| | Extracellular matrix protein expression during mouse detrusor development☆☆☆Presented at the 49th Annual Congress of the British Association of Paediatric Surgeons, Cambridge, England, July 23-26, 2002. Abstract Background/Purpose: Extracellular matrix proteins are implicated in regulating cell proliferation and differentiation. The authors systematically analysed the expression of elastin; collagen types I, III, IV; laminin; and fibronectin during mouse detrusor muscle development, a period during which downregulation of detrusor proliferation and increasing smooth muscle differentiation is known to occur. Methods: Embryonic days 14 (E14) and 18 (E18), and postnatal day 1 (D1) and week 6 (6wk) were examined, a period spanning the inception of the bladder to postnatal maturity. Immunohistochemistry of whole bladders was used to immunolocalise protein expression, and Western blot of dissected detrusor layers was used to semiquantify soluble protein expression. Results: All proteins were detected at all 4 stages. Statistically significant increases were documented for elastin (E14 to 6wk), collagen type I (E18 to 6wk), collagen type III (D1 to 6wk) and laminin (E14 to 6wk). Fibronectin levels were relatively high up to D1, after which levels declined significantly. Collagen type IV levels decreased significantly (E18 to 6wk). Conclusions: The authors postulate that changing levels of laminin and fibronectin have opposing effects on the transition from proliferating primitive mesenchymal cells to differentiated detrusor muscle. Furthermore, changes in collagen type III and elastin may be important for bladder compliance. J Pediatr Surg 38:1-12. Copyright 2003, Elsevier Science (USA). All rights reserved.
In addition to their conventional roles as scaffolds for cells, extracellular matrix proteins (ECM) regulate dynamic functions of cells and tissues.1 ECM is produced, and its arrangement determined, by the cells embedded within it. A complex loop regulating tissue morphology therefore can be envisaged, with cells and their surrounding ECM influencing one another. During normal vertebrate development, ECM proteins direct cell migration, differentiation, and polarity in diverse tissues including developing kidney tubules, mammary epithelia, keratinocytes, osteoblasts, myoblasts, endothelia, and neural crest.2
Regarding mouse detrusor muscle development, previously, we showed a transition from proliferating primitive mesenchymal cells, which express little α smooth muscle actin and no desmin, to a quiescent, differentiated phenotype, with increasing levels of contractile protein.3 Hence, a striking inverse relationship exists between detrusor proliferation and differentiation. We postulated that ECM is important for the switch from proliferation to differentiation during detrusor development. We systematically analysed expression of elastin; collagen types I, III, IV; laminin; and fibronectin during detrusor muscle differentiation in normal mice using immunohistochemistry of whole bladders to localise protein expression to specific layers of the developing bladder and Western blot of dissected detrusor layers to semiquantify levels of soluble protein in the detrusor muscle. Our results show that mouse detrusor morphogenesis is accompanied by complex serial changes in several ECM proteins.
Materials and methods  Chemicals and animals Unless otherwise stated, chemicals were obtained from Sigma-Aldrich Co (Poole, England). We studied normal time-mated, out-bred CD1 mice (Charles Rivers Mouse Farms, Margate, England) with the day of the vaginal plug designated as embryonic day 0. The gestation period of these mice is 20 days. Previous histologic results in CD1 mice3 showed the urinary bladder became a distinct entity from embryonic day 14 (E14). Hence, the stages examined were E14 and embryonic days 18 (E18), and postnatal day 1 (D1) and week 6 (6wk), the latter time correlating with postnatal maturity. Animal procedures were carried out with the approval of the UK Home Office. Urinary bladder histology For histologic studies, a minimum of 4 samples of each sex and stage harvested from 3 to 4 litters were analysed. Tissues were fixed in 4% paraformaldehyde (BDH, Poole, England), embedded in paraffin wax and sections cut at 5 μm. After dewaxing and hydration, slides were placed in 3% potassium dichromate for 1 hour at 60°C. After staining in filtered Celestine blue and hematoxylin for 5 minutes each, slides were placed in filtered chromotope green mixture (0.6% chromotope, 0.3% fast green, 0.6% phosphatungstic acid, and 1% acetic acid) for 15 minutes at room temperature. After 2 cycles of blotting and dehydration, slides were placed in Histoclear and mounted in dextropropoxyphene (BDH). Using this Masson's trichrome stain, collagen stains turquoise, erythrocytes, muscle cell cytoplasm and keratin stain red, and nuclei stain purple.4 Preliminary experiments were performed to optimise the pretreatment regimen for each of the specific ECM antibodies to yield the clearest immunohistochemistry, eg, microwaving in 2.1 g/L of citric acid (pH 6.0) or incubation with proteinase (0.2 mg/mL) or proteinase K (0.02 mg/mL). For collagen I, III, and IV immunohistochemistry sections were dewaxed, hydrated, and permeabilised by microwaving in 2.1 g/L of citric acid (pH 6.0) for 6 minutes at high power. Endogenous peroxidase was quenched with 3% hydrogen peroxide in methanol for 30 minutes at room temperature. Sections were blocked in 10% fetal calf serum (FCS) (GIBCO BRL; Life Technologies, Paisley, England) with 2% bovine serum albumin (BSA) and 0.1% tween-20 (BDH) and incubated in a humidified chamber with goat antihuman type I collagen, goat antihuman type III collagen, or goat antihuman type IV collagen (1310-01, 1330-01 and 1340-01; Southern Biotechnology Associates, Birmingham, AL; all have <10% cross reactivity with other collagen types) diluted 1:10 in blocking solution at 4°C overnight. Omission of the primary antibody and substitution with blocking solution acted as a negative control. After incubation with peroxidase-conjugated rabbit antigoat immunoglobulin (P0449; DAKO, Glostrup, Denmark) for 30 minutes at room temperature, reaction with diaminobenzidine for 10 minutes (collagen IV) to 30 minutes (collagen I and III) produced a brown positive signal. To immunolocalise elastin, laminin, and fibronectin, slides were dewaxed, hydrated, treated with proteinase (0.2 mg/mL) for 10 minutes at 37°C, and their endogenous peroxidase quenched by exposure to 3% hydrogen peroxide in methanol for 30 minutes at room temperature. After blocking with 10% FCS with 2% BSA and 0.1% tween-20 for 10 minutes at room temperature, slides were incubated in a humidified chamber with rabbit antirat elastin (1:400, AB2039; Chemicon Int, Temecula, CA; <0.1% cross reactivity with collagen types I, II, III, V), rabbit antimouse laminin (1:400, AB2034; Chemicon; <0.1% cross reactivity with collagen I to IV and fibronectin) or rabbit antimouse fibronectin (1:800, AB2033; Chemicon; <0.1% cross reactivity with collagen I, III, IV and laminin) at 4°C overnight (or blocking solution as a negative control). After incubation with biotin-conjugated goat antirabbit immunoglobulin (1:200, AP132B; Chemicon) for 30 minutes at room temperature, slides were reacted with streptavidin-conjugated horseradish peroxidase (StreptABComplex K0377; DAKO) for 30 minutes at room temperature and exposed to diaminobenzidine for 60 seconds (fibronectin) to 120 seconds (elastin and laminin). Sections were counterstained in hematoxylin, dehydrated, and mounted in dextropropoxyphene. Slides were examined on a Zeiss Axioplan 2 Microscope (Carl Zeiss, Oberkochen, Germany), photographed on a scanning camera (Laser Optik System, Jenoptik, GmBH, Germany) and imported into Adobe Photoshop. Detrusor Western blot For each of the developmental stages examined, 4 separate blots were analysed for each sex (ie, a total of 8 at each stage). E14 embryos were sexed according to the appearance of their gonads; at this stage the testicular cords can be visualised under a dissecting microscope, whereas the ovary appears homogenous. The sex of later stages was determined by the anogenital distance, which is greater in males. For each lane, the detrusor muscle layer from, on average, 16 E14, 7 E18, 8 D1 or 8 6wk animals were pooled to provide sufficient protein for Western blot. The detrusor smooth muscle cell/mesenchymal layer above the trigone was dissected and homogenised in radioimmunoprecipitation assay buffer (150 mmol/L sodium chloride, 1% nonidet P-40, 0.5% deoxycholic acid, 0.1% sodium dodecyl sulphate, 50 mmol/L Tris [pH 8.0]) containing protease inhibitors (30 μL/mL of 2.2 mg/mL aprotinin, 10 μL/mL of 10 μg/mL phenylmethylsulfonyl fluoride, 10 μL/mL of sodium orthovanadate) at 4°C. After centrifugation at 16,600 g at 4°C for 30 minutes, supernatants were collected and their protein concentration measured (BCA protein assay, Pierce, Rockford, IL). The collagen in the supernatants consists of soluble collagen, primarily newly synthesised collagen, and procollagen, leaving behind the cross-linked collagen, which is difficult to dissolve. Depending on the antibody to be used, 10 to 20 μg of protein per lane was denatured at 98°C for 10 minutes and separated on 6% or 8% sodium dodecyl sulphate polyacrylamide electrophoresis gels, depending on the size of proteins to be analysed. To minimise the variability of experimental conditions, 4 gels, each containing one male and one female sample from each of the stages, were run in parallel. Coomassie blue staining confirmed that similar levels of protein had been loaded in each lane (data not shown). Proteins were transferred to nitrocellulose membranes (Amersham Pharmacia Biotech, Little Chalfont, Buckinghamshire, England) by electroblotting (Bio Rad, Hemel Hempstead, Hertfordshire, England). Ponceau S staining confirmed uniform transfer of the proteins (data not shown) and Rainbow markers (Amersham Pharmacia Biotech) indicated protein sizes. To aid analysis of the collagen western blots, 0.02 to 0.1 μg of purified protein (rat tail collagen type I; First Link UK, Brierley Hill, England; human placenta collagen type III, CC054; Chemicon; Engelbreth-Holm-Swarm mouse sarcoma collagen type IV, C0543; Sigma-Aldrich Co) was loaded as a positive control in one lane of each gel. Blots were blocked for 2 hours at room temperature with 5% (wt/vol) fat-free milk powder, 2% BSA, and 0.2% tween-20 in phosphate buffered saline (PBS, pH 7.4) and were incubated with goat antihuman type I collagen, goat antihuman type III collagen, goat antihuman type IV collagen, rabbit antirat elastin, rabbit antimouse laminin, or rabbit antimouse fibronectin diluted 1:1000 in blocking solution at 4°C overnight. Subsequently, blots were incubated with peroxidase-conjugated rabbit antigoat or goat antirabbit immunoglobulin diluted 1:2000 in blocking solution for 30 minutes at room temperature. Bands were visualised by chemiluminescence (Amersham Pharmacia Biotech) and measured using the Phoretix 1D program (Phoretix International, Newcastle upon Tyne, England). Western blots for collagen type I yielded 2 bands, measurements of which were amalgamated into a total chemiluminescence value for each sex and developmental stage. Statistical analysis of Western blot chemiluminescence Because the data sets were consistent with a normal distribution, means and standard errors of the mean (SEM) were calculated for each set of chemiluminescence measurements at each time point and sex (n = 4). Using the SPSS 8.0 for Windows program, the significance of any difference between males and females at each age was calculated using the independent-sample t test with 95% confidence. Because no significant difference between the sexes could be found for any of the proteins, the data for males and females at each time-point were amalgamated (n = 8). Univariate analysis of variance weighted for differences in the luminescence of the 4 simultaneous-run blots examined the significance of changes in protein expression during development. The Bonferroni test explored differences between individual age-points; this test adjusts the significance level for the fact that multiple comparisons are made. Results There were no significant differences between male and female bladders at any of the developmental time-points on Masson's trichrome or on immunohistochemistry and Western blotting for the specific ECM proteins. For immunohistochemistry, omission of primary antibodies resulted in no colour reaction (data not shown). Masson's trichrome At the inception of the urinary bladder on E14 (Fig 1A and B), Masson's trichrome staining showed a delicate lattice of collagen in the mesenchyme between the detrusor muscle and the inner, more condensed, lamina propria.
In the detrusor layer itself, collagen staining was sparse ( Fig 1B). As the organ grew from E18 to 6wk, the collagen lattice extended across the entire lamina propria and became progressively denser ( Fig 1C-H). Collagen staining also increased in the detrusor layer, concurrent with a progressive organisation of detrusor cells into distinct muscle bundles and layers. In the young adult bladders, muscle bundles were packed tightly and surrounded prominently by collagen ( Fig 1G and H). At this stage, the lamina propria consisted of an inner compact layer of collagen closely associated with the urothelium and an outer layer rich in blood vessels. Expression of specific extracellular matrix proteins in the developing detrusor Elastin immunostaining was present in lamina propria and urothelium but was absent in the detrusor muscle at E14 (Fig 2A).
At E18 and D1, elastin immunolocalised to the detrusor muscle, blood vessel walls, and was seen in a fine reticular pattern in the developing lamina propria ( Fig 2B and C). In mature bladders, elastin was prominent at the interface between layers of detrusor muscle ( Fig 2D). Elastin was seen as a single band at 55 kDa on Western blotting of isolated detrusor layer ( Fig 2E). Levels were minimal at E14 and rose steadily during detrusor development ( P = .001; Fig 2F). Immunostaining did not find a significant signal for collagen type I at E14 (Fig 3A).
From E18 to postnatal maturity, staining became prominent in the lamina propria ( Fig 3B-D). In the mature detrusor muscle, collagen type I staining was confined to the margins of the muscle bundles ( Fig 3D). Western blotting of dissected detrusor layers ( Fig 3E) yielded 2 bands for soluble collagen type I at 110 and 140 kDa at all ages and for the positive control, rat tail collagen type I. This technique detected collagen type I expression in the detrusor layer from the earliest developmental stage, in contrast to immunohistochemistry, possibly reflecting superior antigen availability on western blotting using this antibody. Analysis of variance showed that the level of expression of this protein changed significantly through development ( P = .034; Fig 3F). After an insignificant fall in the level of collagen type I from E14 to E18 ( P = .161), the level increased significantly from E18 to 6wk ( P = .035). Immunohistochemistry for collagen type III showed a positive signal with cytoplasmic “speckles” in the detrusor muscle at E14, E18 and D1 (Fig 4A-C).
Postnatally, the staining pattern changed, with a loss of cytoplasmic “speckles” and more prominent staining of the perimeter of muscle bundles in the young adult bladders ( Fig 4D). Western blotting for soluble collagen type III yielded a single band at 140 kDa for the samples and the positive control, human placenta collagen type III. The band for collagen type III was strongest at 6wk ( Fig 4E). Analysis of variance confirmed a significant rise in soluble collagen type III levels from D1 to 6wk ( P < .001; Fig 3F). At E14 (Fig 5A), collagen type IV immunostaining was relatively weak but appeared speckled in the detrusor muscle cytoplasm at E14, E18, and D1 (Fig 5A-C).
In the young adult bladder, collagen type IV resulted in a “honeycomb” pattern of immunostaining in the detrusor muscle ( Fig 5D). Western blotting showed a major band at 240 kDa for the detrusor layer samples and the positive control, Engelbreth-Holm-Swarm mouse sarcoma collagen type IV ( Fig 5E). Levels of soluble collagen type IV changed significantly during detrusor muscle development ( P = .026) with a decline from E18 to 6wk ( P = .019). Laminin was present in all layers of the bladder on immunohistochemistry from the earliest stage and highlighted the basal lamina of the urothelium and blood vessels (Fig 6A-C).
In the young adult bladder, laminin staining produced a honeycomb pattern in the detrusor muscle ( Fig 6D). On Western blotting of dissected detrusor layers, a single band at 250 kDa was detected ( Fig 6E). Analysis of variance showed steadily increased levels of laminin during development ( P < .001; Fig 6F). On immunohistochemistry, fibronectin localised to the lamina propria, blood vessel walls and, to a lesser extent, to the detrusor layer of the developing bladder (Fig 7A-C).
In the mature bladder, fibronectin primarily outlined blood vessels ( Fig 7D). Western blotting of isolated detrusor layer resulted in a band at 260 kDa ( Fig 7E). As assessed by immunoblotting, levels of fibronectin at first increased from E14 to D1 ( P = .028) and then fell to low levels by 6wk ( P = .002; Fig 7F). Discussion The developmental changes in ECM protein expression in the detrusor layer, as assessed by Western blots, are summarised in Fig 8, against a background of the previously observed transition from an embryonic proliferative phenotype to a quiescent differentiated smooth muscle phenotype.3 These data show that the morphologic growth of the mouse detrusor muscle is accompanied by complex serial changes in the expression of ECM proteins. For the interpretation of collagen Western blots, it is important to bear in mind the difficulty of dissolving cross-linked collagen protein. Hence, our detrusor layer samples for Western blot contain soluble collagen, primarily newly synthesised collagen, and procollagen, rather than the full complement. The elasticity of organs subjected to repeated deformation, including the bladder, resides in elastic fibres, the most abundant component of which is elastin.1 Cartwright and Snow5 have shown that normal human bladder compliance resides in the detrusor muscle layer of the bladder. Using a transgenic mouse that overexpressed rat elastin, Lemack et al6 showed greater bladder compliance versus normal adult controls; furthermore, increased elastin expression in transgenic mice protected against bladder dysfunction induced by partial outflow obstruction. Our own results show increasing levels of elastin protein in the mouse detrusor muscle layer from its inception to maturity, suggesting the potential for improved compliance during development. In normal human bladder development, an increasing number of elastic fibres has been documented between the muscle bundles of the detrusor in the second half of gestation.7 The importance of elastin for normal human bladder function is emphasised by the association of elastin gene deletions in Williams syndrome with bladder dysfunction, including urinary frequency, incontinence, and detrusor instability.8 Interestingly, the cell membrane receptor for elastin is linked through a G protein-phospholipase C-IP3 mediated relay to the regulation of intracellular calcium,9 suggesting that this matrix protein also may affect detrusor cell physiology in as yet undefined ways. Collagen type I accounts for 90% of the body's collagen.4 In this study, western blotting of dissected detrusor layers detected soluble collagen type I from E14, in contrast to collagen type I immunohistochemistry, which did not show a significant signal in this layer at E14, despite optimising pretreatments for antigen exposure. This difference may be explained by superior antigen availability on Western blotting for this primary antibody. Immunohistochemistry using a different anticollagen type I antibody (rabbit antimouse collagen type I, AB765; Chemicon) did show staining at E14 in the lamina propria and detrusor muscle (data not shown); however, this antibody was unsuitable for Western blots. During mouse detrusor muscle development, we observed an insignificant fall in the level of soluble collagen type I from E14 to E18, followed by a significant increase from E18 to 6wk (Fig 8). As in maturing bovine bladders10 and children's bladders,11 collagen type I immunolocalised intensely to the lamina propria and around detrusor muscle bundles during mouse bladder development. Recently, Herz et al12 provided preliminary evidence that collagen type I may modulate detrusor cell biology because damaged collagen type I was mitogenic for rat detrusor smooth muscle cells in vitro. We found that collagen type III is present in all mouse bladder layers from the inception of the discrete organ. Detrusor protein levels were relatively low before birth but rose significantly between D1 and 6wk when the immunohistochemical pattern showed prominent staining around muscle bundles. Prominent immunostaining also has been observed proximate to detrusor muscle bundles in normal human11, 13 and normal bovine bladders.10, 14 The conformation of collagen type III changes from randomly orientated coils to parallel-orientated thin long extended fibres with bladder distension, and the fibres appear to connect muscle bundles in the 25% distended state.14 In noncompliant children's bladders, several studies have reported increased collagen type III immunostaining in and around detrusor muscle bundles.11, 13 We speculate that a specific content for collagen type III exists for optimal bladder function, with increases in collagen type III beyond that point perhaps resulting in reduced compliance or contractility. In our mouse model, these questions only can be answered by further experiments, eg, to measure compliance at different developmental stages and in obstructive uropathy in relation to collagen levels. In contrast to collagen types I and III which form fibrils, collagen type IV molecules assemble into flexible, multilayered sheets and are a major constituent of basal lamina, which underlie epithelial sheets and surround individual muscle, fat, and Schwann cells.1 In our study, immunostaining at 6wk results in a honeycomb pattern in the detrusor muscle, which we believe represents collagen type IV around detrusor cells; others have observed similar appearances in human studies.11, 13 We observed that levels of soluble collagen type IV in the detrusor layer tended to fall between E18 to 6wk. The biological relevance of this pattern remains unclear. Laminin, another major component of basal laminae, is a heterotrimeric glycoprotein, which possesses binding domains for collagen type IV as well as various cell surface receptors including integrins, nidogen, and dystroglycan.15 The binding of laminin to cell surface receptors appears essential for the normal development of, for example, skeletal muscle16 and kidney tubules.15 As for collagen type IV, we found that laminin immunolocalised in a honeycomb pattern in the young adult mouse detrusor muscle, consistent with it being part of basal laminae. Interestingly, emerging evidence indicates that binding to dystroglycan and integrin are important for laminin matrix assembly,17 supporting the theory that cells and their surrounding ECM influence one another. In our study, a steadily rising level of laminin expression occurs with the transition from cellular proliferation to detrusor muscle differentiation.3 In vitro skeletal muscle differentiation is similarly accompanied by increased laminin levels,18 and the differentiation of cultured murine skeletal myoblasts, as assessed by expression of myosin and desmin in myotubes, is actively enhanced by laminin.19 Fibronectin glycoprotein dimers can bind collagen, heparin, integrin, and fibronectin itself.20 Assembly of fibronectin into filaments requires interaction with integrins and molecules of large apparent molecular mass (LAMMs) on the cell surface. Fibronectin is abundant especially in embryonic and regenerating tissues.20 In vitro skeletal muscle differentiation is accompanied by decreased fibronectin levels,18 and culturing skeletal myoblasts on fibronectin stimulates proliferation of undifferentiated precursors and dedifferentiation of more mature myoblasts into myofibroblastlike cells.19 In our current study, detrusor layer fibronectin expression was relatively high up to the first day of postnatal life, but fell to low levels by 6wk, by which time the detrusor muscle cells achieve maturity. In the future, it would be informative to examine the effects of fibronectin and laminin substrates on mouse detrusor muscle differentiation; for example, does fibronectin enhance the proliferative, immature phenotype, and does laminin downregulate proliferation and induce α smooth muscle actin and desmin expression? Acknowledgements The authors thank Dr P. 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Arterioscler Thromb Vasc Biol. 1998;18:1363–1370. MEDLINE Nephro-Urology Unit, Institute of Child Health, University College London, London, England ☆ This work was supported by Action Research, the Royal College of Surgeons, and the Kidney Research Aid Fund. ☆☆ Address reprint requests to Professor A.S. Woolf, Nephro-Urology Unit, Institute of Child Health, 30 Guilford St, London WC1N 1EH, England. PII: S0022-3468(02)63008-8 doi:10.1053/jpsu.2003.50000 © 2003 Published by Elsevier Inc. | 1 of 31  |
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