© 2000 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
Effect of Human Scavenger Receptor Class A Overexpression in Bone Marrow–Derived Cells on Cholesterol Levels and Atherosclerosis in ApoE-Deficient Mice
From the Division of Biopharmaceutics, Leiden/Amsterdam Center for Drug Research (M.V.E., N.H., Th.J.C.), and the Department of Human Genetics (M.P.J.D.W., M.H.H.), Leiden University Medical Center, Sylvius Laboratories, Leiden University (The Netherlands); TNO Prevention and Health, Gaubius Laboratories (L.M.H.), Leiden, The Netherlands; and SmithKline Beecham Pharmaceuticals, (P.H.E.G.) NFSP(N), Harlow, Essex, UK.
Correspondence to M. Van Eck, MSc, Division of Biopharmaceutics, Sylvius Laboratories, Leiden University, PO Box 9503, 2300 RA Leiden, The Netherlands. E-mail M.Eck{at}LACDR.LeidenUniv.nl
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Abstract—In the arterial wall, scavenger receptor class A (SRA) is implicated in pathological lipid deposition. In contrast, in the liver, SRA is suggested to remove modified lipoproteins from the circulation, thereby protecting the body from their pathological action. The role of SRA on bone marrow–derived cells in lipid metabolism and atherogenesis was assessed in vivo by transplantation of bone marrow cells overexpressing human SRA (MSR1) to apoE-deficient mice. In vitro studies with peritoneal macrophages from the transplanted mice showed that macrophage scavenger receptor function, as measured by cell association and degradation studies with acetylated LDL, was
3-fold increased on overexpression of MSR1 in bone
marrow–derived cells as compared with control mice. Despite the
increased macrophage scavenger receptor function in vitro, no
significant effect of MSR1 overexpression in bone marrow–derived cells
on the in vivo atherosclerotic lesion development was found. In
addition to arterial wall macrophages, liver
sinusoidal Kupffer cells also overexpress MSR1 after bone marrow
transplantation, which may scavenge atherogenic particles more
efficiently from the blood compartment. Introduction of bone marrow
cells overexpressing human MSR1 in apoE-deficient mice induced a
significant reduction in serum cholesterol levels of
20% (P<0.001, 2-way ANOVA) as the result of a
decrease in VLDL cholesterol. It is suggested that the
reduction in VLDL cholesterol levels is due to increased
clearance of modified lipoproteins by the overexpressed MSR1 in Kupffer
cells of the liver, thereby protecting the arterial wall
against the proatherogenic action of modified lipoproteins.
Key Words: scavenger receptor • atherosclerosis • hyperlipidemia • macrophages • bone marrow transplantation
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Introduction |
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An early stage in the development of atherosclerosis is the formation of foam cells that arise from macrophages and smooth muscle cells caused by the excessive uptake of cholesteryl ester–rich lipoproteins.1 2 Several lipoprotein receptors have been characterized that might be involved in the uptake of lipoproteins by these cell types, including the LDL receptor, the VLDL receptor, LDL receptor–related protein (LRP), and several types of scavenger receptors.3 4
Scavenger receptor class A (SRA) was the first scavenger receptor that was identified and the molecular cloning of this receptor disclosed 2 isoforms, type I and type II, which are derived from alternative splicing of a single gene product.5 6 7 8 SRA is a trimeric glycoprotein that consists of 6 different domains, including the isoform-specific C-terminal domain and the collagen-like domain, which is involved in ligand binding.9 SRA mediates the uptake and degradation of a broad range of negatively charged ligands and modified lipoproteins, including oxidized and acetylated LDL (OxLDL and AcLDL).10 11 In contrast to the LDL receptor, SRA is not downregulated by intracellular cholesteryl ester accumulation and might therefore play an important role in foam cell formation.5 The expression of SRA in the vessel wall is highly induced during cholesterol feeding and the induction of atherosclerotic lesion formation.3 Immunohistochemical studies in humans, rabbits, and mice demonstrated SRA in atherosclerotic lesions, where it is primarily expressed by macrophages.12 13 However, some studies also suggest that smooth muscle cells do express SRA.14 15 16
Recently, SRA-deficient mice have been generated by targeted disruption
of the SRA gene.17 On an apoE-deficient
background, it was found that SRA deficiency results in a reduction of
60% of spontaneously developed atherosclerosis,
providing evidence that SRA is of prime importance in the development
of atherosclerosis, at least in apoE-deficient
mice.
In addition to arterial wall macrophages, liver sinusoidal Kupffer cells and endothelial cells also express SRA.18 19 Expression of SRA in the liver is thought to form a major protection system of the body by scavenging atherogenic particles from the blood compartment, thereby reducing the accumulation of modified lipoproteins in the interstitial space of the vessel wall.20 21 In apoE-/- mice, SRA deficiency increased serum cholesterol levels by 46%.17 Apparently this increase in serum cholesterol levels did not facilitate atherosclerotic lesion development, possibly as the result of the absence of SRA in macrophages in the arterial wall.
In the present work, we wanted to establish whether overexpression of SRA exclusively in macrophages will influence serum cholesterol levels and the susceptibility to atherosclerosis in apoE-deficient mice. To investigate the effect of macrophage SRA overexpression on atherogenesis, we generated apoE-deficient mice overexpressing human SRA (MSR1) solely in bone marrow–derived cells by transplantation of bone marrow–overexpressing MSR1 to apoE-deficient mice. Macrophages isolated from these transplanted animals show an increased SRA activity as compared with macrophages from control transplanted mice. Surprisingly, despite this increased uptake of modified lipoproteins by macrophages in vitro, no significant effect of MSR1 overexpression in bone marrow–derived cells on in vivo atherosclerotic lesion development could be demonstrated. Analysis of the effect on lipid levels elucidated that VLDL cholesterol levels in the circulation of these mice are reduced, implicating that MSR1 overexpression in macrophages reduces the availability of modified lipoproteins for atherosclerotic lesion formation in the arterial wall.
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Methods |
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Animals
Transgenic mice overexpressing the human scavenger receptor class A gene (MSR1) were generated as previously described by oocyte microinjection of a 180-kb yeast artificial chromosome containing the MSR1 with 60-kb and 40-kb flanking sequences at the 5' and 3' end, respectively, thereby including all native regulatory elements as described previously.22 ApoE-deficient (apoE-/-) mice, hybrids between C56Bl/6 (haplotype H-2b) and 129 Sv (haplotype H-2b) strains (N3 generation of back-crosses to C57Bl/6), were used as recipients for bone marrow transplantations (BMT).23
The animals were housed and bred at the animal facility of the Sylvius Laboratories in Leiden, The Netherlands. Mice used for BMT experiments were housed in sterilized filter-top cages and fed sterilized regular chow diet (SRM-A) containing 5.7% fat (Hope Farms, Woerden, The Netherlands). Drinking water was supplied with antibiotics (83 mg/L ciprofloxacin and 67 mg/L polymyxin B sulfate) and 6.5 g/L sucrose.
Animal procedures were performed at the Sylvius Laboratories of the Leiden/Amsterdam Center for Drug Research in accordance with the national laws. All experimental protocols were approved by the Ethics Committee for Animal Experiments of Leiden University.
Irradiation and BMT
To induce bone marrow aplasia, female (age, 5 to 6 weeks)
apoE-/- mice were exposed to a single dose
of 13 Gy (0.28 Gy/min, 200 kV, 4 mA) total body irradiation by means of
an Andrex Smart 225 Röntgen source (Andrex Radiation Products
AS) with a 4-mm aluminum filter 1 day before the transplantation, as
described previously.24 25 Bone marrow cell suspensions
were isolated by flushing the femurs and tibias from male
apoE-/- and
apoE-/- MSR1 mice with PBS. Single-cell
suspensions were prepared by passing the cells through a 30-µm nylon
gauze. Irradiated recipients received 1.0x107
bone marrow cells by intravenous injection into the tail
vein.
Polymerase Chain Reaction Analysis
Bone marrow and liver tissue was isolated from apoE-deficient
mice transplanted with apoE-/- or
apoE-/- MSR1 bone marrow at 12 weeks after
BMT. DNA, isolated from these tissues, was subsequently tested by
polymerase chain reaction for the presence of the transgene, with the
use of the following MSR1-specific primers: forward
5'-GAAGATGCTGGAGTCACTTGC-3' and reverse
5'-TGGAGCCAATTACTGGTATGC-3'.
Isolation and Modification of Lipoproteins
Human LDL was isolated from healthy volunteers as described by
Redgrave et al.26 After density
ultracentrifugation, LDL (1.019 g/mL<d<1.063 g/mL)
was collected and dialyzed against PBS/1 mmol/L EDTA. Protein
content was determined according to Lowry et al,27 with
bovine serum albumin (BSA) used as an internal standard.
For isolation of apoE-deficient ß-VLDL, apoE-deficient mice were fasted overnight and blood was subsequently collected under anesthesia by puncture of the orbital plexus. After density gradient ultracentrifugation according to Redgrave et al,26 the top fraction (d<1.006 g/mL), containing apoE-deficient ß-VLDL, was isolated and dialyzed against PBS/1 mmol/L EDTA. Protein content was determined according to Lowry et al,27 with BSA used as an internal standard.
LDL was acetylated according to Basu et al.28 Lipoproteins were oxidatively modified by incubation of 0.2 mg/mL protein with 11 µmol/L CuSO4 for 20 hours at 4°C. Radiolabeling of the lipoproteins with 125I at pH 10.0 was performed according to McFarlane,29 modified as described earlier.30
In Vitro Studies With Peritoneal Macrophages
Five days after intraperitoneal injection of
3% Brewer’s thioglycollate medium, macrophages were harvested
by lavage of the peritoneal cavity with 10 mL sterilized PBS. The
isolated macrophages were washed 3 times with sterilized PBS
and plated in 24-well plates at a density of
0.5x106 cells/500 µL in DMEM, supplemented
with 10% (wt/vol) bovine calf serum, 2 mmol/L
L-glutamine, 100 µg/mL streptomycin, and 100 IU/mL
penicillin. After 4 hours, nonadhering cells were removed by washing.
At 2 days after isolation, cells were incubated with the indicated
concentrations of 125I-acetylated
(Ac)LDL, 125I-oxidized (Ox)LDL, or
125I-Oxß-VLDL. After 3 hours at 37°C, cells
were washed and lysed in 0.1 mol/L NaOH, and the cell protein content
was determined according to Lowry et al27 to determine the
cell association per milligram of cell protein. Degradation
products in the medium were measured by addition of 0.4 mL 35%
trichloroacetic acid to 0.5 mL medium. After incubation at 4°C for 30
minutes, 0.25 mL of 0.7 mol/L AgNO3 was added,
samples were centrifuged for 5 minutes at 16 000g,
and the radioactivity was determined in the supernatant.
To study the effect of MSR1 overexpression in oxidized ß-VLDL–induced foam cell formation, macrophages from MSR1-transgenic and wild-type mice were isolated as described above. At 2 days after the isolation, the cells were incubated with the indicated amounts of oxidized ß-VLDL in DMEM/2% BSA for 24 hours at 37°C. After 24 hours, cells were washed and fixed with 2.2% PBS-buffered formaldehyde, and accumulated lipids were stained with 0.5% oil red O in propylene glycol. Oil red O staining was quantified with a light microscope connected to a full-color video camera and running Leica Qwin Imaging Software.
Histological Analysis of Hearts and Aortas
for Atherosclerosis
To analyze the development of
atherosclerosis, transplanted mice were killed at 12
weeks after BMT. Hearts and aortas were perfused in situ with
oxygenated Krebs buffer (37°C, 100 mm Hg) for 20 to
30 minutes through a cannula in the left ventricle, followed by
perfusion with 3.7% neutral-buffered formalin (Formal-fixx, Shandon
Scientific Ltd) for 30 minutes.31 32 Hearts and aortas
were excised and stored in formalin.
To evaluate the development of atherosclerotic lesions, the aortas were separated from the hearts. Hearts were bisected at the level of the atria, and the base of the heart plus aortic root were taken for analysis. Cryostat 10-µm cross sections of the aortic root were made and stained with oil red O (BDH Ltd). The atherosclerotic lesion area in the sections was quantified with a light microscope connected to a full-color video camera and Optimas 6.1 image analysis software (BioScan). The mean atherosclerotic lesion area for each individual mouse was calculated (in µm2) from 10 sections, starting at the appearance of the tricuspid valves, as described previously.31 32
The perfusion-fixed aortas were used to analyze atherosclerosis development over the whole length of the aorta. After dissection of the adventitial fat, the aorta was stained for vascular lipids with oil red O. The presence of oil red O–positive lesions was subsequently studied with the use of a stereomicroscope.
Serum Cholesterol and Triglyceride Analysis
After an overnight fasting period (15 hours), 100 µL blood
was drawn from each individual mouse by tail bleeding. The
concentrations of total cholesterol and
triglycerides in serum were determined by enzymatic
procedures (Boehringer Mannheim). Precipath (standardized
serum; Boehringer Mannheim) was used as an internal
standard.
The distribution of cholesterol over the different lipoproteins in serum was determined by loading of 30 µL serum from each mouse onto a Superose 6 column (3.2x30 mm, Smart-system, Pharmacia). Serum was fractionated at a constant flow rate of 50 µL/min, with the use of PBS. Total cholesterol content of the effluent was determined enzymatically.
Statistical Analysis
Statistically significant differences in repeated measurements
were tested by means of ANOVA in time, and individual comparisons were
made by means of the unpaired Student’s t test (Instat
Graphpad software). A value of P<0.05 was regarded as
significant.
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Results |
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To gain insight into the in vivo effect of MSR1 overexpression in bone marrow–derived cells on atherosclerotic lesion development, female recipient apoE-deficient mice were transplanted with apoE-deficient bone marrow overexpressing MSR1 after lethal irradiation.
Detection of MSR1 Expression After BMT
Repopulation of bone marrow and liver with donor-derived cells was
determined by polymerase chain reaction analysis of the
expression of MSR1 in bone marrow and liver at 12 weeks after BMT. As
expected, no MSR1 DNA was detected in mice that received syngeneic bone
marrow from apoE-/- mice. In mice
transplanted with apoE-/- MSR1 bone
marrow, an MSR1-specific band of 350 bp appeared in DNA from bone
marrow (data not shown), indicating that MSR1 bone marrow cells had
successfully repopulated the recipient mice. Furthermore, MSR1
expression was also demonstrated in the liver, indicating that the
Kupffer cells of the liver also were replaced (data not shown).
Effect of Human Macrophage Scavenger Receptor Class A
Overexpression in Bone Marrow–Derived Cells on Modified Lipoprotein
Cell Association and Degradation
To demonstrate that the successful repopulation of the
transplanted mice with MSR1 bone marrow resulted in a functionally
overexpressed MSR1, thioglycollate-elicited macrophages were
isolated from both transplantation groups at 12 weeks after BMT.
Subsequently, the cell association and degradation of
acetylated LDL, a high-affinity ligand for the scavenger
receptor, by these macrophages was determined. The cell
association and degradation of AcLDL was highly increased in
macrophages from apoE-deficient mice transplanted with
apoE-/- MSR1 bone marrow as compared with
macrophages from control transplanted mice, indicating that
macrophages indeed functionally overexpress MSR1 and therefore
were replaced successfully. The maximal cell association was 2.7-fold
increased from 828±170 ng/mg in macrophages (m
) from
apoE-/-
apoE-/-
mice to 2261±348 ng/mg in m from
apoE-/-
MSR1apoE-/- animals (Figure 1A
). The maximal degradation capacity was
4-fold increased (Figure 1B). For comparison, the association
and degradation of AcLDL by m
, isolated from the donor strains of
mice, was 1.5-fold and 4.4-fold increased in m overexpressing MSR1
as compared with control mice (data not shown). The comparable
association and degradation rates of AcLDL by macrophages
isolated from the transplanted mice and from the donor strains of mice
thus indicate that the replacement of tissue macrophages by
cells from donor origin is complete. This is in accordance with
previously published data by our group showing that after BMT, the
degree of chimerism is complete in bone marrow and
spleen.25
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) or
apoE-/- MSR1 (•) bone marrow. Values are
mean±SD of macrophages of 3 individual mice. *Significant
difference (P<0.01) vs macrophages from control
transplanted mice.
In the apoE-deficient mice that were used as recipients for our BMT
studies, ß-VLDL is the most predominant lipoprotein. Oxidatively
modified ß-VLDL in the subendothelial space of the
arterial wall will presumably be involved in
atherosclerotic lesion formation in these mice. Therefore, the effect
of MSR1 overexpression in macrophages on the association and
degradation of oxidatively modified apoE-deficient ß-VLDL was
established. To discriminate between any possible species differences
in lipoprotein recognition by the human scavenger receptor, the
association and degradation of oxidatively modified apoE-deficient
murine ß-VLDL was also compared with oxidatively modified human LDL
(Figure 2). Oxidative modification of
apoE-deficient ß-VLDL induced a 3.5-fold increase in the association
to macrophages as compared with native apoE-deficient ß-VLDL
(data not shown). Overexpression of MSR1 in macrophages induced
a 33% and 32% increase in the association and degradation of oxidized
apoE-deficient ß-VLDL, respectively. A similar effect was observed on
the association (29% increase) and degradation (21% increase) of
oxidized human LDL, indicating that the overexpressed human MSR1
recognizes human as well as murine modified lipoproteins. The effect of
MSR1 overexpression in macrophages on the uptake of OxLDL is
less pronounced as compared with the effect on AcLDL, which is probably
caused by the presence of additional receptors for OxLDL besides
scavenger receptor class A.33 To demonstrate that the
increased metabolism of oxidized apoE-deficient ß-VLDL by
macrophages from MSR1 transgenic mice results in increased
cholesterol accumulation, macrophages were
analyzed for in vitro foam cell formation by staining of the
accumulated lipids with oil red O. Determination of the oil red O
staining per cell area revealed that lipid accumulation in
macrophages from MSR1-transgenic mice was 1.6-fold increased as
compared with macrophages from wild-type mice, indicating that
macrophages overexpressing human MSR1 are more prone to convert
into foam cells on incubation with murine lipoproteins (Figure 3).
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Effect of Human Macrophage Scavenger Receptor Class A
Overexpression in Bone Marrow–Derived Cells on Susceptibility to
Atherosclerosis
To gain insight into the effect of human macrophage
scavenger receptor class A overexpression in bone marrow–derived cells
on susceptibility to atherosclerosis, we
analyzed the atherosclerotic lesion area in the aortic root of
the transplanted mice at 12 weeks after BMT.
Representative photomicrographs of lipid-rich
atherosclerotic lesions in the aortic root are shown in Figure 4. Despite the increased association of
modified lipoproteins in vitro, no significant difference in the mean
atherosclerotic lesion area could be demonstrated between
apoE-/-
MSR1apoE-/- mice
(2.94±0.85x105
µm2; n=6) as compared with
apoE-/-apoE-/-
animals (3.83±0.69x105
µm2; n=7) (Figure 5).
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In addition to quantitative analysis of atherosclerosis in multiple sections in the aortic root, atherosclerosis development was also studied qualitatively over the total length of the aorta (data not shown). The anatomic distribution of atherosclerotic lesions was identical in both transplantation groups. Lesions were present in the inner curvature of the aortic arch and at branch points with the carotid arteries in both groups of mice. Furthermore, along the descending aorta, atherosclerotic lesions were mainly observed near the orifice of the smaller branching arteries.
Effect of Human Macrophage Scavenger Receptor Class A
Overexpression in Bone Marrow–Derived Cells on Serum Lipid
Levels
In addition to arterial wall macrophages,
liver sinusoidal Kupffer cells overexpress MSR1 after BMT. Because
expression of SRA in the liver is suggested to form a major protection
system of the body by scavenging atherogenic particles from the blood
compartment, the effect of MSR1 overexpression in bone marrow–derived
cells on serum lipid levels was determined. The effect of
transplantation of apoE-deficient mice with bone marrow from
apoE-deficient mice
(apoE-/-apoE-/-)
and apoE-deficient mice overexpressing the human SRA
(apoE-/-
MSR1apoE-/-) on serum
cholesterol levels is depicted in Figure 6. Even on a normal chow diet,
apoE-deficient mice demonstrate a marked
hyperlipidemia. Introduction of bone marrow–derived
cells overexpressing MSR1 in apoE-deficient mice induced a significant
reduction in serum cholesterol concentrations of 20%
(P<0.001, 2-way ANOVA) in the weeks after BMT. No
significant effect of BMT on serum triglyceride levels was
found (data not shown). Analysis of the distribution of
cholesterol over the different lipoprotein fractions at 4
weeks after BMT revealed that the reduction in serum
cholesterol levels caused by overexpression of MSR1 on bone
marrow–derived cells in apoE-deficient mice was caused by a
significant (P<0.05) reduction in VLDL
cholesterol (Figure 7).
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Discussion |
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SRA is implicated in the pathological lipid deposition and subsequent transformation of macrophages to foam cells.1 2 Recently, it has been demonstrated that targeted disruption of the SRA gene in apoE-deficient mice results in a 60% decrease in the mean atherosclerotic lesion area, indicating that SRA is of prime importance for the development of atherosclerosis in these mice.17 Several lipoprotein receptors have been characterized that might be involved in the formation of foam cells, including the LDL receptor, the VLDL receptor, LRP, and several types of scavenger receptors.3 4 However, when apoE, a high-affinity ligand for the LDL receptor, the VLDL receptor, and LRP is absent, the relative contribution of the scavenger receptors to foam cell formation is expected to be most evident. The data of the present study demonstrate that atherosclerotic lesion formation in apoE-deficient mice is not significantly affected by human scavenger receptor class A overexpression in bone marrow–derived cells, despite an increased cell association and degradation of modified lipoproteins in vitro by macrophages. These data therefore indicate that the increased capacity of the SRA to metabolize modified lipoproteins does not result in the anticipated increase in atherosclerotic lesion formation. In contrast to the decreased susceptibility of apoE-deficient mice on targeted disruption of the SRA gene, De Winther et al34 recently demonstrated that SRA deficiency on an apoE3-Leiden background results in even more severe lesions.17 34 In addition, we now demonstrate that overexpression of MSR1 in bone marrow–derived cells does not induce an increase in atherosclerotic lesion development, indicating that the role of SRA in atherogenesis is complex.
After BMT, not only arterial wall macrophages but
also liver sinusoidal Kupffer cells are replaced by cells of donor
origin. Because expression of SRA in the liver is thought to form a
major protection system of the body by scavenging atherogenic particles
from the blood compartment, the effect of introduction of bone
marrow–derived cells overexpressing MSR1 into apoE-deficient mice on
serum lipid levels was determined. Overexpression of MSR1 in bone
marrow–derived cells induced a significant reduction in serum
cholesterol levels. The observed reduction in serum
cholesterol in apoE-/-
MSR1apoE-/- mice was confined to the
VLDL-sized fraction, implicating an involvement of SRA in the clearance
of VLDL-sized lipoproteins. Suzuki et al17 demonstrated
that disruption of the SRA gene in apoE-deficient mice
results in significantly higher serum cholesterol
levels.
In apoE-deficient mice, it was shown that plasma lipid
peroxidation is enhanced because of increased VLDL and LDL lipid
peroxidation.35 36 Furthermore, high titers of
autoantibodies recognizing epitopes of oxidized lipoproteins were
demonstrated in these mice.37 Therefore, it is most likely
that the decrease in VLDL cholesterol levels in
apoE-/-
MSR1apoE-/- mice as compared with
apoE-/-apoE-/-
animals is due to uptake of oxidized VLDL by the overexpressed MSR1 on
macrophages. Especially macrophages of the liver,
Kupffer cells, may be involved in this process because these cells
quantitatively form the major removal site for modified
lipoproteins.38 Wölle et al39 showed
that overexpression of bovine SRA type I in the mouse liver
hepatocytes suppressed the diet-induced high levels of
apoB-containing lipoproteins and led to an enhanced biliary secretion
of cholesterol and bile acids. Together with the data of
Suzuki et al17 and our present data, it is evident
that scavenger receptor expression in the liver influences serum
cholesterol levels, at least in apoE-deficient mice.
In conclusion, MSR1 overexpression in bone marrow–derived cells in apoE-deficient mice does not affect atherosclerotic lesion development, whereas VLDL cholesterol levels were significantly reduced. We suggest that the overexpression of MSR1 in bone marrow–derived cells reduces serum VLDL cholesterol levels by increasing the clearance of oxidatively modified lipoproteins through the overexpressed MSR1 in Kupffer cells of the liver, thereby protecting the arterial wall against the proatherogenic action of these oxidized lipoproteins.
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Acknowledgments |
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The PhD studentship of M. Van Eck was supported by SmithKline Beecham Pharmaceuticals, United Kingdom. M.H. Hofker is an Established Investigator of the Netherlands Heart Foundation. The authors wish to thank Dr G.M. Benson for helpful assistance with image analysis of atherosclerotic lesions.
Received June 24, 1999; accepted July 11, 2000.
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