Human Bladder Smooth Muscle Cells Research Articles

Biological function of bladder smooth muscle cells regulated by multi-modal biomimetic stress

Previous studies have shown that growth arrest, dedifferentiation, and loss of original function occur in cells after multiple generations of culture, which are attributed to the lack of stress stimulation. To investigate the effects of multi-modal biomimetic stress (MMBS) on the biological function of human bladder smooth muscle cells (HBSMCs), a MMBS culture system was established to simulate the stress environment suffered by the bladder, and HBSMCs were loaded with different biomimetic stress for 24 h. Then, cell growth, proliferation and functional differentiation were detected. The results showed that MMBS promoted the growth and proliferation of HBSMCs, and 80 cm H 2O pressure with 4% stretch stress were the most effective in promoting the growth and proliferation of HBSMCs and the expression level of α-smooth muscle actin and smooth muscle protein 22-α. These results suggest that the MMBS culture system will be beneficial in regulating the growth and functional differentiation of HBSMCs in the construction of tissue engineered bladder.

Long non-coding RNA H19 mediates the miR-29b/transforming growth factor-β1/Drosophila mothers against decapentaplegic 3 signalling pathway to promote bladder fibrosis in diabetic rats.

Diabetic bladder fibrosis is a common comorbidity. Altered expression of some long non-coding RNAs (LncRNAs) has been associated with bladder fibrosis. LncRNA H19 has been reported to regulate bladder cancer through miR-29b. However, the action mechanism of LncRNA H19 in bladder fibrosis is unclear. In vitro, human bladder smooth muscle cells (HBSMCs) were cultured with transforming growth factor-β1 (TGF-β1) for 48h to construct cell model of bladder fibrosis. HBSMCs were then transfected with si-LncRNA H19, si-NC, miR-29b-mimic, mimic-NC, or miR-29b-inhibitor. In vivo, Sprague-Dawley (SD) rats were given a high-sucrose-high-fat (HSHF) diet for 4weeks and injected with streptozotocin (STZ, 50mg/kg) to induce bladder fibrosis model in diabetic rats, followed by injection of lentiviral particles knocking down LncRNA H19 expression, empty vector, or miR-29b-inhibitor, respectively. LncRNA H19 was up-regulated in TGF-β1-induced HBSMC fibrosis and STZ-induced diabetic rat bladder fibrosis, whereas miR-29b was down-regulated. si-LncRNA H19 reduced blood glucose levels and improved histopathological damage of bladder tissue in rats. In addition, si-LncRNA H19 or miR-29b-mimic increased the expression of E-cadherin, but decreased the expression of N-cadherin, vimentin, fibronectin (FN) in bladder tissues, and HBSMCs. si-LncRNA H19 reduced TGF-β1/p-drosophila mothers against decapentaplegic 3 (Smad3) protein in HBSMCs and in rat bladder tissues, while miR-29b-inhibitor reversed the effect of si-LncRNA H19. This study indicated that LncRNA H19 may inhibit bladder fibrosis in diabetic rats by targeting miR-29b via the TGF-β1/Smad3 signalling pathway.

Melatonin serves as a novel treatment in bladder fibrosis through TGF-β1/Smad and EMT.

Melatonin (MEL) is an indole amine molecule primarily produced in the pineal gland. Melatonin has been shown in numerous studies to have antifibrotic effects on the kidney, liver, and other organs. However, it is still unclear how melatonin works in bladder fibrosis. We explored how melatonin affects animals with bladder fibrosis and the underlying mechanisms. MEL was used to treat human bladder smooth muscle cells (HBdSMCs) after they were stimulated with transforming growth factor-β1 (TGF-β1) in vitro. Proteomic analysis and bioinformatic analysis of the altered expression of these proteins were subsequently performed on HBdSMCs from the different processing methods. To construct an in vivo bladder fibrosis model, we injected protamine sulfate (PS) and lipopolysaccharide (LPS) twice a week into the rat bladder for six weeks. After two weeks of PS/LPS treatment, the mice in the treatment group were treated with MEL (20 mg/kg/d) for 4 weeks. Finally, we detected the expression of fibrosis markers from different perspectives. The TGF-β1/Smad pathway and epithelial-mesenchymal transition (EMT) in cell and bladder tissues were also identified. Further proteomic analysis was also performed. In vitro, we found that TGF-β1 treatment enhanced the expression of the fibrosis markers collagen III and α-SMA in HBdSMCs. E-cadherin expression decreased while the TGF-β1/Smad pathway was activated. Vimentin and N-cadherin expression was also elevated at the same time. Similar findings were observed in the LPS group. After MEL treatment, the expression of collagen III and α-SMA decreased, the expression of E-cadherin increased, and the expression of vimentin and N-cadherin also decreased. According to our quantitative proteomics analysis, CCN1 and SQLE may be important proteins involved in the development of bladder fibrosis. MEL decreased the expression of these genes, leading to the relief of bladder fibrosis. Bioinformatics analysis revealed that the extracellular space structure related to metabolic pathways, actin filament binding, and stress fibers can serve as a pivotal focus in the management of fibrosis. Melatonin attenuates bladder fibrosis by blocking the TGF-β1/Smad pathway and EMT. CCN1 appears to be a possible therapeutic target for bladder fibrosis.

Decellularized Scaffolds with Double-Layer Aligned Microchannels Induce the Oriented Growth of Bladder Smooth Muscle Cells: toward Urethral and Ureteral Reconstruction.

There is a great clinical need for regenerating the urinary tissue. Native urethra and ureter have bidirectional aligned smooth muscle cells (SMCs) layers, which plays a pivotal role for micturition and transport the urine and inhibit reflux. Up to now, urinary scaffolds have not been designed to induce the native-mimicking aligned arrangement of SMCs. In this study, we engineered a tubular decellularized extracellular matrix (dECM) with an intact internal layer and bidirectional aligned microchannels in the tubular wall, which was realized by the subcutaneous implantation of a template, followed by the removal of the template, and decellularization. The dense and intact internal layer effectively increased the leakage pressure of the tubular dECM scaffolds. The rat-derived dECM scaffolds with three different sizes of microchannels were fabricated by tailoring the fiber diameter of the templates. The rat-derived dECM scaffolds exhibiting microchannels of ∼ 65μm showed suitable mechanical properties, good ability to induce the bidirectional alignment and growth of human bladder SMCs (HBdSMC) and elevated higher functional protein expression in vitro. These data indicated that rat-derived tubular dECM scaffolds manifesting double-layer aligned microchannel may be promising candidates to induce the native-mimicking regeneration of SMCs in urethra and ureter reconstruction. This article is protected by copyright. All rights reserved.

Inflammatory Expression Profiles in Bladder Exstrophy Smooth Muscle: Normalization Over Time

ObjectiveTo test the hypothesis that phenotypes in bladder exstrophy result from alterations in detrusor smooth muscle cell (SMC) gene expression. MethodsWe generated primary human bladder smooth muscle cell lines from patients with classic bladder exstrophy (CBE) undergoing newborn closure (n = 6), delayed primary closure (n = 5), augmentation cystoplasty (n = 6), and non-CBE controls (n = 3). Gene expression profiles were then created using RNA sequencing and characterized using gene set enrichment analysis (GSEA). ResultsWe identified 308 differentially expressed genes in bladder exstrophy SMC when compared to controls, including 223 upregulated and 85 downregulated genes. Bladder exstrophy muscle cell lines from newborn closure and primary delayed closures shared expression changes in 159 genes. GSEA analysis revealed increased expression in the inflammatory response and alteration of genes for genitourinary development in newborn and delayed closure SMC. However, these changes were absent in SMC from older exstrophy patients after closure. ConclusionBladder exstrophy SMC demonstrate gene expression changes in the inflammatory response and genitourinary development. However, gene expression profiles normalized in exstrophy SMC from older patients after closure, suggesting a normalization of exstrophy SMC over time. Our in vitro findings regarding the normalization of exstrophy SMC gene expression following bladder closure suggest that the development of poor detrusor compliance in bladder exstrophy has a complex multifactorial etiology. Taken together, our findings suggest that alterations in SMC gene expression may explain abnormalities in the exstrophy bladder seen prior to and immediately after closure and suggest that surgical closure may allow exstrophy SMC to normalize over time.

YAP-mediated mechanotransduction in urinary bladder remodeling: Based on RNA-seq and CUT&Tag.

Yes-associated protein (YAP) is an important transcriptional coactivator binding to transcriptional factors that engage in many downstream gene transcription. Partial bladder outlet obstruction (pBOO) causes a massive burden to patients and finally leads to bladder fibrosis. Several cell types engage in the pBOO pathological process, including urothelial cells, smooth muscle cells, and fibroblasts. To clarify the function of YAP in bladder fibrosis, we performed the RNA-seq and CUT&Tag of the bladder smooth muscle cell to analyze the YAP ablation of human bladder smooth muscle cells (hBdSMCs) and immunoprecipitation of YAP. 141 differentially expressed genes (DEGs) were identified through RNA-seq between YAP-knockdown and nature control. After matching with the results of CUT&Tag, 36 genes were regulated directly by YAP. Then we identified the hub genes in the DEGs, including CDCA5, CENPA, DTL, NCAPH, and NEIL3, that contribute to cell proliferation. Thus, our study provides a regulatory network of YAP in smooth muscle proliferation. The possible effects of YAP on hBdSMC might be a vital target for pBOO-associated bladder fibrosis.

Emodin inhibits bladder inflammation and fibrosis in mice with interstitial cystitis by regulating JMJD3.

Interstitial cystitis/bladder pain syndrome (IC/BPS) is a devastating urological chronic pelvic pain condition. In search of a potential treatment, we investigated the effect of emodin on IC/BPS inflammation and fibrosis, and explore the potential mechanism. An experimental model of interstitial cystitis was induced by cyclophosphamide, and human bladder smooth muscle cells were treated with lipopolysaccharide to establish the cell model in vitro. In both models, inflammation- and fibrosis-related indexes were measured after emodin administration. Furthermore, the specific antagonists were used to dig for the mechanisms underlying the response to emodin treatment. Emodin significantly ameliorated management of cystitis, reduced the amount of inflammatory cytokines (tumor necrosis factor-α, monocyte chemoattractant protein-1, interleukin-1β, interleukin-8, and interleukin-6) in models, as well as reducing the synthesis of fibrosis marker including collagen1, collagen3, vimentin, fibronectin and α-smooth muscle actin. Further mechanism studies demonstrated that emodin inhibited inflammatory reaction and fibrosis through blocking lysine-specific demethylase 6B (JMJD3) expression via JAK/STAT, NF-κB and TGF-β/SMAD pathways. Our study reveals the critical role of emodin-JMJD3 signaling in interstitial cystitis by regulating inflammation, fibrosis, and extracellular matrix deposition in cells and tissues, and these findings provide an avenue for effective treatment of patients with cystitis.

Tailoring Hydrogel Composition and Stiffness to Control Smooth Muscle Cell Differentiation in Bioprinted Constructs.

Reliable in vitro cellular models are needed to study the phenotypic modulation of smooth muscle cells (SMCs) in health and disease. The aim of this study was to optimize gelatin methacrylate (GelMA)/alginate hydrogels for bioprinting three-dimensional (3D) SMC constructs. Four different hydrogel groups were prepared by mixing different concentrations (% w/v) of GelMA and alginate: G1 (5/1.5), G2 (5/3), G3 (7.5/1.5), and G4 (7.5/3). GelMA 10% was used as control (G5). A circular structure containing human bladder SMCs was fabricated by using an extrusion-based bioprinter. The effects of the mixing ratios on printability, viability, proliferation, and differentiation of the cells were investigated. Rheological analysis showed that the addition of alginate significantly stabilized the change in mechanical properties with temperature variations. The group with the highest GelMA and alginate concentrations (G4) exhibited the highest viscosity, resulting in better stability of the 3D construct after crosslinking. Compared to other hydrogel compositions, cells in G4 maintained high viability (> 80%), exhibited spindle-shaped morphology, and showed a significantly higher proliferation rate within an 8-day period. More importantly, G4 provided an optimal environment for the induction of a SMC contractile phenotype, as evidenced by significant changes in the expression of marker proteins and morphological parameters. Adjusting the composition of GelMA/alginate hydrogels is an effective means of controlling the SMC phenotype. These hydrogels support bioprinting of 3D models to study phenotypic smooth muscle adaptation, with the prospect of using the constructs in the study of therapies for the treatment of urethral strictures.

YAP/Smad3 promotes pathological extracellular matrix microenviroment-induced bladder smooth muscle proliferation in bladder fibrosis progression.

Fibrosis is a chronic inflammation process with excess extracellular matrix (ECM) deposition that cannot be reversed. Patients suffer from bladder dysfunction caused by bladder fibrosis. Moreover, the interactive mechanisms between ECM and bladder fibrosis are still obscure. Hence, we assessed the pivotal effect of Yes‐associated protein (YAP) on the proliferation of bladder smooth muscle in fibrosis process. We identified that stiff ECM increased the expression and translocation of YAP in the nucleus of human bladder smooth muscle cell (hBdSMC). Sequencings and proteomics revealed that YAP bound to Smad3 and promoted the proliferation of hBdSMC via MAPK/ERK signaling pathway in stiff ECM. Moreover, CUT and TAG sequencing and dual‐luciferase assays demonstrated that Smad3 inhibited the transcription of JUN. The YAP inhibitor CA3 was used in a partial bladder outlet obstruction (pBOO) rat model. The results showed that CA3 attenuated bladder smooth muscle proliferation. Collectively, YAP binding with Smad3 in the nucleus inhibited the transcription of JUN, and promoted the proliferation of bladder smooth muscle through the MAPK/ERK signaling pathway. The current study identified a novel mechanism of mechanical force induced bladder fibrosis that provided insights in YAP‐associated organ fibrosis.

Influence of pole‐like kinase 1 on controlling contractility of the urinary tract and gene activated patterns of human bladder smooth muscle cells

BackgroundPole like kinase (PLK)‐mediated promotion of proliferation and smooth muscle contraction in the lower urinary tract has been recently suggested and may offer putative targets and novel compounds for treatments of lower urinary tract symptoms. However, PLK function for contractility of smooth muscle and proliferation of bladder smooth muscle cells are unknown and the specificity of PLK inhibitors has been questioned.MethodsHere, human bladder smooth muscle cells (hBSMCs) inhibited with five PLK inhibitors (TAK960, MLN0905, Wortmannin, GW843682X, Centrinone‐B) and hBSMCs cultured in smooth muscle medium were compared. hBSMCs characteristics according to criteria set by the International Society of Cellular Therapy were confirmed by flow cytometry. Proliferation and viability were assessed by CCK‐8 assay. Contractions of human ureterovesical junction smooth muscle from donation after circulatory death renal transplantation were studied in an organ bath, where they were induced by the α1‐adrenoceptor agonists noradrenaline, phenylephrine, and electric field stimulation. Transcriptome sequencing of hBSMCs were obtained using NEBNext®UltraTM RNA Library Prep Kit for Illumina®. Gene set enrichment analysis was performed using the Kyoto Encyclopedia of Genes and Genomes and gene ontology terms.ResultsViability and proliferation of hBSMCs were reduced in a concentration‐ and time‐dependent manner. TAK960 inhibits adrenergic contractions of human ureterovesical junction smooth muscle, and proliferation of hBSMCs. No significant differences in other PLK inhibitors combined with control were found. Compared to hBSMCs in control medium, 323 upregulated genes and 160 downregulated genes were significantly altered by TAK960 24hours 500nM, respectively (FDR=0.05, FC=1.5). Genes most prominently and significantly upregulated by PLK inhibiting condition were IFI30, GBP4, VTN, SCG2, OAS2, RRAD, TNFSF10, KRT18, SFN, meanwhile, downregulated was CCL11 (log2FC ≥1.5 or ≤ ‐1.5). The most significantly relative signaling pathway included cytokine‐cytokine receptor interaction, systemic lupus erythematosus and transcriptional misregulation cancer (enrichment factor ≥0.05). Accession to cite for these sequences read archive data is PRJNA714246.ConclusionOur findings point to a certain role of PLK‐1 in of proliferation in hBSMCs and contractility in ureterovesical junction smooth muscle, which may be of relevance in lower urinary tract symptom. PLK‐1 inhibitors block these effects and show shared and divergent effects. PLK‐1 regulation of bladder smooth muscle growth and contraction may include genomic and nongenomic mechanisms.

Simplified, High-throughput Analysis of Single-cell Contractility using Micropatterned Elastomers

Cellular contractile force generation is a fundamental trait shared by virtually all cells. These contractile forces are crucial to proper development, function at both the cellular and tissue levels,and regulate the mechanical systems in the body. Numerous biological processes are force-dependent, including motility, adhesion, and division of single-cells, as well as contraction and relaxation of organs such as the heart, bladder, lungs, intestines, and uterus. Given its importance in maintaining proper physiological function, cellular contractility can also drive disease processes when exaggerated or disrupted. Asthma, hypertension, preterm labor, fibrotic scarring, and underactive bladder are all examples of mechanically driven disease processes that could potentially be alleviated with proper control of cellular contractile force. Here, we present a comprehensive protocol for utilizing a novel microplate-based contractility assay technology known as fluorescently labeled elastomeric contractible surfaces (FLECS), that provides simplified and intuitive analysis of single-cell contractility in a massively scaled manner. Herein, we provide a step-wise protocol for obtaining two six-point dose-response curves describing the effects of two contractile inhibitors on the contraction of primary human bladder smooth muscle cells in a simple procedure utilizing just a single FLECS assay microplate, to demonstrate proper technique to users of the method. Using FLECS Technology, all researchers with basic biological laboratories and fluorescent microscopy systems gain access to studying this fundamental but difficult-to-quantify functional cell phenotype, effectively lowering the entry barrier into the field of force biology and phenotypic screening of contractile cell force.

Simplified, High-throughput Analysis of Single-cell Contractility using Micropatterned Elastomers.

Cellular contractile force generation is a fundamental trait shared by virtually all cells. These contractile forces are crucial to proper development, function at both the cellular and tissue levels,and regulate the mechanical systems in the body. Numerous biological processes are force-dependent, including motility, adhesion, and division of single-cells, as well as contraction and relaxation of organs such as the heart, bladder, lungs, intestines, and uterus. Given its importance in maintaining proper physiological function, cellular contractility can also drive disease processes when exaggerated or disrupted. Asthma, hypertension, preterm labor, fibrotic scarring, and underactive bladder are all examples of mechanically driven disease processes that could potentially be alleviated with proper control of cellular contractile force. Here, we present a comprehensive protocol for utilizing a novel microplate-based contractility assay technology known as fluorescently labeled elastomeric contractible surfaces (FLECS), that provides simplified and intuitive analysis of single-cell contractility in a massively scaled manner. Herein, we provide a step-wise protocol for obtaining two six-point dose-response curves describing the effects of two contractile inhibitors on the contraction of primary human bladder smooth muscle cells in a simple procedure utilizing just a single FLECS assay microplate, to demonstrate proper technique to users of the method. Using FLECS Technology, all researchers with basic biological laboratories and fluorescent microscopy systems gain access to studying this fundamental but difficult-to-quantify functional cell phenotype, effectively lowering the entry barrier into the field of force biology and phenotypic screening of contractile cell force.

Secretomes of human pluripotent stem cell-derived smooth muscle cell progenitors upregulate extracellular matrix metabolism in the lower urinary tract and vagina

BackgroundAdult mesenchymal stem cells (MSCs) have been studied extensively for regenerative medicine; however, they have limited proliferation in vitro, and the long culture time induces cell senescence. MSCs also contribute to tissue repair through their paracrine function. In this study, we sought to examine the paracrine effects of human smooth muscle cell progenitors (pSMC) on the urethra and adjacent vagina of stress urinary incontinence rodents. We use human pluripotent stem cell (PSC) lines to derive pSMCs to overcome the issue of decreased proliferation in tissue culture and to obtain a homogenous cell population.MethodThree human PSC lines were differentiated into pSMCs. The conditioned medium (CM) from pSMC culture, which contain pSMC secretomes, was harvested. To examine the effect of the CM on the extracellular matrix of the lower urinary tract, human bladder smooth muscle cells (bSMCs) and vaginal fibroblasts were treated with pSMC-CM in vitro. Stress urinary incontinence (SUI) was induced in rats by surgical injury of the urethra and adjacent vagina. SUI rats were treated with pSMC-CM and monitored for 5 weeks. Urethral pressure testing was performed prior to euthanasia, and tissues were harvested for PCR, Western blot, and histological staining. Kruskal-Wallis one-way ANOVA test and Student t test were used for statistical comparisons.ResultspSMC-CM upregulated MMP-2, TIMP-2, collagen, and elastin gene expression, and MMP-9 activity in the human bladder and vaginal cells consistent with elastin metabolism modulation. pSMC-CM treatment in the SUI rat improved urethral pressure (increase in leak point pressure compared to intact controls, p < 0.05) and increased collagen and elastin expression in the urethra and the adjacent vagina.ConclusionConditioned media from smooth muscle cell progenitors derived from human pluripotent stem cells improved urethral leak point pressure and collagen and elastin content in the SUI rat. These findings suggest a novel therapeutic potential for PSC-based treatments for SUI and pelvic floor disorders where tissues are affected by collagen, elastin, and smooth muscle loss.

Inhibition of LIM kinase reduces contraction and proliferation in bladder smooth muscle

Overactive bladder (OAB) is the most bothersome symptom in lower urinary tract symptoms (LUTS). Current pharmacologic treatment aims to inhibit detrusor contraction; however, shows unsatisfied efficacy and high discontinuation rate. LIM kinases (LIMKs) promote smooth muscle contraction in the prostate; however, their function in the bladder smooth muscle remains unclear. Here, we studied effects of the LIMK inhibitors on bladder smooth muscle contraction and proliferation both in vitro and in vivo experiments. Bladder expressions of LIMKs are elevated in OAB rat detrusor tissues. Two LIMK inhibitors, SR7826 and LIMKi3, inhibit contraction of human detrusor strip, and cause actin filament breakdown, as well as cell proliferation reduction in cultured human bladder smooth muscle cells (HBSMCs), paralleled by reduced cofilin phosphorylation. Silencing of LIMK1 and LIMK2 in HBSMCs resulted in breakdown of actin filaments and decreased cell proliferation. Treatment with SR7826 or LIMKi3 decreased micturition frequency and bladder detrusor hypertrophy in rats with bladder outlet obstruction. Our study suggests that LIMKs may promote contraction and proliferation in the bladder smooth muscle, which could be inhibited by small molecule LIMK inhibitors. LIMK inhibitors could be a potential therapeutic strategy for OAB- related LUTS.

The role of interleukin-6/interleukin-6 receptor signaling in the mechanical stress-induced extracellular matrix remodeling of bladder smooth muscle

Extracellular matrix (ECM) remodeling is strongly associated with pathological changes induced by bladder outlet obstruction (BOO). In this study, we investigated the role of interleukin-6 (IL-6) in mechanical stretch-induced ECM remodeling of bladder smooth muscle. To construct a BOO animal model, the urethras of female Sprague–Dawley rats were partially ligated. In addition, increased hydrostatic pressure and mechanical stretching were applied to human bladder smooth muscle cells (HBSMCs) as an in vitro model. The expression of rat inflammatory genes was analyzed using DNA microarrays. We used quantitative RT-PCR (qRT-PCR) and immunohistochemical staining to detect IL-6 in the bladder smooth muscle of rats. To determine the specificity of IL-6, small interfering ribonucleic acid (siRNA) transfection and IL-6 receptor inhibitor (SC144) were applied to HBSMCs. qRT-PCR with siRNA transfection was also used to determine the specificity of downstream signaling. Moreover, western blotting was conducted to verify the expression results. In the animal model, the expression of ECM components and inflammatory genes was significantly upregulated. The expression of IL-6 was increased at both the mRNA level and the protein level in BOO rats. In vitro, hydrostatic pressure, and mechanical stretching both promoted MMP7 and MMP11 expression. Additionally, downregulation of collagen III occurred in both the hydrostatic pressure group and the mechanical stretch group. However, the expression of fibronectin exhibited opposing patterns between the hydrostatic pressure and mechanical stretch groups. The application of targeted siRNA transfection and an inhibitor (SC144) that targeted IL-6 significantly reversed the changes in MMP7 and MMP11 under mechanical stress and partially increased the expression of collagen III and fibronectin. In summary, IL-6 participated in the ECM remodeling of HBSMCs under mechanical stress, indicating that IL-6 may play an essential role in BOO.

β-Adrenoceptor regulates contraction and inflammatory cytokineexpression of human bladder smooth muscle cells via autophagy under pathological hydrostatic pressure.

Abnormal intravesical pressure created by partial bladder outlet obstruction (PBOO) triggered the progression from chronic inflammation to fibrosis, initiating structural and functional alterations of bladder. To elucidate the underlying mechanisms of contraction and inflammatory response, we investigated the isolated human bladder smooth muscle cells (hBSMC) under pathological hydrostatic pressure (HP) mimicking the in vivo PBOO condition. hBSMCs were subjected to HP of 200 cm H2 O to explore the contraction and inflammatory cytokineexpression of hBSMC treated with β-adrenoceptors (ADRBs) and/or autophagy signaling pathway agonists and/or antagonists. We showed that pathological HP induced the release of the proinflammatory cytokines, including monocyte chemotactic protein-1, regulated upon activation normal T cell expressed and secreted factor, and interleukin-6. HP downregulated ADRB2 and ADRB3 expression, which was consistent with the results of the PBOO rat model. ADRB2 or autophagy activation repressed pathological HP-induced proinflammatory cytokineproduction. ADRB2, ADRB3 or autophagy activation ameliorated the HP-enhanced contraction. The increased contraction and autophagy activity by ADRB2 agonist under HP conditions were reversed by pretreatment with antagonists of adenosine monophosphate-activated protein kinase (AMPK). The present study provides evidence that the ADRB3 agonist suppresses hBSMC contraction under pathological HP conditions. Moreover, the ADRB2 agonist negatively regulates the contraction and inflammatory response of hBSMCs through AMPK/mTOR-mediated autophagy under pathological HP. These findings provide a theoretical basis for potential therapeutic strategies for patients with PBOO.

Rac1 silencing, NSC23766 and EHT1864 reduce growth and actin organization of bladder smooth muscle cells

AimsRacGTPase-mediated proliferation and smooth muscle contraction in the lower urinary tract has been recently suggested and may offer putative targets for treamtment of lower urinary tract symptoms. However, RacGTPase function for proliferation of detrusor smooth muscle cells is unknown and the specificity of Rac inhibitors has been questioned. Here, we examined effects of Rac1 knockdown and of the Rac inhibitors NSC23766 and EHT1864 in human bladder smooth muscle cells (hBSMCs). Main methodsRac1 expression was silenced by shRNA expression. Effects of silencing and Rac inhibitors were assessed by CCK-8 assay, EdU staining, RT-PCR, colony formation assay, flow cytometry, and phalloidin staining. Key findingsSilencing of Rac1 expression reduced the viability (up to 83% compared to scramble shRNA) and proliferation (virtually completely in proliferation assay), increased apoptosis (124%) and the number of dead cells (51%), and caused breakdown of actin organization (56% reduction of polymerized actin compared to scramble shRNA). Effects on proliferation, viability, and actin organization were mimicked by NSC23766 and EHT1864, while both compounds showed divergent effects on cell death (32-fold increase of dead cells by EHT1864, but not NSC23766). Effects of NSC23766 and EHT1864 on viability of hBSMCs were not altered by Rac1 knockdown. SignificanceRac1 promotes proliferation, viability, and cytoskeletal organization, and suppresses apoptosis in bladder smooth muscle cells, which may be relevant in overactive bladder or diabetes-related bladder dysfunction. NSC23766 and EHT1864 mimick these effects, but may act Rac1-independently, by shared and divergent effects.

Regulation of Cellular Stress Signaling in Bladder Ischemia.

IntroductionThe etiology of lower urinary tract symptoms in patients with non-obstructed non-neurogenic bladder remains largely unknown. Clinical studies divulged a significant correlation between reduced bladder blood flow and low bladder compliance. Animal models of bladder ischemia displayed structural modifications, characterized by loss of smooth muscle cells and accumulation of connective tissue in the bladder wall. The underlying mechanisms contributing to structural damage in bladder ischemia remain largely elusive. We previously reported that structural modifications in bladder ischemia correlate with upregulated stress proteins and cell survival signaling, suggesting the potential role of cellular stress in ischemic damage. However, stress response molecules and downstream pathways eliciting bladder damage in ischemia remain largely undetermined.MethodsUsing a rat model of bladder ischemia along with a cell culture hypoxia model, we investigated stress signaling molecules in the ischemic bladder tissues and hypoxic bladder smooth muscle cells.ResultsOur data suggest simultaneous upregulation of two major cellular stress-sensing molecules, namely apoptosis signal-regulating kinase 1 (ASK1) and caspase-3, implying degenerative insult via stress signaling pathway in bladder ischemia. Consistent with bladder ischemia, incubation of cultured human bladder smooth muscle cells at low oxygen tension increased both ASK1 and caspase-3 expression, insinuating hypoxia as an essential factor in ASK1 and caspase-3 upregulation. Gene deletion of ASK1 by ASK1 siRNA in cultured smooth muscle cells prevented caspase-3 upregulation by hypoxia, suggesting caspase-3 regulation by ASK1 under the ischemic/hypoxic conditions. Upregulation of ASK1 and caspase-3 in rat bladder ischemia and human bladder smooth muscle cell hypoxia was associated with subcellular structural modifications consistent with the initial stages of apoptotic insult.ConclusionOur data suggest that stress sensing by ASK1 and caspase-3 may contribute to subcellular structural damage and low bladder compliance. The ASK1/caspase-3 pathway may provide therapeutic targets against cellular stress and degenerative responses in bladder ischemia.

Biofabrication of a Functional Tubular Construct from Tissue Spheroids Using Magnetoacoustic Levitational Directed Assembly.

In traditional tissue engineering, synthetic or natural scaffolds are usually used as removable temporal support, which involves some biotechnology limitations. The concept of "scaffield" approach utilizing the physical fields instead of biomaterial scaffold has been proposed recently. In particular, a combination of intense magnetic and acoustic fields can enable rapid levitational bioassembly of complex-shaped 3D tissue constructs from tissue spheroids at low concentration of paramagnetic agent (gadolinium salt) in the medium. In the current study, the tissue spheroids from human bladder smooth muscle cells (myospheres) are used as building blocks for assembling the tubular 3D constructs. Levitational assembly is accomplished at low concentrations of gadolinium salts in the high magnetic field at 9.5T. The biofabricated smooth muscle constructs demonstrate contraction after the addition of vasoconstrictive agent endothelin-1. Thus, hybrid magnetoacoustic levitational bioassembly is considered as a new technology platform in the emerging field of formative biofabrication. This novel technology of scaffold-free, nozzle-free, and label-free bioassembly opens a unique opportunity for rapid biofabrication of 3D tissue and organ constructs with complex geometry.

131 - Rac1 inhibition by EHT1864, NSC23766 or by knockdown of Rac1 expression reduces proliferation and contraction of human bladder smooth muscle cells

131 - Rac1 inhibition by EHT1864, NSC23766 or by knockdown of Rac1 expression reduces proliferation and contraction of human bladder smooth muscle cells