The role of inflammatory cytokines and ERK1/2 signaling in chronic prostatitis/chronic pelvic pain syndrome with related mental health disorders.

Mental health disorders(MHD) in chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) have been widely studied. However, the underlying role of inflammatory cytokines and their associated signaling pathways have not been investigated. Here, we report the potential role of cytokines and associated signaling pathways in CP/CPPS patients with MHD and in a CP/CPPS animal model. CP/CPPS patients (n = 810) and control subjects (n = 992) were enrolled in this case-control multicenter study, and serum cytokine levels were measured. Male Sprague-Dawley rats received multiple intracutaneous injections of an immuno-agent along with a pertussis-diphtheria-tetanus triple vaccine for autoimmune CP/CPPS development. The results revealed that, in CP/CPPS patients with significant MHD, elevated IL-1α, IL-1ß, IL-4, IL-13, and TNF-α serum levels were observed. The above five cytokines in CP/CPPS rats were significantly elevated in prostate tissue (p < 0.05), and IL-1ß levels were elevated in serum and cerebrospinal fluid. In behavioral tests, CP/CPPS rats showed anxiety- and depression-like symptoms, and impaired spatial and associative memory performance (p < 0.05). In the CP/CPPS group, ERK1/2 phosphorylation levels were increased in the amygdala and nucleus accumbens, and decreased in the hippocampus, but not caudate nucleus. Thus, prostate-derived cytokines, especially IL-1ß, cross the blood brain barrier and may lead to enhanced ERK1/2 signaling in several brain areas, possibly underlying induction of CP/CPPS-related MHD.

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.

Prospects of stem cell treatment in benign urological diseases.

Stem cells (SCs) are undifferentiated cells that are capable of self-renewal and differentiation and that therefore contribute to the renewal and repair of tissues. Their capacity for division, differentiation, and tissue regeneration is highly dependent on the surrounding environment. Several preclinical and clinical studies have utilized SCs in urological disorders. In this article, we review the current status of SC use in benign urological diseases (erectile dysfunction, Peyronie disease, infertility, and urinary incontinence), and we summarize the results of the preclinical and clinical trials that have been conducted.

Lobe-specific expression of phosphodiesterase 5 in rat prostate.

OBJECTIVE: To investigate the level and location of phosphodiesterase 5 (PDE5) expression in rat prostate. METHODS: The ventral, dorsal, and lateral lobes of rat prostate were examined for PDE5 expression by Western blotting. Intact rat urogenital complex, including the urinary bladder and accessory reproductive glands, was examined for PDE5 expression by immunohistochemistry. Individual prostatic lobes were further examined by immunofluorescence for expression of PDE5, α-smooth muscle actin, and rat endothelial cell antigen. RESULTS: Western blot analysis showed that PDE5 was expressed at a significantly lower level in dorsal lobe (DL) than in ventral lobe (VL) or lateral lobe (LL). Immunohistochemistry and immunofluorescence analyses showed that PDE5 was expressed in both acinar epithelium and periacinar smooth muscle. However, although similar levels of smooth muscle PDE5 expression were observed in all 3 prostatic lobes, significantly lower level of epithelial PDE5 expression was found in DL compared with VL or LL. In prostatic blood vessels, PDE5 expression was clearly visible in the endothelium but not as easily detectable in the smooth muscle. CONCLUSION: PDE5 was expressed in the acinar epithelium and periacinar smooth muscle of rat prostate. However, the epithelial PDE5 expression was significantly less in DL than in VL or LL. Regardless, the acinar wall, not the blood vessel wall, is the predominant PDE5 expression site in rat prostate.

Stem cell treatment of erectile dysfunction.

Erectile Dysfunction (ED) is a common disease that typically affects older men. While oral type-5 phosphodieserase inhibitors (PDE5Is) represent a successful first-line therapy, many patients do not respond to this treatment leading researchers to look for alternative treatment modalities. Stem cell (SC) therapy is a promising new frontier for the treatment of those patients and many studies demonstrated its therapeutic effects. In this article, using a Medline database search of all relevant articles, we present a summary of the scientific principles behind SCs and their use for treatment of ED. We discuss specifically the different types of SCs used in ED, the methods of delivery tested, and the methods attempted to enhance SC therapy effect. In addition, we review the current preclinical literature on SC therapy for ED and present a summary of its findings in addition to the single clinical trial published.

Presence of stem/progenitor cells in the rat penis.

Tissue resident stem cells are believed to exist in every organ, and their identification is commonly done using a combination of immunostaining for putative stem cell markers and label-retaining cell (LRC) strategy. In this study, we employed these approaches to identify potential stem cells in the penis. Newborn rats were intraperitoneally injected with thymidine analog, 5-ethynyl-2-deoxyuridine (EdU), and their penis was harvested at 7 h, 3 days, 1 week, and 4 weeks. It was processed for EdU stains and immunofluorescence staining for stem cell markers A2B5, PCNA, and c-kit. EdU-positive cells were counted for each time point and co-localized with each stem cell marker, then isolated and cultured in vitro followed by their characterization using flowcytometry and immunofluorescence. At 7 h post-EdU injection, 410 ± 105.3 penile corporal cells were labeled in each cross-section (∼28%). The number of EdU-positive cells at 3 days increased to 536 ± 115.6, while their percentage dropped to 25%. Progressively fewer EdU-positive cells were present in the sacrificed rat penis at longer time points (1 and 4 weeks). They were mainly distributed in the subtunic and perisinusoidal spaces, and defined as subtunic penile progenitor cells (STPCs) and perisinusoidal penile progenitor cells (PPCs). These cells expressed c-kit, A2B5, and PCNA. After culturing in vitro, only ∼0.324% corporal cells were EdU-labeled LRCs and expressed A2B5/PCNA. Therefore, labeling of penis cells by EdU occurred randomly, and label retaining was not associated with expression of c-kit, A2B5, or PCNA. The penile LRCs are mainly distributed within the subtunic and perisinusoidal space.

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.

Intra-articular transplantation of porcine adipose-derived stem cells for the treatment of canine osteoarthritis: A pilot study.

AIM: To test whether intra-articular injection of porcine adipose-derived stem cells (ADSCs) can treat canine osteoarthritis (OA). METHODS: To enroll in this study dogs must have stifle joint OA that had lasted ≥ 3 mo and been treated with OA medication without significant improvement. Three dogs fulfilled these criteria and were thus subjects for ADSCs treatment. ADSCs were isolated from abdominal adipose tissue of a 2-mo-old female Yorkshire pig. Their stem cell marker expression was examined by immunofluorescence staining. For treatment, 5 million ADSCs were injected into the diseased joint of each dog. In the next 48 h, the patient was observed for signs of inflammatory and allergic reactions. The patient was then discharged to the owner and, at 2, 6, and 12 wk, followed up with orthopedic assessment, owner questionnaire, X-ray imaging, and force-plate gait analysis. RESULTS: Porcine ADSCs expressed mesenchymal stem cell markers CD90 and CD105. Injection of porcine ADSCs into canine stifle joints did not cause any inflammatory or allergic reactions. Orthopedic evaluation found improvements in two dogs, particularly at the longest time point. Owners' evaluation found increased capacity and decreased pain in all three dogs' activities such as walking and running. Radiographic evaluation did not find statistically significant differences before and after treatment. Force-plate analysis found significant improvements in all three dogs after treatment. CONCLUSION: Xenotransplantation of ADSCs for the treatment of OA is feasible. Further studies are needed to validate this novel treatment modality, which can then be implemented for the routine treatment of OA in veterinary medicine.

Conversion of adipose-derived stem cells into natural killer-like cells with anti-tumor activities in nude mice.

Efforts to develop peripheral blood-derived nature killer (NK) cells into therapeutic products have been hampered by these cells' low abundance and histoincompatibility. On the other hand, derivation of NK-like cells from more abundant cell sources such as embryonic stem cells (ESCs) and umbilical cord blood (UCB) requires the selection of rare CD34+ cells. Thus, we sought to convert adipose-derived stem cells (ADSCs), which are abundant and natively CD34+, into NK-like cells. When grown in hematopoietic induction medium, ADSCs formed sphere clusters and expressed hematopoietic markers CD34, CD45, and KDR. Further induction in NK cell-specific medium resulted in a population of cells that expressed NK cell marker CD56, and thus termed ADSC-NK. Alternatively, the hematopoietically induced ADSCs were transduced with NK cell-specific transcription factor E4BP4 prior to induction in NK cell-specific medium. This latter population of cells, termed ADSC-NKE, expressed CD56 and additional NK cell markers such as CD16, CD94, CD158, CD314, FasL, and NKp46. ADSC-NKE was as potent as NK leukemia cell NKL in killing breast cancer cell MCF7 and prostate cancer cells DU145, PC3, LnCap, DuPro, C4-2 and CWR22, but exhibited no killing activity toward normal endothelial and smooth muscle cells. In nude mice test ADSC-NKE was able to significantly delay the progression of tumors formed by MCF7 and PC3. When injected into immunocompetent rats, ADSC-NKE was detectable in bone marrow and spleen for at least 5 weeks. Together, these results suggest that ADSCs can be converted into NK-like cells with anti-tumor activities.

Tunica albuginea allograft: a new model of LaPeyronie's disease with penile curvature and subtunical ossification.

The pathophysiology of LaPeyronie's disease (PD) is considered to be multifactorial, involving genetic predisposition, trauma, inflammation and altered wound healing. However, these factors have not yet been validated using animal models. In this study, we have presented a new model obtained by tunica albuginea allograft. A total of 40, 16-week-old male rats were used. Of these, 8 rats served as controls and underwent a 10 × 2-mm-wide tunical excision with subsequent autografting, whereas the remaining 32 underwent the same excision with grafting of the defect with another rat's tunica. Morphological and functional testing was performed at 1, 3, 7 and 12 weeks after grafting. Intracavernous pressure, the degree of penile curvature and elastic fiber length were evaluated for comparison between the allograft and control groups. The tissues were obtained for histological examination. The penile curvature was significantly greater in the allografted rats as compared with the control rats. The erectile function was maintained in all rats, except in those assessed at 12 weeks. The elastin fiber length was decreased in the allografted tunica as compared to control. SMAD2 expression was detected in the inner part of the allograft, and both collagen-II- and osteocalcin-positive cells were also noted. Tunica albuginea (TA) allograft in rats is an excellent model of PD. The persistence of curvature beyond 12 weeks and the presence of ossification in the inner layer of the TA were similar to those observed in men with PD. Validation studies using this animal model would aid understanding of the PD pathophysiology for effective therapeutic interventions.

Stem-cell therapy for erectile dysfunction.

INTRODUCTION: Stem cells (SCs) have been investigated for the treatment of erectile dysfunction (ED). AREAS COVERED: This review covers key disease targets and all 33 preclinical studies, including their use of SC types, animal models, transplantation routes, and outcome assessment methods. EXPERT OPINION: In the past one and half years there have been more stem-cell-for-erectile-dysfunction studies than the prior 8 years combined. These new studies tend to use combinatory treatment approaches by modifying or supplementing SCs with angiogenic or neurotrophic genes or proteins. However, when considering all risks and benefits, these combinatory approaches do not seem more advantageous than single-SC approaches. Another trend is the choice of transplantation routes other than the standard intracavernous (IC) injection. However, with the exception of intravenous injection, these new transplantation approaches are more cumbersome than IC injection and yet offer no evidence of producing better outcomes. In contrast to these variations, a consensus among these studies is the suggestion that paracrine action, as opposed to cellular differentiation, is the principal therapeutic mechanism. In conclusion, IC injection of a single SC type should be the choice protocol for initial clinical trials, and this is clearly the case with two clinical trials that are currently recruiting patients.

Commonly used mesenchymal stem cell markers and tracking labels: Limitations and challenges.

Early observations that cultured mesenchymal stem cells (MSCs) could be induced to exhibit certain characteristics of osteocytes and chondrocytes led to the proposal that they could be transplanted for tissue repair through cellular differentiation. Therefore, many subsequent preclinical studies with transplanted MSCs have strived to demonstrate that cellular differentiation was the underlying mechanism for the therapeutic effect. These studies generally followed the minimal criteria set by The International Society for Cellular Therapy in assuring MSC identity by using CD70, CD90, and CD105 as positive markers and CD34 as a negative marker. However, the three positive markers are co-expressed in a wide variety of cells, and therefore, even when used in combination, they are certainly incapable of identifying MSCs in vivo. Another frequently used MSC marker, Stro-1, has been shown to be an endothelial antigen and whether it can identify MSCs in vivo remains unknown. On the other hand, the proposed negative marker CD34 has increasingly been shown to be expressed in native MSCs, such as in the adipose tissue. It has also helped establish that MSCs are likely vascular stem cells (VSCs) that reside in the capillaries and in the adventitia of larger blood vessels. These cells do not express CD31, CD104b, or α-SMA, and therefore are designated as CD34+CD31-CD140b-SMA-. Many preclinical MSC transplantation studies have also attempted to demonstrate cellular differentiation by using labeled MSCs. However, all commonly used labels have shortcomings that often complicate data interpretation. The ß-gal (LacZ) gene as a label is problematic because many mammalian tissues have endogenous ß-gal activities. The GFP gene is similarly problematic because many mammalian tissues are endogenously fluorescent. The cell membrane label DiI can be adsorbed by host cells, and nuclear stains Hoechst dyes and DAPI can be transferred to host cells. Thymidine analog BrdU is associated with loss of cellular protein antigenicity due to harsh histological conditions. Newer thymidine analog EdU is easier to detect by chemical reaction to azide-conjugated Alexa fluors, but certain bone marrow cells are reactive to these fluors in the absence of EdU. These caveats need to be taken into consideration when designing or interpreting MSC transplantation experiments.

Defining vascular stem cells.

Mesenchymal stem cells (MSCs) exist in most adult tissues and have been located near or within blood vessels. Although "perivascular" has been commonly used to describe such locations, increasing evidence points at the vessel wall as the exact location. Thus, "vascular stem cells (VSCs)" is recommended as a more accurate term for MSCs. Furthermore, 2 cell populations, namely pericytes and adventitial progenitor cells (APCs), are the likely VSCs. The pericyte evidence relies on the so-called pericyte-specific markers, but none of these markers is pericyte specific. In addition, pericytes appear to be too functionally diverse and sophisticated to have a large differentiation capacity. On the other hand, APCs are more naïve functionally and, therefore, more akin to being VSCs. In vitro, these cells spontaneously differentiate into pericytes, and can be induced to differentiate into vascular cells (endothelial and smooth muscle cells) and mesenchymal cells (e.g., bone, cartilage, and fat). In vivo, indirect evidence also points to their ability to differentiate into mesenchymal cells of their native tissue (e.g., fat). Moreover, they possess a large paracrine capacity and, therefore, can help maintain tissue homeostasis by encouraging the replication and differentiation of mesenchymal cells locally. These proposed in vivo functions are areas of interest for future research on VSCs.

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.

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.

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.

Effects of high glucose on human cavernous endothelial cells.

OBJECTIVE: To obtain experimental evidence for a causal effect of high glucose (HG) on cavernous endothelial dysfunction. METHODS: Cavernous tissues were obtained from patients undergoing surgery for penile prosthesis implantation. Endothelial cells were isolated by binding to anti-CD31 antibody, followed by magnetic capture. Their endothelial identity was verified by flow cytometry and immunofluorescence staining for endothelial markers CD31, von Willebrand factor, and endothelial nitric oxide synthase, and by their ability to form tube-like structures in matrigel (tube formation) and to endocytose acetylated low-density lipoprotein (low-density lipoprotein uptake). The cells were then cultured under normal glucose (NG) (5 mM) or HG (25 mM) conditions, followed by analysis for endothelial gene expression, function, proliferation, apoptosis, and mitochondrial fragmentation. RESULTS: Human cavernous endothelial cell (HCEC) strains were established and determined to be nearly 100% pure endothelial cells. In the HG culture condition, HCECs expressed approximately 50% less CD31, von Willebrand factor, and endothelial nitric oxide synthase, but nearly twice as much collagen IV compared with HCECs grown in NG medium. HG also suppressed low-density lipoprotein uptake and tube formation by approximately 50%. HCECs grew significantly slower in the high-glucose medium than in the NG medium. Approximately 3 times as many cells exhibited apoptosis in the HG medium as in the NG medium. Approximately 4 times as many cells contained fragmented mitochondria in the HG medium as in the NG medium. CONCLUSION: HG caused a decrease in endothelial proliferation, function, and marker expression. It also caused an increase in endothelial collagen IV expression, apoptosis, and mitochondrial fragmentation. Together, these HG-induced changes in cavernous endothelial cells provide an explanation for hyperglycemia's detrimental effects on erectile function.