( : ) : : : : : :
,,, ;,,,,,,,, :,, 1
I ------------------------------------------------------- 3 4 II A ----------------------------------------------- 5-8 B ------------------------------------ 8-9 C --------------------- 10-23 III -------------------------------------------------------------- 24-28 IV ------------------------------------------------------------------ 29 V ( )--------------------------------- 30-32 2
I, (spinal bifia), (sacral agenesis), (cerebral palsy), tether cord,, (myelomeningocele),, ;, ;,,,, ventriculoperitoneal shunt ;,,, ;,,,,,,,, 3
: 1, 2 3, 4
II 2000 9 28 2001 12 30,, Stuart B Bauer 1 ( ),,,,, ;, 2,, :, ; :, denervation, Michael R Freeman, Stuart B Bauer Mary Kelly, Michael R Freeman, Rosalyn Adam,, A (Urodynamics), ;,,,,,,,, (Uroflowmetry, 5
UFR) (cystometrography, CMG), Xylocaine jelly, 7 Fr ( ) ( ),,,, 15 cc, 37 o C ;, (hyper-reflexia) 15, detrusor instability; (hypertonicity) ;, ( : 2, (kg) X 7; 2, ( ) +2 X30),, 15 cc, ;,, meperidine (1 mg/kg body weight) EMLA ( ),, cholinergic drug : Bethanechol; anticholinergic drug: Oxybutynin (Ditropan), Detrol; sympathomimetic drug: Pseudoephedrine 2 (electromyography, EMG) 24-gauge needle 6
electrode, ( ) ( ),, : 1, denervation degeneration 2, Crede Valsalva, bulbocavernosus (anocutaneous ) ; ;, reflex circle, ( lower motor neuron disease) 3,, (detrusor-sphincter dys-synergia, DSD), (upper motor neuron disease),,,,,,, (MRI), tethering cord,,,,,,,, (urethral pressure profile, UPP) ;,, ;, 6 ( ) 7
, 5,, (EMG), (Ultrasonography), (Voiding Cystourethrography, VCUG) B (DSD),,,, (Bauer SB: Early evaluation and management of children with spina bifida In King LR, ed: Urologic Surgery in Neonates and Young Infants Philadelphia, WBSaunders Company, 1988, pp252-264),, (EMG), (hyper-reflexia or hypertonicity), anticholinergic, Oxybutynin hydrochloride, 1mg/kg body weight Q12h 5 mg tid, 1 mg, Detrol ; (DSD), (Clean Intermittent Catheterization, CIC), 4 6 ;, Pseudoephedrine ;,,,, nelaton tube, (vesicoureteral reflux) :, hypertonicity, poor compliance 8
, oxybutynin ;,,,, ;,,,,, 5, ;,, 40 40,,,,, sympathomimetic drug: ephedrine pseudoephedrine,, Young-Dees-Leadbetter procedure Kropp urethral Lengthening procedure Kropp modification (Salle, Ppork) fascial sling procedure bulking agent injection,,,,, tissue engineering, 9
C PDGF-Stimulated DNA Synthesis in Human Bladder Smooth Muscle Cells Occurs Independently of Erk-MAPK Pathway Activation (Hong-Lin Cheng, Rosalyn M Adam, Stuart B Bauer, and Michael R Freeman The Urologic Laboratory, Department of Urology, Children s Hospital and Harvard Medical School, Boston MA 02115) Abstract Introduction and objectives: Bladder smooth muscle cells (SMC) exposed to mechanical forces increase expression of peptide growth factors Excessive mechanical deformation can result in which expression and activity of growth factors are upregulated In the current study we have examined the relative potencies of known SMC mitogens in promoting DNA synthesis in human bladder SMC and have characterized signal transduction pathway activation downstream of growth factor stimulation Materials and Methods: Confluent and quiescent primary cultured human bladder SMC were exposed to different doses of HB-EGF, PDGF or FGF-2 for 24 h The extent of DNA synthesis was determined by uptake of radiolabeled thymidine into acid-precipitable material To identify the signaling pathway(s) responsible for induction of DNA synthesis, cells were pretreated with pathway-selective pharmacological inhibitors prior to addition of growth factors and assay for DNA synthesis Pathway activation was also assessed by immunoblot analysis, following growth factor and/or inhibitor treatment Total cell lysates were fractionated by SDS- PAGE, blotted to nitrocellulose and immunoblotted with antibodies to the non- 10
phosphorylated and phosphorylated forms of p38 SAPK2, Akt and p44/p42 Erk Results: PDGF was found to be the most potent stimulator of DNA synthesis in human bladder SMC, promoting a ~5-fold increase in thymidine incorporation over baseline HB-EGF and FGF-2 were relatively ineffective as mitogens in this assay The PDGF-mediated DNA synthesis response was demonstrated to be specific since it was blocked in a dose-dependent manner with the PDGF receptor tyrosine kinase inhibitor, AG 1296 The inhibitors SB203580 and LY294002, which block signaling through p38sapk2 and PI-3 kinase respectively, were also able to ablate the PDGFstimulated DNA synthesis response whereas the MEK1 inhibitor PD98059, which blocks Erk/MAPK activation, did not affect PDGF-induced thymidine incorporation Involvement of the p38sapk2 and PI-3 kinase pathways was confirmed by immunoblot analysis using antibodies to the activated (phosphorylated) forms of p38 and Akt, which demonstrated increased phosphorylation of these signaling intermediates in response to PDGF-treatment Furthermore, the PDGF-induced upregulation of phosphorylation of p38 and Akt was decreased in the presence of doses of SB203580 and LY294002, respectively, known to block the DNA synthesis response Conclusions: These data demonstrate for the first time that PDGF is a potent mitogen for human bladder SMC The DNA synthesis response to PDGF in bladder SMC is mediated by signaling through the p38sapk2 and PI-3 kinase pathways, but is independent of Erk-MAPK pathway activation 11
Introduction The primary force impinging on cells of the bladder wall is that of mechanical deformation Distension of the bladder wall during cycles of bladder filling and emptying under normal conditiones enables storage of urine at low pressure In cases of outlet obstruction, bladder wall distends beyond its normal physiologic limits leading to the iniation of molecular mechanisms that attempt to compensate for the pressure increase One demonstrated outcome of obstruction is an increase in bladder mass which results from increased smooth muscle cell (SMC) mass (hypertrophy) and number (hyperplasia) [Levin et al, 1984; Levin et al, 1990; Levin et al, 1995; Monson et al, 1994, 1995; Buttyan et al, 1992, 1994; Chen et al, 1994] Although the mechanisms underlying the growth response of the bladder wall to distension have not been fully defined, changes in peptide growth factor expression and activity at the local tissue level are known to occur Altered expression of several growth factors and other molecules in response to mechanical stimuli has been demonstrated, including heparin-binding EGF-like growth factor (HB-EGF) [Park et al,1998; Nguyen et al, 1999; Borer et al, 1999], basic fibroblast growth factor (bfgf), TGF 1 [Buttyan et al, 1992] and cyclooxygenase-2 (COX-2)[Park et al, 1997] These data suggest that SMC exposed to mechanical forces may regulate their own proliferation through the elaboration of growth regulatory molecules, thereby contributing to the SMC hypertrophy and/or hyperplasia observed following outlet obstruction Platelet-derived growth factor (PDGF) is a major mitogen for connetive tissue cells, smooth muscle cells and certain other cell types The PDGF isoforms, binding to and activating two structurally related protein kinase receptors, -receptor and 12
-receptor, exert their cellular effects Mechanical force increases PDGF-B and PDGF receptor expression in vasucular smooth muscle (VSM) cells; And PDGF-B chain plays an important role in the proliferation of VSM cells We have previously demonstrated that neonatal rat bladder SMC exposed to cyclic mechanical deformation in culture undergo DNA synthesis which is mediated in part by selective upregulation of signaling through the p38sapk2 mitogen-activated protein kinase (MAPK) pathway [Park et al, 1998; Nguyan et al, 2001] Significantly, signaling through the Erk-MAPK pathway, which is known to mediate DNA synthesis in many cell types, was found to be dispensable for the stretch-induced DNA synthesis response in rat bladder SMC In the present study, we have identified PDGF as a potent stimulator of DNA synthesis in primary culture human bladder SMC Consistent with our previous findings with rat bladder SMC exposed to mechanical deformation, PDGF-induced DNA synthesis was found to be mediated through selective upregulation of p38sapk2 activity, but occurs independently of Erk/MAPK activation Materials and Methods Reagents Dulbecco s modification of Eagle s medium (Cellgro); human PDGF-BB, HB- EGF, FGF-2 (R&D Systems); AG1296, PD98059, LY294002, SB203580 (Calbiochem); Phospho-Akt antibody, Akt antibody, Phospho-38 antibody, p38 antibody, Phospho-p44/42 MAPK antibody, p44/42 MAPK antibody (Cell signaling) Cell Culture 13
Human bladder smooth muscle cells (SMC) were isolated and propagated in culture as previously described [Atala et al 1993; Borer et al, 1999] Cells were maintained in Dulbecco s modification of Eagle s medium(dmem) supplemented with 10% fetal bovine serum(fbs), penicillin (100 U/ml) and streptomycin (100 µg/ml) at 37 C in a humidified atmosphere of 95% air/5% CO 2 Experiments were performed on cells between passages 2 and 5 DNA Synthesis Assay Cells were seeded at a density of 15 X 10 4 cells/well in 24-well dishes and incubated for 72 hrs in DMEM/10% FBS Cells were then treated for 24 hrs with appropriate growth factors in DMEM and inhibitors over a range of doses 2 hrs before; cells were labeled for the last 12 hrs of the treatment period with 1 µci/well methyl- 3 H-thymidine At the end of the incubation period, cells were fixed with 15% tricholoracetic acid (TCA), washed with methanol and allowed to air dry Then cells were solubilized with 03N NaOH/1% SDS and, prior to analysis using a Rackbeta liquid scintillation counter (LKB Wallac, Gaithersburg, MD), the resulting solution mixed with Optifluor scintillant Immunoblot Analysis Cells were seeded at a density of 1 X 10 5 cells /well in 6-well plates and incubated for 3 days in DMEM/10% FBS till 80% confluence; then serum deprived for 48 hrs Cells were pretreated without or with 20 µm SB203580, 10 µm LY294002 or DMSO (vehicle control) for 1 hr, followed by stimulation with 025 nm PDGF-BB for indicated times Cell were harvested in lysis buffer (625mM Tris-Cl ph 68, 10% glycerol, 2% SDS, 1mM Na 3 VO 4 ) and the protein concentration determined using the Micro BCA assay (Pierce Chemical Co) Equivalent amounts of protein were 14
fractionated on 12% SDS-PAGE gels, the protein transferred to nitrocellulose membrane and the membranes probed with antibodies to the non- phosphorylated and phosphorylated forms of p38sapk2 and Akt Results PDGF-B was found to potently stimulate DNA synthesis, as measured by methyl- 3 H-thymidine incorporation in human bladder SMC In contrast basic FGF/FGF-2 and heparin-binding EGF-like growth factor (HB-EGF) were much less potent as mitogen for bladder SMC (fig1) To delineate which signal tranduction pathways may be responsible for mediating the observed DNA synthesis response to PDGF-B treatment, cells were pretreated for 2 hours with pathway-specific inhibitors prior to stimulation with PDGF-B The inhibitors used were as follows: AG1296, a specific inhibitor of the PDGF-B receptor tyrosine kinase; PD98059, an inhibitor of MEK1, specific for the Erk-MAPK pathway; SB203580, an inhibitor of p38-sapk2 phosphorylation and pathway activation; and LY294002, an inhibitor of PI-3-kinase Dose-dependent inhibition of PDGF-stimulated DNA synthesis in bladder SMC was observed by pretreatment of cells with AG1296, SB203580 and LY294002, but not with PD98059 (fig2) These data implicate the p38sapk2 and PI-3-kinase pathways in mediating the DNA synthesis response to PDGF and suggest that the Erk-MAPK pathway is not required for cell-cycle traverse in human bladder SMC To confirm the effects of the inhibitors observed in vitro, total cell lysates were prepared from cells pretreated with the effective dose of the inhibitors, and stimulated with 025 nm PDGF-B for different time point Cells treated with PDGF-B but not 15
exposed to inhibitors served as controls Lysates were fractionated by SDS-PAGE, transferred to nitrocellulose membrane and membranes were probed with antibodies to the phosphorylated and non-phosphrylated forms of Erk1/Erk2, p38sapk2, and Akt which is downstream of PI-3-kinase PDGF-B was found to stimulate phosphorylation of both p38 and Akt in a time-dependent manner, albeit with differing kinetics Increased p38 phosphorylation was observed as early as 10 mins following PDGF-B treatment with peak phosphorylation observed at 60 mins Akt displayed modest basal phosphorylation which was increased markedly within 5 mins of PDGF-B stimulation and peaked at 30 mins Controls displayed little or no significant phosphorylation of either p38 or Akt (fig 3A, fig 4A) In cells exposed to inhibitors of the p38sapk2 or PI-3-kinase pathways, a decrease in PDGF-B stimulated phosphorylation of the appropriate effector was observed SB203580 (20µM) was found to reduce p38 phosphorylation to baseline or close to baseline at all time points tested The PI-3-kinase inhibitor, LY294002 (10µM), also reduced PDGF-stimulated Akt phosphorylation, although substantial Akt phosphorylation remained especially at the later time points (fig 3B, fig 4B) To confirm that the Erk-MAPK pathway was functional in human bladder SMC and that the PD98059 was active in this cell type, SMC were pretreated without or with different doses of PD98059 for 30 mins and stimulated for 15 mins with 50 ng/ml FGF-2, previously demonstrated to be a bladder SMC mitogen Lysates were prepared as described and blotted with antibodies to the phosphorylated and nonphosphorylated forms of Erk FGF-2 was found to potently stimulate phosphorylation of Erk1/Erk2 and this phosphorylation was inhibited in a dose-dependent manner with increasing doses of PD98059 (fig5) These data confirmed that the Erk-MAPK kinase 16
pathway was indeed functional in human bladder SMC, and was activatable by the appropriate ligand Furthermore the dataindicate that PD98059 was a potent inhibitor of Erk phosphorylation, and therefore the Erk-MAPK pathway at the doses employed in the DNA synthesis Discussion Previous studies in this laboratory and by others have demonstrated altered growth factor expression and DNA synthesis in response to application of mechanical force in bladder smooth muscle cells Basic FGF, keratinocyte growth factor (KGF)/FGF-7 and HB-EGF are known to be upregulated in SMC exposed to mechanical deformation in in vitro and in vivo model systems To determine whether increasede growth factor expression was implicated in the DNA synthesis response in SMC, we performed an initial screen of several know SMC mitogens to compare their relative potency in stimulating uptake of thymidine in primary culture human bladder SMC As anticipated, bfgf and HB-EGF stimulated DNA synthesis in bladder SMC, however they displayed modest potency relative to PDGF-BB which was the most potent factor tested PDGF has long been known as an extremely potent mitogen for vascular SMC, however its activity on bladder SMC proliferation has not been investigated In view of its ability to stimulate DNA synthesis in bladder SMC, PDGF-BB may implicated in the increase in mass of the bladder wall following outlet obstruction PDGF has not been directly implicated in the hypertrophic or hyperplastic response of the bladder wall to outlet obstruction with the proliferative response largely attributed to members of the FGF, EGF-like and IGF growth factor families However PDGF-B gene expression is known to be regulated by shear-stress in certain cell types such as endothelial cells PDGF-B released from such cells in response to mechanical stimuli may then act in a paracrine manner on underlying 17
SMC to achieve its effects Although PDGF has not been identified as a mechanicallyregulated molecule in bladder SMC in vivo or in vitro, it is possible that PDGF is elaborated by the epithelial component of the bladder wall, which is also exposed to mechanical deformation, where it can participate in paracrine stimulation of the associated SMC [Li et al, Hypertens Res 20 217-23; Wilson et al, Hypertension 31, 170-5; Wilson et al, J Cell Biol 123, 741-7; Sumpio J Surg Res 44, 696-701; Sumpio J Vasc Surg 10, 570-1; Omer et al, 1992 Am J Physiol/Renal 263, R1284-90; Sadoshima et al, JBC 267, 10551-60] 18
Figure 1 19
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III, 1960 ventriculoatrial shunt (hy drocephalus), (menigomyelocele),,,,,,,, ;, ;,, Wu et al,,,,, ;, (Folic acid) ;,,,, 1,,,,, ;,, 2, ; ; ; 1970 1980, 7,, 26
, ;,,, 1972 Lapides (clean intermittent catheteri zation, CIC),, ;,,, Lapides, 218, ;, 33,, ; Joseph, 33, 16,, 3,,,, Lindehall et al 87, 11,, ; Cass et al 84,,, 41 (49%), 14 (17%), 8 (9%), 7 (8%), 27
14 (17%); hyper-reflexia hypertonicity, oxybutynin hydrocholoride (Ditropan ),, oxybutynin hydrocholoride,,,,,,,, : ;, : : ;,,, bulking agent Teflon, Collagen, fascial sling, ;,,, 28
, 1980, Mitrofanoff, (appendicovesicostomy), ; 10 12Fr,, Continence rates,,,, Mitrofanoff, ;,,,, ;, 1990 Malone et al Mitrofanoff, antegrade continence enema (ACE procedure) enema,,,,,, 12 Fr,,, ;,,,,,, ;,, 29
,,,, ;,,,, tethering cord,, 30
IV 1 2 3 31