A Modification of Apolipoprotein B Accounts for Most of the
Induction of Macrophage Growth by Oxidized Low Density
Lipoprotein*
Jason S.
Martens
,
Marilee
Lougheed
,
Antonio
Gómez-Muñoz, and
Urs P.
Steinbrecher§
From the Division of Gastroenterology, Department of Medicine, The
University of British Columbia, Vancouver, British Columbia V5Z
4E3, Canada
 |
ABSTRACT |
It has recently been shown that macrophage
proliferation occurs during the progression of atherosclerotic lesions
and that oxidized low density lipoprotein (LDL) stimulates macrophage
growth. Possible mechanisms for this include the interaction of
oxidized LDL with integral plasma membrane proteins coupled to
signaling pathways, the release of growth factors and autocrine
activation of growth factor receptors, or the potentiation of mitogenic
signal transduction by a component of oxidized LDL after
internalization. The present study was undertaken to further elucidate
the mechanisms involved in the growth-stimulating effect of oxidized
LDL in macrophages. Only extensively oxidized LDL caused significant
growth stimulation, whereas mildly oxidized LDL, native LDL, and acetyl
LDL were ineffective. LDL that had been methylated before oxidation (to
block lysine derivatization by oxidation products and thereby prevent
the formation of a scavenger receptor ligand) did not promote growth,
even though extensive lipid peroxidation had occurred. The growth
stimulation could not be attributed to lysophosphatidylcholine
(lyso-PC) because incubation of oxidized LDL with fatty acid-free
bovine serum albumin resulted in a 97% decrease in lyso-PC content but
only a 20% decrease in mitogenic activity. Similarly, treatment of
acetyl LDL with phospholipase A2 converted more than
90% of the initial content of phosphatidylcholine (PC) to lyso-PC, but
the phospholipase A2-treated acetyl LDL was nearly 10-fold
less potent than oxidized LDL at stimulating growth.
Platelet-activating factor receptor antagonists partly inhibited growth
stimulation by oxidized LDL, but platelet-activating factor itself did
not induce growth. Digestion of oxidized LDL with phospholipase
A2 resulted in the hydrolysis of PC and oxidized PC but did
not attenuate growth induction. Native LDL, treated with autoxidized
arachidonic acid under conditions that caused extensive modification of
lysine residues by lipid peroxidation products but did not result in
oxidation of LDL lipids, was equal to oxidized LDL in potency at
stimulating macrophage growth. Albumin modified by arachidonic acid
peroxidation products also stimulated growth, demonstrating that LDL
lipids are not essential for this effect. These findings suggest that
oxidatively modified apolipoprotein B is the main growth-stimulating
component of oxidized LDL, but that oxidized phospholipids may play a
secondary role.
 |
INTRODUCTION |
Macrophage-derived foam cells play an important role in the early
stages of atherosclerosis (1). Cultured macrophages have been shown to
take up chemically modified low density lipoproteins (LDLs)1 such as oxidized LDL
and acetyl LDL, resulting in foam cell formation (2, 3). Oxidized LDL
exhibits many other potentially atherogenic actions in vitro
(2), including direct chemoattractant activity for circulating
monocytes (4), induction of monocyte chemotactic protein-1 expression
(5), and enhancement of the expression of endothelial adhesion
molecules VCAM-1 (vascular cell adhesion molecule-1), ICAM-1
(intercellular adhesion molecule 1), and P-selectin (6-9). These
effects would tend to increase the number of macrophages within the
arterial intima at sites where oxidized LDL was present.
An additional biological action of oxidized LDL that could increase the
number of macrophages within the intima is a mitogenic effect on
monocytes or macrophages. It is noteworthy that macrophages have been
shown to be the predominant cell type expressing proliferating cell
nuclear antigen in atherosclerotic lesions, even in lesions containing
mostly smooth muscle-derived cells (10, 11). Oxidized LDL has been
reported to be mitogenic for cultured mouse peritoneal macrophages and
human monocyte-derived macrophages (12-14). In these studies, the
mitogenic effect of oxidized LDL was mediated by protein kinase C (15)
and was attributed to lysophosphatidylcholine (lyso-PC) that is
generated during LDL oxidation through enzymatic hydrolysis of PC by
platelet-activating factor (PAF) acetylhydrolase, an enzyme that is
normally associated with LDL (16, 17). It has recently been proposed
that this stimulation of macrophage growth involves induction by
lyso-PC of synthesis and secretion of GM-CSF by macrophages (18). The
levels of GM-CSF produced by cultured macrophages in response to
oxidized LDL were several orders of magnitude lower than those required
to produce an increase in cell number. However, anti-GM-CSF almost
completely blocked growth induction by oxidized LDL, and it was
therefore suggested that GM-CSF is a "first signal" that is
necessary but not sufficient for macrophage growth (18).
Oxidized LDL has also been shown to be mitogenic for bovine and rat
vascular smooth muscle cells (19). In these cells, the mitogenic effect
of oxidized LDL was attributed to an oxidized phospholipid with
PAF-like bioactivity and was blocked with PAF receptor antagonists,
suggesting that oxidized LDL may also stimulate growth directly through
PAF receptor activation. The present studies were done to clarify the
mechanism by which oxidized LDL stimulates macrophage growth.
 |
MATERIALS AND METHODS |
Carrier-free Na125I was purchased from Mandel
Scientific (Guelph, Ontario, Canada). Methyl[3H]thymidine
was purchased from Amersham (Cleveland, OH). RPMI 1640 medium and
gentamicin were from Canadian Life Technologies (Burlington, Ontario,
Canada). Defined fetal bovine serum was supplied by Professional
Diagnostics (Edmonton, Alberta, Canada). Crotalus atrox
venom phospholipase A2, 4-[2-aminoethyl]benzenesulfonyl fluoride (Pefabloc),
2,3-bis[2-methoxy-4nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide (XTT), N-methyldibenzopyrazine methyl sulfate, trinitrobenzenesulfonic acid, arachidonic acid, linoleic acid, bovine serum albumin, and butylated hydroxytoluene were purchased from Sigma. GM-CSF and WEB 2086 were from Boehringer Mannheim.
1-Palmitoyl-2-[6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]caproyl]phosphatidylcholine (C6NBD- PC), 1-palmitoyl-2-arachidonyl PC,
1-palmitoyl-2-linoleoyl PC, and PAF were obtained from Avanti Polar
Lipids (Birmingham, AL). 2-(5-oxovaleroyl) PC was a gift from Dr. Judy
Berliner (Department of Pathology, University of California at Los
Angeles, Los Angeles, CA). The Limulus Amebocyte Lysate assay was from
BioWhittaker Inc. (Walkersville, MD). L-659,989 was a gift from Dr.
John Chabala (Merck Sharpe & Dohme Research, Rahway, NJ). Other
chemicals were from Fisher Scientific or VWR Canlab (Edmonton, Alberta, Canada).
Lipoprotein Isolation and Labeling--
LDL (d
1.019-1.063 g/cm3) and HDL3 (d
1.125-1.21 g/cm3) were isolated by sequential
ultracentrifugation of EDTA-anticoagulated plasma obtained from healthy
normolipidemic volunteers (20). Radioiodination was performed using a
modification of the iodine monochloride method (16) and yielded
specific radioactivities between 100-200 cpm/ng LDL protein.
Iodination was performed before the modification of LDL.
Lipoprotein and Phospholipid Modification--
The concentration
of EDTA in LDL preparations was reduced before oxidation by dialysis
against Dulbecco's phosphate-buffered saline (PBS) containing 10 µM EDTA. Conditions for Cu2+ oxidation of LDL
were as follows: incubation of 200 µg/ml LDL in PBS with 5 µM CuSO4 at 37 °C for 2-28 h (16, 21).
Further oxidation was inhibited by the addition of 200 µM
EDTA and 50 µM butylated hydroxytoluene. Acetylation of
LDL was performed by the sequential addition of acetic anhydride (22).
Seven aliquots each of 1 µl of acetic anhydride were added at 15-min
intervals to 4 mg of LDL in 2 ml of ice-cold 50% saturated sodium
acetate. Methylation of LDL was done by the sequential addition of a
total of 40 µl of 2% formalin to 2 mg of LDL in ice-cold PBS
containing 80 mM NaCNBH3 (16). Lyso-PC was
removed from oxidized LDL by incubating 200 µg/ml oxidized LDL with
10 mg/ml fatty acid-free bovine serum albumin for 18 h at 20 °C
in sterile PBS (pH 7.4). A control incubation with oxidized LDL was
performed with the same volume of PBS without bovine serum albumin.
Both oxidized LDL samples were re-isolated by ultracentrifugation for
20 h at 10 °C (d < 1.210 g/cm3)
(23). Phospholipase A2 digestion of LDL was performed by
adding 5 units of phospholipase A2 in 0.2 ml of 0.1 M Tris-HCl with 10 mM CaCl2 to 1.5 mg of native, acetylated, or oxidized LDL in 1 ml of PBS and incubating
at 37 °C for 2 h (4). The reaction was then stopped by the
addition of 10 mM EDTA and refrigeration. To inactivate PAF
acetylhydrolase, LDL (0.4 mg/ml) in PBS or 5% FBS in RPMI 1640 medium
was treated with 0.1 mM Pefabloc for 30 min at 37 °C
(24). This concentration of Pefabloc consistently inhibited
98% of
PAF acetylhydrolase activity. PAF or 2-(5-oxovaleroyl) PC was
incorporated into Pefabloc-treated native or acetyl LDL by incubating
200 nmol of phospholipid with 1.5 mg of LDL containing 50 µM butylated hydroxytoluene at 37 °C for 4 h.
Lipoproteins were dialyzed against PBS containing 10 µM
EDTA and sterile filtered. TLC and phosphorus assay demonstrated an
association of 55 nmol PAF/mg LDL and 90 nmol 2-(5-oxovaleroyl) PC/mg
LDL. Oxidized 2-arachidonyl PC or 2-linoleoyl PC was obtained by
exposing 50 mg of neat phospholipid to air for 3 days at 37 °C.
These conditions resulted in more than one-half of the initial PC
migrating as a series of bands more polar than PC and overlapping the
lyso-PC and PAF zones. The yellow-brown product was dissolved in 1 ml
of ethanol, and then 20-µl aliquots (corresponding to 1 mg or 1.2 µmol of oxidized PC) were briefly sonicated in 1 ml of PBS until
optically clear. Each aliquot was then incubated for 5 h at
37 °C with 1.5 mg of native LDL or acetyl LDL and 0.5 ml of
lipoprotein-deficient serum as a source of phospholipid transfer
activity. The LDL was then re-isolated by ultracentrifugation.
Modification of LDL, HDL, or albumin with fatty acid peroxidation
products was performed as described previously (25). In brief, 3 mg
(10-13 µmol) of fatty acid were heated in an air atmosphere for
72 h, dissolved in PBS, and incubated with 1 mg of LDL protein in
the presence of 10 µM EDTA and 50 µM
butylated hydroxytoluene for 16 h. Excess water-soluble
peroxidation products were then removed by dialysis. Endotoxin levels
in native or modified LDL preparations were consistently less than 100 pg/mg LDL protein.
Cell Culture--
Resident peritoneal macrophages were collected
from male CD-1 mice by peritoneal lavage with ice-cold
Ca2+-free Dulbecco's PBS. Cells were resuspended in RPMI
1640 medium supplemented with 10% fetal bovine serum and adjusted to
1 × 105 cells/ml for XTT assay, 5 × 105 cells/ml for [3H]thymidine incorporation
assay, and 5 × 104 cells/ml for cell counting. For
the XTT and thymidine incorporation assays, 0.1 ml of cell suspension
was added per well to 96-well tissue culture plates. For cell number
determinations, 1.0 ml of cell suspension was added per well to 24-well
tissue culture plates (Falcon, Lincoln Park, NJ). Macrophages were
incubated overnight at 37 °C in a humidified atmosphere of 5%
CO2 in air. Nonadherent cells were then removed by gentle
washing with medium. For analysis of oxidized LDL-induced cell growth,
macrophages were cultured in 0.1 ml of RPMI 1640 medium containing 5%
fetal bovine serum with lipoproteins for 4 days without a medium
change. In some experiments, 0.1 mM Pefabloc was included
in the growth medium to inactivate serum PAF acetylhydrolase.
Lipoprotein uptake and degradation experiments were performed as
described previously (26). Scavenger receptor class A type I/II
(SR-AI/II) knockout mice were obtained from Dr. T. Kodama (University
of Tokyo, Tokyo, Japan). The description of the construct and the
phenotypic characterization of homozygous knockout mice have been
reported elsewhere (26-28). For experiments using SR-AI/II-deficient
macrophages, the cells were obtained and cultured in the exact manner
described above.
XTT Growth Assay--
Macrophage growth was determined by the
XTT formazan method. This assay is based on cellular reduction of XTT
by mitochondrial dehydrogenase to an orange formazan product that can
be measured spectrophotometrically and correlates well with the cell
number (29). Briefly, 50 µl of XTT solution (1 mg/ml XTT and 25 µM N-methyldibenzopyrazine methyl sulfate in RPMI 1640 medium) were added to each well and incubated for 4.5 h at
37 °C. Absorbance at 450 nm was then measured with a multiwell
spectrophotometer. There is a linear correlation between macrophage
cell number and XTT formazan formation ranging from 2 × 103 to 5 × 104 cells/well (30).
Tritiated Thymidine Incorporation Assay--
Macrophage growth
was also determined by the incorporation of [3H]thymidine
into cellular DNA. Briefly, 10 µl of 20 uCi/ml
methyl[3H]thymidine (80 Ci/mmol) were added to each well
of 96-well plates for the last 24 h of each experiment. The medium
was then aspirated, and cells were washed with ice-cold 10%
trichloroacetic acid to precipitate DNA and remove unincorporated
label. Cells were dissolved in 0.5% SDS with 0.3 N NaOH to
hydrolyze the acid-insoluble material. Radioactivity was analyzed using
a liquid scintillation counter.
Cell Counting--
Macrophage cell number was determined during
culture using inverted phase-contrast microscopy by counting the number
of cells within four random fields of view (0.40 mm2) from
two separate wells.
Analytic Procedures--
Protein determination was done by the
method of Lowry et al. (31) in the presence of 0.05% sodium
deoxycholate to minimize turbidity. Bovine serum albumin was used as
the standard. Methylation of LDL increases its chromogenicity in the
Lowry assay by 143% (32); hence, the assay results for methylated LDL
were corrected by this factor. Lipoprotein electrophoresis was done
using a Corning apparatus and Universal agarose film in 50 mM barbital buffer (pH 8.6). Bovine serum albumin was added
to lipoprotein samples before electrophoresis to ensure reproducible
migration distances. Lipoprotein bands were visualized by staining with
Fat Red. Electrophoretic mobilities are expressed relative to that of
native LDL. For phospholipid analysis, lipids were extracted from 100 µg of LDL protein using chloroform/methanol (2:1), and the chloroform
phase was evaporated under nitrogen. Phospholipids were separated by
thin-layer chromatography on Silica Gel 60 using
chloroform/methanol/water (65:35:7) and visualized with iodine vapor.
For some analyses, a better separation of PAF from PC and lyso-PC was
achieved by development with chloroform/methanol/water (50:35:7). Bands
corresponding to PAF, lyso-PC, and PC were scraped from the plates and
assayed for phosphorus content as described previously (33).
Inactivation of PAF acetylhydrolase was determined by measuring the
hydrolysis of C6NBD-PC as described previously (17).
Briefly 10 nmol of C6NBD-PC were incubated at 37 °C for 1 h in 1 ml of PBS with 50 µg of LDL or 5% serum. The
fluorescence of C6NBD fatty acid was measured in the
aqueous phase after chloroform/methanol extraction.
Statistical Analyses--
The differences between growth curves
were evaluated by analysis of variance (ANOVA) with Tukey's method for
multiple comparisons.
 |
RESULTS |
Oxidized LDL Induces Macrophage Cell Growth--
To verify that
oxidized LDL was capable of stimulating the proliferation of mouse
peritoneal macrophages, cells were incubated with 30 µg/ml native or
modified LDL in 5% FBS. In situ cell counts were done every
2 days for up to 10 days. As shown in Fig.
1A, oxidized LDL caused a
progressive increase in cell number, comparable to that seen with 10 ng/ml GM-CSF. Neither native nor acetyl LDL increased the macrophage
cell number. Cells incubated with oxidized LDL showed a substantial
increase in size as well as a change to a more spindle-shaped
morphology with elongated cytoplasmic extensions. Concentrations of
oxidized LDL greater than 40 µg/ml appeared to be cytotoxic, as
reflected by membrane blebbing, cell detachment, and a decrease in the
proportion of cells that excluded trypan blue. Fig. 1B shows
that the increase in cell number was accompanied by a 5-fold increase
in thymidine incorporation in macrophages treated with 30 µg/ml
oxidized LDL for 6 days, whereas acetyl LDL had no effect.

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Fig. 1.
Macrophage growth induction by oxidized LDL.
A, mouse peritoneal macrophages were cultured for 10 days in
24-well plates in RPMI 1640 medium containing 5% FBS and 30 µg/ml
native LDL ( ), acetyl LDL ( ), oxidized LDL ( ), 10 ng/ml GM-CSF
( ), or medium alone ( ). Every 2 days, cells were counted as
described under "Materials and Methods." *, p 0.01 versus any of the three controls. B,
macrophages were incubated in RPMI 1640 medium containing 5% FBS with
either 30 µg/ml acetyl LDL ( ), oxidized LDL ( ), or medium alone
( ) for the indicated number of days. One day before the end of each
experiment, cells were pulsed with 0.2 uCi of
[3H]thymidine. The radioactivity incorporated into the
cells in the ensuing 24 h was determined by liquid scintillation
counting as described under "Materials and Methods." The values
shown represent the mean ± standard error of quadruplicate
determinations from three experiments. *, p 0.01
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Macrophage Growth Is Dependent on the Degree of LDL
Oxidation--
For convenience, subsequent studies of the effect of
native or modified LDL on the growth of murine resident peritoneal
macrophages were done with the XTT reduction assay (30). The XTT assay
is sensitive enough to be applied to cells grown in 96-well plates and
correlates well with the cell number (30). Concentrations of oxidized
LDL as low as 2.5 µg/ml produced significant growth activity, and
maximal stimulation was obtained at about 30 µg/ml (data not shown).
To examine the effect of differing degrees of LDL oxidation on
macrophage growth, LDL samples with defined incremental degrees of
oxidation were prepared by oxidizing 200 µg/ml LDL with 5 µM CuSO4 at 37 °C for various times
ranging from 2 to 24 h. Of note, LDL that was oxidized for 15 h was only 30-40% as potent in stimulating growth as LDL oxidized for
24 h (Fig. 2), even though the
extent of apoB modification at 15 h was sufficient to allow near
maximal binding and uptake of the oxidized LDL particle (Table
I). This suggests that changes to LDL
lipids or apoB associated with very extensive oxidation are required
for growth stimulation. Table I also shows that the PC content of LDL
decreased progressively during oxidation, falling to one-half of the
initial value after 24 h of oxidation. Sphingomyelin content
remained constant under these conditions. About 70% of the lost PC was
converted to lyso-PC, and the rest presumably remains as oxidized PC.
Two factors contribute to the incomplete conversion of oxidized PC to
lyso-PC: (a) some oxidized PC species contain long-chain
polar acyl derivatives that are poor substrates for PAF acetylhydrolase
(17), and (b) PAF acetylhydrolase is inactivated during LDL
oxidation, and very little activity remains beyond 10 h of
oxidation.

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Fig. 2.
Effect of the extent of LDL oxidation on
macrophage cell growth. Macrophages were cultured in 96-well
plates for 4 days without a medium change in RPMI 1640 medium with 5%
FBS containing the indicated concentrations of LDL oxidized by exposure
to copper for 2 ( ), 5 ( ), 10 ( ), 15 ( ), or 24 ( ) h.
Fifty µl of XTT solution were added to each well for 4.5 h at
37 °C, and the formation of XTT formazan product was detected with a
microplate spectrophotometer at 450 nm. The values shown represent the
mean ± standard error of quadruplicate determinations from three
experiments. *, p 0.0001.
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Table I
Phospholipid analysis and electrophoretic mobilities of modified LDLs
LDL was oxidized with 5 µM copper for 2, 5, 10, 15, and
24 h at 37 °C. To remove lyso-PC, oxidized LDL (200 µg/ml)
was incubated with 10 mg/ml fatty acid-free bovine albumin for 24 h at 20 °C and re-isolated by ultracentrifugation. A mock incubation
and re-isolation of oxidized LDL was performed without albumin. Native
or acetyl LDL (1.5 mg) was incubated with 5 units/ml phospholipase
A2 for 2 h at 37 °C and re-isolated from PLA2
by repeated washes with PBS in ultrafiltration membrane cones. All
oxidized LDL samples were oxidized for 24 h unless noted
otherwise. The PC and lyso-PC contents and electrophoretic mobility
relative to the native LDL of each lipoprotein were determined as
described under "Materials and Methods." Each value represents the
mean ± standard error of duplicate determinations from three
separate experiments.
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Macrophage Growth Stimulation Is Dependent on Internalization of
the Oxidized LDL Particle--
SR-AI/II is responsible for about 30%
of the uptake of oxidized LDL by murine macrophages (26). Hence, if
oxidized LDL uptake is essential for growth stimulation in macrophages,
one would expect a proportionate decrease in growth stimulation in
SR-AI/II-deficient macrophages compared with controls. On the other
hand, if uptake was not required or at least was not rate limiting,
then one would expect no difference between controls and SR-AI/II
knockouts in growth stimulation by oxidized LDL. Fig.
3 shows that there was indeed less growth
stimulation by oxidized LDL in SR-AI/II-deficient macrophages compared
with controls. The magnitude of the decrease in growth stimulation
(about one-third) is similar in magnitude to the decrease in oxidized
LDL uptake by these cells. Hence, it is likely that SR-AI/II is not
essential for growth stimulation but simply provides an additional
pathway for the internalization of oxidized LDL. Similar results were
reported previously by Sakai et al. (14), although this
group concluded that SR-AI/II was necessary for growth stimulation. To
determine the effect of blocking internalization via all scavenger
receptors, we exploited a previous observation from our laboratory that
methylation of LDL lysine residues before oxidation prevents their
modification by lipid peroxidation products, and that oxidized methyl
LDL is not internalized by macrophage scavenger receptors (25). As
shown in Fig. 4, the total uptake of
oxidized methyl-LDL was reduced by 74% compared with that of control
oxidized LDL, and the effect on macrophage growth was attenuated by
84%. These results suggest that lysine modification by lipid
peroxidation products (and presumably the internalization of oxidized
LDL by macrophages) is necessary in order to elicit a growth
stimulatory response.

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Fig. 3.
Growth-stimulating effects of oxidized LDL on
SR-AI/II-deficient macrophages. Macrophages from control mice
(closed symbols) or SR-AI/II-deficient mice (open
symbols) were incubated for 4 days in medium with 5% FBS together
with the indicated concentrations of native LDL (squares),
acetyl LDL (circles), or oxidized LDL
(triangles). Cell growth was then measured by the XTT assay.
Each value represents the mean ± standard error of quadruplicate
determinations from three experiments. *, p 0.01.
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Fig. 4.
Effect of methylation of LDL before oxidation
on macrophage cell growth. A, macrophages were incubated at
37 °C with oxidized 125I-LDL ( ) or
125I-LDL that had been methylated before oxidation ( ).
After 5 h, the media were assayed for 125I-LDL
degradation products and cell-associated radioactivity. Results are
expressed as µg LDL internalized (sum of cell-associated and degraded
LDL)/mg protein. Methylation of LDL blocked 87% of LDL lysine residues
as judged by trinitrobenzenesulfonic acid reactivity. B,
macrophages were incubated for 4 days with the same preparations of
oxidized 125I-LDL ( ) or 125I-LDL that had
been methylated before oxidation ( ) that were used in A.
Cell growth was then measured by XTT assay as described in the legend
to Fig. 2.
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The Role of Lyso-PC in Oxidized LDL-induced Macrophage Cell
Growth--
To test the hypothesis advanced by Sakai et al.
(13) that lyso-PC accounts for the growth stimulatory effect of
oxidized LDL, we tested the effect of oxidized LDL that had been
depleted of lyso-PC by incubation with fatty acid-free bovine serum
albumin. As shown in Fig. 5A,
macrophage growth stimulation by oxidized LDL after treatment with
albumin was about 80% compared with that of control oxidized LDL, even
though albumin treatment had removed more than 97% of the lyso-PC
(Table I). Because the incubation with albumin and the subsequent
re-isolation may have caused changes other than simply removing
lyso-PC, a second experiment was performed in which the formation of
lyso-PC from oxidized PC during LDL oxidation was prevented by
inactivating PAF acetylhydrolase with Pefabloc (24). It has been
previously shown that when PAF acetylhydrolase activity is inhibited,
oxidation of LDL results in the accumulation of oxidized PC compounds,
some of which contain short-chain polar acyl fragments in the sn-2
position (34). When LDL was treated with Pefabloc before oxidation, the
formation of lyso-PC was less than 10% of that of the control oxidized
LDL, although the amount of PC consumed (oxidized) was the same.
However, Pefabloc pretreatment did not reduce the ability of oxidized
LDL to stimulate growth (data not shown). These results indicate that
lyso-PC cannot account for more than a small part of the growth
stimulatory effect of oxidized LDL. However, we could not exclude the
possibility that lyso-PC might be capable of stimulating growth under
some conditions. To test this, we treated native and acetyl LDL with
phospholipase A2 and then determined their effects on
macrophage growth. Table I shows that treatment of native or acetyl LDL
with PLA2 resulted in the conversion of more than 90% of
PC to lyso-PC. Oxidized LDL contained 60% less lyso-PC than
PLA2-treated acetyl LDL but was 7- to 10-fold more potent
than PLA2-treated acetyl LDL in inducing macrophage growth
(Fig. 5B). When expressed as the amount of lyso-PC delivered
to the cytosol, the difference is even greater because the rate of
uptake of acetyl LDL and extensively oxidized LDL by macrophages is the
same, but the degradation of oxidized LDL is much less efficient than
that of acetyl LDL (35). PLA2-treated native LDL had no
effect on growth, indicating that lipoprotein internalization and not
simply the transfer of lyso-PC to the plasma membrane was required for
growth stimulation.

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Fig. 5.
Lyso-PC does not account for the ability of
oxidized LDL to induce macrophage growth. A, macrophages
were incubated for 4 days with the indicated concentrations of native
LDL ( ), oxidized LDL ( ), oxidized LDL stripped of lyso-PC by
incubation with fatty acid-free bovine serum albumin ( ), or oxidized
LDL incubated in parallel with PBS alone ( ). Cell growth was
measured by the XTT reduction assay. Each value represents the
mean ± standard error of quadruplicate determinations from four
experiments. *, p 0.05. B, native or
acetylated LDL containing large amounts of lyso-PC are less effective
than oxidized LDL in stimulating macrophage growth. Macrophages were
incubated for 4 days with the indicated concentrations of native LDL
( ), acetyl LDL ( ), oxidized LDL ( ), PLA2-treated
native LDL ( ), or PLA2-treated acetyl LDL ( ). Cell
growth was then measured by the XTT assay. Each value represents the
mean ± standard error of quadruplicate determinations from four
experiments. *, p < 0.0001. Phospholipid composition
of these LDL samples is shown in Table I.
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Macrophage Growth Stimulation by Oxidized LDL Is Partially
Inhibited by PAF Receptor Antagonists, but PAF Itself Does Not Induce
Growth--
Heery et al. (19) reported that the growth of
smooth muscle cells is stimulated by oxidized PC through activation of
the PAF receptor. Because the results described above might be
explained by oxidized phospholipids causing the growth stimulation in
macrophages, the PAF receptor seemed to be a good candidate to test as
a mediator of this effect. Accordingly, macrophages were treated with
the PAF receptor inhibitors L-659,989 or WEB 2086 during incubation with oxidized LDL. Fig. 6A
shows that L-659,989 blocked 50% of the macrophage growth stimulation
by oxidized LDL. There was no morphologic evidence of cytotoxicity at
concentrations of up to 25 µM, and this drug had no
effect on macrophage growth stimulation induced by 10 nM
GM-CSF. However, only 20% of the macrophage growth stimulation by
oxidized LDL was blocked by 20 µM WEB 2086. These inhibitor concentrations are sufficient to completely block the effect
of PAF on neutrophil adhesion (19). PAF alone added to macrophages at
concentrations of up to 1 µM had no effect on growth stimulation (data not shown). The lack of effect of PAF cannot be
attributed to its hydrolysis to lyso-PAF, because serum PAF acetylhydrolase present in the growth medium had been inactivated by
pretreatment with 0.1 mM Pefabloc, and the stability of PAF during incubation was verified by thin-layer chromatography. Because internalization of oxidized LDL is necessary to elicit a growth stimulatory response in macrophages, the growth effect of PAF that had
been incorporated into Pefabloc-treated acetyl LDL was also tested. No
increase in growth was observed with PAF incorporated into acetyl LDL
compared with untreated acetyl LDL (Fig. 6B). These
observations are compatible with the hypothesis that macrophage growth
stimulation is due to oxidized phospholipids that do not act via the
PAF receptor, but possibly through a novel receptor that is partly
inhibited by L-659,989.

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Fig. 6.
Partial inhibition of oxidized LDL-induced
macrophage growth by PAF receptor antagonists. A,
macrophages were preincubated for 30 min with varying concentrations of
the PAF receptor antagonists L-659,989 ( ) or WEB 2086 ( ), and
then 30 µg/ml oxidized LDL was added for 4 days in the continued
presence of each antagonist. As a control for nonspecific toxicity,
parallel incubations of L-659,989 were carried out with macrophages
incubated with 10 ng/ml GM-CSF ( ). Cell growth was measured by the
XTT assay. Each value represents the mean ± standard error of
triplicate determinations from two experiments. *, p 0.005. B, macrophages were incubated with the indicated
concentrations of oxidized LDL ( ), Pefabloc-treated acetyl LDL
( ), or Pefabloc-treated acetyl LDL containing 55 nmol PAF/mg LDL
protein ( ). Residual PAF acetylhydrolase activity in
Pefabloc-treated acetyl LDL was less than 1%. Analysis of medium
containing PAF-enriched acetyl LDL by TLC at the end of the incubation
verified that PAF had not been degraded.
|
|
Oxidized Phosphatidylcholines Account for Only a Small Part of the
Growth Stimulatory Effect of Oxidized LDL--
To directly test the
hypothesis that oxidized PC might be the cause of growth, we tested the
effect of autoxidized 2-arachidonyl PC, 2-linoleoyl PC, or
2-(5-oxovaleroyl) PC, alone or after incorporation into native LDL or
acetyl LDL. Fig. 7 shows that the
autoxidized phospholipids were capable of stimulating growth to a
modest extent when incorporated into native LDL or acetyl LDL, but
their potency was much less than that of oxidized LDL. Acetyl LDL
containing 2-(5-oxovaleroyl) PC or 2-(5-oxovaleroyl) PC alone did not
stimulate growth (data not shown). To determine whether oxidized PCs
accounted for a significant part the growth-stimulating effect of
oxidized LDL, the latter was incubated with PLA2 under
conditions that caused a near complete hydrolysis of intact and
oxidized PC and then compared with untreated oxidized LDL.
Surprisingly, PLA2 digestion not only failed to block the
growth effect of oxidized LDL, but the phospholipolyzed oxidized LDL
actually caused 15% more growth than untreated oxidized LDL at
concentrations from 10 to 30 µg protein/ml (data not shown). This
indicated that oxidized PC could not be the main factor in oxidized LDL
that stimulates growth.

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|
Fig. 7.
Effect of oxidized PC on growth.
Autoxidized 2-linoleoyl PC or 2-arachidonyl PC were incorporated into
native LDL or acetyl LDL as described under "Materials and
Methods." The indicated concentration of modified LDL was then
incubated with macrophages for 4 days, and macrophage growth was
measured using the XTT assay. Oxidized LDL, ; acetyl LDL modified
with oxidized 2-arachidonyl PC, ; native LDL modified with
2-arachidonyl PC, ; acetyl LDL modified with oxidized 2-linoleoyl
PC, ; native LDL modified with oxidized 2-linoleoyl PC, ; acetyl
LDL, . The range of values for incorporation of oxidized PC into LDL
was 420-558 nmol/mg LDL protein.
|
|
LDL Modified by Fatty Acid Oxidation Products Is as Potent as
Copper-oxidized LDL at Stimulating Macrophage Growth--
We next
tested the possibility that an oxidative modification of apoB might be
responsible for the growth-inducing effect of oxidized LDL. The
experiment with methylated LDL described in Fig. 5 was initially
regarded as evidence that receptor-mediated LDL uptake was required for
growth induction. However, this could also be interpreted as a
requirement for lysine modification by peroxidation products. To test
this more directly, we derivatized LDL apoB with fatty acid
peroxidation products under conditions in which LDL lipids themselves
could not be oxidized (25). Such LDL has the same phospholipid
composition as native LDL but shows a marked increase in
electrophoretic mobility as a result of lysine derivatization and is a
ligand for several classes of scavenger receptors (25, 26). Data in
Fig. 8A show that LDL modified with oxidation products derived from linoleic acid or arachidonic acid
was comparable in potency to oxidized LDL in stimulating growth. To
evaluate the extent of modification by fatty acid peroxidation products
necessary to stimulate growth, 1 mg of LDL was treated with oxidation
products from 0.2, 0.4, 0.8, or 1.2 mg of arachidonic acid. The
resulting LDLs had electrophoretic mobilities that were 1.3-, 2.7-, 3.6-, and 4.4-fold that of native LDL, respectively. As judged by these
changes in electrophoretic mobility (25), we estimate that
derivatization of at least 15% of lysine residues was required for
detectable growth induction. Surprisingly, bovine albumin and human
high density lipoprotein modified by arachidonic acid oxidation
products also stimulated growth, although the effect on growth was
linear with concentration as opposed to saturable, as with modified LDL
(Fig. 8B). Nevertheless, the results in Fig. 8 are
compelling evidence that an oxidative modification of apoB rather than
oxidized lipid(s) per se accounts for most of the growth
induction by oxidized LDL.

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|
Fig. 8.
Stimulation of macrophage growth by oxidation
product-modified LDL. A, LDL was modified with fatty acid
peroxidation products as described under "Materials and Methods."
This procedure results in derivatization of LDL lysine residues with
reactive aldehydes, but there is no oxidation of LDL lipids.
Macrophages were incubated for 4 days with the indicated concentrations
of oxidized LDL ( ), LDL modified with oxidation products derived
from 1.2 µg of linoleic acid ( ), or LDL modified with oxidation
products derived from 0.2 ( ), 0.4 ( ), 0.8 ( ), or 1.2 mg of
arachidonic acid ( ), and then cell growth was measured with the XTT
assay. B, bovine serum albumin and human high density
lipoprotein were modified as described above with arachidonic acid
oxidation products. Macrophages were incubated as described above with
the indicated concentrations of native albumin ( ), albumin modified
with oxidation products ( ), high density lipoprotein ( ), or high
density lipoprotein modified by oxidation products ( ), and then cell
growth was measured. Similar results were obtained in two replicate
experiments.
|
|
 |
DISCUSSION |
Sakai et al. (13) were the first to describe the
stimulation of macrophage growth by oxidized LDL. This group concluded that the effect was due to lyso-PC and required lipoprotein
internalization by SR-AI/II (13, 14). Although these interpretations
conflict with ours, the actual data are in general agreement.
Specifically, we confirmed their findings that PLA2-treated
acetyl LDL can mimic the effect of oxidized LDL and that macrophages
lacking scavenger receptors are less responsive to oxidized LDL than
control cells. However, we found that the removal of more than 97% of
lyso-PC from oxidized LDL did not substantially reduce its effect on
growth stimulation and that oxidized LDL was almost an order of
magnitude more potent than PLA2-treated acetyl LDL, which
contained twice as much lyso-PC per particle as oxidized LDL. Of note,
a recent study from the Japanese group also reported that oxidized LDL was more potent than PLA2-treated acetyl LDL at stimulating
macrophage growth (18). Hence, the data from both laboratories indicate that the effect of oxidized LDL on growth cannot be attributed entirely
to lyso-PC. The difference in findings with SR-AI/II-deficient mice
appear to be minor: Sakai et al. (14) reported that about 50-70% of oxidized LDL uptake and a similar proportion of the growth
induction were mediated by SR-AI/II, whereas we found that only about
30% of oxidized LDL uptake and 30% of growth induction were mediated
by this receptor. This discrepancy could be explained by differences in
oxidized LDL preparations between laboratories or by the different
genetic backgrounds of the SR-AI/II-deficient transgenic mice that were used.
Heery et al. (19) analyzed the effects of oxidized LDL on
the growth of arterial smooth muscle cells. They found that oxidized LDL (but not native LDL) induced smooth muscle cell growth, and that
this effect was mimicked by PAF and could be blocked by the PAF
receptor antagonists WEB 2086 and L-659,989. Furthermore, it was shown
that growth stimulation was associated with a lipid fraction
intermediate in polarity between PC and PAF, consistent with oxidized
PC. The biological activity of this component was eliminated by PAF
acetylhydrolase, and, in fact, active fractions could only be recovered
from LDLs that had been pretreated with diisopropylfluorophosphate to
inactive PAF acetylhydrolase before oxidation. These observations in
smooth muscle cells differ substantially from our findings in
macrophages in that PAF itself did not induce growth in macrophages,
and WEB 2086 had only a minimal inhibitory effect on growth. Oxidized
PC itself was found to cause some growth induction in macrophages, but
because the growth effect of oxidized LDL could not be abolished by
PLA2, and the growth was mimicked by LDL modified with
fatty acid oxidation products (and containing no oxidized PC), we
concluded that protein modification was predominantly responsible for
growth induction.
To date, there has been no detailed characterization of the structure
of the oxidized phospholipids in oxidized LDL that are responsible for
PAF receptor activation. Stremler et al. (36, 37) identified
1-palmitoyl-2-[5-oxovaleroyl] PC in oxidized LDL and showed that this
compound is a substrate for PAF acetylhydrolase and can increase
thymidine incorporation in smooth muscle cells (19). However, the
maximal effect of 2-[5-oxovaleroyl] PC was only a 33% increase over
control, whereas PAF and oxidized LDL more than doubled thymidine
incorporation. This suggests that there may be compounds more potent
than 2-[5-oxovaleroyl] PC that account for most of the growth
stimulation. Indeed, in our studies in macrophages, 2-[5-oxovaleroyl]
PC had no effect on growth. Our finding that the active components
could not be removed by preincubation of oxidized LDL with albumin
suggests that the active fraction is not a highly polar PC derivative,
but part of the growth-promoting activity could be due to a
condensation product of oxidized PC or a form with a residue longer
than 5-oxovaleroate at the 2 position. One such PC oxidation product
could be F2-isoprostanoyl PC, which has been shown to be
formed during nonenzymatic oxidation of LDL (38). Alternatively, it
could be a derivative of 2-linoleoyl PC that cannot generate a
derivative containing a short-chain aldehyde.
Reconciliation of the findings in smooth muscle cells with those in
macrophages requires that there are two pathways for growth induction:
one active in smooth muscle cells that involves the PAF receptor and
PAF-like forms of oxidized phosphatidylcholine, and a second pathway
that involves a modification of apoB components present only in very
extensively oxidized LDL and mediated by a different and possibly novel
receptor. Additional studies are required to define the domain of
oxidized apoB that is involved in growth stimulation and to determine
whether this is mediated by a cell surface receptor or is a consequence
of the internalization and processing of modified apoB. Once the
stimulatory component and its receptor have been identified, it should
be possible to clarify the signal transduction pathways involved in
macrophage growth stimulation. Evidence has been presented to implicate
phosphatidylinositol 3-kinase, protein kinase C, and protein
tyrosine kinases (15, 30), but there are discrepancies between
laboratories in the relative importance of these pathways in growth
induction by oxidized LDL.
Although there is strong evidence that oxidatively modified LDL is
present in atherosclerotic lesions (39-41), it has not been proven
that LDL in early or developing lesions in man is oxidized to the
extent that we found to be necessary for macrophage growth induction
in vitro (42). However, even mildly oxidized LDL is capable
of inducing colony-stimulating factor gene expression in endothelial
cells (43), and macrophage colony-stimulalting factor together with
other endothelial-derived factors are synergistic stimuli for
macrophage growth (44). Furthermore, induction of macrophage
colony-stimulating factor has been demonstrated in foam cell
macrophages in human and rabbit atherosclerotic lesions (45). Hence, it
is possible that a lesser degree of oxidation, comparable to what is
present in early lesions in vivo, might have a stimulatory
effect on macrophage growth in vivo.
The ultimate importance of in situ proliferation of
macrophages in the artery wall during the development of
atherosclerotic lesions is unknown. However, the present findings
provide further insights into a possible mechanistic explanation for
the macrophage proliferation in atherosclerotic lesions demonstrated by
immunocytochemical studies (10, 11) and suggest an additional role for
oxidized LDL in the pathogenesis of atherosclerosis.
 |
FOOTNOTES |
*
This study was supported by Grant MT8630 from the Medical
Research Council of Canada.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
These authors contributed equally to this study.
§
To whom correspondence should be addressed: Dept. of Medicine, The
University of British Columbia, 3300 - 950 West 10th Ave., Vancouver,
British Columbia V5Z 4E3, Canada. Tel.: 604-875-5862; Fax:
604-875-5447; E-mail: usteinbr{at}unixg.ubc.ca.
 |
ABBREVIATIONS |
The abbreviations used are:
LDL, low density
lipoprotein;
PAF, platelet-activating factor;
ApoB, apolipoprotein B;
lyso-PC, lysophosphatidylcholine;
PC, phosphatidylcholine;
GM-CSF, granulocyte macrophage colony-stimulating factor;
XTT, 2,3-bis[2-methoxy-4-nitro-5sulfophenyl]-2H-tetrazolium-5-carboxanilide;
PBS, phosphate-buffered saline;
C6NBD-PC, 1-palmitoyl-2-[6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]caproyl]phosphatidylcholine;
HDL, high density lipoprotein;
SR-AI/II, scavenger receptor class A
type I/II;
PLA2, phospholipase A2.
 |
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