Chronic Administration of Sildenafil Modified the Impaired VEGF System and Improved the Erectile Function in Rats with Diabetic Erectile Dysfunction

IntroductionMen frequently develop diabetic erectile dysfunction (DMED), as a result of endothelial dysfunction. DMED patients often have reduced efficacy with phosphodiesterase type 5 inhibitors therapy.AimTo determine whether chronic sildenafil administration can modify the impaired vascular endothelial growth factor (VEGF) system and improve the erectile function in rats with diabetic erectile dysfunction.MethodsA group of Sprague Dawley rats (n = 30) with DMED were induced by intraperitoneal injection of streptozotocin (40 mg/kg) and screened by subcutaneous injection of Apomorphine (100 mg/kg). They were then exposed to either vehicle or sildenafil (prescribed in our hospital, 5 mg/kg and 10 mg/kg, respectively) for 10 weeks. An additional nondiabetic and age-matched control group (n = 10) was also allocated and given the routine diet for the same period. Assessments were performed to both groups at 36 hours after the last dose of sildenafil. Penile intracavernous pressure (ICP), mean arterial pressure (MAP), penile tissue morphology, immunohistologic analysis, and Western blot analysis of VEGF, VEGFR1, and eNOS were determined.Main Outcome MeasureFunctional, morphological, and proteomical changes on penile structures by the chronic Sildenafil (5 mg/kg and 10 mg/kg, respectively) administration were determined.ResultsA significant increase of ICP, ICP/MAP ratio, and area under the curve were observed in the both groups treated by sildenafil (5 mg/kg and 10 mg/kg, respectively), compared with the DMED rats without receiving Sildenafil. Immunohistochemical staining of their penile tissue showed a decrease in VEGF, VEGFR1, and eNOS staining in the controlled group compared with an improvement in the chronic sildenafil administration group. Western blot analysis demonstrated exactly the same results.ConclusionWe demonstrated that daily sildenafil administration can restore the impaired VEGF system in the penis of DMED rats and progressively improve both erectile function and endothelial function, suggesting a potential general mechanism of improved signaling through the VEGF/eNOS signaling cascade. Liu G, Sun X, Dai Y, Zheng F, Wang D, Huang Y, Bian J, and Deng C. Chronic administration of sildenafil modified the impaired VEGF system and improved the erectile function in rats with diabetic erectile dysfunction.     

Effect of the Novel BKCa Channel Opener LDD175 on the Modulation of Corporal Smooth Muscle Tone

IntroductionThe BK_Ca channel has been reported to play an important role in erectile function. Recently, novel BK_Ca channel activator, LDD175, was introduced.AimThis study aims to investigate whether LDD175 relaxes corporal smooth muscle (CSM) via BK_Ca channel activation.MethodsAfter isolation of CSM strip from a male rabbit model, contraction studies using organ bath was performed. Isolating human tissue and cell cultures, electrophysiological studies were done via whole-cell patch-clamp recording.Main Outcome MeasuresVasodilatory effects of LDD175 were evaluated by cumulative addition ranging from 10⁻⁷ to 10⁻⁴ M in corpus cavernosal strips after precontraction with 10⁻⁵ M phenylephrine via organ bath system. Using cultured human CSM cells, patch-clamp recording was performed. Erectile function was measured by in vivo rat cavernous nerve stimulation.ResultsLDD175 caused an endothelium-independent relaxation of corporal tissues, and this effect was abolished by pretreatment with iberiotoxin. The relaxation effect of 10⁻⁴ M LDD175 was greater than that of 10⁻⁶ M udenafil (54.0 ± 3.1% vs. 34.5 ± 3.9%, P < 0.05); 10⁻⁵ M LDD175 with 10⁻⁶ M udenafil caused a greater relaxation effect on strips than 10⁻⁵ M LDD175 or 10⁻⁶ M udenafil alone (50.7%, 34.1%, vs. 20.7%, respectively, P < 0.001). In patch-clamp recordings, LDD175 increased K⁺ currents in a dose-dependent manner, and washout of LDD175 or the addition of iberiotoxin fully reversed the increase. Intravenous LDD175 improved erectile function measured by area under the curve (AUC) of the intracavernosal pressure (ICP)/arterial blood pressure (ABP) ratio (1,612.1 ± 135.6 vs. 1,093.7 ± 123.1, P < 0.05). There was no difference between 10 mg/kg LDD175 and 1 mg/kg udenafil regarding maximal ICP, maximal ICP/ABP ratio, and the AUC of the ICP/ABP ratio (P > 0.05).ConclusionsLDD175 leads to an endothelium-independent relaxation of erectile tissue, primarily through the opening of BK_Ca channels. The results suggest that LDD175 might be a new candidate treatment for erectile dysfunction. Sung HH, Choo SH, Han DH, Chae MR, Kang SJ, Park C-S, So I, Park JK, and Lee SW. Effect of the novel BK_Ca channel opener LDD175 on the modulation of corporal smooth muscle tone. J Sex Med 2015;12:29–38.     

A Protein Tyrosine Kinase Inhibitor,Imatinib Mesylate (Gleevec), Improves Erectile and Vascular Function Secondary to a Reduction of Hyperglycemia in Diabetic Rats

IntroductionErectile dysfunction (ED) afflicts 50% of diabetic men, many of whom experience poor results with phosphodiesterase type 5 inhibitors. The protein tyrosine kinase (PTK) inhibitor imatinib (Gleevec, Novartis Pharmaceuticals, Basel, Switzerland) has therapeutic potential in diabetic men by maintaining β-cell function.AimTo determine if imatinib has a beneficial effect on erectile and vascular function in diabetic rats.MethodsMale Sprague-Dawley rats were divided into six groups: (i) control; (ii) imatinib (50 mg/kg, daily gavage)-treated control; (iii) diabetic; (iv) preventive imatinib (8 weeks); (v) reversal imatinib (4 weeks untreated diabetes and 4 weeks of treatment); and (vi) insulin (8 weeks)-treated diabetic rats.Main Outcome MeasuresAfter 8 weeks, all groups underwent cavernosal nerve stimulation and measurements of intracavernosal pressure (ICP) and mean arterial pressure (MAP). Contractile and relaxation responses were evaluated using isolated strips of corpus cavernosum smooth muscle (CCSM) and aorta.ResultsDiabetic rats exhibited a 32% decrease in weight and fivefold increase in blood glucose levels. Imatinib-treated diabetic rats gained weight and partially improved blood glucose levels. Diabetic rats displayed a decrease in ICP/MAP. While maximum electrical field stimulation- and acetylcholine (ACh)-induced relaxations in CCSM strips from the diabetics were reduced, preventive imatinib or insulin treatment normalized ICP/MAP ratios and improved relaxation responses. ACh responses in diabetic aortas were diminished by 50.1% and restored by imatinib. While contractile responses to phenylephrine in diabetic CCSM were not altered, there was a significant enhancement (59.4 %) in the aortic contractile response in diabetic rats, which was restored by imatinib and insulin treatment.ConclusionsIn diabetic rats, prolonged therapy with imatinib improves diabetes-related ED and vascular function, which may involve normalization of high glucose levels and restoration of PTK activation. Future studies are needed to elaborate on the actions of imatinib on diabetic vascular complications. Gur S, Kadowitz PJ, and Hellstrom WJG. A protein tyrosine kinase inhibitor, imatinib mesylate (Gleevec), improves erectile and vascular function secondary to a reduction of hyperglycemia in diabetic rats.     

Nitric Oxide‐Releasing Polymeric Microspheres Improve Diabetes‐Related Erectile Dysfunction

IntroductionWe have used a long‐acting nitric oxide (NO)‐releasing polymer to develop injectable biodegradable microspheres capable of localized NO release over prolonged periods of time.AimThe aim of this study was to evaluate the therapeutic potential of these microspheres for diabetes‐related erectile dysfunction (ED) in the rat model.MethodsNO‐releasing microspheres were incubated in physiologic buffer, and in vitro NO release was measured using a Griess assay. To ensure no migration, microspheres were fluorescently tagged and injected into the corpus cavernosum of adult rats, and fluorescent imaging was performed weekly for 4 weeks, at which point rats were sacrificed. To assess physiologic efficacy, diabetes was induced in 40 rats using streptozotocin (STZ), whereas 10 rats were kept as age‐matched controls. Diabetic rats were divided into four groups: no treatment, sildenafil, NO‐releasing microspheres, and combination therapy. For each rat, the cavernosal nerve (CN) was stimulated at various voltages, and intracavernosal pressure (ICP) and mean arterial pressure (MAP) were measured via corpus cavernosum and carotid artery catheterization, respectively. Long‐term efficacy was determined by injecting diabetic rats with microspheres and measuring erectile response at predetermined intervals for up to 5 weeks.Main Outcome MeasuresErectile response was determined via calculation of mean peak ICP/MAP and area under curve (AUC) for each experimental group.ResultsUnder physiologic conditions in vitro, microspheres continued NO release for up to 4 weeks. Fluorescent imaging revealed no detectable signal in tissues besides cavernosal tissue at 4 weeks postinjection. Upon CN stimulation, peak ICP/MAP ratio and AUC of diabetic rats improved significantly (P < 0.05) in microsphere and combination therapy groups compared with no treatment and sildenafil groups. In long‐term efficacy studies, microspheres augmented the effect of sildenafil for 3 weeks following injection (P < 0.05).ConclusionsNO‐releasing microspheres significantly improved erectile response in diabetic rats for 3 weeks and hence offer a promising approach to ED therapy, either as monotherapy or combination therapy. Soni SD, Song W, West JL, and Khera M. Nitric oxide‐releasing polymeric microspheres improve diabetes‐related erectile dysfunction. J Sex Med 2013;10:1915‐1925.     

Vardenafil and resveratrol synergistically enhance the nitric oxide/cyclic guanosine monophosphate pathway in corpus cavernosal smooth muscle cells and its therapeutic potential for erectile dysfunction in the streptozotocin-induced diabetic rat: preliminary findings

IntroductionPhosphodiesterase type 5 (PDE5) inhibitors are very effective agents for erectile dysfunction; however, specific patient populations are hard to treat. The efficacy of PDE5 inhibitors is limited because a minimum amount of nitric oxide (NO) is necessary. Resveratrol, a plant polyphenol, is reported to activate endothelial NO synthase (eNOS) through activation of sirtuin 1. We previously reported that human corpus cavernosal smooth muscle cells (CCSMCs) express eNOS and synthesize cyclic guanosine monophosphate (cGMP) via the NO/cGMP pathway.AimTo investigate the ability of resveratrol and/or vardenafil to increase cGMP in an in vitro model using CCSMCs and to improve erectile function in an in vivo rat model of streptozotocin (STZ)‐induced diabetes.MethodsCCSMCs were treated with resveratrol and/or vardenafil. Twenty male Sprague‐Dawley rats were randomly divided into five groups (N = 4 in each group): age‐matched controls, diabetic controls, and diabetic rats treated with resveratrol, vardenafil, or both in combination for the last 4 weeks of an 8‐week period of diabetes induction.Main Outcome MeasuresIntracellular cGMP measurement, intracovernous pressure (ICP)/mean arterial pressure (MAP) ratio, and smooth muscle/collagen ratio.ResultsIntracellular cGMP level was elevated by resveratrol treatment in CCSMCs. The combination treatment of resveratrol and vardenafil had a synergistic effect. Diabetic rats showed impairment of erectile function. Treatment with either resveratrol or vardenafil improved ICP/MAP ratio, and combination therapy with resveratrol and vardenafil had a synergistic effect in improvement of ICP/MAP.ConclusionsTreatment with either resveratrol or vardenafil elevated cGMP level in CCSMCs and improved erectile function in STZ‐induced diabetic rats. Furthermore, a synergistic effect was observed in vitro and in vivo. Resveratrol or combination therapy of resveratrol and vardenafil can improve erectile function in which NO release is impaired, although further study is needed to confirm the results. Fukuhara S, Tsujimura A, Okuda H, Yamamoto K, Takao T, Miyagawa Y, Nonomura N, and Okuyama A. Vardenafil and resveratrol synergistically enhance the nitric oxide/cyclic guanosine monophosphate pathway in corpus cavernosal smooth muscle cells and its therapeutic potential for erectile dysfunction in the streptozotocin‐induced diabetic rat: Preliminary findings.     

The Selectivity and Potency of the New PDE5 Inhibitor TPN729MA

IntroductionTPN729MA is a newly developed phosphodiesterase type 5 inhibitor (PDE5i) for the treatment of erectile dysfunction, which offers potential for greater selectivity and longer duration of action than PDE5i in current clinical use.AimWe investigated the in vitro inhibitory potency and selectivity of TPN729MA on PDE isozymes, and its efficacy in animal models.MethodsThe inhibition of 11 human recombinant PDEs by TPN729MA, sildenafil, and tadalafil were determined using radioimmunoassay. The effect of TPN729MA and sildenafil on intracavernous pressure (ICP), blood pressure (BP), and ICP/BP ratio were determined in a rat model of erection induced by electric stimulation and in a dog model of erection induced by sodium nitroprusside injection.Main Outcome MeasuresThe main outcome measures were IC₅₀ of TPN729MA, sildenafil, and tadalafil for PDE1-PDE11; maximum ICP; BP and ICP/BP ratio.ResultsThe IC₅₀ of TPN729MA, sildenafil, and tadalafil for PDE5 was 2.28, 5.22, and 2.35 nM, respectively. TPN729MA showed 248, 366, 20, and 2671-fold selectivity against PDE1, PDE4, PDE6, and PDE11, respectively. TPN729MA showed excellent selectivity against PDE2, 3, 7, 8, 9, and 10 (>10,000-fold). In the rat model of erection, TPN729MA (5.0 and 2.5 mg/kg), but not sildenafil, significantly increased the maximum ICP compared with vehicle. Significantly increased ICP/BP was observed in the TPN729MA (5.0 mg/kg) group at all time points, in the TPN729MA (2.5 mg/kg) group at 75, 90, 105, and 120 minutes time points, and in sildenafil group at 75 and 90 minutes time points compared with vehicle. In the dog model of erection, TPN729MA and sildenafil significantly increased ICP and ICP/BP but showed no significant effect on BP compared with vehicle.ConclusionsTPN729MA is a potent PDE5i with a balanced selectivity profile. TPN729MA shows excellent in vitro and in vivo potency, and a longer effect on erectile function than sildenafil in animal model. Wang Z, Zhu D, Yang X, Li J, Jiang X, Tian G, Terrett NK, Jin J, Wu H, He Q, Yang B, and Shen J. The selectivity and potency of the new PDE5 Inhibitor TPN729MA. J Sex Med 2013;10:2790–2797.     

Nebivolol Dilates Human Penile Arteries and Reverses Erectile Dysfunction in Diabetic Rats through Enhancement of Nitric Oxide Signaling

IntroductionTraditional beta-blockers have sometimes been associated with erectile dysfunction (ED). Nebivolol is a cardioselective β₁-adrenoceptor antagonist that promotes vasodilation through a nitric oxide (NO)-dependent mechanism.AimWe evaluated the effects of nebivolol on the NO/cyclic guanosine monophosphate (cGMP) signaling pathway, on erectile function and dysfunction, and in human penile vascular tissues.MethodsErectile response to cavernosal nerve electrical stimulation in control and diabetes-induced ED rats were evaluated, along with serum nitrite/nitrate (NOx) concentration and plasma/tissue cGMP levels. Endothelium-dependent and sildenafil-induced relaxation of isolated human corpus cavernosum (HCC) and human penile resistance arteries (HPRA) were also determined.Main Outcome MeasuresThe effects of nebivolol on erectile function and dysfunction and on NO/cGMP-mediated responses.ResultsTreatment with nebivolol significantly potentiated erectile response in control rats, regardless of its effects on blood pressure. Nebivolol increased NOx and plasma cGMP by 3-fold and 2.75-fold, respectively, and significantly augmented the elevation of plasma cGMP produced by sildenafil. Nebivolol enhanced endothelium-dependent and sildenafil-induced relaxations of HCC tissue, and produced endothelium-dependent vasodilation of HPRA. Nebivolol, but not atenolol, significantly improved erectile response in diabetic rats (51.6%, 53.2%, and 87.1% of response at 3 Hz in nondiabetic rats, for vehicle-treated, atenolol-treated, and nebivolol-treated diabetic rats, respectively); after sildenafil administration, ED was completely reversed in nebivolol-treated diabetic rats (69.6% and 112% for diabetic rats treated with sildenafil and nebivolol plus sildenafil, respectively). Accordingly, nebivolol restored systemic NOx levels and cGMP content in penile tissue from these animals.ConclusionsNebivolol in vivo activated the NO/cGMP pathway, enhanced erectile response and reversed ED in diabetic rats. Moreover, nebivolol in vitro potentiated NO/cGMP-mediated relaxation of human erectile tissues. These effects may account for the low incidence of ED in nebivolol-treated hypertensive patients. Nebivolol therefore may have utility in the treatment of ED, particularly ED associated with diabetes. Angulo J, Wright HM, Cuevas P, González-Corrochano R, Fernández A, Cuevas B, La Fuente JM, Gupta S, and de Tejada IS. Nebivolol dilates human penile arteries and reverses erectile dysfunction in diabetic rats through enhancement of nitric oxide signaling.     

Effect of Mucuna pruriens (Linn.) on oxidative stress-induced structural alteration of corpus cavernosum in streptozotocin-induced diabetic rat

IntroductionErectile dysfunction is one of the major secondary complications of diabetes. Mucuna pruriens (M. pruriens), a leguminous plant identified for its antidiabetic, aphrodisiac, and fertility enhancing properties, has been the choice of Indian traditional medicine.AimThe objective of the present study was to analyze the efficacy of M. pruriens on free radicals‐mediated penile tissue alterations in hyperglycemic male rats.MethodsMale albino rats were divided as group I (sham) control, group II (STZ) diabetes‐induced (streptozotocin 60 mg/kg of body weight [bw] in 0.1 M citrate buffer), group III (STZ + MP) diabetic rats administered with 200 mg/kg bw of ethanolic extract of M. pruriens seed, group IV (STZ + SIL) diabetic rats administered with 5 mg/kg bw of sildenafil citrate, group V (sham + MP) administered with 200 mg/kg bw of extract alone, and group VI (sham + SIL) administered with 5 mg/kg bw of sildenafil citrate. The M. pruriens and sildenafil citrate were given (gavage) once daily for a period of 60 days. At the end of 60 days, the animals were sacrificed and subjected to analysis of reactive oxygen species levels, enzymic and nonenzymic antioxidant levels, levels of NOx, histological, and histomorphometrical study of penile tissue.Main Outcome MeasuresRemedial use of M. pruriens seed extract on diabetes‐induced erectile tissue damage.ResultsSignificantly high levels of oxidative stress and low levels of antioxidants in the penile tissue seem to contribute to the increased collagen deposition and fibrosis of erectile tissue in STZ rats. Relatively, there was increased damage in STZ + SIL group. Supplementation of M. pruriens in STZ + MP group has revealed the potency to overcome oxidative stress, and good preservation of penile histoarchitecture.ConclusionThe ethanolic extract of M. pruriens seed significantly recovered or protected erectile tissue from the oxidative stress‐induced degeneration by its antioxidant potentials. These findings propound to serve mankind by the treatment of diabetes‐induced erectile dysfunction. Suresh S and Prakash S. Effect of Mucuna pruriens (Linn.) on oxidative stress‐induced structural alteration of corpus cavernosum in streptozotocin‐induced diabetic rat. J Sex Med 2011;8:1943–1956.     

Intracavernous transplantation of bone marrow-derived mesenchymal stem cells restores erectile function of streptozocin-induced diabetic rats

IntroductionErectile dysfunction (ED) is a frequent complication of diabetes mellitus. The efficacy of common ED therapies is low for diabetes‐associated ED.AimTo explore the effects of transplantation of bone marrow‐derived mesenchymal stem cells (BM‐MSCs) on improving erectile function of streptozocin (STZ)‐induced diabetic rats.MethodsMale Sprague Dawley rats were injected either with STZ to induce diabetes or with citrate buffer as controls. Rat BM‐MSCs were harvested and labeled with CM‐DiI (Chloromethylbenzamido derivatives of 1,1′‐dioctadecyl‐3,3,3′,3′‐tetramethylindocarbocyanine perchlorate), and then transplanted into corporal cavernosum of STZ‐induced diabetic rats. Four weeks after transplantation, all rats were analyzed for erectile function and penile histology.Main Outcome MeasuresErectile function was evaluated by the ratio between intracavernous pressure (ICP) and mean arterial pressure (MAP) during electrostimulation of cavernous nerve. Fate of transplanted BM‐MSCs was identified using immunofluorescence staining. Smooth muscle and endothelium in corpora cavernosum were assessed using immunohistochemistry.ResultsAfter BM‐MSCs transplantation, the ICP/MAP ratio was increased significantly compared with diabetic controls. Content of smooth muscle and endothelium in corporal cavernosa of BM‐MSCs transplanted rats was significantly increased compared to diabetic controls. Immunofluorescence analysis demonstrated that CM‐DiI‐labeled BM‐MSCs could stay in corporal cavernosa for at least 4 weeks and some of them expressed von Willebrand Factor, CD31, calponin, or α‐smooth muscle actin, cells markers for endothelial cells or smooth muscle cells, respectively.ConclusionIntracavernous transplantation of BM‐MSCs had beneficial effects on erectile function of diabetic rats and increased the content of endothelium and smooth muscle in corporal cavernosum. Qiu X, Lin H, Wang Y, Yu W, Chen Y, Wang R, and Dai Y. Intracavernous transplantation of bone marrow‐derived mesenchymal stem cells restores erectile function of streptozocin‐induced diabetic rats.     

Oxaliplatin,an Anticancer Agent,Causes Erectile Dysfunction in Rats due to Endothelial Dysfunction

BackgroundChemotherapeutics, one of the standard treatment options for cancer worldwide, have various adverse effects, including erectile dysfunction (ED).AimTo investigate erectile function in an animal model after administration of the anticancer agent oxaliplatin (L-OHP).MethodsMale Wistar/ST rats were divided into 2 groups: L-OHP rats (n = 21), which were intravenously administered L-OHP (4 mg/kg; twice a week for 4 weeks), and Control rats (n = 21), which were injected with the same volume of 5% glucose solution, using the same dosing schedule. At the end of the study period, erectile function was evaluated by measuring intracavernous pressure (ICP) and mean arterial pressure (MAP) after cavernous nerve stimulation (n = 9–10). Endothelial function was evaluated with an isometric tension study using corpus cavernosum strips (n = 11). Western blot analysis was used to assess neuronal nitric oxide (nNOS) and endothelial NO synthase (eNOS) protein levels (n = 7). Real-time quantitative polymerase chain reaction (qRT-PCR) was used to assess the expression of inflammation- and oxidative stress-related markers (nicotinamide adenine dinucleotide phosphate oxidase-1, p22^phox, interleukin [IL]-6, and nuclear factor-kappa B) (n = 6). Statistical significance was determined using the Student's t-test.OutcomesThe L-OHP group had a significantly lower ICP:MAP ratio than the control group (P < .05). Compared to the Control group, the L-OHP group exhibited significantly lower responses to ACh and eNOS protein levels and significantly higher inflammatory biomarker levels.Clinical TranslationThe results based on this animal model indicate that use of the anticancer agent L-OHP should be considered as a risk factor for ED occurring via reduction of NO bioavailability in humans; our results provide possible treatment strategies for maintaining the erectile function of cancer survivors.Strengths and limitationsOur study showed that the anticancer agent L-OHP has the propensity to cause ED in rats. A major limitation of this study is the lack of an established cure for ED associated with L-OHP and the lack of clinical evidence.ConclusionsL-OHP causes ED in rats via reduction of NO bioavailability caused by endothelial dysfunction.Kataoka T, Mori T, Suzuki J, et al. Oxaliplatin, an Anticancer Agent, Causes Erectile Dysfunction in Rats due to Endothelial Dysfunction. J Sex Med 2021;18:1337–1345.     

Chronic vardenafil treatment improves erectile function via structural maintenance of penile corpora cavernosa in rats with acute arteriogenic erectile dysfunction

IntroductionChronic phosphodiesterase type 5 inhibitor treatment may be useful in reversing erectile dysfunction (ED). However, the mechanisms of this improvement remain unknown.AimThe aim of this article was to determine the mechanisms of the improvement by chronic vardenafil treatment for acute arteriogenic ED in rats.MethodsEight‐week‐old male Wistar‐ST rats were divided into four groups: sham‐operated rats (Control group) and rats with acute arteriogenic ED induced by ligating bilateral internal iliac arteries (Ligation group), subsequently treated with low‐dose (0.4 mg/kg/day; VL group) or high‐dose (4.0 mg/kg/day; VH group) vardenafil for 20 days from 1 week after ligature.Main Outcome MeasuresErectile function was assessed based on changes of intracavernous pressure (ICP) followed by electrostimulation of the cavernous nerves and was evaluated by the area under the curve of ICP/area under the curve of mean arterial pressure (area of ICP/MAP). Transforming growth factor (TGF)‐β₁, vascular endothelial growth factor‐A, endothelial nitric oxide synthase (eNOS), inducible NOS, and neuronal NOS mRNA expression levels in penile corpus cavernosum were determined by real‐time PCR. Western blotting for TGF‐β₁ protein levels and Masson trichrome staining of penile tissues were performed in each at group 4 weeks after surgery.ResultsIn the VH group, area of ICP/MAP was significantly improved when compared with the Ligation group (P < 0.01). The smooth muscle (SM)/collagen ratio in the VH group was significantly higher than in the Ligation group (P < 0.05), and was comparable with that in the Control group. TGF‐β₁ mRNA and protein levels in the VH group were significantly lower when compared with the Ligation group (P < 0.05).ConclusionsChronic vardenafil administration ameliorates impairment of penile hemodynamics and maintains normal SM to collagen ratio in cavernous tissues after acute arterial injury in rats. Hotta Y, Hattori M, Kataoka T, Ohno R, Mikumo M, Maeda Y, and Kimura K. Chronic vardenafil treatment improves erectile function via structural maintenance of penile corpora cavernosa in rats with acute arteriogenic erectile dysfunction. J Sex Med 2011;8:705–711.     

Intracavernosal OnabotulinumtoxinA Exerts a Synergistic Pro-Erectile Effect When Combined With Sildenafil in Spontaneously Hypertensive Rats

BackgroundBotulinum toxin A (BTX-A) has a variety of uses in medicine. Some evidence suggests that intracavernosal (ic) BTX-A injection administered in addition to phosphodiesterase type 5 inhibitors (PDE5-Is) could effectively treat erectile dysfunction (ED) in insufficient responders to PDE5-Is.AimTo provide experimental pharmacological evidence for the use of onabotulinumtoxinA ic alone or in combination with PDE5-Is for difficult-to-treat ED. We thus compared the effects of BTX-A ic alone and BTX-A ic combined with PDE5-I iv, and a placebo treatment ic or iv.MethodsErectile function was evaluated following cavernous nerve electrical stimulation (6 V, 1-millisecond pulse, 45-second duration) at different frequencies (0, 2, 3, 4, 5, 7.5, and 10 Hz) in 4 groups (n = 8 / group) of anesthetized, spontaneously hypertensive rats, a robust animal model of ED of vascular origin. Rats were treated by onabotulinumtoxinA 10U or saline ic 1 week prior to erectile function testing and sildenafil (0.3 mg/kg) or saline iv 4 minutes prior to testing. Frequency-response curves were compared with a 2 way ANOVA.OutcomesBoth onabotulinumtoxinA ic, and sildenafil iv significantly improved erectile responses in spontaneously hypertensive rats, however the effect was greatly amplified when the treatments were combined.ResultsIntracavernosal pressure and/or mean arterial pressure ratios were significantly increased by sildenafil and onabotulinumtoxinA ic versus the control condition. OnabotulinumtoxinA 10U ic combined with sildenafil iv significantly potentiated erectile responses. Area under the curve and/or mean arterial pressure ratio increased by 19% with sildenafil iv, by 15% with onabotulinumtoxinA ic and by 58% with the combined treatment following cavernous nerve electrical stimulation at 6V, 1 ms, 10 Hz: these stimulation parameters elicited the maximal erectile response.Clinical TranslationThese data provide a pharmacological rationale for the combined administration of onabotulinumtoxinA ic and sildenafil iv since the effects of both treatments were potentiated when their administration was combined.Strengths & LimitationsFirst evidence of a synergistic pro-erectile effect of BTX-A combined with PDE5-I, however the mechanism behind the pro-erectile effect of BTX-A ic remains hypothetical.ConclusionsThese results support further studies into the mechanisms behind the pro-erectile effect of BTX-A ic, as well as multicenter randomized control trials to evaluate the safety and efficacy of BTX-A ic combined with sildenafil for difficult-to-treat ED.Giuliano F., Joussain C., Denys P., et al. Intracavernosal OnabotulinumtoxinA Exerts a Synergistic Pro-Erectile Effect When Combined With Sildenafil in Spontaneously Hypertensive Rats. J Sex Med 2022;19:899–906.     

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

IntroductionOne of the proposed mechanisms responsible for diabetes-related erectile dysfunction (ED) is overactivity of RhoA/ROCK signaling, as seen in experimental models of chemical diabetes.AimBecause statins may interfere with RhoA/Rho-kinase (ROCK) signaling through the reduction of geranyl-geranyl pyrophosphate (GGPP), required for RhoA activation, we investigated whether atorvastatin ameliorated diabetes-related ED.MethodsStreptozotocin-induced (8 weeks) diabetic rats and alloxan-induced (8 weeks) diabetic rabbits received atorvastatin (5 mg/kg daily) for the last 2 weeks. In vitro contractility studies were conducted in the rabbit model. In the rat model, sildenafil effect on electrical stimulation (ES)-induced erection was investigated. Atorvastatin action was also analyzed using human fetal penile smooth muscle cells (hfPSMCs) exposed to low (5 mM), high (22 mM), and very high (40 mM) glucose.Main Outcome MeasuresAtorvastatin effect on hyperglicemia-induced RhoA/ROCK signaling was evaluated using the ROCK inhibitor Y-27632 in both animal models and by analyzing functional effects downstream to RhoA activation in hfPSMCs.ResultsIn both diabetic models, atorvastatin did not affect glycemia, lipid plasma levels, and the hypogonadal state. In diabetic rats, atorvastatin ameliorated the erectile response to the ES of the cavernous nerve and normalized sildenafil effect on erectile function, strongly decreased by diabetes. In penile tissue from diabetic animals, atorvastatin completely restored the diabetes-induced hypersensitivity to Y-27632 and prevented RhoA membrane translocation/activation. In hfPSMCs, high glucose significantly increased not only membrane RhoA expression, but also ROCK activity (increased phosphorylation of the ROCK substrate myosin phosphatase target subunit 1) and several RhoA-dependent functions such as proliferation, migration, and smooth muscle-related gene expression. Atorvastatin restored all the high-glucose-induced effects, an action specifically reverted by GGPP.ConclusionAtorvastatin improves diabetes-related ED and restores sildenafil responsiveness, most probably by inhibiting RhoA/ROCK signaling, which underlies several high-glucose-induced derangements in penile smooth muscle cell commitment. Morelli A, Chavalmane AK, Filippi S, Fibbi B, Silvestrini E, Sarchielli E, Zhang X-H, Vignozzi L, Vannelli GB, Forti G, and Maggi M. Atorvastatin ameliorates sildenafil-induced penile erections in experimental diabetes by inhibiting diabetes-induced RhoA/Rho-kinase signaling hyperactivation. J Sex Med 2009;6:91–106.     

P144, A TGF‐β1 Antagonist Peptide,Synergizes with Sildenafil and Enhances Erectile Response via Amelioration of Cavernosal Fibrosis in Diabetic Rats

IntroductionPatients with diabetes exhibit more severe erectile dysfunction (ED) and are less responsive to first‐line oral phosphodiesterase type 5 inhibitor (PDE5i). It has been suggested that increased collagen deposition and reduced smooth muscle content in the corpus cavernosum are important mechanisms for diabetes‐associated ED and that transforming growth factor‐β1 (TGF‐β1) is a potent fibrotic factor responsible for the structural alterations in the corpus cavernosum.AimsThe aims of this study are to determine whether activation of TGF‐β1 and its downstream pathways is responsible for the reduced efficacy of the PDE5is in diabetic ED via abnormalities in cavernosal structures and to investigate the synergistic effects of the TGF‐β1 antagonist P144 and sildenafil on erectile response.MethodsSix weeks after inducting diabetes with streptozotocin in male Sprague‐Dawley rats, age‐matched control and diabetic rats were treated with vehicle, sildenafil, or P144 alone or in combination for 4 weeks, respectively.Main Outcome MeasuresIntracavernous pressure, dynamic infusion cavernosometry, and histological and molecular alterations of the corpus cavernosum were analyzed.ResultsDiabetic rats exhibited a decreased erectile response, severe corporal veno‐occlusive dysfunction (CVOD), and structural alterations including cavernosal fibrosis and decreased smooth muscle content. Expression and activation of TGF‐β1 and its downstream Smad and non‐Smad pathways increased in diabetic rats. Treatment with sildenafil showed modest effect on erectile response and a less suppressive effect on CVOD, cavernosal fibrosis, and molecular alterations. Treatment with P144 had lower effect on erectile response, even greatly improved the histological and molecular alterations and CVOD than sildenafil. The combined treatment with P144 and sildenafil effectively restored erectile response, CVOD, and histological and molecular alterations.ConclusionAn insufficient suppressive effect of sildenafil on cavernosal fibrosis, severe CVOD, and TGF‐β1 pathways was implicated in reduced efficacy of the PDE5i in diabetic ED. Treatment with P144 synergized sildenafil and significantly increased erectile response by the potential antifibrotic activity. Li WJ, Wang H, Zhou J, Li B, Zhang J, Lu M, and Wang Z. P144, a TGF‐β1 antagonist peptide, synergizes with sildenafil and enhances erectile response via amelioration of cavernosal fibrosis in diabetic rats. J Sex Med 2013;10:2942–2951.     

AGE-Breaker ALT-711 Plus Insulin Could Restore Erectile Function in Streptozocin-Induced Type 1 Diabetic Rats

IntroductionThe interaction between advanced glycation end-products (AGEs) and its receptors for AGEs (RAGEs) elicits oxidative stress and mediates the development of erectile dysfunction (ED). ALT-711, an AGE cross-link breaker, has the therapeutic potential for ED but has been less intensively investigated.AimThe aim of this study was to investigate the effects of an AGEs breaker 3-phenacyl-4,5-dimethylthiazolium chloride (ALT-711) plus insulin on erectile function in streptozocin (STZ)-induced type 1 diabetic rats.MethodsFifty 8-week-old Sprague-Dawley rats were randomly distributed into five groups: normal control (C), diabetic (D), insulin-treated diabetic (D + I), ALT-711-treated diabetic (D + ALT-711) and insulin plus ALT-711-treated diabetic (D + I + ALT-711) rats. Diabetes was induced by a single intraperitoneal injection of STZ. Eight weeks after induction of diabetes, ALT-711 was administered by intraperitoneal injection. Two to six units of intermediate-acting insulin were utilized to achieve normal levels of glycemic control. After treatment for 6 weeks, erectile function was determined via measurement of intracavernous pressures (ICPs) following electrostimulation of the cavernous nerve. The deposition of AGEs, expression of RAGEs, superoxide dismutase activity, and lipid peroxidation were measured. We also evaluated penile histological changes such as smooth muscle contents, endothelial cells contents, and apoptotic activity.Main Outcome MeasuresThe main outcome measures were the ratio of ICP/mean arterial pressure (MAP), penile endothelial cells, smooth muscle cells, neuronal nitric oxide synthase, AGE and RAGE expression, malondialdehyde concentration, SOD activity, and apoptosis index.ResultsDiabetic rats demonstrated significantly reduced ICP/MAP ratio, penile endothelial cells, smooth muscles cells, increased AGEs and RAGE expression, and increased apoptosis. Insulin and ALT-711 monotherapy partially restored erectile function and histological changes. However, the combination therapy group showed erectile parameters and components similar to those in C. ALT-711-treated group demonstrated less deposition of AGEs and lower expression of RAGE than those in insulin-treated group.ConclusionThese results suggest that although insulin can effectively control glycemic levels, it does not completely alter the pathological changes in erectile tissues. Better efficacy could be expected with tight glycemic control plus ALT-711, an AGEs cross-link breaker. The combination therapy might have the potential to eliminate metabolic memory by down-regulating the AGEs–RAGE oxidative stress axis. Wang L, Tian W, Uwais Z, Li G, Li H, Guan R, Gao Z, and Xin Z. AGE-breaker ALT-711 plus insulin could restore erectile function in streptozocin-induced type 1 diabetic rats. J Sex Med 2014;11:1452–1462.     

Investigation of the Effects of the Level of Glycemic Control on Erectile Function and Pathophysiological Mechanisms in Diabetic Rats

IntroductionPoor glycemic control is associated with erectile dysfunction (ED); however, differences in ED according to the level of glycemic control have been poorly investigated.AimThe aim of this paper is to investigate the change in erectile function according to the level of glycemic control and to clarify the pathophysiological mechanism of diabetes-associated ED.MethodsStreptozotocin was injected into 55 male Sprague-Dawley rats classified into four groups: control (group 1), diabetes with multiple insulin injections (group 2), diabetes with a single injection (group 3), and untreated diabetes (group 4). Daily insulin injections in groups 2 and 3 were administered for 4 weeks after 10 weeks of diabetic induction.Main Outcome MeasuresThe main outcome measures are the anova or Kruskal–Wallis tests to evaluate glycosylated hemoglobin (HbA1c), testosterone levels, the ratios of intracavernosal pressure to mean arterial pressure (ICP/MAP), area under the ICP curve to MAP (AUC/MAP), and changes in cavernous tissue and protein expression related to Rho kinase and nitric oxide pathways.ResultsHbA1c levels were different between pairs of groups. Group 4 showed the lowest erectile parameters and group 2 showed near normal level. No differences in erectile parameters were found between groups 1 and 2 or between groups 3 and 4, except the ratio of AUC to MAP for group 1 was significantly higher than that of group 2 (20 Hz stimulation). Decrease in erectile function of group 2 was related to decreased expression of nitrergic nitric oxide synthase or decreased testosterone level compared with group 1. Groups 2 and 3 showed significant differences in erectile parameters, which were associated with difference in apoptotic index. Groups 3 and 4 showed no differences in erectile parameters, although these groups had significant differences in apoptotic index, smooth muscle component, and protein expression ratios of phosphorylated to total myosin phosphatase target subunit 1, endothelial nitric oxide synthase, and Akt.ConclusionsImprovement in glycemic control assists recovery from diabetes-associated ED; however, only tight glycemic control can provide recovery from ED to a near normal status. Cho SY, Chai JS, Lee SH, Park K, Paick J-S, and Kim SW. Investigation of the effects of the level of glycemic control on erectile function and pathophysiological mechanisms in diabetic rats. J Sex Med 2012;9:1550–1558.     

Apocynin Improves Erectile Function in Diabetic Rats through Regulation of NADPH Oxidase Expression

IntroductionDiabetes is a risk factor for erectile dysfunction (ED). The proposed mechanisms responsible for diabetic ED are associated with an increase in reactive oxygen species (ROS) production, overactivity of RhoA/ROCK signaling pathway and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, as seen in experimental models of diabetic rats.AimThe aim of this study was to investigate whether NADPH oxidase inhibitor apocynin can ameliorate Streptozotocin (STZ)‐induced diabetes‐related ED by reducing the ROS production and inhibiting the activity of RhoA/ROCK signaling pathway.MethodsThe diabetic rats were treated with and without the NADPH oxidase inhibitor apocynin.Main Outcome MeasuresErectile responses were evaluated by determining mean arterial blood pressure (MAP) and intracavernosal pressure (ICP) with electrical stimulation of the cavernous nerve. Levels of mRNA expression were measured by real‐time polymerase chain reaction (RT‐PCR). Levels of protein expression were examined by Western Blot. ROS production was measured by dihydroethidium (DHE) staining and thiobarbituric acid reactive substances assay.ResultsThe ratio of Maximum ICP‐to‐MAP (MaxICP/MAP) was significantly decreased in diabetic ED rats, compared to that of age‐matched control rats (P < 0.05). Apocynin improved erectile function of diabetic rats (P < 0.05). Expression levels of RhoA (cytosol), nNOS and eNOS were reduced, compared to those of control rats (P < 0.05). Apocynin significantly elevated their expression levels in diabetic rats (P < 0.05). Expression levels of ROCK1, RhoA (membrane fraction), p‐MYPT1 and NADPH oxidase subunits p47^phox and p67^phox were increased in diabetic rats when compared to those of control rats (P < 0.05), and it was observed that apocynin significantly reduced their expression levels in diabetic rats (P < 0.05). ROS production was increased in diabetic rats when compared to that of control rats (P < 0.05), the effect of apocynin was a reduction in the ROS production in diabetic rats (P < 0.05).ConclusionNADPH oxidase inhibitor apocynin can ameliorate diabetes‐related ED by reducing the ROS production and inhibiting the activity of RhoA/ROCK signaling pathway. Li M, Zhuan L, Wang T, Rao K, Yang J, Yang J, Quan W, Liu J, and Ye Z. Apocynin improves erectile function in diabetic rats through regulation of NADPH oxidase expression. J Sex Med 2012;9:3041–3050.     

Effects of Low‐Energy Shockwave Therapy on the Erectile Function and Tissue of a Diabetic Rat Model

IntroductionLow‐energy shockwave therapy (LESWT) has been shown to improve erectile function in patients suffering from diabetes mellitus (DM)‐associated erectile dysfunction (ED). However, the underlying mechanism remains unknown.AimThe aim of this study is to investigate whether LESWT can ameliorate DM‐associated ED in a rat model and examine the associated changes in the erectile tissues.MethodsNewborn male rats were intraperitoneally injected with 5‐ethynyl‐2‐deoxyuridine (EdU; 50 mg/kg) for the purpose of tracking endogenous mesenchymal stem cells (MSCs). Eight weeks later, eight of these rats were randomly chosen to serve as normal control (N group). The remaining rats were injected intraperitoneally with 60 mg/kg of streptozotocin (STZ) to induce DM. Eight of these rats were randomly chosen to serve as DM control (DM group), whereas another eight rats were subject to shockwave (SW) treatment (DM+SW group). Each rat in the DM+SW group received 300 shocks at energy level of 0.1 mJ/mm² and frequency of 120/minute. This procedure was repeated three times a week for 2 weeks. Another 2 weeks later, all 24 rats were evaluated for erectile function by intracavernous pressure (ICP) measurement. Afterward, their penile tissues were examined by histology.Main Outcome MeasuresErectile function was measured by ICP. Neuronal nitric oxide synthase (nNOS)‐positive nerves and the endothelium were examined by immunofluorescence staining. Smooth muscle and MSCs were examined by phalloidin and EdU staining, respectively.ResultsSTZ treatment caused a significant decrease in erectile function and in the number of nNOS‐positive nerves and in endothelial and smooth muscle contents. These DM‐associated deficits were all partially but significantly reversed by LESWT. MSCs (EdU‐positive cells) were significantly more numerous in DM+SW than in DM rats.ConclusionLESWT can partially ameliorate DM‐associated ED by promoting regeneration of nNOS‐positive nerves, endothelium, and smooth muscle in the penis. These beneficial effects appear to be mediated by recruitment of endogenous MSCs. Qiu X, Lin G, Xin Z, Ferretti L, Zhang H, Lue TF, and Lin C‐S. Effects of low‐energy shockwave therapy on the erectile function and tissue of a diabetic rat model. J Sex Med 2013;10:738–746.     

Erectile Dysfunction in Young Non-Obese Type II Diabetic Goto-Kakizaki Rats is Associated with Decreased eNOS Phosphorylation at Ser1177

IntroductionDiabetes mellitus (DM) is a risk factor for erectile dysfunction (ED). Although type 2 DM is responsible for 90–95% diabetes cases, type 1 DM experimental models are commonly used to study diabetes-associated ED.AimGoto-Kakizaki (GK) rat model is relevant to ED studies since the great majority of patients with type 2 diabetes display mild deficits in glucose-stimulated insulin secretion, insulin resistance, and hyperglycemia. We hypothesized that GK rats display ED which is associated with decreased nitric oxide (NO) bioavailability.MethodsWistar and GK rats were used at 10 and 18 weeks of age. Changes in the ratio of intracavernosal pressure/mean arterial pressure (ICP/MAP) after electrical stimulation of cavernosal nerve were determined in vivo. Cavernosal contractility was induced by electrical field stimulation (EFS) and phenylephrine (PE). In addition, nonadrenergic-noncholinergic (NANC)- and sodium nitroprusside (SNP)-induced relaxation were determined. Cavernosal neuronal nitric oxide synthase (nNOS) and endothelial nitric oxide synthase (eNOS) mRNA and protein expression were also measured.Main Outcome MeasureGK diabetic rats display ED associated with decreased cavernosal expression of eNOS protein.ResultsGK rats at 10 and 18 weeks demonstrated impaired erectile function represented by decreased ICP/MAP responses. Ten-week-old GK animals displayed increased PE responses and no changes in EFS-induced contraction. Conversely, contractile responses to EFS and PE were decreased in cavernosal tissue from GK rats at 18 weeks of age. Moreover, GK rats at 18 weeks of age displayed increased NANC-mediated relaxation, but not to SNP. In addition, ED was associated with decreased eNOS protein expression at both ages.ConclusionAlthough GK rats display ED, they exhibit changes in cavernosal reactivity that would facilitate erectile responses. These results are in contrast to those described in other experimental diabetes models. This may be due to compensatory mechanisms in cavernosal tissue to overcome restricted pre-penile arterial blood supply or impaired veno-occlusive mechanisms. Carneiro FS, Giachini FRC, Carneiro ZN, Lima VV, Ergul A, Webb RC, and Tostes RC. Erectile dysfunction in young non-obese type II diabetic Goto-Kakizaki rats is associated with decreased eNOS phosphorylation at Ser¹¹⁷⁷.     

Oral l‐Citrulline Supplementation Improves Erectile Function in Rats with Acute Arteriogenic Erectile Dysfunction

IntroductionOral l‐citrulline supplementation increases serum l‐arginine levels more efficiently than l‐arginine itself and increases nitric oxide (NO) production.AimTo investigate whether oral l‐citrulline supplementation improves erectile function in rats with acute arteriogenic erectile dysfunction (ED).MethodsWe divided 8‐week‐old male Wistar‐ST rats into 3 groups: sham‐operated rats (control group), arteriogenic ED rats who underwent ligation of both internal iliac arteries (ligation group), and arteriogenic ED rats receiving oral 2% l‐citrulline water supplementation (citrulline group). Citrulline water was given to arteriogenic ED rats for 3 weeks from 1 week after surgery. Erectile function was evaluated by maximum intracavernous pressure/mean arterial pressure (ICP/MAP) ratios via cavernous nerve stimulation at 4 weeks after surgery. Then, the penises were resected, stained with Masson's trichrome, and observed microscopically. Serum nitrogen oxides (NOx) levels were measured by high‐performance liquid chromatography. Bonferroni's multiple t‐test was used for statistical analysis.Main Outcome MeasuresThe main outcome measures were changes in ICP/MAP, smooth muscle (SM)/collagen ratios, and NOx levels following l‐citrulline supplementation.ResultsThe ICP/MAP ratio in the ligation group was significantly lower than that in the control group (P < 0.05), denoting ED. The ICP/MAP ratio of the citrulline group was significantly higher than that of the ligation group (P < 0.05), indicating ED amelioration. Levels of NOx in the ligation group were significantly lower than in the control group (P < 0.05), while those in the citrulline group were significantly higher than in the ligation group (P < 0.05). SM/collagen ratios in the ligation group were significantly lower than in the control group (P < 0.05), while ratios in the citrulline group were significantly higher than those in the ligation group (P < 0.05).ConclusionsOral l‐citrulline supplementation improved ICP/MAP and SM/collagen ratios and increased NOx. Therefore, oral l‐citrulline supplementation might be a useful novel therapy for acute arteriogenic ED. Shiota A, Hotta Y, Kataoka T, Morita M, Maeda Y, and Kimura K. Oral l‐citrulline supplementation improves erectile function in rats with acute arteriogenic erectile dysfunction. J Sex Med 2013;10:2423–2429.