© 2000 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
Determinants of Bioactivity of Oxidized Phospholipids
Specific Oxidized Fatty Acyl Groups at the sn-2 Position
From the Departments of Medicine (G.S., N.L., J.W.W., H.L., C.R., A.D.W., A.M.F., J.A.B.) and Pathology (J.A.B.) and the Pasarow Mass Spectrometry Laboratory (K.F.F.) and Department of Psychiatry and Biobehavioral Sciences and Neuropsychiatric Institute, University of California, Los Angeles, and the Department of Pathology (D.C.S.), Wake Forest University School of Medicine, Winston-Salem, NC. Norbert Leitinger is currently at the Department of Vascular Biology and Thrombosis Research, University of Vienna, Vienna, Austria.
Correspondence to Ganesamoorthy Subbanagounder, PhD, Department of Medicine/Cardiology, 47-123, Center for the Health Sciences, UCLA Medical Center, Los Angeles, CA 90095-1679. E-mail gsubbanagounder{at}mednet.ucla.edu
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Abstract |
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Abstract—We previously described 3 bioactive oxidation products of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (PAPC) containing oxovaleroyl (POVPC), glutaroyl (PGPC), and epoxyisoprostane (PEIPC) groups at the sn-2 position that were increased in minimally modified/oxidized low density lipoprotein (MM-LDL) and rabbit atherosclerotic lesions. We demonstrated specific and contrasting effects of POVPC and PGPC on leukocyte-endothelial interactions and described an effect of PEIPC on monocyte binding. The major purpose of the present study was to determine the effects of structural changes on the bioactivities of these 3 lipids. We demonstrate herein that the group at the sn-2 position determines the specific bioactivity and that the substitution of stearoyl for palmitoyl at the sn-1 position or ethanolamine for choline at the sn-3 position of the phospholipid did not alter bioactivity. Oxidized PAPC, oxidized 1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine, and oxidized 1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphorylethanolamine stimulated monocyte binding and inhibited lipopolysaccharide-induced expression of the neutrophil-binding molecule E-selectin. Furthermore, all oxovaleroyl phospholipids but not the glutaroyl phospholipids induced monocyte binding without an increase in vascular cell adhesion molecule-1 (VCAM-1) expression and inhibited lipopolysaccharide-induced E-selectin expression. In contrast, glutaroyl phospholipids but not oxovaleroyl phospholipids stimulated E-selectin and VCAM-1 expression. We further demonstrate that all parts of the phospholipid molecules are required for these bioactivities. Hydrolysis with phospholipase (PL) A1, PLA2, and PLC strongly reduced the bioactivities of POVPC, PGPC, and mixed isomers of PEIPC. PLD had a smaller but still significant effect. The effects of POVPC and PEIPC could be abolished by sodium borohydride treatment, indicating the importance of the reducible groups (carbonyl and epoxide) in these molecules. In summary, these studies identify 6 new bioactive, oxidized phospholipids that are increased in MM-LDL and, where measured, in atherosclerotic lesions. They thus suggest that a family of phospholipid oxidation products containing oxovaleroyl, glutaroyl, and epoxyisoprostane at the sn-2 position play an important role in the regulation of leukocyte-endothelial interactions, bioactivity being in part controlled by several types of phospholipid hydrolases.
Key Words: atherosclerosis • oxidized phospholipids • monocyte-endothelial interactions • E-selectin • phospholipases
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Introduction |
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Minimally oxidized LDL (MM-LDL), in contrast to native LDL, has been shown to stimulate endothelial cells to increase monocyte-endothelial interactions.1 Monocytes are the major inflammatory cells in the atherosclerotic lesion and have been shown to be important mediators of lesion initiation and progression.2 3 An important characteristic of atherosclerotic lesions and other chronic, inflammatory lesions is that they contain essentially no neutrophils.4 Our group has shown that the major bioactive lipids in MM-LDL were derived from oxidation of arachidonoyl phospholipids.5 6 We have identified 3 bioactive, oxidized phospholipids present in oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (PAPC) as 1-palmitoyl-2-oxovaleroyl-sn-glycero-3-phosphorylcholine (POVPC), 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphorylcholine (PGPC), and 1-palmitoyl-2-(5,6-epoxyisoprostane E2)-sn-glycero-3-phosphorylcholine (PEIPC).7 8 The levels of these lipids were increased in atherosclerotic lesions in rabbit aortas, and natural antibodies to these oxidized phospholipids were present in the sera of apo E–null mice.8 9
We have observed several differences in action between POVPC and PGPC. POVPC induced monocyte binding and connecting segment-1 fibronectin expression, increased the levels of cAMP, and inhibited lipopolysaccharide (LPS)-induced E-selectin expression and neutrophil binding. The action of POVPC was inhibited by the platelet-activating factor receptor antagonist WEB 2086.10 11 In contrast, PGPC induced both monocyte and neutrophil binding and vascular cell adhesion molecule-1 (VCAM-1) and E-selectin expression, had no effect on connecting segment-1 or cAMP, and was not inhibited by WEB 2086.10 11 We further demonstrated that the effects of POVPC dominated in mixtures of the active compounds.
This report examines the molecular requirements for the specific actions of POVPC, PGPC, and PEIPC. We also identify new bioactive phospholipids with oxovaleroyl, glutaroyl, and epoxyisoprostane groups, demonstrating their role in the regulation of endothelial-leukocyte interactions. We determined that the levels of these lipids are increased in MM-LDL and atherosclerotic lesions.
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Methods |
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Materials
Dimyristoyl-sn-glycero-3-phosphorylcholine (DMPC), dimyristoyl-sn-glycero-3-phosphorylethanolamine (DMPE), BHT, PMSF, soybean lipoxygenase, phospholipase (PL) A2 (from Naja naja venom), PLC (type IX, from Bacillus cereus), and PLD (type I, from cabbage) were obtained from Sigma Chemical Co. PLA1 (lipase from Rhizopus arrhizus) was obtained from Boehringer Mannheim. PAPC, 1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (SAPC), and heart bovine PE fraction were obtained from Avanti Polar Lipids. Glutaric acid and NaBH4 were obtained from Aldrich Chemical Co.
Lipid and Lipoprotein Modification
LDL (1.019 to 1.069 g/mL) was isolated from the sera of
normal blood donors by density gradient
ultracentrifugation as described
previously13 and stored at 4°C until use within 1 to 4
weeks of isolation. To prepare MM-LDL, LDL was pretreated with PMSF
(3 mmol/L) and incubated at 1 milligram protein per milliliter in
PBS at 37°C with Sepharose-bound soybean lipoxygenase
(20 000 U) by using a previously described method.6 PMSF
did not affect the levels of native and oxidized phospholipids measured
in native LDL (data not shown). To isolate SAPE, bovine heart PE
fraction was treated with 0.5N HCl for 15 minutes, extracted with
chloroform/methanol (2:1, vol/vol), and then dried under
argon.12 The residue was resuspended in chloroform and
subjected to normal-phase coupled high-performance liquid
chromatography–mass spectrometry (HPLC/MS, described
below) to provide enriched
1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphorylethanolamine
(SAPE; charge-to-mass ratio [m/z] 768.5). PAPC, SAPC, and
SAPE were auto-oxidized as described previously.8
POVPC, PGPC, SOVPC, and SGPC were prepared by ozonolysis of PAPC and
SAPC by using the previously reported method.10 14 PEIPC
was isolated from oxidized PAPC, and epoxyisoprostane (the oxidized
fatty acid present in PEIPC) was generated by
PLA2 hydrolysis of PEIPC as described
previously.7 The concentration of bacterial endotoxin in
each treatment solution was <20 pg/mL.
Analysis of Phospholipids
Normal-phase HPLC/MS analysis for oxidized PAPC and
oxidized SAPC was performed as described previously,8 and
oxidized SAPE was fractionated with acetonitrile/methanol/water
(88:4:8, vol/vol/vol) containing 1 mmol/L ammonium acetate. PE
derivatives were less stable than the corresponding PC derivatives and
thus, were used for testing immediately after isolation and
quantification. An API III triple-quadrupole biomolecular mass
analyzer (Perkin-Elmer Sciex Instruments) was used for mass
analysis and quantification of PC derivatives as described
previously.8 For flow-injection analysis in the
negative mode, phospholipids were dissolved in 100 µL of
methanol/water (50:50, vol/vol) containing 10 mmol/L ammonium
acetate and analyzed in the same solvent. For quantitative
analysis, DMPC at 1 µg/10 mg tissue, 10 µg/mg lipoprotein
was used as an internal standard for PC derivatives, and DMPE at 1
µg/mg lipoprotein was used as an internal standard for PE
derivatives.15 To validate the use of DMPC as an internal
standard for quantitative analyses, we correlated the levels of
various phospholipids (PAPC, oxidized PAPC, POVPC, PGPC, and mixed
isomers of PEIPC) determined by electrospray ionization–mass
spectometry analysis, and their levels determined by
phosphorous assay.16 The values obtained by the 2 methods
differed by <5% for all chemical species except PEIPC, which differed
by <10%, thus demonstrating reliable quantification of these lipids
with DMPC. Similarly, we used DMPE as an internal standard for the
analysis of PE derivatives.
PL Hydrolysis and NaBH4 Reduction of Oxidized
Phospholipids
Oxidized phospholipids were hydrolyzed by various PLs by using a
previously reported method with some modifications.7 17 To
standardize the hydrolysis conditions, PAPC, oxidized PAPC, and PGPC
were treated with different amounts of enzymes (1, 5, and 10 U/mL, with
units defined by the supplier for the standard substrates) for
different times (30 minutes and 1, 6, and 18 hours). On the basis of
these studies, oxidized phospholipids were treated with 10 U/mL of PLs
(PLA1, PLA2, PLC, or PLD)
in PBS containing 5 mmol/L CaCl2 at pH 5.6,
7.0, 7.3, and 5.6, respectively). The samples were incubated at 37°C
for 1 hour for PLA2 or overnight for
PLA1, PLC, and PLD7 17 to achieve
70% to 100% hydrolysis. For NaBH4 reduction,
oxidized phospholipids were treated with NaBH4
(50 mmol/L) in PBS or acetonitrile for 30 minutes7 8
to achieve 90% reduction. Phospholipid hydrolysis and
NaBH4 reduction were monitored by the
disappearance of starting phospholipids and/or the formation of
hydrolysis or reduced products.
Cell Culture, Monocyte Adhesion Assay, and E-Selectin
ELISA
Monocyte binding assays were performed as described
previously1 with human aortic endothelial
cells (HAECs) and human monocytes, except that the cells were treated
overnight with 0.8 mg/mL lipoprotein-deficient serum and binding was
performed in lipoprotein-deficient serum to reduce basal HAEC
activation. The cells were then rinsed twice with medium, and binding
of human monocytes to HAECs was performed essentially as described
previously.1 VCAM-1 and E-selectin expression was measured
on HAECs in 96-well dishes as described previously with the use of 10%
fetal bovine serum during the incubation with oxidized
phospholipids.10
Preparation of Lipids From Rabbit Aortas and Lipoproteins
The rabbits used in this study were a subset of animals used in
another study reported elsewhere.18 Some rabbits (n=4)
were studied while they consumed a standard chow diet, whereas other
rabbits (n=6) were studied after 22 weeks on a semipurified,
atherogenic diet with casein, sucrose, and butter as sources of
protein, carbohydrate, and fat, respectively.18 At the end
of treatment with the atherogenic diet, the rabbits were exsanguinated
after ketamine hydrochloride anesthesia (6
mg/kg body weight). Aortas were removed, cleaned of adventitial tissue
(at 4°C), weighed, frozen at -70°C under argon, and stored
protected from light.18 Total lipids and phospholipids
were isolated from LDL, MM-LDL, and rabbit aortas as described
previously.8
Statistical Analysis
Data were analyzed by 1-way ANOVA, probability values
were calculated by using StatView (Abacus Concepts, Inc), and
P<0.05 was considered statistically significant.
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Results |
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Comparison of Oxidation Products in Oxidized PAPC, Oxidized SAPC, and Oxidized SAPE
We first tested the hypothesis that oxidation of SAPC and SAPE would produce oxidation products similar to those in oxidized PAPC. Auto-oxidation of PAPC generated a complex mixture of products, including 3 bioactive, oxidized phospholipids, POVPC (m/z 594.3), PGPC (m/z 610.2), and mixed isomers of PEIPC (m/z 828.5).7 8 Here, we report that auto-oxidation of SAPC and SAPE also resulted in a similar addition of molecular oxygen or fragmentation of the arachidonate moiety at the sn-2 position of the phospholipid (online Figures I through III; please see www... ). These results suggest that the changes at the sn-1 and sn-3 groups did not affect the oxidation patterns of arachidonoyl phospholipids. Although the m/z values of the derivatives in oxidized SAPC and oxidized SAPE suggested that they represented oxovaleroyl, glutaroyl, and epoxyisoprostane phospholipids, we confirmed this identification by using tandem mass spectrometry. Tandem mass spectrometry analyses of phospholipids generated characteristic fragment ions corresponding to the sn-1, sn-2 fatty acid groups, and an ion of lysophospholipid.8 For example, the ion corresponding to 1-stearoyl-2-oxovaleroyl-sn-glycero-3-phosphorylcholine (SOVPC, MH+ ion with m/z 622.3) in oxidized SAPC (online Figure
II; please
see www... ) produced an
(M-CH3)- ion at
m/z 606.3 in the negative mode, which gave characteristic
fragments at m/z 115 (oxovalerate), 283.1 (stearate), and
508.2 [(M-CH3)- ion of
lysophospholipid]. This result confirmed that the ion at
m/z 622.3 in oxidized SAPC was SOVPC. Similar
analyses for ions corresponding to bioactive, oxidized
phospholipids in oxidized SAPC and oxidized SAPE gave the
characteristic sn-2 fragments at m/z 115
(oxovalerate), 131 (glutarate), or 349 (epoxyisoprostane) along with
ions for sn-1 fatty acids and lysophospholipids. These
analyses confirmed the structure of molecules in oxidized SAPC
as SOVPC (m/z 622.3), SGPC (m/z 638.2), and mixed
isomers of SEIPC (m/z 856.6) and in oxidized SAPE as SOVPE
(m/z 580.2), SGPE (m/z 596.2), and mixed isomers
of SEIPE (m/z 814.6).
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Effect of Oxidized Phospholipids on Monocyte Binding
Treatment of endothelial cells with oxidized
arachidonoyl phospholipids (PAPC, SAPC, and SAPE at 5 to 50 µg/mL)
for 4 hours at 37°C induced binding of monocytes in a dose-dependent
fashion. The corresponding native arachidonoyl phospholipids (50
µg/mL) were inactive (Figure 1
). We
fractionated oxidized SAPC and oxidized SAPE by normal-phase HPLC/MS
and tested the fractions for their ability to stimulate
endothelial cells to bind monocytes. The fractions
containing the molecules with m/z values corresponding to
oxovaleroyl, glutaroyl, and epoxyisoprostane derivatives (determined by
reconstructed selective-ion chromatograms) stimulated
endothelial cells to bind monocytes. The bioactivity of
oxidized phospholipids present in oxidized SAPC and oxidized SAPE
was further confirmed by dose-response studies with known amounts of
isolated and synthetic molecules. SOVPC, SGPC, and mixed isomers of
SEIPC (m/z 856.6) were isolated from oxidized SAPC, and
fractions that were enriched in SOVPE, SGPE, and mixed isomers of SEIPE
(m/z 814.5) were isolated from oxidized SAPE and tested for
their ability to induce monocyte binding. These isolated molecules
showed a dose-dependent increase in monocyte binding. Synthetic SOVPC
and SGPC also showed a similar dose-dependent increase in monocyte
binding similar to the corresponding isolated molecules (Figure 2).
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Effect of Oxidized Phospholipids on E-Selectin and VCAM-1
Expression
As was previously reported for oxidized PAPC, oxidized SAPC, and
oxidized SAPE, all (5 to 50 µg/mL) effectively inhibited the action
of LPS on E-selectin expression in a dose-dependent fashion, whereas
the corresponding native arachidonoyl phospholipids (50 µg/mL) did
not have any significant effect (Figure 3). Oxovaleroyl phospholipids alone
caused a similar inhibition. POVPC, SOVPC, and SOVPE (1 to 5 µg/mL)
dose-dependently inhibited LPS-induced E-selectin expression (online
Figure IV; please see www... ). In contrast, PGPC was previously
shown to induce E-selectin, VCAM-1 expression, and binding of
polymorphonuclear neutrophils to HAECs.10 Here,
we demonstrate that PGPC, SGPC, and SGPE all induced E-selectin and
VCAM-1 on HAECs (Figure 4). However,
oxovaleroyl phospholipids alone did not induce significant levels of
VCAM-1 and E-selectin on HAECs (data not shown). These results showed
that the changes at the sn-1 and sn-3 positions
did not affect the ability of oxovaleroyl phospholipids to inhibit LPS
action and glutaroyl phospholipids to stimulate E-selectin or VCAM-1
expression.
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Comparison of Phospholipids in LDL and MM-LDL
POVPC, SOVPC, PGPC, SGPC, mixed isomers of PEIPC, and mixed
isomers of SEIPC were increased by 2- to 11-fold in MM-LDL
phospholipids compared with native LDL phospholipids (the Table
, columns 1 and 2). The levels of SOVPE,
SGPE, and mixed isomers of SEIPE were increased by 3- to 5-fold in
MM-LDL lipids compared with native LDL lipids. The levels of bioactive
PC derivatives detected were 
10- to 25-fold higher than those of the
corresponding PE derivatives. The bioactive, oxidized phospholipids
represent a small fraction of the arachidonoyl phospholipid
oxidation products (5% to 10%). However, this amount is
sufficient to account for the bioactivity of MM-LDL.
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Comparison of Phospholipid Levels in Normal Rabbit Aorta and Rabbit
Atherosclerotic Lesions
Cholesterol, PAPC, and SAPC were all increased in the
rabbits fed the atherogenic diet. The levels of bioactive, oxidized
phospholipids, POVPC, PGPC, mixed isomers of PEIPC, SOVPC, SGPC, and
mixed isomers of SEIPC were increased by 3- to 6-fold in animals fed
the atherogenic diet (the Table , columns 3 and 4). These levels
correspond to 116, 62, and 85 µg/mL POVPC, PGPC, and mixed isomers
of PEIPC, respectively (by approximate conversion of wet weight to
volume). This level is roughly 10 to 20 times that required to
activate the endothelium.
Effects of PLs and NaBH4 on the Bioactivity of
Oxidized Phospholipids
To further identify the structural motifs of oxidized
phospholipids that are important for their bioactivity, we tested the
effects of chemical modification and enzymatic digestion. Treatment of
oxidized phospholipids with NaBH4 reduced the
oxygenated groups such as aldehydes, ketones, and
epoxides.7 8 The specific PLs PLA1,
PLA2, PLC, and PLD were shown to selectively
hydrolyze the sn-1, sn-2, and
sn-3 groups and the phosphate-choline bond,
respectively19 (online Figure V; please see www...
). In the present study, we tested the effects of PL and
NaBH4 treatments on the bioactivity of POVPC,
PGPC, and mixed isomers of PEIPC by a monocyte binding assay (Figure 5). Treatment of these 3 active lipids
with PLA1, PLA2, and PLC
completely abolished their bioactivity, whereas PLD partially reduced
their bioactivity. NaBH4 treatment abolished the
activity of POVPC and mixed isomers of PEIPC; however, the activity of
PGPC was unaltered (Figure 5). These studies suggest that the
aldehyde group in POVPC and reducible groups in PEIPC (carbonyl and
epoxide) are important for their bioactivity. Taken together, these
studies show that the sn-1, sn-2, and
sn-3 groups are essential for their effects on monocyte
binding; however, hydrolysis with PLD only partially reduced activity.
The ability of POVPC (5 µg/mL) to inhibit LPS-induced E-selectin
expression was strongly reduced by all 4 PLs and
NaBH4 (online Figure VI; please see www... ).
Similarly, PGPC- (5 µg/mL) induced E-selectin expression was also
significantly reduced by all 4 PLs; however,
NaBH4 had no significant effect (online Figure
VI; please see www... ).
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The common structural motifs for the bioactive, oxidized phospholipids are the presence of oxovaleroyl, glutaroyl, and an epoxyisoprostane at the sn-2 position; hence, we examined the effects of the isolated sn-2 fatty acids on monocyte adhesion. Oxovaleric acid (1 to 10 µmol/L, fatty acid from POVPC), glutaric acid (1 to 10 µmol/L, fatty acid from PGPC), and epoxyisoprostane (1 to 3 µmol/L, fatty acid from PEIPC) did not induce monocyte binding at the concentrations at which the corresponding intact, oxidized, phospholipids are maximally active (data not shown).
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Discussion |
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These studies were designed to (1) examine the effects of structural changes and hydrolytic enzymes on the actions of bioactive, oxidized phospholipids and (2) to quantify the levels of these lipids in MM-LDL and in atherosclerotic lesions. In previous studies, we identified 3 oxidized phospholipids primarily responsible for the activity of oxidized PAPC.7 8 Although PAPC is the major arachidonoyl phospholipid in native human LDL, SAPC and SAPE are also present. We now provide evidence for the formation of bioactive, oxidized phospholipids from the oxidation of SAPC and SAPE and for their increased levels in MM-LDL (Figure 1
and the
Table).
In the present study, we have presented evidence that the
sn-2 position of the bioactive, oxidized phospholipids is
the main determinant of the type of activity shown by the lipid. The
changes in saturated fatty acid at the sn-1 position and in
the head group at the sn-3 position did not affect
bioactivity (Figures 1 and 2). We have demonstrated that
oxovaleroyl-, glutaroyl-, and epoxyisoprostane-containing phospholipids
all stimulated endothelial cells to bind monocytes
(Figure 2). Oxovaleroyl phospholipids were effective in
inhibiting LPS induced E-selectin expression (online Figure IV;
please see www... ). In contrast, glutaroyl phospholipids induced
the surface expression of E-selectin and VCAM-1 in HAECs (Figure 4). Thus, 2 types of oxidized phospholipids, whether derived
from PAPC, SAPC, or SAPE, have quite different effects, and their
relative levels could control the recruitment of specific leukocytes at
the sites of inflammation.
The present study demonstrates that all 3 groups, sn-1,
sn-2 and sn-3, of bioactive, oxidized
phospholipids are important for bioactivities (Figure 5). Our group has
previously shown that the 2 hydrolytic enzymes, platelet-activating
factor–acetyl hydrolase and paraoxonase, were able to destroy the
bioactivity of oxidized phospholipids.5 6
Studies with NaBH4 demonstrate that the
reducible functional groups (carbonyl and epoxide) are essential for
bioactivity. However, certain changes at the sn-3 position
(PC to PE) do not alter their activity, and cleavage of the choline
moiety of PC by PLD partially reduced its bioactivity (Figures 4 and 5). The past and present studies show that neither
lysophospholipids6 nor the sn-2 fatty
acids in the free form were active in inducing monocyte binding.
Lysophosphatidic acid has been shown to exert a variety of biological
responses, including stimulation of endothelial cells
to bind monocytes and neutrophils and to induce E-selectin and VCAM-1
expression.20 The formation of lysophosphatidic acid
and/or lysophosphatidic acid analogues after treatment of oxidized
phospholipids with PLD may explain the partial, and not complete, loss
of bioactivity (Figure 5).
Our present and past data demonstrate that the bioactive, oxidized phospholipids responsible for stimulating monocyte binding and inhibiting neutrophil binding are increased in atherosclerotic lesions. The presence of oxidized LDL in rabbit and human atherosclerotic lesions21 and increased levels of total phospholipids and lysophosphatidylcholine in rabbit lesions22 have been previously demonstrated. In the present study, the levels of oxidized phospholipids in individual aortas were highly correlated with the accumulation of cholesterol and unoxidized phospholipids (data not shown). One interpretation of this finding is that increased levels of phospholipid oxidation products in the vessel wall are due to the increase in phospholipid, which serves as the oxidative substrate. However, a number of other mechanisms may explain the increased oxidation observed. In general, our results suggest that these oxidized phospholipids in the vessel wall could serve as specific markers of oxidative injury in vivo.
In summary, these results identify 6 new bioactive, oxidized phospholipids that have specific effects on leukocyte–endothelial cell interactions. We have demonstrated that the specificity is determined by the sn-2 groups of oxidized phospholipids. The levels of all 6 bioactive, oxidized phospholipids are increased in MM-LDL, and the levels of at least 3 bioactive, oxidized phospholipids are increased in atherosclerotic lesions, suggesting a role for these newly identified bioactive, oxidized phospholipids in atherogenesis. Finally, these results demonstrate the importance of the integrity of the sn-1, sn-2, and sn-3 positions in the activation of endothelial cells.
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Acknowledgments |
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This work was supported by US Public Health Service grant No. HL 30568, the W.M. Keck Foundation (to K.F.F.), and the Laubish Fund. We would like to thank Dr Mohamad Navab for helpful suggestions.
Received March 30, 2000; accepted June 19, 2000.
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