Arteriosclerosis, Thrombosis, and Vascular Biology. 1999;19:2909-2917
(Arteriosclerosis, Thrombosis, and Vascular Biology. 1999;19:2909.)
© 1999 American Heart Association, Inc.
Lipoprotein-Associated Phospholipase A2, Platelet-Activating Factor Acetylhydrolase, Is Expressed by Macrophages in Human and Rabbit Atherosclerotic Lesions
Presented in part as preliminary results at the 69th Scientific Sessions of the American Heart Association, New Orleans, La, November 1013, 1996, and published in abstract form (Circulation. 1996;94[suppl I]:I-585).
Tomi Häkkinen;
Jukka S. Luoma;
Mikko O. Hiltunen;
Colin H. Macphee;
Kevin J. Milliner;
Lisa Patel;
Simon Q. Rice;
David G. Tew;
Kari Karkola;
Seppo Ylä-Herttuala
From A.I. Virtanen Institute (T.H., J.S.L., M.O.H., S.Y.-H.) and the
Department of Medicine (J.S.L., S.Y.-H.), University of Kuopio, Kuopio,
Finland; the Departments of Vascular Biology (C.H.M., K.J.M., L.P.), Gene
Expression Sciences (S.Q.R.), and Molecular Recognition (D.G.T.), SmithKline
Beecham Pharmaceuticals, Harlow, Essex, UK; and Provincial State Office of
Eastern Finland (K.K.), Kuopio, Finland.
Correspondence to Dr Seppo Ylä-Herttuala, MD, PhD, A.I. Virtanen Institute, University of Kuopio, PO Box 1627, FIN-70211 Kuopio, Finland. E-mail Seppo.YlaHerttuala{at}uku.fi
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Abstract
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AbstractWe studied the
expression of lipoprotein-associated
phospholipase A
2
(Lp-PLA
2), an enzyme capable of hydrolyzing
platelet-activating
factor (PAF), PAF-like phospholipids, and
polar-modified phosphatidylcholines,
in human and rabbit
atherosclerotic lesions. Oxidative modification
of low-density
lipoprotein, which plays an important role in
atherogenesis, generates
biologically active PAF-like modified
phospholipid derivatives with
polar fatty acid chains. PAF is
known to have a potent proinflammatory
activity and is inactivated
by its hydrolysis. On the other
hand, lysophosphatidylcholine
and oxidized fatty acids released from
oxidized low-density
lipoprotein as a result of Lp-PLA
2
activity are thought to be
involved in the progression of
atherosclerosis. Using combined
in situ hybridization
and immunocytochemistry, we detected Lp-PLA
2 mRNA and
protein in macrophages in both human and rabbit atherosclerotic
lesions.
Reverse transcriptasepolymerase chain reaction
analysis
indicated an increased expression of
Lp-PLA
2 mRNA in human atherosclerotic
lesions. In addition,

6-fold higher Lp-PLA
2 activity was detected
in
atherosclerotic aortas of Watanabe heritable
hyperlipidemic
rabbits compared with normal aortas from
control rabbits. It
is concluded that (1) macrophages in both
human and rabbit atherosclerotic
lesions express Lp-PLA
2,
which could cleave any oxidatively
modified phosphatidylcholine
present in the lesion area, and
(2) modulation of
Lp-PLA
2 activity could lead to antiatherogenic
effects in
the vessel wall.
Key Words: platelet-activating factor atherogenesis oxidized LDL macrophages real-time fluorescence polymerase chain reaction
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Introduction
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Oxidative modification of LDL, monocyte migration into
the vessel
wall, subsequent macrophage activation, and foam
cell formation
are key events in the pathogenesis of
atherosclerosis.
1 2 Several
studies have
demonstrated that one of the earliest events in
LDL oxidation is the
hydrolysis of oxidatively modified phosphatidylcholines,
which
generates lysophosphatidylcholine (lyso-PC) and oxidized
fatty
acids.
3 4 5 6 7 8 9 This hydrolysis of oxidized
phosphatidylcholines
within LDL is mediated by the
lipoprotein-associated phospholipase
A
2
(Lp-PLA
2), also known as a
platelet-activating factor (PAF)
acetylhydrolase, which has been
cloned and characterized previously.
3 6 9 The
arterial wall also contains other types of secreted
group
II phospholipase A
2, which may play a role in
this process.
10 11 12 13 14 Although lyso-PC itself is a potent
biological
effector molecule able to stimulate monocyte
5
and T-lymphocyte
chemotaxis,
15 induce adhesion
molecules
7 16 and various growth
factors,
17
and impair vascular relaxation,
18 19 the oxidized
fatty
acids liberated together with lyso-PC may also possess
relevant
biological activity. Given its many biological properties,
lyso-PC,
together with the enzyme responsible for its generation,
Lp-PLA
2,
has been postulated to play a causal
role in inflammation
20 and
atherosclerosis.
21 In addition to lyso-PC
formation, it
has been established that biologically active PAF-like
polar
phospholipids are formed during the LDL oxidation
process.
22 23 On the other hand, the transient appearance
of these PAF-like
phospholipids has been postulated to be due to their
hydrolysis
and subsequent inactivation by
Lp-PLA
2.
24 Thus, in the context
of
atherogenesis, the enzyme Lp-PLA
2 would appear to
have a
dual role, one that is proinflammatory (generation of lyso-PC)
and
another that is anti-inflammatory (degradation of PAF-like
phospholipids).
To explore further the role of Lp-PLA2 in
atherogenesis, we have investigated whether the enzyme is expressed in
human and rabbit atherosclerotic lesions. Previous work has shown that
in addition to its being distributed among plasma lipoprotein fractions
(predominantly LDL in humans),25 an important cellular
source appears to be macrophages.26 The results of
the present study show that lesion macrophages express
Lp-PLA2 mRNA and protein and that
Lp-PLA2 enzyme activity is increased in rabbit
atherosclerotic lesions.
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Methods
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Tissue Samples
Human aortic samples were obtained from 8 medicolegal
autopsies
(4 males and 4 females, aged 29 to 73 years; cause of death,
traffic
accidents, suicide, and gun shot wounds) 4 to 12 hours
postmortem.
Rabbit aortic samples were dissected from aortic arch and
thoracic
aorta of Watanabe heritable hyperlipidemic
(WHHL) rabbits (aged
6 to 12 months) maintained on normal chow and New
Zealand White
(NZW) rabbits (aged 4 to 6 months) fed a 0.5%
cholesterol diet
for 8 weeks (Table

). Plasma
cholesterol levels for the WHHL
and NZW rabbits were 20 to
23 and 11 to 17 mmol/L, respectively.
The animals were killed
under intravenous fentanyl-fluanisone
(0.3 mL/kg, Hypnorm,
Jansen Pharmaceuticals) and midazolam (1
mg/kg, Dormicum, Hoffman-La
Roche) anesthesia. Tissue samples
were removed,
immersion-fixed for 4 hours in formal sucrose
(4%
paraformaldehyde and 15% sucrose containing 50
µmol/L
BHT and 1 mmol/L EDTA), and rinsed in 15%
sucrose/50 µmol/L
BHT/1 mmol/L EDTA for 12
hours.
27 Serial paraffin-embedded
7- to 10-µm sections
were used for the assays. Atherosclerotic
lesions were classified
according to Stary et al
28 into normal
areas, type I
(initial lesions), type II (fatty streaks), type
III (intermediate
lesions), type IV (atheroma), and type V
(fibroatheroma,
calcified and smooth muscle cellrich
plaques) lesions.
All human studies were approved by the Ethics
Committee of the
University of Kuopio, and animal studies were approved
by the
Experimental Animal Committee of the University of Kuopio.
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Table 1. Human and Rabbit Atherosclerotic Samples Used for In Situ
Hybridization (ISH) and Immunocytochemistry (ICC) Studies
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In Situ Hybridization and Immunocytochemistry
Whole-length 1.4-kb human
Lp-PLA2 antisense and sense riboprobes were
synthesized using T7 and T3 polymerase in the presence of
[33P]UTP (NEN Life Science Products) from a
pBluescript II KS plasmid (Stratagene). In situ hybridizations were
performed on pretreated tissue sections (1x106
cpm per section) as described.27 The final wash was with
0.1x SSC at 53°C for 30 minutes. The slides were dipped in Kodak
NTB-2 nuclear track emulsion (Eastman-Kodak) and exposed for 4 weeks.
Nonhybridizing sense probes were used as
controls.27 29
Serial paraffin-embedded sections were used for immunocytochemistry
with the following antibodies: mouse monoclonal antibody (mAb) against
human macrophages (CD68, dilution 1:150; Dako); mouse mAb
against rabbit macrophages (RAM-11, dilution 1:50; Dako), mouse
mAb against muscle
- and
-actin (HHF35, dilution 1:50; Enzo
Diagnostics), guinea pig polyclonal antisera against
malondialdehyde-modified LDL (MAL-2, dilution 1:1000),30
and highly specific mAbs (2C10 and 3H2, dilution 1:50) against human
Lp-PLA2 purified to homogeneity.9
The preparation and specificity of these monoclonal antibodies has been
described in detail elsewhere.30A Briefly, no
cross-reactivity was noted with 3 different varieties of human
recombinant PLA2: 14-kDa
PLA2, 85-kDa PLA2, and a
recently described related serine-dependent PLA2.
Both 14-kDa PLA2 and 85-kDa
PLA2 are calcium-dependent
arachidonic acidselective enzymes,31
whereas the serine-dependent enzyme is calcium independent and has 40%
amino acid identity with
Lp-PLA2.32
An avidin-biotin-horseradish peroxidase system (Histostain-Plus Kit,
Zymed Laboratories) was used for signal detection according to
manufacturers instructions with either diaminobenzidine or aminoethyl
carbazole as color substrates. When immunocytochemistry was combined
with in situ hybridization to facilitate the simultaneous
detection of Lp-PLA2 mRNA and protein on the same
section, the immunostaining step was performed after
the in situ hybridization.29 33 Irrelevant class- and
species-matched immunoglobulins and incubations without the primary
antibody were used as controls for the
immunostainings.27
Micrographs were taken by a digital camera (SenSys KAF1400-G2,
Photometrics Ltd), processed with digital imageprocessing software
(Image-Pro Plus, Media Cybernetics), and printed using a sublimation
printer (Kodak DS 8650, Eastman-Kodak).
Reverse TranscriptasePolymerase Chain Reaction
Human atherosclerotic plaque mRNAs were isolated from pooled
human samples consisting of type II lesions, type IV lesions, and type
V lesions by use of the Fast Track mRNA isolation kit (Invitrogen) and
reverse-transcribed by use of the cDNA synthesis kit (Invitrogen)
according to the manufacturers instructions. All other RNAs were
isolated by use of Trizol reagent (GIBCO BRL) and were
reverse-transcribed from DNase I (GIBCO BRL)treated total RNA by use
of Superscript II and random hexamer primers (GIBCO BRL) according to
the manufacturers instructions. A series of standards were also
prepared by performing a 4-fold serial dilution of total RNA from a
6-day primary culture of human monocytederived macrophages in
the range 2 µg to 0.12 ng RNA per reverse transcriptase (RT)
reaction.
cDNA samples (5 µL of each) were analyzed for
expression of Lp-PLA2 and the housekeeping gene
GAPDH by a real-time quantitative reverse transcriptasepolymerase
chain reaction (RT-PCR) by use of the fluorescent TaqMan 5'
nuclease assay. TaqMan assay oligonucleotide primers
and probes were designed using Primer Express software, version 1.0 (PE
Biosystems). Each TaqMan hydrolysis probe consisted of the
fluorescent reporter dye 6-carboxyfluorescein
(FAM), covalently linked to the 5' end of the
oligonucleotide, and the quencher dye
6-carboxytetramethylrhodamine (TAMRA), attached to the 3' end via a
linker group (PE Biosystems).
PCRs (5'>3' nuclease assay) were performed in MicroAmp Optical 96-Well
Reaction Plates with Optical Caps (PE Biosystems) by use of the ABI
PRISM 7700 Sequence Detection System for thermal cycling and real-time
fluorescence measurements (PE Biosystems). Each 25-µL
reaction consisted of 1x TaqMan Universal PCR Master Mix (10
mmol/L Tris-HCl [pH 8.3], 50 mmol/L KCl, 10 mmol/L EDTA, 60
nmol/L passive reference dye 1 [6-carboxy-X-rhodamine], 0.2
mmol/L dATP, 0.2 mmol/L dCTP, 0.2 mmol/L dGTP, 0.4
mmol/L dUTP, 5.5 mmol/L MgCl2, 8% glycerol,
0.625 U AmpliTaq Gold DNA polymerase, and 0.25 U AmpErase uracil
N-glycosylase), 300 nmol/L forward primer, 300 nmol/L
reverse primer, 100 nmol/L TaqMan quantification probe, and 5 µL
template with a 20 µL mineral oil overlay (Promega). The forward and
reverse primers for Lp-PLA2 were
5'-CCACCCAAATTGC-ATGTGC-3' and 5'-GCCAGTCAAAAGGATAAACCACAG-3',
respectively. The forward and reverse primers for GAPDH were
5'-GCCAAGGTCATCCATGACAAC-3' and 5'-GGGGCCATC-CACAGTCTTC-3',
respectively. The TaqMan probe sequences (FAM-5'>3'-TAMRA) for
Lp-PLA2 and GAPDH were
5'-TTCTGCCTCTGCGGCTGCCTG-3' and 5'-CTCATGACCACA-GTCCATGCCATCACT-3',
respectively. Reaction conditions were as follows: 50°C for 2
minutes, 95°C for 10 minutes, and then 40 cycles at 95°C for 15
seconds and 60°C for 1 minute. Emitted fluorescence for each
reaction well was measured every cycle during both the denaturation and
annealing/extension phases, and amplification plots were constructed
using the ABI PRISM 7700 Sequence Detection System software, version
1.6 (PE Biosystems).
Subsequent analysis was performed on the data output from the
Sequence detector software by use of Microsoft Excel. In brief, the
arbitrary quantity values generated for Lp-PLA2
expression by Sequence Detector (values were generated by comparison of
the fluorescence generated by each sample with a standard curve
of known quantities) were divided by those obtained for each sample for
GAPDH. This gave a normalized value for the expression level of
Lp-PLA2 in each sample. These values were then
divided by the lowest value obtained (that of the aortic smooth muscle
cells, which was set to 1) to give a fold increase value for each
sample.
As additional controls, monocytes and lymphocytes were included in the
RT-PCR analysis. Monocytes and lymphocytes were isolated from
human blood by countercurrent centrifugal elution with a minor
modification.34 Monocytes were obtained at 95% purity;
lymphocytes, at 100% purity. The macrophage sample was
generated by culturing monocytes for 4 days in RPMI supplemented with
2% human serum and 2 mM glutamine. The monocyte, macrophage,
and lymphocyte samples used in the analysis were from the same
donor. Aortic smooth muscle cells were also included in the assay. The
cells were primary smooth muscle cells from a human donor that were
made quiescent in SmGM-2 medium (Clonetics) over a 2-day period.
Analysis of Rabbit Aortic Lp-PLA2
Activity
WHHL rabbits with a Half-Lop (H/LOP) background (Froxfield Farms
Ltd, Hampshire, UK) were used to investigate
Lp-PLA2 activity in aortic atherosclerotic
lesions. Male WHHL rabbits were compared with sex- and age-matched
nondiseased control rabbits, which were either H/LOP or NZW rabbits
maintained on normal chow. Rabbits were killed with an overdose of
anesthetic, and aortas were immediately removed. Aortic samples were
washed at 4°C in a homogenizing buffer (mmol/L: Tris
50 [pH 8], CHAPS 10, EGTA 2, and EDTA 2, along with 1 µg/mL each of
leupeptin, antipain, and pepstatin-A), and 3x0.5-cm sections at the
very beginning of the ascending aorta were removed, frozen in liquid
nitrogen, and stored at -70°C until analyzed. To measure
aortic PLA2 activity, each slice of aorta was
first homogenized in 1 mL of
homogenization buffer by use of a mortar and pestle
on ice. The homogenate was then removed to Eppendorf tubes
and microfuged for 20 minutes at 4°C. Supernatants (20 µL), which
contained all the PLA2 activity (data not shown),
were then assayed using 50 µmol/L PAF as a substrate exactly as
outlined previously.9 Assays were repeated in the presence
of 300 nmol/L SB-222657, a potent and selective
Lp-PLA2 inhibitor,35 to
demonstrate what proportion of the total activity was attributable to
Lp-PLA2. Activities obtained from the 3 sections
were averaged for each rabbit, and protein content was determined by a
modified Lowry method.36
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Results
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In situ hybridization analysis with human
Lp-PLA
2 antisense
riboprobe showed that
Lp-PLA
2 mRNA expression was localized
over the
macrophage-rich regions in all types of human atherosclerotic
lesions
(type I to V lesions according to the classification of Stary
et
al
28 ). Two representative examples of
Lp-PLA
2 mRNA expression
in human type I and II
lesions (diffuse intimal thickening and
fatty streak, respectively) are
seen in Figures 1C

and 2A

. In
the type I lesion, the thickened
intima consists mostly of smooth
muscle cells (Figure 1B

) and
connective tissue, among which
are scattered solitary
macrophages showing a strong signal (Figure
1C

). In type
II lesion intima there are, among smooth muscle
cells (Figure 2D

), a streak of macrophage foam cells (Figure
2C

), which show a positive hybridization signal (Figure 2A

).
An example of Lp-PLA
2 mRNA expression
in an advanced type IV
lesion (atheroma) is seen in Figure 3A

. This lesion has a macrophage
foam
cellrich cap. The micrograph shows the shoulder area
of the
lesion with scattered macrophage foam cells expressing
Lp-PLA
2.
No hybridization signal was seen with
the corresponding sense
probe (Figure 2B

and 3B

).

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Figure 1. Expression of Lp-PLA2 in type I human
atherosclerotic lesion (diffuse intimal thickening). In situ
hybridization autoradiography and
immunostainings of serial sections (panels A to E) are
shown. A, Antibody specific for CD68 (KP1, dilution 1:150). B, Antibody
specific for muscle actin (HHF35, dilution 1:50). C, Combined in situ
hybridization and immunocytochemistry. Arrows indicate
CD68+ cells (KP1, dilution 1:150) expressing
Lp-PLA2 mRNA (positive black grains in the
autoradiography emulsion overlap the brown
immunostain). D, Antibody specific for Lp-PLA2
protein (2C10, dilution 1:50). An arrow indicates a positively stained
(red) cell. E, Nonimmune control for the
immunostaining. Original magnification x19.9 (panels
A, B, and E) and x39.8 (panels C and D).
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Figure 2. Expression of Lp-PLA2 in type II human
atherosclerotic lesion (fatty streak). In situ hybridization
autoradiography and immunostainings of
serial sections (panels A to F) are shown. A, In situ hybridization
with a [33P]UTP-labeled antisense riboprobe for
Lp-PLA2 (positive cells showing bright signal). B,
Nonhybridizing Lp-PLA2 sense probe. C, Antibody specific
for CD68 (KP1, dilution 1:150). D, Antibody specific for muscle actin
(HHF35, dilution 1:50). E, Nonimmune control for the
immunostaining. F, Antibody specific for
Lp-PLA2 protein (2C10, dilution 1:50). Positive cells are
shown in red. Insert shows a low-magnification view of the section.
Hematoxylin counterstain was used. Panels A and B were taken with
polarized light epiluminescence. An asterisk identifies the same
location of intimal foam cells. Original magnification x19.9 (panels A
to E), x59.7 (panel F), and x9.9 (insert in panel F).
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Figure 3. Expression of Lp-PLA2 in the shoulder
area of type IV human atherosclerotic lesion. In situ hybridization
autoradiography and immunostainings of
serial sections (panels A to F) are shown. A, In situ hybridization
with a [33P]UTP-labeled antisense riboprobe for
Lp-PLA2 (positive cells showing bright signal). B,
Nonhybridizing Lp-PLA2 sense probe. C, Antibody specific
for CD68 (KP1, dilution 1:150). D, Antibody specific for muscle actin
(HHF35, dilution 1:50). E, Antibody specific for Lp-PLA2
protein (2C10, dilution 1:50). Positive cells are shown in red. Insert
shows a low-magnification view of the lesion. F, Nonimmune control for
the immunostainings. Hematoxylin counterstain was used.
Panels A and B were taken with polarized light epiluminescence.
Original magnification x19.9 (panels A to D and panel F), x59.7
(panel E), and x9.9 (insert in panel E).
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Immunostaining of serial sections with monoclonal
antibody 2C10 against Lp-PLA2 protein showed
positive staining in the same areas where Lp-PLA2
mRNA was detected (Figures 1D
, 2F
, and 3E
and the Table
).
By use of a combined in situ hybridization and immunocytochemical
analysis, macrophages (CD68 mAbs) were clearly
identified as the source of Lp-PLA2 mRNA (Figure 1C
). Immunostainings with an antibody for
oxidized LDL (MAL-2) showed a positive signal in the same areas that
were positive for the antibody 2C10 (results not shown). Medial smooth
muscle cells showed no detectable hybridization signal or
immunostaining for Lp-PLA2.
RT-PCR analysis from human type II, type IV, and type V lesions
confirmed the induced expression of Lp-PLA2 mRNA
in atherosclerotic lesions (Figure 4
). It
is also clear from the RT-PCR analysis that macrophages
and lymphocytes are the major source of Lp-PLA2
expression, in view of the fact that only a very low level of
expression was found in human aortic smooth muscle cells (Figure 4
).

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Figure 4. RT-PCR analysis of Lp-PLA2
expression in human type II, type IV, and type V lesions in cultured
human plasma leukocytes and human arterial smooth muscle
cells (SMCs). mRNA was isolated from all samples, transcribed to
first-strand cDNA, and used for real-time fluorescence RT-PCR
analysis as described in Methods. Results from duplicate
determinations are expressed in relation to the expression in primary
aortic SMCs, whose value was set to 1.
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Positive in situ hybridization (data not shown) and immunocytochemistry
for Lp-PLA2 were detected in WHHL rabbit and NZW
rabbit atherosclerotic lesions. Examples of advanced
macrophage-rich atherosclerotic plaques from rabbit aorta are
seen in Figure 5
.
Immunostainings for Lp-PLA2
protein with monoclonal antibody 3H2 (Figure 5
, panels B and F)
colocalized with macrophages (Figure 5
, panels A and E).
As with the human lesions, no positive Lp-PLA2
signal was detected in medial smooth muscle areas (Figure 5
, panels C and G).

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Figure 5. Lp-PLA2 protein in rabbit
atherosclerotic lesion. Serial sections of a WHHL (panels A to D) and
NZW (panels E to H) rabbit atherosclerotic lesion. A and E, Antibody
specific for rabbit macrophages (RAM11, dilution 1:50). B and
F, Antibody specific for Lp-PLA2 protein (3H2, dilution
1:50). Positive cells are shown in red. C and G, Antibody specific for
muscle actin (HHF35, dilution 1:50). D and H, Nonimmune controls for
the immunostainings. An asterisk indicates the same
intimal smooth muscle cell area in panels E to H. Original
magnification x19.9.
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Compared with aortas from age- and sex-matched control rabbits,
extracts from diseased aortas of the WHHL rabbits were shown to contain
increased PLA2 activity (Figure 6
). The identity of the increased
PLA2 activity was confirmed as
Lp-PLA2, in view of the fact that all of the
elevated PLA2 activity could be inhibited by
preincubation of the extract with
Lp-PLA2specific inhibitor
SB-222657. From these findings, it was also demonstrated that whereas
in control rabbits
60% of the aortic PAF-hydrolyzing activity could
be attributed to Lp-PLA2, this proportion was
increased to 90% in aortas from diseased rabbits (Figure 6
).
This actually represents a >6-fold increase in
Lp-PLA2 activity in atherosclerotic lesions from
WHHL rabbits compared with aortas from the control rabbits.

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Figure 6. Lp-PLA2 activity is elevated in rabbit
atherosclerotic lesions. Upper aortic segments from sex- and
age-matched WHHL (n=6), normal H/LOP (n=4), and normal NZW (n=4)
rabbits were homogenized and assayed for PLA2
activity, in the absence or presence of 300 nmol/L SB-222657, which is
a selective Lp-PLA2 inhibitor as outlined in
Methods. PAF-AH indicates PAF acetylhydrolase. Data represent
the mean±SD.
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 |
Discussion
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Lp-PLA
2 is found to be predominantly
associated with LDL in
human plasma,
25 37 and it is also
known that macrophages secrete
Lp-PLA
2
activity in cell culture.
20 26 How much
macrophages
contribute to blood levels of
Lp-PLA
2 is presently unknown.
More recently,
it has been speculated that the level of Lp-PLA
2
in
the blood is completely dependent on the rate of lipoprotein
clearance.
38 In this model, blood
Lp-PLA
2 levels will be lower when the
rate of
lipoprotein, particularly LDL, removal is high, and
the opposite is
true when the clearance rate of lipoproteins
is low. Consistent
with this notion is the observation that
small dense LDL, a lipoprotein
pool that is slowly metabolized
and very atherogenic,
39 is
actually enriched with
Lp-PLA
2.
40
Lp-PLA2 would appear to play an important role in
inflammatory reactions. On one hand, this enzyme is capable of
hydrolyzing and inactivating PAF and related oxidized or polar
phospholipids, whereas on the other hand, it has the capacity for
generating large quantities of 2 proinflammatory lipid mediators,
lyso-PC and free oxidized fatty acids, after the hydrolysis of oxidized
phosphatidylcholines. Which of these activities predominate in
atherogenesis remains unknown.21 23 In the present
study, we show that Lp-PLA2 is expressed in
lesion macrophages and that Lp-PLA2
enzyme activity is 6-fold higher in WHHL rabbit atherosclerotic
arteries than in control rabbit arteries. Thus, the expression and
enzyme activity of Lp-PLA2 are increased in
atherogenesis, which is characterized by a microenvironment of high
oxidative stress and the presence of oxidized LDL.41 In
situ hybridization and RT-PCR were used to confirm arterial
expression of Lp-PLA2, which cannot be
distinguished from LDL or plasma-derived Lp-PLA2
on the basis of immunocytochemistry or enzyme activity
analyses. Also, simultaneous in situ hybridization
and cell typing by immunocytochemistry were used to confirm that
macrophages are the source of the enzyme in atherosclerotic
lesions.
Oxidized LDL plays an important role in the pathogenesis of
atherosclerosis.2 Oxidized LDL is
present in atherosclerotic lesions in vivo,41 and at
least part of the proinflammatory effects of oxidized LDL are mediated
by lyso-PC.3 4 5 6 7 Indeed, several studies have indicated
that elevated levels of lyso-PC are found in atherosclerotic
lesions.42 43 Lp-PLA2 may be a key
enzyme responsible for the increased formation of lyso-PC in
atherosclerotic lesions, in view of the fact that oxidative
modification of LDL generates substrates for the enzyme. Thus,
expression of Lp-PLA2 in activated
macrophages will probably lead to the release in
atherosclerotic lesions of lyso-PC and free oxidatively modified fatty
acids in potentially large quantities. Several biological activities
have been assigned to increased lyso-PC content, such as
chemoattractant activity for human monocytes,5
endothelial dysfunction,18 44 induction of
the expression of endothelial leukocyte adhesion
molecules,7 and increased expression of
platelet-derived growth factor and heparin-binding epidermal growth
factorlike proteins.17 Thus, although preventing the
proposed biological activities of PAF-like substances,
Lp-PLA2 could augment the atherosclerotic process
by releasing into the microenvironment increased concentrations of
lyso-PC and oxidatively modified free fatty acids from oxidized LDL.
It has been shown previously that Lp-PLA2 is able
to inhibit LDL oxidation in vitro.8 On the other hand,
others have not been able to confirm these
observations.6 9 32 44 Whether
Lp-PLA2 activity is primarily proatherogenic or
antiatherogenic remains to be elucidated. The final test will come from
evaluating potent and selective inhibitors of the enzyme in
animal models of atherosclerosis. It appears that
Lp-PLA2 expression is clearly derived from
monocyte/macrophages and lymphocytes, whereas group II
secretory phospholipase A2 is highly expressed in
smooth muscle cells in both normal and atherosclerotic
arteries.13 Also, group II phospholipase
A2 can cleave normal unmodified LDL
phospholipids, whereas Lp-PLA2 requires oxidation
to generate a substrate.32 Thus,
Lp-PLA2 is closely associated to the inflammatory
aspects of atherogenesis and oxidation of LDL. Even though
Lp-PLA2 can be anti-inflammatory under certain
conditions, such as in a rat foot pad model after exogenous PAF
application,20 lyso-PC and free oxidized fatty acids in
atherosclerotic lesions can substantially amplify the pathological
process and cause chronic monocyte/macrophage-dominated
inflammation, which is typical of atherosclerosis, in
view of the fact that the arterial wall contains much
higher concentrations of LDL than most other
physiological compartments.45 46
Increased expression of Lp-PLA2 in lesion
macrophages suggests that modulation of the enzyme activity
could become a potential target for the development of antiatherogenic
therapy in the vessel wall.
 |
Acknowledgments
|
|---|
This study was supported by grants from the Finnish Foundation
for
Cardiovascular Research, the Sigrid Juselius
Foundation, and
the Finnish Academy. The authors also thank Mervi
Nieminen for
excellent technical assistance, Dr Theresa Reape for the
supply
of smooth muscle cells, and Marja Poikolainen for preparing
the
manuscript.
Received September 28, 1998;
accepted May 28, 1999.
 |
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