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INTRODUCTION |
One feature of developing atherosclerotic plaques is accumulation
of cholesterol in plaque monocyte-macrophages. Epidemiological studies
have linked the development of atherosclerotic plaques with levels of
low density lipoprotein
(LDL),1 the major carrier of
plasma cholesterol. Evidence suggests that macrophage cholesterol
originates in part from circulating LDL that enters the blood vessel
wall. However, native LDL itself fails to induce cholesterol
accumulation in macrophages. Because of this finding, it has been
surmised that LDL must undergo changes that increase its uptake by macrophages.
Modifications to LDL that aggregate the LDL also promote LDL
accumulation by macrophages (1-8). Recently, we showed that LDL
aggregated through vortexing or treatment with phospholipase C caused
its uptake by a unique endocytic pathway in monocyte-macrophages (9).
These aggregated LDLs induced and entered a labyrinth of
surface-connected membrane bound compartments (SCC) within the
macrophage. Because this endocytosis pathway results in uptake and
storage of material within compartments that remain open to the
extracellular space, we have named this endocytic process "patocytosis" from the Latin, patere, meaning to lie
open. LDL entry into SCC does not depend on the LDL receptor.
Aggregated LDL that enters SCC is mostly stored rather than degraded.
Unesterified cholesterol-rich liposomes are a prominent component of
lesion lipid (10-14). These unique cholesterol-rich particles are
found in early developing atherosclerotic lesions and their appearance
precedes foam cell development within lesions (14-16). Previously, we
showed that treatment of LDL with cholesterol esterase converts the
0.02-µm LDL particles into
0.1-µm liposomes (17). LDL-derived
liposomes are similar in size and chemical composition to liposomes
found in atherosclerotic lesions. Both LDL-derived liposomes and lesion
liposomes show a high molar ratio of unesterified cholesterol to
phospholipid (>2:1) and a high percentage (>75%) of their
cholesterol is unesterified. Like aggregation of LDL, conversion of LDL
into liposomes potentially exposes lipid and protein domains normally
hidden in native LDL. We now report that cholesterol esterase mediated
conversion of LDL into liposomes is an LDL modification that stimulates
patocytosis and foam cell formation.
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MATERIALS AND METHODS |
Preparation of Lipoproteins and LDL Liposomes--
Human LDL
prepared with density gradient centrifugation was obtained from
PerImmune (Rockville, MD) (18). LDL-derived liposomes were produced
essentially as described previously (17). Briefly, LDL was sequentially
treated with trypsin, soybean trypsin inhibitor, and then with
cholesterol esterase to hydrolyze the cholesteryl ester core of LDL.
Treatment with trypsin was found earlier to be necessary before
cholesterol esterase could hydrolyze LDL cholesteryl esters. This
treatment converts LDL that normally has about 25% of its cholesterol
in unesterified form into unesterified cholesterol-rich liposomes that
have >90% of their cholesterol in unesterified form (see Ref. 17 for
the characterization of these LDL liposomes). It should be noted that
trypsin treatment of LDL does not eliminate its binding to the LDL
receptor (19). LDL liposomes were purified by gel filtration as
described earlier with the exception that the elution buffer was
Ca2+- and Mg2+-free Dulbecco's
phosphate-buffered saline containing 0.1% disodium EDTA. The purified
LDL liposomes were sterilized by filtration through polysulfone
0.45-µm (pore-size) filters (Gelman Sciences, Ann Arbor, MI) into
sterile polypropylene tubes and stored at 4 °C.
LDL liposomes were also prepared from lipid extracted from LDL. In this
case lipid was extracted from LDL using 2:1 chloroform/methanol as
described by Folch et al. (20). Then the solvent was
evaporated under a stream of nitrogen. One-ml of buffer containing 0.15 M NaCl, 50 mM Tris-HCl (pH 7.2), and 0.5 mM EDTA was added to the lipid that was then sonicated
under nitrogen for 3 h at 51 °C with 50 watts power (Branson
Sonifier 250, Danbury, CT). This dispersed the lipid into particles as
described previously for preparation of synthetic LDL-sized lipid
particles (21). LDL liposomes were then prepared from these lipid
particles as was described above for native LDL.
In one type of experiment purified LDL liposomes (at a cholesterol
concentration of 1300 nmol/ml) were further treated 2 h at
37 °C with papain (0.5 mg/ml) followed by leupeptin (0.2 mg/ml for
30 min) (both from Boehringer-Mannheim) to neutralize the papain. These
protease-treated LDL liposomes were used to learn whether a protein
component of the liposomes was required for macrophage uptake.
The preparation of microcrystalline cholesterol and acetylated LDL were
described in Ref. 22. Rabbit
-very low density lipoprotein was
obtained from Biomedical Technology (Stoughton, MA) prepared as in Ref.
23 from plasma of New Zealand White rabbits fed a 1% cholesterol diet
for 5 weeks.
Incubation of Macrophages with LDL Liposomes and Potential
Inhibitors of LDL Liposome Uptake--
Human monocyte-derived
macrophages were cultured as described previously except that 2 × 106 monocytes/well were initially seeded into 12-well
(22-mm diameter) culture plates (Plastek C, MatTek Corp., Ashland, MA)
(22). Two-week-old monocyte-macrophage cultures were rinsed 3 times with RPMI 1640 medium and incubated for the indicated times at 37 °C
in RPMI 1640 medium with the indicated concentrations of liposomes
expressed as nanomole of total cholesterol/ml of medium.
Potential inhibitors of liposome uptake that were tested included
cytochalasin D, nocodazole, polyinosinic acid (all from Sigma), C7
mouse anti-LDL receptor monoclonal antibody (purified from supernatant
of cell line number 1691-CRL, American Type Culture Collection,
Manassas, VA) (24), and isotype-matched control monoclonal
IgG2b antibody (catalog number 50330, ICN, Aurora, OH), and
receptor-associated protein that inhibits lipoprotein binding to the
LDL receptor-related protein (25). The role of heparan sulfate
proteoglycans in LDL liposome uptake was tested by first treating
macrophages for 3 h at 37 °C without or with 50 mM
chlorate plus 80 units/ml heparinase I (Sigma). Then these macrophages
were rinsed and incubated 1 day with either 200 nmol/ml LDL liposomes
or 100 µg/ml rabbit
-very low density lipoprotein without or with
50 mM chlorate (26, 27).
In one experiment, purified apoB (PerImmune) (28) was incubated with
monocyte-macrophage cultures. First, the apoB (0.5 mg), which contained
detergents, was solubilized in saline. Then the detergents were removed
by exhaustive dialysis against saline. The now insoluble apoB was
centrifuged, resuspended in RPMI 1640 at a concentration of 1 mg/ml,
dispersed by direct sonication (15 s), and then incubated 5 h with
monocyte-macrophages to learn whether apoB could stimulate patocytosis.
Two other insoluble proteins, collagen IV and fibrin (catalog numbers
C5533 and F5386, respectively, obtained from Sigma) were each
resuspended directly into RPMI 1640 culture medium at a concentration
of 1 mg/ml, sonicated 15 s, and incubated with
monocyte-macrophages also at a concentration of 1 mg/ml. Following
incubations, macrophage cultures were analyzed for their cholesterol
contents or for ultrastructural changes as described below.
Assay of Cholesterol Content of Monocyte-Macrophages and LDL
Liposomes--
Macrophages were rinsed, harvested, and processed as
described previously (29). Unesterified and esterified cholesterol contents of macrophages and LDL liposomes were determined enzymatically according to the fluorometric method of Gamble et al. (30). The mean ± S.E. (when shown) were determined from three culture wells for each data point.
Electron Microscopy--
Extracellular and intracellular
membranes were differentiated with ruthenium red according to the
method of Luft (31). Monocyte-macrophage cultures were ruthenium
red-stained and prepared for electron microscopy as described
previously (22).
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RESULTS |
LDL Liposomes Caused Substantial Macrophage Cholesterol
Accumulation--
Conversion of LDL into liposomes with cholesterol
esterase treatment greatly increased LDL uptake by human
monocyte-macrophages. Native LDL did not cause much cholesterol
accumulation in macrophages (Table I and
Fig. 1a). Only when LDL was
converted to liposomes (i.e. was treated with trypsin
followed by cholesterol esterase) did LDL induce substantial
cholesterol accumulation in the macrophages. With this treatment, the
formed LDL liposomes (that contained >90% of their cholesterol in
unesterified form) induced a 3-5-fold increase in macrophage
cholesterol content. Treatments of LDL with either trypsin or
cholesterol esterase alone showed little effect on cholesterol
accumulation (Table I).
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Table I
Effect of treating LDL with cholesterol esterase on macrophage
cholesterol accumulation
Macrophages were incubated 2 days with 200 nmol of cholesterol/ml of
LDL treated as indicated. 1.25 mg of LDL in 2.5 ml of 50 mM
Tris saline buffer (pH 7.2) containing 1 mM Na2
EDTA was treated as indicated sequentially (17) with trypsin (95 units/ml for 2 h), soybean trypsin inhibitor (STI) (0.25 mg/ml for
30 min), and cholesterol esterase (CEase) (55 units/ml for 2 h).
Then, the treated LDL samples were exhaustively dialyzed against
phosphate-buffered saline and added directly to macrophage cultures
without isolating the treated LDL.
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Fig. 1.
Effect of LDL liposome concentration and time
of incubation on macrophage cholesterol accumulation. In
a, macrophages were incubated for 2 days with the indicated
concentrations of LDL liposomes. , indicates macrophage total
cholesterol content for LDL incubated at a cholesterol concentration of
200 nmol/ml. In b, macrophages from a different culture were
incubated with 200 nmol/ml of LDL liposome cholesterol for the
indicated number of days. After incubations, cells were rinsed and
their cholesterol contents were determined as described under
"Materials and Methods." TC, total cholesterol;
EC, esterified cholesterol; UC, unesterified
cholesterol.
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Macrophage cholesterol accumulation during a 2-day incubation was
maximal when macrophages were incubated with LDL liposomes at a
concentration of 150-200 nmol of cholesterol/ml (Fig. 1a). During such incubations, macrophage cholesterol accumulation varied but
could reach levels more than 400 nmol/mg cell protein with greater than
60% of cholesterol esterified. Incubation of macrophages with LDL
liposomes for up to 5 days showed that most of the increase in
macrophage cholesterol content occurred during the first 2 days of
incubation (Fig. 1b). The lag in total cholesterol
accumulation at 1 day occurred in 2 out of 3 time course experiments.
Cholesterol was esterified during the entire 5-day incubation. In
another experiment not shown, the acyl-CoA:cholesterol acyltransferase inhibitor, S58-035, completely blocked esterification of cholesterol during a 3-day incubation of macrophages with LDL liposomes (200 nmol
of cholesterol/ml), but did not decrease total cholesterol accumulation.
LDL liposomes induced macrophage cholesterol accumulation comparable to
the levels induced by acetylated LDL and microcrystalline cholesterol
(Table II). The amount of cholesteryl
ester synthesized by macrophages incubated with LDL liposomes and
microcrystalline cholesterol was similar although more total
cholesterol accumulated in macrophages incubated with microcrystalline
cholesterol. Different LDL liposome preparations induced similar levels
of cholesterol accumulation when incubated with macrophages from the
same culture. On the other hand, the degree of cholesterol accumulation
varied with different macrophage cultures (see comparable data for the 2-day incubations in Fig. 1, a and b).
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Table II
Macrophage cholesterol accumulation induced by LDL liposomes compared
with other lipid particles
Macrophages were incubated 4 days with 100 µg of protein/ml of
acetylated LDL or with 200 or 520 nmol of cholesterol/ml of LDL
liposomes and microcrystalline cholesterol, respectively. The 200 nmol/ml of LDL liposome cholesterol was equivalent to about 75 µg/ml
of LDL protein. Following incubations, macrophages were rinsed and
analyzed for their cholesterol contents.
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LDL Liposomes Induced and Entered Macrophage Surface-connected
Compartments--
Experiments were carried out to determine whether
LDL liposomes could induce and enter macrophage SCC as we previously
reported occurs with aggregated LDL and microcrystalline cholesterol
(22). Macrophages were incubated with LDL liposomes for 1 day and then processed for electron microscopy with ruthenium red, an electron dense
stain that labels cellular membranes. Ruthenium red does not penetrate
the plasma membrane of aldehyde-fixed cells. Thus, in aldehyde-fixed
cells, ruthenium red stains only cellular membranes that are in
continuity with the extracellular space. Fig.
2 shows that LDL liposomes induced and
entered macrophage SCC that were ruthenium red stained. Ruthenium red
also stained the LDL liposomes contained within the SCC. Many
non-membrane bound lipid droplets accumulated in the cytoplasm of
macrophages incubated with LDL liposomes reflecting esterification of
LDL liposome cholesterol.

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Fig. 2.
Ruthenium red staining of LDL
liposome-induced surface-connected compartments. Macrophage
cultures were incubated with 100 nmol/ml LDL liposome cholesterol for 1 day. Then, cells were rinsed, fixed with 2% glutaraldehyde, and then
exposed to 0.15% ruthenium red during additional fixation with first
fresh glutaraldehyde and then 2% osmium tetroxide. Following fixation,
cells were embedded in epon plastic and thin sectioned, but not
counterstained. The membranes of SCC (indicated by arrows)
stained with ruthenium red. Also, note that the SCC are filled with LDL
liposomes (also shown in inset) that also stained with
ruthenium red. N, nucleus. Magnification is × 5000 for
main photomicrograph and × 46,000 for the inset.
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Like phagocytosis, patocytosis (the SCC endocytic process) is inhibited
by cytochalasin D, an agent that disrupts actin microfilaments, but not
by nocodazole, an agent that disrupts microtubules. When macrophages
were incubated with LDL liposomes in the presence of cytochalasin D,
LDL liposomes were observed attached to the plasma membrane but did not
enter macrophages and no SCC formed (data not shown). Thus,
cytochalasin D blocked uptake of LDL liposomes by macrophages, but not
their binding to the macrophage surface. As a result, cytochalasin D
decreased but did not eliminate macrophage cholesterol accumulation
induced by LDL liposomes (Fig. 3). Some increase in macrophage unesterified cholesterol content apparently occurred due to binding of LDL liposomes to the macrophage plasma membrane. Cytochalasin D decreased unesterified cholesterol
accumulation by 26%. However, this inhibitor decreased esterified
cholesterol accumulation to a greater extent at 88%, suggesting that
uptake into SCC and possibly some additional
actin-dependent process (32) was necessary for
esterification of LDL liposome cholesterol.

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Fig. 3.
Effect of cytochalasin D on LDL
liposome-induced macrophage cholesterol accumulation. Macrophage
cultures were incubated with 200 nmol/ml LDL liposome cholesterol for 1 day without and with 4 µg/ml cytochalasin D. Controls received 2 µl/ml dimethyl sulfoxide, the solvent in which cytochalasin D was
dissolved. Then, the cells were rinsed and analyzed for their
cholesterol contents.
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Apolipoprotein B-mediated Uptake of LDL Liposomes into
SCC--
The capacity of LDL liposomes to induce cholesterol
accumulation depended on its protein component. This was shown by
exposing LDL liposomes to the protease, papain, then neutralizing the
papain with leupeptin, and finally incubating these protease-treated LDL liposomes with macrophages. Papain treatment of the LDL liposomes significantly decreased macrophage cholesterol accumulation (Table III). If papain was neutralized with
leupeptin before exposing the LDL liposomes to the papain, papain did
not effectively decrease macrophage uptake of LDL liposomes. This
showed that leupeptin inhibition of papain was sufficient to prevent
any substantial effect of the added papain on macrophage receptors
involved in uptake of the LDL liposomes. Incubation of macrophages with
LDL liposomes prepared from extracted LDL lipid also did not cause any
macrophage cholesterol accumulation (data not shown). This further
demonstrated the importance of apoB in mediating LDL liposome uptake by
macrophages.
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Table III
Effect of treating LDL liposomes with protease on macrophage
cholesterol accumulation
LDL liposomes (1300 nmol of cholesterol/ml) were treated as indicated
with 0.5 mg/ml papain and 0.2 mg/ml leupeptin. Then macrophages were
incubated 2 days with the treated LDL liposomes (200 nmol of
cholesterol/ml). Following incubations, macrophages were rinsed and
analyzed for their cholesterol contents.
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The above results suggested that apoB, the major protein component of
the LDL liposomes mediated their uptake into macrophage SCC. We
directly tested this conclusion by incubating macrophages with purified
apoB, a protein that is insoluble in aqueous buffers such as culture
medium. Macrophages were incubated with 1 mg/ml insoluble apoB and then
examined by electron microscopy for the presence of SCC. ApoB induced
SCC in most macrophages and an amorphous ruthenium red-stained material
(consistent with apoB protein) accumulated within the SCC (Fig.
4). On the other hand, incubation of
macrophages with the same concentration of two other insoluble proteins, collagen and fibrin, did not induce SCC.

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Fig. 4.
Ruthenium red staining of apoB-induced
surface-connected compartments. Macrophages were incubated with 1 mg/ml purified insoluble apoB for 5 h. Then, the cells were
prepared for electron microscopy with ruthenium red staining as
described in the legend to Fig. 2. Arrows indicate ruthenium
red-stained SCC that are filled with amorphous apoB protein that also
stained with ruthenium red (also shown in inset).
N, nucleus. Magnification is × 5000 for main
photomicrographs and × 23,000 for the inset.
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Uptake of LDL Liposomes Did Not Depend on LDL, LDL Receptor-related
Protein, or Polyinosinic Acid-inhibitable Scavenger
Receptors--
Uptake of LDL liposome cholesterol did not depend on
the LDL receptor. Cholesterol-enrichment of macrophages decreases
macrophage expression of the LDL receptor (33). However, macrophage
cholesterol-enrichment did not decrease cholesterol accumulation
induced by LDL liposomes. When macrophages were first incubated with
acetylated LDL for 2 days, their cholesterol content doubled (from
80 ± 1 to 160 ± 2 nmol of cholesterol/mg of cell protein)
(Table IV). Nevertheless, a subsequent
2-day incubation with LDL liposomes of these and other macrophages that
were not cholesterol-enriched produced similar increments in macrophage
cholesterol content (167 ± 4 and 170 ± 7 nmol of
cholesterol/mg of cell protein, respectively). Additional findings
indicated that LDL receptors were not involved in LDL liposome uptake.
Incubation of LDL liposomes (200 nmol/ml) with macrophages for 2 days
in the presence of 100 µg/ml of an anti-LDL receptor monoclonal
antibody (C7) (24) did not decrease macrophage cholesterol
accumulation. Also, methylation of apoB, which blocks apoB binding to
macrophage LDL receptors (34), did not interfere with apoBs capacity to
induce and enter macrophage SCC.
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Table IV
Effect of macrophage cholesterol enrichment on subsequent LDL
liposome-induced macrophage cholesterol accumulation
Macrophages were incubated without or with acetylated LDL (AcLDL) (50 µg of protein/ml) for 2 days to cholesterol-enrich the macrophages.
Then, the macrophages were rinsed and incubated without and with
liposomes (200 nmol of cholesterol/ml) for 2 days. Following this
incubation, macrophages were analyzed for their cholesterol contents.
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Other potential lipoprotein receptors also did not mediate macrophage
uptake of LDL liposomes. Polyinosinic acid (2.5 mg/ml), a scavenger
receptor inhibitor (35) failed to inhibit LDL liposome-induced macrophage cholesterol accumulation. However, polyinosinic acid did
inhibit by >90% acetylated LDL-induced macrophage cholesterol accumulation (both the LDL liposomes and acetylated LDL were incubated with macrophages for 2 days at a total cholesterol concentration of 200 nmol/ml). Receptor-associated protein (1 µM), an
inhibitor of LDL receptor-related protein and other LDL receptor family members (25), showed no effect on LDL liposome-induced macrophage cholesterol accumulation.
Macrophage uptake of LDL liposomes may not depend on a macrophage
cell-surface protein. Exposure of macrophages to trypsin (20 µg/ml
for 30 min at 37 °C) followed by soybean trypsin inhibitor (30 µg/ml for 30 min) actually increased macrophage cholesterol accumulation during a 1-day incubation with LDL liposomes (200 nmol of
cholesterol/ml) and cycloheximide (20 µg/ml) added to block protein
synthesis (165 ± 3 nmol/mg cell protein for sham-treated macrophages and LDL liposomes without cycloheximide compared with 211 ± 11 nmol/mg cell protein for trypsin-treated macrophages and
LDL liposomes plus cycloheximide). On the other hand, as expected for a
protein receptor-mediated uptake process, trypsin alone, cycloheximide
alone, and trypsin plus cycloheximide treatments significantly
decreased acetyl-LDL-induced (100 µg/ml) macrophage cholesterol
accumulation (140 ± 1 with sham treatments, 132 ± 2 with
trypsin alone, 121 ± 5 with cycloheximide alone, and 114 ± 4 nmol cholesterol/mg cell protein with combined trypsin and cycloheximide treatments).
Macrophage uptake of apoB-containing lipoproteins in some instances can
be mediated by cell surface proteoglycans (26, 36-38). However,
digestion of macrophages with heparinase I (80 units/ml, Sigma)
followed by incubation of macrophages for 1 day with LDL liposomes (200 nmol/ml) in the presence of 50 mM chlorate (an inhibitor of
proteoglycan sulfation (27)) failed to reduce macrophage cholesterol
accumulation. On the other hand, as recently reported for pigeon
peritoneal macrophages (26), heparinase I treatment did decrease by
70% macrophage cholesterol accumulation induced by rabbit
-very low
density lipoprotein (100 µg of protein/ml) in the same experiment.
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DISCUSSION |
Previously we showed that microcrystalline cholesterol and
aggregated LDL enter SCC that form in human monocyte-macrophages during
incubation with these lipid particles (22). Uptake of the lipid
particles into SCC is an actin-dependent process we call
patocytosis. Patocytosis is distinct from phagocytosis in which
vacuoles pinch off from the plasma membrane. We now show that liposomes
derived from cholesterol esterase-treated LDL also are accumulated by
macrophages through patocytosis. Uptake of LDL liposomes was
predominantly mediated by the apoB component rather than the lipid
component of the LDL liposomes. This was indicated by the findings
that: 1) protease treatment of the LDL liposomes decreased most
macrophage cholesterol accumulation; 2) LDL liposomes prepared from LDL
lipid extracts caused no macrophage cholesterol accumulation; and 3)
purified apoB could induce and enter macrophage SCC.
Sufficient hydrolysis of LDL cholesteryl ester may be important for the
conversion of LDL into a lipid particle that can cause macrophage
cholesterol accumulation. In two previous studies, it was reported that
cholesterol esterase treatment of LDL did not increase and even
decreased LDL uptake by macrophages. In one of these studies (39), LDL
was treated with cholesterol esterase in the presence of trypsin rather
than first treating LDL with trypsin and then neutralizing the trypsin
with a trypsin inhibitor as in our study. In this earlier study, it is
likely that there was little or no hydrolysis of LDL cholesteryl ester due to trypsin-mediated degradation of any added cholesterol esterase. While the LDL cholesteryl ester content was not monitored in this earlier study, electron microscopy showed that trypsin and cholesterol esterase treatment of LDL did not produce liposomes. This suggests that
LDL cholesteryl ester hydrolysis was minimal. In the other study
(40), only about 33% of LDL cholesteryl ester was hydrolyzed in contrast to the almost complete hydrolysis of LDL cholesteryl ester in our study. It is likely that the low level of
cholesteryl ester hydrolysis in both these earlier studies was not
sufficient to form liposomes from the LDL, and thus possibly not
sufficient to permit macrophage binding of cryptic apoB domains or to
create larger liposomal lipid particles with multiple copies of apoB
(discussed below).
Because of the greater size of LDL liposomes compared with native LDL,
more than one LDL particle should contribute to each LDL liposome.
Thus, even if not greatly aggregated, LDL liposomes (like aggregated
LDL) should be multivalent with respect to apoB, whereas LDL is known
to have only one copy of apoB per LDL particle (41). A lipid particle
with a multivalent apoB ligand may have the capacity to initiate
receptor cross-linking and cell signaling pathways that could trigger
uptake into SCC.
ApoB is unique among the apolipoproteins in that it is very
hydrophobic, a property that accounts for its insolubility in aqueous
buffer. In this regard, we have not observed SCC when macrophages were
incubated, for example, with
apoE,2 a soluble
apolipoprotein that mediates LDL receptor uptake of
-very low
density lipoprotein into surface-connected tubules of mouse peritoneal
macrophages (42, 43). The other lipid particle that we find enters SCC
is microcrystalline cholesterol, another hydrophobic material. Studies
are in progress to learn whether hydrophobicity is a requirement for
stimulation of the SCC endocytic pathway.
Whatever apoB characteristics are required to stimulate the SCC
pathway, uptake did not occur through apoB-mediated binding of LDL
liposomes to LDL, LDL receptor-related protein, or polyinosinic acid-inhibitable scavenger receptors. This was shown by a lack of
inhibition by C7 monoclonal antibody, receptor-associated protein, and
polyinosinic acid, respectively. Also, uptake of LDL liposome cholesterol was not down-regulated by cholesterol enrichment of the
macrophages, previously shown to down-regulate LDL receptors in human
monocyte-macrophages (33). Exposure of macrophages to enzymes that
digest proteins or proteoglycans did not decrease LDL liposome uptake,
suggesting that LDL liposome uptake was mediated by some other type of molecule.
Cholesterol-rich liposomes accumulate in the extracellular spaces of
atherosclerotic plaques often surrounding macrophage foam cells and are
present within membranous compartments of these macrophages (44).
Recently, it was shown that monocyte-macrophages secrete bile
salt-stimulated cholesterol esterase under certain conditions and that
a similar enzyme is present within human aorta (45, 46). Thus, it is
possible that LDL liposomes form from LDL in atherosclerotic plaques
and contribute to macrophage cholesterol accumulation in these plaques.
In conclusion, conversion of LDL to cholesterol-rich liposomes produces
a lipid particle that is taken up by human monocyte-macrophages converting them into foam cells (greater than 60% of the cholesterol in these macrophages was esterified). Like aggregated LDL, the LDL
liposomes entered macrophages by a non-LDL receptor pathway that caused
accumulation of the lipid particles in macrophage SCC. The major
component of LDL, apoB, mediated uptake of the LDL liposomes, and could
by itself induce and enter macrophage SCC. It remains controversial
whether uptake of lipoprotein-derived cholesterol by macrophages in
atherosclerosic plaques causes the accumulation of this cholesterol
within plaques, or whether uptake of this lipid is secondary and
represents a macrophage function to remove this lipid from the plaques
(44). In any case, learning that apoB can direct entry of LDL-derived
liposomes and possibly other forms of modified LDL into SCC is
important to our understanding of macrophage function in atherogenesis.