© 1997 American Heart Association, Inc.
Articles |
Expression of Phospholipase A2 Isoforms in Human Normal and Atherosclerotic Arterial Wall
From the Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm (L.S.E., P.L, H-E.C.); and the Departments of Surgery (A.D., U.H.) and Rheumatology (J.F.), Karolinska Hospital, Stockholm, Sweden.
Correspondence to Liselotte Schäfer Elinder, King Gustaf V's Research Institute, Department of Medicine, Karolinska Hospital, 17176 Stockholm, Sweden.
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Abstract |
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Abstract LDL particles must be modified in the arterial wall to be taken up by macrophages at an excessive rate, leading to foam cell formation. Phospholipase A2 (PLA2) has been shown to modify LDL particles in vitro by degrading its phospholipids, resulting in enhanced uptake by macrophages. Reaction products of PLA2 are lysophospholipids and nonesterified fatty acids (mainly arachidonic acid), which are precursors of potent inflammatory mediators and which have been found in atherosclerotic regions of the arterial wall. To elucidate the expression of PLA2 in normal and diseased arteries, frozen tissue sections of human nonatherosclerotic mesenteric artery and carotid plaques were examined by immunohistochemistry using specific antibodies against secretory PLA2 types I and II and cytosolic PLA2 (85 kd). Secretory PLA2 type I was not detected. High expression of secretory PLA2 type II was found throughout the media in both normal and atherosclerotic artery specimens, in which smooth muscle cells dominated. Cytosolic PLA2 was found exclusively in diseased artery, mainly in the intima in regions with an inflammatory infiltrate consisting of macrophages and smooth muscle cells. Furthermore, both normal and atherosclerotic artery possessed substantial PLA2 activity. It is suggested that secretory PLA2 type II could play an important role in early atherogenesis because it is present in the preatherosclerotic arterial wall, where it may lead to LDL modification, foam cell formation, and activation of immune mechanisms.
Key Words: phospholipase A2 • atherosclerosis • arterial wall
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Introduction |
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PLA2 catalyzes the hydrolysis of the sn-2 fatty acyl chain of glycerophospholipids to yield nonesterified fatty acids and lysophospholipids. Lysophospholipids and products of arachidonic acid, ie, prostaglandins and leukotrienes, participate in many vital cellular functions, including signal transduction, inflammation, and smooth muscle cell proliferation.1 2 3 PLA2s make up a family of enzymes that can be divided into low-molecular-mass enzymes (14 kd; types I and II), which are cell associated or extracellular, and high-molecular-mass enzymes (85 to 110 kd), which are localized in the cytosol.2 4 Recently it was reported that sPLA2-I has biological activities that are receptor mediated and unrelated to its catalytic function. Specific receptors for this enzyme have been found in rat vascular smooth muscle and endothelial cells.5 6 sPLA2-II has been found in inflammatory fluids, suggesting that it participates in local and systemic inflammatory responses.7 Its proinflammatory and cytotoxic effects are thought to be due to hydrolysis of membrane phospholipids and generation of proinflammatory lipid mediators.3 cPLA2 (85 kd) is expressed in various types of cells, such as monocytes, macrophage-like cell lines, platelets, arterial smooth muscle cells, and immature B lymphocytes.3 8 9 10 11 cPLA2 has a strong preference for phospholipids with arachidonic acid in the sn-2 position, suggesting that cPLA2 may be involved in eicosanoid production.3 8 Endothelial cell-derived eicosanoids like prostaglandins I2 and E2 play important roles in the maintenance of vascular tone, in platelet aggregation, in endothelial reactivity,12 and in smooth muscle cell proliferation.1 13 Furthermore, PLA2 is involved in the synthesis of platelet-activating factor, another inflammatory lipid mediator thought to play a role in atherogenesis.14 Thus, the activation of PLA2s may serve as a key step in a cascade of reactions leading to the production of mediators involved in atherogenesis and inflammation. Clinical studies have implicated PLA2s in the pathogenesis of disorders of the cardiovascular, gastrointestinal, and pulmonary systems and of the skin.15 However, the evidence linking PLA2 to atherosclerosis is very limited. Lysophosphatidylcholine is found in association with oxidized LDL16 and shows many of the atherogenic effects of oxidized LDL in vitro.16 17 18 19 20 21 22 23 24 25 High levels of lysophosphatidylcholine have been found in the intima and inner media of atherosclerotic aorta from hypercholesterolemic squirrel monkey26 and in association with LDL particles extracted from human atherosclerotic lesions.27 Because lysophosphatidylcholine as well as arachidonic acid are products of PLA2-catalyzed reactions, we studied the expression of PLA2 by immunohistochemistry in the normal and diseased human vessel wall. This report deals with the expression of sPLA2-I (pancreatic type), sPLA2-II (platelet type), and the 85-kd cPLA2.
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Methods |
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Preparation of Tissue
Atherosclerotic lesions were obtained from 10 patients undergoing carotid endarterectomy for symptomatic carotid artery disease. All examined specimens consisted of advanced atherosclerotic lesions (type VI according to the classification of Stary et al28 ) with a thin fibrous cap, a necrotic core, and signs of previous plaque rupture with deposits of fibrin and signs of thrombus formation at various stages. Apparently normal artery specimens were obtained from mesenteric vessels in six patients undergoing colorectal cancer surgery. The tissue samples used for immunohistochemistry were placed in cold Dulbecco's PBS and transported to the laboratory. Within 1 hour the sample was rinsed in cold PBS, embedded in OCT compound, and snap-frozen in isopentane at -20°C. The tissue was sectioned into 6-µm sections in a cryostat at -20°C. The sections were mounted on chrome-gelatine-coated slides, air-dried for 1 hour, and stored at -70°C until staining.
Immediately after removal the arterial samples to be analyzed for PLA2 activity were placed in DMEM supplemented with 50 µg/mL L-ascorbic acid, 50 µg/mL streptomycin, 50 IU/mL penicillin, and 0.1% bovine serum albumin. Within 1 hour the samples were taken to the laboratory, cleared from blood, and rinsed in cold PBS. Carotid specimens consisted of media and intima. Normal mesenteric artery specimens were rinsed as above, and the adventitia was stripped off.
Immunological Reagents
The following PLA2 antibodies were used: monoclonal
anti-human PLA2-I, from mouse-mouse hybrid cells (clone
2.223.288, Boehringer Mannheim, Mannheim, Germany); monoclonal
anti-human PLA2-II, from mouse-mouse hybrid cells (clone
4A1, Boehringer Mannheim). According to the supplier, there is
no cross-reactivity between the type I and type II antibodies;
monoclonal mouse anti-human cPLA2 (clone M3-1) was a kind
gift from Dr Ruth Kramer (Lilly Research Laboratories, Indianapolis,
Ind., USA). Isotype-matched mouse IgG clone MOPC31 c, used as a control
antibody, was obtained from Ancell Corporation (Bayport, Minn., USA).
The following antibodies, all from DAKO A/S (Glostrup, Denmark), were
used as cell-specific markers: anti-
-actin (1A4) for smooth muscle
cells, anti-CD14 (TUK4), anti-CD68 (PG-M1) for monocytes and
macrophages, and factor VIII-related antigens (anti-von
Willebrand factor).
Staining for PLA2
The cryostat sections were fixed at 4°C in acetone/water (1:1
vol/vol) for 30 seconds, followed by 3 minutes in 100% acetone.
After air-drying and rehydration in PBS, the slides were blocked in
2.5% normal goat serum in PBS for 1 hour at 20°C. All incubations
were performed in a humid chamber. The sections were then covered with
the relevant antibody or negative control antibody at a dilution of 10
µg/mL in the blocking solution. The sections were incubated
for 1 hour at 20°C and washed three times in PBS. Staining was
performed by covering the sections with a solution of goat
anti-mouse-IgG coupled to alkaline phosphatase diluted 1:50 in blocking
solution. The sections were incubated for 1 hour and washed three times
in PBS. The color was developed with
5-bromo-4-chloro-3-indolylphosphate/nitroblue tetrazolium tablets
according to the supplier's instructions. Levamisole was added to the
substrate solution at a concentration of 1.8 mg/mL to inhibit
endogenous alkaline phosphatase activity. Positive staining
resulted in a blue color. After washing in alcohol and xylene, the
slide was mounted in Mountex.
Staining for Cell-Specific Markers
After fixation of the tissue sections as described above,
endogenous peroxidase activity was blocked for 1 hour at
20°C in the dark in a humid chamber with 1% hydrogen peroxide and
2% sodium azide in PBS, followed by three washes in PBS. The
cell-specific antibodies were dissolved in PBS with 1% bovine serum
albumin and 0.02% sodium azide, and the slides were incubated
overnight. After three washes in PBS, blocking was performed in PBS
with 1% normal horse serum for 15 minutes. Incubation with the
biotin-labeled secondary antibody (biotinylated horse anti-mouse
antibody) in blocking solution was performed for 30 minutes. An
avidin-biotin horseradish peroxidase complex was dissolved in PBS
according to the supplier's instructions. The slides were incubated in
this solution for 60 minutes. The color was developed in
3-amino-9-etylcarbazol for 15 minutes, resulting in a red color. The
slides were counterstained for 2 minutes in Mayer's hematoxylin and
for 20 seconds in eosin and mounted in glycerine gelatin.
Assay of PLA2 Activity
The arterial specimens were
homogenized in 2 to 3 mL of ice-cold
homogenization buffer consisting of 20
mmol/L Tris-HCl (pH 7.5), 2 mmol/L EGTA, 1
mmol/L EDTA, 1 mmol/L PMSF, 20 µg/mL
soybean trypsin inhibitor, 0.1 mg/mL bacitracin,
0.5 mmol/L benzamidine, and 20 µmol/L
leupeptin. Cell debris was pelleted by centrifugation
at 10 000xg for 10 minutes at 4°C. PLA2
activity was assayed with a 1:1 mixture of the substrates 1-palmitoyl
2-(1-14C)arachidonyl-phosphatidylcholine and 1-palmitoyl
2-(1-14C)arachidonyl-phosphatidylethanolamine (120 nCi/mL)
at a concentration of 4 µM. This substrate is preferred by
cPLA2. Another substrate that is preferred by
sPLA2s,
(5,6,8,9,11,12,14,15-3H)arachidonyl-labeled
Esherichia coli membrane (11 nmol Pi/mL, 120
nCi/mL), was also used. The substrate was suspended in assay buffer
(80 mmol/L glycine, pH 9.0, 5 mmol/L
CaCl2, and 1 mg/mL human serum albumin
[essentially fatty acid free]) and sonicated for 10 minutes at 4°C.
The assay was initiated by adding 100 µL of supernatant to 400 µL
of assay buffer and carried out for 30 minutes at 37°C. Enzyme
activity was assayed with and without 5 mmol/L
dithiotreitol, which inhibits the activity of sPLA2. A
trifluoromethyl ketone analog of arachidonic acid
(AACOCF3) dissolved in ethanol was used as a specific
inhibitor of cPLA2 activity, and
4-bromophenacyl bromide was used to inhibit sPLA2. The
reaction was terminated by adding 2 vol of ice-cold methanol containing
0.5% acetic acid and 40 µmol/L stearic acid, followed by
vigorous vortexing. Precipitated proteins were removed by
centrifugation at 800xg for 10 minutes, and
the supernatant was applied to a disposable 100-mg C18
reverse-phase column. The column was washed once with water, followed
by 25% methanol in water, and nonesterified fatty acids were eluted
with 500 µL of methanol. Arachidonic acid was
analyzed by HPLC. The Radial-Pak cartridge (5x100 mm) was
packed with 4-µm C18 particles. The mobile phase was
methanol/water/trifluoroactic acid (85:15:0.007), and the flow
rate was 1.2 mL/min. The arachidonic acid peak was
identified by the retention time of an authentic standard.
Radioactivity was detected with a ß-RAM HPLC flow-through monitor
system. Quantitation was performed by integration of the peak area and
external standardization. The protein concentration of the samples was
measured with a protein assay kit (Bio Rad Laboratories).
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Results |
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Immunohistochemistry
Expression of sPLA2-I, sPLA2-II, and cPLA2 was investigated in frozen arterial tissue sections. sPLA2-I protein was not detected, in either normal artery or atherosclerotic lesions (data not shown). Fig 1
shows representative
examples of the results of immunostaining of an
atherosclerotic lesion. Fig 1, A, shows an overview of the tissue
section of study. Intima and media were separated by a necrotic core,
indicated by a star. Intense staining for sPLA2-II was
detected throughout the media and in streaks in the intima, indicated
by arrows (Fig 1, B). This staining pattern appeared to coincide with
that for -actin, demonstrating smooth muscle cells (Fig 1, C), and
to a lesser extent for macrophages (Fig 1, D). Positive
staining for cPLA2 was found throughout intimal plaques in
streaks or lumps, as well as in medial streaks (small arrows) (Fig 1, E). Such cPLA2-positive areas were rich in both smooth
muscle cells (Fig 1, C) and macrophages (Fig 1, D). Therefore,
both cell types were probably involved in synthesizing
cPLA2. The control stain performed with an isotype-matched
mouse IgG antibody was negative (Fig 1, F). Occasional staining of the
endothelium was seen with all the monoclonal
anti-PLA2 antibodies, including the control IgG, indicating
that this was nonspecific (not shown).
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Results of immunostaining of a normal artery are shown
in Fig 2. It is apparent from Fig 2, A,
stained with anti-von Willebrand factor, that the
endothelium was intact and that there was no intimal
thickening, indicating that this artery was indeed undiseased. Smooth
muscle cells were the dominating cell type throughout the media, as
indicated by the presence of -actin (Fig 2, B). Intense staining for
sPLA2-II was observed throughout the media (Fig 2, C). The
diffuse appearance of the sPLA2-II stain compared with the
-actin stain could be due to a predominantly extracellular location
of the enzyme. A few macrophages were present in the outer
media closest to the adventitia (Fig 2, D). The stains for
cPLA2 (Fig 2, E) and the control isotype-matched IgG (Fig 2, F) were negative.
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In control immunoblots (data not shown) using the
sPLA2-II antibody, a band with a molecular weight of 14 kd
was detected in platelets. With cultured smooth muscle cells, no
band was detected at the molecular weight of -actin (42 kd),
indicating the absence of cross-reactivity between the
sPLA2-II antibody and -actin. With the cPLA2
antibody, U937 cells and monocytes showed a band at 85 kd.
PLA2 Activity
PLA2 activity was determined in normal mesenteric
artery specimens and carotid atherosclerotic lesions.
Homogenates of arterial wall specimens were
incubated with a 1:1 mixture of
phosphatidylcholine-phosphatidylethanolamine or E. coli
membranes. Both substrates were labeled with radioactive
arachidonic acid (see "Methods"). The detection
limit of released arachidonic acid was 150 cpm. It was
found that PLA2 activity was nonlinearly associated with
sample amount, indicating the presence of endogenous
phospholipid substrate in the arterial specimens.
Therefore, a quantitative comparison of PLA2 activity
between normal and atherosclerotic artery is not easily performed. For
this reason, PLA2 activity was not expressed in absolute
terms but rather per 100 µL of supernatant. When the
phosphatidylcholine-phosphatidylethanolamine substrate was used, the
activity in representative specimens of normal and
atherosclerotic artery was 2314±154 and 2899±416 cpm/100
µL·min-1, respectively. With the E. coli
membrane substrate, the activities were almost equal in normal and
atherosclerotic samples, namely 1642±46 and 1705±176 cpm/100
µL·min-1, respectively. The PLA2
activity in control samples boiled for 2 minutes was zero.
The molecular nature of the PLA2 isoforms giving rise to
arachidonic acid release was further explored with use
of enzyme inhibitors. A trifluoromethyl ketone analog of
arachidonic acid (AACOCF3) is four orders
of magnitude less potent as an inhibitor of
sPLA2,29 whereas 4-bromophenacylbromide
inhibits sPLA2.2 The relative inhibition of
PLA2 activity by these two substances is shown in Fig 3. The phospholipid substrate,
1-palmitoyl 2-(1-14C)arachidonyl-phosphatidylcholine and
1-palmitoyl 2-(1-14C)arachidonyl-phosphatidylethanolamine,
is preferred by cPLA2. AACOCF3, at a
concentration of 10 µM, inhibited the total activity by 55% (Fig 3, A). With the E. coli membrane substrate,
4-bromophenacylbromide at 100 µM reduced the total PLA2
activity by 77% (Fig 3, B).
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Discussion |
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The identification of proinflammatory PLA2s in the arterial wall may be of major importance in the understanding of the inflammatory component of atherosclerosis. The present data show that sPLA2-II was expressed strongly in the media of both normal artery and atherosclerotic lesions. This finding was supported by partial inhibition of total PLA2-II activity in homogenized arterial wall samples by the sPLA2 inhibitor 4-bromophenacylbromide. The area of staining corresponded to that of
-actin, suggesting that the
cellular origin of this enzyme was mainly smooth muscle cells.
sPLA2-I was not detected in any of the artery specimens
examined. It was not possible to establish whether any of the
PLA2s were expressed by the endothelium
because occasional endothelial staining with the
control IgG was also observed. These findings are in contrast to recent
findings of Menschikowski et al,30 who investigated the
expression of sPLA2-II in atherosclerotic plaques taken
from various sites, including the carotid artery, by
immunohistochemistry with monoclonal antibodies. Those investigators
reported expression of sPLA2-II exclusively in areas with
macrophage-derived foam cells and not in normal arteries.
One major difference between the two studies is that we used frozen
tissue sections, whereas they used formalin-fixed sections. When the
latter approach was used in the beginning of our investigations, the
stains for all three PLA2s turned out negative (unpublished
data). This method of tissue preservation probably changes the epitope
recognized by the monoclonal anti-PLA2 antibody, thereby
reducing its affinity. Expression of sPLA2-II has been
shown to occur in tissue homogenates from the digestive
tract, cartilage, the parotid gland, and prostate.31 By
immunohistochemistry, sPLA2-II has been detected in
placental tissue.32 In agreement with our results, the
enzyme was expressed most strongly by vascular smooth muscle cells and
to a lesser extent by endothelial cells and connective
tissue fibroblasts. cPLA2 protein was found mainly in the atherosclerotic intima in regions with an inflammatory infiltrate and to a much lesser extent in the media. These areas consisted mainly of macrophages and intimal smooth muscle cells. However, the morphology of cryosections is generally less well preserved than that of formalin-fixed specimens. Therefore, the degree of colocalization cannot be pinpointed with certainty to one specific cell type. Macrophages are a known source of this enzyme, where it plays an important role in arachidonic acid release and eicosanoid synthesis during inflammation,3 and recently, cPLA2 was also demonstrated in cultured human arterial smooth muscle cells.10 Thus, intimal smooth muscle cells of the synthetic proliferative type,33 as well as macrophages, were the most likely cellular sources of cPLA2. The presence of cPLA2 in arterial plaque was confirmed by use of the specific cPLA2 inhibitor AACOCF3, which reduced the total PLA2 activity by more than 50%.
It was previously shown that PLA2-modified LDL particles
are taken up and degraded at an enhanced rate by macrophages,
leading to foam cell formation.34 35 It is therefore
possible that the presence of sPLA2-II in the normal
preatherosclerotic arterial wall may be of importance for
initiation of inflammatory reactions and fatty streak formation. The
enzyme has a high affinity for cell surface-bound heparan sulfate
proteoglycans,36 like LDL.37
sPLA2-II prefers to act on glycerophospholipids in an
aggregated form,3 like in LDL particles. Therefore, there
is a high probability for an interaction to occur between LDL and
sPLA2-II in the subendothelial space in
hyperlipidemia, where the number of LDL particles is
elevated. The reaction products of both sPLA2-II and
cPLA2 are lysophospholipids and arachidonic
acid, which are precursors of potent inflammatory mediators like
platelet-activating factor and eicosanoids. These substances could
initiate, sustain, and potentiate inflammatory reactions during all
stages of atherosclerosis development by attracting and
activating immune-competent cells.16 17 18 19 20 21 22 23 24 25 Cytokines
like TNF-, IL-1, and IL-6 may amplify this sequence of events
because they increase the expression of both
sPLA2-II36 and
cPLA2.38 39 Our findings could be of special
relevance for patients undergoing maintenance hemodialysis for
chronic renal failure. This treatment results in the release of high
levels of inflammatory cytokines in the blood. Recently it was
shown that plasma PLA2 activity of unknown origin is also
increased in these patients.40 Results of several studies
suggest that these patients have accelerated
atherosclerosis.41 It may therefore be
speculated that a cytokine-mediated stimulation of
PLA2 expression in the arterial wall could
aggravate lipid deposition by enhancing LDL modification, foam cell
formation, and inflammation, leading to the rapid progression of
atherosclerosis seen in these patients.
What, then, may the physiological function of a proinflammatory enzyme like sPLA2-II be in the normal arterial wall? It has been suggested that its role in the intestinal mucosa is to defend the organism against infections by attacking bacteria and producing inflammatory eicosanoids.31 This could also be true in the arterial wall because the specific cytokines produced during certain systemic bacterial infections42 upregulate the expression of sPLA2-II. In conclusion, the present findings of proinflammatory PLA2s in the human arterial wall open up new perspectives on atherogenesis by integrating the lipid and the inflammatory components of this disease.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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Dr Torgny Svenberg, Dr Anette von Rosen, and Dr Jesper Swedenborg at the Department of Surgery, Karolinska Hospital, are gratefully acknowledged for supplying the artery specimens. The excellent technical assistance of Pernilla Nyberg at the Department of Rheumatology is also greatly appreciated. This study was supported by funds from the Swedish Medical Research Council (grants 03X-07135 and 09100), King Gustaf V's 80-Year Fund, the Swedish Heart and Lung Foundation, the Karolinska Institute, Lars Hiertas Minne, Sigurd and Elsa Goljes Minne, and the Loo and Hans Ostermans Fund.
Received April 18, 1996; accepted November 25, 1996.
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