© 2000 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
Macrophage Cholesterol Metabolism, Apolipoprotein E, and Scavenger Receptor AI/II mRNA in Atherosclerosis-Susceptible and -Resistant Mice
From the Division of Medicine, Geriatric Unit (G.F., A.B.-Y., S.B.); the Lipid Research Laboratory, Division of Medicine, Hadassah University Hospital (Y.D., G.H., Y.S.); and the Department of Experimental Medicine, Hebrew University-Hadassah Medical School (M.B.-N., O.S.), Jerusalem, Israel.
Correspondence to Prof Y. Stein, Lipid Research Laboratory, Division of Medicine, Hadassah University Hospital, PO Box 12220, Jerusalem 91120, Israel. E-mail ystein{at}hadassah.org.il
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Abstract |
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Abstract—Female mice known to be susceptible (C57BL) and resistant (C3H and BALB/c) to diet-induced atherosclerosis were studied. Feeding of a cholate-containing atherogenic diet for 1 month resulted in an increase in plasma total cholesterol, little or no change in total phospholipids and high density lipoprotein (HDL) cholesterol, and a fall in HDL phospholipid, which was most pronounced in the C57BL strain. In elicited macrophages, cholesterol esterification was lower with acetylated low density lipoprotein (acLDL) and higher with ß-very low density lipoprotein (ß-VLDL) in C57BL than in C3H or BALB/C strains. In resident macrophages, acLDL enhanced cholesterol esterification more than did rabbit ß-VLDL. With acLDL, more apolipoprotein E (apoE) was recovered in all macrophage cultures. In macrophages from chow-fed mice, most apoE was in the medium, whereas in mice fed an atherogenic diet, half of the apoE was in the cells. ApoE protein was highest in macrophages from BALB/c mice fed an atherogenic diet; an increase in apoE mRNA occurred in BALB/c and C3H macrophages. Scavenger receptor AI/II mRNA was significantly higher in macrophages from atherosclerosis-resistant mice. Thus, higher HDL phospholipid and plasma apoE levels (reported by others), together with high macrophage scavenger receptor AI/II mRNA, could inhibit accretion of cholesterol in the vessel wall in the 2 resistant strains.
Key Words: macrophages • cholesteryl ester • apolipoprotein E • scavenger receptors • atherosclerosis
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Introduction |
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Ever since Anitschkow1 introduced cholesterol feeding as a method to induce atherosclerosis in rabbits, this approach has been used in other species. Mice,2 hamsters,3 and pigeons4 were among the preferred animals because of their small size, but they proved to be more resistant than rabbits to cholesterol enrichment of the basic diet. Cholate addition to the diet promoted the development of hypercholesterolemia and in some strains of mice induced aortic atherosclerosis.2 These studies have provided evidence that certain strains of mice are resistant to diet-induced atherosclerosis. The accepted mechanism responsible for this trait was the difference in HDL levels in response to the diet.5 Thus, in the C57BL/6 strain, diet-induced hypercholesterolemia was accompanied by a reduction in plasma HDL, whereas lesser reduction occurred in atherosclerosis-resistant strains, such as C3H and BALB/c.6 Searching for additional differences in propensity to develop atherosclerosis, another study focused on some aspects of lipid metabolism in mouse macrophages.7 Induction by the atherogenic diet (AD) of acyl coenzyme A:cholesterol acyl transferase (ACAT) was found in homogenates of macrophages derived from C57BL/6 but not from C3H mice, whereas macrophage neutral cholesteryl ester (CE) hydrolase in C3H mice was twice that in C57BL/6 mice.7 In the present study, we compare cholesterol esterification and hydrolysis in intact macrophages from 3 strains of mice, C57BL, C3H, and BALB/c.
Another aspect of macrophage involvement in lipid metabolism concerns the role of scavenger receptor (SR) types AI and II (SR-AI/II) and apoE in lipoprotein uptake and cholesterol efflux. These parameters have been investigated recently in 2 strains of rabbits, which differ in their susceptibility to diet-induced atherosclerosis.8 It appears that macrophages isolated from rabbits with low atherosclerotic response to cholesterol feeding have a higher expression of SR-AI/II and, to some extent, of apoE than those derived from atherosclerosis-prone rabbits.8 Therefore, in the present study, we also compared the expression of apoE and SR-AI/II in the 3 strains of mice on chow diets and on ADs, which to the best of our knowledge has not been reported so far.
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Methods |
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Animals
Two-month-old female mice (C57BL/6, C3H, and BALB/c) were housed in rooms at constant temperature with a 12-hour light-dark cycle and fed a pelleted chow diet or a high fat (15%), high cholesterol (1.25%), sodium cholate (0.5%) diet (Teklad 88051), designated "atherogenic."
Lipoproteins
LDLs were isolated from human plasma according to Havel et
al.9 Acetylation of LDL was performed
according to Fraenkel-Conrat.10
Studies in Cell Culture
Peritoneal macrophages obtained by peritoneal lavage
with PBS were designated "resident." Peritoneal macrophages
isolated 4 days after intraperitoneal injection of
thioglycollate were designated "elicited."11
Macrophages (1x106) were seeded in 30-mm
Petri dishes or 0.5x106 cells per well in
12-well multiwells in DMEM containing 10% FBS. The nonadherent cells
were removed after 2 hours, and the adherent macrophages were
cultured for 24 hours in DMEM containing 10% FBS. To label cells with
[3H]cholesterol, the latter
was added to serum-containing medium (1 µCi/mL). To study the
expression of SR-AI/II mRNA, apoE mRNA, and secretion of apoE, the
cells were incubated for 24 hours in serum-free medium containing 1%
Nutridoma (Boehringer-Mannheim) without or with
acetylated LDL (acLDL, 50 µg protein/mL).
Isolation of Cellular RNA
Total cellular RNA was isolated by the procedure of
Cox,12 with some modification as previously
described.13 The integrity of all RNA samples was
determined by electrophoresis on a 1.2% agarose gel containing 2.2
mol/L formaldehyde.
Quantification of ApoE
Cellular and secreted apoE were quantified in cultures
maintained for 24 hours in a serum-free medium with or without acLDL.
The secreted apoE reported is that present in the medium after 24
hours of incubation. The media were collected and, after the addition
of protease inhibitors, centrifuged to eliminate
suspended cells. The apoE was precipitated from the medium by the
addition of 50 µg/mL of fumed silica (Sigma Chemical Co) and
centrifugation at 13 000g for 10 minutes.
Each pellet was washed 3 times with sterile water and dissolved in
gel-loading buffer. Cellular apoE was extracted from the cells with the
use of STEN buffer (50 mmol/L Tris-Cl, pH 7.6, containing 150
mmol/L NaCl, 2 mmol/L EDTA, 1% Nonidet P-40, 20 mmol/L
phenylmethylsulfonyl fluoride, and 5 µg/mL leupeptin).
Samples were electrophoresed on 10% polyacrylamide gels
containing SDS, as described.14 The proteins were
transferred to nitrocellulose paper by blotting and treated with an
anti-rat apoE polyclonal antiserum (1:5000 dilution, provided by Dr
R.E. Pitas, Gladstone Institutes, San Francisco, Calif). The
nitrocellulose immunoblot was then incubated with goat
anti-rabbit secondary antibody conjugated to horseradish peroxidase
(1:10 000 dilution, Jackson ImmunoResearch). After a washing to remove
unbound antibody, the immunocomplex was detected by use of an ECL kit
(Amersham Corp), according to the manufacturer’s instructions.
Quantification of the level of cellular and secreted apoE was
determined by densitometric scanning (Bio-Rad Multi-Analyst PC, version
1.1).
Estimation of Macrophage SR-AI/II and ApoE mRNA by
RT-PCR
The 2-step procedure is based on initial reverse transcription
(RT) of RNA to cDNA, followed by amplification of cDNAs by polymerase
chain reaction (PCR), as described below. cDNAs were prepared from
serial dilutions (0.5, 1, 1.5, and 2 µg) of total cellular RNA by
mixing with 200 ng of a random hexamer primer, heating to 65°C for 4
minutes, and slowly cooling to 25°C to anneal the primer. The
reaction mixture (total volume 50 µL) consisted of 50 mmol/L
Tris-Cl (pH 8.32), 140 mmol/L KCl, 10 mmol/L
MgCl2, 4 mmol/L dNTP, 4 mmol/L
dithiothreitol, and 40 µm avian myeloblastosis virus RT. The
reactions were carried out at 42°C for 2 hours.
PCRs for the expression of SR-AI/II were prepared in a final volume of
20 µL, and they contained 5 µL of cDNA reaction mixture prepared
from 1 µg RNA, 23 mmol/L dNTP, 18.87 mmol/L
(NH4)2SO4,
76 mmol/L Tris (pH 8.8), 7.67 mmol/L
MgCl2, 11.36 mmol/L dithiothreitol, 193
µg/mL BSA, 11.36% dimethyl sulfoxide, 10 µCi of
[
-32P]dCTP (1 Ci=37 GBq), and 250 ng each of
oligonucleotide primers A (5'-TGA CAC TGC TTG ATG TTC
AAC TCC 3') and B (5'-TCA TGG GCT CCA CTA CCA CCA AC 3'). Primers C
(5'-TGG ATG ACG ATA TCG CTG CGC TCG 3') and D (5'-GGT GCT CCT CAG GGG
CCA CACG 3'), specific for ß-actin mRNA, were included in the
reaction mixtures. This gene serves as a reporter for normalizing cDNA
input. The reaction mixture was heated to 95°C for 7 minutes, and Taq
polymerase (Boehringer-Mannheim, 1 µL per reaction) was added
at 88°C. cDNA was amplified by use of a PCR thermal cycler
(Minicycler, MJ Research). The reaction mixture was heated to 95°C
for 7 minutes, and this was followed by 22 cycles consisting of 1.5
minutes for denaturation at 94°C, 1 minute for annealing at 62°C,
and 1 minute for extension at 72°C, with a step cycle for 7 minutes
at 72°C for 1 cycle.
PCRs for apoE were prepared in a final volume of 20 µL and contained the same reaction mixture as for the SRs. The oligonucleotide primers for the apoE amplification were E (5'-GGA CAT ATGAC GGA AGT AAA GGC 3') and F (5'-TGT CTT CCA CTA TTG GCT CGA ACC 3'). Primers C and D, specific for ß-actin mRNA, were included in the reaction mixtures. The reaction mixture was heated to 95°C for 10 minutes, and this was followed by 22 cycles consisting of 1.5 minutes for denaturation at 94°C, 1 minute for annealing at 65°C, and 1 minute for extension at 72°C, with a step cycle for 7 minutes at 72°C for 1 cycle. Twenty-two cycles were used, because SR, apoE, and ß-actin products from 1 µg RNA were in the linear range of the PCR; ie, efficacy of the PCR for apoE, SR, and ß-actin was identical and, hence, allowed normalization and comparison of the products obtained.
The amplified products were subsequently analyzed on an 8% polyacrylamide gel. The radioactive bands, corresponding to ß-actin (304 bp), apoE (609 bp), or SR (535 bp), were determined in a Bio Imaging Analyzer BAS 1000 (Fuji) and were expressed as percentage of photo-stimulated luminescence units.
Cholesterol Esterification and Hydrolysis of
CE
To study cholesterol esterification and CE
hydrolysis in cultured macrophages, the cells were incubated
for 24 hours in medium containing 10% FBS and
[3H]cholesterol (1 µCi/mL). The
medium was removed and replaced by the same medium containing acLDL (50
µg protein/mL), and the cells were incubated for 5 or 24 hours. To
prepare 3H-labeled acLDL,
[3H]cholesterol (20 µCi) in
acetone was added (2 µL/mL) to medium containing acLDL (1 mg
protein/mL) and lipoprotein-deficient serum (2 mg protein/mL) and was
left for 16 hours. The [3H]acLDL (50 µg
protein/mL) was added to serum-free medium and incubated with cells for
5 hours. ß-VLDL (density 1.006 to 1.019 g/mL) obtained from
cholesterol-fed rabbits was also used instead of acLDL to
induce cholesterol esterification. Thereafter, the medium
was removed, and the cells were washed for 15 minutes at 37°C in
medium containing 0.5% BSA, 15 minutes in serum-free medium, and then
in PBS. At that time, triplicate wells were terminated and served for
the determination of total and CE radioactivity. For estimation of CE
hydrolysis, macrophages that had been exposed to acLDL for 24
hours were incubated in medium that contained lipoprotein-deficient
serum, ACAT inhibitor (5 µg/mL), and
liposomes.8 In experiments with resident peritoneal
macrophages from chow-fed or AD-fed mice, an alternate protocol
was also used in which unlabeled cells were incubated with
[3H]acLDL. After 5 or 24 hours, the cells were
washed with PBS and scraped with methanol, and lipids were extracted
according to Folch et al.15 Cell protein was determined on
the precipitate, and separation of
[3H]cholesterol and
[3H]CE was performed as described in Reference
8 .
Materials
Culture medium and FBS were obtained from GIBCO.
[7(n)-3H]Cholesterol
was from Amersham. All reagents and dioleoylphosphatidylcholine
were of analytical grade and were obtained from Sigma. ACAT
inhibitor, compound 58-035, was generously provided by
Sandoz (East Hanover, NJ).
Statistical Evaluation
The results are presented as mean±SE. The difference
between groups was tested by the Student t test.
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Results |
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Plasma Lipid Values
Macrophages investigated in the present study were derived from 3 strains of female mice fed either chow or an AD for 1 month. Plasma lipids (10 mice per group) from mice on chow or AD were significantly lower in the C57BL mice (P<0.01) than in the C3H or BALB/c mice (Table 1
). On
the AD, the percent change from chow was computed for 4 lipid
parameters, ie, total cholesterol (TC), HDL
cholesterol (HDL-C), total phospholipids (T-PL), and HDL
phospholipids (HDL-PL, Figure). The
relative increase in TC and HDL-C on AD was similar in the 3 strains.
With respect to phospholipids, a 22% decrease was found in T-PL in the
C57BL mice, there was no change in the C3H mice, and there was a slight
increase in the BALB/c mice. The difference in response to the AD was
more marked with respect to HDL-PL, so that although in C57BL there was
a 66% decrease, it was only 29% and 17% in C3H and BALB/c,
respectively (Figure). The effect of AD on HDL-C and HDL-PL was
expressed as percentage of TC and T-PL, respectively. For the AD, the
percentage of HDL-C decreased from 75% to 24% in C57BL, from 88% to
29% in C3H, and from 82% to 26% in BALB/c. For the chow diet, the
percentage of HDL-PL ranged between 91% and 94% for all 3 strains,
whereas for the AD, it decreased most markedly in C57BL (to 41%) and
only to 66% or 69% in C3H and BALB/c, respectively.
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CE Metabolism in Peritoneal Macrophages
In elicited macrophages incubated with
[3H]cholesterol, the extent of
cellular labeling (dpm/µg cell protein) was not significantly
different among the 3 strains for both diets. In macrophages
exposed to acLDL for 5 or 24 hours, the percentage of
cholesterol esterification did not vary between the chow
diet and AD. After 5 hours on the chow diet,
[3H]CE was 19±1.8% of total radioactivity in
C57BL/6 mice and 26±1.5% and 27±1.6% in C3H and BALB/c mice,
respectively. After 24 hours, these values were 21±2.2% in C57BL/6
and 30±1.5% and 36±2% in C3H and BALB/c mice, respectively. The
higher percentage of CE in C3H and BALB/c mice was statistically
significant (P<0.01), and similar results were obtained
with macrophages isolated after feeding of the AD. Hydrolysis
of cellular [3H]CE, determined after 5 or 24
hours of incubation, was similar in macrophages derived from
the 3 strains on both diets (data not shown). The extent of
cholesterol esterification by resident macrophages
incubated with acLDL, labeled with
[3H]cholesterol for 5 hours, was
similar in the 3 strains, irrespective of whether the cells were
derived from mice fed chow or an AD (data not shown). We also compared
the extent of CE formation with acLDL and ß-VLDL in resident and
elicited macrophages (Table 2).
ß-VLDL induced less CE formation than did acLDL in resident
macrophages in the 3 strains. However, in elicited
macrophages, ß-VLDL induced more cholesterol
esterification than did acLDL in cells derived from C57BL mice (Table 2).
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Macrophage ApoE
Chow Diet
ApoE was determined in resident macrophages from mice on a
chow diet or AD. Macrophages from chow-fed mice were incubated
for 24 hours with or without acLDL, and apoE was found predominantly in
the medium in the 3 strains studied (Table 3). The apoE recovered in cells plus
medium was highest in C57BL mice. In macrophages from C57BL
mice incubated with acLDL, there was a 2-fold increase in
cell-associated and medium apoE, and similar results were observed in
BALB/c macrophages. In cells derived from C3H mice, the
addition of acLDL resulted in a 5-fold increase of apoE in the culture
medium.
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Atherogenic Diet
When the macrophages were derived from mice fed the AD,
the total amount of apoE recovered in C3H (without acLDL) and in BALB/c
mice with and without acLDL was increased. In C57BL
macrophages, the total amount of apoE recovered did not
increase with the feeding of AD. There was a change in the distribution
of apoE between cells and medium in all the strains, with and without
the addition of acLDL, which resulted in an increase in the cellular
association of apoE in macrophages derived from mice fed the AD
(Table 3
).
Expression of ApoE and SR-AI/II mRNA
ApoE mRNA was determined in resident macrophages derived
from mice on a chow diet and those on the AD, in the absence and
presence of acLDL. Incubation with acLDL for 24 hours resulted in a
significant increase in the expression of apoE mRNA in the 3 strains of
mice (Table 4), in accord with the
increase in apoE (Table 3). Expression of apoE mRNA in the
presence of acLDL in macrophages from chow-fed mice showed no
difference between the strains. Macrophages from the
resistant strains, ie, C3H and BALB/c, responded to AD with a
significant increase in apoE mRNA, with the BALB/c being the highest,
whereas no change occurred in C57BL macrophages (Table 4).
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The effect of acLDL on macrophage SR-AI/II mRNA was studied in
chow- and AD-fed mice (Table 5). In the
presence of acLDL, no significant difference was seen in
macrophages derived from chow-fed mice, whereas a significant
increase of SR-AI/II mRNA was observed in all 3 strains fed the AD.
Comparison of SR-AI/II mRNA in the presence of acLDL showed a higher
expression in C3H and BALB/c mice than in C57BL mice fed the AD (Table 5).
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Discussion |
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In the present study, we compared certain plasma lipid parameters in 3 strains of mice: C57BL, which is well known to develop atherosclerosis when fed a cholate-, cholesterol-, and fat-containing diet, and C3H and BALB/c, which are resistant to atherosclerosis.2 16 17 These and other studies5 6 revealed a relationship between low HDL and susceptibility to high HDL and resistance to atherosclerosis. In analogy to the above reports, we found differences in plasma levels of HDL-C in response to the AD. However, so far, little or no mention has been made of the effect of AD on plasma and HDL-PL. Therefore, we also focused on plasma phospholipids, and our results are, in general, compatible with the recently published data by Albers et al.18 In the present study, although the AD caused a similar decrease in HDL-C, the decrease in HDL-PL was significantly greater in C57BL than in the C3H or BALB/c female mice. In tissue culture studies, efflux of cellular cholesterol is dependent on phospholipid concentration in the medium19 20 21 ; therefore, the finding of lower plasma HDL-PL may be important for the propensity of the C57BL mice to develop atherosclerosis on the AD.
Cholesterol metabolism in thioglycollate-elicited macrophages from atherosclerosis-prone and -resistant mice was compared by using ß-VLDL to induce cholesterol accumulation.7 The salient findings were that in macrophages from C57BL mice fed AD, there was induction of ACAT, determined in cell homogenates, and that neutral CE hydrolase was low compared with the level in C3H mice.7 Presently, we studied the induction of cholesterol esterification in intact macrophages, with acLDL. Feeding of the AD did not result in higher cholesterol esterification in elicited and resident macrophages from the C57BL strain. In elicited but not in resident macrophages, cholesterol esterification in the presence of acLDL was higher in C3H and BALB/c than in C57BL macrophages. In addition, although in elicited C57BL macrophages, ß-VLDL induced more cholesterol esterification than did acLDL, this was not so in macrophages from C3H and BALB/c mice. Moreover, in resident macrophages, ß-VLDL induced less cholesterol esterification than did acLDL, even though the concentration of ß-VLDL cholesterol was >5-fold higher.
The role of apoE in the prevention of atherosclerosis has been extensively studied in transgenic mice.22 23 24 Even small amounts of macrophage-derived apoE can attenuate atherogenesis in apoE-deficient mice.25 26 27 Therefore, we investigated macrophage apoE in the 3 strains of mice, and in accord with previous findings,28 the addition of acLDL resulted in a significant increase in apoE. Feeding of the AD affected mainly the distribution between medium and cell-associated apoE, which became equal, as reported for a human apoE-producing J774 cell line.29 The higher recovery of apoE in the cell-associated form might have been due to the diet-induced increase in cell surface proteoglycans, which were shown to trap secreted apoE.29 The increase in apoE in the presence of acLDL was also accompanied by an increase in apoE mRNA in the 3 strains, as has been shown previously for peritoneal macrophages derived from Swiss-Webster mice.28 In the present study, when the mice were fed the AD, this effect was more pronounced in macrophages from C3H and BALB/c strains. The finding of no significant difference in apoE between macrophages derived from AD-fed C57BL and C3H mice was unexpected, inasmuch as plasma levels of apoE were reported to be 50% higher on the low fat diet, and on the AD they were twice as high in C3H than in C57BL mice.30
In macrophages from the 3 strains of mice fed the AD, there was a significant increase in SR-AI/II mRNA after exposure to acLDL. Upregulation of macrophage SR-AI/II mRNA in mice was observed after intraperitoneal injection of LDL and acLDL.31 The choice of SR-AI/II as a candidate for resistance to atherosclerosis in C3H and BALB/c mice, in spite of diet-induced hypercholesterolemia, was based on recently published studies. Thus, treatment with macrophage colony–stimulating factor, which augments the expression of macrophage SR-A mRNA,32 33 was shown to prevent the development of atherosclerosis in Watanabe heritable hyperlipidemic rabbits.34 In addition, dexamethasone, which upregulates macrophage SR-A,35 attenuated atherosclerosis in cholesterol-fed rabbits.36 We have also recently reported that in a strain of rabbits that shows a low atherosclerotic response to diet-induced hypercholesterolemia, the expression of SR-AI/AII mRNA in macrophages was significantly higher than in rabbits with high atherosclerotic response.8 The protective role of macrophage SR (MSR) in atherogenesis was illustrated also in apoE3 Leiden (E3L, a human dysfunctional apoE variant) transgenic mice.37 When these mice were crossed with MSR-A– deficient mice, the E3L-MSR-A-/- mice developed more severe lesions compared with E3L- MSR+/+ mice.37 High functional expression of SR in human monocyte–derived macrophages was also observed in healthy old men (mean age 84 years) compared with young men (18 to 24 years).38 In that study, it was also shown that the macrophages from healthy octogenarians had higher apoE mRNA. It seems of interest to cite a recent study dealing with the effect of genetic background on atherogenesis in apoE-/- mice.39 Thus, apoE-/- mice with an FVB background developed markedly less atherosclerosis than did apoE-/- mice with a C57BL background. However, strain intercross examination of the F2 generation showed a wide range of lesion development but no correlation between lesion area and plasma lipoproteins, such as HDL, apoA-I, apoA-II, or apoJ. The authors suggested that genetic differences in macrophages or in the vessel wall components could be responsible for resistance to atherosclerosis.
In conclusion, there are several salient findings that may explain the resistance of the 2 mouse strains to diet-induced atherosclerosis. The higher HDL-PL and plasma apoE levels, together with high macrophage SR-AI/II mRNA, could inhibit the accretion of cholesterol in the vessel wall.
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Acknowledgments |
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This study was supported in part by a grant from the Ridgefield Foundation (G.F.) and the Mario Shapiro Fund (Y.S.). The excellent assistance of J. Hollander is gratefully acknowledged.
Received December 16, 1999; accepted April 13, 2000.
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