© 1999 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
Role of Group II Secretory Phospholipase A2 in Atherosclerosis
2. Potential Involvement of Biologically Active Oxidized Phospholipids
From the Departments of Medicine (N.L., A.D.W., S.Y.H., B.I., J.-H.Q., J.H., M.N., A.M.F., A.J.L., J.A.B.), Pathology (N.L., J.A.B.), Psychiatry and Biobehavioral Sciences and the Neuropsychiatric Institute (K.M.F.), University of California, Los Angeles; Chrysalis DNX Transgenic Sciences (D.S.G.), Princeton, NJ; and the Department of Medicine (F.C.d.B.), University of Kentucky, Lexington. Current address for Norbert Leitinger, Department of Vascular Biology and Thrombosis Research, Schwarzspanierstrasse 17, A1090 Vienna, University of Vienna, Austria.
Correspondence to Judith A Berliner, 13-239 CHS, Departments of Pathology and Medicine, Los Angeles, CA 90095-1732. E-mail jberline{at}pathology.medsch.ucla.edu
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Abstract—Secretory nonpancreatic phospholipase A2 (group II sPLA2) is induced in inflammation and present in atherosclerotic lesions. In an accompanying publication we demonstrate that transgenic mice expressing group II sPLA2 developed severe atherosclerosis. The current study was undertaken to determine whether 1 mechanism by which group II sPLA2 might contribute to the progression of inflammation and atherosclerosis is by increasing the formation of biologically active oxidized phospholipids. In vivo measurements of bioactive lipids were performed, and in vitro studies tested the hypothesis that sPLA2 can increase the accumulation of bioactive phospholipids. We have shown previously that 3 oxidized phospholipids derived from the oxidation of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (PAPC) stimulated endothelial cells to bind monocytes, a process that is known to be an important step in atherogenesis. We now show that these 3 biologically active phospholipids are significantly increased in livers of sPLA2 transgenic mice fed a high-fat diet as compared with nontransgenic littermates. We present in vitro evidence for several mechanisms by which these phospholipids may be increased in sPLA2 transgenics. These studies demonstrated that polyunsaturated free fatty acids, which are liberated by sPLA2, increased the formation of bioactive phospholipids in LDL, resulting in increased ability to stimulate monocyte-endothelial interactions. Moreover, sPLA2-treated LDL was oxidized by cocultures of human aortic endothelial cells and smooth muscle cells more efficiently than untreated LDL. Analysis by electrospray ionization–mass spectrometry revealed that the bioactive phospholipids, compared with unoxidized PAPC, were less susceptible to hydrolysis by human recombinant group II sPLA2. In addition, HDL from the transgenic mice and human HDL treated with recombinant sPLA2 in vitro failed, in the coculture system, to protect against the formation of biologically active phospholipids in LDL. This lack of protection may in part relate to the decreased levels of paraoxonase seen in the HDL isolated from the transgenic animals. Taken together, these studies show that levels of biologically active oxidized phospholipids are increased in sPLA2 transgenic mice; they also suggest that this increase may be mediated by effects of sPLA2 on both LDL and HDL.
Key Words: mice, transgenic • lipid peroxidation • LDL oxidation • inflammation • spectrum analysis, mass • phospholipids
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Introduction |
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It is shown in an accompanying paper that fatty streak formation is increased in sPLA2 transgenic mice on a high-fat diet.1 There is considerable evidence that oxidative modification of LDL is directly related to the development of the atherosclerotic lesion.2 3 4 5 6 7 Treatment of endothelial cells with minimally oxidized LDL (LDL that has undergone antioxidant depletion and oxidation of arachidonic acid-containing phospholipids, with little or no protein modification) increased monocyte binding and transmigration in cocultures of endothelial cells and smooth muscle cells.4 8 The major lipids responsible for the biological activity of minimally oxidized LDL (MM-LDL) are derived from the oxidation of arachidonate-containing phospholipids9 such as 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (PAPC). Three active lipids accounted for the major bioactivity in MM-LDL and oxidized PAPC (OxPAPC): 1-palmitoyl-2-(5)oxovaleroyl-sn-glycero-3-phosphorylcholine (POVPC, m/z 594.3 (M+H+)), 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphorylcholine (PGPC, m/z 610.2 (M+H+)), and a molecule with m/z 828.6 (M+H+).10 We demonstrated that these bioactive phospholipids were increased in fatty streak lesions from cholesterol-fed rabbits and they were significantly less abundant when the atherosclerotic diet was supplemented with antioxidants such as vitamin E and probucol.10 Natural antibodies against these phospholipids were found in the serum of apoE null mice, which develop advanced atherosclerotic lesions owing to massive hypercholesterolemia.10 11
Both the liver and the arterial wall are sites of lipid accumulation when mice are fed an atherogenic diet. Moreover, in recombinant inbred strains derived from C57BL/6J and C3H/HeJ mice, hepatic inflammatory gene induction and aortic fatty streak development cosegregated, indicating similar pathophysiology in these tissues.12 In these same recombinant inbred strains, levels of POVPC, PGPC, and the molecule with m/z 828.6 in livers also cosegregated with lesion development.13 In the present study we determined whether levels of biologically active oxidized phospholipids were increased in sPLA2 transgenics and performed in vitro tests of the hypothesis that sPLA2 accelerates their accumulation.
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Methods |
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Materials
L-
-1-Palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine
was obtained from Avanti Polar Lipids Inc. or Sigma Chemical Co.
Soybean lipoxygenases (SLO),
sPLA2 (Naja naja), butylated
hydroxytoluene (BHT), dimyristoylphosphatidylcholine (DMPC), palmitic
acid, stearic acid, oleic acid, linoleic acid,
arachidonic acid, and endotoxin-free gelatin,
tissue-culture grade, were obtained from Sigma. Aminopropyl solid phase
extraction columns were obtained from J.T. Baker. Chloroform, methanol,
isopropanol, and ethyl ether (all HPLC grade or better) were obtained
from Fisher Scientific. Tissue culture media, serum, and supplements
were obtained from sources previously reported.8
Transwells and chamber slides were obtained from Costar. The
fluorescent probe DiI
(1,1'-dioctadecyl-3,3,3'-tetramethyl-indo-carbocyanine perchlorate) was
purchased from Molecular Probes. Human recombinant
sPLA2 was a generous gifts from Dr J. Browning,
Biogen. The method of preparation of sPLA2 has
been previously described.14 Briefly, recombinant
sPLA2 was purified from medium of Chinese hamster ovary
(CHO) cells transfected with an expression vector encoding human
sPLA2.
Animals
sPLA2 transgenic mice were obtained from
Chrysalis DNX Transgenic Sciences, Princeton, NJ, and had been
produced as described previously.15 The atherogenic diet
contained 1.25% cholesterol, 15.75% fat, and 0.5% sodium
cholate (TD 90221, Harlan-Teklad). After 12 weeks on the high-fat diet,
the mice were sacrificed by cervical dislocation after the
administration of isoflurane. All animal procedures were conducted
according to the regulations of the University of California Animal
Research Committee.
Lipoprotein Isolation and Modification
Human LDL (d=1.019 to 1.069 g/mL) and HDL (d=1.069 to 1.210
g/mL) were isolated from the sera of healthy blood donors by density
gradient ultracentrifugation as
described16 and used within 1 to 2 weeks of isolation.
Concentration of LDL is expressed in terms of protein content as
determined according to the method of Lowry et al.17 The
concentration of endotoxin in solutions tested in biological assays was
less than 50 pg/mL (determined by a chromogenic assay),
which is 40-fold less than that required to induce
monocyte-endothelial interactions. LDL was modified
using purified SLO and PLA2, according to Sparrow
et al.18 To remove enzymes from the reaction by
centrifugation, SLO and PLA2 were
separately bound to CNBr-activated Sepharose beads as
described.18 LDL (1 mg/mL) was modified by incubation with
SLO (5000 U/mL) alone, SLO+PLA2 (20 U/mL), or
SLO+fatty acids (187 mmol/L) at 37°C for 24 hours or at 4°C
for 48 hours.19 The enzymes were removed by
centrifugation, BHT (100 µmol/L) and EDTA
(0.3 mmol/L) were added, and the modified LDL was stored at 4°C
and used within 2 weeks.
Mouse HDL were isolated from pooled plasma by fast protein liquid chromatography,20 in the absence of EDTA to avoid inactivation of paraoxonase. HDL were incubated at concentrations of 2 mg protein/mL with recombinant human sPLA2 (0.5 µg/mL) for 16 hours, subsequently isolated again, and then added to the cocultures at a final concentration of 250 to 350 µg/mL.
Cell Culture
Human aortic endothelial cells (HAEC) and rabbit
aortic endothelial cells (RAEC) were cultured as
described.8 Human aortic smooth muscle cells (HASMC) were
isolated as previously described.21 Cocultures of HAEC and
HASMC were grown, and monocyte binding and transmigration experiments
were performed in transwells or chamber slides as previously
described.21 In all experiments, HAEC and HASMC from the
same donor were used at passages 4 to 6. Blood monocytes were obtained
from a large pool of healthy donors by modification of the Recalde
procedure as described previously.22
Monocyte Adhesion Assay
Binding of human monocytes to endothelial cells
was performed essentially as described previously.8 HAEC
or RAEC were treated with MM-LDL (125 to 200 µg/mL) for 4 hours at
37°C. Treatment media were removed, the cells were rinsed twice with
medium, and a suspension of human monocytes (2 to
3x105/well) was added for 12 minutes. Unbound
monocytes were removed, and the number of bound monocytes was
determined microscopically.
Monocyte Transmigration Assay
These assays were performed essentially as described
previously.21 Cocultures were treated with native LDL (250
to 350 mg/mL) in the absence or presence of various test compounds for
24 hours. The culture supernatants were subsequently transferred to
untreated cocultures and were incubated for an additional 24 hours.
Monocytes were labeled with the fluorescent probe DiI at 4°C
for 10 minutes, washed, and resuspended in medium-199 at the desired
cell density. Labeled monocytes were added to the treated cocultures at
2x105 cells/cm2 and were
incubated for 60 minutes at 37°C. The medium containing nonadherent
cells was removed, and the cell layers were washed to remove loosely
adherent monocytes. The cocultures were fixed on polycarbonate
membranes with 10% neutral buffered formalin at room temperature for
24 hours and were mounted on glass slides. The number of monocytes in
the subendothelial space (beneath the
endothelial cells) in a minimum of 9 fields was
determined under 500x magnification and fluorescence
microscopy.
Measurement of Lipid Hydroperoxides
Lipid hydroperoxide formation was measured using the method of
Auerbach et al.23
Lipid Extraction
Lipids were extracted from modified LDL and from mouse
livers using a modification of the method of Bligh and
Dyer.24 Liver tissue samples were homogenized
in chloroform/methanol (2:1) containing 0.01% BHT for 5 minutes at
0°C. After addition of water, liver suspensions were vortexed for 2
minutes and centrifuged at 1200g for 15 minutes at
4°C (Beckman J-6B; rotor, Beckman JS-4.2). In separate studies
chloroform/methanol/BHT was added to modified LDL in PBS, and these
suspensions were vortexed for 2 minutes. The chloroform phase was
carefully removed and 5 vol of chloroform was added to the residual
aqueous phase. The mixture was vortexed and centrifuged as
described above, and the chloroform phase was pooled with the previous
extract. Phospholipid recovery typically ranged between 96% and 98%
as determined by spiking samples with known quantities of
1-palmitoyl-2-[14C]arachidonoyl-sn-glycero-3-phosphorylcholine.
Solid Phase Extraction
Phospholipids, free fatty acids, and neutral lipids were
separated by the method of Kaluzny et al.25 using
aminopropyl solid phase extraction chromatography.
Total lipid extracts were dried under nitrogen, resuspended in 200 µL
of chloroform, and applied to the aminopropyl columns, which had been
washed with 3 mL of methanol and preconditioned with 3 mL of hexane.
Neutral lipids were eluted with 3 mL of chloroform/2-propanol (2:1),
free fatty acids with 3 mL of 3% acetic acid in ethyl ether, and
phospholipids with 3 mL of methanol. When phospholipids were used in
tissue culture experiments, acetic acid retained in the columns was
removed with pure ethyl ether before phospholipid elution. Lipid
fractions were dried under nitrogen and either used immediately or
resuspended in chloroform containing 0.01% BHT, covered with argon,
and stored at -44°C.
Substrate Specificity Assay
OxPAPC was produced by exposing dry PAPC to air for 3 days.
Oxidation products, as well as residual unoxidized PAPC, were
present in these preparations. Liposomes were produced by
sonicating OxPAPC for 10 minutes in Tris buffer, pH 7.4. Fifty
microliters containing 500 µg of OxPAPC liposomes was incubated with
either human recombinant sPLA2 (1 µg), snake
venom sPLA2 (Naja naja; 5 U), or
buffer alone for 90 minutes at room temperature in Tris buffer
containing 2 mmol/L Ca2+. The reaction was
stopped and lipids were extracted by adding 200 µL
chloroform/methanol (2:1)+0.01% BHT. After vortexing and
centrifugation for 10 minutes, 100 µL of the
chloroform phase was transferred into a glass tube, and 500 ng of
internal standard (DMPC) was added to each tube.26
Samples were dried under a stream of nitrogen and redissolved in
acetonitrile/water/formic acid (50:50:0.1; vol/vol/vol) and
analyzed by electrospray ionization–mass spectrometry
(ESI-MS).
Electrospray Ionization–Mass Spectrometry
An API III triple-quadrupole biomolecular mass analyzer
(Perkin-Elmer Sciex Instruments) was used for mass analysis of
phospholipids. Flow-injection analysis was performed as
described previously.10 For quantitative measurements,
DMPC was used as an internal standard.26 Mass spectrometry
of individual phospholipid molecular species represents the
most sensitive, discriminating, and direct method to assess alterations
in phospholipid molecular species in biological tissues.26
In contrast to previously used techniques like fast atom bombardment
mass spectrometry, which results in extensive fragmentation of
individual phospholipid ions, electrospray ionization results in the
efficient production of molecular ions of phospholipids with
negligible fragmentation.27 Therefore, ESI-MS offers a
sensitive and reliable tool for quantitative phospholipid
analysis in biological tissues. For quantitative measurements
we were assuming that possible differential effects of phospholipid
oxidation on extraction recovery, ionization efficiency, and molecular
ion yield were negligibly small in comparison with the quantitative
differences reported. In an extensive examination of phospholipid
quantitation by ESI-MS, Han et al26 reported that the
effects of unsaturation and differential surface characteristics were
minimal. Because any 13C effects on quantitation
were constant within the comparisons made, no corrections of the
molecular ion signal intensities have been performed. Within the range
of concentrations measured, trial experiments indicated that the
response was linear, such that increases in phospholipid content were
reflected in proportional increases in phospholipid to internal
standard ratio.
Statistical Analysis
Statistical analysis was performed using 1-way ANOVA;
probability values <0.05 were considered statistically
significant.
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Results |
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Evaluation of Phospholipids by ESI-MS
Previous studies have shown that group II sPLA2 was expressed to a significantly greater extent in livers15 and aortas1 of sPLA2 transgenic mice when compared with nontransgenic littermates. Liver was used as a surrogate for aorta as validated in previous studies in Bl-6 mice.12 Phospholipids were separated from a total lipid extract obtained from livers of female sPLA2 transgenic mice and nontransgenic littermates maintained for 12 weeks on an atherogenic diet. Analysis of phospholipids was performed by ESI-MS, and quantification of the different phospholipid species was carried out by addition of DMPC as an internal standard, as described in the Methods. The levels of native phospholipids, 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (PLPC, m/z 758) and PAPC, m/z 782, in livers of transgenic female mice were not significantly different from the levels in livers of nontransgenic (Figure 1A
).
In contrast, the levels of POVPC and PGPC were significantly increased
in transgenic animals versus nontransgenics (Figure 1B;
P<0.003 for POVPC, P<0.014 for PGPC). The
bioactive molecule with m/z 828.6 was not increased in the
transgenic compared with nontransgenic animals (Figure 1B).
Interestingly, the levels of lysophosphatidylcholine (LPC)
(sn-1=palmitic acid (16:0), m/z 496, and
sn-1=stearic acid (18:0), m/z 524 were not
significantly different between transgenic and nontransgenic animals
(Figure 1A).
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Effect of sPLA2 Products on LDL Phospholipid
Oxidation
Our group and others have shown that treatment of LDL with
sPLA2 from Naja naja combined with SLO
leads to the formation of conjugated dienes and bioactive
phospholipids.18 19 On the basis of these findings,
we hypothesized that treatment of LDL with sPLA2
led to a limited liberation of fatty acids that, when oxidized, could
cause oxidation of arachidonate-containing phospholipids,
leading to the formation of bioactive phospholipids. To test this
hypothesis, LDL was incubated without additions, or in the presence of
SLO and linoleic acid. The formation of conjugated dienes was increased
2- to 3-fold in the phospholipid fraction of the treated LDL compared
with the untreated LDL (Figure 2A). An
approximately 10-fold increase in the level of absorbance at 270 nm, an
indication for the levels of bioactive phospholipids,9 was
also observed (Figure 2B). The treated LDL preparation was
active in stimulating endothelial cells to bind
monocytes, further confirming the presence of biologically active
phospholipids (Figure 2C). Arachidonic acid had
similar effects (data not shown). To further test the hypothesis that
oxidized fatty acids play a role in the formation of bioactive
phospholipids, LDL was treated with oxidized linoleic acid in the
absence of other additives. This treatment also stimulated the
formation of bioactive phospholipids (monocyte binding increased by
2.4±0.3-fold). Oxidized fatty acids alone in the absence of LDL had no
effect on monocyte binding (data not shown). Several lines of evidence
suggest that lipoxygenase may play a significant role
in the oxidation of LDL by cells.28 Free polyunsaturated
fatty acids (PUFAs) are better substrates than esterified PUFAs for
some mammalian lipoxygenases. We tested the hypothesis
that treatment of LDL with sPLA2 would increase
the ability of cells to oxidize LDL and thereby form bioactive
phospholipids. Pretreatment of LDL with different concentrations of
human recombinant sPLA2 and subsequent incubation
for 24 hours with cocultures of HAEC and HASMC resulted in increased
formation of lipid hydroperoxides (Figure 3A) and increased bioactivity (Figure 3B).
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Substrate Specificity of sPLA2
It was clear from the above studies that
sPLA2 could both hydrolyze phospholipids and lead
to the formation of bioactive phospholipids. We hypothesized that these
2 observations were compatible with the idea that other phospholipids
in OxPAPC were better substrates for sPLA2 than
the bioactive oxidized phospholipids. To assess the substrate
specificity among the various species of oxidized phospholipids
present in OxPAPC, a human recombinant sPLA2
and snake venom sPLA2 were used. Liposomes
consisting of OxPAPC (which contained small amounts of native PAPC)
were incubated with the enzymes and analyzed by ESI-MS. The
bioactive molecules POVPC (m/z 594.3), PGPC (m/z
610.2), and m/z 828.6 were poorly hydrolyzed by the
treatment with human recombinant sPLA2, whereas
unoxidized PAPC (m/z 782.4) was effectively hydrolyzed
(Figure 4 and the Table). After treatment
with recombinant sPLA2, the level of LPC was
increased to a greater extent than could be accounted for by hydrolysis
of native PAPC. This suggested that the excess LPC was because of
hydrolysis of other oxidized phospholipids present in OxPAPC.
Although the reduction of the >100 peaks (seen by MS) is not
necessarily discernible in Figure 4, the collective
production of LPC from these lipids is highly significant. The
snake venom sPLA2 showed a preference for POVPC
(m/z 594.3) and unoxidized PAPC (m/z 782.4) among
the phospholipids present in OxPAPC. Representative
mass spectra are shown in Figure 4 and the results of these
experiments are summarized in the Table.
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HDL From sPLA2 Transgenic Mice Fail to Protect Against
LDL Oxidation
HDL has been shown in vitro to prevent LDL oxidation and
accumulation of bioactive lipids.4 This protective HDL
function may also play an important role in fatty streak formation. We
examined the protective effect of HDL from sPLA2
transgenic mice and their nontransgenic littermates using a coculture
system in which cell-induced LDL oxidation stimulates the secretion of
monocyte chemotactic protein-1 (MCP-1): levels of MCP-1 are assessed as
monocyte chemotaxis and transmigration in the coculture.29
As previously reported, the addition of HDL along with LDL to such
cocultures inhibited the formation of biologically active oxidized
LDL.29 The HDL isolated from the plasma of
sPLA2 transgenic mice did not protect against
LDL-induced monocyte transmigration, whereas HDL from nontransgenic
littermates was protective (Figure 5A).
In fact, the HDL from the transgenic mice significantly promoted
LDL-oxidation and monocyte transmigration in the coculture. The HDL
from the sPLA2 transgenics contained one-third
the paraoxonase activity of HDL from nontransgenics. To further study
the sPLA2-mediated effects on HDL, we performed
additional in vitro experiments (Figure 5B). First, we
preincubated human HDL with sPLA2 for 3 hours
with gentle mixing; the HDL was then isolated again and added to the
coculture together with human LDL. Monocyte transmigration in the
coculture containing sPLA2-treated HDL was
increased 2-fold as compared with the addition of LDL alone (no HDL)
and 
3-fold compared with the addition of LDL and sham-treated HDL.
Therefore, sPLA2-treated HDL not only failed to
protect against LDL oxidation but also exhibited proinflammatory
capacity.
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Discussion |
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Previous studies reported in the accompanying manuscript1 showed that atherosclerosis was increased in sPLA2 transgenic mice. In these studies and those of others, group II sPLA2 was shown to be present in atherosclerotic lesions.1 30 31 A major goal of these studies was to test the hypothesis that sPLA2 could increase the levels of biologically active oxidized phospholipids, previously shown to have atherogenic effects.10 The importance of bioactive lipids in the development of atherosclerosis is suggested by the findings that animals injected with MDA-LDL32 or oxidized LDL33 before cholesterol feeding developed antibodies to the lipids and were partially protected against lesion development during cholesterol feeding. A role for these bioactive phospholipids in other inflammatory processes is suggested by the presence of antibodies to the bioactive phospholipids in a number of diseases of chronic inflammation.34
Because of the limited amount of fatty streak lesion material available
for the study we compared the levels of bioactive phospholipids in
livers of transgenic and nontransgenic female mice. The results of
previous studies, showing that pathological changes in livers of Bl-6
mice fed an atherogenic diet parallel those in the
aorta,12 make the liver an appropriate target for study in
these animals. We found that the levels of the native phospholipids
PLPC and PAPC were not increased in the livers of the transgenic mice
compared with the nontransgenics (Figure 1A
). Somewhat
surprisingly, levels of lysophosphatidylcholine were also not increased
(Figure 1A), perhaps because of high levels of lysophospholipase
in the liver.35 However, levels of the biologically active
oxidized phospholipids POVPC and PGPC were highly significantly
elevated (Figure 1B) in the transgenics compared with the
nontransgenics (Figure 1B).
Our in vitro studies suggest that 1 mechanism by which group II
sPLA2 accelerates atherosclerosis
is by increasing the levels of biologically active oxidized
phospholipids. We present studies suggesting that
sPLA2 may lead to more generation and less
destruction of active lipids. The hypothetical scheme for the increased
formation of bioactive phospholipids in sPLA2
transgenic animals is shown in Figure 6.
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We and others have previously shown that treatment of LDL with SLO and
PLA2 increases oxidation, resulting in the
formation of bioactive LDL.18 19 Treatment of LDL with
sPLA2 led to a limited release of
PUFAs.36 We now show that treatment of LDL with free PUFAs
(which are products of PLA2 activity) in the
presence of SLO or with oxidized PUFA causes oxidation of
PUFA-containing phospholipids and thereby formation of bioactive
phospholipids (Figure 2). Others have shown that free PUFAs are
much better substrates for mammalian lipoxygenases than
are phospholipid-bound PUFAs.37 Furthermore, PUFA radicals
can act essentially as catalysts to propagate lipid oxidation. Evidence
that sPLA2 enhances the formation of biologically
active phospholipids in cellular systems is provided by our studies in
cell culture. Treatment of LDL with sPLA2 led to
formation of bioactive phospholipids and hydroperoxides (Figure 3). Free fatty acids have been shown to diffuse spontaneously
across phospholipid bilayers, described as a flip-flop
mechanism.38 Recently, active transport in addition to
passive diffusion of free fatty acids across cell membranes has been
postulated. It has been shown that carrier-mediated uptake of fatty
acids in hepatocytes follows an inwardly directed
transmembrane proton gradient and is stimulated by the presence of
H+ at the outer surface of the plasma
membrane.39 Lower pH levels, which have been measured at
sites of inflammation, would therefore increase the uptake of free
fatty acids into cells. Because the uptake of free fatty acids into
mitochondria for subsequent ß-oxidation is highly regulated,
accumulating free fatty acids would be a likely substrate for oxidizing
enzymes, resulting in increased levels of lipid hydroperoxides. These
hydroperoxides could then leave the cells seeding LDL, resulting in the
formation of biologically active oxidized phospholipids.
LDL may not be the only source of bioactive oxidized phospholipids in
sPLA2 transgenic animals. In vivo,
sPLA2 may also liberate PUFAs from other
lipoproteins, dying erythrocytes, or membranes of other cells leading
to formation of bioactive lipids. Others have shown that whereas normal
cell membranes are not good substrates for sPLA2,
membranes from damaged cells may become good substrates.40
Although our studies demonstrated that the bioactive phospholipids were
not good substrates for human recombinant sPLA2,
they also demonstrated that hydrolysis of other oxidized phospholipids
in OxPAPC resulted in a significant increase in LPC after treatment
(Figure 4). Others have demonstrated that, in oxidized
phospholipid vesicles that contain phosphatidyl ethanolamine as well as
phosphatidyl choline, there is significant hydrolysis of oxidized
phospholipids by snake venom sPLA2, caused by
increased Ca2+ binding affinity and activation of
the enzyme.41 42 The present studies have focused on
the role of sPLA2 in the formation of 3 important
bioactive phospholipids. However, increased levels of
sPLA2 may also increase the levels of other
bioactive phospholipids such as lysophosphatidic acid. Fourcade et
al43 showed that sPLA2 plays an
important role in producing lysophosphatidic acid and in the release
from the cell membrane in the form of microvesicles.
Our results suggest that sPLA2 may also increase
the levels of biologically active oxidized phospholipids by altering
HDL. We show that HDL isolated from the transgenic animals had lost its
protective activity. (Figure 5A). In addition, we show that in
vitro sPLA2-treated HDL also had lost its
protective activity and even enhanced monocyte transmigration induced
by cell-modified LDL (Figure 5B). HDL from
sPLA2 transgenic mice had lower levels of
paraoxonase,1 an enzyme that can degrade bioactive
phospholipids.9 Therefore loss of paraoxonase may be 1
mechanism by which the HDL protective mechanism is compromised,
resulting in increased levels of bioactive phospholipids. However,
other changes in sPLA2-treated HDL, such as
increased cholesterol delivery to cells, shown to occur
with other lipases,44 may represent another
important mechanism.
In summary, we have presented evidence that the increase in atherosclerosis in sPLA2 transgenic mice may relate to the ability of sPLA2 to release PUFAs, which catalyze the formation of bioactive phospholipids. These bioactive phospholipids are poorly degraded by sPLA2, and the loss of protective activity of HDL in transgenic animals, perhaps in part caused by decreased paraoxonase activity, may further enhance their accumulation.
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Acknowledgments |
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This work was supported by National Institute of Health grants HL30568 (A.J.L., A.M.F., J.A.B.), AG10886 (F.C.d.B.), the Council for Tobacco Research (F.C.d.B.), and Veterans Administration Research Funds (F.C.d.B.).
Received January 12, 1998; accepted October 5, 1998.
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References |
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1. Ivandic B, Castellani L, Wang XP, Qiao JH, Mehrabian M, Navab M, Fogelman AM, Grass DS, Swanson ME, de Beer MC, de Beer F, Lusis AJ. Role of group II secretory PLA2 in atherosclerosis, 1: increased atherogenesis and altered lipoproteins in transgenic mice expressing group II phospholipase A2. Arterioscler Thromb Vasc Biol. 1999;19:1284–1290.
2. Steinberg D, Parthasarathy S, Carew TE, Khoo JC, Witztum JL. Beyond cholesterol: modifications of low-density lipoprotein that increase its atherogenicity. N Engl J Med. 1989;320:915–924.[Medline] [Order article via Infotrieve]
3. Witztum JL, Steinberg D. Role of oxidized low density lipoprotein in atherogenesis. J Clin Invest. 1991;88:1785–1792.
4.
Navab M, Berliner JA, Watson AD, Hama SY, Territo MC,
Lusis AJ, Shih DM, Van Lenten BJ, Frank JS, Demer LL, Edwards PA,
Fogelman AM. The yin and yang of oxidation in the development of the
fatty streak: a review based on the 1994 George Lyman Duff Memorial
Lecture. Arterioscl Thromb Vasc Biol. 1996;16:831–842.
5. Witztum JL. The oxidation hypothesis of atherosclerosis. Lancet. 1994;344:793–795.[Medline] [Order article via Infotrieve]
6.
Berliner JA, Navab M, Fogelman AM, Frank JS, Demer LL,
Edwards PA, Watson AD, Lusis AJ. Atherosclerosis: basic
mechanisms: oxidation, inflammation, and genetics.
Circulation. 1995;91:2488–2496.
7. Berliner JA, Heinecke JW. The role of oxidized lipoproteins in atherogenesis. Free Radic Biol Med. 1996;20:707–727.[Medline] [Order article via Infotrieve]
8. Berliner JA, Territo MC, Sevanian A, Ramin S, Kim JA, Bamshad B, Esterson M, Fogelman AM. Minimally modified low density lipoprotein stimulates monocyte endothelial interactions. J Clin Invest. 1990;85:1260–1266.
9. Watson AD, Berliner JA, Hama SY, La Du BN, Faull KF, Fogelman AM, Navab M. Protective effect of high density lipoprotein associated paraoxonase. J Clin Invest. 1995;96:2882–2891.
10.
Watson, AD, Leitinger N, Navab M, Faull KF,
Hörrkö S, Witztum JL, Palinski W, Schwenke D, Salomon RG,
Sha W, Subbanagounder G, Fogelman AM, Berliner JA. Structural
identification by mass spectrometry of oxidized phospholipids in
minimally oxidized low density lipoprotein that induce
monocyte/endothelial interactions and evidence for
their presence in vivo. J Biol Chem. 1997;272:13597–13607.
11. Hörkkö S, Miller E, Dudl E, Reaven P, Curtiss LK, Zvaifler NJ, Terkeltaub R, Pierangeli SS, Branch DW, Palinski W, Witztum JL. Antiphospholipid antibodies are directed against epitopes of oxidized phospholipids: recognition of cardiolipin by monoclonal antibodies to epitopes of oxidized low density lipoprotein. J Clin Invest. 1996;98:815–825.[Medline] [Order article via Infotrieve]
12. Liao F, Andalibi A, Qiao JH, Allayee H, Fogelman AM, Lusis AJ. Genetic evidence for a common pathway mediating oxidative stress, inflammatory gene induction, and aortic fatty streak formation in mice. J Clin Invest. 1994;94:877–884.
13. Berliner JA, Leitinger N, Watson AD, Huber J, Fogelman AM, Navab M. Oxidized lipids in atherogenesis: formation, destruction and action. Thromb Haemost. 1997;78:195–199.[Medline] [Order article via Infotrieve]
14. Cirino G, Cicala C, Sorrentino L, Maiello FM, Browning JL. Recombinant secreted phospholipase A2 induces a cynovitis-like inflammation in the rat air pouch. J Rheumatol. 1994;21:824–829.[Medline] [Order article via Infotrieve]
15. Grass DS, Felkner RH, Chiang MY, Wallace RE, Nevalainen TJ, Bennet CF, Swanson ME. Expression of human group II PLA2 in transgenic mice results in epidermal hyperplasia in the absence of inflammatory infiltrate. J Clin Invest. 1996;97:2233–2241.[Medline] [Order article via Infotrieve]
16. Havel RJ, Eder HA, Bragdon JH. The distribution and chemical composition of ultracentrifugally separated lipoproteins of human serum. J Clin Invest. 1955;43:1345–1353.
17.
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein
measurement with the folin phenol reagent. J Biol Chem. 1951;193:265–275.
18. Sparrow CP, Parthasarathy S, Steinberg D,. Enzymatic modification of low density lipoprotein by purified lipoxygenase plus phospholipase A2 mimics cell-mediated oxidative modification. J Lipid Res. 1988;29:745–753.[Abstract]
19. Parhami F, Fang ZT, Fogelman AM, Andalibi A, Territo MC, Berliner JA. Minimally modified low density lipoprotein-induced inflammatory responses in endothelial cells are mediated by cyclic adenosine monophosphate. J Clin Invest. 1993;92:471–478.
20.
Warden CH, Qiao JH, Castellani LW, Lusis AJ.
Atherosclerosis in transgenic mice overexpressing
apolipoprotein A-II. Science. 1993;261:469–472.
21. Navab M, Hough GP, Stevenson LW, Drinkwater DC, Kaks H, Fogelman AM. Monocyte migration into the subendothelial space of a coculture of adult human aortic endothelial and smooth muscle cells. J Clin Invest. 1988;82:1853–1863.
22. Fogelman AM, Sykes K, Van Lenten BJ, Territo MC, Berliner JA. Modification of the Recalde method for the isolation of human monocytes. J Lipid Res. 1988;29:1243–1247.[Abstract]
23. Auerbach BJ, Kiely JS, Cornicelli JA. A spectrophotometric microtiter-based assay for the detection of hydroperoxy derivatives of linoleic acid. Anal Biochem. 1992;20:375–380.
24. Bligh EG, Dyer WJ. A rapid method of total lipid extraction and purification. Can J Biochem Physiol. 1959;37:911–917.
25. Kaluzny MA, Duncan LA, Merritt MV, Epps DE. Rapid separation of lipid classes in high yield and purity using bonded phase columns. J Lipid Res. 1985;26:135–140.[Abstract]
26. Han X, Gubitosi-Klug RA, Collins BJ, Gross RW. Alterations in individual molecular species of human platelet phospholipids during thrombin stimulation: electrospray ionization mass spectrometry-facilitated identification of the boundary conditions for the magnitude and selectivity of thrombin-induced platelet phospholipid hydrolysis. Biochemistry. 1996;35:5822–5832.[Medline] [Order article via Infotrieve]
27.
Han X, Gross RW. Electrospray ionization mass
spectroscopic analysis of human erythrocyte plasma membrane
phospholipids. Proc Natl Acad Sci U S A. 1994;91:10635–10639.
28.
Parthasarathy S, Wieland E, Steinberg D. A role for
endothelial cell lipoxygenase in the
oxidative modification of low-density lipoproteins. Proc Natl
Acad Sci U S A. 1989;86:1046–1050.
29. Navab M, Imes SS, Hama SY, Hough GP, Ross LA, Bork RW, Valente AJ, Berliner JA, Drinkwater DC, Laks H, Fogelman AM. Monocyte transmigration induced by modification of low density lipoprotein in co-cultures of human aortic wall cells is due to induction of monocyte chemotactic protein 1 synthesis and is abolished by high density lipoprotein. J Clin Invest. 1991;88:2039–2046.
30. Menschikowski M, Kasper M, Lattke P, Schiering A, Schiefer S, Stockinger H, Jaross W. Secretory group II phospholipase A2 in human atherosclerotic plaques. Atherosclerosis. 1995;118:173–181.[Medline] [Order article via Infotrieve]
31.
Hurt-Camejo E, Andersen S, Standal R, Rosengren B,
Sartipy P, Stadberg E, Johansen B. Localization of nonpancreatic
secretory phospholipase A2 in normal and
atherosclerotic arteries. Arterioscler Thromb Vasc Biol. 1997;17:300–309.
32.
Palinski W, Miller E, Witztum JL. Immunization of low
density lipoprotein (LDL) receptor-deficient rabbits with homologous
malondialdehyde-modified LDL reduces atherogenesis. Proc Natl
Acad Sci U S A. 1995;92:821–825.
33.
Ameli S, Hultgardh-Nilsson A, Regnstrom J, Calara F,
Yano J, Cercek B, Shah PK, Nilsson J. Effect of immunization with
homologous LDL and oxidized LDL on early
atherosclerosis in hypercholesterolemic
rabbits. Arterioscl Thromb Vasc Biol. 1996;16:1074–1079.
34. Palinski W, Hörkkö S, Miller E, Steinbrecher UP, Powell HC, Curtiss LK, Witztum JL. Cloning of monoclonal autoantibodies to epitopes of oxidized lipoproteins from apolipoprotein E-deficient mice: demonstration of epitopes of oxidized low density lipoprotein in human plasma. J Clin Invest. 1996;98:800–814.[Medline] [Order article via Infotrieve]
35.
Sugimoto H, Hayashi H, Yamashita S. Purification, cDNA
cloning and regulation of lysophospholipase from rat liver.
J Biol Chem. 1996;271:7705–7711.
36. de Beer FC, de Beer MC, van der Westerhuyzen DR, Castellani LW, Lusis AJ, Swanson ME, Grass DS. Secretory non-pancreatic phospholipase A2: influence on lipoprotein metabolism. J Lipid Res. 1997;38:2232–2239.[Abstract]
37. Yamamoto S, Suzuki H, Ueda N. Arachidonate 12-lipoxygenase. Prog Lipid Res. 1997;36:23–41.[Medline] [Order article via Infotrieve]
38. Kleinfeld AM, Chu P, Romero C. Transport of long-chain native fatty acids across lipid bilayer membranes indicates that transbilayer flip-flop is rate limiting. Biochemistry. 1997;36:14146–14158.[Medline] [Order article via Infotrieve]
39. Elsing C, Kassner A, Stremmel W. Effect of surface and intracellular pH on hepatocellular fatty acid uptake. Am J Physiol. 1996;271:1067–1073.
40. Sevanian A, Wratten ML, McLeod LL, Kim E. Lipid peroxidation and phospholipase A2 activity in liposomes composed of unsaturated phospholipids: a structural basis for enzyme activation. Biochim Biophys Acta. 1988;961:316–327.[Medline] [Order article via Infotrieve]
41. Sevanian A, Kim E. Phospholipase A2 dependent release of fatty acids from peroxidized membranes. Free Radic Biol Med. 1985;1:263–271.
42. Salgo MG, Corongiu FP, Sevanian A. Enhanced interfacial catalysis and hydrolytic specificity of phospholipase A2 toward peroxidized phosphatidylcholine vesicles. Arch Biochem Biophys. 1993;304:123–132.[Medline] [Order article via Infotrieve]
43. Fourcade O, Simon MF, Viode C, Rugani N, Leballe F, Ragab A, Fournie B, Sarda L, Chap H. Secretory phospholipase A2 generates the novel lipid mediator lysophosphatidic acid in membrane microvesicles shed from activated cells. Cell. 1995;80:919–927.[Medline] [Order article via Infotrieve]
44. Bamberger M, Lund-Katz S, Phillipis M, Rothblat G. Mechanism of the hepatic lipase induced accumulation of HDL cholesterol by cells in culture. Biochemistry. 1985;24:3693–3701[Medline] [Order article via Infotrieve]
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|
|
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|
|
|
|
|
|
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|
|
|
|
 |
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|
|
|
|
|
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|
|
|
|
|
|
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|
|
|
|
|
|
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|
|
|
|
|
|
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|
|
|
|
|
|
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|
|
|
|
|
|
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|
|
|
|
|
|
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|
|
|
|
|
|
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|
|
|
|
|
|
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|
|
|
|
|
|
M. Murakami, R. S. Koduri, A. Enomoto, S. Shimbara, M. Seki, K. Yoshihara, A. Singer, E. Valentin, F. Ghomashchi, G. Lambeau, et al. Distinct Arachidonate-releasing Functions of Mammalian Secreted Phospholipase A2s in Human Embryonic Kidney 293 and Rat Mastocytoma RBL-2H3 Cells through Heparan Sulfate Shuttling and External Plasma Membrane Mechanisms J. Biol. Chem., March 23, 2001; 276(13): 10083 - 10096. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Boren, A. Lookene, E. Makoveichuk, S. Xiang, M. Gustafsson, H. Liu, P. Talmud, and G. Olivecrona Binding of Low Density Lipoproteins to Lipoprotein Lipase Is Dependent on Lipids but Not on Apolipoprotein B J. Biol. Chem., July 13, 2001; 276(29): 26916 - 26922. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Reddy, S. Hama, V. Grijalva, K. Hassan, R. Mottahedeh, G. Hough, D. J. Wadleigh, M. Navab, and A. M. Fogelman Mitogen-activated Protein Kinase Phosphatase 1 Activity Is Necessary for Oxidized Phospholipids to Induce Monocyte Chemotactic Activity in Human Aortic Endothelial Cells J. Biol. Chem., May 11, 2001; 276(20): 17030 - 17035. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. M. Stulnig, J. Huber, N. Leitinger, E.-M. Imre, P. Angelisova, P. Nowotny, and W. Waldhausl Polyunsaturated Eicosapentaenoic Acid Displaces Proteins from Membrane Rafts by Altering Raft Lipid Composition J. Biol. Chem., September 28, 2001; 276(40): 37335 - 37340. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. M. Shih, Y.-R. Xia, X.-P. Wang, E. Miller, L. W. Castellani, G. Subbanagounder, H. Cheroutre, K. F. Faull, J. A. Berliner, J. L. Witztum, et al. Combined Serum Paraoxonase Knockout/Apolipoprotein E Knockout Mice Exhibit Increased Lipoprotein Oxidation and Atherosclerosis J. Biol. Chem., June 2, 2000; 275(23): 17527 - 17535. [Abstract] [Full Text] [PDF]
|
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