Department of Anatomy, Physiological Sciences, and Radiology, College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina 27606
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ABSTRACT |
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Fibroblast growth factor
(FGF)-2, which stimulates DNA synthesis by type II cells in the lung,
has been shown to be regulated by transforming growth factor
(TGF)-1, an important inflammatory cytokine, in vascular epithelium.
The goal of this study was to determine if FGF-2 production by alveolar
type II cells is modulated by TGF-
1 or FGF-1, which also stimulates
DNA synthesis by type II cells. Isolated rat type II cells were exposed
to 0-40 ng/ml of TGF-
1 or 0-500 ng/ml of FGF-1 in
serum-free medium for 1-5 days. With a specific
immunoassay, significant increases of FGF-2 protein in type II cell
lysates to levels above those in control cells were achieved after 1 day of exposure to 100 ng/ml of FGF-1 and after 3 days of treatment
with 8 ng/ml of TGF-
1. Similarly, transcripts for FGF-2 were
dramatically increased above those in control cells with TGF-
1 or
FGF-1, as were those for FGF receptor-1. These results demonstrate
important regulatory links between FGF-2 and both TGF-
1 and FGF-1 in
the alveolar epithelium that could contribute to the regulation of
normal cell turnover, development, and the repair processes after
injury in the lung.
basic fibroblast growth factor; acidic fibroblast growth factor; alveolar epithelium; alveolar injury; pulmonary fibrosis; transforming growth factor
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INTRODUCTION |
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NORMAL LUNG FUNCTION
depends on an intact alveolar epithelium, which is composed of alveolar
type I and type II cells. The latter, in addition to regulating of
surfactant metabolism and ion transport, functions to maintain stable
cell populations under homeostatic conditions and to restore the
integrity of the alveolar epithelium after alveolar epithelial damage
by their capacity to proliferate and differentiate (1, 7).
Numerous growth factors, including members of the fibroblast growth
factor (FGF) family (17, 20), hepatocyte growth factor
(20), transforming growth factor (TGF)-
(25), and epidermal growth factor (21, 25),
have been found to stimulate DNA synthesis by type II cells or growth
of fetal rat pulmonary epithelium. Among these, FGF-2 is particularly
interesting because it has multiple biological activities in vivo and
in vitro, including angiogenesis, mitogenesis, cellular
differentiation, and repair of tissue injury (4). In the
lung, FGF-2 has been immunolocalized in isolated alveolar type II cells
(29) and in basement membranes including those in the
alveolus (28). Little is known of its regulation in the lung, but its spectrum of effects and localization make it a likely focus for autocrine/paracrine linkage(s) between the growth factors produced by alveolar macrophages, alveolar epithelium, and fibroblasts and the modulation of type II cell behavior.
TGF- and its isoforms have been shown to play multiple roles in
regulating growth and differentiation in various cells and tissues,
especially after injury (23). In the pulmonary alveolus, TGF-
has been shown to be produced by local activated macrophages, and increased levels have been detected in lung lavage fluid after lung
injury (32). A recent study (18) showed that
TGF-
stimulated the production of selected extracellular matrix
(ECM) components by alveolar type II cells. In lung fibroblasts,
TGF-
alone can increase expression of FGF-2 transcripts but not of
protein (12). A specific relationship between TGF-
and
FGF-2 has not been established in alveolar epithelial cells.
FGF-1 and -2 are closely related growth factors with 55% homology (2). They both have a broad spectrum of tissue distribution, target cells, and biological activities. The roles of FGF-1 and -2 are well understood (15), and a previous study (17) has demonstrated that both can stimulate DNA synthesis in isolated type II cells. A recent study (13) showed that exogenous FGF-2 can stimulate FGF-1 production in retinal pigmented epithelial cells. FGF-1 can induce gene expression of platelet-derived growth factor and nerve growth factor genes (8) but has yet to be linked to any regulation of FGF-2. The potential for growth factor interactions in the distal lung is further supported by the immunodetection of FGF-1 in type II cells (28) and macrophages (3) in normal lungs and by the expression of FGF-1 protein (3, 28) and mRNA transcripts (3) in alveolar epithelial cells and macrophages after injury.
Because TGF-1 and FGF-1 are found in injured lung (3,
32) and FGF-2 is a stimulant of DNA synthesis in isolated type II cells, we postulated that TGF-
1 and/or FGF-1 could act indirectly in epithelial repair through the modulation of FGF-2 production in type
II cells. To test this hypothesis, primary cultures of isolated adult
rat alveolar type II cells were treated with different concentrations
of TGF-
1 and/or FGF-1 at selected times postisolation, and the
changes in FGF-2 protein expression and mRNA were examined. The results
indicated that FGF-2 expression by isolated rat type II cells can be
modulated by TGF-
1 and FGF-1.
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MATERIALS AND METHODS |
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Cell Preparation
Briefly, rat type II pneumocytes were isolated from pathogen-free, 200- to 250-g Fischer CDF rats (Charles River Laboratories, Wilmington, MA) according to the procedure of Dobbs (7) with some minor modifications (31). Isolated cells were suspended in a concentration of 100,000 cells/ml with DMEM supplemented with 10% fetal bovine serum, 100 U/ml of penicillin, 100 µg/ml of streptomycin, and 50 µg/ml of gentamicin.Cell Culture and Treatment
Standard culture dishes, 24-well plates, or chamber slides (Nunc, Naperville, IL) were coated with 0.06 µg/mm2 of type I collagen (Sigma, St. Louis, MO) as described previously (31). The suspended cells were seeded at a density of 2 × 104 cells/cm2 and allowed to attach and spread overnight at 37°C in 5% CO2. After 24 h, the attached cells were washed once with DMEM and exposed to serum-free, hormonally defined medium with and without treatment with 100 ng FGF-1/ml, 8 ng TGF-FGF-2 Detection and Protein Assay
Conditioned culture medium samples were collected directly from the cell culture dishes. The attached cells were harvested by scraping followed by low-speed centrifugation. The cell pellet from each dish was washed once with 1× PBS and resuspended with 100 µl of 50 mM Tris · HCl (pH 7.4), 1 mM MgCl2, 0.85% NaCl, and 1.5% NP-40 lysing buffer. Insoluble cellular debris was removed by high-speed centrifugation (12,000 g for 10 min). The resulting cell lysate and conditioned culture medium samples were stored at[3H]Thymidine-DNA Incorporation Assay
After 24 and 48 h of culture in [3H]thymidine-containing medium, treated cells were washed and processed as described previously (22). Radioactivity of the cell lysates was measured with an LKB 1219 scintillation counter (Wallac, Turku, Finland). Results were analyzed as previously described (31).RT-PCR
RNA preparation. Type II cells grown in culture dishes were quickly washed once with cold 1× PBS, lysed, and extracted with TRI Reagent (Molecular Research Center, Cincinnati, OH) according to the instructions of the manufacturer. After extraction, RNA samples were treated with RQ1 DNase (Promega, Madison, WI) at 37°C for 30 min to remove residual DNA and extracted again with TRI Reagent. The RNA was quantified by GeneQuant (Pharmacia, Piscataway, NJ), and its integrity was confirmed by electrophoresis on a denatured agarose gel containing formaldehyde (27).
Preparation of primers and competitive template standards.
Based on gene sequences of rat -actin, FGF-2, FGF receptor (FGFR)-1,
and FGFR-2, the paired conventional forward and reverse primers and
competitive template (CT) primers (Table
1) were selected with Oligo software and
synthesized by Retrogen (San Diego, CA). CT primers were designed
according to previous strategies (5a). Each was ~40
nucleotides in length, with ~20 nucleotides in the 5'-end identical
to the corresponding conventional reverse primers and ~20 nucleotides
in the 3'-end corresponding to an internal region of the opposite
strand of the PCR target sequence. CT standards were synthesized and
quantified as described by others (34). Briefly, the
forward primer and CT primer of each target gene were used to amplify
CT standards from cDNA known to express the relevant gene by standard
PCR. Generated CT standards were purified with Wizard PCR Preps
(Promega), quantified by GeneQuant and gel electrophoresis, and
compared with pGEM size markers (Promega). The molarity of each CT
standard was calculated as described by Willey et al.
(34). Because the 3' sequence of the designed CT primer
corresponded to internal regions of the conventional PCR target
sequence and the 5'-end to the conventional reverse primer sequence,
the synthesized CT standards were shorter than the conventional PCR
target fragments and could be further amplified by PCR with
conventional forward-reverse primers. Therefore, they were
competitively amplified with native genes when added to a conventional
PCR, and the copy number of native genes was determined according to
the amount of CT standard added and the ratio of native gene product
and CT product (34).
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RT reaction and quantitative PCR assay.
Two micrograms of total RNA from each sample were used for cDNA
synthesis. The RT reaction was carried out as described previously (37) with random hexamer primers and AMV reverse
transcriptase (Promega). RT products were stored at 20°C for later use.
Immunohistochemical Analysis of FGF-2
Type II cells on culture slides were fixed with 5% formaldehyde-PBS for 10 min at room temperature. After they were washed, the slides were reacted with 2% horse serum-PBS for 30 min at room temperature and then incubated with rabbit anti-FGF-2 polyclonal antibody (1:500; Sigma) at 4°C overnight. As a control, the same concentration of normal rabbit serum was used. The following day, the cells were washed with PBS, and FGF-2 was detected with a fluorescein-labeled anti-rabbit antiserum (2 µg/ml in PBS; Vector Laboratories, Burlingame, CA). Staining was observed and recorded with an Olympus Optimax fluorescent microscope.Data Analysis
All experiments were performed in triplicate or quadruplicate and were repeated at least three times. Statistical means ± SE were developed for the various treatment groups. Data were also expressed as percent of control (untreated or growth factor-free medium) so that %control = (mean value of treatment/mean value of untreated control) × 100. Each experimental group was compared with its control group by paired t-test. Differences were considered significant at P < 0.05. ![]() |
RESULTS |
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Type II Cell Culture and Its FGF-2 Production
Twenty-four hours after being plated, the attached cells were observed as isolated cells or as clusters of up to 10 cells (at ~30-40% confluence). Approximately 90-95% of the cells were confirmed to be type II alveolar cells as indicated by the abundance of typical lamellar bodies in their cytoplasm. After a change to serum-free medium, cells gradually grew to 50-60% confluence by 24 h (day 1) and 90-100% confluence by 72 h (day 3).Immunofluorescent localization of FGF-2 protein in type II cells was
proven in both serum-free and growth factor-treated type II cells,
although FGF-1- and TGF-1-treated cells tended to have a higher
degree of reactivity (Fig. 1,
a-c). Immunofluorescence was not detectable in
specimens treated with normal serum (Fig. 1d). FGF-2 protein
was also confirmed in cell lysates, but not in conditioned medium, by
ELISA assay, which demonstrated that type II cells cultured in
serum-free medium without growth factor stimulation produce ~40 pg
FGF-2/ mg protein.
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Modulation of FGF-2 Production
After preliminary tests, dose-response experiments were conducted for 24 h (1 day) for FGF-1 and 72 h (3 days) for TGF-Both cell lysate samples and conditioned culture medium samples were
analyzed for FGF-2 content by ELISA. Assay results of type II cell
lysate samples showed that both TGF-1 and FGF-1 treatments
stimulated FGF-2 production in isolated rat type II cells
in a dose-dependent fashion (Figs. 2 and
3). The maximum response to
TGF-
1 was achieved with 8 ng/ml on day 3 (Fig. 2) and
with 100~500 ng/ml for FGF-1 on day 1 (Fig. 3). Dose
dependency was demonstrated by the lack of an effect on FGF-2 content
in cultures that received <0.5 ng/ml of TGF-
1 or 1 ng/ml of FGF-1, whereas cultures that received 0.5-8 ng/ml of TGF-
1 and
10-100 ng/ml of FGF-1 showed sharp increases in FGF-2 protein in
type II cells. A time course study demonstrated that treatment with 8 ng TGF-
1/ml increased FGF-2 to 76.7 ± 10.8 pg/mg protein on day 3 (181% of untreated control cells; Fig.
4) and that FGF-2 remained elevated on
day 5. FGF-2 production by cells treated with 100 ng/ml of
FGF-1 peaked on day 1 with 197 ± 34 pg/mg protein (537% of untreated control cells) and then steadily declined
thereafter as seen on days 3 and 5 (Fig. 4).
However, growth factor-treated type II cells still produced
substantially greater amounts of FGF-2 than nontreated cells on
day 5 (FGF-1 treatment) and on day 7 (TGF-
1
treatment; data not shown). On day 1, the FGF-2 levels were
40 ± 6.3 pg/mg protein for TGF-
1-treated cells and 36.7 ± 6.4 pg/mg protein for untreated control cells, respectively (Fig.
4). When 8 ng of TGF-
1 and 100 ng of FGF-1 were combined to treat
type II cells, the highest yield of FGF-2 was 188 ± 40 pg/mg
protein (day 1), which was not statistically different from FGF-1 alone but was higher than TGF-
1 alone (Fig. 4).
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In all conditioned culture medium samples, FGF-2 was not detectable by the ELISA assay used. This could have been a result of either the sensitivity of the ELISA kit (0.5 pg FGF-2/ml minimum) or the low level of free FGF-2 present in the medium.
The biological effects of TGF-1 and FGF-1 were further
examined by changes in total protein amount and thymidine incorporation into DNA. Results indicated substantial increases in thymidine incorporation (189% of control level on day 1 and 274% on
day 2 for TGF-
1; 367% of control level on day
1 and 671% on day 2 for FGF-2; Fig.
5). Similarly, total cellular protein was
increased (145% of control level for TGF-
1 and 173% of control
level for FGF-1 on day 2; Fig.
6).
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TGF-1 and FGF-1 Modulation of FGF-2 and FGFR-1 Gene
Expression
Quantitative RT-PCR analysis demonstrated that both TGF-1 and FGF-1
treatments can substantially increase the expression of FGF-2 mRNA and
FGFR-1 mRNA in type II cells (Figs. 7 and
8). The response to both growth
factors was very rapid at the mRNA level. The expression of FGFR-1 and
FGF-2 mRNAs peaked within 24 h after treatment. By comparison,
FGFR-1 mRNA expression peaked earlier than FGF-2 mRNA and declined to
normal levels after 24 h. However, FGF-2 mRNA levels peaked at
24 h and returned to normal levels after 72 h.
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As shown in Figs. 7 and 8, the largest increases in FGF-2 mRNA were
940 ± 100% of untreated control level for TGF-1 and
1,310 ± 67% for FGF-1 after 1 day. The largest increase of
FGFR-1 mRNA was ~500% in both growth factor treatment groups after
12 h of treatment. However, no significant changes in FGFR-2 mRNA
were observed in either group of growth factor-treated cells (data not shown).
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DISCUSSION |
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Type II cell proliferation and differentiation are crucial processes in normal alveolar epithelial turnover, development, and repair after injury. The events that modulate these processes are poorly understood but likely involve a variety of key growth factors produced by type II and other lung cells. Previous studies by Sannes and colleagues (28, 29) suggested that FGF-2, which stimulates thymidine incorporation by type II cells, is present in both isolated type II cells and the basement membranes of the lung. The purpose of this study was to define specific conditions that modify FGF-2 production by type II cells.
TGF- is a multifunctional factor that plays a central role in the
regulation of cell growth and differentiation, having either stimulatory or inhibitory effects depending on the particular cellular
context of its action. It has been shown to be mitogenic for
osteoblasts and Schwann cells but to inhibit proliferation of
lymphocytes and endothelial cells (23). Previous reports indicated that TGF-
inhibited DNA synthesis in neonatal and adult rabbit type II cells (25) but not in rat type II cells
(33). TGF-
has been shown to be linked to FGF-2 in two
distinct ways: 1) FGF-2 has been shown to activate latent
TGF-
(tissue bound) through its regulation of plasminogen activator
and its inhibitor in vascular endothelium (9) and
2) TGF-
has been demonstrated to directly increase the
expression of FGF-2 in lung fibroblasts (12). Such a
relationship could be highly relevant to the alveolar epithelium in
which the progenitor cells (i.e., type II cells) respond to FGF-2 as a
mitogen. Previous studies have demonstrated that TGF-
stimulated
biosynthesis of ECM in alveolar type II cells (18) and the
gene expression of FGF-2 in lung cells (12). Given this
profile, we reasoned that TGF-
could act to modulate other type II
cell responses to produce certain growth factors. FGF-2 was a
compelling target because of its known effects on DNA synthesis in type
II cells, which is a key element in the reepithelialization of the
alveolus after injury (1, 14). Accordingly, we studied the
effects of TGF-
1 and FGF-1 on the production of FGF-2 and its
receptors in vitro by examining the expression of FGF-2 and FGFRs
through the detection of their specific mRNA and FGF-2 protein. The
results indicated that alveolar type II cells possess the components of
an FGF-2 autocrine loop (i.e., expression of FGF-2 and its receptor).
Such a loop could be initiated by exogenous growth factors such as
exposure to FGF-1 and/or TGF-
1, which could, in turn, lead to an
increase in the mRNA level of FGF-2 and its FGFR-1. Under the
conditions used in the present study, both TGF-
1 and FGF-1 were
found to stimulate DNA synthesis as reflected in thymidine uptake and
protein production and clearly acted to upregulate FGF-2 and
FGFR-1 mRNA expression. Although it is not clear how much of
the effect on DNA synthesis is direct or indirect (e.g., effects of
FGF-2), the data support the notion that TGF-
1 is capable of
influencing or modulating epithelial proliferative and reparative
events after injury. This may be a key relationship that, in effect,
defines the resolution of repair in alveolar regions. Furthermore,
FGF-2 has been immunolocalized in the alveolar basement membrane (ABM)
(28), and its detection is diminished 7 days after
oxygen-induced fibrosis (29). Presumably this is due to
its release from the ABM by proteolytic events attendant to
inflammation (26). Such release could be expected to be
important in initial reepithelialization events in the alveolus as type
II cells divide and differentiate into type I cells. As reparative
processes continue, the presence of TGF-
in the alveolar environment
could act on type II cells in two clearly distinctive ways, both
involving biosynthesis of FGF-2 and FGFR-1: 1) facilitate end-stage repair by stimulating further reepithelialization and 2) stimulate biosynthesis of new ABM, including FGF-2 and
other connective tissue components (30). In both cases,
TGF-
would be acting to return alveolar structure-function
relationships to normal.
Such mechanisms could be further promoted by additional growth factors known to be present and active in alveolar regions, such as FGF-1, which has a broad range of biological activity that includes modulation of growth factor production (8). However, its effect on members of its own growth factor family has not been widely appreciated. The present study showed that FGF-1 can modulate the production of FGF-2 protein and its FGFR-1 gene. These results, combined with previous data (13), indicate that there is a potentially important regulatory link between FGF-1 and -2, which demonstrates that growth factors in the same family can regulate each other to promote biosynthetic activity and proliferation. In this way, FGF-1 could act to amplify its own activity through upregulation of a powerful related mitogen and its receptors.
FGFs interact with their target cells via a dual-receptor system
consisting of a cell surface heparan sulfate proteoglycan (low-affinity
receptor) and a transmembrane receptor linked to tyrosine kinase
(high-affinity receptor) (11). There are at least four
types of receptors (FGFR-1 to -4) with intrinsic tyrosine kinase
activity, all derived from separate genes (11). The
mechanism for differential expression of these receptor genes has not
been clearly defined. FGF-2 has been reported to bind to both FGFR-1 and -2 with high affinity (6). Endogenous FGF-1 has been
shown to stimulate FGFR-1 gene expression in retinal pigmented
epithelium (13). In human liver myofibroblasts, TGF-1
treatment stimulates both FGFR-1 and -2 gene transcription
(24). However, in the present study, the FGFR-1 message
level was increased with TGF-
1 or FGF-1 treatment. This difference
suggested the possibility of a FGFR preference in different cell types
and that in type II cells, FGFR-1 may play a more important role in
FGF-2 binding and signal transduction. In support of this notion, whole
animal studies on rats exposed to 85% oxygen indicated that expression of both FGF-2 and FGFR-1 was elevated in lungs after 6 days of exposure
(5). Because both TGF-
1 and FGF-1 are known to be elevated in models of alveolar damage and in injured lung (3, 32), it is possible that their presence, at least in part,
accounts for the increased expression of FGF-2 mRNA and protein and
FGFR-1 mRNA.
Many growth factors, including those of the FGF family, bind to components of the ECM (15). When tissue environments are damaged or altered during inflammatory events, these latent growth factors can be proteolytically liberated from the ECM and thus influence local proliferative and reparative events (9). It is not clear from the results of the present study whether the biosynthesized FGF-2 is actually released from type II cells. The FGF-2 produced by rat type II cells was only found in cell lysates (which would include ECM) but not in conditioned medium. This could be due to the low level of FGF-2 in the medium or the limitations of the assay (0.5 pg FGF-2/ml minimum). It could also be a result of the fact that at least some of the detectable FGF-2 was associated with biosynthesized ECM. This issue was not addressed by the current work but could be resolved by immunolocalization of FGF-2 at the level of the confocal or electron microscopy or by appropriate biosynthetic labeling studies.
The increase in DNA synthesis observed in type II cells treated with
TGF-1 was somewhat surprising, given the results of previously
published studies (25, 33). It has been demonstrated that
manipulation of experimental conditions can elicit dramatically different responses from isolated type II cells (25). For
example, insulin-like growth factor I increased type II cell
numbers when the cells were plated at high density but had no effect at
lower density. FGF-1, on the other hand, has both stimulatory and
inhibitory effects on human type II cells depending on the serum
concentration in the culture medium. In addition to the obvious species
and age differences when studies are compared, the more subtle
differences such as cell density, serum concentration, or the matrix
substrata on which the cells are cultured can greatly affect which
growth factors elicit a type II cell response and its magnitude
(25). It is possible that the lower cell density
cultures (2 × 104 cells/cm2) used in the
present study were necessary to achieve the result of DNA synthesis in
adult rat type II cells, whereas the higher density cell cultures of
neonatal rabbit type II cells (25) did not. In contrast,
low cell density alone may not be sufficient to facilitate a
TGF-
1-stimulated DNA synthesis as evidenced in earlier work
(25) with lower cell densities than those employed here.
Interestingly, Ryan et al. (25) used uncoated
plates in their culture conditions, so it could be argued that
substrata could play an equally important role. Although this
discussion does not address the distinction between DNA synthesis and
cell proliferation in type II cells, it does emphasize the importance of those conditions, probably multiple in nature, which favor the
capacity of TGF-
1 to stimulate FGF-2 production, which, in turn,
could account for the observed increase in DNA synthesis. In vivo, this
could translate into modest but potentially important increases in type
II cell numbers during repair after injury.
In conclusion, the data presented here demonstrate that TGF-1 and
FGF-1 stimulate gene and protein expression of FGF-2 and its receptor
in type II cells in vitro. However, in vivo, the presence of TGF-
1
and FGF-1 after injury or during an inflammatory event could modulate
type II cells to increase the expression of FGF-2 and its receptor,
which in turn, could act as an autocrine or paracrine growth factor to
increase type II cell numbers. Such a relationship could constitute a
significant mechanism for influencing proliferative and biosynthetic
events that are critical for the proper resolution of tissue injury in
the alveolus.
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ACKNOWLEDGEMENTS |
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We thank Dr. Pi-Wan Cheng (University of Nebraska Medical Center, Omaha, NE) for helpful discussions.
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FOOTNOTES |
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This study was supported by National Heart, Lung, and Blood Institute Grant HL-44497 and a grant from the state of North Carolina.
Address for reprint requests and other correspondence: P. L. Sannes, Dept. of Anatomy, Physiological Sciences, and Radiology, College of Veterinary Medicine, North Carolina State Univ., 4700 Hillsborough St., Raleigh, NC 27606 (E-mail: philip_sannes{at}ncsu.edu).
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 10 May 1999; accepted in final form 15 June 2000.
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