Chronic Cigarette Smoking Impairs Erectile Function through Increased Oxidative Stress and Apoptosis, Decreased nNOS, Endothelial and Smooth Muscle Contents in a Rat Model.

Cigarette use is an independent risk factor for the development of erectile dysfunction (ED). While the association between chronic smoking and ED is well established, the fundamental mechanism(s) of cigarette-related ED are incompletely understood, partly due to no reliable animal model of smoking-induced ED. The present study was designed to validate an in vivo rat model of chronic cigarette-induced ED. Forty 12-week old male Sprague-Dawley rats were divided into 4 groups. Ten rats served as control group and were exposed only to room air. The remaining 30 rats were passively exposed to cigarette smoke (CS) for 4 weeks (n = 10), 12 weeks (n = 10), and 24 weeks (n = 10). At the 24-week time point all rats were assessed with intracavernous pressure (ICP) during cavernous nerve electrostimulation. Blood and urine were collected to measure serum testosterone and oxidative stress, respectively. Corporal tissue was assessed by Western blot for neuronal nitric oxide synthase (nNOS). Penile tissues were subjected to immunohistochemistry for endothelial, smooth muscle, and apoptotic content. Mean arterial pressure (MAP) was significantly higher in 24-week cigarette exposed animals compared to the control animals. Mean ICP/MAP ratio and cavernosal smooth muscle/endothelial contents were significantly lower in the 12- and 24-week rats compared to control animals. Oxidative stress was significantly higher in the 24-week cigarette exposed group compared to control animals. Mean nNOS expression was significantly lower, and apoptotic index significantly higher, in CS-exposed animals compared to control animals. These findings indicate that the rat model exposure to CS increases apoptosis and oxidative stress and decreases nNOS, endothelial and smooth muscle contents, and ICP in a dose dependent fashion. The rat model is a useful tool for further study of the molecular and cellular mechanisms of CS-related ED.

Novel therapeutic approach for neurogenic erectile dysfunction: effect of neurotrophic tyrosine kinase receptor type 1 monoclonal antibody.

BACKGROUND: Erectile dysfunction (ED) is a major health issue in aged populations, and neurogenic ED is particularly difficult to treat. Novel therapeutic approaches are needed for treatment of neurogenic ED of peripheral origin. OBJECTIVE: To investigate the therapeutic effects of a neurotrophic tyrosine kinase receptor type 1 monoclonal antibody (TrkA-mAb) on erectile function and sexual behavior in a rat model of cavernous nerve injury (CNI). DESIGN, SETTING, AND PARTICIPANTS: In one experiment, 84 male rats were randomly assigned to seven groups. The groups underwent either CNI or sham surgery, subsequent injection into the major pelvic ganglion (IMPG) of phosphate-buffered saline (PBS), an immunoglobulin G (IgG) control, or TrkA-mAb, and then intracavernosal (IC) injection of either PBS or varying TrkA-mAb concentrations immediately after surgery and then 1 wk later. Erectile function was assessed and histologic/molecular analyses were performed at 6 wk after surgery. In a second experiment, 36 male rats were randomly divided into three groups. The groups underwent CNI or sham surgery and then IC injection of PBS, IgG, or TrkA-mAb immediately after surgery and for 5 wk thereafter. At 6 wk after surgery, the performance of the rats in sexual behavior tests was videotaped. INTERVENTION: CNI or sham surgery; IMPG of PBS, IgG, or TrkA-mAb; IC injection of PBS or TrkA-mAb. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The intracavernous pressure response to cavernous nerve electrostimulation was measured and midpenile cross-sections were histologically examined. Western blotting (WB) of cavernous tissue protein was performed. Rats were assessed for chasing, mounting, intromission, and ejaculation behaviors during sexual behavior tests. The data were analyzed using one-way analysis of variance followed by the Tukey-Kramer t test. RESULTS AND LIMITATIONS: Recovery of erectile function of varying degrees was observed in the TrkA-mAb groups. TrkA-mAb treatment significantly suppressed tyrosine hydroxylase-positive nerve fibers in the corpus cavernosum and enhanced neuronal nitric oxide synthase-positive fibers in the dorsal nerve. The ratio of smooth muscle to collagen in the corpus cavernosum was significantly improved in TrkA-mAb treatment groups compared to PBS vehicle and IgG control groups. WB confirmed these biological changes. There was a nonsignificant increase in the average number of intromissions and ejaculations in the TrkA-mAb group. The study limitations include small sample size, variability in sexual behavior, lack of data on the neuromuscular mechanism involved, and lack of information of the role of neurotrophins or cytokines in regeneration. CONCLUSIONS: TrkA-mAb successfully inhibits sympathetic nerve regeneration, leads to parasympathetic nerve regeneration, and has therapeutic effects on ED and sexual behavior disorder in a rat model of CNI. PATIENT SUMMARY: This report provides strong evidence that a neurotrophic tyrosine kinase receptor type 1 monoclonal antibody (TrkA-mAb) inhibits sympathetic nerve regeneration, leads to parasympathetic nerve regeneration, and has therapeutic effects on erectile dysfunction and sexual behavior disorder in a rat model of cavernous nerve injury. The results raise the possibility that human patients with neurogenic erectile dysfunction may respond to TrkA-mAb in a manner that parallels the response seen in our rodent study.

Effects of intravenous injection of adipose-derived stem cells in a rat model of radiation therapy-induced erectile dysfunction.

INTRODUCTION: Radiation therapy (RT) for prostate cancer is frequently associated with posttreatment erectile dysfunction (ED). AIM: To investigate whether injection of adipose-derived stem cells (ADSCs) can ameliorate RT-associated ED. METHODS: Thirty male rats were divided into three groups. The control + phosphate-buffered saline (PBS) group received tail-vein injection of PBS. The radiation + PBS group received radiation over the prostate and tail-vein injection of PBS. The radiation + ADSC group received radiation over the prostate and tail-vein injection of ADSCs, which were labeled with 5-ethynyl-2-deoxyuridine (EdU). Seventeen weeks later, erectile function was evaluated by intracavernous pressure (ICP) in response to electrostimulation of cavernous nerves (CNs). Penile tissue and major pelvic ganglia (MPG) were examined by immunofluorescence (IF) and EdU staining. MAIN OUTCOME MEASURES: Erectile function was measured by ICP. Protein expression was examined by IF, followed by image analysis and quantification. RESULTS: Radiation over the prostate caused a significant decrease in erectile function and in the expression of neuronal nitric oxide synthase (nNOS) in penis and MPG. Cavernous smooth muscle (CSM) but not endothelial content was also reduced. Injection of ADSCs significantly restored erectile function, nNOS expression, and CSM content in the irradiated rats. EdU-positive cells were visible in MPG. CONCLUSIONS: Radiation appears to cause ED via CN injury. ADSC injection can restore erectile function via CN regeneration.

Both immediate and delayed intracavernous injection of autologous adipose-derived stromal vascular fraction enhances recovery of erectile function in a rat model of cavernous nerve injury.

BACKGROUND: Intracavernous injection of cultured adipose-derived stem cells (ADSCs) effectively restores erectile function in cavernous nerve (CN)-injured rats when administered at the time of injury. However, culturing exposes ADSCs to the risk of contamination and dedifferentiation. OBJECTIVE: Explore the effect of uncultured autologous adipose-derived stromal vascular fraction (SVF) on improving erectile function in a rat model of CN injury when administered at the time of injury or 4 wk after injury. DESIGN, SETTING, AND PARTICIPANTS: Eighty-nine male Sprague Dawley rats were randomly divided into four groups. CN injury or sham surgery was performed at the start of the study, and rats were treated with either SVF or vehicle. Functional testing and histologic analysis were performed 12 wk after CN crush or sham surgery. INTERVENTION: We used intracavernous injection of saline immediately after CN crush (n=23), intracavernous injection of SVF immediately after CN crush (n=17), intracavernous injection of SVF 4 wk after CN crush (n=23), or sham surgery (n=26). OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: We studied intracavernous pressure (ICP) response to CN electrostimulation and performed histologic examination of midpenile cross-sections. Data were analyzed using one-way analysis of variance followed by the Tukey-Kramer test. RESULTS AND LIMITATIONS: Both immediate and delayed treatment with SVF resulted in a significantly increased ICP-to-mean arterial pressure ratio compared with the vehicle-treated group. Both immediate and delayed treatment with SVF significantly increased expression of neuronal nitric oxide synthase and neurofilament in dorsal penile nerves compared to the vehicle group. Furthermore, the smooth muscle-to-collagen ratio within the corpus cavernosum was significantly improved in both of the SVF groups compared to vehicle-treated rats. The main limitation of the study is the lack of determination of the SVF components. CONCLUSIONS: Uncultured autologous SVF injected immediately or 4 wk after CN crush improved erectile function, promoted nerve regeneration, and prevented fibrosis of the corpus cavernosum following CN injury.

Recruitment of intracavernously injected adipose-derived stem cells to the major pelvic ganglion improves erectile function in a rat model of cavernous nerve injury.

BACKGROUND: Intracavernous (IC) injection of stem cells has been shown to ameliorate cavernous-nerve (CN) injury-induced erectile dysfunction (ED). However, the mechanisms of action of adipose-derived stem cells (ADSC) remain unclear. OBJECTIVES: To investigate the mechanism of action and fate of IC injected ADSC in a rat model of CN crush injury. DESIGN, SETTING, AND PARTICIPANTS: Sprague-Dawley rats (n=110) were randomly divided into five groups. Thirty-five rats underwent sham surgery and IC injection of ADSC (n=25) or vehicle (n=10). Another 75 rats underwent bilateral CN crush injury and were treated with vehicle or ADSC injected either IC or in the dorsal penile perineural space. At 1, 3, 7 (n=5), and 28 d (n=10) postsurgery, penile tissues and major pelvic ganglia (MPG) were harvested for histology. ADSC were labeled with 5-ethynyl-2-deoxyuridine (EdU) before treatment. Rats in the 28-d groups were examined for erectile function prior to tissue harvest. MEASUREMENTS: IC pressure recording on CN electrostimulation, immunohistochemistry of the penis and the MPG, and number of EdU-positive (EdU+) cells in the injection site and the MPG. RESULTS AND LIMITATIONS: IC, but not perineural, injection of ADSC resulted in significantly improved erectile function. Significantly more EdU+ ADSC appeared in the MPG of animals with CN injury and IC injection of ADSC compared with those injected perineurally and those in the sham group. One day after crush injury, stromal cell-derived factor-1 (SDF-1) was upregulated in the MPG, providing an incentive for ADSC recruitment toward the MPG. Neuroregeneration was observed in the group that underwent IC injection of ADSC, and IC ADSC treatment had beneficial effects on the smooth muscle/collagen ratio in the corpus cavernosum. CONCLUSIONS: CN injury upregulates SDF-1 expression in the MPG and thereby attracts intracavernously injected ADSC. At the MPG, ADSC exert neuroregenerative effects on the cell bodies of injured nerves, resulting in enhanced erectile response.

Stem cell therapy for erectile dysfunction: a critical review.

Erectile dysfunction (ED) is a prevailing health problem that seriously impacts quality of life. Current treatment options are less effective for patients having cavernous nerve (CN) injury or diabetes mellitus-related ED. These 2 types of ED are thus the main focus of past and current stem cell (SC) therapy studies. In a total of 16 studies so far, rats were exclusively used as disease models and SCs were mostly derived from bone marrow, adipose tissue, or skeletal muscle. For tracking, SCs were labeled with LacZ, green fluorescent protein, 4',6-diamidino-2-phenylindole, DiI, bromodeoxyuridine, or 5-ethynyl-2-deoxyuridine, some of which might have led to data misinterpretation. SC transplantation was done exclusively by intracavernous (IC) injection, which has been recently shown to have systemic effects. Functional assessment was done exclusively by measuring increases of IC pressure during electrostimulation of CN. Histological assessment usually focused on endothelial, smooth muscle, and CN contents in the penis. In general, favorable outcomes have been obtained in all trials so far, although whether SCs had differentiated into specific cell lineages remains controversial. Recent studies have shown that intracavernously injected SCs rapidly escaped the penis and homed into bone marrow. This could perhaps explain why intracavernously injected SCs had systemic antidiabetic effects and prolonged anti-ED effects. These hypotheses and the differentiation-versus-paracrine debate require further investigation.

Pentoxifylline promotes recovery of erectile function in a rat model of postprostatectomy erectile dysfunction.

BACKGROUND: Cavernous nerve (CN) injury during radical prostatectomy (RP) causes CN degeneration and secondary penile fibrosis and smooth muscle cell (SMC) apoptosis. Pentoxifylline (PTX) is a phosphodiesterase inhibitor that further inhibits multiple cytokine pathways involved in nerve degeneration, apoptosis, and fibrosis. OBJECTIVES: To evaluate whether PTX enhances erectile function in a rat model of CN injury. DESIGN, SETTING AND INTERVENTIONS: Forty male Sprague-Dawley rats underwent CN crush injury and were randomized to oral gavage feeding of phosphate-buffered saline (vehicle) or PTX 25, PTX 50, or PTX 100 mg/kg per day. Ten animals underwent sham surgery and received vehicle treatment. Treatment continued for 28 d, followed by a wash-out period of 72 h. An additional eight rats underwent resection of the major pelvic ganglion (MPG) for tissue culture and examination of direct effects of PTX on neurite sprouting. MEASUREMENTS: Intracavernous pressure recording on CN electrostimulation, immunohistologic examination of the penis and the CN distal to the injury site, and length of neurite sprouts in MPG culture. RESULTS: Daily oral gavage feeding of PTX resulted in significant improvement of erectile function compared to vehicle treatment in all treated groups. After treatment with PTX 50 and PTX 100 mg/kg per day, the expression of neuronal nitric oxide synthase in the dorsal penile nerve was significantly higher than in vehicle-treated rats. Furthermore, PTX treatment prevented collagen deposition and SMC loss in the corpus cavernosum. In the CN, signs of Wallerian degeneration were ameliorated by PTX treatment. MPG culture in medium containing PTX resulted in a significant increase of neurite length. CONCLUSIONS: PTX treatment following CN injury in rats improved erectile recovery, enhanced nerve regeneration, and preserved the corpus cavernosum microarchitecture. The clinical availability of this compound merits application in penile rehabilitation studies following RP in the near future.

Injections of adipose tissue-derived stem cells and stem cell lysate improve recovery of erectile function in a rat model of cavernous nerve injury.

INTRODUCTION: Erectile dysfunction (ED) remains a major complication after radical prostatectomy. The use of adipose tissue-derived stem cells (ADSCs) has shown promising results for the treatment of ED. However, the mechanisms of action for stem cell therapy remain controversial, with increasing evidence pointing to paracrine pathways. AIM: To determine the effects and to identify the mechanism of action of ADSC and ADSC-derived lysate in a rat model of cavernous nerve (CN) crush injury. METHODS: Thirty-two male Sprague-Dawley rats were randomly divided into four equal groups: one group underwent sham operation, while three groups underwent bilateral CN crush. Crush-injury groups were treated at the time of injury with intracavernous injection of ADSC, lysate, or vehicle only (injured controls). Erectile function was assessed by CN electrostimulation at 4 weeks. Penile tissue was collected for histology. MAIN OUTCOME MEASURES: Intracavernous pressure increase upon CN stimulation; neuronal nitric oxide synthase (nNOS) content in the dorsal penile nerve; smooth muscle content, collagen content, and number of apoptotic cells in the corpus cavernosum. RESULTS: Both ADSC and lysate treatments resulted in significant recovery of erectile function, as compared with vehicle treatment. nNOS content was preserved in both the ADSC and lysate group, with significantly higher expression compared with vehicle-treated animals. There was significantly less fibrosis and a significant preservation of smooth muscle content in the ADSC and lysate groups compared with injured controls. The observed functional improvement after lysate injection supports the hypothesis that ADSCs act through release of intracellular preformed substances or by active secretion of certain biomolecules. The underlying mechanism of recovery appears to involve neuron preservation and cytoprotection by inhibition of apoptosis. CONCLUSIONS: Penile injection of both ADSC and ADSC-derived lysate can improve recovery of erectile function in a rat model of neurogenic ED.

Erectogenic and neurotrophic effects of icariin, a purified extract of horny goat weed (Epimedium spp.) in vitro and in vivo.

INTRODUCTION: Epimedium species (aka horny goat weed) have been utilized for the treatment of erectile dysfunction in Traditional Chinese Medicine for many years. Icariin (ICA) is the active moiety of Epimedium species. AIM: To evaluate the penile hemodynamic and tissue effects of ICA in cavernous nerve injured rats. We also studied the in vitro effects of ICA on cultured pelvic ganglia. METHODS: Rats were subjected to cavernous nerve injury and subsequently treated for 4 weeks with daily gavage feedings of a placebo solution of normal saline and Dimethyl sulfoxide (DMSO) vs. ICA dissolved in DMSO at doses of 1, 5, and 10 mg/kg. A separate group underwent a single dose of ICA 10 mg/kg 2 hours prior to functional testing. Functional testing with cavernous nerve stimulation and real-time assessment of intracavernous pressure (ICP) was performed at 4 weeks. After functional testing, penile tissue was procured for immunohistochemistry and molecular studies. In separate experiments, pelvic ganglia were excised from healthy rats and cultured in the presence of ICA, sildenafil, or placebo culture media. MAIN OUTCOME MEASURE: Ratio of ICP and area under the curve (AUC) to mean arterial pressure (MAP) during cavernous nerve stimulation of subject rodents. We also assayed tissue expression of neuronal nitric oxide synthase (nNOS), eNOS: endothelial nitric oxide synthase (eNOS), calponin, and apoptosis via immunohistochemistry and Western blot. Serum testosterone and luteinizing hormone (LH) were assayed using enzyme-linked immunosorbant assay (ELISA). Differential length of neurite outgrowth was assessed in cultured pelvic ganglia. RESULTS: Rats treated with low-dose ICA demonstrated significantly higher ICP/MAP and AUC/MAP ratios compared with control and single-dose ICA animals. Immunohistochemistry and Western blot were revealing of significantly greater positivity for nNOS and calponin in penile tissues of all rats treated with ICA. ICA led to significantly greater neurite length in cultured specimens of pelvic ganglia. CONCLUSION: ICA may have neurotrophic effects in addition to known phosphodiesterase type 5 inhibiting effects.

The effect of intracavernous injection of adipose tissue-derived stem cells on hyperlipidemia-associated erectile dysfunction in a rat model.

INTRODUCTION: Hyperlipidemia has been associated with erectile dysfunction (ED) via damage to the cavernous endothelium and nerves. Adipose tissue-derived stem cells (ADSC) have been shown to differentiate into endothelial cells and secrete vasculotrophic and neurotrophic factors. AIM: To assess whether ADSC have therapeutic effects on hyperlipidemia-associated ED. METHODS: Twenty-eight male rats were induced to develop hyperlipidemia with a high-fat diet (hyperlipidemic rats, HR). Ten additional male rats were fed a normal diet to serve as controls (normal rats, NR). Five months later, all rats were subjected to ADSC isolation from paragonadal fat. The cells were cultured for 1 week, labeled with 5-ethynyl-2'-deoxyuridine (EdU), and then injected autologously into the corpus cavernosum of 18 HR. The remaining 10 HR rats were injected with phosphate buffered saline (PBS). At 2 and 14 days post-transplantation, four rats in the HR + ADSC group were sacrificed for tracking of the transplanted cells. At 28 days post-transplantation, all remaining rats were analyzed for serum biochemistry, erectile function, and penile histology. MAIN OUTCOME MEASURES: Erectile function was assessed by intracavernous pressure (ICP) measurement during electrostimulation of the cavernous nerve. Cavernous nerves, endothelium, and smooth muscle were assessed by immunohistochemistry. RESULTS: Serum total cholesterol and low-density lipoprotein levels were significantly higher in HR than in NR. High-density lipoprotein level was significantly lower in HR than in NR. Mean ICP/mean arterial pressure ratio was significantly lower in HR + PBS than in NR + PBS or HR + ADSC. Neuronal nitric oxide synthase (nNOS)-positive nerve fibers and endothelial cells were fewer in HR + PBS than in HR + ADSC. Smooth muscle content was significantly higher in both HR groups than in NR. CONCLUSIONS: Hyperlipidemia is associated with abnormalities in both the nerves and endothelium. Treatment with ADSC ameliorates these adverse effects and holds promise as a potential new therapy for ED.

Treatment of erectile dysfunction in the obese type 2 diabetic ZDF rat with adipose tissue-derived stem cells.

INTRODUCTION: Erectile dysfunction (ED) is a major complication of type 2 diabetes, and many diabetic men with ED are refractory to common ED therapies. AIM: To determine whether autologous adipose tissue-derived stem cells (ADSCs) injected into the penis of impotent type 2 diabetic rats improve erectile function. MAIN OUTCOME MEASURES: Blood glucose levels, intracavernous pressure (ICP) increase upon cavernous nerve (CN) electrostimulation, and immunohistochemistry. METHODS: Twenty-two male Zucker diabetic fatty (ZDF) rats were used. At 22 weeks of age, all the animals underwent unilateral CN electrostimulation and ICP measurement to confirm impotence. Paragonadal adipose tissue was harvested to procure ADSCs. The impotent animals were randomized to ADSC treatment and sham control groups. At 23 weeks of age, the treatment group animals underwent a penile injection of 1 million ADSCs; the control group animals received vehicle only. Erectile function studies were repeated at 26 weeks of age, followed by tissue harvest. RESULTS: The rats developed diabetes within the first 10 weeks of age. At 22 weeks of age, 20 out of the 22 rats presented with ED. The post-treatment ICP increase during CN stimulation and ICP increase/mean arterial pressure were significantly higher in the treatment group compared with controls. Three weeks after injection into the corpus cavernosum, only a small number of BrdU-labeled ADSCs was detectable within corporal tissue of the treatment group. There was a significant increase in neuronal nitric oxide synthase (nNOS) in the penile dorsal nerve and in the number of endothelial cells in the corpora cavernosa of the rats in the treatment group. CONCLUSION: Autologous ADSCs injected into the penis were effective to improve erectile function and to alter the microarchitecture of the corpus cavernosum. Since the number of ADSCs retained in the corpus cavernosum is very small, we postulate that their paracrine function, not trans-differentiation to smooth muscle or endothelial cells, is responsible for the improvement in penile function.

Intracavernous growth differentiation factor-5 therapy enhances the recovery of erectile function in a rat model of cavernous nerve injury.

INTRODUCTION: Neurogenic erectile dysfunction remains a serious complication in the postprostatectomy population. Effective protective and regenerative neuromodulatory strategies are needed. AIM: To determine the effect of growth differentiation factor-5 (GDF-5) on erectile function and its mechanism in a rat model of cavernous nerve (CN) injury. MAIN OUTCOME MEASURES: Erectile function was assessed by CN electrostimulation at 4 weeks. Penile tissues were examined by real-time polymerase chain reaction (PCR) and immunohistochemical analyses. METHODS: Forty-eight male Sprague-Dawley rats were randomly divided into six equal groups: one group underwent sham operation (uninjured controls), while five groups underwent bilateral CN crush. Crush-injury groups were treated at the time of injury with intracavernous injection of a slow-release suspension of liquid microparticles containing no GDF-5 (vehicle), 0.4 microg (low concentration), 2 microg (intermediate concentration), or 10 microg GDF-5 (high concentration). One untreated group served as injured controls. RESULTS: GDF-5 enhanced erectile recovery and significantly increased intracavernous pressure in the low and intermediate-concentration groups vs. injured controls. Low-concentration GDF-5 demonstrated the best functional preservation, as the intracavernous pressure increase in this group did not differ significantly from uninjured controls. A dose-response relationship was confirmed for the effects of GDF-5 in penile tissue. Low-concentration GDF-5 showed better preservation of the penile dorsal nerves and antiapoptotic effects in the corpus cavernosum (P < 0.05 vs. injured controls). Although high concentration GDF-5 did not confer meaningful erectile recovery, this dose was more effective at decreasing transforming growth factor-beta than low-concentration GDF-5. CONCLUSIONS: Intracavernous injection of low (0.4 microg) or intermediate-concentration GDF-5 (2 microg) was effective in preserving erectile function in a rat model of neurogenic erectile dysfunction. The underlying mechanism appears to involve neuron preservation and antiapoptosis.

Molecular Yin and Yang of erectile function and dysfunction.

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.

Effects of icariin on phosphodiesterase-5 activity in vitro and cyclic guanosine monophosphate level in cavernous smooth muscle cells.

OBJECTIVES: To investigate the effect of icariin on the cyclic guanosine monophosphate (cGMP)-hydrolytic activity of phosphodiesterase-5 (PDE5) isoforms and the cGMP levels in cavernous smooth muscle cells treated with sodium nitroprusside (SNP). METHODS: PDE5 isoforms (PDE5A1, A2, and A3) were isolated from sf9 insect cells infected with baculoviruses carrying PDE5 isoform cDNA. Icariin was isolated from Epimedii herba. Varying amounts (10(-6) to 10(-11) M) of icariin or zaprinast were added to reaction mixtures containing PDE5 isoforms and cGMP. The inhibitory effects of icariin and zaprinast were analyzed by GraphPad Software and are expressed as concentration that inhibits 50% (IC50) values. Cavernous smooth muscle cells were isolated from 3-month-old rats, treated with icariin (100 and 200 microM) or zaprinast (200 microM) for 15 minutes, and then with 10 microM SNP for 30, 60, 120, 240, and 360 minutes. The cells were then analyzed for the cGMP concentration using an enzyme immunoassay system. RESULTS: Icariin inhibited PDE5A1, A2, and A3 with an IC50 value of 1.0, 0.75, and 1.1 microM, respectively. The corresponding IC50 values for zaprinast were 0.33, 0.23, and 0.32 microM. Icariin consistently outperformed the control (SNP-only treatment) in maintaining greater cGMP levels, particularly at the greater concentration of 200 microM. In contrast, zaprinast at 200 microM did better than the control only at 60 and 360 minutes. CONCLUSIONS: Icariin was inhibitory to all three PDE5 isoforms with similar IC50 values, which were approximately three times greater than those for zaprinast. Icariin was able to enhance cGMP levels in SNP-treated cavernous smooth muscle cells.

Improving erectile function by silencing phosphodiesterase-5.

PURPOSE: We investigated whether phosphodiesterase-5 (PDE5) can be down-regulated by a specific small interfering RNA (siRNA) and whether this would improve erectile function. MATERIALS AND METHODS: A PDE5 siRNA encoding oligonucleotide was inserted into pSUPER-retro vector, resulting in the siRNA expressing construct, pPDE5-silencer. The construct was packaged into oncoretroviral particles and then transduced into rat cavernous smooth muscle cells (CSMCs). Cells were examined for PDE5 expression by reverse transcriptase-polymerase chain reaction, Western blotting and immunofluorescence microscopy. Cells were then treated with 10 microM sodium nitroprusside (SNP) and examined for cyclic guanosine monophosphate (cGMP) at 0, 10, 30, 60 and 240 minutes. The siRNA expressing cassette was transferred from the oncoretrovirus to lentivirus, which was then injected into rat penises. Three months later erectile function was examined by electrostimulation and PDE5 expression in cavernous smooth muscle was determined by immunohistochemistry. RESULTS: CSMCs transfected with pPDE5-silencer (CSMC plus siRNA) showed an 88.2% decrease in PDE5 compared with CSMCs transfected with control vector (CSMCs plus vector). Within 10 minutes of SNP treatment cells (CSMCs, CSMCs plus vector and CSMCs plus siRNA) showed similar sharp increases in cGMP. However, while cGMP levels in CSMCs and CSMCs plus vector returned to almost baseline in 1 hour, the cGMP level in CSMCs plus siRNA remained high even 4 hours after SNP treatment. Rats injected with the siRNA expressing lentivirus showed increased electrostimulated erectile function, as measured by peak intracorporeal pressure and the intracorporeal pressure increase, compared with rats injected with control lentivirus. PDE5 expression was decreased in the siRNA treated cavernous smooth muscle. CONCLUSIONS: PDE5 expression could be decreased by siRNA, resulting in prolonged cGMP accumulation and improved erection.

The effect of neural embryonic stem cell therapy in a rat model of cavernosal nerve injury.

OBJECTIVE: To isolate embryonic stem cells that have differentiated along the neuronal cell line, and to assess whether injecting these neural stem cells into the corpus cavernosum influences cavernosal nerve regeneration and functional status. MATERIALS AND METHODS: Embryonic neural stem cells were obtained; 26 male Sprague-Dawley rats were divided into four groups: five had a sham operation; eight (controls) had a bilateral cavernosal nerve crush and injection of culture medium into the corpora cavernosa; four had an injection of neural embryonic stem (NES) cells into the major pelvic ganglion (MPG); and nine had bilateral cavernosal nerve crush and injection of NES cells into the corpora cavernosa. Erectile response was assessed by cavernosal nerve electrostimulation at 3 months, and penile tissue samples were evaluated histochemically for nitric oxide synthase (NOS)-containing fibres, tyrosine hydroxylase and neurofilament staining. RESULTS: The groups injected with NES cells into the MPG and corpora cavernosa had significantly higher intracavernosal pressures than the control group. Immunohistochemical staining also revealed differences in the quality of the NOS-containing nerve fibres. Neurofilament staining was significantly better in the experimental groups injected with NES cells. CONCLUSION: We were able to isolate embryonic stem cells that had differentiated along the neural cell line and, using these NES cells intracavernosally, showed improved erectile function in a rat model of neurogenic impotence.

[Gene expression profiles and effects of transforming growth factor-beta1 intervention in Peyronie's disease].

OBJECTIVE: To demonstrate molecular insight into the pathology of Peyronie's disease (PD). A preliminary profile of differential gene expression between the PD plaque and control tunica albuginea was obtained with DNA microarrays. Also, to investigate the effect of intervention in PD cells, transforming growth factor-beta1 (TGF-beta1) was recruited to treat PD cell lines. METHODS: Three PD plaques and control tunica albugineas were constructed and studied. cDNA probes were prepared from RNA isolated from those cells and hybridized with the Clontech Atlas 3.6 Array. Relative changes of greater than 2.0 defined up-regulation and down-regulation, respectively. The expression of selected individual gene MCP-1 and the effect of TGF-beta1 on MCP-1 were analyzed by reverse transcriptase-polymerase chain reaction. RESULTS: Some up-regulated genes in the PD plaque detected by the Clontech assay were screened, one of them was monocyte chemotactic protein. One involved the pathogenesis of PD as a downstream gene and responded to the TGF-beta1 treatment but not CTGF. The results were also confirmed by TR-PCR in all the types of cell. CONCLUSIONS: The cell lines from plaque tissue and normal tunica from men with PD were successfully established. The findings indicate a potential role for MCP-1 over expression in the pathogenesis of PD as a downstream gene regulated by some genes and could be a new therapeutic target in PD. The information may allow a better understanding of the basic mechanisms involved in the etiology and pathogenesis of PD. Furthermore, it may permit some strategies of therapeutic interventions combine routine methods with Chinese herbal medicine.

The effect of vascular endothelial growth factor on a rat model of traumatic arteriogenic erectile dysfunction.

PURPOSE: We tested the hypothesis that intracavernous injection of vascular endothelial growth factor (VEGF) can restore erectile function in a rat model of traumatic arteriogenic erectile dysfunction. MATERIALS AND METHODS: Exploration of bilateral internal iliac arteries was performed in 50, 3-month-old male rats. A total of 44 rats underwent bilateral ligation of the internal iliac arteries and 6 that underwent exploration only served as the sham operated group. Minutes later intracavernous injection of phosphate buffered saline (PBS) plus bovine serum albumin in 16 rats, 2 microg. VEGF plus PBS plus BSA in 12 and 4 microg. VEGF plus PBS plus BSA in 16 was performed. At weeks 1, 2 and 6 about a third of the rats in each group underwent electrostimulation of the cavernous nerves to assess erectile function and were then sacrificed. Penile tissues were collected for histochemical and electron microscopy examinations. RESULTS: No impairment of erectile function was noted in sham operated rats. Immediately after arterial ligation all rats showed little or no erectile response to neurostimulation. In PBS treated rats modest recovery of erectile function was noted at week 6. Significant recovery of erectile function was noted in VEGF treated rats at weeks 1 and 2 in the 4 microg. group only and at week 6 in the 2 and 4 microg. groups. Neuronal nitric oxide synthase staining showed a reduction in neuronal nitric oxide synthase positive nerve fibers in the dorsal or intracavernous nerves at week 1. Moderate recovery of neuronal nitric oxide synthase positive nerve fibers was noted in the 2 and 4microg. VEGF treated groups but not in the PBS treated group. Electron microscopy revealed no pathological change in sham operated rats. In dorsal nerves the atrophy of myelinated and nonmyelinated nerve fibers was noted in ligated plus PBS treated rats. Partial recovery was observed in VEGF treated rats. Scattered atrophic smooth muscle cells were seen in PBS and occasionally in VEGF treated rats but not in the sham operated group. The most dramatic findings in VEGF treated rats were hypertrophy and hyperplasia of the endothelial cells, especially those lining the small capillaries. CONCLUSIONS: Ligation of bilateral internal iliac arteries produced a reliable animal model of traumatic arteriogenic erectile dysfunction. Intracavernous injection of VEGF minutes after arterial ligation facilitated the recovery of erectile function.