© 1996 American Heart Association, Inc.
Articles |
Angiotensin II–Modified LDL Is Taken Up by Macrophages Via the Scavenger Receptor, Leading to Cellular Cholesterol Accumulation
From The Lipid Research Laboratory, Rambam Medical Center, Rappaport Institute for Research in the Medical Sciences, The Bruce Rappaport Technion Faculty of Medicine, Haifa, Israel.
Correspondence to Dr Shlomo Keidar, The Lipid Research Laboratory, Rambam Medical Center, Rappaport Institute for Research in the Medical Sciences, The Bruce Rappaport Technion Faculty of Medicine, Haifa 31096, Israel.
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Abstract |
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Abstract The incidence of myocardial infarction is significantly higher in hypertensive patients with increased plasma concentration of angiotensin (Ang) II. Ang II was shown to bind to LDL in vitro, and in the present study we showed its binding to LDL in vivo. Ang II (10-7 mol/L) was incubated with LDL for 3 hours at 37°C, followed by reseparation of the modified lipoprotein (Ang II–LDL) and its incubation with J-774 A.1 macrophages. Binding of Ang II to LDL significantly increased the lipoprotein protein degradation (by 25%) and its cell association (by 75%) compared with nontreated LDL. Unlike Ang II–LDL, both Ang I–LDL and Ang III–LDL were taken up by macrophages similar to native LDL. The lipid composition and size of Ang II–LDL were similar to those of native LDL, and it was not aggregated. Ang II–LDL was not oxidized, as the contents of malondialdehyde and peroxides were not different from those found in native LDL. On heparin-Sepharose column chromatography, Ang II–LDL was eluted in the void volume, like acetylated LDL (Ac-LDL) and unlike native LDL, which binds to heparin. The cellular degradation of Ang II-125I–labeled LDL by J-774 A.1 macrophages was studied in the presence of a 50-fold excess of nonlabeled native LDL, Ang II–LDL, Ac-LDL, or oxidized LDL (Ox-LDL). Whereas native LDL had no effect on the degradation of Ang II-125I–LDL by the macrophages, Ac-LDL, Ox-LDL, and Ang II–LDL reduced the cellular uptake of the lipoprotein by 77%, 82%, and 87%, respectively. Similarly, fucoidin but not free Ang II reduced macrophage degradation of the labeled Ang II–LDL. We conclude that Ang II can modify LDL to a form that is not oxidized or aggregated but is still taken up at an enhanced rate by macrophages via the scavenger receptor.
Key Words: angiotensin • LDL • macrophages • scavenger receptor
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Introduction |
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Hypertensive patients with elevated plasma levels of Ang II were shown to have a fivefold increase in the incidence of MI compared with hypertensive patients with normal or decreased levels of Ang II.1 In addition, several studies2 3 have recently shown a reduced incidence of recurrent MI and unstable angina in post-MI patients treated with ACE inhibitors. This class of drugs has also demonstrated an inhibitory effect on the progression of atherosclerosis in Watanabe heritable hyperlipidemic rabbits,4 cholesterol-fed cynomolgus monkeys,5 cholesterol-fed rabbits,6 and minipigs.7
Macrophage cholesterol accumulation is an early event in atherogenesis,8 and it probably results from an increased uptake of modified LDL via the macrophage scavenger receptor, which unlike the LDL receptor is not regulated by the cellular cholesterol content.9 10
A major class of scavenger receptors to which Ox-LDL binds is the Ac-LDL receptor. In addition to Ox-LDL, the Ac-LDL receptor binds several other chemically or enzymatically modified forms of LDL, leading to cellular cholesterol accumulation and foam cell formation.9
Recently, we have demonstrated that Ang II increases the uptake of LDL by arterial wall macrophages.11 As Ang II is produced by these cells,12 its effect on macrophage-LDL interactions can possibly affect macrophage cholesterol accumulation and foam cell formation. We have recently shown13 14 that Ang II stimulates macrophage lipid peroxidation and by so doing can lead to cell-mediated oxidation of LDL and the formation of atherogenic Ox-LDL. Since Ang II specifically binds to LDL,11 it can possibly produce another modified form of LDL, Ang II–modified LDL (Ang II–LDL). If this hypothesis can be proven, then Ang II can possibly affect macrophage foam cell formation not only by the formation of Ox-LDL13 but also by the formation of Ang II–LDL. The present study was undertaken to discover the possible formation of Ang II–LDL and assess the interaction of this modified LDL with macrophages. The results of this study show that Ang II binds to LDL both in vitro and in vivo and that the resulting Ang II–modified LDL is taken up by macrophages via the scavenger receptor at an enhanced rate.
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Methods |
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Cells
The J-774 A.1 murine macrophage-like cell line was purchased from American Type Culture Collection. The cells were plated at 2.5x105 cells per 16-mm dish in DMEM supplemented with 10% fetal calf serum, penicillin, streptomycin, and glutamine and were fed every 3 days and used for experiments within 7 days of plating. Human skin fibroblasts were grown in monolayers, used between the 5th and 20th population doubling, and maintained in a humidified 5% CO2 incubation at 37°C in stock flasks containing DMEM and 10% fetal calf serum, penicillin, streptomycin, and glutamine. For experimentation, confluent monolayers of cells were dissociated with 0.05% trypsin and 0.02% EDTA solution in saline and seeded at 3x104 cells into 35-mm petri dishes.15
Human monocyte–derived macrophages were prepared from the blood of fasting normolipidemic subjects by density gradient centrifugation.16 Twenty milliliters of blood anticoagulated with sodium heparin (final concentration, 10 U/mL) was layered over 15 mL Ficoll-Paque (Pharmacia). After centrifugation at 500g for 30 minutes at 23°C, the mixed mononuclear cell band was removed by aspiration, and the cells were washed twice at 4°C in DMEM supplemented with L-glutamine (final concentration, 2 mmol/L), 100 U/mL penicillin, and 100 µg/mL of streptomycin. The cells were plated at 3x105 monocytes per 16-mm dish (Primaria Brand, Falcon Labware) in the same medium (0.5 mL) containing 20% autologous serum. After 2 hours of incubation at 37°C in 5% CO2/95% air, nonadherent cells were removed by three washes with serum-free medium. The cells were placed in fresh medium containing 20% autologous serum, fed twice weekly, and used for experiments after 7 days in culture.
Lipoprotein Preparation and Characterization
LDL was prepared
from human plasma (in 1 mmol/L EDTA) drawn from
fasted normolipidemic volunteers. LDL (d=1.019 to 1.063
g/mL) was prepared by discontinuous density gradient
ultracentrifugation as described
previously.17 The lipoprotein was washed at
d=1.063 g/mL and dialyzed against 150 mmol/L NaCl and 1
mmol/L EDTA, pH 7.4. LDL was then sterilized by filtration and was used
within 2 weeks. In one experiment LDL was also separated from
apoE-deficient mice 24 hours after the injection of
125I-labeled Ang II. ApoE-deficient mice were kindly
provided to us by Dr Jan Breslow, Rockefeller University, New York, NY.
Gene targeting in mouse embryonic stem cells was used to create mice
that lack apoE. Blood was collected from the retro-orbital plexus
under anesthesia with ether into Eppendorf tubes with 1
mmol/L Na2 EDTA.18 The protein content of the
lipoproteins was determined with the Folin phenol
reagent.19 Cholesterol was analyzed by
the ferrous chloride assay,20 triglycerides by
enzymatic assay,21 and phospholipids by the method of
Bartlett.22 Vitamin E content of the lipoproteins was
analyzed by the bathophenanthroline–ferric chloride
assay.23
Ang II–modified LDL (Ang II–LDL) was prepared by the addition of 10-7 mol/L Ang II (pure angiotensin II was obtained from Sigma Chemical Co) to 1 mg of LDL protein per milliliter (10-7 mol Ang II/2x10-6 mol LDL apoB-100) and incubated for 3 hours at 37°C. Then the modified lipoprotein was reseparated by ultracentrifugation at d=1.210 g/mL. Ang II–LDL was then dialyzed against 150 mmol/L saline and 1 mmol/L EDTA at 4°C for 18 hours.
Plasma and LDL content of Ang II were determined by radioimmunoassay.24
LDL and Ang II were radioiodinated by the iodine monochloride method.25 LDL was also radiolabeled with 1 mCi/mL of 3[H]cholesteryl linoleate to label its cholesteryl ester moiety, as previously described.26
LDL was acetylated by repeated additions of acetic anhydride to 5 mg of protein per milliliter of LDL diluted 1:1 (vol/vol) with saturated ammonium acetate at 4°C.9 Acetic anhydride was added at a 40-fold molar excess with regard to total lysines in LDL, and the modification was confirmed by electrophoresis on cellulose acetate at pH 8.6 in barbital buffer.27
Ox-LDL was prepared by dialysis of LDL against PBS overnight, followed by its incubation with 10 µmol/L CuSO4 for 18 hours at 37°C. Oxidation was terminated by refrigeration and the addition of 0.1 mmol/L EDTA. The degree of LDL oxidation was determined by analysis of MDA equivalents by using the thiobarbituric acid reactive substance assay,28 and LDL peroxides were measured by their capacity to convert iodide to iodine, measured at 365 nm.29
SDS-PAGE was performed with 3% to 20% gradient gel with mercaptoethanol as a reducing agent.30 Electrophoresis was performed at a constant current (5 mA) for 16 hours at 25°C. The gels were stained with 0.1% Coomassie brilliant blue R and destained with 10% acetic acid. Nondenaturing polyacrylamide gradient gel electrophoresis of the lipoprotein was performed on 3% to 20% gels to compare their relative sizes and assess possible aggregation of the lipoprotein.31 Lipoprotein aggregation was also measured in samples of 125I-labeled LDL and Ang II-125I–labeled LDL in the original samples as well as in precipitates obtained after centrifugation (10 000g for 10 minutes) of these lipoproteins. Lipoprotein aggregation was then calculated as the percent of the precipitate radioactivity of the total LDL radioactivity.32 Free lysine amino groups in LDL were estimated with TNBS. LDL (50 µg protein) was mixed with 1 mL of 4% NaHCO3 and 50 µL of 0.1% TNBS at pH 8.4 and heated for 1 hour at 37°C. The absorbance at 340 nm was then measured.33
Macrophage Metabolism of LDL
Cellular Lipoprotein
Protein Degradation
Cellular degradation of LDL and Ang II–LDL
(as well as Ang
I–LDL and Ang III–LDL) was measured after incubation of the cells
(1x106 per 16-mm dish) with various amounts (25 to
100 µg of protein per milliliter) of the
radioiodinated lipoprotein in serum-free DMEM
containing 0.2% BSA. The hydrolysis of LDL protein was assayed in the
incubation medium by measurement of trichloroacetic acid–soluble,
noniodide radioactivity. Cell-free LDL degradation was minimal and
subtracted from total degradation. The cell layer was washed three
times with PBS and extracted by 1 hour of incubation at room
temperature with 0.1N NaOH for measurement of cellular protein content
as well as cell-associated
125I-lipoproteins.34
Lipoprotein-Mediated Cellular Cholesterol
Esterification and Accumulation
Lipoprotein cholesterol uptake was
also estimated by
measurement of the stimulation of 3[H]oleate
incorporation into CE.35 Cells were incubated with LDL or
Ang II–LDL (25 µg of protein per milliliter) for 18 hours at
37°C.
Then the cell layer was washed and 3[H]oleate complexed
with albumin (2.7 mmol/L, 83 mmol oleate/mg albumin, 10
µCi/mL) was added to the macrophages and further incubated
for 2 hours at 37°C. At the end of the incubation, cellular lipids
were extracted with hexane/isopropanol (3:2, vol/vol), and the CE was
separated by TLC using hexane/ether/acetic acid (130:30:1.5,
vol/vol/vol). The CE spot was scraped into vials containing 4 mL
scintillation fluid and counted in a ß-scintillation
counter.
Cellular cholesterol mass was determined after macrophage incubation with 100 µg of protein per milliliter of LDL or Ang II–LDL for 18 hours at 37°C. Cellular lipids were extracted with hexane/isopropanol (3:2, vol/vol) and determined by the ferrous chloride assay.20
Cellular Uptake of
the Lipoprotein
Cholesterol
J-774 A.1 macrophages were incubated with
3[H]CE-labeled LDL or Ang
II-3[H]CE–labeled LDL (25 µg of protein per
milliliter) for 18 hours at 37°C. The cells were then washed three
times with ice-cold PBS, and solubilization of cells was obtained
by the addition of 0.1 N NaOH for 2 hours at room temperature. Then an
aliquot of the cells (100 µL) was transferred to scintillation vials
containing 4 mL scintillation fluid and counted in a
ß-scintillation counter.25
Heparin-Binding Characteristics of the Lipoproteins
LDL and
Ang II–LDL were submitted to heparin-Sepharose affinity
chromatography. Sepharose complexed with heparin
(Affi-gel Heparin, Bio-Rad Labs) was packed into a minicolumn
(1.5x8 cm). The column was equilibrated with 0.05 mol/L NaCl and 2
mmol/L phosphate buffer (pH 7.4) containing 0.01% of NaN3
per volume). One milligram of lipoprotein protein was applied onto the
column, and elution was begun at a flow rate of 30 mL/h. The fractions
were monitored at 280 nm in a spectrophotometer for analysis of
their protein content. After the unbound fraction was eluted (and the
absorbance at 280 nm had decreased back to baseline values), the
retained fraction (bound) was eluted with 0.8 mol/L
NaCl.36 37 Recovery of the lipoprotein protein ranged
between 80% and 86%. In experiments where the lipoprotein was labeled
in its CE of 3[H]CE-LDL, the radioactivity in the
fractions was counted in a ß-scintillation counter.
Statistical Analysis
Statistical analysis was performed using
the nonpaired
Student t test. Results are given as mean±SD.
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Results |
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Enhanced Macrophage Uptake of Ang II–LDL
We have previously shown that 125I-labeled Ang II specifically binds to LDL after their incubation for 1 hour at 37°C and reseparation of the lipoprotein.11 After LDL (10 µg of protein per milliliter, 2x10-8 mol/L) incubation with Ang II (10-7 mol/L) for 1 hour at 37°C and reseparation of the lipoprotein by ultracentrifugation (d=1.210/mL), analysis of Ang II in this LDL by radioimmunoassay revealed the presence of 250±35 pg of Ang II/mg LDL protein.
We have also
analyzed the binding capabilities of Ang II to LDL
in vivo in both mice and humans. In mice that were injected with 800
ng/mL of Ang II, 16±4 pg of the Ang II was found to be associated with
1 mg of plasma LDL 24 hours after the injection (n=3). We have used
apoE-deficient transgenic mice, which are characterized by a high
plasma concentration of VLDL and LDL.18 38 LDL was
isolated from these mice by density
ultracentrifugation.17 Of the total
radioactivity that was injected as 125I-Ang II, 11±3% was
present in the plasma 24 hours after the injection, and 25±5% of
this plasma-associated radioactivity was bound to LDL. Furthermore,
in two hypertensive patients, radioimmunoassay analysis
revealed the presence of 4.0±0.4 pg of Ang II/mg LDL protein, and this
represents about 10% of the total plasma Ang II concentration
(39.2±7.3 pg of Ang II per milliliter, n=2). These data clearly
demonstrate that Ang II binds to LDL both in vitro and in vivo. Upon
incubation of 1 mg of protein per milliliter
(2x10-6 mol/L) of
3[H]CE-LDL with Ang II
(10-7 mol/L) for 3 hours at 37°C, the
formation of a modified LDL (Ang II–LDL), with respect to its
interaction with macrophages, was noted. This Ang II–LDL ([25
µg of protein per milliliter] obtained after reseparation of the
lipoprotein by ultracentrifugation at
d=1.210 g/mL), when incubated with J-774 A.1
macrophages (1x106 cells per 16-mm dish)
for 18 hours at 37°C, was taken up 61% more than the native LDL (Fig
1A
). Cellular degradation and cell association of Ang
II-125I–labeled LDL were increased by 29% and 56%,
respectively, compared with native LDL (Fig 1B and
1C). The increased
macrophage uptake of Ang II–LDL was shown to be related to
enhanced binding of this lipoprotein, as the binding of Ang II–LDL to
the cells (analyzed at 4°C) increased by 75% compared with
native LDL (Fig 1D). Ang II–LDL also increased the
cellular
cholesterol esterification rate by 30% compared with LDL
(Fig 1E). To further characterize the effect of Ang
II–LDL on
macrophage lipoprotein uptake, total cholesterol
mass was also determined after cell incubation with LDL (100 µg of
protein per milliliter) for 16 hours at 37°C. The cellular
cholesterol mass increased by 16% after macrophage
incubation with Ang II–LDL compared with the effect of native LDL (Fig
1F).
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With increasing concentrations of Ang II to modify
the LDL,
macrophage uptake of 3[H]CE-labeled LDL (25 µg
of protein per milliliter) significantly increased in a
dose-dependent fashion. Each increased Ang II concentration
resulted in a significant (P<.01) increment in cellular
uptake of the lipoprotein compared with the lower Ang II concentration
by up to 95% when an Ang II concentration of
10-6 mol/L was used (Fig 2).
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Analysis
of macrophage degradation of Ang
II-125I–labeled LDL over lipoprotein concentrations, which
range between 10 and 50 µg of protein per milliliter, revealed in the
J-774 A.1 macrophage cell line and also in human
monocyte–derived macrophages 18% to 32% and 41% to
61%, respectively, increased cellular degradation of the modified
lipoprotein compared with native LDL (Table 1). Free
radioiodinated Ang II was also found to bind and to be
internalized by J-774 A.1 macrophages (data not shown).
Macrophage degradation of Ang II-125I–LDL was then
compared with that of Ang-I125I–LDL and Ang
III-125I–LDL (similarly prepared as described for Ang
II–LDL). On preliminary experimentation,
radioiodinated Ang I and Ang III (the precursor and
product of Ang II, respectively) were found to bind to LDL (data
not shown). Only Ang II–LDL and not Ang I–LDL or Ang III–LDL
demonstrated an increased degradation rate by J-774 A.1
macrophages compared with native LDL (Fig 3),
suggesting the specificity of Ang II in the formation of modified
LDL.
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Physicochemical Composition of Ang II–LDL
Analysis of
Ang II–LDL composition revealed no
significant changes in the lipoprotein content of
cholesterol, phospholipids, or triglycerides
compared with native LDL (Table 2). The LDL protein
concentration was also not affected by its incubation with Ang II
(1.0±0.2 versus 0.9±0.2 mg of protein per milliliter in the
absence
and presence of Ang II, respectively; n=3). In addition, no differences
in the composition of the phospholipid subclass could be found by TLC
analysis of the lipoprotein phospholipids (data not shown).
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Ang
II–modified LDL was not aggregated, as shown by
analysis of the radioactivity in the precipitate.
High-speed centrifugation (10 000g for
10 minutes) of 125I-labeled LDL and Ang
II-125I–LDL revealed the presence (in the precipitate) of
4.5±1.1% and 5.8±1.4% of the total radioactivity, respectively
(n=3). TNBS reactivity (which measures the content of free
-amino lysine groups on the LDL apoB-100) of Ang II–LDL was
similar to that of native LDL, and electrophoresis of Ang II–LDL on
cellulose acetate showed mobility similar to that of native LDL (data
not shown). The size of Ang II–LDL was unchanged compared with native
LDL, as determined by nondenatured gradient gel electrophoresis.
Ang
II–LDL was not oxidized, as the MDA content in Ang II–LDL was
1.78±0.23 nmol/mg compared with a value of 2.25±0.17 nmol/mg LDL
protein in native LDL. Similarly, peroxide content in LDL and Ang
II–LDL was 17.1±3.3 and 16.3±3.7 nmol/mg LDL protein,
respectively.
Analysis of Ang II–LDL on SDS-PAGE revealed that the apoB-100
was not fragmented (data not shown), suggesting that Ang II–LDL was
indeed not oxidized. The content of vitamin E in LDL and Ang II–LDL
was 1.30±0.15 and 1.25±0.14 µg/mg of LDL protein,
respectively. To
further characterize Ang II–LDL, the modified lipoprotein was
subjected to heparin-Sepharose column chromatography,
as heparin binds the positively charged amino acid residues in the
lipoprotein apoB-100. The heparin-binding capacity of Ang II–LDL
(Fig 4A) was compared with that of native LDL (Fig
4B),
Ac-LDL (Fig 4C), and Ox-LDL (Fig 4D). Using 1 mg
of lipoprotein
protein, we monitored the heparin-binding characteristics in the
eluted fractions by protein analysis at 280 nm (Fig 4). Fig
4
shows that on using Ang II–LDL, Ac-LDL, or Ox-LDL, 65%, 87%, or 93%
of the lipoprotein protein, respectively, was found in the unbound
fraction, whereas on using native LDL, only 4% of the lipoprotein
protein was found in the unbound fraction. Most of the LDL particles
(96%) were bound to the heparin column. Free 125I–
labeled Ang II did not bind to the heparin column, as 98±2% of the
loaded material eluted in the void volume of the column.
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The effect of
using increasing Ang II concentrations in the preparation
of Ang II–LDL was then studied. The heparin-binding
characteristics of Ang II–LDLs that were prepared by incubation of
3[H]CE-LDL (1 mg of protein per milliliter) with
increasing concentrations of Ang II (10-7
to 10-6 mol/L) are illustrated in Fig 5A
through 5D. Whereas native LDL almost completely binds
to the heparin column (Fig 5A), an Ang II dose-dependent
increment
in the relative content of the unbound fraction was noted. Only 34% of
the Ang II–LDL was found in the unbound fraction at Ang II
concentration of 10-7 mol/L (Fig 5B),
whereas up to 94% of the total radioactivity eluted as an unbound
fraction at 10-6 mol/L of Ang II (Fig 5D).
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Macrophage Receptor Responsible for the Enhanced Cellular
Uptake of Ang II–LDL
To analyze the macrophage receptor
responsible for
the enhanced cellular uptake of Ang II–LDL, the degradation of Ang
II-125I–LDL (10 µg of protein per milliliter) was
studied in the presence of a 25- or 50-fold excess concentration of
nonlabeled LDL, Ac-LDL, Ox-LDL, or Ang II–LDL. The nonlabeled Ox-LDL,
Ac-LDL, and Ang II–LDL at a 50-fold excess concentration reduced Ang
II-125I–LDL cellular degradation by 77%, 82%, and 87%,
respectively (Fig 6). Nonlabeled LDL, however, at both
25- and 50-fold excess concentrations over that of Ang
II-125I–LDL did not affect the degradation of Ang
II-125I–LDL by the macrophages (Fig 6). The
addition of fucoidin (50 µg/mL) (which binds to the
macrophage scavenger receptor) to the macrophages, in
the presence of 10 µg/mL of Ang II-125I–LDL, inhibited
cellular degradation of Ang II-125I–LDL by 91% (from
354±20 to 32±7 ng/mg cell protein), whereas free Ang II had no
effect (333±17 ng of LDL protein degraded·mg cell
protein-1·5
h-1).
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In human skin fibroblasts, which do not possess the scavenger receptor but only the LDL receptor, the degradation of native LDL (25 µg of protein per milliliter) was 881±30 ng·mg cell protein-1·5 h-1, whereas the cellular degradation rates of Ac-LDL and Ang II–LDL were only 54±9 and 140±48 ng·mg cell protein-1·5 h-1, respectively, suggesting that the LDL receptor is not involved in the cellular uptake of Ang II–LDL.
Ang II–LDL Susceptibility to Oxidation by the
Macrophages
To analyze the possibility that on incubation of Ang
II–LDL with macrophages the cells can oxidize the lipoprotein
(and thus may enhance its uptake via a scavenger receptor), LDL or Ang
II–LDL (25 µg of protein per milliliter) was incubated with J-774
A.1 macrophages for 18 hours at 37°C followed by
analysis of the MDA and peroxide content in the medium. The
content of MDA in Ang II–LDL and native LDL was 2.0±0.2 and
2.3±0.1
nmol/mg lipoprotein protein, respectively (n=3). The peroxide content
in Ang II–LDL and LDL was 31±3.5 and 26.5±1.5 nmol/mg
lipoprotein
protein, respectively. These results suggest that Ang II–LDL is not
oxidized and that the macrophages did not oxidize this modified
LDL under the experimental conditions used in the present study.
These results were further supported by the inability of the
antioxidant vitamin E (25 µmol/L) to affect macrophage
degradation of Ang II-125I–LDL (25 µg of protein per
milliliter) either when added during LDL incubation with Ang II or
during the incubation of Ang II-125I–LDL with the
macrophages (Table 3).
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Discussion |
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TopAbstractIntroductionMethods Results Discussion References |
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The present study shows that Ang II can modify LDL to a form that is taken up via the macrophage scavenger receptor at an enhanced rate compared with native LDL. Acetylation of LDL causes the loss of its binding capacity to heparin. In the present study, Ang II binding to LDL was also shown to reduce the binding of LDL to heparin in a dose-dependent manner. Heparin binds to the LDL surface by its interaction with the positively charged amino acid residues in apoB-100, and at least four unique heparin-binding domains have been identified in the LDL apoB-100.39 It might be that the interaction of Ang II with some heparin-binding domains in the LDL apoB-100 prevented the modified lipoprotein's ability to bind to the LDL receptor and enabled the modified lipoprotein to interact with the macrophage scavenger receptor. Modification of the LDL amino acids histidine, lysine, or arginine was shown to cause a significant loss in its heparin-binding capacity,40 suggesting that these basic amino acids are involved in the lipoprotein binding to heparin. It is also possible that Ang II binding to the LDL apoB-100 basic amino acids can change the conformation of the LDL molecule and thus its heparin-binding capability. The binding of lipoproteins to macrophages is dependent on the charge (total and local) as well as conformation of the lipoprotein particle. Binding of Ang II to the LDL surface can affect both the local charge and conformation of the receptor(s). The scavenger receptors demonstrated very broad but by no means totally indiscriminate binding specificity.41 Scavenger receptors bind a wide variety of negatively charged ligands. Ang II may also react with phospholipid amino groups, producing a negatively charged lipoprotein surface that mediates binding to the scavenger receptor.42 Recently it was shown that on incubation of Ang II with rat mesangial cells, which possess scavenger receptors, a substantial increment in the uptake of LDL by these cells was observed.43 The involvement of the scavenger receptor and not the LDL receptor in the uptake of Ang II–LDL by macrophages is supported by the following data: (1) Human skin fibroblasts (which lack the scavenger receptor) are unable to take up Ang II–LDL; (2) nonlabeled Ox-LDL, Ac-LDL, and fucoidin, which bind to the scavenger receptor but not native LDL, which binds to the LDL receptor, significantly compete with Ang II-125I–labeled LDL for its degradation by macrophages; and (3) Ang II–LDL, like Ac-LDL and Ox-LDL but unlike native LDL, does not bind to heparin.
Ang II was recently shown to be a potent activator of lipid peroxidation in macrophages14 and was also shown to stimulate the production of superoxide anions from smooth muscle cells.44 Ang II–LDL, however, was not oxidized, and the following data strengthen this conclusion: (1) The content of MDA and peroxide in Ang II–LDL was not significantly different from that of native LDL; (2) the apoB-100 of the modified lipoprotein was not fragmented (as shown on SDS-PAGE); (3) the electrophoretic mobility of Ang II–LDL on cellulose acetate was similar to that of native LDL; (4) after 5 hours of incubation of Ang II–LDL with macrophages, the modified lipoprotein did not contain an increased content of MDA or peroxides; (5) the addition of the antioxidant vitamin E during the preparation of Ang II–LDL or during the incubation of the modified lipoprotein with macrophages had no significant effect on the degradation of Ang II–LDL; and (6) Ang II–LDL was not aggregated, as determined by centrifugation as well as gradient gel electrophoresis analysis, in contrast to the tendency of Ox-LDL to form aggregates.32 It is therefore suggested that the increased macrophage uptake of Ang II–LDL was not caused by oxidation of this modified LDL. Thus, dose-dependent effects of Ang II on LDL binding to heparin and on the uptake of Ang II–LDL by macrophages, as well as the ineffectiveness of Ang I and Ang III (the precursor and product of Ang II, respectively) suggest a specific effect of Ang II on the extent of LDL modification and its biological activity in the cells.
The stimulatory effect of Ang II on the transfer of LDL across the endothelial barrier into the vessel wall was recently studied.45 46 It was suggested45 that the effect of Ang II to increase the uptake of LDL by the rat aorta is independent of the pressor effect of Ang II. In contrast, Nielsen et al46 demonstrated that Ang II increases the flux of LDL from the plasma into the arterial wall via an increase in blood pressure. The results of the present study, demonstrating the formation of Ang II–LDL (on LDL incubation with Ang II) that is taken up by the cells at an increased rate, may explain this phenomenon by the possible in vivo formation of Ang II–LDL. With respect to the in vivo significance of the present results, although it was not possible to demonstrate enhanced macrophage uptake of Ang II–LDL on using Ang II concentration as low as 10-11 mol/L, it is suggested that increased local concentration of Ang II in areas of the arterial wall can cause in vivo effects similar to those shown in the present in vitro study. A recent study47 confirmed the differences in the internal and plasma compartments for Ang II and Ang-I and demonstrated that while Ang I concentration in normal rat plasma was over threefold higher than plasma Ang II, this ratio was reversed in the kidney.
The vascular wall has been shown to be equipped with all the enzymes necessary to synthesize Ang II.12 Local generation of Ang II from Ang I was demonstrated in the canine coronary artery, and ACE inhibitors abolished this formation.48 ACE is induced in injured vessels and expressed mainly in the intima. The degree of neointimal formation showed greater correlation with tissue ACE than plasma ACE,49 and both Ang II binding sites and ACE were demonstrated in the early atherosclerotic plaque.50 These results suggest that tissue ACE and the local synthesis of Ang II may play a major role in neointimal formation. Although plasma concentrations of Ang II in hypertensive patients were reported to be substantially lower than those used in our in vitro studies, in the local subcellular space Ang II concentrations are much higher than those in the plasma. In addition, other vasoactive substances, such as bradykinin, which is present in areas of the atherosclerotic plaque, may increase the effect of Ang II; thus, a lower local concentration of Ang II may exert its deleterious effect.
Ang II may exert its atherogenic effects1 2 via several different mechanisms, leading to the formation of modified forms of lipoproteins,51 52 53 54 which were previously shown to enhance atherosclerosis. The present study suggests the contribution of Ang II to LDL modification in the arterial wall via the formation of Ang II–modified LDL, which is taken up by the cells at an increased rate via the macrophage scavenger receptor and can thus cause foam cell formation.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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The authors acknowledge the excellent technical aid of Judith Oikinine, the manuscript typing by Ilana Cohen, and the mice study by Dr Tony Hayek.
Received May 18, 1995; accepted September 28, 1995.
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References |
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TopAbstractIntroductionMethods Results Discussion References |
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1. Alderman MH, Madhavan S, Ooi WL, Cohen H, Sealey JE, Laragh JH. Association of the renin-sodium profile with the risk of myocardial infarction in patients with hypertension. N Engl J Med. 1991;324:1098-1104. [Abstract]
2. Pfeffer MA, Braunwald E, Moye LA. Effect of captopril on mortality and morbidity in patients with left ventricular dysfunction after myocardial infarction: results of the survival and ventricular enlargement trial. N Engl J Med. 1992;327:669-677. [Abstract]
3. Yusuf S, Pepine CJ, Garces-Pouleur H, Salem D, Kostis J, Benedict C, Rousseau M, Bourassa M, Pitt B. Effect of captopril on myocardial infarction and unstable angina in patients with low ejection fractions. Lancet. 1992;340:1173-1178. [Medline] [Order article via Infotrieve]
4.
Chobanian AV, Haudenschild CC, Nickerson C, Drago
R. Antiatherogenic effect of captopril in the Watanabe heritable
hyperlipidemic rabbit.
Hypertension. 1990;15:327-331.
5. Aberg G, Ferrer P. Effects of captopril on atherosclerosis in cynomolgus monkeys. J Cardiovasc Pharmacol. 1990;15:S65-S72.
6. Schuh JR, Blehm DJ, Friedrich GE, McMahon EG, Blaine EH. Differential effects of renin-angiotensin system blockade on atherogenesis in cholesterol-fed rabbits. J Clin Invest. 1993;91:1453-1458.
7.
Charpiot P, Rolland PH, Friggi A, Piquet P, Scalbert
E, Bodard H, Barlatier A, Latrille V, Tranier P, Mercier C, Luccioni R,
Calof R, Garcon D. ACE inhibition with perindopril and
atherogenesis-induced structural and functional changes in minipig
arteries. Arterioscler Thromb. 1993;13:1125-1138.
8. Steinberg D, Parthasarathy S, Carew TE, Khoo JC, Witztum JL. Beyond cholesterol: modifications of low density lipoprotein that increase its atherogenicity. N Engl J Med. 1989;320:915-924. [Medline] [Order article via Infotrieve]
9. Brown MS, Goldstein JL. Lipoprotein metabolism in the macrophage: implications for cholesterol deposition in atherosclerosis. Annu Rev Biochem. 1983;52:223-261. [Medline] [Order article via Infotrieve]
10. Aviram M. Effect of lipoproteins and platelets on macrophages' cholesterol metabolism. In: Harris JR, ed. Blood Cell Biochemistry, Vol 2: Megakaryocytes, Platelets, Macrophages and Eosinophils. New York, NY: Plenum Publishing Corporation; 1991:179-208.
11. Keidar S, Kaplan M, Shapira C, Brook JG, Aviram M. Low density lipoprotein isolated from patients with essential hypertension exhibits increased propensity for oxidation and enhanced uptake by macrophages: a possible role for angiotensin II. Atherosclerosis. 1994;107:71-84.[Medline] [Order article via Infotrieve]
12. Dzau VJ. Circulating versus local renin-angiotensin system in cardiovascular homeostasis. Circulation. 1988;7(suppl I):I-4-I-13.
13. Keidar S, Kaplan M, Hoffman A, Brook JG, Aviram M. Angiotensin II stimulates macrophage-mediated lipid peroxidation of low density lipoproteins. Atherosclerosis. 1995;115:201-215. [Medline] [Order article via Infotrieve]
14.
Keidar S, Brook JG, Aviram M.
Angiotensin II enhances lipid peroxidation of low density
lipoprotein. News Physiol Sci. 1993;8:245-248.
15.
Basu SK, Goldstein JL, Anderson RGW, Brown MS.
Degradation of ionized low density lipoprotein and regulation of
cholesterol metabolism in homozygous familial
hypercholesterolemic fibroblasts.
Proc Natl Acad Sci U S A. 1976;73:3178-3182.
16. Aviram M. Low density lipoprotein and scavenger receptor activities are modulated by secretory products derived from cells of the arterial wall. Metabolism. 1989;38:445-449. [Medline] [Order article via Infotrieve]
17. Aviram M. Plasma lipoprotein separation by discontinuous density gradient ultracentrifugation in hyperlipidemic patients. Biochem Med. 1983;30:111-118. [Medline] [Order article via Infotrieve]
18. Plump AS, Smith J, Hayek T, Aalto-Setala K, Walsh A, Verstuyft J, Rubin EM, Breslow JL. Severe hypercholesterolemia and atherosclerosis in apo E-deficient mice created by homologous recombinant ES cells. Cell. 1992;71:343-353. [Medline] [Order article via Infotrieve]
19.
Lowry OH, Rosenbrough NJ, Farr JL, Randall RJ.
Protein measurement with the Folin phenol reagent.
J Biol Chem. 1951;193:265-275.
20. Chiamori N, Henry RJ. Study of ferric chloride method for determination of total cholesterol and cholesterol ester. Am J Clin Pathol. 1959;31:305-309. [Medline] [Order article via Infotrieve]
21. Wieland O. Eine enzymatische method zur bestimmung von glycerine. Biochem Z. 1957;324:313-317.
22.
Bartlett GR. Phosphorus assay in column
chromatography. J Biol
Chem. 1959;234:466-472.
23. Hashim SA, Schutringer GR. Rapid determination of tocopherol in macro- and micro-quantities of plasma. Am J Clin Nutr. 1966;19:137-144.[Medline] [Order article via Infotrieve]
24. Emanuel RL, Cain JP, Williams GH. Double antibody radioimmunoassay of renin activity and angiotensin II in human peripheral plasma. J Lab Clin Med. 1973;81:632-640. [Medline] [Order article via Infotrieve]
25. Bilheimer DW, Eisenberg S, Levy RI. The metabolism of very low density lipoprotein protein, I: preliminary in vitro and in vivo observations. Biochim Biophys Acta. 1972;260:212-221. [Medline] [Order article via Infotrieve]
26. Maor I, Aviram M. Oxidized low density lipoprotein leads to macrophage accumulation of unesterified cholesterol as a result of lysosomal trapping of the lipoprotein hydrolyzed cholesteryl ester. J Lipid Res. 1994;35:803-819. [Abstract]
27. Colf B, Verheyden Y. Electrophoresis and Sudan black staining of lipoprotein on gelatinized cellulose acetate. Clin Chim Acta. 1967;18:325-326.
28. Buege JA, Aust SD. Microsomal lipid peroxidation. Methods Enzymol. 1978;52:302-310. [Medline] [Order article via Infotrieve]
29. El-Saadani M, Esterbauer H, El-Sayad M, Goher M, Nasser AY, Jurgens G. A spectrophotometric assay for lipid peroxides in serum lipoproteins using a commercially available reagent. J Lipid Res. 1989;30:627-630. [Abstract]
30. Swaney JB, Kueh KS. Separation of apolipoproteins by an acrylamide-gradient sodium dodecyl sulfate gel electrophoresis system. Biochim Biophys Acta. 1976;446:561-565. [Medline] [Order article via Infotrieve]
31. Krauss RM, Burke DJ. Identification of multiple subclasses of plasma low density lipoprotein in normal humans. J Lipid Res. 1982;23:97-104. [Abstract]
32.
Hoff HF, Whitaker TE, O'Neil J. Oxidation
of low density lipoprotein leads to particle aggregation and altered
macrophage recognition. J Biol
Chem. 1992;267:602-609.
33.
Steinbrecher V, Witztum JL, Parthasarathy S, Steinberg
D. Decrease in reactive amino groups during oxidation or
endothelial cell modification of LDL.
Arteriosclerosis. 1987;7:135-143.
34.
Aviram M, Dankner G, Brook JG. Platelet
secretory products increase LDL oxidation, enhance its uptake by
macrophages and reduce its fluidity.
Arteriosclerosis. 1990;10:559-563.
35. Aviram M, Fuhrman B, Keidar S, Maor I, Rosenblat M, Dankner G, Brook JG. Platelet modified low density lipoprotein induces macrophage cholesterol accumulation and platelet activation. J Clin Chem Clin Biochem. 1989;27:3-12. [Medline] [Order article via Infotrieve]
36. Keidar S, Goldberg AC, Cook K, Bateman J, Schonfeld G. A high carbohydrate-fat free diet alters the proportion of heparin-bound VLDL in plasma and the expression of VLDL–apo ß-100 epitopes. Metabolism. 1990;39:281-288. [Medline] [Order article via Infotrieve]
37.
Keidar S, Brook JG, Rosenblat M, Fuhrman B, Dankner G,
Aviram M. Involvement of the macrophage low density
lipoprotein receptor-binding domains in the uptake of oxidized low
density lipoprotein. Arterioscler Thromb. 1992;12:484-493.
38. Hayek T, Oiknine J, Brook JG, Aviram M. Increased plasma lipoprotein lipid peroxidation in apo E-deficient mice. Biochem Biophys Res Commun. 1994;201:1567-1574. [Medline] [Order article via Infotrieve]
39. Cardin AD, Randall CJ, Hirose N, Jackson RL. Physical-chemical interaction of heparin and human plasma low-density lipoproteins. Biochemistry. 1987;26:5513-5518. [Medline] [Order article via Infotrieve]
40. Pan YT, Kruski AW, Elbein AD. Binding of [3H]heparin to human plasma low density lipoprotein. Arch Biochem Biophys. 1978;189:231-240. [Medline] [Order article via Infotrieve]
41.
Krieger M, Acton S, Ashkenas J, Pearson A, Penman M,
Resnick D. Molecular fly paper, host defense, and
atherosclerosis: structure, binding properties, and
functions of macrophage scavenger receptor.
J Biol Chem. 1993;268:4569-4572.
42. Sambrano GR, Steinberg D. Recognition of oxidatively damaged and apoptotic cells by an oxidized low density lipoprotein receptor on mouse peritoneal macrophages: role of membrane phosphatidylserine. Proc Natl Acad Sci U S A. 1995;92:1395-1400.
43. Coritsidis G, Rifici V, Gupta S, Rie J, Shan Z, Neugarten J, Schlondorff D. Preferential binding of oxidized LDL to rat glomeruli in vivo and cultured mesangial cells in vitro. Kidney Int. 1991;39:858-866. [Medline] [Order article via Infotrieve]
44.
Griendling KK, Minieri CA, Ollerenshaw JD, Alexander
RW. Angiotensin II stimulates NADH and NADPH oxidase
activity in cultured vascular smooth muscle cells.
Circ Res. 1994;74:1141-1148.
45.
Cardona-Sanclemente LE, Medina R, Born GVR.
Effect of increasing doses of angiotensin II infused into
normal and hypertensive Wistar rats on low density lipoprotein and
fibrinogen uptake by aortic walls. Proc Natl Acad Sci
U S A. 1994;91:3285-3288.
46.
Nielsen LB, Stender S, Kjeldsen K, Nordestgaard
BG. Effect of angiotensin II and enalapril on
transfer of low-density lipoprotein into aortic intima in
rabbits. Circ Res. 1994;75:63-69.
47.
Allan DR, McKnight JA, Kifor I, Coletli CM, Hollenberg
NK. Converting enzyme inhibition and renal tissue
angiotensin II in the rat.
Hypertension. 1994;24:516-522.
48. Cockcroft JR, Keogh BE, Webb DJ, Ritter JM, Taylor KM. Responses to angiotensin with and without angiotensin-converting enzyme inhibition in the canine left anterior descending coronary artery. Am J Cardiol. 1993;71:1498-1499. [Medline] [Order article via Infotrieve]
49. Rakugi H, Kim D-K, Krieger JE, Wang DS, Dzau DS, Pratt RE. Induction of angiotensin converting enzyme in the neointima after vascular injury: possible role in restenosis. J Clin Invest. 1994;93:339-346.
50. Zambetis BM, Dusting GJ, Mendelsohn FA, Richardson K. Autoradiographic localization of angiotensin converting enzyme and angiotensin II binding sites in early atheroma-like lesions in rabbit arteries. Clin Exp Pharmacol Physiol. 1991;18:337-340. [Medline] [Order article via Infotrieve]
51. Aviram M, Rosenblat M. Macrophage-mediated oxidation of extracellular low density lipoprotein requires an initial binding of the lipoprotein to its receptor. J Lipid Res. 1994;35:385-398. [Abstract]
52. Aviram M, Bierman EL, Oram JF. High density lipoprotein stimulates sterol translocation between intracellular and plasma membrane pools in human monocyte-derived macrophages. J Lipid Res. 1989;30:65-76. [Abstract]
53.
Aviram M, Keidar S, Brook JG. Reduced uptake of
cholesterol esterase-modified low density lipoprotein
by macrophages. J Biol Chem. 1991;266:11567-11574.
54.
Aviram M. Low density lipoprotein modification
by cholesterol oxidase induces enhanced uptake and
cholesterol accumulation in cells. J
Biol Chem. 1992;267:218-225.
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