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At least 19 recordsLinked to original sources
PubMed1982

Urothelium-specific antibody and lectin surface mapping of bladder urothelium.

Coupled ligand-colloidal gold complexes were found to provide a convenient approach for the localization of scanning electron microscopy of cell surface membrane antigens and lectin-binding sites on bladder urothelium and for the immunocytochemical identification of urothelial cell populations at different stages of differentiation. The ligands used to prove the membrane were a urothelium-specific rabbit antibody raised to a urothelial membrane-associated antigen (UMA), and two lectins: Concanavalin A (Con A) and peanut agglutinin (PNA). A complex luminal surface distribution pattern was demonstrated by the UMA antigen related to the stage of urothelial cell maturation and differentiation. UMA could be detected on the surface of immature and early differentiating intermediate cells, but was absent from the late differentiation stage, becoming re-expressed as the cells matured and was found in greatest abundance on the terminally differentiated superficial cells. It was absent on cells in benign hyperplasia of the urothelium. Cellular and regional differences in lectin binding to the urothelial cell surface was suggested with Con A receptors localized uniformly over the superficial cells, and PNA receptors confined to linear arrays or occasional clusters over the apical surface but evenly dispersed over the lateral surface of these cells.

Animals↗
PubMed1989

The morphology of normal human bladder urothelium.

A comprehensive study of human bladder urothelium was undertaken to define the normal histological and fine structural features of this tissue. Urothelial biopsies from consenting male and female patients undergoing diagnostic or review cystoscopies were analysed. In 31 patients there was an apparently normal urothelium lining the bladder, and in 3 patients the trigone appeared normal. Normal urothelium was not observed to contain lymphocytes, lymphoid follicles, mast cells, Brunn's nests or cysts. No mitoses were seen despite examining about 50,000 urothelial cells. Trigonal and bladder urothelium normally consisted of 3 and 3-6 cell layers, respectively, but they shared the same basic architecture of basal, intermediate and superficial (or surface) cell types. The urothelium possessed a regular, polarised architecture of increasing morphological complexity and differentiation from base to surface. Occasional, slender, cytoplasmic projections were observed to reach the basal lamina from the intermediate cell layer, but not from the surface cell layer. Human urothelium should therefore be considered a stratified, not a pseudostratified, epithelium. The nuclear shape in cross-section was indented in the basal layer, and rounded in the superficial layer. Correspondingly, chromatin configurations of urothelial nuclei were evenly and finely granular in the superficial layer and condensed in the basal layer, suggesting a greater degree of transcriptional activity in the former. Intermediate cell nuclei assumed intermediate degrees of shape and chromatin configuration. Prominent nucleoli were found in the nuclei of all cell layers. Both basal and intermediate cell nuclei and superficial cell nuclei contained characteristic nuclear bodies. Urothelial cells of all layers were connected by interdigitations of cytoplasmic processes and by desmosomes. Clusters of mitochondria were seen throughout the urothelium. Elaborate Golgi membranes and rough endoplasmic reticulum, although rare in the basal layer, were observed in the remainder of the urothelium. Large, prominent lysosomes were identified with the electron microscope and histochemically in the surface layer. The superficial aspect of the urothelium was lined, at least in some regions, by an asymmetric luminal membrane. Tight junctions linked adjacent urothelial surface cells. Such junctions were not observed anywhere else in the urothelium. Fine cytoplasmic filaments, probably of the intermediate type, were most conspicuous in the surface layer. Overall, normal human bladder urothelium is arranged in increasing complexity from base to surface.

Adolescent↗
PubMed2006

Bladder tissue formation from cultured bladder urothelium.

Tissue recombination is a powerful method to evaluate the paracrine-signaling events that orchestrate the development of organs using the in vivo environment of a host rodent. Studies have reported the successful generation of primary cultures of rodent bladder urothelium, but none have reported their use to recapitulate bladder tissue with tissue recombination. We propose that primary cultured bladder urothelium, when recombined with inductive embryonic bladder mesenchyme, will form bladder tissue in a recombination model. Adult rat bladders were isolated and urothelium obtained. Sheets of bladder urothelium were re-suspended in collagen and maintained in tissue culture. After expansion (>20 passages), the urothelium was recombined with embryonic day-14 mouse bladder mesenchyme, then grafted beneath the renal capsule of immunocompromised mouse hosts. Grafts were harvested after 28 days. Control grafts were performed with bladder mesenchyme alone, cultured bladder urothelium alone, and collagen matrix alone. Final tissues were evaluated with staining and immunohistochemistry (H&E, Gomori's trichrome, broad-spectrum uroplakin, and smooth muscle actin alpha and gamma). Immunocytochemistry on cultured urothelium for broad-spectrum keratin, vimentin, and broad-spectrum uroplakin confirmed pure populations, void of mesenchymal contaminants. Staining of recombinant grafts demonstrated bladder tissue with mature urothelium and stromal differentiation. Control tissues were void of bladder tissue formation. We have successfully demonstrated that a chimeric bladder is formed from primary cultured bladder urothelium recombined with embryonic bladder mesenchyme. This is a powerful new tool for investigating the molecular mechanisms of bladder development and disease. Future applications may include the in vitro genetic manipulation of urothelium and examining those effects on growth and development in an in vivo environment.

Animals↗
PubMed2003

Targeting urothelium: ex vivo assay standardization and selection of internalizing ligands.

PURPOSE: With the goal of targeting the human bladder using phage display technology we designed and tested a tissue binding assay on intact urothelium ex vivo. This approach may form the molecular basis for clinical development of peptide or peptidomimetic guided intravesical compounds. MATERIALS AND METHODS: We screened 2 phage display random peptide libraries on human urothelium. Select peptides were tested for their binding ability to human urothelium, 2 human transitional cell carcinoma cell lines and a nontransitional cell carcinoma cell line. Next we standardized an ex vivo binding assay, validated binding of selected phage to whole urothelium, and evaluated whether receptor mediated internalization into urothelium derived cells occurred. Finally we tested if the presence of the glycosaminoglycan layer had any effect on the binding of the urothelium targeted phage. RESULTS: Phage selected and recovered in the screening were isolated and sequenced. Displayed peptide sequences were searched against online protein databases. Five classes of peptide motifs were characterized based on their ability to bind to normal urothelium but not to control cell lines. Remarkable consistency and reproducibility were observed in the ex vivo binding assays. Two classes of peptide motifs sharing the sequence Ile/Leu-Ser-Gly-Leu bound to normal urothelium and to 2 transitional cell carcinoma cells but not to nontransitional cell carcinoma cells in a glycosaminoglycan independent manner and mediated internalization into cells of urothelial origin. CONCLUSIONS: We introduce a strategy for screening combinatorial peptide libraries on bladder mucosa, a standard model for ex vivo intact urothelium binding assays and a panel of urothelium binding peptides that may be suitable for translation into targeted intravesical therapy applications.

Carcinoma, Transitional Cell↗
PubMed1996

A pharmacological and histochemical study of hamster urethra and the role of urothelium.

1. Electrical field stimulation (EFS) of circular strips of hamster proximal urethra caused frequency-dependent relaxations at raised tone. Phentolamine (10(-6) M), propranolol (10(-6) M) and atropine (10(-6) M) were present throughout the experiment. Neurogenic relaxation was attenuated by L-NG-nitroarginine methyl ester (L-NAME) (10(-4) M), was restored by L-arginine (3 x 10(-3) M) but not by D-arginine (3 x 10(-3) M) and completely blocked by tetrodotoxin (10(-6) M). Neurogenic relaxation was also reduced by suramin (10(-4) M) and totally blocked by suramin together with L-NAME. Strips of hamster urethra devoid of urothelium showed little, if any, relaxant response to EFS. 2. An immunohistochemical study showed nitric oxide synthase-immunoreactive nerves in the smooth muscle layers and in the lamina propria, just beneath the urothelium, but no nitric oxide synthase (NOS) staining in the urothelial layer. 3. Noradrenaline elicited a significantly greater contraction in strips without urothelium than in control strips. L-NAME (10(-4) M) did not affect noradrenaline-induced contraction in both control and urothelium-free strips. The contractile response to acetylcholine was not dependent on the presence or absence of urothelium. Nevertheless the response induced by exogenous acetylcholine (10(-3) M) was increased by L-NAME (10(-4) M), both in intact and in urothelium-free strips. 4. Prostaglandin E2 (10(-8)-5 x 10(-6) M) and 2-methyl-thio-ATP (10(-9)-10(-5) M) relaxed proximal urethra. Suramin (10(-4) M) significantly inhibited the relaxation induced by 2-methyl-thio-ATP. The amplitude of these responses was not significantly different between intact and urothelium-free strips and was not blocked by L-NAME (10(-4) M). 5. These results suggest that nitric oxide (NO) is the principal transmitter involved in the non-adrenergic, non-cholinergic (NANC) relaxation of hamster proximal urethra possibly together with another inhibitory transmitter released from nerves. NO can be released from nerves located in the circular smooth muscle layer and in the lamina propria rather than in the urothelium. The reduced neurogenic relaxation in urothelium-free preparations suggests that a NO-dependent inhibitory factor is released from the urothelium. In addition, ATP and prostaglandin E2 may be involved, together with NO, in the urethra during micturition.

Animals↗
PubMed2000

In vitro engineering of human stratified urothelium: analysis of its morphology and function.

PURPOSE: Gastric or intestinal patches, commonly used for reconstructive cystoplasty, may induce severe metabolic complications. The use of bladder tissues reconstructed in vitro could avoid these complications. We compared cellular differentiation and permeability characteristics of human native with in vitro cultured stratified urothelium. MATERIALS AND METHODS: Human stratified urothelium was induced in vitro. Morphology was studied with light and electron microscopy and expression of key cellular proteins was assessed using immunohistochemistry. Permeability coefficients were determined by measuring water, urea, ammonia and proton fluxes across the urothelium. RESULTS: As in native urothelium the stratified urothelial construct consisted of basal membrane and basal, intermediate and superficial cell layers. The apical membrane of superficial cells formed villi and glycocalices, and tight junctions and desmosomes were developed. Immunohistochemistry showed similarities and differences in the expression of cytokeratins, integrin and cellular adhesion proteins. In the cultured urothelium cytokeratin 20 and integrin subunits alpha6 and beta4 were absent, and symplekin was expressed diffusely in all layers. Uroplakins were clearly expressed in the superficial umbrella cells of the urothelial constructs, however, they were also present in intermediate and basal cells. Symplekin and uroplakins were expressed only in the superficial cells of native bladder tissue. The urothelial constructs showed excellent viability, and functionally their permeabilities for water, urea and ammonia were no different from those measured in native human urothelium. Proton permeability was even lower in the constructs compared to that of native urothelium. CONCLUSIONS: Although the in vitro cultured human stratified urothelium did not show complete terminal differentiation of its superficial cells, it retained the same barrier characteristics against the principal urine components. These results indicate that such in vitro cultured urothelium, after being grown on a compliant degradable support or in coculture with smooth muscle cells, is suitable for reconstructive cystoplasty.

Cell Differentiation↗
PubMed2006

Qualitative and quantitative expression profile of muscarinic receptors in human urothelium and detrusor.

PURPOSE: We compared the complete spectrum of receptor subtypes expressed by human detrusor and its primary culture with the expression profile in a human urothelium immortalized cell line, and in fresh urothelium tissue and its primary cell culture. MATERIALS AND METHODS: The levels of mRNA expressed for receptor subtypes M1 through M5 were determined with reverse transcriptase-polymerase chain reaction and quantitative polymerase chain reaction in total RNA extracted individually from different human bladder specimens, including fresh tissue of human urothelium and detrusor, and their respective primary cultures, as well as from the UROtsa cell line. RESULTS: All 5 muscarinic receptors were detected in fresh human bladder tissue by reverse transcriptase-polymerase chain reaction RNA. The same was true in separated urothelium and detrusor tissue except for the lack of the M5 receptor transcript. Receptor subtype mRNA expression in the UROtsa cell line paralleled expression in fresh human bladder. Quantitative polymerase chain reaction data further corroborated these results and showed comparable mRNA expression for M2 and M3 in primary detrusor cultures. Primary cultures also had a decreased copy number of receptor genes than native tissue. The decrease was even more pronounced in primary urothelium culture and the UROtsa cell line in the presence of high calcium. M2 and M3 receptors were also detected in urothelium and detrusor by immunoreactivity. CONCLUSIONS: We identified all 5 existing muscarinic receptor subtypes in detrusor and urothelium, and transcripts levels of M2 and M3 were comparable in detrusor. These results support an alternative site of action in urothelium for anti-muscarinic drugs. Urothelial receptors should be considered in the design of future drugs for overactive bladder.

Adolescent↗
PubMed2006

Alterations of M(2)-muscarinic receptor protein and mRNA expression in the urothelium and muscle layer of the streptozotocin-induced diabetic rat urinary bladder.

Diabetes associated alterations of M2-muscarinic receptors (M2-mAChR) in the urothelium and muscle layer of the urinary bladder were studied using streptozotocin (STZ)-induced diabetic rats. Male Wistar rats were divided into two groups; group I: normal control rats; group II: STZ-induced diabetic rats, 2 weeks after induction. The bladder was divided into urothelium and muscle layer by microdissection. Tissue M2-mAChR protein levels were measured by Western blotting. Expression of the mRNA that encoded M2-mAChR was estimated using the method of reverse transcription combined with polymerase chain reaction (RT-PCR). M2-mAChR protein and mRNA expressions were found in both the urothelium and muscle layer of the rat urinary bladder. In control rats, the M2-mAChR protein expression ratio in the urothelium and muscle layer was 1:1.66; that for mRNA was 1:0.97. Two weeks after induction of diabetes, the M2-mAChR mRNA expression in the urothelium and muscle layer were significantly increased by 44.4% (P<0.01, n=8) and 28.6% (P<0.01, n=8), respectively. Correspondently, the bladder M2-mAChR protein levels were significantly increased by 33.3% (P<0.001, n=8) in the urothelium and 25.3% (P<0.01, n=8) in the muscle layer of the diabetic rats. In conclusion, M2-mAChR mRNA and protein are expressed in both the urothelium and muscle layer of the rat urinary bladder. STZ-induced diabetes increases mRNA and protein expression of the M2-mAChR in the urothelium as well as the muscle layer.

Animals↗
PubMed2005

Intrinsic expression of Th2 cytokines in urothelium of congenital ureteropelvic junction obstruction.

BACKGROUND: The cytokine-producing ability of urothelium, a urinary tract barrier between urine and underlying connective tissue, may exacerbate the pathogenesis of congenital ureteropelvic junction obstruction (UPJ-O) disease. A role for urothelium in human urinary tract obstruction has rarely been described. In this study, we investigated the immunopathologic characteristics of, and cytokine production by, urothelium in children with congenital UPJ-O. METHODS: Twenty-four children with congenital UPJ-O who had received pyeloplasty were enrolled. Morphologic abnormalities and pathologic and inflammatory changes of UPJ-O segments were studied. Expression of cytokines and chemokines in urothelium was investigated and compared with control tissue by immunohistochemistry (IHC), in situ hybridization (ISH), and urinary enzyme-linked immunosorbent assay (ELISA). RESULTS: Atypical or simple hyperplasia of the urothelium with evidence of Ki67 over-expression was found frequently in UPJ-O. There were variable degrees of inflammatory cell and eosinophil infiltration. Augmented expression of IL-5 and eotaxin detected by IHC and ISH, and enhanced degranulation of tissue mast cells were observed in the urothelium of UPJ-O segments. IL-4, IFN-gamma, and TNF-alpha were undetectable. Significantly higher levels of urinary IL-5, IFN-gamma, and eotaxin were detected in urine collected from the obstructed kidney. Among these, high urinary IL5 and/or IFN-gamma levels were associated with more severe obstructive uropathy (P < 0.05). CONCLUSION: Urothelium, like intestinal and respiratory epithelia, plays an active role in immunoregulation and may contribute to exacerbation of the pathogenesis of congenital UPJ-O. Many eosinophil-associated disease cytokines, which can lead to the degranulation of mast cells, are predominant regulators in UPJ-O urothelium.

Adolescent↗
PubMed2001

Restoration of the rat urothelium after cyclophosphamide treatment.

Processes leading to the recovery of a normal three-layered urothelium from a hyperplastic urothelium induced by cyclophosphamide (CP) treatment in rats have been investigated. A single intraperitoneal (ip) dose of CP caused extensive loss of cells from urothelium, but the remaining cells started to express epidermal growth factor receptor (EGFR) in their plasma membranes. On day 2 after CP injection, proliferating cell nuclear antigen (PCNA) immunohistochemistry showed a rapid increase in positively stained nuclei, from which a hyperplastic urothelium developed, composed of undifferentiated cells expressing EGFR over the entire plasma membrane. Subsequently, EGFR gradually disappeared from the apical plasma membrane but remained in the basolateral membranes. After day 6, PCNA-positive nuclei in all cell layers decreased, except in basal cells. Apoptotic cells were detectable by the TUNEL assay at day 2, and increased in number in all layers of the hyperplastic urothelium until day 10, returning to the control levels by day 14. Electron microscopic evidence showed that apoptotic cells were either pinched off into the bladder lumen or phagocytosed by the neighbouring urothelial cells. Thus, the urothelium responds to the damage by intense proliferation for a week, resulting in an undifferentiated hyperplastic state. Differentiation of superficial cells then begins and damaged cells are gradually removed by apoptosis until the three-layered urothelium is fully restored by two weeks following CP treatment.

Animals↗
PubMed2005

Effect of rilmakalim on detrusor contraction in the presence and absence of urothelium.

Openers of K(ATP) channels are known to inhibit KCl-, carbachol- and also electrically induced contractions in detrusor muscle strips from various species. Contractions of isolated strips of urinary bladder are usually of higher amplitude when the urothelium has been removed. This has been explained by the release of an urothelium-derived relaxing factor. In this study we examined whether intact urothelium may modulate the effect of the selective KATP channel opener rilmakalim. Contractile responses to 85 mM KCl and 10 microM carbachol were measured in detrusor strips from mouse, pig and man. In the presence of an intact urothelium, contractions were significantly reduced in strips from all three species investigated. In preparations with urothelium rilmakalim reduced KCl contractions with similar potency and efficacy [-logIC50 (M) 4.6 to 5.1; Effmax reduction to 14-30% of control]. However, in urothelium-denuded strips rilmakalim was more potent in pig (-logIC50 5.5) than in mouse and man (-logIC50 4.7 and 4.4, respectively). The order of potency for rilmakalim to suppress carbachol-induced contractions was pig (-logIC50 6.7)>man (5.8)>mouse (4.7); contractions were significantly more reduced in pig (Effmax reduction to 11+/-2%, n=10) and in mouse (21+/-2%, n=8) than in human detrusor (55+/-5%, n=5). The presence of urothelium did not affect the concentration-response curves for rilmakalim, with the exception of KCl-induced contractions in pig. Only the rilmakalim-induced relaxation of carbachol-mediated contractions in pig were prevented by the KATP channel blocker glibenclamide. We conclude that with this one exception, the responses to rilmakalim in detrusor contractions were not mediated by KATP channel opening.

Animals↗
PubMed2000

Urothelium-derived inhibitory factor(s) influences on detrusor muscle contractility in vitro.

The function of the bladder urothelium in modulating contractile responses of the underlying detrusor smooth muscle to muscarinic stimulation has been examined in the pig bladder. Saturation curves for [3H]-QNB binding demonstrated a greater muscarinic receptor density in the urothelium than in the detrusor smooth muscle. The presence of an intact urothelium on isolated bladder strips inhibited contractions induced by carbachol but not KCl. Contractions of a urothelium-denuded muscle strip were inhibited in the presence of a second bladder strip with an intact urothelium, but not if the second strip was denuded. The urothelium-induced inhibition of contractions was not prevented in the presence of L-NOARG, methylene blue, indomethacin, propranolol, suramin, TEA or apamin. The data suggest the presence of a diffusable, urothelium-derived inhibitory factor, which could not be identified but appears to be neither nitric oxide, a cyclo-oxygenase product, a catecholamine, adenosine, GABA nor an EDHF sensitive to apamin.

Animals↗
PubMed2003

Inhibition of human detrusor contraction by a urothelium derived factor.

PURPOSE: Stimulating muscarinic receptors in pig bladder urothelium causes the release of a diffusable factor that inhibits contractions of the underlying detrusor muscle. We investigated whether the contractions of human detrusor strips elicited by the muscarinic agonist carbachol, electrical field stimulation, KCl or the neurokinin receptor agonist neurokinin A are affected by the urothelium. MATERIALS AND METHODS: Paired intact and urothelium denuded muscle strips were placed in modified gassed Tyrode's solution at 37C. Cumulative concentration-response curves to carbachol or KCl were constructed. In other tissues the strips were stimulated electrically (1 to 40 Hz) with trains of square wave pulses 20 seconds in duration at 5-minute intervals. RESULTS: Cholinergic contractions evoked by electrical field stimulation at 10 and 30 Hz or by carbachol were significantly inhibited in the presence of an intact urothelium. Contractions elicited by KCl and by 10 microM neurokinin A were not modified by the urothelium. The urothelium mediated inhibition of contractions induced by carbachol was not affected by 300 microM L-NG-nitroarginine, 1 microM ODQ (1H-[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one), 1 microM propranolol or 5 microM indomethacin. CONCLUSIONS: Muscarinic agonists stimulate the release of an inhibitory factor from the human urothelium. The factor is distinct from nitric oxide and it persists in the presence beta-adrenoceptor blockade or cyclooxygenase inhibition.

Aged↗
PubMed2006

Non-neuronal cholinergic system in human bladder urothelium.

OBJECTIVES: To evaluate the non-neuronal cholinergic system in the isolated human bladder. Recently, several reports have demonstrated that non-nerve-evoked acetylcholine (ACh) release may contribute to various pathophysiologic conditions. METHODS: Human bladders were obtained from 15 patients. Bladder strips with or without urothelium were suspended in an organ bath filled with Krebs-Henseleit solution. ACh release was measured using microdialysis and high-performance liquid chromatography. The contribution of urothelium to non-nerve-evoked ACh release was tested, and the effects of age and elevation of the resting tension (0 to 40 mN) on ACh release were also evaluated. Furthermore, choline acetyltransferase (ChAT) immunohistochemical staining of bladder was performed. RESULTS: Immunohistochemistry showed marked ChAT-positive staining in the urothelium and suburothelial region. Tetrodotoxin-insensitive non-nerve-evoked ACh release occurred and was significantly greater in strips with than without urothelium. The non-nerve-evoked ACh release from strips with urothelium increased with age, and the positive correlation between age and release was significant. Stretching of the strips caused increases in non-nerve-evoked ACh release that was significantly greater in strips with than without urothelium. The stretch-induced release of non-nerve-evoked ACh increased with age. CONCLUSIONS: These data have demonstrated that a non-neuronal cholinergic system is present in the human bladder and that the urothelium/suburothelium partially releases ACh. We found a significant age-related and stretch-induced increase in non-nerve-evoked ACh release. This suggests that the non-neuronal cholinergic system may contribute to the physiology and pathophysiology of human bladder function.

Acetylcholine↗
PubMed2002

Urothelium derived inhibitory factor and cross-talk among receptors in the trigone of the bladder of the pig.

PURPOSE: In the dome of the bladder of the pig muscarinic receptor stimulation has been shown to release a factor from the urothelium that exerts an inhibitory effect on the underlying smooth muscle. We examined whether the urothelium in the trigone of the bladder also releases this factor, identified which receptors stimulate its release and investigated possible cross-talk among these receptor systems in the trigone. MATERIALS AND METHODS: Paired longitudinal strips of pig bladder were isolated, the urothelium was removed from 1 strip per pair and tissues were set up in gassed Krebs solution at 37C. Cumulative concentration-response curves to carbachol, phenylephrine or histamine were constructed. In some tissues a second phenylephrine curve was constructed in the presence of 1 microM. carbachol or 1 microM. histamine. In a further group of tissues the second phenylephrine curve was constructed in the presence of 1 microM. carbachol and 1 microM. atropine or 1 microM. histamine and 100 nM. mepyramine. RESULTS: In the presence of an intact urothelium contractile responses to carbachol and histamine but not to phenylephrine were depressed. In the presence of 1 microM. carbachol or 1 microM. histamine the responses of intact urothelium strips to phenylephrine were significantly depressed. This effect was absent in the presence of atropine and mepyramine, respectively. CONCLUSIONS: Carbachol and histamine induce the release of a urothelium derived inhibitory factor in the bladder trigone. The factor appears to mediate cross-talk between these systems and the alpha-adrenoceptor system in this region of the bladder.

Animals↗
PubMed1999

Expression of transforming growth factor beta1 and its receptors in normal human urothelium and human transitional cell carcinomas.

Previous studies indicated that transforming growth factor beta1 (TGFbeta1) is expressed by normal urothelial cells and exerts regulatory autocrine functions in urothelial maintenance and wound healing. However, little is known about the expression patterns of TGFbeta1 and its receptors in bladder tumors. Therefore, we studied the protein and mRNA localization of TGFbeta1 and TGFbeta receptor types I and II (TGFbetaRI and TGFbetaRII) in normal human urothelium and transitional cell carcinomas (TCCs) of different grades and stages. Expression of TGFbeta1 and its receptors was examined by immunocytochemistry and mRNA in situ hybridization in normal urothelium and TCCs using a semiquantitative method. By immunocytochemistry, the expression of TGFbeta1 and TGFbetaRII was higher in superficial and basal cell layers of normal urothelium than in the intermediate layer. A similar localization was seen in superficial TCCs. TGFbetaRI was mainly present in basal and intermediate cell layers of normal urothelium and superficial TCCs. In contrast, in muscle invasive TCCs, all tumor cells stained intensely for all three proteins. No correlation was found between immunostaining and TCC grade. In situ hybridization pointed out that all cell layers in normal urothelium exhibit similar TGFbeta1 mRNA levels. Elevated TGFbeta1 mRNA levels were noted in TCCs irrespective of grade or stage. In conclusion, these data indicate that in normal urothelium TGFbeta1, TGFbetaRI, and TGFbetaRII expression depend on maturation and differentiation. This pattern is particularly lost in muscle invasive TCCs, in which the expression of the three proteins is enhanced. These data suggest autocrine TGFbeta1 mechanisms in human TCC cells that may be more pronounced in muscle invasive TCC cells.

Activin Receptors, Type I↗
PubMed2005

Rat urothelium: improved techniques for serial cultivation, expansion, freezing and reconstitution onto acellular matrix.

PURPOSE: The rat has been a cost-effective model for the evaluation of bladder development, cancer and stromal-epithelial interactions. Serial cultivation of rat urothelium has been difficult. We developed a reliable protocol for the harvest, serial cultivation and cryopreservation of rat urothelium. We investigated the differentiation markers of in vivo bladder urothelium compared with cells reconstituted onto an acellular bladder matrix. MATERIALS AND METHODS: Epithelial harvest techniques using trypsin and collagenase were compared. Medium and conditions were optimized for serial culture and growth characteristics were calculated. Cultured cells were cryopreserved, and then recovered and grown on acellular bladder matrices. Morphology and markers of differentiation were compared between normal bladder and engineered grafts using scanning electron microscopy (SEM) and immunohistochemistry. RESULTS: Atraumatic enzymatic removal of urothelium with trypsin yielded more cells with greater viability than collagenase. Cells could be reliably grown beyond 10 passages using fibroblast conditioned medium and a 3T3 feeder layer during initial passages. Cryopreserved cells were successfully recovered and incorporated onto acellular matrices. Immunostaining and SEM of engineered grafts demonstrated early markers of differentiation, such as surface microvilli and cytokeratin 17, on polygonal cells with typical tight junctions. CONCLUSIONS: Rat urothelium can be reliably grown using fibroblast conditioned medium and a 3T3 feeder layer during primary culture. Serially passaged cells can survive cryopreservation and they are able to reconstitute epithelium on an acellular bladder matrix. Cells that are incorporated into the matrix express markers of early differentiation and demonstrate typical morphological characteristics by SEM. These culture techniques and this in vitro organoid model should facilitate the use of rat urothelium.

Animals↗
PubMed2004

Urothelium regeneration using viable cultured urothelial cell sheets grafted on demucosalized gastric flaps.

OBJECTIVE: To evaluate urothelium regeneration by grafting viable cultured urothelial cell sheets, harvested from temperature-responsive culture surfaces, on demucosalized gastric flaps in a dog model. MATERIALS AND METHODS: Viable urothelium was obtained from eight beagle dogs by partial cystectomy. Harvested urothelial cells were seeded on temperature-responsive culture dishes modified with the thermally sensitive polymer, poly(N-isopropylacrylamide). Urothelial cells cultured for 3 weeks generated contiguous urothelial cell sheets that were noninvasively harvested with no enzymatic treatment from these dishes, by reducing culture temperature. Urothelial cell sheets were autografted onto surgically demucosalized gastric flaps. Three weeks after autografting the dogs were killed and the gastric flaps with the urothelial cell sheets were examined. Cell and tissue characteristics were compared between these urothelial cell sheet-grafted gastric flaps and native urothelium. Ultrafine structures were also examined by electron microscopy. RESULTS: Five of the eight urothelial cell sheet-grafted flaps showed viable urothelial regeneration. Urothelial cell sheets attached spontaneously to demucosalized tissue surfaces completely, with no suture or fixing, and developed into a stratified viable epithelium very similar to native urothelium. Regenerated urothelium remained unstained by antiproton pump antibody, which typically stains epithelial cells positively in gastric mucosal layers. On three of the eight flaps where there were severe haematomas, grafted cell sheets were not adherent and there was no urothelial regeneration. CONCLUSIONS: Urothelial cell sheets were autografted onto dog demucosalized gastric flaps successfully, with no suture or fixation, generating a multilayered urothelium in vivo. The novel intact cell-sheet grafting method rapidly produces native-like epithelium in vivo. This versatile technology should prove useful in urinary tract tissue engineering and surgical reconstruction.

Animals↗