Primary Bladder Smooth Muscle Cells Research Articles

TGFβ2 mediates oxidative stress–induced epithelial-to-mesenchymal transition of bladder smooth muscle

Bladder outlet obstruction (BOO) is the primary clinical manifestation of benign prostatic hyperplasia, the most common urinary system disease in elderly men, and leads to associated lower urinary tract symptoms. Although BOO is reportedly associated with increased systemic oxidative stress (OS), the underlying mechanism remains unclear. The elucidation of this mechanism is the primary aim of this study. A Sprague–Dawley rat model of BOO was constructed and used for urodynamic monitoring. The bladder tissue of rats was collected and subjected to real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR), histological examination, and immunohistochemical staining. Through bioinformatics prediction, we found that transforming growth factor β2 (TGFβ2) expression was upregulated in rats with BOO compared with normal bladder tissue. In vitro analyses using primary bladder smooth muscle cells (BSMCs) revealed that hydrogen peroxide (H2O2) induced TGFβ2 expression. Moreover, H2O2 induced epithelial-to-mesenchymal transition (EMT) by reducing E-cadherin, an endothelial marker and CK-18, a cytokeratin maker, and increasing mesenchymal markers, including N-cadherin, vimentin, and α-smooth muscle actin (α-SMA) levels. The downregulation of TGFβ2 expression in BSMCs using siRNA technology alleviated H2O2-induced changes in EMT marker expression. The findings of the study indicate that TGFβ2 plays a crucial role in BOO by participating in OS-induced EMT in BSMCs.

Exosomes from human urine-derived stem cells carry NRF1 to alleviate bladder fibrosis via regulating miR-301b-3p/TGFβR1 pathway.

Bladder outlet obstruction (BOO) is a common disease that always make the bladder develops from inflammation to fibrosis. This study was to investigate the effect of exosomes from human urine-derived stem cells (hUSCs) on bladder fibrosis after BOO and the underlying mechanism. The BOO mouse model was established by inserting a transurethral catheter, ligation of periurethral wire, and removal of the catheter. Mouse primary bladder smooth muscle cells (BSMCs) were isolated and treated with TGFβ1 to mimic the bladder fibrosis model in vitro. Exosomes from hUSCs (hUSC-Exos) were injected into the bladder of BOO mice and added into the culture of TGFβ1-induced BSMCs. The associated factors in mouse bladder tissues and BSMCs were detected. It was confirmed that thetreatment of hUSC-Exosalleviated mouse bladder fibrosis and down-regulated fibrotic markers (a-SMA and collagen III) in bladder tissues and TGFβ1-induced BSMCs. Overexpression of NRF1 in hUSC-Exos further improved the effects of hUSC-Exos on bladder fibrosis both in vivo and in vitro. TGFβR1 was a target of NRF1 and miR-301b-3p, and miR-301b-3p was atarget of NRF1. It was next characterized that hUSC-Exos carried NRF1 to up-regulate miR-301B-3p, thereby reducing TGFβR1level. Our results illustrated that hUSC-Exos carried NRF1 to alleviate bladder fibrosis through regulating miR-301b-3p/TGFβR1 pathway.

VEGF-incorporated PVDF/collagen nanofibrous scaffold for bladder wall regeneration and angiogenesis

A common problem encountered with tissue-engineering constructs is the lack of adequate angiogenesis in newly produced tissue. We compared biocompatibility and expression level of apoptotic and angiogenesis-related gene markers in the primary bladder smooth muscle cells (HBdSMC) and human umbilical vein endothelial cells (HUVEC) when grown on the intact and VEGF-loaded scaffolds that were made of polyvinylidene fluoride and collagen. It was found an increased protein adsorption, cell attachment, and viability in VEGF-loaded scaffold compared to the intact scaffold. In addition, apoptotic and angiogenesis-related genes downregulation and upregulation were also detected in HBdSMC and HUVEC while grown on the VEGF-loaded scaffold, respectively.

MiR-133 modulates TGF-β1-induced bladder smooth muscle cell hypertrophic and fibrotic response: Implication for a role of microRNA in bladder wall remodeling caused by bladder outlet obstruction

Bladder outlet obstruction (BOO) evokes urinary bladder wall remodeling significantly, including the phenotype shift of bladder smooth muscle cells (BSMCs) where transforming growth factor-beta1 (TGF-β1) plays a pivotal role given the emerging function of modulating cellular phenotype. miR-133 plays a role in cardiac and muscle remodeling, however, little is known about its roles in TGF-β1-induced BSMC hypertrophic and fibrotic response. Here, we verified BOO induced bladder wall remodeling and TGF-β1 expression mainly located in bladder endothelium. Furthermore, we uncovered miR-133a/b expression profile in BOO rats, and then explored its regulated effects on BSMCs' phenotypic shift. Our study found that miR-133 became down-regulated during rat bladder remodeling. Next, we sought to examine whether the expression of miR-133 was down-regulated in primary BSMCs in response to TGF-β1 stimulation and whether forced overexpression of miR-133 could regulate profibrotic TGF-β signaling. We found that stimulation of BSMCs with exogenous TGF-β1 of increasing concentrations resulted in a dose-dependent decrease of miR-133a/b levels and transfection with miR-133 mimics attenuated TGF-β1-induced α-smooth muscle actin, extracellular matrix subtypes and fibrotic growth factor expression, whereas it upregulated high molecular weight caldesmon expression compared with the negative control. Also, downregulation of p-Smad3, not p-Smad2 by miR-133 was detected. Additionally, miR-133 overexpression suppressed TGF-β1-induced BSMC hypertrophy and proliferation through influencing cell cycle distribution. Bioinformatics analyses predicted that connective tissue growth factor (CTGF) was the potential target of miR-133, and then binding to the 3′-untranslated region of CTGF was validated by luciferase reporter assay. These results reveal a novel regulator for miR-133 to modulate TGF-β1-induced BSMC phenotypic changes by targeting CTGF through the TGF-β–Smad3 signaling pathway. A novel antifibrotic functional role for miR-133 is presented which may represent a potential target for diagnostic and therapeutic strategies in bladder fibrosis.

A bilayered hybrid microfibrous PLGA–Acellular matrix scaffold for hollow organ tissue engineering

Various synthetic and natural biomaterials have been used for regeneration of tissues and hollow organs. However, clinical outcome of reconstructive procedures remained challenging due to the lack of appropriate scaffold materials, supporting the needs of various cell types and providing a barrier function required in hollow organs. To address these problems, we have developed a bilayered hybrid scaffold comprising unique traits of polymeric microfibers and naturally derived acellular matrices and tested its potential for hollow organ regeneration in a rat bladder model. Hybrid scaffolds were fabricated by electrospinning of PLGA microfibers directly onto the abluminal surface of a bladder acellular matrix. Stability of this bilayered construct was established using modified spinning technique. The resulting 3-dimensional framework provided good support for growth, attachment and proliferation of primary bladder smooth muscle cells. Histological analysis in vivo at 4 and 8 weeks post implantation, revealed regeneration of bladder tissue structures consisting of urothelium, smooth muscle and collagen rich layers infiltrated with host cells and micro vessels. Furthermore, hybrid scaffolds maintained normal bladder capacity, whereas BAM recipients showed a significant distension of the bladder. These results demonstrate that this adaptable hybrid scaffold supports bladder regeneration and holds potential for engineering of bladder and other hollow organs.

1965 DOWN-REGULATION OF LARGE CONDUCTANCE, CALCIUM-ACTIVATED K+ CHANNEL GENE EXPRESSION IN MICE AND HUMAN DETRUSOR SMOOTH MUSCLE FOLLOWING PARTIAL BLADDER OUTLET OBSTRUCTION: EFFECTS OF STRETCH AND HYPOXIA

You have accessJournal of UrologyUrodynamics/Incontinence/Female Urology: Non-Neurogenic Voiding Dysfunction1 Apr 20121965 DOWN-REGULATION OF LARGE CONDUCTANCE, CALCIUM-ACTIVATED K+ CHANNEL GENE EXPRESSION IN MICE AND HUMAN DETRUSOR SMOOTH MUSCLE FOLLOWING PARTIAL BLADDER OUTLET OBSTRUCTION: EFFECTS OF STRETCH AND HYPOXIA Ettickan Boopathi, Stephen Zderic, Sankar Addya, Paolo Fortina, Alan Wein, and Samuel Chacko Ettickan BoopathiEttickan Boopathi Glenolden, PA More articles by this author , Stephen ZdericStephen Zderic Philadelphia, PA More articles by this author , Sankar AddyaSankar Addya Philadelphia, PA More articles by this author , Paolo FortinaPaolo Fortina Philadelphia, PA More articles by this author , Alan WeinAlan Wein Philadelphia, PA More articles by this author , and Samuel ChackoSamuel Chacko Glenolden, PA More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2012.02.2124AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Large-conductance voltage- and calcium-activated potassium (BK) channels have been shown to play a role in detrusor overactivity (DO). Recently, our group demonstrated that the expression of BKα and the accessory β1 subunit was decreased in partial bladder outlet obstruction (PBOO)-induced DO in the rabbit model for PBOO and in men with BPH-induced PBOO. The loss of these proteins contributed to the spontaneous contractions and DO. Here we show that the expression of BKα and BKβ was downregulated in the detrusor smooth muscle (DSM) from 14-day obstructed murine bladders both at mRNA and protein levels. Since PBOO is associated with bladder wall stretch and hypoxia, we ask whether stretch or hypoxia contributes to the downregulation of BKα and BKβ expression. Data show that stretch and hypoxia down-regulate BKα and BKβ protein expression in a time dependent manner in cultured human primary bladder myocytes. METHODS Male C57Bl/6 mice were surgically obstructed and kept for 14 days. Sham-operated mice served as a control. Bladders were excised; urothelium scarped off with a scalpel, and the serosa was removed. DSM obtained from PBOO and sham control animals were used for microarray and western blotting. For stretch and hypoxia experiments, human primary bladder smooth muscle cells were seeded on type I collagen-coated silicone sheeting membrane at a density of 1 × 105 per well in M199 medium containing 10 percent FBS and grown to near confluence. Cells were rendered quiescent by incubation in medium containing 1 percent FBS for 48 hours and then subjected to cyclic stretch and 1 percent hypoxia independently for 24 and 48 hours. RESULTS Expression of BKα and BKβ in the DSM were decreased in 14-day obstructed groups both at the mRNA and protein levels as shown by microarray and immunoblotting compared to that of sham-operated animals. There was a significant time dependent downregulation of BKα and BKβ expression in stretch and hypoxia compared to un-stretched cells and cells grown in normal oxygen level. CONCLUSIONS These results confirm that the BKα and BKβ gene expression is downregulated in obstructed murine bladder smooth muscle. Based on our experimental data from cell culture experiments, mechanical stretch and hypoxia in the obstructed bladder are the initiating factors for the down regulation of BKα and BKβ gene expression in the DSM in PBOO observed in animal models and men. © 2012 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 187Issue 4SApril 2012Page: e793 Advertisement Copyright & Permissions© 2012 by American Urological Association Education and Research, Inc.MetricsAuthor Information Ettickan Boopathi Glenolden, PA More articles by this author Stephen Zderic Philadelphia, PA More articles by this author Sankar Addya Philadelphia, PA More articles by this author Paolo Fortina Philadelphia, PA More articles by this author Alan Wein Philadelphia, PA More articles by this author Samuel Chacko Glenolden, PA More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Nuclear Fusion‐Independent Smooth Muscle Differentiation of Human Adipose‐Derived Stem Cells Induced by a Smooth Muscle Environment

Human adipose-derived stem cells hASC have been isolated and were shown to have multilineage differentiation capacity. Although both plasticity and cell fusion have been suggested as mechanisms for cell differentiation in vivo, the effect of the local in vivo environment on the differentiation of adipose-derived stem cells has not been evaluated. We previously reported the in vitro capacity of smooth muscle differentiation of these cells. In this study, we evaluate the effect of an in vivo smooth muscle environment in the differentiation of hASC. We studied this by two experimental designs: (a) in vivo evaluation of smooth muscle differentiation of hASC injected into a smooth muscle environment and (b) in vitro evaluation of smooth muscle differentiation capacity of hASC exposed to bladder smooth muscle cells. Our results indicate a time-dependent differentiation of hASC into mature smooth muscle cells when these cells are injected into the smooth musculature of the urinary bladder. Similar findings were seen when the cells were cocultured in vitro with primary bladder smooth muscle cells. Chromosomal analysis demonstrated that microenvironment cues rather than nuclear fusion are responsible for this differentiation. We conclude that cell plasticity is present in hASCs, and their differentiation is accomplished in the absence of nuclear fusion.

Polyesterurethane foam scaffold for smooth muscle cell tissue engineering

Reconstruction of the genitourinary tract, using engineered urological tissues, requires a mechanically stable biodegradable and biocompatible scaffold and cultured cells. Such engineered autologous tissue would have many clinical implications. In this study a highly porous biodegradable polyesterurethane-foam, DegraPol ® was evaluated with tissue engineered human primary bladder smooth muscle cells. The cell–polymer constructs were characterized by histology, scanning electron microscopy, immunohistochemistry and proliferation assays. Smooth muscle cells grown on DegraPol ® showed the same morphology as when grown on control polystyrene surface. Positive immunostaining with alpha smooth muscle actin indicated the preservation of the specific cell phenotype. Micrographs from scanning electron microscopy showed that the cells grew on the foam surface as well as inside the pores. In addition they grew as cell aggregates within the foam. The smooth muscle cells proliferated on the Degrapol ®; however, proliferation rate decreased due to apoptosis with time in culture. This study showed that Degrapol ® has the potential to be used as a scaffold.

BIOLOGICAL VASCULARIZED MATRIX FOR BLADDER TISSUE ENGINEERING: MATRIX PREPARATION, RESEEDING TECHNIQUE AND SHORT-TERM IMPLANTATION IN A PORCINE MODEL

We generated a vascularized, autologous, reseeded bladder substitute and evaluated immediate vascularization and perfusion of the graft after implantation to the recipient organism in a porcine model. Acellular matrix was processed from porcine small bowel segments by subsequent mechanical, chemical and enzymatic decellularization, preserving the jejunal arteriovenous pedicles. In 2 separate steps the matrix was reseeded with primary bladder smooth muscle cells (SMCs) and urothelial cells (UCs), and its vascular structures were resurfaced with endothelial progenitor cells (EPCs). To evaluate graft perfusion short-term implantation was performed. The acellular scaffold was successfully repopulated with multilayers of ingrowing SMCs and superficial UCs. After reseeding the jejunal arteriovenous pedicles with EPCs and cultivation for 3 weeks the larger vessels as well as the intramural scaffold capillary network were repopulated with cell monolayers expressing endothelial specific proteins. Perfusion stagnation and implant thrombosis occurred within 30 minutes after the implantation of acellular scaffolds not reseeded with EPCs. In the EPC reseeded group the vascular system revealed intact perfusion and no relevant thrombus formation was observed after 1 or 3 hours. The current study of successful SMC and UC reseeding, vessel resurfacing with EPCs and short-term vascular patency represents the promising in vitro and in vivo basis for further evaluation of this biological vascularized matrix in chronic long-term large animal implantation experiments.

Differential expression of SM22 isoforms in myofibroblasts and smooth muscle cells from rabbit bladder.

The E-11 and 1-B8 monoclonal antibodies raised to the smooth muscle (SM)-specific SM22 protein from pig stomach were used to study the in vivo and in vitro phenotypic characteristics of myofibroblasts (MF) and SM cells (SMC) from the bladder detrusor muscle and serosal thickening of male rabbit. The 22-kDa SM22 band found in the SM extract appeared to be composed of distinct isoforms when examined in non-equilibrium two-dimensional gel electrophoresis (2D-EF): alpha (the most basic), beta, gamma, and delta (the most acidic) in the ratio of 34(alpha):23(beta):36(gamma):8(delta). Western blots of 2D-electrophoresed bladder extracts treated with E-11 and 1-B8 showed that alpha, beta, and delta, but not gamma isoforms were labeled with E-11, whereas alpha, beta, and gamma isoforms were stained with 1-B8. This differential immunoreactivity was not influenced by phosphorylation. The tissue distribution of SM22 immunostaining was heterogeneous in the bladder SM and serosal thickening developed as a consequence of partial outflow obstruction of the urinary bladder. At the cellular level, the 1-B8 and E-11 antibodies stained the SMC in a "diffuse" (the whole cytoplasm) and "honeycomb" (the peripheral cytoplasm) manner, whereas MF immunostaining was quite homogeneous. The two antibodies also reacted with cultured primary bladder SMC and MF grown in low serum conditions showing a heterogeneous SM22 cell distribution but an identical subcellular localization, i.e., the actin-containing filamentous network, distinguishable in part from that found in vivo. The immunocytochemical, Western blotting and 2D-EF patterns of MF from thickened serosa indicated that the gamma isoform alone is expressed in this tissue. This SM22 variant appeared before the completion of the cellular transition from MF to fully differentiated SMC. This pattern is reminiscent of bladder ontogenesis where SM22 expression in the developing bladder wall precedes that of SM myosin. Taken together these data suggest that: (i) SM22 isoforms are differently assorted in MF vs. SMC; (ii) SM22 is a SMC-lineage marker inasmuch as its expression occurs in an experimental condition characterized by a time-related cell phenotypic transition from MF to SMC, and (iii) cell conversion ability of serosal cells in the adult might take place via the reactivation of a specific "foetal" gene programme.