- Email: [email protected]
Effect of Sustained Hydrostatic Pressure on Rat Bladder Smooth Muscle Cell Function
Bladder Physiology Effect of Sustained Hydrostatic Pressure on Rat Bladder Smooth Muscle Cell Function Margaret Rebecca Drumm, Brittany D. York, and Jiro Nagatomi OBJECTIVES
METHODS
RESULTS
CONCLUSIONS
To test a hypothesis that bladder smooth muscle cells (BSMCs) shift their phenotype from contractile to synthetic in response to elevated hydrostatic pressure. Although mechanical stimuli are needed for development of the bladder, the exact mechanisms for this process are poorly understood. Rat BSMCs were exposed to 7.5 cm H2O of hydrostatic pressure in custom-made columns to a maximum of 48 hours. After exposure to pressure, the smooth muscle cells were fixed, stained, and imaged to quantify cell morphology and proliferation. Additionally, Western blotting was used to quantify extracellular signal-regulated kinase (ERK½) activation as well as phenotype marker proteins, ␣-smooth muscle actin, and SM-22. Compared with the control, BSMCs exposed to hydrostatic pressure exhibited a more spread morphology after 4 hours and the expression of activated ERK½ was a maximum of two-fold at 1.5-3 hours. Moreover, cell density of BSMCs exposed to hydrostatic pressure exhibited an increase after 48 hours when compared with their respective controls. In contrast, ␣-smooth muscle actin and SM-22 expression was similar in the control and in cells exposed to hydrostatic pressure for 48 hours. The morphologic and proliferative changes of BSMC in response to hydrostatic pressure possibly indicate a phenotypic shift from contractile to synthetic. Moreover, the activation of ERK½ intracellular signaling pathway may represent a potential mechanism for the pressure-induced BSMC proliferation. The comparable levels of contractile proteins observed in both control and pressure group BSMCs suggest that not all the phenotype markers are regulated concomitantly by a single stimulus. UROLOGY 75: 879 – 885, 2010. © 2010 Elsevier Inc.
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he cells of the bladder are constantly subjected to mechanical stimuli, such as hydrostatic pressure and stretch, during filling and voiding cycles. Although appropriate mechanical stimuli are needed for growth and development of the urinary bladder,1,2 the abnormal mechanical environment created by various voiding dysfunctions, for example, because of spinal cord injury3,4 or obstruction due to benign prostate hypertrophy,5 can cause cellular alterations that can jeopardize the integrity of the bladder. These cellular-level changes include smooth muscle hypertrophy and hyperplasia, as well as increases or decreases in the extracellular matrix (ECM) synthesis, which all can alter bladder tissue architecture and compliance,3,6,7 thereby compromising the function of the organ. It has been well documented that smooth muscle cells (SMCs) have the potential to undergo phenotypic changes from contractile to synThe funding for this research was provided by Clemson University (to J.N.). M.R.D. was supported partly by funding from South Carolina Space Grant Consortium. From the Department of Bioengineering, Rhodes Engineering Research Center, Clemson University, Clemson, South Carolina Reprint requests: Jiro Nagatomi, Ph.D., Department of Bioengineering, 501 Rhodes Engineering Research Center, Clemson University, Clemson, SC 296340905. E-mail: [email protected] Submitted: May 15, 2009, accepted (with revisions): August 20, 2009
© 2010 Elsevier Inc. All Rights Reserved
thetic, the 2 extreme ends of the spectrum which are characterized by cell proliferation rate, cell morphology, expression levels of contractile (eg, ␣-smooth muscle actin, SM-22), and ECM proteins (eg, collagen, elastin).6,8-12 In comparison with contractile SMCs that exist in normal physiological environments, synthetic SMCs exhibit increased cell proliferation,6,8-10 decreased expression of contractile proteins,6,8 and ECM remodeling.8,11,12 On the basis of these, we hypothesize that mechanical stimuli trigger SMC phenotypic changes, which, in turn, result in the long-term alterations of bladder tissue. Previously, the effects of mechanical stimuli on bladder cell behavior have been studied by several investigators using in vitro culture models. For example, when exposed to cyclic stretch (20% maximum, 0.1 Hz) for a maximum of 48 hours, bladder smooth muscle cells (BSMCs) in vitro exhibited increased cell size,13,14 cell numbers,15,16 and stretch-regulated gene expression (eg, heparin-binding EGF-like growth factor17 and the cysteine-rich protein Cyr6118) as compared with the control. Moreover, activation of several signaling pathways such as PI3K/Akt,16 p38,17 and extracellular signal-regulated kinase (ERK½)19 has been shown to mediate cyclic 0090-4295/10/$34.00 doi:10.1016/j.urology.2009.08.050
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stretch and the cellular-level events. Although these studies provided valuable information, exposure of SMCs to cyclic stretch only partially simulates the in vivo mechanical environment. Thus, further investigation is necessary to advance our understanding of the relationship between mechanical forces and bladder SMC phenotype shift. In addition to stretch, the cells of the bladder are constantly subjected to transmural hydrostatic pressure. More specifically, BSMCs that reside within the wall tissue are subjected to a pressure gradient that exists between the inside and outside of the bladder (⌬P ⫽ intravesical pressure ⫺ intra-abdominal pressure). Because many previous in vivo studies demonstrated that urinary diversion (removal of pressure load) leads to bladder atrophy,20-22 it can be argued that pressure is an important stimulus for the health of the bladder. A seminal in vitro study by Haberstroh et al23 exposed ovine BSMCs to pure hydrostatic pressure (4, 6, and 8.5 cm H2O), without stretch, for a maximum of 7 days and observed a significant increase in cell proliferation after 5 days, which was abrogated in the presence of heparin-binding epidermal growth factor (HB-EGF) inhibitor, CRM197.1 Their results provided the first evidence that BSMCs were sensitive to elevated hydrostatic pressure and that the response was mediated, at least in part, through HB-EGF.23 Although it might be concluded from these findings that hydrostatic pressure triggers phenotypic shift that leads to long-term tissue-level changes, cell proliferation represents only one of the SMC phenotype markers. Synthetic SMCs exhibit decreased expression of smooth muscle-specific proteins6,8 and a spread-out morphology,6,24 whereas contractile SMCs exhibit an elongated morphology. Moreover, it is still unknown which down-stream pathways mediate hydrostatic pressure and the growth factor-induced BSMC proliferation. The present study, therefore, aimed to test the hypothesis that hydrostatic pressure triggers several biological events that are pertinent to phenotype expression of BSMC. We specifically quantified cell morphology, cell proliferation, ERK½ activation (part of mitogenactivated protein kinase signaling pathway triggered by EGF), and contractile protein expression (␣-actin and SM22) after exposure of rat BSMC to sustained hydrostatic pressure.
MATERIAL AND METHODS BSMC Culture BSMCs were isolated from female Sprague-Dawley rats (250350 g) according to previously reported techniques.6,25 Briefly, the urothelium and mucosa were mechanically removed from the smooth muscle layer, which was finely diced and digested in sterile RPMI medium 1640 (Invitrogen; Carlsbad, CA) supplemented with 0.1% collagenase (Sigma-Aldrich, St. Louis, MO) and 0.5% trypsin EDTA (Invitrogen), with gentle stirring under standard culture conditions (ie, 37°C in a humidified atmosphere of 95% air and/or 5% CO2) for 30 minutes. The digested tissue was then filtered through a 100-m cell strainer 880
(BD Biosciences, Bedford, MA) and centrifuged at 1200 rpm for 3 minutes. The supernatant was discarded and the cell pellet was resuspended in RPMI supplemented with 10% fetal bovine serum (FBS; HyClone, Logan, UT) and 1% penicillin-streptomycin (Invitrogen). The cells were cultured under standard cell culture conditions with medium change every 3 to 4 days. The phenotype of BSMCs was determined by the expression of ␣-smooth muscle actin, SM-22, and h-caldesmon. Cells with passages of 8 or lower were used in all experiments.
Exposure of BSMC to Sustained Hydrostatic Pressure BSMCs were seeded at a predetermined density (10 000 or 200 000 cells per coverslip for morphologic or molecular analysis, respectively) on sterile glass coverslips (18 mm in diameter, product no. 48382-041: VWR) in RPMI supplemented with 10% FBS and 1% penicillin-streptomycin. After 24 hours, the media were replaced with low-serum media (1% FBS), and the cells were incubated for 24 hours before exposure to hydrostatic pressure. In specified experiments, BSMCs were incubated with 10 M meiosis-specific serine threonine protein kinase ½ (MEK½) inhibitor (U0126; Cell Signaling Technology, Danvers, MA) for 2 hours before pressure application. The cells that were incubated with low serum RPMI served as control for the inhibitor experiments. After another media change with fresh low-serum media, these cells were exposed to the sustained hydrostatic pressure using vertical columns filled with medium to the height of 7.5 cm of the column, creating hydrostatic pressure on the BSMCs seeded below as reported in the published data1,23,26 for a maximum of 48 hours. The cells maintained under atmospheric pressure (normal culture mediumheight ⫽ 0.3 cm) for the duration of experiments were used as the control.
Analysis of Cell Morphology and Proliferation At the end of each prescribed time period, BSMCs were fixed in freshly prepared 2% paraformaldehyde (Sigma-Aldrich) and stained with rhodamine-phalloidin (Invitrogen) and DAPI (Invitrogen) for actin filaments and cell nuclei, respectively. A fluorescence microscope (Nikon Instruments, Melville, NY) and digital camera (Q-Imaging: Surrey, BC, Canada) were used to image the BSMCs. For each sample, 12 images were taken in each quadrant of the coverslip. The cell morphology was analyzed by calculating the aspect ratio (major axis: minor axis) of 16 randomly selected cells in each image using Image-Pro Plus 5.0 software (Media Cybernetics, Inc., Bethesda, MD). The cell proliferation was quantified by counting all stained cell nuclei in each of the 12 image fields (0.577 mm2) and averaging. The data were reported as the number of cells per square millimeter.
Protein Collection and Western Blot Analysis At the end of the prescribed time periods (30, 90, and 180 minutes for signaling molecules and 24 and 48 hours for phenotypic marker proteins), BSMCs were removed from the coverslips by scraping in PBS. The cell suspension was centrifuged at 300 g for 7 minutes at 4°C and the cell pellet was resuspended in 150 L of complete cell extraction buffer: 5 mL of cell extraction buffer (CEB; Invitrogen), 250 L of protease inhibitor cocktail (Sigma-Aldrich), and 17 L of 0.3 M stock solution of phenylmethylsulfonyl fluoride (Sigma-Aldrich). The cell suspension was then incubated on ice with periodic vortexing for 30 minutes. The lysates were clarified with centrifuUROLOGY 75 (4), 2010
Figure 1. Cell morphology of rat BSMC exposed to sustained hydrostatic pressure (7.5 cm H2O) for 4 hours. At the end of experiments, BSMCs were fixed and stained with rhodamine-phalloidin and DAPI for actin filaments and nuclei, respectively. Cells maintained under control (no pressure) conditions (A) and cells exposed to pressure (B). The arrows indicate examples of major and minor axis measurements used to calculate the aspect ratios (magnification ⫽ 100 x). Histograms of aspect ratios of BSMCs maintained under controlled conditions (C) and exposed to sustained hydrostatic pressure for 4 hours (D). Data are mean ⫾ SD, analyzed using t test between 2 groups; *P ⬍.05; n ⫽ 3; 384 cells per group per experiment.
gation at 14 000 rpm at 4°C for 10 minutes and prepared for protein quantification or stored at ⫺80°C. The protein concentration in each sample was quantified using a protein assay kit based on the Bradford method27 and following the manufacturer’s instructions (Bio-Rad, Hercules, CA). Total protein samples (5-20 g protein) collected from cultures were separated using sodium dodecyl sulfate–polyacrylamide gel electrophoresis on a 12% polyacrylamide gel in Tris/ glycine/SDS buffer (Bio-Rad) at 200 V for 1 hour. Separated protein bands on the acrylamide gels were transferred to polyvinylidene difluoride (Bio-Rad) membranes at 100 V for 1 hour. The polyvinylidene difluoride membranes containing the protein bands were blocked for 1 hour and incubated with primary antibody for the protein of interest (␣-SMA, SM-22, GAPDH, total ERK½, and phosphorylated-ERK1/2) at an appropriate dilution overnight at 4°C. The membranes were incubated with secondary antibodies in the presence of 1 L streptactin-HRP (Bio-Rad) at room temperature for 1 hour before chemiluminescent labeling, following the manufacturers’ instructions (Immun-Star HRP Substrate Kit; Bio-Rad). Images of the labeled membranes were taken using FluorChemSP (Alpha Innotech, San Leandro, CA). The intensity of each protein band was analyzed to detect changes in protein expression using spot densitometry using AlphaEase FC software (Alpha Innotech, San Leandro, CA). The expression of phosphorylated ERK½ was normalized by the expression of total ERK½ to determine the level of ERK½ activation. The expression of ␣-smooth muscle actin and SM-22 was normalized by the GAPDH expression in each sample. UROLOGY 75 (4), 2010
Statistical Analysis All experiments were run in duplicate and were repeated at a minimum of 3 separate times. Numerical data were analyzed using analysis of variance (ANOVA) and Tukey test in SigmaPlot software (Systat Software, Chicago, IL); values of P ⬍.05 were considered significant.
RESULTS BSMC Morphology Under Sustained Hydrostatic Pressure The BSMCs exposed to sustained hydrostatic pressure (7.5 cm H2O) for 4 hours exhibited an overall shift from an elongated morphology to a more rounded morphology within the population (Figs. 1A and B). Aspect ratios of these cells were calculated and plotted in histograms (Figs. 1C and D) to quantitatively analyze the morphologic changes in response to sustained hydrostatic pressure. When compared with the control, BSMCs that were exposed to sustained hydrostatic pressure (7.5 cm H2O) for 4 hours exhibited an increase in number of cells within the population, with an aspect ratio between 1.0 and 2.0 (rounded morphology) and a decrease in cells with an aspect ratio of 4.0 or higher (elongated morphology) (Figs. 1C and D). 881
Figure 2. Activation of ERK½ in rat BSMCs after sustained hydrostatic pressure for 4 hours. Spot densitometry was used to quantify the ratio of phosphorylated ERK½ expression and total ERK½ expression. The fold increase was calculated from the ratio of phosphorylated ERK½ and total ERK½ expression for each time point. Representative chemiluminescence images for ERK½ activation (A); ERK½ activation in BSMCs after sustained hydrostatic pressure from 4 independent experiments (B). Average ERK½ activation in BSMC exposed to sustained hydrostatic pressure (C). Data are mean ⫾ SD, analyzed using ANOVA; *P ⬍ .05 when compared with the control; **P ⬍.05 when compared with the 30-minute group; n ⫽ 3 of 4 experiments.
ERK½ Activation in BSMCs Under Sustained Hydrostatic Pressure In the present study, the activation of ERK½ in BSMCs exposed to sustained hydrostatic pressure for a maximum of 3 hours was quantified through Western blotting and spot densitometry of chemiluminescence imaging (Fig. 2A). When compared with the control, BSMCs exposed to sustained hydrostatic pressure exhibited a maximum of a 2-fold increase in the activation of ERK½ at 30, 90, and 180 minutes. In 3 of the 4 experiments conducted (samples 1-3), the peak activation of ERK½ occurred at the 180 minutes time point, but in 1 other experiment (sample 4), ERK½ activation occurred earlier and peaked at 90 minutes (Fig. 2B). The mean increase in activation of ERK½ in samples 1-3 in response to sustained hydrostatic pressure for 180 minutes is significantly (P ⬍.05; n ⫽ 3) greater (1.64-fold) than that of the control (baseline, at 0 minute) and at 30 minutes (Fig. 2C).
BSMC Proliferation Under Sustained Hydrostatic Pressure The proliferative response of BSMCs to sustained hydrostatic pressure (7.5 cm H2O) for 48 hours was quantified through cell density (cells/mm2) measurements (Fig. 3). The cell density of BSMCs subjected to hydrostatic pressure for 24 hours was similar to the no pressure control at the same time point (data not shown). However, the cell density in BSMCs subjected to hydrostatic pressure for 48 hours exhibited a significant (P ⬍.05; n ⫽ 5) increase when compared with the control after 48 hours (Fig. 3). In the presence of MEK½ inhibitor, this increase in the cell density was absent (Fig. 3). 882
Figure 3. Proliferative response of BSMCs exposed to sustained hydrostatic pressure for 48 hours. At the end of experiments, cells were fixed and stained with DAPI for nuclei, which were then counted using ImagePro software. Data are mean ⫾ SD, analyzed using ANOVA and paired t test; *P ⬍.05 when compared with the control; n ⫽ 3-5.
Contractile Protein Expression in BSMC Under Sustained Hydrostatic Pressure BSMCs pretreated with MEK½ inhibitor, U0126, were exposed to hydrostatic pressure (7.5 cm H2O) for 48 hours and the expression of contractile proteins ␣-smooth muscle actin (␣-SMA) and SM-22 were quantified. When compared with the control (no pressure in the absence of U0126), BSMC expression of ␣-SMA and SM-22 was similar in all other groups (no pressure in the presence of U0126, and cells exposed to sustained hydrostatic pressure, both in the absence and presence of U0126) at 48 hours (Fig. 4). UROLOGY 75 (4), 2010
Figure 4. Expression of contractile proteins, ␣-SMA and SM22, in rat BSMCs after exposure to sustained hydrostatic pressure for 48 hours. Spot densitometry was used to quantify contractile protein expression and normalized to GAPDH protein expression. (A) Representative western blot spot densitometry for ␣-SMA and SM22 protein expression; (B) Expression of contractile-marker proteins ␣-SMA and (C) SM22 after exposure to hydrostatic pressure and in the presence of a MEK½ inhibitor. Data are mean ⫾ SD; analyzed using ANOVA; n ⫽ 4.
COMMENT The present study investigated the effect of elevated, but physiological-level, hydrostatic pressure on BSMC phenotype in vitro. The experimental setup we used was adapted from previously published studies that examined the effects of hydrostatic pressure on BSMCs1,23 and vascular endothelial cells;26 and this design allowed application of pressure to cultured cells with minimal variation in pH, pO2, and pCO2.26 The results of the present study demonstrated that BSMCs subjected to sustained hydrostatic pressure for 4 hours showed a population shift in morphology from elongated to more rounded morphology. This was evidenced by more cells (48%) exhibiting an aspect ratio of 1.0 to 2.0 and fewer cells (6.5%) exhibiting an aspect ratio of ⬎ 4.0 after exposure to pressure, when compared with the control (40%, 11%, respectively), suggesting a possible phenotypic shift from contractile to synthetic. Although these are the first observations of BSMC morphologic response to pressure, others have investigated and reported changes in SMC morphology in response to mechanical stimuli. For example, when vascular SMC were subjected to uniaxial stretch for a maximum of 40 minutes, these cells exhibited more polarized cell morphology in the direction of the stretch.28 More recently, our laboratory reported that BSMCs within 3D collagen constructs exposed to sustained tension for 48 hours exhibited significantly higher aspect ratios, suggesting a more contractile phenotype, than that of the control.6 These findings imply that morphologic responses of SMCs depend on the type of mechanical stimuli applied; stretching results in a shift toward more contractile, whereas exposure to hydrostatic pressure results in a shift toward more synthetic phenotype of SMC. In addition to the cell morphology change, BSMCs subjected to sustained hydrostatic pressure for 3 hours UROLOGY 75 (4), 2010
exhibited a maximum of 2-fold increase in ERK½ activation (Fig. 2). Moreover, exposure of BSMCs to sustained hydrostatic pressure (7.5 cm H2O) for 48 hours led to a significant increase in cell proliferation when compared with the control (Fig. 3). When BSMCs were pretreated with MEK½ inhibitor, U0126, however, the cell proliferation under hydrostatic pressure was similar to that of the control (Fig. 3), suggesting that pressureinduced BSMC proliferation is mediated through the ERK½ pathway. These results added another level of details to the previous observations that pressure-induced BSMC proliferation was mediated by HB-EGF.23 As previously reported with a lung epithelial cell model,29 it is possible that elevated hydrostatic pressure triggers HBEGF shedding and/or release from BSMC surfaces, which in turn activates the EGF receptor and a cascade of events that include ERK½ activation and cell proliferation. Although the present study provided some explanation, further investigation is necessary to determine the exact mechanism for pressure mechanotransduction by BSMC. Currently, involvement of the ERK pathway in BSMC mechanotransduction is somewhat controversial. For example, one group previously reported that p38/SAPK, but not ERK½ mediates the effect of cyclic stretch (25% at 0.1 Hz for 30 minutes to a maximum of 24 hours) on BSMC proliferation and HB-EGF mRNA expression.17 In contrast, another group reported a very rapid (within 5 minutes) ERK½ activation in BSMCs exposed to static and cyclic stretch (5%-10%, 0.1 Hz).19 These authors also reported a significant increase in BSMC proliferation in response to cyclic stretch, which was significantly reduced in the presence of ERK½ inhibitor, demonstrating the involvement of early, transient ERK activation in this event.19 However, in 3 of the 4 experiments of the 883
present study, BSMC exhibited activation of ERK½ at the 180-minute time point under sustained hydrostatic pressure, whereas 1 specimen exhibited this peak activation at 90 minutes (Fig. 2). Despite this variation in peak activation time, possibly because of the cell batch differences, our results to date certainly demonstrate that BSMCs exhibit ERK½ activation in response to sustained hydrostatic pressure, but no earlier than in 90 minutes. On the basis of these, it can be speculated that BSMCs respond more readily to mechanical environments of pathologic conditions such as cyclic stretch, whereas prolonged exposure to physiological-level pressure induce similar responses. The results of the present study indicated that, when compared with the control, expression of ␣-SMA and SM-22 was similar in the BSMCs exposed to sustained hydrostatic pressure after 48 hours. Expression of these contractile proteins has been previously used as an index of a SMC phenotypic shift; synthetic SMC express less of these proteins compared with contractile SMC. To date, however, the exact relationship between contractile protein expression and other events such as cell proliferation is still partially understood. For example, when compared with the control, vascular SMC exhibited an increase in cell proliferation, but similar ␣-SMA expression in response to PDGF (5.0 ng/mL) in a 2D monolayer culture.10 When these cells were cultured in 3D collagen gels; however, exposure to the same level of PDGF led to both an increase in cell proliferation and a decrease in ␣-SMA expression.10 These findings suggest that cell proliferation and ␣-SMA expression of SMCs are not governed by a single pathway, but may be regulated synergistically by the growth factor signaling and physical environments (2D vs 3D).10 Moreover, another group reported that the stretch-induced upregulation of contractile proteins, ␣-SMA, calponin, and SM-22 in vascular SMCs was blocked by inhibiting p38 activation, but not by inhibiting ERK½.30 These results provide further support that although ERK½ activation in SMC may lead to increased cell proliferation, these cellular events are independent from contractile protein expression. Although these previous findings provide some explanations for the lack of contractile protein changes in BSMC under hydrostatic pressure, it is also possible that we did not observe any differences because the time course of the present study was limited to 48 hours by the lowserum content in cultures. Further investigation, therefore, is necessary to elucidate the relationship between mechanical stimuli and BSMC phenotypic marker protein expression.
CONCLUSIONS The results of the present in vitro study provided the evidence that BSMCs exposed to physiological-level hydrostatic pressure for prolonged periods exhibited morphologic and proliferative changes that possibly indicate a phenotypic shift from contractile to synthetic. The 884
comparable levels of contractile proteins observed in both control and pressure group BSMCs suggest that not all the phenotype markers are regulated concomitantly by a single stimulus. Moreover, the activation of ERK½ intracellular signaling pathway under hydrostatic pressure observed in the present study may represent a potential mechanism for mechanically induced BSMC proliferation observed in the present and other studies.19 These findings add cellular and/or molecular level knowledge to the current understanding that although appropriate mechanical stimuli are needed for growth and development of the urinary bladder,1,2 excessive mechanical forces created by various voiding dysfunctions3-5 can alter bladder tissue architecture and compliance.3,6,7 Acknowledgment. The authors thank Dr Bruce Gao, Department of Bioengineering, Clemson University, for providing the rats used as the source of bladder cells. References 1. Haberstroh KM, Kaefer M, Retik AB, et al. The effects of sustained hydrostatic pressure on select bladder smooth muscle cell functions. J Urol. 1999;162:2114-2118. 2. Maizels M. Normal and anomalous development of the urinary tract. In: Walsh PC, Retik AB, Vaughan ED Jr, Wein AJ, eds. Campbell’s Urology,7th ed. Philadelphia, PA: W.B. Saunders; 1998: 1577. 3. Korossis S, Bolland F, Ingham E, et al. Review: tissue engineering of the urinary bladder: considering structure-function relationships and the role of mechanotransduction. Tissue Eng. 2006;12:635-644. 4. Watanabe T, Rivas DA, Chancellor MB. Urodynamics of spinal cord injury. Urol Clin North Am. 1996;23:459-473. 5. Chancellor MB, Rivas DA, Huang B, et al. Micturition patterns after spinal trauma as a measure of autonomic functional recovery. J Urol. 1994;151:250-254. 6. Roby T, Olsen S, Nagatomi J. Effect of sustained tension on bladder smooth muscle cells in three-dimensional culture. Ann Biomed Eng. 2008;36:1744-1751. 7. Stover J, Nagatomi J. Cyclic pressure stimulates DNA synthesis through the PI3K/Akt signaling pathway in rat bladder smooth muscle cells. Ann Biomed Eng. 2007;35:1585-1594. 8. Worth NF, Rolfe BE, Song J, et al. Vascular smooth muscle cell phenotypic modulation in culture is associated with reorganisation of contractile and cytoskeletal proteins. Cell Motil Cytoskeleton. 2001;49:130-145. 9. Sobue K, Hayashi K, Nishida W. Molecular mechanism of phenotypic modulation of smooth muscle cells. Horm Res. 1998;50(suppl 2):15-24. 10. Stegemann JP, Nerem RM. Altered response of vascular smooth muscle cells to exogenous biochemical stimulation in two- and three-dimensional culture. Exp Cell Res. 2003;283:146-155. 11. Adelow CA, Frey P. Synthetic hydrogel matrices for guided bladder tissue regeneration. Methods Mol Med. 2007;140:125-140. 12. Backhaus BO, Kaefer M, Haberstroh KM, et al. Alterations in the molecular determinants of bladder compliance at hydrostatic pressures less than 40 cm H2O. J Urol. 2002;168:2600-2604. 13. Karim O, Cendron M, Mostwin J, et al. Developmental alterations in the fetal lamb bladder subjected to partial urethral obstruction in utero. J Urol. 1993;150:1060-1063. 14. Peters C, Vasavada S, Dator D, et al. The effect of obstruction on the developing bladder. J Urol. 1992;148:491-496. 15. Orsola A, Adam RM, Peters CA, et al. The decision to undergo DNA or protein synthesis is determined by the degree of mechanical deformation in human bladder muscle cells. Urology. 2002;59: 779-783.
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16. Adam RM, Roth JA, Cheng H, et al. Signaling through PI3K/Akt mediates stretch and PDGF-BB-dependent DNA synthesis in bladder smooth muscle cells. J Urol. 2003;169:2388-2393. 17. Nguyen HT, Adam RM, Bride SH, et al. Cyclic stretch activates p38 SAPK2-, ErbB2-, and AT1-dependent signaling in bladder smooth muscle cells. Am J Physiol Cell Physiol. 2000;279:C1155C1167. 18. Tamura I, Rosenbloom J, Macarak E, et al. Regulation of Cyr61 gene expression by mechanical stretch through multiple signaling pathways. Am J Physiol Cell Physiol. 2001;281:C1524-C1532. 19. Halachmi S, Aitken KJ, Szybowska M, et al. Role of signal transducer and activator of transcription 3 (STAT3) in stretch injury to bladder smooth muscle cells. Cell Tissue Res. 2006;326:149-158. 20. Jayanthi VR, McLorie GA, Khoury AE, et al. The effect of temporary cutaneous diversion on ultimate bladder function. J Urol. 1995;154:889-892. 21. Machado MG, Yoo JJ, Atala A. Defunctionalized bladders: effects before and after refunctionalization in an animal model. J Urol. 2000;164:1002-1007. 22. Krabill KA, Sung HW, Tamura T, et al. Factors influencing the structure and shape of stenotic and regurgitant jets: an in vitro investigation using Doppler color flow mapping and optical flow visualization. J Am Coll Cardiol. 1989;13:1672-1681.
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23. Haberstroh KM, Kaefer M, Bizios R. Inhibition of pressure induced bladder smooth muscle cell hyperplasia using CRM197. J Urol. 2000;164:1329-1333. 24. Rensen SS, Doevendans PA, van Eys GJ. Regulation and characteristics of vascular smooth muscle cell phenotypic diversity. Neth Heart J. 2007;15:100-108. 25. Kropp BP, Zhang Y, Tomasek JJ, et al. Characterization of cultured bladder smooth muscle cells: assessment of in vitro contractility. J Urol. 1999;162:1779-1784. 26. Acevedo AD, Bowser SS, Gerritsen ME, et al. Morphological and proliferative responses of endothelial cells to hydrostatic pressure: role of fibroblast growth factor. J Cell Physiol. 1993;157:603-614. 27. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of proteindye binding. Anal Biochem. 1976;72:248-254. 28. Katsumi A, Milanini J, Kiosses WB, et al. Effects of cell tension on the small GTPase Rac. J Cell Biol. 2002;158:153-164. 29. Tschumperlin DJ, Dai G, Maly IV, et al. Mechanotransduction through growth-factor shedding into the extracellular space. Nature. 2004;429:83-86. 30. Qu M-J, Liu B, Wang H-Q, et al. Frequency-dependent phenotype modulation of vascular smooth muscle cells under cyclic mechanical strain. J Vasc Res. 2007;44:345-353.
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METHODS
RESULTS
CONCLUSIONS
To test a hypothesis that bladder smooth muscle cells (BSMCs) shift their phenotype from contractile to synthetic in response to elevated hydrostatic pressure. Although mechanical stimuli are needed for development of the bladder, the exact mechanisms for this process are poorly understood. Rat BSMCs were exposed to 7.5 cm H2O of hydrostatic pressure in custom-made columns to a maximum of 48 hours. After exposure to pressure, the smooth muscle cells were fixed, stained, and imaged to quantify cell morphology and proliferation. Additionally, Western blotting was used to quantify extracellular signal-regulated kinase (ERK½) activation as well as phenotype marker proteins, ␣-smooth muscle actin, and SM-22. Compared with the control, BSMCs exposed to hydrostatic pressure exhibited a more spread morphology after 4 hours and the expression of activated ERK½ was a maximum of two-fold at 1.5-3 hours. Moreover, cell density of BSMCs exposed to hydrostatic pressure exhibited an increase after 48 hours when compared with their respective controls. In contrast, ␣-smooth muscle actin and SM-22 expression was similar in the control and in cells exposed to hydrostatic pressure for 48 hours. The morphologic and proliferative changes of BSMC in response to hydrostatic pressure possibly indicate a phenotypic shift from contractile to synthetic. Moreover, the activation of ERK½ intracellular signaling pathway may represent a potential mechanism for the pressure-induced BSMC proliferation. The comparable levels of contractile proteins observed in both control and pressure group BSMCs suggest that not all the phenotype markers are regulated concomitantly by a single stimulus. UROLOGY 75: 879 – 885, 2010. © 2010 Elsevier Inc.
T
he cells of the bladder are constantly subjected to mechanical stimuli, such as hydrostatic pressure and stretch, during filling and voiding cycles. Although appropriate mechanical stimuli are needed for growth and development of the urinary bladder,1,2 the abnormal mechanical environment created by various voiding dysfunctions, for example, because of spinal cord injury3,4 or obstruction due to benign prostate hypertrophy,5 can cause cellular alterations that can jeopardize the integrity of the bladder. These cellular-level changes include smooth muscle hypertrophy and hyperplasia, as well as increases or decreases in the extracellular matrix (ECM) synthesis, which all can alter bladder tissue architecture and compliance,3,6,7 thereby compromising the function of the organ. It has been well documented that smooth muscle cells (SMCs) have the potential to undergo phenotypic changes from contractile to synThe funding for this research was provided by Clemson University (to J.N.). M.R.D. was supported partly by funding from South Carolina Space Grant Consortium. From the Department of Bioengineering, Rhodes Engineering Research Center, Clemson University, Clemson, South Carolina Reprint requests: Jiro Nagatomi, Ph.D., Department of Bioengineering, 501 Rhodes Engineering Research Center, Clemson University, Clemson, SC 296340905. E-mail: [email protected] Submitted: May 15, 2009, accepted (with revisions): August 20, 2009
© 2010 Elsevier Inc. All Rights Reserved
thetic, the 2 extreme ends of the spectrum which are characterized by cell proliferation rate, cell morphology, expression levels of contractile (eg, ␣-smooth muscle actin, SM-22), and ECM proteins (eg, collagen, elastin).6,8-12 In comparison with contractile SMCs that exist in normal physiological environments, synthetic SMCs exhibit increased cell proliferation,6,8-10 decreased expression of contractile proteins,6,8 and ECM remodeling.8,11,12 On the basis of these, we hypothesize that mechanical stimuli trigger SMC phenotypic changes, which, in turn, result in the long-term alterations of bladder tissue. Previously, the effects of mechanical stimuli on bladder cell behavior have been studied by several investigators using in vitro culture models. For example, when exposed to cyclic stretch (20% maximum, 0.1 Hz) for a maximum of 48 hours, bladder smooth muscle cells (BSMCs) in vitro exhibited increased cell size,13,14 cell numbers,15,16 and stretch-regulated gene expression (eg, heparin-binding EGF-like growth factor17 and the cysteine-rich protein Cyr6118) as compared with the control. Moreover, activation of several signaling pathways such as PI3K/Akt,16 p38,17 and extracellular signal-regulated kinase (ERK½)19 has been shown to mediate cyclic 0090-4295/10/$34.00 doi:10.1016/j.urology.2009.08.050
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stretch and the cellular-level events. Although these studies provided valuable information, exposure of SMCs to cyclic stretch only partially simulates the in vivo mechanical environment. Thus, further investigation is necessary to advance our understanding of the relationship between mechanical forces and bladder SMC phenotype shift. In addition to stretch, the cells of the bladder are constantly subjected to transmural hydrostatic pressure. More specifically, BSMCs that reside within the wall tissue are subjected to a pressure gradient that exists between the inside and outside of the bladder (⌬P ⫽ intravesical pressure ⫺ intra-abdominal pressure). Because many previous in vivo studies demonstrated that urinary diversion (removal of pressure load) leads to bladder atrophy,20-22 it can be argued that pressure is an important stimulus for the health of the bladder. A seminal in vitro study by Haberstroh et al23 exposed ovine BSMCs to pure hydrostatic pressure (4, 6, and 8.5 cm H2O), without stretch, for a maximum of 7 days and observed a significant increase in cell proliferation after 5 days, which was abrogated in the presence of heparin-binding epidermal growth factor (HB-EGF) inhibitor, CRM197.1 Their results provided the first evidence that BSMCs were sensitive to elevated hydrostatic pressure and that the response was mediated, at least in part, through HB-EGF.23 Although it might be concluded from these findings that hydrostatic pressure triggers phenotypic shift that leads to long-term tissue-level changes, cell proliferation represents only one of the SMC phenotype markers. Synthetic SMCs exhibit decreased expression of smooth muscle-specific proteins6,8 and a spread-out morphology,6,24 whereas contractile SMCs exhibit an elongated morphology. Moreover, it is still unknown which down-stream pathways mediate hydrostatic pressure and the growth factor-induced BSMC proliferation. The present study, therefore, aimed to test the hypothesis that hydrostatic pressure triggers several biological events that are pertinent to phenotype expression of BSMC. We specifically quantified cell morphology, cell proliferation, ERK½ activation (part of mitogenactivated protein kinase signaling pathway triggered by EGF), and contractile protein expression (␣-actin and SM22) after exposure of rat BSMC to sustained hydrostatic pressure.
MATERIAL AND METHODS BSMC Culture BSMCs were isolated from female Sprague-Dawley rats (250350 g) according to previously reported techniques.6,25 Briefly, the urothelium and mucosa were mechanically removed from the smooth muscle layer, which was finely diced and digested in sterile RPMI medium 1640 (Invitrogen; Carlsbad, CA) supplemented with 0.1% collagenase (Sigma-Aldrich, St. Louis, MO) and 0.5% trypsin EDTA (Invitrogen), with gentle stirring under standard culture conditions (ie, 37°C in a humidified atmosphere of 95% air and/or 5% CO2) for 30 minutes. The digested tissue was then filtered through a 100-m cell strainer 880
(BD Biosciences, Bedford, MA) and centrifuged at 1200 rpm for 3 minutes. The supernatant was discarded and the cell pellet was resuspended in RPMI supplemented with 10% fetal bovine serum (FBS; HyClone, Logan, UT) and 1% penicillin-streptomycin (Invitrogen). The cells were cultured under standard cell culture conditions with medium change every 3 to 4 days. The phenotype of BSMCs was determined by the expression of ␣-smooth muscle actin, SM-22, and h-caldesmon. Cells with passages of 8 or lower were used in all experiments.
Exposure of BSMC to Sustained Hydrostatic Pressure BSMCs were seeded at a predetermined density (10 000 or 200 000 cells per coverslip for morphologic or molecular analysis, respectively) on sterile glass coverslips (18 mm in diameter, product no. 48382-041: VWR) in RPMI supplemented with 10% FBS and 1% penicillin-streptomycin. After 24 hours, the media were replaced with low-serum media (1% FBS), and the cells were incubated for 24 hours before exposure to hydrostatic pressure. In specified experiments, BSMCs were incubated with 10 M meiosis-specific serine threonine protein kinase ½ (MEK½) inhibitor (U0126; Cell Signaling Technology, Danvers, MA) for 2 hours before pressure application. The cells that were incubated with low serum RPMI served as control for the inhibitor experiments. After another media change with fresh low-serum media, these cells were exposed to the sustained hydrostatic pressure using vertical columns filled with medium to the height of 7.5 cm of the column, creating hydrostatic pressure on the BSMCs seeded below as reported in the published data1,23,26 for a maximum of 48 hours. The cells maintained under atmospheric pressure (normal culture mediumheight ⫽ 0.3 cm) for the duration of experiments were used as the control.
Analysis of Cell Morphology and Proliferation At the end of each prescribed time period, BSMCs were fixed in freshly prepared 2% paraformaldehyde (Sigma-Aldrich) and stained with rhodamine-phalloidin (Invitrogen) and DAPI (Invitrogen) for actin filaments and cell nuclei, respectively. A fluorescence microscope (Nikon Instruments, Melville, NY) and digital camera (Q-Imaging: Surrey, BC, Canada) were used to image the BSMCs. For each sample, 12 images were taken in each quadrant of the coverslip. The cell morphology was analyzed by calculating the aspect ratio (major axis: minor axis) of 16 randomly selected cells in each image using Image-Pro Plus 5.0 software (Media Cybernetics, Inc., Bethesda, MD). The cell proliferation was quantified by counting all stained cell nuclei in each of the 12 image fields (0.577 mm2) and averaging. The data were reported as the number of cells per square millimeter.
Protein Collection and Western Blot Analysis At the end of the prescribed time periods (30, 90, and 180 minutes for signaling molecules and 24 and 48 hours for phenotypic marker proteins), BSMCs were removed from the coverslips by scraping in PBS. The cell suspension was centrifuged at 300 g for 7 minutes at 4°C and the cell pellet was resuspended in 150 L of complete cell extraction buffer: 5 mL of cell extraction buffer (CEB; Invitrogen), 250 L of protease inhibitor cocktail (Sigma-Aldrich), and 17 L of 0.3 M stock solution of phenylmethylsulfonyl fluoride (Sigma-Aldrich). The cell suspension was then incubated on ice with periodic vortexing for 30 minutes. The lysates were clarified with centrifuUROLOGY 75 (4), 2010
Figure 1. Cell morphology of rat BSMC exposed to sustained hydrostatic pressure (7.5 cm H2O) for 4 hours. At the end of experiments, BSMCs were fixed and stained with rhodamine-phalloidin and DAPI for actin filaments and nuclei, respectively. Cells maintained under control (no pressure) conditions (A) and cells exposed to pressure (B). The arrows indicate examples of major and minor axis measurements used to calculate the aspect ratios (magnification ⫽ 100 x). Histograms of aspect ratios of BSMCs maintained under controlled conditions (C) and exposed to sustained hydrostatic pressure for 4 hours (D). Data are mean ⫾ SD, analyzed using t test between 2 groups; *P ⬍.05; n ⫽ 3; 384 cells per group per experiment.
gation at 14 000 rpm at 4°C for 10 minutes and prepared for protein quantification or stored at ⫺80°C. The protein concentration in each sample was quantified using a protein assay kit based on the Bradford method27 and following the manufacturer’s instructions (Bio-Rad, Hercules, CA). Total protein samples (5-20 g protein) collected from cultures were separated using sodium dodecyl sulfate–polyacrylamide gel electrophoresis on a 12% polyacrylamide gel in Tris/ glycine/SDS buffer (Bio-Rad) at 200 V for 1 hour. Separated protein bands on the acrylamide gels were transferred to polyvinylidene difluoride (Bio-Rad) membranes at 100 V for 1 hour. The polyvinylidene difluoride membranes containing the protein bands were blocked for 1 hour and incubated with primary antibody for the protein of interest (␣-SMA, SM-22, GAPDH, total ERK½, and phosphorylated-ERK1/2) at an appropriate dilution overnight at 4°C. The membranes were incubated with secondary antibodies in the presence of 1 L streptactin-HRP (Bio-Rad) at room temperature for 1 hour before chemiluminescent labeling, following the manufacturers’ instructions (Immun-Star HRP Substrate Kit; Bio-Rad). Images of the labeled membranes were taken using FluorChemSP (Alpha Innotech, San Leandro, CA). The intensity of each protein band was analyzed to detect changes in protein expression using spot densitometry using AlphaEase FC software (Alpha Innotech, San Leandro, CA). The expression of phosphorylated ERK½ was normalized by the expression of total ERK½ to determine the level of ERK½ activation. The expression of ␣-smooth muscle actin and SM-22 was normalized by the GAPDH expression in each sample. UROLOGY 75 (4), 2010
Statistical Analysis All experiments were run in duplicate and were repeated at a minimum of 3 separate times. Numerical data were analyzed using analysis of variance (ANOVA) and Tukey test in SigmaPlot software (Systat Software, Chicago, IL); values of P ⬍.05 were considered significant.
RESULTS BSMC Morphology Under Sustained Hydrostatic Pressure The BSMCs exposed to sustained hydrostatic pressure (7.5 cm H2O) for 4 hours exhibited an overall shift from an elongated morphology to a more rounded morphology within the population (Figs. 1A and B). Aspect ratios of these cells were calculated and plotted in histograms (Figs. 1C and D) to quantitatively analyze the morphologic changes in response to sustained hydrostatic pressure. When compared with the control, BSMCs that were exposed to sustained hydrostatic pressure (7.5 cm H2O) for 4 hours exhibited an increase in number of cells within the population, with an aspect ratio between 1.0 and 2.0 (rounded morphology) and a decrease in cells with an aspect ratio of 4.0 or higher (elongated morphology) (Figs. 1C and D). 881
Figure 2. Activation of ERK½ in rat BSMCs after sustained hydrostatic pressure for 4 hours. Spot densitometry was used to quantify the ratio of phosphorylated ERK½ expression and total ERK½ expression. The fold increase was calculated from the ratio of phosphorylated ERK½ and total ERK½ expression for each time point. Representative chemiluminescence images for ERK½ activation (A); ERK½ activation in BSMCs after sustained hydrostatic pressure from 4 independent experiments (B). Average ERK½ activation in BSMC exposed to sustained hydrostatic pressure (C). Data are mean ⫾ SD, analyzed using ANOVA; *P ⬍ .05 when compared with the control; **P ⬍.05 when compared with the 30-minute group; n ⫽ 3 of 4 experiments.
ERK½ Activation in BSMCs Under Sustained Hydrostatic Pressure In the present study, the activation of ERK½ in BSMCs exposed to sustained hydrostatic pressure for a maximum of 3 hours was quantified through Western blotting and spot densitometry of chemiluminescence imaging (Fig. 2A). When compared with the control, BSMCs exposed to sustained hydrostatic pressure exhibited a maximum of a 2-fold increase in the activation of ERK½ at 30, 90, and 180 minutes. In 3 of the 4 experiments conducted (samples 1-3), the peak activation of ERK½ occurred at the 180 minutes time point, but in 1 other experiment (sample 4), ERK½ activation occurred earlier and peaked at 90 minutes (Fig. 2B). The mean increase in activation of ERK½ in samples 1-3 in response to sustained hydrostatic pressure for 180 minutes is significantly (P ⬍.05; n ⫽ 3) greater (1.64-fold) than that of the control (baseline, at 0 minute) and at 30 minutes (Fig. 2C).
BSMC Proliferation Under Sustained Hydrostatic Pressure The proliferative response of BSMCs to sustained hydrostatic pressure (7.5 cm H2O) for 48 hours was quantified through cell density (cells/mm2) measurements (Fig. 3). The cell density of BSMCs subjected to hydrostatic pressure for 24 hours was similar to the no pressure control at the same time point (data not shown). However, the cell density in BSMCs subjected to hydrostatic pressure for 48 hours exhibited a significant (P ⬍.05; n ⫽ 5) increase when compared with the control after 48 hours (Fig. 3). In the presence of MEK½ inhibitor, this increase in the cell density was absent (Fig. 3). 882
Figure 3. Proliferative response of BSMCs exposed to sustained hydrostatic pressure for 48 hours. At the end of experiments, cells were fixed and stained with DAPI for nuclei, which were then counted using ImagePro software. Data are mean ⫾ SD, analyzed using ANOVA and paired t test; *P ⬍.05 when compared with the control; n ⫽ 3-5.
Contractile Protein Expression in BSMC Under Sustained Hydrostatic Pressure BSMCs pretreated with MEK½ inhibitor, U0126, were exposed to hydrostatic pressure (7.5 cm H2O) for 48 hours and the expression of contractile proteins ␣-smooth muscle actin (␣-SMA) and SM-22 were quantified. When compared with the control (no pressure in the absence of U0126), BSMC expression of ␣-SMA and SM-22 was similar in all other groups (no pressure in the presence of U0126, and cells exposed to sustained hydrostatic pressure, both in the absence and presence of U0126) at 48 hours (Fig. 4). UROLOGY 75 (4), 2010
Figure 4. Expression of contractile proteins, ␣-SMA and SM22, in rat BSMCs after exposure to sustained hydrostatic pressure for 48 hours. Spot densitometry was used to quantify contractile protein expression and normalized to GAPDH protein expression. (A) Representative western blot spot densitometry for ␣-SMA and SM22 protein expression; (B) Expression of contractile-marker proteins ␣-SMA and (C) SM22 after exposure to hydrostatic pressure and in the presence of a MEK½ inhibitor. Data are mean ⫾ SD; analyzed using ANOVA; n ⫽ 4.
COMMENT The present study investigated the effect of elevated, but physiological-level, hydrostatic pressure on BSMC phenotype in vitro. The experimental setup we used was adapted from previously published studies that examined the effects of hydrostatic pressure on BSMCs1,23 and vascular endothelial cells;26 and this design allowed application of pressure to cultured cells with minimal variation in pH, pO2, and pCO2.26 The results of the present study demonstrated that BSMCs subjected to sustained hydrostatic pressure for 4 hours showed a population shift in morphology from elongated to more rounded morphology. This was evidenced by more cells (48%) exhibiting an aspect ratio of 1.0 to 2.0 and fewer cells (6.5%) exhibiting an aspect ratio of ⬎ 4.0 after exposure to pressure, when compared with the control (40%, 11%, respectively), suggesting a possible phenotypic shift from contractile to synthetic. Although these are the first observations of BSMC morphologic response to pressure, others have investigated and reported changes in SMC morphology in response to mechanical stimuli. For example, when vascular SMC were subjected to uniaxial stretch for a maximum of 40 minutes, these cells exhibited more polarized cell morphology in the direction of the stretch.28 More recently, our laboratory reported that BSMCs within 3D collagen constructs exposed to sustained tension for 48 hours exhibited significantly higher aspect ratios, suggesting a more contractile phenotype, than that of the control.6 These findings imply that morphologic responses of SMCs depend on the type of mechanical stimuli applied; stretching results in a shift toward more contractile, whereas exposure to hydrostatic pressure results in a shift toward more synthetic phenotype of SMC. In addition to the cell morphology change, BSMCs subjected to sustained hydrostatic pressure for 3 hours UROLOGY 75 (4), 2010
exhibited a maximum of 2-fold increase in ERK½ activation (Fig. 2). Moreover, exposure of BSMCs to sustained hydrostatic pressure (7.5 cm H2O) for 48 hours led to a significant increase in cell proliferation when compared with the control (Fig. 3). When BSMCs were pretreated with MEK½ inhibitor, U0126, however, the cell proliferation under hydrostatic pressure was similar to that of the control (Fig. 3), suggesting that pressureinduced BSMC proliferation is mediated through the ERK½ pathway. These results added another level of details to the previous observations that pressure-induced BSMC proliferation was mediated by HB-EGF.23 As previously reported with a lung epithelial cell model,29 it is possible that elevated hydrostatic pressure triggers HBEGF shedding and/or release from BSMC surfaces, which in turn activates the EGF receptor and a cascade of events that include ERK½ activation and cell proliferation. Although the present study provided some explanation, further investigation is necessary to determine the exact mechanism for pressure mechanotransduction by BSMC. Currently, involvement of the ERK pathway in BSMC mechanotransduction is somewhat controversial. For example, one group previously reported that p38/SAPK, but not ERK½ mediates the effect of cyclic stretch (25% at 0.1 Hz for 30 minutes to a maximum of 24 hours) on BSMC proliferation and HB-EGF mRNA expression.17 In contrast, another group reported a very rapid (within 5 minutes) ERK½ activation in BSMCs exposed to static and cyclic stretch (5%-10%, 0.1 Hz).19 These authors also reported a significant increase in BSMC proliferation in response to cyclic stretch, which was significantly reduced in the presence of ERK½ inhibitor, demonstrating the involvement of early, transient ERK activation in this event.19 However, in 3 of the 4 experiments of the 883
present study, BSMC exhibited activation of ERK½ at the 180-minute time point under sustained hydrostatic pressure, whereas 1 specimen exhibited this peak activation at 90 minutes (Fig. 2). Despite this variation in peak activation time, possibly because of the cell batch differences, our results to date certainly demonstrate that BSMCs exhibit ERK½ activation in response to sustained hydrostatic pressure, but no earlier than in 90 minutes. On the basis of these, it can be speculated that BSMCs respond more readily to mechanical environments of pathologic conditions such as cyclic stretch, whereas prolonged exposure to physiological-level pressure induce similar responses. The results of the present study indicated that, when compared with the control, expression of ␣-SMA and SM-22 was similar in the BSMCs exposed to sustained hydrostatic pressure after 48 hours. Expression of these contractile proteins has been previously used as an index of a SMC phenotypic shift; synthetic SMC express less of these proteins compared with contractile SMC. To date, however, the exact relationship between contractile protein expression and other events such as cell proliferation is still partially understood. For example, when compared with the control, vascular SMC exhibited an increase in cell proliferation, but similar ␣-SMA expression in response to PDGF (5.0 ng/mL) in a 2D monolayer culture.10 When these cells were cultured in 3D collagen gels; however, exposure to the same level of PDGF led to both an increase in cell proliferation and a decrease in ␣-SMA expression.10 These findings suggest that cell proliferation and ␣-SMA expression of SMCs are not governed by a single pathway, but may be regulated synergistically by the growth factor signaling and physical environments (2D vs 3D).10 Moreover, another group reported that the stretch-induced upregulation of contractile proteins, ␣-SMA, calponin, and SM-22 in vascular SMCs was blocked by inhibiting p38 activation, but not by inhibiting ERK½.30 These results provide further support that although ERK½ activation in SMC may lead to increased cell proliferation, these cellular events are independent from contractile protein expression. Although these previous findings provide some explanations for the lack of contractile protein changes in BSMC under hydrostatic pressure, it is also possible that we did not observe any differences because the time course of the present study was limited to 48 hours by the lowserum content in cultures. Further investigation, therefore, is necessary to elucidate the relationship between mechanical stimuli and BSMC phenotypic marker protein expression.
CONCLUSIONS The results of the present in vitro study provided the evidence that BSMCs exposed to physiological-level hydrostatic pressure for prolonged periods exhibited morphologic and proliferative changes that possibly indicate a phenotypic shift from contractile to synthetic. The 884
comparable levels of contractile proteins observed in both control and pressure group BSMCs suggest that not all the phenotype markers are regulated concomitantly by a single stimulus. Moreover, the activation of ERK½ intracellular signaling pathway under hydrostatic pressure observed in the present study may represent a potential mechanism for mechanically induced BSMC proliferation observed in the present and other studies.19 These findings add cellular and/or molecular level knowledge to the current understanding that although appropriate mechanical stimuli are needed for growth and development of the urinary bladder,1,2 excessive mechanical forces created by various voiding dysfunctions3-5 can alter bladder tissue architecture and compliance.3,6,7 Acknowledgment. The authors thank Dr Bruce Gao, Department of Bioengineering, Clemson University, for providing the rats used as the source of bladder cells. References 1. Haberstroh KM, Kaefer M, Retik AB, et al. The effects of sustained hydrostatic pressure on select bladder smooth muscle cell functions. J Urol. 1999;162:2114-2118. 2. Maizels M. Normal and anomalous development of the urinary tract. In: Walsh PC, Retik AB, Vaughan ED Jr, Wein AJ, eds. Campbell’s Urology,7th ed. Philadelphia, PA: W.B. Saunders; 1998: 1577. 3. Korossis S, Bolland F, Ingham E, et al. Review: tissue engineering of the urinary bladder: considering structure-function relationships and the role of mechanotransduction. Tissue Eng. 2006;12:635-644. 4. Watanabe T, Rivas DA, Chancellor MB. Urodynamics of spinal cord injury. Urol Clin North Am. 1996;23:459-473. 5. Chancellor MB, Rivas DA, Huang B, et al. Micturition patterns after spinal trauma as a measure of autonomic functional recovery. J Urol. 1994;151:250-254. 6. Roby T, Olsen S, Nagatomi J. Effect of sustained tension on bladder smooth muscle cells in three-dimensional culture. Ann Biomed Eng. 2008;36:1744-1751. 7. Stover J, Nagatomi J. Cyclic pressure stimulates DNA synthesis through the PI3K/Akt signaling pathway in rat bladder smooth muscle cells. Ann Biomed Eng. 2007;35:1585-1594. 8. Worth NF, Rolfe BE, Song J, et al. Vascular smooth muscle cell phenotypic modulation in culture is associated with reorganisation of contractile and cytoskeletal proteins. Cell Motil Cytoskeleton. 2001;49:130-145. 9. Sobue K, Hayashi K, Nishida W. Molecular mechanism of phenotypic modulation of smooth muscle cells. Horm Res. 1998;50(suppl 2):15-24. 10. Stegemann JP, Nerem RM. Altered response of vascular smooth muscle cells to exogenous biochemical stimulation in two- and three-dimensional culture. Exp Cell Res. 2003;283:146-155. 11. Adelow CA, Frey P. Synthetic hydrogel matrices for guided bladder tissue regeneration. Methods Mol Med. 2007;140:125-140. 12. Backhaus BO, Kaefer M, Haberstroh KM, et al. Alterations in the molecular determinants of bladder compliance at hydrostatic pressures less than 40 cm H2O. J Urol. 2002;168:2600-2604. 13. Karim O, Cendron M, Mostwin J, et al. Developmental alterations in the fetal lamb bladder subjected to partial urethral obstruction in utero. J Urol. 1993;150:1060-1063. 14. Peters C, Vasavada S, Dator D, et al. The effect of obstruction on the developing bladder. J Urol. 1992;148:491-496. 15. Orsola A, Adam RM, Peters CA, et al. The decision to undergo DNA or protein synthesis is determined by the degree of mechanical deformation in human bladder muscle cells. Urology. 2002;59: 779-783.
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16. Adam RM, Roth JA, Cheng H, et al. Signaling through PI3K/Akt mediates stretch and PDGF-BB-dependent DNA synthesis in bladder smooth muscle cells. J Urol. 2003;169:2388-2393. 17. Nguyen HT, Adam RM, Bride SH, et al. Cyclic stretch activates p38 SAPK2-, ErbB2-, and AT1-dependent signaling in bladder smooth muscle cells. Am J Physiol Cell Physiol. 2000;279:C1155C1167. 18. Tamura I, Rosenbloom J, Macarak E, et al. Regulation of Cyr61 gene expression by mechanical stretch through multiple signaling pathways. Am J Physiol Cell Physiol. 2001;281:C1524-C1532. 19. Halachmi S, Aitken KJ, Szybowska M, et al. Role of signal transducer and activator of transcription 3 (STAT3) in stretch injury to bladder smooth muscle cells. Cell Tissue Res. 2006;326:149-158. 20. Jayanthi VR, McLorie GA, Khoury AE, et al. The effect of temporary cutaneous diversion on ultimate bladder function. J Urol. 1995;154:889-892. 21. Machado MG, Yoo JJ, Atala A. Defunctionalized bladders: effects before and after refunctionalization in an animal model. J Urol. 2000;164:1002-1007. 22. Krabill KA, Sung HW, Tamura T, et al. Factors influencing the structure and shape of stenotic and regurgitant jets: an in vitro investigation using Doppler color flow mapping and optical flow visualization. J Am Coll Cardiol. 1989;13:1672-1681.
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23. Haberstroh KM, Kaefer M, Bizios R. Inhibition of pressure induced bladder smooth muscle cell hyperplasia using CRM197. J Urol. 2000;164:1329-1333. 24. Rensen SS, Doevendans PA, van Eys GJ. Regulation and characteristics of vascular smooth muscle cell phenotypic diversity. Neth Heart J. 2007;15:100-108. 25. Kropp BP, Zhang Y, Tomasek JJ, et al. Characterization of cultured bladder smooth muscle cells: assessment of in vitro contractility. J Urol. 1999;162:1779-1784. 26. Acevedo AD, Bowser SS, Gerritsen ME, et al. Morphological and proliferative responses of endothelial cells to hydrostatic pressure: role of fibroblast growth factor. J Cell Physiol. 1993;157:603-614. 27. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of proteindye binding. Anal Biochem. 1976;72:248-254. 28. Katsumi A, Milanini J, Kiosses WB, et al. Effects of cell tension on the small GTPase Rac. J Cell Biol. 2002;158:153-164. 29. Tschumperlin DJ, Dai G, Maly IV, et al. Mechanotransduction through growth-factor shedding into the extracellular space. Nature. 2004;429:83-86. 30. Qu M-J, Liu B, Wang H-Q, et al. Frequency-dependent phenotype modulation of vascular smooth muscle cells under cyclic mechanical strain. J Vasc Res. 2007;44:345-353.
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