Vascular Biology |
From the A.I. Virtanen Institute (P.L., S.Y.-H.) and the Department of Medicine (S.Y.-H.), University of Kuopio, Kuopio, Finland, and the Kumamoto University School of Medicine (M.T.), Kumamoto, Japan.
Correspondence to Seppo Ylä-Herttuala, MD, PhD, A.I. Virtanen Institute, University of Kuopio, PO Box 1627, Neulaniementie 2, FIN-70211 Kuopio, Finland. E-mail Seppo.Ylaherttuala{at}uku.fi
| Abstract |
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Key Words: atherogenesis gene transfer retrovirus oxidized LDL
| Introduction |
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Although the importance of SR-A activity in macrophages has been clearly established, the role of SR-A in nonmacrophage cell lines remains unclear. Although cultured rabbit fibroblasts and smooth muscle cells (SMCs) express SR-A activity on stimulation with phorbol esters,13 no or very limited SR-A expression has been detected in SMCs in human, Watanabe heritable hyperlipidemic rabbit, or cholesterol-fed New Zealand White rabbit atherosclerotic lesions.6 8 9 Also, SR-A expression in atherosclerotic lesions of apoE3-Leiden transgenic mice has been detected in macrophages but not in SMCs.10 However, in cholesterol-fed New Zealand White rabbits, in which the arterial wall was damaged by balloon denudation, SR-A immunoreactivity was detected in a small number of proliferating, intimal SMCs.14 15 Whether expression of the SR-A in SMCs and fibroblasts in vivo leads to foam cell formation and other proatherogenic changes remains unknown.
Retrovirus-mediated gene transfer can be used for stable transfection of eukaryotic cell lines in which gene expression is driven by a retroviral long-terminal repeat (LTR) promoter.16 Cell lines transfected by retroviral techniques have proved valuable for the analysis of transgene functions because they can lead to long-lasting, high-level expression of the transgene that is not regulated by natural promoters. Retroviruses also open up possibilities for in vivo gene transfer with prolonged transgene expression in target tissues.16
The aim of the study was to test the hypothesis that SR-A activity leads to proatherogenic changes in nonmacrophage cell lines. It was found that SR-A expression leads to foam cell formation, enhanced cell death, and apoptosis in stably transfected SMCs and fibroblasts.
| Methods |
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2 and PA317 mouse fibroblast cell lines,20 21 rat
fibroblast cell line F209,17 human embryonal kidney 293GP
cell line,22 and rabbit aortic SMC line RAASMC
(established in our laboratory from a newborn New Zealand White rabbit
aortic explant19 ) were used in the study. Cells were
verified as SMCs by
-actin immunostaining. Cells
were maintained in Dulbeccos modified Eagles medium (DMEM, Gibco)
at 37°C in a 5% CO2 atmosphere. The growth
medium was supplemented with 10% FCS, 100 U/mL penicillin, 100 µg/mL
streptomycin, and 2 mmol/L L-glutamine (Gibco).
Neomycin-resistant cells were maintained in the same medium
containing neomycin analogue G418 (400 µg/mL, Sigma). Peritoneal
macrophages were harvested from the peritoneal cavity of
8-week-old male mice.23 Macrophages were cultured
in RPMI containing 10% FBS and used for the experiments within 36
hours after isolation.
Production of Retroviruses and Stable Cell Lines
MMLV Retroviruses
For MMLV retroviruses, the ecotropic packaging cell line
220 was transfected with retroviral plasmids by using a
standard calcium phosphate precipitation method. Viruses were harvested
from
2 cells after 48 hours and used for infection of amphotropic
PA317 packaging cells21 as follows. On day 0, PA317 cells
were plated at 5x105 cells/10-cm dish. On day 1,
monolayers were incubated for 16 hours with filtered (0.45 µm)
2 supernatants in the presence of 8 µg/mL Polybrene (Sigma). On
day 2, the medium was replaced with G418-containing medium. After 10 to
12 days of selection, G418-resistant clones were isolated and
expanded.19
Pseudotyped Retroviruses
Pseudotyped retroviral vector pLSRNL containing the G
glycoprotein of vesicular stomatitis virus (VSV-G) was
produced in human kidney 293GP cells cotransfected with MMLV
gag and pol genes.24 293GP
cells were transfected by using a standard calcium phosphate
coprecipitation with 50 µg of pLSRNL DNA. Stable clones expressing
SR-A were selected by using 600 µg/mL geneticin. A stable producer
clone was selected for the production of pseudotyped pLSRNL
virus. To generate pseudotyped virus, the producer clone was
transfected with pHCMV-G expression plasmid as
described.24 Fresh medium containing 10% FBS was changed
after 8 to 12 hours of transfection. Pseudotyped viruses generated from
the transfected cells were collected 24, 48, and 72 hours after the
change of medium. Collected supernatants were filtered (0.45 µm)
and subjected to ultracentrifugation in a Beckman SW-28
rotor at 50 000g (25 000 rpm) at 4°C for 120 minutes.
The pellets were resuspended overnight at 4°C in 200 µL of 0.1x
Hanks/1% sucrose in PBS and combined before titration and
storage.25 The concentrated viruses containing either
SR-A or LacZ were used to transduce RAASMCs. A multiplicity of
infection of 10 to 100 viruses was used to transduce RAASMCs in the
presence of 8 µg/mL Polybrene for 16 hours. After 48 hours the cells
were trypsinized, and selection was started with 600 µg/mL geneticin.
After 10 to 12 days of selection, G418-resistant SMC clones
were isolated and expanded.19
Determination of Virus Titers
MMLV- and VSV-Gpseudotyped viruses were titrated on F209 cells
in the presence of Polybrene (8 µg/mL).19 Titers of the
best MMLV producer clones for pLSRNL, pLSrRNL, and
pLZRNL were 7x104, 2x103,
and 8x104 colony-forming units (cfu)/mL,
respectively. Titers of the unconcentrated, pseudotyped viruses pLSRNL
and pLZRNL were 104 to 106
cfu/mL. After concentration the titers were 106
to 107 cfu/mL.
Northern and Southern Blot Analyses
Poly(A)+ mRNA was isolated from the clones by an SDS/proteinase
K method and used for Northern blot analysis.26
Hybridizations were carried out overnight at 42°C with
106 counts per minute per milliliter of
random-primed 32P-labeled bovine SR-A probe,
neomycin probe, or ß-actin probe.26 Genomic DNA was
isolated from stably transfected clones and from untransduced control
cells by proteinase K treatment for Southern blot
analysis.27 Genomic DNA (30 µg) was digested
with SduI, BspTI, and Mva1269I
restriction enzymes (Fermantas, Finnzymes), and the fragments were
separated in 0.8% agarose gel, blotted onto a nylon membrane, and
hybridized with the SR-A probe. Autoradiography was
used for signal detection.
Immunostaining
Cells were plated on glass chamber slides and were grown in 10%
lipid-deficient serum/DMEM. Cells were fixed with 4%
paraformaldehyde for 10 minutes at 20°C,
permeabilized in 0.2% Triton X-100/PBS for 10 minutes,
and washed with PBS. Thereafter, the cells were blocked in 3% BSA/PBS
for 2 hours and incubated with SRKO 4 antibody15 for 1
hour and subsequent incubation with biotinylated horse anti-mouse IgG
(Vector Laboratories) for an additional 30 minutes. An
avidin-biotinhorseradish peroxidase system (Vector Laboratories) was
used for immunostaining. The nuclei were stained with
hematoxylin. Cells incubated without the primary antibody were used
as controls.
Immunoelectron Microscopy
To observe the localization of SR-A and SR-Amediated
endocytosis by immunoelectron microscopy, cell samples of untransduced
and transduced PA317 fibroblasts were incubated for 48 hours in the
presence of acetylated (ac) LDL (5 µg/mL) and fixed for 10
minutes with 0.05% glutaraldehyde in 0.165 mol/L
cacodylate buffer, pH 7.4. Immunostaining was carried
out for 1 hour with an anti-bovine SR-A IgG-D2.28 The
samples were then incubated for 1 hour with an anti-mouse
immunoglobulin [F(ab')2] conjugated with
peroxidase (Amersham). Between each staining step, washing with PBS
containing 0.005% saponin (Sigma) was performed as previously
described29 to facilitate permeabilization of the cells.
Peroxidase activity was visualized with 3,3'-diaminobenzidine as a
substrate. After diaminobenzidine staining, the cells were postfixed
with 1% OsO4 for 60 minutes, dehydrated through
a graded series of ethanols, embedded in Epon 812, and sectioned
serially. Ultrathin sections were observed with an H-7500 electron
microscope (Hitachi) without counterstaining.
Analysis of Lipoprotein Metabolism
Fluorescence Microscopy and Flow Cytometry Analysis
to Measure DiI-acLDL Uptake
LDL (d=1.019 to 1.063 g/mL) from fresh, human plasma
was isolated by ultracentrifugation as
described.30 LDL was labeled with fluorescent
DiI
(1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine
perchlorate; Molecular Probes) as described31 and then
acetylated. For competition studies, LDL was acetylated
with acetic anhydride32 and oxidized by overnight
incubation with copper ions (20 µmol/L).23 For the
uptake assay of fluorescent lipoproteins, cells were incubated
at 37°C on chamber slides with growth medium containing 10 µg/mL
DiI-acLDL for 4 to 8 hours in the presence or absence of acLDL, oxLDL,
or polyinosinic acid (Sigma). Cells were either analyzed
by flow cytometry or fixed with 0.05% glutaraldehyde
or 4% paraformaldehyde (10 minutes, 20°C) for
fluorescence microscopy.33
For flow cytometry analysis, untransduced cells and stably transduced clones were grown in 10% FBS/DMEM and trypsinized 2 days before the assay. In some experiments, cells were stimulated with 50 ng/mL phorbol 12-myristate 13-acetate (PMA) for 24 hours. Cultured fibroblasts or SMCs (1 to 2x106 cells) were washed with PBS and incubated with 10 µg/mL DiI-acLDL in 10% lipid-deficient serum/DMEM at 37°C for 5 hours. For competition assays, a 25-fold excess of acLDL, oxLDL, and polyinosinic acid was used. Immediately after the incubations, the cells were placed on ice and washed twice with ice-cold PBS. Cells were harvested by trypsinization and suspended at a concentration of 106 cells/mL in 2% FBS/PBS. Cells (5x103 to 104) were analyzed by fluorescence-activated cell sorter (FACS, Becton Dickinson, research software version B 2/88) and an argon-ion laser (514-nm excitation) and a 580-nm filter. The data collected from the cells were analyzed with Consort 30 software.
Degradation Assay
For the degradation assay, LDL was iodinated with
carrier-free Na125I,23
acetylated with acetic anhydride,32 and oxidized
by overnight incubation with copper ions (20
µmol/L).23 Modified, iodinated LDL was used
for the degradation analyses at 5 to 10 µg/mL. A 25-fold
excess of unlabeled acLDL or oxLDL was used in the competition assays.
After the cells were washed, the amounts of
125I-labeled, acid-soluble material in the medium
(degradation) and in the cells (cell-associated activity) were
determined.23 Values obtained from cell-free wells were
subtracted before calculating the results. Protein concentrations were
determined by the method of Lowry et al.34
Lipid Staining
Cells (5x103 to
104) were preincubated on chamber slides for 2
days in 10% lipid-deficient serum/DMEM. On the third day, the medium
was removed and fresh medium with 100 to 360 µg/mL acLDL was added.
For competition analysis, a 5-fold excess of polyinosinic acid
was used. After a 1- to 3-day incubation, the cells were washed with
1x PBS, fixed with 1.25% glutaraldehyde, and stained
with oil red O for 15 minutes, followed by hematoxylin counterstaining.
Cells were then washed in tap water, and lipid staining was evaluated
by light microscopy.
Apoptosis and Cell Death Assay
Annexin V (annexin VFITC apoptosis detection kit,
Genzyme) was used for identification of the cells undergoing an early
phase of apoptosis. Annexin V binds to exposed
phosphatidylserine in the outer leaflet of the
plasma membrane due to disruption of phospholipid asymmetry
characteristic of early apoptotic events. Because translocation
of phosphatidylserine to the external cell surface
also occurs during necrosis, annexin VFITC was used in conjunction
with the vital dye propidium iodide (PI) to distinguish
apoptotic cells (annexin+/PI-) from necrotic cells
(annexin-/PI+) or those cells that had already died due to
apoptosis (annexin+/PI+). Uptake of the fluorescent
dyes was measured from 104 cells by flow
cytometry. The protocol was standardized by using etoposide (8
µmol/L) and okadaic acid (5 nmol/L) treatments, which are known to
cause apoptosis in cultured cells.35 36
| Results |
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To document the integration of the SR-A cDNA into the genome of the transduced cells, Southern blot analysis of several clones was performed (data not shown). After digestion of the clones with BspTI, a 5-kb band was obtained in all but 2 clones, thus verifying integration of the complete retroviral cassette. Three smaller bands were present in the 2 aberrant clones. The reason for the additional bands in these clones remains unknown, but it may be due to rearrangements in the retroviral cassette during the integration step (data not shown). Thus, retroviral gene transfer does not always lead to a properly oriented functional expression cassette in the transduced cells.
Immunocytochemistry and Immunoelectron Microscopy
The presence of SR-A in the transduced cells was studied by
immunocytochemistry. Protein was located in the cytoplasm and was
concentrated in areas near the nucleus (Figure 3
). The presence of SR-A in the
transduced mouse fibroblasts was studied by immunoelectron microscopy.
A positive reaction for SR-A was detected in the transduced cells
(Figures 4A
and 4B
). At higher
magnification, the reaction for SR-A was observed on the plasma
membrane condensed at coated-pitlike structures and in some
intracellular structures similar to endocytotic vesicles (Figure 4C
). These findings suggest, but do not prove, participation of
the transduced SR-A in a functional endocytotic pathway. No positive
reaction was observed in the untransduced control cells (Figure 4D
).
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Fluorescence Microscopy and Flow Cytometry Analysis
of DiI-acLDL Uptake
As analyzed by fluorescence microscopy, transduced
RAASMC clones efficiently internalized DiI-acLDL, whereas untransduced
cells did not express any detectable SR-A activity (Figure 5
). In the transduced cells, DiI-acLDL
was detected in spherical vesicles that were evenly distributed
throughout the cytoplasm (Figure 5
).
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Transduced fibroblasts and SMCs analyzed by flow cytometry
showed functional SR-A receptors on the cell surface. No uptake of
DiI-acLDL was detected in the untransduced control cells (Figure 6A
) unless they were stimulated. SMC
clone 14 showed 60% to 80% higher DiI-acLDL uptake activity than the
untransduced control SMCs, and the uptake was totally inhibited by a
40-fold excess of unlabeled acLDL or polyinosinic acid. Cytochalasin D
was used to inhibit phagocytosis, but it had no effect on the uptake of
DiI-acLDL by the transduced SMCs (data not shown). Flow cytometry
analysis also revealed altered morphology of the
SR-Atransduced SMCs compared with untransduced, control SMCs. The
transduced cells were bigger and more granular than the control
cells (Figure 6B
). PMA (50 ng/mL) pretreatment for 24
hours increased the binding of DiI-acLDL in LacZ-transduced control
cells, but only a small increase was seen in SR-Atransduced cells
after PMA treatment (Figure 6C
).
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Degradation Assays
To demonstrate that the transduced SR-A was functional,
degradation rates of 125I-labeled acLDL were
studied. Degradation activity was measured in 8 fibroblast
clones and 2 SMC clones (Figure 7
). Six fibroblast clones had SR-A in the
right orientation (clones 5, 10, 14, 16, 21, and 23) and 2
clones (11 and 17) in the reverse orientation (derived from
pLSrNL). Degradation of
125I-acLDL in the best-transduced clones (5, 16,
21, and 14) having SR-A in the right orientation was 9- to 22-fold
higher than in the control cells. The degradation activity of the
untransduced control cells, SR-A clones 17, 11, and 23, and in
LacZ-transduced cells was <10 ng/mg cell protein, whereas
corresponding values for the best-transduced clones 5, 16, 21, and 14
were 216±59, 126±96, 114±44, and 86±11 ng/mg cell protein,
respectively (Figure 7A
). Displacement assays with a 25-fold
excess of unlabeled acLDL showed a 42% decrease in the degradation of
125I-acLDL in the most active clone 5. For
comparison, the degradation of 125I-acLDL in
mouse peritoneal macrophages was
2.5 to 8 µg/mg cell
protein (data not shown). Two transduced SMC clones (clones 12 and 14)
showed 6- to 8-fold increased degradation of
125I-acLDL (Figure 7B
). Degradation was
effectively competed by unlabeled acLDL, oxLDL, and polyinosinic acid.
In long-term cultures, the degradation rates of the transfected clones
were somewhat lower but remained stable for at least 9 months (data not
shown). Average degradation activities of the transduced clones were
487 ng/mg cell protein for clone 14 and 293 ng/mg cell protein for
clone 12. A 25-fold excess of the unlabeled acLDL or polyinosinic acid
decreased the degradation to 20% and 10% of the control values,
respectively, whereas oxLDL decreased the degradation to 40% of the
controls (Figure 7B
). The degradation of
125I-oxLDL was 2-fold higher in SMC clone 14
compared with untransduced SMCs (data not shown), indicating an
increased uptake of oxLDL via SR-A.
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Lipid Staining
Clear differences were seen in lipid loading after oil red O
staining in the transduced and untransduced fibroblasts and SMCs
(Figure 8
). Incubation with 100 µg/mL
acLDL led to foam cell formation in SR-Atransduced fibroblasts,
whereas 360 µg/mL acLDL was needed to cause foam cell formation in
the SR-Atransduced SMCs. Some differences were observed in the
distribution pattern of lipid vesicles in fibroblasts and SMCs after
incubation with 100 µg/mL acLDL. In fibroblasts, the lipid vesicles
were mostly seen in perinuclear areas, whereas in SMCs the lipid
vesicles were mostly located in the periphery of the cell (Figure 8
).
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Apoptosis and Cell Death
The effects of oxLDL on apoptosis and cell death were
measured in SR-Atransduced SMCs and control cells by flow cytometry.
OxLDL induced apoptosis and cell death in SMCs during a 48- to
72-hour incubation at concentrations of 200 to 500 µg/mL. A similar
apoptotic response was seen in fibroblasts with less amount of
oxLDL (50 to 200 µg/mL). After a 72-hour incubation in the presence
of 200 µg/mL oxLDL, 18% of the SR-Atransduced SMCs were in early
apoptosis (annexin+/PI-) compared with 7% in the control
cells (Figure 9A
). The proportion of late
apoptotic (annexin+/PI+) cells was also moderately increased.
Untreated control and transduced SMCs were primarily annexin-/PI-,
indicating that they were viable and not undergoing enhanced
apoptosis or cell death. Incubation with acLDL or native LDL
did not induce any significant apoptosis (data not shown). A
48-hour incubation with a higher concentration of oxLDL (500 µg/mL)
increased annexin V positivity in the SR-Atransduced SMCs by 3.6-fold
compared with the control cells (Figure 9B
). In the transduced
SMCs, a 4.3-fold increase in PI+ but annexin- cells (ie, necrotic cell
death) and a 2.9-fold increase in the control cells were seen when
compared with untreated control cells, indicating that the higher
concentration of oxLDL also induced cell death without
apoptosis.
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| Discussion |
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The role of SR-A in the development of macrophage-derived foam cells is well established. In human, rabbit, and mouse arteries, SR-A receptors are expressed in lesion macrophages.6 7 8 9 10 Because oxLDL is present in atherosclerotic lesions,23 38 it is conceivable that SR-A could lead to lipid accumulation and foam cell formation. The role of SR-A in atherogenesis has been confirmed in SR-A genedisrupted mice, which develop 60% less atherosclerosis when bred with apoE-knockout mice compared with control mice.11 Also, marked variability in the induction of SR-A activity has been reported in 2 strains of rabbit that differ in their susceptibility to atherosclerosis.39
Inconsistent data exist regarding the role of SR-A in nonmacrophage cell lines. Immunohistochemical studies suggest that SR-A is expressed in some SMCs in the balloon-injured, hypercholesterolemic rabbit neointima,14 40 whereas in other studies, SR-A mRNA or immunoreactive protein has not been identified in human, rabbit, or mouse lesion SMCs.6 8 9 10 However, in vitro rabbit and human SMCs express SR-A after PMA stimulation. Endothelial cells are known to take up acLDL, and SR-A immunoreactivity has been detected in the endothelium with the use of anti-peptide antibodies,41 whereas no such reactivity has been found in other studies.6 8 9 10 40 It is possible that conflicting results are at least partly due to differences in the specificity of reagents used for the studies.
We have demonstrated that functional SR-A activity can be stably expressed in SMC and fibroblast cell lines. Transfection leads to foam cell formation with both acLDL and oxLDL. Even though human atherosclerotic lesions contain a significant portion of SMC-derived foam cells, it has been difficult to generate SMC foam cells in vitro.32 42 Accordingly, the consequences of foam cell formation to SMC biology have been difficult to study. Stably transfected cell lines can be used as models of foam cell formation and lipid accumulation in vitro. The capacity of transfected SMCs to take up acLDL or oxLDL was much lower than that of macrophages, which suggests that SMC-derived foam cells might not develop as rapidly as macrophage-derived foam cells. However, human lesions develop slowly during the first 3 decades of life,43 44 and it is conceivable that even a low SR-A activity could lead to a gradual oxLDL accumulation in arterial SMCs over several years. Increased oxLDL accumulation without foam cell formation may also alter SMC functions through various effects of oxidized lipids on transcription factors and gene expression.45 46 47 48 Other lipoprotein receptors than SR-A and uptake mechanisms may also contribute to lipid uptake in lesion SMCs.4
It was found that after incubation with oxLDL, the stably transduced SMC lines were more susceptible to apoptosis and cell death, which together with foam cell formation could contribute to atheroma formation and lesion development. These findings are in line with recent reports that oxLDL can cause apoptosis in cultured cells and that apoptotic cell death occurs in human and experimental atherosclerotic plaques.45 46 47 49 50 Susceptibility to apoptosis in response to oxLDL incubation was increased in the SR-Atransduced cells, from 1.5- to 2.6-fold, in comparison with the untransduced cells, which is in line with a 2-fold increase in the degradation of 125I-oxLDL seen in the transduced cells. OxLDL caused more apoptosis compared with acLDL, despite the fact that an 8-fold increase in the rate of 125I-acLDL degradation was seen in the transduced SMCs. However, it is impossible to fully distinguish the degree of apoptosis caused by the SR-Amediated uptake of oxLDL from that of the nonSR-Amediated uptake. The results also suggest that the role of SR-A in cell death is indirect and due to an increased uptake of oxLDL. It is important to note that the increased rate of apoptosis was not associated with an increased rate of replication (P.L. et al, unpublished observations, 1998).51
Even though apoptosis has been shown to occur in plaques and not in fatty streaks, SMCs within human fatty streaks express Bax, a proapoptotic protein of the Bcl-2 family52 that increases the susceptibility of cells to apoptosis.53 The expression of Bax can activate a common pathway of apoptosis by triggering a mitochondrial cytochrome c release.54 It has also been shown that apoptosis in vascular SMCs can be induced through caspase-3 activation and Bcl-2 downregulation by lipid peroxides, which are present in oxLDL,55 and that oxidative stress response elements are present in the SR-A promoter of human SMCs.56
In conclusion, our results show that SR-A expression in SMCs and fibroblasts leads to proatherogenic changes by increasing lipid accumulation and predisposing cells to apoptosis. In addition to the significant role of SR-A in macrophages, these receptors may participate in atherogenesis by predisposing nonmacrophage cells to proatherogenic changes. It is possible that modulation of SR-A activity may be useful for the prevention of proatherogenic changes in vivo.
| Acknowledgments |
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Received August 27, 1998; accepted April 21, 1999.
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