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.     

Targeting NADPH Oxidase Decreases Oxidative Stress in the Transgenic Sickle Cell Mouse Penis

IntroductionSickle cell disease (SCD) is a state of chronic vasculopathy characterized by endothelial dysfunction and increased oxidative stress, but the sources and mechanisms responsible for reactive oxygen species (ROS) production in the penis are unknown.AimsWe evaluated whether SCD activates NADPH oxidase, induces endothelial nitric oxide synthase (eNOS) uncoupling, and decreases antioxidants in the SCD mouse penis. We further tested the hypothesis that targeting NADPH oxidase decreases oxidative stress in the SCD mouse penis.MethodsSCD transgenic (sickle) mice were used as an animal model of SCD. Hemizygous (hemi) mice served as controls. Mice received an NADPH oxidase inhibitor apocynin (10 mM in drinking water) or vehicle. Penes were excised at baseline for molecular studies. Markers of oxidative stress (4‐hydroxy‐2‐nonenal [HNE]), sources of ROS (eNOS uncoupling and NADPH oxidase subunits p67^phox, p47^phox, and gp91^phox), and enzymatic antioxidants (superoxide dismutase [SOD]1, SOD2, catalase, and glutathione peroxidase‐1 [GPx1]) were measured by Western blot in penes.Main Outcome MeasuresSources of ROS, oxidative stress, and enzymatic antioxidants in the SCD penis.ResultsRelative to hemi mice, SCD increased (P < 0.05) protein expression of NADPH oxidase subunits p67^phox, p47^phox, and gp91^phox, 4‐HNE‐modified proteins, induced eNOS uncoupling, and reduced Gpx1 expression in the penis. Apocynin treatment of sickle mice reversed (P < 0.05) the abnormalities in protein expressions of p47^phox, gp91^phox (but not p67^phox) and 4‐HNE, but only slightly (P > 0.05) prevented eNOS uncoupling in the penis. Apocynin treatment of hemi mice did not affect any of these parameters.ConclusionNADPH oxidase and eNOS uncoupling are sources of oxidative stress in the SCD penis; decreased GPx1 further contributes to oxidative stress. Inhibition of NADPH oxidase upregulation decreases oxidative stress, implying a major role for NADPH oxidase as a ROS source and a potential target for improving vascular function in the SCD mouse penis. Musicki B, Liu T, Sezen SF, and Burnett AL. Targeting NADPH oxidase decreases oxidative stress in the transgenic sickle cell mouse penis. J Sex Med 2012;9:1980–1987.     

TNF‐α, Erectile Dysfunction,and NADPH Oxidase‐Mediated ROS Generation in Corpus Cavernosum in High‐Fat Diet/Streptozotocin‐Induced Diabetic Rats

IntroductionPatients with diabetes‐associated erectile dysfunction (ED) are characterized by an increase in circulating tumor necrosis factor‐alpha (TNF‐α). However, no study has indicated whether and how TNF‐α plays a role in the pathogenesis of ED associated with diabetes.AimWe examined the effects and potential mechanism of infliximab (INF), a chimeric monoclonal antibody to TNF‐α, on reactive oxygen species (ROS) generation in corpus cavernosum and ED in diabetic rats.MethodsFour groups of male rats were used: age‐matched normal controls; diabetic rats induced by a high‐fat diet (HFD) combined with a single streptozotocin (STZ) injection (35 mg/kg body weight, intraperitoneal [i.p.]); nondiabetic rats receiving INF (5 mg/kg body weight/week, i.p.), and diabetic rats receiving INF. Erectile function was assessed with electrical stimulation of the cavernous nerve after 8 weeks. The blood and penile tissues were harvested for plasma biochemical determinations, serum TNF‐α measurement, penile ROS detection, and molecular assays of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunits, endothelial nitric oxide synthase (eNOS), phospho‐eNOS, and neural nitric oxide synthase (nNOS) in the penis.Main Outcome MeasuresThe effect of INF on HFD/STZ‐induced diabetic ED and NADPH oxidase‐mediated ROS generation was studied in diabetic corpus cavernosum.ResultsUntreated diabetic rats displayed significantly decreased erectile parameters, and increased plasma TNF‐α levels, penile ROS production, p47^phox and gp91^phox expression compared with nondiabetic controls. INF neutralized TNF‐α and significantly reduced ED in diabetic rats, in which marked decreases in p47^phox and gp91^phox expression and ROS generation in corpus cavernosum were noted. The ratio of phospho‐eNOS to eNOS and expression of nNOS in the penis were significantly increased in INF‐treated vs. untreated diabetic rats.ConclusionsIncreased TNF‐α expression associated with diabetes contributes to ED by promoting NAPDH oxidase‐mediated ROS generation in corpus cavernosum. INF protects against diabetic ED by neutralizing TNF‐α. Long T, Liu G, Wang Y, Chen Y, Zhang Y, and Qin D. TNF‐α, erectile dysfunction, and NADPH oxidase‐mediated ROS generation in corpus cavernosum in high‐fat diet/streptozotocin‐induced diabetic rats. J Sex Med 2012;9:1818–1831.     

Icariin Combined with Breviscapine Improves the Erectile Function of Spontaneously Hypertensive Rats

IntroductionThe impaired erectile response in spontaneously hypertensive rats (SHR) is caused by increased signaling of RhoA/Rho‐kinase and decreased signaling of nitric oxide (NO). Icariin improves erectile function via upregulating multitargets in NO/cyclic guanosine monophosphate (NO/cGMP) pathway, which breviscapine accomplishes by downregulating RhoA/Rho‐kinase pathway.AimTo investigate the effect and mechanism of icariin combined with breviscapine on the erectile function of SHR.MethodsFive 12‐week‐old male Wistar‐Kyoto (WKY) rats and 20 age‐matched male SHR were evenly randomized into WKY rats control group, SHR control group, icariin‐treated group, breviscapine‐treated group, and combined treatment group treated by vehicle, icariin, breviscapine, and icariin plus breviscapine, respectively, by gavage for four successive weeks. Maximum intracavernosal pressure/mean arterial pressure (ICPmax/MAP) and the expression of endothelial nitric oxide synthase (eNOS), neuronal nitric oxide synthase (nNOS), phosphodiesterase type 5 inhibitors (PDE5), and Rho‐associated, coiled‐coil containing protein kinase 1 and 2 (ROCK1 and ROCK2) in the cavernous tissues were determined.ResultsThe ICPmax/MAP in the combined treatment group was significantly increased compared with SHR control group, icariin‐treated group, and breviscapine‐treated group. The expression of eNOS and nNOS was significantly higher in the combined treatment group than in SHR control group, icariin‐treated group, and breviscapine‐treated group (P < 0.05). The expression of PDE5 was significantly lower in the icariin‐treated group than in SHR control group (P < 0.05). The expression of ROCK1 was significantly lower in the combined treatment group than in other groups (P < 0.05). The expression of ROCK2 was significantly higher in SHR control group than in WKY rats control group, icariin‐treated group, and combined treatment group (P < 0.05). Among these groups, the expression of eNOS and nNOS was the strongest, and ROCK1 was the lowest in WKY rats control group.ConclusionIcariin combined with breviscapine has synergistic effects on erectile function of SHR through different signal pathways. Li Y, Jiang J, He Y, Jiang R, Liu J, Fan Z, and Cheng Y. Icariin combined with Breviscapine improves the erectile function of spontaneously hypertensive rats. J Sex Med 2014;11:2143‐2152.     

Hypercholesterolemia-Induced Erectile Dysfunction: Endothelial Nitric Oxide Synthase (eNOS) Uncoupling in the Mouse Penis by NAD(P)H Oxidase

IntroductionHypercholesterolemia induces erectile dysfunction (ED) mostly by increasing oxidative stress and impairing endothelial function in the penis, but the mechanisms regulating reactive oxygen species (ROS) production in the penis are not understood.AimsWe evaluated whether hypercholesterolemia activates nicotinamide adenine dinucleotide phosphate (NAD[P]H) oxidase in the penis, providing an initial source of ROS to induce endothelial nitric oxide synthase (eNOS) uncoupling and endothelial dysfunction resulting in ED.MethodsLow-density-lipoprotein receptor (LDLR)–null mice were fed Western diet for 4 weeks to induce early-stage hyperlipidemia. Wild type (WT) mice fed regular chow served as controls. Mice received NAD(P)H oxidase inhibitor apocynin (10 mM in drinking water) or vehicle. Erectile function was assessed in response to cavernous nerve electrical stimulation. Markers of endothelial function (phospho [P]-vasodilator-stimulated-protein [VASP]-Ser-239), oxidative stress (4-hydroxy-2-nonenal [HNE]), sources of ROS (eNOS uncoupling and NAD[P]H oxidase subunits p67^phox, p47^phox, and gp91^phox), P-eNOS-Ser-1177, and eNOS were measured by Western blot in penes.Main Outcome MeasuresThe main outcome measures are the molecular mechanisms of ROS generation and endothelial dysfunction in hypercholesterolemia-induced ED.ResultsErectile response was significantly (P < 0.05) reduced in hypercholesterolemic LDLR-null mice compared with WT mice. Relative to WT mice, hypercholesterolemia increased (P < 0.05) protein expressions of NAD(P)H oxidase subunits p67^phox, p47^phox and gp91^phox, eNOS uncoupling, and 4-HNE-modified proteins, and reduced (P < 0.05) P-VASP-Ser-239 expression in the penis. Apocynin treatment of LDLR-null mice preserved (P < 0.05) maximal intracavernosal pressure, and reversed (P < 0.05) the abnormalities in protein expressions of gp67^phox and gp47^phox, 4-HNE, P-VASP-Ser-239, and eNOS uncoupling in the penis. Apocynin treatment of WT mice did not affect any of these parameters. Protein expressions of P-eNOS-Ser-1177 and total eNOS were unaffected by hypercholesterolemia.ConclusionActivated NAD(P)H oxidase in the penis is an initial source of oxidative stress resulting in eNOS uncoupling, thus providing a mechanism of eNOS uncoupling and endothelial dysfunction in hypercholesterolemia-induced ED. Musicki B, Liu T, Lagoda GA, Strong TD, Sezen SF, Johnson JM, and Burnett AL. Hypercholesterolemia-induced erectile dysfunction: Endothelial nitric oxide synthase (eNOS) uncoupling in the mouse penis by NAD(P)H oxidase.     

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.     

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.     

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.     

Superoxide Anion Production by NADPH Oxidase Plays a Major Role in Erectile Dysfunction in Middle‐Aged Rats: Prevention by Antioxidant Therapy

Introduction.Prevalence of erectile dysfunction (ED) increases progressively with aging, but the ED pathophysiology at its early stages is still poorly investigated.Aim.This study aimed to evaluate the functional and molecular alterations of erectile function at middle age, focusing on the contribution of oxidative stress in erectile tissue for the ED.Methods.Young (3.5‐month) and middle‐aged (10‐month) male Wistar rats were used. Rat corpus cavernosum (RCC) was dissected free and mounted in 10‐mL organ baths containing Krebs solution. Intracavernosal pressure (ICP) in anesthetized rats was evaluated.Main Outcome Measures.Concentration–response curves to endothelium‐dependent and endothelium‐independent agents, as well as to electrical field stimulation (EFS), were obtained in RCC strips. Measurement of cyclic guanosine monophosphate (cGMP) and expressions of neuronal nitric oxide synthase (nNOS) and endothelial NOS (eNOS), gp91^phox and superoxide dismutase‐1 (SOD‐1) expressions in RCC were evaluated.Results.ICP was significantly reduced in middle‐aged compared with young rats. RCC relaxations to acetylcholine (10^?8 to 10^?2 M), sodium nitroprusside (10^?8 to 10^?2 M), sildenafil (10^?9 to 10^?5 M), BAY 41‐2272 (10^?9 to 10^?5 M), and EFS (4–32 Hz) were decreased in middle‐aged group, which were nearly normalized by apocynin (NADPH oxidase inhibitor; 10^?4 M) or SOD (75 U/mL). Prolonged treatment with apocynin (85 mg/rat/day, 4 weeks) also restored the impaired relaxations in middle‐aged rats. Relaxations to 8‐bromoguanosine 3′,5′‐cyclic monophosphate sodium salt (8‐Br‐cGMP; 10^?8 to 3 × 10^?4 M) remained unchanged between groups. Basal and stimulated cGMP production were lower in middle‐aged group, an effect fully restored by apocynin and SOD. Protein expression of nNOS and phosphorylated eNOS (p‐eNOS) (Ser‐1177) reduced, whereas gp^91phox mRNA expression increased in RCC from middle‐aged rats.Conclusions.ED in middle‐aged rats is associated with decreased NO bioavailability in erectile tissue due to upregulation of NADPH oxidase subunit gp91^phox and downregulation of nNOS/p‐eNOS. Antioxidant therapies may be a good pharmacological approach to prevent ED at its early stages.     

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.     

Valproic Acid Prevents Penile Fibrosis and Erectile Dysfunction in Cavernous Nerve‐Injured Rats

IntroductionBilateral cavernous nerve injury (BCNI) causes profound penile changes such as apoptosis and fibrosis leading to erectile dysfunction (ED). Histone deacetylase (HDAC) has been implicated in chronic fibrotic diseases.AimsThis study will characterize the molecular changes in penile HDAC after BCNI and determine if HDAC inhibition can prevent BCNI‐induced ED and penile fibrosis.MethodsFive groups of rats (8–10 weeks, n = 10/group) were utilized: (i) sham; (ii and iii) BCNI 14 and 30 days following injury; and (iv and v) BCNI treated with HDAC inhibitor valproic acid (VPA 250 mg/kg; 14 and 30 days). All groups underwent cavernous nerve stimulation (CNS) to determine intracavernosal pressure (ICP). Penile HDAC3, HDAC4, fibronectin, and transforming growth factor‐β1 (TGF‐β1) protein expression (Western blot) were assessed. Trichrome staining and the fractional area of fibrosis were determined in penes from each group. Cavernous smooth muscle content was assessed by immunofluorescence to alpha smooth muscle actin (α‐SMA) antibodies.Main Outcome MeasuresWe measured ICP; HDAC3, HDAC4, fibronectin, and TGF‐β1 protein expression; penile fibrosis; penile α‐SMA content.ResultsThere was a voltage‐dependent decline (P < 0.05) in ICP to CNS 14 and 30 days after BCNI. Penile HDAC3, HDAC4, and fibronectin were significantly increased (P < 0.05) 14 days after BCNI. There was a slight increase in TGF‐β1 protein expression after BCNI. Histological analysis showed increased (P < 0.05) corporal fibrosis after BCNI at both time points. VPA treatment decreased (P < 0.05) penile HDAC3, HDAC4, and fibronectin protein expression as well as corporal fibrosis. There was no change in penile α‐SMA between all groups. Furthermore, VPA‐treated BCNI rats had improved erectile responses to CNS (P < 0.05).ConclusionHDAC‐induced pathological signaling in response to BCNI contributes to penile vascular dysfunction. Pharmacological inhibition of HDAC prevents penile fibrosis, normalizes fibronectin expression, and preserves erectile function. The HDAC pathway may represent a suitable target in preventing the progression of ED occurring post‐radical prostatectomy. Hannan JL, Kutlu O, Stopak BL, Liu X, Castiglione F, Hedlund P, Burnett AL, and Bivalacqua TJ. Valproic acid prevents penile fibrosis and erectile dysfunction in cavernous nerve‐injured rats. J Sex Med 2014;11:1442–1451.     

Change of erectile function and responsiveness to phosphodiesterase type 5 inhibitors at different stages of streptozotocin-induced diabetes in rats

IntroductionIt has been suggested that risk of erectile dysfunction (ED) increases with duration of diabetes and phosphodiesterase type 5 inhibitors (PDE5I) are not as effective in treatment of diabetes-associated ED. However, few studies have investigated time-dependent change in erectile function during the course of diabetes.AimTo investigate time-dependent change in erectile function and responsiveness to PDE5I in streptozotocin-induced diabetic rats and to understand the pathophysiology of diabetic ED.Main Outcome MeasuresAt 6, 8, 10, 12, and 14 weeks after diabetic induction, erectile function was assessed by cavernous nerve stimulation before and after administration of DA-8159, a novel PDE5I. Penile tissue was assessed for apoptosis with immunohistochemistry. Protein expression of Rho-kinase 2 (ROCK2), myosin phosphatase targeting subunit 1 (MYPT1), and endothelial nitric oxide synthase (eNOS) was evaluated by Western blot.MethodsStreptozotocin was injected into 50 8-week-old male Sprague-Dawley rats, which were then classified into five diabetic groups according to the observation period.ResultsDiabetic rats maintained normal erectile responses until 6 weeks of diabetes. Following 8 weeks, the rats showed lower erectile responses at higher frequencies of nerve stimulation, which were normalized to control by administration of DA-8159. In contrast, erectile responses were significantly decreased in 10-week diabetic rats, and administration of DA-8159 resulted in partial recovery of normal responses. At more than 12 weeks, rats demonstrated severe deterioration of erectile function, which did not fully respond to PDE5I. Corporal apoptosis was significantly increased after 10 weeks. Upregulation of ROCK2 was found at 6 weeks, and was followed by an increase of MYPT1 phosphorylation. Phosphorylation of eNOS showed marked suppression at 6 weeks and remained lower during the experimental period.ConclusionsImpairment of erectile function was followed by decreased responsiveness to PDE5I during the course of diabetes. The RhoA/ROCK pathway played an important role in diabetes-associated ED. Cho SY, Park K, Paick J-S, and Kim SW. Change of erectile function and responsiveness to PDE5 (Type 5 phosphodiesterase) inhibitors at different stages of streptozotocin-induced diabetes in rats.     

Poly(ADP‐Ribose) Polymerase Inhibition Improves Erectile Function in Diabetic Rats

IntroductionErectile dysfunction (ED) is a common and hard‐to‐treat complication of diabetes mellitus (DM). Multiple lines of evidence have shown that poly(ADP‐ribose) polymerase (PARP) activation plays an important role in neurovascular dysfunction in diabetes, which is the crucial mechanism for diabetic ED.AimTo investigate the preventive benefit of a PARP inhibitor in a rat model of ED induced by diabetes.MethodsEstablished streptozotocin‐diabetic male Sprague‐Dawley rats were given PJ‐34, a selective PARP inhibitor, by oral gavage at a dose of 10 mg/kg twice daily for 8 weeks. Erectile responses under electrical stimulation of the cavernous nerve, PARP activity and reactive oxygen species (ROS) production were measured. Nitric oxide synthase (NOS) isoforms were evaluated by Western blot and real‐time quantitative PCR. Nuclear factor‐kappa B activition and apoptosis in corpus cavernosa (CC) were also investigated.Main Outcome MeasuresThe effects of PARP inhibition on the development of diabetic ED were determined.ResultsDiabetes markedly attenuated the erectile responses (intracavernosal pressure/mean systemic arterial blood pressure) and these were partially prevented by PJ‐34 treatment. Promoted oxidative stress associated PARP activation was found in CC from vehicle‐treated diabetic rats. PJ‐34 blocked PARP activity and the diabetes‐associated ROS generation. Decreased expression and activity of constitutive NOS (cNOS), including endothelial NOS (eNOS) and neuronal NOS (nNOS), associated with enhanced inducible NOS (iNOS) expression and activity were observed in vehicle‐treated diabetic rats. Although PJ‐34 had no effect on eNOS expression, it significantly prevented the decrease in nNOS expression and cNOS activity, and inhibited iNOS expression and activity in diabetic rats. PARP blockade by PJ‐34 to some extent prevented diabetes‐associated apoptosis and NF‐κB activation.ConclusionsOur results indicate that PARP activation plays an important role in the pathogenesis of diabetic ED and PARP inhibition may be a promising strategy to prevent development of diabetic ED. Wan ZH, Li WZ, Li YZ, Chen L, Li GH, Hu WF, Peng S, Yu JJ, and Guo F. Poly(ADP‐Ribose) polymerase inhibition improves erectile function in diabetic rats.     

Erectile Dysfunction Precedes Coronary Artery Endothelial Dysfunction in Rats Fed a High‐Fat,High‐Sucrose,Western Pattern Diet

IntroductionIt is suggested that erectile dysfunction (ED) may be an early risk factor for cardiovascular disease.AimThe goal of this study was to determine whether development of ED precedes the onset of coronary artery endothelial dysfunction in response to a Western diet (WD), thereby establishing whether the WD differentially impacts the endothelium in a time‐dependent manner. Additionally, a goal was to determine if diet‐induced ED is reversible with intracavernosal sepiapterin treatment.MethodsMale Sprague‐Dawley rats were fed a WD for 4, 8, or 12 weeks, or a control diet for 8 weeks. Erectile function was evaluated by measuring the mean arterial pressure (MAP) and intracavernosal pressure (ICP) in response to electrical field stimulation of the cavernosal nerve near the major pelvic ganglion, in the absence and presence of sepiapterin. Coronary artery endothelial function was evaluated ex vivo with cumulative doses of acetylcholine (ACh) applied to segments of the left anterior descending coronary artery preconstricted with serotonin.Main Outcome MeasuresErectile function was assessed as the ICP response to electrical field stimulation (EFS), normalized to MAP. Coronary artery endothelial function was assessed as the effective concentration producing 50% of a maximal response (EC₅₀) of the ACh response.ResultsThe ICP/MAP response to EFS was significantly attenuated following both 8 and 12 weeks of the WD compared with the control diet (P < 0.05). Sepiapterin treatment augmented the ICP/MAP response in all WD groups (P < 0.05). The coronary artery EC₅₀ of the ACh response was not different from control following 4 or 8 weeks but was significantly elevated following 12 weeks of the WD (P < 0.01).ConclusionsThese data suggest that erectile function is reduced prior to coronary artery endothelial function in response to the WD. Improvement of erectile function with sepiapterin in WD rats indicates that nitric oxide synthase uncoupling is a key mechanism in diet‐induced ED. La Favor JD, Anderson EJ, Hickner RC, and Wingard CJ. Erectile dysfunction precedes coronary artery endothelial dysfunction in rats fed a high‐fat, high‐sucrose, Western pattern diet. J Sex Med 2013;10:694–703.     

Internet-Based Brief Sex Therapy for Heterosexual Men with Sexual Dysfunctions: A Randomized Controlled Pilot Trial

IntroductionInternet-based sex therapy for men with erectile dysfunction has been advocated as an easily accessible and cost-effective treatment.AimTo test whether Internet-based sex therapy is superior to waiting list.MethodsInternet-based therapy was administered to heterosexual men with erectile dysfunction or premature ejaculation, without face-to-face contact, in a waiting-list controlled design, with pre-, post-, and follow-up measurements at 3 and 6 months posttreatment. Treatment was based on the sensate-focus model of Masters and Johnson, and supplemented with cognitive restructuring techniques.Main Outcome MeasuresSelf-reported improvement of sexual functioning, erectile functioning (men with ED), premature ejaculation (men with PE), sexual desire, overall sexual satisfaction, and sexual self-confidence.ResultsNinety-eight men participated (58 ED, 40 PE). Sexual functioning was much or somewhat improved in 40 participants (48%). In participants with ED, a near significant effect of treatment was found (P = 0.065), with higher levels of sexual desire (P < 0.05) and sexual self-confidence (P = 0.05) in treated men, in addition to improved erectile functioning (P = 0.01) and overall sexual satisfaction (P < 0.001) in both groups. In participants with PE, treatment was not superior to waiting list. In participants with ED, erectile functioning (P < 0.05) and overall sexual satisfaction (P = 0.002) improved significantly. In participants with PE, latency to ejaculation (P < 0.001), sexual desire (P < 0.05), and overall sexual satisfaction (P < 0.05) improved significantly from baseline to posttreatment, with no further changes at both follow-ups. Sexual self-confidence in men with PE remained unchanged during treatment until follow-up at 3 months posttreatment, and then was found to be improved at 6-months follow-up (P < 0.05).ConclusionInternet-based sex therapy for male erectile dysfunction was efficacious for male erectile disorder. For men with premature ejaculation, however, treatment was not superior to waiting list. van Lankveld JJDM, Leusink P, van Diest S, Gijs L, and Slob AK. Internet-based brief sex therapy for heterosexual men with sexual dysfunctions: A randomized controlled pilot trial. J Sex Med 2009;6:2224–2236.     

Diminazene Protects Corpus Cavernosum Against Hypercholesterolemia‐Induced Injury

IntroductionAngiotensin‐converting enzyme 2 (ACE2) is a key enzyme of the renin angiotensin system, which breaks down angiotensin II and forms angiotensin‐(1–7). In erectile tissues, it has been documented that angiotensin II contributes to the development of erectile dysfunction (ED), while treatment with angiotensin‐(1–7) improves penile erection. However, the expression and function of ACE2 in erectile tissues have never been investigated.AimHere, we examined the expression of ACE2 in erectile tissues and its actions against hypercholesterolemia‐induced corpus cavernosum (CC) injury.MethodsHypercholesterolemic apolipoprotein E knockout (ApoE^−/−) mice, a well‐known model of ED, were treated with diminazene aceturate (DIZE), an ACE2 activator compound, or vehicle for 3 weeks. Reactive oxygen species (ROS), collagen content, and protein expression of ACE2, neuronal nitric oxide synthase (nNOS), endothelial nitric oxide synthase (eNOS), nicotinamide adenine dinucleotide phosphate‐oxidase (NADPH) subunits were evaluated in the penis of DIZE‐treated and untreated ApoE^−/− mice. Functional studies were performed in CC strips.Main Outcome MeasuresACE2 expression and its role in modulating nitric oxide (NO)/ROS production and fibrosis within the CC of hypercholesterolemic mice were the main outcome measures.ResultsACE2 was expressed in smooth muscle and endothelial cells of mouse CC. Interestingly, ACE2 was downregulated in penis of hypercholesterolemic mice with ED, suggesting a protective role of ACE2 on the CC homeostasis. In accordance with that, pharmacological ACE2 activation by DIZE treatment reduced ROS production and NADPH oxidase expression, and elevated nNOS and eNOS expression and NO bioavailability in the penis of ApoE^−/− mice. Additionally, DIZE decreased collagen content within the CC. These beneficial actions of DIZE on the CC were not accompanied by improvements in atherosclerotic plaque size or serum lipid profile.ConclusionACE2 is expressed in erectile tissue and its reduction is associated with hypercholesterolemia‐induced ED. Additionally, treatment with DIZE improved hypercholesterolemia‐induced CC injury, suggesting ACE2 as a potential target for treating ED. Fraga‐Silva RA, Costa‐Fraga FP, Montecucco F, Sturny M, Faye Y, Mach F, Pelli G, Shenoy V, da Silva RF, Raizada MK, Santos RAS, and Stergiopulos N. Diminazene protects corpus cavernosum against hypercholesterolemia‐induced injury. J Sex Med 2015;12:289–302.     

Effects of Chronic Vardenafil Treatment Persist after End of Treatment in Rats with Acute Arteriogenic Erectile Dysfunction

IntroductionIn our previous study, chronic vardenafil treatment improved erectile function soon after the end of the treatment in rats with acute arteriogenic erectile dysfunction (ED).AimThe aim of this study is to evaluate whether the effects of chronic vardenafil treatment persist after the end of treatment using rats with acute arteriogenic ED.MethodsRats were randomly divided into three groups: (i) control; (ii) ligation; and (iii) vardenafil + no treatment. Rats in the ligation and vardenafil + no treatment groups underwent ligature of the bilateral internal iliac arteries to induce acute arteriogenic ED and were subsequently treated with vehicle or vardenafil (4.0 mg/kg/day), respectively, for 3 weeks. Subsequently, all rats were kept for a further 2 weeks with no treatment. Rats in the control group underwent sham surgery.Main Outcome MeasuresErectile function was assessed by changes in intracavernous pressure (ICP). Smooth muscle (SM)/collagen ratios in corpus cavernosum were analyzed by Masson trichrome staining. Transforming growth factor‐β₁ (TGF‐β₁) mRNA and protein levels in corpus cavernosum (CC) were, respectively, evaluated by real‐time polymerase chain reaction (PCR) analysis and Western blotting analysis.ResultsICP/mean arterial pressure (MAP) in the ligation group remained significantly lower than that in control group (P < 0.01). Despite no treatment for 2 weeks, ICP/MAP in the var + no treatment group remained significantly higher than that in ligation group (P < 0.05). SM/collagen ratio in the ligation group remained significantly lower when compared with the control group (P < 0.01). The ratio in the var + no treatment group remained significantly higher when compared with the ligation group at 2 weeks after the end of treatment (P < 0.05). TGF‐β₁ mRNA and protein levels did not differ among the groups.ConclusionsThe effects of chronic vardenafil treatment on erectile function and penile structure persist, even after the end of treatment, in acute arteriogenic ED rats. Hotta Y, Ohno R, Kataoka T, Mikumo M, Takahata Y, Ohno M, Maeda Y, and Kimura K. Effects of chronic vardenafil treatment persist after end of treatment in rats with acute arteriogenic erectile dysfunction. J Sex Med 2012;9:1799–1805.     

Intravenous Preload of Mesenchymal Stem Cells Rescues Erectile Function in a Rat Model of Cavernous Nerve Injury

IntroductionWe evaluated the potential preventive effects and mechanisms of intravenously preloaded mesenchymal stem cells (MSCs) for erectile dysfunction (ED) in a cavernous nerve (CN) injury model.MethodsMale Sprague–Dawley (SD) rats were used for this study. Rats were randomized into two groups. One group was intravenously preloaded with MSCs (1.0 × 10⁶ cells in 1 mL total fluid volume) and the other was infused with medium alone (1 mL Dulbecco's modified Eagle's medium [DMEM]) for sham control, respectively. Crushed CN injury was induced immediately after infusion. The surgeon was blind to the experimental conditions (MSC or medium).Main Outcome MeasuresTo assess erectile function, we measured the intracavernous pressure (ICP) and arterial pressure (AP) at 1 hour and 2 weeks after CN injury. After measuring the initial ICP/AP of pre‐injury (normal) male SD rats, they were randomized into the two groups and infused with MSCs or medium. PKH26‐labelled MSCs were used for tracking. To investigate the mRNA expression levels of neurotrophins in the major pelvic ganglia (MPG), we performed real‐time quantitative real‐time polymerase chain reaction.ResultsThe reduction of ICP/AP and area under the curve of ICP (ICP‐AUC) in the MSC group was significantly lower than in the DMEM group (P < 0.05; P < 0.05) at 1 hour. The ICP/AP and ICP‐AUC at 2 weeks post‐injury in the MSC group was significantly higher than in the DMEM group (P < 0.01; P < 0.05). The preloaded PKH26‐labelled MSCs were detected in the MPG and CN using confocal microscopy indicating homing of the cells to the injured nerve and ganglia. Glia cell‐derived neurotrophic factor (GDNF) and neurturin, which are important neurotrophic factors for erection, had expression levels in MPG significantly higher in the MSC group than in the DMEM group (P < 0.01, 0.05).ConclusionIntravenous preload of MSCs before a CN injury may prevent or reduce experimental ED. Takayanagi A, Sasaki M, Kataoka‐Sasaki Y, Kobayashi K, Matsuda Y, Oka S, Masumori N, Kocsis JD and Honmou O. Intravenous preload of mesenchymal stem cells rescues erectile function in a rat model of cavernous nerve injury. J Sex Med 2015;12:1713–1721.