Sphingomyelinase Converts Lipoproteins From
Apolipoprotein E Knockout Mice Into Potent Inducers of
Macrophage Foam Cell Formation
Sudhir Marathe, Yunsook Choi, Andrew R. Leventhal, Ira Tabas
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Abstract—The apoE knockout (E0) mouse is one of the most widely used animal models of atherosclerosis, and there may
be similarities to chylomicron remnant–induced atherosclerosis in humans. Although the lesions of these mice contain
large numbers of cholesteryl ester (CE)-laden macrophages (foam cells), E0 plasma lipoproteins are relatively weak
inducers of cholesterol esterification in macrophages. Previous in vivo work has suggested that arterial wall
sphingomyelinase (SMase) may promote atherogenesis in the E0 mouse, perhaps by inducing subendothelial lipoprotein
aggregation and subsequent foam cell formation. The goal of the present study was to test the hypothesis that the
modification of E0 lipoproteins by SMase converts these lipoproteins into potent inducers of macrophage foam cell
formation. When d⬍1.063 E0 lipoproteins were pretreated with SMase and then incubated with E0 macrophages,
cellular CE mass and stimulation of the cholesterol esterification pathway were increased ⬇5-fold compared with
untreated lipoproteins. SMase-treated E0 lipoproteins were more potent stimulators of cholesterol esterification than
either E0 lipoproteins in the presence of lipoprotein lipases or oxidized E0 lipoproteins. The uptake and degradation of
SMase-treated E0 lipoproteins by macrophages were saturable and specific and substantially inhibited by partial
proteolysis of cell-surface proteins. Uptake and degradation were diminished by an anti-apoB antibody and by
competition with human Sf 100-400 hypertriglyceridemic VLDL, raising the possibility that a receptor that recognizes
apoB-48 might be involved. In conclusion, SMase-modification of E0 lipoproteins, a process previously shown to occur
in lesions, may be an important mechanism for foam cell formation in this widely studied model of atherosclerosis.
Moreover, the findings in this report may provide important clues regarding the atherogenicity of chylomicron remnants
in humans. (Arterioscler Thromb Vasc Biol. 2000;20:2607-2613.)
Key Words: sphingomyelinase 䡲 lipoproteins 䡲 macrophages 䡲 foam cells 䡲 apolipoprotein E knockout mice
T
he apoE knockout (E0) mouse develops extensive atherosclerosis and has become one of the most widely used
animal models to explore atherogenesis in vivo.1–3 It is
presumed that the remnant-like particles that accumulate in
the plasma of these mice are atherogenic and induce cholesteryl ester (CE)-laden macrophages (foam cells) formation,1,2 yet previous studies have shown that these plasma
lipoproteins can induce only modest CE accumulation in
cultured macrophages.4 – 6 The answer to this dilemma undoubtedly lies in the overall hypothesis that these lipoproteins
become modified in the arterial wall to a form with
increased ability to induce macrophage foam cell formation. Previous work has shown that the addition of 5
␮g/mL lipoprotein lipase (LpL) or the oxidation of E0
lipoproteins can increase the ability of the particles to
induce foam cell formation in cultured macrophages.4,5
Whether there is sufficient LpL or sufficiently extensive
lipoprotein oxidation in E0 lesions to facilitate these
processes in vivo remains to be determined.
See page 2509
A lipoprotein modification that is known to occur in
lesions and that greatly enhances the ability of LDL to induce
CE accumulation in macrophages is lipoprotein aggregation.7–12 One possible inducer of subendothelial lipoprotein
aggregation is arterial wall sphingomyelinase (SMase).13–19
SMase treatment of lipoproteins results in aggregation and
fusion of the particles, and aggregated LDL isolated from
human lesions, but not monomeric lesional LDL or native
plasma LDL, is enriched in ceramide, a marker of SMase
action on the lipoproteins.14 A particular species of SMase
called secretory SMase (S-SMase) is the best candidate for
this enzyme because it is the only known extracellular SMase
in mammals, it is secreted by cultured arterial wall cells, and it
is found in normal endothelium and especially in atherosclerotic
intima.17 The role of S-SMase in the E0 mouse may be
particularly important for the following reasons: (1) lipoproteins
from the lesions of E0 mice are aggregated (Maor et al20 and
unpublished data from our researchers); (2) lipoproteins isolated
Received May 22, 2000; revision accepted September 1, 2000.
From the Departments of Medicine (S.M., Y.C., A.R.L., I.T.) and Anatomy and Cell Biology (I.T.), Columbia University, New York, NY.
Correspondence to Ira Tabas, MD, PhD, Department of Medicine, Columbia University, 630 West 168th St, New York, NY 10032. E-mail
[email protected]
© 2000 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol. is available at http://www.atvbaha.org
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Figure 1. S-SMase–treated d⬍1.063 lipoproteins from E0 mice are potent inducers of cholesterol esterification in macrophages. A,
J774 macrophages were incubated in medium alone (No LPs) or in medium containing 50 ␮g/mL untreated d⬍1.063 lipoproteins from
E0 mice (E0 LPs), E0 lipoproteins treated with S-SMase, or the same solution containing S-SMase but no lipoproteins. After a 24-hour
incubation, the cells were assayed for CE mass. B, Peritoneal macrophages from C57 mice were incubated in medium alone (No LPs)
or in medium containing 50 ␮g/mL untreated d⬍1.063 lipoproteins from E0 mice (E0 LPs), E0 lipoproteins treated with bacterial SMase,
oxidized E0 lipoproteins, or E0 lipoproteins in the presence of 5 ␮g/mL LpL (E0 LPs/LpL). After incubation for 18 hours, the cells were
assayed for the incorporation of [14C]oleate into cholesteryl [14C]oleate.
from the lesions of E0 mice are enriched in ceramide14; (3) E0
lesions stain abundantly for immunoreactive SMase16; (4) E0
lipoproteins are among the most susceptible lipoproteins to the
action of S-SMase, probably because these lipoproteins uniquely
have a high sphingomyelin-to-phospholipid ratio15,21; and, most
important, (5) E0 mice on a SMase knockout background have
smaller lesions than E0 mice with normal SMase.18
In light of these findings, we hypothesize that the hydrolysis of subendothelial E0 lipoproteins by arterial wall SMase
converts these particles to a form that can induce macrophage
foam cell formation. To test this hypothesis, we compared the
ability of native and SMase-treated E0 lipoproteins to induce
CE accumulation and to stimulate the cholesterol esterification pathway in macrophages. Our results show that the
SMase-aggregated E0 lipoproteins are indeed potent inducers
of macrophage foam cell formation. Moreover, our results
indicate that a substantial portion of the cellular uptake of
these aggregated particles involves the interaction of apoB
with a cell-surface receptor that is uniquely competed by
human hypertriglyceridemic VLDL.
Methods
Materials
The Falcon tissue culture plasticware used in these studies was
purchased from Fisher Scientific Co. Tissue culture media and other
tissue culture reagents were obtained from GIBCO BRL. FBS was
obtained from Hyclone Laboratories. LpL was purified from bovine
milk as previously described22 and provided by Kirsten D. Mazany
and Dr Kevin J. Williams. The source of S-SMase was serum-free
conditioned medium harvested from DG44 Chinese hamster ovary
cells stably transfected with human acid SMase cDNA.13,23 Bacterial
SMase from Bacillus cereus, chondroitin ABC lyase from Proteus
vulgaris, heparitinase from Flavobacterium heparinum, and trypsin
from porcine pancreas (type IX, 13 000 U/mg) were purchased from
Sigma Chemical Co. Goat anti-murine apoB was prepared and
characterized as described previously24 and provided by Drs Kevin J.
Williams and Daniel Levine. Receptor-associated protein (RAP) was
provided by Dr Dudley Strickland. Mouse IgG2a and anti-mouse
CD12/CD32 Fc receptor antibody were obtained from Chemicon and
PharMingen, respectively. All other chemicals and reagents were
obtained from Sigma Chemical Co, and all organic solvents were
obtained from Fisher Scientific Co.
Native and Modified Lipoproteins
The VLDL and LDL fraction from the plasma of chow-fed 4- to
8-month-old female or male E0 mice was prepared through preparative ultracentrifugation (d⬍1.063 g/mL).15 S-SMase treatment of
the lipoproteins was carried through the incubation of 50 ␮L (5 ␮g)
lipoproteins in 100 mmol/L HEPES, pH 7.2, 100 ␮mol/L ZnCl2 with
85 ␮L DG44 conditioned media for 16 hours at 37°C.15 For
treatment with bacterial SMase, 1 mg/mL lipoproteins was incubated
with 50 mU/mL B cereus SMase for 4 hours at 37°C in PBS
containing 2 mmol/L MgCl2.12 LDL (d⫽1.020 to 1.063 g/mL) was
isolated from fresh human plasma through preparative ultracentrifugation and acetylated as described previously.25,26 Oxidation was
carried out by incubation of the E0 lipoproteins (1 mg/mL) with 5
␮mol/L CuSO4 for 18 hours at 37°C, followed by the addition of
1 mmol/L EDTA and dialysis against 150 mmol/L NaCl, 0.3 mmol/L
EDTA. The lipoproteins were labeled with 125I with the use of
Iodogen-coated tubes (Pierce) and Na[125I] (NEN Life Science
Products)27; the labeled lipoproteins had a specific activity of 250 to
400 cpm/ng protein and were used within 3 weeks of iodination. Sf
100-400 VLDL from a man with a plasma triglyceride level of 1320
mg/dL was obtained through preparative NaBr ultracentrifugation
(d⬍1.006 g/mL), followed by discontinuous NaCl gradient centrifugation.28 Trypsinized hypertriglyceridemic VLDL, which was generously provided by Drs Sandra Gianturco and William Bradley, was
prepared through the incubation of hypertriglyceridemic VLDL with
10 mg/mL trypsin (Worthington 3⫻ crystallized) for 2 hours at
37°C, followed by reisolation through ultracentrifugation.29
Cells
J774.A1 macrophages (American Type Culture Collection) were
maintained in spinner culture in DMEM, 10% (v/v) FBS containing
Marathe et al
Sphingomyelinase, Foam Cells, and ApoE Knockout Mice
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Figure 2. The uptake and degradation of
SMase-treated d⬍1.063 lipoproteins from E0
mice represent a saturable, specific process.125Ilabeled d⬍1.063 lipoproteins from E0 mice were
treated with bacterial SMase and then incubated with mouse peritoneal macrophages at
the indicated concentrations for 5 hours. The
media were then assayed for 125I-lipoprotein
degradation.
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50 U/mL penicillin, 50 U/mL streptomycin, and 2 mmol/L glutamine. The medium was replaced with fresh medium each day. Mouse
peritoneal macrophages from 25- to 35-g female C57BL6/J mice and
from various gene-targeted mice were harvested from the peritoneum 3
days after the intraperitoneal injection of 40 ␮g concanavalin A in 0.5
mL PBS.30 ApoE, LDLR, and class A scavenger receptor knockout
mice were obtained from Jackson Laboratories, and CD36 knockout
mice were kindly provided by Drs Maria Febbraio and Roy Silverstein
(Weill Medical College of Cornell University, New York, NY). The
cells were plated onto 22-mm dishes in DMEM containing 10% (v/v)
FBS, 20% (v/v) L-cell conditioned medium (LCM), 100 U/mL penicillin, 100 ␮g/mL streptomycin, and 292 ␮g/mL glutamine and were used
within 3 days.
Cellular Assays
Lipid extracts of the cells were assayed for total and free cholesterol
mass with gas-liquid chromatography.12 Cellular cholesterol esterification was assayed by measuring the incorporation of [14C]oleate
into cellular cholesteryl [14C]oleate.22 Degradation of the 125I-labeled
lipoproteins was determined from the [125I] cpm of TCA-soluble,
non– chloroform-extractable material in the medium.22 Unless indicated otherwise, results are given as mean⫾SEM (n⫽3).
Results
Treatment of d<1.063 Lipoproteins From E0 Mice
With SMase Markedly Increases Their Ability to
Induce Foam Cell Formation
To address the hypothesis that SMase-induced aggregation may
contribute to foam cell formation in E0 mice, we incubated J774
macrophages with d⬍1.063 lipoproteins isolated directly from
the plasma of E0 mice or with these lipoproteins after treatment
with S-SMase, the enzyme proposed to induce lipoprotein
aggregation in atherosclerotic lesions.17 Note that J774 macrophages do not synthesize apoE31 and thus provide a model for
foam cell formation in apoE-deficient macrophages. In confirmation of previous results,15,21 S-SMase treatment led to visible
lipoprotein aggregation. As shown in Figure 1A, the untreated,
monomeric plasma lipoproteins caused some degree of CE
accumulation in the macrophages, but the loading induced by
S-SMase–aggregated lipoproteins was 5-fold higher. S-SMase
alone had no effect, indicating that it was the aggregated
lipoproteins and not residual SMase activity that caused the CE
loading.
To determine whether the increase in CE accumulation
involved stimulation of the acyl-CoA:cholesterol acyltrans-
ferase pathway, mouse peritoneal macrophages were incubated with monomeric or SMase-aggregated d⬍1.063 E0
lipoproteins in the presence of [14C]oleate; for this and the
following experiments, soluble bacterial SMase was used,
which has the same effect on lipoproteins as human
S-SMase.12,15 As shown in Figure 1B (first 3 columns),
the SMase-aggregated lipoproteins led to a 5- to 6-fold higher
increase in incorporation of the labeled oleate into cholesteryl
[14C]oleate, indicating acyl-CoA:cholesterol acyltransferase–
mediated cholesterol esterification and not simply cellular
accumulation of undegraded lipoprotein CE. Importantly,
SMase-aggregated d⬍1.063 E0 lipoproteins were also excellent inducers of cholesterol esterification in peritoneal macrophages from E0 mice (143⫾1.2 nmol cholesteryl
[14C]oleate 䡠 mg–1 䡠 18 h–1 versus 107⫾1.0 in macrophages
from wild-type mice). In addition, the uptake and degradation
of SMase-treated 125I-E0 lipoproteins were similar in macrophages from wild-type versus E0 mice (1050⫾39 versus
1012⫾25 ng degraded 䡠 mg–1 䡠 5 h–1). Thus, apoE secretion by
macrophages is not necessary for the uptake and processing
of SMase-modified E0 lipoproteins.
Others have proposed that E0 lipoprotein oxidation or LpL,
acting as a bridging molecule, may facilitate E0 lipoprotein
uptake by macrophages.4,5 As shown in Figure 1B, both of these
perturbations indeed increased cholesterol esterification compared with untreated lipoproteins, but neither was as potent as
SMase-induced aggregation. The addition of LpL to SMaseaggregated E0 lipoproteins, or SMase aggregation of oxidized
E0 lipoproteins, did not increase cholesterol esterification above
that seen with SMase-aggregated lipoproteins (data not shown).
The Uptake and Degradation of d<1.063 E0
Lipoproteins by Macrophages Involve an
Interaction Between One or More Cell Surface
Proteins and ApoB
The uptake and degradation of SMase-treated d⬍1.063 E0
lipoproteins showed evidence of saturability, as demonstrated
by the concentration curve in Figure 2. Moreover, uptake and
degradation were competed 78% by unlabeled SMase-treated
E0 lipoproteins (data not displayed). These data are consistent
with the notion that uptake of the lipoproteins is a receptor-
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Figure 3. The interaction of macrophages with
SMase-treated d⬍1.063 E0 lipoproteins
involves the interaction of a cell-surface protein
with apoB and is competed by htgVLDL. A,
Control mouse peritoneal macrophages were
incubated in medium containing 50 ␮g/mL 125Ilabeled E0 d⬍1.063 lipoproteins for 2 hours
and then assayed for 125I-lipoprotein cell association. To effect partial proteolysis of cellsurface proteins, macrophages were incubated
for 15 minutes at 37°C with 250 ␮g/mL trypsin
and then rinsed with PBS and incubated with
250 ␮g/mL soybean trypsin/chymotrypsin inhibitor plus 2 ␮mol/L cycloheximide for 5 minutes
at 37°C. The cells were then rinsed again with
PBS and incubated with medium containing 50
␮g/mL soybean trypsin/chymotrypsin inhibitor,
2 ␮mol/L cycloheximide, and 50 ␮g/mL 125Ilabeled d⬍1.063 lipoproteins from E0 mice.
After a 2-hour incubation, the cells were
assayed for 125I-lipoprotein cell association. B,
Macrophages were incubated with 50 ␮g/mL
125
I-labeled E0 d⬍1.063 lipoproteins for 5 hours
in the presence of either 0.4 ␮g/mL nonimmune
goat IgG or goat anti-murine apoB IgG plus
blockers of macrophage Fc receptors (25
␮g/mL concentration of anti-mouse CD12/
CD32 Fc receptor antibody and 50 ␮g/mL concentration of mouse IgG2a). After 5 hours of
incubation, 125I-lipoprotein degradation was
assayed. C, Peritoneal macrophages were incubated with 10 ␮g/mL SMase-treated 125Ilipoproteins from E0 mice alone or in the presence of 100 ␮g/mL unlabeled LDL, oxidized
LDL, or human htgVLDL. After 5 hours of incubation, 125I-lipoprotein degradation was
assayed.
mediated process. To determine whether a cell-surface protein was involved, cells were treated with a limiting concentration of trypsin to effect partial hydrolysis of cell-surface
proteins without otherwise damaging the cells; cycloheximide was added to prevent synthesis on new receptor protein.
As shown in Figure 3A, trypsin treatment resulted in ⬇50%
inhibition of the association of SMase-treated E0 lipoproteins
with macrophages. In a parallel experiment, trypsin treatment
of macrophages inhibited the cell association of acetyl-LDL,
a lipoprotein known to interact with cell-surface receptor
proteins,32 by 60% (data not shown). Thus, the lack of
complete inhibition of SMase-treated E0 lipoprotein uptake
by trypsin most likely represents incomplete hydrolysis of
cell-surface proteins under the conditions of this experiment.
The major protein on E0 lipoproteins is apoB-48.1,2 Figure
3B shows the results of an experiment in which a polyclonal
antibody that recognizes murine apoB (both B-100 and B-48)
was tested for its ability to inhibit the uptake and degradation
of SMase-treated E0 lipoproteins. As shown, the anti-apoB
antibody inhibited uptake by 40% compared with a similar
incubation with a nonimmune IgG. The lack of complete
inhibition could be due to the roles of other proteins on the
aggregated E0 lipoproteins, partial uptake by Fc receptors
despite our attempts to block these receptors (see Methods),
or less-than-complete antibody binding to the apoB on the
lipoproteins. In summary, at least a substantial portion of the
interaction of macrophages with SMase-treated E0 lipoproteins involves the interaction of apoB on the lipoproteins with
a cell-surface receptor on the macrophages.
Preliminary Investigation Into Possible Receptors
That Mediate the Uptake of SMase-Treated E0
Lipoproteins by Macrophages
Although the LDL receptor would not be expected to mediate
the uptake of SMase-treated E0 lipoproteins, we tested this
possibility by comparing the degradation of SMase-treated
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I-E0 lipoproteins in macrophages from wild-type versus
LDL receptor knockout mice. As predicted, the degradation
values were similar: 1050⫾39 versus 999⫾33 ng䡠mg–1䡠5 h–1.
We next considered the idea that the particles might become
modified (eg, by oxidation) to a form recognized by macrophage
class A or B scavenger receptors. However, the degradation of
SMase-treated E0 lipoproteins was similar in macrophages from
wild-type mice (1496⫾99 ng 䡠 mg–1 䡠 5 h–1), scavenger receptor
A knockout mice (1583⫾77 ng 䡠 mg–1 䡠 5 h–1), and CD36 knockout mice (1582⫾124 ng 䡠 mg–1 䡠 5 h–1). Finally, the treatment of
macrophages with chondroitin ABC lyase plus heparitinase had
no effect on lipoprotein uptake (data not shown; see Tabas et
al22). Thus, neither members of the LDL receptor family,
members of the scavenger receptor family, nor cell-surface
glycosaminoglycans appear to mediate the interaction of macrophages with SMase-treated E0 lipoproteins.
Gianturco et al28 and Bradley et al33 demonstrated that a
cell-surface protein on macrophages recognizes apoB-48 on
Sf 100-400 VLDL from hypertriglyceridemic subjects (htgVLDL). Although the physiological role of this cell-surface
protein is not yet known, we considered the possibility that it
or another receptor for apoB-48 –rich lipoproteins may also
recognize the apoB-48 on SMase-treated E0 lipoproteins.
Figure 3C shows a competitive inhibition experiment in
which the abilities were compared of unlabeled LDL, oxidized LDL, and Sf 100-400 htgVLDL to block the uptake and
degradation of SMase-treated 125I-E0 lipoproteins. In corroboration of the data above, neither LDL nor oxidized LDL was
a competitor. htgVLDL, however, was a potent competitor,
and similar results were obtained with trypsinized htgVLDL
(data not shown), which is a particularly potent ligand for the
macrophage protein that recognizes apoB-48.28 htgVLDL
also inhibited the uptake of SMase-aggregated E0 lipoproteins by macrophages from apoE knockout mice (60% inhibition) and from LDL receptor knockout mice (70% inhibition), indicating that the portion of the interaction that was
inhibitable by htgVLDL involved neither apoE nor the LDL
receptor. Importantly, htgVLDL was a relatively poor competitor of degradation of monomeric 125I-labeled E0 lipoproteins (only 12.6% inhibition) and of 125I-acetyl-LDL (no
inhibition), indicating a lack of toxic effects of htgVLDL and
specificity of competition for SMase-aggregated E0 lipoproteins. Thus, the macrophage receptor activity that mediates
the uptake of SMase-treated E0 lipoproteins is uniquely
competed by Sf 100-400 human htgVLDL.
Discussion
The major objective of the present study was to suggest a new
hypothesis as to how macrophage foam cells form in the
widely studied E0 mouse model of atherosclerosis. Lipoproteins isolated directly from the plasma of these mice induce
only a small amount of CE accumulation in macrophages
compared with modified lipoproteins,4 – 6 and therefore these
particles in their native state are unlikely to induce the
massive CE accumulation seen in actual atherosclerotic
lesions from these mice. In fact, the answer to this important
dilemma is undoubtedly multifactorial. In addition to SMaseinduced aggregation, others have shown, and we have verified, that the addition of LpL or oxidation of E0 particles can
increase cholesterol esterification in macrophages. SMase
was the most potent inducer (Figure 1B), and the addition of
2611
LpL to SMase-induced aggregates or the oxidation of E0
lipoproteins before SMase aggregation did not further enhance cholesterol esterification. Moreover, preliminary in
vivo studies that show E0 mice without SMase have smaller
lesions support a role for SMase in the promotion of atherogenesis in E0 mice.18 Nevertheless, the lesions of E0 mice
probably contain a variety of modified particles, and each
may contribute to macrophage foam cell formation.
Regarding other possible mechanisms of foam cell formation in E0 mice, Hayek et al34 proposed that HDL from E0
mice is a relatively poor inducer of cholesterol efflux from
macrophages, so it is possible that this factor also contributes
to macrophage CE accumulation in this model. The role of
macrophage-secreted apoE is uncertain. The hypothesis that
macrophage-secreted apoE promotes cholesterol efflux has
been supported by the results of three in vivo studies:
transplantation of E0 bone marrow into wild-type mice led to
increased lesion development compared with transplantation
of wild-type marrow into these mice,35 and macrophagetargeted expression of apoE in E0 mice, or transplantation of
apoE-expressing bone marrow into E0 mice, led to a reduction in early lesion size.36,37 In another study, however, the
hypothesis that macrophage-secreted apoE might contribute
to particle uptake was supported by the finding that the
transplantation of E0 bone marrow into wild-type mice (the
same strategy of the first study mentioned above) led to a
reduction in early lesion size.38 In the present study, the
internalization of E0 particles and the stimulation of cholesterol esterification were similar in macrophages from E0 mice
versus those from wild-type mice.
We also presented some initial findings related to how
SMase-aggregated E0 lipoproteins are recognized by macrophages. The data in Figures 2 and 3A strongly suggest that a
cell-surface protein mediates at least a substantial portion of
this interaction. A substantial portion of lipoprotein uptake
could be inhibited by an antibody that recognizes murine
apoB, and because most of the apoB on E0 lipoproteins is
apoB-48, these data suggest an important role for this protein.
The ability of native and trypsinized Sf 100-400 htgVLDL to
inhibit the uptake of SMase-aggregated E0 lipoproteins is
consistent with this idea, because a macrophage cell-surface
protein that recognizes these VLDLs has been shown to bind
apoB-48.28,33 The actual physiological function of this particular cell-surface protein, however, has not yet been elucidated, and its possible role in foam cell formation in E0 mice
must await gene knockout studies.
There are several fundamental aspects of atherogenesis that
are addressed by the findings in this report. First, the E0
mouse has been one of the most widely used models of
atherosclerosis, and the presence of large numbers of macrophages with massive CE accumulation in the lesions of these
mice is one of the most important characteristics of the
model. Thus, the knowledge of how foam cells form in this
model is critical for studies that address mechanisms of
atherogenesis in E0 mice as well as for those that explore
genetic, pharmacological, and dietary interventions to prevent
lesions or to reduce lesion size. Second, the principle that
modification of plasma lipoproteins in the subendothelium of
developing lesions is necessary for foam cell formation is
directly applicable to LDL-induced atherosclerosis in animals
and humans.9,39,40 In particular, native plasma LDL is a weak
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inducer of CE loading in macrophages, but aggregated LDL,
which is prominently found in atherosclerotic lesions, is a
potent inducer of cholesterol esterification in macrophages.10 –12 Because aggregated LDL from human lesions
shows evidence of SMase hydrolysis,14 the findings in the
present report suggest that the E0 model may be a reasonable
model with which to explore the role of SMase in human
atherogenesis. Third, although apoE deficiency is an extremely rare cause of human atherosclerosis, functional mutations in this protein can lead to a more common disease,
namely, familial dysbetalipoproteinemia (type III hyperlipoproteinemia).41,42 Type III VLDL from most patients is only
a modest inducer of CE accumulation in macrophages,43,44 so
the mechanisms of foam cell formation in the lesions of these
subjects may share characteristics with foam cell formation in
E0 mice. Finally, the particles that accumulate in E0 mice
share some properties with those of chylomicron remnants in
humans,1,2 which are thought to be atherogenic and which
may be quite abundant in the postprandial state.45 Interestingly, human chylomicron remnants, unlike those that accumulated in fat-fed rabbits or dogs,46 are rather weak inducers
of cholesterol esterification in cultured macrophages.47,48
Thus, modification in the arterial wall by SMase may represent one mechanism that links these particles to atherosclerosis and heart disease in humans.
Acknowledgments
This study was supported by National Institutes of Health grant
HL-56984 (Dr Tabas) and a research grant from Berlex Biosciences
(Dr Tabas). We thank Kirsten D. Mazany and Dr Kevin J. Williams
for providing the LpL, Drs Kevin Williams and Daniel Levine for
providing the anti-apoB antibody, Drs Sandra Gianturco and William
Bradley for providing the trypsinized hypertriglyceridemic VLDL,
Dr Ed Schuchman for providing the DG44 cells, Dr Dudley
Strickland for providing the RAP, and Drs Maria Febbraio and Roy
Silverstein for providing the CD36 knockout mice.
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Sphingomyelinase Converts Lipoproteins From Apolipoprotein E Knockout Mice Into
Potent Inducers of Macrophage Foam Cell Formation
Sudhir Marathe, Yunsook Choi, Andrew R. Leventhal and Ira Tabas
Arterioscler Thromb Vasc Biol. 2000;20:2607-2613
doi: 10.1161/01.ATV.20.12.2607
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