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.

Long-term effects of phytoestrogen daidzein on penile cavernosal structures in adult rats.

OBJECTIVES: Daidzein is a soy isoflavone with estrogenic activity present in plant-based food items and health foods and used as an alternative therapy for cancer and cardiovascular diseases. The present study was designed to investigate the effects of daidzein on the cavernosal components, including smooth muscle cells, collagen fibers, and elastic fibers, that are the key structures fundamental for erection. METHODS: A total of 30 adult male Sprague-Dawley rats were equally divided into a normal control group, three experimental groups, and one positive control group. The three experimental groups were given daidzein at a dose of 2, 20, and 100 mg/kg body weight daily, and the positive control group received 0.1 mg diethylstilbestrol per animal daily for 90 days. The collagen fibers and elastic fibers in the corpora cavernosa were measured using histochemical or immunohistochemical techniques, and their relative contents were evaluated quantitatively or semiquantitatively. RESULTS: The relative content of collagen fibers in the corpus cavernosa in rats treated with low-dose daidzein (2 mg/kg) was not significantly different from that of controls, as was the case for the smooth muscle and elastic fiber content (all P >0.05). However, the relative content of the collagen fibers was significantly increased in rats treated with a medium dose (20 mg/kg) and a high dose (100 mg/kg) of daidzein. The smooth muscle cell and elastic fiber content was reduced significantly compared with that of the controls (all P <0.01). Similar alterations were observed in the diethylstilbestrol-treated rats. CONCLUSIONS: These results suggest that daidzein, if ingested in a relatively large amount, could induce histopathologic alterations in the penile cavernosal structures characterized by an increase in the collagen content and a reduction in smooth muscle cell and elastic fiber content, which might be suggestive of erectile dysfunction.

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.

T-type (alpha1G) low voltage-activated calcium channel interactions with nitric oxide-cyclic guanosine monophosphate pathway and regulation of calcium homeostasis in human cavernosal cells.

INTRODUCTION: Nitric oxide-cyclic guanosine monophosphate (NO-cGMP)-mediated relaxation of cavernosal smooth muscle during erection is accompanied by a decrease in intracellular calcium concentrations ([Ca2+](i)). However, it is not known whether and how an increase in [Ca2+](i) is responsible for (i) initiating smooth muscle contraction/detumescence following relaxation; and (ii) maintaining the penis in a flaccid state under nonstimulating conditions. AIM: To elucidate (i) the mechanism(s) of [Ca2+](i) homeostasis regulation in human cavernosal smooth muscle cells (HCSMC); and (ii) how NO-cGMP interacts with such [Ca2+](i) homeostasis. METHODS: We evaluated the expression and function of both T-type and L-type Ca2+ channels in HCSMC by employing selective probes/inhibitors using various cellular and molecular techniques (e.g., reverse transcriptase and real-time polymerase chain reaction, cell proliferation assay, fura-2 Ca2+ fluorescence spectroscopy, enzyme-linked immuno-absorbent assay (ELISA)). MAIN OUTCOME MEASURE: We have demonstrated for the first time significant interactions of NO-cGMP with the T-type (alpha1G) Ca2+ channel in HCSMC. RESULTS: Our results suggest that in addition to NO-induced rapid and transient decrease in [Ca2+](i) that results in smooth muscle relaxation, NO-cGMP also enhanced mRNA expression of the T-type (alpha1G) Ca2+ channel resulting in delayed elevation of [Ca2+](i). This could be abolished by a selective T-channel blocker, NNC 55-0396. Another unique finding of this study is that dose-dependent HCSMC proliferation in vitro by NO is associated with the activation of the T-type (alpha1G) Ca2+ channel that regulates [Ca2+](i) homeostasis in these cells. CONCLUSIONS: Human cavernosal cells express T-type (alpha1G) Ca2+ channels that are involved in maintaining [Ca2+](i) homeostasis and regulation of NO-cGMP-induced smooth muscle relaxation-contraction responsible for penile erection, flaccidity, and tonicity. Targeting these Ca2+ channels may (i) associate various comorbidities with the onset of erectile dysfunction; (ii) provide a biochemical basis for differences between therapeutic profiles of various phosphodiesterase type 5 inhibitors, especially in nonresponders to current therapy; and (iii) provide biochemical basis in understanding mechanism(s) of drug tolerance.

Endothelium-dependency of yohimbine-induced corpus cavernosum relaxation.

Development and maintenance of penile erection requires the relaxation of the smooth muscle cells in the cavernous bodies and is essentially mediated by nitric oxide (NO). The penile flaccid state is conversely maintained by the alpha adrenergic neuroeffector system and by other vasoconstrictors, such as endothelin-1 (ET-1). In this study we examined the mechanisms involved in yohimbine-induced relaxation in human and rabbit corpora cavernosa (CC). We essentially found that yohimbine not only blocks contractions induced by adrenergic agonists, but also by non-adrenergic substances, such as ET-1. This effect was unrelated to antagonism at the level of ET receptors, because yohimbine did not affect ET-1-induced increase in intracellular calcium in isolated CC cells. Conversely, our data suggest that yohimbine counteracts ET-1-induced contractions by interfering with NO release from the endothelium. In fact, yohimbine-induced CC relaxation was inhibited by the mechanical removing of the endothelium and by blocking NO formation or signalling via guanylate cyclase and cGMP formation. Conversely, yohimbine activity was strongly increased by inhibiting cGMP degradation. In an experimental model of hypogonadism, performed on rabbits by chronic treatment with a long-lasting GnRH agonist, the relaxant yohimbine activity was also decreased, but completely restored by androgen supplementation. This effect was evident only in preparations in which the main source of NO was present (endothelium) or in which NO formation was not impaired by L-NAME. Our data indicate that the relaxant effect of yohimbine is both endothelium and androgen-dependent. This might justify the lack of efficacy of this drug in treatment of some form of organic erectile dysfunction.

[Effect of Na2SeO3 on the damages of genetic materials induced by MNNG in children's foreskin fibroblasts in vitro].

In order to study the effect of selenium on anticarcinogenesis, micronuclei (MN) and chromosome aberrations (CA) were used as the indexes to reflect the damages on the genetic materials induced by MNNG in children's foreskin fibroblasts in vitro. In the MN test, the final concentrations of Na2SeO3 were 10(-7), 10(-6), 10(-5) and 10(-4) M and MNNG, 10(-5)M. In the CA test, Na2SeO3 were used in 10(-7), 10(-6) and 10(-5)M and MNNG, 10(-6)M as the final concentrations. Relative to the time of MNNG treatment, the cells were exposed to Na2SeO3 4 hours before and at the same time as with the carcinogen. The results showed that the MN% (number of cells out of one thousand MN) was reduced from 4.31 +/- 0.91% to 1.55 +/- 0.54% and 1.54 +/- 0.54% (P less than 0.05), respectively. The CA% (the percentage of the cell with CA) was reduced from 86 +/- 7% to 34 +/- 9% and 33 +/- 9% (P less than 0.05), respectively. However there was no like results when the cells were treated with Na2SeO3 and MNNG simultaneously. Na2SeO3 had no significant protective effects on the cells when the concentration was 10(-7)M. If the dose was 10(-4)M or more, Na2SeO3 became toxic to the cells. The results suggested that the protection of Na2SeO3 on the damages of genetic materials induced by MNNG be time and dose dependent.