Bladder Smooth Muscle Cells Research Articles

Activation of NFKB-JMJD3 signaling promotes bladder fibrosis via boosting bladder smooth muscle cell proliferation and collagen accumulation

Chronic cystitis is characterized by the hyperplasia and fibrosis of the bladder wall as well as attenuated compliance of the bladder. To further unravel its underlying molecular mechanism, the role of NFκB-JMJD3 signaling pathway in cystitis induced bladder fibrosis was investigated. Jmjd3 and Col1/3 expression was detected in a cystitis mouse model that was developed by intraperitoneal injection of cyclophosphamide (CYP). Human bladder smooth muscle cells (hBSMCs) were stimulated in vitro with lipopolysaccharide (LPS), and the cell proliferation and collagen accumulation were detected using EdU, CCK8, flow cytometry, qPCR, western blotting and immunofluorescence assays. Furthermore, the effects of NFκB and JMJD3 on cell proliferation and collagen accumulation were investigated using its selective antagonists, JSH23 and GSK-J4, respectively. CYP induced cystitis significantly increased Jmjd3, Col1 and Col3 expression in the bladder muscle cells. Furthermore, LPS stimulation markedly activated NFκB signaling and elevated JMJD3 expression in hBSMCs, and the activation of NFκB-JMJD3 signaling significantly promoted cell proliferation and collagen accumulation by upregulating CCND1 and COL1/3 expression, respectively. Our study reveals the critical role of NFκB-JMJD3 signaling in cystitis induced bladder reconstruction by regulating hBSMC proliferation and extracellular matrix (ECM) deposition, and these findings provide an avenue for effective treatment of patients with cystitis.

Improved bladder smooth muscle cell differentiation of the mesenchymal stem cells when grown on electrospun polyacrylonitrile/polyethylene oxide nanofibrous scaffold.

Reconstruction of the bladder wall plays an important role in improving its function in patients with urinary bladder dysfunction. Tissue engineering has been trying to introduce biocompatible nanofibers as scaffolds for bladder wall matrix substitutes. In this study a composite nanofibrous scaffold was fabricated from polyacrylonitrile (PAN) and polyethylene oxide (PEO) blend by electrospinning method and then its morphological and mechanical characteristics was evaluated by scanning electron microscopy (SEM), tensile, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Then smooth muscle cell (SMC) differentiation supportive capacity of PAN-PEO nanofibers was investigated by culturing of human adipose tissue-derived mesenchymal stem cells (AT-MSCs) on this scaffold and then its differentiation potential in different groups was investigated using SMC-related gene and protein markers. SEM and MTT results demonstrated that PAN-PEO supported AT-MSCs attachment, growth and proliferation, especially at early times after cell seeding. The obtained results from real-time reverse transcription polymerase chain reaction revealed that collagen-I-α1, collagen-III-α1, α-smooth muscle actin (α-SMA), calponin1, SM22α, caldesmon1, elastin, and myosin heavy chain (MHC) genes were expressed in AT-MSCs cultured on PAN-PEO significantly higher than those stem cells that cultured on the culture plate as a control. In addition α-SMA and MHC proteins were also expressed in AT-MSCs cultured on PAN-PEO significantly higher than control. According to the results PAN-PEO nanofibrous scaffold showed a positive AT-MSCs-seeded PAN-PEO has a great promising potential to use in bladder tissue engineering applications.

Responses of bladder smooth muscle to the stretch go through extracellular signal-regulated kinase (ERK)/p90 ribosomal S6 protein kinase (p90RSK)/Nuclear factor-κB (NF-κB) Pathway.

The present study was designed to study changes and its potential mechanisms in human bladder smooth muscle subjected to stretch. Bioinformatics analyses including differential expression analysis, overrepresentation enrichment analysis, principal component analysis, and weighted gene coexpression network analysis were used to analyze a microarray dataset (GSE47080) of partial bladder outlet obstruction (pBOO) in rat to find the potential changes of gene expressions. Bladder from pBOO model and human bladder smooth muscle cells (HBSMCs) subjected to sustained prolonged stretch were collected for Western blot analysis, real-time polymerase chain reaction, and fluorescence analysis to verify the changes of gene expressions and preliminarily study the potential role of signaling pathway regulation in treatment of pBOO. The bioinformatics analysis showed that chronic obstruction activated mitogen-activated protein kinase pathway and changed cytoskeleton structure in bladder smooth muscle. In in vivo experiments in mice, pBOO was verified by cystometry. Partial BOO activated the extracellular signal-regulated kinase (ERK)/p90 ribosomal S6 protein kinase (p90RSK)/nuclear factor-κB (NF-κB) signaling pathway in DM. The messenger RNA (mRNA) expressions of contractile phenotypic proteins increased after pBOO. In in vitro experiments of HBSMCs, mechanical stretch activated ERK/p90RSK/NF-κB in HBSMCs in a time-dependent manner. The mRNA expressions of α-smooth muscle actin and SM22 also increased and filamentous actin (F-actin) polymerization was enhanced as well. Inhibition of ERK/p90RSK/NF-κB pathway reversed mechanical stretch-induced changes of contractile phenotypic expression and F-action polymerization. Continuous stretch increases expressions of contractile phenotypic proteins and promotes the polymerization of F-actin. This process partially goes through ERK/p90RSK/NF-κB pathway.

Efficacy and Safety of Basic Pharmacotherapy of Chronic Abacterial Prostatitis

A basic complex has been proposed for the treatment of chronic abacterial prostatitis (CAP), including a nonsteroidal anti-inflammatory drug to limit the effects of prostaglandins; selective alpha adrenergic blocker for modulation of the bladder overactivity and smooth muscle cells of the prostate; Serenoa repens palm fruit extract for prolonged and safe antiinflammatory and anti-sclerotic effects.The objective: evaluation of the efficacy and safety of basic pharmacotherapy of chronic abacterial prostatitis.Materials and methods. The study involved 32 patients with CAP. For the assessment of the patient’s condition, a questionnaires (NIH-CPSI, PHQ-9, GAD-7) and uroflowmetry were used. The ELISA revealed changes in the concentration of testosterone, dihydrotestosterone (DHT), estradiol and prostate-specific antigen (PSA) in the blood, as well as cytokine ejaculate. As a basic treatment, we used a combination of diclofenac sodium (Dicloberl retard, 100 mg/day, orally, 2 weeks), a tamsulozin with modified release (Flosin, 0.4 mg/day, orally, 1 month), an extract of palm fruit Serenoa repens (Prostamol Uno, 320 mg/day, orally, 6 months), regardless of the presence and severity of pain and dysuric manifestations in patients. The reduction of NIH-CPSI by 6 points, PHQ-9 by 6 points, GAD-7 by 5 points were considered as clinically significant.Results. Significant favorable changes in the symptoms of prostatitis, depression and anxiety were observed after 2 weeks and persisted throughout the observation period (6 months). There was a statistically significant increase in testosterone levels, a decrease in blood DHT and PSA, as well as a significant decrease in proinflammatory cytokines in the ejaculate (IL-1β, TNF-α, IL-8), along with an increase in the antiinflammatory cytokine (IL-10). The clinical efficacy of treatment for reducing prostatitis symptoms was 69%, depression symptoms – 75%, anxiety – 84%. Side effects of therapy: ejaculation disorders were observed in 78%, epigastric pain – 10%, orthostatic hypotension – 6%, which did not prevent all patients from completing the treatment.Conclusion. The basic pharmacotherapy was quite effective in reducing the symptoms of prostatitis, associated psychological disorders, was safe and can be recommended for patients with СAP.

535 - Large-conductance voltage- and calcium-activated potassium channels regulate contraction function of human bladder smooth muscle cells under hydrostatic pressure

535 - Large-conductance voltage- and calcium-activated potassium channels regulate contraction function of human bladder smooth muscle cells under hydrostatic pressure

Bladder smooth muscle cell differentiation of the human induced pluripotent stem cells on electrospun Poly(lactide-co-glycolide) nanofibrous structure

Smooth muscle cell (SMC) regeneration plays an important role in retrieving the bladder-wall functionality and it can be achieved by a proper cell-co-polymer constructed by tissue engineering. Human induced pluripotent stem cells (iPSCs), which can be specifically prepared for the patient, was considered as cells in this study, and Poly(lactide-co-glycolide) (PLGA) as a most interesting polymer in biomedical applications was applied to the scaffold fabrication by electrospinning. After scaffold characterization, SMC differentiation potential of the human iPSCs was investigated while cultured on the PLGA nanofibrous scaffold by evaluation of the SMC related important gene and protein markers. Alpha-smooth muscle actin (ASMA), Smooth muscle 22 alpha (SM-22a) as two early SMC markers were significantly up regulated either two and three weeks after differentiation induction in human iPSCs cultured on PLGA compared to those cells cultured on the tissue culture polystyrene (TCPS). But Calponin-1, Caldesmon1 and myosin heavy chain (MHC) expression differences in human iPSCs cultured on PLGA and TCPS were significant only three weeks after differentiation induction based on its lately expression in the differentiation process. ASMA and MHC proteins were also considered for evaluation by immunocytochemistry on differentiated iPSCs whereas results showed higher expression of these proteins in stem cells grown on PLGA compared to the TCPS. According to the results, human iPSCs demonstrated a great SMC differentiation potential when grown on PLGA and it could be considered as a promising cell-co-polymer for use in bladder tissue engineering.

Wnt-GSK3β/β-Catenin Regulates the Differentiation of Dental Pulp Stem Cells into Bladder Smooth Muscle Cells.

Smooth muscle cell- (SMC-) based tissue engineering provides a promising therapeutic strategy for SMC-related disorders. It has been demonstrated that human dental pulp stem cells (DPSCs) possess the potential to differentiate into mature bladder SMCs by induction with condition medium (CM) from bladder SMC culture, in combination with the transforming growth factor-β1 (TGF-β1). However, the molecular mechanism of SMC differentiation from DPSCs has not been fully uncovered. The canonical Wnt signaling (also known as Wnt/β-catenin) pathway plays an essential role in stem cell fate decision. The aim of this study is to explore the regulation via GSK3β and associated downstream effectors for SMC differentiation from DPSCs. We characterized one of our DPSC clones with the best proliferation and differentiation abilities. This stem cell clone has shown the capacity to generate a smooth muscle layer-like phenotype after an extended differentiation duration using the SMC induction protocol we established before. We further found that Wnt-GSK3β/β-catenin signaling is involved in the process of SMC differentiation from DPSCs, as well as a serial of growth factors, including TGF-β1, basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), hepatocyte growth factor (HGF), platelet-derived growth factor-homodimer polypeptide of B chain (BB) (PDGF-BB), and vascular endothelial growth factor (VEGF). Pharmacological inhibition on the canonical Wnt-GSK3β/β-catenin pathway significantly downregulated GSK3β phosphorylation and β-catenin activation, which in consequence reduced the augmented expression of the growth factors (including TGF-β1, HGF, PDGF-BB, and VEGF) as well as SMC markers (especially myosin) at a late stage of SMC differentiation. These results suggest that the canonical Wnt-GSK3β/β-catenin pathway contributes to DPSC differentiation into mature SMCs through the coordination of different growth factors.

Issue Highlights

IUBMB LifeVolume 71, Issue 2 p. 151-151 Issue HighlightsFree Access Issue Highlights First published: 09 January 2019 https://doi.org/10.1002/iub.2007AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinked InRedditWechat 152 Lung Inflammation and Disease: A Perspective on Microbial Homeostasis and Metabolism Roberto Mendez, Sulagna Banerjee, Sanjoy K. Bhattacharya, Santanu Banerjee The lung microbiome is significantly smaller than the gut microbiome, both in terms of diversity and biomass. The lower respiratory tract microbiome is constantly replenished by oral and upper respiratory tract microbiota and yet maintains a unique microbial signature. In this review, we provide a perspective on the role of these bacteria in health and disease. We touch upon the known challenges associated with lung microbiome studies and discuss various environmental factors and diseases where commensals are known to play a major role. Finally, we discuss the influence of bacterial metabolites in homeostasis and disease-induced microbial perturbations. 235 MicroRNA-101 Protects Bladder of BOO from Hypoxia- Induced Fibrosis by Attenuating TGF-β- smad2/3 Signaling Baimei He, Qiong Chen, Dongbo Zhou, Lijing Wang, Zhaoqian Liu Patients with benign prostatic hyperplasia often suffer from frequent micturition, the urgency of urination, the urine pain. Even the obstruction was removed, the symptom often remains. Studies have found it may be associated with the fibrosis of bladder after obstruction. Our study found hypoxia promoted the expression of ECM in bladder smooth muscle cells, and the expression of microRNA-101 was up-regulated in the bladder after obstruction. MicroRNA-101 restrained the smad2/3 signaling pathway through depressing the expression of TGFβR1. MicroRNA-101 is emerging as an anti-fibrotic miRNA in the remodeling of bladder after bladder outlet obstruction. 283 Synergistic Antifungal Activity of Isoquercitrin: Apoptosis and Membrane Permeabilization Related to Reactive Oxygen Species in Candida albicans Suhyun Kim, Eun-Rhan Woo, Dong Gun Lee Isoquercitrin (ISO) at non-antifungal concentration has synergistic effect with amphotericin B (AMB) and fluconazole (FLC). These combinations inhibited the activity of superoxide dismutase and lead to disruption of antioxidant system. The collapse of the antioxidant system highly increased reactive oxygen species (ROS) level. Their abnormal concentrations of ROS caused apoptosis in cells treated with ISO and FLC. In contrast, ROS had different effect on the cells treated with ISO and AMB by inducing membrane permeabilization. REFERENCES 1Mendez, R., Banerjee, S., Bhattacharya, S.K., and Banerjee, S. Lung Inflammation and Disease: A Perspective on Microbial Homeostasis and Metabolism. IUBMB Life. 2019, 71, 152– 165. Wiley Online LibraryCASPubMedWeb of Science®Google Scholar 2Wang, N., Duan, L., Ding, J., Cao, Q., Qian, S., Shen, H., and Qi, J. MicroRNA-101 Protects Bladder of BOO from Hypoxia-Induced Fibrosis by Attenuating TGF-β- smad2/3 Signaling. IUBMB Life. 2019, 71, 235– 243. Wiley Online LibraryGoogle Scholar 3 Kim, S., Woo, E.-R., and Lee, D.G. Synergistic Antifungal Activity of Isoquercitrin: Apoptosis and Membrane Permeabilization Related to Reactive Oxygen Species in Candida albicans. IUBMB Life. 2019, 71, 283– 292. Wiley Online LibraryCASPubMedWeb of Science®Google Scholar Volume71, Issue2February 2019Pages 151-151 ReferencesRelatedInformation

The KV 7 channel activator retigabine suppresses mouse urinary bladder afferent nerve activity without affecting detrusor smooth muscle K+ channel currents.

KV 7 channels are a family of voltage-dependent K+ channels expressed in many cell types, which open in response to membrane depolarization to regulate cell excitability. Drugs that target KV 7 channels are used clinically to treat epilepsy. Interestingly, these drugs also cause urinary retention, but it was unclear how. In this study, we focused on two possible mechanisms by which retigabine could cause urinary retention: by decreasing smooth muscle excitability, or by decreasing sensory nerve outflow. Urinary bladder smooth muscle had no measurable KV 7 channel currents. However, the KV 7 channel agonist retigabine nearly abolished sensory nerve outflow from the urinary bladder during bladder filling. We conclude that KV 7 channel activation likely affects urinary bladder function by blocking afferent nerve outflow to the brain, which is key to sensing bladder fullness. KV 7 channels are voltage-dependent K+ channels that open in response to membrane depolarization to regulate cell excitability. KV 7 activators, such as retigabine, were used to treat epilepsy but caused urinary retention. Using electrophysiological recordings from freshly isolated mouse urinary bladder smooth muscle (UBSM) cells, isometric contractility of bladder strips, and ex vivo measurements of bladder afferent activity, we explored the role of KV 7 channels as regulators of murine urinary bladder function. The KV 7 activator retigabine (10μM) had no effect on voltage-dependent K+ currents or resting membrane potential of UBSM cells, suggesting that these cells lacked retigabine-sensitive KV 7 channels. The KV 7 inhibitor XE-991 (10μM) inhibited UBSM K+ currents; the properties of these currents, however, were typical of KV 2 channels and not KV 7 channels. Retigabine inhibited voltage-dependent Ca2+ channel (VDCC) currents and reduced steady-state contractions to 60mM KCl in bladder strips, suggesting that reduction in VDCC current was sufficient to directly affect UBSM function. To determine if retigabine altered ex vivo bladder sensory outflow, we measured afferent activity during simulated transient contractions (TCs) of the bladder wall. Simulated TCs caused bursts of afferent activity that were nearly abolished by retigabine. The effects of retigabine were blocked by co-incubation with XE-991, suggesting specific activation of KV 7 channels on afferent nerves. These results indicate that retigabine primarily affects urinary bladder function by inhibiting TC generation and afferent nerve activity, which are key to sensing bladder fullness. Any direct inhibition of UBSM contractility is likely to be from non-specific effects on VDCCs and KV 2 channels.

Injecting RNA interference lentiviruses targeting the muscarinic 3 receptor gene into the bladder wall inhibits neurogenic detrusor overactivity in rats with spinal cord injury.

To investigate the effects of injecting RNA interference (RNAi) lentiviruses targeting the muscarinic 3 (M3 ) receptor gene into the bladder wall on bladder activity in rats with spinal cord injury (SCI). Four M3 RNAi lentiviruses were constructed and used to infect primary cultured bladder smooth muscle cells (BSMCs). Western blotting and quantitative reverse transcription polymerase chain reaction (qRT-PCR) were performed to determine the optimal RNAi lentivirus with the highest interference efficiency. Female Wistar rats were subjected spinal cord transection at T9-10 and randomly divided into three groups (n = 8), namely, blank control, negative control, and experimental groups, and injected into the bladder wall with saline, negative control shRNA, and M3 RNAi lentiviruses, respectively, 1 week after spinal cord transection. The normal rats were used as normal control group. Urodynamic parameters and bladder tissues were evaluated in the different groups. An M3 RNAi lentivirus with the highest interference efficiency (78.9%) was constructed and identified. Three weeks after injecting M3 RNAi lentiviruses into the bladder wall, Western blotting and qRT-PCR showed that the M3 receptor was significantly downregulated in the experimental group. Cystometric evaluation suggested that downregulating M3 receptor expression could substantially decrease basal pressure, residual volume, and non-voiding contraction number, increase intercontraction interval, and significantly improve bladder compliance in rats with SCI. Injecting RNAi lentiviruses targeting the M3 receptor gene into the bladder wall could effectively inhibit neurogenic detrusor overactivity (NDO) due to SCI. Thus, this approach may be a potential treatment for NDO in SCI.

Pharmacological characterization of DA-8010, a novel muscarinic receptor antagonist selective for urinary bladder over salivary gland

Several antimuscarinics have been commonly used for overactive bladder patients, but dry mouth as a major anticholinergic side effect remains a shortcoming to limit long-term use. The aim of this study was to elucidate the pharmacological properties of DA-8010, a novel muscarinic receptor antagonist selective for urinary bladder over salivary gland. DA-8010 exhibited a high binding affinity for human muscarinic M3 receptor with pKi of 8.81 ± 0.05 and great potencies for human M3 receptor and rat bladder preparation. The potency of DA-8010 for bladder smooth muscle cells was 3.6-fold higher than that for salivary gland cells isolated from mice. Intravenous administration of DA-8010 dose-dependently inhibited rhythmic urinary bladder contractions induced by distension in rats, indicating the most potent activity (ID30 = 0.08 mg/kg) among the antimuscarinics tested. Taken together with the inhibitory effects of DA-8010 and other antimuscarinics on carbachol-induced salivary secretion in rats, the in vivo functional selectivity of DA-8010 for urinary bladder over salivary gland was 3.1-fold, 3.2-fold and 5.2-fold greater than those observed for solifenacin, oxybutynin and darifenacin, respectively. Furthermore, oral administration of DA-8010 in mice resulted in more selective and persistent binding for muscarinic receptors in the bladder rather than in the submaxillary gland, in comparison with other antimuscarinics. These findings suggest that DA-8010 is a potent muscarinic M3 receptor antagonist to be highly selective for bladder over salivary gland, which might be a promising agent with greater efficacy and less dry mouth in the treatment of overactive bladder.

Nexilin/NEXN controls actin polymerization in smooth muscle and is regulated by myocardin family coactivators and YAP

Nexilin, encoded by the NEXN gene, is expressed in striated muscle and localizes to Z-discs, influencing mechanical stability. We examined Nexilin/NEXN in smooth muscle cells (SMCs), and addressed if Nexilin localizes to dense bodies and dense bands and whether it is regulated by actin-controlled coactivators from the MRTF (MYOCD, MKL1, MKL2) and YAP/TAZ (YAP1 and WWTR1) families. NEXN expression in SMCs was comparable to that in striated muscles. Immunofluorescence and immunoelectron microscopy suggested that Nexilin localizes to dense bodies and dense bands. Correlations at the mRNA level suggested that NEXN expression might be controlled by actin polymerization. Depolymerization of actin using Latrunculin B repressed the NEXN mRNA and protein in bladder and coronary artery SMCs. Overexpression and knockdown supported involvement of both YAP/TAZ and MRTFs in the transcriptional control of NEXN. YAP/TAZ and MRTFs appeared equally important in bladder SMCs, whereas MRTFs dominated in vascular SMCs. Expression of NEXN was moreover reduced in situations of SMC phenotypic modulation in vivo. The proximal promoter of NEXN conferred control by MRTF-A/MKL1 and MYOCD. NEXN silencing reduced actin polymerization and cell migration, as well as SMC marker expression. NEXN targeting by actin-controlled coactivators thus amplifies SMC differentiation through the actin cytoskeleton, probably via dense bodies and dense bands.

Digitally Tunable Microfluidic Bioprinting of Multilayered Cannular Tissues.

Despite advances in the bioprinting technology, biofabrication of circumferentially multilayered tubular tissues or organs with cellular heterogeneity, such as blood vessels, trachea, intestine, colon, ureter, and urethra, remains a challenge. Herein, a promising multichannel coaxial extrusion system (MCCES) for microfluidic bioprinting of circumferentially multilayered tubular tissues in a single step, using customized bioinks constituting gelatin methacryloyl, alginate, and eight-arm poly(ethylene glycol) acrylate with a tripentaerythritol core, is presented. These perfusable cannular constructs can be continuously tuned up from monolayer to triple layers at regular intervals across the length of a bioprinted tube. Using customized bioink and MCCES, bioprinting of several tubular tissue constructs using relevant cell types with adequate biofunctionality including cell viability, proliferation, and differentiation is demonstrated. Specifically, cannular urothelial tissue constructs are bioprinted, using human urothelial cells and human bladder smooth muscle cells, as well as vascular tissue constructs, using human umbilical vein endothelial cells and human smooth muscle cells. These bioprinted cannular tissues can be actively perfused with fluids and nutrients to promote growth and proliferation of the embedded cell types. The fabrication of such tunable and perfusable circumferentially multilayered tissues represents a fundamental step toward creating human cannular tissues.

DNA Methylation Reduces the Yes-Associated Protein 1/WW Domain Containing Transcription Regulator 1 Pathway and Prevents Pathologic Remodeling during Bladder Obstruction by Limiting Expression of BDNF

Chronic bladder obstruction and bladder smooth muscle cell (SMC) stretch provide fibrotic and mechanical environments that can lead to epigenetic change. Therefore, we examined the role of DNA methylation in bladder pathology and transcriptional control. Sprague-Dawley female rats underwent partial bladder obstruction by ligation of a silk suture around the proximal urethra next to a 0.9-mm steel rod. Sham operation comprised passing the suture around the urethra. After 2 weeks, rats were randomized to normal saline or DNA methyltransferase inhibitor, 5-aza-2-deoxycytidine (DAC) at 1 mg/kg, three times/week intraperitoneally. After 6 weeks, bladders were weighed and divided for histology and RNA analysis by high-throughput real-time quantitative PCR arrays. DAC treatment during obstruction invivo profoundly augmented brain-derived neurotrophic factor (BDNF) expression compared with the obstruction with vehicle group, which was statistically correlated with pathophysiologic parameters. BDNF, cysteine rich angiogenic inducer 61 (CYR61), and connective tissue growth factor (CTGF) expression clustered tightly together using Pearson's correlation analysis. Their promoters were associated with the TEA domain family member 1 (TEAD1) and Yes-associated protein 1/WW domain containing transcription regulator 1 pathways. Interestingly, DAC treatment increased BDNF expression in bladder SMCs (P<0.0002). Stretch-induced BDNF was inhibited by the YAP/WWTR1 inhibitor verteporfin. Verteporfin improved the SMC phenotype (proliferative markers and SMC marker expression), in part by reducing BDNF. Expression of BDNF is limited by DNA methylation and associated with pathophysiologic changes during partial bladder outlet obstruction and SMC phenotypic change invitro.

Determining integrity of bladder innervation and smooth muscle function 1 year after lower spinal root transection in canines.

To assess bladder smooth muscle function and innervation after long-term lower spinal root transection in canines. Thirteen female mixed-breed hound dogs underwent bladder decentralization, which included transection of all sacral dorsal and ventral roots caudal to L7 and hypogastric nerves, bilaterally (n = 3); all sacral roots and hypogastric nerves plus transection of L7 dorsal roots, bilaterally (n = 4); or a sham operation (n = 6). At a year after initial surgery, bladder function was assessed in vivo by stimulation of the pelvic plexus. The bladder tissue was harvested for ex vivo smooth muscle contractility studies. Remaining bladder was evaluated for nerve morphology immunohistochemically using neuronal marker PGP9.5, apoptotic activity using terminal deoxynucleotidyl transferase dUTP nick end labeling, and histopathology using a hematoxylin and eosin stain. Sacral root decentralization did not reduce maximum strength of pelvic plexus stimulation-induced bladder contraction, although long-term sacral dorsal and ventral root plus L7 dorsal root transection significantly decreased contraction strength. Electric field stimulation-induced contractions of the detrusor from all decentralized animals were preserved, compared to controls. Viable nerves and intramural ganglia were visualized in the bladder wall, regardless of group. There was no difference in amount of apoptosis in bladder smooth muscle between groups. Bladder smooth muscle cells maintain their function after long-term bladder decentralization. While pelvic plexus-induced bladder contractions were less robust at 1 year after lower spinal root transection, the absence of atrophy and preservation of at least some nerve activity may allow for successful surgical reinnervation after long-term injury.

Abstract 5035: Identification of novel therapeutics of bladder cancer cells through upregulation of SPARC expression in using 2D cell cultures and 3D bladder organoids

Abstract We have recently reported the tumor suppressor effect of SPARC in urinary bladder cancer. We have shown that the loss of SPARC in bladder cancer resulted accelerated bladder carcinogenesis and metastasis multiple pathways involving cell cycle deregulation, inflammation and angiogenesis. We have also reported that SPARC protein expression significantly decreased in the cancerous compartment in advanced human bladder cancer as well as in carcinogen-induced murine urothelial cancer. However, the mechanism of the downregulation of SPARC expression in cancer in general and in bladder cancer in particular is still unraveled. The purpose of our study is to identify therapeutic candidates as regulators of SPARC expression in bladder cancer. We developed an in vitro 3-D bladder model system “bladder organoids” that comprises the normal and cancerous urothelial cell, bladder smooth muscle cells, fibroblasts and endothelial cells. We differentially expressed fluorescent SPARC promoter reports in the cancerous and non-cancerous cells to screen for the differential expression of SPARC in each cell type using FDA-approved and natural product drug libraries and to determine the cytotoxicity on cancerous vs non-cancerous cells. Real time monitoring of changes in SPARC expression was done by measuring fluorescence intensity using IncuCyte® ZOOM and InCell Analyzer 1000 Live Imaging Analysis Systems. We identified drugs that consistently exhibited dose-dependent augmentation or down-regulation of SPARC expression in cancer cells. Our screens using the identified novel up-regulators of SPARC expression that differentially reduce the viability of cancer cells and spare non-cancer cells in the organoids. Our efforts produced a platform that mimics bladder cancer microenvironment that can be adapted for throughput screening for targeted personalized therapies. Citation Format: Chirayu M. Patel, Cristina Ivan, Anthony Atala, Neveen Said. Identification of novel therapeutics of bladder cancer cells through upregulation of SPARC expression in using 2D cell cultures and 3D bladder organoids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5035.

Tumor Necrosis Factor-α Initiates miRNA-mRNA Signaling Cascades in Obstruction-Induced Bladder Dysfunction

Bladder outlet obstruction (BOO) and the ensuing clinical lower urinary tract dysfunction are common in elderly patients. BOO is accompanied by urodynamic changes in bladder function and leads to organ fibrosis and ultimately loss of contractility. Comprehensive transcriptome analysis of bladder samples from human patients with different urodynamically defined phenotypes of BOO revealed tumor necrosis factor (TNF)-α as the top upstream signaling pathway regulator. Herein, we validated next-generation sequencing and pathway analysis in cell-based models using bladder smooth muscle and urothelial cells exposed to TNF-α. miRNA profiling and transcriptome analysis of TNF-α-treated bladder smooth muscle cells revealed striking similarities with human BOO. Using a comparative approach, TNF-specific and TNF-independent pathways were delineated in human biopsy specimens. Concomitant down-regulation of smooth muscle cell-specific miRNAs and smooth muscle markers after TNF-α treatment was in accordance with the loss of contractility in humans in advanced obstruction-induced bladder remodeling. The expression levels of four abundant TNF-regulated miRNAs were modulated; the compensatory up-regulation of miR-199a-5p reduced NF-κB signaling. Essential hubs of TNF-α signaling pathways mitogen-activated protein kinase kinase kinase (apoptosis signal-regulating kinase 1) and inhibitor of nuclear factor κ B kinase subunit β (IκB kinase β) were targeted by miR-199a-5p. miR-199a-5p might be part of a negative feedback loop, reducing the impact of TNF, whereas its down-regulation in acontractile bladders from BOO patients advances the disease. The compensatory up-regulation of miR-199a-5p together with TNF-α inhibition may be therapeutically beneficial.

Increased autophagy contributes to impaired smooth muscle function in neurogenic lower urinary tract dysfunction.

To explore whether autophagy plays a role in the remodeling of bladder smooth muscle cells (SMCs) in children with neurogenic lower urinary tract dysfunction (NLUTD), we investigated the effect of autophagy in NLUTD in the paediatric population. Bladder biopsies were taken from children with NLUTD and healthy donors as controls. Samples were labeled with the SMC markers calponin, smoothelin, and the autophagy proteins LC3, ATG5, and Beclin1. The contractile ability of bladder derived SMCs was investigated. ATG5 gene and protein was upregulated in NLUTD muscle tissue compared to normal bladder. NLUTD muscle exhibited a punctated immunostaining pattern for LC3 in a subset of the SMCs, confirming the accumulation of autophagosomes. Pronounced elevation of ATG5 in the SMC in NLUTD tissue was associated with a downregulation of the key contractile proteins smoothelin and calponin. Pharmacological blocking of autophagy completely stopped the cells growth in normal bladder SMCs. Inhibition of autophagy in the NLUTD SMCs, with already elevated levels of ATG5, resulted in a reduction of ATG5 protein expression to the basal level found in normal controls. Our study suggests that autophagy is an important factor affecting the remodeling of SMCs and the alteration of functionality in bladder smooth muscle tissue in the NLUTD. Since autophagy can be influenced by oral medication, this finding might lead to novel strategies preventing the deterioration of NLUTD muscle.

MP09-14 ESTABLISHMENT OF IMMORTALIZED HUMAN BLADDER SMOOTH MUSCLE CELLS

You have accessJournal of UrologyBladder & Urethra: Anatomy, Physiology & Pharmacology I1 Apr 2018MP09-14 ESTABLISHMENT OF IMMORTALIZED HUMAN BLADDER SMOOTH MUSCLE CELLS Masaki Yoshida, Naohiro Hashimoto, Hisae Nishii, and Masanori Nomiya Masaki YoshidaMasaki Yoshida More articles by this author , Naohiro HashimotoNaohiro Hashimoto More articles by this author , Hisae NishiiHisae Nishii More articles by this author , and Masanori NomiyaMasanori Nomiya More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2018.02.340AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Smooth muscle cell (SMC) cultures are an excellent tool for the study of cellular events and molecular mechanisms by which the phenotypic plasticity is controlled. Although many SMC systems have been previously analyzed, human bladder cell biology has not been fully evaluated. Therefore we attempted to establish the immortalized human bladder smooth muscle cells. METHODS In immortalization of human smooth muscle cells, primary cultured human bladder smooth muscle cells derived from a 68 year old man were transduced with recombinant lentiviruses and retroviruses encoding human cyclin D1, human mutant CDK4, and human telomerase. Immunoblotting analysis were performed using primary antibodies included mouse monoclonal antibodies for calponin, α-smooth muscle actin (α-SMA), ?-smooth muscle actin (γ-SMA) and rabbit monoclonal antibodies for muscarinic acetylcholine receptor M2 and M3 and rabbit polyclonal antibodies for smooth muscle myosin heavy chain 11 (MYH11). In addition, immunofluorescence analyses for connexin 43 were also performed. Live imaging of intracellular calcium evaluated with Calcium Kit-Fluo 4 after stimulation with high potassium medium and carbachol. Smooth muscle contraction were also evaluated live imaging of F-actin using SiR-A. RESULTS We have established an immortalized human bladder SMC line designated as hBS11. hBS11 cells exhibited continuous cell proliferation for more than 50 populations under optimized culture conditions. The cell cycle interval was 18.0±3.2h. A chromosome analysis of the cells revealed a normal 46XY diploid karyotype. hBS11 cells expressed smooth muscle differentiation marker proteins, including MYH11, α-SMA, γ-SMA, and calponin. The connexin 43 signals were also detected as spotty signals on the surface of hBS11 cells. M2 and M3 receptors expressed in differentiated hBS11 cells. Both the cholinergic receptor agonist calbachol and a high concentration of extracellular potassium increased intracellular calcium in differentiated hBS11 cells. Differentiated hBS11 cells stimulated by calcium ionophore (A23187) exhibited contractility. CONCLUSIONS The present study demonstrates that the hBS11 cell maintains plasticity of the differentiation that a smooth muscle cell originally has. Immortalized hBS11 cell provides useful analytical system for elucidation of the pathophysiological mechanism of lower urinary tract dysfunction and the development of the new treatment options, in addition to the elucidation of molecular differentiation mechanism, reproduction mechanism, and the function control mechanism of the smooth muscle cell. © 2018FiguresReferencesRelatedDetails Volume 199Issue 4SApril 2018Page: e111-e112 Advertisement Copyright & Permissions© 2018MetricsAuthor Information Masaki Yoshida More articles by this author Naohiro Hashimoto More articles by this author Hisae Nishii More articles by this author Masanori Nomiya More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Store operated calcium channels are associated with diabetic cystopathy in streptozotocin‑induced diabetic rats.

Store operated calcium channels (SOCCs) have been suggested to play a critical role in many diabetic complications. Diabetic cystopathy (DCP) is common in patients with diabetes, but the role of SOCCs in DCP is still unclear. The aim of the present study was to investigate the role of SOCCs in DCP with streptozocin (STZ)-induced diabetic rats. Specifically, the authors investigated whether SOCCs were altered in streptozocin (STZ)-induced diabetic rats and, if so, how this may contribute to the contraction of bladder detrusor strips and the intracellular Ca2+ concentration of bladder smooth muscle cells in diabetic rats. Cyclopiazonic acid (CPA, 10 µM) and SKF-96365 (10 µM) were used to activate and inhibit SOCCs respectively, to research the effects of SOCCs on the contraction of the bladder detrusor strips in normal and STZ-induced diabetic rats at the 4th, 8th and 12th week after the diabetic rat model was established. The changes of intracellular Ca2+ were also evaluated under confocal microscopy with pretreated Fluo-4AM. In addition, the expressions of Orai1 and STIM1 were detected by reverse transcription-quantitative polymerase chain reaction and western blotting at different time points. According to the results, the contractive frequency of diabetic bladder muscle strips was higher than that of controls in the 4th and 8th week. The increased fluorescence intensity was detected after using CPA and SKF-96365 in diabetic groups. The expressions of Orai1 and STIM1 changed in a time-dependent manner.