© 1997 American Heart Association, Inc.
Articles |
Localization of Nonpancreatic Secretory Phospholipase A2 in Normal and Atherosclerotic Arteries
Activity of the Isolated Enzyme on Low-Density Lipoproteins
the Wallenberg Laboratory for Cardiovascular Research, Sahlgrenska University Hospital, Goteborg University, Sweden (E.H.-C., B.R., P.S.); the UNIGEN Center for Molecular Biology, Norwegian University of Science and Technology, Trondheim, Norway (S.A., R.S., B.J.); and the Department of Gynecology, Ostra Hospital, Gothenburg, Sweden (E.S.).
Correspondence to Eva Hurt-Camejo, PhD, Wallenberg Laboratory, Fack 16, Sahlgrenska University Hospital, Gothenburg 41 345, Sweden. E-mail eva.hurt@wlab.wall.gu.se.
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Secretory nonpancreatic type II phospholipase A2 (snpPLA2) hydrolyzes fatty acids at the sn-2 position in phospholipids releasing free fatty acids (FFAs) and lysophospholipids. These products may act as intracellular second messengers or can be further metabolized into proinflammatory lipid mediators. The presence of snpPLA2 in extracellular fluids and serum during inflammation has suggested a role of the enzyme in this process. However, the presence of snpPLA2 in a variety of normal tissues suggests that snpPLA2 may also have physiological functions. Atherosclerosis appears to have an inflammatory component. Here we report on the snpPLA2 localization in normal and atherosclerotic lesions and on the properties of the isolated enzyme. A strong snpPLA2 immunoreactivity was observed in the arterial media that was colocalized with
-actin–positive vascular smooth muscle cells (SMCs) in both normal and atherosclerotic vessels. In aortic atherosclerotic lesions, snpPLA2 was observed colocalized with CD68-positive macrophages and HHF-35–positive SMCs and extracellularly in the lipid core. snpPLA2 was isolated from human normal arteries and from aorta with lesions. The enzyme was isolated by acid extraction of normal arterial tissues followed by immunoaffinity chromatography. The purified snpPLA2 had an expected molecular weight of 14 kD by polyacrylamide gel electrophoresis and appeared as a single band in immunoblotting. The enzymatic activity was followed by measuring release of fatty acids from phospholipid liposomes or LDL as substrates. The enzymatic activity was inhibited with two specific inhibitors for human snpPLA2: (1) monoclonal antibody 187 and (2) LY311727, a synthetic selective inhibitor. The mRNA for snpPLA2 was detected with reverse transcriptase polymerase chain reaction. These results indicate that snpPLA2 is present in human arteries and that it is able to hydrolyze phospholipids in LDL. The results support the hypothesis that snpPLA2 can release proinflammatory lipids at places of LDL deposition in the arterial wall.
Key Words: atherosclerosis • vascular smooth muscle cells • inflammation • macrophages
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Extracellular snpPLA2 is a 14-kD, calcium-dependent enzyme that hydrolyzes phospholipids at the sn-2 position, yielding FFA and lysophospholipids.1 These products may either act as intracellular second messengers or be further metabolized into proinflammatory lipid mediators, including eicosanoids and platelet-activating factor and lysophosphatidic acid.2 3 Polyunsaturated FFAs are susceptible to free radical–mediated oxidation.4 Lysophosphatidylcholine, on the other hand, appears in vitro to trigger different cellular proinflammatory processes; ie, migration of monocytes, expression of growth factors and adhesion molecules,5 6 7 induction of cell proliferation,8 9 inhibition of endothelium-dependent relaxation,10 and mediation of the expression of scavenger receptors in SMCs.11
The cDNA for snpPLA2 encodes a 144–amino acid protein including a 20–amino acid signal peptide. This indicates that snpPLA2 is a secreted enzyme. The enzyme is a very basic (pK 10.5) protein, has seven disulfide bridges, binds to SO4-GAG, is not selective for the type of fatty acid in the sn-2 position, and has a requirement for millimolar concentrations of calcium. Some of these characteristics indicate that snpPLA2 will be active in the extracellular milieu. The ability of snpPLA2 to bind SO4-GAG could be a mechanism for immobilizing cell-secreted and circulating enzyme in the extracellular matrix. This, together with the remarkable stability of the enzyme, suggests that snpPLA2 could remain active for prolonged periods in the extracellular environment of tissues.12
Evidence of a strong correlation between elevated levels of extracellular snpPLA2 and pathological conditions characterized by inflammation have been reported.13 14 While this concept has been challenged,15 16 additional in vivo and in vitro results suggest that extracellular snpPLA2 may be associated with the inflammatory response. Some of the most relevant findings indicate that extracellular snpPLA2 stimulates hyperemia associated with sites of chronic inflammation17 and that exogenously added snpPLA2 enhances prostaglandin generation18 and monocyte adhesion in endothelial cells.19 In addition, several proinflammatory cytokines, IL-1, IL-6, and TNF-, upregulate snpPLA2 mRNA expression and protein secretion in different rat cells20 21 22 and in human hepatoma cell line.23 The enzyme snpPLA2 is also found in a variety of tissues.24 This suggests that, in addition to its association with the inflammatory response, snpPLA2 may play a role in physiological functions, such as normal phospholipid turnover25 and the repair of lipid peroxidation damage.26
The snpPLA2 acts optimally in aggregated phospholipids such as a monolayer.1 The surface of LDL particles consists of a monolayer of phospholipids and cholesterol with an embedded apoB-100 chain.27 LDL phospholipids are hydrolyzed when the lipoprotein is incubated with PLA2.28 These observations suggest that LDL particles may act as physiological substrates for extracellular snpPLA2. A key event of early atherogenesis is presently believed to be the accumulation of apoB-100–containing lipoproteins in the arterial intima. We and others have shown that the interaction of LDL with extracellular matrix chondroitin-sulfate proteoglycans appears to contribute to this accumulation of lipoproteins in atherogenesis (see References 29 and 30).
On the basis of these data, we hypothesize that owing to their capacity to bind to glycosaminoglycans, snpPLA2 and LDL may be found colocalized extracellularly in the arterial wall. This colocalization may facilitate the action of the enzyme on LDL-phospholipids, releasing proinflammatory factors, lysophospholipids, and FFAs at sites of LDL deposition in the arterial wall. This mechanism may contribute to the inflammatory response that accompanies atherosclerosis. To explore this hypothesis, we decided to investigate (1) the presence of snpPLA2 in human arteries and (2) the hydrolysis of LDL by snpPLA2 isolated from human arteries.
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Reagents
Mouse mAb BF1 (subclass IgG2b) against human recombinant snpPLA2, kindly provided by Jeffrey Browning, Biogen Inc, Cambridge, Mass,15 and mAb PLA 185 (IgG1) against snpPLA2, purified from human placenta (Stoner et al16 ) were used to detect snpPLA2 in tissue sections and in immunoblottings. As a control antibody, we used mAb TWAR (subclass IgG2a) against Chlamydia pneumoniae, kindly provided by Are Dalen, Faculty of Medicine, Norwegian University of Technology Science, Trondheim, Norway. Biotinylated rabbit anti-mouse IgG, FITC-conjugated streptavidin, unspecific antibody IgG1, and peroxidase-conjugated goat immunoglobulin to mouse IgG were from Dakopatts AB. Mouse mAb against human snpPLA2 isolated from sperm (subclass IgG1k), from Upstate Biotechnology Incorporated, was used in Western blots and immunoaffinity chromatography. Mouse mAb 187 (IgG) against human recombinant snpPLA2 was kindly provided by Robert Marcus, Hoffmann-La Roche Inc, Nutley, NJ.16 This last mAb was used for inhibition of snpPLA2 activity. Cells were identified by using mouse mAb anti-muscle actin HHF-35 (Enzo Diagnostics Inc); mAb against
-smooth muscle actin (Sigma Chemical Co); and mAb against human macrophages CD68 (Dakopatts AB). Salts and buffer substances were of analytical grade and were purchased from Merck. Protease inhibitors were purchased from Sigma Chemical Co. Polyacrylamide gel reagents, nitrocellulose membranes (0.45 µm), horseradish peroxide color development 4CN were obtained from Bio-Rad Laboratories. Gene Amp RNA PCR kit was from Perkin-Elmer Corp. Nusieve GTG agarose was from FMC Bioproducts. DNA 1-kb ladder was from Bethesda Research Laboratories. LY311727 (H386/76, ASTRA-Hassle) is a specific inhibitor of snpPLA2 that was structure-based designed at Lilly Research Laboratories.32
Human Tissue
Atherosclerotic arteries and nonatherosclerotic arteries were obtained at autopsy or surgery. Uterine arteries were obtained from women <40 years of age undergoing hysterectomy (n=25). The arteries were immediately dissected from the excised uterus, rinsed free from blood at room temperature and under sterile conditions in cell culture medium as described.33 The arteries were dried with filter paper, weighed, and stored at -70°C until extraction or cut in 0.5- to 1.0-cm rings for histological and immunohistological study. All tissue samples for immunohistochemistry were embedded in optimum cutting temperature compound (OCT, Tissue-Tek, Miles Laboratories) and snap-frozen in liquid nitrogen. Freshly cut frozen sections of 5-µm thickness were collected on poly-L-lysine–coated slides and allowed to dry 1 hour and stored at -20°C. Immediately before staining, the sections were fixed in cold acetone for 5 minutes.
Immunohistochemistry
Single-label immunofluorescence staining of tissue was performed on human atherosclerotic (n=8) and nonatherosclerotic (n=4) arteries as previously described.34 The mAb BF1, mAb PLA185, and control antibody TWAR were diluted to 
30 µg/mL in PBS with 1.2% fraction V BSA (Sigma Chemical Co) and 2% human serum. The cell-specific monoclonal antibody CD68, which recognizes human macrophages,35 was used at a titer of 1:100; mAb HHF-35, which recognizes muscle cell actin and therefore is specific for smooth muscle cells when used in this context,36 was used at a titer of 1:35. The mAb against -actin was used at a titer of 1:1400. Biotinylated rabbit anti-mouse IgG and FITC/streptavidin were diluted (1:40 and 1:50, respectively) in PBS with 1.2% BSA.
Extraction of snpPLA2 From Human Arteries
Each snpPLA2 extraction (n=3) was done with 5 g total wet tissue from six to eight uterine arteries stored at -70°C. The arteries were minced and homogenized for 15 seconds with a Polytron PT 20 at speed 3 at 4°C in 50 mmol/L Tris pH 8.0 buffer containing 1 mmol/L EDTA, 1 mmol/L EGTA, 10 mmol/L benzamidine, 50 nmol/L soybean trypsin inhibitor, 769 nmol/L aprotinin, 2 µmol/L pepstatin A, 100 µmol/L leupeptin, and 5 µmol/L AEBSF. The homogenate was centrifuged at 2000g for 10 minutes at 4°C. The fatty surface layer and pellet debris were discarded. The clear extract was mixed with an equal volume of 0.18 mol/L H2SO437 containing 500 mmol/L NaCl, incubated 1 hour in ice, centrifuged at 10 000 rpm for 15 minutes, and then dialyzed against 50 mmol/L Tris pH 8.0 buffer containing 500 mmol/L NaCl, 5 µmol/L AEBSF, 10 mmol/L benzamidine, and 1.2 µmol/L leupeptin (binding buffer). An immunoaffinity column was prepared by binding mAb against human sperm snpPLA2 to cyanogen bromide–activated Sepharose (Pharmacia) according to the manufacturer's instructions. The column was equilibrated with binding buffer and the dialyzed arterial extract was passed through. After washing the column, the bound material was eluted with 50 mmol/L glycine buffer (pH 2.7) containing 500 mmol/L NaCl. Fractions of 1 mL were collected in tubes containing 30 µL of 1 mol/L Tris buffer to increase the pH of the fractions. PLA2 activity was determined in the collected fractions by measuring the release of FFA from the hydrolysis of liposomes, as described below. The fractions containing snpPLA2 activity were pooled and dialyzed against water containing 1 µmol/L AEBSF at 4°C for 24 hours with at least two changes. The dialyzed sample was lyophilized and stored at -20°C.
Immunoblotting
SDS–polyacrylamide gel electrophoresis with a 4% stacking gel was run under nonreducing conditions.38 The amount of protein sample applied in the gel is indicated in the figure legends. After transferring the gels, the membranes were immunoblotted with different mAbs against human snpPLA2: mAb against human sperm snpPLA2, mAb BF1, and mAb PLA185. The conditions of the immunoblotting were according to Upstate Biotechnology Incorporated protocols. As molecular-weight markers we used rainbow-colored proteins, 2350 to 46 000 kD, from Amersham. As a positive control, we used human recombinant snpPLA2 isolated from the cell-cultured medium of a Chinese hamster ovary cell line transfected with the gene for human snpPLA2 (CHO-snpPLA2).39
PCR Amplification Procedures
Total RNA was isolated from human uterine artery (0.5 g) and from CHO-snpPLA2–transfected cells.40 The oligonucleotides, designed from the determined cDNA sequence for human snpPLA2,41 were(1) upper primer, 5'position 136: 5'ATG AAG ACC CTC CTA CTG TTG 3' and (2) lower primer, 3'position 458: 5'GCA GCA GCC TTA TCA CAC TCA C 3'. The expected length of the amplified product is 344 bp. Total RNA (1, 10, and 100 ng) was reverse transcribed into cDNA and then amplified by PCR.42 The exponential phase of amplification was obtained with an initial incubation at 95°C for 2 minutes and then 33 cycles between 95°C and 64°C for 1 minute each. An additional extension reaction at 64°C for 7 minutes was included after cycle 33. Samples were analyzed by agarose gel electrophoresis containing ethidium bromide staining.
Lipoprotein
LDL (d=1.019 to 1.063) was isolated by differential ultracentrifugation from plasma from normolipidemic healthy fasted male volunteers. Before use, the LDL was equilibrated with PD-10 columns (Pharmacia Biotech Norden) in the appropriate buffer always containing 10 µmol/L BHT.43
Enzymatic Activity of snpPLA2 Extracted From Arteries
The enzymatic activity of arterial snpPLA2 eluted from the immunoaffinity chromatography was determined by measuring the release of FFA from the hydrolysis of two types of substrates: PC-liposomes or phospholipids in LDL. The liposomes were prepared by dissolving 10 mg L--phosphatidylcholine,ß-oleoyl-
-palmitoyl (Sigma Chemical Co) in 200 µL 4% nonidet P-40, 2% deoxycholic acid (sodium salt) and completed to 2 mL with 120 mmol/L Tris-HCl, pH 8.0, containing 12 mmol/L CaCl2 and 0.1 mmol/L EDTA. For measurement of the enzymatic activity, we used a colorimetric method for the quantitation of nonesterified FFA (NEFA C Kit, Wako Chemicals GmbH). Sample aliquots of 50 µL containing 320 ng protein from snpPLA2 were transferred to wells in a 96-multiwell plate preheated to 37°C. Then, 30 µL of PC-liposome preparation containing 150 µg phosphatidylcholine (2.5 mmol/L final concentration) or 80 to 100 µg LDL (1 mmol/L phospholipids final concentration) was added to the samples and incubated at 37°C. The plates in which liposomes were used as substrate were incubated 30 minutes, and the plates in which LDL was the substrate were incubated overnight. After incubation, the enzymatic reaction was stopped and the FFA detected by measuring absorbance at 550 nm following the manufacturer's procedure. A calibration curve with different concentrations of FFA and a positive control with human recombinant snpPLA2 were included.
Inhibition of snpPLA2 Activity by Monoclonal Antibody and Selective Inhibitor
For inhibition of snpPLA2 activity, 10 µg/mL mAb 187 or unspecific IgG antibody was incubated with 6 µg/mL protein of isolated human arterial snpPLA2 or 1 µg/mL protein of human recombinant snpPLA2 in a buffer consisting of 120 mmol/L Tris-HCl, pH 8.0, containing 12 mmol/L CaCl2 and 0.1 mmol/L EDTA. After 1 hour at 37°C, 50 µL of the incubation mixtures was transferred to wells in a 96-multiwell plate containing PC-liposomes or LDL as substrates in the concentrations indicated before. The measurement of released FFA was performed as described above. The enzymatic activity of snpPLA2 was also inhibited by incubation with 1 or 10 µmol/L of the specific inhibitor LY311727 with 6 µg/mL snpPLA2 (429 nmol/L) before addition of substrate. Protein was determined by the Bradford procedure, using bovine -globulin as the standard (Bio-Rad protein assay, Bio-Rad Laboratories).
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snpPLA2 in Human Nonatherosclerotic Arteries
In this study, human uterine arteries were used as control or "nonatherosclerotic" arteries. No signs of macroscopic lesion, intimal thickening, or lipid deposition were observed in these arteries.44 When snpPLA2 was detected by using a mAb against human snpPLA2 (BF1), a strong immunofluorescence staining was observed that was colocalized with SMCs in the arterial media (Fig 1
). These SMCs stained positively with antibody against -actin (data not shown). Endothelial cells stained weakly or not at all. Some positive staining was also observed in the adventitia. To further corroborate the presence of the enzyme in the arteries, we isolated snpPLA2 from these arteries by strong acid extraction (90 mmol/L H2SO4)37 45 and further immunoaffinity chromatography. The purified human arterial snpPLA2 was characterized by polyacrylamide gel electrophoresis and immunoblotting. Results from immunoblottings (n=3) showed a positive single band with a molecular weight of 14 kD similar to the band observed with human recombinant snpPLA2 used as a positive control (Fig 2).
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PCR Amplification of snpPLA2 mRNA
Immunohistochemistry in tissue sections and Western blot analysis of tissue extracts showed the presence of snpPLA2 in human arteries. We wanted to extend these results and use PCR to screen for the presence of snpPLA2 mRNA in the tissue. Fig 3 shows the PCR product of snpPLA2 mRNA from human uterine artery and from CHO-snpPLA2–transfected cells as the positive control. The size of the snpPLA2 amplified product was 344 bp. These results support the immunodetection of snpPLA2 and indicate that cells from the vascular wall are able to synthesize snpPLA2.
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snpPLA2 in Human Atherosclerotic Arteries
Fig 4 shows serial sections of an abdominal aortic atherosclerotic lesion characterized by a notably thickened intima and a still-intact arterial media (Fig 4a). Cell composition of this lesion shows CD68-positive macrophages present only in the thick intima (Fig 4c) and HH-35–positive SMCs present both in the intima and media (Fig 4d). The HH-35–positive cells in the intima show a spindle-shaped morphology and were localized mainly in the subendothelial region of the lesion. The snpPLA2 protein detected with the mAb BF1 was present all over the lesion (Fig 4b) and followed the immunofluorescent pattern obtained with HH-35–positive SMCs (Fig 4d). However, more immunofluorescent staining was observed with snpPLA2 than with HH-35 in the intima. As observed in normal arteries, the snpPLA2 staining was particularly strong in association with SMCs in the media of atherosclerotic lesions. Similar results were observed in atherosclerotic lesions from carotid arteries and with mAb PLA185 (data not shown). The control antibody showed only weak fluorescence, and only the autofluorescence from the elastic tissue in the media was observed (Fig 4f).
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Besides SMCs and some CD68-positive macrophages, other positive snpPLA2 immunostaining in the intima may also be colocalized with extracellular matrix. Since snpPLA2 is a small molecule that strongly binds to GAGs, its distribution in the intima may reflect the distribution of GAGs. However, preliminary experiments suggest that double immunohistochemistry will not allow discrimination between a homogeneous colocalization and differential distribution of the enzyme and GAGs. It appears that immunohistochemistry with gold-labeled antibodies and electron microscopy will be required to explore these two alternatives.
In the intima, snpPLA2 immunofluorescence staining was found associated with cells of similar morphology to the HHF-35 -actin–positive spindle-shaped SMCs (Fig 5a through 5d). Some round cells found in the same sections did not show immunoreaction with the antibodies against snpPLA2. Similar results were obtained with mAb PLA 185 against human snpPLA2 purified from placenta (data not shown). One reason for this dissimilar expression of snpPLA2 between cells of the same type may be different states of cell differentiation in the arterial lesions and different concentrations of cytokines known to modulate snpPLA2 expression in vitro. In vitro human arterial SMCs express snpPLA2 only after cell differentiation is induced by culturing the cells in nonproliferative conditions in sera-free medium for >7 days (data not shown).
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snpPLA2 in Advanced Lesions
Fig 6 shows a serial section of an advanced lesion in the abdominal aorta. The objective of this picture was to localize the muscular media in the section, characterized by the positivity toward -actin, and to indicate its strong reaction to antibodies toward snpPLA2. When snpPLA2 was detected with mAb BF1, a strong staining was observed all over the advanced lesion (Fig 6b). Similar to normal arteries (Fig 1
) and early atherosclerotic lesions (Fig 4b), a strong immunostaining was observed in the media of the advanced plaques colocalized with HHF-35–positive cells. In the thick intima, snpPLA2 immunoreactivity was colocalized with CD68-positive macrophages (Fig 6c). The cells in the lipid core were mainly CD68-positive macrophages and some -actin–positive SMCs overlying the lipid core on the shoulder and the luminal side. In the lipid core of advanced lesions, snpPLA2 immunoreactivity was particularly localized at the shoulder of the lipid core (Fig 6b). snpPLA2 staining was also localized inside the lipid core and in the region between the lipid core and the media, where neither SMCs nor CD68 macrophages were detected (Fig 6c). These results indicated that both macrophages and SMCs in advanced lesions contained immunoreactive snpPLA2 and that snpPLA2 may also be found extracellularly. A positive band of 14 kD molecular weight corresponding to snpPLA2 was also detected by immunoblotting with mAbs PLA 185 and BF1 in extract from 150 mg wet tissue from human aorta with atherosclerotic lesions (data not shown).
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snpPLA2 Associated With Foam-like Cells
Fig 7 shows sections of an early lesion. Fig 7a was stained to demonstrate lipid accumulation. Red-colored lipid droplets are localized mainly on the luminal side of the intima. A section from this area at low magnification was used for snpPLA2 detection and is shown at higher magnification in Fig 7b. Immunoreactivity with the snpPLA2-specific antibody BF1 showed as a strong fluorescence within cells and diffuse staining around the cells. These cells containing snpPLA2 showed lipid inclusions in histological sections (Fig 7c and 7e) and had a morphology similar to the CD68-positive macrophages (Fig 7e and 7f). These observations suggest that snpPLA2 is present in lipid-laden macrophages.
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Selective Inhibition of snpPLA2 Activity
The inhibitor mAb 18716 and the selective inhibitor LY31172732 were useful reagents for corroborating the identity of snpPLA2 purified from human arteries. The incubation with 10 µg/mL of mAb 187 resulted in >80% inhibition of the purified human arterial snpPLA2 and human recombinant snpPLA2 compared with the incubation with an unspecific antibody (Fig 8). The selective inhibitor LY311727 had a strong inhibitory effect on human arterial snpPLA2 (Fig 9). Both reagents, the mAb 187 and LY311727, also inhibited the hydrolysis of LDL phospholipids by human arterial snpPLA2 (Figs 8 and 9). The effect of LY311727 on human arterial snpPLA2 was stronger when using PC-liposomes than when using LDL as substrate. This occurrence may be due to differences in the mole fraction content of phospholipids between the two substrates or differences in the affinity of snpPLA2 for LY311727 and LDL. These results confirmed the identity of the 14-kD immunoreactive protein purified from human nonatherosclerotic arteries as snpPLA2. These experiment also indicated that arterial snpPLA2 can hydrolyze the phospholipids present in LDL particles, thus suggesting that LDL may function as a physiological substrate for snpPLA2.
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The results presented demonstrate that snpPLA2 is present in both nonatherosclerotic and atherosclerotic arteries. In nonatherosclerotic arteries, the most intense staining was observed associated with
-actin–positive SMCs in the media; no snpPLA2 was detected in the intima. In atherosclerotic arteries, snpPLA2, as well as SMCs, was present in both the media and intima, colocalized with CD68-positive macrophage-derived foam cells as well as extracellularly. Consequently, one may expect that the total amount of snpPLA2 may be increased in human atherosclerotic lesions. Recently, Menschikowski and coworkers46 reported the presence of snpPLA2 associated only with some macrophages/foam cells and in some regions with calcification and cell necrosis. No snpPLA2 was found associated with SMCs either in normal or atherosclerotic human arteries. The immunoreactivity that these investigators obtained with antibodies against snpPLA2 was lower than that obtained by us. These discrepancies between their results and ours may be explained by the use of different tissue fixation methods and different antibodies. A number of reports suggest a correlation between elevated levels of snpPLA2 and several inflammatory diseases.47 48 49 However, the mechanisms of increased snpPLA2 in the inflammatory response are not yet clearly understood. The levels of extracellular snpPLA2 appear to be regulated through both secretion of already synthesized enzyme and modulation of the gene expression. In vitro studies using cultured rat aortic SMCs demonstrated that cytokines as well as bacterial lipopolysaccharide upregulate snpPLA2 expression, whereas glucocorticoids downregulate it.50 One hypothesis is that the elevation of circulating snpPLA2 may be a consequence of the synthesis of snpPLA2 in vascular smooth muscle, saturation of these tissues, and release of snpPLA2 into the circulation.15 48
Atherosclerosis shares many characteristics with the inflammatory process. Additionally, a high incidence of atherosclerosis and high mortality from cardiovascular diseases have been reported in patients with chronic inflammatory diseases who have prolonged periods of high extracellular snpPLA2 activity.51 Inflammatory cytokines, including IL-1 and TNF-, can regulate genes in vascular wall cells and macrophages that are involved in atherogenesis.52 Several in vitro studies indicate that cytokines such as IL-1, IL-6, and TNF can stimulate a number of animal21 53 and human18 23 cell types to release snpPLA2. Furthermore, extracellular snpPLA2 increases T-lymphocyte response. This observation suggests that the activation of lymphocytes by snpPLA2 may create a positive feedback mechanism, sustaining chronic inflammation through cytokine secretion.54 T lymphocytes are present in human atherosclerotic lesions.55 However, no study has been carried out with human vascular SMCs and human macrophages. Therefore, the question of how the synthesis and secretion of snpPLA2 is regulated by these cells in the context of atherosclerosis remains unclear.
Our findings showing snpPLA2 associated with CD68-positive lipid-laden macrophages and less immunostaining associated with non–lipid-laden CD68-positive macrophages raises the question whether snpPLA2 plays a role in the lipid accumulation that is a hallmark of their morphological transformation into foam cells in atherogenesis. Although its presence in macrophages does not necessarily imply a role for snpPLA2 in this process, PLA2-modified LDL is taken up at an enhanced rate by macrophages.56
We could isolate active snpPLA2 from human normal arterial tissue with sulfuric acid extraction. This extraction procedure is used for cationic proteins that associate to anionic substances such as SO4-GAGs.45 Human snpPLA2 is a cationic protein and binds to S04-GAGs.57 1 The enzyme has three consensus heparin-binding sequences, one in the amino-terminal and two in the carboxyl-terminal region of the protein.37 58 The S04-GAGs of proteoglycan macromolecules found in the extracellular and pericellular compartments are the most negatively charged molecules in human tissues, with a high affinity for proteins that contain clusters of basic amino acids.59 The ability of snpPLA2 to bind SO4-GAG could be a mechanism for immobilizing cell-secreted and circulating enzyme to the extracellular matrix or pericellular proteoglycans.60 61 This characteristic, together with the remarkable stability of the enzyme, suggests that snpPLA2 could remain active for prolonged periods in the extracellular matrix of tissues.12 In vitro studies have demonstrated that the binding to heparin or heparan sulfate GAGs results in inhibition of snpPLA2 activity.57 62 However, binding to chondroitin sulfate GAGs enhances the enzyme's activity.63 These findings may have interesting in vivo consequences, since there are regional differences in the distribution of proteoglycans in the arterial wall. Versican, a large interstitial chondroitin sulfate proteoglycan, is prominent in the tunica media of normal artery and in the intima of early developing atherosclerotic lesions, whereas heparan sulfate proteoglycans are mainly found as cell-surface proteoglycans and in the basement membrane of endothelial cells in the arterial wall.59 Therefore, one may speculate that the activity of snpPLA2 in the arteries may possibly be modulated by the type of GAGs that the enzyme associates with.
LDL accumulates within the arterial wall in sites that are prone to development of atheroma.64 The interaction between SO4-GAGs and LDL has been extensively studied and is generally accepted as being one of the main explanations of LDL retention within the arterial wall.29 30 The concentration of LDL in the intima of a normal artery is approximately twice the serum concentration and can be higher in atherosclerotic lesions.65 One LDL particle contains around 800 molecules of phospholipids. If all the phospholipids present in 1 mg/mL of LDL (2 µmol/L) are hydrolyzed by snpPLA2, then >1 mmol/L lysophospholipids and FFA could be produced. These high concentrations of proinflammatory lipid factors, if rapidly liberated in the arterial intima, may be an important focal factor influencing the functionality of cells present in the arterial wall.2 5 6 8 9 10 11 Indirect evidence indicates that hydrolysis of phosphatidylcholine by PLA2-like activity takes place rapidly once apoB-100 lipoproteins are in the intima. Compared with plasma, apoB-100–containing particles isolated from human and rabbit arterial lesions are relatively enriched in sphingomyelin, a phospholipid that is not a substrate for snpPLA2.31 66 In addition, they have a lower content of phosphatidylcholine, a substrate for snpPLA2, and the phospholipid fraction is impoverished in linoleic acid, the most abundant acyl residue in the sn-2 position of phosphatidylcholine in LDL.67 Furthermore, the concentration of lysophosphatidylcholine in intima plus inner media of atherosclerotic aorta has been reported to be eight times that in comparable control tissue in an animal model.68 In the present study we demonstrated the presence of snpPLA2 in atherosclerotic lesions. We also showed that LDL is susceptible to hydrolysis by snpPLA2 isolated from human arteries. On the basis of these results, we hypothesize that snpPLA2 expressed in atherosclerotic plaques may be proatherogenic by two mechanisms. (1) It may induce local release of relatively high concentrations of FFA and lysophospholipids that may sustain inflammation and affect the functionality of cells at sites of LDL retention in the intima and (2) snpPLA2 can induce changes in the LDL particles, modifying them to a more atherogenic form.56 These mechanisms may be modulated by the colocalization of snpPLA2 and its substrate, LDL, to the SO4-GAGs.
Lysophosphatidylcholine, a product of snpPLA2 activity, has been reported to induce proliferation of SMCs.9 The presence of an intense staining of snpPLA2 in the arterial media colocalized with -actin–positive SMCs raises the question of whether the contact of cells containing snpPLA2 with plasma lipoproteins accumulated in the pericellular space69 may contribute to the arterial hyperplasia that occurs as a consequence of angioplasty applied in humans or balloon injury in experimental animal models.
Recently, a potent and selective inhibitor of human snpPLA2, LY311727, was developed for possible clinical application(s) as an anti-inflammatory agent.32 LY311727 inhibits snpPLA2 by binding irreversibly to the catalytic center of the enzyme. In the present work, we showed that LY311727 in vitro efficiently inhibited the hydrolysis of LDL phospholipids by purified human arterial snpPLA2. These results open the possibility for further in vivo animal studies to test the hypothesis that the hydrolysis of LDL by extracellular snpPLA2 in the arterial wall may play a role in the pathogenesis of atherosclerosis.
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Acknowledgments |
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This work was supported by grants from the Swedish Heart and Lung Foundation (project Nos. 51014 and 51013), the Swedish Society of Medicine (project No. 437.0), the Royal Society of Arts and Sciences in Gothenburg, and the Norwegian Research Council. Mouse mAb BF1 against human recombinant snpPLA2 was kindly provided by Jeffrey Browning, Biogen Inc, Cambridge, Mass; mAb TWAR against Chlamydia pneumoniae by Are Dalen, Faculty of Medicine, Norwegian University of Technology Science, Trondheim, Norway; and mouse mAb 187 against human recombinant snpPLA2 by Robert Marcus, Hoffmann-La Roche Inc, Nutley, NJ. We thank Morten Dalaker for the aortic tissues and Pal Kristian Selbo for valuable technical assistance. We also thank Tommy Sporrong and Lars-Åke Mattsson for their interest in this project.
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Presented in part at the 68th Scientific Sessions of the American Heart Association, Anaheim, California, November 13-16, 1995.
Received January 18, 1996;
revision received June 4, 1996;
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K. Öörni, M. O. Pentikäinen, M. Ala-Korpela, and P. T. Kovanen Aggregation, fusion, and vesicle formation of modified low density lipoprotein particles: molecular mechanisms and effects on matrix interactions J. Lipid Res., November 1, 2000; 41(11): 1703 - 1714. [Abstract] [Full Text]
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K. Kugiyama, Y. Ota, H. Kawano, H. Soejima, H. Ogawa, S. Sugiyama, H. Doi, and H. Yasue Increase in plasma levels of secretory type II phospholipase A2 in patients with coronary spastic angina Cardiovasc Res, July 1, 2000; 47(1): 159 - 165. [Abstract] [Full Text] [PDF]
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M. W. Anthonsen, D. Stengel, D. Hourton, E. Ninio, and B. Johansen Mildly Oxidized LDL Induces Expression of Group IIa Secretory Phospholipase A2 in Human Monocyte-Derived Macrophages Arterioscler Thromb Vasc Biol, May 1, 2000; 20(5): 1276 - 1282. [Abstract] [Full Text] [PDF]
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 U. J. F. Tietge, C. Maugeais, W. Cain, D. Grass, J. M. Glick, F. C. de Beer, and D. J. Rader Overexpression of Secretory Phospholipase A2 Causes Rapid Catabolism and Altered Tissue Uptake of High Density Lipoprotein Cholesteryl Ester and Apolipoprotein A-I J. Biol. Chem., March 31, 2000; 275(14): 10077 - 10084. [Abstract] [Full Text] [PDF]
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P. T. Kovanen and M. O. Pentikainen Secretory Group II Phospholipase A2 : A Newly Recognized Acute-Phase Reactant With a Role in Atherogenesis Circ. Res., March 31, 2000; 86(6): 610 - 612. [Full Text] [PDF]
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P. Sartipy, B. Johansen, K. Gasvik, and E. Hurt-Camejo Molecular Basis for the Association of Group IIA Phospholipase A2 and Decorin in Human Atherosclerotic Lesions Circ. Res., March 31, 2000; 86(6): 707 - 714. [Abstract] [Full Text] [PDF]
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M. Menschikowski, A. Rosner-Schiering, R. Eckey, E. Mueller, R. Koch, and W. Jaross Expression of Secretory Group IIA Phospholipase A2 in Relation to the Presence of Microbial Agents, Macrophage Infiltrates, and Transcripts of Proinflammatory Cytokines in Human Aortic Tissues Arterioscler Thromb Vasc Biol, March 1, 2000; 20(3): 751 - 762. [Abstract] [Full Text] [PDF]
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T. Hakkinen, J. S. Luoma, M. O. Hiltunen, C. H. Macphee, K. J. Milliner, L. Patel, S. Q. Rice, D. G. Tew, K. Karkola, and S. Yla-Herttuala Lipoprotein-Associated Phospholipase A2, Platelet-Activating Factor Acetylhydrolase, Is Expressed by Macrophages in Human and Rabbit Atherosclerotic Lesions Arterioscler Thromb Vasc Biol, December 1, 1999; 19(12): 2909 - 2917. [Abstract] [Full Text] [PDF]
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 K. Kugiyama, Y. Ota, K. Takazoe, Y. Moriyama, H. Kawano, Y. Miyao, T. Sakamoto, H. Soejima, H. Ogawa, H. Doi, et al. Circulating Levels of Secretory Type II Phospholipase A2 Predict Coronary Events in Patients with Coronary Artery Disease Circulation, September 21, 1999; 100(12): 1280 - 1284. [Abstract] [Full Text] [PDF]
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P. Sartipy, G. Camejo, L. Svensson, and E. Hurt-Camejo Phospholipase A2 Modification of Low Density Lipoproteins Forms Small High Density Particles with Increased Affinity for Proteoglycans and Glycosaminoglycans J. Biol. Chem., September 3, 1999; 274(36): 25913 - 25920. [Abstract] [Full Text] [PDF]
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E. Hurt-Camejo, B. Rosengren, P. Sartipy, K. Elfsberg, G. Camejo, and L. Svensson CD44, a Cell Surface Chondroitin Sulfate Proteoglycan, Mediates Binding of Interferon-gamma and Some of Its Biological Effects on Human Vascular Smooth Muscle Cells J. Biol. Chem., July 2, 1999; 274(27): 18957 - 18964. [Abstract] [Full Text] [PDF]
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J. K. Hakala, K. Oorni, M. Ala-Korpela, and P. T. Kovanen Lipolytic Modification of LDL by Phospholipase A2 Induces Particle Aggregation in the Absence and Fusion in the Presence of Heparin Arterioscler Thromb Vasc Biol, May 1, 1999; 19(5): 1276 - 1283. [Abstract] [Full Text] [PDF]
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B. Ivandic, L. W. Castellani, X.-P. Wang, J.-H. Qiao, M. Mehrabian, M. Navab, A. M. Fogelman, D. S. Grass, M. E. Swanson, M. C. de Beer, et al. Role of Group II Secretory Phospholipase A2 in Atherosclerosis : 1. Increased Atherogenesis and Altered Lipoproteins in Transgenic Mice Expressing Group IIa Phospholipase A2 Arterioscler Thromb Vasc Biol, May 1, 1999; 19(5): 1284 - 1290. [Abstract] [Full Text] [PDF]
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N. Leitinger, A. D. Watson, S. Y. Hama, B. Ivandic, J.-H. Qiao, J. Huber, K. F. Faull, D. S. Grass, M. Navab, A. M. Fogelman, et al. Role of Group II Secretory Phospholipase A2 in Atherosclerosis : 2. Potential Involvement of Biologically Active Oxidized Phospholipids Arterioscler Thromb Vasc Biol, May 1, 1999; 19(5): 1291 - 1298. [Abstract] [Full Text] [PDF]
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 D. Beasley COX-2 and cytosolic PLA2 mediate IL-1beta -induced cAMP production in human vascular smooth muscle cells Am J Physiol Heart Circ Physiol, April 1, 1999; 276(4): H1369 - H1378. [Abstract] [Full Text] [PDF]
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P. Sartipy, G. Bondjers, and E. Hurt-Camejo Phospholipase A2 Type II Binds to Extracellular Matrix Biglycan : Modulation of Its Activity on LDL by Colocalization in Glycosaminoglycan Matrixes Arterioscler Thromb Vasc Biol, December 1, 1998; 18(12): 1934 - 1941. [Abstract] [Full Text] [PDF]
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W. Pruzanski, E. Stefanski, F. C. de Beer, M. C. de Beer, P. Vadas, A. Ravandi, and A. Kuksis Lipoproteins are substrates for human secretory group IIA phospholipase A2: preferential hydrolysis of acute phase HDL J. Lipid Res., November 1, 1998; 39(11): 2150 - 2160. [Abstract] [Full Text]
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 L. Thommesen, W. Sjursen, K. Gasvik, W. Hanssen, O.-L. Brekke, L. Skattebol, A. K. Holmeide, T. Espevik, B. Johansen, and A. Lagreid Selective Inhibitors of Cytosolic or Secretory Phospholipase A2 Block TNF-Induced Activation of Transcription Factor Nuclear Factor-{kappa}B and Expression of ICAM-1 J. Immunol., October 1, 1998; 161(7): 3421 - 3430. [Abstract] [Full Text] [PDF]
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M. Romano, E. Romano, S. Bjorkerud, and E. Hurt-Camejo Ultrastructural Localization of Secretory Type II Phospholipase A2 in Atherosclerotic and Nonatherosclerotic Regions of Human Arteries Arterioscler Thromb Vasc Biol, April 1, 1998; 18(4): 519 - 525. [Abstract] [Full Text] [PDF]
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S. L. Schissel, X.-c. Jiang, J. Tweedie-Hardman, T.-s. Jeong, E. H. Camejo, J. Najib, J. H. Rapp, K. J. Williams, and I. Tabas Secretory Sphingomyelinase, a Product of the Acid Sphingomyelinase Gene, Can Hydrolyze Atherogenic Lipoproteins at Neutral pH. IMPLICATIONS FOR ATHEROSCLEROTIC LESION DEVELOPMENT J. Biol. Chem., January 30, 1998; 273(5): 2738 - 2746. [Abstract] [Full Text] [PDF]
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J. M. Upston, P. K. Witting, R. Alleva, and R. Stocker Oxidation of Free Fatty Acids in Low Density Lipoprotein by 15-Lipoxygenase Stimulates Nonenzymic, alpha -Tocopherol-mediated Peroxidation of Cholesteryl Esters J. Biol. Chem., November 28, 1997; 272(48): 30067 - 30074. [Abstract] [Full Text] [PDF]
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H. Peilot, B. Rosengren, G. Bondjers, and E. Hurt-Camejo Interferon-gamma Induces Secretory Group IIA Phospholipase A2 in Human Arterial Smooth Muscle Cells. INVOLVEMENT OF CELL DIFFERENTIATION, STAT-3 ACTIVATION, AND MODULATION BY OTHER CYTOKINES J. Biol. Chem., July 21, 2000; 275(30): 22895 - 22904. [Abstract] [Full Text] [PDF]
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M. Hernandez, L. Fuentes, F. J. Fernandez Aviles, M. S. Crespo, and M. L. Nieto Secretory Phospholipase A2 Elicits Proinflammatory Changes and Upregulates the Surface Expression of Fas Ligand in Monocytic Cells: Potential Relevance for Atherogenesis Circ. Res., January 11, 2002; 90(1): 38 - 45. [Abstract] [Full Text] [PDF]
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