1 Department of Pharmacology, University of Sydney, New South Wales 2006, Australia; and 2 Department of Internal Medicine and Research, University Hospital Basel, 4031 Basel, Switzerland
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ABSTRACT |
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We investigated the chemotactic action of PDGF and urokinase on human airway smooth muscle (HASM) cells in culture. Cells were put in collagen-coated transwells with 8-µm perforations, incubated for 4 h with test compounds, then fixed, stained, and counted as migrated nuclei by microscopy. Cells from all culture conditions showed some basal migration (migration in the absence of stimuli during the assay), but cells preincubated for 24 h in 10% FBS or 20 ng/ml PDGF showed higher basal migration than cells quiesced in 1% FBS. PDGFBB, PDGFAA, and PDGFAB were all chemotactic when added during the assay. PDGF chemotaxis was blocked by the phosphatidyl 3'-kinase inhibitor LY-294002, the MEK inhibitor U-0126, PGE2, formoterol, pertussis toxin, and the Rho kinase inhibitor Y-27632. Urokinase alone had no stimulatory effect on migration of quiescent cells but caused a dose-dependent potentiation of chemotaxis toward PDGF. Urokinase also potentiated the elevated basal migration of cells pretreated in 10% FBS or PDGF. This potentiating effect of urokinase appears to be novel. We conclude that PDGF and similar cytokines may be important factors in airway remodeling by redistribution of smooth muscle cells during inflammation and that urokinase may be important in potentiating the response.
cell migration; asthma; airway remodeling; urokinase-type plasminogen activator
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INTRODUCTION |
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REMODELING OF THE AIRWAY WALL is an important aspect of the pathogenesis of asthma (18, 22, 26, 30, 35). In an extensive microscopic study of human airways, Carroll et al. (2) found the histological thickness of many airway structural components, including smooth muscle, was increased in asthmatic airways. Contributions to the increased smooth muscle mass could result from hypertrophy and/or hyperplasia of cells in existing smooth muscle bundles or from migration of cells to form new smooth muscle bundles, analogous to the formation of neointima in atherosclerosis. One of the cell types likely to contribute to airway remodeling through cell migration is the myofibroblast, which appears in greater numbers in allergen-challenged asthmatic airways (13) and may share a common origin with smooth muscle cells.
In this study we investigated the promigratory effects on human airway smooth muscle (HASM) cells of two proteins, platelet-derived growth factor (PDGF) and urokinase, which have both been reported as important in cell migration but which act via different signal transduction pathways. We further tested the modulation of migration by a range of inhibitors likely to target the signal transduction of PDGF and urokinase and by two drugs commonly used for asthma treatment, formoterol and budesonide.
In an early study on smooth muscle cell migration, Grotendorst et al.
(16) showed that PDGF is chemotactic for rabbit aortic myocytes and that movement across an artificial perforated membrane requires a coating of collagen or fibronectin. PDGF is also chemotactic for canine tracheal myocytes (19), as it is for many
nonsmooth muscle cell types (33). Signal transduction
through PDGF receptors is based on tryosine kinase cascades and binding
of proteins with SH2 domains. Although the PDGF- receptor is
generally reported to be more active in chemotactic signaling than the
PDGF-
receptor, the relative activity of the two receptors may be
cell type and context dependent. Activation of phosphatidyl 3'
(PI3)-kinase and the Ras/Raf/MEK/ERK phosphorylation cascade are
central to PDGF chemotaxis in most cell types. Irani et al.
(21) reported that transient expression of constitutively
active PI3-kinase in a rat smooth muscle cell line is sufficient to
induce migration but not as effective as stimulation by growth factors.
Urokinase and its receptor urokinase-type plasminogen activator (uPAR) are well established as factors influencing cell migration. Migration of HASM cells in vitro was demonstrated by Mukhina et al. (27), who found urokinase to be a chemotactic agent and defined the active protein domain. Urokinase has been identified as a migratory agent (35, 36), but it has also been associated with proliferation rather than migration in wound repair (4). In other reports, the migratory response of cells to urokinase is slow or small (29). Urokinase and uPAR are involved in formation of clusters of proteins at the leading edge of migrating cells: for example, integrins and Src protein tyrosine kinase in rat smooth muscle cells (6) and the protein tyrosine kinases JAK1 and Tyk2 in human vascular smooth muscle cells (7). The signal transduction of urokinase was reported to be via Ras, ERK, and myosin light chain kinase (MLCK) (28) for increasing basal migration of human breast cancer cells.
Cell migration and chemotaxis (migration toward a chemical stimulus) involve coordination of many cell functions (3, 25). In response to a chemotactic stimulus, cells create leading-edge complexes of proteins, which form attachments to the extracellular matrix. Actin polymerization is activated at the leading edge, which is thereby extended, and the cell body and nucleus is pulled forward by myosin contraction. Finally, the trailing edge of the cell must detach from the matrix for the cell to move forward. Many chemotactic and migratory stimuli have been identified, and the signal transduction pathways involved are correspondingly diverse. Cell migration can for instance be initiated by stimulation of both G protein-coupled receptors and tyrosine kinase receptors.
We demonstrate here that all three isoforms of PDGF are chemotactic for
HASM cells in culture and that this effect is mediated via a mechanism
that can be blocked by PI3-kinase inhibition, MEK inhibition, Rho
inhibition, pertussis toxin, PGE2, and the 2-adrenergic receptor agonist formoterol. In contrast,
the glucocorticoid budesonide had no effect on migration or chemotaxis,
and inhibitors of MLCK promoted migration. Surprisingly, urokinase did
not have a chemotactic or migratory action when it was applied alone,
but, rather, it potentiated both chemotaxis toward PDGF and basal
migration of activated cells.
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METHODS |
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Materials. PDGFAA and PDGFBB (human recombinant) were from from ICN Biomedicals (Aurora, OH). PDGFAB was from Upstate Biotechnology (Lake Placid, NY). Urokinase (purified from human urine), tissue plasminogen activator (tPA, single chain from human melanoma cell culture), and collagen (type I from calf skin) were from Sigma (St. Louis, MO). Monoclonal, activated anti-MAPK (clone MAPK-YT) was from Sigma/Fluka. Polyclonal Anti-uPAR antibody was from R&D Systems (Minneapolis, MN). ML-7, ML-9, and W-7 were from Fluka Chemie (Buchs, Switzerland). Pertussis toxin and Y-27632 were from Calbiochem (La Jolla, CA). U-0126 was from Promega (Madison, WI). All other reagents and growth media were of analytical or cell culture grade.
HASM cell culture.
Primary cultures of HASM cells were established as reported previously
by our group (22). The protocol was approved by the Human
Ethical Review Committee of The University of Sydney. In brief,
macroscopically normal human lung was obtained from patients undergoing
lung transplantation or partial resection. Large bronchi (5- to 15-mm
internal diameter) were dissected from the surrounding parenchyma and
dipped in 70% (vol/vol) ethanol in water to kill surface organisms.
ASM bundles, viewed under a dissecting microscope, were dissected from
the bronchi and placed in tissue flasks containing DMEM supplemented
with 1% Fungizone, 1% penicillin-streptomycin, and 10% fetal bovine
serum (FBS). The smooth muscle cells grew to confluence in a humidified
CO2 incubator in 16-24 days and were passaged into
175-cm2 flasks at 7- to 10-day intervals. Pure populations
of smooth muscle cells were confirmed by the presence of positive
staining for -smooth muscle actin.
Cell preparation. Cells from passages 4-7 were seeded into six-well plates at a density of 6 × 104 cells/cm2 in 10% FBS and allowed to settle overnight. For the following 24 h, cells were quiesced in DMEM containing 1% FBS. For preincubation experiments, the quiescence medium was replaced with 10% FBS or 20 ng/ml PDGF (activation media) for 24 h. Cells were lifted with minimal trypsinization for the chemotaxis assay (terminated with 10% FBS), resuspended in DMEM containing 0.1% BSA, and added to the upper compartment of the transwells at 2 × 104 cells/ml.
Cell migration assay. Cell culture membrane inserts with 8-µm pore size, in 48-well plate format (Falcon), were collagen coated by incubation with collagen solution (0.1 mg/ml collagen type I in DMEM, diluted from 1 mg/ml stock in 0.1 M acetic acid) for 1 h. Coated inserts were then air dried for 1 h and washed with DMEM containing 0.1% BSA.
Migration assays were carried out in in 1 ml of DMEM containing 0.1% BSA. Cells were added to the upper compartments, then left to settle for 30 min in a CO2 incubator before treatments were added to the top or bottom compartments. At the end of the assay (4 h), the membranes were fixed in ice-cold methanol. Cells were stained with hematoxylin and eosin, and the upper membrane surface was scraped clean of cells. Membranes were then mounted on slides, and the cells were manually counted as the number of nuclei in a standard central field. Cell nuclei were more reliable than cytoplasms as an assessment of migrated cells, as cytoplasms were often only partially migrated or difficult to distinguish individually.Western blots. Quiesced cells were plated into six-well plates precoated with collagen and left for 1 h to equilibrate. Conditions and medium (0.1% BSA) were as similar to the migration assay as possible. After exposure to stimuli, total cell protein was extracted directly into ice-cold electrophoresis sample buffer (1% SDS, 3.75% glycerol, 15.625 mM Tris · HCl, pH 6.8, 0.001% bromphenol blue, and 100 mM DTT). Samples were heated to 100°C for 5 min and loaded onto 10% polyacrylamide SDS gels for standard elecrophoresis and blotting onto polyvinylidene difluoride membranes. Phospho-ERK was detected by specific antibody coupled to chemiluminescence.
Addition of agents. Pertussis toxin was added to cells 16 h before assay and maintained in the medium during assay. Anti-uPAR neutralizing antibody was added at 0.5 µg/ml 30 min before assay. U-0126 (10 µM, manufacturer's recommended concentration), LY-294002 (50 µM) (30), ML-7 (3 µM), ML-9 (30 µM), W-7 (51 µM), and Y-27632 (25 µM) were added 15 min before assay.
Statistical analysis. The number of cell cultures used in experiments (n) refers to cell cultures derived from individual donors. Results were analyzed for significance by Student's t-test. A P value < 0.05 was considered significant.
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RESULTS |
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Basal cell migration.
The standard preparation for cells in our assay system was 24-h
incubation in 1% FBS medium before trypsinization for the migration
assay, so the cells were in a quiesced state (18). All
cell cultures demonstrated some basal migration in assay medium of
0.1% BSA and the absence of chemotactic agents, an average of 64 cells
per membrane (n = 30). When the cells were preincubated for 24 h in PDGF (20 ng/ml) or 10% FBS and then washed,
subsequent basal migration was elevated (Fig.
1B). It is important to note that cell migration was dependent on precoating of the filters with
collagen I, and cells did not migrate at all when this was omitted.
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Cell migratory agonists. We tested the efficacy of a number of compounds for their ability to stimulate HASM cell migration (Fig. 1A). The most effective agent was PDGFBB at 20 ng/ml, which caused a 12.4 ± 0.8-fold increase over basal migration (P < 0.05, n = 3 cell cultures). The other PDGF dimers, PDGFAA and PDGFAB (20 ng/ml), also stimulated migration (5 ± 0.2- and 3.5 ± 0.3-fold, respectively, P < 0.05, n = 3 and n = 10). The migratory response to PDGF was dose dependent between 0.5 and 10 ng/ml, which gave maximal response for PDGFAA and PDGFBB (data not shown). PDGF stimulus was chemotactic, that is, more than twice as many cells migrated when the PDGF was added to the lower (trans) well of the migration chamber than when the PDGF was added to the same side as the cells.
In addition, we tested two plasminogen activators, tPA and urokinase, and found that although tPA stimulated migration (2.6 ± 0.7, P < 0.05, n = 5), urokinase was not stimulatory (0.88 ± 0.21-fold, n = 8) (Fig. 1A).Urokinase potentiates chemotaxis.
Although urokinase alone was not chemotactic for HASM cells, when added
at a concentration of 10 nM, it potentiated the chemotaxis induced by
PDGF. The potentiation occurred both with PDGFBB (2 ng/ml,
1.9 ± 0.3-fold potentiation over PDGFBB alone,
P < 0.05, n = 6) and with
PDGFAB (5 ng/ml, 2.6 ± 0.04-fold, P < 0.05, n = 3). We tested the dose responsiveness of
urokinase-induced potentiation of PDGFAB-induced chemotaxis
and found it to be active at concentrations higher than 0.1 nM (Fig.
2A). The action of urokinase
appeared to be mediated by the urokinase receptor uPAR, as
preincubation of cells with uPAR-inactivating antibody abolished the
urokinase effect (Fig. 2B). The anti-uPAR did not, however,
affect PDGF chemotaxis itself.
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Time course of PDGF chemotaxis.
We investigated the time course of HASM cell chemotaxis toward PDGF in
two cell cultures (Fig. 3). Analysis of
the migration membranes by microscopy revealed that at 1 h a large
number of cells were in the process of migrating to the far side of the migration membrane, but at that stage we saw only (roughly circular) cytoplasmic extensions through the 8-µm migration pores. Only very
few of these leading cytoplasmic edges included nuclei, and so these
were not counted as migrated cells. In the following (2-4) hours, increasing numbers of cells moved
through the pores with their nuclei, and these cells acquired the
typical elongated shape of HASM cells. Migration of large numbers of
cells (nuclei) did not occur until after 2 h of exposure to a PDGF
gradient, and the rate of migration was maximal between 2 and 3 h.
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Inhibition of cell migration.
To confirm the mechanisms of signal transduction involved in PDGF
chemotaxis, we used LY-294002 (50 µM) to inhibit PI3-kinase and
U-0126 (10 µM) to inhibit MEK and the MAPK pathway. Both inhibitors reduced chemotaxis toward PDGFAB and PDGFBB to
basal levels, although they did not abolish migration (Fig.
4A). Pertussis toxin (50 ng/ml, 12-h preincubation) also strongly inhibited cell migration (toward a PDGF stimulus) to 0.1 ± 0.03 of basal migration
(P < 0.05, n = 4). We also tested the
long-acting 2-adrenergic receptor agonist formoterol and
found that it inhibited PDGF chemotaxis in a dose-dependent manner at
concentrations >10
12 M (Fig. 4B).
PGE2 (10
6 M), another activator of adenylyl
cyclase, strongly inhibited PDGF chemotaxis to less than basal levels
(0.2 ± 0.1-fold of basal, P < 0.05, n = 2). The corticosteroid budesonide
(10
8 M) did not inhibit chemotaxis toward PDGF (1.25 ± 0.45-fold over PDGF alone, P < 0.05, n = 2), nor did it interfere with formoterol inhibition
of PDGF chemotaxis (1.2 ± 0.1-fold over formoterol and PDGF,
P < 0.05, n = 2).
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Signal transduction of urokinase. Following the work of Nguyen et al. (28), we investigated the role of MLCK in urokinase action. Inhibitors of MLCK (3 µM ML-7 and 30 µM ML-9) stimulated migration toward 20 ng/ml PDGFBB (Fig. 4C). We confirmed this unexpected result with a dose-dependence test for ML-7, which stimulated migration at concentrations of 0.9 and 3 µM, but not at higher or lower doses (n = 2, data not shown). A third inhibitor of MLCK, W-7 (51 µM), which acts by inhibition of calmodulin action, completely blocked migration (Fig. 4C), but microscopic analysis during the assay showed that it caused the cells to round up and de-adhere. This process was slow and progressive, and all cells could be dislodged by shaking after 4 h. An alternative activator of myosin light chain is Rho kinase (26), and in our cells inhibition of Rho kinase by Y-27632 (25 µM) inhibited migration (Fig. 4C).
Activation of ERK 1/2 by PDGF and urokinase.
We analyzed phosphorylation (activation) of ERK 1/2 (p44/p42 MAPK) by
Western blot (Fig. 5). PDGFBB
caused transient activation of ERK, but urokinase alone did not
activate it. However, when urokinase was added together with PDGF it
caused prolonged activation of ERK, with significantly higher
phospho-ERK at 40 min.
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DISCUSSION |
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Migration of smooth muscle cells may contribute to airway
remodeling in asthma. In this study we show that all three PDGF isoforms are chemoattractants for HASM cells and that HASM cell migration is potentiated by urokinase. The PDGF-induced chemotaxis was
mediated via both the PI3-kinase and the MEK/ERK pathways, since both
LY-294002 and U-0126 inhibited the cells' response. In addition, the
long-acting 2-agonist formoterol, but not the corticosteroid budesonide, inhibited migratory responses.
Repeated inflammation of asthmatic airways is thought to result in remodeling of the cellular structure of the airway wall. In a study of bronchial biopsies from allergic asthmatic patients, Gizycki et al. (13) found that migratory cells with a contractile phenotype, myofibroblasts, appeared in the subepithelial region during the late response following allergen challenge. These cells could be subepithelial fibroblasts that have assumed the characteristics of muscle cells as shown by their increased expression of smooth muscle actin (31). Alternatively they could be muscle cells that have migrated to lie under the epithelium in much the same way as arterial smooth muscle cells migrate to form a neointima in a remodeled vessel. Similar remodeling occurs in other tissues, and Roche et al. (32) reported a resemblance between asthma and collagenous colitis, both diseases characterized by remodeling, excessive smooth muscle contractility, and mucus secretion.
PDGF has been characterized as a chemoattractant for vascular smooth
muscle cells from both sheep (16) and humans
(34) and also for canine tracheal myocytes
(19). The mechanism of PDGF induction of chemotaxis was
reviewed by Ronnstrand and Heldin (33), who described PDGF
as primarily affecting connective tissue cells. PDGF and its receptor
both occur as isoforms and are active as dimers. PDGFAA
activates only the PDGF -receptor dimer, whereas PDGFAB activates
- and
-receptors, and
PDGFBB activates
-,
-, and
-receptors.
The PDGF
- and
-receptors have a differential role in chemotaxis,
and the
-receptor is usually described as the active form for
chemotaxis, whereas the homodimeric
-form has been found to be
inactive or inhibitory. However, others have reported that, in human
fibroblasts and human vascular smooth muscle cells, all three forms of
the PDGF receptor are active in chemotaxis (11, 20). Our
results show that all three dimers of PDGF are active in chemotaxis of
HASM cells in culture, although their efficacy varied.
PI3-kinase is a primary component in signal transduction of PDGF-induced chemotaxis (33), and here we confirm, using the PI3-kinase inhibitor LY-294002, that migration of HASM cells is reduced to basal levels when PI3-kinase activity is blocked. We also found that U-0126, the inhibitor of MEK and subsequently of ERK 1/2, inhibits PDGF chemotaxis. ERK has been reported to be both a participant and a nonparticipant in chemotaxis (33), and its function may differ between cell types. In mouse fibroblasts, ERK 1/2 signals through its membrane-associated fraction to activate calpain, which facilitates migration by altering cell adhesion (14). Our finding that pertussis toxin inhibited migration in HASM cells agrees with reports from several cell types (5, 10, 17, 23). Although pertussis toxin is mainly used as an inhibitor of G protein-coupled receptors, it has recently been reported to have other actions, for example, to activate ERK 1/2 (12, 37). From the study of Wang et al. (37), it seems likely that the inhibitory effect of pertussis toxin on cell migration is mediated through maintenance of high cAMP concentrations.
PDGF chemotaxis was also inhibited by the long-acting
2-agonist formoterol and by PGE2.
Formoterol has also been reported to inhibit chemotaxis of eosinophils
(8). Our HASM cells were, however, inhibited by nanomolar
concentrations of formoterol, rather than by micromolar concentrations
as in eosinophils.
2-Agonists and PGE2 both
stimulate adenylyl cyclase, and Kohyama et al. (24) found
that fibroblast chemotaxis is inhibited by PGE2, forskolin, and the
2-agonist isoprenaline. Conversely, chemotaxis
of vascular smooth muscle cells is stimulated through inhibition of
adenylyl cyclase (37). In regard to the use of long-acting
2-agonists and glucocorticoids in asthma therapy, it is
relevant to note that a glucocorticoid, budesonide, neither inhibited
PDGF-dependent chemotaxis nor affected the inhibitory action of
formoterol. Therefore, it is likely that the inhibitory action of
formoterol was mediated via an increase in cAMP levels and not via
stimulation of the glucocorticoid receptor (9). PDGF
itself can stimulate cAMP formation in human arterial smooth muscle
cells, via PGE2 synthesis mediated by MAPK activation
(15). This cAMP peak was not, however, sufficient to
"feed back" on MAPK activation. Subsequent research established
that the magnitude (and therefore effectiveness) of PGE2/PKA activation was dependent on the expression level
of cyclooxygenase (COX)-2 in the cells and that where COX-2 expression
was high enough, PDGF-stimulated cAMP peak was indeed inhibitory
(1).
Urokinase is a protease secreted by many cell types as an inactive form and is activated to a serine protease by proteolytic cleavage, which creates a two-chain protein linked though a disulfide bond. The action of urokinase in mesenchymal cell migration is not as clear as that of PDGF, and migration has been reported to be both marginal and slow (29). Urokinase is, however, well established as a migratory stimulus in many cell types and can act directly both through its proteolytic activity and through binding to its cell surface receptor uPAR (27). Much research has concluded that activation of uPAR is the primary mechanism of urokinase stimulation of migration, and our studies with a neutralizing antibody to uPAR confirm this. We found that urokinase, as opposed to the unrelated plasminogen activator tPA, was not itself a migratory stimulus in quiesced cells but potentiated chemotaxis toward PDGF. We also found that cells that were taken directly from a stimulatory medium (i.e., exposed to PDGF) retained an elevated basal migration and that this migration was also potentiated by urokinase. These results suggest that in HASM cells urokinase does not initiate migration but, rather, facilitates it. This is at variance with the findings of Mukhina et al. (27), who reported a direct migratory effect of urokinase in HASM cells. Whether this is related to the fact that they studied tracheal as opposed to bronchial smooth muscle cells is not clear. Our results are, however, in agreement with those of Nguyen et al. (28), who found that urokinase amplifies basal migration. They identified activation of MLCK as a downstream effector of urokinase in breast cancer cells, but our results with the same MLCK inhibitors (ML-7, ML-9, and W-7) showed no inhibition in airway smooth muscle cells, although W-7 caused the cells to de-adhere. These differences in inhibition may reflect the different contractile mechanisms in the cell types. Rho kinase is an alternative activator of MLCK in airway smooth muscle cells in culture (26), where MLCK and Rho kinase appear to activate contraction in different parts of the cell and over different time scales. Indeed, the inhibitor of Rho kinase, Y-27632, blocked migration of our HASM cells. Migration toward PDGF was inhibited to below basal levels, and no difference was seen in the presence of urokinase. ERK has been identified as another downstream effector of urokinase signaling, in breast cancer cells (28). Our data show that, in HASM cells, stimulation was not direct, but urokinase could potentiate stimulation by PDGF. Urokinase alone caused no phosphorylation of ERK 1/2, whereas PDGF by contrast activated ERK. However, urokinase in combination with PDGF caused increased activation of ERK at extended time (40-min stimulation). These results support a model for cell migration in which the amplitude and duration of ERK activation are central signals. Inhibition of ERK activation blocks migration, whereas the effect of urokinase is to potentiate both activation of ERK and migration.
In summary, we have shown that HASM cells in culture migrate in response to all isoforms of PDGF and that this response is mediated via the PI-3 kinase and/or the MEK/ERK pathways. Migration and chemotaxis can be inhibited by interventions associated with elevation in cAMP levels, and PDGF-induced chemotaxis can be markedly potentiated by urokinase. Whether this increased migration and chemotaxis impact on airway remodeling in asthma requires further investigation.
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ACKNOWLEDGEMENTS |
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We thank the transplant and thoracic surgeons, theater staff, and pathologists of the Sydney Central Area Hospitals and the Kantonsspital Basel for invaluable cooperation in obtaining bronchial tissue for our cell cultures.
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FOOTNOTES |
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Address for reprint requests and other correspondence: S. M. Carlin, Pneumologie, Lab 305, Dept. Forschung, Kantonspital Basel, 4031 Basel, Switzerland (E-mail: Stephen.Carlin{at}unibas.ch).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published February 7, 2003;10.1152/ajplung.00092.2002
Received 27 March 2002; accepted in final form 21 January 2003.
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