© 1999 American Heart Association, Inc.
Vascular Biology |
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 10–13, 1996, and published in abstract form (Circulation. 1996;94[suppl I]:I-585).
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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Abstract—We studied the expression of lipoprotein-associated phospholipase A2 (Lp-PLA2), 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-PLA2 activity are thought to be involved in the progression of atherosclerosis. Using combined in situ hybridization and immunocytochemistry, we detected Lp-PLA2 mRNA and protein in macrophages in both human and rabbit atherosclerotic lesions. Reverse transcriptase—polymerase chain reaction analysis indicated an increased expression of Lp-PLA2 mRNA in human atherosclerotic lesions. In addition,
6-fold higher Lp-PLA2 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-PLA2,
which could cleave any oxidatively modified phosphatidylcholine
present in the lesion area, and (2) modulation of
Lp-PLA2 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 A2 (Lp-PLA2), 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 A2, 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 monocyte5 and T-lymphocyte chemotaxis,15 induce adhesion molecules7 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-PLA2, has been postulated to play a causal role in inflammation20 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-PLA2.24 Thus, in the context of atherogenesis, the enzyme Lp-PLA2 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 al28 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 cell–rich
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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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 acid–selective 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 manufacturer’s 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 image–processing software (Image-Pro Plus, Media Cybernetics), and printed using a sublimation printer (Kodak DS 8650, Eastman-Kodak).
Reverse Transcriptase—Polymerase 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 manufacturer’s 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 manufacturer’s 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 monocyte–derived 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 transcriptase—polymerase 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-PLA2 antisense riboprobe showed that Lp-PLA2 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 al28 ). Two representative examples of Lp-PLA2 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-PLA2 mRNA expression
in an advanced type IV lesion (atheroma) is seen in Figure 3A. This lesion has a macrophage
foam cell–rich cap. The micrograph shows the shoulder area of the
lesion with scattered macrophage foam cells expressing
Lp-PLA2. No hybridization signal was seen with
the corresponding sense probe (Figure 2B and 3B).
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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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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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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-PLA2–specific 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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Discussion |
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Lp-PLA2 is found to be predominantly associated with LDL in human plasma,25 37 and it is also known that macrophages secrete Lp-PLA2 activity in cell culture.20 26 How much macrophages contribute to blood levels of Lp-PLA2 is presently unknown. More recently, it has been speculated that the level of Lp-PLA2 in the blood is completely dependent on the rate of lipoprotein clearance.38 In this model, blood Lp-PLA2 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-PLA2.40
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 factor–like 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.
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Acknowledgments |
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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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