Recombinant insulin-like growth factor-1 activates satellite cells in the mouse urethral rhabdosphincter.

BACKGROUND: The goal of this study is to demonstrate the efficacy of a new method for the treatment of urinary incontinence by stimulation of urethral rhabdosphincter satellite cells. We show that satellite cells do exist in the sphincter muscle of retired male mice breeders by staining for c-Met, a satellite cell specific protein. Once activated by recombinant mouse Insulin-like Growth Factor-1(rIgf-1), the satellite cells develop into muscle cells within the rhabdosphincter thereby potentially strengthening it. METHODS: 20 µl (1 µg/µl) of rIgf-1 was surgically injected directly into the urethral wall of retired male mouse breeders. Mice injected with phosphate buffered saline (PBS) were used as controls. 4 weeks later, urethras were harvested and serially-sectioned through the sphincter for routine hematoxylin-eosin staining as well as immunohistochemical staining with satellite cell specific anti-c-Met antibody and proliferation specific anti-Ki-67 antibody. RESULTS: Anti-c-Met antibody positive cells (c-Met+) were identified in the rhabdosphincter. c-Met+ cells increased by 161.8% relative to controls four weeks after rIGF-1 injection. Anti- Ki-67 antibody positive cells were identified and characterized as cells with centrally located nuclei in striated muscle bundles of rIGF-1 treated animals. CONCLUSIONS: Satellite cells in the mouse rhabdosphincter can be activated by rIGF-1 treatment, which subsequently are incorporated into existing skeletal muscle bundles. Using this approach, the rhabdosphincter can be induced to regenerate and potentially strengthen via satellite cell activation and likely improve urinary continence.

Urinary diversion results in marked decreases in proliferation and apoptosis in fetal bladder.

PURPOSE: We sought to determine if bladder cycling is required for remodeling during fetal development. MATERIALS AND METHODS: For this study 5 fetal sheep bladders were harvested after 2 weeks of urinary diversion, initiated at approximately 90 days of gestation. Six unoperated sheep bladders of approximately 105 days of gestational age were used as controls. Dividing cells and cells undergoing apoptosis were quantified by using Ki-67 antibody and TUNEL (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling) assay, respectively. In addition, expression of the antiapoptosis gene, Bcl2, and cell division control protein 42 were quantified by real-time polymerase chain reaction. RESULTS: The thickness of bladder tissue layers is dramatically altered as a consequence of urinary diversion (defunctionalization). The percentage of Ki-67 and TUNEL positive cells in control bladders was 5.8% and 47.1%, respectively. However, in diverted bladders apoptosis and cell mitosis were significantly decreased with essentially 0% of Ki-67 and TUNEL positive cells per microscope field in the mucosa and detrusor muscle layers. In addition, relative mRNA expression of antiapoptotic gene Bcl2 was significantly lower in control than in diverted bladder tissue, while Cdc42 was significantly higher in the detrusor but not in the lamina propria. CONCLUSIONS: Cell mitotic activity and coordinated cell death appear to be involved in growth and remodeling, which are activated by the mechanical input that occurs during bladder cycling. Fetal urinary diversion results in a repression of mitotic and apoptotic activity, limiting normal bladder growth and remodeling.

Beta and gamma-sarcoglycans are decreased in the detrusor smooth muscle cells of the partially obstructed rabbit bladder.

PURPOSE: We evaluated and quantified the levels of sarcoglycans present in the detrusor muscle layer of rabbits with partial bladder outlet obstruction. MATERIALS AND METHODS: Rabbits underwent surgery, as previously described, to partially obstruct the urethra. One, 3, 7 and 14 days after obstruction the detrusor muscle layer was dissected free of the remaining bladder tissue and extracted with detergent to isolate the transmembrane components of the dystroglycan-glycoprotein complex. Several components of the dystroglycan-glycoprotein complex were characterized and quantified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. RESULTS: Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis several bands were noted on gels with a molecular weight (43 and 35 kDa, respectively) corresponding to beta and gamma-sarcoglycan. As obstruction progressed longitudinally, the levels of beta and gamma-sarcoglycan showed progressive decrease at the protein level with beta-sarcoglycan levels recovering at later time points. Bladders with a functional physiology that showed more advanced symptoms of dysfunction had a greater decrease in beta and gamma-sarcoglycan protein. CONCLUSIONS: The levels of beta and gamma-sarcoglycan progressively change with obstruction with greater changes occurring in the levels of gamma-sarcoglycan. It is likely that alterations in the dystroglycan-glycoprotein complex are responsible for some of the changes in muscle physiology that occur as a consequence of obstruction.

Altered extracellular matrix expression in the diverted fetal sheep bladder.

PURPOSE: It is unclear whether filling and emptying are important to bladder development. We tested this in an experimental preparation. MATERIAL AND METHODS: Urinary diversion was performed in 7 fetal lambs at 90 days of gestation and 6 unoperated fetal lambs served as controls. Transmural sections were analyzed for changes in tissue layer thickness and/or composition after 14 days of urinary diversion. Matrix mRNA levels (collagen I and III, and FN) as well as the cytokines/growth factors IGF-1, EGR-1, WT-1 and BCL-2 were quantified by real-time polymerase chain reaction. Hydroxyproline measurements of total collagen and collagen subtype quantification were done by enzyme-linked immunosorbent assay. RESULTS: Diverted fetal bladders showed a 27% and 57% decrease in mucosal and detrusor muscle layer thickness, respectively. In contrast, there was a 270% increase in serosal layer thickness in diverted bladders. The mRNA levels of COL1A1, COL3A1, IGF-1, EGR-1 and the anti-apoptotic gene BCL-2 were increased significantly in the serosal/detrusor layer of diverted bladders. In the mucosa levels of these mRNAs remained unchanged except for those of FN and WT-1, which were significantly decreased and increased, respectively. Total collagen, and type I and III collagen protein levels were significantly increased in diverted bladders. CONCLUSIONS: The lack of mechanical loading in diverted bladders leads to the arrest of detrusor smooth muscle growth, and concurrent fibrosis and thickening of the serosal layer. Changes in the levels of IGF-1, BCL-2 and EGR-1 likely have regulatory roles that affect the smooth muscle phenotype in the detrusor layer.

Smooth muscle trans-membrane sarcoglycan complex in partial bladder outlet obstruction.

The urinary bladder experiences both distension and contraction as a part of the normal filling and emptying cycle. To empty properly, tension generated intracellularly in a smooth muscle cell must be smoothly and efficiently transferred across its sarcolemma to the basement membrane, which mediates its binding to both the extracellular matrix and to other cells. As a consequence of urethral obstruction, the bladder cannot generate appropriate force to contract the organ, thereby leading to inefficient emptying and associated sequelae. In this study, an animal model of urethral obstruction was utilized to study the membrane-associated structures that transfer tension across the sarcolemma of bladder smooth muscle cells. Immunohistochemical localization of key components of the smooth muscle tension transfer apparatus (TTA) was performed utilizing specific antibodies against:(1) the alpha-chains of type IV collagen, a basement membrane component, and (2) beta-sarcoglycan, an integral membrane protein that is a participant in the physical linkage between the cytoskeleton and the basement membrane. We demonstrate, in obstructed animals, that there is a pronounced disruption of the TTA with a physical displacement of these two components that can be demonstrated at the level of the light microscope using scanning confocal microscopy. Electron microscopy further demonstrates significant increases in the size of the junctional plaques between smooth muscle cells.

Functional changes in bladder tissue from type III collagen-deficient mice.

OBJECTIVE: Collagen fibers impart tensile strength and transfer tension from bladder smooth muscle cells. We have previously shown that fibrotic bladders are characterized by an increased type III:type I collagen ratio. To determine the effect of decreased type III collagen on bladder function, type III collagen-deficient mice (COL3A1) were studied physiologically. METHODS: Bladders from wild-type (+/+) and heterozygous (+/-) COL3A1 mice were biochemically characterized to determine total collagen (hydroxyproline analysis) and collagen subtype concentration (cyanogen bromide digestion and ELISA). Alterations in collagen fiber diameter were assessed by electron microscopy. Bladder muscle strips were used to assess physiologic function. RESULTS: Hydroxyproline content decreased in heterozygous bladders, which had 50% less type III collagen. Wild-type bladders had a biphasic distribution of collagen fiber sizes, whereas heterozygous bladder collagen fibers spanned a broad range. Physiologically, there were no differences in contractile responses between wild-type and heterozygotes when stimulated with ATP, carbachol or KCl, indicating normal contraction via purinergic and muscarinic receptors, and in response to direct membrane depolarization. In contrast, tension generation in heterozygotes was decreased after field stimulation (FS), indicating decreased synaptic transmission. Length-tension studies showed that the heterozygote muscle strips generated less tension per unit length, indicating that they were more compliant than wild-type controls. CONCLUSIONS: Critical levels of type III collagen appear to be a requirement for normal bladder tension development and contraction. Our data show that a decrease in the type III:type I collagen ratio, and altered fiber size, results in a more compliant bladder with altered neurotransmitter function.

Transforming growth factor-beta1-induced hypertrophy and matrix expression in human bladder smooth muscle cells.

OBJECTIVES: To determine whether transforming growth factor beta (TGF-beta) could activate hyperplasia, hypertrophy, and altered collagen expression in human detrusor smooth muscle cells (SMCs). METHODS: Human bladder SMCs were treated in vitro with TGF-beta1 and analyzed for changes in both proliferative and hypertrophic responses by cell number and volume measurements, as well as for alterations in extracellular matrix gene and protein expression by Northern blot and enzyme-linked immunosorbent assay. RESULTS: Proliferation of bladder SMCs was refractory to TGF-beta1, whereas the cells became hypertrophic upon TGF-beta1 treatment. The interstitial collagens, types I and III, were increased significantly in TGF-beta1-treated cultures in a dose-dependent manner. These increases were blocked in the presence of TGF-beta1 neutralizing antibody and also when cultures were treated with the protein synthesis inhibitor cycloheximide, indicating that new protein synthesis is necessary for upregulation of the interstitial collagens. Messenger ribonucleic acid transcripts for both the COL1A1 and COL3A1 genes were elevated at 4, 6, and 24 hours in TGF-beta1-treated cultures, preceding the expression of the collagenous protein, showing that TGF-beta1 effects on bladder smooth muscle occur, at least in part, at the transcriptional level. CONCLUSIONS: These results indicate that human bladder SMCs have the potential to mediate both a hypertrophic and fibrotic response upon TGF-beta1 stimulation.

Mast cell chymase is a possible mediator of neurogenic bladder fibrosis.

AIMS: Urinary bladders of patients with myelomeningocele, owing to spina bifida, are often functionally impaired, fibrotic organs. Common to this condition are repeated occurrences of bladder infection and inflammation. Since mast cells have been associated with a fibrogenic response in inflammatory conditions, we investigated the role of mast cell granule product, chymase, as a mediator of myleodysplastic bladder fibrosis. METHODS: Human control and myelodysplastic bladder tissues were stained with Unna's stain and chymase antibody to determine mast cell number and localization. Cell specific localization of collagen mRNAs was determined by in situ hybridization (ISH). In vitro, normal human bladder fibroblasts were treated with recombinant chymase, heparin and inhibitors, and collagen subtype concentration was determined by enzyme linked immunosorbent assay (ELISA). RESULTS: Myelodysplastic bladders were characterized by increased mast cells in the detrusor muscle layer compared to control bladders, as well as mast cell degranulation and increased connective tissue deposition. Both types I and III collagen mRNA localized to fibroblasts surrounding detrusor muscle fascicles, whereas only collagen III mRNA localized to cells within connective tissue infiltrated muscle bundles in myelomeningocele bladder tissue. Chymase treatment of bladder fibroblasts, in vitro, was dose-dependent and resulted in significant increases in both types I and III collagen. Heparin did not alter collagen protein expression, whereas heparin-chymase combination modulated type III collagen expression. Serine protease inhibitor, phenylmethylsulfonlyfluoride, did not inhibit collagen synthesis, whereas denatured chymase resulted in decreased collagenous protein levels. CONCLUSIONS: Bladder fibrosis may be mediated by mast cell chymase stimulation of collagen synthesis.

Compression and tension: differential effects on matrix accumulation by periodontal ligament fibroblasts in vitro.

Human periodontal ligament fibroblasts were subjected to 10% cyclic equibiaxial tensional and compressive forces in vitro. Media supernatants were analyzed for changes in total protein, extracellular matrix proteins type I collagen and fibronectin, as well as MMP expression by gelatin zymography and Western blot. RNA analyses for changes in collagen, MMP-2, and TIMP-2 were carried out by either Real-time PCR and/or Northern blot. Application of compressional forces resulted in decreases in type I collagen and fibronectin protein, Col1A1 RNA, and increases in total protein, MMP-2 protein (latent and active), and MMP-2 RNA. TIMP-2 RNA was unchanged by compressive forces. In contrast, tensional forces increased total protein, type I collagen, Col1A1 RNA, as well as MMP-2 and TIMP-2 RNA. These studies show that cells can perceive two different forms of mechanical stimuli and respond in a differential manner relative to extracellular matrix synthesis and degradation.

Matrix synthesis by bladder smooth muscle cells is modulated by stretch frequency.

The bladder is a physically active organ that undergoes periodic stretching as a part of its normal function. To determine the role that stretching or mechanical deformation may play in altering the synthetic phenotype of bladder wall cells, a series of experiments were carried out to quantify several extracellular matrix (ECM) messenger ribonucleic acids (mRNAs) and their corresponding protein levels after mechanical challenge. Bladder smooth muscle cells were grown on distensible membranes in an apparatus that can reliably and reproducibly subject cells to well-characterized periods of mechanical stretching. For this study, cultured bovine bladder cells were subjected to cyclic mechanical deformation of varying frequencies to determine if this variable altered ECM expression. Using this experimental system, we demonstrated that smooth muscle cells were acutely sensitive to mechanical deformation and showed alteration in the synthesis of the major fibrillar collagens, types I and III. Concomitant analyses of mRNA in these cells show that levels of type I collagen correlate with mRNA levels at all frequencies except at 60 cycles/min, and, thus, type I production appears to be transcriptionally regulated. Interestingly, type III protein levels do not correlate with mRNA measurements except at 20 cycles/min, and, therefore, a different regulatory mechanism likely governs type III production. These studies demonstrate that smooth muscle cell ECM secretory phenotype can be altered by the frequency of mechanical deformation experienced by the cells. These data support the concept that stretching of the bladder wall affects the secretory phenotype of smooth muscle cells and can result in an altered ECM composition.

Correlation of P-cadherin and beta-catenin expression and phosphorylation with carcinogenesis in rat tongue cancer induced with 4-nitroquinoline 1-oxide.

Using biochemical and immunohistochemical techniques, we have investigated P-cadherin, beta-catenin, c-src and c-met protein expression, and phosphorylation of beta-catenin in a rat model of tongue cancer induced with 4-nitroquinoline 1-oxide. Six-week-old male Sprague-Dawley rats were given either normal drinking water (controls) or 50 ppm 4NQO solution as drinking water for 16 and 20 weeks. This treatment produced dysplasia and well-differentiated squamous cell cancer in rat tongues after 16 and 20 weeks, respectively. In controls, P-cadherin and beta-catenin were expressed only in cell membranes of tongue suprabasal epithelial cells, whereas strong reaction to P-cadherin antibody was observed during carcinogenesis, especially in nests of cancer cells. However, dysplastic and cancer cells expressed beta-catenin not only in cell membranes but also in the nuclear and cytoplasmic compartments. During carcinogenesis, immunohistochemical reaction to phosphotyrosine increased gradually. Reaction to the c-src product was strongest at the dysplastic stage and, to the c-met product, at the cancer stage. In addition, western blotting analysis showed a marked increase in the expression of beta-catenin and phosphotyrosine in dysplastic and cancer cells compared with the controls. Using immunoprecipitation and western blotting techniques, we found that phosphorylated beta-catenin gradually increased during carcinogenesis. These experiments demonstrate that cell-cell adhesion in epithelial cells was reduced by phosphorylation of beta-catenin and that beta-catenin overexpression in nuclear and cytoplasmic compartments during carcinogenesis and the production of the c-met product that is associated with the phosphorylation of beta-catenin in tongue cancer.

Response of the fetal sheep bladder to urinary diversion.

PURPOSE: In adults urinary diversion results in bladder atrophy and a rapid decrease in contractile function. Little is known about the effects of urinary diversion on bladder development. In this regard we characterized the responses of fetal sheep bladder strips obtained from animals that underwent urinary diversion. MATERIALS AND METHODS: Urinary diversion was performed on fetal sheep after 90 days of gestation (term 147 days) and bladder tissue was obtained 2 weeks later. Contractile and relaxant responses of full-thickness bladder strips were tested. RESULTS: Bladders from fetal sheep subjected to urinary diversion weighed significantly less than control fetal bladders. Histological studies demonstrated marked connective tissue infiltration and the reorganization of smooth muscle elements. Carbachol stimulated a tonic contraction, while field stimulation administered during the tonic contraction elicited a phasic relaxation or a biphasic response, consisting of an initial relaxation and then a phasic contraction in control and diverted bladders. Contractile responses of defunctionalized strips to carbachol were significantly less than those of control bladder strips. Contractile responses of defunctionalized bladder strips to field stimulation at 1 Hz. were significantly greater than those of control strips. Responses of the 2 sets of fetal bladder strips to higher frequencies were similar, as were the contractile responses to adenosine triphosphate and KCl. Field stimulated relaxations in the presence of carbachol stimulated contraction of defunctionalized bladder strips were significantly greater than those of control strips, while the relaxant responses of each set of fetal bladder strips to isoproterenol and nitroprusside were similar. CONCLUSIONS: Urinary diversion in normal fetal sheep resulted in marked structural changes, reduced carbachol stimulation and increased field stimulation relaxation.

The transforming growth factor-beta-inducible matrix protein (beta)ig-h3 interacts with fibronectin.

Proper growth and development require the orderly synthesis and deposition of individual components of the extracellular matrix (ECM) into well ordered networks. Once formed, the ECM maintains tissue structure and houses resident cells. One ECM component, (beta)ig-h3, is a highly conserved transforming growth factor-beta-inducible protein that has been hypothesized to function as a bifunctional linker between individual matrix components and resident cells. To gain insights into its physiological function, full-length (beta)ig-h3 protein was produced using a baculovirus expression system and purified under native conditions. Human fibroblasts attached and spread on (beta)ig-h3-coated plates and developed actin stress fibers. Purified (beta)ig-h3 binds fibronectin (FN) and type I collagen (Col I) but does not bind gelatin. Using defined fragments of FN, we localized the (beta)ig-h3 recognition region to the gelatin/collagen binding domain present in the N-terminal region of the FN molecule. Our results identify FN and Col I as two ligands of (beta)ig-h3 in the ECM.