© 1999 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
All ApoB-Containing Lipoproteins Induce Monocyte Chemotaxis and Adhesion When Minimally Modified
Modulation of Lipoprotein Bioactivity by Platelet-Activating Factor Acetylhydrolase
From the Division of Endocrinology and Metabolism (C.L., F.S., T.S., S.Hörkkö, J.L.W., P.D.R.), Department of Medicine, University of California, San Diego, and the Division of Cardiology (S.Hama, M.N.), Department of Medicine, University of California, Los Angeles, Calif; the Division of Endocrinology (P.V.S.), Rush Medical College, Chicago, Ill; and the Department of Medicine (M.M.), University of Shizuoka, Shizuoka, Japan.
Correspondence to Peter Reaven, MD, Division of Endocrinology and Metabolism, Department of Medicine, 0682, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0682.
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Abstract—Mildly oxidized LDL has many proinflammatory properties, including the stimulation of monocyte chemotaxis and adhesion, that are important in the development of atherosclerosis. Although ApoB-containing lipoproteins other than LDL may enter the artery wall and undergo oxidation, very little is known regarding their proinflammatory potential. LDL, IDL, VLDL, postprandial remnant particles, and chylomicrons were mildly oxidized by fibroblasts overexpressing 15-lipoxygenase (15-LO) and tested for their ability to stimulate monocyte chemotaxis and adhesion to endothelial cells. When conditioned on 15-LO cells, LDL, IDL, but not VLDL increased monocyte chemotaxis and adhesion
4-fold. Chylomicrons and postprandial remnant particles were also
bioactive. Although chylomicrons had a high 18:1/18:2 ratio, similar to
that of VLDL, and should presumably be less susceptible to oxidation,
they contained (in contrast to VLDL) essentially no
platelet-activating factor acetylhydrolase (PAF-AH) activity.
Because PAF-AH activity of lipoproteins may be reduced in vivo by
oxidation or glycation, LDL, IDL, and VLDL were treated in vitro to
reduce PAF-AH activity and then conditioned on
15-lipoxygenase cells. All 3 PAF-AH–depleted
lipoproteins, including VLDL, exhibited increased stimulation of
monocyte chemotaxis and adhesion. In a similar manner, lipoproteins
from Japanese subjects with a deficiency of plasma PAF-AH activity were
also markedly more bioactive, and stimulated monocyte adhesion nearly
2-fold compared with lipoproteins from Japanese control subjects with
normal plasma PAF-AH. For each lipoprotein, bioactivity resided in the
lipid fraction and monocyte adhesion could be blocked by PAF-receptor
antagonists. These data suggest that the susceptibility of
plasma lipoproteins to develop proinflammatory activity is in part
related to their 18:1/18:2 ratio and PAF-AH activity, and that
bioactive phospholipids similar to PAF are generated during oxidation
of each lipoprotein. Moreover, LDL, IDL, postprandial remnant
particles, and chylomicrons and PAF-AH–depleted VLDL all give rise to
proinflammatory lipids when mildly oxidized.
Key Words: atherosclerosis • lipid peroxidation • platelet-activating factor acetylhydrolase • autoantibodies • LDL oxidation
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Introduction |
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Evidence from both in vitro and in vivo studies suggests that oxidation of LDL may contribute to early atherosclerotic lesion formation.1 2 The extent of LDL oxidation appears to influence which of a variety of potential atherogenic properties of oxidized LDLs predominate at a given point in time.3 When LDL is less oxidized, ie, minimally modified LDL (mm-LDL), it stimulates monocyte chemotaxis, adherence to and transmigration through endothelial cells,3 4 5 6 as well as expression of several growth factors such as macrophage colony-stimulating factor.3 These and other "bioactive" properties of mm-LDL very likely contribute to the development of atherosclerosis. Many of the proinflammatory properties of mm-LDL may result from the formation of oxidized phospholipids.3 7 Several of these oxidized phospholipids have been identified by liquid chromatography/mass spectrometry and at least 1 group of proinflammatory compounds results from oxidative degradation of arachidonic acid within the phospholipid.8 Studies by our laboratory have suggested that oxidation of other phospholipid polyunsaturated fatty acids (PUFAs), such as linoleic acid, may also contribute to the generation of bioactive phospholipids.9
Most investigations of lipoprotein oxidation in relation to atherosclerosis have focused on LDL, which has been isolated by ultracentrifugation over the broad density range of 1.019 to 1.063 g/mL. More recently it has become apparent that small dense subfractions of LDL have enhanced susceptibility to copper-mediated oxidation and may have greater atherogenic potential.10 However, the relevance of in vitro copper-mediated oxidation has been questioned and differences in the susceptibility of LDL subfractions to become "minimally modified" and bioactive have not been studied. There is also increasing evidence that more triglyceride-rich lipoproteins (IDLs, VLDLs, and postprandial remnant particles) may also enter the artery wall and contribute to the lipid accumulation in lesions.11 12 Several of these particles have been previously shown to be susceptible to oxidation in vitro.13 14 15 Because lipid-rich lipoproteins such as IDLs, VLDLs, and chylomicrons as well as their remnant particles may be an important source of PUFA-containing phospholipids, it is conceivable that their oxidation may generate substantial bioactivity. In addition, studies indicate that in humans oxidized lipids in the diet are absorbed by the small intestine and are transported in chylomicrons to the circulation, where they can be incorporated into VLDL by the liver, or perhaps directly transferred to other lipoproteins.16 17 Thus, postprandial lipoproteins may already contain oxidized lipids that could be readily converted to proinflammatory lipids after exposure to mild oxidative stress. The current studies evaluate the susceptibility of various lipoproteins and lipoprotein subfractions to generate proinflammatory particles when exposed to mild oxidative stress, as measured by their ability to stimulate monocyte chemotaxis and monocyte adherence to endothelial cells.
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Methods |
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Lipoprotein Isolation
VLDL (d
1.006 g/mL), IDL (d=1.006
to 1.019 g/mL), LDL (d=1.022 to 1.063 g/mL), as well as
dense LDL (1.040 to 1.063 g/mL) and buoyant LDL (1.026 to 1.032 g/mL)
were isolated by density gradient ultracentrifugation
from pooled human plasma containing a final concentration of gentamicin
0.22 mmol/L, chloramphenicol 0.15 mmol/L,
D-phenylalanyl-L-prolyl-L-arginine
chloromethyl ketone 1 mmol/L, benzamidine 2 mmol/L, and EDTA
300 mmol/L.18 19 After isolation, the
lipoproteins were dialyzed in PBS containing EDTA for 24 hours, stored
at 4°C in the dark, and used within 1 week. EDTA was removed from the
LDL just before each oxidation experiment by dialysis for 20 hours at
4°C in the dark with 2 6-L changes of PBS. For some experiments, we
isolated LDL and VLDL from pooled plasma from several "normal"
Japanese subjects as well as from Japanese subjects with a deficiency
in plasma platelet-activating factor acetylhydrolase (PAF-AH)
(generously provided by Drs Subbaiah and Miwa). The affected
individuals have a recently characterized missense mutation near the
active site of the PAF-AH.20 Chylomicrons were isolated by
overlaying 6 mL of plasma with 6 mL of saline and ultracentrifuging the
mixture for 20 minutes in a SW 41 rotor at 35 000 rpm. The
chylomicrons were washed by repeating the above process 2 additional
times. A postprandial "remnant"-enriched lipoprotein fraction
(1.006<d<1.021 g/mL) was also prepared by density gradient
ultracentrifugation. For chylomicron and remnant
particle isolation, blood was collected from volunteers 4 hours after a
standard high-fat meal. All lipoprotein fractions were checked for
contamination by albumin or other lipoproteins by
acrylamide gel analysis. Chylomicron preparations
contained predominantly ApoB48-containing particles and postprandial
remnant preparations contained primarily ApoB100-containing particles
and some ApoB48-containing particles. Lipopolysaccharide
content of lipoproteins was measured the gel clot method (Sigma
Chemical) and the ApoB100 content of lipoproteins was determined by
using immunoprecipitation analysis (Incstar Corp).
Lipoprotein Oxidation
The formation of conjugated dienes was measured as previously
described,21 by incubating lipoproteins at 150 µg
ApoB100/mL with 5 µmol/L copper sulfate in 1 mL of PBS at
30°C. The absorbance at 234 nm was measured continuously in a Uvikon
951 spectrophotometer. For presentation of conjugated diene
data, the first derivative of the rapid phase of oxidation was
calculated and its intercept with the x axis (lag time)
determined. In some experiments, the extent of copper-mediated lipid
oxidation was determined by the generation of malondialdehyde
products (thiobarbituric acid–reactive substances [TBARS] assay)
by the method of Yagi22 with fluorescence
intensity measured at 553 nm and excitation at 515 nm.
Fatty Acid Determinations
Lipids from lipoprotein samples were extracted by a modification
of the method of Folch et al.23 The fatty acids were
transmethylated and analyzed in a Varian gas
chromatograph Model 3700, equipped with a column of 10% Silar
5CP on a Gas Chrom QII, 100/120 mesh as described.24
Vitamin E Content
-Tocopherol was measured by HPLC as described
previously.18 -Tocopherol acetate was
prepared in 100% ethanol and used as an extraction internal standard
and for standard curve preparation. Actual concentrations of
-tocopherol were determined by measuring absorbance of
prepared solutions and calculating concentrations based on known
spectral data. Calculations were determined from a standard curve of
peak area ratios of sample/internal standard.
Cell Culture Procedures
Murine fibroblasts expressing high levels of intracellular
15-lipoxygenase (15-LO) were established by infection
with a retroviral vector as previously described.25 26 We
previously demonstrated27 that incubation of LDL on these
15-LO cells, but not on control fibroblasts expressing
ß-galactosidase, generates a modified LDL that meets all the criteria
for mm-LDL4 6 and in particular can stimulate
monocyte chemotaxis and adhesion to endothelial
cells.27 28 Although there is controversy regarding the
mechanisms of in vivo modification of lipoproteins, 15-LO cells are
used in this system not to illustrate the importance of 15-LO in
lipoprotein oxidation, but as a reliable method of generating
mm-LDL, as we previously demonstrated. Cells were grown in Dulbecco's
modified essential medium with high glucose (10 mmol/L),
containing 10% FCS and G418 sulfate (50 mg/mL) at 37°C and in 5%
CO2. Fibroblasts were plated on 96-well plates at
35 000 cells per well and grown for 2 days until nearly confluent. The
cells were washed free of serum, and lipoproteins (LDL, IDL, and VLDL)
at 30 to 150 µg ApoB100/mL were then incubated with the fibroblasts
at 37°C for 20 hours in Ham's F-10 media. Chylomicrons and
postprandial remnant particle fractions were incubated on 15-LO cells
based on equal triglyceride concentrations. TBARS were
determined to assess the extent of lipoprotein oxidation and
bioactivity was assessed as described below.
In additional experiments, lipoprotein fractions were modified on endothelial cell/smooth muscle cell cocultures as described by Navab et al.4 5 Human aortic smooth muscle cells were initially plated on 96-well plates at 35 000 cells per well and grown for 2 days until nearly confluent. Then, human umbilical vein or porcine aortic endothelial cells were plated on the smooth muscle cells and grown until confluent. Lipoproteins were then incubated on the coculture at 37°C for 20 hours in RPMI media in the presence of 5% lipoprotein-deficient serum and bioactivity of the supernatant was then assessed.
Human monocytes were isolated from blood collected in 4 mmol/L EDTA. A monocyte-enriched fraction was isolated by density ultracentrifugation at 22°C, using Histopaque 1077 (Sigma Chemical). The cells were then plated in RPMI 1640 (Biowhittaker)+10% homologous serum for 3 hours at 37°C. Nonadherent cells were washed off and the adherent monocytes were released, using PBS containing 0.18% EDTA, and were then washed twice in PBS.29 Monocytes were then frozen in 10% DMSO, 30% serum, and 60% RPMI media and stored in liquid nitrogen until used.
Monocyte Chemotaxis Assay
Assays were performed in a chemotaxis chamber (Neuro Probe Inc)
with a polycarbonate filter (Poretics) of 5-µm pore size separating
the upper and lower wells. The lower wells were filled with 29 µL of
supernatant (diluted 1:5 in 0.1% BSA/Tyrode's salt solution)
from either fibroblast or coculture incubation experiments and the
chambers were treated as previously described by Navab et
al.5 The monocytes that migrated from the upper chamber to
the lower surface of the filter were then counted by using a light
microscope and expressed as cells per high-power field. The results of
at least 4 to 8 wells were averaged for each experimental
condition.
Monocyte Adhesion Assay
The assay, with minimal modifications, was performed as
described by Navab et al.5 Lipoproteins conditioned in
media alone, by fibroblasts in F-10 media, or by
endothelial/smooth muscle cell cocultures in RPMI for
20 hours were transferred to confluent porcine aortic
endothelial monolayers in 96-well tissue culture plates
and the plates were incubated for 4 hours at 37°C. The supernatants
were then removed and the endothelial monolayers washed
twice with RPMI 1640. THP-1 cells (a monocyte-like cell line) were
placed on the endothelial cells at 45 000 cells per
well, and the plates incubated for 20 minutes at 37°C. The suspension
was removed, and the cells were washed vigorously (at least 3 times) to
remove all but the firmly adherent THP-1 cells. The number of adherent
THP-1 cells was determined in 4 high-power fields per well and the
results of 4 to 8 separate wells were averaged for each experiment. In
some experiments, we separated conditioned LDL from the aqueous
supernatant by passing the incubation mixture through a membrane cone
with a molecular weight cutoff of 25 000 (Amicon). Both the retained
lipoproteins (resuspended in Tyrode's salt solution) and ultrafiltrate
were then tested individually for chemotaxis activity. In additional
studies, we extracted the total lipids with
chloroform/methanol30 from supernatants of experiments
where different lipoprotein fractions had been incubated on 15-LO
cells. The lipid extract was then dried under nitrogen and resuspended
in ethanol to test for chemotactic activity as described above. In some
experiments, PAF-receptor antagonists were added at 10
µmol/L in an ethanol/DMSO solution (<1%) to
endothelial monolayers 30 minutes before conditioned
LDL was added. Specific PAF-receptor antagonists (Biomol)
tested included BN52021 and Lau 203 as previously
described.27
Chemiluminescent Immunoassay for Antibody Binding
Supernatants containing conditioned lipoproteins or native
lipoprotein samples were diluted to 10 µg/mL in PBS buffer
(containing 0.27 mmol/L EDTA) and were plated onto 96-well white
round-bottomed high binding Microfluor (Dynex Technologies, Inc)
microtiter plates overnight at 4°C. The wells were washed 4 times
with PBS buffer and blocked with PBS buffer containing 1% BSA for 30
minutes. Natural monoclonal autoantibodies (E0 autoantibodies)
directed against epitopes of oxidized LDL were cloned from
ApoE-deficient mice as previously described.31 E0
autoantibodies were incubated with the plated lipoproteins for 1 hour
at room temperature. The amount of antibody bound was measured with
alkaline phosphatase–labeled goat anti-mouse-IgM (Sigma) (in TBS
buffer containing 1% BSA), using a chemiluminescent technique
previously described.32 Data are expressed in relative
chemiluminescent light units.
Additional Assays
In some experiments, lipoproteins were incubated with serine
esterase inhibitors aminobenzylsulfonyl fluoride
(ABSF) (Sigma) at 25 to 400 µmol/L or phenylmethysulfonyl
fluoride (PMSF) (Sigma) at 3 mmol/L for 60 to 90 minutes
to irreversibly inhibit PAF-AH. Lipoprotein samples were then dialyzed
free of excess inhibitors and EDTA and then conditioned on
15-LO cells as described above.
PAF-AH activity in lipoproteins was measured by the method of Stafforini et al.33 In brief, diluted tritiated PAF (Du Pont–NEN) was incubated with 4 µg of protein from each lipoprotein fraction for 30 minutes before the reaction was stopped by the addition of a 50-µL mixture of acetic acid and sodium acetate. The cleaved tritiated acetate product was separated from the intact PAF substrate by reverse-phase octadecyl silica gel column chromatography. The PAF-AH activity is expressed as nanomoles of PAF hydrolyzed per hour per milligram of protein or per milligram of ApoB100.
The presence of PAF-AH protein in various lipoprotein classes was
demonstrated by western blot analysis. After lipid extraction,
10 µg of protein was resuspended in Tris-glycine reducing buffer,
heated at 70°C for 10 minutes, and run on 4% to 12% Tris-glycine
gradient gels (Novex) in 25 mmol/L Trizma base, 192 mmol/L
glycine, and 1% SDS for 2 hours at 125 V. Samples were transferred to
nitrocellulose paper in Novex transfer units over 16 hours at 4°C at
50 to 100 V in a transfer buffer containing 25 mmol/L Tris,
192 mmol/L glycine, and 20% methanol. Nonspecific binding sites
were blocked with Superblock (Pierce) and 0.1% human serum
albumin and incubated overnight with a 1:500 dilution of rabbit
anti-human LDL PAF-AH34 (a generous gift of Dr Colin
Macphee). After extensive washing, an alkaline phosphatase–conjugated
goat anti-rabbit IgG (diluted 1:10 000) was added and the signal
detected by the addition of alkaline phosphate substrate.
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Composition of Lipoprotein Particles
The compositions of isolated LDL, IDL, and VLDL particles are shown in Table 1
. Data are shown
normalized to mg of ApoB100 unless stated otherwise so that comparisons
between lipoprotein classes may be made per lipoprotein particle.
Although the vitamin E content per particle was significantly greater
in VLDL, the vitamin E content per PUFA was not different between each
lipoprotein. Although PAF-AH activity when expressed per total protein
was greatest in LDL, the level of enzyme activity per ApoB100 protein,
ie, per particle, was similar in LDL, IDL, and VLDL. The presence of
PAF-AH protein in each ApoB-containing lipoprotein was confirmed by
western blot analysis, using a polyclonal antibody to PAF-AH
(Figure 1). The PAF-AH protein band in
HDL was only faintly visible, consistent with the 50- to
100-fold lower specific activity of PAF-AH measured in this
lipoprotein. In contrast, the fatty acid composition between
lipoproteins did vary significantly. The content of 18:1 (as a
percentage of the total fatty acid composition) increased and the
content of 18:2 and 20:4 decreased progressively from LDL to IDL to
VLDL (Table 2). We also isolated buoyant
and dense LDL subfractions and their characteristic features are shown
in Table 3. Dense LDL fractions contained
less lipid and vitamin E per molecule and were smaller, as previously
reported.18
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Susceptibility of Lipoproteins to Copper-Mediated
Oxidation
Susceptibility of LDL, IDL, and VLDL to copper-mediated oxidation
was measured by several different methods. Conjugated diene formation
revealed that based on equal amounts of ApoB100, ie, equal numbers of
particles, LDL consistently oxidized more rapidly than did the
other 2 lipoproteins (Figure 2A). The lag
time for VLDL oxidation was significantly longer than that of other
lipoproteins. The peak level of conjugated diene formation was higher
for both IDL and VLDL compared with LDL, consistent with their
greater lipid content. As shown in Figure 2B, there was also a
similar order of susceptibility to copper-mediated oxidation among the
different lipoprotein fractions as measured by the formation of TBARS.
LDL was first to undergo oxidation, followed by IDL and then VLDL.
Similar patterns of oxidation were seen when oxidation was mediated by
2,2-azobis (2-amidinopropane) dihydrochloride rather than by copper
(data not shown). As previously reported,10 18 dense LDL
particles were more readily oxidized when exposed to copper and had
significantly reduced lag times compared with buoyant LDL (167.6±99.3
versus 252.1±70.8 minutes).
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), IDL (•), and VLDL (
). Shown are
representative experiments.
Bioactivity Generated From Lipoproteins Conditioned on 15-LO
Cells
We previously showed that incubation of LDL with
15-LO–overexpressing fibroblasts mildly oxidizes LDL25 26
and converts it to a mm-LDL that induced greater monocyte
chemotaxis and adhesion.27 To evaluate whether mild
oxidative stress would have similar effects on other lipoproteins, we
conditioned LDL, IDL, and VLDL on 15-LO cells and then compared the
supernatants from these incubations for their chemotactic activity and
induction of monocyte adhesion to endothelial cells.
Figure 3A shows that conditioning LDL and
IDL on 15-LO cells significantly increased their ability to stimulate
monocyte chemotaxis. In contrast, conditioning VLDL on 15-LO cells only
modestly (and not significantly) increased its stimulation of monocyte
chemotaxis. In several additional experiments, HDL was also conditioned
on 15-LO cells, and like VLDL, demonstrated little or no bioactivity
compared with lipoproteins incubated in media alone. Incubation of LDL
and IDL conditioned on 15-LO cells with endothelial
cells markedly increased the subsequent adherence of THP-1 cells to
endothelial cells, whereas conditioned VLDL did not do
so. Lipopolysaccharide was measured in each lipoprotein class
and was consistently below our detection level of 0.5 ng/mL, a
value that does not stimulate monocyte chemotaxis or adhesion in our
assays.
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Recent investigations have highlighted the increased risk for
cardiovascular disease associated with small dense
LDL.35 36 Several mechanisms for this relation have been
proposed, including enhanced susceptibility to oxidation of small dense
LDL.10 18 We therefore also undertook studies to determine
whether small dense LDL subfractions were more readily converted to a
bioactive mm-LDL than were larger buoyant LDL. As shown in Figure 4, after conditioning on 15-LO cells,
dense LDL stimulated greater chemotaxis and monocyte adhesion than did
buoyant LDL.
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Relation Between Lipoprotein Oxidation and Bioactivity
The large differences in bioactivity of LDL and IDL compared with
VLDL after incubation on 15-LO cells could not readily be explained by
differences in oxidation, at least as measured by nonspecific measures
of lipid peroxidation, such as TBARS. Although 15-LO–conditioned IDL
was as bioactive as LDL, its level of TBARS/ApoB100 after conditioning
on 15-LO cells was significantly lower than that of LDL and comparable
with that of VLDL (LDL, 34±10.7 nmol/mg ApoB; IDL, 10.7±8.7 nmol/mg
ApoB; and VLDL, 15.4±10.7 nmol/mg ApoB). This is consistent
with several previous reports where measures of TBARS have not
corresponded to the development of LDL bioactivity after incubation
with 15-LO–overexpressing cells.27 37 One interpretation
of these data is that oxidation is not directly related to the
development of bioactivity. However, inhibition of oxidation by the
addition of EDTA to the incubation mixture or by using
probucol-enriched lipoproteins consistently reduced generation
of lipoprotein-induced bioactivity (data not shown). It is known that
reducing the concentration of LDL exposed to a given concentration of
copper will result in greater oxidation. Therefore, we substantially
reduced the concentration of VLDL incubated with the 15-LO cells from
150 to 30 µg ApoB/mL to promote greater oxidation. This led to an
increase in both TBARS formation and greater stimulation of monocyte
chemotaxis and adhesion (data not shown), suggesting that the decreased
susceptibility of VLDL to oxidation was in part responsible for its
diminished bioactivity (on a per particle basis). A second explanation
of the apparent disassociation between lipid oxidation and bioactivity
is that bioactivity may result from the generation of unique oxidation
products not directly reflected in the TBARS assay. To assess this
possibility, we measured the level of binding to 15-LO–modified
lipoproteins of naturally occurring autoantibodies that have been
cloned from ApoE-deficient mice.31 These antibodies bind
to oxidized phospholipids present in oxidized LDL.8 32
As demonstrated in Figure 5 (inset),
E06 antibody binding to each 15-LO–modified lipoprotein
fraction increased dramatically compared with "native"
lipoproteins. Similar results occurred when several other E0
antibodies that are directed toward LDL phospholipid oxidation epitopes
were used (data not shown). The formation of oxidized phospholipid
epitopes appeared to reflect the inherent susceptibility of these
lipoproteins to copper-mediated oxidation, as well as their
bioactivity. This is demonstrated in Figure 5, which shows the
results of experiments in which LDL, at decreasing concentrations of
ApoB, was incubated with 15-LO fibroblasts. As the LDL concentration
was decreased (presumably enhancing oxidation), there was enhanced
binding of E06 (Figure 5A) that was paralleled by an
increased ability to stimulate monocyte chemotaxis (corrected for
ApoB100 concentration) (Figure 5B). These experiments
demonstrate that generation of oxidized phospholipids occurs in
parallel to the generation of bioactive products and supports the
notion that bioactivity results in part from oxidation of lipoprotein
phospholipids.
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Substantial Bioactivity Resides in the Lipid Fractions of
Lipoproteins Conditioned on 15-LO Cells
Lipid extracts of cell-conditioned LDL and IDL were prepared
and when tested for stimulation of chemotaxis, this fraction contained
>70% of the original bioactivity. When experimental conditions were
modified to enhance cell-mediated oxidation of VLDL (as described
above), this lipid extract was also bioactive (data not shown). In a
similar manner, separating lipoproteins after their incubations on
15-LO cells from the rest of the supernatant by ultrafiltration through
a membrane with a mw cutoff of 25 000 revealed that the bioactivity
was predominantly associated with the lipoprotein particles and not the
aqueous supernatant (data not shown). We and other investigators have
recently shown that generation of bioactivity appears, in part, to
result from the oxidative degradation of lipoprotein
phospholipids.3 7 8 27 At least a portion of these
bioactive substances resemble PAF in structure, and appear to
activate cell adhesion through the PAF receptor. To determine
whether the bioactivity of each 15-LO–conditioned lipoprotein was
related to the generation of PAF-like particles, we added structurally
different PAF-receptor antagonists to the
endothelial cells before and during their incubation
with the lipoproteins. As shown in Figure 6, the PAF-receptor
antagonist Lau 203 completely blocked the ability of
conditioned LDL and IDL to stimulate monocyte adherence. In a similar
manner, it appears that even the modest bioactivity of conditioned VLDL
was also inhibited by the PAF-receptor antagonist. Similar
results occurred with the PAF-receptor antagonist BN52021.
The vehicle for the PAF-receptor antagonists had no effect
on monocyte adhesion nor was the PAF-receptor antagonist
Lau 203 able to reduce the stimulation of monocyte adhesion
induced by addition of tumor necrosis factor or
lipopolysaccharide (data not shown).
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Effect of PAF-AH on Bioactivity of Lipoproteins Conditioned on
15-LO Cells
Lipoproteins contain several different enzymes that may have the
ability to degrade bioactive oxidized phospholipids.3 One
such enzyme that is particularly prominent in ApoB100-containing
lipoproteins is PAF-AH. It has been reported that the majority of
PAF-AH in plasma resides in LDL.38 39 As shown in Figure 1
, we demonstrated the presence of PAF-AH protein in IDL and
VLDL in addition to LDL. To compare the role of PAF-AH activity in
degrading bioactive phospholipids in different ApoB100-containing
lipoproteins, we pretreated LDL, IDL, and VLDL with serine esterase
inhibitors, which irreversibly inhibit PAF-AH, conditioned
them on 15-LO cells, and then repeated the assays of bioactivity. Each
lipoprotein fraction treated with PMSF or ABSF had
lipoprotein-associated PAF-AH activity that was reduced to <20% of
their basal activity. In comparison, lipoproteins conditioned on 15-LO
cells overnight only lose 10% to 15% of their PAF-AH activity. All of
the PAF-AH–depleted lipoproteins, including VLDL, showed enhanced
bioactivity compared with the PAF-AH–replete lipoproteins (Figure 7). In separate experiments, E06
antibody binding to PAF-AH–depleted lipoproteins after their
modification by 15-LO cells was increased to a greater extent than was
binding to 15-LO–conditioned PAF-AH–replete lipoproteins (data not
shown). These data support that lipoproteins depleted of PAF-AH
generate greater amounts of bioactive oxidized phopholipids when mildly
oxidized. Reducing PAF-AH activity in native lipoproteins or those
conditioned in media alone did not enhance their bioactivity (data not
shown).
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Enzyme inhibitors may have other unknown effects on cells
or lipoproteins. Therefore, we compared LDL isolated from plasma from
Japanese subjects who have been identified as having an
"inactivating" mutation in the gene for PAF-AH to LDL from Japanese
control subjects.20 40 The lipid (cholesterol,
triglyceride, and phospholipid) and fatty acid composition
of the lipoproteins as well as the plasma vitamin E levels were not
different in the 2 groups (data not shown). The LDL from individuals
homozygous for this mutation contained no PAF-AH activity, whereas
activity was present in LDL from the control subjects (720
nmol PAF/h/mg of protein). When conditioned on 15-LO cells, LDL
from plasma PAF-AH–deficient subjects stimulated greater monocyte
adhesion than did LDL samples from the control subjects (Figure 8). Treatment of Japanese control LDL
with ABSF completely inactivated PAF-AH, and after
conditioning on 15-LO cells these LDL samples demonstrated bioactivity
equal to PAF-AH–deficient samples. A similar increase in bioactivity
resulted when VLDL, isolated from these same PAF-AH–deficient
patients, was conditioned on 15-LO cells (data not shown). These data
illustrate the potentially important antiinflammatory role of PAF-AH in
all ApoB100-containing lipoproteins.
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Bioactivity of Postprandial Lipoproteins Conditioned on 15-LO Cells
and Smooth Muscle/Endothelial Cell Cocultures
Recent studies have suggested postprandial
hyperlipoproteinemia, particularly elevations
in remnant particles, may contribute to lesion
formation.41 42 43 We therefore evaluated chylomicrons and a
remnant-enriched fraction that were isolated 4 hours after a
high-fat meal. As shown in Table 2, postprandial chylomicrons
were markedly enriched in 18:1 and relatively low in 18:2 and 20:4.
However, the vitamin E/PUFA ratio was very low in this lipoprotein
fraction (3.9±1.9) compared with all other lipoproteins, and PAF-AH
activity was essentially absent. In contrast, the postprandial remnant
particles, predominantly comprised of hepatic derived lipoproteins,
were less enriched with 18:1 but contained significant amounts of
PAF-AH (784±441 nmol PAF degraded · h-1 ·
mg-1 of ApoB100) and vitamin E (13 µg/mg of PUFA).
Because the content of protein in these postprandial lipoprotein
fractions is reduced, and they contain many different apoproteins, it
was difficult to analyze these fractions on a per-particle
basis. Therefore, we selected the quantity of each fraction to incubate
with 15-LO cells based on triglyceride content, and used an
amount that was approximately equal to that previously used for
incubations of VLDL. After exposure to 15-LO cells, chylomicrons
(Figure 9A) and the postprandial
remnant-rich fraction (Figure 9B) both stimulated monocyte
chemotaxis compared with unconditioned samples. It is noteworthy that
when chylomicrons were incubated on the
endothelial/smooth muscle cell coculture, they failed
to stimulate monocyte chemotaxis or adhesion. In contrast, the smaller
lipoproteins in the postprandial remnant-rich fraction were bioactive
when conditioned on either 15-LO cells or on the coculture. Because
lipoproteins must pass into the subendothelial space to
become oxidized in the coculture system, these results suggest that
only the smaller remnant particles, and not the chylomicrons, were able
to pass through the endothelial cell layer of the
coculture during the time frame of these experiments.
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Discussion |
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Evidence now supports the concept that the development of atherosclerosis is in many ways an inflammatory process that is initiated or exacerbated by hyperlipidemia. An early step in this process is the recruitment and adhesion of monocytes to sites of inflammation. Our findings demonstrate that all ApoB100-containing lipoproteins are susceptible to mild oxidation and acquire proinflammatory properties. These results were not dependent on the cell system used to induce mild oxidation, as similar bioactivity was generated when lipoproteins were incubated on smooth muscle/endothelial cell cocultures (data not shown). When compared on a per-particle basis, mm-IDL and mm-LDL are relatively equal in their ability to stimulate monocyte chemotaxis and adhesion. With exposure to greater oxidative stress, VLDL can also acquire proinflammatory activity. Exposure of postprandial lipoproteins to mild oxidative stress also induces their transformation to bioactive particles. Although these experiments only demonstrated that minimally oxidized IDL, VLDL, and postprandial chylomicrons and remnant particles can increase monocyte chemotaxis and adhesion, it is likely that they will also have other proinflammatory properties associated with mm-LDL.3 44
Numerous studies have demonstrated that individuals at increased risk for atherosclerosis, such as individuals with diabetes, familial combined hyperlipidemia, or type III hyperlipidemia, have increased levels of non-LDL ApoB-containing lipoproteins.15 41 45 46 As a result, it has been suggested that remnant lipoproteins, in particular, may be proatherogenic, and in vitro studies have suggested several possible mechanisms by which particles such as IDL or PAF-AH depleted-VLDL may accelerate lesion formation.13 Moreover, animal studies have demonstrated that elevations in remnant particles can induce atherosclerosis.43 47 This has also been supported by studies of atherosclerosis progression in humans.42 48 49 50 Finally, several recent studies have demonstrated the presence of non-LDL lipids and lipoproteins in lesions in the artery wall,11 12 consistent with the concept that lipoproteins larger than LDL, such as IDL and VLDL, may contribute to lesion formation.
Comparisons between lipoprotein fractions revealed several features in common. Measures of bioactivity generally reflected the susceptibility of these lipoproteins to copper-mediated oxidation. For example, compared with IDL and LDL, VLDL was more resistant to transition metal–mediated oxidation and less readily developed bioactivity. When conditions were changed to promote greater oxidation of VLDL, it too developed bioactivity. This provides some support for the relevance of in vitro measurements of copper-mediated lipoprotein oxidation.
Several lines of evidence suggest that at least a major portion of mm-lipoprotein bioactivity results from the generation of oxidized phospholipids, and this seemed true for each class of lipoproteins. In all lipoproteins tested, the majority of the bioactivity resided in the lipid fraction. In addition, during conditioning on 15-LO cells, all ApoB100-containing lipoproteins developed oxidation epitopes that were detected by endogenously produced murine autoantibodies that have been demonstrated to specifically bind oxidized phospholipids.8 32 By using this extremely sensitive and specific immunological assay of "oxidation-specific" epitopes, we were able to document the parallel relation between mild phospholipid oxidation and bioactivity. In addition, as described below, decreases in lipoprotein-associated PAF-AH, an enzyme that appears to degrade biologically active products of phospholipid oxidation, enhances lipoprotein proinflammatory activity. It is noteworthy that the stimulation of monocyte adhesion by each class of lipoprotein could be blocked by PAF-receptor antagonists. These data suggest that the source of bioactivity of each class of lipoprotein is in part related to the development of PAF or PAF-like lipid products. This is consistent with the findings of previous investigators who have demonstrated that bioactivity of mm-LDL appears related to specific products of oxidized phospholipids.3 8 Although our findings are consistent with the concept that PAF or PAF-like lipids increased monocyte chemotaxis through their interaction with the monocyte PAF receptor, our data do not exclude other alternatives.
The susceptibility of ApoB100-containing lipoproteins to undergo oxidation and develop bioactivity emphasizes the potentially important role of PAF-AH in these fractions. In each lipoprotein class, the loss of PAF-AH seemed to increase the level of bioactivity that developed when conditioned on cells. This was perhaps most evident in VLDL, where the decrease of PAF-AH activity by serine esterate inhibitors facilitated its transformation from a mild to a relatively potent inflammatory particle. However, such inhibitors may have nonspecific effects on cells and/or lipoproteins. Indeed, there is evidence that such inhibition of PAF-AH in some studies may reduce bioactivity of oxidized LDL.34
To address these possibilities, we evaluated lipoproteins obtained from Japanese subjects with known mutations in their gene for the plasma form of PAF-AH. These individuals have plasma and lipoproteins that contain no measurable PAF-AH activity. Minimally oxidized LDL containing the inactive PAF-AH consistently induced greater monocyte adhesion. Similar results occurred with VLDL samples isolated from these PAF-AH–deficient subjects. These data support the concept that PAF-AH degrades bioactive oxidized phospholipids within oxidized lipoproteins. However, the exact role and in vivo relevance of plasma PAF-AH awaits studies of PAF-AH depletion and overexpression in animals as well as careful, long-term follow-up of patients with deficiencies of plasma PAF-AH.
Although there were many similarities between lipoprotein fractions in their response to mild oxidative stress, there were also some unique differences. For example, VLDL was in general less easily transformed to a bioactive particle. This may in part have resulted from its increased content of 18:1. We previously demonstrated that lipoproteins24 28 51 and liposomes9 enriched in 18:1 were less easily oxidized and less readily generated chemotactic activity.9 28 Although chylomicrons were also relatively enriched in 18:1, they contained reduced levels of vitamin E and no measurable PAF-AH activity. The absence of these 2 latter components may have rendered them more susceptible to oxidation, thus permitting chylomicrons to more readily develop bioactivity (compared with VLDL) when mildly oxidized.
We also now report that when conditioned on 15-LO fibroblasts, dense LDL developed more bioactivity compared with buoyant LDL. We, and others, have previously reported that dense LDL has greater susceptibility to copper-mediated oxidation compared with buoyant LDL.10 18 These present observations support the hypothesis that dense LDL is more atherogenic than buoyant LDL.35 36 52 In contrast, chylomicron size did appear to influence its susceptibility to oxidation in the coculture system. Whereas this fraction was readily modified by the 15-LO fibroblasts, this was not the case when it was added to the coculture system. Because the coculture uses lipoprotein-deficient serum, which contains sufficient antioxidants to inhibit oxidation in most cases, it has been presumed that lipoprotein modification within the coculture systems requires entry into the proteoglycan-rich matrix between the 2 cell layers.4 5 In this microenvironment, relatively "free" from antioxidants, lipoprotein oxidation can occur. Presumably, chylomicrons are too large to enter the subendothelial space and therefore cannot become minimally modified. This finding is consistent with the reports by Nordestgaard and Zilversmit53 and Van Heek and Zilversmit54 who demonstrated in animal models that elevations in chylomicrons do not enhance atherosclerosis in rabbits.
This study demonstrates that all ApoB-containing lipoproteins acquire proinflammatory activity when minimally oxidized. Factors that can modulate the degree of bioactivity that develops include lipoprotein fatty acid composition, PAF-AH content, and lipoprotein size. These data also support the concept that triglyceride-rich lipoproteins may also be proatherogenic.
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Acknowledgments |
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This work was supported by grants from the National Heart, Lung, and Blood Institute, National Institutes of Health (SCOR-HL-56989), the Paul Beeson Physician Faculty Scholars in Aging Research Award (PDR), and the VA/JDF Diabetes Center Award. We thank Joellen Barnett, Elizabeth Miller, and Richard Elam for excellent technical assistance in the conduct of these studies.
Received July 21, 1998; accepted December 2, 1998.
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