© 2000 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
Mildly Oxidized LDL Induces Expression of Group IIa Secretory Phospholipase A2 in Human Monocyte–Derived Macrophages
From the UNIGEN Center for Molecular Biology (M.W.A., B.J.), Norwegian University of Science and Technology, Trondheim, Norway; and Institut National de la Sante et de la Recherche Medicale (INSERM) Unit 321 (D.S., D.H., E.N.), Lipoproteines et Atherogenese, Hopital de la Pitie, Paris, France.
Correspondence to Berit Johansen, UNIGEN Center for Molecular Biology, Norwegian University of Science and Technology, N-7489 Trondheim, Norway. E-mail Berit.Johansen{at}chembio.ntnu.no
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Abstract |
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Abstract—Phospholipase A2s (PLA2s) constitute a family of enzymes that hydrolyze fatty acids of membrane phospholipids, thus initiating the synthesis of proinflammatory mediators. Various PLA2s have been detected in human atherosclerotic arteries (advanced lesions); however, only the secretory group of PLA2 has been shown to specifically hydrolyze low density lipoprotein (LDL)–associated phospholipids and, as such, may play a potential role in atherogenesis. In the present study, we investigated the expression pattern of group IIa, IV, and V PLA2s in human macrophages, which are the key cells involved in the onset and perpetuation of atherosclerosis. Immunohistochemical staining by double labeling showed that the secretory nonpancreatic PLA2 (snpPLA2) is detectable in macrophages in the intima of early atherosclerotic lesions. Reverse transcription–polymerase chain reaction analysis of RNA extracted from human monocytes clearly showed that expression of group IV PLA2 was enhanced during differentiation into macrophages, with an onset of induction at days 2 to 3 of differentiation. Group V snpPLA2 was constitutively expressed on differentiation, whereas the detection of group IIa snpPLA2 was dependent on both differentiation and subsequent stimulation of macrophages. Indeed, the transcription of group IIa snpPLA2 in macrophages was induced by treatment with minimally modified or mildly oxidized LDL, whereas native, extensively oxidized, or acetylated LDL had no effect. To our knowledge, this is the first report describing induction of group IIa snpPLA2 expression in human monocyte–derived macrophages. The mRNA levels of cytosolic PLA2 group IV and snpPLA2 group V remained unchanged on LDL treatment. Thus, our results show that the expression of distinct PLA2 enzymes is regulated not only during differentiation of monocytes into macrophages but also on exposure of macrophages to distinct LDL species. Consequently, our results indicate a potential role for both cytosolic and secretory PLA2 enzymes in inflammation and in macrophage functions related to atherosclerosis, with a specific role for group IIa snpPLA2 in LDL scavenging.
Key Words: atherosclerosis • phospholipase A2 • LDL • immunohistochemistry • macrophages
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Introduction |
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Atherosclerosis is a chronic inflammatory disease characterized by the early and persistent presence of macrophages. The recruitment of circulating monocytes into the vascular intima and their subsequent transformation into macrophages/foam cells are key elements of the initiation and development of atherosclerosis. Foam cell formation is a result of macrophage scavenging of modified lipoproteins that have undergone oxidative or enzymatic modification in the vascular wall.1 However, the mechanisms by which macrophages are transformed into foam cells are not fully elucidated.
Phospholipase A2 enzymes (PLA2s; EC 3.1.1.4) hydrolyze fatty acids esterified at the sn-2 position of glycerophospholipids, thus generating unsaturated, free fatty acids and lysophospholipids. PLA2s catalyze the rate-limiting step in the formation of biologically active lipids, including prostaglandins, hydroxy fatty acids, leukotrienes, thromboxanes, and platelet-activating factor. The PLA2 enzymes comprise a heterogeneous family of enzymes with distinct enzymatic properties, including substrate specificity and Ca2+ requirement.2 Ten different PLA2s have been described,2 3 and 8 have been found in human tissues, whereas only 3 of them are specifically involved in arachidonic acid release.
The PLA2 enzymes relevant to arachidonic acid release in human tissues may be grouped as secretory or cytosolic (c) enzymes. The cytosolic group encompasses the group IV 85-kDa PLA2s,4 5 6 which are ubiquitously expressed and regulated by micromolar concentrations of Ca2+ and reversible phosphorylation.7 Several distinct secretory PLA2s have been identified2 ; they require millimolar concentrations of calcium for catalytic activity but do not display any specificity for arachidonic acid in the phospholipid sn-2position. The expression of group IIa8 and group V9 secretory nonpancreatic PLA2s (snpPLA2s) and equally of group IV cPLA2 has been shown to be regulated in vitro by proinflammatory cytokines such as tumor necrosis factor and interleukin-1.10 11 Group IIa and V secretory PLA2s have also been reported to be involved in arachidonic acid release during inflammatory conditions.12 13 14 An increased level of secretory PLA2 in the circulation and locally in the tissue has been found in association with various pathological states, such as rheumatoid arthritis, sepsis, infections, lung inflammation, and psoriasis.15 16 17 18
PLA2 enzymes have been detected in atherosclerotic arteries and have been suggested to be involved in the progression of atherosclerosis.19 20 21 22 23 By immunofluorescence studies, the group I22 and IIa21 22 snpPLA2s and the group IV cPLA222 were found in advanced lesions of human atherosclerotic arteries. Other properties of secretory PLA2s relevant to the development of atherosclerosis relate to their ability to hydrolyze phospholipids in LDL.21 24 Also, it has been found that both snpPLA2 and LDL bind to proteoglycans,20 thus increasing the availability of LDL as a substrate for snpPLA2. Indeed, Aviram and Maor25 reported that PLA2-modified LDL was taken up more avidly by macrophages, leading to enhanced formation of foam cells. Additionally, the snpPLA2 may contribute to the inflammatory response by releasing proinflammatory lipid mediators, including lysophosphatidylcholine, lyso–platelet-activating factor, and free fatty acids.
Despite the immunohistochemical detection of PLA2 enzymes in advanced lesions of atherosclerotic arteries, the cellular source of PLA2 detected either extracellularly or intracellularly in the atherosclerotic intima is uncertain. In our previous study,21 we detected, by immunohistochemical methods, the snpPLA2 enzyme in smooth muscle cells in both the media and intima and in foam cells in the intima of advanced atherosclerotic lesions. However, it is still uncertain whether the snpPLA2 detected in foam cells is expressed by them or rather merely associates with LDL that has infiltrated into the intima and becomes endocytosed as a complex with LDL.
In an effort toward understanding the role of various PLA2 enzymes in macrophage function during atherosclerosis, we have undertaken to determine the expression pattern of the proinflammatory (groups IIa, IV, and V) PLA2 enzymes in human monocytes and monocyte-derived macrophages. Subsequently, we investigated the impact of either native, minimally modified, or oxidatively modified LDL on the expression of these enzymes. We report herein for the first time that the expression of group IIa PLA2s in human macrophages is upregulated by a brief treatment with either minimally modified (MM) or mildly oxidized (ox) LDL but not with native, extensively oxidized, or acetylated LDL. In contrast, the mRNA levels of groups IV and V PLA2 remained unchanged, independent of the oxidation status of LDL.
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Materials
Plasma and buffy coats for monocyte isolation were obtained from the Hospital Transfusion Center (Hopital Broussais, Paris, France). RPMI-1640 culture medium and PBS were supplied by BioWhittaker. Pools of human sera (100 to 150 donors) for use in cell culture media were supplied by ATGC Biotechnologies. Nutridoma HU medium was supplied by Boehringer Mannheim. "RNA plus" was from Bioprobe System. Dynazyme DNA polymerase was obtained from Dynazyme (Finnzymes Oy). Moloney murine leukemia virus reverse transcriptase was from Gibco-BRL, and RNasin was from Promega. A mouse monoclonal antibody (mAb), BF1 (subclass IgG2b), kindly provided by Jeff Browning, Biogen Inc, Cambridge, Mass, generated against human recombinant group IIa PLA2 was used to detect group IIa PLA2 in tissue sections. The following antibodies were from Dako: anti-CD68 (clone EBM11), anti-CD14 (clone TÜK4), anti-CD3 (clone T3-4B5), and goat anti-mouse FITC-conjugated IgG. Rabbit anti-mouse TRITC-conjugated IgG1 was purchased from Europa Research Products, and FITC-labeled rabbit anti-mouse IgG2b was from Advanced ChemTech. Control mAb TWAR, subclass IgG2a, generated against Chlamydia pneumoniae, was kindly provided by Are Dalen, Faculty of Medicine, Norwegian University of Science and Technology, Norway. The chromogenic Limulus amebocyte lysate assay for lipopolysaccharide was purchased from Biogenic. The assay kits for lactic dehydrogenase and protein bicinchoninic acid reagents were purchased from Boehringer Mannheim and Pierce Interchim, respectively.
Tissue Specimens
Arteries were obtained at autopsy or surgery. All
pathological samples were obtained according to institutional
guidelines, were quickly frozen in LN2, and were
stored at -80°C until sectioning. Tissue samples were imbedded in
optimum cutting temperature compound (OCT, Tissue-Tek, Miles
Laboratories) and snap-frozen in LN2. Freshly cut
frozen sections of 5-µm thickness were collected on
poly-L-lysine–coated slides and allowed to dry for 1 hour
before storage at -20°C. Immediately before being stained, the
sections were fixed in ice-cold acetone for 5 minutes.
Immunohistochemistry
Tissue sections were stained with mAbs by using an
indirect biotin/streptavidin/FITC technique described
previously.18 21 In direct double FITC/TRITC detection,
the following antibodies were used: mouse mAb BF1 (subtype IgG2b),
recognizing group IIa PLA2, and an mAb specific
for macrophage marker CD68 (subtype IgG1) were used in
double-labeling experiments detected with FITC- or TRITC-conjugated
subtype-specific rabbit polyclonal antibodies diluted 1:25 or 1:20,
respectively, in PBS with 1.2% BSA. Staining with secondary antibodies
alone or with an irrelevant primary mAb TWAR was used as a control for
nonspecific fluorescence. The microscope was equipped with a
double-channel filter set with an excitation range of 450 to 490 nm and
of 510 to 560 nm; barrier filters sensitive at 520 and 590 nm were
included, thus allowing separation of the green (FITC) and red
(TRITC) images.
Characterization of Human Monocyte–Derived Macrophages
Monocytes and macrophages were characterized with
specific antibodies, which were detected by indirect
immunostaining. CD14 and CD68 were used as specific
makers for monocytes and CD3, for lymphocytes. At day 14 of culture,
all adherent cells were CD68-positive and CD3-negative, thus indicating
the absence of T cells.
Isolation, Culture, and Treatment of Human Monocyte–Derived
Macrophages
Human mononuclear cells were isolated from buffy coats of
cells in blood samples drawn from individual healthy donors (20
to 35 mL) as described earlier.26 In brief, buffy coats
diluted with PBS (1:1, vol/vol) were carefully loaded onto a Ficoll
gradient under sterile conditions. After an initial
centrifugation step (20 minutes, 2700g),
monocytes were collected at the interface and then washed 3 times at
room temperature in PBS containing 0.1% EDTA (successively at
1000g, 340g, and 160g for 10 minutes)
and then once in PBS alone at 160g for 10 minutes. Finally,
the cell pellet was resuspended in RPMI-1640 medium containing
gentamicin (40 µg/mL) and glutamine (0.05%). A typical monocyte
preparation yielded 100x106 cells per donor. The
cells were seeded at a density of 3x106 cells
per well in Primaria 6-well dishes. After 45 minutes of adherence, the
cells were washed twice with PBS, and finally fresh medium containing
10% pooled human sera was added. The culture medium was changed after
6 days. At day 14 of culture, monocyte-derived macrophages
(denoted as macrophages) were washed 2 times with PBS and then
incubated for defined time intervals with 100 µg/mL native LDL or
oxLDL in the aforementioned culture medium, to which 1% (vol/vol)
Nutridoma had been added instead of human serum. Incubations were
carried out in a humidified 37°C incubator (95% air atmosphere/5%
CO2). Cell viability was measured by trypan blue
exclusion and by the release of lactic dehydrogenase activity (5% to
10%) into the medium. At the end of the incubation period (6 hours),
cells were washed 2 times with PBS, and the total RNA from
6-well plates was immediately extracted with RNA plus.
Lipoprotein Purification and Chemical Modifications
Normolipidemic plasma of healthy blood donors was used to
isolate LDL by sequential ultracentrifugation. Native
LDLs (d=1.024 to 1.050 g/mL) were centrifuged at
their upper limiting density, thereafter extensively dialyzed at 4°C
against 0.01 mol/L PBS (pH 7.4) deionized with Chelex-100, subjected to
a short (1-hour) dialysis in RPMI-1640, and used immediately in cell
cultures. Aliquots of LDL were taken to check the purity of each
preparation as described elsewhere.27 Protein content
was determined with the use of the bicinchoninic acid assay kit.
Copper-oxidized LDLs (oxLDLs) were prepared after dialysis in PBS
without EDTA by incubating 500 µg of LDL protein per milliliter in
PBS containing 2.5 µmol/L CuCl2 for 2, 6,
or 48 hours at 37°C under sterile conditions. At the end of the
incubation period, oxidations were stopped with EDTA (1 mmol/L),
and LDLs were extensively dialyzed at 4°C, first against PBS (pH 7.4)
and then against RPMI-1640, and subsequently filtered through a
0.22-µm filter (Millipore). The time course of the copper-induced
oxidation of LDL was deduced from the spectrophotometric measurement of
conjugated-diene formation at 234 nm. The electrophoretic mobility was
expressed as that of oxLDL relative to that of native LDL (ie, relative
electrophoretic mobility [REM]).28 Determination of the
content of thiobarbituric acid–reacting substances,
expressed as malondialdehyde equivalents29 in oxLDL,
provided an estimation of the degree of lipid oxidation. Typically,
native LDL preparations contained <1.3 nmol/mg LDL protein (n=2). For
other forms of altered LDL, respective values were as follows: MM-LDL,
5.3±4; 2-hour–oxidized LDL (designated as mildly oxidized LDL),
11.5±5; 6-hour–oxidized LDL (designated as moderately oxidized LDL),
23.4±12; and 48-hour–oxidized LDL (designated as extensively oxidized
LDL), 61±2. Acetylated LDLs were prepared according to the
procedure of Basu et al30 and typically had REM values of
3 to 4. Endotoxin content of all LDL preparations was measured with the
chromogenic Limulus amebocyte lysate kit and was
always <50 pg/100 µg LDL protein.
RNA Isolation
Total RNA was isolated from adherent macrophages in
6-well culture dishes with RNA plus according to the protocol of the
manufacturer. RNA concentrations were spectrophotometrically determined
at 260 nm.
Reverse Transcription (RT) and Polymerase Chain Reaction
(PCR)
First-strand cDNA synthesis was generated from 2 µg of total
RNA by using random hexamers as primers. The RT reaction was performed
in a final volume of 20 µL and contained 5 ng/mL random hexamers,
1 mmol/L dNTP, 2 U/µL RNasin (Promega), and 10 U/µL of Moloney
murine leukemia virus reverse transcriptase (Gibco-BRL). The reaction
was carried out at 37°C for 60 minutes. Human
PLA2 enzymes and ß-actin were amplified from 4
to 6 µL of the cDNA reaction by using specific gene primers. PCRs
were performed in a final volume of 50 µL containing 0.4 mmol/L
of each primer and 1.2 U of Dynazyme DNA polymerase. Primers
synthesized according to known human cDNA sequences were the following:
5'-GAAGTTGAGACCACCCAGCA-3'(forward primer) and
5'-GTTGCATCCTTGGGGGATCCTCTG-3' (reverse primer) for group IIa
snpPLA2; 5'-GAGTTTTGGGCGTTTCTGGT-3' (forward
primer) and 5'-ACGGCAGGTTAAATGTGAGC-3' (reverse primer) for group
IV cPLA2; 5'-GCAACATTCGCACACAGTCC-3' (forward
primer) and 5'-CCCTACCAAGTCCTATGACC-3' (reverse primer) for group V
snpPLA2; and 5'-GAAATCGTGCGTGACATTAAG-3' (forward
primer) and 5'-CTAGAAGCATTTGCGGTGGACGATGGAGGGGCC-3'
(reverse primer) for ß-actin. The PCR consisted of 50 cycles of 50
seconds at 94°C, 50 seconds at 57°C, and 30 seconds at 72°C (for
amplification of PLA2 genes) and 20 cycles of 50
seconds at 94°C, 50 seconds at 65°C, and 30 seconds at 72°C (for
amplification of ß-actin). The PCRs were preceded by 3 minutes
at 94°C.
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Results |
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Cellular Localization of snpPLA2 in Early Intimal Atherosclerotic Lesions
We have recently shown that snpPLA2 is associated with foamlike cells in the lipid core of advanced lesions in atherosclerotic arteries.21 In this study, we stained serial sections of early intimal lesions of human aorta, macroscopically characterized by fatty streak formation, with antibodies directed toward group IIa snpPLA2 and toward the specific macrophage antigen CD68. Morphology and positive anti-CD68 staining easily permitted identification of macrophages (Figure 1A
). Cells of
similar morphology stained positively with antibody specific for
snpPLA2 in the neighboring section (Figure 1B). Double labeling was performed by subtype-specific
TRITC-conjugated antibody recognizing anti-CD68 (Figure 1C) and
FITC-conjugated subtype-specific antibody recognizing
anti-snpPLA2 (Figure 1D), which showed
coexpression of CD68 and snpPLA2 proteins in the
same cell. Thus, the macrophages present in the intimal
region in early lesions of atherosclerotic arteries clearly express
snpPLA2.
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Expression of PLA2s During Differentiation of Human
Monocytes Into Macrophages
For the purpose of this study, we developed an in vitro system
that enabled us to examine the expression of distinct isoforms of
PLA2 in human monocytes and in mature
macrophages. Therefore, we utilized the calibrated RT-PCR
procedure to assess the mRNA levels of different
PLA2 isoforms in human monocytes on
differentiation into macrophages. Monocytes were cultured for 0
to 14 days in the presence of human serum, and the RT-PCR
analyses were performed on total cellular RNA. PCR
amplification of group IIa, IV, and V PLA2s was
performed by using ß-actin as a control. The amplifications were
performed from the same cDNA preparations to allow relative comparisons
between the intensity of different PLA2
transcripts. Figure 2 shows the time
course of expression of PLA2 isoforms in
monocytes and macrophages as evaluated by RT-PCR.
Interestingly, the mRNA level of group IV cPLA2
was increased during differentiation of monocytes into
macrophages, the onset occurring at days 2 to 3 of culture.
Group IIa snpPLA2 was detected in neither
monocytes nor fully differentiated unstimulated macrophages.
Group V snpPLA2 was expressed at a constant but
low level. The RT-PCR mRNA determinations were repeated 3 times with
each RNA preparation obtained from 3 independent donors.
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Expression of PLA2 Isoforms in Macrophages
Treated With LDL
Native and modified LDLs have been shown to influence expression
of genes relevant to atherosclerosis, eg,
macrophage scavenger receptors, monocyte chemotactic protein-1,
and macrophage colony stimulating factor.31 To
investigate whether the exposure of macrophages to LDL
regulated the expression of PLA2 isoforms in
human macrophages, we performed the RT-PCR analysis of
total RNA extracted from macrophages differentiated for 14 days
and subsequently treated for 6 hours with LDLs oxidized to various
extents with copper ions (Figure 3).
Surprisingly, the expression of group IIa PLA2
was now easily detectable in macrophages treated with MM-LDL or
with LDL oxidized for 2 hours (lanes labeled 2 and 3). Although
MM-LDL and mildly oxidized LDL (2-hour oxidation) induced the
expression of group IIa snpPLA2, extensively
oxidized LDL (48-hour oxidation) had no such effect. The amounts of
mRNA of group IV and group V PLA2s were not
changed by the LDL treatments. Similar results were observed in 4
independent macrophage preparations (from 4 different donors),
which were treated with various LDL preparations. To examine the
importance of LDL modification for snpPLA2 mRNA
synthesis, RT-PCR analysis of group IIa expression was
performed on RNA isolated from macrophages treated with
native LDL, 2 species of MM-LDL, and LDL oxidized for 2 or 6
hours (Figure 4). Native LDL (dialyzed in
the presence of EDTA) failed to induce group IIa
snpPLA2 mRNA (lane labeled 2), whereas native LDL
left for 8 days at 4°C (lane labeled 3), MM-LDL (lane labeled 4), and
oxLDL (lanes labeled 5 and 6) significantly increased
snpPLA2 mRNA expression (Figure 4). Thus,
minimal oxidation of LDL (
5 to 16 nmol malondialdehyde per milligram
LDL protein) is a prerequisite for induction of
snpPLA2 mRNA in macrophages.
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The mechanism by which such mildly oxidized LDL affects
snpPLA2 transcription may involve the lipid
and/or the protein moieties of LDL. To address this question, we
compared the effects of native or oxLDL with those of
acetylated LDL on snpPLA2 mRNA induction
in human macrophages. While mildly oxidized LDL produced the
expected induction of group IIa snpPLA2
expression, their acetylated counterparts had no effect on
snpPLA2 mRNA synthesis (Figure 5). Thus, we suggest that the induction
of snpPLA2 transcription is not mediated by the
scavenger receptor A family of LDL scavenger receptors.
Moreover, we suggest that the stimulatory effect of mildly oxidized LDL
on snpPLA2 expression is not a consequence of
massive cholesterol loading of macrophages.
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Discussion |
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Several enzymes of the PLA2 family have been detected in human atherosclerotic lesions; therefore, raising the question of their participation in atherogenesis is relevant. Monocyte-derived macrophages play a key role in the initiation of atherosclerosis owing to their ability to create inflammatory reactions and their evolution toward foam cells. However, the presence and regulation of distinct PLA2 isoforms in human macrophages have received little attention. Previous studies performed on plaques from advanced atherosclerotic lesions have shown that the snpPLA2-positive staining was associated with foam cells derived from macrophages.19 21 However, such colocalization with macrophages could be a result of endocytosis of snpPLA2 trapped by modified LDL. In the intima of arterial tissue, both snpPLA2 and LDL have been found to associate with the same negatively charged glycosaminoglycans. Additionally, the PLA2 enzymatic activity toward LDL is clearly stimulated by such associations.20 One may therefore speculate that there is an association between snpPLA2 and LDL before their eventual endocytosis by macrophages. We therefore undertook this study to examine (1) whether macrophages could be the source of snpPLA2, (2) which other PLA2 isoforms are expressed in macrophages, and (3) how they are regulated on maturation and exposure to LDL. By using specific anti-snpPLA2 antibodies and antibodies toward macrophage antigen CD68, we found that macrophages from the intima of early atherosclerotic lesions contained substantial amounts of immunoreactive snpPLA2, as previously demonstrated for advanced lesions.21 Our results clearly show that snpPLA2 is already present in macrophages at an early stage of atherosclerosis. Thus, snpPLA2 may be involved in the initiation of atherogenesis and may function to accelerate the inflammatory reactions and enhance foam cell formation.
Owing to the existence of several homologous variants of secretory
PLA2, we utilized calibrated RT-PCR
analysis to establish precisely the expression pattern of
PLA2s in human monocyte–derived
macrophages. Expressional analysis by RT-PCR is of
primary importance because of the possibility of cross-reactivity of
antibodies between different groups of secretory
PLA2. Therefore, we investigated the mRNA
expression of group IV cPLA2 by RT-PCR in human
monocytes during differentiation into macrophages and showed
that it was strongly elevated, indicating that
cPLA2 may possess important functions in mature
macrophage. Indeed, cPLA2 has also been
detected in macrophages in atherosclerotic
arteries.22 Additionally, previous results obtained from
macrophage-like cell lines argue in favor of a prominent role
of cPLA2 in macrophage intracellular
arachidonic acid signaling and propagation of
inflammatory reactions,32 33 34 as well as in functional
coupling to the secretory PLA2s leading to enzyme
activation.13 35 36 Nakamura et al37
have shown that treatment of human monocytes with macrophage
colony stimulating factor stimulated expression of
cPLA2 and promoted its own
phosphorylation; thus these authors attributed a role
for cPLA2 in monocyte proliferation and
differentiation. Therefore, our results showing an upregulation of
cPLA2 mRNA expression on differentiation
corroborate the aforementioned results.37 In our study,
the expression of group V snpPLA2 remained
unchanged during differentiation, whereas group IIa
snpPLA2 could not be detected at any stage of
differentiation of human monocytes into macrophages. However,
in 1 set of experiments, we were able to show that
snpPLA2 is expressed to some extent in untreated
macrophages (Figure 4
), possibly due to a brief
proinflammatory exposure of the monocyte donors before blood donation.
Nevertheless, our observations that mildly oxidized LDL increases
expression of group IIa PLA2, independent of
initial snpPLA2 expression levels, remain
valid.
Native and moderately modified LDLs have been shown to exhibit both
stimulatory and inhibitory effects on gene transcription,
eg, on macrophage scavenger receptors,38
platelet-activating factor receptor,26 granulocyte
macrophage colony stimulating factor,39 monocyte
chemotactic protein-1,40 41 and cellular adhesion
molecules.42 43 To examine whether LDL modulated the
expression of PLA2 genes, human monocyte–derived
macrophages were incubated with various LDL preparations
displaying different degrees of oxidation, and
PLA2 mRNA levels were assessed by RT-PCR.
Transcription of the group IV and V PLA2s was
unaffected by the LDL treatments. However, an interesting finding was
that group IIa snpPLA2 mRNA was strongly induced
in macrophages by MM-LDL and mildly oxidized LDL (Figure 3). Native LDL (protected from oxidation with EDTA and used
immediately after preparation) did not induce
snpPLA2 group IIa expression, whereas native LDL
left for 8 days at 4°C augmented its expression, possibly owing to
increased oxidation on storage (Figure 4). MM-LDL and mildly
oxidized LDL stimulated snpPLA2 expression to a
greater extent than did LDL oxidized in the presence of copper ions.
This indicates that a slight oxidative modification of LDL particles is
necessary to stimulate the expression of group IIa
snpPLA2.
Treatment of macrophages with acetylated or extensively
oxidized LDL did not affect mRNA levels of group IIa
PLA2, suggesting that the LDL-regulated
expression of group IIa PLA2s is not mediated by
the scavenger receptor A family of scavenger receptors. The results
presented herein were obtained by treatment of
macrophages with LDL for 6 hours. During this 6-hour incubation
period, we did not detect any significant increase in the cholesteryl
ester content (as assessed in a classic colorimetric
assay; data not shown), thus suggesting that massive
cholesterol loading of macrophages was not involved
in the induction of group IIa snpPLA2 mRNA.
Additionally, the preparations of native LDL failed to induce
snpPLA2, suggesting that the classic LDL receptor
was not involved, either. However, we may not exclude the implication
that CD36, a member of the scavenger receptor B family44
of scavenger receptors, is involved in the regulation of group IIa
snpPLA2 transcription. Moreover, the possibility
also exists that non–receptor-mediated pathways, ie, the heparin
sulfate proteoglycan–facilitated45 uptake of LDL, could
be involved in regulating group IIa PLA2 mRNA
synthesis. Uptake of modified LDL by macrophages results in
formation of macrophage foam cells and is a key cellular event
in the early stage of atherosclerosis. Secretory
PLA2s have been suggested to generate LDLs of a
more atherogenic form. We have previously shown that the human
recombinant group IIa PLA2 is able to hydrolyze
LDL in vitro and that the hydrolytic rate is increased 4-fold in the
presence of intimal matrix components like chondroitin sulfate
chains.20 It has additionally been found that
PLA2-treated LDL is taken up more avidly by
macrophages25 and equally, that the
PLA2-treated LDLs enhance release of
macrophage colony stimulating factor from macrophages,
thus inducing macrophage growth, which is probably linked to
the development of atherosclerotic lesions.39 Furthermore,
mild oxidation of LDL leads to an increased susceptibility of LDL
phospholipids to hydrolysis by
PLA2.46 Thus, our data showing that
mildly oxidized LDL (5 to 16 nmol malondialdehyde per milligram LDL
protein) regulates the transcription of the group IIa
snpPLA2 gene are in favor of a prominent role for
snpPLA2 in early atherogenesis.
In conclusion, we suggest that the enzymes of the PLA2 family may possess critical roles in macrophage function during the early stages of atherosclerosis. Group IIa snpPLA2 expression seems to be a specific response of intimal macrophages to their exposure to MM-LDL and mildly oxidized LDL, hence enabling the cells to modify and scavenge LDL accumulating in the intima. Future studies will elucidate the mechanisms implicated in LDL-induced gene expression of group IIa snpPLA2 and will allow identification of the lipoprotein receptor(s) involved.
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Acknowledgments |
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This work was supported by a fellowship from the Norwegian University of Science and Technology (to M.W.A.). The collaboration was made possible by a concerted action grant from the EU Biomed-2 program BMH4-OCT-96-0134 and by grants from NorFA and Nasjonalforeningen, Cardiovascular Program. Partial support from INSERM is also acknowledged. We would like to thank Dr M. John Chapman for stimulating discussion.
Received April 14, 1999; accepted January 8, 2000.
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