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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:2257-2263.)
© 1997 American Heart Association, Inc.

Articles

Expression of Phospholipase A₂ Isoforms in Human Normal and Atherosclerotic Arterial Wall

Liselotte Schäfer Elinder; Alexandra Dumitrescu; Pontus Larsson; Ulf Hedin; Johan Frostegård; ; Hans-Erik Claesson

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.

	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

	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.

	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 P_i/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 sPLA_2. 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).

	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).

View larger version (150K): [in this window] [in a new window]   Figure 1. Results of immunostaining of serial sections of an atherosclerotic carotid lesion from a 66-year-old man. A, Overview photomicrograph of staining with hematoxylin and eosin showing that the media was separated from the intima by a necrotic core (*) (original magnification, x40). The cell-rich area to the left of the necrosis consisted primarily of smooth muscle and inflammatory cells. Farther to the left was the fibrous cap region. B, Monoclonal mouse antibody specific for sPLA2-II showing intense staining of the media (x100). C, Stain for -actin showing smooth muscle cells (x100). D, Enlarged view of stain for macrophages with anti-CD14 (x100). E, Staining with monoclonal mouse antibody specific for cPLA2. Staining is limited primarily to intimal areas rich in smooth muscle cells and macrophages. F, Control stain performed with an isotype-matched mouse IgG (x100).

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.

View larger version (133K): [in this window] [in a new window]   Figure 2. Results of immunostaining of serial sections from the middle colic artery of a 67-year-old man. All photomicrographs were enlarged 200 times. A, Antibody against von Willebrand factor showing intact endothelial cell lining. B, Antibody against -actin showing smooth muscle cells in the media. C, Monoclonal mouse antibody specific for sPLA2-II showing intense staining throughout the media. D, Staining for macrophages with anti-CD-14. E, Staining for cPLA2 with mouse monoclonal antibody was negative. F, Control stain using isotype-matched mouse IgG.

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).

View larger version (16K): [in this window] [in a new window]   Figure 3. Relative inhibition of total PLA2 activity in carotid plaque. PLA2 activity was assayed in triplicate in the supernatant of homogenized arterial wall specimens with the addition of a trifluoromethyl ketone analog of arachidonic acid (AACOCF3), a specific cPLA2 inhibitor, or 4-bromophenacylbromide (BPB), which inhibits sPLA2, as described in "Methods." The substrates used were a 1:1 mixture (4 µM) of 1-palmitoyl 2-(1-14C)arachidonyl-phosphatidylcholine and 1-palmitoyl 2-(1-14C)arachidonyl-phosphatidylethanolamine (PC/PE) (A) and (5,6,8,9,11,12,14,15-3H)arachidonyl-labeled E. coli membrane (120 nCi or 11 nmol inorganic phosphate) (B). The mean activity in the control sample in A was 5735 cpm/100 µL·min-1, and the mean activity in the control sample in B was 6811 cpm/100 µL·min-1.

	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.

	Selected Abbreviations and Acronyms

 

cPLA2 = cytosolic phospholipase A2 HPLC = high-performance liquid chromatography IgG = immunoglobulin G IL = interleukin PLA2 = phospholipase A2 sPLA2-I = secretory phospholipase A2 type I sPLA2-II = secretory phospholipase A2 type II

	Acknowledgments

 
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.

	References

Top Abstract Introduction Methods Results Discussion References

 

1. Palmberg L, Lindgren JA, Thyberg J, Claesson HE. On the mechanism of induction of DNA synthesis in cultured arterial smooth muscle cells by leukotrienes: possible role of prostaglandin endoperoxide synthase products and platelet-derived growth factor. J Cell Sci. 1991;98:141-149.[Abstract/Free Full Text]
2. Glaser KB, Mobilio D, Chang JY, Senko N. Phospholipase A₂ enzymes: regulation and inhibition. TIPS. 1993;14:92-98.
3. Mukherjee AB, Miele L, Pattabiraman N. Phospholipase A₂ enzymes: regulation and physiological role. Biochem Pharmacol. 1994;48:1-10.[Medline] [Order article via Infotrieve]
4. Dennis EA. Diversity of group types, regulation, and function of phospholipase A₂. J Biol Chem. 1994;269:13057-13060.[Free Full Text]
5. Hanasaki K, Arita H. Characterization of a high affinity binding site for pancreatic-type phospholipase A₂ in the rat. J Biol Chem. 1992;267:6414-6420.[Abstract/Free Full Text]
6. Kishino J, Ohara O, Nomura K, Kramer RM, Arita H. Pancreatic-type phospholipase A₂ induces group II phospholipase A₂ expression and prostaglandin biosynthesis in rat mesangial cells. J Biol Chem. 1994;269:5092-5098.[Abstract/Free Full Text]
7. Kramer RM, Hession C, Johansen B, et al. Structure and properties of a human non-pancreatic phospholipase A₂. J Biol Chem. 1989;264:5768-5775.[Abstract/Free Full Text]
8. Clark JD, Lin LL, Kriz RW, et al. A novel arachidonic acid-selective cytosolic PLA₂ contains a Ca²⁺-dependent translocation domain with homology to PKC and GAP. Cell. 1991;65:1043-1051.[Medline] [Order article via Infotrieve]
9. Kramer RM, Roberts EF, Manetta JV, Hyslop PA, Jakubowski J. Thrombin-induced phosphorylation and activation of Ca²⁺-sensitive cytosolic phospholipase A₂ in human platelets. J Biol Chem. 1993;268:26796-26804.[Abstract/Free Full Text]
10. Graves LM, Bornfeldt KE, Sidhu JS, et al. Platelet-derived growth factor stimulates protein kinase A through a mitogen-activated protein kinase-dependent pathway in human arterial smooth muscle cells. J Biol Chem. 1996;271:505-511.[Abstract/Free Full Text]
11. Feltenmark S, Runarsson G, Larsson P, Jacobsson PJ, Björkholm M, Claesson HE. Diverse expression of cytosolic phospholipase A₂, 5-lipoxygenase and prostaglandin H synthase 2 in acute pre-B-lymphocytic leukemia cells. Br J Haematol. 1995;90:585-594.[Medline] [Order article via Infotrieve]
12. Hajjar DP, Pommerantz KB. Signal transduction in atherosclerosis: integration of cytokines and the eicosanoid network. FASEB J. 1992;6:2933-2941.[Abstract]
13. Palmberg L, Claesson HE, Thyberg J. Leukotrienes stimulate initiation of DNA synthesis in cultured arterial smooth muscle cells. J Cell Sci. 1987;88:151-159.[Abstract/Free Full Text]
14. Evangelou AM. Platelet-activating factor (PAF): implications for coronary heart and vascular diseases. Prostaglandins Leukot Essent Fatty Acids. 1994;50:1-28.[Medline] [Order article via Infotrieve]
15. Pruzanski W, Vadas P. Phospholipase A₂—a mediator between proximal and distal effectors of inflammation. Immunol Today. 1991;12:143-146.[Medline] [Order article via Infotrieve]
16. Quinn M, Parthasarathy S, Steinberg D. Lysophosphatidylcholine: a chemotactic factor for human monocytes and its potential role in atherogenesis. Proc Natl Acad Sci U S A. 1988;85:2805-2809.[Abstract/Free Full Text]
17. Kume N, Cybulsky MI, Gimbrone MA. Lysophosphatidylcholine, a component of atherogenic lipoproteins, induces mononuclear leukocyte adhesion molecules in cultured human and rabbit endothelial cells. J Clin Invest. 1992;90:1138-1144.
18. Locher R, Weisser B, Mengden T, Brunner C, Vetter W. Lysolecithin actions on vascular smooth muscle cells. Biochem Biophys Res Commun. 1992;183:156-162.[Medline] [Order article via Infotrieve]
19. Mangin EL, Kugiyama K, Nguy JH, Kerns SA, Henry PD. Effects of lysolipids and oxidatively modified low density lipoprotein on endothelium-dependent relaxation of rabbit aorta. Circ Res. 1993;72:161-166.[Abstract/Free Full Text]
20. McMurray H, Parthasarathy S, Steinberg D. Oxidatively modified low density lipoprotein is a chemoattractant for human T lymphocytes. J Clin Invest. 1993;92:1004-1008.
21. Yokote K, Morisaki N, Zenibayashi M, et al. The phospholipase-A₂ reaction leads to increased monocyte adhesion of endothelial cells via the expression of adhesion molecules. Eur J Biochem. 1993;217:723-729.[Medline] [Order article via Infotrieve]
22. Asaoka Y, Yoshida K, Sasaki Y, et al. Possible role of secretory group II phospholipase A₂ in T-lymphocyte activation: implication in propagation of inflammatory reaction. Proc Natl Acad Sci U S A. 1993;90:716-719.[Abstract/Free Full Text]
23. Kume N, Gimbrone MA. Lysophosphatidylcholine transcriptionally induces growth factor gene expression in cultured human endothelial cells. J Clin Invest. 1994;93:907-911.
24. Nakano T, Raines E, Abraham J, Klagsbrun M, Ross R. Lysophosphatidylcholine upregulates the level of heparin-binding epidermal growth factor-like growth factor mRNA in human monocytes. Proc Natl Acad Sci U S A. 1994;91:1069-1073.[Abstract/Free Full Text]
25. Morisaki N, Yokote K, Takahashi K, et al. Role of phospholipase A₂ in expression of the scavenger pathway in cultured aortic smooth muscle cells stimulated with phorbol 12-myristate 13-acetate. Biochem J. 1994;303:247-253.
26. Portman OW, Alexander M. Lysophosphatidylcholine concentrations and metabolism in aortic intima plus inner media: effect of nutritionally induced atherosclerosis. J Lipid Res. 1969;10:158-165.[Abstract]
27. Ylä-Herttuala S, Palinski W, Rosenfeld ME, et al. Evidence for the presence of oxidatively modified low density lipoprotein in atherosclerotic lesions of rabbit and man. J Clin Invest. 1989;84:1086-1095.
28. Stary HC, Chandler AB, Dinsmore RE, et al. A definition of advanced types of atherosclerotic lesions and a histological classification of atherosclerosis. Circulation. 1995;92:1355-1374.[Abstract/Free Full Text]
29. Street IP, Lin HK, Laliberté F, et al. Slow- and tight-binding inhibitors of the 85-kd human phospholipase A₂. Biochemistry. 1993;32:5935-5940.[Medline] [Order article via Infotrieve]
30. Menschikowski M, Kasper M, Lattke P, et al. Secretory group II phospholipase A₂ in human atherosclerotic plaques. Atherosclerosis. 1995;118:173-181.[Medline] [Order article via Infotrieve]
31. Nevalainen T, Haapanen TJ. Distribution of pancreatic (group I) and synovial-type (group II) phospholipase A₂ in human tissues. Inflammation. 1993;17:453-464.[Medline] [Order article via Infotrieve]
32. Andersen S, Sjursen W, Logreid A, Austgulen R, Johansen B. Immunohistologic detection of non-pancreatic phospholipase A₂ (type II) in human placenta and its possible involvement in normal parturition at term. Prostaglandins Leukot Essent Fatty Acids. 1994;51:19-26.[Medline] [Order article via Infotrieve]
33. Kocher O, Gabbiani G. Cytoskeletal features of normal and atheromatous human arterial smooth muscle cells. Hum Pathol. 1986;17:875-880.[Medline] [Order article via Infotrieve]
34. Aviram M, Maor I. Phospholipase A₂-modified LDL is taken up at enhanced rate by macrophages. Biochem Biophys Res Commun. 1992;185:465-472.[Medline] [Order article via Infotrieve]
35. Sparrow CP, Parthasarathy S, Steinberg D. Enzymatic modification of low density lipoprotein by purified lipoxygenase plus phospholipase A₂ mimics cell-mediated oxidative modification. J Lipid Res. 1988;29:745-753.[Abstract]
36. Suga H, Murakami M, Kudo I, Inoue K. Participation in cellular prostaglandin synthesis of type-II phospholipase A₂ secreted and anchored on cell-surface heparan sulfate proteoglycan. Eur J Biochem. 1993;218:807-813.[Medline] [Order article via Infotrieve]
37. Hurt E, Bondjers G, Camejo G. Interaction of LDL with human arterial proteoglycans stimulates its uptake by human monocyte-derived macrophages. J Lipid Res. 1990;31:443-454.[Abstract]
38. Lin LL, Lin AY, DeWitt DL. Interleukin-1 induces the accumulation of cytosolic phospholipase A₂ and the release of prostaglandin E₂ in human fibroblasts. J Biol Chem. 1992;267:23451-23454.[Abstract/Free Full Text]
39. Hoeck WG, Ramesha CS, Chang DJ, Fan N, Heller RA. Cytoplasmic phospholipase A₂ activity and gene expression are stimulated by tumor necrosis factor: dexamethasone blocks the induced synthesis. Proc Natl Acad Sci U S A. 1993;90:4475-4479.[Abstract/Free Full Text]
40. Vishwanath BS, Fux CA, Uehlinger DE, Frey BM, Franson RC, Frey FJ. Haemodialysis activates phospholipase A₂ enzyme. Nephrol Dial Transplant. 1996;11:109-116.[Abstract/Free Full Text]
41. Rossi A, Bonfante L, Giacomini A, et al. Carotid artery lesions in patients with nondiabetic chronic renal failure. Am J Kid Dis. 1996;27:58-66.[Medline] [Order article via Infotrieve]
42. Mendall MA, Patel P, Ballam L, Strachan D, Northfield TC. C reactive protein and its relation to cardiovascular risk factors: a population based cross sectioal study. BMJ. 1996;312:1061-1065.[Abstract/Free Full Text]

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