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Diabetes impairs endothelium-dependent relaxation of human penile vascular tissues mediated by NO and EDHF
BBRC Biochemical and Biophysical Research Communications 312 (2003) 1202–1208 www.elsevier.com/locate/ybbrc
Diabetes impairs endothelium-dependent relaxation of human penile vascular tissues mediated by NO and EDHF Javier Angulo,a,b Pedro Cuevas,b Argentina Fern andez,b Sonia Gabancho,a,b a a Antonio Allona, Antonio Martın-Morales, Ignacio Moncada,a Sebasti an Videla,c and I~ nigo S aenz de Tejadaa,b,* a
Fundaci on para la Investigaci on y el Desarrollo en Andrologıa. Madrid 28304, Spain b Departamento de Investigaci on, Hospital Ram on y Cajal, Madrid, Spain c Departamento de Investigaci on Clınica, Laboratorios Dr. Esteve, Barcelona, Spain Received 4 November 2003
Abstract Standard treatments for erectile dysfunction (ED) (i.e., PDE5 inhibitors) are less effective in diabetic patients for unknown reasons. Endothelium-dependent relaxation (EDR) of human corpus cavernosum (HCC) depends on nitric oxide (NO), while in human penile resistance arteries (HPRA) endothelium-derived hyperpolarizing factor (EDHF) and NO participate. Here we show that diabetes significantly reduced EDR induced by acetylcholine (ACh) in HCC and HPRA. Relaxation attributed to EDHF was also impaired in HPRA from diabetic patients. The PDE5 inhibitor, sildenafil (10 nM), reversed diabetes-induced endothelial dysfunction in HCC, but not in HPRA. Calcium dobesilate (DOBE; 10 lM) fully reversed diabetes-induced endothelial dysfunction in HPRA by specifically potentiating the EDHF-mediated component of EDR. Impairment by diabetes of NO and EDHF-dependent responses precluded the complete recovery of endothelial function in HPRA by sildenafil. This could explain the poor clinical response to PDE5 inhibitors of diabetic men with ED and suggests that a pharmacological approach that combines enhancement of NO/cGMP and EDHF pathways could be necessary to treat ED in many diabetic men. Ó 2003 Elsevier Inc. All rights reserved. Keywords: Human corpus cavernosum; Human penile resistance arteries; Diabetes; Erectile dysfunction; Endothelium-derived hyperpolarizing factor; Calcium dobesilate; Sildenafil
The endothelium is an important contributor to penile smooth muscle relaxation (both arterial and trabecular), and therefore, penile erection. Diabetes is associated with endothelial dysfunction [1,2] as well as with a high incidence of erectile dysfunction [3]. In fact, in aging and other vascular diseases associated with endothelial dysfunction such as hypercholesterolemia and hypertension, a high prevalence of erectile dysfunction is also observed [4]. Nitric oxide (NO) is a key mediator of endotheliumdependent relaxation (EDR). However, the existence of an unidentified endothelial factor that promotes smooth muscle hyperpolarization and relaxation and is resistant to NO synthase and cyclooxygenase inhibition has been * Corresponding author. Fax: +34-91-358-50-45. E-mail address: [email protected] (I.S. Tejada).
0006-291X/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2003.11.034
clearly established (see [5] for review) and it has particular functional relevance in small arteries [6,7]. We have recently demonstrated that, while in human corpus cavernosum endothelium-dependent relaxation is mediated by NO, in human penile resistance arteries, in addition to NO, endothelium-derived hyperpolarizing factor (EDHF) contributes significantly to endotheliumdependent relaxation [8]. In patients, in several vascular beds, it has been shown that diabetes impairs endothelium-dependent relaxation mediated by NO [1,2,9], including HCC [10], but alteration by diabetes of EDHF-mediated responses has only been reported in animal models [11,12]. Type 5 phosphodiesterase (PDE5) inhibitors potentiate NO-mediated responses by enhancing intracellular cGMP accumulation and constitute an efficacious oral therapy for the treatment of erectile dysfunction [13–15].
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However, although these compounds show efficacy to treat erectile dysfunction in some diabetic patients, the efficacy is significantly lower than that observed in the general population of impotent patients [16–18], for reasons that are not presently known. Calcium dobesilate (DOBE) potentiates endotheliumdependent relaxation of human penile arteries by specifically enhancing the responses mediated by EDHF [8]. Thus, if diabetes impaired EDHF-mediated responses DOBE could potentially improve vasodilation via this pathway. With these premises, the aims of the present study were to characterize endothelial function in human corpus cavernosum and penile resistance arteries from diabetic and non-diabetic impotent men and to evaluate the effects of enhancing the NO/cGMP pathway with the PDE5 inhibitor sildenafil and of potentiating EDHFmediated responses with DOBE on endotheliumdependent relaxation in penile tissues from these patients.
Materials and methods Human penile tissues. Human penile tissue biopsies were obtained from 18 impotent diabetic (12 with type 1 diabetes and 6 with type 2 diabetes) and 25 non-diabetic impotent men who gave informed consent at the time of penile prosthesis insertion. The study was approved by the local Ethics Committee. Tissues were maintained at 4–6 °C in M-400 solution (composition per 100 ml: mannitol, 4.19 g; KH2 PO4 , 0.205 g; K2 HPO4 3H2 O, 0.97 g; KCl, 0.112 g; and NaHCO3 , 0.084 g) until used, which was between 2 and 16 h from extraction [19]. There were no significant differences in the age of diabetic and non-diabetic patients from whom the biopsies were collected (59.8 1.5 and 58.6 1.8 years, respectively). The presence of co-morbid conditions was similar in the diabetic and non-diabetic groups. Hypertension was present in four diabetic patients (22%) and four non-diabetic patients (16%), hypercholesterolemia was present in five diabetic patients (28%) and four non-diabetic patients (16%), smoking habit was present in 10 diabetic patients (56%) and 11 non-diabetic patients (44%), and neurological alterations were present in three diabetic patients (17%) and two non-diabetic patients (8%) (p > 0:05 for all comparisons). Vascular reactivity of human penile resistance arteries. Penile small arteries, helicine arteries (lumen diameter 150–400 lm), which are the terminal branches of deep penile arteries, were dissected by carefully removing the adhering trabecular tissue, and arterial ring segments (2 mm long) were subsequently mounted on two 40 lm wires on microvascular double Halpern–Mulvany myographs (J.P. Trading, Aarhus, Denmark) for isometric tension recordings. The vessels were allowed to equilibrate for 30 min in physiological salt solution (PSS) of the following composition (mM): NaCl 119, KCl 4.6, CaCl2 1.5, MgCl2 1.2, NaHCO3 24.9, glucose 11, KH2 PO4 1.2, and EDTA 0.027 at 37 °C continuously bubbled with 95% O2 /5% CO2 mixture to maintain a pH of 7.4. Passive tension and internal circumference of vascular segments when relaxed in situ under a transmural pressure of 100 mmHg (L100 ) were determined. The arteries were then set to an internal circumference equivalent to 90% of L100 , at which the force development was close to maximal [20]. The preparations were then exposed to 125 mM Kþ (KPSS, equimolar substitution of NaCl for KCl in PSS) and the contractile response was measured. The arteries were contracted with 1 lM norepinephrine (80% of KPSS induced contraction approximately) and relaxation responses were evaluated by cumulative additions of compounds to the chambers.
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Experiments with human corpus cavernosum tissue. Strips of corpus cavernosum tissue (3 3 7 mm) were immersed in 8 ml organ chambers containing PSS, maintained at 37 °C, and aerated with 5% CO2 /95% O2 , pH 7.4. Each tissue strip was incrementally stretched to optimal isometric tension, as determined by maximal contractile response to 1 lM phenylephrine [21]. Tissues were contracted with 0.5–3 lM phenylephrine (80% of KPSS induced contraction) and relaxation responses were evaluated by cumulative additions of compounds to the chambers. Drugs and materials. Phenylephrine, norepinephrine (arterenol), acetylcholine, indomethacin, and NG -nitro-L -arginine (L -NNA) were obtained from Sigma Chemical (St. Louis, MO). Calcium dobesilate (calcium dihydroxy-2,5 benzenesulfonate, Doxium) was provided by Dr. Esteve Laboratories (Barcelona, Spain). Sildenafil was a gift from Nitromed (Bedford, MA). For in vitro experiments, all drugs were dissolved in deionized water, except for sildenafil which was dissolved at 10 mM concentration in DMSO. The subsequent dilutions were made in deionized water. Data analysis. Relaxation responses are expressed as percentage of total relaxation (loss in tone) induced by the addition of 0.1 mM papaverine HCl to the chambers at the end of the experiment. All data are expressed as means SE. Complete concentration–response curves were obtained and compared by a two-factor analysis of variance (ANOVA) statistical test using StatView software for Apple computers.
Results Effects of diabetes on endothelium-dependent relaxation of human corpus cavernosum and penile resistance arteries Acetylcholine (ACh; 1 nM–10 lM) produced concentration-dependent relaxation of HCC and HPRA. The responses to ACh were significantly attenuated in tissues from diabetic patients when compared to the responses in tissues from non-diabetic patients (Figs. 1A and B). We have previously identified that in HPRA, but not in HCC, there is a significant component in the relaxation response to ACh that is resistant to NO synthase (NOS) and cyclooxygenase (COX) inhibition and is attributed to EDHF [8]. In HPRA, ACh-induced relaxation, in the presence of the NOS inhibitor, NG -nitroL -arginine (L -NNA; 100 lM), and the inhibitor of COX, indomethacin (5 lM), was significantly attenuated in tissues from diabetic patients compared to responses in tissues from non-diabetic patients (Fig. 1C). Comparison of effects of PDE5 inhibition with sildenafil on endothelium-dependent relaxation in tissues from diabetic and non-diabetic patients Inhibition of PDE5 activity with sildenafil (10 nM) significantly potentiated ACh-induced relaxation in HCC from non-diabetic as well as from diabetic patients (Fig. 2). Furthermore, in the presence of sildenafil, the responses to ACh were normalized as there were no longer differences in endothelium-dependent relaxation of diabetic and non-diabetic HCC (Fig. 2). Sildenafil (10 nM) was also able to clearly potentiate ACh-induced relaxations in HPRA from non-diabetic patients (Fig. 3). In contrast, sildenafil (10 nM) did not significantly
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Fig. 1. Effects of diabetes on the endothelium-dependent relaxation induced by acetylcholine (ACh; 1 nM–10 lM) on phenylephrine-contracted human corpus cavernosum strips (A) and norepinephrine-contracted human penile resistance arteries in the absence (B) or the presence (C) of combined inhibition of NO synthase and cyclooxygenase with NG -nitro-L -arginine (L -NNA; 100 lM) and indomethacin (INDO; 5 lM), respectively. Data are expressed as means SEM of the percentage of total relaxation induced by 0.1 mM papaverine. n indicates the number of patients from whom the tissues were collected for the experiments. *** indicates p < 0:005 vs no diabetes by a two-factor ANOVA test.
Fig. 2. Effects of the treatment with sildenafil (10 nM) on the endothelium-dependent relaxation induced by acetylcholine (ACh; 1 nM– 10 lM) on phenylephrine-contracted human corpus cavernosum strips from non-diabetic and diabetic patients. Data are expressed as means SEM of the percentage of total relaxation induced by 0.1 mM papaverine. n indicates the number of patients from whom the tissues were collected for the experiments. *** indicates p < 0:005 vs responses in non-diabetic tissues and yy p < 0:01, yyy p < 0:005 vs respective untreated tissues by a two-factor ANOVA test.
enhance endothelium-dependent relaxation of HPRA from diabetic patients (Fig. 3). After sildenafil treatment, ACh-induced relaxation in diabetic HPRA still showed attenuated responses when compared to nondiabetic tissues (Fig. 3). Furthermore, endothelium-dependent relaxation attributable to EDHF in diabetic HPRA, after NOS and COX inhibition, was unaffected
Fig. 3. Effects of the treatment with sildenafil (10 nM) on the endothelium-dependent relaxation induced by acetylcholine (ACh; 1 nM– 10 lM) on norepinephrine-contracted human penile resistance arteries from non-diabetic and diabetic patients. Data are expressed as means SEM of the percentage of total relaxation induced by 0.1 mM papaverine. n indicates the number of patients from whom the tissues were collected for the experiments. ** indicates p < 0:01 and ***p < 0:005 vs responses in non-diabetic arteries and yyy indicates p < 0:005 vs respective untreated arteries by a two-factor ANOVA test.
by sildenafil (Fig. 4). Thus, sildenafil was not able to reverse diabetes-induced endothelial dysfunction in HPRA from diabetic men. Comparison of the effects of calcium dobesilate on endothelium-dependent relaxation of human penile resistance arteries from diabetic and non-diabetic patients Treatment with DOBE (10 lM) did not significantly modify ACh-induced relaxation of HCC from diabetic
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Fig. 4. Effects of the treatment with sildenafil (10 nM) on the endothelium-dependent relaxation induced by acetylcholine (ACh; 1 nM– 10 lM) on norepinephrine-contracted human penile resistance arteries from diabetic patients in the presence of L -NNA (100 lM) plus indomethacin (INDO; 5 lM). Data are expressed as means SEM of the percentage of total relaxation induced by 0.1 mM papaverine. n indicates the number of patients from whom the tissues were collected for the experiments.
Fig. 5. Effects of the treatment with calcium dobesilate (DOBE 10 lM) on the endothelium-dependent relaxation induced by acetylcholine (ACh; 1 nM–10 lM) on phenylephrine-contracted human corpus cavernosum strips from non-diabetic and diabetic patients. Data are expressed as means SEM of the percentage of total relaxation induced by 0.1 mM papaverine. n indicates the number of patients from whom the tissues were collected for the experiments. *** indicates p < 0:005 vs responses in non-diabetic tissues by a two-factor ANOVA test.
or non-diabetic patients (Fig. 5). However, DOBE (10 lM) significantly potentiated ACh-induced relaxation of HPRA from non-diabetic and diabetic patients (Fig. 6). This potentiating effect was more pronounced
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Fig. 6. Effects of the treatment with calcium dobesilate (DOBE 10 lM) on the endothelium-dependent relaxation induced by acetylcholine (ACh; 1 nM–10 lM) on norepinephrine-contracted human penile resistance arteries from non-diabetic and diabetic patients. Data are expressed as means SEM of the percentage of total relaxation induced by 0.1 mM papaverine. n indicates the number of patients from whom the tissues were collected for the experiments. *** indicates p < 0:005 vs responses in non-diabetic arteries and yyy indicates p < 0:005 vs respective untreated arteries by a two-factor ANOVA test.
Fig. 7. Effects of the treatment with calcium dobesilate (DOBE 10 lM) on the endothelium-dependent relaxation induced by acetylcholine (ACh; 1 nM–10 lM) on norepinephrine-contracted human penile resistance arteries from non-diabetic and diabetic patients in the presence of L -NNA (100 lM) plus indomethacin (INDO; 5 lM). Data are expressed as means SEM of the percentage of total relaxation induced by 0.1 mM papaverine. n indicates the number of patients from whom the tissues were collected for the experiments. *** indicates p < 0:005 vs control responses in non-diabetic arteries and yyy indicates p < 0:005 vs respective untreated arteries by a two-factor ANOVA test.
in HPRA from diabetic patients. Indeed, diabetes-induced endothelial dysfunction was reversed by DOBE, since responses to ACh in HPRA from diabetic patients
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after treatment with DOBE were better than those in non-diabetic HPRA without DOBE treatment and comparable to those in HPRA from non-diabetic patients treated with DOBE (Fig. 6). DOBE (10 lM) also specifically enhanced the endothelium-dependent relaxation resistant to NOS and COX inhibition in HPRA from non-diabetic and diabetic patients (Fig. 7). Furthermore, treatment with DOBE fully reversed diabetes-induced impairment of EDHF-mediated relaxation of HPRA (Fig. 7).
Discussion The prevalence of severe ED in diabetic men is almost threefold higher than in the general population [3]. Diabetes mellitus is a main independent risk factor for ED in epidemiological studies [4]. The pathophysiology of diabetic ED could, at least in part, be related to the presence of endothelial dysfunction as would suggest the high prevalence of ED in other diseases associated with endothelial dysfunction such as hypertension, hypercholesterolemia, or aging [4]. Furthermore, in patients with type 2 diabetes the presence of ED correlated with endothelial dysfunction [22]. Here we show that endothelium-dependent relaxation is impaired in both human penile corpus cavernosum (HCC) and penile resistance arteries (HPRA) from diabetic patients. Defective endothelium-dependent relaxation of diabetic HCC has been previously reported [10] and the present study shows that diabetes also impairs endothelium-dependent relaxation in HPRA. However, we have recently reported substantially different mechanisms in the endothelium-dependent relaxation of HPRA and HCC. While in HCC tissue endothelium-dependent relaxation is essentially mediated by nitric oxide (NO), endothelium-derived hyperpolarizing factor (EDHF) is responsible for an important component of acetylcholine-induced relaxation of HPRA [8]. This observation is consistent with the results obtained in horse penile resistance arteries [23] and with the relevant role of relaxations mediated by EDHF in other human resistance vessels [6,7]. The contribution of EDHF to endothelium-dependent relaxation of HPRA led us to evaluate the impact of diabetes on EDHF-mediated responses in these vessels. A significant finding of the present study is that endothelium-dependent relaxation, specifically attributable to EDHF, is attenuated in HPRA from diabetic patients. In diabetic rats, defective EDHF-mediated relaxation has been reported in mesenteric [11] and renal [12] microvasculature, and it has been suggested that diabetes has a greater impact on endothelial function in arteries with EDHF-mediated responses than in those depending almost exclusively on NO action [24]. However, the influence of diabetes on EDHF-mediated responses in
human vasculature has not been reported. The understanding of the impairment induced by diabetes on endothelial function in HCC and HPRA is crucial to plan therapeutic strategies for its reversal. Inhibition of PDE5 activity in human penile smooth muscle is able to treat many men with erectile dysfunction [13–15]. PDE5 inhibition enhances the NO/cGMP pathway by inhibiting cGMP hydrolysis, causing its accumulation in penile smooth muscle, which facilitates its relaxation [25,26] and therefore, penile erection [27]. However, although sildenafil has been shown to be efficacious for treating ED in some diabetic patients, the percentage of efficacy in this population is clearly reduced when compared to non-diabetic men [16,28,29]. Indeed, the existence of ED and concomitant diabetes is a prognostic factor for a poor response to sildenafil [30]. Lower efficacy of PDE5 inhibition in diabetic patients with ED has also been reported with the new PDE5 inhibitors tadalafil [17] and vardenafil [18]. The mechanisms responsible for this poor clinical response are not understood. Here we show that the PDE5 inhibitor sildenafil normalizes endothelium-dependent relaxation in diabetic HCC when compared to responses in non-diabetic HCC. This observation agrees with previous studies performed in diabetic rabbits: the PDE5 inhibitor, zaprinast, produced similar relaxant responses in corpus cavernosum from diabetic and non-diabetic rabbits [31] and the treatment with sildenafil enhanced SNP- and electrical field stimulation-induced relaxation of diabetic rabbit corpus cavernosum and restored cGMP levels in this tissue [32]. However, sildenafil was not effective in HPRA. This suggests that enhancement of the NO/cGMP pathway with sildenafil is not enough to reverse the impairment of endothelium-dependent relaxation in diabetic HPRA. This is likely due to the fact that an important component of endothelium-dependent relaxation in HPRA involves a different pathway, EDHF, which, as we show in this study, is also impaired by diabetes and it is unaffected by sildenafil. Although other factors could participate, the lack of complete recovery of endothelium-dependent relaxation in penile arteries from diabetic patients with PDE5 inhibition could explain the lower clinical efficacy of these agents in diabetic men with ED. Dobesilate is an angioprotective agent that has been shown to be effective in the treatment of diabetic retinopathy [33]. In addition, DOBE is able to potentiate endothelium-dependent relaxation of rabbit aorta [34] and diabetic rat aorta [35]. Different mechanisms have been proposed to explain the effects of DOBE, including its ability to increase endothelial NO synthase activity [36] to reduce aldose reductase activity [37], and its antioxidant capacity in vitro [38] and in vivo [39]. We have previously demonstrated that DOBE specifically enhances endothelium-dependent relaxation of HPRA attributed to EDHF while having no effect on
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endothelium-dependent relaxation of HCC where EDHF has no role [8]. In the present study, we observed enhancement of endothelium-dependent and EDHFmediated relaxation by DOBE in penile arteries from diabetic and non-diabetic patients. Furthermore, DOBE was able to eliminate the differences between endothelium-dependent relaxation of diabetic and non-diabetic arteries, showing its capability to reverse diabetes-induced endothelial dysfunction in HPRA. The results obtained with DOBE are consistent with a recent study showing that chronic treatment with evening primrose oil did not improve endothelium-dependent relaxation of corpus cavernosum from diabetic rats while this treatment recovered endothelial function in mesenteric resistance vessels from these animals by enhancing EDHF-mediated responses [40], in a similar way that DOBE differentially affects endothelial function in HCC and HPRA. In conclusion, diabetes impairs endothelium-dependent relaxation of HCC, where only NO is involved, and in HPRA, where EDHF and NO participate. Indeed, we report that EDHF-mediated relaxation is impaired in penile arteries from diabetic men. Sildenafil, that potentiates the NO/cGMP pathway, restores endothelial function in corpus cavernosum tissue from diabetic patients but has no significant effects in penile arteries from these patients. EDHF-mediated responses are potentiated by DOBE, which completely recovers endothelial function in penile arteries from diabetic patients. These observations could explain the poor clinical response to PDE5 inhibitors in diabetic men with ED and we hypothesize that the combination of DOBE and a PDE5 inhibitor could be beneficial in the oral treatment of diabetic ED.
Acknowledgments We thank Rocıo Gonzalez-Corrochano for her technical assistance. This work was partially supported by a Grant from Laboratorios Dr. Esteve.
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Diabetes impairs endothelium-dependent relaxation of human penile vascular tissues mediated by NO and EDHF Javier Angulo,a,b Pedro Cuevas,b Argentina Fern andez,b Sonia Gabancho,a,b a a Antonio Allona, Antonio Martın-Morales, Ignacio Moncada,a Sebasti an Videla,c and I~ nigo S aenz de Tejadaa,b,* a
Fundaci on para la Investigaci on y el Desarrollo en Andrologıa. Madrid 28304, Spain b Departamento de Investigaci on, Hospital Ram on y Cajal, Madrid, Spain c Departamento de Investigaci on Clınica, Laboratorios Dr. Esteve, Barcelona, Spain Received 4 November 2003
Abstract Standard treatments for erectile dysfunction (ED) (i.e., PDE5 inhibitors) are less effective in diabetic patients for unknown reasons. Endothelium-dependent relaxation (EDR) of human corpus cavernosum (HCC) depends on nitric oxide (NO), while in human penile resistance arteries (HPRA) endothelium-derived hyperpolarizing factor (EDHF) and NO participate. Here we show that diabetes significantly reduced EDR induced by acetylcholine (ACh) in HCC and HPRA. Relaxation attributed to EDHF was also impaired in HPRA from diabetic patients. The PDE5 inhibitor, sildenafil (10 nM), reversed diabetes-induced endothelial dysfunction in HCC, but not in HPRA. Calcium dobesilate (DOBE; 10 lM) fully reversed diabetes-induced endothelial dysfunction in HPRA by specifically potentiating the EDHF-mediated component of EDR. Impairment by diabetes of NO and EDHF-dependent responses precluded the complete recovery of endothelial function in HPRA by sildenafil. This could explain the poor clinical response to PDE5 inhibitors of diabetic men with ED and suggests that a pharmacological approach that combines enhancement of NO/cGMP and EDHF pathways could be necessary to treat ED in many diabetic men. Ó 2003 Elsevier Inc. All rights reserved. Keywords: Human corpus cavernosum; Human penile resistance arteries; Diabetes; Erectile dysfunction; Endothelium-derived hyperpolarizing factor; Calcium dobesilate; Sildenafil
The endothelium is an important contributor to penile smooth muscle relaxation (both arterial and trabecular), and therefore, penile erection. Diabetes is associated with endothelial dysfunction [1,2] as well as with a high incidence of erectile dysfunction [3]. In fact, in aging and other vascular diseases associated with endothelial dysfunction such as hypercholesterolemia and hypertension, a high prevalence of erectile dysfunction is also observed [4]. Nitric oxide (NO) is a key mediator of endotheliumdependent relaxation (EDR). However, the existence of an unidentified endothelial factor that promotes smooth muscle hyperpolarization and relaxation and is resistant to NO synthase and cyclooxygenase inhibition has been * Corresponding author. Fax: +34-91-358-50-45. E-mail address: [email protected] (I.S. Tejada).
0006-291X/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2003.11.034
clearly established (see [5] for review) and it has particular functional relevance in small arteries [6,7]. We have recently demonstrated that, while in human corpus cavernosum endothelium-dependent relaxation is mediated by NO, in human penile resistance arteries, in addition to NO, endothelium-derived hyperpolarizing factor (EDHF) contributes significantly to endotheliumdependent relaxation [8]. In patients, in several vascular beds, it has been shown that diabetes impairs endothelium-dependent relaxation mediated by NO [1,2,9], including HCC [10], but alteration by diabetes of EDHF-mediated responses has only been reported in animal models [11,12]. Type 5 phosphodiesterase (PDE5) inhibitors potentiate NO-mediated responses by enhancing intracellular cGMP accumulation and constitute an efficacious oral therapy for the treatment of erectile dysfunction [13–15].
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However, although these compounds show efficacy to treat erectile dysfunction in some diabetic patients, the efficacy is significantly lower than that observed in the general population of impotent patients [16–18], for reasons that are not presently known. Calcium dobesilate (DOBE) potentiates endotheliumdependent relaxation of human penile arteries by specifically enhancing the responses mediated by EDHF [8]. Thus, if diabetes impaired EDHF-mediated responses DOBE could potentially improve vasodilation via this pathway. With these premises, the aims of the present study were to characterize endothelial function in human corpus cavernosum and penile resistance arteries from diabetic and non-diabetic impotent men and to evaluate the effects of enhancing the NO/cGMP pathway with the PDE5 inhibitor sildenafil and of potentiating EDHFmediated responses with DOBE on endotheliumdependent relaxation in penile tissues from these patients.
Materials and methods Human penile tissues. Human penile tissue biopsies were obtained from 18 impotent diabetic (12 with type 1 diabetes and 6 with type 2 diabetes) and 25 non-diabetic impotent men who gave informed consent at the time of penile prosthesis insertion. The study was approved by the local Ethics Committee. Tissues were maintained at 4–6 °C in M-400 solution (composition per 100 ml: mannitol, 4.19 g; KH2 PO4 , 0.205 g; K2 HPO4 3H2 O, 0.97 g; KCl, 0.112 g; and NaHCO3 , 0.084 g) until used, which was between 2 and 16 h from extraction [19]. There were no significant differences in the age of diabetic and non-diabetic patients from whom the biopsies were collected (59.8 1.5 and 58.6 1.8 years, respectively). The presence of co-morbid conditions was similar in the diabetic and non-diabetic groups. Hypertension was present in four diabetic patients (22%) and four non-diabetic patients (16%), hypercholesterolemia was present in five diabetic patients (28%) and four non-diabetic patients (16%), smoking habit was present in 10 diabetic patients (56%) and 11 non-diabetic patients (44%), and neurological alterations were present in three diabetic patients (17%) and two non-diabetic patients (8%) (p > 0:05 for all comparisons). Vascular reactivity of human penile resistance arteries. Penile small arteries, helicine arteries (lumen diameter 150–400 lm), which are the terminal branches of deep penile arteries, were dissected by carefully removing the adhering trabecular tissue, and arterial ring segments (2 mm long) were subsequently mounted on two 40 lm wires on microvascular double Halpern–Mulvany myographs (J.P. Trading, Aarhus, Denmark) for isometric tension recordings. The vessels were allowed to equilibrate for 30 min in physiological salt solution (PSS) of the following composition (mM): NaCl 119, KCl 4.6, CaCl2 1.5, MgCl2 1.2, NaHCO3 24.9, glucose 11, KH2 PO4 1.2, and EDTA 0.027 at 37 °C continuously bubbled with 95% O2 /5% CO2 mixture to maintain a pH of 7.4. Passive tension and internal circumference of vascular segments when relaxed in situ under a transmural pressure of 100 mmHg (L100 ) were determined. The arteries were then set to an internal circumference equivalent to 90% of L100 , at which the force development was close to maximal [20]. The preparations were then exposed to 125 mM Kþ (KPSS, equimolar substitution of NaCl for KCl in PSS) and the contractile response was measured. The arteries were contracted with 1 lM norepinephrine (80% of KPSS induced contraction approximately) and relaxation responses were evaluated by cumulative additions of compounds to the chambers.
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Experiments with human corpus cavernosum tissue. Strips of corpus cavernosum tissue (3 3 7 mm) were immersed in 8 ml organ chambers containing PSS, maintained at 37 °C, and aerated with 5% CO2 /95% O2 , pH 7.4. Each tissue strip was incrementally stretched to optimal isometric tension, as determined by maximal contractile response to 1 lM phenylephrine [21]. Tissues were contracted with 0.5–3 lM phenylephrine (80% of KPSS induced contraction) and relaxation responses were evaluated by cumulative additions of compounds to the chambers. Drugs and materials. Phenylephrine, norepinephrine (arterenol), acetylcholine, indomethacin, and NG -nitro-L -arginine (L -NNA) were obtained from Sigma Chemical (St. Louis, MO). Calcium dobesilate (calcium dihydroxy-2,5 benzenesulfonate, Doxium) was provided by Dr. Esteve Laboratories (Barcelona, Spain). Sildenafil was a gift from Nitromed (Bedford, MA). For in vitro experiments, all drugs were dissolved in deionized water, except for sildenafil which was dissolved at 10 mM concentration in DMSO. The subsequent dilutions were made in deionized water. Data analysis. Relaxation responses are expressed as percentage of total relaxation (loss in tone) induced by the addition of 0.1 mM papaverine HCl to the chambers at the end of the experiment. All data are expressed as means SE. Complete concentration–response curves were obtained and compared by a two-factor analysis of variance (ANOVA) statistical test using StatView software for Apple computers.
Results Effects of diabetes on endothelium-dependent relaxation of human corpus cavernosum and penile resistance arteries Acetylcholine (ACh; 1 nM–10 lM) produced concentration-dependent relaxation of HCC and HPRA. The responses to ACh were significantly attenuated in tissues from diabetic patients when compared to the responses in tissues from non-diabetic patients (Figs. 1A and B). We have previously identified that in HPRA, but not in HCC, there is a significant component in the relaxation response to ACh that is resistant to NO synthase (NOS) and cyclooxygenase (COX) inhibition and is attributed to EDHF [8]. In HPRA, ACh-induced relaxation, in the presence of the NOS inhibitor, NG -nitroL -arginine (L -NNA; 100 lM), and the inhibitor of COX, indomethacin (5 lM), was significantly attenuated in tissues from diabetic patients compared to responses in tissues from non-diabetic patients (Fig. 1C). Comparison of effects of PDE5 inhibition with sildenafil on endothelium-dependent relaxation in tissues from diabetic and non-diabetic patients Inhibition of PDE5 activity with sildenafil (10 nM) significantly potentiated ACh-induced relaxation in HCC from non-diabetic as well as from diabetic patients (Fig. 2). Furthermore, in the presence of sildenafil, the responses to ACh were normalized as there were no longer differences in endothelium-dependent relaxation of diabetic and non-diabetic HCC (Fig. 2). Sildenafil (10 nM) was also able to clearly potentiate ACh-induced relaxations in HPRA from non-diabetic patients (Fig. 3). In contrast, sildenafil (10 nM) did not significantly
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Fig. 1. Effects of diabetes on the endothelium-dependent relaxation induced by acetylcholine (ACh; 1 nM–10 lM) on phenylephrine-contracted human corpus cavernosum strips (A) and norepinephrine-contracted human penile resistance arteries in the absence (B) or the presence (C) of combined inhibition of NO synthase and cyclooxygenase with NG -nitro-L -arginine (L -NNA; 100 lM) and indomethacin (INDO; 5 lM), respectively. Data are expressed as means SEM of the percentage of total relaxation induced by 0.1 mM papaverine. n indicates the number of patients from whom the tissues were collected for the experiments. *** indicates p < 0:005 vs no diabetes by a two-factor ANOVA test.
Fig. 2. Effects of the treatment with sildenafil (10 nM) on the endothelium-dependent relaxation induced by acetylcholine (ACh; 1 nM– 10 lM) on phenylephrine-contracted human corpus cavernosum strips from non-diabetic and diabetic patients. Data are expressed as means SEM of the percentage of total relaxation induced by 0.1 mM papaverine. n indicates the number of patients from whom the tissues were collected for the experiments. *** indicates p < 0:005 vs responses in non-diabetic tissues and yy p < 0:01, yyy p < 0:005 vs respective untreated tissues by a two-factor ANOVA test.
enhance endothelium-dependent relaxation of HPRA from diabetic patients (Fig. 3). After sildenafil treatment, ACh-induced relaxation in diabetic HPRA still showed attenuated responses when compared to nondiabetic tissues (Fig. 3). Furthermore, endothelium-dependent relaxation attributable to EDHF in diabetic HPRA, after NOS and COX inhibition, was unaffected
Fig. 3. Effects of the treatment with sildenafil (10 nM) on the endothelium-dependent relaxation induced by acetylcholine (ACh; 1 nM– 10 lM) on norepinephrine-contracted human penile resistance arteries from non-diabetic and diabetic patients. Data are expressed as means SEM of the percentage of total relaxation induced by 0.1 mM papaverine. n indicates the number of patients from whom the tissues were collected for the experiments. ** indicates p < 0:01 and ***p < 0:005 vs responses in non-diabetic arteries and yyy indicates p < 0:005 vs respective untreated arteries by a two-factor ANOVA test.
by sildenafil (Fig. 4). Thus, sildenafil was not able to reverse diabetes-induced endothelial dysfunction in HPRA from diabetic men. Comparison of the effects of calcium dobesilate on endothelium-dependent relaxation of human penile resistance arteries from diabetic and non-diabetic patients Treatment with DOBE (10 lM) did not significantly modify ACh-induced relaxation of HCC from diabetic
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Fig. 4. Effects of the treatment with sildenafil (10 nM) on the endothelium-dependent relaxation induced by acetylcholine (ACh; 1 nM– 10 lM) on norepinephrine-contracted human penile resistance arteries from diabetic patients in the presence of L -NNA (100 lM) plus indomethacin (INDO; 5 lM). Data are expressed as means SEM of the percentage of total relaxation induced by 0.1 mM papaverine. n indicates the number of patients from whom the tissues were collected for the experiments.
Fig. 5. Effects of the treatment with calcium dobesilate (DOBE 10 lM) on the endothelium-dependent relaxation induced by acetylcholine (ACh; 1 nM–10 lM) on phenylephrine-contracted human corpus cavernosum strips from non-diabetic and diabetic patients. Data are expressed as means SEM of the percentage of total relaxation induced by 0.1 mM papaverine. n indicates the number of patients from whom the tissues were collected for the experiments. *** indicates p < 0:005 vs responses in non-diabetic tissues by a two-factor ANOVA test.
or non-diabetic patients (Fig. 5). However, DOBE (10 lM) significantly potentiated ACh-induced relaxation of HPRA from non-diabetic and diabetic patients (Fig. 6). This potentiating effect was more pronounced
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Fig. 6. Effects of the treatment with calcium dobesilate (DOBE 10 lM) on the endothelium-dependent relaxation induced by acetylcholine (ACh; 1 nM–10 lM) on norepinephrine-contracted human penile resistance arteries from non-diabetic and diabetic patients. Data are expressed as means SEM of the percentage of total relaxation induced by 0.1 mM papaverine. n indicates the number of patients from whom the tissues were collected for the experiments. *** indicates p < 0:005 vs responses in non-diabetic arteries and yyy indicates p < 0:005 vs respective untreated arteries by a two-factor ANOVA test.
Fig. 7. Effects of the treatment with calcium dobesilate (DOBE 10 lM) on the endothelium-dependent relaxation induced by acetylcholine (ACh; 1 nM–10 lM) on norepinephrine-contracted human penile resistance arteries from non-diabetic and diabetic patients in the presence of L -NNA (100 lM) plus indomethacin (INDO; 5 lM). Data are expressed as means SEM of the percentage of total relaxation induced by 0.1 mM papaverine. n indicates the number of patients from whom the tissues were collected for the experiments. *** indicates p < 0:005 vs control responses in non-diabetic arteries and yyy indicates p < 0:005 vs respective untreated arteries by a two-factor ANOVA test.
in HPRA from diabetic patients. Indeed, diabetes-induced endothelial dysfunction was reversed by DOBE, since responses to ACh in HPRA from diabetic patients
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after treatment with DOBE were better than those in non-diabetic HPRA without DOBE treatment and comparable to those in HPRA from non-diabetic patients treated with DOBE (Fig. 6). DOBE (10 lM) also specifically enhanced the endothelium-dependent relaxation resistant to NOS and COX inhibition in HPRA from non-diabetic and diabetic patients (Fig. 7). Furthermore, treatment with DOBE fully reversed diabetes-induced impairment of EDHF-mediated relaxation of HPRA (Fig. 7).
Discussion The prevalence of severe ED in diabetic men is almost threefold higher than in the general population [3]. Diabetes mellitus is a main independent risk factor for ED in epidemiological studies [4]. The pathophysiology of diabetic ED could, at least in part, be related to the presence of endothelial dysfunction as would suggest the high prevalence of ED in other diseases associated with endothelial dysfunction such as hypertension, hypercholesterolemia, or aging [4]. Furthermore, in patients with type 2 diabetes the presence of ED correlated with endothelial dysfunction [22]. Here we show that endothelium-dependent relaxation is impaired in both human penile corpus cavernosum (HCC) and penile resistance arteries (HPRA) from diabetic patients. Defective endothelium-dependent relaxation of diabetic HCC has been previously reported [10] and the present study shows that diabetes also impairs endothelium-dependent relaxation in HPRA. However, we have recently reported substantially different mechanisms in the endothelium-dependent relaxation of HPRA and HCC. While in HCC tissue endothelium-dependent relaxation is essentially mediated by nitric oxide (NO), endothelium-derived hyperpolarizing factor (EDHF) is responsible for an important component of acetylcholine-induced relaxation of HPRA [8]. This observation is consistent with the results obtained in horse penile resistance arteries [23] and with the relevant role of relaxations mediated by EDHF in other human resistance vessels [6,7]. The contribution of EDHF to endothelium-dependent relaxation of HPRA led us to evaluate the impact of diabetes on EDHF-mediated responses in these vessels. A significant finding of the present study is that endothelium-dependent relaxation, specifically attributable to EDHF, is attenuated in HPRA from diabetic patients. In diabetic rats, defective EDHF-mediated relaxation has been reported in mesenteric [11] and renal [12] microvasculature, and it has been suggested that diabetes has a greater impact on endothelial function in arteries with EDHF-mediated responses than in those depending almost exclusively on NO action [24]. However, the influence of diabetes on EDHF-mediated responses in
human vasculature has not been reported. The understanding of the impairment induced by diabetes on endothelial function in HCC and HPRA is crucial to plan therapeutic strategies for its reversal. Inhibition of PDE5 activity in human penile smooth muscle is able to treat many men with erectile dysfunction [13–15]. PDE5 inhibition enhances the NO/cGMP pathway by inhibiting cGMP hydrolysis, causing its accumulation in penile smooth muscle, which facilitates its relaxation [25,26] and therefore, penile erection [27]. However, although sildenafil has been shown to be efficacious for treating ED in some diabetic patients, the percentage of efficacy in this population is clearly reduced when compared to non-diabetic men [16,28,29]. Indeed, the existence of ED and concomitant diabetes is a prognostic factor for a poor response to sildenafil [30]. Lower efficacy of PDE5 inhibition in diabetic patients with ED has also been reported with the new PDE5 inhibitors tadalafil [17] and vardenafil [18]. The mechanisms responsible for this poor clinical response are not understood. Here we show that the PDE5 inhibitor sildenafil normalizes endothelium-dependent relaxation in diabetic HCC when compared to responses in non-diabetic HCC. This observation agrees with previous studies performed in diabetic rabbits: the PDE5 inhibitor, zaprinast, produced similar relaxant responses in corpus cavernosum from diabetic and non-diabetic rabbits [31] and the treatment with sildenafil enhanced SNP- and electrical field stimulation-induced relaxation of diabetic rabbit corpus cavernosum and restored cGMP levels in this tissue [32]. However, sildenafil was not effective in HPRA. This suggests that enhancement of the NO/cGMP pathway with sildenafil is not enough to reverse the impairment of endothelium-dependent relaxation in diabetic HPRA. This is likely due to the fact that an important component of endothelium-dependent relaxation in HPRA involves a different pathway, EDHF, which, as we show in this study, is also impaired by diabetes and it is unaffected by sildenafil. Although other factors could participate, the lack of complete recovery of endothelium-dependent relaxation in penile arteries from diabetic patients with PDE5 inhibition could explain the lower clinical efficacy of these agents in diabetic men with ED. Dobesilate is an angioprotective agent that has been shown to be effective in the treatment of diabetic retinopathy [33]. In addition, DOBE is able to potentiate endothelium-dependent relaxation of rabbit aorta [34] and diabetic rat aorta [35]. Different mechanisms have been proposed to explain the effects of DOBE, including its ability to increase endothelial NO synthase activity [36] to reduce aldose reductase activity [37], and its antioxidant capacity in vitro [38] and in vivo [39]. We have previously demonstrated that DOBE specifically enhances endothelium-dependent relaxation of HPRA attributed to EDHF while having no effect on
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endothelium-dependent relaxation of HCC where EDHF has no role [8]. In the present study, we observed enhancement of endothelium-dependent and EDHFmediated relaxation by DOBE in penile arteries from diabetic and non-diabetic patients. Furthermore, DOBE was able to eliminate the differences between endothelium-dependent relaxation of diabetic and non-diabetic arteries, showing its capability to reverse diabetes-induced endothelial dysfunction in HPRA. The results obtained with DOBE are consistent with a recent study showing that chronic treatment with evening primrose oil did not improve endothelium-dependent relaxation of corpus cavernosum from diabetic rats while this treatment recovered endothelial function in mesenteric resistance vessels from these animals by enhancing EDHF-mediated responses [40], in a similar way that DOBE differentially affects endothelial function in HCC and HPRA. In conclusion, diabetes impairs endothelium-dependent relaxation of HCC, where only NO is involved, and in HPRA, where EDHF and NO participate. Indeed, we report that EDHF-mediated relaxation is impaired in penile arteries from diabetic men. Sildenafil, that potentiates the NO/cGMP pathway, restores endothelial function in corpus cavernosum tissue from diabetic patients but has no significant effects in penile arteries from these patients. EDHF-mediated responses are potentiated by DOBE, which completely recovers endothelial function in penile arteries from diabetic patients. These observations could explain the poor clinical response to PDE5 inhibitors in diabetic men with ED and we hypothesize that the combination of DOBE and a PDE5 inhibitor could be beneficial in the oral treatment of diabetic ED.
Acknowledgments We thank Rocıo Gonzalez-Corrochano for her technical assistance. This work was partially supported by a Grant from Laboratorios Dr. Esteve.
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