Antioxidant Mechanism of Xiaojin Pill () for Treatment of Peyronie's Disease in Rats Based on Matrix Metalloproteinases.

OBJECTIVE: To evaluate the effects of Xiaojin Pill () in the treatment of Peyronie's disease (PD) in a rat model. METHODS: Twenty-four male Sprague-Dawley rats were randomly divided into four groups with 6 in each: sham operation, PD model, vehicle control and Xiaojin Pill groups. The rats in the sham operation group received penile tunica albsginea (TA) injection with 50 µL vehicle, while the rats in the other 3 groups received 50 µL penile TA injection of 50 µg transforming growth factor (TGF)-ß1. Forty-two days after the injection, rats in the vehicle control and Xiaojin Pill groups received 0.5 mL water and Xiaojin Pill solution (107 mg/kg of body weight), respectively by gavage for 28 days, while those in the sham operation and PD model groups did not receive any intervention. After intervention, the expressions of matrix metalloproteinase 2/9 (MMP2/9), nitric oxidesynthase (NOS), superoxide dismutase (SOD) and malondialdehyde (MDA) were measured. RESULTS: Rats in the PD model and vehicle control groups presented obvious fibrosis in corpus cavernosum (CC) and demonstrated a significantly increased expressions of MMP2 and MMP9 in the CC compared with the sham operation group (all P<0.01). In contrast, the expressions of MMP2 and MMP9 in the Xiaojin Pill group were significantly down-regulated (both P<0.01). In addition, the levels of NOS and MDA in CC were significantly increased while the activity of SOD was decreased in the PD model and vehicle control groups compared with the sham operation group (all P<0.01). After Xiaojin Pill treatment, the levels of MDA, NOS and SOD appeared to be corrected (all P<0.01). CONCLUSIONS: Xiaojin Pill could reduce fibrosis in the CC by decreasing the expressions of MMPs, NOS and MDA, and by increasing the activity of SOD. Therefore, Xiaojin Pill might be a therapeutic option for PD.

A loss of estrogen signaling in the aromatase deficient mouse penis results in mild hypospadias.

Hypospadias is the abnormal opening of the urethra on the underside of the penis and occurs in approximately 1/125 live male births worldwide. The incidence rate of hypospadias has dramatically increased over the past few decades. This is now attributed, at least in part, to our exposure to endocrine-disrupting chemicals (EDCs) which alter the hormonal signals required for development of the penis. In humans androgens are the main drivers of fusion of the urethral folds to form the urethra within the shaft of the penis, a process required for termination of the urethra in its normal location at the tip of the penis. However, recent research has suggested that estrogen also plays a role in this process. To better understand how EDCs impact urethral development it is essential that we understand the normal function of hormones during development of the penis. To define the role of estrogen in urethral development we examined development of the penis in the aromatase (Cyp19a1) Knockout (ArKO) mouse strain in which endogenous estrogen production is completely ablated. We found that the ArKO penis had a mild hypospadias phenotype. The developing ArKO postnatal penis displayed an early disruption in preputial development, which likely causes the mild hypospadias observed in adults. Using qPCR, we found altered expression of keratin genes and key urethral patterning genes in response to the disrupted estrogen signaling. The hypospadias phenotype was almost identical to that reported for the estrogen receptor α (ERα) knockout confirming that ERα is the predominant receptor for mediating estrogen action during development of the mouse penis. Our results show that estrogen is required for normal prepucial development and placement of the mature urethral opening at the distal aspect of the penis. We also identified several genes which are potential downstream targets of estrogen during normal urethral closure. With this knowledge, we can now better understand how anti-estrogenic as well as estrogenic EDCs disrupt urethral closure to cause mild hypospadias in both mice and humans.

Effect of p-coumaric acid on the erectogenic enzyme activities and non-protein thiol level in the penile tissue of normal and doxorubicin-induced oxidative stress male rat.

This study investigated effect of p-coumaric acid (PCA) on erectogenic enzyme activity and non-protein thiol level in the penile tissue of normal and doxorubicin (DOX)-induced oxidative stress male rat. Sixty-four (64) adult male rats weighing between 170 and 180 g were used for this work. After 14 days of acclimatisation, the rats were divided into eight groups (n = 8). Rats were orally pre-treated with PCA dose dependently (50 and 100 mg/kg body weight [b.w.t]) and vitamin E (100 mg/kg b.w.t) for 14 days before induction with a single dose of DOX (15 mg/kg b.w.t, via i.p.). The result revealed that arginase, acetylcholinesterase (AChE), angiotensin-I-converting enzyme (ACE), phosphodiesterase-5 (PDE-5), adenosine monophosphohydrolase (AMPdase) activities were significantly (p < 0.05) higher in the DOX-induced rats as against the control, which was significantly p < 0.05) higher when compared to normal rats treated with PCA. PCA also improved non-protein thiol level in the penile tissue of both normal and DOX-induced rats. Hence, this study revealed that PCA is capable of causing inhibitory effects on the activities of enzymes, associated with oxidative stress-induced erectile dysfunction (ED) and could also be used as an aphrodisiac agent in the management/treatment of ED.

Chronic administration of LIMK2 inhibitors alleviates cavernosal veno-occlusive dysfunction through suppression of cavernosal fibrosis in a rat model of erectile dysfunction after cavernosal nerve injury.

We evaluated whether chronic administration of LIMK2-inhibitors could improve erectile function by alleviating CVOD through suppressing cavernosal fibrosis in a rat model of cavernosal nerve crush-injury (CNCI). Forty-two 12-week-old rats were equally categorized into the three groups: sham-surgery (S), CNCI (I), and CNCI treated with LIMK2-inhibitors (L). The L-group was treated with daily intraperitoneal injection of LIMK2-inhibitors (10.0 mg/kg) for 30-days after surgery. Erectile function was assessed using dynamic-infusion-cavernosometry (DIC). Penile tissue was processed for Masson's-trichrome staining, Western-blotting, and double immunofluorescence. The I-group showed significantly higher maintenance and drop rates as well as lower papaverine response, compared to the S-group. Chronic inhibition of LIMK2 in the L-group significantly improved the DIC parameters compared to those in the I-group, although the parameters were not completely restored to normal control values. Also, the I-group showed a reduced smooth muscle (SM)-to-collagen ratio, decreased immunohistochemical staining for α-SM-actin, increased number of fibroblasts positive for phosphorylated Cofilin, increased LIMK2/Cofilin phosphorylation and increased protein expression of Collagen-1 or Fibronectin, compared to the S-group. The L-group showed significant improvements in SM/collagen ratio and the deposition of Collagen-1 or Fibronectin compared to the I-group, although not completely normalized. According to the densitometry and confocal microscopy results, the L-group showed restoration of LIMK2/Cofilin phosphorylation and amount of fibroblasts positive for phosphorylated Cofilin to the normal control value. In conclusion, chronic inhibition of LIMK2 can improve CVOD and ED by alleviating cavernosal fibrosis via normalizing the LIMK2/Cofilin pathway.

Antifibrotic Synergy Between Phosphodiesterase Type 5 Inhibitors and Selective Oestrogen Receptor Modulators in Peyronie's Disease Models.

BACKGROUND: Peyronie's disease (PD) is a fibrotic disorder of the penile tunica albuginea, characterised by the formation of a localised fibrous plaque that can lead to deformity and erectile dysfunction. Nonsurgical therapeutic options for PD are limited in efficacy and safety. Myofibroblasts are key cells in the pathogenesis of PD, and inhibition of myofibroblast transformation has been suggested as a therapeutic option. OBJECTIVE: To identify potential drugs using a novel phenotypic assay and then to test them using in vitro and in vivo models of PD. DESIGN, SETTING, AND PARTICIPANTS: We have developed and validated a phenotypic screening assay that measures myofibroblast transformation, by which we tested 21 compounds that were suggested to be efficacious in treating PD. The successful hits from this assay were further tested using in vitro and in vivo models of PD. RESULTS AND LIMITATIONS: The new assay was able to detect transforming growth factor-ß1-induced myofibroblast transformation. Using this assay, phosphodiesterase type 5 inhibitors (PDE5i) and selective oestrogen receptor modulators (SERMs) were identified to significantly inhibit myofibroblast transformation. A PDE5i (vardenafil) and an SERM (tamoxifen) inhibited myofibroblast transformation, collagen gel contraction, and extracellular matrix production in a synergistic fashion. In a rat model of PD, the antifibrotic effect of the combination of vardenafil and tamoxifen was greater than that of each drug alone. This study is limited by not providing a molecular mechanism for the proposed synergy. CONCLUSIONS: This is the first demonstration of a synergistic activity between a PDE5i and an SERM discovered through a phenotypic screening approach. Future clinical trials using a combination of these drugs should be considered during the active phase of PD, given the early evidence of benefit in both in vitro and in vivo models. PATIENT SUMMARY: This report suggests that the combination of a phosphodiesterase type 5 inhibitor and a selective oestrogen receptor modulator may be efficacious in treating Peyronie's disease in its active phase.

African crocus (Curculigo pilosa) and wonderful kola (Buchholzia coriacea) seeds modulate critical enzymes relevant to erectile dysfunction and oxidative stress.

Background The seeds of African crocus (AC) (Curculigo pilosa) and wonderful kola (WK) (Buchholzia coriacea) are commonly used in folklore medicine in managing erectile dysfunction (ED) without the full understanding of the possible mechanism of actions. This study investigated and compared the effects of aqueous extracts from the seeds of AC and WK on arginase and acetylcholinesterase (AChE) activities and some pro-oxidant [FeSO4 and sodium nitroprusside (SNP)]-induced lipid peroxidation in rat penile homogenate in vitro. Method Aqueous extracts of AC and WK were prepared, and their effects on arginase and AChE activities as well as FeSO4- and SNP-induced lipid peroxidation in rat penile homogenate were assessed. Furthermore, phenolic constituents of the extract were determined using high-performance liquid chromatography coupled with diode-array detector (HPLC-DAD). Results Both extracts exhibited concentration-dependent inhibition on arginase (AC, IC50=0.05 mg/mL; WK, IC50=0.22 mg/mL) and AChE (AC, IC50=0.68 mg/mL; WK, IC50=0.28 mg/mL) activities. The extracts also inhibited FeSO4- and SNP-induced lipid peroxidation in rat penile homogenate. HPLC-DAD analysis revealed the presence of phenolic acids (gallic, caffeic, ellagic and coumaric acids) and flavonoids (catechin, quercetin and apigenin) in AC and WK. AC had higher arginase inhibitory and antioxidative activities but lower AChE inhibitory properties when compared with WK. Conclusions These effects could explain the possible mechanistic actions of the seeds in the management/treatment of ED and could be as a result of individual and/or synergistic effect of the constituent phenolic compounds of the seeds.

TNF-α antagonism with etanercept enhances penile NOS expression, cavernosal reactivity, and testosterone levels in aged rats.

Erectile dysfunction (ED) has been reported to be associated with inflammation. This study investigated the effects of tumor necrosis factor alpha (TNF-α) inhibitor etanercept on penile neuronal nitric oxide synthase (nNOS) and endothelial nitric oxide synthase (eNOS) expressions, testosterone concentrations, neurogenic and endothelium-dependent relaxations of corpus cavernosum (CC), and circulating and cavernosal levels of inflammatory markers in aged rats. Animals were separated into control, aged, and etanercept-treated aged groups. Aged rats displayed significantly increased serum and cavernosal TNF-α, C-reactive protein (CRP), monocyte chemoattractant protein-1 (MCP-1) and intercellular adhesion molecule (ICAM-1) levels, and decreased penile nNOS and eNOS expressions and serum testosterone levels compared with controls. In etanercept-treated aged group, NOS expressions were similar to that of the control group. The circulating and cavernosal concentrations of TNF-α, CRP, MCP-1, ICAM-1, and testosterone were also normalized by etanercept. Neurogenic and endothelium-dependent relaxant responses significantly decreased in aged rats and etanercept treatment markedly improved these relaxation responses. Our findings indicate that aging decreases penile NOS expression, neurogenic and endothelium-dependent relaxations of CC, and also suppresses serum testosterone levels by inducing inflammatory response that may contribute to the development of ED. TNF-α antagonism may be a novel strategy to treat aging-associated ED.

Priapism in a Fabry disease mouse model is associated with upregulated penile nNOS and eNOS expression.

Fabry disease is a glycosphingolipidosis caused by deficient activity of α-galactosidase A; it is one of a few diseases that are associated with priapism, an abnormal prolonged erection of the penis. The goal of this study was to investigate the pathogenesis of Fabry disease-associated priapism in a mouse model of the disease. We found that Fabry mice develop late-onset priapism. Neuronal nitric oxide synthase (nNOS), which was predominantly present as the 120-kDa N-terminus-truncated form, was significantly upregulated in the penis of 18-month-old Fabry mice compared to wild type controls (~fivefold). Endothelial NOS (eNOS) was also upregulated (~twofold). NO level in penile tissues of Fabry mice was significantly higher than wild type controls at 18 months. Gene transfer-mediated enzyme replacement therapy reversed abnormal nNOS expression in the Fabry mouse penis. The penile nNOS level was restored by antiandrogen treatment, suggesting that hyperactive androgen receptor signaling in Fabry mice may contribute to nNOS upregulation. However, the phosphodiesterase-5A expression level and the adenosine content in the penis, which are known to play roles in the development of priapism in other etiologies, were unchanged in Fabry mice. In conclusion, these data suggested that increased nNOS (and probably eNOS) content and the consequential elevated NO production and high arterial blood flow in the penis may be the underlying mechanism of priapism in Fabry mice. Furthermore, in combination with previous findings, this study suggested that regulation of NOS expression is susceptible to α-galactosidase A deficiency, and this may represent a general pathogenic mechanism of Fabry vasculopathy.

[Immediate and delayed intracavernous injection of bone marrow mesenchymal stem cells to improve erectile function in rats with cavernous nerve injury].

OBJECTIVE: To explore the effects of immediate and delayed intracavernous injection of bone marrow mesenchymal stem cells (BM-MSCs) on neurogenic erectile dysfunction (NED) induced by bilateral cavernous nerve injury in Sprague-Dawley (SD) rats. METHODS: BM-MSCs isolated from male SD rats were cultured and identified. Twenty-eight 8-week-old male SD rats were randomly divided into four groups, sham operation, NED model control, BM-MSCs immediate, and BM-MSCs delayed, and NED models were established in the latter three groups by crushing the bilateral cavernous nerves. The rats in the sham operation and model control groups were injected intracavernously with placebo while those in the latter two with BM-MSCs immediately or 2 weeks after modeling. At 12 weeks after operation, the penile function of the rats was assessed according to the penile intracavernous pressure (ICP), mean arterial pressure (MAP), and ICP/MAP ratio obtained from different groups of rats. Then, all the animals were sacrificed and the penile cavernosal tissue collected for histological analysis. RESULTS: At 12 weeks after modeling, both ICP and ICP/MAP were significantly increased in the BM-MSCs immediate and delayed groups as compared with those in the model control (P <0.05), and so were the ratio of smooth muscle to collagen (P <0.05) and the smooth muscle content in the corpus cavernosum (P <0.05), and the number of neurofilament (NF)-positive nerve fibers (P <0.05) and the expression of neuronal nitric oxide synthase (nNOS) in the dorsal nerves of the midshaft penis (P <0.05). CONCLUSIONS: Intracavernous injection of BM-MSCs can improve erectile function in rats with bilateral cavernous nerve injury by elevating the smooth muscle-collagen ratio and smooth muscle content in the corpus cavernosum and thus preventing its fibrosis as well as by increasing the number of NF-positive nerve fibers and expression of nNOS in the penile dorsal nerves.

[Molecular mechanisms of androgens regulating the eNOS expression in rat corpus cavernosum].

OBJECTIVE: To investigate whether androgens can regulate the expression of eNOS in rat corpus cavernosum through AKT3, PIK3CA, CALM, and CAV1 and influence erectile function. METHODS: Thirty-six 8-week-old male SD rats were randomly divided into groups A (4-week control), B (6-week control), C (4-week castration), D (6-week castration), E (4-week castration + testosterone replacement), and F (6-week castration + testosterone replacement). Both the testis and epididymis were removed from the rats in groups C, D, E and F, and on the second day after surgery, the animals of groups E and F were subcutaneously injected with testosterone propionate at 3 mg per kg of the body weight qd alt while all the others with isodose oil instead. At 4 weeks (for groups A, C and E) and 6 weeks (for groups B, D and F) after treatment, we detected the maximum intracavernous pressure (ICPmax), the mean carotid arterial pressure (MAP) and their ratio (ICPmax/MAP), measured the level of serum testosterone (T), and determined the expressions of eNOS, P-eNOS, AKT3, PIK3CA, CALM and CAV1 in the corpus cavernosum by Western blot and immunohistochemistry. RESULTS: No statistically significant differences were observed in the body weight and MAP among different groups. The serum T level and ICPmax/MAP were remarkably lower in groups C and D than in the other four groups (P<0.01) as well as in groups E and F than in A and B (P<0.05) but exhibited no significant differences either between E and F or between A and B. Immunohistochemistry showed that eNOS and P-eNOS were mainly expressed in the vascular endothelial cell membrane and cavernous vascular lumen, while AKT3, PIK3CA, CALM and CAV1 chiefly in the vascular endothelial cell cytoplasm and membrane, with a few in the smooth muscle cells. Western blot analysis manifested that the expressions of eNOS, P-eNOS, AKT3, PIK3CA, CALM and CAV1 were markedly lower in groups C and D than in A, B, E and F (P<0.01) as well as in D than in C (P<0.05) but those in groups E and F did not showed any significant difference from those in A and B, nor E from F or A from B. CONCLUSIONS: Androgens can improve erectile function by upregulating the expressions of AKT3, PIK3CA, CALM and CAV1 protein molecules and activating eNOS after its phosphorylation, though the exact molecular mechanisms are yet to be further studied.

[Expression of nNOS and ultrastructural changes in the penile tissue of rats with prolactinoma-induced erectile dysfunction].

OBJECTIVE: To study the expression of nNOS and ultrastructural changes in the penile tissue of rats with prolactinoma-induced erectile dysfunction (ED). METHODS: We established the model of prolactinoma in 20 male Westar rats by peritoneal injection of diethylstilbestrol (DES) and treated the control rats with normal saline (n = 10) or sterilized arachis oil (n = 10). After 8 weeks, we performed the apomorphine test and measured the weight of the pituitary gland and the levels of serum prolactin (PRL) and testosterone (T) to confirm the successful construction of the prolactinoma-induced ED model. Then we determined the expression of nNOS in the penile tissue by immunohistochemistry and examined the ultrastructural changes of the penile cavernosum under the transmission electron microscope. RESULTS: The prolactinoma-induced ED model was successfully established in 15 rats. The weight of the pituitary gland was significantly increased in the rats treated with DES as compared with the normal saline and sterilized arachis oil controls ([46.7 ± 15.5] vs [11.7 ± 2.4] and [12.4 ± 2.3] mg, both P < 0.05). The level of serum PRL was markedly higher while that of T remarkably lower in the former than in the latter two groups ([1,744.9 ± 304.5] vs [11.5 ± 2.4] and [10.6 ± 1.9] ng/ml, both P < 0.0l; [1.54 ± 0.46] vs [3.11 ± 1.08] and [3.04 ± 1.11] ng/ml, both P < 0.05). The rate of penile erection was significantly reduced in the prolactinoma-induced ED model rats in comparison with the normal saline and arachis oil controls (16.7% vs 100% and 87.5%, both P < 0.05), and so was the expression of nNOS in the penile tissue (0.024 ± 0.011 vs 0.066 ± 0.019 and 0.058 ± 0.021, both P < 0.05). Transmission electron microscopy manifested significant ultrastructural changes in the endothelial and smooth muscle cells of the cavernous tissue in the prolactinoma-induced ED models. CONCLUSION: The ultrastructural changes of the penile cavernous tissue and the reduced expression of nNOS in penile tissue may be the most important mechanisms of prolactinoma-induced ED in rats.

[Expressions of CSE and CBS in the penile corpus cavernosum of hyperglycemia rats and their implications].

OBJECTIVE: To investigate the impact of hyperglycemia on the hydrogen sulfide (H2S) signaling pathway in rat penile tissue and its relationship with erectile function. METHODS: Twenty healthy male Sprague Dawley (SD) rats aged 8 weeks were randomly divided into groups A (4-week healthy control), B (4-week diabetes mellitus model), C (6-week healthy control) and D (6-week diabetes mellitus model). The rats in groups B and D were injected intraperitoneally with streptozotocin at 50 mg/kg to induce diabetes mellitus, while those in groups A and C with the same volume of normal saline. The animals were killed at 4 (groups A and B) and 6 weeks (groups C and D) after treatment for measurement of the maximal intracavernous pressure/mean arterial blood pressure (ICP(max)/MAP) by electrostimulation, determination of the H2S concentration in the plasma and penile tissue, and detection of the expressions of cystathionine-beta-synthetase (CBS) and cystathionine-gamma-lyase (CSE) in the penile corpus cavernosum by immunohisto- chemistry and Western blot. RESULTS: With electrostimulation of the pelvic ganglia at 5V and 7 V, ICP(max)/MAP was significantly reduced in groups B (0.19 +/- 0.03 and 0.29 +/- 0.04) and D (0.14 +/- 0.04 and 0.25 +/- 0.04) as compared with A (0.46 +/- 0.07 and 0.68 +/- 0.09) and C (0.43 +/- 0.07 and 0.65 +/- 0.16) (P < 0.05). No statistically significant differences were found in the level of serum testosterone either between groups A and B ([469.19 +/- 126.46] ng/dl vs [359.08 +/- 60.06] ng/dl, P > 0.05) or between C and D ([470.44 +/- 209.28] ng/dl vs [297.01 +/- 96.58] ng/dl, P > 0.05). Groups B and D showed remarkable reduction in the H2S concentration (P < 0.05) and the expressions of CBS and CSE (P < 0.05) in comparison with A and C, and the CBS and CSE expressions were even more significantly decreased in D than in B (P < 0.05). CONCLUSION: The reduced concentration of H2S and decreased expressions of CBS and CSE in the penile corpus cavernosum of the diabetic rats suggested that the H2S signaling pathway might be involved in hyperglycemia-induced erectile dysfunction.

Platelet-derived growth factor regulation of type-5 phosphodiesterase in human and rat penile smooth muscle cells.

INTRODUCTION: Relaxation of cavernous smooth muscle cells (SMCs) is a key component in the control of the erectile mechanism. SMCs can switch their phenotype from a contractile differentiated state to a proliferative and dedifferentiated state in response to a change of local environmental stimuli. Proliferation and contraction are both regulated by the intracellular second messengers cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), which are degraded by phosphodiesterases (PDEs). The most abundant PDE present in corpora cavernosa is the electrolytic cGMP-specific phosphodiesterase type 5 (PDE5). AIM: We investigated the cellular localization of PDE5 in in vitro cultured corpora cavernosa cells and the effect of mitogenic stimulation on PDE5 expression. METHODS: Biochemical ad molecular techniques on cultured SMCs from human and rat penis. MAIN OUTCOME MEASURES: We studied the ability of the quiescent SMC phenotype vs. the proliferating phenotype in modulation of PDE5 expression. RESULTS: We demonstrated that PDE5 is localized in the cytoplasm, in the perinuclear area, and in discrete cytoplasmic foci. As previously demonstrated in human myometrial cells, the cytoplasmic foci may correspond to centrosomes. In corpora cavernosa, PDE5 protein levels are strongly regulated by the mitotic activity of the SMCs, as they were increased in quiescent cultures. In contrast, treatment with platelet-derived grow factor (PDGF), one of the most powerful mitogenic factors for SMCs, reduces the expression of PDE5 after 24 hours of treatment. CONCLUSION: We found that PDGF treatment downregulates PDE5 expression in proliferating SMCs, suggesting that PDE5 may represent one of the markers of the contractile phenotype of the SMCs of corpora cavernosa.

[Expressions of CSE and CBS in the corpus cavernosum of spontaneous hypertensive rats].

OBJECTIVE: To investigate the expressions of cystathionine gamma-lyase (CSE) and cystathionine beta-synthase (CBS) in the corpus cavernosum of spontaneous hypertensive rats (SHR) and their relationship with erectile dysfunction. METHODS: This study included 10 male SHRs and 10 healthy male Wistar-Kyoto (WKY) rats as controls, all aged 12 weeks. We applied a series of electric stimuli to the major pelvic ganglions of the rats, observed changes in the ratio of intracavernosal to mean arterial blood pressure (ICP/MAP), measured the levels of serum testosterone (T) and endogenous H2S, and determined the expressions of CSE and CBS in the corpus cavernosum by Western blot and immunohistochemistry. RESULTS: No obvious difference was found in the serum T level between the two groups. Compared with the WKY rats, the SHRs showed significant reduction in the ICP/MAP ratio, the contents of plasma H2S ([21.92 +/- 2.75] micromol/L vs [10.49 +/- 1.35] micromol/L, P < 0.05) and endogenous corpus cavernosal H2S ([87.67 +/- 2.12] nmol/mg prot vs [52.60 +/- 3.44] nmol/mg prot, P < 0.05), the level of endogenous H2S synthesis ([4.35 +/- 0.32] nmol/mg per min vs [1.14 +/- 0.07] nmol/mg per min, P < 0.05) and the expressions of CBS and CSE (P < 0.05). Immunohistochemistry showed that CSE and CBS were distributed mainly in the smooth muscle cells and vascular endothelial cells of the corpus cavernosum. The ICP/MAP ratio was highly positively correlated with the expressions of CSE (r = 0.977, P < 0.05) and CBS (r = 0.955, P < 0.05) in the corpus cavernosal tissue. CONCLUSION: Hypertension inhibits endogenous H2S synthesis by suppressing the expressions of CSE and CBS in the corpus cavernosum, which might be related with hypertension-induced reduction of erectile function.

Abnormal lipid metabolism down-regulates adenosine triphosphate synthase ß-subunit protein expression in corpus cavernosum smooth muscle in vitro and in vivo.

Metabolic syndrome is closely related to erectile dysfunction (ED), and hyperlipidaemia is considered a major risk factor for ED. Adenosine triphosphate (ATP) synthase is believed to play an important role in metabolic syndrome; it has been hypothesised that ATP synthase contributes to ED development. We have verified this hypothesis using primary cultured human corpus cavernosum smooth muscle (HCCSM) cells treated with excessive free fat acid (FFA) and a high-fat diet (HFD) mouse model. Our results showed that high fatty factors could cause lipid accumulation in HCCSM cells, which could result in abnormal lipid metabolism, such as high levels of triglycerides, cholesterol and glucose in the HFD mice. There was a remarkable down-regulation of ATP synthase and p-Akt after in vivo and in vitro excessive FFA treatments. These results indicated that abnormal lipid metabolism could induce ATP synthase down-regulation via the Akt phosphorylation pathway and that ATP synthase may be a target of lipotoxicity in corpus cavernosum smooth muscle cells.

Intracavernous delivery of clonal mesenchymal stem cells restores erectile function in a mouse model of cavernous nerve injury.

INTRODUCTION: Recently, much attention has focused on stem cell therapy; bone marrow-derived stem cells (BMSCs) are one of the most studied mesenchymal stem cells used in the field of erectile dysfunction (ED). However, a major limitation for the clinical application of stem cell therapy is the heterogeneous nature of the isolated cells, which may cause different treatment outcomes. AIM: We investigated the effectiveness of mouse clonal BMSCs obtained from a single colony by using subfractionation culturing method (SCM) for erectile function in a mouse model of cavernous nerve injury (CNI). METHODS: Twelve-week-old C57BL/6J mice were divided into four groups: sham operation group, bilateral CNI group receiving a single intracavernous (IC) injection of phosphate-buffered saline (20 µL) or clonal BMSCs (3 × 10(5) cells/20 µL), and receiving a single intraperitoneal (IP) injection of clonal BMSCs (3 × 10(5) cells/20 µL). MAIN OUTCOME MEASURES: The clonal BMSC line was analyzed for cell-surface epitopes by using fluorescence-activated cell sorting and for differentiation potential. Two weeks after CNI and treatment, erectile function was measured by electrically stimulating the cavernous nerve. The penis was harvested for histologic examinations and Western blot analysis. RESULTS: Clonal BMSCs expressed cell surface markers for mesenchymal stem cells and were capable of differentiating into several lineages, including adipogenic, osteogenic, and chondrogenic cells. Both IC and IP injections of clonal BMSCs significantly restored cavernous endothelial and smooth muscle content, and penile nNOS and neurofilament content in CNI mice. IC injection of clonal BMSCs induced significant recovery of erectile function, which reached 90-100% of the sham control values, whereas IP injection of clonal BMSCs partially restored erectile function. CONCLUSION: We established a homogeneous population of mouse clonal BMSCs using SCM; clonal BMSCs successfully restored erectile function in CNI mice. The homogeneous nature of clonal mesenchymal stem cells may allow their clinical applications.

Akita spontaneously type 1 diabetic mice exhibit elevated vascular arginase and impaired vascular endothelial and nitrergic function.

BACKGROUND: Elevated arginase (Arg) activity is reported to be involved in diabetes-induced vascular endothelial dysfunction. It can reduce L-arginine availability to nitric oxide (NO) synthase (NOS) and NO production. Akita mice, a genetic non-obese type 1 diabetes model, recapitulate human diabetes. We determined the role of Arg in a time-course of diabetes-associated endothelial dysfunction in aorta and corpora cavernosa (CC) from Akita mice. METHODS AND RESULTS: Endothelium-dependent relaxation, Arg and NOS activity, and protein expression levels of Arg and constitutive NOS were assessed in aortas and CC from Akita and non-diabetic wild type (WT) mice at 4, 12 and 24 wks of age. Systolic blood pressure (SBP) was assessed by tail cuff. In aorta and CC, Akita mice exhibited a progressive impairment of vascular endothelial and nitrergic function increased Arg activity and expression (Arg1 in aorta and both Arg1 and Arg2 in CC) compared with that of age-matched WT mice. Treatment of aorta and CC from Akita mice with an Arg inhibitor (BEC or ABH) reduced diabetes-induced elevation of Arg activity and restored endothelial and nitrergic function. Reduced levels of phospho-eNOS at Ser(1177) (in aorta and CC) and nNOS expression (in CC) were observed in Akita mice at 12 and 24 wks. Akita mice also had decreased NOS activity in aorta and CC at 12 and 24 wks that was restored by BEC treatment. Further, Akita mice exhibited moderately increased SBP at 24 wks and increased sensitivity to PE-induced contractions in aorta and sympathetic nerve stimulation in CC at 12 and 24 wks. CONCLUSIONS: Over 24 wks of diabetes in Akita mice, both aortic and cavernosal tissues exhibited increased Arg activity/expression, contributing to impaired endothelial and nitrergic function and reduced NO production. Our findings demonstrate involvement of Arg activity in diabetes-induced impairment of vascular function in Akita mouse.

[Expressions of cystathionine-beta-synthase and cystathionine-gamma-lyase in the corpus cavernosum smooth muscle of castrated rats].

OBJECTIVE: To study the expressions of cystathionine-beta-synthase (CBS) and cystathionine-gamma-lyase (CSE) in the corpus cavernosum smooth muscle of castrated rats and their roles in erectile dysfunction after castration. METHODS: We randomly assigned 40 eight-week-old male SD rats to groups A (2-week sham-operation), B (4-week sham-operation), C (2-week castration) and D (4-week castration). We determined the level of serum testosterone (T) and the expressions of CBS and CSE in the corpus cavernosum smooth muscle of the rats after operation using immunohistochemistry and RT-PCR. RESULTS: The T level was significantly decreased in groups C ([11.85 +/- 6.73] nmol/L) and D ([1.96 +/- 1.23] nmol/L) as compared with A ([89.65 +/- 17.13] nmol/L) and B ([106.75 +/- 19.68] nmol/L) (P < 0.05). CBS and CSE were expressed in all groups of rats, but the relative expressions of CBS and CSE mRNA were significantly lower in groups C (0.93 +/- 0.14 and 0.87 +/- 0.20) and D (0.79 +/- 0.17 and 0.71 +/- 0.12) than in A (2.13 +/- 0.65 and 1.93 +/- 0.15) and B (2.07 +/- 0.53 and 1.89 +/- 0.45) (P < 0. 05), so were the optical density values (IA) of the CBS and CSE proteins, 130.35 +/- 23.56 and 93.56 +/- 36.64 in group C and 80.29 +/- 29.65 and 58.56 +/- 19.95 in group D, as compared with 310.57 +/- 130.56 and 269.56 +/- 116.76 in group A and 349.68 +/-112.35 and 298.35 +/- 100.76 in group B (P < 0.05). The androgen level was positively correlated with the expressions of CBS and CSE in the corpus cavernosum smooth muscle of the rats. CONCLUSION: Androgen regulates erectile function via the expressions of CBS and CSE.

Imatinib mesylate (Gleevec) induces human corpus cavernosum relaxation by inhibiting receptor tyrosine kinases (RTKs): identification of new RTK targets.

OBJECTIVE: To evaluate the effect of the tyrosine kinase inhibitor imatinib mesylate (Gleevec) on human corpus cavernosum (HCC) smooth muscle tone. METHODS: HCC were obtained from 18 erectile dysfunction (ED) patients undergoing penile prosthesis surgery. The effects of imatinib in HCC strips were investigated in the presence of various inhibitors. The human phosphoreceptor protein tyrosine kinase (PTK) array (Proteome Profiler Array) detected changes in receptor phosphorylation before and after imatinib. Immunohistochemistry was used to localize phosphorylated c-kit (CD117/stem cell factor) in HCC smooth muscle cells. RESULTS: Phenylephrine-induced contraction in HCC was significantly inhibited by imatinib (97.7% ± 2.3%). l-nitro-arginine methyl ester (l-NAME) or guanylyl cyclase inhibitor [1H-1,2,4] oxadiazolo [4,3-a]quinoxalin-1-one (ODQ) alone did not reverse the effect of imatinib, but suppressed this response in combination (18.0% ± 0.6%). The K(+) channel blockers (apamin and tetraethyl ammonium) decreased the imatinib-induced relaxation by 64% and 51%, respectively. PTK microarray analysis of 42 different phospho-receptor tyrosine kinases showed 14 were clearly activated in HCC. Imatinib treatment significantly inhibited phosphorylation of PTKs. A high level of CD117/c-kit-positive immunostaining was detected in untreated HCC smooth muscle, but not in treated HCC. CONCLUSION: Imatinib caused HCC smooth muscle relaxation in vitro mediated by nitric oxide/guanosine monophosphate signaling, involving the large-conductance Ca(2+)-activated K(+)-channels (BK(Ca)) or by inhibiting the upregulated PTK pathway. These results suggest that imatinib may also benefit erectile dysfunction patients who are not responsive to phosphodiesterase-5 inhibitors.

Taurine enhances the sexual response and mating ability in aged male rats.

It has been demonstrated that taurine is abundant in male reproductive organs, and can be biosynthesized by testis, but the taurine concentration will reduce with aging. The levels of serum LH, T, NOS, and NO were found to be obviously increased by taurine supplementation in aged rats in our previous study. In addition, aging will result in a significant decline in sexual response and function, which may be attributed to the androgen deficiency. Furthermore, NO has been proposed as a crucial mediator of penile erection. That makes us hypothesize that there is potential relationship between taurine decline and erection dysfunction in aged males. So the primary aim of the present study was to investigate the effect of taurine on male sexuality in rats. Taurine was offered in water to male aged (20 months old) rats for 110 days. The effects of taurine on the sexual response, mating ability, levels of serum reproductive hormones, and penile NOS and NO levels were investigated. The results showed that taurine can significantly reduce the EL and ML; obviously increase the ERF, MF, IF, and EJF; stimulate the secretion of GnRH, LH, and T; and elevate penis NOS and NO level in aged rats. The results indicated that taurine can enhance the sexual response and mating ability in aged male rats by increasing the level of testosterone and NO, but the exact mechanism of which needs to be further investigated.