Departments of 1 Pediatrics, 2 Microbiology, and 4 Cell Biology, University of Alabama at Birmingham, Birmingham, Alabama 35294; 3 Section of Pulmonary Diseases and Critical Care Medicine, Department of Medicine, Tulane University, New Orleans, Louisiana, 70112; and 5 The Ruth and Billy Graham Children's Medical Center, Asheville, North Carolina 28801
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ABSTRACT |
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Fibroblasts are heterogeneous with respect to
surface markers, morphology, and participation in fibrotic responses.
This study was undertaken to determine whether
Thy-1 and
Thy-1+ rat lung fibroblasts, which
have distinct and relevant phenotypes, differ in their proliferative
responses to platelet-derived growth factor (PDGF) isoforms.
Homogeneous populations of
Thy-1
and
Thy-1+ fibroblasts were found to
proliferate equally in the presence of PDGF-BB, but PDGF-AA-mediated
proliferation occurred only in Thy-1
cells. This
differential activity correlated with significantly higher expression
of PDGF-
receptor in
Thy-1
fibroblasts as shown
by immunoblotting, immunofluorescence, and Northern blotting. There was
a rapid increase in c-myc mRNA in Thy-1
but not in
Thy-1+ fibroblasts on stimulation
with PDGF-AA and PDGF-BB. The PDGF-
receptor, which mediates
signaling by all PDGF isoforms, has been implicated in numerous
clinical and experimental forms of fibrosis and regulates lung
morphogenesis. Differential expression of the PDGF-
receptor
supports distinct roles for
Thy-1
and
Thy-1+ fibroblast populations in
developmental and fibrotic processes in the lung.
cell surface molecules; rodent; proliferation
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INTRODUCTION |
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FIBROSIS OFTEN FOLLOWS INJURY or inflammation in
tissues and is thought to represent exaggeration or aberration of
normal wound-healing events. Fibroblasts within a fibrogenic milieu
clearly differ from those in normal tissues. For example, fibroblasts isolated from lungs with active fibrotic disease or from scleroderma lesions produce increased amounts of collagen (15, 29).
"Fibrosis" cells have increased proliferative capacity, are
capable of anchorage-independent growth, and are morphologically
distinct (21, 27, 29). Evidence accumulated over the last decade
suggests that the "fibrotic" phenotype arises from selective
recruitment or expansion of a subset of fibroblasts with the potential
for a more vigorous fibrogenic response rather than by uniform
activation of all the resident mesenchymal cells within an inflamed
tissue. Differences among subsets of fibroblasts have been identified
on the basis of surface markers, cytoskeletal arrangement, lipid
content, and cytokine profile (18). The best-characterized experimental
model of fibroblast heterogeneity is based on surface expression of the
Thy-1 glycoprotein. Morphological and secretory differences between
Thy-1-positive (Thy-1+) and
Thy-1-negative (Thy-1)
mouse and rat lung fibroblasts have shown that these subpopulations share some of the morphological criteria that distinguish normal cells
from fibrotic ones. Thy-1
but not Thy-1+ fibroblasts express
Ia, the rodent major histocompatibility complex class II antigen, after
interferon-
stimulation and are capable of producing interleukin
(IL)-1 (16, 19, 20). Although the exact function of Thy-1 is unknown,
its structural similarity to immunoglobulin suggests an important role
in cell-cell and cell-substrate interactions (7).
Cytokines and growth factors, such as transforming growth factor
(TGF)-, tumor necrosis factor-
, IL-1
, and platelet-derived growth factor (PDGF), have been demonstrated in elevated concentrations in clinical and experimental fibrogenic disorders of the lung parenchyma and airways (10, 14). The proliferative responses of
fibroblasts to both TGF-
and IL-1 are thought to be indirectly mediated by autocrine stimulation via PDGF, a powerful fibroblast mitogen and chemoattractant (13, 22). Thus the PDGF system appears to
be an important regulator of fibrotic responses to multiple mediators.
The TGF-
and IL-1 responses occur largely through the interaction of
the AA homodimer of PDGF with the
form of the PDGF receptor
(PDGFR-
). PDGF A and B isoforms form homo- and heterodimers. PDGF-AA
signals only through the
form of the receptor, whereas AB and BB
isoforms can bind to both
- and
-receptor subtypes. The
PDGF-AA/PDGFR-
pathway has been shown to mediate the mitogenic
response of lung fibroblasts in both human scleroderma and rodent
bleomycin injury (17, 24) as well as in other fibrotic conditions,
including dermal keloid scarring (9) and in vitro asbestos-induced
fibroblast mitogenesis (12). Because the
-receptor is
capable of binding any PDGF isoform, it can process signals via all
three ligands, and thus its regulation affects nearly all PDGF-mediated
cellular responses. Because of the possible distinct roles of
Thy-1
and
Thy-1+ cells in fibrotic
conditions, we examined whether these cell types differ in their
responses to PDGF. As a result, we have identified differential
expression of PDGFR-
and differential signaling in response to PDGF
isoforms in these fibroblast subpopulations.
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METHODS |
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Materials. The following
cDNAs were used for Northern hybridization: a rat PDGFR- cDNA
corresponding to the full-length coding sequence (a generous gift from
Randall R. Reed, Johns Hopkins University School of Medicine,
Baltimore, MD) was subcloned to generate a probe including the first
1,718 bases, which encode for the extracellular portion of the
receptor; the PDGFR-
cDNA was the generous gift from Michael Pech
(Hoffman-La Roche, Basel, Switzerland); and cDNA for murine c-Myc was
kindly provided by Dr. Andrew Kraft [University of Alabama at
Birmingham, Birmingham, AL (UAB)]. A cDNA fragment derived from murine
18S rRNA (American Type Culture Collection, Manassas, VA) or a 700-bp
fragment of rat cyclophilin cDNA, kindly provided by Dr. Etty
Benveniste (UAB), were used as loading controls. The recombinant human
(h) PDGF-AA and recombinant hPDGF-BB were obtained from R&D Systems
(Minneapolis, MN). The following antibodies were employed in the
Western blotting and immunofluoresence analyses: rabbit polyclonal
anti-hPDGFR-
and anti-hPDGFR-
(Santa Cruz Biotechnology, Santa
Cruz, CA); FITC-conjugated mouse anti-mouse/rat Thy-1.1; and mouse IgG
isotype standard (PharMingen, San Diego, CA). Secondary antibodies as indicated were obtained from Molecular Probes (Eugene, OR) and Kirkegaard and Perry Laboratories (Gaithersburg, MD). Control rabbit
antiserum [anti-human epithelial sodium channel-
(hENaC-
)] was a kind gift from Dr. Kevin Kirk (UAB). Tissue
culture plastic ware was obtained from Becton Dickinson Labware
(Franklin Lakes, NJ) or Nunc (Naperville, IL). Fetal bovine serum (FBS)
was purchased from Sigma (St. Louis, MO), and minimum essential medium
(MEM) and Ham's F-12 medium were from Mediatech (Herndon, VA).
Reagents (electrophoresis grade) used to prepare the buffers were
purchased from Fisher Scientific (Pittsburgh, PA) unless otherwise stated.
Cell culture. The preparation of Lewis rat lung fibroblasts
and their sorting into
Thy-1 and
Thy-1+ populations has been
described in detail (16). We used cells that have undergone <15
culture passages from isolation, and we confirmed the presence or
absence of Thy-1 staining in >95% of cells by flow cytometry (as
described in Ref. 16) every second or third culture passage.
Fibroblasts were screened for
Mycoplasma contamination with a
PCR-based kit (Stratagene, La Jolla, CA). The cells were seeded into
appropriate culture dishes and grown in MEM with 10% FBS until ~90%
confluent, then rendered quiescent by culturing in MEM with 0.4% FBS
for 48 h. The monolayers were washed with serum-free medium (SFM), and
SFM containing mediators of interest was added for the times indicated.
Proliferation assay. Fibroblasts were plated at 15,000 cells/250 µl in 24-well plates and allowed to attach overnight. The monolayers were washed twice with defined SFM (SFDM; Ham's F-12 medium and 0.25% albumin; Sigma) and rendered quiescent in SFDM plus insulin-transferrin-selenium (10 µg/ml; Life Technologies, Gaithersburg, MD) for 48 h. Growth factors were added as indicated in medium with 0.4% FBS for 8 h, after which [3H]thymidine (Amersham, Arlington Heights, IL) was added to a final concentration of 5 µCi/ml for an additional 16 h (24 h total). The wells were gently aspirated, washed three times with MEM, and placed on ice. The monolayers were treated with ice-cold 5% trichloroacetic acid (TCA) for 15 min., washed, and solubilized in prewarmed (37°C) buffer (0.2N NaOH and 0.1% SDS) for 30 min at 37°C before scintillation counting. Each condition was assayed in triplicate. The wells with medium containing either 0.4 or 10% FBS were used to determine [3H]thymidine uptake in quiescence and log-phase growth, respectively. The counts per minute obtained for cells grown in 0.4% FBS alone were averaged and subtracted from all experimental values and are expressed in arbitrary units, with the average counts per minute in wells exposed to 10% FBS alone set at 100.
Western immunoblotting. Whole cell
protein lysates from Thy-1
and Thy-1+ fibroblasts (10 µg/lane) prepared in the presence of protease inhibitors were
separated by SDS-PAGE with 10% acrylamide gels. The protein was
electroblotted onto polyvinylidine fluoride membranes and probed with
antibodies to PDGFR-
or -
as indicated. The membranes were probed
with an appropriate horseradish peroxidase-conjugated secondary
antibody, and the labeled proteins were detected with enhanced
chemiluminescence. Some membranes were stripped of the initial
antibodies by incubation in 0.0625 M Tris-Cl, pH 6.8, 2% SDS, and 0.1 M
-mercaptoethanol at 68°C for 30 min. The membranes were then
washed in Tris-buffered saline and exposed to film to determine removal
of the original signal before being reblocked and probed with a
different antibody.
Immunofluorescence. Monolayers of
Thy-1,
Thy-1+, and unsorted Lewis
fibroblasts were grown to near confluence on 22 × 22-mm glass
coverslips and then rendered quiescent. The coverslips were blocked
with 50% normal goat serum (NGS; Sigma)-PBS and incubated with
anti-Thy-1.1-FITC diluted 1:20 in 50% NGS-PBS for 1 h at 4°C. The
coverslips were washed with PBS, again blocked with 50% NGS-PBS, and
incubated with anti-PDGFR-
or anti-PDGFR-
diluted 1:50 in
blocking buffer for 1 h at 4°C. The coverslips were washed with PBS
and fixed in 3% formaldehyde (transmission electron microscopy grade; Tousimis, Rockville, MD)-PBS for 45 min at room
temperature. The coverslips were washed with PBS, blocked with 5%
NGS-PBS, and incubated with goat anti-rabbit IgG-Texas Red-X (Molecular Probes) diluted 1:80 in 5% NGS-PBS for 40 min at 37°C. Controls for antibody specificity were mouse IgG1,
-FITC
(anti-trinitrophenol; PharMingen) at 1:20 (control for anti-Thy-1) and
rabbit antiserum (anti-hENaC-
) at 1:50, followed by goat anti-rabbit
IgG-Texas Red-X at 1:80. All cells were also stained with Hoechst 33258 (Molecular Probes). The coverslips were mounted on glass microscope slides and examined with an Olympus IX70 inverted epifluorescence microscope. Images were acquired with a SenSys cooled charge-coupled device, high-resolution, monochromatic digital camera (Photometrics, Tucson, AZ) and analyzed with IP Lab Spectrum software (Signal Analytics, Fairfax, VA).
Northern blotting. Quiescent or
proliferating fibroblast monolayers were lysed with a commercial RNA
isolation buffer (Ultraspec, Biotecx, Houston, TX), and total RNA was
prepared according to the manufacturer's instructions. Fifteen
micrograms of total RNA in denaturation buffer were added to each well
of a 1.2% agarose gel and electrophoretically separated overnight. RNA
was transferred to a nylon membrane (Immobilon-N, Millipore, Bedford,
MA) by capillary action. Prehybridization solution and hybridization
diluent consisted of 6× saline-sodium phosphate-EDTA, 5×
Denhardt's solution, 1% SDS, and 20 µg/ml of salmon sperm DNA. The
membranes were prehybridized for 3-5 h at 62°C and hybridized
overnight at 62°C with cDNA probes labeled with
[-32P]dCTP with
random primers. The hybridized membranes were washed with 2×
saline-sodium citrate and 0.5% SDS, then with 0.2× saline-sodium citrate and 0.5% SDS at 37 and 62°C, respectively. The resulting hybridized signal was detected by autoradiography and quantified with
either a densitometric scanner (Bio-Rad, Hercules, CA) or image
phosphor analysis (PhosphorImager and ImageQuant, Molecular Dynamics,
Sunnyvale, CA).
Data analysis. To test for differences in signal from mediator-exposed samples and controls in Western and Northern blotting or differences in proliferation, a one-way ANOVA or paired Student's t-test was performed on the interval data generated by scanning of autoradiographs or storage phosphor scanning of hybridized membranes or on data calculated from measured counts per minute in proliferation assays. Where significant differences were noted, Dunnett's multiple comparisons procedure was employed to test for differences at particular mediator concentrations. Significance was accepted at a P value of <0.05 for all analyses.
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RESULTS |
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Differential mitogenic response to
PDGF-AA. PDGF isoforms appear to have distinct roles in
fibrotic responses. We therefore determined the response of
Thy-1 and
Thy-1+ fibroblasts to PDGF-AA and
PDGF-BB with
[3H]thymidine
incorporation as an indicator of proliferation. Figure 1 demonstrates that there is a
concentration-dependent proliferative response of
Thy-1
, but not of
Thy-1+, lung fibroblasts to
PDGF-AA, which is maximal at 5 ng/ml
(P = 0.00006 vs.
Thy-1+). In contrast, both cell
populations respond to PDGF-BB; however, the
Thy-1
cells have slightly
higher responses than the Thy-1+
fibroblasts (42 vs. 9% compared with response to 10% FBS,
P = 0.001 at 1 ng/ml of PDGF-BB; 104 vs. 69%, P = 0.01 at 5 ng/ml).
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Differential expression of PDGFR-
protein. The contrasting responses of
Thy-1
and
Thy-1+ cells to PDGF-AA suggested
a potential difference in PDGFR-
expression in these fibroblast
populations. Western blotting was therefore used to establish the
relative expression of PDGFR-
and PDGFR-
in
Thy-1
and
Thy-1+ cells (Fig.
2). In quiescent monolayers (0.4% FBS),
the PDGFR-
signal was, on average, 11 times higher in
Thy-1
than in
Thy-1+ cell lysates
(P = 0.01;
n = 3 monolayers). In
overexposed Western blots, a small amount of PDGFR-
protein is
visible in Thy-1+ lanes (data not
shown). The blots were stripped and reprobed with PDGFR-
antiserum,
and the two populations were found to express nearly equivalent
PDGFR-
levels (Fig. 2B),
demonstrating that differences in PDGFR-
are not based on unequal
protein loading. The levels of either receptor subunit did not change
significantly with the addition of 10% FBS, although serum withdrawal
caused a transient elevation in PDGFR-
in
Thy-1
, but not in
Thy-1+, cells, which was maximal
(~2-fold) at 6 h and returned to baseline at 24 h (data not shown).
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Differences in PDGFR-
mRNA. To verify the differential expression of
PDGFR-
, total RNA was isolated from
Thy-1
and
Thy-1+ cells and subjected to
Northern analysis. Figure 3 shows
representative autoradiographs of a membrane probed with
- and
-receptor cDNAs. Although both cell types have PDGFR-
message,
the levels in Thy-1
fibroblasts are on average 3.3-fold higher than in cells expressing Thy-1. Conversely, the level of PDGFR-
is slightly (1.4-fold) higher
in Thy-1+ cells.
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Immunofluorescent visualization of
PDGFRs. To determine whether differences in PDGFR-
level occur in mixed (unsorted) populations of fibroblasts and whether
there is significant variation in PDGFR-
expression within sorted
populations, monolayers were examined by digital confocal microscopy
after direct immunofluorescent staining with Thy-1 antibody and
indirect staining with PDGFR-
and PDGFR-
antisera. Representative
photomicrographs are shown in Fig. 4.
Appropriate control antibodies demonstrate negligible levels of
nonspecific staining (Fig. 4A). In
primary, unsorted Lewis rat lung fibroblast monolayers, Thy-1 staining
(FITC-labeled antibody; Fig. 4B,
green) was confined to a subset of the cells. This surface-staining
pattern with punctate areas of increased intensity is characteristic of
many glycosylphosphatidylinositol-linked surface molecules (26).
PDGFR-
staining (Texas Red-X-labeled secondary antibody; Fig. 4,
red) is also seen only in a subset of cells in a mixed population, and
there is almost no colocalization of red and green fluorescence except
in areas where cells overlap. Consistent with Fig. 2, sorted
Thy-1+ fibroblasts (Fig. 4,
D and
F) show staining with anti-Thy-1, very little staining for PDGFR-
, and uniform staining for PDGFR-
. Thy-1
fibroblasts (Fig. 4,
C and
E) do not stain with anti-Thy-1, and most express both PDGF receptors. A combination of surface and perinuclear staining is observed for both PDGFR-
and PDGFR-
, consistent with published work on PDGFR immunostaining (25). In
subconfluent, sorted monolayers, cells staining positive for Thy-1
and/or PDGFR-
were counted in eight separate fields on two separate
coverslips for each cell type. Sixty-six percent of
Thy-1
cells stained
positive for PDGFR-
vs. 0% of
Thy-1+ cells
(P = 0.0007; data not shown). Although
not all Thy-1
cells
demonstrate PDGFR-
immunostaining, it is clear that the differences
seen in Western blotting were not due to very high levels of PDGFR-
expression in only a small subset of
Thy-1
cells. The
visualization of PDGFR-
staining in an unsorted population of rat
lung fibroblasts (Fig. 4B) shows
that differential PDGFR-
expression is not induced by culturing
Thy-1
and
Thy-1+ cells separately.
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Differences in intracellular signaling after
stimulation with PDGF-AA. We measured
c-myc mRNA as an indicator of
intracellular signaling activated on stimulation of the cell
populations with PDGF isoforms. Expression of the early-response gene
c-myc is characteristic of entry into
the cell cycle (11). We exposed quiescent fibroblast monolayers
(n = 3) to PDGF-AA or -BB in SFM and
prepared total RNA, which was then used to prepare Northern blots that
were hybridized with a murine c-myc
probe. Initial experiments to determine the time course demonstrated
rapid induction, with elevated c-myc
levels at 15 min, peaking at 30 min, and remaining elevated at 1 h
(data not shown). Figure 5 demonstrates
increased c-myc mRNA in
Thy-1, but not in
Thy-1+, cells exposed to PDGF-AA
(2 and 5 ng/ml) and -BB (1 and 2 ng/ml).
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DISCUSSION |
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The data presented demonstrate a clear difference in the proliferative
response of Thy-1 and
Thy-1+ rat lung fibroblasts to
PDGF-AA, a growth factor that is central to parenchymal remodeling in
the lung. Using a combination of experimental approaches, we have shown
that the preferential proliferation of lung fibroblasts lacking surface
Thy-1 is based on increased expression of the PDGFR-
subunit. These
findings demonstrate an important and relevant functional difference
between these fibroblast subsets and support the independent roles of
these two populations in fibrotic responses.
The divergent proliferative responses of the two subpopulations is
striking in that there is no discernible response of the Thy-1+ cells to PDGF-AA. This is
not based on an inability of
Thy-1+ fibroblasts to respond to a
proliferative stimulus because cells respond normally to PDGF-BB. The
lack of any proliferative response to the AA isoform despite the
presence of a small amount of -receptor in
Thy-1+ cells may indicate that
there is a threshold of receptor expression necessary for signaling
(22). The slightly stronger response of the
Thy-1
cells to the BB
isoform compared with that of the
Thy-1+ cells at 1 and 5 ng/ml is
consistent with the known ability of PDGF-BB to signal through both
- and
-receptors. Interestingly, in terms of
c-myc induction, limited PDGF response
was seen with either isotype in
Thy-1+ cells. Others (6) have
demonstrated that there are differences in the sequence of
intracellular signals generated through activation of the two PDGFR
subtypes, suggesting that PDGF-AA and -BB initiate cell cycle traverse
via separate mechanisms. Our data from immunoblotting, Northern
blotting, and immunofluoresence indicate that the differences we
describe in signaling are likely due to differing levels of
-receptor expression.
A subset of fibroblasts uniquely responsive to PDGF-AA may play an
important role in pulmonary fibrogenesis. Fibroblasts isolated from
lung fibrotic lesions have an increased proliferative capacity but are
not transformed (4). This likely results from autocrine growth
signaling in which the PDGFR- plays an important role. A number of
other clinical and experimental conditions have been described in which
fibroblasts respond differentially to the PDGF-AA isoform, including
scleroderma lung and skin lesions, keloid scars, rodent
bleomycin-induced lung injury, and asbestos-induced mitogenesis (9, 12,
17, 24). Increased expression of PDGFR-
has been demonstrated in all
of these examples, consistent with our findings in
Thy-1
cells. The
-receptor plays an important role in regulating cellular responses
to PDGF. Not only is it capable of eliciting signals in response to all
three PDGF isoforms, but the PDGFR-
also has been shown to modulate
activities mediated by the PDGFR-
(5, 8). Notably, the latter study
demonstrated functional PDGFR-
responses in only a subset of normal
fibroblasts (8). To date, no work has correlated the expression of
PDGFR-
to that of Thy-1.
It remains to be determined whether the presence or absence of the
Thy-1 molecule influences the unique characteristics of the two
populations described herein or whether Thy-1 is a "surrogate" marker for subpopulations that differ in other significant respects. In
NIH/3T3 fibroblasts, loss of Thy-1 surface expression on transformation by viral oncoproteins is associated with anchorage-independent growth
and increased major histocompatibility complex II expression (1, 23).
Thus it appears that the presence or absence of Thy-1 impacts
significantly on the ability of mesenchymal cells to interact with
matrix, regulate proliferation, and participate in immune or
inflammatory responses. It has been demonstrated that disruption of
cell-matrix interactions may be more important than cytokine/growth
factor signals in regulating PDGFR expression (2). Enhanced expression
of PDGFR- has been described under anchorage-independent conditions
in normal rat kidney fibroblasts, conditions in which only the AA and
AB isoforms (which signal through the
-receptor) are mitogenic (28).
After injury, there is a marked disruption of normal tissue matrices.
Such a change in "cellular context" may create an environment
into which PDGFR-
-positive Thy-1
fibroblasts are
recruited and induced to proliferate, thus positioning them to direct
subsequent repair or scarring. The potential of PDGFR-
-positive
fibroblasts to alter lung architecture has been underscored recently by
a study (3) of the PDGF-AA null mouse, a homozygous lethal
mutation that lacks normal pulmonary alveolar development
related to loss of PDGFR-
-positive myofibroblasts.
We have demonstrated that expression of PDGFR- in rat lung
fibroblasts is inversely correlated with expression of Thy-1. Whether
via its effect on cell cytoskeletal arrangement and matrix interaction,
regulation of cytokine and growth factor profiles, or mechanisms yet to
be elucidated, Thy-1 expression seems to regulate cellular properties
that are central to fibrogenic responses. Further characterization of
the role of fibroblast Thy-1 display will likely shed important new
light on the pathophysiology of fibrosis.
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ACKNOWLEDGEMENTS |
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We thank Shawn Williams for invaluable assistance with digital confocal microscopy and image processing and Simon Jones and Sandra Hagood for critical reading of the manuscript.
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FOOTNOTES |
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This work was supported by National Heart, Lung, and Blood Institute Grants HL-03239 (to J. S. Hagood) and HL-03374 (to J. A. Lasky) and American Lung Association Grant RG183N (to J. A. Lasky).
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: J. S. Hagood, 1918 University Blvd, 689 MCLM, Birmingham, AL 35294 (E-mail: jhagood{at}peds.uab.edu).
Received 15 December 1998; accepted in final form 25 February 1999.
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