Effects of Losartan,HO‐1 Inducers or HO‐1 Inhibitors on Erectile Signaling in Diabetic Rats

IntroductionActivation of the renin‐angiotensin system which is common in diabetes mellitus might affect heme oxygenase (HO‐1) gene expression.AimAssessment of the effects of administration of angiotensin II (Ang II) receptor antagonist (losartan) with HO‐1 inducer or inhibitor on erectile signaling in diabetic rats.Materials and MethodsSeventy male rats were divided equally into seven groups; healthy controls, streptozotocin‐induced diabetic rats, rats on citrate buffer, diabetic rats on losartan, diabetic rats on HO‐1 inducer (cobalt protoporphyrin [CoPP]), diabetic rats on losartan and CoPP, and diabetic rats on losartan and HO‐1 inhibitor (stannus mesoporphyrin [SnMP]).Main Outcome MeasureHO enzyme activity, HO‐1 gene expression, cyclic guanosine monophosphate (cGMP) assay, intracavernosal pressure (ICP), and cavernous tissue sinusoids surface area.ResultsHO‐1 gene expression, HO enzymatic activity, and cGMP were significantly decreased in the cavernous tissue of diabetic rats. These parameters were significantly elevated with the use of CoPP that restored the normal control levels of HO enzyme activity. Administration of losartan exhibited a significant enhancing effect on these parameters compared with the diabetic group, but not restored to the control levels, whereas administration of CoPP combined with losartan led to the restoration of their normal levels. ICP demonstrated significant decline in diabetic rats. The use of CoPP and/or losartan led to its significant improvement compared with diabetic rats. Administration of either losartan and/or CoPP led to a significant increase in the cavernous sinusoids surface area of diabetic rats. Administration of losartan with SnMP significantly decreased the enhancing effect of losartan on the studied parameters.ConclusionThe decline in erectile function in diabetes mellitus could be attributed to the downregulation of HO‐1 gene expression. HO‐1 induction added to Ang II receptor antagonist could improve erectile function. Abdel Aziz MT, El Asmer MF, Mostafa T, Atta H, Mahfouz S, Fouad H, Rashed L, Sabry D, Hassouna A, Abdel Aziz AT, Senbel A, and Demery A. Effects of losartan, HO‐1 inducers or HO‐1 inhibitors on erectile signaling in diabetic rats.     

Caspase Signaling in ED Patients and Animal Models

BackgroundCurrent treatments for erectile dysfunction (ED) are ineffective in prostatectomy and diabetic patients due to cavernous nerve (CN) injury, which causes smooth muscle apoptosis, penile remodeling, and ED. Apoptosis can occur via the intrinsic (caspase 9) or extrinsic (caspase 8) pathway.AimWe examined the mechanism of how apoptosis occurs in ED patients and CN injury rat models to determine points of intervention for therapy development.Methods and OutcomesImmunohistochemical and western analyses for caspase 3-cleaved, caspase-8 and caspase-9 (pro and active forms) were performed in corpora cavernosal tissue from Peyronie’s, prostatectomy and diabetic ED patients (n = 33), penis from adult Sprague Dawley rats that underwent CN crush (n = 24), BB/WOR diabetic and control rats (n = 8), and aged rats (n = 9).ResultsCaspase 3-cleaved was observed in corpora cavernosa from Peyronie’s patients and at higher abundance in prostatectomy and diabetic tissues. Apoptosis takes place primarily through the extrinsic (caspase 8) pathway in penis tissue of ED patients. In the CN crushed rat, caspase 3-cleaved was abundant from 1–9 days after injury, and apoptosis takes place primarily via the intrinsic (caspase 9) pathway. Caspase 9 was first observed and most abundant in a layer under the tunica, and after several days was observed in the lining of and between the sinuses of the corpora cavernosa. Caspase 8 was initially observed at low abundance in the rat corpora cavernosa and was not observed at later time points after CN injury. Aged and diabetic rat penis primarily exhibited intrinsic mechanisms, with diabetic rats also exhibiting mild extrinsic activation.Clinical translationKnowing how and when to intervene to prevent the apoptotic response most effectively is critical for the development of drugs to prevent ED, morphological remodeling of the corpora cavernosa, and thus, disease management.Strengths and limitationsAnimal models may diverge from the signaling mechanisms observed in ED patients. While the rat utilizes primarily caspase 9, there is a significant flux through caspase 8 early on, making it a reasonable model, as long as the timing of apoptosis is considered after CN injury.ConclusionsApoptosis takes place primarily through the extrinsic caspase 8 dependent pathway in ED patients and via the intrinsic caspase 9 dependent pathway in commonly used CN crush ED models. This is an important consideration for study design and interpretation that must be taken into account for therapy development and testing of drugs, and our therapeutic targets should ideally inhibit both apoptotic mechanisms.Martin S, Harrington DA, Ohlander S, et al. Caspase Signaling in ED Patients and Animal Models. J Sex Med 2021;18:711–722.     

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.     

Adrenomedullin and Angiopoietin‐1 Additively Restore Erectile Function in Diabetic Rats: Comparison with the Combination Therapy of Vascular Endothelial Growth Factor and Angiopoietin‐1

IntroductionErectile dysfunction (ED) is a major health problem. We have shown that adrenomedullin (AM) restores erectile function in diabetic rats.AimThe aim of this study is to explore a better treatment for ED, we examined whether combination of AM and angiopoietin‐1 (Ang‐1) was more effective to treat ED than treatment with AM alone or Ang‐1 alone. We also compared the effect of the combination therapy with that of treatment with vascular endothelial growth factor‐A (VEGF‐A).MethodsMale Wistar rats were injected with streptozotocin (STZ) to induce diabetes. Adenoviruses expessing AM (AdAM), Ang‐1 (AdAng‐1), and VEGF‐A (AdVEGF‐A) 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 the 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 α‐smooth muscle actin (SMA), VE‐cadherin and type I collagen was assessed by Western blot analysis.ResultsInfection with AdAM plus AdAng‐1 more effectively restored erectile function than infection with AdAM alone or AdAng‐1 alone. This combination therapy restored erectile function to a level similar to that observed in the age‐matched Wistar rats. Expression of SMA and VE‐cadherin increased more significantly in the AdAM plus AdAng‐1‐treated group than in the AdAM‐ or AdAng‐1‐treated group. Although AdVEGF‐A infection restored erectile function significantly, it also caused enlargement of the trabeculae of the cavernous body, aberrant angiogenesis, and overproduction of type I collagen.ConclusionsThese results suggested that combination therapy with AM and Ang‐1 potently restored erectile function and normal morphology of the cavernous body compared with VEGF‐A administration. This combination therapy will be useful to treat ED patients with a severely damaged cavernous body. Nishimatsu H, Suzuki E, Nomiya A, Niimi A, Suzuki M, Fujimura T, Fukuhara H, and Homma Y. Adrenomedullin and angiopoietin‐1 additively restore erectile function in diabetic rats: Comparison with the combination therapy of vascular endothelial growth factor and angiopoietin‐1. J Sex Med **;**:**–**.     

Effect of Intracavernous Administration of Angiopoietin‐4 on Erectile Function in the Streptozotocin‐Induced Diabetic Mouse

IntroductionErectile dysfunction (ED) is a highly prevalent complication of diabetes, and the severity of endothelial dysfunction is one of the most important factors in reduced responsiveness to oral phosphodiesterase type 5 inhibitors.AimTo study the effects of human angiopoietin‐4 (Ang‐4) protein on erectile function in diabetic mice.MethodsDiabetes was induced by intraperitoneal injection of streptozotocin into 8‐week‐old C57BL/6J male mice. At 8 weeks after the induction of diabetes, the animals were divided into four groups: control nondiabetic mice and diabetic mice receiving two successive intracavernous injections of phosphate buffered saline (days ?3 and 0), a single intracavernous injection of Ang‐4 protein (day 0), or two successive intracavernous injections of Ang‐4 protein (days ?3 and 0).Main Outcome MeasuresOne week after treatment, we measured erectile function by electrical stimulation of the cavernous nerve. The penis was harvested and stained with hydroethidine or antibodies to Ang‐4, platelet/endothelial cell adhesion molecule‐1, and phosphorylated endothelial nitric oxide synthase (eNOS). We also determined the differential expression of Ang‐4 in cavernous tissue in the control and diabetic mice. The effect of Ang‐4 protein on the phosphorylation of Tie‐2, Akt, and eNOS was determined in human umbilical vein endothelial cells (HUVECs) by Western blot.ResultsThe cavernous expression of Ang‐4 was downregulated in diabetic mice; Ang‐4 was mainly expressed in endothelial cells. Local delivery of Ang‐4 protein significantly increased cavernous endothelial content, induced eNOS phosphorylation, and decreased the generation of superoxide anion and apoptosis in diabetic mice. Ang‐4 protein strongly increased the phosphorylation of Tie‐2, Akt, and eNOS in HUVECs. Repeated intracavernous injections of Ang‐4 induced significant restoration of erectile function in diabetic mice (87% of control values), whereas a single intracavernous injection of Ang‐4 protein elicited modest improvement.ConclusionsCavernous endothelial regeneration by use of Ang‐4 protein may have potential for the treatment of vascular disease‐induced ED, such as diabetic ED. Kwon M‐H, Ryu J‐K, Kim WJ, Jin H‐R, Song K‐M, Kwon K‐D, Batbold D, Yin GN, Koh GY, and Suh J‐K. Effect of intracavernous administration of angiopoietin‐4 on erectile function in the streptozotocin‐induced diabetic mouse. J Sex Med 2013;10:2912–2927.     

Angiotensin II Increases Corpus Cavernosal Contractility and Oxidative Stress in Partial Bladder Outlet Obstructed Rabbits: Relevance to Erectile Dysfunction

IntroductionWe investigated the effect angiotensin II (Ang II), a corpus cavernosal smooth muscle (CCSM) constrictor peptide, has on tissue taken from rabbits following chronic partial bladder outlet obstruction (PBOO), as this model is characterized by an increase in corpus cavernosal collagen deposition and a marked reduction and impaired relaxation of CCSM cells.AimTo determine the interaction between Ang II and nitric oxide (NO) and the development of oxidative stress (OS) in a rabbit model of chronic PBOO.MethodsCorpus cavernosal tissue was obtained from 12 sham‐operated and 20 PBOO rabbits. Organ bath studies determined Ang II/NO interaction on CCSM function using losartan (AT1 receptor antagonist), sodium nitroprusside (SNP, NO donor), electrical field stimulation (EFS), and vardenafil (phosphodiesterase type 5 inhibitor). The role of OS in the Ang II response was also determined using diphenylene iodonium chloride (DPI), the nicotinamide adenine dinucleotide phosphate oxidase inhibitor, which inhibits superoxide production and superoxide dismutase (SOD, the enzyme that accelerates the breakdown of superoxide).Main Outcome MeasureAction of Ang II and AT1 receptor antagonist, as well as SOD and DPI on CCSM function.ResultsAng II caused a dose‐dependent contraction of CCSM strips that was enhanced in PBOO rabbits and inhibited by losartan, DPI, and SOD. CCSM relaxation induced by SNP/EFS was impaired in this model and improved by vardenafil and losartan.ConclusionsThese findings imply that the increased Ang II contractile response is a pathological consequence of PBOO and that AT1 receptor inhibition may be a therapeutic approach to treat ED associated with PBOO.     

An Oral Formulation of Angiotensin‐(1‐7) Reverses Corpus Cavernosum Damages Induced by Hypercholesterolemia

IntroductionThe renin angiotensin system plays a crucial role in erectile function. It has been shown that elevated angiotensin‐II levels contribute to the development of erectile dysfunction (ED). Oppositely, angiotensin‐(1‐7) (Ang‐[1‐7]) mediates penile erection by activation of receptor Mas. Recently, we have developed a formulation based on Ang‐(1‐7) inclusion in cyclodextrin (CyD) [Ang‐(1‐7)‐CyD], which allows for the oral administration of Ang‐(1‐7).AimIn the present study, we evaluated the effects of chronic treatment with Ang‐(1‐7)‐CyD on penile fibrosis, oxidative stress, and endothelial function in hypercholesterolemic mice.MethodsApolipoprotein(Apo)E^?/? mice fed a Western‐type diet for 11 weeks received Ang‐(1‐7)‐CyD or vehicle during the final 3 weeks. Collagen content and reactive oxygen species (ROS) production within the corpus cavernosum were evaluated by Sirius red and dihydroethidium staining, respectively. Protein expression of neuronal nitric oxide synthase (nNOS) and endothelial nitric oxide synthase (eNOS), nicotinamide adenine dinucleotide phosphate (NADPH) subunits (p67‐phox and p22‐phox), and AT1 and Mas receptors in the penis was assessed by Western blotting. Nitric oxide (NO) production was measured by Griess assay in the mice serum. Cavernosal strips were mounted in an isometric organ bath to evaluate the endothelial function.Main Outcome MeasuresThe effect of Ang‐(1‐7)‐CyD treatment on penile fibrosis, oxidative stress, and endothelial function in hypercholesterolemia‐induced ED.ResultsAng‐(1‐7)‐CyD treatment reduced collagen content in the corpus cavernosum of ApoE^?/? mice. This effect was associated with an attenuation of ROS production and a diminished expression of NADPH. Furthermore, Ang‐(1‐7)‐CyD treatment augmented the expression of nNOS and eNOS in the penis and elevated vascular NO production. Importantly, these effects were accompanied by an improvement in cavernosal endothelial function.ConclusionLong‐term treatment with Ang‐(1‐7)‐CyD reduces penile fibrosis associated with attenuation of oxidative stress. Additionally, cavernosal endothelial function in hypercholesterolemic mice was markedly improved. These results suggest that Ang‐(1‐7)‐CyD might have significant therapeutic benefits for the treatment of erectile dysfunction. Fraga‐Silva RA, Costa‐Fraga FP, Savergnini SQ, De Sousa FB, Montecucco F, da Silva D, Sinisterra RD, Mach F, Stergiopulos N, da Silva RF, and Santos RAS. An oral formulation of angiotensin‐(1–7) reverses corpus cavernosum damages induced by hypercholesterolemia. J Sex Med 2013;10:2430–2442.     

Intracavernous Delivery of Synthetic Angiopoietin-1 Protein as a Novel Therapeutic Strategy for Erectile Dysfunction in the Type II Diabetic db/db Mouse

IntroductionPatients with erectile dysfunction (ED) associated with type II diabetes often have impaired endothelial function and tend to respond poorly to oral phosphodiesterase type 5 inhibitors. Therefore, neovascularization is a promising strategy for curing diabetic ED.AimTo determine the effectiveness of a soluble, stable, and potent angiopoietin-1 (Ang1) variant, cartilage oligomeric matrix protein (COMP)-Ang1, in promoting cavernous angiogenesis and erectile function in a mouse model of type II diabetic ED.MethodsSixteen-week-old male db/db mice (in which obesity and type II diabetes are caused by a mutation in the leptin receptor) and control C57BL/6J mice were used and divided into four groups (N = 14 per group): age-matched controls; db/db mice receiving two successive intracavernous injections of phosphate-buffered saline (PBS) (days ?3 and 0; 20 µL); db/db mice receiving a single intracavernous injection of COMP-Ang1 protein (day 0; 5.8 µg/20 µL); and db/db mice receiving two successive intracavernous injections of COMP-Ang1 protein (days ?3 and 0; 5.8 µg/20 µL).Main Outcome MeasuresTwo weeks later, erectile function was measured by electrical stimulation of the cavernous nerve. The penis was then harvested and stained with antibodies to platelet/endothelial cell adhesion molecule-1 (PECAM-1) (endothelial cell marker), phosphohistone H3 (PH3, a nuclear protein indicative of cell proliferation), phospho-endothelial nitric oxide synthase (eNOS), and eNOS. Penis specimens from a separate group of animals were used for cyclic guanosine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP) quantification.ResultsLocal delivery of COMP-Ang1 protein significantly increased eNOS phosphorylation and cGMP and cAMP expression compared with that in the group treated with PBS. Repeated intracavernous injections of COMP-Ang1 protein completely restored erectile function and cavernous endothelial content through enhanced cavernous neoangiogenesis as evaluated by PECAM-1 and PH3 immunohistochemistry and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling assay, whereas a single injection of COMP-Ang1 protein elicited partial improvement.ConclusionCavernous neovascularization using recombinant Ang1 protein is a novel therapeutic strategy for the treatment of ED resulting from type II diabetes. Jin H-R, Kim WJ, Song JS, Piao S, Tumurbaatar M, Shin SH, Choi MJ, Tuvshintur B, Song K-M, Kwon M-H, Yin GN, Koh GY, Ryu J-K, and Suh J-K. Intracavernous delivery of synthetic angiopoietin-1 protein as a novel therapeutic strategy for erectile dysfunction in the type II diabetic db/db mouse.     

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.     

Impact of systemically active neurohumoral factors on the erectile response of the rat

IntroductionMean arterial pressure (MAP) and specific regulation of penile blood flow are the primary determinants of an erection. While this concept is well recognized, the differential relationship between systemically acting vasoactive factors on arterial pressure and erectile responses is not well described.AimThe aim of this study was to determine how the modification of systemic levels of neurohumoral factors impacts on the magnitude and efficiency of the erectile response.Main Outcome MeasuresThe main outcome measures for this study are changes in MAP and intracavernosal pressure (ICP) following electrostimulation of the cavernous nerve.MethodsAnesthetized adult, male Sprague‐Dawley rats were catheterized for measuring MAP (carotid), ICP, and drug administration (vena cava). Erections were induced via cavernous nerve electrostimulation. Vasoactive drug infusions were used to produce changes in MAP levels including: hexamethonium, angiotensin II (ANGII) ± hexamethonium, methoxamine ± hexamethonium, losartan, MAHMA NONOate, and terbutaline.ResultsIn general, ICP and MAP were linearly correlated regardless of treatment. Hexamethonium markedly dropped MAP and proportionately decreased the magnitude of the erectile response. ANGII or methoxamine given to hexamethonium‐pretreated or untreated rats increased MAP similarly, but produced contrasting effects on erectile responses. ANGII‐induced pressor responses were associated with increased erectile responses whereas all methoxamine treatments markedly decreased erectile responses. Depressor changes with losartan or terbutaline, but not MAHMA NONOate, also impacted negatively on the efficiency of the erectile responses at lower arterial pressures.ConclusionsIn general, the magnitude of the erectile responses was found to be dependent upon the level of MAP, although the mechanism by which arterial pressure was changed impacted substantially on the characteristics of the relationship. The major finding was that circulation‐wide α‐adrenoceptor stimulation was extremely deleterious to erectile responses whereas global stimulation of ANG II receptors was actually proerectile. Overall, the results indicate that neurohumoral specificity in systemic hemodynamic control is also critical in establishing the optimal erectile environment in rats. MacKenzie LD, Heaton JPW, and Adams MA. Impact of systemically active neurohumoral factors on the erectile response of the rat. J Sex Med 2011;8:2461–2471.     

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.     

Metformin Treatment Improves Erectile Function in an Angiotensin II Model of Erectile Dysfunction

IntroductionIncreased angiotensin II (AngII) levels cause hypertension, which is a major risk factor for erectile dysfunction (ED). Studies have demonstrated that increased AngII levels in penile tissue are associated with ED. A recent study showed that metformin treatment restored nitric oxide synthase (NOS) protein expression in penile tissue in obese rats; however, whether metformin treatment can be beneficial and restore erectile function in a model of ED has not yet been established.AimThe goal of this study was to test the hypothesis that AngII induces ED by means of increased corpus cavernosum contraction, and that metformin treatment will reverse ED in AngII-treated rats.MethodsMale Sprague-Dawley rats were implanted with mini-osmotic pumps containing saline or AngII (70 ng/minute, 28 days). Animals were then treated with metformin or vehicle during the last week of AngII infusion.Main Outcome MeasuresIntracavernosal pressure; corpus cavernosum contraction and relaxation; nNOS protein expression; extracellular signal-regulated kinase (ERK1/2), AMP-activated protein kinase (AMPK), and eNOS protein expression and phosphorylation.ResultsAngII-induced ED was accompanied with an increase in corpus cavernosum contractility, decreased nitrergic relaxation, and increased ERK1/2 phosphorylation. Metformin treatment improved erectile function in the AngII-treated rats by reversing the increased contraction and decreased relaxation. Metformin treatment also resulted in an increase in eNOS phosphorylation at ser1177.ConclusionsMetformin treatment increased eNOS phosphorylation and improved erectile function in AngII hypertensive rats by reestablishing normal cavernosal smooth muscle tone. Labazi H, Wynne BM, Tostes R, and Webb RC. Metformin treatment improves erectile function in an angiotensin II model of erectile dysfunction. J Sex Med 2013;10:2154–2164.     

Changes in the Penile Arteries of the Rat after Fractionated Irradiation of the Prostate: A Pilot Study

IntroductionExternal beam radiotherapy for prostate cancer leads to erectile dysfunction in 36%–43% of patients. The underlying mechanism is largely unknown, although some clinical studies suggest that the arterial supply to the corpora cavernosa is responsible. Two animal experimental studies reported on the effects of a single fraction of prostate irradiation on the penile structures. However, irradiation in multiple fractions is more representative of the actual clinical treatment.AimThe present prospective, controlled study was initiated to investigate the effect of fractionated prostate irradiation on the arteries of the corpora cavernosa.Main Outcome MeasuresHistological evaluation of the penile tissue in comparison with control rats at 2, 4, and 9 weeks after irradiation.MethodsThe prostate of twelve rats was treated with external beam radiation in 5 daily fractions of 7.4 gray. Three control rats were treated with sham irradiation. Prostatic and penile tissue was evaluated for general histology (hematoxylin–eosin). The penile tissue was further evaluated after combined staining for collagen (resorcin fuchsin) and α-smooth muscle actin (SMA) (Biogenex).ResultsThe prostate showed adequate irradiation with fibrosis occurring at 9 weeks after irradiation. The corpora cavernosa showed arteries that had developed loss of smooth muscle cells expressing SMA, thickening of the intima, and occlusions. All the control rats maintained normal anatomy.ConclusionThis is the first animal experimental study that demonstrates changes in the arteries of the corpora cavernosa after fractionated irradiation to the prostatic area. The preliminary data suggests that erectile dysfunction after radiotherapy might be caused by radiation damage to the arterial supply of the corpora cavernosa. van der Wielen GJ, Vermeij M, de Jong BWD, Schuit M, Marijnissen J, Kok DJ, van Weerden WM, and Incrocci L. Changes in the penile arteries of the rat after fractionated irradiation of the prostate: A pilot study. J Sex Med 2009;6:1908–1913.     

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

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

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.     

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.     

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.     

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.     

Effects of icariin on improving erectile function in streptozotocin-induced diabetic rats

IntroductionIcariin has been shown to improve penile hemodynamics in animal models of erectile dysfunction from cavernous nerve injury and castration. The effects of icariin on penile hemodynamics in diabetic animals remain to be determined. Transforming growth factor β1 (TGFβ1) has been implicated in the pathogenesis of diabetes‐related erectile dysfunction.AimThe aim of this study was to investigate the effects of icariin in the penis of streptozotocin (STZ)‐induced diabetic rat.MethodsTwo‐month‐old Sprague–Dawley male rats received one‐time intraperitoneal (IP) STZ (60 mg/kg) or vehicle injection after a 16‐hour fast. Three days later, the STZ‐induced diabetic rats were randomly divided into four groups and were treated with daily gavage feedings of a 50:50 mix of normal saline and dimethyl sulfoxide (DMSO) or icariin dissolved in DMSO at doses of 1, 5, and 10 mg/kg for 3 months. A positive control group underwent IP injection of saline followed by daily gavage of saline/DMSO solution. Treatment was stopped 1 week prior to functional assay and euthanasia.Main Outcome MeasurePenile hemodynamics was assessed by electrical stimulation of the cavernous nerves with real‐time intracavernous pressure (ICP) measurement. After euthanasia, penile tissue was studied using immunohistochemistry, Western blot, and enzyme‐linked immunosorbent assay (ELISA) to assess the nitric oxide‐cyclic guanosine monophosphate (NO‐cGMP) and TGFβ1/Smad2 signaling pathway.ResultsDiabetes attenuated ICP response in control animals. Untreated diabetic animals had decreased smooth muscle/collagen ratio and endothelial cell content in the corpora cavernosa; treatment with icariin partially attenuating these effects. Icariin‐treated animals also had a significantly greater expression of nicotinamide adenine dinucleotide phosphate‐positive nerves and the endothelial cell markers, von Willebrand factor (vWF), and platelet endothelial cell adhesion molecule‐1 (PECAM). TGFβ1/Smad2 signaling pathway was down‐regulated in the penis from icariin‐treated models relative to what was observed in negative control animals.ConclusionIcariin treatment preserved penile hemodynamics, smooth muscle and endothelial integrity, and neuronal nitric oxide synthase expression in the penis of diabetic rats. Down‐regulation of TGFβ1/Smad2 signaling pathway might mediate this effect. Liu T, Xin H, Li W‐R, Zhou F, Li G‐Y, Gong Y‐Q, Gao Z‐Z, Qin X‐C, Cui W‐S, Shindel AW, and Xin Z‐C. Effects of icariin on improving erectile function in streptozotocin‐induced diabetic rats. J Sex Med 2011;8:2761–2772.