© 1999 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
Scavenger Receptor Activity Is Increased in Macrophages From Rabbits With Low Atherosclerotic Response:
Studies in Normocholesterolemic High and Low Atherosclerotic Response Rabbits
From the Institute of Clinical Chemistry, University Hospital Grosshadern (D.T., R.B., J.T.), Munich, Germany; the Lipid Research Laboratory (O.S., Y.S.), Division of Medicine, Hadassah University Hospital, Jerusalem, Israel; and the Department of Experimental Animal Research (K.N.), University Hospital Göttingen, Göttingen, Germany.
Correspondence to Joachim Thiery Institute for Clinical Chemistry, University Hospital Grosshadern, Marchioninistrasse 15, 81366 Munich, Germany. E-mail thiery{at}klch.med.uni-muenchen.de
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Abstract |
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Abstract—We have previously described 2 strains of New Zealand White rabbits with a high (HAR) or low (LAR) atherosclerotic response to hypercholesterolemia. In the present study, we focused on class A scavenger receptor (SR-A) activity and ApoE expression in macrophages from both rabbit strains. These parameters play a crucial role in maintaining cholesterol homeostasis in the arterial wall and may be involved in the development of atherosclerosis. SR activity, as measured by uptake of DiI-labeled acetylated LDL, was significantly higher in macrophages from LAR rabbits (2177±253 ng/mg cell protein) than in macrophages from HAR rabbits (1153±200 ng/mg cell protein). The higher SR activity was caused by a greater number of SRs (apparent Vmax, 4100 ng/mg in LAR and 1980 ng/mg in HAR rabbits). The high SR activity in macrophages from LAR rabbits was associated with a significantly higher expression of SR-A mRNA compared with macrophages from HAR rabbits. However, the latter finding could not be explained by differences in the activity of transcription factor–activating protein 1 (AP-1), which was comparable in macrophages from both strains of rabbits. Because under certain circumstances SR-A mRNA expression is regulated in parallel with ApoE expression, we also evaluated this parameter. Although ApoE mRNA was 74% higher in macrophages from LAR rabbits, the difference did not reach statistical significance. In conclusion, the increased expression of SR-A in macrophages in the presence of adequate amounts of ApoE may play a role in attenuating atherosclerosis in LAR rabbits.
Key Words: atherosclerosis • scavenger receptor • ApoE • hypercholesterolemia • rabbit macrophages
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Introduction |
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In our laboratory, 2 strains of New Zealand White rabbits were developed in which the atherosclerotic response to hypercholesterolemia was either high (HAR) or low (LAR).1 This difference in the propensity to atherosclerosis development, in view of similar plasma cholesterol and lipoprotein levels as well as their composition, seems to be genetically determined because it is still evident after 11 generations.1 In a previous study, we compared several putative factors that could influence lesion induction at the level of the arterial wall.2 No difference was seen in the enhancement of monocyte adhesion to arterial endothelial cells with ß-VLDL derived from LAR and HAR rabbits.2 However, in the aortae of HAR rabbits, in which more atherosclerotic lesions were found than in the aortae of LAR rabbits, there was more expression of vascular cell adhesion molecule-1 (VCAM-1), but only associated with the lesions.3 In addition, the peroxidative capacity of macrophages and smooth muscle cells (SMCs) of both rabbit strains toward LDL was similar.2 However, the responsiveness of cultivated aortic SMCs to induction of scavenger receptor (SR) activity when challenged with either phorbol 12-myristate 13-acetate (PMA) or platelet-derived growth factor-BB was significantly lower in LAR than in HAR rabbits.2
Since during the initial stages of atherogenesis cholesteryl esters accumulate primarily in macrophages, the aim of the present study was to compare certain aspects of cholesterol metabolism in macrophages derived from normocholesterolemic LAR and HAR rabbits. Because the propensity to develop atherosclerosis in pigeons has been attributed to impaired hydrolysis of cholesteryl ester in macrophages,4 we also examined this parameter in LAR and HAR rabbits. We studied the uptake of 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI)-labeled acetylated LDL (DiI-acLDL) by cultured macrophages and the plasma clearance of intravenously injected DiI-acLDL. In addition, we also compared the expression of class A scavenger receptor type I and II (SR-AI/II) and ApoE in peritoneal macrophages isolated from the 2 strains of rabbits.
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Methods |
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Animals
The rabbits used in the present study are from the 2 strains established previously.1 One strain was designated LAR, with a low atherosclerotic response to cholesterol feeding; the other strain, HAR, had a high atherosclerotic response. The rabbits were kept on regular chow diet and used when 12 months old. Animal care and experimental procedures involving animals conformed to the position of the American Heart Association on research animal use and were approved by the Regierung von Oberbayern.
Isolation of Rabbit Peritoneal Macrophages
For isolation of peritoneal macrophages, rabbits were
anesthetized with ketamine-HCl/xylazine-HCl and
were injected with 40 mL of mineral oil, through a 16-gauge catheter.
After 3 days, the rabbits were injected intravenously with
500 U heparin and then given an overdose of pentobarbital. The
peritoneal cavity was washed with 1000 mL of a 154 mmol/L NaCl
solution. The cell suspension was collected through a catheter and
placed in 50 mL Falcon tubes containing EDTA (0.1 g/L). All the
solutions used contained 100 µg/mL of gentamycin. The tubes were
centrifuged at 200g for 10 minutes, the supernatant
discarded, and the cell pellet resuspended in 10 mL of Hanks' balanced
salt solution containing 0.1 g/L EDTA. Cell suspensions from 4 tubes
were pooled and recentrifuged as above. The cell pellet was
washed with DMEM (Sigma) and resuspended in DMEM containing 10% FBS;
2x106 cells were plated on 35-mm culture dishes.
After 2 hours, the nonadherent cells were removed and the cell layer
washed with serum-free medium. The cells were cultivated in DMEM
containing 10% FBS.
Lipoprotein Isolation and Labeling
LDL was prepared from freshly drawn CPD plasma from
normolipidemic human blood donors. To prevent oxidation, 0.1 g/L EDTA
was present during the whole isolation procedure. Plasma density
was adjusted to 1.063 g/mL with crystalline NaCl. After
ultracentrifugation at 150 000g for 24
hours at 10°C, the supernatant was collected, pooled, and adjusted
with distilled water to d=1.019 g/mL. It was
recentrifuged as before and the infranatant was collected and
adjusted to d=1.063 g/mL. LDL (d=1.019 to 1.063
g/mL) was collected from the supernatant after
recentrifugation (150 000g, 24 hours,
10°C). LDL preparations were extensively dialyzed against 5
mmol/L Tris, 154 mmol/L NaCl, 0.1 g/L EDTA, pH 7.4, at 4°C.
LDL was labeled with the fluorescent probe DiI (Molecular Probes) as previously described.5 LDL and DiI-LDL were acetylated according to Basu et al.6 All lipoprotein preparations were stored at 4°C in sterile containers after filtration sterilization (0.45 µm). Protein concentrations were determined by the method of Lowry et al7 by using BSA as a standard. Total cholesterol was determined by using an enzymatic procedure (Boehringer Mannheim).
Clearance of DiI-acLDL From Plasma In Vivo
The rabbits were anesthetized with ketamine-HCl
and xylazine-HCl, and 1 mg of DiI-acLDL protein (1 mL) was
injected into the marginal ear vein. Blood was drawn from the artery of
the contralateral ear before and 1, 2, 3, 4, 5, 7, 10, 15, 20, 30, 45,
and 60 minutes after injection. Fluorescence intensity was
measured in 1:20 dilutions of plasma with 154 mmol/L NaCl by using
a microtiter plate fluorescence reader (Fluorolite 1000,
Dynatech) with emission and excitation wavelengths set at 520 and 580
nm, respectively. The fluorescence data were expressed as
percentages of the first 1-minute time point (initial
concentration).8 All results were corrected for
autofluorescence of the plasma isolated before injection of
DiI-acLDL. The t1/2 of DiI-acLDL in the
circulation was calculated based on the disappearance of DiI
fluorescence from plasma, using the best fit of a
monoexponential function through all the data points
determined.8
Determination of SR Activity
SR activity was determined 18 hours after isolation of
peritoneal macrophages. The cells were incubated with 10 µg
DiI-acLDL protein/mL at 37°C for 5 hours. The specificity of
DiI-acLDL uptake was determined in the presence of 50-fold excess of
unlabeled lipoproteins. In some experiments, the
concentration-dependent uptake was measured in the presence of 5 to 50
µg DiI-acLDL protein/mL. After incubation with DiI-acLDL, the cells
were washed twice with 0.4% BSA in PBS and 3x with PBS and
were lysed with 0.1 mol/L NaOH/0.1% SDS as previously
described.5 Fluorescence intensity was determined
with a microtiter plate fluorescence reader (Fluorolite 1000,
Dynatech) with emission and excitation wavelengths set at 520 and 580
nm, respectively. The results were expressed as nanograms of
cell-associated DiI-acLDL per milligram of cell protein.5
Scatchard plot analysis was used for the calculation of
Km and Vmax.
All incubations were performed in triplicate, and values agreed within
10% of the mean.
Northern Blot Analysis
Total RNA was extracted from 3x107
freshly isolated peritoneal macrophages, using the monophasic
phenol-guanidine isothiocyanate TRIZOL reagent (Life
Technologies). RNA (15 µg) was electrophoresed on 1% agarose gels
containing 2.2 mol/L formaldehyde, transferred to a Nytran-N membrane
(Schleicher & Schuell), and cross-linked by UV irradiation. The
membranes were prehybridized for 2 hours at 42°C, followed by an
overnight hybridization with a fragment of rabbit class A SR cDNA
common to both type I and type II receptors or a cDNA probe
specific for rabbit ApoE (Table
),
radiolabeled with 32P by random-priming
(Multiprime, Amersham). The membranes were washed and exposed to
storage phosphor screens. In addition, the membranes were stripped and
rehybridized with a rabbit ß-actin probe to control for any variation
in RNA loading (Table). The blots were quantified by
phosphorimage densitometry (Storm 860, Molecular Dynamics). SR-A and
ApoE mRNA were expressed relative to ß-actin mRNA.
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Preparation of Nuclear Extracts
Nuclear extracts were prepared from 7x106
freshly isolated rabbit peritoneal macrophages as described by
Schreiber et al12 with modifications. In brief, the
cells were washed once with 10 mL of ice-cold PBS and resuspended in
250 µL of ice-cold buffer containing 10 mmol/L HEPES, pH 7.9,
10 mmol/L KCl, 0.1 mmol/L EDTA, 1 mmol/L DTT, 1
mmol/L PMSF, 1% protease inhibitor cocktail, and 0.4%
NP-40. One milliliter of protease inhibitor cocktail
consisted of 500 µg of antipain, 500 µg of aprotinin, 500 µg of
leupeptin, 50 µg of pepstatin, 750 µg of bestatin, 400 µg of
phosphoramidon, and 500 µg of trypsin
inhibitor (Boehringer Mannheim). The cell
suspension was pipetted up and down, to lyse the cells, transferred to
an Eppendorf tube, incubated on ice for 10 minutes, and
centrifuged at 12 000g for 5 minutes (4°C). The
pellet was resuspended carefully in the buffer described above, and
centrifuged again at 12 000g for 5 minutes at
4°C. The nuclear pellet was resuspended in 100 µL of buffer
containing 20 mmol/L HEPES, pH 7.9, 0.4 mol/L NaCl, 1 mmol/L
EDTA, 1 mmol/L DTT, 1 mmol/L PMSF, and 1% protease
inhibitor cocktail and rocked vigorously on a shaking
platform for 15 minutes. After centrifugation
(12 000g, 5 minutes, 4°C) the supernatant containing the
nuclear extracts was frozen at -70°C. Protein content of the nuclear
extracts was determined with the Bradford assay (Coomassie Plus,
Pierce).
DNA Binding Assay
Double-stranded activating protein-1 (AP-1) consensus
oligonucleotide (Promega) was labeled with
[
-32P]ATP (Amersham), using T4
polynucleotide kinase and purified on G-25 columns
(ProbeQuant G-25 microcolumn, Pharmacia). For DNA binding assays, 0.8
µg of nuclear protein was used and diluted with buffer containing
20 mmol/L HEPES, pH 7.9, 0.4 mol/L NaCl, 1 mmol/L EDTA,
1 mmol/L DTT, 1 mmol/L PMSF, and 1% protease
inhibitor cocktail on ice to give a final volume of 9 µL
per sample. After addition of 2 µL H2O and 3
µL 5x EMSA-binding buffer [containing 20% glycerol, 5 mmol/L
MgCl2, 2.5 mmol/L EDTA, 2.5 mmol/L DTT,
250 mmol/L NaCl, 50 mmol/L Tris-HCl, pH 7.5, 0.25 mg/mL
poly(dI–dC) · poly(dI–dC)], the mixture was incubated on ice
for 10 minutes. The radiolabeled AP-1 probe (50 000 cpm/µL) was then
added and the mixture was incubated on ice for another 30 minutes.
After addition of 2 µL of 50% glycerol, the samples were loaded on a
nondenaturing 5% acrylamide/bisacrylamide
(29:1) gel. DNA–protein complexes were fractionated by electrophoresis
at 20 V (4°C) for 16 hours in 0.25x Tris–borate–EDTA buffer. Gels
were dried, exposed to storage phosphor screens, and quantified by
phosphorimage analysis (Storm 860, Molecular Dynamics).
[3H]Cholesteryl Ester Hydrolysis
Peritoneal macrophages were incubated between days 1 and
2 after plating for 24 hours in DMEM containing 10% FBS,
[3H]cholesterol 1.5 µCi/mL
(Amersham), and acLDL 50 µg of protein/mL. Thereafter, the cells were
washed as described above, triplicate dishes were terminated to
ascertain the 0' time value of [3H]cholesteryl
ester, and the other dishes were incubated for 6 to 48 hours in
serum-free medium containing 40 µg/mL apoproteins derived from
delipidation of HDL,13 and 20 µg/mL sonicated
dioleoylphosphatidylcholine liposomes (Sigma). At the end of
incubation, the cells were washed with PBS and scraped with 1 mL 50%
methanol and 2x1 mL 100% methanol. After addition of an equal volume
of chloroform, the lipids were extracted and analyzed by thin
layer chromatography by using chloroform/ethyl acetate
(95:5, vol/vol) to separate
[3H]cholesterol from
[3H]cholesteryl ester.
Statistical Analysis
The results are presented as mean±SEM values. The
statistical significance of difference between groups was evaluated
with Student's t test.
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Results |
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The prominent difference between HAR and LAR rabbits is the much lesser accumulation of cholesteryl esters in the aorta of the LAR rabbits after cholesterol feeding. Because different rates of cholesteryl ester hydrolysis might have contributed to such a result, we examined the rate of cholesteryl ester hydrolysis in peritoneal macrophages. Cells were cultured in medium containing [3H]cholesterol and acLDL for 24 hours. The percentage of label recovered in cholesteryl ester was determined at the end of 24-hour pulse and during 48-hour chase in the presence of complexes of HDL apoproteins and phosphatidylcholine liposomes in the medium. Results obtained with macrophages of 3 HAR and 3 LAR rabbits are shown in Figure 1
. Within the first 6 hours there was no
loss of [3H]cholesteryl ester. Between 6 and 48
hours, the decrease of [3H]cholesteryl ester
was linear and there was no difference between the cells of HAR and LAR
rabbits. This finding did not support the notion that altered
cholesteryl ester hydrolysis could contribute to the difference in the
atherosclerotic response to cholesterol feeding of HAR and
LAR rabbits.
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In the next experiments, we compared SR activity as determined by the
uptake of acLDL in peritoneal macrophages from HAR and LAR
rabbits. Figure 2 shows the results
obtained from cells of 13 LAR and 12 HAR rabbits. Macrophages
from LAR rabbits had a significantly higher specific uptake of acLDL
than macrophages from HAR rabbits. There was no difference in
nonspecific uptake. In several experiments, saturation kinetics of
uptake of acLDL by peritoneal macrophages from HAR and LAR
rabbits were analyzed (Figure 3).
In experiments with macrophages from LAR rabbits, the apparent
Vmax was significantly higher whereas no
difference was found in Km or slope. Thus,
the higher uptake of acLDL by macrophages isolated from LAR
rabbits is apparently caused by a higher receptor number rather than by
an increase in affinity.
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To determine whether the high expression of SRs in macrophages
of LAR rabbits is regulated at the level of transcription, we
determined SR-A mRNA in macrophages of rabbits from each group
by Northern blot analysis (Figure 4a). The membranes were exposed to probes
complementary to sequences in the collagen-like domain of the SR and
thus the 3 major bands of SR-AI and II, namely, 1.8, 2.8, and 3.8 kb,
could be visualized. A significantly higher signal was obtained with
mRNA isolated from LAR rabbits. The membranes were rehybridized with a
rabbit ß-actin cDNA probe (Figure 4b). The intensity of the
bands was quantified and the data were expressed as SR-A mRNA relative
to ß-actin mRNA. The results (mean±SE values of 3 animals per
strain) show a significantly higher expression of SR-AI/II in
the macrophages of LAR rabbits, a finding that is
consistent with the higher uptake of DiI-acLDL by these
cells.
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SR gene expression is regulated via a signal transduction pathway
involving AP-1 among other transcription factors. To learn more about
the regulatory processes involved in the SR activity and expression in
HAR and LAR macrophages, we studied AP-1 activity in these
cells. It is noteworthy that we found a time-dependent effect, with low
AP-1 in freshly isolated peritoneal macrophages and a
substantial increase of AP-1 activity during the first 2 hours of
cultivation at 37°C, when the cells became adherent to culture
dishes. AP-1 activity was highest after 24 hours; however, there was no
significant difference between AP-1 activity in macrophages
from both rabbit strains (Figure 5).
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To learn whether a difference in SR activity between LAR and HAR
rabbits could also be detected in vivo, we determined the clearance of
DiI-acLDL from plasma after intravenous injection in 6 LAR
and 6 HAR rabbits. Figure 6 depicts a
representative experiment in a pair of LAR and HAR
rabbits in which the decay curves of fluorescence intensity are
superimposed. There was no difference in the
t1/2 of DiI-acLDL clearance between the HAR and
LAR rabbits, suggesting that there is no difference in hepatic SR
activity in the 2 strains of rabbits.
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Under certain circumstances, such as in the presence of
macrophage colony–stimulating factor (M-CSF), high expression
of SR mRNA in macrophages was accompanied by high expression of
ApoE.14 Therefore, we also examined the expression of ApoE
in macrophages of LAR and HAR rabbits. Membranes were
hybridized with a rabbit ApoE cDNA probe, stripped of the probe, and
rehybridized with a rabbit ß-actin probe. The expression of ApoE in
macrophages of LAR rabbits was 74% higher than in HAR rabbits,
but the difference did not reach statistical significance (Figure 7).
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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The aim of this study was to explore if certain factors of macrophage function involved in cholesterol balance are related to the attenuated atherosclerotic response of LAR rabbits to hypercholesterolemia. Because of the paucity of blood monocytes in normocholesterolemic rabbits, we chose to study peritoneal macrophages as substitutes for "lesion macrophages." Investigations in pigeons that differed in their susceptibility to develop atherosclerosis when fed cholesterol-rich diets have shown an impairment in cholesteryl ester hydrolysis in peritoneal macrophages derived from the susceptible birds.4 In the present study, we did not find any difference in the rates of hydrolysis of cholesteryl ester by macrophages derived from the 2 strains of rabbits. Thus, the mechanism responsible for the difference in aortic cholesterol accumulation found in the above-mentioned pigeons4 is apparently not operative in the LAR and HAR rabbits.
The major new finding of the present study is that the SR activity
in macrophages of normocholesterolemic LAR
rabbits was consistently higher than in the HAR rabbits. At
first glance such a result might seem contradictory to our previous
findings, which showed that induction of SR in aortic SMCs of LAR
rabbits was highly attenuated when compared with HAR
rabbits.2 However, there are significant differences in SR
regulation in SMCs and in macrophages. First, the SR in
macrophages is expressed constitutively and its activity is
high, whereas in SMCs this receptor is hardly expressed unless
stimulated.15 16 17 18 Second, the regulation of SR in
macrophages and SMCs is rather different, especially in
response to various effectors. Thus, although platelet secretory
products were shown to inhibit SR activity in
macrophages,19 20 they potently stimulate SR
activity in rabbit and human SMCs.17 Among the active
factors in the platelet secretory products was transforming
growth factor-ß1, which enhanced SR activity in SMCs 
2-fold, and
when combined with platelet-derived growth factor-BB and epidermal
growth factor, the activation was 12-fold.17 On the
other hand, transforming growth factor-ß1 was shown to inhibit SR
activity in THP-1 macrophages.21 The other
cytokines that have an opposite influence on SR in
macrophages and SMCs are tumor necrosis factor-
and
interferon- (IFN-). Both cytokines cause a reduction in
SR expression in macrophages22 23 24 but upregulate
the SR expression in SMCs.25
Recently, IFN- has been implicated to potentiate
atherosclerosis in ApoE knockout mice.26
In that study, IFN- receptor knockout mice were crossed with ApoE
knockout mice and fed a western diet for 12 weeks. There was a
significant reduction in atherosclerotic lesion size and a 60%
reduction in lesion lipid accumulation in these double knockout mice.
In the above-mentioned study,26 disruption of the IFN-
receptor gene also resulted in marked induction of hepatic ApoA4 and an
increase in plasma phospholipid–ApoA4–rich particles, which are
potentially atheroprotective.27 One could also expect in
these double knockout mice an increase in macrophage SR
activity because of the absence of IFN- receptor.
Augmentation of SR expression in macrophages, which results in an increase in the number of SR, was reported after treatment with macrophage colony–stimulating factor (M-CSF)14 28 or dexamethasone.29 It is noteworthy that M-CSF prevented atherosclerosis in Watanabe Heritable Hyperlipidemic rabbits,30 without reducing plasma cholesterol levels, and dexamethasone treatment suppressed atherosclerosis in cholesterol fed rabbits.31 These 2 independent studies30 31 provided evidence, contrary to the prevailing opinion, that an increase in SR-A activity in macrophages can be associated with attenuation of atherosclerosis. On the other hand, accelerated atherosclerosis, which was ascribed to an increase in macrophage SR activity, was found in mice lacking tumor necrosis factor receptor p55.32
The role of the macrophage SR in atherosclerosis was further investigated in a recent publication in which the creation of SR-AI/II knockout mice was reported by Suzuki et al.33 When these mice were fed a high-fat diet containing 1.25% cholesterol for 11 weeks, plasma cholesterol levels rose to >300 mg/dL in both SR-A knockouts and controls, but no data concerning development of atherosclerosis were given. When SR-A knockout mice were crossed with ApoE knockout mice, the lesion size in the female double knockout mice at 5 months of age was significantly smaller than in ApoE knockout mice. However, in the double knockout mice, plasma cholesterol levels were 50% higher than in the ApoE knockout mice. Thus, there could have been an increase in size of the atherogenic lipoprotein particles in the double knockout mice, in which case one might expect a reduction in atherosclerosis despite higher plasma cholesterol levels.34 The latter possibility is further strengthened by the finding in a recent study35 in which SR-A knockout mice were crossed with ApoE3Leiden transgenic mice. In these mice fed an atherogenic diet, serum cholesterol, triglycerides, and ApoE levels were the same as in controls (SR-A +/+; ApoE3Leiden). However, contrary to expectations, SR-A deficiency did not result in reduction of atherosclerosis in this mouse model and even more severe complex lesions were found in the SR-A -/-; ApoE3Leiden mice.35
In another study with SR-AI/II knockout mice it was reported that the serum decay and liver uptake were the same in control and knockout mice irrespective whether a high or low dose of acLDL was injected.36 The authors concluded that additional receptors (other than SR-AI/II) participate in the uptake of acLDL in the liver. Presently, there is no difference in the plasma decay when HAR and LAR rabbits are injected with DiI-acLDL, and thus our findings would agree with these conclusions.36
How do we envisage the putative role of high SR expression of LAR rabbit macrophages in attenuation of atherosclerosis? Because SR expression is not downregulated by cholesterol, a macrophage expressing more SR could be better equipped to metabolize modified lipoproteins efficiently. In human monocytes stimulated with M-CSF, an increase in macrophage SR occurred in parallel with a rise in ApoE mRNA,14 a major participant in reverse cholesterol transport. The importance of macrophage ApoE in the reduction of atherosclerosis (without reduction of plasma cholesterol levels)37 accrued from studies on ApoE-deficient mice that were transplanted with normal bone marrow. Moreover, macrophage-derived ApoE, which comprised <7% of normal plasma levels, restored the cholesterol efflux capacity of ApoE-deficient plasma.38 The conclusion of the latter study was that low-dose expression of macrophage ApoE protected ApoE-deficient mice from atherosclerosis.38 Moreover, increased atherosclerosis was found in irradiated mice, transplanted with ApoE null bone marrow from ApoE knockout mice, compared with mice transplanted with normal bone marrow.39 In the present study, ApoE mRNA in macrophages of LAR rabbits was 1.7-fold higher than in those of HAR rabbits, but the difference did not reach statistical significance (P=0.09). Thus, it appears that in the presence of normal or moderately increased ApoE, macrophages with high SR activity could contribute to lesser cholesterol accumulation in the aortae of LAR rabbits.
In a recent study on human monocyte–derived macrophages, higher uptake of modified lipoproteins and higher expression of ApoE mRNA were found in healthy old subjects than in young individuals.40 These results supported the notion that higher expression of SR and ApoE in monocyte-derived macrophages could be beneficial, because these old males (mean age, 84 years) had no clinical coronary heart disease. In another study, a remarkable pedigree of a Canadian family was described, in which 2 members had planar xanthomas and 7 members showed overexpression of SR mRNA in monocytes and macrophages.41 Premature coronary heart disease was not found in any member of the family. In fact, not 1 in the whole family of 13 members, 31 to 90 years of age, died of coronary heart disease, although in 6, plasma cholesterol levels were >200 mg/dL.41 The authors suggest that the overexpression of SR and CD36 in monocytes may have a protective effect on atherogenesis; this is in accord with the findings of our study.
In conclusion, the increased expression of SR-A in macrophages in the presence of adequate amounts of ApoE may play a role in attenuation of atherosclerosis in LAR rabbits. This postulate could be in accord with the accepted protective role of SR-A in host defense against lethal endotoxic shock induced by Gram-negative bacterial infections.42 43
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Acknowledgments |
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This study was supported by a grant from the Deutsche Forschungsgemeinschaft (Th 374, 2-2), a fellowship award from the International Atherosclerosis Society to Dr Joachim Thiery, and the Mario Shapiro Fund, the Hebrew University, Jerusalem, to Dr Yechezkiel Stein. We are indebted to Ulrike Haas and Wolfgang Wilfert for their skillful technical assistance. We thank Dr Peter Lohse for synthesis of the primers used in this study. We greatly appreciate the help of Dr Michelle Mietus-Snyder for setting up molecular techniques as well as her expert advice.
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Footnotes |
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Dedicated to Professor Dr Dietrich Seidel on the occasion of his 60th birthday.
Received August 24, 1998; accepted October 9, 1998.
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References |
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