Download Decrease in Reactive Amino Groups during Oxidation or Endothelial

Decrease in Reactive Amino Groups during Oxidation or
Endothelial Cell Modification of LDL
Correlation with Changes in Receptor-Mediated Cataboiism
Urs P. Steinbrecher, Joseph L. Witztum, Sampath Parthasarathy, and Daniel Steinberg
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The monocyte/macrophage appears to be the precursor of many of the lipid-laden
cells in atherosclerotic lesions, but the mechanism by which these cells accumulate
cholesterol to become foam cells remains unclear. We have previously reported that
cultured endothelial cells can modify low density lipoprotein (LDL) in a manner that
leads to rapid uptake by the acetyl LDL receptor of macrophages. This modification
involves free radical-induced peroxidation of LDL and is accompanied by many
changes in the physicochemical properties of LDL including increased electrophoretic mobility, increased density, decreased content of esterified cholesterol, hydrolysis
of phosphatidylcholine, and fragmentation of apolipoprotein B. Under conditions
highly favorable to oxidation, a similar modification can occur even in the absence of
cells. In the present studies, oxidation of LDL simply by exposure t o 5 / i M C u + +
resulted in a modification that was indistinguishable from that produced by endothelial cells. Moreover, it was demonstrated that LDL oxidation by either method is accompanied by a marked decrease in amino group reactivity, comparable to that seen with
the chemical modifications of LDL that lead to recognition by the acetyl LDL receptor.
Inhibitors of proteolytic enzymes did not reduce fragmentation of apolipoprotein B
during oxidation. The rate of cataboiism of intravenously injected oxidized LDL in
guinea pigs was very rapid, and over 80% of the degradation occurred in the liver.
These studies demonstrate that all of the changes associated with endothelial cell
modification of LDL can be attributed to oxidation. The cells can, however, promote
oxidation under conditions where it would otherwise occur very slowly. Modification
of LDL by endothelial cells or 5 /iM C u + + results in a marked decrease in LDL amino
group reactivity that correlates with accelerated LDL clearance via the acetyl LDL
receptor and decreased clearance by the classical LDL receptor in cultured cells and
in vivo. (Arteriosclerosis 7:135-143, March/April 1987)
the acetyl LDL receptor is that they increase the net negative charge of LDL by derivatization of the epsilon amino
groups of lysine residues of apolipoprotein B (apo B). Cultured endothelial cells and smooth muscle cells can also
modify LDL in a manner that leads to an increase in net
negative charge and an increased rate of degradation by
macrophages.8 Other properties of this biologically modified LDL include increased density, a decreased content of
esterified cholesterol, and hydrolysis of up to 50% of LDL
phosphatidylcholine to lysophosphatidylcholine.8"11
We recently showed that modification of LDL by endothelial cells involves free radical peroxidation and is catalyzed by redox-active metal ions such as C u + + present in
culture media.12 In these studies it was also demonstrated
that although modification did not occur under standard
incubation conditions in cell-free dishes, an apparently
similar modification could be achieved in the absence of
cells under conditions favorable to oxidation, for example
by incubating LDL in F-10 medium supplemented with 5
/*M Cu + + . These findings suggested that oxidation in itself
was sufficient to cause the changes associated with endothelial cell modification. However, considerable variability
in the extent of modification was encountered with different
media supplemented with the same concentration of
Cu + +
In the present report we describe conditions for repro-
C
ultured macrophages possess several distinct types
of surface binding sites that mediate the uptake of
various normal or modified lipoproteins.1"3 One of these,
termed the acetyl LDL or "scavenger" receptor, binds low
density lipoproteins (LDL) which have been modified by
acetylation, acetoacetylation, carbamylation, or treatment
with malondialdehyde.4"7 The potential importance of this
receptor lies in the fact that it does not appear to be regulated by cellular cholesterol content, and can thus more readily lead to foam cell formation.1 A common characteristic of
these chemical modifications that lead to recognition by
From the Department of Medicine, University of British Columbia, Vancouver, British Columbia, Canada and the Department of
Medicine, University of California at San Diego, La Jolla, California.
These studies were supported by Grant MA-8630 from the
Medical Research Council of Canada, Grant 84-01 from the British Columbia Health Care Research Foundation, and SCOR
Grant HL-14197 from the NHLBI. Urs P. Steinbrecher is a Scholar
of the Medical Research Council of Canada, and Joseph L. Witztum is an Established Investigator of the American Heart Association.
Address for reprints: Urs P. Steinbrecher, Department of Medicine, UBC Health Sciences Centre Hospital, Department of Medicine, Room F-137, 2211 Wesbrook Mall, Vancouver, British Columbia, V6T 1W5, Canada.
Received June 9, 1986; revision accepted October 23, 1986.
135
136
ARTERIOSCLEROSIS V O L 7, No 2, MARCH/APRIL 1987
dudbly obtaining LDL with any desired degree of oxidation
simply by exposure to 5 ixM Cu + + In phosphate-buffered
saline. Variability due to medium components such as amino acids, antloxidants, or other metal ions is thereby eliminated. Characterization of oxidized LDL obtained by this
method revealed that the number of reactive amino groups
In LDL decreases markedly with oxidation. A similar decrease in amino groups was also found in endothelial-cell
modified LDL. The loss of reactive amino groups is accompanied by decreased degradation of LDL in vitro and in
vivo via the LDL receptor, and increased degradation via
the acetyl LDL receptor. These results are the first indication that the altered catabolism of oxidized LDL or endothelial-cell modified LDL may be explainable at least in part by
alterations of lysine amino groups.
Methods
12S
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Carrier-free Na l and Na131l were obtained from Amersham Corporation (Arlington Heights, Virginia) or New
England Nuclear (Lachine, Quebec). Tetrachbrodiphenylglycoluril (lodo-Gen) was from Pierce Chemical Corporation (Rockford, Illinois). Iodine monochloride, 2-thiobarbituric add, bovine albumin, Tween-20, aprotinin, fucoidin,
dextran sulfate, polyinosinic acid, polycytidylic acid, and
tetramethoxypropane were from Sigma (St. Louis, Missouri). Acetic anhydride was from BDH Chemical (Toronto,
Ontario) and sodium acetate, from Fisher Scientific (Vancouver, British Columbia). Trinitrobenzenesulfonic add
was purchased from Aldrich Chemical Company (Milwaukee, Wisconsin) and recrystallized before use. Enzymatic
assay kits for free cholesterol, total cholesterol, and triglycerides were supplied by Boehringer Mannheim Corporation (Dorval, Quebec). Polyvinylchloride 96-well microtitration plates were from Becton Dickinson (Mlssissauga,
Ontario). Ultrafiltratlon membrane cones (CF25, 25,000
molecular weight cutoff) were purchased from Amicon
Corporation (Danvers, Massachusetts). Goat antiserum to
guinea pig IgG was obtained from Utton Bionetics (Kensington, Maryland). Fetal bovine serum, goat serum,
Ham's F-10 medium, alpha minimal essential medium
(MEM), and gentamicin were from Gibco (Mlssissauga,
Ontario). Female CD-1 mice were supplied by Simonsen
Laboratories (Gilroy, California) or Charles River Breeding
Laboratories (Wilmington, Massachusetts). Male Hartley
guinea pigs were from Charles River or from the University
of British Columbia animal care colony.
Llpoproteln Isolation and Labelling
Plasma from fasting normal human subjects was collected Into EDTA (1 mg/ml), and LDL (d = 1.019-1.063 g/ml)
was isolated by sequential ultracentrtfugation. For routine
cell culture experiments, LDL was radioiodinated using a
modification of the iodine monochloride method of McFarlane13 to specific radioactivities ranging from 50 to 175
cpm/ng. For studies of tissue sites of catabolism in vivo,
LDL was labelled with radioiodinated tyramine cellobiose
as described by Pittman and colleagues.14 Briefly, tyramine was coupled to cellobiose by reductive amination
with NaCNBH3, radioiodinated using tetrachlorodiphenyl-
glycoluril, and linked to LDL with cyanuric chloride. The
extent of derivatization was 1-2 moles of iodotyramine
cellobiose per mole of apo B (assuming Mr 500,000), and
the specific radioactivity ranged from 60 to 162 cpm/ng.
Protein was determined by the Lowry method, using bovine albumin as the standard.15
Before use in experiments, LDL was dialyzed against
PBS containing a very low concentration of EDTA (10 pM).
This concentration was sufficient to inhibit spontaneous
oxidation even on prolonged storage, but was tow enough
to permit subsequent oxidation either by cells or CuSO4.
Cultured Cells
The rabbit aortic endothellal cells used in these studies
were from a line established and characterized by Buonassisi and coworkers.18 The cells were grown In Ham's F10 medium containing 15% fetal bovine serum and plated
in 60 mm plastic culture dishes and used when confluent.
Resident mouse peritoneal macrophages were obtained
as previously described.8"11 Female CD-1 mice were killed
with ether, and the peritoneum was lavaged with C a + + and Mg ++ -free Dulbecco's PBS. Peritoneal cells were
suspended in alpha MEM containing 10% fetal bovine serum and gentamicin sulfate (50 ^ig/ml) and seeded in 12well tissue culture plates at 1.5 x 10s cells per dish. Nonadherent cells were removed 1 hour after plating and the
adherent macrophages were used in experiments on the
following day. Normal human skin fibroblasts obtained
from a preputial biopsy were grown in DME medium containing 10% fetal bovine serum. Cells from the 9th to 16th
passage were plated in 6-well plastic culture plates. When
they reached 70% confluency, the medium was replaced
with DME containing 2.5 mg/ml lipoprotein-deficient serum, and the cells were used in degradation studies 24
hours later.
Modification of Llpoprotelns
Endothelial cell-modified LDL was prepared by adding 2
ml of serum-free F-10 medium containing 100 to 200 /ig/ml
LDL protein to each 60 mm dish of endothelial cells, and
incubating for 24 hours at 37°C. Parallel control incubations of LDL in cell-free dishes were done in every experiment. LDL (100 to 200 /tg protein/ml) was oxidized in the
absence of cells by exposure to 5 pM CuSO4 In EDTA-free
PBS at 37°C. Control incubations were done in the presence of 200 /uM EDTA without CuSO4. The extent of oxidation could be varied reproducibly simply by varying the
incubation period between 3 and 24 hours. Oxidation was
arrested by refrigeration and addition of 200 fiM EDTA and
40 (iM butylated hydroxytoluene. Except where otherwise
indicated, modified and control lipoproteins were reisolated by ultracentrifugation at d = 1.15 g/ml before further
analysis. To rule out the possibility that oxidized LDL was
further altered during this additional ultracentrifugation
step, in some experiments LDL was reisolated using ultrafiltration membrane cones with a 25,000 M, cutoff. Oxidized LDL reisolated with this method, which requires less
than 1 hour, was indistinguishable from ultracentrifugally
reisolated LDL in all the analyses described below.
DECREASED AMINO GROUPS IN OXIDIZED LDL
Cell Culture Studies
Various concentrations of lipoprotein were incubated
with mouse peritoneal macrophages or human skin fibroblasts for 5 hours at 37°C in a humidified CO2 incubator.
With conventionally radtoiodinated lipoproteins, degradation products were assayed as trichloroacetic acid-soluble
nonkxJide radioactivity.11 In experiments with iodotyramine
celloblose-labelled LDL, the total cell content of radioactivity at the end of the incubation period was determined as a
measure of uptake and degradation over the 5-hour incubatjon since the label from degraded LDL does not escape
into the medium at an appreciable rate.14
Animal Studies
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Plasma disappearance rate and tissue sites of catabolism of Intravenously injected normal and oxidized LDL
were determined In guinea pigs using the trapped label"
technique developed by Prttman and colleagues.14 This
method is based on the facts that most mammalian cells
are unable to metabolize certain disaccharides such as
sucrose or celloblose, and that these sugars do not readily
cross lysosomal membranes. When cells take up and degrade a protein that has been coupled to labelled sucrose
or cellobiose, the sugar remains trapped in the lysosomes,
serving as a cumulative marker of the number of molecules
of labelled protein degraded by the cell.
Guinea pigs were anesthetized with ether, and 60 nC\ of
oxidized 12SI tyramine cellobiose (TC) LDL was injected
into an exposed jugular vein. Some animals also received
30 jiCI of native 131I TC-LDL. Serial blood samples were
obtained by cardiac puncture with the animals under ether
anesthesia; aliquots of plasma were counted in a LKB
1282 gamma spectrometer. The fractional catabolic rates
of the injected tracers were calculated from the plasma
decay curves using an iterative curve-peeling program.17
When most of the injected radioactivity had been cleared
from the circulation (24 hours for normal or lightly oxidized
LDL; 1 hour for extensively oxidized LDL), the animals
were anesthetized with ketamine (30 mg/kg), fentanyl
(0.08 mg/kg), and droperidol (4 mg/kg), perfused for 10
minutes via the internal jugular vein with 200 ml Hank's
buffered salt solution, and exsanguinated. Individual organs and tissues were dissected out, weighed, and counted. Skin was assumed to be 18% of body weight, muscle
36%, fat 9%, and bone marrow 1 %. All other organs and
tissues were counted in toto. Radioactivity in the gut contents was assumed to represent biliary excretion and was
included in the estimation of degradation by the liver.18
Analytic Methods
Lipoprotein electrophoresis was done using a Coming
apparatus and Universal agarose film in 50 mM barbital
buffer (pH 8.6). Bovine albumin at a final concentration of
20 mg/ml was added to dilute lipoprotein samples to ensure reproducible migration distances. Sucrose density
gradient ultracentrifugation was performed as previously
described.8 Proteins were analyzed on 3% to 12% polyacrylamide gradient gels in the presence of sodium dodecyl sulfate (SDS). Free amino groups on LDL were estimat-
Steinbrecher et al.
137
ed using trinitrobenzenesulfonic acid (TNBS).19 LDL (25 to
50 fig protein) was mixed with 1 ml 4% NaHCO3 (pH 8.4)
and 50 ii\ 0.1% TNBS; this was heated for 1 hour at 37°C,
and then the absorbance at 340 nm was recorded. Concentration of amino groups was determined by a reference
to a valine standard. Lipid peroxide was estimated as the
fluorescent reaction product with thiobarbituric acid (TBA),
using freshly diluted tetramethoxypropane as a standard.11 Free cholesterol, total cholesterol, and triglycerides
were determined using enzymatic kits according to the
manufacturer's Instructions except that the volumes of all
reagents were reduced by half. For phospholipid analysis,
LDL was extracted using chloroform/methanol.20 Phospholipids were separated by thin-layer chromatography on
silica gel G using chloroform/methanol/water (65:35:7),
and the bands were visualized with iodine vapor. Lysophosphatidylcholine and phosphatldylcholine zones were
scraped into test tubes, digested with HCIO4, and assayed
for phosphorus content.21
Apo B Immunoreactlvlty
Antisera to apo B were obtained from guinea pigs that
were hyperimmunized with human LDL as previously reported.22 Competition studies were performed as described using 96-well polyvinylchloride microtftration
plates coated with 50 ngAvell of human LDL. To each well
was added 25 ^1 of a 1:10,000 dilution of antiserum and 25
IJL\ of buffer containing varying amounts of competitor. After
overnight incubation at 10°C, the wells were washed four
times. Bound antibody was quantified using 12SI goat antiguinea pig IgG. Commercially obtained antiserum to guinea pig IgG was partially purified by salt fractionatlon and
was radtoiodinated to a specific activity of 6,000 to 10,000
cpm/ng using tetrachlorodiphenylglycoluril.23 A saturating
amount of this second antibody was added to each well
and was incubated for 4 hours at room temperature. The
wells were then washed, isolated, and counted in a gamma
spectrometer.
Ethical approval for phlebotomy of human volunteers
was obtained from the appropriate committees at the University of California at San Diego and the University of
British Columbia. Animal experiments were approved by
the Animal Care Coordinator of the University of British
Columbia.
Results
Physical and Chemical Characterization of
Copper-Oxidized LDL
Oxidation of LDL by exposure to Cu + + resulted in
changes in density and lipid composition very similar to
those previously reported for endothellal-cell modified
LDL.8-10 The data shown in Table 1 indicate that there was
a major decrease in the total cholesterol in oxidized LDL,
whereas the free cholesterol was only slightly reduced. A
moderate decrease in triglyceride was noted as well. The
apparent decrease in cholesterol may have been due to
oxidation of LDL cholesterol with consequent failure to
react with the cholesterol esterase or oxidase in the assay
kit, because the lost cholesterol was not recovered in the
medium after removal of the oxidized LDL (not shown).
138
ARTERIOSCLEROSIS VOL 7, No 2, MARCH/APRIL 1987
Table 1. Composition of Cu "-Oxidized LDL and Endothellal Cell-Modified LDL
LDL
Control LDL
LDL oxidized 6 hours
LDL oxidized 20 hours
Endothelial cell-modified LDL
Density
(g/cm3)
1.033
1.042
1.070
1.070
Total
cholesterol
Free
cholesterol
Trtglyceride
Total
phospholipid
(/tmol P/mg
protein)
Lyso PC/PC
molar ratio
0.23 ±0.1
0.17±0.01
0.12 + 0.04
0.18 (0.198)*
1.04±0.12
1.12±0.03
1.00 ±0.04
0.95
0.11 ±0.04
0.21 ±0.06
0.59 ±0.2
0.76
(mg lipid/mg protein)
1.4±0.1
1.1 ±0.2
0.83 ±0.2
1.1 (0.83)*
0.39 ±0.05
0.35 + 0.06
0.35 ±0.1.
0.29 (0.47)*
Reisolated control LDL, oxidized LDL, or endothelial cell-modified LDL were analyzed for protein, total and free cholesterol, triglycerlde,
total lipid phosphorus, phosphatidytchollne (PC), and lysophosphatktytcholine (lyso PC) as described in Methods. Endothelial cell-modified
LDL was the product of a 20-hour incubation with cells. Control LDL was Incubated for 20 hours with 200 jtM EDTA In the absence of Cu + + .
Values for control and oxidized LDL are means + so of at least four determinations; the values for endothelial cell-modified LDL are from
a single experiment.
'Numbers in parentheses are data reported by Henricksen and colleagues9 presented here for comparison.
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Table 1 also shows that significant hydrolysis of phosphatjdylcholine to lysophosphatidylcholine occurred during
oxidation.
When normal human LDL (100 to 200 fig protein/ml)
was incubated in PBS containing 5 fiM CuSO4 for varying
time intervals, there was a time-dependent increase in its
mobility on agarose gel electrophoresis, and this was accompanied by a striking decrease in TNBS reactivity (Figure 1). Although the TNBS reactivity of LDL correlates well
with measurements of intact lyslne residues by amino acid
analysis,24 as much as 10% of the TNBS reactivity of LDL
could theoretically be attributable to phosphatJdylethanolamine and phosphatidylserine. Therefore, it is possible that
modification of these phospholipids could account for part
of the decrease in TNBS reactivity. This decrease in TNBS
reactivity was observed with or without LDL reisolation,
Indicating that it was probably not due simply to the release
of lysine-rich fragments from apo B, but rather that the
lysine amino groups were either modified or made inaccessible to the TNBS reagent. After ultracentrtfugal reisolation and washing, a 45% decrease in TNBS reactivity
was also found with endothellal-cell modification of LDL.
Table 2 shows that a time-dependent increase in the
content of TBA-reactive substances (a measure of lipid
peroxide) was found in the unfractionated oxidation mixtures. However, very little TBA-reactive material was detected in the reisolated LDL, and more than 90% of the
TBA-reactive material in the unfractionated mixtures
passed through an ultrafiltratjon membrane with a nominal
molecular weight cutoff of 25,000. These findings suggest
Substances
In
Cu + + Table 2. TBA-Reactive
OxWIzed LDL and Endothellal Cell-Modified LDL
LDL
.2
.4
.6
.8
Relative electrophoretic mobility
1 0
Figure 1. Correlation of LDL electrophoretic mobility and TNBS
reactivity with duration of oxidation. LDL (100 to 200 /ig protein
per ml) was incubated at 3 7 X in PBS containing 5 jtM CuSO4 for
3, 6, or 20 hours. Further oxidation was inhibited by refrigeration
and the addition of EDTA and BHT. Control LDL was Incubated
without CuS0 4> and with EDTA and BHT. Allquots were taken for
agarose gel electrophoresis and estimation of reactive amino
groups using TNBS. In some experiments, LDL was reisolated by
ultracentrifugation and assayed for protein before analysis. Electrophoretic mobility of LDL is expressed as migration relative to
bovine albumin (2.5 cm In this system). TNBS reactivity per mg
protein is shown as a percentage of that of nonoxidlzed control
LDL (o), and each point is the mean of duplicate determinations.
The results shown represent pooled data from nine separate experiments. The correlation between etectrophoreUc mobility and
TNBS reactivity was significant (r = 0.93, p < 0.001).
Native LDL
LDL oxidized 1 hour
LDL oxidized 3 hours
LDL oxkfized 6 hours
LDL oxkfized 20 hours
Endothefial cell-modified
LDL
TBA
reacTBA reacTBA reactivity
tivity of
tivity k)
unfractjonLDL after in ultraated medium reisolation
filtrate
(nmol MDA/ (nmol MDA/ (%of
mg protein) mg protein) total)
1.2±0.3
11.3±0.2
27.1 ±3.0
29.3 ±3.8
32.7 ±3.4
NO
33.6 ±5.9
++
NO
NO
NO
3.1 ±0.4
2.4 ±0.7
1.0 ±0.6
90%
93%
97%
2.1 + 1.5
94%
LDL was modified by exposure to C u
for varying time Intervals or by incubation with endothelial cells for 20 hours as described in Methods. An aliquot of the oxidation mixture or endothelial cell culture supernatant was assayed for total TBA-reactive
substances. LDL was then reisolated by ultracentrifugation or ultrafittration and assayed for protein and TBA reactivity. In experiments where LDL was reisolated by uttrafiltration, the TBA reactivity in the filtrate was also measured, and is expressed as a percent
of the total TBA reactivity before filtration.
Values shown are means ± so of duplicates from at least two
experiments.
no = not determined.
DECREASED AMINO GROUPS IN OXIDIZED LDL
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that TBA-reactive products (presumably derived from peroxidized fatty acids) were released from LDL into the aqueous phase. This loss of fatty acids from phospholipids
(Table 1), and perhaps from cholesterol esters and triglycerides may contribute to the increase in density observed with oxidation of LDL.
Major alterations in apollpoprotein B were also found to
accompany oxidation. Denaturing polyacrytamide gel
electrophoresis indicated rather extensive fragmentation
of apo B-100 even after modest degrees of oxidation, consistent with findings previously reported by others. 2526
This fragmentation could be due to direct oxidative scission of peptide bonds, or alternatively, to activation of a
proteolytjc enzyme by oxidation.26 To determine which of
these mechanisms accounts for apoproteln degradation
during oxidation, LDL was incubated with CuSO4 in the
presence of various proteolytic Inhibitors. Phenylmethyl
sulfonyl fluoride, /V-ethylmaleimide, benzamidine, and diisopropytfluorophosphate all failed to Inhibit proteolysis
when added at a concentration of 1 mM. Soybean trypsin
Inhibitor, hirudin, aprotinin, and pepstatln were also ineffective. Diazoacetyl norleucine methyl ester, p-hydroxymercuribenzoate, and drthiobis nltrobenzoic add (all 1
mM) inhibited proteolysis but also Inhibited lipid peroxidatJon. These results favor the explanation of direct freeradical mediated peptide bond scission. The recovery of
protein in the reisolated oxidized LDL by Lowry assay or by
radioiodine recovery (when 12SI LDL was oxidized) was
usually 60% to 90%, indicating that most of the apoprotein
fragments remained associated with LDL.
To evaluate the effects of the oxidation-related changes
on apolipoprotein B Immunoreactivity, incubations of unlabelled LDL with CuSO4 were done under conditions identical to those described above. Native LDL, and LDL oxidized for 3, 6, or 20 hours were then tested for their ability
to bind a specific antiserum to human apo B using a solidphase radioimmunoassay. The results of a typical experiment are illustrated in Figure 2. Even though marked fragmentation of LDL protein was demonstrated by
SDS/PAGE, the competition curves of LDL oxidized for 3,
6 or 20 hours were superimposable with that of native LDL,
indicating that immunoreactivity was not destroyed by
oxidation.
1
Steinbrecher et al.
139
10
Competitor concentration (^g/ml)
Figure 2. Immunoreactivity of apo B in oxidized LDL. As described, 96-well flexible microtitration plates were coated with native LDL. Varying amounts of unlabelled native or modified LDL
competitor in 25 y\ of buffer and 25 iA of guinea pig antiserum to
human LDL (final dilution 1:20,000) were added to each well and
these were incubated for 16 hours. The amount of antibody bound
to the wells was quantified using 125I goat anti-guinea pig IgG.
Results are expressed as amount of radioactivity bound as a
function of the concentration (In /Ag/ml) of competitor. EHndIng in
the absence of competitor was 4750 com per well. Each point Is
the mean of duplicate wells. The competition curves of native LDL
(o), LDL oxidized for 3 hours (•), 6 hours (A), and 16 hours (•)
were superimposable, Indicating no loss of immunoreactivtty after
oxidation even though each oxidized LDL showed extensive fragmentation of apo B by SDS/PAQE.
peritoneal macrophages. Only oxidized LDLs with electrophoretlc mobility greater than 0.55 relative to bovine albumin showed increased uptake in macrophages. The results shown in Figure 4 indicate that the amount of high
affinity saturable degradation by these cells increased pro-
10
Effects of Oxidation of LDL on Subsequent
Degradation by Flbroblasts and Macrophages
The effects of varying degrees of oxidation on the ability
of LDL to interact with the LDL receptor was evaluated
through LDL degradation experiments in cultured normal
human skin fibroblasts. Figure 3 shows that after 3 hours of
oxidation the high affinity component of degradation by
these cells was almost completely abolished. In this experiment, the TNBS reactivity of the LDL oxidized for 3
hours was 15% less than that of native LDL and the electrophoretic mobility relative to bovine albumin was 0.48.
These results are consistent with previous studies indicating that modification of as few as 5% of lysine residues of
LDL can affect recognition by the fibroblast LDL receptor.27
To determine the extent of oxidation required to permit
recognition by the acetyl LDL receptor, LDL with varying
degrees of oxidation was incubated with cultured mouse
0
10
20
30
LDL concentration (pg/ml)
10
50
Figure 3. Degradation of native and Cu ++ -oxidized LDL by
cultured normal human fibroblasts. Subconfluent cultures of fibroblasts were incubated for 24 hours In DME medium containing
2.5 mg/ml llpoprotein-deficient serum to stimulate expression of
LDL receptors. The indicated concentrations of radioiodinated native LDL or LDL oxidized to varying degrees by exposure to Cu + +
were then added. Agarose gel electrophoresis was done to assess the extent of oxidation of each preparation. After 6 hours of
incubation, the content of trichloroacerJc acid-soluble nonlodide
radioactivity in the medium was determined. Results are presented as ng of LDL protein degraded per mg cell protein in 6 hours.
Each point represents the mean of results from duplicate dishes.
Native LDL, relative electrophoretic mobility (R() 0.28 (o); oxidized
LDL with R, 0.48 (•), oxidized LDL with R, 0.56 (A), and oxidized
LDL with R, 0.76 (•).
140
ARTERIOSCLEROSIS V O L 7, No 2, MARCH/APRIL 1987
100
\
80
o
o
[\
60
40
JO
10
20
30
40
SO
LDL concentration (pig/ml)
++
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Figure 4. Degradation of native and C u -oxidized LDL by
cultured mouse peritoneal macrophages. Resident macrophages
were harvested from female CD-1 mice by peritoneal lavage.
Cells were plated In a-MEM containing 10% fetal bovine serum,
and used for experiments the next day. The Indicated concentrations of radioiodlnated LDL were then added in serum-free medium, and after 6 hours of incubation, the content of trichloroacetic
acid-soluble nontodlde radioactivity in the medium was measured.
The results are presented as M9 LDL protein degraded per mg cell
protein in 6 hours; each point represents the mean of results from
duplicate incubations. Native LDL, R, 0.30 (o), oxidized LDL with
R, 0.68 (•), R, 0.76 (A), and R, 0.96 (•).
gresslvely as the extent of oxidation was increased above
this threshold value. To define the specificity of the high
affinity uptake and degradation process for oxidized LDL,
we examined the effects of various competitors on degradation of labelled extensively oxidized LDL. Unlabelled
Cu ++ -oxidlzed LDL or acetyl LDL competed effectively for
125
I oxidized LDL degradation, but native LDL did not (Figure 5). Dextran sulfate, fucoidin, and polyinoslnic acid
have been found to compete for acetyl LDL binding and
degradation via the scavenger receptor, but polycytidylic
acid does not.28 Competition results with these negatively
charged substances against oxidized LDL showed a very
similar pattern (Figure 6). About 75% of degradation was
inhibitable with these competitors, suggesting that other
pathways made only a minor contribution to degradation of
oxidized LDL by mouse peritoneal macrophages.
20
Competitor c
30
ntrotion (
Figure 5. Specificity of the macrophage receptor for C u + + oxldlzed LDL Resident mouse peritoneal macrophages were prepared as described in the legend to Figure 4. "*I-LDL was oxidized for 20 hours (R| 0.76) and added to duplicate dishes of cells
at a concentration of 10 /xg protein/ml together with varying concentrations of unlabelled native LDL (o), LDL oxidized for 20 hours
(•), or acetyt-LDL (A). After Incubation for 6 hours, the medium
was assayed for trichloroacetic acid-soluble nonkxiide radioactivity. The results are expressed as /xg LDL protein degraded per mg
cell protein in 6 hours.
mined. As discussed in the Methods section, the labelled
tyramine cellobiose entering with LDL remains trapped in
the tissue and provides a cumulative measure of LDL
degradation.
Figure 7 shows representative plasma decay curves of
oxidized and native LDL; catabollc rates calculated from
the decay curves are presented in Table 3. Even with
minimal degrees of oxidation, there was a two to fivefold
increase in the plasma clearance rate, while more extensively oxidized LDL was removed from the circulation within minutes. These same LDL preparations were tested for
uptake by cultured fibroblasts, and a marked decrease in
uptake was observed even with limited oxidation, analogous to the results shown in Figure 3. When incubated with
cultured peritoneal macrophages, only the most extensively oxidized LDL showed a marked increase in the rate of
uptake. Analysis of the tissue sites of catabolism of these
LDLs (Table 4) reveals that as the degree of oxidation
increased, the fractional uptake by tissues rich in LDL receptors (e.g., the adrenals and gonads) decreased. Con-
Catabollsm of Oxidized LDL In Vivo In Guinea
Pigs
To determine whether the altered receptor recognition of
oxidized LDL described above would affect in vivo catabclism, we directly compared the plasma clearance rate and
the tissue sites of catabolism of homologous native LDL
with LDL that had undergone varying degrees of oxidation.
Guinea pig LDL was labelled with 125I tyramine cellobiose,
and aliquots were oxidized by exposure to 5 /xM C u + + for
3, 6, or 20 hours. Native guinea pig LDL labelled with 131I
tyramine cellobiose was used as a reference. Oxidized
and native LDL were Injected simultaneously Into the jugular vein, and repeated samplings of plasma were then
made for measurement of radioactivity. When most of the
isotope had been cleared from plasma, the animals were
perfused with Hank's buffered salt solution, and then the
radioactivity in individual tissues and organs was deter-
2
4
6
6
10
Competitor concentration (ng/mt]
Figure 6. Competition for macrophage degradation of C u + + oxldlzed LDL by negatively charged compounds. Experimental
conditions were Identical to those in Figure 5, except that the
competitors used were dextran sulfate (A), fucoidin (•), polyinosinic acid (•), and polycytidylic add (o).
DECREASED AMINO GROUPS IN OXIDIZED LDL
20
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Figure 7. Plasma decay curves of native and Cu + +-oxidized
LDL in guinea pigs. Guinea pig LDL was labelled with 125l-tyramine cellobiose, as described In Methods, oxidized by Incubation
with 5 /iM C u + + for 3, 6, or 20 hours, and then injected Intravenously into guinea pigs together with native guinea pig LDL labelled with 1^1l-tyramine cellobiose. Serial plasma samples were
obtained by cardiac puncture, and were counted In a two-channel
gamma spectrometer. Representative plasma radioactivity
curves are shown for native LDL (o), and for LDL oxidized for 3
hours (•), 6 hours (A), and 20 hours (•). Similar results were
obtained in other animals Injected with the same labels (Table 3).
comltantly, uptake by the liver increased. It has been previously shown that Intravenously injected acetyl LDL and
EC-modified LDL are cleared almost entirely by the hepatic sinusoidal endothelial cells, as these cells appear to
express a high level of acetyl LDL receptor activity In
vivo. 29 ' M It seems likely that these cells account for the
increased fractional uptake of oxidized LDL by the liver in
the present study as well but this was not directly
demonstrated.
Discussion
The results presented above indicate that all of the
changes in physical properties, composition, and biologic
behavior of LDL induced by incubation with endothelial
cells are mimicked when LDL is oxidized by exposure to
Cu + + in the absence of cells under the conditions described In the Methods section. In conjunction with our
previous observation that antioxidants inhibit endothelial
cell-induced modification, these results suggest that endothelial cells modify LDL primarily or exclusively by somehow promoting its oxidation. It appears likely that the modifications produced by other cells, such as aortic smooth
muscle cells or leukocytes, are on the same basis. 931 " 33
Recent studies by Heinecke and colleagues34 suggest that
the mechanism by which cells oxidize LDL involves superoxide secretion into the medium.
In the present studies, It is demonstrated for the first time
that the TNBS reactivity of LDL decreases markedly during
oxidation or endothelial cell modification. As noted above,
the total TNBS reactivity of LDL may include contributions
from phospholipids, and thus the decrease in lysine epsilon amino groups could be somewhat less than the decrease in total TNBS reactivity. Nevertheless, the correla-
Steinbrecher et al.
141
tions of altered biologic activity with degree of oxidation
and proportion of amino groups modified agree well with
predictions based on the behavior of various chemically
modified LDLs.8'7' ^ **•M With cultured mouse peritoneal
macrophages, a progressive Increase in LDL degradation
with increasing degree of LDL oxidation was seen rather
than the abrupt "threshold" reported by Haberiand and
colleagues7 for degradation of malondialdehyde-treated
LDL in human monocyte-macrophages. It has not yet been
determined if this is attributable to a difference in the nature of the modified LDLs or to the different types of macrophages. The decrease in TNBS reactivity in oxidized or
endothelial-cell modified LDL cannot be due to derivatization of lysine by malondialdehyde, as the malondlaldehyde
content (TBA-reactivity) of oxidized LDL or endothelial-cell
modified LDL10 is less than 5% of that of malondialdehydemodifled LDL.5'24 However, it is entirely possible that lysines were derivatized by other aldehydes derived from
peroxidation of LDL lipids.
Changes affecting lipid components during LDL oxidation may also be important. The increased content of lysophosphatidylchollne would be expected to markedly alter
the molecular ordering of the phospholipids In the outer
monolayer of LDL, and oxidation of core lipids could also
influence the structure and stability of the particle. The
oxidized fatty acids that are released may themselves
have important biologic effects; for example, lipid hydroperoxides can Influence the production of prostaglandlns
by cultured fibroblasts.36 Alternatively, the peroxides themselves could become substrates for eicosanoid-produdng
enzymes located on the surface of endothelial cells.37 The
prostaglandin or leukotriene products that might result
would have profound effects on vascular tone, permeability, platelet function, and leukocytes. Oxidized sterols
could affect membrane function or cholesterol synthesis.38
The present studies indicate that oxidation of LDL converts it to a form that is recognized by the acetyl LDL
receptor of macrophages, and therefore could potentially
contribute to foam cell formation. However, it remains to be
determined whether LDL oxidation plays any role In foam
cell formation in vivo. There are abundant antioxidant defenses in blood including vitamin E, ascorbate, carotene,
peroxidases, superoxide dismutase, and cerutoplasmin.
Even if LDL oxidation were to occur in plasma, it appears
from the results described above that the oxidized LDL
would be rapidly cleared by the liver, and hence would be
Table 3. Fractionated Catabollc Rates (FCR) of
Cu++-Oxldlzed LDL In Guinea Pigs
LDL
Native LDL (n = 5)
LDL oxidized 3 hours (n = 2)
LDL oxidized 6 hours (n = 2)
LDL oxidized 20 hours (n == 2)
FCR
(pools/hr)
0.07 ±0.02
0.14,0.15
0.53,0.48
>10,>10
Turnover studies In guinea pigs of native and Cu ++ -oxidized
guinea pig LDL were carried out as described in Methods. Because of the very rapid clearance of 20-hour oxidized LDL, only a
minimum estimate of FCR was possible.
Results for native LDL represent mean ± so, but for oxidized
LDLs Individual values are given.
142
ARTERIOSCLEROSIS VOL 7, No 2, MARCH/APRIL 1987
Table 4. Tissue Sites of Catabollsm of Native and Cu"-Oxidized LDL
LDL
Native LDL (n = 5)
LDL oxidized 3 hours (n =: 2)
LDL oxidized 6 hours (n =: 2)
LDL oxidized 20 hours (n = 2)
Liver
Adrenals
Testes
Spleen
Marrow
52.7 ±0.6
71.5,72.9
81.8,80.8
81.9,83.5
4.2 ±1.0
1.0,1.2
0.18,0.20
0.07,0.06
0.3 ±0.09
0.15,0.15
0.04,0.03
0.02,0.04
1.3 ±0.2
1.1,1.0
0.5,0.8
2.5,0.4
9.3 ±2.5
8.5,10.9
8.6,8.5
4.7,7.2
Guinea pigs were Injected intravenously with native or oxidized LDL labelled with radioiodinated tyramlne cellobiose which remains
trapped in the degrading tissues. After most of the isotope had been cleared from plasma the content of radioactivity in all tissues was
measured.
Results for each tissue are expressed as the percentage of total recovered radioactivity in all tissues. Values for liver catabolism were
calculated including radioactivity in gut contents with the assumption that this represents biliary excretion.
Data for native LDL are means ± so, but for oxidized LDLs, the Individual values are given.
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unlikely to contribute to atherogenesis. On the other hand,
there Is evidence that llpld peroxidation does indeed occur
in vivo,39"41 and that products of lipid peroxidation can be
found in atherosclerotic lesions.39 Once LDL has entered
the artery wall, conditions would appear to be more favorable for LDL oxidation, as the concentration of LDL in the
arterial intima is high relative to that of other plasma proteins,42 and the residence time of LDL may be prolonged
due to interactions with matrix substances.43 Perhaps
most Important, LDL in the intimal space is in close proximity to endothelial cells and smooth muscle cells, both of
which can promote LDL oxidation. These factors might
well allow LDL oxidation to occur in this location; if macrophages were also present, these cells could further contribute to oxidation by releasing additional free radical intermediates, or could internalize the oxidized lipoproteins
and become foam cells.
Finally, it is possible that oxidation of LDL in the artery
wall may contribute to atherogenesis in ways other than
foam cell formation. Some potential effects of fatty acid
peroxides and oxidized sterols have been discussed
above, but in addition, oxidized LDL has been shown to be
toxic to cultured endothelial cells.44""46 This toxicity is associated with the lipid fraction of oxidized LDL, and is diminished if serum or HDL is present. 4647 In early atheromatous lesions, foam cells are sometimes found tightly
apposed to the abluminal surface of the endothelium.48 If
LDL in that microenvironment underwent oxidation, it
could lead to endothelial cell damage and the lifting off of
cells overlying a fatty streak as described by Gerrity49 and
by Fagglotto et al. 50
Acknowledgments
Marilee Lougheed, Diane Thorpe, Dana Daggett Hill, Loma Joy,
and Richard Elam provided expert technical assistance. The tyramlne cellobiose labelling studies were facilitated by assistance
and advice from Ray Plttman. Manuscript preparation was skillfully accomplished by Jean Wong and Wendy Semko.
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Decrease in reactive amino groups during oxidation or endothelial cell modification of LDL.
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U P Steinbrecher, J L Witztum, S Parthasarathy and D Steinberg
Arterioscler Thromb Vasc Biol. 1987;7:135-143
doi: 10.1161/01.ATV.7.2.135
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