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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Effects of Intravenous Injection of Adipose‐Derived Stem Cells in a Rat Model of Radiation Therapy‐Induced Erectile Dysfunction

IntroductionRadiation therapy (RT) for prostate cancer is frequently associated with posttreatment erectile dysfunction (ED).AimTo investigate whether injection of adipose‐derived stem cells (ADSCs) can ameliorate RT‐associated ED.MethodsThirty male rats were divided into three groups. The control + phosphate‐buffered saline (PBS) group received tail‐vein injection of PBS. The radiation + PBS group received radiation over the prostate and tail‐vein injection of PBS. The radiation + ADSC group received radiation over the prostate and tail‐vein injection of ADSCs, which were labeled with 5‐ethynyl‐2‐deoxyuridine (EdU). Seventeen weeks later, erectile function was evaluated by intracavernous pressure (ICP) in response to electrostimulation of cavernous nerves (CNs). Penile tissue and major pelvic ganglia (MPG) were examined by immunofluorescence (IF) and EdU staining.Main Outcome MeasuresErectile function was measured by ICP. Protein expression was examined by IF, followed by image analysis and quantification.ResultsRadiation over the prostate caused a significant decrease in erectile function and in the expression of neuronal nitric oxide synthase (nNOS) in penis and MPG. Cavernous smooth muscle (CSM) but not endothelial content was also reduced. Injection of ADSCs significantly restored erectile function, nNOS expression, and CSM content in the irradiated rats. EdU‐positive cells were visible in MPG.ConclusionsRadiation appears to cause ED via CN injury. ADSC injection can restore erectile function via CN regeneration. Qiu X, Villalta J, Ferretti L, Fandel TM, Albersen M, Lin G, Dai Y, Lue TF, and Lin C‐S. Effects of intravenous injection of adipose‐derived stem cells in a rat model of radiation therapy‐induced erectile dysfunction. J Sex Med 2012;9:1851–1858.