© 2000 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
The Pathogenesis of Foam Cell Formation
Modified LDL Stimulates Uptake of Co-Incubated LDL Via Macropinocytosis
From the Department of Pathology, Wake Forest University School of Medicine of Wake Forest University, Medical Center Boulevard, Winston-Salem, NC.
Correspondence to Nancy L. Jones, PhD, Wake Forest University School of Medicine, Medical Center Blvd, Winston-Salem, NC 27157-1092. E-mail njones{at}wfubmc.edu
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Abstract |
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Abstract—Previously, modified LDLs were shown to stimulate macropinocytosis in pigeon macrophages. Simultaneous intracellular trafficking of LDL and AcLDL, differentially labeled with colloidal gold, was done to determine whether uptake of LDL, which does not cause foam cell formation, was internalized via a separate route from AcLDL, which stimulates foam cell formation. AcLDL and LDL were followed at either low (12 µg/mL) concentrations near the saturation of high affinity binding sites or high (50 to 150 µg/mL) lipoprotein concentrations used to induce foam cell formation. The colloidal gold distribution and percentage of co-labeling as observed by transmission electron microscopy were determined for organelles involved with coated-pit endocytosis or macropinocytosis. LDL simultaneously incubated with AcLDL on macrophages at the low concentration was predominately internalized via coated-pit endocytosis. AcLDL was internalized via both coated-pit endocytosis and macropinocytosis at low concentration. At higher lipoprotein concentrations (50 to 150 µg/mL), AcLDL continued to be internalized via macropinocytosis. Interestingly, a significant portion of the co-incubated LDL, at high concentrations, also trafficked via macropinocytosis. LDL internalized by macropinosomes at high lipoprotein concentrations suggests that AcLDL-stimulated macropinocytosis might increase uptake of co-incubated lipoproteins. When 125I-LDL was incubated with cold AcLDL, LDL degradation at 37°C doubled, without a corresponding increase in cell association or total binding of LDL at 4°C. These studies suggest that modified LDL-stimulated macropinocytosis is a mechanism for increased degradation of co-incubated LDL potentially leading to foam cell formation.
Key Words: lipoproteins • endocytosis • macropinocytosis • foam cell formation
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Introduction |
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There is a generally accepted scenario for macrophage foam cell formation. Initially, lipoproteins are internalized by coated-pit endocytosis. These internalized lipoproteins use receptors that are not down-regulated by increases in cellular cholesterol concentrations. One such unregulated receptor is the scavenger receptor(s).1 2 The receptor-mediated internalized lipoproteins are then delivered to lysosomes where the cholesteryl ester is hydrolyzed to free cholesterol by the action of acid cholesterol esterase.3 Free cholesterol, perhaps transported to the plasma membrane,4 is re-esterified by acyl coenzyme A: cholesterol acyltransferase (ACAT) and stored as cholesterol ester in intracellular lipid droplets. The cholesterol ester can be hydrolyzed by neutral cholesterol esterases, and the free cholesterol can be released from the cell if an acceptor is available. An imbalance in the amount of cholesterol retained within the cell and the amount released to extracellular acceptors is believed to cause foam cell formation.
Uptake of native LDL alone does not cause in vitro foam cell formation.5 In short-term experiments, macrophages internalize ample LDL, but the cholesterol is unable to stimulate esterification.6 Uptake of LDL, and subsequently internalized cholesterol, leads to down- regulation of LDL receptors. An apparent quandary with the foam cell formation literature is that LDL, a reliable risk factor for predicting heart disease, does not cause macrophage foam cell formation in vitro. The presence of modified lipoproteins within the artery, and in vitro macrophage foam cell formation by uptake of these modified lipoproteins, has been suggested to explain this disparity.7
Pigeon blood monocytes have been cultured in vitro as a model of monocyte-derived foam cells. Like human macrophages,3 they can be stimulated by endocytosis of either ßVLDL, AcLDL, or OxLDL to accumulate cholesterol esters resulting in a foam cell-like appearance,8 9 10 whereas native LDL does not cause cholesterol ester accumulation. Pigeon AcLDL is internalized in part by coated-pit endocytosis. However, AcLDL also stimulates macropinocytosis and is internalized in part by macropinocytosis.11 12 This stimulation of macropinocytosis by modified LDLs suggests an intriguing hypothesis for foam cell formation; the presence of some modified LDL may affect the internalization or intracellular trafficking of co-incubated LDL normally internalized by coated-pit endocytosis. The present study used simultaneous labeling methodology to determine whether LDL co-incubation with AcLDL was internalized via macropinocytosis.
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Methods |
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Macrophage Culture and Lipoproteins
White Carneau pigeon monocyte derived macrophages and adherent THP-1 cells were cultured for 7 days, as previously described.13 14 Hypercholesterolemic LDL (d
1.006 and d
1.080) was isolated from White Carneau
pigeons15 and acetylated by the method of Basu et
al16 and moderately oxidized with
CuSO4.11 Lipoproteins (20 to 50
µg/mL) were conjugated to colloidal gold (small, 14 or 17 nm; large,
32 or 37 nm) and prepared as described in detail in the online
publication
(http://atvb.ahajournals.org/cgi/content/full/20/3/773/DC1).
Simultaneous Trafficking of Lipoproteins
Macrophages were incubated at either low (12.5 µg/mL)
or high (50 to 150 µg/mL) equal protein concentrations of AcLDL and
LDL. Cells were washed twice with ice-cold phosphate buffered saline
(PBS) and then incubated with labeled lipoproteins for 2 hours at
4°C. The cells were then washed twice with PBS at 4°C. Media
prewarmed to 37°C was added, and the cells were incubated at 37°C
for <1, 5, 10, 30, 60, and 120 minutes. Cells were fixed with 2.5%
glutaraldehyde, removed from the plates by scraping,
and embedded in Spurr’s for standard, transmission electron
microscopy. Silver thin sections (60 to 80 nm) were visualized at 80
keV in a Philips EM-400 transmission electron microscopy. Three
experiments were done for the low (12.5 µg/mL) concentrations of
lipoproteins, and 3 were done for the high (50 to 150 µg/mL)
concentrations.
The colloidal gold distribution was determined for the following organelles of macrophages: plasma membrane, clathrin-coated pits and vesicular profiles, early endosomes, macropinosomes, spherical late endosomes, late endosomal-prelysosomal tubuloreticular compartments (LEPTs) and lysosomes. The morphologies used to classify the organelles are published on line (X). The percentage gold distribution was determined for each time point by totaling the gold labeling in each organelle class for 11 micrographs (7000X) divided by the total label in all organelles.
Analysis of Co-Labeling
The co-labeling of all organelle types, except the plasma
membrane, was analyzed by the percentage of the labeled
lipoprotein in the co-labeled organelles. The lipoprotein in the
co-labeled organelles was divided by the total lipoprotein in each
organelle type. The percentage of lipoprotein in the co-labeled
organelles did not differ with time. Therefore, data were averaged for
each organelle despite time. The nonparametric
Wilcoxon paired t test was used to
analyze significant differences between the lipoprotein pairs
for each organelle. To determine whether the co-labeling of AcLDL or
LDL was different between the following 2 organelle types, LEPTs and
lysosomes, data were analyzed by a
nonparametric unpaired t test (Instat Version
3.00, GraphPad Software Inc). Nonparametric multiple
comparisons were made by the Kruskal-Wallis analysis to
determine whether the co-labeling of organelles was different among the
organelle types. When the null hypothesis was rejected, significant
differences at the P<0.05 level between organelles were
determined using a nonparametric Tukey-type comparison.
AcLDL Affect on Binding, Internalization, and Degradation of
LDL
Macrophages were prechilled for 30 minutes at 4°C,
before binding at 4°C for 2 hours. To determine whether AcLDL would
affect the binding and degradation of LDL, a constant concentration of
125I-LDL (50 µg/mL) was added in the presence
of either no other lipoprotein (control), AcLDL (12 µg/mL), or LDL
(12 µg/mL). A 50-fold excess of unlabeled LDL (2.5 mg/mL) was added
to determine nonspecific binding. After washing the macrophages
3 times with ice-cold PBS, the binding at 4°C was determined by
measuring the cell-associated radioactivity after digestion of the
cells with 1 N NaOH. The protein concentration was determined by the
method of Lowry.
For degradation and internalization studies, macrophages were incubated with 125I-LDL (100 µg/mL) with either OxLDL, LDL, or AcLDL (20 µg/mL) for 2 or 5 hours to determine whether AcLDL could alter the uptake and degradation of co-incubated LDL. Macrophages were preincubated with Ringer’s Buffer (155 mmol/L NaCl, 5 mmol/L KCl, 1 mmol/L MgCl2, 2 mmol/L Na2HPO4, 10 mmol/L glucose, 10 mmol/L Hepes, pH 7.2, 0.5 mg/mL bovine serum albumin) for 30 minutes at 37°C. Degradation was determined by measuring the trichloroacetic acid–soluble, non-iodide radioactivity in the media. The cell layer was washed extensively and digested with 1N NaOH overnight. An aliquot was counted in a Beckman gamma counter to determine cell-associated 125I-radioactivity, and another aliquot was used to determine protein concentration. Counts were adjusted by subtracting degradation of lipoproteins on plates with no cells. The results were expressed as amount bound, cell-associated, and degraded per µg cell protein.
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Results |
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Low Concentration Trafficking of AcLDL+LDL
LDL is internalized by coated-pit endocytosis, whereas AcLDL is internalized by both coated-pit endocytosis and macropinocytosis in pigeon macrophages.12 We hypothesize that ligands internalized by macropinocytosis will merge with ligands internalized via coated-pit endocytosis at the LEPT (please see Figure
I, published
online at
http://atvb.ahajournals.org/cgi/content/full/20/3/773/DC1). In the
present study, a time course of simultaneous
trafficking was done for LDL and AcLDL conjugated to different sized
colloidal gold particles. Macrophages were incubated with
lipoproteins at 12.5 µg/mL to examine the high affinity uptake of
lipoproteins at <1, 5, 10, 30, 60, and 120 minutes (Methods).
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The plasma membrane surface was highly convoluted and ruffled at both
low and high concentrations of lipoproteins (Figures 1
and 2). Approximately 70% of AcLDL
and LDL was on the plasma membrane at time zero at low concentrations
(Figure 3A). Correspondingly, at <1
minute, 30% of AcLDL and LDL was in early endocytic organelles such as
clathrin-coated pits and vesicular profiles, early endosomes, and
macropinosomes. From 5 to 10 minutes, the percent distribution of
lipoproteins on the plasma membrane increased slightly (Figure 3A). This elevation of lipoprotein, AcLDL, and LDL on the plasma
membrane at 10 minutes might reflect unruffling of the plasma membrane.
With time, the percentage of AcLDL and LDL on the plasma membrane
progressively decreased until only 18% remained on the plasma membrane
at 120 minutes (Figure 3E).
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), macropinosomes (
),
spherical endosomes (
), late endosomal-prelysosomal
tubuloreticular compartment (LEPT) (
), and lysosomes
(
). The time course is plotted for (A) LDL at low concentrations,
(B) AcLDL at low concentrations, (C) LDL at high concentrations, and
(D) AcLDL at high concentrations. Data for the plasma membrane and pits
and vesicular profiles are shown separately in E and F, respectively.
The percentage distribution for AcLDL (•) and LDL (
AcLDL and LDL were present in coated-pits, endosomes, and
macropinosomes at early times (Figure 1). AcLDL was more
frequently seen in macropinosomes than LDL. Organelles
consistent with only coated-pit endocytosis are clathrin-coated
pits, early endosomes, and spherical endosomes. Organelles
consistent only with macropinocytosis are macropinosomes
(Figure I published online at
http://atvb.ahajournals.org/cgi/content/full/20/3/773/DC1). The
distribution of AcLDL and LDL were compared in these organelles to
determine whether uptake was via coated-pit endocytosis or
macropinocytosis. At <1 through 10 minutes, the distribution of LDL
was consistently increased in pits and vesicular profiles,
endosomes, and spherical endosomes compared with the distribution of
AcLDL (Table). The distribution of LDL was 7-, 3-, and 3-fold
increased versus AcLDL in pits, endosomes, and spherical endosomes,
respectively. Conversely, the distribution of AcLDL in macropinosomes
was 7-fold greater than for LDL (Table and Figure 3). At
5 and 10 minutes, the distribution of AcLDL was significantly greater
than LDL in macropinosomes with 32% and 17% of AcLDL compared with
7% and 1% of LDL, respectively (Figure 3).
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Time Course (Low Concentration)
Generally, LDL moved more rapidly through the endocytic organelles
than AcLDL (Figure 3). The time course for maximal distribution
of LDL was 0 to 10 minutes for coated pits, 0 to 10 minutes for early
endosomes, 5 to 10 minutes for spherical endosomes, 30 to 120 minutes
for LEPT, and 120 minutes for lysosomes. The distribution of
LDL peaked in macropinosomes at 30 minutes. The time course for maximal
distribution of AcLDL was 0 to 10 minutes for coated pits, 10 to 30
minutes for early endosomes, 10 to 60 minutes for spherical endosomes,
60 to 120 minutes for LEPT, and 120 minutes for lysosomes.
AcLDL was present in macropinosomes through 30 minutes.
Co-Trafficking (Low Concentration)
The simultaneous incubation of lipoprotein pairs for
this experiment was designed to detect if lipoproteins trafficked
through the same endocytic organelles or whether these pathways remain
separate. The quantitative data support the hypothesis that most of LDL
trafficks via coated-pit endocytosis with little LDL present in
macropinosomes (Figure 3A). A significant portion of AcLDL is
present both in macropinosomes and endosomes, indicating that AcLDL
trafficks in part by coated-pit endocytosis and in part by
macropinocytosis (Figure 3B). The co-labeling data also support
this conclusion. At low concentrations of LDL+AcLDL, trafficking of
AcLDL was more segregated from LDL in the coated-pit endocytic pathway.
AcLDL had a significantly lower extent of co-labeling than LDL in
clathrin-coated pits, early endosomes, and spherical endosomes (Figure 4A). These data indicate that at low
concentrations, 60% of AcLDL trafficks through separate early
endosomes and spherical endosomes. Conversely, 75% of LDL
co-trafficked with AcLDL in early endosomes and spherical
endosomes.
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Clathrin-coated pits and vesicular profiles had 2% to 10% co-labeling, which was less co-labeling than the rest of the endocytic pathway at low lipoprotein concentrations. These data are consistent with a previous study using this methodology15 and other data indicating early endosomes as the sites of fusion between multiple vesicles; by that mechanism, the lipoproteins are joined and exhibited consistently higher co-labeling.17 18 19 20 LEPT had greater co-labeling with 75% co-labeling for both AcLDL and LDL. The most plausible explanation for these results is that ligands internalized via macropinocytosis fused with ligands internalized via endocytosis at the LEPTs. Lysosomes had a slightly lower extent of co-labeling with 48% and 61% of AcLDL and LDL co-trafficking. This may reflect sorting via the LEPT to subpopulations of lysosomes.
In macropinosomes, AcLDL had a significantly lower extent of co-labeling than LDL (82% of LDL co-trafficked with AcLDL compared with only 62% of AcLDL). However, AcLDL was twice as likely to co-traffick with LDL via macropinocytosis (macropinosomes) than coated-pit endocytosis (coated-pits, early endosomes, and spherical endosomes). The greatest extent of co-labeling in any organelle occurred in macropinosomes; if LDL was present in macropinosomes, 82% of the time AcLDL was also present. Previously, the greatest extent of co-trafficking possible with this co-trafficking methodology at this lipoprotein concentration was 80% to 90%.15 Therefore, 100% of LDL co-trafficked with AcLDL. This suggests that for LDL to traffick in macropinosomes, AcLDL must be present.
High Concentration Trafficking of AcLDL+LDL
At high AcLDL concentrations, the hypothesis is that the pathway
associated with foam cell formation, either coated-pit endocytosis or
macropinocytosis, will have a greater distribution of AcLDL. LDL at
high concentrations should be primarily internalized by coated-pit
endocytosis, unless AcLDL-stimulated macropinocytosis influences LDL
uptake. A time course of simultaneous trafficking was done
for LDL+AcLDL at concentrations of lipoproteins that stimulate foam
cell formation 50 to 150 µg/mL.
At <1 minute, the plasma membrane surface was highly convoluted and
ruffled (Figure 2A). At high concentrations, 65% of AcLDL and
LDL was on the plasma membrane at <1 minute (Figure 3). At 5
minutes, the distribution of both AcLDL and LDL on the plasma membrane
dropped to 40%. At 10 minutes, the distribution of lipoproteins on the
plasma membrane increased to 50% (Figure 3). This dramatic drop
at 5 minutes and subsequent increase at 10 minutes probably reflects
membrane ruffling activity. Some of AcLDL on membrane ruffles might
have appeared internalized within the cell; when the membrane ruffles
unfolded, an elevation of lipoproteins on the plasma membranes at 10
minutes resulted. With time, the distribution of AcLDL and LDL
decreased on the plasma membrane until 30 minutes. After that, the
distribution of lipoproteins on the plasma membrane remained stable or
perhaps slightly increased with 35% of AcLDL and LDL remaining
associated with the plasma membrane at 120 minutes (Figure 2).
This elevation in the distribution of lipoproteins on the plasma
membrane at these later times may reflect regurgitation
from macropinosomes. Time-lapse phase contrast microscopy showed that
during AcLDL incubation, both nonfusion of membrane ruffles and
regurgitation of macropinosomes
occurred.12
AcLDL and LDL were present in coated-pits, endosomes, and
macropinosomes at early times (Figure 2). The distribution of
AcLDL and LDL was compared in organelles consistent with
coated-pit endocytosis versus macropinocytosis to determine whether
AcLDL and LDL were taken up by coated-pit endocytosis versus
macropinocytosis (Table). LDL was 5- and 2-fold increased versus
AcLDL in the percent distribution in pits and vesicles and spherical
endosomes, respectively. LDL and AcLDL had the same distribution to
early endosomes with 17% and 20%, respectively, in early endosomes at
<1 to 10 minutes. This was a greater distribution of AcLDL and LDL to
the early endosomes than at low concentrations.
At <1 to 10 minutes, the same proportion of AcLDL and LDL (24%) was
present in macropinosomes (Table). Comparing trafficking of
LDL at 5 and 10 minutes showed 28% and 27% of LDL trafficked in
macropinosomes at high concentrations compared with 7% and 1% of LDL
at low concentration. The same distribution of AcLDL trafficked in
macropinosomes at high and low concentrations. These data indicated
that when AcLDL is present at high concentrations, a significant
portion of LDL trafficks via macropinocytosis. At high concentrations
of AcLDL, there was a shift toward more trafficking via
macropinocytosis. The total volume of AcLDL present in
macropinosomes over the entire time course was elevated, whereas the
distribution of AcLDL in coated pits and vesicles and spherical
endosomes were decreased (Figure 3).
Time Course (High Concentration)
The time course for maximal distribution of LDL at high
concentrations was 0 to 5 minutes for pits and vesicular profiles, 0 to
10 minutes for early endosomes, 10 to 30 minutes for spherical
endosomes, 10 to 60 minutes for LEPT, and 120 minutes for
lysosomes. The time course for maximal distribution of AcLDL at
high concentrations was 10 minutes for pits and vesicles, 0 to 10
minutes for early endosomes, 60 to 120 minutes for spherical endosomes,
10 to 60 minutes for LEPT, and 120 minutes for lysosomes
(Figures 3). A greater proportion of the input lipoprotein
(15%) reached the lysosomes at low versus high concentrations
of lipoproteins. This difference in distribution to the
lysosome could be accounted for by the distribution of
lipoproteins remaining associated with the plasma membrane at 120
minutes. Approximately 18% versus 35% of the input lipoprotein
remained associated with the plasma membrane at low versus high
concentrations. The time course of residence in macropinosomes doubled
at high concentrations; AcLDL and LDL were present in
macropinosomes for 60 minutes versus 30 minutes at low
concentration.
Co-Trafficking (High Concentration)
The quantitative data suggest that at high concentrations, AcLDL
continued to be internalized via coated-pit endocytosis and
macropinocytosis. However, LDL was now internalized in part via
macropinocytosis as well as coated-pit endocytosis. The co-labeling
data suggest that at high lipoprotein concentrations, no segregation of
AcLDL trafficking from LDL trafficking occurred (Figure 4B). At
high concentrations, the extent of co-labeling was greater than at low
concentrations in clathrin-coated pits with 30% to 50%, but greater
co-labeling was seen in the rest of the organelles. The percentage
distribution in coated pits, early endosomes, and spherical endosomes
were 61% and 56% for AcLDL and 72% and 53% for LDL, respectively.
Macropinosomes had the greatest extent of co-labeling with 75% and
85% for AcLDL and LDL, respectively. Interestingly, nearly all of the
lipoproteins were mixed in the LEPT compartment, with 92% of AcLDL and
88% of LDL in co-labeled organelles. Lysosome co-labeling was
less than the LEPT compartment with 62% of AcLDL and 51% of LDL in
co-labeled lysosomes. Previously, the LEPT compartment has been
designated as an organelle where fusion and segregation into distinct
lysosomes occurs.21 This suggests that these
lipoproteins are sorted to different lysosomes.
Lipoprotein Binding Sites
Previously, AcLDL was shown to bind predominately to membrane
ruffles and smooth portions of the membrane, whereas LDL bound to
microvilli.11 At both low and high concentrations, AcLDL
bound predominately to membrane ruffles and smooth areas of the plasma
membrane. However, when LDL was incubated with AcLDL, at both low and
high concentrations, LDL bound predominately to membrane ruffles
(please see Figure II, published on-line at
http://atvb.ahajournals.org/cgi/content/full/20/3/773/DC1). The
shift of LDL binding to membrane ruffles probably reflects the
stimulation of membrane ruffling stimulated by AcLDL.11 12
Enhanced Degradation of LDL Co-Incubated With AcLDL and
OxLDL
Co-incubation of LDL with AcLDL caused a shift in the
intracellular trafficking of LDL. Potentially, this could result in
internalization of LDL via fluid-phase or adsorptive endocytosis during
AcLDL stimulated macropinocytosis. To determine whether AcLDL affects
the degradation of co-incubated LDL, 125I-LDL was
incubated with cells in the presence of AcLDL. First, the binding was
determined for 125I-LDL (50 µg/mL) alone and
with co-incubated cold AcLDL or LDL (12 µg/mL). The total binding and
specific binding of LDL were reduced with AcLDL and LDL present in
pigeon macrophages (Figure 5) and
THP-1 cells (data not shown). In some experiments, nonspecific binding
of LDL increased, which may be due to trapping in AcLDL stimulated
membrane ruffles (data not shown).
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To determine whether AcLDL-stimulated macropinocytosis could increase
the uptake and subsequent degradation of LDL,
125I-LDL was incubated with macrophages
at 100 µg/mL, a concentration above saturation of high affinity
receptors. Cold AcLDL (20 µg/mL) was added to stimulate
macropinocytosis during incubation of 125I-LDL. A
control condition was co-incubation with cold LDL (20 µg/mL). The
125I-LDL in the cell associated fraction (Figures 5B and 5C) was not increased with AcLDL or LDL in pigeon
macrophages at 2 hours (data not shown) or 5 hours (Figure 5B). However, the degradation of 125I-LDL
was increased when AcLDL was present (Figure 5B).
OxLDL also stimulates macropinocytosis in pigeon
macrophages.12 OxLDL also increased the
degradation of 125I-LDL, with no affect on the
125I-LDL in the cell associated fraction (Figure 5C). OxLDL also stimulated the degradation of
125I-LDL in THP-1 macrophages. In THP-1
cells, OxLDL decreased the cell association of
125I-LDL (Figure 5D). Incubation
125I-LDL with cold AcLDL also decreased the cell
association but had no affect on the degradation of co-incubated
125I-LDL in THP-1 macrophages (data not
shown).
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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The time course for AcLDL and LDL internalization at low concentrations via receptor-mediated endocytosis is similar to other examples of coated-pit endocytosis reported in the literature. Interaction of AcLDL and LDL with receptors leads to uptake via coated-pit endocytosis at the base of microvilli on the plasma membrane. The contents of multiple pits and their derivative endosome vesicles fuse with each other by <1 to 10 minutes.17 18 19 20 These early endosomes pass on their contents or mature to spherical endosomes by 5 to 10 minutes. Multiple spherical endosomes may fuse with the LEPTs from 10 to 60 minutes.22 Lysosomes are then matured from the LEPTs.
At high concentrations, a similar time course is seen for coated-pit endocytosis of LDL and AcLDL. In contrast, the residence of both AcLDL and LDL in macropinosomes was doubled to 60 minutes at the higher concentration. Another difference between high and low concentrations was the increase in the distribution of both AcLDL and LDL on the plasma membrane at 120 minutes.
Most internalization studies use lipoproteins near the saturation of high affinity binding sites. However, foam cell formation requires higher concentrations of AcLDL. These studies speculated that at high lipoprotein concentrations, AcLDL would be segregated from LDL trafficking and more AcLDL would be internalized via macropinocytosis. At high concentrations, most AcLDL trafficked via macropinosomes. However, unexpectedly, LDL also trafficked through macropinosomes. If LDL is co-incubated with AcLDL at concentrations near the saturation of the high affinity receptor (low concentrations), LDL predominately trafficks through coated-pit endocytosis with little trafficking in macropinosomes. As shown here, at concentrations above the saturation of the high affinity receptor, a large portion of LDL trafficks through macropinsomes. Subsequent studies showed AcLDL has a similar influence on trafficking of Pigeon ßVLDL. ßVLDL co-incubated with AcLDL shifted from 5.8% to 8.4% of ßVLDL in macropinosomes at low versus high lipoprotein concentrations. As a control, ßVLDL and LDL were co-incubated at high or low concentrations. Both Pigeon ßVLDL and LDL had only 1% to 2% traffick in macropinosomes (data not shown).
The method for entry into macropinosomes of co-incubated ligands is not known. Earlier studies indicated that modified LDLs stimulate fluid-phase uptake in pigeon macrophages.12 However, adsorptive endocytosis cannot be ruled out. The ultrastructural methodology with 4°C loading, washing, and subsequent incubation at 37°C would not indicate true fluid-phase uptake but rather adsorptive endocytosis. Recently glycosaminoglycans and proteoglycans have been shown to contribute to the catabolism of OxLDL.23 24 One study showed a cooperation between glycosaminoglycans and cell-surface scavenger receptors (SR-As). Digestion of heparan sulfate and chondroitin sulfate could reduce degradation by 40%.23 This affect was not seen at 10 µg/mL but only at concentrations >25 µg/mL. Perhaps some modified LDL is internalized by attachment to the plasma membrane via glycosaminoglycans, but stimulation of macropinocytosis by interaction with the scavenger receptor is necessary. Interestingly, pigeon AcLDL-specific binding is saturable, but total binding was linearly dependent on concentration, similar to fluid phase uptake.25
Although the ultrastructural methodology favored adsorptive endocytosis, some label was trapped during the 4°C incubation. It could be possible that some LDL and AcLDL became trapped within pockets formed from membrane folds, which would be in the fluid-phase. Ruthenium red stained macrophages showed that at early times, some AcLDL was in the lumen of ruthenium red macropinosomes. And at 10 minutes, at both high and low concentrations, the distribution of the lipoproteins on the plasma membrane increased relative to the distribution at 5 minutes presumably from unruffling activity. The biochemical studies showed that total binding of LDL with AcLDL in our studies was actually decreased. In the internalization and degradation experiments, we did not wash the cells. Therefore, the increase in degradation of LDL co-incubated with AcLDL was more likely due to fluid-phase uptake rather than nonspecific binding and adsorptive endocytosis although the later two can be involved.
The following model is presented for modified LDLs stimulation
of foam cell formation via macropinocytosis (Figure 6). AcLDL binding to macrophages
stimulates membrane ruffling and macropinocytosis via interaction with
a scavenger receptor(s). This modified LDL stimulation of
macropinocytosis results in an increase of fluid-phase uptake and
membrane internalization. When other lipoproteins are co-incubated with
modified LDLs, if receptors are available, these co-incubated ligands
will still be predominately internalized via receptor-mediated
endocytosis in coated pits. At ligand concentrations above the
saturation of high affinity sites, significant portions of the
co-incubated lipoproteins are internalized in macropinosomes,
presumably by fluid-phase or adsorptively endocytosed via nonspecific
binding, low affinity-high capacity binding, and/or proteoglycan bound
ligands due to increased membrane internalization during membrane
ruffling.
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The atherogenic potential of macropinocytosis remains to be determined. First, macropinocytosis may be atherogenic due to large capacity uptake via fluid-phase or a stimulation of membrane internalization of nonspecific and low affinity binding of ligand. This uptake would be dependent on the scavenger receptor(s) and independent of cholesterol content in the cell. However, a net increase in cell association of 125I-LDL with AcLDL and OxLDL did not occur. Some experiments at 1 and 2 hours showed an increased cell association of LDL with modified LDLs present. The inconsistency could be due to a transient cell association and subsequent regurgitation. Macropinocytosis may also allow differential trafficking of cholesterol, with perhaps increased access to ACAT. Macropinosomes, compared with clathrin-coated and noncoated pits, are long-lived (up to 30 to 60 minutes). Previously, retention of lipoproteins on the plasma membrane and in surface tubules for entry into macrophages was suggested to be associated with a stimulation of ACAT.26 The surface tubules for entry into macrophages was an environment in which partial degradation of ßVLDL occurred. Macropinocytosis may also stimulate modification of LDL by prolonged contact with the macrophage surface and/or regurgitation. Sequestration of lipoproteins within tightly closed membrane ruffles and regurgitation from macropinosomes may be environments that enhance modification of LDL by macrophages. Recently, it was shown that cyclic adenosine monophosphate could selectively promote regurgitation of macropinosomes.27 Conditions may exist in the artery wall that would selectively promote uptake or regurgitation of lipoproteins. The increase in degradation without a consistent increase in cell association of 125I-LDL suggests either degradation in the membrane ruffled areas or regurgitation of macropinosomes.
An apparent quandary in the foam cell formation literature is that, although high LDL plasma concentration is a reliable risk factor for predicting heart disease, LDL uptake by macrophages in vitro does not cause macrophage foam cell formation. The data from this study suggest an intriguing hypothesis; the presence of some modified LDL can affect the intracellular trafficking and potentially the metabolism of unmodified LDL. This would suggest that not all of the arterial LDL would need to be modified for foam cell formation to occur with high concentrations of LDL. Modified LDLs stimulated an increase in the total degradation of LDL at 37°C, without increasing the total binding at 4°C. These new findings present a novel working hypothesis: first, macropinocytosis rather than coated-pit endocytosis is associated with foam cell formation; second, that nonreceptor mediated endocytosis and low affinity uptake is an important pathway for foam cell formation; and third, that modified LDL stimulation of macropinocytosis causes increased degradation of co-internalized lipoproteins.
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Acknowledgments |
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This work was supported by National Institutes of Health grant HL-41990 and a Venture Grant from the Wake Forest University School of Medicine (to N.L.J.). William A. Hooker and Marie K. Plyler contributed the bulk of the technical assistance on these studies. Revill R. Mallory did the map wheel quantification for the ten minute time point. We thank Mary Ann Hayner for manuscript preparation. We also thank the MICROMED staff of Wake Forest University School of Medicine for IVEM technical assistance and EM printing.
Received May 25, 1999; accepted October 15, 1999.
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