Departments of 1 Surgery and 3 Medicine and Therapeutics, Mater Misericordiea Hospital, Conway Institute of Biomolecular and Biomedical Research, University College, Dublin 7, Ireland; and 2 Department of Reproductive and Surgical Sciences, Royal Victoria Infirmary, University of Newcastle, Newcastle-upon-Tyne NE2 4HH, United Kingdom
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ABSTRACT |
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Our understanding of the pathophysiology
of the overactive bladder is poor. It has been proposed that localized
contractions result in the abnormal stretching of bladder smooth
muscle. We hypothesize that stretch regulates the cellular processes
that determine tissue size. The purpose of this study was to
investigate the effect of stretch on apoptosis, proliferation,
cell hypertrophy, and growth factor production in human bladder smooth
muscle cells in vitro. Normal human detrusor muscle was obtained from
patients undergoing radical cystectomy for invasive bladder cancer, and primary cultures were established. Cells were mechanically stretched on
flexible plates at a range of pressures and times. Apoptosis was assessed by propidium iodide incorporation and flow cytometry. Radiolabeled thymidine and amino acid incorporation were used to assess
proliferation and cell hypertrophy. ELISA and RT-PCR were used to
assess growth factor production. Mechanical stretch inhibits
apoptosis in a time- and dose-dependent manner and was associated with increases in the antiapoptotic proteins heat shock protein-70 and cIAP-1. Stretch also increases smooth muscle cell proliferation and hypertrophy, but hypertrophy is the more dominant response. These changes were associated with increases in IGF-1 and
basic FGF and a decrease in transforming growth factor-1. Mechanical
stretch regulates apoptosis, proliferation, and cell hypertrophy in human bladder smooth muscle cells.
overactive bladder; apoptosis; heat shock proteins; hypertrophy
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INTRODUCTION |
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RECENTLY, THE PREVALENCE OF overactive bladder has been estimated at one in six of all adults aged >40 years (19). The effect of this is made all the more significant when the detrimental influence of bladder overactivity on all aspects of quality of life are taken into account (18). The pathological changes that occur in the overactive bladder are well characterized, consisting of detrusor smooth muscle (SM) hypertrophy, increased extracellular matrix production, and cholinergic denervation (4). These findings are considered common to most conditions leading to detrusor instability (4). Although there is a strong association among outlet obstruction (17), neurological disease (1), and bladder overactivity, little is known about the events that lead to these changes or the underlying mechanisms involved.
A theory by Coolsaet et al. (9) proposes that local bladder wall contractions, termed micromotions, which are synonymous with intrinsic activity, cause stretching of the adjacent SM (9). Not only may the sensation of bladder stretch initiate urgency, but in the presence of an overactive bladder, this spontaneous activity will spread throughout the bladder because of an increase in electromechanical coupling (12). Drake et al. (10) developed this concept further and described their model of peripheral autonomous modules. Essentially, a module is a circumscribed area of detrusor defined by its individual intramural bladder ganglia. Synchronized modular activity brings about bladder emptying, whereas unsynchronized activity causes stretching of the relaxed SM.
We hypothesize that mechanical stretch of bladder SM regulates detrusor size in bladder overactivity. We intend to establish primary human bladder SM cell (hBSMC) cultures, subject them to mechanical stretch, and assess their response in terms of apoptosis, proliferation, and cellular hypertrophy and to identify possible underlying mechanisms involved.
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MATERIALS AND METHODS |
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Materials.
RPMI-1640, penicillin and streptomycin solution, amphotericin B,
L-glutamine, PBS, HEPES (1 M), and FCS were purchased from GIBCO Life Technologies, (Cambridge, UK). The following primary antibodies were used: -SM actin, including fluorescently labeled antibody (Sigma); c-IAP-1 (R&D Systems, Oxford, UK); Bcl-2 (Signal Transduction Laboratories), and heat shock protein (HSP)27 and HSP70
(Stressgen Biotech). The secondary antibody used was mouse horseradish
peroxidase conjugated (Signal Transduction Laboratories). Sigma
supplied all other chemicals unless otherwise stated.
Primary hBSMC culture. Normal detrusor tissue was obtained from patients undergoing radical cystectomy for muscle invasive bladder cancer, with both ethical approval and the patients' consent. Tissue was isolated from macroscopically normal areas of bladders from five separate patients. Whole bladder was transferred to the Pathology Department, where a suitable full thickness biopsy of normal bladder was obtained and maintained in normal buffered culture media without additional oxygenation. This tissue was assessed and deemed normal by the attending pathologist and was then processed (within 30 min). The biopsy was washed three times with sterile PBS, the urothelial and serosal layers were dissected off, and the remaining muscle layer was chopped until it was too small to bisect any further. The muscle tissue was then transferred to 10 ml of disaggregation medium (RPMI-1640, 0.1% collagenase type XI, 0.1% trypsin inhibitor type 2-S, and 0.05% hyaluronidase types 3 and 2-S), maintained in a water bath at 37°C, and agitated at 100 rpm overnight (12-16 h). The resulting cell suspension was washed twice by centrifugation at 400 g for 8 min at 21°C in RPMI. The pellet was resuspended in 10 ml of complete culture media (RPMI-1640, 10% heat-inactivated FCS, 10 mM HEPES, 100 mg/ml penicillin/streptomycin, 100 mg/ml amphotericin B, and 0.02 mM glutamine) and added to a 75-cm2 sterile tissue culture flask. The preparation was cultured in an incubator (Forma Scientific) at 37°C in 5% CO2-95% air. The medium in the flask was changed every 48 h. Cells were cultured until the flask became a fully confluent monolayer (~3 wk). Passaging of cells was performed by enzymatic dissociation (0.05% trypsin). Cells were subcultured onto elastomer-based, collagen-coated, flexible six-well culture plates (Flexercell) at a density of 1 × 104 cells/well and allowed to adhere and grow over a 4-day period. Twenty-four hours before all experiments, the medium was changed from 10 to 0.5% FCS to induce quiescence. Primary cultures were maintained and passaged no greater than five times. Cultured cells were not pooled because experiments were performed on cells derived from individual bladders at any one time.
Characterization of cultured cells.
Light microscopy was used to assess the morphological characteristics
of the cultured SM cells. Whole cell lysates were collected from cells
(see Western blotting), and Western blot analyses were performed. The expression of -SM actin and SM myosin confirmed the
SM origin of the cells. Similarly, cells were suspended in FITC
(flourescein-labeled) monoclonal antibody to
-SM actin (5 µl/1 × 106 cells) and assessed with a Flow
Cytometry (Falcon/Becton Dickinson, Cambridge, UK). With this
technique, a population of cells expressing
-SM actin could be
isolated and evaluated.
In vitro mechanical stretch. hBSMCs were seeded onto flexible six-well culture plates as described previously. The Flexercell Strain Unit (FX-2000, Flexcell, McKeesport, PA), a computer-driven and vacuum-assisted mechanical stretch device, was used. Treatment plates were subjected to stretch at 3, 6, 12, 24, and 48 h. Control (rigid) plates were not stretched. Initially, the effects of mechanical stretch were examined at 0, 7.22, 12.37, and 18.47% stretch (40, 80, and 120 cmH2O, respectively). These parameters reflect in vivo voiding pressures under both normal (40 cmH2O) and pathological (80-120 cmH2O) conditions. The effect of mechanical stretch on the parameters examined was maximal at 12.37% stretch, so all subsequent experiments were performed at this degree of stretch. The detrusor is a unitary SM with slow-wave rhythm activity. Cultured cells were subjected to continuous 30-s cycles consisting of 20 s of stretch and 10 s of relaxation.
Assessment of apoptosis and viability.
Once the stretch protocol was complete, cells were enzymatically
removed (0.05% trypsin) from their collagen substrate and were
centrifuged at 300 g for 10 min. Cells were then resuspended in 200 µl of culture medium containing FITC monoclonal antibody to
-SM actin (5 µl/1 × 106 cells), incubated on ice
for 15 min, and then centrifuged at 300 g for 6 min. Cells
were then gently resuspended in 500 µl of hypotonic fluorochrome
solution [50 µg/ml propidium iodide (PI), 3.4 mM sodium citrate, 1 mM Tris, 0.1 mM EDTA, and 0.1% Triton X-100; no Triton X-100 was
included when assessing cell viability] and then stored in the dark at
4°C for 15-30 min before analysis with a Coulter XL
cytofluorometer (Falcon/Becton Dickinson). A minimum of 5,000 events were collected and analyzed. Apoptotic cell nuclei were
distinguished from normal nuclei by their hypodiploid DNA. The rates of
apoptosis and viability in SM cells were calculated on the flow
cytometer by obtaining the percentage of
-SM actin-positive cells
that did not incorporate PI compared with the
-SM actin-positive cells that did (see Fig. 3).
Western blotting.
Total cellular protein was extracted and pooled by using Nonidet P-40
(NP-40) protein isolation solution (0.5% NP-40, 10 mM Tris, pH 8.0, 60 mM KCl, 1 mM EDTA, pH 8.0, 1 mM DTT, 10 mM PMSF, and 1 µM leupeptin,
pepstatin, and aprotinin). Protein content was measured by the Bradford
Assay protein assay kit (Bio-Rad). Western blotting was performed as
previously described by using primary antibodies directed against
-SM actin, c-IAP-1, HSP27 and HSP70, and Bcl-2 (8).
Cytokine detection.
Cell culture supernatants were collected at the corresponding times and
stored at 80°C. Cytokine production from control and stretched
cells released into the supernatant was assayed with ELISA kits (R&D
Systems). The manufacturer guidelines were strictly adhered to at all
times. The corresponding cellular protein was also collected at the
corresponding time points, and the results were expressed as picograms
per microgram of protein.
RT-PCR.
Total RNA was isolated from control and treated hBSMCs by using TRIzol
reagent (GIBCO Life Technologies) according to the manufacturer's
protocol. The extracted RNA was dissolved in
diethylpyrocarbonate-treated water and then quantified by measuring the
absorbance at 260 nm. RNA (5 µg) was resolved on 1.5%
formaldehyde-agarose gel to assess RNA integrity. One microgram of
total RNA was used to synthesize the first-strand cDNA by using random
hexamers. PCR amplification of the cDNA template was performed in a
thermal cycler (Perkin-Elmer 7700). Probe and primer sequences for
transforming growth factor-1 (TGF-
1) and the endogenous control
18S rRNA were purchased from Applied Biosystems as a predeveloped assay
reagent. RT-PCR was performed on an ABI PRISM 7700 sequence detection
system (Applied Biosystems). Cycling conditions were as follows:
step 1, 2 min at 50°C; step 2, 10 min at
95°C; step 3, 15 s at 95°C; step 4, 1 min at 60°C; and repeat step 3, ×40 cycles. Probes were
labeled with 5' FAM and 3' TAMRA as a quencher, with the exception of the ribosomal probe, which was labeled with 5' VIC to facilitate dual
reporter assay. The manufacturer's guidelines were strictly adhered to
at all times.
Radiolabeled thymidine and amino acid studies. Radiolabeled [H3]thymidine or leucine (0.25 µCi/1 × 104 cells) was added to alternative wells 24 h before mechanical stretch. In the measurement of leucine incorporation, a modified protocol was used as described. In addition, the standard culture medium (not leucine-free) was used for these experiments. All culture plates, irrespective of their time point, were removed from stretch at the same time (t = 0) so that all cells were exposed to the radioisotope for the same period. Once stretching was complete, the culture medium was removed and each well was washed three times with PBS. Cells were lysed with 2% SDS solution, and the lysate was added to 9 ml of scintillation fluid (Ultima Gold, Sigma). Beta isotope emission was assessed by using a 2-min protocol on a beta counter (Packard, Tri-Carb).
Statistical analysis. Statistical analysis was carried out by using ANOVA-one-way ANOVA with Student's-Newman correction. Significance was assumed for values of P < 0.05. Results are expressed as means ± SD.
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RESULTS |
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Characterization of hBSMC cultures.
Cultured cells were examined to confirm their SM cell origin and their
differentiated state. Cell morphology demonstrated SM cell
characteristics, namely, an ellipsoid shape with tapered ends and a
single, centrally placed nucleus. Cells were tightly packed and
formed a "hill and valley" appearance in culture (Fig. 1A). Western blotting
confirmed expression of the SM cell markers -SM actin and SM myosin
on cell lysates derived from cells in each passage used (Fig.
1B). Flow cytometry demonstrates that ~99% of cultured
cells were viable
-SM actin-expressing cells, suggesting minimal
contamination (see Fig. 3).
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Effect of mechanical stretch on apoptosis.
hBSMCs had a high basal rate of spontaneous apoptosis, even in
optimium conditions. Table 1 displays our
initial rates of spontaneous apoptosis at the various time
points and degrees of mechanical stretch. At the lower degree of
stretch, apoptosis is reduced at 24 h only. Mechanical
stretch at 12.37% (80 cmH2O) induced a time-dependent
decrease in spontaneous rates of apoptosis (Fig.
2A). Spontaneous
apoptosis was significantly decreased as early as 6 h
(P = 0.026) and continued at 12, 24, and 48 h of stretch (P = 0.036, 0.0001, and 0.015, respectively).
Figure 2B confirms that there is no alteration in cell
viability with mechanical stretch. Representative samples of the flow
cytometry data are demonstrated in Fig.
3. At the higher degree of stretch, there was no significant change in apoptotic rates.
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Effect of mechanical stretch on cell proliferation and hypertrophy.
Mechanical stretch stimulated proliferation in hBSMCs (Fig.
5). Thymidine incorporation was
significantly increased at 3 h of stretch (P = 0.021), representing a 13.6% increase in proliferation compared with
control. Maximal increases in proliferation were seen at 6 h of
stretch (P = 0.0001), representing a 20.5% increase in
proliferation. Thymidine incorporation remained significantly increased
at 12 h (P = 0.032) compared with control.
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Effect of mechanical stretch on growth factor production.
To study possible mediators for the alterations in apoptosis,
proliferation, and hypertrophy, the effect of mechanical stretch on
growth factor production was evaluated. Although hBSMCs are known to
produce a number of cytokines, we focused on three growth factors known
to mediate the effects of stretch in animal models (6,
24). The initial response of the SM cells in terms of growth
factor production is to increase both IGF-1 (P = 0.006 at 6 h) and basic FGF (bFGF) levels (P = 0.006 at
3 h) in response to cellular stretch. IGF-1 (Fig.
7A) and bFGF levels (Fig.
7B) then become significantly reduced at 48 h. This
reduction may be related to the limitations of the in vitro model
established, wherein cells may adapt to long-term stretch. In contrast,
TGF-1 levels are seen to decrease significantly at 6 and 24 h
(P = 0.005 and 0.02, respectively) in response to
mechanical stretch (Fig. 7C1). There was a trend toward a
recovery of the TGF-
1 levels as mechanical stretch is continued to
48 h, which again may reflect the ability of the cells to adapt to
stretch. By using RT-PCR techniques, the alteration in TGF-
1 protein
was demonstrated to be as a direct result of a decrease in TGF-
1
mRNA production in response to mechanical stretch (Fig.
7C2). VEGF is a potent angiogenic agent, and its production
is increased in vascular SM cells undergoing mechanical stretch
(22). The initial response of hBSMCs to mechanical stretch
is to increase VEGF production (data not shown) at 3 and 6 h
compared with control (350 ± 5.9, 531 ± 11.5, and 639 ± 0.2 pg/µg protein at 0, 3, and 6 h, respectively). This
suggests that mechanical stretch may stimulate angiogenesis in SM
cells.
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DISCUSSION |
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Apoptosis, proliferation, and cell hypertrophy are the three processes that regulate tissue size. We have demonstrated, by using human bladder tissue, that mechanical deformation regulates all three processes in vitro. The accumulative effect in vivo would be increased tissue size. The inhibition of apoptosis in detrusor muscle by mechanical stretch has not been documented previously, although apoptosis has been shown to have a role in the regression of the hypertrophied rabbit bladder (23). The potential role of apoptosis in bladder overactivity may provide us with new therapeutic targets. The high basal rate of spontaneous apoptosis in our cells is in contrast to the low rates of apoptosis seen in vivo. This may be explained by the dynamic environment that the cells are subjected to in vivo. It is possible that by removing these cells from their dynamic environment in the bladder, we induce apoptosis, which is then reversed in our model of in vitro stretch.
The absence of the antiapoptotic protein Bcl-2 in hBSMCs was not expected, because its role in vascular SM cell apoptosis is well described (11). A direct comparison of apoptotic rates and underlying mechanisms between a vascular and a bladder SM cell culture would help to clarify this situation. Other antiapoptotic mechanisms were examined. Mechanical stretch is a cellular stress, so it is not surprising that the expression of HSP70 is regulated by stretch. Similar results were obtained by Chen et al. (5) in their rabbit model of acute overdistension, suggesting that stretch may be the common mechanism in stimulating HSP70. We have also demonstrated the regulation of cIAP-1 by mechanical stretch. Although HSP70 inhibits the formation of the apoptosome (procaspase-9, Apaf-1, and cytochrome c), cIAP-1 is known to bind to and inhibit procaspase-3, a terminal effector cell death protease, and thus prevent cell death (25).
This study demonstrates that hBSMCs increase both proliferation and cell size in response to mechanical stretch. This is in agreement with a recent study by Orsola et al. (20), who also demonstrated that over a 24-h period, the increase in hBSMC size is greater than the increase in proliferation, at 12% stretch. They also examined the effect of 6 and 20% stretch and concluded that the decision to undergo proliferation or hypertrophy is dependent on the degree of stretch. Increased detrusor thickness is well described in the overactive bladder, but neither the stimulus nor the relative contributions of hyperplasia or hypertrophy are known in vivo (16). Karim et al. (15) have previously demonstrated both detrusor hyperplasia and hypertrophy in the obstructed guinea pig bladder and suggested a biphasic response, whereby hyperplasia precedes hypertrophy. Park et al. (21) have also demonstrated an increase in rat bladder SM cells in response to mechanical stretch. We also noted an early increase in proliferation and a more sustained hypertrophic response in our in vitro model of detrusor stretch. It was not possible to quantify a change in cell numbers in response to stretch because of the opaque culture well, but proliferation in the otherwise slow-growing hBSMCs was significantly enhanced by mechanical stretch.
This study also demonstrates the regulation of growth factors in hBSMCs by mechanical stretch. bFGF is an SM mitogen with antiapoptotic effects and is upregulated early in the bladder's response to distension (6). IGF-1 is also a SM cell mitogen with antiapoptotic effects, and its expression in obstructed rat bladder SM is increased (7). The increased production of both growth factors in response to mechanical stretch in human tissue may be mediating the alterations in apoptosis, proliferation, and hypertrophy in a paracrine or autocrine fashion, although inhibition studies would be needed to clarify this.
TGF-1 is a multifunctional cytokine that stimulates fibroblast
proliferation, regulates the synthesis of matrix components, stimulates
SM cell apoptosis, and is regulated by bladder distension (6). We have demonstrated decreases in TGF-
1 at both
the mRNA and the protein levels in response to stretch, and this may
reduce its proapoptotic effects. The effect of mechanical stretch
on VEGF production in vascular SM cells has been noted previously (22). We have demonstrated that mechanical stretch
significantly increases VEGF production by hBSMCs. The regulation of
VEGF by mechanical stretch in bladder SM does provide a possible
mechanism for the increased microvasculature seen in hypertrophied SM
(e.g., overactive bladder) (3, 13). It also
suggests that mechanical stretch may have effects on other tissues in
the bladder and that SM may mediate these secondary effects. In
addition, VEGF has recently been demonstrated to be a survival factor
in malignant cells (Bouchier-Hayes D., personal communication) and may
even mediate the antiapoptotic effects of mechanical stretch. The
accumulative effect of altered cytokine expression is increased cell
survival and stimulation of cell number and size.
We have utilized a previously validated model for examining the effect of mechanical stretch on bladder SM cells (21). hBSMCs were maintained in culture up to their fifth passage, wherein they maintained their morphology, their proliferative capacity, and their SM marker expression. To provide a deformable matrix for the cells to adhere to, the cells were subcultured onto collagen (type 1)-coated elastomer membranes, similar to their in vivo situation. The negative pressure applied to the base of the well subjects the adherent cells to a reproducible degree of mechanical deformation. It is not known to what degree bladder SM is stretched in vivo, but the pressures applied to the base of the plates does correspond to clinical urodynamic parameters of intravesical pressure, and as mentioned previously, the detrusor possesses slow-wave activity, justifying the pressure and time variables selected.
In conclusion, mechanical stretch of bladder SM adjacent to contracting modules has been proposed as a mechanism for the overactive bladder. We have demonstrated, by using hBSMCs, that mechanical stretch does regulate processes central to tissue size, such as apoptosis, proliferation, and cell hypertrophy.
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ACKNOWLEDGEMENTS |
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The authors acknowledge the technical assistance of Drs. Amanda O'Neill, Ronan Coffey, and Ophelia Blake. We also acknowledge the assistance of Chanel Watson and her colleagues in the procurement of suitable tissue.
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FOOTNOTES |
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This work was supported by the British Urological Foundation/Pfizer Scholarship 2001/2002 (D. Galvin) and in part by Mater College.
Address for reprint requests and other correspondence: R. W. G. Watson, Dept. of Surgery, Mater Misericordiae Hospital, 47 Eccles St., Dublin 7, Ireland (E-mail: research{at}profsurg.iol.ie).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
August 6, 2002;10.1152/ajprenal.00168.2002
Received 1 May 2002; accepted in final form 15 July 2002.
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