Identification of PDGFRα Positive Populations of Interstitial Cells in Human and Guinea Pig Bladders

Identification of PDGFR␣ Positive Populations of Interstitial Cells in Human and Guinea Pig Bladders Kevin P. Monaghan,* Louise Johnston* and Karen D. McCloskey† From the Centre for Cancer Research and Cell Biology, Queen’s University, Belfast, United Kingdom

Purpose: The bladder wall comprises a complex array of cells, including urothelium, smooth muscle, nerves and interstitial cells. Interstitial cells have several subtypes based on site, morphology and differential expression of markers such as anti-vimentin and anti-KIT. We examined whether a subpopulation of interstitial cells immunopositive for PDGFR␣ exists in human and guinea pig bladders. Materials and Methods: Human and guinea pig bladder tissues were processed for immunohistochemistry and examined by bright field or confocal microscopy. Whole mount tissues and paraffin sections were labeled with antibodies to PDGFR␣, vimentin, KIT and PGP9.5. Protein expression was assessed by Western blot. Results: PDGFR␣⫹ cells were present in human and guinea pig bladders. In the guinea pig PDGFR␣⫹ cells had a branched stellate morphology and formed networks in the lamina propria. In human and guinea pig detrusors PDGFR␣⫹ cells were elongated on the boundary of smooth muscle bundles or were seen as groups of stellate cells in the interbundle spaces. PDGFR␣⫹ cells were located close to nerves labeled by PGP9.5. Double labeling revealed that PDGFR␣⫹ cells were a subgroup of the vimentin⫹ population. A significant proportion of PDGFR␣⫹ cells were also KIT⫹. Bands corresponding to PDGFR␣, KIT and vimentin proteins were detected on Western blot. Conclusions: To our knowledge this study is the first to identify PDGFR␣⫹/KIT⫹ cells in the bladder lamina propria and detrusor layers. These cells are a subgroup of the vimentin⫹ population, showing the complexity of bladder interstitial cells. PDGFR␣⫹ cells are apparently structurally associated with intramural nerves, indicating integration with bladder control mechanisms.

Abbreviations and Acronyms FLC ⫽ fibroblast-like cell HRP ⫽ horseradish peroxidase IC ⫽ interstitial cell ICC ⫽ IC of Cajal PBS ⫽ phosphate buffered saline PDGF ⫽ platelet-derived growth factor PDGFR ⫽ PDGF receptor PGP9.5 ⫽ protein gene product 9.5 Submitted for publication October 28, 2011. Study received approval from the Office of Research Ethics Committee Northern Ireland. Supported by a grant from the European Union, FP7, INComb. * Equal study contribution. † Correspondence: Centre for Cancer Research and Cell Biology, 97 Lisburn Rd., Queen’s University, Belfast, BT9 7BL, Northern Ireland, United Kingdom (telephone: ⫹44 2890 972386; e-mail: [email protected]).

Key Words: urinary bladder; interstitial cells of Cajal; receptors, plateletderived growth factor; humans; guinea pigs BLADDER ICs represent a heterogeneous group of cells that has been studied for more than a decade. Methods of identifying bladder IC have depended heavily on the mesenchymal cell marker anti-vimentin and the ICC marker anti-KIT, which show that complex IC arrays exist in the bladder wall layers. Although differences in nomenclature have been adopted by different research groups, such as ICCs,

Cajal-like cells, ICC-like cells, IC and myofibroblasts, the literature corroborates the view that specialized cells exist in the bladder that are distinct from smooth muscle cells and neurons.1,2 Several bladder IC classes have been characterized, including 1) suburothelial ICs at the base of the urothelium,3 2) lamina propria ICs in the space between urothelium and de-

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trusor,4 – 6 3) intramuscular ICs on the boundary of detrusor smooth muscle bundles,6,7 4) interbundle ICs in the spaces between smooth muscle bundles6,7 and 5) subserosal ICs on the outer adventitial/serosal surface of the bladder.8 Also, perivascular ICs are associated with microvessels in the bladder wall.9,10 Subpopulations of bladder ICs have distinct morphologies depending on site. They have been reported in mouse, rat, guinea pig, pig and human bladders. The complexity of the IC population in the bladder wall is shown by the fact that not all ICs may be labeled with the same markers. For example, KIT antibodies label comparatively fewer cells than antivimentin, which labels a remarkably larger group of cells that includes ICs and fibroblasts. Anti-KIT is a reliable marker of gut ICCs while there is substantial evidence that KIT⫹ cells exist in the lamina propria and detrusor layers of the bladder.6 –9,11–14 These KIT⫹ cells appear to be distinct from mast cells in morphology and the lack of immunoreactivity for mast cell tryptase.9 The observation that only a subpopulation of ICs is positive for KIT suggests that additional cell types exist in the vimentin positive cellular networks of the bladder wall. The identification of PDGFR␣⫹/KIT- cells in the gut,15 which are considered to be fibroblast-like cells that were previously characterized ultrastructurally,16 raises the question of whether PDGFR␣⫹ cells might exist in the bladder. In gut tissue PDGFR␣⫹ cells were found adjacent to KIT positive ICC networks in all gut regions and they were located close to enteric nerves. Moreover, physiological evidence of the role of PDGFR␣⫹ cells in murine gastrointestinal smooth muscle tissue indicates that they have a key role in the mediation of inhibitory responses to purines, such as adenosine triphosphate and adenosine diphosphate.17 We examined whether a population of PDGFR␣⫹ cells exists in human and guinea pig bladders, and

compared PDGFR␣⫹ cell populations with vimentin⫹ and KIT⫹ cells.

MATERIALS AND METHODS Tissue Collection Male guinea pigs weighing 250 to 500 gm were sacrificed by cervical dislocation in accordance with Schedule 1 of United Kingdom Home Office regulations. Bladders were removed, opened longitudinally from neck to dome and pinned to a Sylgard® dissecting dish. The mucosal and detrusor layers were separated by sharp dissection to leave 2 whole mount tissue preparations. Two human samples from the bladder body were obtained from patients undergoing reconstructive surgery who were clinically considered to have normal bladder activity with informed consent and ethical approval from the Office of Research Ethics Committee Northern Ireland.

Fluorescence Confocal Microscopy Tissues were fixed in 4% paraformaldehyde, washed in PBS and blocked in 1% bovine serum albumin containing 0.05% Triton-X 100 before incubation in primary antibodies for 24 hours (see table). After washing in PBS tissues were incubated in fluorescent secondary antibodies, washed in PBS and mounted on glass slides with coverslips. Slides were viewed by epifluorescence microscopy and regions of interest were imaged by a C1 confocal microscope mounted on an E90i upright microscope running EZ-C1 software (Nikon®). Fluorophores were excited with a 405 nm laser diode, the 488 nm line of an argon ion laser or a green HeNe laser at 543 nm. Resulting fluorescence was collected to photomultiplier tubes by appropriate emission filter sets. For double and multiple labeling fluorophores were imaged sequentially to minimize bleed through and experiments were repeated on tissue from at least 3 animals. Images represent projections of Z-stacks reconstructed in EZ-C1 and Photoshop®. Control tissues were prepared by omitting primary antibody to assess the specificity of the secondary antibody or omitting all antibodies to assess autofluorescence. Nuclei were counterstained with DAPI (Sigma-Aldrich®) in several tissues to reveal the cellular arrangement. Murine

Primary and secondary antibodies Primary Antibody (product No.)

Supplier

Host

Dilution

Anti-PDGFR␣ (AF1062)

R&D Systems®

Goat

1:200

Anti-PGP9.5 (Z5116) Anti-KIT (PC34)

Dako Merck

Rabbit Rabbit

1:200 1:200

Anti-KIT (A4502) Anti-vimentin (V2258)

Dako Sigma-Aldrich

Rabbit Mouse

1:1,000 1:200

Western blot: Anti-␤-actin (V2228) Anti-vimentin (V2258) Anti-PDGFR␣ (AF1062)

Sigma-Aldrich

Mouse

R&D Systems

Goat

1:10,000 1:1,000 1:1,000

Secondary Antibody

Supplier

Alexa 568 anti-goat, anti-goat HRP ⫹ DAB Alexa 488 anti-rabbit Alexa 488 anti-rabbit, anti-rabbit HRP ⫹ DAB Goat anti-rabbit HRP Alexa 488 anti-mouse, antimouse HRP ⫹ DAB Goat anti-mouse HRP

Invitrogen™, Santa Cruz Biotechnology, Santa Cruz, California

Donkey anti-goat HRP

Dilution 1:200

Invitrogen, Dako

1:200 1:200

Santa Cruz Biotechnology Invitrogen, Dako

1:2,000 1:200

Santa Cruz Biotechnology

1:2,000

Santa Cruz Biotechnology

1:2,000

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fibroblast J2-3T3 cells served as a positive control for PDGFR␣ antibody.

DAB Immunohistochemistry Human and guinea pig bladder samples were fixed in 8% neutral buffered formalin and processed for wax histology. Sections (3 ␮m) were deparaffinized in xylene, rehydrated in alcohol, washed in tap water and heated in a pressure cooker in tris-ethylenediaminetetraacetic acid (pH 9.0) for antigen retrieval. Endogenous peroxidase activity was blocked with 3% H2O2. Sections were washed, blocked with 1% bovine serum albumin in tris buffered saline and incubated in primary antibodies overnight. Sections were then washed in tris buffered saline, incubated in secondary antibody horseradish peroxidase conjugate (1:200), washed and stained with DAB and substrate chromogen system (Dako, Glostrup, Denmark) before counterstaining with hematoxylin using an Autostainer (Leica®). They were viewed by bright field microscopy. Control slides were prepared by omitting primary antibody.

Western Blot Samples of full-thickness guinea pig bladder were homogenized using a dounce homogenizer in 1 ml ice cold RIPA buffer composed of PBS containing 1% NP-40, 0.5% sodium deoxycholate and 0.5% sodium dodecyl sulfate, supplemented with a Complete Protease Inhibitor Tablet (Roche, Basel, Switzerland) in 10 ml lysis buffer. Whole cell lysates were obtained by centrifuging the resultant homogenate at 10,000 ⫻ gravity for 10 minutes at 4C and extracting the supernatant. Protein concentration was determined by the Bradford assay with bovine albumin globulin (Sigma-Aldrich) as the standard. Protein lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on precast 4% to 15% gradient gels (BioRad®) according to manufacturer instructions with 40 ␮g protein sample per lane. Separated proteins were transferred to nitrocellulose membranes at 300 mA for 2 hours. Nitrocellulose blots were blocked for 1 hour using 5% milk in PBS-0.2%Tween at room temperature. They were incubated with primary antibody at 4C overnight, followed by 1-hour incubation in the appropriate HRP conjugated secondary antibody. Membranes were washed with PBS-0.2% Tween after primary and secondary antibody incubations (see table). Bands were revealed by chemiluminescence. Signal was detected on an Alpha Innotech FluorChem® SP imaging system.

RESULTS PDGFR␣ⴙ Cells In histological sections. Sections of human and guinea pig bladder were labeled with anti-PDGFR␣ and stained with DAB. PDGFR␣⫹ cells were identified in the detrusor with a similar pattern of distribution in humans and guinea pigs (fig. 1, A and B). PDGFR␣⫹ cells were found on the edge of smooth muscle bundles or in interstitial spaces between the bundles. Labeling sections from the same sample with anti-KIT or anti-vimentin revealed KIT⫹ or vimentin⫹ cells of comparable morphology at loca-

Figure 1. Sections of human bladder were labeled with antibodies and visualized with DAB staining (brown areas) (A, C, E and G). Nuclei were counterstained with hematoxylin. PDGFR␣⫹ cells (arrows) were present in interstitial spaces between smooth muscle bundles (SM) and on bundle boundaries. Cells immunopositive for KIT (C) and vimentin (E) were also present. Sections of guinea pig bladder (B, D, F and H) also contained PDGFR␣⫹ (B), KIT⫹ (D) and vimentin⫹ (F) cells. Control sections for secondary antibody used for PDGFR␣ labeling in human (G) and guinea pig (H) bladders did not show nonspecific DAB staining. Scale bar applies to all images.

tions similar to those of PDGFR␣⫹ cells, ie at the edge of smooth muscle bundles or in interbundle spaces (fig. 1, C to F). Control tissues prepared by omitting the primary antibody PDGFR␣ did not contain nonspecific binding of DAB secondary antibody (fig. 1, G and H). In bladder whole mounts. Confocal imaging of whole mount sheet preparations from 10 guinea pig bladders labeled with anti-PDGFR␣ revealed the morphological arrangement of immunopositive cells and their association with other bladder wall components. Figure 2, A and B show typical networks of stellate PDGFR␣⫹ cells in lamina propria preparations. Where bundles of muscularis mucosal smooth muscle

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Figure 2. On whole mount sheet of guinea pig bladder lamina propria labeled with anti-PDGFR␣ immunopositive cells had stellate multipolar morphology and formed networks in lamina propria layer (A to C). Note small smooth muscle bundle of muscularis mucosa (MM) with 2 associated PDGFR␣⫹ cells (arrows) on boundary (C). In the detrusor muscularis PDGFR␣⫹-cells formed 2 subpopulations (D to F), including elongated cells with side branches (arrows) on smooth muscle bundle (SM) boundary, which were visible with background fluorescence, and stellate cells (asterisks) in interstitial spaces between bundles.

were present, PDGFR␣⫹ cells were associated with the bundles, running in parallel with the bundle axis (fig. 2, C). In detrusor the PDGFR␣⫹ cells were elongated with branched processes or stellate. These morphologies are similar to those reported for vimentin⫹ and KIT⫹ cells in mouse, human and guinea pig bladders. Elongated immunopositive cells were on the boundary of smooth muscle bundles, visible on micrographs due to low level background fluorescence (fig. 2, D and E). Stellate cells were located in the interstitial space between smooth muscle bundles (fig. 2, F). Minimal fluorescence was present in controls. Relationship with KITⴙ or vimentinⴙ cells. To assess whether there was overlap in cell populations labeled with the broad marker anti-vimentin and PDGFR␣⫹ double labeling protocols were performed. The vimentin and PDGFR␣ staining patterns were distinctly different. Vimentin staining was filamentous and PDGFR␣ staining was comparatively punctate (fig. 3, A to D), consistent with staining of intermediate filaments and membrane receptors, respectively. Lamina propria PDGFR␣⫹ cells were also immunopositive for vimentin but vimentin-/ PDGFR␣⫹ cells were not observed. Vimentin⫹/ PDGFR␣- cells, which were present in most examined micrographs, represent a separate cell popu-

lation. PDGFR␣⫹ cells associated with the muscularis mucosa or with microvessels also expressed vimentin (fig. 3, E and F). Two populations of vimentin⫹ cells were also found in the detrusor, that is vimentin⫹/PDGFR␣and vimentin⫹/PDGFR␣⫹ cells. Figure 4 shows the overlap between vimentin and PDGFR␣ labeling with cells on the boundary of smooth muscle bundles and interbundle cells positive for each label. There were also cells that stained for vimentin but only weakly for anti-PDGFR␣, revealing complexity in the detrusor vimentin⫹ interstitial cell population. We previously reported that bladder KIT⫹ cells represent a subpopulation of the vimentin⫹ population.6 Thus, we examined whether PDGFR␣⫹ cells co-expressed KIT or represented a different class of interstitial cell. Double labeling protocols in lamina propria showed remarkable overlap in KIT and PDGFR␣ expression with most PDGFR␣⫹ cells also KIT⫹ (fig. 5, A to C). Occasional KIT⫹ cells that were not apparently PDGFR␣⫹ were noted in the lamina propria and conversely some PDGFR␣⫹ cells had unconvincing immunoreactivity for KIT. Similar experiments on detrusor tissue showed significant overlap in KIT⫹ and PDGFR␣⫹ labeling with cells immunopositive for KIT plus PDGFR␣ (fig. 5, D to F).

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Figure 3. Preparations co-labeled with anti-vimentin (green areas) and anti-PDGFR␣ (red areas) reveal 2 lamina propria cell populations (A to D). One group co-expressed vimentin and PDGFR␣ (arrows), and other expressed vimentin only (asterisks). PDGFR␣⫹/vimentin⫹ cells were found on boundary of muscularis mucosa (MM) smooth muscle (E) and were associated with mucosal blood vessels (BV) (F).

To assess whether PDGFR␣⫹ cells were structurally associated with nerves, as previously reported for KIT⫹/vimentin⫹ cells, mucosal and detrusor tissues were co-labeled with PDGFR␣ and the panneuronal marker PGP9.5. PDGFR␣⫹ cells were located close to nerve fibers (fig. 6). Structural associations between PDGFR␣⫹ cells and nerve fibers or nerve varicosities were often identified in the lamina propria and the detrusor, respectively. Within the resolution limits of light microscopy structural associations are interpreted as physically close, ie up to 0.25 ␮m. Transmission electron microscopy is required to determine whether the associations between nerves and PDGFR␣⫹ cells indicate a neuroeffector junction, ie 10 to 100 nm. Protein Expression PDGFR␣ expression was tested in guinea pig bladder tissue and in J2-3T3 cells as the positive control using Western blot (fig. 7, A). Bands corresponding to 180 kDa were labeled with anti-PDGFR␣ in bladder tissue and J2-3T3 cells. This indicated that 1) PDGFR␣ is expressed in bladder tissue and 2) the anti-PDGFR␣ used was specific to PDGFR␣, consistent with the findings of Iino et al,16 who used the same antibody. In a separate set of experiments bands corresponding to vimentin (58 kDa), KIT (145 kDa) and PDGFR␣ (180 kDa) were labeled with the appropriate antibodies with the housekeeping gene

␤-actin (42 kDa) as a positive control (fig. 7, B). Western blot was performed using tissue from 3 bladders in independent experiments.

DISCUSSION These findings indicate the presence of PDGFR␣⫹ cells in human and guinea pig bladders. Western blot confirmed the protein expression of PDGFR␣, KIT and vimentin in bladder tissue. The sites of PDGFR␣⫹ cells in guinea pig and human tissues are reminiscent of those previously reported for KIT⫹/ vimentin⫹ bladder ICs.6,9 PDGFR␣⫹ cells were found as 1) networks in the lamina propria, 2) discrete, elongated cells associated with blood vessels, 3) discrete, elongated cells on the boundary of muscularis mucosa and 4) detrusor smooth muscle bundles, and 5) in the interbundle spaces. PDGFR␣⫹ cells were closely associated with nerves, consistent with previous findings for KIT⫹ interstitial cells in mice, guinea pigs and humans, which were closely associated with PGP9.5 or vesicular acetylcholine transporter (cholinergic) labeled nerves.6,9,14 Recently a group reported PDGFR␣⫹ cells in the mouse bladder. Together with our findings PDGFR␣⫹ cells have now been identified in mouse, human and guinea pig bladders.16 A main objective of our research was to investigate whether PDGFR␣⫹ cells represent a new class of bladder ICs or whether they are components of

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PDGFR␣ POSITIVE INTERSTITIAL CELLS IN HUMAN AND GUINEA PIG BLADDERS

Figure 4. Detrusor preparations co-labeled with anti-PDGFR␣ (red areas) and anti-vimentin (green areas) also revealed 2 cell populations (A to C). One group co-expressed vimentin and PDGFR␣ (arrows), and other expressed vimentin only (asterisk). Note higher magnification (D to F) of inset (C). Scale bars apply to A to C and D to F, respectively.

the KIT⫹/vimentin⫹ cell populations. Results revealed remarkable overlap between PDGFR␣ and KIT labeling with most PDGFR␣⫹ cells also immunopositive for KIT. Occasional KIT⫹ cells were not also PDGFR␣⫹. Likewise most if not all PDGFR␣⫹ cells were also vimentin⫹, a finding consistent with previous studies showing that KIT⫹ cells were also vimentin⫹. These findings indicate that KIT⫹ bladder ICs can also be labeled with PDGFR␣⫹ antibodies. Given the reported difficulty with labeling bladder ICs with KIT antibodies, which are thought to be due to species differences and the susceptibility of KIT epitopes to damage by fixatives or processing protocols, anti-PDGFR␣ represents an additional marker for bladder ICs that appears to work well in paraformaldehyde fixed tissues. The finding that anti-PDGFR␣ and anti-KIT labeled the same bladder cells is in contrast to what was reported for gastrointestinal tissue. Iino et al described PDGFR␣⫹ cells in the gut that had no overlap with neighboring KIT⫹ cells.16 They concluded that PDGFR␣⫹ cells were FLCs, which were previously characterized ultrastructurally.17,18 The observation that KIT⫹ ICCs are distinct from PDGFR␣⫹ FLCs is supported by recent physiological data showing PDGFR␣⫹-FLC function in the relay of purinergic signals to gastrointestinal smooth muscle.19 Moreover, gut PDGFR␣⫹ cells had similar sites and quantities in wild-type and mutant W/Wv

gastrointestinal tissue but KIT⫹ populations were decreased or altogether absent in W/Wv gut tissue. PDGF was discovered in 1974 as a serum factor that stimulates vascular smooth muscle cell proliferation.19 This growth factor is involved in diverse physiological and pathophysiological conditions, and it acts as a mitogen and chemotactic factor.20,21 PDGF has key roles in embryonic development and wound healing. However, its roles in normal physiology, including maintenance of vascular tone, platelet aggregation and homeostatic regulation of interstitial tissue pressure,22 are less well understood. PDGF is expressed by cells such as fibroblasts, vascular smooth muscle, neurons, macrophages and platelets, and it has several isoforms, including PDGF-AA, BB, AB, CC and DD.21 These ligands act on the specific receptor tyrosine kinases PDGFR␣ and PDGFR␤, which are reportedly expressed by numerous cell types, including platelets, fibroblasts, vascular smooth muscle, neurons and macrophages. PDGFR␣ receptor, which was of interest in the current study, shows promiscuity in that any PDGF isoform appears to be able to bind to PDGFR␣ dimers.23 Expression levels of PDGF ligands and their receptors change under conditions such as inflammation, hypoxia or mechanical stress, and altered PDGF signaling is implicated in atherosclerosis, organ fibrosis and several malignancies.20,23 Smooth muscle cells and fibroblasts are known to

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Figure 5. Lamina propria preparation labeled with anti-PDGFR␣ (red areas, A) and anti-KIT (green areas, B) shows significant overlap between 2 labels in merged micrograph (C). Arrows indicate double labeled cells. Asterisks indicate PDGFR␣⫹ cells that were not convincingly KIT⫹. Arrowheads indicate occasional KIT⫹ cells not labeled by PDGFR␣. Detrusor preparation labeled with anti-PDGFR␣ (D) and anti-KIT (E) shows significant overlap between 2 labels in merged micrograph (F). Scale bars apply to A to C and D to F, respectively.

respond to PDGF, resulting in vascular wall alterations and fibrotic tissue scars, while PDGFR␣ is involved in mesenchymal cell or fibroblast based pathologies, such as organ fibrosis.23 In the bladder little has been reported on PDGF and its receptors in normal physiology. However, PDGF signaling is associated with remodeling of bladder wall cellular components in response to pathophysiological situations. In chronically ob-

Figure 6. Mucosal preparation co-labeled with anti-PDGFR␣ (red areas) and pan-neuronal marker anti-PGP9.5 (green areas) reveals frequent structural associations between PDGFR␣⫹ cells and nerves (A). In detrusor nerves and PDGFR␣⫹ cells were closely associated (B). Nuclei were counterstained with DAPI to indicate smooth muscle bundle site and orientation.

structed bladders PDGF expression varies during the period when tissue changes from early inflammation to later fibrosis.24 PDGF-A mRNA initially decreased after obstruction but approached baseline by 13 weeks after obstruction, coinciding with an initial decrease in suburothelial myofibroblasts (up to 8 weeks), which was followed by recovery during weeks 8 to 13. The hypoxic conditions induced by obstruction activated hypoxia inducible factor-1␣ and PDGF-A, a mitogen for fibroblasts, resulting in suburothelial myofibroblast proliferation. Our finding that PDGFR␣ receptor is expressed on lamina propria ICs supports these results and may indicate a role for PDGF in the maintenance/control of the lamina propria interstitial cell population. Exposure of cultured human bladder fibroblasts and smooth muscle cells to sustained hydrostatic pressure, analogous with obstruction, resulted in increased expression of PDGF-BB and its receptor in fibroblasts but not in smooth muscle cells.25 Correlation between mechanical stimulation of the bladder wall and PDGF signaling was shown by ex vivo rat bladder wall distention, which led to phosphorylation and activation of PDGFRs, thus leading to downstream signaling via the phosphatidylinositol 3-kinase/Akt pathway.26 In cultured rat bladder smooth muscle cells up-regulation of DNA

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Figure 7. Western blot. Note PDGFR␣ and ␤-actin expression as bands at 180 and 42 kDa, respectively, in positive control J2-3T3 cells and guinea pig bladder tissue (A). Guinea pig bladder tissue expressed PDGFR␣, KIT and vimentin proteins with ␤-actin as positive control (B).

synthesis was observed in response to PDGF-BB stimulation via the phosphatidylinositol 3-kinase/ AKT pathway.27 Also, Adam et al noted that mechanical stretch or PDGF-BB altered the expression of 15 genes in oligonucleotide arrays.28 Mechanical (stretch) and growth factor (PDGF-BB) evoked signals use common transcriptional regulators to induce gene expression alterations. Studies of dysfunctional bladder, including obstructed8,29 and spinal cord injured tissues,30 show changes in interstitial cell subpopulations that correlated with bladder pathophysiology and smooth muscle hypertrophy. Our finding that these ICs express PDGFR␣ is consistent with the known roles of PDGF ligand and receptor signaling in tissue remodeling. While to our knowledge this has not yet been shown for the bladder, it seems feasible that PDGFR␣⫹ ICs would be particularly susceptible to aberrant PDGF signaling under conditions of mechanical stress or hypoxia, which are characteristic of dysfunctional bladder tissue.

CONCLUSIONS Human and guinea pig bladders contain PDGFR␣⫹ IC populations that are also immunopositive for KIT and vimentin. PDGFR␣⫹ ICs are found in the mucosal lamina propria and detrusor smooth muscle layers, close to intramural nerves. The ability of anti-PDGFR␣ to label bladder ICs further characterizes their phenotype and provides an additional marker of these cells. The finding that bladder ICs express PDGFR␣ may help explain the mechanisms underlying reported changes in IC expression in the pathophysiological bladder.

ACKNOWLEDGMENTS Dr. D. Patel provided J2-3T3 cells. Drs. D. Patel and S. McDade assisted with Western blots. Dr. S. Woolsey provided human bladder samples. G. McGregor and K. Arthur, Tissue Core Technology Unit, Queen’s University, provided technical assistance.

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