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1.  Porcine Bladder Urothelial, Myofibroblast, and Detrusor Muscle Cells: Characterization and ATP Release 
ATP is released from the bladder mucosa in response to stretch, but the cell types responsible are unclear. Our aim was to isolate and characterize individual populations of urothelial, myofibroblast, and detrusor muscle cells in culture, and to examine agonist-stimulated ATP release. Using female pig bladders, urothelial cells were isolated from bladder mucosa following trypsin-digestion of the luminal surface. The underlying myofibroblast layer was dissected, minced, digested, and cultured until confluent (10–14 days). A similar protocol was used for muscle cells. Cultures were used for immunocytochemical staining and/or ATP release investigations. In urothelial cultures, immunoreactivity was present for the cytokeratin marker AE1/AE3 but not the contractile protein α-smooth muscle actin (α-SMA) or the cytoskeletal filament vimentin. Neither myofibroblast nor muscle cell cultures stained for AE1/AE3. Myofibroblast cultures partially stained for α-SMA, whereas muscle cultures were 100% stained. Both myofibroblast and muscle stained for vimentin, however, they were morphologically distinct. Ultrastructural studies verified that the suburothelial layer of pig bladder contained abundant myofibroblasts, characterized by high densities of rough endoplasmic reticulum. Baseline ATP release was higher in urothelial and myofibroblast cultures, compared with muscle. ATP release was significantly stimulated by stretch in all three cell populations. Only urothelial cells released ATP in response to acid, and only muscle cells were stimulated by capsaicin. Tachykinins had no effect on ATP release. In conclusion, we have established a method for culture of three cell populations from porcine bladder, a well-known human bladder model, and shown that these are distinct morphologically, immunologically, and pharmacologically.
PMCID: PMC3113165  PMID: 21713125
ATP; porcine; bladder; urothelium; myofibroblast; detrusor; ultrastructure
2.  Gene expression of muscarinic, tachykinin, and purinergic receptors in porcine bladder: comparison with cultured cells 
Urothelial cells, myofibroblasts, and smooth muscle cells are important cell types contributing to bladder function. Multiple receptors including muscarinic (M3/M5), tachykinin (NK1/NK2), and purinergic (P2X1/P2Y6) receptors are involved in bladder motor and sensory actions. Using female pig bladder, our aim was to differentiate between various cell types in bladder by genetic markers. We compared the molecular expression pattern between the fresh tissue layers and their cultured cell counterparts. We also examined responses to agonists for these receptors in cultured cells. Urothelial, suburothelial (myofibroblasts), and smooth muscle cells isolated from pig bladder were cultured (10–14 days) and identified by marker antibodies. Gene (mRNA) expression level was demonstrated by real-time PCR. The receptor expression pattern was very similar between suburothelium and detrusor, and higher than urothelium. The gene expression of all receptors decreased in culture compared with the fresh tissue, although the reduction in cultured urothelial cells appeared less significant compared to suburothelial and detrusor cells. Cultured myofibroblasts and detrusor cells did not contract in response to the agonists acetylcholine, neurokinin A, and β,γ-MeATP, up to concentrations of 0.1 and 1 mM. The significant reduction of M3, NK2, and P2X1 receptors under culture conditions may be associated with the unresponsiveness of cultured suburothelial and detrusor cells to their respective agonists. These results suggest that under culture conditions, bladder cells lose the receptors that are involved in contraction, as this function is no longer required. The study provides further evidence that cultured cells do not necessarily mimic the actions exerted by intact tissues.
PMCID: PMC3842897  PMID: 24348420
smooth muscle; urothelium; suburothelium; myofibroblasts; muscarinic receptors; tachykinin receptors; purinergic receptors; cell culture
3.  A study of cytokeratin 20 immunostaining in the urothelium of neuropathic bladder of patients with spinal cord injury 
BMC Urology  2002;2:7.
Normal urothelium is characterised by terminally differentiated superficial cells, which express cytokeratin 20 in the cytoplasm. In contrast, cultured human stratified urothelium, which does not undergo complete terminal differentiation of its superficial cells, does not express cytokeratin 20. If spinal cord injury (SCI) affects urothelial differentiation or induces squamous or other metaplastic change undetected by histological analysis, the superficial urothelial cells of the neuropathic bladder might be expected to show absence of immunostaining for cytokeratin 20.
Patients and Methods
We studied immunostaining for cytokeratin 20 in bladder biopsies taken from 63 consecutive SCI patients. Immunostaining was performed on paraffin-embedded tissue using a mouse monoclonal antibody (clone: Ks20.8).
Of 63 biopsies, the epithelium was scarce in two. Eight biopsies showed squamous metaplasia and immunostaining for cytokeratin 20 was absent in all the eight biopsies. Of the remaining 53 cases, in which the umbrella cell layer of the urothelium was intact, immunostaining for cytokeratin 20 was seen only in ten biopsies.
Superficial cells in the transitional epithelium showed immunostaining for cytokeratin 20 in 10 of 53 bladder biopsies taken from SCI patients. The reasons for this could be either that there is an underlying metaplasia or that changes in the neuropathic bladder affect urothelial differentiation. Taken with evidence from other systems, such as loss of cytokeratin 20 expression from static organ cultures of urothelial tissue, this might suggest that other factors, such as impairment of voluntary voiding in SCI patients, could affect expression of markers such as cytokeratin 20.
PMCID: PMC125297  PMID: 12147174
4.  A Novel Model of Urinary Tract Differentiation, Tissue Regeneration, and Disease: Reprogramming Human Prostate and Bladder Cells into Induced Pluripotent Stem Cells 
European Urology  2013;64(5):753-761.
Primary culture and animal and cell-line models of prostate and bladder development have limitations in describing human biology, and novel strategies that describe the full spectrum of differentiation from foetal through to ageing tissue are required. Recent advances in biology demonstrate that direct reprogramming of somatic cells into pluripotent embryonic stem cell (ESC)-like cells is possible. These cells, termed induced pluripotent stem cells (iPSCs), could theoretically generate adult prostate and bladder tissue, providing an alternative strategy to study differentiation.
To generate human iPSCs derived from normal, ageing, human prostate (Pro-iPSC), and urinary tract (UT-iPSC) tissue and to assess their capacity for lineage-directed differentiation.
Design, setting, and participants
Prostate and urinary tract stroma were transduced with POU class 5 homeobox 1 (POU5F1; formerly OCT4), SRY (sex determining region Y)-box 2 (SOX2), Kruppel-like factor 4 (gut) (KLF4), and v-myc myelocytomatosis viral oncogene homolog (avian) (MYC, formerly C-MYC) genes to generate iPSCs.
Outcome measurements and statistical analysis
The potential for differentiation into prostate and bladder lineages was compared with classical skin-derived iPSCs. The student t test was used.
Results and limitations
Successful reprogramming of prostate tissue into Pro-iPSCs and bladder and ureter into UT-iPSCs was demonstrated by characteristic ESC morphology, marker expression, and functional pluripotency in generating all three germ-layer lineages. In contrast to conventional skin-derived iPSCs, Pro-iPSCs showed a vastly increased ability to generate prostate epithelial-specific differentiation, as characterised by androgen receptor and prostate-specific antigen induction. Similarly, UT-iPSCs were shown to be more efficient than skin-derived iPSCs in undergoing bladder differentiation as demonstrated by expression of urothelial-specific markers: uroplakins, claudins, and cytokeratin; and stromal smooth muscle markers: α-smooth-muscle actin, calponin, and desmin. These disparities are likely to represent epigenetic differences between individual iPSC lines and highlight the importance of organ-specific iPSCs for tissue-specific studies.
IPSCs provide an exciting new model to characterise mechanisms regulating prostate and bladder differentiation and to develop novel approaches to disease modelling. Regeneration of bladder cells also provides an exceptional opportunity for translational tissue engineering.
Take Home Message
Induced pluripotent stem cells derived from human prostate and urinary tract cells can differentiate back into prostate and bladder cells. These cells provide a convenient ready-to-access model that offers enormous potential in clinical regenerative medicine and disease modelling.
PMCID: PMC3819995  PMID: 23582880
Prostate; Bladder; Differentiation; Pluripotent; Stem cells; Tissue engineering; Ureter; Urothelium; Androgen receptor; POU5F1 (formerly OCT4); SOX2; KLF4; MYC (formerly cMYC); NANOG
5.  Evaluation of Silk Biomaterials in Combination with Extracellular Matrix Coatings for Bladder Tissue Engineering with Primary and Pluripotent Cells 
PLoS ONE  2013;8(2):e56237.
Silk-based biomaterials in combination with extracellular matrix (ECM) coatings were assessed as templates for cell-seeded bladder tissue engineering approaches. Two structurally diverse groups of silk scaffolds were produced by a gel spinning process and consisted of either smooth, compact multi-laminates (Group 1) or rough, porous lamellar-like sheets (Group 2). Scaffolds alone or coated with collagen types I or IV or fibronectin were assessed independently for their ability to support attachment, proliferation, and differentiation of primary cell lines including human bladder smooth muscle cells (SMC) and urothelial cells as well as pluripotent cell populations, such as murine embryonic stem cells (ESC) and induced pluripotent stem (iPS) cells. AlamarBlue evaluations revealed that fibronectin-coated Group 2 scaffolds promoted the highest degree of primary SMC and urothelial cell attachment in comparison to uncoated Group 2 controls and all Group 1 scaffold variants. Real time RT-PCR and immunohistochemical (IHC) analyses demonstrated that both fibronectin-coated silk groups were permissive for SMC contractile differentiation as determined by significant upregulation of α-actin and SM22α mRNA and protein expression levels following TGFβ1 stimulation. Prominent expression of epithelial differentiation markers, cytokeratins, was observed in urothelial cells cultured on both control and fibronectin-coated groups following IHC analysis. Evaluation of silk matrices for ESC and iPS cell attachment by alamarBlue showed that fibronectin-coated Group 2 scaffolds promoted the highest levels in comparison to all other scaffold formulations. In addition, real time RT-PCR and IHC analyses showed that fibronectin-coated Group 2 scaffolds facilitated ESC and iPS cell differentiation toward both urothelial and smooth muscle lineages in response to all trans retinoic acid as assessed by induction of uroplakin and contractile gene and protein expression. These results demonstrate that silk scaffolds support primary and pluripotent cell responses pertinent to bladder tissue engineering and that scaffold morphology and fibronectin coatings influence these processes.
PMCID: PMC3567020  PMID: 23409160
6.  Human Umbilical Mesenchymal Stem Cells-Seeded Bladder Acellular Matrix Grafts for Reconstruction of Bladder Defects in a Canine Model 
PLoS ONE  2013;8(11):e80959.
The goal of this study was to explore the feasibility of utilizing human umbilical mesenchymal stem cells (HUMSCs)-seeded Bladder acellular matrix graft (BAMG) for bladder reconstruction in a canine model.
Methodology/Principal Findings
HUMSCs were isolated from newborn umbilical cords and identified by flow cytometry. Partial cystectomy was performed in the experimental and control group. Bladder defects were repaired with HUMSCs-BAMG in the experimental group and repaired with unseeded-BAMG in control group. The implanted grafts were harvested after surgery. H&E and immunohistochemistry staining were performed to evaluate the regeneration of the bladder defect. Primary cultured HUMSCs displayed typical fibroblast morphology with spindle-shaped. Flow cytometry indicated that these cells were positive for CD105 (97.3%) and CD44 (99%), but negative for CD34 (2.8%), CD31 (2.1%), and CD45 (1.7%). Immunohistochemistry staining showed that a multilayered urothelium and well-developed smooth muscle were observed at 12 weeks in experiment group. In contrast, multilayered urothelial tissues were also observed at 12 weeks in group B, but well-developed smooth muscle bundles were observed.
Our preliminary results demonstrate that UMSC-seeded BAMGs are superior to unseeded BAMGs to promote the regeneration of bladder defects. Our findings indicated that HUMSCs may be a potential cell source for bladder tissue engineering.
PMCID: PMC3835736  PMID: 24278354
7.  Expression and function of rat urothelial P2Y receptors 
The control and regulation of the lower urinary tract (LUT) is partly mediated by purinergic signaling. This study investigated the distribution and function of P2Y receptors in the rat urinary bladder. Application of P2Y agonists to rat urothelial cells evoked increases in intracellular calcium; the rank order of agonist potency (pEC50 ± S.E.M.) was ATP (5.10 ± 0.07)>UTP (4.91 ± 0.14)>UTPγS (4.61 ± 0.16) = ATPγS (4.70 ± 0.05) > 2MeSADP = NECA = ADP (<3.5). The rank order potency for these agonists indicates that urothelial cells functionally express P2Y2/P2Y4 receptors, with a relative lack of contribution from other P2Y or adenosine receptors. Real-time PCR, western blotting and immunocytochemistry, confirmed the expression of P2Y2, and to a lesser extent P2Y4 in the urothelium. Immunocytochemical studies revealed expression of P2Y2 staining in all layers of the urothelium, with relative absence of P2Y4. P2Y2 staining was also present in sub-urothelial nerve bundles and underlying detrusor smooth muscle. Addition of UTP and UTPγS was found to evoke ATP release from cultured rat urothelial cells. These findings indicate that cultured rat urothelial cells functionally express P2Y2/P2Y4 receptors. Activation of these receptors could have a role in autocrine and paracrine signaling throughout the urothelium. This could lead to the release of bioactive mediators such as additional ATP, nitric oxide and acetylcholine, which can modulate the micturition reflex by acting on sub-urothelial myofibroblasts and/or pelvic afferent fibers.
PMCID: PMC2760272  PMID: 18216148
Purinergic receptors; urinary bladder; epithelium; lower urinary tract
8.  Cytokeratin 15 Marks Basal Epithelia in Developing Ureters and Is Upregulated in a Subset of Urothelial Cell Carcinomas 
PLoS ONE  2013;8(11):e81167.
The mammalian ureter contains a water-tight epithelium surrounded by smooth muscle. Key molecules have been defined which regulate ureteric bud initiation and drive the differentiation of ureteric mesenchyme into peristaltic smooth muscle. Less is known about mechanisms underlying the developmental patterning of the multilayered epithelium characterising the mature ureter. In skin, which also contains a multilayered epithelium, cytokeratin 15 (CK15), an acidic intermediate filament protein, marks cells whose progeny contribute to epidermal regeneration following wounding. Moreover, CK15+ precursor cells in skin can give rise to basal cell carcinomas. In the current study, using transcriptome microarrays of embryonic wild type mouse ureters, Krt15, coding for CK15, was detected. Quantitative polymerase chain reaction analyses confirmed the initial finding and demonstrated that Krt15 levels increased during the fetal period when the ureteric epithelium becomes multilayered. CK15 protein was undetectable in the ureteric bud, the rudiment from which the ureter grows. Nevertheless, later in fetal development, CK15 was immunodetected in a subset of basal urothelial cells in the ureteric stalk. Superficial epithelial cells, including those positive for the differentiation marker uroplakin III, were CK15-. Transformation-related protein 63 (P63) has been implicated in epithelial differentiation in murine fetal urinary bladders. In wild type fetal ureters, CK15+ cells were positive for P63, and p63 homozygous null mutant ureters lacked CK15+ cells. In these mutant ureters, sections of the urothelium were monolayered versus the uniform multilayering found in wild type littermates. Human urothelial cell carcinomas account for considerable morbidity and mortality. CK15 was upregulated in a subset of invasive ureteric and urinary bladder cancers. Thus, in ureter development, the absence of CK15 is associated with a structurally simplified urothelium whereas, postnatally, increased CK15 levels feature in malignant urothelial overgrowth. CK15 may be a novel marker for urinary tract epithelial precursor cells.
PMCID: PMC3832456  PMID: 24260555
9.  Purinergic and muscarinic modulation of ATP release from the urothelium and its paracrine actions 
The urothelium is a newly recognized sensory structure that detects bladder fullness. Pivotal to this sensory role is the release of ATP from the urothelium. However, the routes for urothelial ATP release, its modulation by receptor-mediated pathways, and the autocrine/paracrine role of ATP are poorly understood, especially in native tissue. We examined the action of key neurotransmitters: purinergic and muscarinic agonists on ATP release and its paracrine effect. Guinea pig and human urothelial mucosa were mounted in a perfusion trough; superfusate ATP was measured using a luciferin-luciferase assay, and tissue contractions were recorded with a tension transducer. Intracellular Ca2+ was measured in isolated urothelial cells with fura-2. The P2Y agonist UTP but not the P2X agonist α,β-methylene-ATP generated ATP release. The muscarinic agonist carbachol and the M2-preferential agonist oxotremorine also generated ATP release, which was antagonized by the M2-specific agent methoctramine. Agonist-evoked ATP release was accompanied by mucosal contractions. Urothelial ATP release was differentially mediated by intracellular Ca2+ release, cAMP, exocytosis, or connexins. Urothelium-attached smooth muscle exhibited spontaneous contractions that were augmented by subthreshold concentrations of carbachol, which had little direct effect on smooth muscle. This activity was attenuated by desensitizing P2X receptors on smooth muscle. Urothelial ATP release was increased in aging bladders. Purinergic and muscarinic agents produced similar effects in human urothelial tissue. This is the first demonstration of specific modulation of urothelial ATP release in native tissue by purinergic and muscarinic neurotransmitters via distinct mechanisms. Released ATP produces paracrine effects on underlying tissues. This process is altered during aging and has relevance to human bladder pathologies.
PMCID: PMC3920053  PMID: 24285497
Urothelium; ATP release; paracrine effect; purinergic; muscarinic; neurotransmitters; sensory
10.  Bladder cancer cell in co-culture induces human stem cell differentiation to urothelial cells through paracrine FGF10 signaling 
FGF10 is required for embryonic epidermal morphogenesis including brain development, lung morphogenesis, and initiation of limb bud formation. In this study, we investigated the role of FGF10 as a lead induction factor for stem cell differentiation toward urothelial cell. To this end, human multi-potent stem cell in vitro system was employed. Human amniotic fluid stem cells were co-cultured with immortalized bladder cancer lines to induce directed differentiation into urothelial cells. Urothelial markers, uroplakin II, III and cytokeratin 8, were monitored by RT-PCR, immunocytochemistry and western blot analysis. Co-cultured stem cells began to express uroplakin II, III and cytokeratin 8. Targeted FGF10 gene knock down from bladder cancer cells abolished the directed differentiation. In addition, when FGF10 downstream signaling was blocked with the mek inhibitor, the co-culture system lost the capacity to induce urothelial differentiation. Exogenous addition of recombinant FGF10 protein promoted stem cell differentiation into urothelium cell lineage. Together, this report suggests that paracrine FGF10 signaling stimulates the differentiation of human stem cell into urothelial cells. Current study provides insight into the potential role of FGF10 as a lead growth factor for bladder regeneration and its therapeutic application for bladder transplantation.
PMCID: PMC3951840  PMID: 23949743
Human amniotic fluid stem cell; Fibroblast growth factor 10; Urothelium; Co-culture; Bladder cancer cells
11.  Clonal, Self-Renewing and Differentiating Human and Porcine Urothelial Cells, a Novel Stem Cell Population 
PLoS ONE  2014;9(2):e90006.
Although urothelial progenitor-like cells have been described in the human urinary tract, the existence of stem cells remains to be proven. Using a culture system that favors clonogenic epithelial cell growth, we evaluated and characterized clonal human urothelial cells. We isolated human urothelial cells that were clonogenic, capable of self-renewal and could develop into fully differentiated urothelium once re-implanted into the subcapsular space of nude mice. In addition to final urothelial cell differentiation, spontaneous formation of bladder-like microstructures was observed. By examining an epithelial stem cell signature marker, we found p63 to correlate with the self-renewal capacity of the isolated human urothelial clonal populations. Since a clinically relevant, long-term model for functional reconstitution of human cells does not exist, we sought to establish a culture method for porcine urothelial cells in a clinically relevant porcine model. We isolated cells from porcine ureter, urethra and bladder that were clonogenic and capable of self-renewal and differentiation into fully mature urothelium. In conclusion, we could isolate human and porcine cell populations, behaving as urothelial stem cells and showing clonogenicity, self-renewal and, once re-implanted, morphological differentiation.
PMCID: PMC3935977  PMID: 24587183
12.  Urothelium-derived Sonic hedgehog promotes mesenchymal proliferation and induces bladder smooth muscle differentiation 
Induction of smooth muscle differentiation from bladder mesenchyme depends on signals that originate from the urothelium. We hypothesize Sonic hedgehog (Shh) is the urothelial signal that promotes bladder mesenchymal proliferation and induces bladder smooth muscle differentiation.
Pregnant FVB mice were euthanized on embryonic day (E) 12.5 and fetal bladders were harvested. Two experimental protocols were utilized: Bladder mesenchyme (BLM) was isolated by incubating intact bladders (IB) in 0.02 M EDTA and then removing the urothelium by microdissection. IB and BLM were cultured in Shh-deficient media or BLM was cultured in Shh-supplemented (480 nM) media for 72 h.IB were cultured for 72 h in media containing different concentrations of Shh (0, 48, and 480 nM).
Specimens were sized by serial sectioning. Cell counts were performed after trypsin digestion. Immunohistochemistry was performed to detect smooth muscle-specific protein expression. α-Actin expression was quantified using Western blot.
All specimens were viable at 72 h. BLM cultured without Shh survived but did not grow or undergo smooth muscle differentiation. IB cultured without Shh and BLM cultured with Shh grew and expressed smooth muscle proteins at 72 h. IB cultured with Shh were larger and contained more cells than IB cultured without Shh (all p <0.05). Increasing Shh concentration from 48 to 480 nM did not change bladder size, cell counts, or the level of α-actin expression. Prior to culture, IB did not express α-actin. After culture of IB in Shh-deficient media, α-actin was detected throughout the mesenchyme except in the submucosal layer. The IB submucosa was thinner after culture with 48 nM Shh and smooth muscle completely obliterated the submucosa after culture with 480 nM Shh.
In fetal mouse bladders, urothelium-derived Shh is necessary for mesenchymal proliferation and smooth muscle differentiation. Shh concentration affects mesenchymal proliferation and patterning of bladder smooth muscle.
PMCID: PMC3712847  PMID: 20227816
Bladder; Urothelium; Sonic hedgehog; Smooth muscle differentiation
13.  P2X2 and P2X3 Receptor Expression in Postnatal and Adult Rat Urinary Bladder and Lumbosacral Spinal Cord 
P2X receptors mediate the effects of ATP in micturition and nociception. During postnatal maturation, a spinobulbospinal reflex and voluntary voiding replace primitive voiding reflexes. This may involve changes in neuroactive compounds and receptors in bladder reflex pathways. We examined P2X2 and P2X3 receptors in bladder and spinal cord from postnatal (P0-P36) and adult Wistar rats. Western blot of whole bladders for P2X2 and P2X3 expression was performed. Immunostaining for P2X2 and P2X3 receptors in urothelium and detrusor smooth muscle whole-mounts and spinal cord sections was examined. Western blot demonstrated an age-dependent decrease (R2= 0.96, p ≤ 0.005) in P2X2 receptor expression in bladder whereas P2X3 receptor expression in bladder peaked (p ≤ 0.005) from P14-P21. P2X2-immunoreactivity (IR) was present in urothelial cells, suburothelial plexus, detrusor smooth muscle and serosa at birth with staining in urothelial cells and serosa being most predominant. With increasing postnatal age, the intensity of P2X2-IR decreased in urothelial cells but increased in suburothelial plexus. P2X3-IR increased in urothelial cells and suburothelial plexus with postnatal age whereas staining in detrusor and serosa remained relatively constant. At birth, P2X3-IR was present in the dorsal horn (DH), lateral collateral pathway (LCP), and dorsal commissure. With increasing age, P2X3-IR was restricted to superficial DH and LCP. P2X2-IR was present in ependyme cells (S-100-IR) of the central canal as early as P2. These studies demonstrate plastic expression of P2X2 and P2X3 receptors in bladder and spinal cord during early postnatal development at times coincident with appearance of mature voiding patterns.
PMCID: PMC1305916  PMID: 15947072
postnatal development; micturition reflexes; sacral parasympathetic nucleus; dorsal commissure; dorsal horn
14.  Cajal–like interstitial cells as a novel target in detrusor overactivity treatment: true or myth? 
The Cajal–like intestitial cells (ICCs) act as a pacemaker and are responsible for generating smooth muscle activity in the gastrointestinal tract (GI). Interstitial cells that resemble ICCs in the GI have been identified in the urinary bladder.
Materials and methods
This review is based on a systemic literature research. The medline/pubmed, scopus, embase, and Web of Science databases were browsed in order to identify original and review articles, as well as editorials relating to cajal–like cells, urinary bladder, detrusor overactivity, overactive bladder, glivec, etc. The controlled vocabulary of the Medical Subject Headings (MeSH) database was used to ensure the sensitivity of the searches. 40 papers met the criteria and were used for this review.
Cajal cells lie in close proximity to the muscle cells, autonomic nerve endings, and urothelial cells. There is increasing evidence that ICCs play role in urinary tract dysfunction development (e.g. detrusor overactivity, primary obstructive megaureter, congenital ureteropelvic junction obstruction, etc.). ICCs may be responsible for generating electrical potentials and induction of detrusor muscle contractions. Novel pathomechanisms of detrusor overactivity development have been postulated, as follows: 1) the disturbance of spontaneous contractility caused by altered signal transduction of ICCs between nerves and detrusor muscle cells, and 2). the alteration in signal transduction between urothelium and afferent nerve endings via suburothelial ICCs. The c–kit receptor is not only a detection marker of these cells, but may also play a crucial role in the control of bladder function.
Cajal cells in urinary bladder suggest that the c–kit receptor may provide a novel target for treating detrusor overactivity. The review presents the current knowledge of ICCs, its role in urinary bladder function, and potential novel therapeutic strategy.
PMCID: PMC3992455  PMID: 24757530
cajal cells; overactive bladder; treatment; glivec
15.  Substratum Topography Modulates Corneal Fibroblast to Myofibroblast Transformation 
These findings clearly demonstrate that substratum nanotopography inhibits TGFβ-induced corneal myofibroblast differentiation and suggests that structural features of this scale help to stabilize the keratocyte and fibroblast phenotypes in vivo.
The transition of corneal fibroblasts to the myofibroblast phenotype is known to be important in wound healing. The purpose of this study was to determine the effect of topographic cues on TGFβ-induced myofibroblast transformation of corneal cells.
Rabbit corneal fibroblasts were cultured on nanopatterned surfaces having topographic features of varying sizes. Cells were cultured in media containing TGFβ at concentrations ranging from 0 to 10 ng/mL. RNA and protein were collected from cells cultured on topographically patterned and planar substrates and analyzed for the myofibroblast marker α-smooth muscle actin (αSMA) and Smad7 expression by quantitative real time PCR. Western blot and immunocytochemistry analysis for αSMA were also performed.
Cells grown on patterned surfaces demonstrated significantly reduced levels of αSMA (P < 0.002) compared with planar surfaces when exposed to TGFβ; the greatest reduction was seen on the 1400 nm surface. Smad7 mRNA expression was significantly greater on all patterned surfaces exposed to TGFβ (P < 0.002), whereas cells grown on planar surfaces showed equal or reduced levels of Smad7. Western blot analysis and αSMA immunocytochemical staining demonstrated reduced transition to the myofibroblast phenotype on the 1400 nm surface when compared with cells on a planar surface.
These data demonstrate that nanoscale topographic features modulate TGFβ-induced myofibroblast differentiation and αSMA expression, possibly through upregulation of Smad7. It is therefore proposed that in the wound environment, native nanotopographic cues assist in stabilizing the keratocyte/fibroblast phenotype while pathologic microenvironmental alterations may be permissive for increased myofibroblast differentiation and the development of fibrosis and corneal haze.
PMCID: PMC3317421  PMID: 22232431
16.  Urothelial beta3 adrenergic receptors in the rat bladder 
Neurourology and Urodynamics  2011;30(1):144-150.
To investigate the distribution of beta-3 adrenergic receptors (β3ARs) in the rat bladder and to examine the contribution of urothelial β3ARs to agonist-induced suppression of bladder reflexes and relaxation of smooth muscle.
Bladder tissue was collected from 8-10 months old female SD rats. In some samples, the urothelium was surgically separated from the smooth muscle. The expression and localization of βAR mRNA and β3AR protein were determined using RT-PCR and immmunohistochemistry. Contractile responses to the specific β3AR agonists TAK-677 and BRL37344 were measured in bladder strips with or without the urothelium. The contribution of urothelial β3AR to the micturition reflex was assessed in continuous cystometry in urethane anesthetized rats using intravesical delivery of β3AR agonists.
RT-PCR detected mRNA of all βARs in urothelium and smooth muscle. Immunostaining detected β3ARs throughout the urothelium, in the smooth muscle, myofibroblast-like cells, and in the peripheral nerves. Ovariectomy did not change the distribution of β3ARs in any bladder structure. Intravesical administration of TAK-677 and BRL37344 (1 - 5×10-4 M) decreased voiding frequency and amplitude of bladder contractions. In bladder strips in vitro both β3AR agonists (10-12 - 10-4 M) relaxed the smooth muscle in a concentration-dependent manner to the same extent in strips with and without the urothelium.
In addition to their presence in bladder smooth muscle, β3ARs are present in the urothelium where their activation may alter reflex voiding via release of factor(s) that act on non-myocyte structures including the afferent and/or efferent nerves to influence bladder contractility.
PMCID: PMC3395322  PMID: 21046653
urethane anesthesia; bladder strips; immunohistochemistry; cystometry; intravesical application; RT-PCR; BRL37344; TAK-677
17.  Role of gap junctions in spontaneous activity of the rat bladder 
Increased gap junction expression in lamina propria myofibroblasts and urothelial cells may be involved in detrusor overactivity, leading to incontinence. Immunohistochemistry was used to compare connexin (Cx) 26, 43, and 45 expression in the bladders of neonatal, adult, and spinal cord-transected rats, while optical imaging was used to map the spread of spontaneous activity and the effects of gap junction blockade. Female adult Sprague-Dawley rats were deeply anesthetized, a laminectomy was performed, and the spinal cord was transected (T8/T9). After 14 days, their bladders and those of age-matched adults (4 mo old) and neonates (7-21 day old) were excised and studied immunohistochemically using frozen sections or optically using whole bladders stained with voltage- and Ca2+-sensitive dyes. The expression of Cx26 was localized to the urothelium, Cx43 to the lamina propria myofibro-blasts, and Cx45 to the detrusor smooth muscle. While the expression of Cx45 was comparable in all bladders, the expression of Cx43 and Cx26 was increased in neonate and transected animals. In the bladders of adults, spontaneous activity was initiated at multiple sites, resulting in a lack of coordination. Alternatively, in neonate and transected animals spontaneous activity was initiated at a focal site near the dome and spread in a coordinated fashion throughout the bladder. Gap junction blockade (18β-glycyrrhetinic acid, 1 μM) abolished this coordinated activity but had no effect on the uncoordinated activity in adult bladders. These data suggest that coordinated spontaneous activity requires gap junction upregulation in urothelial cells and lamina propria myofibroblasts.
PMCID: PMC3037091  PMID: 17581924
lamina propria myofibroblasts; optical mapping; urothelium; voltage-and Ca2+-sensitive dyes
18.  P53 and Cancer-Associated Sialylated Glycans Are Surrogate Markers of Cancerization of the Bladder Associated with Schistosoma haematobium Infection 
Bladder cancer is a significant health problem in rural areas of Africa and the Middle East where Schistosoma haematobium is prevalent, supporting an association between malignant transformation and infection by this blood fluke. Nevertheless, the molecular mechanisms linking these events are poorly understood. Bladder cancers in infected populations are generally diagnosed at a late stage since there is a lack of non-invasive diagnostic tools, hence enforcing the need for early carcinogenesis markers.
Methodology/Principal Findings
Forty-three formalin-fixed paraffin-embedded bladder biopsies of S. haematobium-infected patients, consisting of bladder tumours, tumour adjacent mucosa and pre-malignant/malignant urothelial lesions, were screened for bladder cancer biomarkers. These included the oncoprotein p53, the tumour proliferation rate (Ki-67>17%), cell-surface cancer-associated glycan sialyl-Tn (sTn) and sialyl-Lewisa/x (sLea/sLex), involved in immune escape and metastasis. Bladder tumours of non-S. haematobium etiology and normal urothelium were used as controls. S. haematobium-associated benign/pre-malignant lesions present alterations in p53 and sLex that were also found in bladder tumors. Similar results were observed in non-S. haematobium associated tumours, irrespectively of their histological nature, denoting some common molecular pathways. In addition, most benign/pre-malignant lesions also expressed sLea. However, proliferative phenotypes were more prevalent in lesions adjacent to bladder tumors while sLea was characteristic of sole benign/pre-malignant lesions, suggesting it may be a biomarker of early carcionogenesis associated with the parasite. A correlation was observed between the frequency of the biomarkers in the tumor and adjacent mucosa, with the exception of Ki-67. Most S. haematobium eggs embedded in the urothelium were also positive for sLea and sLex. Reinforcing the pathologic nature of the studied biomarkers, none was observed in the healthy urothelium.
This preliminary study suggests that p53 and sialylated glycans are surrogate biomarkers of bladder cancerization associated with S. haematobium, highlighting a missing link between infection and cancer development. Eggs of S. haematobium express sLea and sLex antigens in mimicry of human leukocytes glycosylation, which may play a role in the colonization and disease dissemination. These observations may help the early identification of infected patients at a higher risk of developing bladder cancer and guide the future development of non-invasive diagnostic tests.
Author Summary
Epidemiological studies associate infection with S. haematobium, an endemic parasitic flatworm in Africa and the Middle East, with the development of bladder cancer. Nevertheless, little molecular evidence exists supporting this association. This work draws attention to the common molecular pathways underlying these two events, highlighting a potentially unreported link between infection and cancer development. It has been demonstrated that a panel of biomarkers commonly associated with aggressive forms of bladder cancer is also present in non-malignant tissues infected with the parasite. This may offer a means of early identification of people with this parasitic infection who are at risk of developing of bladder cancer, and may guide the establishment of non-invasive diagnostic tests. Furthermore, we observed that parasite eggs mimic the molecular nature of human cells, providing a possible mechanism of immune escape and persistent infection. Such knowledge is considered pivotal to develop novel therapeutic strategies.
PMCID: PMC4263606  PMID: 25502795
19.  Heparin-binding EGF-like growth factor is an autocrine growth factor for human urothelial cells and is synthesized by epithelial and smooth muscle cells in the human bladder. 
Journal of Clinical Investigation  1997;99(5):1028-1036.
The epidermal growth factor receptor (HER1) has been implicated in regenerative growth and proliferative diseases of the human bladder epithelium (urothelium), however a cognate HER1 ligand that can act as a growth factor for normal human urothelial cells (HUC) has not been identified. Here we show that heparin-binding EGF-like growth factor (HB-EGF), an activating HER1 ligand, is an autocrine regulator of HUC growth. This conclusion is based on demonstration of HB-EGF synthesis and secretion by primary culture HUC, identification of HER1 as an activatable HB-EGF receptor on HUC surfaces, stimulation of HUC clonal growth by HB-EGF, inhibition of HB-EGF-stimulated growth by heparin and of log-phase growth by CRM 197, a specific inhibitor of HB-EGF/HER1 interaction, and identification of human urothelium as a site of HB-EGF precursor (proHB-EGF) synthesis in vivo. ProHB-EGF expression was also detected in the vascular and detrusor smooth muscle of the human bladder. These data suggest a physiologic role for HB-EGF in the regulation of urothelial proliferation and regeneration subsequent to mucosal injury. Expression of proHB-EGF is also a feature of differentiated vascular and detrusor smooth muscle in the bladder. Because proHB-EGF is known to be the high affinity diphtheria toxin (DT) receptor in human cells, synthesis of the HB-EGF precursor by human urothelium also suggests the possibility of using the DT-binding sites of proHB-EGF as an in vivo target for the intraluminal treatment of urothelial diseases.
PMCID: PMC507911  PMID: 9062361
20.  Characterization of the Early Proliferative Response of the Rodent Bladder to Subtotal Cystectomy: A Unique Model of Mammalian Organ Regeneration 
PLoS ONE  2012;7(10):e47414.
Subtotal cystectomy (STC; surgical removal of ∼75% of the rat urinary bladder) elicits a robust proliferative response resulting in complete structural and functional bladder regeneration within 8-weeks. The goal of these studies was to characterize the early cellular response that mediates this regenerative phenomenon, which is unique among mammalian organ systems. STC was performed on eighteen 12-week-old female Fischer F344 rats. At 1, 3, 5 and 7-days post-STC, the bladder was harvested 2-hours after intraperitoneal injection of bromodeoxyuridine (BrdU). Fluorescent BrdU labeling was quantified in cells within the urothelium, lamina propria (LP), muscularis propria (MP) and serosa. Cell location was confirmed with fluorescently co-labeled cytokeratin, vimentin or smooth muscle actin (SMA), to identify urothelial, interstitial and smooth muscle cells, respectively. Expression of sonic hedgehog (Shh), Gli-1 and bone morphogenic factor-4 (BMP-4) were evaluated with immunochemistry. Three non-operated rats injected with BrdU served as controls. Less than 1% of cells in the bladder wall were labeled with BrdU in control bladders, but this percentage significantly increased by 5-8-fold at all time points post-STC. The spatiotemporal characteristics of the proliferative response were defined by a significantly higher percentage of BrdU-labeled cells within the urothelium at 1-day than in the MP and LP. A time-dependent shift at 3 and 5-days post-STC revealed significantly fewer BrdU-labeled cells in the MP than LP or urothelium. By 7-days the percentage of BrdU-labeled cells was similar among urothelium, LP and MP. STC also caused an increase in immunostaining for Shh, Gli-1 and BMP-4. In summary, the early stages of functional bladder regeneration are characterized by time-dependent changes in the location of the proliferating cell population, and expression of several evolutionarily conserved developmental signaling proteins. This report extends previous observations and further establishes the rodent bladder as an excellent model for studying novel aspects of mammalian organ regeneration.
PMCID: PMC3470577  PMID: 23077610
21.  β–Adrenergic receptor subtype expression in myocyte and non-myocyte cells in human female bladder 
Cell and tissue research  2010;342(2):295-306.
β3-Adrenergic receptor agonists are currently under clinical development for the treatment of overactive bladder, a condition that is prevalent in postmenopausal women. These agents purportedly relax bladder smooth muscle through a direct action at the myocyte β3-receptor. The aim of this study was to examine the expression of the individual beta-adrenergic receptors in full thickness sections from ageing human female bladder. We obtained a series of rabbit polyclonal antibodies generated against each of the three β-adrenergic receptors, and validated their receptor specificity in CHOK1 cells expressing each of the individual receptors. Immunostaining for β1, β2, and β3 were each more prominent in the urothelium than in the detrusor, with all receptors expressed in the same cell types, indicating co-expression of all three receptors throughout the urothelium in addition to the detrusor. Staining of all receptors was also observed in suburothelial myofibroblast-like cells, intramural ganglion cells, and in Schwann cells of intramural nerves. The β3-receptor in the human urothelium appears to be functional, as two different selective β3-receptor agonists, TAK677 and BRL37344, stimulate cAMP formation in UROtsa cells. Densitometry analysis indicates a persistent expression of all receptors throughout the bladder with increasing age, with the exception of the β2-receptor in the urothelium of the trigone, which appears to decrease slightly in older women. These data indicate that β3-receptor expression is maintained with age, but may function in concert with other β-receptors. Activation of the myocyte receptor may be influenced by action on non-myocyte structures including the intramural ganglion cells and myofibroblasts.
PMCID: PMC3113530  PMID: 20953633
Overactive bladder; β-adrenergic receptors; Immunohistochemistry; Urothelium; Age
22.  Cytokine Effects on Gap Junction Communication and Connexin Expression in Human Bladder Smooth Muscle Cells and Suburothelial Myofibroblasts 
PLoS ONE  2011;6(6):e20792.
The last decade identified cytokines as one group of major local cell signaling molecules related to bladder dysfunction like interstitial cystitis (IC) and overactive bladder syndrome (OAB). Gap junctional intercellular communication (GJIC) is essential for the coordination of normal bladder function and has been found to be altered in bladder dysfunction. Connexin (Cx) 43 and Cx45 are the most important gap junction proteins in bladder smooth muscle cells (hBSMC) and suburothelial myofibroblasts (hsMF). Modulation of connexin expression by cytokines has been demonstrated in various tissues. Therefore, we investigate the effect of interleukin (IL) 4, IL6, IL10, tumor necrosis factor-alpha (TNFα) and transforming growth factor-beta1 (TGFβ1) on GJIC, and Cx43 and Cx45 expression in cultured human bladder smooth muscle cells (hBSMC) and human suburothelial myofibroblasts (hsMF).
Methodology/Principal Findings
HBSMC and hsMF cultures were set up from bladder tissue of patients undergoing cystectomy. In cytokine stimulated cultured hBSMC and hsMF GJIC was analyzed via Fluorescence Recovery after Photo-bleaching (FRAP). Cx43 and Cx45 expression was assessed by quantitative PCR and confocal immunofluorescence. Membrane protein fraction of Cx43 and Cx45 was quantified by Dot Blot. Upregulation of cell-cell-communication was found after IL6 stimulation in both cell types. In hBSMC IL4 and TGFβ1 decreased both, GJIC and Cx43 protein expression, while TNFα did not alter communication in FRAP-experiments but increased Cx43 expression. GJ plaques size correlated with coupling efficacy measured, while Cx45 expression did not correlate with modulation of GJIC.
Our finding of specific cytokine effects on GJIC support the notion that cytokines play a pivotal role for pathophysiology of OAB and IC. Interestingly, the effects were independent from the classical definition of pro- and antiinflammatory cytokines. We conclude, that connexin regulation involves genomic and/or post-translational events, and that GJIC in hBSMC and hsMF depend of Cx43 rather than on Cx45.
PMCID: PMC3107230  PMID: 21674053
23.  Roles of estrogen receptor α and β in modulating urothelial cell proliferation 
Endocrine-related cancer  2008;15(1):351-364.
We reported previously that both subtypes of estrogen receptors, ERα and ERβ, are expressed by human urothelial cells and mediate estrogen-induced cell proliferation in these cells. The aim of this study was to determine the extent to which each ER subtype contributes to urothelial cell proliferation and their possible involvement in the regulation of the cell cycle. We compared the expression of ERα and ERβ mRNAs and protein quantitatively in primarily cultured human bladder urothelial cells obtained from six individuals with three immortalized urothelial (E6, E7, and UROtsa) and two bladder cancer cell lines (HTB-9 and T24). We found that all these cells express similar levels of ERβ, but immortalized and cancer cells express much higher amounts of ERα than primary cells. Higher levels of ERα mRNA were also observed in the biopsies of bladder transitional cell carcinoma compared with sample from the same bladder unaffected by tumor. Using the ERα-selective agonist PPT, the ERβ-selective agonist DPN, and specific small interfering RNA against ERα or ERβ, we found that ERβ predominantly mediates estrogen-induced G1/S transition and cell proliferation in the primary urothelial cells. By contrast, ERα predominantly mediates estrogen-induced G1/S transition and cell proliferation in bladder cancer cell lines. Furthermore, we found that 17β-estradiol (E2) rapidly induces phosphorylation of extracellular signal-regulated kinases, but U0126, a mitogen-activated protein kinase kinase (MEK) inhibitor, does not affect E2-induced urothelial cell proliferation. E2 up-regulated cyclin D1 and cyclin E expression in both the primary and bladder cancer cells, and the cancer cells have higher cyclin D1 and cyclin E expression during G0/G1 phases. Our data suggest that estrogen exerts its effects through different ER subtypes in urothelial cells. Increased expression of ERα may contribute to early induction of cyclin D1 and cyclin E during the cell cycle in bladder cancer cells.
PMCID: PMC3513362  PMID: 18310301
24.  Telocytes subtypes in human urinary bladder 
Urinary bladder voiding is a complex mechanism depending upon interplay among detrusor, urothelium, sensory and motor neurons and connective tissue cells. The identity of some of the latter cells is still controversial. We presently attempted to clarify their phenotype(s) in the human urinary bladder by transmission electron microscopy (TEM) and immunohistochemistry. At this latter aim, we used CD34, PDGFRα, αSMA, c-Kit and calreticulin antibodies. Both, TEM and immunohistochemistry, showed cells that, sharing several telocyte (TC) characteristics, we identified as TC; these cells, however, differed from each other in some ultrastructural features and immunolabelling according to their location. PDGFRα/calret-positive, CD34/c-Kit-negative TC were located in the sub-urothelium and distinct in two subtypes whether, similarly to myofibroblasts, they were αSMA-positive and had attachment plaques. The sub-urothelial TC formed a mixed network with myofibroblasts and were close to numerous nerve endings, many of which nNOS-positive. A third TC subtype, PDGFRα/αSMA/c-Kit-negative, CD34/calret-positive, ultrastructurally typical, was located in the submucosa and detrusor. Briefly, in the human bladder, we found three TC subtypes. Each subtype likely forms a network building a 3-D scaffold able to follow the bladder wall distension and relaxation and avoiding anomalous wall deformation. The TC located in the sub-urothelium, a region considered a sort of sensory system for the micturition reflex, as forming a network with myofibroblasts, possessing specialized junctions with extracellular matrix and being close to nerve endings, might have a role in bladder reflexes. In conclusions, the urinary bladder contains peculiar TC able to adapt their morphology to the organ activity.
PMCID: PMC4244015  PMID: 25139461
lamina propria; sub-urothelium and submucosa; detrusor; mast cells; myofibroblasts; CD34; PDGFRα; c-Kit; αSMA; calreticulin
25.  Loss of the Urothelial Differentiation Marker FOXA1 Is Associated with High Grade, Late Stage Bladder Cancer and Increased Tumor Proliferation 
PLoS ONE  2012;7(5):e36669.
Approximately 50% of patients with muscle-invasive bladder cancer (MIBC) develop metastatic disease, which is almost invariably lethal. However, our understanding of pathways that drive aggressive behavior of MIBC is incomplete. Members of the FOXA subfamily of transcription factors are implicated in normal urogenital development and urologic malignancies. FOXA proteins are implicated in normal urothelial differentiation, but their role in bladder cancer is unknown. We examined FOXA expression in commonly used in vitro models of bladder cancer and in human bladder cancer specimens, and used a novel in vivo tissue recombination system to determine the functional significance of FOXA1 expression in bladder cancer. Logistic regression analysis showed decreased FOXA1 expression is associated with increasing tumor stage (p<0.001), and loss of FOXA1 is associated with high histologic grade (p<0.001). Also, we found that bladder urothelium that has undergone keratinizing squamous metaplasia, a precursor to the development of squamous cell carcinoma (SCC) exhibited loss of FOXA1 expression. Furthermore, 81% of cases of SCC of the bladder were negative for FOXA1 staining compared to only 40% of urothelial cell carcinomas. In addition, we showed that a subpopulation of FOXA1 negative urothelial tumor cells are highly proliferative. Knockdown of FOXA1 in RT4 bladder cancer cells resulted in increased expression of UPK1B, UPK2, UPK3A, and UPK3B, decreased E-cadherin expression and significantly increased cell proliferation, while overexpression of FOXA1 in T24 cells increased E-cadherin expression and significantly decreased cell growth and invasion. In vivo recombination of bladder cancer cells engineered to exhibit reduced FOXA1 expression with embryonic rat bladder mesenchyme and subsequent renal capsule engraftment resulted in enhanced tumor proliferation. These findings provide the first evidence linking loss of FOXA1 expression with histological subtypes of MIBC and urothelial cell proliferation, and suggest an important role for FOXA1 in the malignant phenotype of MIBC.
PMCID: PMC3349679  PMID: 22590586

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