1 McDonald Research Laboratory, University of British Columbia, Vancouver, British Columbia V6Z 1Y6, Canada; and 2 Section of Pulmonary and Critical Care Medicine, Department of Medicine, Division of Biological Sciences, University of Chicago, Chicago, Illinois 60637
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ABSTRACT |
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Our laboratory recently demonstrated
the pattern of cell surface glycosylation of nonsecretory central
airway epithelium (Dorscheid DR, Conforti AE, Hamann KJ, Rabe KF, and
White SR. Histochem J 31: 145-151, 1999), but the role
of glycosylation in airway epithelial cell migration and repair is
unknown. We examined the functional role of cell surface carbohydrates
in wound repair after mechanical injury of 1HAEo human
airway epithelial and primary bronchial epithelial monolayers. Wound
repair stimulated by epidermal growth factor was substantially attenuated by 10
7 M tunicamycin (TM), an N-glycosylation
inhibitor, but not by the inhibitors deoxymannojirimycin or
castanospermine. Wound repair of 1HAEo
and primary airway
epithelial cells was blocked completely by removal of cell surface
terminal fucose residues by
-fucosidase. Cell adhesion to collagen
matrix was prevented by TM but was only reduced ~20% from control
values with prior
-fucosidase treatment. Cell migration in Blind
Well chambers stimulated by epidermal growth factor was blocked by
pretreatment with TM but
-fucosidase pretreatment produced no
difference from control values. These data suggest that cell surface
N-glycosylation has a functional role in airway epithelial cell
adhesion and migration and that N-glycosylation with terminal
fucosylation plays a role in the complex process of repair by
coordination of certain cell-cell functions.
epithelium; tunicamycin; fucosidase; cell migration; cell adhesion; cell spreading
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INTRODUCTION |
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COMPLEX CARBOHYDRATE STRUCTURES attached to cell surface proteins and lipids have functional roles in cell adhesion via integrin proteins (24, 29), proliferation (3, 26a), and growth potential (26, 30) in several cell types. Glycoconjugate expression is an essential element for migration of corneal epithelial cells in surface repair (28). The expression of terminal sugars in the epithelium of central airways has been associated with blood group antigen expression of complex carbohydrates (27), and several diseases, including asthma, have been associated with specific combinations of ABO or Lewis antigens (18). More recently, it has been noted that human cells express a class of proteins known as selectins that recognize specific carbohydrate structures (11). The general carbohydrate composition of selectin ligands includes fucose, galactose, and sialic acid residues. Functions associated with this receptor family include lymphocyte homing, inflammatory cell migration, metastasis, hemopoietic cell maturation (13), and coordination of repair of endothelial cell layers (38). P-selectin, which recognizes specific carbohydrate structures, is similarly required in endothelial cell migration during repair (9). Many cellular functions that utilize the selectin family do not yet have an identified ligand core structure.
Airway epithelial cells are known to have specific surface glycosylation patterns. Secretory cells express a number of O- and N-linked glycoproteins as demonstrated by lectin histochemistry (1, 2, 27, 31). These can differ substantially based on location in the large and small airways (1, 31). Our laboratory (7) has recently characterized cell surface glycosylation in nonsecretory cells of central human airway epithelium and airway epithelial cell lines that utilize lectin-binding patterns. In that study (7), galactose- or galactosamine-specific lectins labeled basal epithelial cells and cell lines derived from basal cells. Lectins specific for several different carbohydrate structures bound columnar epithelial cells, and certain fucose-specific lectins labeled subsets of the airway epithelial cells. These differences may be relevant in various cellular functions of these cells.
The airway epithelium is a target of inflammatory and physical stimuli in obstructive airway diseases such as asthma and bronchopulmonary dysplasia. The epithelium provides a physical barrier to the external environment and regulates several key metabolic functions of airways including fluid and ion transport to the airway lumen, mucociliary clearance, and regulation of airway diameter. Repair of a damaged epithelium may be a necessary part of restoring airway function to its normal state and may thus play a role in chronic airway remodeling as seen in asthma. Repair generally involves several steps including spreading of epithelial cells at the margin followed by migration of distant cells into the damaged region and, finally, proliferation of new epithelial cells (10, 19).
The purpose of this study was to determine if cell surface glycosylation mediated the repair of airway epithelium after mechanical injury. Human airway epithelial cell lines and primary bronchial epithelial cells grown in monolayer culture were studied with the use of time-lapse videomicroscopy after mechanical injury. Stimulated repair was studied in the presence of specific inhibitors of glycosylation or in the presence of endo- and exoglycosidases. Cell migration in Blind Well chambers and adhesion to collagen-coated plates were studied in the presence of these inhibitors and enzymes. Our data demonstrate that N-glycosylation of cell surface glycoprotein molecules, along with terminal fucose residues, regulate both monolayer repair and migration of epithelial cells in culture.
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METHODS |
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Materials.
Bovine serum albumin (BSA), trypsin, epidermal growth factor (EGF),
hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF),
human placental collagen IV, and EDTA were obtained from Sigma (St.
Louis, MO). Fetal calf serum (FCS) was obtained from HyClone (Logan,
UT) and was heat denatured before use. Tunicamycin (TM),
deoxymannojirimycin (DJM), -fucosidase, endoglycosidase H,
O-glycosidase, sialidase,
-galactosidase,
amyloglucosidase, N-acetyl-
-D-glucosaminidase, and
castanospermine (CSP) were obtained from Boehringer Mannheim
(Indianapolis, IN). The biotinylated lectins Aleuria
aurantia agglutinin (AAA), Ulex europaeus
agglutinin (UEA)-I, Lens culinaris agglutinin (LCA),
and Pisum sativum agglutinin (PSA) were obtained from EY
Laboratories (San Mateo, CA). Streptavidin-horseradish peroxidase was
obtained from DAKO (Carpinteria, CA).
Cell culture.
1HAEo cells are SV40-transformed normal human airway
epithelial cells that have been characterized previously (5,
14) and express multiple surface carbohydrate markers of primary
basal airway epithelial cells (7). Cells were grown on
collagen IV-coated flasks or plates in Eagle's modified essential
medium (MEM) containing 10% FCS, 2 mM L-glutamine, 100 µg/ml of streptomycin, and 100 U/ml of penicillin G (medium
A) and incubated at 37°C in 5% CO2. Cells were
passaged into matrix-coated six-well plates for wound repair
experiments. Primary normal human bronchial epithelial (NHBE) cells
were purchased from Clonetics (Walkersville, MD). These cells were
derived from a single donor and were supplied as first-passage cells.
Cells were placed in defined medium (Clonetics) containing 5 µg/ml of
insulin, 0.5 µg/ml of human EGF, 10 mg/ml of transferrin, 6.5 µg/ml
of triiodothyronine, 0.5 mg/ml of epinephrine, and 2 ml/l of bovine
pituitary extract. Cells were subcultured and used between
passages 3 and 7. Experiments were done in the same manner as those conducted with the 1HAEo
cell line
except that cells were kept in defined medium and not in 10% FCS, and
wound repair was performed at ~70% confluence.
Monolayer wound repair assay. Our laboratory has described this method previously (20-22). Cells were grown until confluent in medium A and then placed in 2 ml of medium B (medium A without FCS). A small wound (~1 mm2) was made in the confluent monolayer with a rubber stylet, and epithelial cells were removed without disturbing the underlying matrix. Wound closure was documented serially for 24 h starting immediately after wound creation. Microscope images were photographed with a Sony Iris charge-coupled device camera (Sony, Rolling Meadows, IL) on a Nikon Diaphot inverted stage microscope. Video images were digitized with a Macintosh computer and Apple Video Player software (Apple Computer, Cupertino, CA). Analysis of perimeter length and area of the remaining wound in each image was performed with NIH Image software (Wayne Rasband, National Institutes of Health, Bethesda, MD).
In each experiment, one well was used as a negative control (no EGF, inhibitor, or glycosidase), and one well was treated with 15 ng/ml of EGF, which has previously been demonstrated to be a potent accelerant for epithelial monolayer wound closure (20). In six experiments, cells were treated concurrently with both EGF and 10Lectin histochemistry.
Cell monolayers grown on two-chamber slides were treated for 12 or
24 h with -fucosidase (100 mU/ml), CSP (10
5 M),
or DJM (10
3 M); fixed with 4% paraformaldehyde at room
temperature (RT) for 4 h; and subsequently dehydrated in
sequential alcohol baths. Endogenous peroxidases were quenched by
incubation in 0.3% H2O2 for 30 min. Slides
were blocked in HEPES buffer (10 mM HEPES, 150 mM NaCl, 0.1 mM
MgCl2, and 0.1 mM CaCl2 at pH 7.2) containing 0.1% BSA before incubation for 60 min at RT with biotinylated lectins
(5 µg/ml) in HEPES buffer without BSA. Slides were rinsed and
incubated in 1:300 streptavidin-horseradish peroxidase for 20 min at
RT. Diaminobenzidine augmented with nickel plus a 1:1,000 dilution of
30% H2O2 was applied for 7 min followed by a
rinse in distilled, deionized water. Slides were counterstained with hematoxylin and mounted for viewing. Control slides were processed, but
the lectin was omitted. Specificity of the reactions for fucose was
verified with the use of HEPES buffer containing 50 mM fucose during
incubation for the AAA and UEA-1 lectins, 100 mM glucose for LCA
lectin, and 50 mM mannose for the PSA lectin.
Cell death determination.
Cells were grown until confluent in medium A and then
incubated for 24 h in 2 ml of medium B, 2 ml of
medium B plus 15 ng/ml of EGF, or concurrently with EGF plus
the addition of either 5 × 107 M TM or 250 mU/ml of
-fucosidase. Cells were lysed in the culture wells by the addition
of 500 µl of hypotonic propidium iodide (PI) solution (50 µg/ml of
PI and 0.1% Triton X-100 in 0.1% sodium citrate), followed by flow
cytometric analysis of the intact nuclei with a Becton Dickinson
FACScan flow cytometer. At least 5,000 nuclei were examined for each
sample to determine the proportion of nonviable nuclei or cell death.
Cell death is expressed as the percentage of total counted nuclei
(cells) that demonstrated <2 N chromosomal content on flow cytometry
by PI staining.
Cell adhesion assay.
Cells were grown until confluent in medium A and then
incubated overnight with medium B alone or with either
5 × 107 M TM or 250 mU/ml of
-fucosidase. The
next day, after treatment with 0.02% EDTA for 20 min at 37°C, the
cells were collected with a rubber policeman and replated on collagen
IV-coated plates. Cells previously incubated in medium B
alone were continued in medium B, and cells previously
incubated with either inhibitor were continued in either medium
B alone or medium B plus the same concentration of
inhibitor. Plates were washed gently after 2, 6, or 24 h to remove
nonadherent cells, and adherent cells were collected and counted with a
Coulter counter.
Cell chemotaxis assay.
Our laboratory previously described this assay (21, 22).
Briefly, cells were grown until confluent in medium A and
then incubated overnight with medium B with and without the
inhibitors TM (5 × 107 M) or
-fucosidase (250 mU/ml). Cells were collected in medium B and adjusted to
106 cells/ml. Chemotaxis assays were performed with 48-well
Blind Well chambers (NeuroProbe, Cabin John, MD). In order, 26 µl of medium B containing 0-100 ng/ml of EGF were placed in
the bottom wells of the chamber, a gelatin-coated 8-µm-pore
polycarbonate filter (Poretics, Livermore, CA) was placed over the
bottom wells, and the upper plate was placed securely on top with 50 µl of the cell suspension placed in each of the top wells. The
chamber was then incubated at 37°C in 5% CO2 for 6 h, an incubation period that elicits maximal migration (21,
22). The filter was then removed, with the adherent cells on the
top of the filter removed by gentle scraping in 3% acetic acid. The
cells on the bottom of the filter were then fixed in methanol with
Malachite Green (0.2 mg/l) overnight. The next day, filters were
stained with traditional methylene blue stain (0.47 g of methylene
blue, 0.44 g of azure A, 4.0 g of dibasic sodium phosphate,
and 5.0 g of monobasic potassium phosphate per 1.0 liter of
distilled, deionized water). Migration through the filter was measured
as the total number of cells counted in 10 high-power fields (HPF) with
a Nikon light microscope at ×400 magnification.
Data analysis. Wound closure is expressed either as area (µm2) or as a percentage of area at time 0. In previous videomicroscopy experiments with cell monolayers (22), intraobserver variability was <2%, and interobserver variability was <4% for all measurements. Cell adhesion is expressed as the percentage of cells adhered for a particular condition compared with the total number of cells initially plated. Cell chemotaxis is expressed as the number of cells migrating per 10 HPF. Comparisons between multiple groups were made by analysis of variance; when significant differences were found, further comparisons were made by Fisher's least significant difference test. Comparisons between two groups were made by paired Student's t-test. Bonferroni's correction was made as appropriate for multiple comparisons. Differences were considered significant when P < 0.05.
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RESULTS |
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Effect of glycosylation inhibitors on epithelial cell monolayer
wound repair.
Mean initial wound area for the 136 1HAEo cell
monolayers used in this study was 949,380 ± 37,330 µm2. Initial wound area and perimeter for monolayers
within each experimental series were equivalent and consistent. Control
monolayers closed modestly over 24 h, whereas wounds in monolayers
treated with 15 ng/ml of EGF closed substantially in the same time
period. In control and EGF-only experiments pooled across experimental series, the remaining wound area after 24 h was 64 ± 5% in
control cultures (n = 35) and 24 ± 4% in
EGF-treated wounds (P < 0.0001; n = 31).
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Effect of the exoglycosidase -fucosidase treatment on expression
of terminal fucose residues by epithelial cells.
The lectins AAA and UEA-1 bind specific and differing fucose linkages
(23, 37) and label basal airway epithelial cells in situ
(7). Both lectins had bound to the 1HAEo
cell monolayers before
-fucosidase treatment. Treatment with
-fucosidase for 12 h inhibited subsequent binding of the AAA lectin to 1HAEo
cell monolayers (Fig.
6, right). In contrast,
binding of UEA-1 was not affected by exoglycosidase exposure (data not
shown).
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Effect of glycosylation inhibitors on epithelial cell viability.
Treatment of 1HAEo cell monolayers for 24 h with 15 ng/ml of EGF plus either TM or
-fucosidase elicited no difference in cell viability compared with control monolayers as determined by
hypotonic PI analysis (Table 2).
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Effect of glycosylation inhibitors on epithelial cell adhesion.
Both TM and -fucosidase blocked 1HAEo
cell adhesion.
Pretreatment with 5 × 10
7 M TM blocked adhesion to
collagen IV almost completely (Table 3).
The reduction in cell adhesion was similar in control cells and in
cells treated with EGF and was not reversed by the removal of TM during
the adhesion period (Table 3). Similarly, pretreatment with 250 mU/ml
of
-fucosidase blocked adhesion to collagen IV, although the extent
of the reduction of adhesion with
-fucosidase treatment was markedly
less than that generated by TM (Table 4). As in the experiments with TM, the reduction in cell adhesion was
similar in EGF-treated and control cells and was not reversed by
removal of
-fucosidase during the adhesion period (Table 4).
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Effect of glycosylation inhibitors on epithelial cell chemotaxis.
EGF elicited migration of 1HAEo cells over 6 h in
chemotaxis chambers. Migration through chemotaxis filters 6 h
after treatment with 30 ng/ml of EGF was 18.0 ± 3.9 vs. 5.2 ± 0.9 cells/10 HPF for cells not treated with EGF (n = 5 cultures/group; P = 0.01). Pretreatment with 5 × 10
7 M TM for 12 h blocked subsequent
EGF-stimulated migration completely (Fig.
7). Cell migration through chemotaxis
filters 6 h after treatment with 30 ng/ml of EGF after
pretreatment with 5 × 10
7 M TM was 3.7 ± 0.7 cells/10 HPF (P = 0.03; n = 3 cultures)
vs. cells treated with EGF alone. In contrast, pretreatment with
-fucosidase did not abolish the subsequent concentration response to
EGF (Fig. 7). In these experiments, cell migration through chemotaxis
filters 6 h posttreatment with 30 ng/ml of EGF after pretreatment
with 250 mU/ml of
-fucosidase was 26.1 ± 10.9 cells/10 HPF
(P = NS vs. cells treated with EGF alone;
n = 4).
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DISCUSSION |
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Our laboratory (7) recently characterized cell surface glycosylation in nonsecretory cells of central human airway epithelium and airway epithelial cell lines with the use of lectins that bind specific glycosyl residues. Galactose-specific and galactosamine-specific lectins labeled basal epithelial cells and cell lines derived from basal cells, and fucose-binding lectins specifically labeled subsets of airway epithelial cells. These differences may be relevant in several functions of these cells such as repair after injury. Our data in this study demonstrate that N-glycosylated glycoproteins, particularly those with a terminal fucose residue, participate in the adhesion and migration of airway epithelial cells and facilitate closure of epithelial wounds in monolayer culture.
Treatment of monolayers with TM inhibited wound repair in a
concentration-dependent manner such that treatment with
106 M TM blocked wound repair significantly within
12 h (Fig. 1). TM prevents nascent peptide N-glycosylation in the
Golgi body. As such, pretreatment of the monolayers with TM before
wound creation blocked migration and closure completely. This suggests
the need for N-glycoproteins to facilitate repair. The
inhibition of closure was not the result of cell death as demonstrated
by PI staining for cell viability. However, alteration in integrin
N-glycosylation elicited by TM treatment decreased the function of
these receptors in cell adhesion and migration (3, 25,
33). Integrins are associated with "firm adhesion" of
epithelial cells to matrix proteins (8), and TM treatment
of airway epithelial cells produces a near total loss of adherence to
collagen IV (Table 3). Integrins facilitate a portion of wound closure
of damaged airway epithelial cell monolayers (35); the
loss of adhesion mediated by changes in N-glycosylation could result in
decreased wound closure rates. To the extent that integrins facilitate
repair, this remains only a partial explanation of the TM-inhibited
closure rates.
Treatment with either endoglycosidases to cleave discrete distal
portions of mature polysaccharides or inhibitors to prevent specific
modifications to nascent high-mannose N-linked structures did not slow
repair. The requirement of N-linked glycoproteins for certain cellular
processes has been described (13, 24, 25, 33), but often,
the identification of a more specific glycoconjugate has not been
possible. Treatment of cell monolayers with CSP and DJM altered the
cell surface carbohydrate profiles as determined by lectin-binding
patterns (Fig. 3C). These altered patterns were consistent
with appropriate enzymatic inhibition, whereas terminal fucosylation
remained intact. This suggests that the presence of any N-glycosyl
chain can serve as a substrate for fucosyltransferases and result in
terminal fucosylation. That neither CSP nor DJM inhibited wound closure
suggests that the intact terminal fucose linkages facilitated the
epithelial wound repair. The removal of fucose residues by the action
of -fucosidase, as identified by the lectin AAA, corresponded to the
absolute inhibition of monolayer wound repair (Figs. 4 and 6).
Lectin histochemical surveys of the intact human airway demonstrate the
expression of specific fucose linkages on subsets of airway epithelial
cells (7). The cell monolayers used in this study express
terminal fucose. Although some morphological features of the
1HAEo cells are not the same as the pseudostratified
columnar epithelium seen in vivo (34), the monolayers are
similar to those described in other studies of epithelial cell function
(26) and have multiple characteristics of primary airway
epithelial cells (5, 7, 14). Additionally, confirmation of
significant wound inhibition was demonstrated in primary isolated NHBE
cells in an attempt to more closely model wound repair by in vivo
epithelial cells. To determine if fucose residues on cell surface
structural proteins expressed by these cells were involved in cell
migration and spreading in vivo, monolayers were treated with the
exoglycosidase
-fucosidase. For both the 1HAEo
cell
line and NHBE primary cells, the remaining wound area 24 h after
treatment with both EGF and 250 mU/ml of
-fucosidase was
substantially greater than that in monolayers treated with EGF alone
(Fig. 4). Impairment of wound repair after removal of terminal fucose
residues did not result from cellular toxicity because viability and
cell death were unchanged at all concentrations of
-fucosidase
compared with those in control monolayers. Similarly, the use of
heat-denatured and inactivated
-fucosidase did not impair wound
repair. One past study (18) demonstrated that patients with severe asthma have a deficiency of terminal fucose in their blood
group surface antigens. Combined with our data, this suggests that
defects in epithelial repair in asthma patients may be due, in part, to
such a deficiency.
The EGF receptor can be fucosylated (6). The particular
carbohydrate structure determines the affinity of the receptor for the
ligand. Loss of the fucose residue reduces receptor affinity for EGF.
To demonstrate that the wound repair inhibition is not an EGF
receptor-specific phenomenon, we examined the effect of -fucosidase
treatment during wound repair accelerated by two other growth factors,
PDGF and HGF, the function of which is not regulated by such structures
(6). As for EGF,
-fucosidase treatment inhibited repair
stimulated by these growth factors.
Of note is the effect of -fucosidase on epithelial cell adhesion and
migration. Although the effect on wound closure was profound,
-fucosidase treatment elicited only a slight reduction in two
processes thought to be significant components of repair. Exposure to
-fucosidase has not been demonstrated to alter integrin function.
Intact integrins function to mediate "hard adhesion" of cells to a
protein matrix. Repair after injury is a complex event that may require
participation of molecules or processes not otherwise utilized during
the study, specifically, adhesion or migration. Cell-to-cell or "soft
adhesion" and migration of one cell with respect to another is a
possible explanation for the inability of
-fucosidase to inhibit
either migration or adhesion as separate processes while inhibiting
epithelial wound repair. A human family of lectins, selectins, bind to
a fucose-containing ligand and have been demonstrated to be responsible
for cell-cell and not cell-matrix mobility (29). The
involvement of selectins is possible because the loss of fucose and the
selectin ligand are coordinate with wound repair inhibition. Treatment
with
-fucosidase during repair removes AAA lectin binding but not
that for UEA-1. UEA-1 binds
-1,4-fucose linkages (23),
whereas the lectin AAA binds
-1,3- and
-1,2-fucose linkages that
include the sialyl-Lex structures (37), a
classic ligand for selectins. Evaluation of selectin function during
repair of airway epithelium requires further study.
One potential limitation of our study was the use of a simple matrix, collagen IV, on which our cells were grown. Duplication of the basement membrane with a variety of matrix proteins such as fibronectin, laminins, collagens, proteoglycans, entactin, and nidogen (16) may permit multiple interactions of different glycosylated proteins with the elements of a complex matrix compared with this single matrix protein. Our data demonstrate a role for specific N-glycosylations in the complex functions of epithelial cell migration and adhesion during wound repair over collagen.
In summary, we demonstrated that N-glycosylated proteins participate in the airway epithelial cell processes of adhesion and migration. N-glycosylated proteins and proteins with terminal fucose residues mediate wound closure of airway epithelial cells and repair of epithelial cell monolayers. These data suggest that molecules not previously characterized with reference to cell surface glycosylation have a role in airway epithelial cell migration and repair.
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ACKNOWLEDGEMENTS |
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We thank Dr. Dieter Gruenert (University of California, San Francisco) for providing the human airway epithelial cells used in this study. We thank Amber Conforti and Amy Mann for assistance.
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FOOTNOTES |
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This work was supported by National Heart, Lung, and Blood Institute Grants HL-51853 and HL-60531. D. Dorscheid was supported by National Heart, Lung, and Blood Institute National Research Service Award HL-07605 and is a Parker B. Francis Fellow in Pulmonary Research.
Portions of this paper were presented at the international conference of the American Thoracic Society in San Francisco, California, in May 1997.
Address for reprint requests and other correspondence: D. Dorscheid, Rm. 292 Burrard Bldg., St. Paul's Hospital, 1081 Burrard St., Vancouver, BC V6Z 1Y6, Canada (E-mail: ddorscheid{at}mrl.ubc.ca).
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.
Received 24 February 2000; accepted in final form 15 May 2001.
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