Chronic Treatment with an Oral Rho‐Kinase Inhibitor Restores Erectile Function by Suppressing Corporal Apoptosis in Diabetic Rats

IntroductionIt has been suggested that the up‐regulation of the contractile RhoA/Rho‐kinase (ROCK) signaling pathway is one of the important mechanisms for diabetes‐associated erectile dysfunction (ED). However, the exact role of RhoA/ROCK signaling in the pathogenesis of diabetes‐related ED has not been fully delineated.AimTo determine whether the RhoA/ROCK pathway is involved in the regulation of corporal apoptosis and whether administration of insulin or fasudil, a specific ROCK inhibitor, could ameliorate ED in streptozotocin‐induced diabetic rats.Main Outcome MeasuresAt 16 weeks after diabetes induction, erectile function was assessed by cavernous nerve stimulation. Penile tissue was assessed for apoptosis with terminal deoxynucleotidyl transferase‐mediated 2′‐deoxyuridine 5′‐triphosphate (dUTP) nick end labeling assay. Expression of myosin phosphatase target subunit 1 (MYPT1), protein kinase B (Akt), and phospho‐endothelial nitric oxide synthase (eNOS) were evaluated by Western blot. Immunohistochemical study was carried out for smooth muscle alpha‐actin, B‐cell leukemia/lymphoma 2 (Bcl‐2), and Bcl‐2‐associated X Protein (Bax). Activity of caspase‐3 and phosphatase and tensin homolog deleted on chromosome ten (PTEN) was also determined.MethodsMale Sprague‐Dawley rats (8 weeks old) were randomly divided into four groups: age‐matched controls, diabetic controls, and diabetic rats treated with insulin (10 U/day, subcutaneous injection) or fasudil (30 mg/kg/day, oral) for the last 4 weeks of the 16 weeks after diabetes induction.ResultsDiabetic rats showed impairment of erectile function, increased MYPT1 phosphorylation, and corporal apoptosis. Expression of phospho‐Akt, phospho‐eNOS, and Bcl‐2 were decreased, whereas activity of PTEN and caspase‐3 and expression of Bax were increased. Treatment with fasudil normalized these molecular and histologic alterations, and restored erectile function. Insulin treatment showed similar effects to those of fasudil, however, the effects were smaller than fasudil.ConclusionsThis study indicates that up‐regulation of the penile RhoA/ROCK pathway in diabetic rats enhances corporal apoptosis via the PTEN/Akt pathway resulting in ED, which could be prevented by chronic treatment with fasudil. Li WJ, Park K, Paick J‐S, and Kim SW. Chronic treatment with an oral rho‐kinase inhibitor restores erectile function by suppressing corporal apoptosis in diabetic rats.     

Effect of Chronic Administration of PDE5 Combined with Glycemic Control on Erectile Function in Streptozotocin‐Induced Diabetic Rats

IntroductionChronic treatment with phosphodiesterase type 5 inhibitors (PDE5) is effective in an animal model of diabetes‐induced erectile dysfunction (DMED). In addition, recent research indicates that glycemic control can restore DMED.AimsWe evaluated the effect of chronic administration of PDE5 combined with glycemic control on DMED.MethodsSprague‐Dawley rats (8 weeks old) were divided into five groups (n = 10 each): normal control (C), diabetes (DM), DM treated with insulin (DM‐I), DM treated with PDE5 (DM‐P), and DM treated with insulin and PDE5 (DM‐I + P). Rats in the diabetic groups received an injection of streptozotocin (45 mg/kg). After 10 weeks of induced diabetes, the DM‐I group was treated with a daily injection of neutral protamine Hagedorn, and the DM‐P group was treated with a daily dosage of 20 mg/kg PDE5 (DA‐8159) for 4 weeks. The DM‐I + P group was treated with both treatments simultaneously. After 14 weeks of induced diabetes, an evaluation of erectile function and histological and biochemical markers of corporal tissue was performed.Main Outcome MeasuresErectile function and histological and biochemical markers in corporal tissue.ResultsRats in the DM group showed markedly lower erectile parameters than those in the C group, whereas rats in the DM‐I and DM‐P groups showed intermediate erectile function between the DM and C groups. Rats in the DM‐I + P group showed restored erectile function, comparable with group C. A comparison of apoptotic index, expression of the endothelial marker, and phosphorylation of endothelial nitric oxide synthase and Akt displayed a similar pattern with the results from cavernosometry (DM < DM‐I = DM‐P < DM‐I + P = C, P < 0.05). The distribution of phosphorylated myosin phosphatase target subunit 1 was in the reverse order.ConclusionsChronic administration of PDE5 or glycemic control with insulin resulted in restoration of overt DMED. The combination of both treatments was superior to monotherapy with insulin or PDE5. Choi WS, Kwon OS, Cho SY, Paick J‐S, and Kim SW. Effect of chronic administration of PDE5 combined with glycemic control on erectile function in streptozotocin‐induced diabetic rats. J Sex Med 2015;12:600–610.     

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.     

Hydroxyl Fasudil,an Inhibitor of Rho Signaling,Improves Erectile Function in Diabetic Rats: A Role for Neuronal ROCK

IntroductionThe pathogenesis of diabetic erectile dysfunction (ED) includes neuropathy, but the molecular basis for neurogenic ED is incompletely understood. The RhoA/ROCK pathway has been implicated in diabetic neuropathy and in ED, but its role in diabetic neurogenic ED is not known.AimsThe aim of this study was to determine whether hydroxyl fasudil, a ROCK inhibitor, affects diabetic neuropathy‐related ED.MethodsType 1 diabetes mellitus was induced in male rats by streptozotocin (75 mg/kg, intraperitoneally). After 8 weeks, diabetic rats were administered hydroxyl fasudil, a selective ROCK inhibitor (10 mg/kg/day, intraperitoneally) or vehicle, for 4 weeks. Age‐matched control, nondiabetic, rats were treated intraperitoneally for 4 weeks with saline. At week 12, after a 2 day washout, neuro‐stimulated erectile function was evaluated. Major pelvic ganglia (MPG) were collected for Western blot analysis of RhoA, ROCK‐1, ROCK‐2, phospho (P)‐AKT (Ser⁴⁷³), and P‐phosphatase and tensin homolog (P‐PTEN) (Ser³⁸⁰/Thr^382/383).Main Outcome MeasuresEffect of ROCK inhibitor hydroxyl fasudil on erectile function and ROCK/P‐AKT/P‐PTEN pathway in the MPG of diabetic rats.ResultsErectile response was significantly (P < 0.05) reduced in diabetic rats compared with nondiabetic rats and was preserved (P < 0.05) in diabetic rats treated with hydroxyl fasudil. In diabetic rats, RhoA and ROCK‐2 protein expressions in MPG were increased (P < 0.05) and remained increased in hydroxyl fasudil‐treated rats. P‐AKT (Ser⁴⁷³) expression was decreased (P < 0.05), whereas P‐PTEN (Ser³⁸⁰/Thr^382/383) expression was increased (P < 0.05) in MPG of diabetic rats compared with nondiabetic rats, and both were reversed (P < 0.05) in diabetic rats treated with hydroxyl fasudil.ConclusionImproved erectile function and restored P‐AKT and P‐PTEN in the MPG with hydroxyl fasudil treatment suggest the role of Rho signaling via PTEN/AKT pathway in neurogenic diabetic ED. Sezen SF, Lagoda G, Musicki B, and Burnett AL. Hydroxyl fasudil, an inhibitor of Rho signaling, improves erectile function in diabetic rats: A role for neuronal ROCK. J Sex Med 2014;11:2164‐2171.     

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.     

STIM/Orai Inhibition as a Strategy for Alleviating Diabetic Erectile Dysfunction Through Modulation of Rat and Human Penile Tissue Contractility and in vivo Potentiation of Erectile Responses

BackgroundStromal interaction molecule (STIM)/Orai calcium entry system appears to have a role in erectile dysfunction (ED) pathophysiology but its specific contribution to diabetic ED was not elucidated.AimTo evaluate STIM/Orai inhibition on functional alterations associated with diabetic ED in rat and human penile tissues and on in vivo erectile responses in diabetic rats.MethodsRat corpus cavernosum (RCC) strips from nondiabetic (No DM) and streptozotocin-induced diabetic (DM) rats and human penile resistance arteries (HPRA) and corpus cavernosum (HCC) from ED patients undergoing penile prosthesis insertion were functionally evaluated in organ chambers and wire myographs. Erectile function in vivo in rats was assessed by intracavernosal pressure (ICP) responses to cavernous nerve electrical stimulation (CNES). Expression of STIM/Orai elements in HCC was determined by immunofluorescence and immunoblot.Main Outcome MeasuresFunctional responses in RCC, HCC and HPRA and STIM/Orai protein expression in HCC. In vivo erectile responses to CNES.ResultsInhibition of Orai channels with YM-58483 (20 µM) significantly reduced adrenergic contractions in RCC but more effectively in DM. Thromboxane-induced and neurogenic contractions were reduced by STIM/Orai inhibition while defective endothelial, neurogenic and PDE5 inhibitor-induced relaxations were enhanced by YM-58483 (10 µM) in RCC from DM rats. In vivo, YM-58483 caused erections and attenuated diabetes-related impairment of erectile responses. YM-58483 potentiated the effects of PDE5 inhibition. In human tissues, STIM/Orai inhibition depressed adrenergic and thromboxane-induced contractions in ED patients more effectively in those with type 2 diabetes. Diabetes was associated with increased expression of Orai1 and Orai3 in ED patients.Clinical TranslationTargeting STIM/Orai to alleviate diabetes-related functional alterations of penile vascular tissue could improve erectile function and potentiate therapeutic effects of PDE5 inhibitors in diabetic ED.Strengths and LimitationsImproving effects of STIM/Orai inhibition on diabetes-related functional impairment was evidenced in vitro and in vivo in an animal model and validated in human tissues from ED patients. Functional findings were complemented with expression results. Main limitation was low numbers of human experiments due to limited human tissue availability.ConclusionsSTIM/Orai inhibition alleviated alterations of functional responses in vitro and improved erectile responses in vivo in diabetic rats, potentiating the effects of PDE5 inhibition. STIM/Orai inhibition was validated as a target to modulate functional alterations of human penile vascular tissue in diabetic ED where Orai1 and Orai3 channels were upregulated. STIM/Orai inhibition could be a potential therapeutic strategy to overcome poor response to conventional ED therapy in diabetic patients.Sevilleja-Ortiz A, El Assar M, García-Gómez B, et al. STIM/Orai Inhibition as a Strategy for Alleviating Diabetic Erectile Dysfunction Through Modulation of Rat and Human Penile Tissue Contractility and in vivo Potentiation of Erectile Responses. J Sex Med 2022;19:1733–1749.     

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.     

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.     

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.     

The Novel Antioxidant,AC3056 (2,6-di-t-butyl-4-((Dimethyl-4-Methoxyphenylsilyl)Methyloxy)Phenol), Reverses Erectile Dysfunction in Diabetic Rats and Improves NO-mediated Responses in Penile Tissue from Diabetic Men

IntroductionDiabetes is associated with a high incidence of erectile dysfunction (ED) and poor response to standard treatments. Oxidative stress could be relevant in the pathophysiology of diabetic ED.AimTo evaluate the effects of the antioxidant, AC3056 (2,6-di-t-butyl-4-((dimethyl-4-methoxyphenylsilyl)methyloxy)phenol), on diabetic ED.MethodsErectile responses to cavernosal nerve electrical stimulation were determined in streptozotocin-induced diabetic rats. Relaxation of human corpus cavernosal (HCC) tissue and penile resistance arteries (HPRA) from human cavernosal specimens was evaluated in organ chambers and myographs, respectively.Main Outcome MeasuresThe influence of AC3056 on erectile responses, lipid peroxidation, and nitrite plus nitrate serum content, and nuclear factor-κB (NF-κB) expression in penile tissue, in diabetic rats, and on endothelium-dependent and neurogenic relaxation of HCC and HPRA from diabetic patients was determined.ResultsEight weeks of diabetes caused ED in rats that was prevented by oral AC3056 (0.3% w/w in rat chow) when given from the induction of diabetes. AC3056 also prevented the diabetes-induced elevation of serum thiobarbituric acid-reactive substances (TBARS), the reduction of serum nitric oxide (NO) derivatives, and the increase of NF-κB expression. Acute oral administration of AC3056 (450 mg/kg) partially reversed ED in 8-week diabetic rats. Complete reversion of ED was achieved after 3 days of treatment with 0.3% AC3056. This effect remained after 5 weeks of treatment, but it disappeared after withdrawing for 1 week. Erectile function in diabetic rats was inversely related to serum TBARS. AC3056- (30 µM) reversed endothelial dysfunction in diabetic HCC and enhanced endothelium-dependent relaxation in diabetic HPRA and significantly potentiated neurogenic relaxation of both tissues. The reduced cGMP content in HCC from diabetic patients after exposure to acetylcholine (10 µM) was corrected by AC3056 (30 µM).ConclusionsThese results suggest that oxidative stress has a relevant role in pathophysiology of diabetic ED and provide a rationale for the use of antioxidant therapy in the treatment of ED in diabetes. Angulo J, Peiró C, Cuevas P, Gabancho S, Fernández A, González-Corrochano R, La Fuente JM, Baron AD, Chen KS, and Sáenz de Tejada I. The novel antioxidant, AC3056 (2,6-di-t-butyl-4-([dimethyl-4-methoxyphenylsilyl] methyloxy) phenol), reverses erectile dysfunction in diabetic rats and improves NO-mediated responses in penile tissue from diabetic men. J Sex Med 2009;6:373–387.     

Activated Rho Kinase Mediates Diabetes‐Induced Elevation of Vascular Arginase Activation and Contributes to Impaired Corpora Cavernosa Relaxation: Possible Involvement of p38 MAPK Activation

IntroductionActivated RhoA/Rho kinase (ROCK) has been implicated in diabetes‐induced erectile dysfunction. Earlier studies have demonstrated involvement of ROCK pathway in the activation of arginase in endothelial cells. However, signaling pathways activated by ROCK in the penis remain unclear.AimWe tested whether ROCK and p38 MAPK are involved in the elevation of arginase activity and subsequent impairment of corpora cavernosal (CC) relaxation in diabetes.MethodsEight weeks after streptozotocin‐induced diabetes, vascular functional studies, arginase activity assay, and protein expression of RhoA, ROCK, phospho‐p38 MAPK, p38 MAPK, phospho‐MYPT‐1^Thr850, MYPT‐1 and arginase levels were assessed in CC tissues from nondiabetic wild type (WT), diabetic (D) WT (WT + D), partial ROCK 2^+/? knockout (KO), and ROCK 2^+/? KO + D mice.Main Outcome MeasuresThe expression of RhoA, ROCK 1 and 2, phosphorylation of MYPT‐1^Thr850 and p38 MAPK, arginase activity/expression, endothelial‐ and nitrergic‐dependent relaxation of CC was assayed.ResultsDiabetes significantly reduced maximum relaxation (E_max) to both endothelium‐dependent acetylcholine (WT + D: E_max; 61 ± 4% vs. WT: E_max; 75 ± 2%) and nitrergic nerve stimulation. These effects were associated with increased expression of active RhoA, ROCK 2, phospho‐MYPT‐1^Thr850, phospho‐p38 MAPK, arginase II, and activity of corporal arginase (1.6‐fold) in WT diabetic CC. However, this impairment in CC of WT + D mice was absent in heterozygous ROCK 2^+/? KO + D mice for acetylcholine (E_max: 80 ± 5%) and attenuated for nitrergic nerve‐induced relaxation. CC of ROCK 2^+/? KO + D mice showed much less ROCK activity, did not exhibit p38 MAPK activation, and had reduced arginase activity and arginase II expression. These findings indicate that ROCK 2 mediates diabetes‐induced elevation of arginase activity. Additionally, pretreatment of WT diabetic CC with inhibitors of arginase (ABH) or p38 MAPK (SB203580) partially prevented impairment of ACh‐ and nitrergic nerve‐induced relaxation and elevation of arginase activity.ConclusionROCK 2, p38 MAPK and arginase play key roles in diabetes‐induced impairment of CC relaxation.     

Nebivolol Potentiates the Efficacy of PDE5 Inhibitors to Relax Corpus Cavernosum and Penile Arteries from Diabetic Patients by Enhancing the NO/cGMP Pathway

IntroductionThe efficacy of oral pharmacotherapy for erectile dysfunction (ED) (i.e., type 5 phosphodiesterase [PDE5] inhibitors) is significantly reduced in diabetic patients. Nebivolol is a selective β₁‐blocker used for treating hypertension that has been shown to increase the efficacy of sildenafil to reverse ED in diabetic rats.AimTo evaluate the effects of nebivolol on the efficacy of the PDE5 inhibitors, sildenafil, tadalafil, and vardenafil to relax human corpus cavernosum (HCC) and vasodilate human penile resistance arteries (HPRA) from diabetic patients with ED (DMED). The influence of nebivolol on the capacity of these three PDE5 inhibitors to stimulate cyclic guanosine monophosphate (cGMP) production in HCC was also evaluated.MethodsHCC and HPRA were obtained from organ donors without ED (NEND; n = 18) or patients with diabetes undergoing penile prosthesis implantation (DMED; n = 19). Relaxations of HCC strips and HPRA to sildenafil, tadalafil, and vardenafil were evaluated in organ chambers and wire myographs. cGMP content in HCC was determined by ether extraction and quantification by ELISA.Main Outcome MeasuresEffects of nebivolol on PDE5 inhibitor‐induced relaxation of HCC, vasodilation of HPRA and cGMP accumulation in HCC.ResultsTreatment with nebivolol (1 μM) significantly potentiated sildenafil‐, tadalafil‐ and vardenafil‐induced relaxations of HCC and vasodilations of HPRA from both NEND and DMED. Enhancement of relaxant capacity by nebivolol resulted in reversion of the impairment of PDE5 inhibition‐induced responses in DMED and it was accompanied by enhancing the ability of PDE5 inhibitors to increase cGMP in HCC restoring reduced cGMP levels in HCC from DMED.ConclusionsNebivolol potentiated the capacity of PDE5 inhibitors to relax vascular structures of erectile tissue from diabetic patients by enhancing the nitric oxide (NO)/cGMP pathway in these tissues. These effects suggest a potential therapeutic utility of nebivolol as an adjunct to PDE5 inhibitors for the treatment of ED associated with diabetes. Martínez‐Salamanca JI, La Fuente JM, Cardoso J, Fernández A, Cuevas P, Wright HM, and Angulo J. Nebivolol potentiates the efficacy of PDE5 inhibitors to relax corpus cavernosum and penile arteries from diabetic patients by enhancing the NO/cGMP pathway. J Sex Med 2014;11:1182–1192.     

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.     

Farnesoid X receptor activation improves erectile function in animal models of metabolic syndrome and diabetes

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

Losartan,an Angiotensin Type I Receptor,Restores Erectile Function by Downregulation of Cavernous Renin-Angiotensin System in Streptozocin-Induced Diabetic Rats

IntroductionThe high incidence of erectile dysfunction (ED) in diabetes highlights the need for good treatment strategies. Recent evidence indicates that blockade of the angiotensin type I receptor (AT1) may reverse ED from various diseases.AimTo explore the role of cavernous renin-angiotensin system (RAS) in the pathogenesis of diabetic ED and the role of losartan in the treatment of diabetic ED.MethodsThe AT1 blocker (ARB) losartan (30 mg/kg/d) was administered to rats with streptozocin (65 mg/kg)-induced diabetes. Erectile function, cavernous structure, and tissue gene and protein expression of RAS in the corpora cavernosa were studied.Main Outcome MeasureWe sought to determine the changes of cavernous RAS in the condition of diabetes and after treatment with losartan.ResultsRAS components (angiotensinogen, [pro]renin receptor, angiotensin-converting enzyme [ACE], and AT1) were expressed in cavernosal tissue. In diabetic rats, RAS components were upregulated, resulting in the increased concentration of angiotensin II (Ang II) in the corpora. A positive feedback loop for Ang II formation in cavernosum was also identified, which could contribute to overactivity of cavernous RAS in diabetic rats. Administration of losartan blocked the effect of Ang II, downregulated the expression of AT1 and Ang II generated locally, and partially restored erectile function (losartan-treated group revealed an improved intracavernous pressure/mean systemic arterial pressure ratio as compared with the diabetic group (0.480 ± 0.031 vs. 0.329 ± 0.020, P < 0.01). However, losartan could not elevate the reduced smooth muscle/collagen ratio in diabetic rats.ConclusionsThe cavernous RAS plays a role in modulating erectile function in corpora cavernosa and is involved in the pathogenesis of diabetic ED. ARB can restore diabetic ED through downregulating cavernous RAS. Yang R, Yang B, Wen Y, Fand F, Cui S, Lin G, Sun Z, Wang R, and Dai Y. Losartan, an angiotensin type I receptor blocker, restores erectile function by down-regulation of cavernous renin-angiotensin system in streptozocin-induced diabetic rats. J Sex Med 2009;6:696–707.     

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.     

Intracavernous Injection of Human Umbilical Cord Blood Mononuclear Cells Improves Erectile Dysfunction in Streptozotocin-Induced Diabetic Rats

IntroductionErectile dysfunction (ED) worsens in men with diabetes. Human umbilical cord blood (HUCB), because of its widespread availability and low immunogenicity, is a valuable source for stem cell-based therapies.AimTo determine the effect of intracavernous injection of HUCB mononuclear cells (MNCs) on ED in rats with diabetes induced by streptozotocin.MethodsThirty adult male Sprague-Dawley rats were equally divided into three groups: (i) control, (ii) diabetes induced by streptozotocin (35 mg/kg intravenously for 8 weeks), and (iii) diabetic rats treated with MNCs (1 × 10⁶ cells by intracavernosal injection). The HUCB-MNCs isolated by the Ficoll-Hypaque technique were obtained from eight healthy donors and administered to diabetic rats after 4 weeks.Main Outcome MeasuresThe ratio of intracavernosal pressure to mean arterial pressure ratio; the protein expression of endothelial and neuronal markers, such as von Willebrand factor, neuronal nitric oxide synthase, hypoxia-inducible factor-1α, and vascular endothelium growth factor; and the relative area of smooth muscle to collagen using western blotting and Masson trichrome staining were determined.ResultsDiabetic rats demonstrated a significantly decreased ratio of intracavernosal pressure to mean arterial pressure (0.26 ± 0.04; P < .01) and treatment with MNCs restored erectile function in diabetic rats (0.67 ± 0.05) compared with control rats (0.56 ± 0.02). In bath studies, neurogenic relaxant and contractile responses were significantly decreased in diabetic cavernosal tissues, which were restored by treatment. The ratio of smooth muscle to collagen was partly recovered by treatment, whereas von Willebrand factor levels were not altered in any group. Neuronal nitric oxide synthase and vascular endothelium growth factor levels were decreased, which were not restored by treatment. Increased hypoxia-inducible factor-1α protein expression in the diabetic group was completely normalized in MNC-treated diabetic samples.ConclusionThese results suggest that HUCB-MNC treatment can enhance the recovery of erectile function and promote numerous activities such the contribution of the hypoxia-inducible factor-1α and von Willebrand factor pathway to the neurogenic erectile response of diabetic rats. HUCB-MNCs in the healing process could involve an adaptive regenerative response and appear to be a potential candidate for cell-based therapy in ED of men with diabetes. It is evident that HUCB could provide a realistic therapeutic modality for the treatment of diabetic ED.     

Adrenomedullin mediates adipose tissue-derived stem cell-induced restoration of erectile function in diabetic rats

IntroductionErectile dysfunction (ED) is a major health problem. It is known that diabetic patients are more refractory to common treatments for ED.AimTo explore the better treatment for ED, we examined the effects of adipose‐derived stem cells (ASC) on ED using a diabetic rat model. We also analyzed the cytokines produced by ASC and implicated in ASC‐induced restoration of erectile function.MethodsMale Wistar rats were injected with streptozotocin (STZ) to induce diabetes. ASC or adenoviruses were injected into the penis 6 weeks after STZ administration. Erectile function, penile histology and protein expression were analyzed 4 weeks after the injection of ASC or adenoviruses.Main Outcome MeasuresIntracavernous pressure and mean arterial pressure were measured to evaluate erectile function. The morphology of the penis was analyzed by Elastica van Gieson stain and immunohistochemistry. The expression of proteins specific for vascular endothelial cells (VEC) was assessed by Western blot analysis.ResultsASC restored erectile function especially when they were cultured in medium containing growth factors for VEC. This restoration was associated with improvement in the histology of the cavernous body, and increased expression of VEC markers such as VE‐cadherin and endothelial nitric oxide synthase (eNOS). When the expression of adrenomedullin (AM), a vasoactive peptide originally isolated from human pheochromocytoma tissue, was knocked down, the effect of ASC on ED was significantly diminished. Knockdown of AM was associated with decreased expressions of VE‐cadherin and eNOS. Furthermore, overexpression of AM induced by adenovirus infection significantly improved erectile function in these diabetic rats. Overexpression of AM was associated with increased expressions of VE‐cadherin and eNOS.Conclusions.These results suggested that ASC have the potentials to restore erectile function and that AM produced by ASC plays a major role in the restoration of erectile function. Nishimatsu H, Suzuki E, Kumano S, Nomiya A, Liu M, Kume H, and Homma Y. Adrenomedullin mediates adipose tissue‐derived stem cell‐induced restoration of erectile function in diabetic rats. J Sex Med 2012;9:482–493.