EZH2 and matrix co-regulate phenotype and KCNB2 expression in bladder smooth muscle cells.

BACKGROUND: Partial bladder outlet obstruction (PBO) is a widespread cause of urinary dysfunction and patient discomfort, resulting in immense health care costs. Previously, we found that obstruction is associated with altered regulation of epigenetic machinery and altered function. Here we examined if PBO and chronic bladder obstructive disease (COBD) affect epigenetic marks in a proof of principle gene and explored mechanisms of its epigenetic regulation using in vitro models. METHODS: Archival obstruction tissues from COBD had been created in 200-250 g female Sprague-Dawley rats by surgical ligation of the urethra for 6 weeks, followed by removal of the suture and following animals for 6 more weeks. Obstruction (PBO) is the 6-week ligation only. Sham ligations comprise passing the suture behind the urethra. Histone3 lysine27 trimethylation (H3K27me3) was studied by immunostaining and Chromatin immunoprecipitation (ChIP)/PCR. The interaction of matrix with KCNB2 regulation was studied in human bladder SMC plated on damaged matrix and native collagen and treated with vehicle or UNC1999. Cells were analyzed by immunostaining for cell phenotype, and western blotting for KCNB2, H3K27me3 and EZH2. Effects of conditioned media from these cells were also examined on cell phenotype. siRNA against KCNB2 was examined for effects on cell phenotype and gene expression by RT-qPCR. RESULTS: H3K27me3 increased by immunofluorescence during PBO, and by ChIP/PCR during COBD in the CpG Island (CGI) as well as 350 bp upstream. Obstruction vs. sham also showed an increase in H3K27me3 deposition. In SMC in vitro, EZH2 inhibition restored KCNB2 expression and partially restored SMC phenotype. CONCLUSIONS: Regulation of KCNB2 at the promoter demonstrated dynamic changes in H3K27me3 during COBD and obstruction. In vitro models suggest that matrix plays a role in regulation of EZH2, H3K27me3 and KCNB2, which may play a role in the regulation of smooth muscle phenotype in vivo.

Spontaneous urinary bladder regeneration after subtotal cystectomy increases YAP/WWTR1 signaling and downstream BDNF expression: Implications for smooth muscle injury responses.

Rodents have the capacity for spontaneous bladder regeneration and bladder smooth muscle cell (BSMC) migration following a subtotal cystectomy (STC). YAP/WWTR1 and BDNF (Brain-derived neurotrophic factor) play crucial roles in development and regeneration. During partial bladder outlet obstruction (PBO), excessive YAP/WWTR1 signaling and BDNF expression increases BSMC hypertrophy and dysfunction. YAP/WWTR1 and expression of BDNF and CYR61 were examined in models of regeneration and wound repair. Live cell microscopy was utilized in an ex vivo model of STC to visualize cell movement and division. In Sprague-Dawley female rats, STC was performed by resection of the bladder dome sparing the trigone, followed by closure of the bladder. Smooth muscle migration and downstream effects on signaling and expression were also examined after scratch wound of BSMC with inhibitors of YAP and BDNF signaling. Sham, PBO and incision (cystotomy) were comparators for the STC model. Scratch wound in vitro increased SMC migration and expression of BDNF, CTGF and CYR61 in a YAP/WWTR1-dependent manner. Inhibition of YAP/WWTR1 and BDNF signaling reduced scratch-induced migration. BDNF and CYR61 expression was elevated during STC and PBO. STC induces discrete genes associated with endogenous de novo cell regeneration downstream of YAP/WWTR1 activation.

Persistent myopathy despite release of partial obstruction: in vivo reversal of dysfunction and transcriptional responses using rapamycin.

Current and potential medical therapy for obstruction-induced myopathic bladder dysfunction (from benign prostatic hyperplasia or posterior urethral valves) focuses on symptoms. The persistent tissue pathology and dysfunction after release of obstruction is often deemed irreversible without any systematic therapeutic approaches. As rapamycin can attenuate bladder smooth muscle hypertrophy and dysfunction during the genesis of partial obstruction in vivo, we tested whether rapamycin could improve persistent function after release of obstruction (de-obstruction or REL). Female Sprague-Dawley rat bladders were partially obstructed (PBO) by suturing around both the urethra and a para-urethral steel rod, then removing the rod. One day prior to release of obstruction (preREL), voiding parameters and residual urine volume of preREL+future rapa, preREL+future veh groups were recorded. Release of obstruction (REL) was performed by suture removal following 6 weeks of PBO. For 4 more weeks after the de-obstruction, REL animals were randomized to rapamycin (REL+rapa) or vehicle (REL+veh). PBO for 6 weeks were used as positive controls. In shams, the urethra was exposed, but no suture tied. Voiding parameters and residual urine volume were measured prior to sacrifice of sham and REL+veh or REL+rapa, and PBO. Rapamycin efficacy was tested by pair-wise comparison of changes in individual voiding data from preREL+future veh or preREL+future rapa versus REL+veh or REL+rapa, respectively, as well as by comparisons of REL+veh to REL+rapa groups. Bladders were weighed and processed for a high-throughput QPCR array, and histopathology. Bladder/body mass ratios with PBO increased significantly and remained higher in the release phase in REL+veh animals. REL+rapa versus REL+veh improved residual volumes and micturition fractions toward sham levels. Three genes encoding extracellular proteins, BMP2, SOD3, and IGFBP7, correlated with functional improvement by Pearson's correlations. The promoters of these genes showed enrichment for several motifs including circadian E-boxes. While obstruction and REL augmented CLOCK and NPAS2 expression above sham levels, rapamycin treatment during release significantly blocked their expression. This experimental design of pharmaco-intervention during the de-obstruction phase revealed a novel pathway dysregulated during the clinically relevant treatment phase of obstructive bladder myopathy.

DNA Methylation Reduces the Yes-Associated Protein 1/WW Domain Containing Transcription Regulator 1 Pathway and Prevents Pathologic Remodeling during Bladder Obstruction by Limiting Expression of BDNF.

Chronic bladder obstruction and bladder smooth muscle cell (SMC) stretch provide fibrotic and mechanical environments that can lead to epigenetic change. Therefore, we examined the role of DNA methylation in bladder pathology and transcriptional control. Sprague-Dawley female rats underwent partial bladder obstruction by ligation of a silk suture around the proximal urethra next to a 0.9-mm steel rod. Sham operation comprised passing the suture around the urethra. After 2 weeks, rats were randomized to normal saline or DNA methyltransferase inhibitor, 5-aza-2-deoxycytidine (DAC) at 1 mg/kg, three times/week intraperitoneally. After 6 weeks, bladders were weighed and divided for histology and RNA analysis by high-throughput real-time quantitative PCR arrays. DAC treatment during obstruction in vivo profoundly augmented brain-derived neurotrophic factor (BDNF) expression compared with the obstruction with vehicle group, which was statistically correlated with pathophysiologic parameters. BDNF, cysteine rich angiogenic inducer 61 (CYR61), and connective tissue growth factor (CTGF) expression clustered tightly together using Pearson's correlation analysis. Their promoters were associated with the TEA domain family member 1 (TEAD1) and Yes-associated protein 1/WW domain containing transcription regulator 1 pathways. Interestingly, DAC treatment increased BDNF expression in bladder SMCs (P < 0.0002). Stretch-induced BDNF was inhibited by the YAP/WWTR1 inhibitor verteporfin. Verteporfin improved the SMC phenotype (proliferative markers and SMC marker expression), in part by reducing BDNF. Expression of BDNF is limited by DNA methylation and associated with pathophysiologic changes during partial bladder outlet obstruction and SMC phenotypic change in vitro.

Non-invasive voiding assessment in conscious mice.

OBJECTIVE: To review available options of assessing murine bladder function and to evaluate a non-invasive technique suitable for long-term recording. METHODS: We reviewed previously described methods to record rodent bladder function. We used modified metabolic cages to capture novel recording tracings of mouse micturition. We evaluated our method in a pilot study with female mice undergoing partial bladder outlet obstruction or sham operation, respectively; half of the partial obstruction and sham group received treatment with an S6K-inhibitor, targeting the mTOR pathway, which is known to be implicated in bladder response to obstruction. RESULTS: Our non-invasive method using continuous urine weight recording reliably detected changes in murine bladder function resulting from partial bladder outlet obstruction or treatment with S6K-inhibitor. We found obstruction as well as treatment with S6K-inhibitor to correlate with a hyperactive voiding pattern. CONCLUSIONS: While invasive methods to assess murine bladder function largely disturb bladder histology and intrinsically render post-cystometry gene expression analysis of questionable value, continuous urine weight recording is a reliable, inexpensive, and critically non-invasive method to assess murine bladder function, suitable for a long-term application.

Nerve-sparing Mid-urethral Obstruction (NeMO) in Female Small Rodents.

Partial bladder outlet obstruction (pBOO) has a high prevalence, causes significant patient burden, and immense health care costs. The most common animal model to investigate bladder remodeling in pBOO are female rodents undergoing partial obstruction at the proximal urethra. Variability in the degree of obstruction and animal mortality are major concerns with proximal obstruction. Furthermore, dissecting around the proximal urethra and bladder neck jeopardizes bladder innervation. We developed a nerve-sparing mid-urethral obstruction (NeMO) model for pBOO avoiding the disadvantages of the traditional model. We approached the urethra just inferior to the pubic symphysis, which obviated the need for laparotomy as well as for dissection in this area; also, the striated urethral sphincter remained untouched. We performed NeMO in female Sprague-Dawley rats (12 obstructions, 6 sham animals) as well as in female C57/bl6 mice (20 obstructions, 18 sham animals). After two weeks, we evaluated bladder function, bladder mass, and body mass. We had no mortalities among obstructed- or sham-operated female rats; as described for the traditional proximal pBOO-method, we tied the suture around the proximal urethra and a temporarily placed 0.9 mm metal rod. NeMO induced an 85% increase in bladder mass after two weeks, average residual urine volume was 0.4 mL in partially obstructed rats while only 0.03 mL in sham animals. In mice, we tested 3 sizes of cannulas that we placed along the urethra when tying the suture. We found that using a 27-gauge cannula resulted in over 50% animal mortality; placing the 25-gauge cannula did not yield the desired response in increasing bladder mass; utilizing a 26-gauge cannula yielded favorable results with minimal animal mortality (1/8) yet a significant 2-fold increase in bladder mass.

Uropathogenic E. coli (UPEC) Infection Induces Proliferation through Enhancer of Zeste Homologue 2 (EZH2).

UNLABELLED: Host-pathogen interactions can induce epigenetic changes in the host directly, as well as indirectly through secreted factors. Previously, uropathogenic Escherichia coli (UPEC) was shown to increase DNA methyltransferase activity and expression, which was associated with methylation-dependent alterations in the urothelial expression of CDKN2A. Here, we showed that paracrine factors from infected cells alter expression of another epigenetic writer, EZH2, coordinate with proliferation. Urothelial cells were inoculated with UPEC, UPEC derivatives, or vehicle (mock infection) at low moi, washed, then maintained in media with Gentamycin. Urothelial conditioned media (CM) and extracellular vesicles (EV) were isolated after the inoculations and used to treat naïve urothelial cells. EZH2 increased with UPEC infection, inoculation-induced CM, and inoculation-induced EV vs. parallel stimulation derived from mock-inoculated urothelial cells. We found that infection also increased proliferation at one day post-infection, which was blocked by the EZH2 inhibitor UNC1999. Inhibition of demethylation at H3K27me3 had the opposite effect and augmented proliferation. CONCLUSION: Uropathogen-induced paracrine factors act epigenetically by altering expression of EZH2, which plays a key role in early host cell proliferative responses to infection.

Assessment of local structural disorders of the bladder wall in partial bladder outlet obstruction using polarized light imaging.

Partial bladder outlet obstruction causes prominent morphological changes in the bladder wall, which leads to bladder dysfunction. In this paper, we demonstrate that polarized light imaging can be used to identify the location of obstruction induced structural changes that other imaging modalities fail to detect. We induced 2-week and 6-week partial outlet obstruction in rats, harvested obstructed bladders, then measured their retardances while distended to high pressures and compared them to controls. Our results show that the retardance of the central part of the ventral side (above the ureters) closer to the urethra can be used as a potential metric of the distending bladder obstruction.

Reduced flow after tubularized incised plate urethroplasty--increased fibrogenesis, elastin fiber loss or neither?

PURPOSE: Low urinary flow rates are common after tubularized incised plate urethroplasty but the etiology remains unclear and may be related to low urethral compliance due to abnormal collagen concentrations and/or fewer elastic fibers in the healed urethral plate. We hypothesized that inserting a preputial mucosal graft over the dorsal raw area after the midline incision may avoid scarring and improve urethral compliance. MATERIALS AND METHODS: Adult rabbits were submitted to tubularized incised plate urethroplasty with or without inlay preputial graft according to a previously described protocol. Tissular concentrations of collagens I, III, IV, VI, VIII and XIII were measured. Histomorphometric analysis was used to quantify elastic fibers in the urethra. Tubularized incised plate urethroplasty with and without inlay preputial graft was compared to normal rabbit urethras (controls). RESULTS: mRNA concentrations for collagens I, II and XIII were similar between controls and operated rabbits. The proportions between collagens I and III were 1.05, 0.87 and 1.21, respectively, in controls and animals undergoing tubularized incised plate urethroplasty with and without inlay preputial graft. mRNA concentrations for collagen IV and collagens VI/VIII tended to be higher and lower, respectively, in the operated urethras, despite showing statistical significance only for collagen VIII in animals undergoing tubularized incised plate urethroplasty with inlay preputial graft vs controls (p=0.02). The operated animals did not demonstrate a reduced number of elastic fibers in the urethral tissues compared to controls. CONCLUSIONS: Elastic fiber number and distribution were similar between tubularized incised plate urethroplasty cases and controls, suggesting that decreased concentrations of elastic fibers do not explain the reduced urethral compliance after tubularized incised plate urethroplasty. The raw area determined by the dorsal urethral incision regenerated after standard tubularized incised plate urethroplasty, while cicatrization with fibrosis occurred in correspondence to the grafted areas after tubularized incised plate urethroplasty with inlay preputial graft.

Phenotypic switching induced by damaged matrix is associated with DNA methyltransferase 3A (DNMT3A) activity and nuclear localization in smooth muscle cells (SMC).

Extracellular matrix changes are often crucial inciting events for fibroproliferative disease. Epigenetic changes, specifically DNA methylation, are critical factors underlying differentiated phenotypes. We examined the dependency of matrix-induced fibroproliferation and SMC phenotype on DNA methyltransferases. The cooperativity of matrix with growth factors, cell density and hypoxia was also examined. Primary rat visceral SMC of early passage (0-2) were plated on native collagen or damaged/heat-denatured collagen. Hypoxia was induced with 3% O2 (balanced 5% CO2 and 95% N2) over 48 hours. Inhibitors were applied 2-3 hours after cells were plated on matrix, or immediately before hypoxia. Cells were fixed and stained for DNMT3A and smooth muscle actin (SMA) or smooth muscle myosin heavy chain. Illumina 450 K array of CpG sites was performed on bisulfite-converted DNA from smooth muscle cells on damaged matrix vs native collagen. Matrix exquisitely regulates DNMT3A localization and expression, and influences differentiation in SMCs exposed to denatured matrix +/- hypoxia. Analysis of DNA methylation signatures showed that Matrix caused significant DNA methylation alterations in a discrete number of CpG sites proximal to genes related to SMC differentiation. Matrix has a profound effect on the regulation of SMC phenotype, which is associated with altered expression, localization of DNMTs and discrete changes DNA methylation.

Rapamycin attenuates bladder hypertrophy during long-term outlet obstruction in vivo: tissue, matrix and mechanistic insights.

PURPOSE: Previous molecular studies showed that the mTOR inhibitor rapamycin prevents bladder smooth muscle hypertrophy in vitro. We investigated the effect of rapamycin treatment in vivo on bladder smooth muscle hypertrophy in a rat model of partial bladder outlet obstruction. MATERIALS AND METHODS: A total of 48 female Sprague-Dawley® rats underwent partial bladder outlet obstruction and received daily subcutaneous injections of rapamycin (1 mg/kg) or vehicle commencing 2 weeks postoperatively. A total of 36 rats underwent sham surgery and received rapamycin or vehicle. Rats were sacrificed 3, 6 and 12 weeks after surgery. Before sacrifice, voiding was observed in a metabolic cage for 24 hours. Bladder-to-body weight in gm bladder weight per kg body weight and post-void residual urine were assessed. We evaluated Col1a1, Col3a1, Eln and Mmp7 mRNA expression and histology. Two-factor ANOVA and the post hoc t test were applied. RESULTS: Bladder outlet obstruction caused a significant increase in bladder weight in all obstructed groups. Three weeks postoperatively (1 week of treatment) there was no difference in the bladder-to-body weight ratio in the obstructed group. However, at 6 and 12 weeks (4 and 10 weeks of treatment, respectively) the bladder-to-body weight ratio of rats with obstruction plus rapamycin was significantly lower than that of rats with obstruction plus vehicle. Post-void residual urine volume after 6 and 12 weeks of obstruction was lower in obstructed rats with rapamycin compared to that in obstructed rats with vehicle. Rapamycin decreased the obstruction induced expression of Col1a1, Col3a1, Eln and Mmp7. CONCLUSIONS: Rapamycin prevents mechanically induced hypertrophy in cardiovascular smooth muscle. In vivo mTOR inhibition may attenuate obstruction induced detrusor hypertrophy and help preserve bladder function.

Optical assessment of tissue anisotropy in ex vivo distended rat bladders.

Microstructural remodelling in epithelial layers of various hollow organs, including changes in tissue anisotropy, are known to occur under mechanical distension and during disease processes. In this paper, we analyze how bladder distension alters wall anisotropy using polarized light imaging (followed by Mueller matrix decomposition). Optical retardance values of different regions of normal rat bladders under different distension pressures are derived. Then optical coherence tomography is used to measure local bladder wall thicknesses, enabling the calculation of the tissue birefringence maps as a measure of the tissue anisotropy. Selected two-photon microscopy is also performed to better understand the compositional origins of the obtained anisotropy results. The dome region of the bladder shows maximum birefringence when the bladder is distended to high pressures, whereas the ventral remains roughly isotropic during distension. In addition, the average anisotropy direction is longitudinal, along the urethra to dome. The derived wall anisotropy trends are based on birefringence as an intrinsic property of the tissue organization independent of its thickness, to aid in understanding the structure-functions relation in healthy bladders. These new insights into the wall microstructure of ex vivo distending bladders may help improve the functionality of the artificially engineered bladder tissues.

Uropathogenic E. coli infection provokes epigenetic downregulation of CDKN2A (p16INK4A) in uroepithelial cells.

Host cell and bacterial factors determine severity and duration of infections. To allow for bacteria pathogenicity and persistence, bacteria have developed mechanisms that modify expression of host genes involved in cell cycle progression, apoptosis, differentiation and the immune response. Recently, Helicobacter pylori infection of the stomach has been correlated with epigenetic changes in the host genome. To identify epigenetic changes during Escherichia coli induced urinary tract infection (UTI), we developed an in vitro model of persistent infection of human uroepithelial cells with uropathogenic E. coli (UPEC), resulting in intracellular bacteria colonies. Cells inoculated with FimH-negative E. coli (N-UPEC) that are not internalized and non-inoculated cells were used as controls. UPEC infection significantly induced de novo methyltransferase (DNMT) activity (12.5-fold P=0.002 UPEC vs non-inoculated and 250-fold P=0.001 UPEC vs N-UPEC inoculated cells) and Dnmt1 RNA expression (6-fold P=0.04 UPEC vs non-inoculated cells) compared with controls. DNMT1 protein levels were significantly increased in three uroepithelial cell lines (5637, J82, HT-1197) in response to UPEC infection as demonstrated by confocal analysis. Real-time PCR analysis of candidate genes previously associated with bacteria infection and/or innate immunity, revealed UPEC-induced downregulation of the tumor suppressor gene CDKN2A (3.3-fold P=0.007 UPEC vs non-inoculated and 3.3-fold P=0.001 UPEC vs N-UPEC) and the DNA repair gene MGMT (9-fold P=0.03 UPEC vs non-inoculated). Expression of CDH1, MLH1, DAPK1 and TLR4 was not affected. Pyrosequencing of CDKN2A and MGMT CpG islands revealed increased methylation in CDKN2A exon 1 (3.8-fold P=0.04 UPEC vs N-UPEC and UPEC vs non-inoculated). Methylation of MGMT was not affected. UPEC-induced methylation of CDKN2A exon 1 may increase bladder cancer and presage UTI risk, and be useful as a biological marker for UTI susceptibility or recurrence.

Mammalian target of rapamycin (mTOR) induces proliferation and de-differentiation responses to three coordinate pathophysiologic stimuli (mechanical strain, hypoxia, and extracellular matrix remodeling) in rat bladder smooth muscle.

Maladaptive bladder muscle overgrowth and de-differentiation in human bladder obstructive conditions is instigated by coordinate responses to three stimuli: mechanical strain, tissue hypoxia, and extracellular matrix remodeling.( 1,2) Pathway analysis of genes induced by obstructive models of injury in bladder smooth muscle cells (BSMCs) identified a mammalian target of rapamycin (mTOR)-specific inhibitor as a potential pharmacological inhibitor. Strain-induced mTOR-specific S6K activation segregated differently from ERK1/2 activation in intact bladder ex vivo. Though rapamycin's antiproliferative effects in vascular smooth muscle cells are well known, its effects on BSMCs were previously unknown. Rapamycin significantly inhibited proliferation of BSMCs in response to mechanical strain, hypoxia, and denatured collagen. Rapamycin inhibited S6K at mTOR-sensitive phosphorylation sites in response to strain and hypoxia. Rapamycin also supported smooth muscle actin expression in response to strain or hypoxia-induced de-differentiation. Importantly, strain plus hypoxia synergistically augmented mTOR-dependent S6K activation, Mmp7 expression and proliferation. Forced expression of wild-type and constitutively active S6K resulted in loss of smooth muscle actin expression. Decreased smooth muscle actin, increased Mmp7 levels and mTOR pathway activation during in vivo partial bladder obstruction paralleled our in vitro studies. These results point to a coordinate role for mTOR in BSMCs responses to the three stimuli and a potential new therapeutic target for myopathic bladder disease.

The bladder extracellular matrix. Part I: architecture, development and disease.

From the earliest studies with epithelial cells implanted into detrusor muscle to later experiments on smooth muscle in defined collagen gels, cell niche and extracellular matrix (ECM) have been clearly shown to orchestrate cellular behavior and fate whether quiescent, migratory, or proliferative. Normal matrix can revert transformed cells to quiescence, and damaged matrix can trigger malignancy or dedifferentiation. ECM influence in disease, development, healing and regeneration has been demonstrated in many other fields of study, but a thorough examination of the roles of ECM in bladder cell activity has not yet been undertaken. Structural ECM proteins, in concert with adhesive proteins, provide crucial structural support to the bladder. Both structural and nonstructural components of the bladder have major effects on smooth muscle function, through effects on matrix rigidity and signaling through ECM receptors. While many ECM components and receptors identified in the bladder have specific known functions in the vascular smooth musculature, their function in the bladder is often less well defined. In cancer and obstructive disease, the ECM has a critical role in pathogenesis. The challenge in these settings will be to find therapies that prevent hyperproliferation and encourage proper differentiation, through an understanding of matrix effects on cell biology and susceptibility to therapeutics.

The bladder extracellular matrix. Part II: regenerative applications.

Bladder regeneration is a long-sought goal that could provide alternatives to cystoplasty using non-urological tissues. Regeneration might be achieved in different ways, such as seeding matrices with stem cells or conventional cells, or repopulation of the matrix by the body's own reservoir of cells. Consideration of how the extracellular matrix directs cell behavior will be crucial to the success of regenerative therapies.

Mechanotransduction of extracellular signal-regulated kinases 1 and 2 mitogen-activated protein kinase activity in smooth muscle is dependent on the extracellular matrix and regulated by matrix metalloproteinases.

Excessive wall stretch of distensible hollow organs in cardiovascular and urinary systems can activate matrix metalloproteinases (MMPs), thereby releasing matrix neoepitopes and growth factor ligands, leading to ERK1/2 activation. However, the role of MMPs in mechanotransduction of ERK1/2 signaling in the bladder is unknown. We examined bladders undergoing sustained distension over time, which provides a novel platform for smooth muscle mechanotransduction studies. Bladder distension ex vivo caused increased proliferation and MMP activity. Conditioned medium from distended compared with undistended bladders induced proliferation in bladder smooth muscle cells (BSMCs). When conditioned medium from distended bladders was used to proteolyze collagen type I matrices, matrices augmented BSMC proliferation, which was inhibited if bladders were distended in presence of broad-spectrum MMP inhibitors. Distension of ex vivo bladders also induced ERK1/2 phosphorylation in situ, which was dependent on MMP activity in the intact bladder. Similarly, stretching BSMCs in vitro induced increases in ERK1/2 activation and ERK1/2-dependent proliferation under discrete mechanical conditions, and distension conditioned medium itself induced MMP-dependent ERK1/2 activation in BSMCs. Overall, stretch-induced proliferation and ERK1/2 signaling in bladder tissue and BSMCs likely depend on secreted MMP activity. Identification of intermediaries between MMPs and ERK1/2 may elaborate novel mechanisms underlying mechanotransduction in bladder smooth muscle.

Role of signal transducer and activator of transcription 3 (STAT3) in stretch injury to bladder smooth muscle cells.

Excessive stretch of the bladder can lead to wall thickening including the growth of bladder smooth muscle cells (BSMC). Only three phospho-proteins (JNK, p38, and PI3K) have been previously shown to participate in stretch-induced BSMC growth. CD1 mouse bladders were hyper- or non-distended by our ex vivo bladder distention model and screened, by a commercial screening method, for phosphorylated signaling proteins. This uncovered a factor previously unexamined for its role in bladder stretch injury: signal transducer and activator of transcription 3 (STAT3). STAT3 was assessed for its role in mitogen- and stretch-induced BSMC proliferation. Proliferation was assessed by 3H-thymidine incorporation/cell counting in response to mitogenic stimulation or to stretch on silastic collagen or carboxyl-coated membranes. JAK2, upstream of STAT3, was inhibited by AG490 (2 microM). Ex vivo distention of bladders activated a discrete number of kinases, including two MAPK pathways (JNK and ERK2) and STAT3. STAT3 signaling was activated during hyperdistention of intact bladder and by stretch and mitogenic treatments of BSMC in vitro. JAK2/STAT3 inhibition by AG490 blocked mitogen- and stretch-induced BSMC proliferation. Thus, BSMC stretch responses may involve the recruitment of both growth factor and mechanically induced BSMC growth responses integrated by a common signaling pathway, STAT3.

Integrins expressed with bladder extracellular matrix after stretch injury in vivo mediate bladder smooth muscle cell growth in vitro.

PURPOSE: It is unknown how bladder smooth muscle cells sense extrinsic mechanical stimuli. The integrins are a large versatile family of transmembrane mechanoreceptors that transduce extracellular matrix (ECM) alterations into the cell, thereby, regulating proliferation, differentiation and ECM synthesis. To our knowledge we provide the first evidence that the integrins may be involved in responses to whole bladder distention and bladder smooth muscle cell stretch. MATERIALS AND METHODS: Bladders from 100 to 120 gm. rats were stretched to 40 cm. H2O for 5 minutes. Five to 96 hours after distention whole bladder mRNAs were isolated for analysis of temporal expression of collagen and integrin genes. Separately quiescent primary culture bladder smooth muscle cells from 1-day-old Sprague-Dawley rats were stretched cyclically for 4 hours. Relative expression of select integrin subunit mRNAs was assessed by semiquantitative reverse transcriptase-polymerase chain reaction. Integrin blockade with asparagine-glycine-arginine peptides was used to determine the role of integrins in stretch induced proliferation and the cell cycle in bladder smooth muscle cells. RESULTS: Within 24 hours bladder distention stimulated collagen expression 2-fold (type I) and 5-fold (type III). Collagen levels beyond 24 hours were 8-fold (type I) and 2-fold (type III) greater than in controls, revealing an inverse temporal type I-to-III ratio beyond 24 hours. Coordinate alterations were observed in integrin and collagen expression. In vitro bladder smooth muscle cell integrin beta1, beta3 and alphav subunit expression was increased by mechanical stretch 2.5, 3.8 and 5-fold, respectively, while alpha1 expression decreased. Asparagine-glycine-arginine peptide inhibition of integrin function significantly inhibited stretch induced bladder smooth muscle cell proliferation and exit from the G2/M phase of the cell cycle. CONCLUSIONS: To our knowledge these results demonstrate for the first time that that bladder distention initiates dynamic alterations in ECM expression. The ability of integrin blockade to suppress stretch induced bladder smooth muscle cell proliferation and the coordinate changes in bladder ECM and integrin expression suggest that integrins mediate key responses to mechanical stimuli in the bladder. Furthermore, cell cycle analysis of resting and stretched bladder smooth muscle cells revealed novel avenues for the examination of integrin and stretch regulation of bladder smooth muscle cell growth.