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1.  Activated Rho Kinase Mediates Diabetes-Induced Elevation of Vascular Arginase Activation and Contributes to Impaired Corpora Cavernosa Relaxation: Possible Involvement of p38 MAPK Activation 
The journal of sexual medicine  2013;10(6):1502-1515.
Activated 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.
We tested whether ROCK and p38 MAPK are involved in the elevation of arginase activity and subsequent impairment of corpora cavernosal (CC) relaxation in diabetes.
Eight weeks after streptozotocin-induced diabetes, vascular functional studies, arginase activity assay, and protein expression of RhoA, ROCK, phospho-p38 MAPK, p38 MAPK, phospho-MYPT-1Thr850, 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 Measures
The expression of RhoA, ROCK 1 and 2, phosphorylation of MYPT-1Thr850 and p38 MAPK, arginase activity/expression, endothelial- and nitrergic-dependent relaxation of CC was assayed.
Diabetes significantly reduced maximum relaxation (Emax) to both endothelium-dependent acetylcholine (WT + D: Emax; 61 ± 4% vs. WT: Emax; 75 ± 2%) and nitrergic nerve stimulation. These effects were associated with increased expression of active RhoA, ROCK 2, phospho-MYPT-1Thr850, 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 (Emax: 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.
ROCK 2, p38 MAPK and arginase play key roles in diabetes-induced impairment of CC relaxation.
PMCID: PMC3807104  PMID: 23566117
Diabetes; Rho Kinase; p38 MAPK; Arginase
2.  Erectile Dysfunction in Young Non-Obese Type II Diabetic Goto-Kakizaki Rats is Associated with Decreased eNOS Phosphorylation at Ser1177 
The journal of sexual medicine  2010;7(11):3620-3634.
Diabetes mellitus (DM) is a risk factor for erectile dysfunction (ED). Although type 2 DM is responsible for 90–95% diabetes cases, type 1 DM experimental models are commonly used to study diabetes-associated ED.
Goto-Kakizaki (GK) rat model is relevant to ED studies since the great majority of patients with type 2 diabetes display mild deficits in glucose-stimulated insulin secretion, insulin resistance, and hyperglycemia. We hypothesized that GK rats display ED which is associated with decreased nitric oxide (NO) bioavailability.
Wistar and GK rats were used at 10 and 18 weeks of age. Changes in the ratio of intracavernosal pressure/mean arterial pressure (ICP/MAP) after electrical stimulation of cavernosal nerve were determined in vivo. Cavernosal contractility was induced by electrical field stimulation (EFS) and phenylephrine (PE). In addition, nonadrenergic-noncholinergic (NANC)- and sodium nitroprusside (SNP)-induced relaxation were determined. Cavernosal neuronal nitric oxide synthase (nNOS) and endothelial nitric oxide synthase (eNOS) mRNA and protein expression were also measured.
Main Outcome Measure
GK diabetic rats display ED associated with decreased cavernosal expression of eNOS protein.
GK rats at 10 and 18 weeks demonstrated impaired erectile function represented by decreased ICP/MAP responses. Ten-week-old GK animals displayed increased PE responses and no changes in EFS-induced contraction. Conversely, contractile responses to EFS and PE were decreased in cavernosal tissue from GK rats at 18 weeks of age. Moreover, GK rats at 18 weeks of age displayed increased NANC-mediated relaxation, but not to SNP. In addition, ED was associated with decreased eNOS protein expression at both ages.
Although GK rats display ED, they exhibit changes in cavernosal reactivity that would facilitate erectile responses. These results are in contrast to those described in other experimental diabetes models. This may be due to compensatory mechanisms in cavernosal tissue to overcome restricted pre-penile arterial blood supply or impaired veno-occlusive mechanisms.
PMCID: PMC3022479  PMID: 20807325
Goto-Kakizaki; Type 2 Diabetes Mellitus; Erectile Dysfunction; Corpus Cavernosum
3.  Characterization of Erectile Function in Elastin Haploinsufficicent Mice 
The journal of sexual medicine  2011;8(11):3075-3085.
Elastin fibers confer passive recoil to many tissues including the lung, skin, and arteries. In the penis, elastin is present in sinusoids, arterioles, and in the tunica albuginea. Although decreased penile elastin has been reported in men with erectile dysfunction, the exact role of elastin in physiologic processes integral to erection remains speculative.
The aim of this study was to characterize erectile function in elastin-deficient mice.
Elastin haploinsufficient mice (Eln+/−) and aged match Eln+/+ (Wt) mice were used. Cavernosum was removed from some mice for quantification of elastin, collagen, and smooth muscle actin. Ex vivo assessment of contractile force generation was performed by myography. In vivo assessment of intracorporal pressure normalized to mean arterial pressure in response to electrical stimulation of the cavernosal nerve was measured. Veno-occlusive function was determined by cavernosography.
Main Outcome Measures
The main outcome measures of this study were the in vitro and in vivo assessment of cavernosal vasoreactivity, veno-occlusive function and erection in mice deficient in elastin.
Eln+/− mice exhibited ~33% less penile elastin than Wt mice, with no change in collagen. Cavernosal tissue from Eln+/− mice has a significantly heightened contractile response, explained in part by increased smooth muscle cell content. Veno-occlusive function was significantly altered in Eln+/− mice. Interestingly, erectile function was impaired only at submaximal voltage (1 V) stimulation (there was no impairment during the higher 2-V stimulus).
Eln+/− mice display a cavernosal phenotype consistent with developmental changes attributable to the loss of elastin. These alterations confer a degree of altered erectile function that is able to be overridden by maximal stimulatory input. Altogether, these data suggest that elastin is important for erectile function.
PMCID: PMC3765001  PMID: 21883953
Matrix; Animal Models; Sexual DysfunctionIntroduction
4.  Adenosine Actions are Preserved in Corpus Cavernosum from Obese and Type II Diabetic db/db Mouse 
The journal of sexual medicine  2008;5(5):1156-1166.
Erectile dysfunction (ED) in diabetes is associated with autonomic neuropathy and endothelial dysfunction. Whereas the nonadrenergic-noncholinergic (NANC)/neurogenic nitric oxide pathway has received great attention in diabetes-associated ED, few studies have addressed sympathetic overactivity.
To test the hypothesis that adenosine-induced inhibition of adrenergic-mediated contractile responses in mouse corpus cavernosum is impaired in the presence of diabetes.
The db/db (obesity and type II diabetes caused by a leptin receptor mutation) mouse strain was used as a model of obesity and type II diabetes, and standard procedures were performed to evaluate functional cavernosal responses.
Main Outcome Measures
Increased cavernosal responses to sympathetic stimulation in db/db mice are not associated with impaired prejunctional actions of adenosine.
Electrical field stimulation (EFS)-, but not phenylephrine (PE)-, induced contractions are enhanced in cavernosal strips from db/db mice in comparison with those from lean littermates. Direct effects of adenosine, 2-chloro-adenosine, A1 receptor agonist C-8031 (N6 cyclopentyladenosine), and sodium nitroprusside are similar between the strips from lean and db/db mice, whereas relaxant responses to acetylcholine and NANC stimulation are significantly impaired in the cavernosal strips from db/db mice. 5′-Iodotubercidin (adenosine kinase inhibitor) and dipyridamole (inhibitor of adenosine transport), as well as the A1 agonist C-8031, significantly and similarly inhibit contractions induced by stimulation of adrenergic nerves in the cavernosal strips from lean and db/db mice.
Results from this study suggest that corpora cavernosa from obese and diabetic db/db mice display altered neural-mediated responses that would favor penile detumescence, i.e., increased contractile response to adrenergic nerve stimulation and decreased relaxant responses upon activation of NANC nerves. However, increased cavernosal responses to adrenergic nerve stimulation are not due to impaired negative modulation of sympathetic neurotransmission by adenosine in this diabetic model.
PMCID: PMC3016713  PMID: 18221284
Adenosine; Obesity; Diabetes; Sympathetic Neurotransmission; Animal Models
5.  Effects of ethanol on the tonicity of corporal tissue and the intracellular Ca2+ concentration of human corporal smooth muscle cells 
Asian Journal of Andrology  2010;12(6):890-898.
Heavy alcohol consumption is associated with an increased risk of erectile dysfunction (ED); however, the acute effects of ethanol (EtOH) on penile tissue are not fully understood. We sought to investigate the effects of EtOH on corporal tissue tonicity, as well as the intracellular Ca2+ concentration ([Ca2+]i) and potassium channel activity of corporal smooth muscle. Strips of corpus cavernosum (CC) from rabbits were mounted in organ baths for isometric tension studies. Electrical field stimulation (EFS) was applied to strips precontracted with 10 μmol L−1 phenylephrine as a control. EtOH was then added to the organ bath and incubated before EFS. The [Ca2+]i levels were monitored by the ratio of fura-2 fluorescence intensities using the fura-2 loading method. Single-channel and whole-cell currents were recorded by the conventional patch-clamp technique in short-term cultured smooth muscle cells from human CC tissue. The corpus cavernosal relaxant response of EFS was decreased in proportion to the concentration of EtOH. EtOH induced a sustained increase in [Ca2+]i in a dose-dependent manner, Extracellular application of EtOH significantly increased whole-cell K+ currents in a concentration-dependent manner (P < 0.05). EtOH also increased the open probability in cell-attached patches; however, in inside-out patches, the application of EtOH to the intracellular aspect of the patches induced slight inhibition of Ca2+-activated potassium channel (KCa) activity. EtOH caused a dose-dependent increase in cavernosal tension by alterations to [Ca2+]i. Although EtOH did not affect KCa channels directly, it increased the channel activity by increasing [Ca2+]i. The increased corpus cavernosal tone caused by EtOH might be one of the mechanisms of ED after heavy drinking.
PMCID: PMC3739069  PMID: 20852651
alcohol; Ca2+-activated potassium channel; corporal smooth muscle; penile erection
The journal of sexual medicine  2009;6(Suppl 3):279-285.
Hypertension is closely associated with erectile dysfunction (ED) as it has been observed in many experimental models of hypertension. Additionally, epidemiological studies show that approximately a third of the hypertensive patients have ED.
To test the hypothesis that the two-kidney one-clip (2K-1C) rat model of hypertension displays normal erectile function due to increased nitric oxide (NO) production in the penis.
Ganglionic-induced increase in intracavernosal pressure (ICP)/mean arterial pressure (MAP) ratio was used as an index of erectile function in 2K-1C and in normotensive sham-operated (SHAM) anesthetized rats. Cavernosal strips from hypertensive and normotensive rats were used for isometric tension measurement. The contraction induced by alpha-adrenergic agonist phenylephrine, and the relaxation induced by the nitric oxide (NO) donor sodium nitroprusside (SNP) and by the Rho-kinase inhibitor Y-27632 were performed in the absence and in the presence of the nitric oxide synthase (NOS) inhibitor L-NNA.
Changes in ICP/MAP induced by ganglionic stimulation were not different between 2K-1C and SHAM rats. The contractile response induced by phenylephrine as well as the relaxation induced by SNP or the Y-27632 were similar in cavernosal strips from both groups. However, in the presence of L-NNA, the relaxation induced by Y-27632 was significantly impaired in 2K-1C compared to SHAM).
These data suggest that hypertension and ED could be dissociated from high levels of blood pressure in some animal models of hypertension. Erectile function in 2K-1C hypertensive rats is maintained in spite of the increased Rho-kinase activity by increased NO signaling.
PMCID: PMC2716695  PMID: 19267850
Two kidney-one clip hypertension; Rho-kinase; Nitric Oxide; Erectile Function
7.  Sonic Hedgehog Protein Is Decreased and Penile Morphology Is Altered in Prostatectomy and Diabetic Patients 
PLoS ONE  2013;8(8):e70985.
Erectile dysfunction (ED) is a debilitating medical condition and current treatments are ineffective in patients with cavernous nerve (CN) injury, due to penile remodeling and apoptosis. A critical regulator of penile smooth muscle and apoptosis is the secreted protein sonic hedgehog (SHH). SHH protein is decreased in rat prostatectomy and diabetic ED models, SHH inhibition in the penis induces apoptosis and ED, and SHH treatment at the time of CN injury suppresses smooth muscle apoptosis and promotes regeneration of erectile function. Thus SHH treatment has significant translational potential as an ED therapy if similar mechanisms underlie ED development in patients. In this study we quantify SHH protein and morphological changes in corpora cavernosal tissue of control, prostatectomy and diabetic patients and hypothesize that decreased SHH protein is an underlying cause of ED development in prostatectomy and diabetic patients. Our results show significantly decreased SHH protein in prostatectomy and diabetic penis. Morphological remodelling of the penis, including significantly increased apoptotic index and decreased smooth muscle/collagen ratio, accompanies declining SHH. SHH signaling is active in human penis and is altered in a parallel manner to previous observations in the rat. These results suggest that SHH has significant potential to be developed as an ED therapy in prostatectomy and diabetic patients. The increased apoptotic index long after initial injury is suggestive of ongoing remodeling that may be clinically manipulatable.
PMCID: PMC3743882  PMID: 23967143
8.  The effect of mirodenafil on the penile erection and corpus cavernosum in the rat model of cavernosal nerve injury 
Impotence is one of the common complications after the radical prostatectomy. One of the main reasons of this complication is due to the dysfunction of the veins in corpus cavernosum. Recent studies have shown that the erectile function is improved after the long-term therapy of phosphodiesterase type 5 inhibitor among patients with post-prostatectomy erectile dysfunction. In this study, we evaluated the effects of mirodenafil on the penile erection and corpus cavernosum tissues in the rat model of cavernosal nerve injury. Rats were divided into four groups: (1) control group, (2) bilateral cavernosal nerve injury group, (3) mirodenafil 10 mg therapy group after the nerve injury and (4) mirodenafil 20 mg therapy group after the nerve injury. After we identified the nerve from the pelvic nerve complex on the lateral side of the prostate, the rats in the control group were sutured without causing any nerve injury and in other groups we damaged the nerve by compressing it with a vessel clamp. Then, 10 and 20 mg kg−1 of mirodenafil were orally administered to two experimental groups. After 8 weeks, the intracavernosal pressure (ICP) was recorded. The immunohistochemical staining and western blot were performed, and the effect of mirodenafil on the expression of cyclic guanosine monophosphate (cGMP) was evaluated through enzyme-linked immunosorbent assay. The ICP of nerve-injured group was decreased compared with the control group; however, the ICP of the mirodenafil-administered groups was improved compared with the nerve-injured group. The Masson's trichrome staining confirmed that the smooth muscle (SM) component was increased in the mirodenafil-administered groups. The nitric oxide synthase expression and cGMP of mirodenafil-administered groups was increased compared with the nerve-injured group. Long-term therapy of mirodenafil may improve the erectile function after the radical prostatectomy by preserving the SM content and inhibiting the fibrosis of the corpus cavernosum.
PMCID: PMC2959156  PMID: 20861845
mirodenafil; corpus cavernosum; erectile dysfunction
9.  Chronic Oral Administration of the Arginase Inhibitor 2(S)-amino-6-boronohexanoic Acid (ABH) Improves Erectile Function in Aged Rats 
Journal of andrology  2012;33(6):1169-1175.
Arginase expression and activity have been noted to be heightened in conditions associated with erectile dysfunction, including aging. Previously, arginase inhibition by chronic administration of the arginase inhibitor 2-(S)-amino-6-boronohexanoic acid (ABH) has been shown to improve endothelial dysfunction in aged rats. The objective of this study was to assess whether chronic oral ABH administration affects cavernosal erectile function. Rats were divided into 4 groups: young control, young treated with arginase inhibitor, aged control, and aged treated with arginase inhibitor. Arginase activity was measured and presented as a proportion of young untreated rats. In vivo erectile responses to cavernous nerve stimulation were measured in all cohorts. The cavernous nerve was stimulated with a graded electrical stimulus, and the intracavernosal/ mean arterial pressure ratios and total intracavernosal pressure were recorded. Arginase activity was elevated in the aged rats compared with young controls; however, arginase activity was significantly decreased in aged rats treated with ABH. With the addition of ABH, erectile responses improved in the aged rats (P < .05). Oral inhibition of arginase with ABH results in improved erectile function in aged rats, resulting in erectile hemodynamics similar to young rats. This represents the first documentation of systemic arginase inhibition positively affecting corporal cavernosal function.
PMCID: PMC4029113  PMID: 22492840
Erectile dysfunction; nitric oxide; penile erection; aging
10.  The selective rho-kinase inhibitor Azaindole-1 has long lasting erectile activity in the rat 
Urology  2013;81(2):465.e7-465.e14.
To investigate the effects of the selective Rho-kinase (ROCK) inhibitor azaindole-1 on erectile function under physiologic and pathophysiologic conditions in the rat.
The effect of intracavernosal (i.c.) injections of azaindole-1 on change in ICP, ICP/MAP, AUC, and response duration were investigated in the anesthetized rat under control conditions and when NANC neurotransmission and cholinergic function or sGC were inhibited or after cavernosal nerve crush injury.
The i.c. injections of azaindole-1 produced dose-related increases in ICP/MAP and AUC that were long lasting at the highest doses studied when compared with the prototypical ROCK-inhibitor fasudil. Erectile responses were not altered by 7-NI and atropine in doses that reduced the response to cavernosal nerve stimulation by 86%, indicating that they were independent of NO release by cavernosal nerves or activation of muscarinic receptors in the corpora cavernosa. Erectile responses to azaindole-1 were not altered by the sGC inhibitor ODQ in a dose that attenuated responses to the NO donor SNP indicating that they were independent of an action on sGC. The erectile response to ic injections of azaindole-1 or Y-27632 which was reported to be NO/cGMP- dependent were not attenuated after cavernosal nerve crush injury.
The present studies indicate azaindole-1 has long lasting erectile activity that is independent of NO release, muscarinic receptor, or sGC activation or the integrity of the cavernosal nerves.
PMCID: PMC3564057  PMID: 23374844
Azaindole-1; selective Rho-kinase inhibitor; erectile dysfunction; oxidative stress; impaired cavernosal nerve function
11.  The effects of combined free radical scavenger and sildenafil therapy on age-associated erectile dysfunction: An animal model 
Urology Annals  2014;6(4):314-320.
Aging results in erectile dysfunction that is partially attributed to decreased nitric oxide (NO) and increased free radical generation. Vitamin E enhances endothelial cell function and acts as a free radical scavenger; however, its benefits on erectile function in the elderly are unknown.
The aim of the following study is to determine if Vitamin E alone, or in combination with the phosphodiesterase 5 inhibitor sildenafil, may improve erectile function and the NO signaling in a cohort of aged (13-15 month old) rats.
Materials and Methods:
Male Sprague-Dawley rats (n = 28) were divided based upon age into young (4-5 months old, n = 7) and aged (13-15 months old, n = 21) cohorts. Aged rats were treated with Vitamin E, sildenafil or a combination of both. Penile cavernosal and dorsal nerve tissues were evaluated for neuronal nitric oxide synthase (nNOS) and caveolin-1 expression. Erectile function was assessed through intra-cavernous pressure (ICP) recordings.
nNOS and cavoelin-1 were significantly decreased in aged rats compared with young controls. In aged rats, both Vitamin E and sildenafil partially recovered nNOS expression but when combined, a synergistic elevation in nNOS was observed. The significant decreases in ICP recorded in aged rats were improved with sildenafil; however, Vitamin E did not yield any additional improvements in ICP.
Diminished levels of nNOS and caveolin-1 are found in aged rats. When combined with sildenafil, Vitamin E synergistically increased nNOS expression. Since biochemical gains were not realized physiologically, other contributing factors likely exist.
PMCID: PMC4216537  PMID: 25371608
Aging; antioxidant; erectile dysfunction; impotence; nitric oxide; sildenafil; vitamin E
12.  Emerging Role for TNF-α in Erectile Dysfunction 
The journal of sexual medicine  2010;7(12):3823-3834.
A role for cytokines in the pathophysiology of erectile dysfunction (ED) has emerged. Cytokines induce genes that synthesize other peptides in the cytokine family and several mediators, such as prostanoids, leukotrienes, nitric oxide, bradykinin, reactive oxygen species, and platelet-activating factor, all of which can affect vascular function. Consistent with the fact that the cavernosal tissue is a complex extension of the vasculature, risk factors that affect the vasculature have been shown to affect cavernosal function as well. Accordingly, the penile tissue has been recognized as an early sentinel for atherosclerosis that underlies coronary artery disease and cardiovascular diseases (CVD).
To review the literature pertaining to the role of tumor necrosis factor-alpha (TNF-α) in ED.
PubMed search for pertinent publications on the role of cytokines, particularly TNF-α, in CVD and ED.
Main Outcome Measures
Clinical and experimental evidence demonstrates that TNF-α may play a role in ED.
TNF-α has been shown to play an important role in CVD, mainly due to its direct effects on the vasculature. In addition, high levels of TNF-α were demonstrated in patients with ED. In this review, we present a short description of the physiology of erection and the cytokine network. We focus on vascular actions of TNF-α that support a role for this cytokine as a potential candidate in the pathophysiology of ED, particularly in the context of CVD. A brief overview of its discovery, mechanisms of synthesis, receptors, and its main actions on the systemic and penile vasculature is also presented.
Considering that ED results from a systemic arterial defect not only confined to the penile vasculature, implication of TNF-α in the pathophysiology of ED offers a humoral linking between CVD and ED.
PMCID: PMC3031865  PMID: 20345734
Erectile Dysfunction; TNF-α; Cytokines; Cardiovascular Disease; Coronary Artery Disease
Bisphenol A (BPA), a suspected reproductive biohazard and endocrine disruptor released from plastics is associated with erectile dysfunction (ED) in occupationally exposed workers. However, in rats, despite the induction of hypogonadism, apoptosis of the penile corporal smooth muscle, fat infiltration into the cavernosal tissue, and changes in global gene expression with the intraperitoneal administration of high dose BPA, ED was not observed.
We investigated whether BPA administered orally rather than intraperitoneally to rats for longer periods and lower doses will lead to ED.
Main Outcomes Measures
ED, histological, and biochemical markers in rat penile tissues.
2.5-month old rats were given drinking water daily without and with BPA at 1 and 0.1 mg/kg/day. Two months later, erectile function was determined by cavernosometry (DIC) and electrical field stimulation (EFS) and serum levels of testosterone (T), estradiol (E2), and BPA were measured. Penile tissue sections were assayed by Masson (smooth muscle (SM)/collagen), Oil Red O (fat), TUNEL (apoptosis), immunohistochemistry for Oct 4 (stem cells), and α-SM actin/ calponin (SM and myofibroblasts), applying quantitative image analysis. Other markers were assayed by western blots. DNA microarrays/microRNA assays defined transcription profiles.
Orally administered BPA did not affect body weight, but: 1) decreased serum T and E2; 2) reduced the EFS response and increased the DIC drop rate; 3) increased within the corporal tissue the presence of fat, myofibroblasts and apoptosis; 4) lowered the contents of SM and stem cells, but not nerve terminals; and 5) caused alterations of the transcriptional profiles for both mRNA and microRNAs within the penile shaft.
Long-term exposure of rats to oral BPA,caused a moderate corporal veno-occlusive dysfunction (CVOD), possibly due to alterations within the corporal tissue that pose gene transcriptional changes related to inflammation, fibrosis and epithelial/ mesenchymal transition (EMT).
PMCID: PMC4098849  PMID: 24305612
Erectile dysfunction; fibrosis; corporal veno-occlusive dysfunction; epithelial mesenchymal transition; microRNA
14.  Dimethylarginine dimethylaminohydrolase in rat penile tissue: reduced enzyme activity is responsible for erectile dysfunction in a rat model of atherosclerosis 
Asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase (NOS), is mainly metabolized by NG,NG-dimethylarginine dimethylaminohydrolase (DDAH). We investigated whether altered cavernosal ADMA–DDAH metabolism might cause impairment of erection in rat model of atherosclerosis (AS). Male Sprague–Dawley rats (3 months old) were divided into an AS group and a normal control (Con) group (n=20 in each group). The AS rats received AS-prone treatment (6 weeks of 1% cholesterol diet plus early 2 weeks of NG-nitro-L-arginine methyl ester (3 mg ml−1 per day) treatment). After 6 weeks, rats underwent cavernosometry measuring the maximal intracavernosal pressure/mean arterial pressure (ICP/MAP) ratios as a surrogate marker of erectile function. The amount of cavernosal ADMA was assessed by immunoblot analysis and correlated with the ICP/MAP. Isoform-specific DDAH expression was compared by immunohistochemistry. Cavernosal DDAH and NOS activity were measured. Cavernosal malondialdehyde levels were assayed to determine the degree of lipid peroxidation. Compared to the controls, the AS rats had signs of impaired erectile function. Higher cavernosal ADMA was observed in the AS rats. The cavernosal ADMA had a moderately negative correlation with the ICP/MAP. Immunohistochemistry revealed the expression of both isoforms was not affected by the presence of AS. However, significantly diminished DDAH as well as NOS activity was observed in the AS group. In addition, elevated cavernosal malondialdehyde levels were noted in the AS rats. Our study showed that decreased cavernosal DDAH activity is the cause of cavernosal ADMA accumulation leading to reduced cavernosal NOS activity and impairment of erectile function.
PMCID: PMC2834501  PMID: 19603041
erectile dysfunction; atherosclerosis; rats; asymmetric dimethylarginine; dimethylarginine dimethylaminohydrolase
15.  Increased cavernosal relaxation by Phoneutria nigriventer toxin, PnTx2-6, via activation at NO/cGMP signaling 
Erectile dysfunction mechanisms in diabetic patients are multifactorial and often lead to resistance to current therapy. Animal toxins have been used as pharmacological tools to study penile erection. Human accidents involving the venom of Phoneutria nigriventer spider are characterized by priapism. We hypothesize that PnTx2-6 potentiates cavernosal relaxation in diabetic mice by increasing cGMP. This effect is nNOS dependent. Cavernosal strips were contracted with phenylephrine (10−5 M) and relaxed by electrical field stimulation (EFS, 20V, 1–32 Hz) in the presence or absence of PnTx2-6 (10−8 M).Cavernosal strips from nNOS and eNOS knocaut (KO) mice, besides nNOS inhibitor (10−5M), were used to evaluate the role of this enzyme in the potentiation effect evoked by PnTx2-6. Tissue cGMP levels were determined after stimulation with PnTx2-6 in presence or absence of L-NAME (10−4M) and ω-conotoxin GVIA (10−6M), an N-type calcium channel inhibitor. Results showed PnTx2-6 enhanced cavernosal relaxation in diabetic mice (65%) and eNOS KO mice, but not in nNOS KO mice. The toxin effect in the cavernosal relaxation was abolished by nNOS inhibitor. cGMP levels are increased by PnTx2-6, however L-NAME abolished this enhancement as well as ω-conotoxin GVIA. We conclude PnTx2-6 facilitates penile relaxation in diabetic mice through a mechanism dependent on nNOS, probably via increasing NO/cGMP production.
PMCID: PMC3253321  PMID: 21975567
Phoneutria nigriventer; erectile dysfunction; PnTx2-6 toxin; NO; cGMP
16.  Adenosine Deaminase Enzyme Therapy Prevents and Reverses the Heightened Cavernosal Relaxation in Priapism 
The journal of sexual medicine  2010;7(9):3011-3022.
Priapism featured with painful prolonged penile erection is dangerous and commonly seen in sickle cell disease (SCD). The preventive approaches or effective treatment options for the disorder are limited because of poor understanding of its pathogenesis. Recent studies have revealed a novel role of excess adenosine in priapism caused by heightened cavernosal relaxation, and therefore present an intriguing mechanism-based therapeutic possibility.
The aim of this study was to determine the therapeutic effects of adenosine deaminase (ADA) enzyme therapy to lower adenosine in priapism.
Both ADA-deficient mice and SCD transgenic (Tg) mice display priapism caused by excessive adenosine. Thus, we used these two distinct lines of mouse models of priapism as our investigative tools. Specifically, we treated both of these mice with different dosages of polyethylene glycol–modified ADA (PEG–ADA) to reduce adenosine levels in vivo. At the end points of the experiments, we evaluated the therapeutic effects of PEG–ADA treatment by measuring adenosine levels and monitoring the cavernosal relaxation.
Main Outcome Measures
Adenosine levels in penile tissues were measured by high-performance liquid chromatography, and cavernosal relaxation was quantified by electrical field stimulation (EFS)-induced corporal cavernosal strip (CCS) assays.
We found that lowering adenosine levels in penile tissues by PEG–ADA treatment from birth in ADA-deficient mice prevented the increased EFS-induced CCS relaxation associated with priapism. Intriguingly, in both ADA-deficient mice and SCD Tg mice with established priapism, we found that normalization of adenosine levels in penile tissues by PEG–ADA treatment relieved the heightened EFS-induced cavernosal relaxation in priapism.
Our studies have identified that PEG–ADA is a novel, safe, and mechanism-based drug to prevent and correct excess adenosine-mediated increased cavernosal relaxation seen in two independent priapic animal models, and suggested its therapeutic possibility in men suffering from priapism.
PMCID: PMC2906644  PMID: 19845544
Adenosine Signaling; Priapism; Novel Therapies; Pharmacologic Treatment of Priapism
The journal of sexual medicine  2008;6(2):415-428.
Corporal veno-occlusive dysfunction (CVOD), which usually is associated with a loss of smooth muscle cells (SMC) and an increase in fibrosis within the corpora cavernosa, can be induced by an injury to the cavernosal nerves. The corporal tissue expresses inducible nitric oxide synthase (iNOS), presumably as an anti-fibrotic and SMC-protective response.
We studied the temporal relationship in the corpora between the expression of iNOS, other histological and biochemical changes, and the development of CVOD, after bilateral cavernosal nerve resection (BCNR) in the rat.
Rats underwent either BCNR or sham operation. Cavernosometry was performed 1, 3, 7, 15, 30, and 45 days (n=8/groups) after surgery. Penile tissue sections were subjected to Masson trichrome staining for SMC and collagen, and immunodetection for alpha smooth muscle actin, iNOS, neuronal NOS (nNOS), endothelial NOS (eNOS), proliferating cell nuclear antigen (PCNA), and TUNEL. Quantitative western blot analysis was done in homogenates.
Main outcome measures
Time course on the development of fibrosis and CVOD
Following BCNR, CVOD was detectable 30 days later and it became more pronounced by 45 days. In contrast, the SMC/collagen ratio in the BCNR corpora was reduced at 7 days and bottomed at 30 and 45 days, due in part to the reduction of SMC, presumably caused by an increase in apoptosis peaking at 3 days. PCNA also peaked at 3 days but then decayed. nNOS was reduced early (3-7 days) and disappeared at 30 days, whereas eNOS was not affected. iNOS was induced at day 3, and steadily increased peaking at 30 days.
CVOD develops in the BCNR rat as a result of the early loss of corporal SMC by the neuropraxia-induced apoptosis, which the initial cell replication response cannot counteract, followed by fibrosis. The time course of iNOS induction supports the antifibrotic role of iNOS.
PMCID: PMC2756287  PMID: 19138364
fibrosis; erectile dysfunction; smooth muscle; nerve sparing; radical prostatectomy; penis; nitric oxide; cGMP; collagen; inducible nitric oxide synthase; apoptosis
18.  Transcription of G-protein coupled receptors in corporal smooth muscle is regulated by sialorphin (an endogenous neutral endopeptidase inhibitor) 
The Journal of urology  2008;180(2):760-766.
Several reports have suggested the rat Vcsa1 gene is down-regulated in models of erectile dysfunction (ED). Vcsa’s protein product, sialorphin, is an endogenous neutral endopeptidase (NEP), and its down-regulation could result in prolonged activation of G-protein activated signaling pathways by their peptide agonists. We investigated if down- regulation of Vcsa1 could result in adaptive change in the expression of G-protein coupled receptors (GPCR).
Materials and Methods
Gene expression in cultured rat corporal smooth muscle cells (CSM) following treatment with siRNA directed against Vcsa1 or the NEP gene was analyzed using microarray and quantitative RT-PCR. In rats Vcsa1 is one of the most down-regulated genes following bilateral transection of the cavernosal nerves. Using that animal model, we also investigated whether the down-regulation of Vcsa1 is accompanied by similar changes in gene expression observed in the CSM cells where Vcsa1 was knocked-down in vitro.
Microarray analysis and quantitative RT-PCR demonstrated that CSM cells treated in vitro with siRNA against Vcsa1 resulted in up-regulation of GPCR as a functional group. In contrast, treatment of CSM cells that lowered NEP activity resulted in decreases in GPCR expression. These results suggest that the peptide product of Vcsa1, sialorphin, can effect GPCR expression by acting on NEP. In animals with bilaterally transected cavernous nerves the reduced expression of Vcsa1 is accompanied by increased GPCR expression in cavernosal tissue.
These experiments suggest that the mechanism by which Vcsa1 modulates erectile function is partly mediated through changes in GPCR expression.
PMCID: PMC2744426  PMID: 18554633
erectile dysfunction; G-protein coupled receptors; neutral endopeptidase inhibitor; sialorphin; Vcsa1
19.  Penile revascularization—contemporary update 
Asian Journal of Andrology  2012;15(1):5-9.
Contemporary therapies for erectile dysfunction are generally targeted towards older men and universally engage pharmacological and/or device related treatment options. Penile revascularization, using microvascular arterial bypass surgical techniques, is a non-pharmacological, non-device-related, and reconstructive surgical strategy for men with erectile dysfunction that was first described by Dr Vaclav Michal in 1973. Contemporary penile revascularization attempts to ‘cure' pure arteriogenic erectile dysfunction in young men with arterial occlusive pathology in the distal internal pudendal, common penile or proximal cavernosal artery secondary to focal endothelial injury from blunt pelvic, perineal or penile trauma. A microvascular anastomosis is fashioned between the donor inferior epigastric and recipient dorsal penile artery. Increased perfusion pressure is theoretically communicated to the cavernosal artery via perforating branches from the dorsal artery. This article will review the history, indications and pathophysiology of blunt trauma-induced focal arterial occlusive disease in young men with erectile dysfunction, current surgical techniques utilized and results of surgery. Contemporary use of penile revascularization is a logical and wanted therapeutic option to attempt to reverse erectile dysfunction in young men who have sustained blunt pelvic, perineal or penile trauma.
PMCID: PMC3739123  PMID: 23241636
erectile dysfunction; microvascular arterial bypass surgery; penile revascularization; traumatic arterial occlusive disease; vascular reconstructive surgery
20.  The Effects of Trimetazidine and Sildenafil on Bilateral Cavernosal Nerve Injury Induced Oxidative Damage and Cavernosal Fibrosis in Rats 
The Scientific World Journal  2014;2014:970363.
Aim. The aim of this study was to compare the effects of sildenafil and trimetazidine on bilateral cavernosal nerve injury-induced oxidative damage and fibrotic changes in cavernosal tissue in rat model. Material and Methods. A total of 32 male Sprague-Dawley rats were randomly divided into 4 groups; each group consist 8 rats (control, BCI, BCI + TMZ, and BCI + sildenafil groups). Tissue superoxide dismutase (SOD), malondialdehyde (MDA), and protein carbonyl (PC) levels were determined biochemically and distribution of cavernosal fibrosis density among groups was performed histopathologically. Results. Tissue SOD levels in BCI group were significantly lower than the control group (P < 0.05). Tissue MDA and PC levels in BCI group were significantly higher than the control group (P < 0.05). TMZ and sildenafil administration significantly increased tissue SOD levels (P < 0.05) and reduced tissue MDA and PC levels (P < 0.05). Histologically, the degree of cavernosal fibrosis and collagen density was higher in BCI group in comparison to control, TMZ-treated, and sildenafil-treated groups. Conclusion. BCI caused oxidative damage and increased cavernosal fibrosis in rat penis. TMZ and sildenafil treatment decreased oxidative damage and reduced the degree of fibrosis in penile tissue due to BCI.
PMCID: PMC3977563  PMID: 24782681
21.  Metabolic Syndrome and Erectile Dysfunction 
Diabetes Care  2011;34(8):1875-1877.
To study the relation between metabolic syndrome (MS), cavernosal morphological vasculopathy, and peripheral vascular alterations (carotid and femoral wall) in patients with erectile dysfunction.
A total of 207 patients and 50 control subjects were evaluated for cardiovascular risk factors, physical examination, reproductive hormones, ultrasound analysis of cavernosal, carotid and femoral arteries (intima-media thickness), and cavernosal flow measurement (peak systolic velocity).
A total of 28% of patients had MS, and they presented with a high prevalence of cavernosal alterations (70.3%) and systemic vascular impairment (59.3%), whereas patients with cavernosal alterations (44%) showed the higher prevalence of MS (48.9%). The number of MS components was related to the prevalence of penile vasculopathy. However, multivariate analysis showed that MS is not an independent predictor for cavernosal vasculopathy.
Patients with cavernosal vasculopathy have an increased cardiometabolic risk, and screening for MS components might identify individuals with a higher risk for cavernosal and systemic atherosclerosis.
PMCID: PMC3142025  PMID: 21730283
22.  Modulation of Penile Erection in Rabbits by Mondia Whitei: Possible Mechanism of Action 
Mondia whitei root was evaluated to validate its anecdotal use and determine its possible mode of action in the management of erectile dysfunction. Rabbits were administered with daily oral doses of 100–400 mg kg−1 crude ethanolic extract of M. whitei and sildenafil (50 mg kg−1) as positive control for 6 weeks. Cavernosal tissue NOS activity and levels of NO and cGMP, and NOS and PDE protein expressions were investigated. The effect of the crude extract, chloroform and petroleum ether fractions in vitro on cavernosal tissue NOS activity and levels of NO and cGMP at 0.01 and 0.10 mg g−1 tissue were also investigated. Results indicate that the crude extract increased NOS activity by 7% at 200 mg kg−1 with corresponding increases in NO (88%) and cGMP (480%) levels. No significant changes in these measurements were observed with the 100 and 400 mg kg−1 doses whilst sildenafil slightly reduced them (15.9–37.5%). NOS and PDE protein expressions in test animals were not different from controls. Pre-incubation of cavernosal tissue in vitro with the crude extract of M. whitei and its chloroform fraction markedly increased NOS activity (26–132%) and levels of NO (25%) and cGMP (50–400%) at 0.01 mg g−1 tissue but these were reduced to near control levels when their concentrations were increased to 0.10 mg g−1 tissue whilst the petroleum ether fraction had no effect. These findings suggest that M. whitei may influence erectile function through activation/stimulation of NOS with corresponding increases in tissue NO and cGMP levels and that certain chemical constituents present in the chloroform fraction may be responsible for biological activity.
PMCID: PMC3025616  PMID: 21461152
Erectile dysfunction; nitric oxide; cGMP; sildenafil; organic fractions
23.  Transplantation of Muscle-Derived Stem Cells into the Corpus Cavernosum Restores Erectile Function in a Rat Model of Cavernous Nerve Injury 
Korean Journal of Urology  2011;52(5):359-363.
Muscle-derived stem cells (MDSCs) harvested from skeletal muscles have the advantage of providing easier access and do not pose the immunogenic risks of embryonic stem cells. We investigated the effect of intracavernosal transplantation of MDSCs on erectile function in rats with bilateral cavernous nerve injury.
Materials and Methods
Adult male white rats underwent experimentation in 3 groups: group I, sham operation; group II, bilateral cavernous nerve injury; group III, bilateral cavernous nerve injury with MDSC injection. MDSCs were harvested from the femoral muscle of rats and were then injected into the cavernosum. Survival of MDSCs and measurement of erectile function was studied after 4 weeks. We checked the intracavernosal pressure (ICP) and obtained penile tissue. The expression of cyclic guanosine monophosphate (cGMP) was analyzed.
Four weeks after transplantation, PKH-26-labeled MDSCs were identified in the cavernosal tissues of group III. Peak ICP and the drop rate of group II were 52±8.7 mmHg and 34±6.5 mmHg/min, respectively, whereas peak ICP and the drop rate of group III were 97±15.6 mmHg and 17±4.9 mmHg/min, respectively, showing that erectile function improved after MDSC transplantation (p<0.05). The expression of cGMP was significantly lower in group II (21.9±5.8 fmol/well) than in group I and group III (70.2±10.3 and 58.9±10.5 fmol/well, respectively).
In a cavernous nerve injury rat model, intracavernosal transplantation of MDSCs showed acceptable survival of MDSCs as well as improvement of erectile function.
PMCID: PMC3106171  PMID: 21687398
Erectile dysfunction; Stem cells; Transplantation
24.  Does kidney transplantation onto the external iliac artery affect the haemodynamic parameters of the cavernosal arteries? 
Asian Journal of Andrology  2011;14(4):621-625.
Reduced cavernosal arterial inflow has been hypothesized to be the likely cause of erectile dysfunction after kidney transplants in recipients revascularized through end-to-end anastomosis to the internal iliac artery, suggesting that end-to-side anastomosis at the external iliac artery is preferable. The aim of this study was to prospectively evaluate the effect of the use of the external iliac artery on erectile function, hormone profiles and penile blood flow by evaluating changes in penile colour Doppler ultrasound parameters in a consecutive series of 22 recipients before and after end-to-side external iliac artery transplantation. The mean International Index of Erectile Function–Erectile Function (IIEF–EF) domain score decreased significantly 3 months after transplant (18.09±6.33 vs. 22.50±7.09, P=0.01). The reduction in peak systolic velocity (PSV) was significant for the cavernous artery homolateral to the side of transplant (42.60±18.77 vs. 52.01±19.91, P=0.01). The mean postoperative end diastolic velocity (EDV) did not differ significantly from the preoperative value (P=0.74). No statistical differences were found in the serum levels of testosterone or prolactin. Kidney grafts anastomosed at the external iliac artery produced significant (P=0.01) reductions in arterial inflow at the homolateral cavernosal artery that remained above the normal threshold. Whether these haemodynamic changes can explain the worsening of postoperative erectile function remains to be proven.
PMCID: PMC3720083  PMID: 22198628
cavernosal arteries; Doppler; erectile dysfunction; external iliac artery; kidney transplant
25.  Analysis of Erectile Responses to Imatinib in the Rat 
Urology  2013;82(1):10.1016/j.urology.2013.04.009.
To investigate the erectile and cardiovascular responses to the tyrosine kinase inhibitor imatinib in the rat.
The effect of intracavernosal injection of imatinib on the intracavernosal pressure (ICP), ICP/mean arterial pressure (MAP) ratio, area under the curve, and duration of the increase in ICP and the effect of intravenous injection of imatinib on the MAP, cardiac output, and total peripheral resistance were investigated. The effect of the nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester on the responses to imatinib was investigated.
Intracavernosal injection of imatinib produced significant dose-related increases in the ICP, ICP/MAP ratio, area under the curve, and duration of the increase in ICP and decreases in the MAP. The erectile responses to imatinib were rapid in onset and short in duration. The erectile responses to imatinib were not significantly altered by NG-nitro-L-arginine methyl ester or cavernosal nerve crush injury, and imatinib was significantly less potent than the nitric oxide donor sodium nitroprusside in inducing erection. Intravenous injection of imatinib produced significant dose-related decreases in the MAP without significantly changing the cardiac output, and imatinib was significantly less potent than sodium nitroprusside in decreasing the MAP. Systemic vascular resistance was decreased in a significant dose-related manner, and the vaso-dilator responses to imatinib were not altered by NG-nitro-L-arginine methyl ester.
The present results have indicated that imatinib has significant erectile and systemic vasodilator activity in the rat that is not dependent on nitric oxide release. Another tyrosine kinase inhibitor, nilotinib, also increased the ICP and decreased the MAP in the rat. These data suggest that tyrosine kinases might play a constitutive role in maintaining penile tumescence and the baseline vasoconstrictor tone in the peripheral vascular bed.
PMCID: PMC3810015  PMID: 23806406

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