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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.  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
3.  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
4.  TNF-α knockout mice have increased corpora cavernosa relaxation 
The journal of sexual medicine  2009;6(1):115-125.
Erectile dysfunction (ED) is considered an early clinical manifestation of vascular disease and an independent risk factor for cardiovascular events associated with endothelial dysfunction and increased levels of pro-inflammatory cytokines. Tumor necrosis factor-alpha (TNF-α), a pro-inflammatory cytokine, suppresses endothelial nitric oxide synthase (eNOS) expression.
Considering that nitric oxide (NO) is of critical importance in penile erection, we hypothesized that blockade of TNF-α actions would increase cavernosal smooth muscle relaxation through an increase in NOS expression.
In vitro organ bath studies were used to measure cavernosal reactivity in wild type and TNF-α knockout (TNF-α KO) mice and NOS expression was evaluated by western blot. In addition, spontaneous erections (in vivo) were evaluated by videomonitoring the animals (30 min.). Collagen and elastin expression were evaluated by Masson trichrome and Verhoff-van Gieson stain reaction, respectively.
Main Outcome Measures
Corpora cavernosa from TNF-α KO mice exhibited increased NO-dependent relaxation, which was associated with increased eNOS and neuronal NOS (nNOS) cavernosal expression.
Cavernosal strips from TNF-α KO mice displayed increased endothelium-dependent [97.4±5.3 vs Control: 76.3±6.3, %] and nonadrenergic-noncholinergic (NANC) [93.3±3.0 vs Control: 67.5±16.0; 16 Hz] relaxation compared to control animals. These responses were associated with increased protein expression of eNOS and nNOS (p<0.05). Sympathetic-mediated [0.69±0.16 vs Control: 1.22±0.22; 16 Hz] as well as phenylephrine-induced contractile responses [1.6±0.1 vs Control: 2.5±0.1, mN] were attenuated in cavernosal strips from TNF-α KO mice. Additionally, corpora cavernosa from TNF-α KO mice displayed increased collagen and elastin expression. In vivo experiments demonstrated that TNF-α KO mice display increased number of spontaneous erections.
Corpora cavernosa from TNF-α KO mice display alterations that favor penile tumescence, indicating that TNF-α plays a detrimental role in erectile function. A key role for TNF-α in mediating endothelial dysfunction in ED is markedly relevant since we now have access to anti-TNF-α therapies.
PMCID: PMC2843140  PMID: 19170842
tumor necrosis factor alpha; corpus cavernosum; endothelial nitric oxide synthase; neuronal nitric oxide synthase; mouse
5.  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
6.  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
7.  A Unique Case of Penile Necrotizing Fasciitis Secondary to Spontaneous Corpus Cavernosal Abscess 
Case Reports in Urology  2013;2013:576146.
Corpus cavernosal abscess and necrotizing fasciitis occur rarely, and precipitating factors can usually be elicited with careful history and examination. Whilst both conditions share common risk factors such as diabetes mellitus, this is the first reported case of penile necrotizing fasciitis secondary to spontaneous corpus cavernosal abscess in an otherwise healthy patient. A 32-year-old man presented with 4-day history of swollen, painful penis, with ultrasound confirming corpus cavernosal abscess. Biopsies were taken and the cavity aspirated, but, despite intravenous antibiotics, he developed penile necrotizing fasciitis necessitating open cavernostomy and debridement. The overlying skin defect healed by secondary intention, but the patient experienced persistent postoperative erectile dysfunction, so he was referred for penile prosthesis insertion.
PMCID: PMC3808107  PMID: 24198996
8.  Penile Reconstruction for a Case of Genital Lymphoedema Secondary to Proteus Syndrome 
ISRN Urology  2011;2011:431536.
To our knowledge penile lymphoedema secondary to Proteus syndrome has not previously been reported. Hence we report a case of a 16-year-old male who was referred with features of right hemi-hypertrophy and severe lymphoedema affecting his scrotum and penis. He had previously undergone scrotal reduction surgery at the age of 13, but had since developed worsening penile oedema. His main concern was that of cosmetic appearance prior to sexual debut, and he also complained of erectile dysfunction. An MRI confirmed gross oedema of the penile skin, but normal underlying cavernosal structure, and no other anatomical abnormality. Under general anaesthesia, the entire diseased penile skin was excised. Two full thickness skin grafts were harvested from the axillae, and grafted onto the dorsal and ventral penile shaft respectively. A compressive dressing and urinary catheter was applied for 7 days. Follow-up at 4 months confirmed complete graft take with minimal scarring, and the patient was very satisfied with the cosmetic outcome. He had also noticed a recovery in erectile activity, and feels psychologically and physically more prepared for sexual relations.
PMCID: PMC3197011  PMID: 22084799
9.  Transient Distal Penile Corporoglanular Shunt as an Adjunct to Aspiration and Irrigation Procedures in the Treatment of Early Ischemic Priapism 
Korean Journal of Urology  2013;54(6):394-398.
Ischemic priapism, a compartment syndrome, requires urgent treatment in order to nourish the corpora cavernosa. As the first step, aspiration of blood and irrigation of the cavernosal bodies is performed to prevent fibrotic activity and secure erectile capability. During aspiration, there are risks of cardiovascular side effects of adrenergic agonists. We aimed to evaluate a transient distal penile corporoglanular shunt technique in place of aspiration and irrigation techniques for treatment of early ischemic priapism.
Materials and Methods
A transient distal penile shunt was applied to 15 patients with early ischemic priapism between January 2011 and May 2012. Priapism duration, history, causes, pain, and any prior management of priapism were assessed in all patients. A complete blood count and penile Doppler ultrasonography were performed, which showed attenuated blood flow in the cavernosal artery. A sterile closed system blood collection set, which has two needles and tubing, was used for the transient distal penile shunt.
Ten of 15 patients with early ischemic priapism were successfully treated with this transient shunt technique. No additional procedures were needed after the resolution of rigidity in the 10 successfully treated patients.
The transient nature of this technique is an advantage over aspiration and irrigation in the treatment of early ischemic priapism. Our results indicate that the technique can be offered for patients with an ischemic priapism episode of no more than 7 hours.
PMCID: PMC3685640  PMID: 23789049
Penile disease; Penile erection; Priapism; Urological surgical procedures
10.  Treatment with CB2 Agonist JWH-133 Reduces Histological Features Associated with Erectile Dysfunction in Hypercholesterolemic Mice 
Hypercholesterolemia is one of the most important risk factors for erectile dysfunction, mostly due to the impairment of oxidative stress and endothelial function in the penis. The cannabinoid system might regulate peripheral mechanisms of sexual function; however, its role is still poorly understood. We investigated the effects of CB2 activation on oxidative stress and fibrosis within the corpus cavernosum of hypercholesterolemic mice. Apolipoprotein-E-knockout mice were fed with a western-type diet for 11 weeks and treated with JWH-133 (selective CB2 agonist) or vehicle during the last 3 weeks. CB2 receptor expression, total collagen content, and reactive oxygen species (ROS) production within the penis were assessed. In vitro corpus cavernosum strips preparation was performed to evaluate the nitric oxide (NO) bioavailability. CB2 protein expression was shown in cavernosal endothelial and smooth muscle cells of wild type and hypercholesterolemic mice. Treatment with JWH-133 reduced ROS production and NADPH-oxidase expression in hypercholesterolemic mice penis. Furthermore, JWH-133 increased endothelial NO synthase expression in the corpus cavernosum and augmented NO bioavailability. The decrease in oxidative stress levels was accompanied with a reduction in corpus cavernosum collagen content. In summary, CB2 activation decreased histological features, which were associated with erectile dysfunction in hypercholesterolemic mice.
PMCID: PMC3835849  PMID: 24302957
11.  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
12.  Comparison of the involvement protein kinase C in agonist-induced contractions in mouse aorta and corpus cavernosum 
European journal of pharmacology  2008;590(0):363-368.
Protein kinase C (PKC) is involved in the regulation of vascular smooth muscle contraction. However, the role of PKC in erectile function is poorly understood. This study investigated whether PKC mediates agonist-induced contractions in mouse penile tissue (corpora cavernosa). We also compared the effects of PKC activators and inhibitors on contractile responses in mouse corpus cavernosum with those in mouse aorta. Aortic rings and corpus cavernosal strips from C57BL/6J mice was isolated, mounted in the organ bath for isometric tension recording. Our data showed that a PKCα/β selective inhibitor, Gö6976 (10 µM), inhibited phenylephrine and 9,11-dideoxy-11α,9α-epoxymethanoprostaglandin F2α (U46619, a thromboxane mimetic)-induced contractions in mouse aorta, reducing the maximum contraction from 123 ± 2% of KCl-induced maximum contraction to 7 ± 2% and 13 ± 1%, respectively. A non-selective PKC inhibitor, chelerythrine (30 µM), also significantly reduced phenylephrine-and U46619-induced maximum contractions in mouse aorta. However, Gö6976 and chelerythrine had no significant effects on phenylephrine-and U46619-induced contractions in corpus cavernosum. Furthermore, a PKC activator, phorbol-12,13-dibutyrate (0.1 µM), significantly increased contractions in aorta (208 ± 14% of KCl-induced maximum contraction) but failed to cause contractions in corpus cavernosum at 1 and 10 µM. Western blot analysis data suggested that protein expression of PKC was similar in aorta and corpus cavernosum. Taken together, our data indicate that PKC does not have a significant role in agonist-induced contractions in mouse corpus cavernosum, whereas it mediates the contractile response to agonists in the aorta.
PMCID: PMC3939724  PMID: 18614166
aorta; penis; protein kinase C; smooth muscle; erectile dysfunction; corpus cavernosum
13.  Arginase II Deletion Increases Corpora Cavernosa Relaxation in Diabetic Mice 
The journal of sexual medicine  2010;8(3):722-733.
Diabetes-induced erectile dysfunction involves elevated arginase (Arg) activity and expression. Because nitric oxide (NO) synthase and Arg share and compete for their substrate L-arginine, NO production is likely linked to regulation of Arg. Arg is highly expressed and implicated in erectile dysfunction.
It was hypothesized that Arg-II isoform deletion enhances relaxation function of corpora cavernosal (CC) smooth muscle in a streptozotocin (STZ) diabetic model.
Eight weeks after STZ-induced diabetes, vascular functional studies, Arg activity assay, and protein expression levels of Arg and constitutive NOS (using western blots) were assessed in CC tissues from non-diabetic wild type (WT), diabetic (D) WT (WT+D), Arg-II knockout (KO) and Arg-II KO+D mice (N=8–10 per group).
Main Outcome Measures
Inhibition or lack of arginase results in facilitation of CC relaxation in diabetic CC.
Strips of CC from Arg-II KO mice exhibited an enhanced maximum endothelium-dependent relaxation (from 70+3% to 84+4%) and increased nitrergic relaxation (by 55%, 71%, 42%, 42%, and 24% for 1, 2, 4, 8 and 16 Hz, respectively) compared to WT mice. WT+D mice showed a significant reduction of endothelium-dependent maximum relaxation (44+8%), but this impairment of relaxation was significantly prevented in Arg-II KO+D mice (69+4%). Sympathetic-mediated and alpha-adrenergic agent-induced contractile responses also were increased in CC strips from D compared to non-D controls. Contractile responses were significantly lower in Arg-II KO control and D versus the WT groups. WT+D mice increased Arg activity (1.5-fold) and Arg-II protein expression and decreased total and phospho-eNOS at Ser-1177, and nNOS levels. These alterations were not seen in Arg-II KO mice. Additionally, the Arg inhibitor BEC (50 μM) enhanced nitrergic and endothelium-dependent relaxation in CC of WT+D mice.
These studies show for the first time that Arg-II deletion improves CC relaxation in type 1 diabetes.
PMCID: PMC3117078  PMID: 21054801
14.  Correlation between penile cavernosal artery blood flow and retinal vascular findings in arteriogenic erectile dysfunction 
Arteriogenic erectile dysfunction (ED) is a target organ disease of atherosclerosis, and therefore might be a predictor of systemic atherosclerosis. Being systemic, it might be possible to evaluate the extent of atherosclerosis from retinal vascular findings. We investigated the possible correlation between penile cavernosal artery blood flow and retinal vascular findings in patients with arteriogenic ED.
Patients and methods:
Sixty patients with ED were divided according to the peak systolic velocity (PSV) in their penile cavernosal arteries into two groups; Group A included 30 patients with PSV less than 25 cm/sec, and Group B included 30 patients with PSV more than 35 cm/sec. Blood flow in the penile cavernosal artery was measured with color Doppler ultrasonography. All patients were assessed by ocular fundus examination under amydriatic conditions to evaluate retinal vascular atherosclerotic changes using Hyman’s classification.
Evidence of retinal vascular atherosclerotic changes was found in 19 patients (63.3%) in Group A and in 10 patients (33.3%) in Group B.
Our study confirms the possibility of predicting penile arterial vascular status in patients with ED from their retinal vascular findings by using amydriatic simple, practical funduscopy.
PMCID: PMC2946996  PMID: 20922041
erectile dysfunction; atherosclerosis; retinal vascular atherosclerosis
15.  Differential Expression of Nerve Injury-Induced Protein 1 (Ninjurin 1) in In Vivo and In Vitro Models for Diabetic Erectile Dysfunction 
Korean Journal of Urology  2012;53(9):636-642.
Endothelial dysfunction and peripheral neuropathy are important mechanisms responsible for diabetes-induced erectile dysfunction (ED). Nerve injury-induced protein 1 (Ninjurin 1) is known to be related to neuroinflammatory processes and is also reported to induce vascular regression during the developmental period. In the present study, we determined the differential expression of Ninjurin 1 in penile tissue of streptozotocin (STZ)-induced diabetic mice with ED.
Materials and Methods
Diabetes was induced in 8-week-old C57BL/6J mice by intraperitoneal injections of STZ (50 mg/kg for 5 days). Eight weeks later, erectile function was measured by electrical stimulation of the cavernous nerve (n=6 per group). The penis was then harvested for immunohistochemical analysis and Western blot analysis for Ninjurin 1 (n=4 per group). We also determined Ninjurin 1 expression in primary cultured mouse cavernous endothelial cells (MCECs) incubated under the following conditions: normal glucose condition (5 mM), high-glucose condition (30 mM), and high-glucose condition (30 mM)+insulin (1 nM).
The expression of Ninjurin 1 protein was significantly higher in both cavernous endothelial cells and the dorsal nerve bundle of diabetic mice than in those of controls. In the in vitro study in MCECs, Ninjurin 1 expression was also significantly increased by the high-glucose condition and was returned to baseline levels by treatment with insulin.
Regarding the role of Ninjurin 1 in neuropathy and vascular regression, it would be interesting to examine the effects of inhibition of Ninjurin 1 on erectile function in animal models of ED with a vascular or neurogenic cause.
PMCID: PMC3460007  PMID: 23061002
Diabetes mellitus; Endothelium; Erectile dysfunction; Nerve; Ninj1
16.  Loss of Raf Kinase Inhibitor Protein Promotes Cell Proliferation and Migration of Human Hepatoma Cells 
Gastroenterology  2006;131(4):1208-1217.
Background & Aims
The Raf kinase inhibitor protein (RKIP) has been identified as a suppressor of the mitogen-activated protein kinase (MAPK) pathway. Loss of RKIP function promotes tumor metastasis in prostate cancer and melanoma. The IGF-I mediated MAPK cascade is often activated in hepatocellular carcinoma (HCC), but the role of RKIP in the molecular pathogenesis of these tumors is unknown. This study was performed to evaluate the role of RKIP in HCC development.
The levels of RKIP expression in HCC tumor and corresponding peritumoral tissues were determined by immunohistochemistry and Western blot analysis. The underlying mechanisms of RKIP were assessed with immunoblot analysis, Raf kinase activity assay, cell proliferation and migration assays after either overexpression or knockdown of RKIP expression in HCC cell lines.
RKIP expression is downregulated in human HCC compared to adjacent peritumoral tissues. Low RKIP levels were correlated with enhanced extracellular-signal-regulated-kinase (ERK)/MAPK pathway activation. Reconstitution experiments antagonized IGF-I mediated MAPK pathway activation resulting in reduced nuclear accumulation of phospho-ERK. In contrast, knockdown of RKIP expression using siRNA induced activation of the ERK/MAPK pathway. Ectopic expression of RKIP altered HCC cell proliferation and migration.
Our findings indicate that downregulation of RKIP expression is a major factor in activation of the IGF-I/ERK/MAPK pathway during human hepatocarcinogenesis.
PMCID: PMC2593881  PMID: 17030190
17.  COMP-Angiopoietin-1 Promotes Cavernous Angiogenesis in a Type 2 Diabetic Rat Model 
Journal of Korean Medical Science  2013;28(5):725-730.
Cartilage oligomeric matrix protein-angiopoietin-1 (COMP-Ang1) is an angiogenic factor for vascular angiogenesis. The aim was to investigate the effect of an intracavernosal injection of COMP-Ang1 on cavernosal angiogenesis in a diabetic rat model. Male Otsuka Long-Evans Tokushima Fatty (OLETF) rats made up the experimental group (1 yr old) and Long-Evans Tokushima Otsuka (LETO) rats made up the control group. The experimental group was divided into vehicle only, 10 µg COMP-Ang1, and 20 µg COMP-Ang1. COMP-Ang1 was injected into the corpus cavernosum of the penis. After 4 weeks, the penile tissues of the rats were obtained for immunohistochemistry and Western blot analysis. The immunoreactivity of PECAM-1 and VEGF was increased in the COMP-Ang1 group compared with the vehicle only group. Moreover, the expression of PECAM-1 and VEGF was notably augmented in the 20 µg Comp Ang-1 group. In the immunoblotting study, the expression of PECAM-1 and VEGF protein was significantly less in the OLEFT rats than in the control LETO rats. However, this expression was restored to control level after intracavernosal injection of COMP-Ang1. These results show that an intracavernosal injection of COMP-Ang1 enhances cavernous angiogenesis by structurally reinforcing the cavernosal endothelium.
PMCID: PMC3653085  PMID: 23678264
Erectile Dysfunction; Rats, Inbred OLETF; Angiogenesis; Angiopoietin-1
18.  Long-term results of the surgical treatment of Peyronie's disease with Egydio's technique: a European multicentre study 
Asian Journal of Andrology  2011;13(6):842-845.
The long-term outcomes of 157 patients affected by Peyronie's disease (PD) who underwent penile straightening with Egydio's technique between January 2004 and December 2008 are reported. Only patients with PD who were stable for at least 6–12 months prior to surgery were enrolled in this study. Preoperative assessment included a dynamic echo colour Doppler ultrasound scan to evaluate the degree of penile deformity and the peak systolic velocity in the cavernosal arteries and an assessment of erectile function with the administration of the International Index of Erectile Function 5 (IIEF-5) questionnaire. Stretched penile length was recorded pre- and postoperatively. Surgical complications, cosmesis and sexual function, patient satisfaction and postoperative erectile function were assessed postoperatively at 3 months, 1 year and 2 years, respectively. After a median follow-up period of 20 months (range: 12–24 months), we found that mild residual curvature (12%) and glans hypoesthesia (3%) were the only causes of partial dissatisfaction. No rejection of the graft was observed. All patients recovered their ability to penetrate with no difficulty. In addition, an intraoperative average increase of 2.5 cm (range: 1.7–4.1 cm) in stretched penile length was recorded, with all patients engaging in penetrative sexual intercourse. In conclusion, this procedure represents a safe and reproducible technique for the correction of penile curvature resulting from PD and yields excellent cosmetic and functional results.
PMCID: PMC3752552  PMID: 21743482
Egydio's technique; erectile dysfunction; graft; Peyronie's disease
19.  Components of the mitogen-activated protein kinase cascade are activated in hepatic cells by Echinococcus multilocularis metacestode 
AIM: To explore the effect of Echinococcus multilocularis (E. multilocularis) on the activation of mitogen-activated protein kinase (MAPK) signaling pathways and on liver cell proliferation.
METHODS: Changes in the phosphorylation of MAPKs and proliferating cell nuclear antigen (PCNA) expression were measured in the liver of patients with alveolar echinococcosis (AE). MAPKs, MEK1/2 [MAPK/extracellular signal-regulated protein kinase (ERK) kinase] and ribosomal S6 kinase (RSK) phosphorylation were detected in primary cultures of rat hepatocytes in contact in vitro with (1) E. multilocularis vesicle fluid (EmF), (2) E. multilocularis-conditioned medium (EmCM).
RESULTS: In the liver of AE patients, ERK 1/2 and p38 MAPK were activated and PCNA expression was increased, especially in the vicinity of the metacestode. Upon exposure to EmF, p38, c-Jun N-terminal kinase (JNK) and ERK1/2 were also activated in hepatocytes in vitro, as well as MEK1/2 and RSK, in the absence of any toxic effect. Upon exposure to EmCM, only JNK was up-regulated.
CONCLUSION: Previous studies have demonstrated an influence of the host on the MAPK cascade in E. multilocularis. Our data suggest that the reverse, i.e. parasite-derived signals efficiently acting on MAPK signaling pathways in host liver cells, is actually operating.
PMCID: PMC2678582  PMID: 19418584
Echinococcus multilocularis; Hepatic alveolar echinococcosis; Mitogen-activated protein kinase; Host-parasite interactions; Liver
20.  Activation of the MAPK pathway is a common event in uveal melanomas although it rarely occurs through mutation of BRAF or RAS 
British Journal of Cancer  2005;92(11):2032-2038.
In contrast to cutaneous melanoma, there is no evidence that BRAF mutations are involved in the activation of the mitogen-activated protein kinase (MAPK) pathway in uveal melanoma, although there is increasing evidence that this pathway is activated frequently in the latter tumours. In this study, we performed mutation analysis of the RAS and BRAF genes in a panel of 11 uveal melanoma cell lines and 19 primary uveal melanoma tumours. In addition, Western blot and immunohistochemical analyses were performed on downstream members of the MAPK pathway in order to assess the contribution of each of these components. No mutations were found in any of the three RAS gene family members and only one cell line carried a BRAF mutation (V599E). Despite this, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK), ERK and ELK were constitutively activated in all samples. These data suggest that activation of the MAPK pathway is commonly involved in the development of uveal melanoma, but occurs through a mechanism different to that of cutaneous melanoma.
PMCID: PMC2361800  PMID: 15928660
MAPK pathway; uveal melanoma; BRAF; RAS; mutation
21.  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
22.  Resveratrol, an activator of SIRT1, restores erectile function in streptozotocin-induced diabetic rats 
Asian Journal of Andrology  2013;15(5):646-651.
The high incidence of erectile dysfunction (ED) in diabetes highlights a need for effective treatment strategies. Resveratrol, an activator of silent information regulator 2-related enzymes 1 (sirtuin1, SIRT1), has received attention for its valuable effects in cancer, neurodegenerative diseases, longevity and cardiovascular disease. To explore the effects of resveratrol in diabetes-induced ED, resveratrol was administered to rats with streptozocin (65 mg kg−1)-induced diabetes. Erectile function, cavernous structure, tissue protein expression of silent information regulator 2-related enzymes 1 (sirtuin1, SIRT1), p53 and forkhead transcription factor O 3a (FOXO3a), superoxide dismutase (SOD) activity and malondialdehyde (MDA) levels in the corpora cavernosa were studied. We found that SIRT1 was expressed in cavernosal tissue, and it was downregulated in the corpora of diabetic rats. The administration of resveratrol upregulated the expression of SIRT1 and restored erectile function. In contrast, resveratrol downregulated the expression of p53 and FOXO3a, which regulate apoptosis and oxidative stress. Furthermore, the resveratrol-treated group showed an improvement in smooth muscle content, SOD activity and MDA levels when compared with the diabetic group. Therefore, the ability of resveratrol to improve diabetes-induced ED is likely related to its activation of SIRT1 expression, thus causing the suppression of apoptosis and resistance towards oxidative stress.
PMCID: PMC3881649  PMID: 23792339
apoptosis; erectile dysfunction; oxidative stress; resveratrol
23.  Estrogen receptor alpha (ERα) phospho-serine-118 is highly expressed in human uterine leiomyomas compared to matched myometrium 
It is thought that the growth of uterine leiomyomas may be mediated by the interaction of estrogen receptor alpha (ERα) and growth factor pathways and that phosphorylation of ERα at serine 118 (ERα-phospho-Ser118) is important in this interaction. In this study, immunoblotting and immunohistochemistry were used to investigate the expression of ERα-phospho-Ser118, phosphorylated p44/42 mitogen-activated protein kinase (phospho-p44/42 MAPK), and proliferating cell nuclear antigen (PCNA) in human leiomyoma and myometrial tissues during the proliferative and secretory phases of the menstrual cycle. We found that tumors taken from the proliferative phase expressed significantly higher levels of ERα-phospho-Ser118, phospho-p44/42 MAPK, and PCNA compared to patient-matched myometria and had significantly higher ERα-phospho-Ser118 and PCNA expression compared to secretory phase tumors. Also, enhanced colocalization and association of phospho-p44/42 MAPK and ERα-phospho-Ser118 were observed in proliferative phase tumors by confocal microscopy and immunoprecipitation, respectively. These data suggest that ERα-phospho-Ser118 may be important in leiomyoma growth and is possibly phosphorylated by phospho-p44/42 MAPK.
PMCID: PMC2693272  PMID: 18853184
Uterine leiomyoma; Estrogen receptor alpha phosphorylated serine 118; Phosphorylated mitogen-activated protein kinase
24.  Over-expression of the mitogen-activated protein kinase (MAPK) kinase (MEK)-MAPK in hepatocellular carcinoma: Its role in tumor progression and apoptosis 
BMC Gastroenterology  2003;3:19.
Hepatocellular carcinoma (HCC) is one of the most common malignancies in South East Asia. Although activation of the MEK-MAPK is often associated with cellular growth, the role of MEK-MAPK in growth and survival of hepatocarcinoma cells has not been established.
Immuno-histochemistry was used to localize phosphorylated MAPK and MEK1/2 in the tissues. 3-(4,5-Dimethylthiazol-2-y1)-2,5-diphenyltetrazolium bromide (MTT) assay and ELISA were used to determine cell viability and cell proliferation. Deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay was used to detect apoptotic cells. Western blots analysis was performed to determine the levels of proteins involved in the MEK-MAPK and apoptotic pathways. Transfection study was performed to assess the role of MEK-MAPK pathway in growth and survival of liver cancer cells.
We report that phosphorylation of MEK1/2 at Ser217/221 was detected by immuno-histochemistry in 100% (46 of 46) of HCCs examined. A positive signal was localized in the nuclei of hepatocarcinoma cells but not in dysplastic hepatocytes or stromal cells. Over-expression and phosphorylation of MAPK was also detected in 91% (42 of 46) and 69% (32 of 46) of HCCs examined, respectively. The percentage of cells showing positively for phosphorylated MEK1/2 increased with advancing tumor stage. In vitro, treatment of human HepG2 and Hep3B cells with MEK1/2 specific inhibitors U0126 and PD98059 led to growth inhibition and apoptosis. U0126 induced the release of cytochrome c and increased the cleavage of caspase-3, caspase-7, and poly ADP-ribose polymerase (PARP). Inhibition of phosphatidylinositol 3-kinase (PI-3K), c-Jun N-terminal kinase (JNK) and p38 kinase activities caused only a mild apoptosis in HepG2 and Hep3B cells. Activated MEK1-transfected cells were more resistant to UO126-induced apoptosis in vitro and formed larger tumors in SCID mice than mock-transfected cells.
In conclusion, our results demonstrate that MEK-MAPK plays an important role in the growth and survival of liver cancer cells and suggest that blocking MEK-MAPK activity may represent an alternative approach for the treatment of liver cancer.
PMCID: PMC317301  PMID: 12906713
25.  Molecular Yin and Yang of erectile function and dysfunction 
Asian journal of andrology  2008;10(3):433-440.
In regard to erectile function, Yin is flaccidity and Yang erection. In the past decade, research has mostly focused on the Yang aspect of erectile function. However, in recent years, the Yin side is attracting increasingly greater attention. This is due to the realization that penile flaccidity is no less important than penile erection and is actively maintained by mechanisms that play critical roles in certain types of erectile dysfunction (ED); for example, in diabetic patients. In addition, there is evidence that the Yin and Yang signaling pathways interact with each other during the transition from flaccidity to erection, and vice versa. As such, it is important that we view erectile function from not only the Yang but also the Yin side. The purpose of this article is to review recent advances in the understanding of the molecular mechanisms that regulate the Yin and Yang of the penis. Emphasis is given to the Rho kinase signaling pathway that regulates the Yin, and to the cyclic nucleotide signaling pathway that regulates the Yang. Discussion is organized in such a way so as to follow the signaling cascade, that is, beginning with the extracellular signaling molecules (e.g., norepinephrin and nitric oxide) and their receptors, converging onto the intracellular effectors (e.g., Rho kinase and protein kinase G), branching into secondary effectors, and finishing with contractile molecules and phosphodiesterases. Interactions between the Yin and Yang signaling pathways are discussed as well.
PMCID: PMC2893021  PMID: 18385905
erectile function; erectile dysfunction; molecular mechanisms; Rho kinase signaling; cyclic nucleotide signaling; Yin–Yang

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