Urethral musculature and innervation in the female rat.

AIMS: The urethral sphincter and urethral muscle innervation are critically involved in maintaining continence, especially in the female. However, the urethral muscle type and distribution, as well as the urethral nerves are far from being well documented. Our aim was to clearly identify the distribution of urethral striated muscle, smooth muscle, and urethral nerves. METHODS: In a cohort analysis of 3-month-old female Sprague-Dawley rats, cross and longitudinal sections of female rat urethra were extensively investigated using morphological techniques. Urethras were harvested to the sections, in order to provide both global and detailed visions of the urethra. H&E, Masson's Trichrome, phalloidin and immunoflourence stains were used. The cytoarchitecture, nitrergic, and cholinergic innervations were mainly investigated. Different layers of the segments of urethra were traced to draw curve graphs that represent the thickness of each muscle layer of urethral wall. RESULTS: The results showed that the primary peak of striated muscle is in the middle urethra. The inner layer close to mucosa was found to contain longitudinal smooth muscle. Near the bladder orifice, the circular smooth muscle dominates, which becomes thinner distally throughout the rest of urethra. In the middle urethra the vast majority of the urethral muscle are circularly oriented striated muscle cells. Typical nerve endings were present in high power images to show the different characteristic features of nerve innervation. CONCLUSIONS: This study has illustrated the detailed morphological structure and innervations of the normal female rat urethra and can serve as a basis for further study of stress urinary incontinence (SUI).

Estrogen attenuates TGF-ß1 induced elastogenesis in rat urethral smooth muscle cells by inhibiting Smad response elements.

PURPOSE: We investigated the effect and mechanism of estrogen on elastogenesis in urethral smooth muscle cells in vitro. MATERIALS AND METHODS: Urethral smooth muscle cells were isolated from normal adult female rats. For elastogenesis assay cells were treated with TGF-ß1, the potent TGF-ß1 receptor inhibitor SB431542 and estrogen for 2 weeks. Real-time polymerase chain reaction was performed to assay gene expression during this process. Activity of the TGF-ß1 responsive elements CAGA(12)-Luc and GCCG(12)-Luc were also assayed. Estrogen receptor and Smad2/3 interaction was evaluated by immunoprecipitation and Western blot. RESULTS: TGF-ß1 induced elastogenesis in rat urethral smooth muscle cells. This effect was partially blocked by estrogen and completely abrogated by SB431542. SB431542 completely inhibited activation of the Smad2/3 response element CAGA(12)-Luc and estrogen significantly inhibited activation. The Smad1/4 response element GCCG(12)-Luc was not affected by SB431542 treatment but estrogen partially inhibited the activation of GCCG(12)-Luc induced by TGF-ß1. Estrogen receptor bound to Smad 2 and 3 in vitro. CONCLUSIONS: Estrogen attenuated TGF-ß1 induced elastogenesis via binding of its activated receptor to Smad2/3 to inhibit the TGF-ß1 response element in rat urethral smooth muscle cells.

Effects of EdU labeling on mesenchymal stem cells.

BACKGROUND: Thymidine analog 5-ethynyl-2-deoxyuridine (EdU) has recently been used for tracking mesenchymal stem cells (MSCs). In the present study, we tested whether EdU was cytotoxic and whether it interfered with differentiation, cytokine secretion and migration of MSCs. METHODS: EdU labeling was performed by incubating adipose-derived stem cells (ADSCs) with 10(-8) mol/L of EdU for 48 h. Incorporation of EdU was detected by reaction with azide-conjugated Alexa594. The labeled and unlabeled ADSCs were compared for proliferation and apoptosis as determined by CellTiter and comet assays, respectively. They were also compared for neuron-like and endothelial differentiation as determined by morphology, marker expression and function. Comparison of their secreted cytokine profile was performed by cytokine antibody array. Comparison of their response to homing factor SDF-1 was performed by migration assay. RESULTS: EdU was incorporated into the nucleus in approximately 70% of ADSCs. No significant differences in proliferation and apoptosis rates were observed between EdU-labeled and unlabeled ADSCs. Isobutylmethylxanthine induced both EdU-labeled and unlabeled ADSCs to assume a neuron-like morphology and to express ß-III tubulin. Endothelial growth medium-2 (EGM2) induced endothelial differentiation in both EdU-labeled and unlabeled ADSCs, including the ability to uptake low-density lipoprotein and to form capillary-like structures as well as the expression of vWF, eNOS and CD31. EdU-labeled and unlabeled ADSCs exhibited identical secreted cytokine profile and identical migratory response to SDF-1. DISCUSSION: At the recommended dosage of 10(-8) mol/L, EdU is non-toxic to ADSCs. EdU label did not interfere with differentiation, cytokine secretion or migratory response to SDF-1 by ADSCs.

Effects of low-energy shockwave therapy on the erectile function and tissue of a diabetic rat model.

Introduction. Low-energy shockwave therapy (LESWT) has been shown to improve erectile function in patients suffering from diabetes mellitus (DM)-associated erectile dysfunction (ED). However, the underlying mechanism remains unknown. Aim. The aim of this study is to investigate whether LESWT can ameliorate DM-associated ED in a rat model and examine the associated changes in the erectile tissues. Methods. Newborn male rats were intraperitoneally injected with 5-ethynyl-2-deoxyuridine (EdU; 50 mg/kg) for the purpose of tracking endogenous mesenchymal stem cells (MSCs). Eight weeks later, eight of these rats were randomly chosen to serve as normal control (N group). The remaining rats were injected intraperitoneally with 60 mg/kg of streptozotocin (STZ) to induce DM. Eight of these rats were randomly chosen to serve as DM control (DM group), whereas another eight rats were subject to shockwave (SW) treatment (DM+SW group). Each rat in the DM+SW group received 300 shocks at energy level of 0.1 mJ/mm(2) and frequency of 120/minute. This procedure was repeated three times a week for 2 weeks. Another 2 weeks later, all 24 rats were evaluated for erectile function by intracavernous pressure (ICP) measurement. Afterward, their penile tissues were examined by histology. Main Outcome Measures. Erectile function was measured by ICP. Neuronal nitric oxide synthase (nNOS)-positive nerves and the endothelium were examined by immunofluorescence staining. Smooth muscle and MSCs were examined by phalloidin and EdU staining, respectively. Results. STZ treatment caused a significant decrease in erectile function and in the number of nNOS-positive nerves and in endothelial and smooth muscle contents. These DM-associated deficits were all partially but significantly reversed by LESWT. MSCs (EdU-positive cells) were significantly more numerous in DM+SW than in DM rats. Conclusion. LESWT can partially ameliorate DM-associated ED by promoting regeneration of nNOS-positive nerves, endothelium, and smooth muscle in the penis. These beneficial effects appear to be mediated by recruitment of endogenous MSCs. Qiu X, Lin G, Xin Z, Ferretti L, Zhang H, Lue TF, and Lin C-S. Effects of low-energy shockwave therapy on the erectile function and tissue of a diabetic rat model. J Sex Med 2013;10:738-746.

Is CD34 truly a negative marker for mesenchymal stromal cells?

The prevailing school of thought is that mesenchymal stromal cells (MSC) do not express CD34, and this sets MSC apart from hematopoietic stem cells (HSC), which do express CD34. However, the evidence for MSC being CD34(-) is largely based on cultured MSC, not tissue-resident MSC, and the existence of CD34(-) HSC is in fact well documented. Furthermore, the Stro-1 antibody, which has been used extensively for the identification/isolation of MSC, was generated by using CD34(+) bone marrow cells as immunogen. Thus, neither MSC being CD34(-) nor HSC being CD34(+) is entirely correct. In particular, two studies that analyzed CD34 expression in uncultured human bone marrow nucleated cells found that MSC (BMSC) existed in the CD34(+) fraction. Several studies have also found that freshly isolated adipose-derived MSC (ADSC) express CD34. In addition, all of these ADSC studies and several other MSC studies have observed a disappearance of CD34 expression when the cells are propagated in culture. Thus the available evidence points to CD34 being expressed in tissue-resident MSC, and its negative finding being a consequence of cell culturing.

Identification of active and quiescent adipose vascular stromal cells.

BACKGROUND AIMS: Recent studies have demonstrated the existence of both active and quiescent stem cells in bone marrow, hair follicle and intestine. We attempted to identify active and quiescent vascular stromal cells (VSC) in adipose tissue. METHODS: For identification of active VSC, adult rats were injected intraperitoneally with thymidine analog 5-ethynyl-2-deoxyuridine (EdU) and their subcutaneous tissue harvested 3 days later. For identification of quiescent VSC, newborn rats were injected intraperitoneally with EdU and their subcutaneous tissue harvested 9 weeks later. The harvested adipose tissues were examined for the co-localization of EdU with VSC marker CD34, smooth muscle marker SMA, endothelial marker RECA and pericyte marker CD140b. RESULTS: In adult rat adipose tissues harvested 3 days after EdU injection, there were 28.80 ± 8.70 (mean ± SD) EdU+ cells/100 × microscopic field, and approximately 6.2% of cell nuclei were labeled with EdU. The percentages of EdU+ cells expressing the following markers were approximately: 84 for CD34, 5.6 for RECA (rat endothelial marker), 3.7 for SMA and 14.8 for CD140b. In the adipose tissues of newborn rats that were harvested 9 weeks after EdU injection, the percentages of EdU+ cells expressing the following markers were approximately: 76 for CD34, 1.8 for RECA, 0 for SMA and 12.9 for CD140b. In both the short-term (active) and long-term (quiescent) EdU-labeled adipose tissues, the EdU label was consistently co-localized with CD34 and in the proximity of CD140b stain or in the adventitia. CONCLUSIONS: Both active and quiescent VSC expressed CD34 and localized to capillaries and the adventitia of larger blood vessels.

Common pitfalls in some of the experimental studies in erectile function and dysfunction: a consensus article.

INTRODUCTION: Experimental studies investigating physiology of erectile function and pathophysiology erectile dysfunction employ several in vitro and in vivo techniques. As the field of sexual medicine expanding, the proper conduct of such techniques is becoming an even more important necessity than before. AIM: This review article aims to guide scientists, particularly young researchers and new comers in the field, toward employment of these techniques in an appropriate, timely, and competent fashion. METHODS: The authors reviewed the existing available published articles on the following topics: intracavernosal pressure measurements, cavernous nerve injury models, nitric oxide-cyclic guanosine monophosphate pathway, hypertension- and smoking-induced erectile dysfunction models, and stem cells. RESULTS: The authors present a consensus on how to best perform these models and techniques and also highlight the pitfalls. CONCLUSIONS: The authors hope that this article will assist and encourage young scientists in the field and that similar articles covering other important models will be also available to them soon.

Scaffoldless tissue engineering of stem cell derived cavernous tissue for treatment of erectile function.

INTRODUCTION: As one-third of erectile dysfunction (ED) patients do not respond to phosphodiesterase-5 inhibitors, there is great demand for new therapeutic options. Adipose tissue-derived stem cells (ADSCs) represent an ideal source for new ED treatment. AIM: To test if ADSCs can be differentiated into smooth muscle cells (SMCs) and endothelial cells (ECs), if these differentiated cells can be used to engineer cavernous tissue, and if this engineered tissue will remain for long time after implantation and integrate into corporal tissue. METHOD: Rat ADSCs were isolated and differentiated into SMC and ECs. The differentiated cells were labeled with 5-ethynyl-2-deoxyuridine (EdU) and used to construct cavernous tissue. This engineered tissue was implanted in penises of normal rats. The rats were sacrificed after 1 and 2 months; penis and bone marrow were collected to assess cell survival and inclusion in the penile tissues. MAIN OUTCOME MEASURES: The phenotype conversion was checked using morphology, immunocytochemistry (immunohistochemistry [IHC]), and Western blot for SMC and EC markers. The cavernous tissue formation was assessed using rat EC antibody (RECA), calponin, and collagen. The implanted cell survival and incorporation into penis were evaluated with hematoxylin and eosin, Masson's trichrome, and IHC (RECA, calponin, and EdU). RESULTS: The phenotype conversion was confirmed with positive staining for SMC and EC markers and Western blot. The formed tissue exhibited architecture comparable to penile cavernous tissue with SMC and ECs and extracellular matrix formation. The implanted cells survived in significant numbers in the penis after 1 and 2 months. They showed proof of SMC and EC differentiation and incorporation into penile tissue. CONCLUSIONS: The results showed the ability of ADSCs to differentiate into SMC and ECs and form cavernous tissue. The implanted tissue can survive and integrate into the penile tissues. The cavernous tissue made of ADSCs forms new technology for improvement of in vivo stem cell survival and ED treatment.

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.

Mesenchymal stem cell marker Stro-1 is a 75 kd endothelial antigen.

Stro-1 is the best-known mesenchymal stem cell (MSC) marker. However, previous studies have observed its expression in the endothelium. In the present study we performed immunofluorescence (IF) staining for Stro-1, using endothelial marker vWF as reference. In the liver, both proteins were expressed in the endothelium of the central veins and hepatic sinusoids. In the lung, both were expressed in the endothelium of pulmonary blood vessels, but while vWF was absent in the alveolar capillaries, Stro-1 was present. In the kidney, both were expressed in the endothelium of renal arterial branches, but while vWF was strongly expressed in the glomeruli, Stro-1 only scantly. IF staining in cultured endothelial cells also showed extensive overlaps between Stro-1 and vWF. Western blot analysis with Stro-1 antibody detected a single protein band of 75 kd in endothelial cells but not smooth muscle cells, fibroblasts, or B cells. Cancer cell lines PC3, DU145, MCF7, and K562 were also positive. Adipose-derived stem cells (ADSCs) expressed higher levels of Stro-1 when cultured beyond the first passage or when induced to differentiate into endothelial cells. These data, together with previous studies, indicate that Stro-1 is intrinsically an endothelial antigen, and its expression in MSC is probably an induced event.

Cavernous smooth muscle hyperplasia in a rat model of hyperlipidaemia-associated erectile dysfunction.

UNLABELLED: What's known on the subject? and what does the study add? Increased cavernous smooth muscle content has been repeatedly observed in rat models of hyperlipidaemia - associated erectile dysfunction. This study shows that the increased smooth muscle content is due to hyperplasia. OBJECTIVE: • To investigate the structural changes, including possible smooth muscle hyperplasia, in the penis of a hyperlipidaemia-associated erectile dysfunction (ED) animal model. MATERIALS AND METHODS: • Hyperlipidaemia was induced in rats through a high-fat diet. • Penile tissues of normal and hyperlipidaemic rats were stained with Alexa-488-conjugated phalloidin and/or with antibodies against rat endothelial cell antigen, neuronal nitric oxide synthase (nNOS), and collagen type IV (Col-IV) before image and statistical analyses were carried out. • The main outcome measures were the smooth muscle, endothelial, Col-IV and nNOS content of the corpus cavernosum. RESULTS: • Phalloidin intensely stained all smooth muscle in the penis, revealing the circular and longitudinal components of cavernous smooth muscle (CSM). • The CSM content was significantly higher in the hyperlipidaemic than in the normal rats (P < 0.05). • Cell numbers in both circular and longitudinal CSM were significantly higher in the hyperlipidaemic than in the normal rats (P < 0.05). • Cavernous endothelial content was significantly lower in hyperlipidaemic than in normal rats (P < 0.05). • nNOS-positive nerves within the dorsal nerves, around the dorsal arteries, and in the corpora cavernosa were all significantly lower in the hyperlipidaemic than in the normal rats (P < 0.05). CONCLUSIONS: • Hyperlipidaemia is associated with reduced nNOS-positive nerves, reduced endothelium, and increased CSM in the penis. • The increased CSM is attributable to hyperplasia. • These structural changes may explain why hyperlipidaemic men are more likely to develop ED.

Modulation of smooth muscle tonus in the lower urinary tract: interplay of myosin light-chain kinase (MLCK) and MLC phosphatase (MLCP).

OBJECTIVE: To assess and compare the expression and activity of myosin light-chain kinase (MLCK) and MLC phosphatase (MLCP) in rat bladder and urethra. MATERIALS AND METHODS: Bladder and urethral smooth muscles were obtained from 2-month-old female Sprague-Dawley rats. They were analysed by real-time polymerase chain reaction for the mRNA expression of MLCK and myosin phosphatase-targeting subunit of protein phosphatase type 1 (MYPT1, a subunit of MLCP). Levels of MLCK and MYPT1 mRNA expression were determined as a ratio to the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The tissues were also analysed by Western blotting for MLCK and MYPT1 protein expression as a ratio to the expression of ß-actin. A two-step enzymatic activity assay using phosphorylated and dephosphorylated smooth muscle myosin was used to assess MLCK and MLCP activity. RESULTS: MLCK mRNA expression was higher in the bladder than in the urethra [mean (sd) ratio to GAPDH: 0.26 (0.17) vs 0.14 (0.12); P = 0.09]. MYPT1 mRNA expression was significantly higher in the bladder than in the urethra [mean (sd) ratio to GAPDH: 2.31 (1.04) vs 0.56 (0.36); P = 0.001]. Expression of both MLCK and MYPT1 protein was significantly higher in the bladder compared with the urethra [mean (sd) ratio to ß-actin: 1.63 (0.25) vs 0.91 (0.29) and 0.97 (0.10) vs 0.37 (0.29), respectively; both P < 0.001]. Enzymatic assay identified significantly greater MLCK activity in the bladder than in the urethra. While, MLCP activity was lower in the bladder than in the urethra. CONCLUSION: In healthy young female rats, MLCK activity is higher and MLCP activity is lower in the bladder relative to the urethra. These differences probably play a role in modulating the functional differences between bladder and urethral smooth muscle tone.

Adipose tissue-derived stem cells secrete CXCL5 cytokine with neurotrophic effects on cavernous nerve regeneration.

INTRODUCTION: Previously we reported that paracrine actions likely mediated the therapeutic effects of adipose tissue-derived stem cells (ADSCs) on a rat model of cavernous nerve (CN) injury. AIM: To identify potential neurotrophic factors in ADSC's secretion, test the most promising one, and identify the molecular mechanism of its neurotrophic action. METHODS: Rat major pelvic ganglia (MPG) were cultured in conditioned media of ADSC and penile smooth muscle cells (PSMCs). Cytokine expression in these two media was probed with a cytokine antibody array. CXCL5 cytokine was quantified in these two media by enzyme-linked immunosorbent assay (ELISA). Activation of Janus Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) by CXCL5 was tested in neuroblastoma cell lines BE(2)C and SH-SY5Y as well as in Schwann cell line RT4-D6P2T by Western blot. Involvement of CXCL5 and JAK/STAT in ADSC-conditioned medium's neurotrophic effects was confirmed with anti-CXCL5 antibody and JAK inhibitor AG490, respectively. MAIN OUTCOME MEASURES: Neurotrophic effects of ADSC and PSMC-conditioned media were quantified by measuring neurite length in MPG cultures. Secretion of CXCL5 in these two media was quantified by ELISA. Activation of JAK/STAT by CXCL5 was quantified by densitometry on Western blots for STAT1 and STAT3 phosphorylation. RESULTS: MPG neurite length was significantly longer in ADSC than in PSMC-conditioned medium. CXCL5 was secreted eight times higher in ADSC than in PSMC-conditioned medium. Anti-CXCL5 antibody blocked the neurotrophic effects of ADSC-conditioned medium. CXCL5 activated JAK/STAT concentration-dependently from 0 to 50 ng/mL in RT4-D6P2T Schwann cells. At 50 ng/mL, CXCL5 activated JAK/STAT time-dependently, peaking at 45 minutes. AG490 blocked these activities as well as the neurotrophic effects of ADSC-conditioned medium. CONCLUSIONS: CXCL5 was secreted by ADSC at a high level, promoted MPG neurite growth, and activated JAK/STAT in Schwann cells. CXCL5 may contribute to ADSC's therapeutic efficacy on CN injury-induced ED.

Effects of transplantation of adipose tissue-derived stem cells on prostate tumor.

BACKGROUND: Obesity is a risk factor for prostate cancer development, but the underlying mechanism is unknown. The present study tested the hypothesis that stromal cells of the adipose tissue might be recruited by cancer cells to help tumor growth. METHODS: PC3 prostate cancer cells were transplanted into the subcutaneous space of the right flank of athymic mice. One week later, adipose tissue-derived stromal or stem cells (ADSC) or phosphate-buffered saline (PBS, as control) was transplanted similarly to the left flank. Tumor size was monitored for the next 34 days; afterwards, the mice were sacrificed and their tumors harvested for histological examination. The ability of PC3 cells to attract ADSC was tested by migration assay. The involvement of the CXCL12/CXCR4 axis was tested by migration assay in the presence of a specific inhibitor AMD3100. RESULTS: Throughout the entire course, the average size of PC3 tumors in ADSC-treated mice was larger than in PBS-treated mice. ADSC were identified inside the tumors of ADSC-treated mice; CXCR4 expression was also detected. Migration assay indicated the involvement of the CXCL12/CXCR4 axis in the migration of ADSC toward PC3 cells. Capillary density was twice as high in the tumors of ADSC-treated mice than in the tumors of PBS-treated mice. VEGF expression was similar but FGF2 expression was significantly higher in tumors of ADSC-treated mice than in the tumors of PBS-tread mice. CONCLUSION: Prostate cancer cells recruited ADSC by the CXCL12/CXCR4 axis. ADSC helps tumor growth by increasing tumor vascularity, and which was mediated by FGF2.

Adipose tissue-derived stem cells secrete CXCL5 cytokine with chemoattractant and angiogenic properties.

Adipose tissue-derived stem cells (ADSC) secreted CXCL5 cytokine abundantly and higher passaged ADSC up to passage 6 (P6) secreted more CXCL5 than lower passaged ADSC. Higher passaged ADSC also appeared to express higher levels of CXCL5 receptor, i.e., CXCR2. Both CXCL5 and CXCR2 were localized in the tunica intima and tunica adventitia of blood vessels in adipose tissue. Colocalization with CD34 further indicates their association with the putative ADSC in tunica adventitia. Migration assay indicates chemoattractant effects of CXCL5 on ADSC and HUVEC endothelial cells. CXCL5 also enhanced matrigel-based endothelial tube-like formation of HUVEC.

Prominent expression of phosphodiesterase 5 in striated muscle of the rat urethra and levator ani.

PURPOSE: We investigated phosphodiesterase 5 distribution and activity in the urethra. MATERIALS AND METHODS: Rat tissues were examined for phosphodiesterase 5 and alpha-smooth muscle actin expression. Urethral phosphodiesterase 5 activity was examined by tissue bath in the presence of sildenafil (Pfizer, New York, New York). RESULTS: Anti-alpha-smooth muscle actin antibody (Abcam) stained all known smooth muscles in all tested tissues and revealed a few smooth muscle fibers in the levator ani muscle. Anti-phosphodiesterase 5 antibody (Abcam) stained smooth muscle in the penis and bladder but not striated leg muscle. However, it stained predominantly striated muscle in the urethra and the levator ani muscle. In the urethra the amount of phosphodiesterase 5 in striated muscle was 6 times that in smooth muscle. In urethral striated muscle phosphodiesterase 5 expression was localized to Z-band striations. Smooth and striated muscle intermingling was clearly visible on the inner and outer rims of the circularly arranged striated muscle layer. Relaxation of precontracted urethral tissues by sodium nitroprusside (Sigma-Aldrich) was enhanced by sildenafil, indicating phosphodiesterase 5 activity, which was primarily located in the striated muscle according to phosphodiesterase 5 staining. CONCLUSIONS: Despite its presumed smooth muscle specificity phosphodiesterase 5 was predominantly expressed in the striated muscle of the urethra and in the levator ani muscle. Results are consistent with earlier studies in which these striated muscles were developmentally related to smooth muscle. They also suggest that these striated muscles are possibly regulated by phosphodiesterase 5.

Adipose derived stem cells ameliorate hyperlipidemia associated detrusor overactivity in a rat model.

PURPOSE: Adipose tissue derived stem cells can differentiate into muscle and neuron-like cells in vitro. We investigate the usefulness of adipose tissue derived stem cells for overactive bladder in obese hyperlipidemic rats. MATERIALS AND METHODS: Hyperlipidemia was induced in healthy rats by a high fat diet. The resulting obese hyperlipidemic rats were treated with bladder injection of saline, adipose tissue derived stem cells or tail vein injection of adipose tissue derived stem cells. Bladder function was assessed by 24-hour voiding behavior study and conscious cystometry. Bladder histology was assessed using immunostaining and trichrome staining, followed by image analysis. RESULTS: Serum total cholesterol and low density lipoprotein were significantly higher in obese hyperlipidemic rats than in normal rats (p <0.01). The micturition interval was shorter in saline treated obese hyperlipidemic rats than in normal rats, obese hyperlipidemic rats that received adipose tissue derived stem cells via the tail vein and obese hyperlipidemic rats that received adipose tissue derived stem cells by bladder injection (mean +/- SEM 143 +/- 28.7 vs 407 +/- 77.9, 281 +/- 43.9 and 368 +/- 66.7 seconds, respectively, p = 0.0084). Bladder wall smooth muscle content was significantly lower in obese hyperlipidemic rats than in normal animals (p = 0.0061) while there was no significant difference between obese hyperlipidemic groups. Nerve content and blood vessel density were lower in controls than in obese hyperlipidemic rats treated with adipose tissue derived stem cells. CONCLUSIONS: Hyperlipidemia is associated with increased urinary frequency, and decreased bladder blood vessel and nerve density in rats. Adipose tissue derived stem cell treatment ameliorates these adverse effects and holds promise as a potential new therapy for overactive bladder.

Treatment of stress urinary incontinence with adipose tissue-derived stem cells.

BACKGROUND AIMS: Effective treatment for stress urinary incontinence (SUI) is lacking. This study investigated whether transplantation of adipose tissue-derived stem cells (ADSC) can treat SUI in a rat model. METHODS: Rats were induced to develop SUI by postpartum vaginal balloon dilation and bilateral ovariectomy. ADSC were isolated from the peri-ovary fat, examined for stem cell properties, and labeled with thymidine analog BrdU or EdU. Ten rats received urethral injection of saline as a control. Twelve rats received urethral injection of EdU-labeled ADSC and six rats received intravenous injection of BrdU-labeled ADSC through the tail vein. Four weeks later, urinary voiding function was assessed by conscious cystometry. The rats were then killed and their urethras harvested for tracking of ADSC and quantification of elastin, collagen and smooth muscle contents. RESULTS: Cystometric analysis showed that eight out 10 rats in the control group had abnormal voiding, whereas four of 12 (33.3%) and two of six (33.3%) rats in the urethra-ADSC and tail vein-ADSC groups, respectively, had abnormal voiding. Histologic analysis showed that the ADSC-treated groups had significantly higher elastin content than the control group and, within the ADSC-treated groups, rats with normal voiding pattern also had significantly higher elastin content than rats with voiding dysfunction. ADSC-treated normal-voiding rats had significantly higher smooth muscle content than control or ADSC-treated rats with voiding dysfunction. CONCLUSIONS: Transplantation of ADSC via urethral or intravenous injection is effective in the treatment and/or prevention of SUI in a pre-clinical setting.

Transdifferentiation of adipose-derived stem cells into hepatocytes: a new approach.

BACKGROUND: Several studies have demonstrated techniques in differentiating human adipose-derived stem cells (hADSCs) into hepatocytes. Unfortunately, transdifferentiation is inefficient, and the function of these induced hepatocyte-like cells (which we termed 'iHeps') is low compared with that of real hepatocytes. AIMS: We aimed to identify transcriptional deficiencies in iHeps that are critical to hepatocyte development, which may provide insights into improving the efficiency of transdifferentiation. METHODS: hADSCs were differentiated into iHeps, and iHeps were assayed for hepatocyte-like activity. iHeps were then screened for expression of several growth factors, receptors and transcription factors (TFs) critical to liver development using reverse transcription-polymerase chain reaction (RT-PCR). Deficient TFs were transduced into hADSCs and hepatocyte function was reassessed after hepatic differentiation. RESULTS: Differentiation of hADSCs into iHeps resulted in the upregulation of hepatic proteins. However, the levels of expression of hepatocyte-specific proteins in these iHeps were well below those of Huh 7.5 hepatoma cells, used in comparison. Five developmental TFs were notably absent on the RT-PCR screen. Lentiviral transduction of these TFs into hADSCs followed by culture in hepatocyte induction medium resulted in increased albumin expression compared with untransduced hADSCs treated in a parallel fashion. CONCLUSIONS: These five missing TFs are known to regulate hepatocyte differentiation and some are required to establish the competence of the foregut endoderm. Presumably due to their mesenchymal lineage, hADSCs do not express these endodermal TFs and are not fully competent to respond to critical developmental signals. Supplementation of these TFs may induce competency and enhance the differentiation of hADSCs into hepatocytes.

The effect of long-term hormonal treatment on voiding patterns during filling cystometry and on urethral histology in a postpartum, ovariectomized female rat.

OBJECTIVE: To study whether long-term treatment with oestrogen (E(2) ), selective E(2) receptor modulators (SERMs), or growth hormone (GH) can prevent the development of abnormal voiding patterns during filling cystometry (CMG) in a postpartum, ovariectomized (Ovx) female rat. MATERIALS AND METHODS: Immediately after spontaneous delivery, 60 primiparous Sprague-Dawley rats were randomly divided into six equal groups. One group served as uninjured sham controls and five groups underwent intravaginal balloon dilatation. On day seven, previously dilated rats underwent bilateral Ovx and implantation of a subcutaneous hormone-delivery pump. The five treatment groups received normal saline (control), E(2) , raloxifene, levormeloxifene, or GH for 7 weeks. Conscious CMG was performed 7 weeks after Ovx. Urethral sphincter tissue was harvested for elastin immunohistochemistry and real-time polymerase chain reaction of α(1A) -adrenoceptor mRNA. RESULTS: No abnormal voiding patterns were detected in the group treated with GH. The E(2) , raloxifene and levormeloxifene groups had greater detrusor overactivity and urethral relaxation incontinence than control rats. The raloxifene group had a significantly lower baseline bladder pressure and opening pressure. GH-treated rats had higher elastin content in the urethra. Urethral α(1A) -adrenoceptor mRNA concentration was significantly lower in the SERM-treated rats compared with controls. CONCLUSIONS: GH prevents the development of abnormal voiding patterns during filling CMG in a rat model of parturition-induced incontinence; E(2) and SERMs may worsen voiding patterns.