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CARDIOVASCULAR
arKlinik für Innere Medizin III, Kardiologie, Angiologie und Internistische Intensivmedizin, Universitätsklinikum des Saarlandes, Homburg/Saar, Germany (M.Ba., N.S., M.Bö.); and Department of Pharmacology Medical Faculty, Mersin University Campus Yenisehir, Mersin, Turkey (K.B., O.A.)
Received December 20, 2007; accepted March 24, 2008.
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Abstract |
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). Prevalence of ED in Western industrialized countries is approximately 20 to 30% in the general population, but it is rising to more than 50% in patients with high-risk cardiovascular events, indicating a strong association between cardiovascular risk factors, especially hypertension, and erectile function (Feldman et al., 1994; Braun et al., 2000; Baumhäkel and Böhm, 2007; Böhm et al., 2007). Although nitric oxide (NO) plays an important role in the physiology of erectile function, impairment of the endothelial monolayer in the penile arteries and the corpus cavernosum is suggested to be the pathophysiological mechanism (Yavuzgil et al., 2005; Baumhäkel et al., 2006). Moreover, recent trials demonstrated differential effects of antihypertensive treatments on erectile function. Substances inhibiting the renin-angiotensin system are supposed to have beneficial effects on erectile function in vivo as well as in vitro (Fogari et al., 1998, 2001; Duesing, 2003). In contrast, in the last two decades especially, β-receptor blockers were reported to impair erectile function. A meta-analysis of the latest β-receptor blocker trials conducted in the 1990s with 35,000 patients showed a significant increase of sexual dysfunction in patients treated with a β-receptor blocker compared with placebo, but this analysis was limited by poor data management regarding erectile function (Ko et al., 2002). In contrast, recent randomized trials suggested that β-receptor blockade had no effect on erectile function (Franzen et al., 2001; Silvestri et al., 2003). Moreover, previous reports about increased ED may be biased by the psychological effect on patients with knowledge of being treated with β-receptor blockers as demonstrated by Silvestri et al. (2003). Consistent with these findings, a prospective randomized trial in a huge cardiovascular high-risk population did not demonstrate a negative effect of β-receptor blockade on erectile function in multivariate analysis (Böhm et al., 2007).
Considering the strong correlation and pathophysiological link between endothelial and erectile function, substances with beneficial effects on nitric oxide synthase may improve erectile function. Nebivolol, as a third generation β-receptor blocker with NO-releasing effects, was recently shown to improve erectile function in a small trial with hypertensive men, whereas metoprolol did not (Brixius et al., 2007). Herein, possible mechanisms were not identified.
The objective of this study was to determine the effects and possible mechanisms of treatment with the β-receptor blockers, nebivolol and metoprolol, on penile endothelial function in cholesterol-fed apolipoprotein E (ApoE)-/- mice with atherosclerosis.
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Materials and Methods |
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). Chemicals were obtained from Sigma-Aldrich (Munich, Germany). All chemicals were dissolved in distilled water, with the exception of nebivolol-HCl, which was dissolved in dimethyl sulfoxide and then further diluted, but never exceeding, 0.005% in organ bath.
Aortic Ring Preparation and Tension Recording. After excision, the descending thoracic aorta was immersed in Tyrode's solution containing 118 mM NaCl, 2.5 mM CaCl2, 4.73 mM KCl, 1.2 mM MgCl2, 1.2 mM KH2PO4, 2.5 mM NaHCO3, 0.026 mM Na EDTA, and 5.5 mM D-(+)glucose, pH 7.4. Adventitial tissue was carefully removed. Three-millimeter rings were mounted in organ bath chambers filled with the Tyrode's solution described above (37°C, aerated with 95% O2 and 5% CO2) and were attached to a force transducer recording isometric tension. Aortic rings were stretched to a resting tension of 10 mN, which was maintained throughout the experiment. Pharmacologically induced contraction of aortic rings was performed with an 
-agonist, (R)-(-)-phenylephrine-HCl (10 µM). Drugs were added in increasing concentrations to obtain cumulative concentration-response curves for carbachol (carbamylcholine-chloride, 1 nM–100 µM), as an endothelium-dependent relaxing agent, and glyceryl trinitrate (100 nM–100 µM), as a NO donor. The drugs were washed out before adding the next substance. The relaxing effect of carbachol was abolished by adding N-nitro-L-arginine methyl ester (1 µM).
Corpus Cavernosum Preparation and Tension Recording. Penises were removed and immersed in chilled buffer (described above). The glans penis and urethra were excised, and adherent tissues were carefully removed, keeping the tunica albuginea intact. Corpus cavernosal strips (CCS) were suspended in organ bath chambers filled with Tyrode's solution (described above) (37°C, aerated with 95% O2 and 5% CO2) and attached to a force transducer recording isometric tension. CCS were stretched to a resting tension of 3 mN, which was maintained throughout the experiment. After equilibration, cavernosal strips were precontracted with phenylephrine (5 µM). After a steady state of contraction was obtained, drugs were added in increasing concentrations to obtain cumulative concentration-response curves for carbachol (1 nM-1 µM) and glyceryl trinitrate (100 nM–100 µM). The drugs were washed out before adding the next substance. The relaxing effect of carbachol was abolished by adding N-nitro-L-arginine methyl ester (1 µM). Relaxation of the corpora cavernosa mimics erectile function, and decreased relaxation to a stimulus indicates erectile dysfunction (Büyükafar and Un, 2003).
Acute effects of oxidative stress on endothelial function of the corpus cavernosum were assessed in organ bath experiments using CCS of C57BL/6J wild-type mice, as described above. After equilibration of the resting tension (3 mN), tissue was pretreated with H2O2 (100 µM) for 20 min followed by precontraction with phenylephrine- (5 µM) and carbachol-induced relaxation. To test acute antioxidative effects, metoprolol and nebivolol were added in the organ bath chamber 20 min before H2O2. Both metoprolol (100 nM) and nebivolol (100 nM) were used in concentration of 50% β1-receptor binding (Maack et al., 2000, 2001). To control the direct relaxing effects of nebivolol, penile endothelial function was studied in C57BL/6J mice with increasing concentrations of nebivolol (0.1–10 µM) after preconstriction with phenylephrine (5 µM).
Staining Procedures. The aortic sinus and corpora cavernosa were snap-frozen at -80°C and sectioned on a Leica cryostat (10 µm). At least five consecutive sections per animal per staining were used for analysis. To detect atherosclerotic lesions in the aortic sinus, Oil Red O staining was performed as described previously (Laufs et al., 2005). Morphometric analysis was performed with the Lucia Measurement Software 4.6 (Nikon, Melville, NY), measuring the lipid-staining plaque area and related vessel diameter. To assess penile collagen content, sirius-red staining of corpora cavernosal sections was performed. Corpora cavernosal strips were snap-frozen and stored at -80°C. Segments were sectioned on a Leica cryostat (10 µm) that were placed on glass slides and incubated with the Sirius Red agent. Collagen content was quantified using fluorescence microscopy. CCS from each treatment group were processed in parallel, and images were acquired with identical acquisition parameters and stored digitally.
Measurement of Lipid Peroxidation. Aortic tissue/corpus cavernosum was homogenized in phosphate-buffered saline, pH 7.4, containing butylated hydroxytoluene (4 mM). Lipid hydroperoxides were determined using the Lipid Peroxidation Assay Kit II (Calbiochem, Darmstadt, Germany) and expressed as micromoles per milligram of protein (Laufs et al., 2005).
Statistical Analysis. All data are expressed as the mean ± S.E.M. Statistical significance was assumed at p < 0.05. Intergroup differences were assessed with the analysis of variance test using Newman-Keuls post hoc analysis (GraphPad Prism 4.03; GraphPad Software, San Diego, CA).
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Results |
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Aortic/Cavernosal Endothelial Function. Penile endothelial function was determined as relaxation of CCS (two CCS per animal, 9–10 animals per treatment group), and endothelial function was determined by relaxation of aortic rings (AoR) (four AoR per animal, 6–8 animals per treatment group), in all groups. Response to muscarinergic stimulation with carbachol was significantly impaired in the aortic rings of ApoE-/- mice compared with WT mice (Fig. 2). Parallel to endothelial function in the aorta, endothelium-dependent relaxation of corpora cavernosal strips was also significantly decreased in ApoE-/- mice (Fig. 3). Both AoR and CCS revealed an impaired efficacy of muscarinergic stimulation in ApoE-/- mice. In addition, the pD2 value calculated from the concentration-response curves to carbachol was shifted to the right in the CCS of ApoE-/- mice (pD2: WT 6.83 ± 0.06 and ApoE-/- 6.58 ± 0.05, p < 0.01). Relaxation of AoR and CCS related to carbachol was significantly improved in nebivolol but not or only to a less extent, respectively, in metoprolol-treated mice (Figs. 2 and 3). In CCS, both efficacy and potency of muscarinergic stimulation with carbachol were significantly increased in nebivolol-treated mice [pD2: 6.86 ± 0.07 (p < 0.01) versus ApoE-/-]. Potency of carbachol stimulation was not improved in the CCS of mice treated with metoprolol [pD2: 6.75 ± 0.09 (N.S.) versus ApoE-/-]. Endothelium-independent relaxation induced by glyceryl trinitrate of the aortic rings and corpora cavernosa was not significantly different between all groups.
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Vascular/Penile Oxidative Stress. As a global parameter of oxidative stress, lipid peroxidation of the aortic wall (n = 5) and corpora cavernosa (n = 7) was measured. Lipid hydroperoxides were significantly increased in both tissues (aorta and corpora cavernosa) in ApoE-/- mice compared with WT mice with a restoration in nebivolol but not metoprolol-treated mice (Fig. 4).
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Collagen Content. Collagen content as a parameter of fibrotic changes in the corpus cavernosum was calculated as described above. The content of collagen fibers was significantly enhanced in ApoE-/- mice with a restoration in nebivolol and in part in metoprolol-treated ApoE-/- mice (Fig. 7).
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Discussion |
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Erectile function is crucially dependent on nitric oxide synthesis of the endothelial monolayer of the penile arteries and the corpus cavernosum, and it is consequently associated with known cardiovascular risk factors, especially hypertension (Feldman et al., 1994; Yavuzgil et al., 2005). Moreover, ED is suggested to be an early symptom of generalized atherosclerosis preceding major cardiovascular events (Baumhäkel and Böhm, 2007). Thus, cardiovascular evaluation has been recommended for patients with ED, providing the opportunity for optimized preventional treatment, but cardiovascular drugs are often related to a decrease in erectile function, especially in patients with hypertension (Modebe, 1990). In particular, β-receptor blockers have been reported to impair erectile function, but randomized clinical trials could not confirm this effect (Grimm et al., 1997; Rosenkranz and Erdmann, 2001; Böhm et al., 2007). In contrast, a recent trial suggests that β-receptor blocker-related ED may be dependent on knowledge of the drug class of the patient, indicating a major role of psychological factors (Silvestri et al., 2003).
In our study, endothelial function was significantly increased in nebivolol-treated ApoE-/- mice. In metoprolol-treated mice, improvement of endothelial function was significant at a carbachol concentration of 1 µM only. Moreover, atherosclerotic plaque formation in the aortic root as well as reactive oxygen species (ROS) production was decreased or even normalized by nebivolol, whereas metoprolol had less or no effects. These results are consistent with recent findings indicating the beneficial role of nebivolol on endothelial function in different animal models (Oelze et al., 2006; Wolf et al., 2007). Comparable effects could be observed in the penile tissue. Endothelial function of the corpus cavernosum was improved in nebivolol-treated mice, whereas metoprolol treatment improved endothelial function at a carbachol concentration of 30 nM only. This result emphasizes the strong association between aortic and penile endothelial function.
Metoprolol and nebivolol have a similar potency to bind to β1- and β2-adrenergic receptors, but nebivolol, in particular, is suggested to exert NO-releasing effects, probably via reduction of reactive oxygen species, whereas metoprolol is lacking this pleiotropic effect on vascular function (Oelze et al., 2006). Thus, antioxidative activity of nebivolol with a consecutive increase of nitric oxide release may be the mechanism needed to improve erectile function. In penile tissue, only nebivolol, but not metoprolol, reduced oxidative stress significantly. Effects of the treatment with nebivolol on superoxide production in the erectile tissue are consistent with previous reports on vascular tissues and could explain decreased inhibition of endothelial NO synthase activity in the corpora cavernosa as recently demonstrated (Oelze et al., 2006; Reidenbach et al., 2007). Moreover, nebivolol decreased collagen content of the penile tissue to a greater extent than did metoprolol. These effects of nebivolol treatment on structural changes in the penis are in line with recent data comparing nebivolol and amlodipine in hypertensive rats (Toblli et al., 2006). Decrease of fibrotic changes is also probably related to a decrease of oxidative stress.
β-blockers currently used have at least one chiral center, and beneficial cardiovascular effects may reside in the S-enantiomer (Siebert et al., 2008). Nebivolol is a racemic mixture and differs from all β-blockers with a hydroxypropanolamine structure with cardiovascular activity at the R-enantiomer at the hydroxy group. Moreover, hydroxylated nebivolol metabolites could play a role in beneficial effects beyond blood pressure reduction (Siebert et al., 2008). Recent results in rat aorta suggested that nebivolol is in part degraded by its reaction with ROS (de Groot et al., 2004). Thus, antioxidative effects of nebivolol could, at least in part, be explained by acute scavenging of ROS. Therefore, even improvement of penile endothelial function might be dependent on these effects. Preincubation with nebivolol, but not metoprolol, improved diminished endothelial function of the corpus cavernosum after stimulation with ROS. These effects are probably independent of receptor binding, because the nebivolol concentrations used were ineffective in a cumulative concentration response regarding direct relaxing effects. The nebivolol-induced relaxation of penile smooth muscle cells could be demonstrated in concentrations higher than 100 nM. However, these effects are not likely to play a major role in chronic treatment regarding plasma concentrations of nebivolol, which are one order of magnitude less (Selvan et al., 2007). In summary, direct and acute antioxidative effects of nebivolol probably contribute to the presented results, but conclusions about the extent remain speculative.
Thus, for the first time, treatment with β-receptor blockers is shown to improve penile endothelial function as a surrogate of erectile function in an atherosclerotic animal model. Hence, these results support recent clinical trials, which failed to demonstrate negative effects of β-receptor blockade on erectile function in patients with high-risk cardiovascular events (Silvestri et al., 2003; Böhm et al., 2007). In particular, in hypertensive men, nebivolol did not change the frequency of sexual contacts as a surrogate of erectile function, whereas in patients treated with atenolol, the frequency of sexual contacts decreased significantly (Boydak et al., 2005). In contrast, a recent trial in male hypertensive patients comparing metoprolol and nebivolol indicated beneficial effects of nebivolol, but not metoprolol, on erectile function (Brixius et al., 2007). These clinical results are consistent with the presented mechanistic study. Moreover, in clinical trials, the effects of β-receptor blockade on erectile function are, in part, suggested to be dependent on blood pressure reduction. The results of our study demonstrate the effects, which are independent on the decrease of blood pressure, considering similar blood pressure levels in all treatment groups. Despite the absence of blood pressure reduction, metoprolol did not decrease erectile function, which is also an important finding that supports the thesis that selective β-receptor blockade does not affect erectile function. Nebivolol improved erectile function by a decrease of oxidative stress. This effect is probably dependent on increased nitric oxide release and a decreased heart rate, which is known to improve endothelial function (Beere et al., 1984).
In conclusion, treatment with the β-receptor blocker nebivolol improved endothelial function of the corpus cavernosum in ApoE-/- mice. These beneficial effects may be dependent on a reduction of oxidative stress in the corpus cavernosum. In addition, the effects of treatment with nebivolol and metoprolol on endothelial function were comparable in penile and aortic tissue, indicating the strong association of endothelial and erectile function.
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Footnotes |
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: ED, erectile dysfunction; NO, nitric oxide; ApoE, apolipoprotein E; WT, wild-type; CCS, corpora cavernosal strips; AoR, aortic rings; ROS, reactive oxygen species.
Address correspondence to: Dr. Magnus Baumhäkel, Klinik für Innere Medizin III, Universitätsklinikum des Saarlandes, D-66421 Homburg/Saar, Germany. E-mail: magnus{at}baumhaekel.de
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