.6 ± 5 36.7 ± 1.97 41.6 ± 2.8 78.5 ± 4.3 92.2 ± 4.2
1.19 ± 0.03 1.96 ± 0.07***°°°
0.99 ± 0.06 1.85 ± 0.06***°°°
224.7 ± 7.1°°°
221.9 ± 13.3°°°
43.6 ± 2 1,451.1 ± 63.6***°°°
35.8 ± 2 1,675.8 ± 168.4***°°°
85.9 ± 4.1 301.7 ± 24.7***°°°
89.5 ± 2.1
133 ± 3.4°°°
37.5 ± 1.5
43.6 ± 1.5°
60.4 ± 3.9 171.6 ± 27.7**°°°∧ 125.6 ± 4.1°°° 32.9 ± 2∧∧
6.9 ± 0.93
1.5 ± 0.31°°°
1.27 ± 0.34°°°
168.7 ± 12.7
307.6 ± 38.4°°°
477 ± 153°°°
715.7 ± 34.7
517.7 ± 24.3°°°
446.9 ± 31.9°°
3.4 ± 0.1 0.6 ± 0.05
3 ± 0.05°°°
2.84 ± 0.08°°°
0.35 ± 0.04°°° ∧
0.46 ± 0.04 ∧∧
∧∧∧
*P < 0.05, **P < 0.01, ***P < 0.001 vs. baseline; °P < 0.05, °°P < 0.01, °°°P < 0.001 vs. control week 12; P < 0.05, P < 0.01, P < 0.001 vs. HFD week 12. Results are reported as mean ± SEM. iAUC = incremental area under the curve of glucose blood level during oral glucose tolerance test (OGTT). MAP = mean arterial pressure; VAT = visceral adipose tissue
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A
B
C
D
Figure 1 Effect of tadalafil on bladder contractility. (A) Effect of increasing concentrations of carbachol in bladder strips from control (closed circles) and HFD (closed squares) rabbits. Contractile response to carbachol is expressed in % (ordinate) of contraction obtained in bladder strips by KCl 80 mM; abscissa: concentrations of carbachol (0.001–10 μM). (B) Effect of carbachol (10 μM) in bladder strips from control (black bar), HFD (white bar) and tadalafil-treated HFD (gray bar) rabbits. Maximal response to KCl (80 mM), before the addition of carbachol, was taken as 100%. The effect of carbachol was evaluated as percentage of this response. Results were obtained from seven independent experiments and reported as mean ± SEM (C) Relaxation elicited by increasing concentrations of Y-27632 (abscissa) in bladder strips precontracted with 10 μM carbachol from control (N = 13 independent experiments; 9 animals), HFD (N = 10 independent experiments; 6 animals), HFD + tadalafil (N = 7 independent experiments; 7 animals) groups. Relaxant response to Y-27632 is expressed in % (ordinate) of contraction obtained with 10 μM carbachol. Each point represents mean ± SEM of n samples for each experimental group. (D) Relationship between the visceral fat weight (abscissa) and the response to Y-27632 (expressed as area under the curve [AUC] ordinate). This relationship are derived from univariate Spearman’s regression analysis and the relative coefficient of correlation (r), level of significance (P) and number of samples (N) are reported within the panel. Control, black circles; HFD, white squares; HFD + tadalafil, black triangles. *P < 0.05, **P < 0.01 vs. control.
We then tested the effect of tadalafil treatment on bladder tension induced by a fixed dose of carbachol (10 μm). This dose of carbachol (10 μm) was selected, based on the present (see Figure 1A) and a previous study [26], as that one eliciting maximal increase in bladder tension. Carbachol (10 μm) induced a significantly higher response in HFD bladder strips than in control ones (P < 0.05). Tadalafil treatment completely normalized carbachol responsiveness (see Figure 1B). J Sex Med 2014;11:1159–1172
Effect of Tadalafil on RhoA/ROCK Pathway in MetS Bladder In bladder strips precontracted with carbachol, we next evaluated the relaxant response to the selective ROCK inhibitor Y-27632 (Figure 1C). The relative IC50 values (−logIC50) and potency ratios were then calculated, using the program ALLFIT [27]. The amplitude of the response to Y-27632 was normalized to maximal carbachol responsiveness (taken as 100%), because it was significantly different among groups (see before). In control
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rabbits, Y-27632 dose-dependently relaxed carbachol-precontracted bladder strips (−logIC50 = 5.25 +/− 0.1) (Figure 1C). HFD induced a highly significant (P < 0.0001 vs. control), leftward shift of the Y-27632 sigmoidal relationship (−logIC50 = 6.7 +/− 0.08; IC50s potency ratio = 28.654 [10.97– 46.34]). This potentiating effect of HFD on responsiveness to Y-27632 was completely normalized (P = 0.2 vs. control) by tadalafil dosing (−logIC50 = 5.11 +/− 0.1; IC50s potency ratio = 0.7 [0.243–1.224]; Figure 1C). When all the experimental groups were considered, at univariate Spearman’s regression analysis, the responsiveness to Y-27632 (expressed as area under the curve) resulted positively related to the presence of MetS (r = 0.450, P < 0.05, not shown). Among MetS factors, visceral fat accumulation (r = 0.552, P < 0.01; see Figure 1D) and blood pressure (r = 0.453, P < 0.05, not shown) were both positively associated with responsiveness to Y-27632.
Effect of Tadalafil on Bladder Expression of RhoA/ROCK System To further investigate the HFD-induced RhoA/ ROCK pathway activation, we analyzed RhoA intracellular distribution (membrane vs. cytosol), in bladder extracts. As shown in Figure 2, membrane fraction of RhoA (which is indicative of its activation) was significantly increased in HFD rabbits, as compared with control (P < 0.05). Tadalafil dosing completely counteracted this effect, thereby causing cytosolic accumulation (P < 0.01 vs. HFD) (Figure 2). Accordingly, mRNA expression of several HFD-induced [23] and RhoA-dependent myofibroblast markers, (such as αSMA; desmin; transgelin, SM22α; ROCK1, [28] was significantly reduced in the bladder of tadalafil-treated rabbits, when compared with HFD group (Figure 3). Tadalafil treatment reduced also RhoA and ROCK2 mRNA expression; however, without reaching statistical significance (Figure 3). Consistent with a positive effect of tadalafil dosing on HFD-induced bladder alterations, also the mRNAs expression of other myofibroblast transdifferentiation/fibrosis (transforming growth factor β, TGFβ1; fibronectin 1, FN1; metallopeptidase inhibitor 2, TIMP2; matrix metallopeptidase 9, MMP9), inflammatory (cytochrome C oxidase subunit II, COX2, interleukin1β, IL1β; toll-like receptor type 2, TLR2; toll-like receptor type 4, TLR4; six-transmembrane protein of prostate 2, STAMP2; lactoferrin, LACT), and hypoxia (carbonic anhydrase IX, CAIX) markers
Figure 2 Effect of tadalafil on RhoA/ROCK pathway in MetS bladder. Intracellular distribution of RhoA evaluated by Western blot analysis with anti-RhoA primary antibody on membrane and cytosolic fractions obtained from bladder tissue of the indicated experimental groups. The bottom panel shows representative immunoblots with anti-RhoA and anti-β-actin, used as loading control. The top panel shows a graphical representation of densitometry data obtained from membrane/cytosol RhoA, calculated as % of control group and expressed as mean ± SEM from three independent experiments. *P < 0.05 vs. control; P < 0.01 vs. HFD.
were significantly reduced by tadalafil treatment (Figure 3). A significant reduction of estrogen receptor α was also observed (Figure 3). Interestingly, the expression of all these genes was positively associated with PDE type 5 (PDE5) mRNA expression in bladder (see Table 2). In addition, PDE5 mRNA expression in bladder from HFD tended to be increased, without, however, reaching statistical significance (data not shown). No significant changes for PDE5 expression were observed in tadalafil-treated HFD group (data not shown). The increased RhoA activation, observed in HFD bladder, was associated with an increased expression of its downstream effector ROCK1, as evaluated by immunohistochemistry. In control rabbit, ROCK1 protein was prominently immunolocalized in the muscular compartment, whereas urothelial layer was negative (Figure 4A). J Sex Med 2014;11:1159–1172
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Figure 3 Effect of tadalafil treatment on mRNA expression of genes related to inflammation, fibrosis, and steroid receptors in rabbit bladder. Bars represents tadalafil-induced variations of bladder genes, compared with HFD. Data were obtained by quantitative RT-PCR, then were expressed as percentage of HFD (HFD, N = 37, HFD + tadalafil, N = 14). *P < 0.05, **P < 0.01 vs. HFD.
HFD induced an increase in ROCK1 immunopositivity (at computer-assisted quantitative analysis P < 0.001 vs. control, Figure 4D), which was more evident in the smooth muscle bundles and blood vessels of sub-urothelium (Figure 4B). These changes were normalized by tadalafil dosing (Figure 4C, D).
In Vitro Effect of Tadalafil in Bladder Smooth Muscle Cells In order to further investigate the effect of tadalafil on RhoA/ROCK signaling, according to functional and immunohistochemical results (see before), we initiated studies on smooth muscle cells from isolated rabbit bladder (rbBSMC), evaluating two RhoA-mediated cellular functions: cell migration (Figure 5A, B) and mRNA expression of RhoA-dependent smooth muscle genes (Figure 5C). Using the Boyden’s chamber technique, we analyzed rbBSMC migration, as a read-out for RhoA/ROCK activation status (Figure 5A–B). Cell migration was studied in basal J Sex Med 2014;11:1159–1172
conditions and after the addition of PDGF, a wellknown stimulator of RhoA-mediated migration. As expected, PDGF (10 ng/mL, for 5 hours) induced a sustained increase in migration of rbBSMC across filters (188.61 ± 7.1% P < 0.0001 vs. control cells). Pretreatment with tadalafil (100 nM, 24 hours) abrogated PDGF-induced rbBSMC chemotaxis (P < 0.0001 vs. PDGF), to an extent similar to that observed with the RhoA selective inhibitor C3 exoenzyme (1 μg/mL; P < 0.0001 vs. PDGF). Interestingly, tadalafil was also able to reduce rbBSMC basal migration (P = 0.0001 vs. control cells; Figure 5). We next studied the molecular mechanism through which tadalafil blunted PDGF-induced migration. Exposing rbBSMC cells to a PDE-insensitive PKG agonist, Sp-8-Br-PET-cGMP, PDGFinduced migration was significantly reduced, to an extent similar to that observed with tadalafil treatment (Figure 5B). The effects of tadalafil was completely reverted by the PKG inhibitor KT 5823 (10 nM) (Figure 5B).
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Tadalafil Inhibits Bladder RhoA/ROCK Activity Table 2 Association between mRNA expression of PDE5 and genes of fibrosis, inflammation, steroid receptors and NO pathway in rabbit bladder
Fibrosis RhoA ROCK1 ROCK2 αSMA SM22α Desmin Vimentin TGFβ1 FN1 TIMP1 TIMP2 MMP2 MMP9 Inflammation IL1β TLR4 RAGE STAMP2 Steroid receptors AR PR GPR30 ERα NO pathway eNOS PDE11
r
P value
N
0.464 0.487 0.585 0.241 0.475 0.373 0.329 0.510 0.367 0.353 0.528 0.277 0.535
