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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1998;18:1934-1941.)
© 1998 American Heart Association, Inc.

Original Contributions

Phospholipase A₂ Type II Binds to Extracellular Matrix Biglycan

Modulation of Its Activity on LDL by Colocalization in Glycosaminoglycan Matrixes

Peter Sartipy; Göran Bondjers; Eva Hurt-Camejo

From the Wallenberg Laboratory for Cardiovascular Research, Department of Heart and Lung Disease, Göteborg University, Gothenburg, Sweden.

Correspondence to Peter Sartipy, Wallenberg Laboratory, Sahlgrenska University Hospital, S-413 45 Gothenburg, Sweden. E-mail Peter.Sartipy{at}wlab.wall.gu.se

	Abstract

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Abstract—We recently reported the presence of secretory, nonpancreatic phospholipase A₂ type II (snpPLA₂; EC 3.1.1.4) in human atherosclerotic arteries (Hurt-Camejo et al, Arterioscler Thromb Vasc Biol. 1997;17:300–309). SnpPLA₂ may generate the proinflammatory products lysophospholipids and free fatty acids, thus contributing to atherogenesis when acting on low density lipoproteins (LDLs) retained in the arterial wall. Immunohistochemical studies showed that smooth muscle cells (SMCs) in human arterial tissue are the main sources of snpPLA₂. In cultures of human arterial SMCs, snpPLA₂ interacts with versican and smaller heparan/chondroitin sulfate proteoglycans (PGs) secreted as soluble components into the medium. In the present study, we investigated the binding of snpPLA₂ to extracellular matrix (ECM) PGs produced by SMCs. The results show that snpPLA₂ can bind to the ECM at physiological salt concentrations. ECM-bound snpPLA₂ was active, hydrolyzing phosphatidylcholine-containing micelles. Soluble chondroitin-6-sulfate at concentrations >1 µmol/L, but not heparin or heparan sulfate, was able to release ECM-bound snpPLA₂. The PG mainly involved in the binding of snpPLA₂ was identified as biglycan. Perlecan was also present in the ECM synthesized by SMCs, but it contributed less to the binding of snpPLA₂. Experiments with immobilized glycosaminoglycans indicated that snpPLA₂ hydrolyzed 7-fold more LDL phospholipids when the lipoprotein and the enzyme were colocalized in a matrix with chondroitin-6-sulfate compared with one with heparin. These data suggest that retention of snpPLA₂ in ECMs of different composition may modulate the enzymatic activity of snpPLA₂ toward LDL. The results presented in this work support the hypothesis of the potential contribution of snpPLA₂ to atherosclerosis.

Key Words: phospholipase A₂ type II • proteoglycans • vascular cells • LDL • atherosclerosis

	Introduction

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Proteoglycans (PGs) are macromolecules that contain at least 1 glycosaminoglycan (GAG) chain covalently attached to a core protein. The GAGs are sulfated polysaccharides that contribute to many of the properties of PGs, especially to the high density of negative charge that characterizes these molecules. PGs of diverse structure are localized in the extracellular matrix (ECM), pericellularly, and intracellularly. They can have many different functions in the tissues where they reside.¹ Apart from their structural function, PGs also immobilize and modulate the activity of growth factors and cytokines extracellularly through electrostatic interactions. Well-documented examples of such effects concern platelet-derived growth factor,² basic fibroblast growth factor,^{3 4} transforming growth factor-ß,⁵ and interferon-.^{6 7}

Atherosclerosis develops preferentially at sites of intimal thickening characterized by an accumulation of PGs with high affinity for LDL.^{8 9 10} A series of in vivo and in vitro studies support this hypothesis.^{11 12 13 14 15 16 17 18} The interaction with chondroitin sulfate (CS)–rich PGs in the arterial intima contributes to the retention of apoB-containing lipoproteins in the vessel wall. This process may be a first step in a sequence of events leading to further modification of LDL particles in situ, such as oxidation and aggregation.^{19 20}

The LDL particle consists of a neutral lipid core of cholesteryl esters and triglycerides surrounded by a monolayer of amphipathic cholesterol and phospholipids with an embedded apoB-100 molecule. LDL retained in the arterial intima may be modified through the hydrolysis of its phospholipids by extracellular secretory, nonpancreatic, phospholipase A₂ (snpPLA₂).^{21 22} The PLA₂s are enzymes classified into 5 groups that share the property of hydrolyzing the sn-2 fatty acyl ester bond in phospholipids, thus producing free fatty acids (FFAs) and lysophospholipids. Incubation of LDL with snpPLA₂ under physiological conditions results in phospholipid hydrolysis.^{22 23 24} SnpPLA₂ is a strongly cationic protein (pI=10.5) with a total of 23 arginine and lysine residues with high affinity for PGs and GAGs.^{23 25 26} The components in the extracellular environment that associate with the enzyme after secretion have not been characterized, nor has the physiological role of the bound enzyme been elucidated.

The PGs present in the ECM of the arterial wall are synthesized mainly by smooth muscle cells (SMCs) and endothelial cells.⁸ Production of ECM by these cells is considered responsible for much of the intimal expansion seen in atherosclerotic plaque. In human atherosclerotic arteries, snpPLA₂ is present mainly in the ECM compartment associated with collagen fibers and PGs.²⁷ The enzyme is primarily synthesized by SMCs.²⁴ SnpPLA₂ isolated from human arteries is active toward LDL.²⁴ We have previously reported that human arterial SMCs produce and secrete soluble PGs into the cell culture medium. These PGs interact with both snpPLA₂ and LDL.^{23 28} However, the SMCs in vitro also synthesize an ECM that remains tightly adherent to the culture plates after removal of the cells. This matrix may be analogous to the in vivo ECM.^{7 29}

In the present work, we investigated the binding of snpPLA₂ to the ECM synthesized by SMCs in vitro. This model may mimic the ECM organization around SMCs in the arterial wall. The PGs that form part of the ECM were characterized, and their ability to bind snpPLA₂ was studied. The activity of snpPLA₂ on LDL phospholipids appears dependent on the type of GAG to which these 2 proteins are associated.²³ We further investigated this hypothesis by using an in vitro model of an insoluble chondroitin-6-sulfate (C6S) or heparin matrix and found that the matrixes exerted different effects on snpPLA₂-mediated hydrolysis of LDL phospholipids.

	Methods

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Materials
Benzamidine-HCl, Triton X-100, -amino-n-caproic acid, guanidinium-HCl, HEPES, trypsin-EDTA, methotrexate, FFA-free human albumin, cetylpyridinium chloride, ß-oleoyl--palmitoyl-L--phosphatidylcholine, heparin (6500 Da; from porcine intestinal mucosa), and heparan sulfate (7000 Da; from bovine intestinal mucosa) were purchased from Sigma Chemical Co. Hi-Trap Q and Hi-Trap heparin columns and CNBr-activated Sepharose were bought from Pharmacia Biotech AB. Precast 4% to 12% Tris-glycine gels were bought from Novex. Glass Econo-Columns and Affi-prep Hz support were bought from Bio-Rad Laboratories Inc. [³⁵S]Na₂SO₄ (100 mCi/mmol), L-[4,5-³H]leucine (120 Ci/mmol), ECL+Plus Western blotting detection system, and Hyperfilms for autoradiography were purchased from Amersham International. C6S (80 000 Da; from shark cartilage), chondroitinase ABC (ChABC; EC 4.2.2.4), chondroitinase AC (ChAC), and heparitinase I (HS I; EC 4.2.2.8) were bought from Seikagaku Co. Collagen type I and multiwell culture Primaria plates were from Collaborative Biomedical Products. Cell culture vessels, culture media, antibiotics, nonessential amino acids, and FCS were purchased from Flow Laboratories. Dulbecco's PBS was bought from J.R.H. Biosciences Sera-Laboratory. Ready-Safe scintillation cocktail was from Beckman Instruments Inc. Spectra/Por membranes (molecular weight cutoff, 3500) for dialysis were purchased from Spectrum Laboratory Products. The NEFA-C kit was from Wako Chemicals GmbH. Blyscan Proteoglycan and Glycosaminoglycan assay kit was bought from Biocolor Ltd. Anti-human snpPLA₂ monoclonal antibody was purchased from Upstate Biotechnology Inc. Anti-human perlecan antibody (monoclonal) was bought from Zymed Laboratories Inc. Rabbit anti-human decorin antibody (polyclonal) was from Chemicon International Inc. Polyclonal rabbit antiserum against biglycan, LF-51, was kindly provided by Dr Larry W. Fisher (National Institute of Dental Research, Bethesda, Md). Salts and other buffer substances or detergents were of analytical grade and purchased from Merck. All of the water used was filtered through a Milli-Pore Milli-Q system and was of high purity (resistivity >18 M · cm^-1).

Purification of Recombinant SnpPLA₂
Chinese hamster ovary cells transfected with the human gene for snpPLA2 (kindly provided by Dr Berit Johansen, UNIGEN, Trondheim, Norway)³⁰ were cultured in serum-free minimum essential medium (Eagle's -modification), and active snpPLA₂ was isolated from the cell culture medium as described.²³ The purity of each preparation was analyzed by SDS–polyacrylamide gel electrophoresis (PAGE) and Western blotting using monoclonal antibodies against snpPLA₂. Generally, the yield was 300 to 400 µg snpPLA₂ per L of cell culture medium.

Binding of SnpPLA₂ to ECM Synthesized by Human Arterial SMCs
ECM synthesized by human arterial SMCs was prepared in 96-well plates as described.⁷ This procedure has been shown to leave the intact ECM attached to the surface of the well. The amount of GAGs present in the ECM-coated plates was determined by using a Blyscan Proteoglycan and Glycosaminoglycan assay kit and following the protocol suggested by the manufacturer. This assay is based on the complex formation between the dye dimethyl methylene blue and sulfated PGs/GAGs. Reaction between the dye and GAGs at acidic pH produces a complex with an absorbance maximum at 525 nm. For the retention studies, ECM in 96-well plates was equilibrated in HEPES(140) buffer (10 mmol/L HEPES, 140 mmol/L NaCl, 5 mmol/L CaCl₂, and 2 mmol/L MgCl₂), pH 7.4, and snpPLA₂ was added in increasing concentrations as indicated in Figure 1. The matrix was incubated with snpPLA₂ for 2 hours at room temperature and then washed 3 times with HEPES(140), pH 7.4, to remove nonbound snpPLA₂. The remaining ECM-associated snpPLA₂ was determined by ELISA according to the protocol indicated by Upstate Biotechnology Inc, which uses a monoclonal antibody against snpPLA₂. To avoid radiolysis that might induce degradation of snpPLA₂ and also ECM components, we decided to use monoclonal antibodies to measure native snpPLA₂ instead of radiolabeling snpPLA₂.

View larger version (13K): [in this window] [in a new window]   Figure 1. Binding isotherm of snpPLA2 association with human arterial SMC–derived ECM prepared in 96-well culture plates. The matrix was equilibrated in HEPES(140) buffer, pH 7.4, and increasing concentrations of snpPLA2 were added as indicated on the x axis. After incubation for 2 hours at room temperature, nonbound snpPLA2 was washed away, and the snpPLA2 retained in the ECM was determined by ELISA using a monoclonal antibody against snpPLA2. The figure shows a representative experiment performed 3 times. Each data point represents mean± SD (n=4).

Displacement of SnpPLA₂ Bound to ECM
Displacement of ECM-bound snpPLA₂ was carried out by first equilibrating the ECM-coated plates with HEPES(140) buffer, pH 7.4. SnpPLA₂ was then added to each well in a final concentration of 0.1 µmol/L. After incubation for 2 hours at room temperature, the ECM plates were washed with HEPES(140), pH 7.4, 3 times to remove nonbound snpPLA₂. GAGs were added in increasing concentrations as indicated in Figure 2. After incubation at 4°C for 18 hours, the ECM plates were washed with HEPES(140), pH 7.4. The amount of ECM-bound snpPLA₂ was measured by an ELISA as described above.

View larger version (17K): [in this window] [in a new window]   Figure 2. Displacement of snpPLA2 bound to ECM-coated plates. The matrix was prepared in 96-well culture plates and preincubated with 0.1 µmol/L snpPLA2 in HEPES(140) buffer, pH 7.4. After 2-hour incubation at room temperature, nonbound snpPLA2 was washed away, and C6S or heparin was added in HEPES(140), pH 7.4, as indicated on the x axis. After incubation for 18 hours at 4°C the ECM-plates were extensively washed. The displacement of snpPLA2 was determined by measuring the amount of snpPLA2 that remained associated with the ECM by ELISA using a monoclonal antibody against snpPLA2. The figure shows a representative experiment performed 3 times. Each data point represents mean±SD (n=4).

Activity of SnpPLA₂ Immobilized in ECM
To measure the activity of snpPLA₂ immobilized in ECM plates, matrix was prepared in 96-well culture plates and equilibrated in HEPES(140), pH 7.4. SnpPLA₂ was added to the matrix in a final concentration of 0.1 µmol/L. After incubation for 2 hours at room temperature, the ECM plates were washed 3 times with HEPES(140), pH 7.4, to remove nonbound snpPLA₂. Mixed micelles containing phosphatidylcholine were used as the substrate and were prepared by dissolving ß-oleoyl--palmitoyl-L--phosphatidylcholine in 2% sodium deoxycholate and 4% Nonidet P-40 with vortexing. The mixed micelles were then diluted 25 times to a final concentration of 2 mg/mL with HEPES(140), pH 7.4, containing 10 mg/mL FFA-free human albumin. Fifty microliters of diluted micellar solution was added to each ECM-coated well followed by incubation at 37°C for the different time points indicated in Figure 3. The concentration of enzymatically derived FFAs was determined using an NEFA-C kit as described.²³ In these experiments, the concentrations of substrate and snpPLA₂ were high enough to allow the use of a colorimetric assay such as the NEFA-C kit to measure the enzyme activity.

View larger version (12K): [in this window] [in a new window]   Figure 3. Activity of snpPLA2 immobilized in ECM-coated plates. The matrix was prepared in 96-well culture plates and equilibrated with HEPES(140) buffer, pH 7.4. SnpPLA2 was added to each well at a concentration of 0.1 µmol/L, and after incubation for 2 hours at room temperature the nonbound snpPLA2 was removed by repeated washes. The enzymatic activity was determined by measuring the liberated FFAs from mixed micelles containing phosphatidylcholine present in great excess after incubation at 37°C for different time points indicated on the x axis. The control wells without snpPLA2 have been subtracted, and the values in the figure are thus specific snpPLA2 activity. Each data point represents mean±SD of 4 separate incubations. The dashed line indicates linear regression analysis.

Isolation of PGs From ECM
Human arterial SMCs were cultured with [³⁵S]sulfate and [³H]leucine to biosynthetically label PGs as described.²³ To remove peripheral extrinsically associated PGs, the cell culture medium was removed and the cells were washed with Dulbecco's PBS containing 50 µg/mL heparin for 30 minutes at room temperature.³¹ The heparin-containing buffer was removed, and the cells were washed 3 times with Dulbecco's PBS without heparin. The cells were dissolved by 2 extractions, with 5 mL each of buffer containing 1% Triton X-100, 0.15 mol/L NaCl, 10 mmol/L Tris, 5 mmol/L MgCl₂, 2 mmol/L EDTA, 0.255 mmol/L DTT, and 1 µmol/L 4-(2-aminoethyl)(benzenesulfonyl) fluoride (AEBSF), pH 7.2. After incubation for 30 minutes with gentle shaking, the extract was removed and the remaining matrix was washed with Dulbecco's PBS. The ECM was solubilized by 2 extractions with 5 mL each of 8 mol/L urea, 2 mmol/L EDTA, 0.5% Triton X-100, and 20 mmol/L Tris-HCl (pH 7.5) containing protease inhibitors (10 mmol/L EDTA, 1 mg/mL benzamidine-HCl, and 10 mmol/L -amino-n caproic acid). The bottles were left overnight at 4°C before the ECM extract was collected with a cell scraper.

The ECM extract was chromatographed on a Hi-Trap Q (5-mL) column equilibrated with binding buffer containing 8 mol/L urea, 20 mmol/L Tris, 2 mmol/L EDTA, and 0.5% Triton X-100 (pH 7.5) at a flow rate of 5 mL/min. The ³H- and ³⁵S-labeled PG-containing fractions were collected after elution with a linear NaCl gradient (0.25 to 1.5 mol/L NaCl) and dialyzed at 4°C against water containing 1 mmol/L AEBSF and 1 mg/mL benzamidine-HCl. This preparation is referred to as "total ECM-PGs."

Characterization of ECM-PGs
To identify the type of PGs present in the ECM, immunoprecipitation with monoclonal and polyclonal antibodies was carried out as described.³² In brief, protein A–Sepharose 4 Fast Flow was saturated with rabbit anti-mouse IgG antibody followed by incubation with anti-perlecan monoclonal antibody. Equal counts of radiolabeled ECM-PGs were added to the Sepharose gels. The immunocomplexes formed were dissociated by boiling in sample buffer for SDS-PAGE.³³ The Sepharose gels were pelleted by centrifugation, and the supernatants were loaded on a precast 4% to 12% Tris-glycine gel. After electrophoresis, the gel was fixed in 0.1% cetylpyridinium chloride in isopropanol/acetic acid/water, 30:10:60, vol/vol/vol, before vacuum-drying and visualization by autoradiography. A similar procedure was used for the polyclonal antibody against decorin, with the exception that no rabbit anti-mouse IgG antibody was included.

Western blot analysis was done essentially as described³⁴ after removing the GAGs by treatment with ChABC (100 mU/mL) and HS I (40 mU/mL) at 37°C for 24 hours in PBS buffer. The antibodies used were the same as those listed above and a polyclonal rabbit antiserum against biglycan, LF-51, kindly provided by Dr Larry W. Fisher.³⁵ The digested ECM-PGs were separated on a precast 4% to 12% Tris-glycine gel and then blotted to a polyvinylidene difluoride membrane. The membranes were incubated with primary monoclonal antibody (diluted 1:1000) or polyclonal antibodies (diluted 1:10 000). After a 1.5-hour incubation at room temperature, the primary antibodies were removed and the membranes were incubated with the secondary antibodies (diluted 1:25 000) overnight at 4°C. The membranes were then developed using an ECL+Plus Western blotting detection system (Amersham) according to the protocol indicated by the manufacturer.

Affinity Chromatography on a Sepharose-SnpPLA₂ Column
To specifically isolate in the ECM those PGs with an affinity for snpPLA₂, a column was prepared from snpPLA₂ bound to CNBr-activated Sepharose according to the manufacturer's procedure. Affinity chromatography was carried out as described.²³ In parallel, equal counts of ECM-PGs were passed through a control column without snpPLA₂. Retained labeled PGs were eluted with a linear NaCl gradient (20 to 1500 mmol/L). The radioactivity in each fraction was determined by liquid scintillation counting, and the fractions containing labeled PGs were collected.

To determine the relative contribution of different GAGs to the binding, snpPLA₂ affinity–isolated ECM-PGs were treated with ChABC (100 mU/mL) or HS I (40 mU/mL) at 37°C for 18 hours. As a control, ECM-PGs were incubated in the absence of GAG-degrading enzyme in the same way. The digested PG preparations were rechromatographed on the snpPLA₂ affinity column and eluted with a linear NaCl gradient (20 to 1000 mmol/L). Fractions were collected, and the total radioactivity in each fraction was determined by liquid scintillation counting.

PG Characterization by SDS-PAGE
The molecular weight and GAG composition of the snpPLA₂ affinity–isolated PGs were determined by incubating equal aliquots of radiolabeled PGs for 18 hours at 37°C in the presence or absence of specific GAG-degrading enzymes: ChABC (100 mU/mL), HS I (40 mU/mL), or ChAC (100 mU/mL). The PGs were then loaded on a precast 4% to 12% Tris-glycine gel, and SDS-PAGE was run as described previously.³³ The gel was fixed in acetic acid/isopropanol/water, 10:30:60, vol/vol/vol, containing 0.1% cetylpyridinium chloride, and the dried gel was developed by autoradiography.

SnpPLA₂ Hydrolysis of LDL Immobilized in C6S or Heparin Matrixes
Affi-prep Hz support containing 3.6 mg C6S per mL gel was prepared according to the manufacturer's procedure and equilibrated in buffer A containing 10 mmol/L HEPES, 20 mmol/L NaCl, 2 mmol/L MgCl₂, 10 mmol/L CaCl₂, and 10 mg/mL FFA-free human albumin, pH 8.0. Two columns (1 mL each) were prepared, and 1 was used as a control without any snpPLA₂; the other was loaded with 50 µg snpPLA₂. The column was washed with buffer A. No snpPLA₂ activity could be detected in the collected column wash, indicating 100% retention of snpPLA₂. Two milligrams of human LDL (d 1.019 to 1.063) containing 10 µmol/L BHT was passed through each column. Nonbound LDL was determined by measuring cholesterol in the column wash. Of the total amount of added LDL, 80% to 90% was bound to the gels. The flow was stopped and the column outlets were sealed. The GAG columns, containing either LDL only (control column) or LDL plus snpPLA₂, were incubated at 37°C for 20 hours. Enzymatically released FFAs were then eluted with buffer A and collected, while the LDL particles and snpPLA₂ remained bound to the GAG matrixes. The amount of FFAs produced was quantified using the NEFA C-kit. SnpPLA₂ and LDL were eluted from the GAG matrixes with a linear NaCl gradient (20 to 1000 mmol/L), and fractions of 0.5 mL were collected. Each fraction was assayed for cholesterol content according to the CHOD-PAP method³⁶ using cholesterol reagent bought from Boehringer Mannheim. The peak fractions containing cholesterol were further analyzed by agarose gel electrophoresis³⁷ to evaluate any difference in electrophoretic mobility between the snpPLA₂-treated LDL and the LDL eluted from the control column (without snpPLA₂). The elution of snpPLA₂ was determined by monitoring the enzyme activity in the collected fractions by using mixed micelles containing L--phosphatidylcholine as the substrate. In parallel, the same procedure was used with Hi-Trap heparin columns. Both types of GAG columns retained similar amounts of snpPLA₂ and LDL.

	Results

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The SMC-derived ECM–coated plates contained 5.6 ng GAG/mm² with a CS to HS ratio of 4:1. After incubation with snpPLA₂, the amount of ECM-bound snpPLA₂ was determined, and the binding isotherm is shown in Figure 1. The monoclonal antibody used recognizes native snpPLA₂, suggesting that the enzyme is bound in an active form. It was possible to release snpPLA₂ bound to ECM by adding increasing concentrations of C6S, as shown in Figure 2. Displacement of ECM-bound snpPLA₂ was detected after addition of at least 1 µmol/L C6S. Addition of increasing concentrations of C6S resulted in a decrease of the total amount of snpPLA₂ retained in the ECM plate. At 1 mmol/L C6S, only 15% of snpPLA₂ remained bound to the ECM compared with the controls (no addition of C6S). However, the more sulfated and negatively charged GAG heparin did not displace ECM-bound snpPLA₂. Separate experiments using HS gave similar results as with heparin (data not shown).

SnpPLA₂ was active when immobilized in ECM plates as shown in Figure 3. The matrix was preincubated with snpPLA₂ and the activity measured by determination of the concentration of enzymatically liberated FFAs after incubation with mixed micelles containing phosphatidylcholine. The activity was proportional to the incubation time, indicated by the regression line (dashed line).

The PGs present in the ECM were isolated and characterized using immunochemical analysis. We could detect perlecan and biglycan using monoclonal and polyclonal antibodies. The positive immunoprecipitation reaction with anti-perlecan antibodies is shown in Figure 4A (lane 2). Perlecan has a core protein of 400 to 470 kDa with attached GAG chains and is therefore retained at the top of the gel. Lane 4 represents the starting material consisting of total ECM-PGs. The gel pattern displayed, besides perlecan, a smaller PG that was degraded by ChABC and ChAC (data not shown). This PG did not react with anti-decorin antibody (lane 3), and based on its relative molecular weight (200 to 250 kDa) and CS content, it appeared to be biglycan. As a positive control, decorin was immunoprecipitated from the pool of soluble PGs secreted into the cell culture medium, and decorin migrated on gel electrophoresis in the range 100 to 150 kDa (data not shown). Decorin was not detected in the ECM extracts, even after GAG digestion and Western blotting (Figure 4B). On the other hand, the antibodies against biglycan gave a strong reaction after the GAGs were removed and confirmed the presence of biglycan with a core protein of 46 kDa. In addition, perlecan was also detected on Western blot (data not shown). These results indicate that the PGs present in the ECM synthesized by human arterial SMCs in vitro are mainly biglycan and perlecan.

View larger version (33K): [in this window] [in a new window]   Figure 4. A, Immunoprecipitation using anti-human perlecan monoclonal and anti-decorin polyclonal antibodies of total 35S- and 3H-labeled ECM-PGs isolated by ion-exchange chromatography. After dissociation of the formed immunocomplexes, the supernatants were loaded on a 4% to 12% Tris-glycine SDS-PAGE gel. After electrophoresis the gel was fixed, vacuum-dried, and developed by autoradiography. Lane 1, rabbit anti-mouse IgG (nonspecific control); lane 2, anti-perlecan monoclonal antibody; lane 3, anti-decorin polyclonal antibody; and lane 4, total ECM-PGs (starting material for immunoprecipitation). Molecular weight standards are in the left lane. B, Western blotting using anti-biglycan and anti-decorin polyclonal rabbit antiserum as indicated in the figure. The ECM-PGs were digested with ChABC and HS I as described in Methods and separated on a 4% to 12% Tris-glycine gel. The gel was blotted onto a polyvinylidene difluoride membrane and developed with an ECL+Plus Western blotting detection system. Molecular weight standards are indicated in the left part of the figure.

Affinity chromatography was used to isolate the PGs that bound to snpPLA₂. From the total amount of ³⁵S counts per minute loaded on the snpPLA₂-Sepharose, 64% bound to the column. The retained PGs were eluted with a linear NaCl gradient as shown in Figure 5A. The PGs eluted as a single peak at 350 mmol/L NaCl. Only a small fraction of the counts per minute (<8%) was retained in a control column without snpPLA₂. ChABC treatment resulted in a 55% reduction of radioactivity bound to the snpPLA₂ column and also a decrease in affinity, because the elution peak is slightly shifted toward lower NaCl concentrations. HS degradation reduced the amount by 21% (Figure 5B), but no shift in affinity was detected. These results indicate that association of snpPLA₂ is mainly via the GAG moiety of PGs.

View larger version (31K): [in this window] [in a new window]   Figure 5. SnpPLA2 affinity chromatography for specific isolation of ECM-PGs. A, The total ECM-PG preparation was chromatographed in 10 mmol/L HEPES, 20 mmol/L NaCl, 5 mmol/L CaCl2, and 2 mmol/L MgCl2, pH 7.4, on a column with immobilized snpPLA2. The bound radiolabeled PGs were eluted with a linear NaCl gradient as indicated in the figure (dashed line). Fractions of 0.5 mL were collected, and the total counts in each fraction are indicated in the figure. B, After enzymatic degradation of the GAGs with ChABC or HS I, the snpPLA2 affinity–isolated PGs were rechromatographed on the snpPLA2-Sepharose column. The bound PGs were eluted with a linear NaCl gradient (dashed line). Fractions of 0.5 mL were collected and total counts in each fraction were determined by liquid scintillation counting. Ctrl indicates the control incubated in the absence of GAG-degrading enzymes.

The radioactive PGs that bound to the snpPLA₂ column were characterized by SDS-PAGE after specific GAG digestion. As shown in Figure 6, there were 2 distinct PGs that had affinity for snpPLA₂, and they coeluted from the experimental column. A large HSPG was detected that only just entered the polyacrylamide gel. This band disappeared after treatment with HS I (lane 3). Consistent with the large size and GAG composition, we concluded that this PG was perlecan as indicated above. A smaller CSPG also had affinity for snpPLA₂, and this band disappeared after treatment with ChABC (lane 2). The degradation was almost complete after treatment with ChAC (lane 4), suggesting that CS made up most of the GAGs. On the basis of the immunochemical analysis of the total ECM-PGs presented above and the CS component, we concluded that this PG was biglycan.

View larger version (100K): [in this window] [in a new window]   Figure 6. The relative molecular weight and GAG composition of snpPLA2 affinity–isolated PGs was determined by degradation of GAGs by ChABC, HS I, or ChAC followed by SDS-PAGE on a 4% to 12% Tris-glycine gel. After fixing and drying the gel, an autoradiograph was obtained. Radiolabeled molecular weight standards are in the left lane. Lane 1, no enzyme; lane 2, ChABC; lane 3, HS I; and lane 4, ChAC.

To study the specific effect of different GAGs present in the ECM on snpPLA₂ activity, we used a model that consisted of immobilized GAGs in experimental columns. One column contained purified heparin that resembled the HS in perlecan, and the other contained C6S that was similar to the GAGs of biglycan. In this model, it is possible to colocalize the GAGs, the snpPLA₂, and the potential substrate (the LDL), because both LDL and snpPLA₂ bind to GAGs. SnpPLA₂ hydrolyzes only the acyl group bound in the sn-2 position in phospholipids and produces FFAs and lysophospholipids.³⁸ The activity of snpPLA₂ was monitored by measuring the amount of LDL-derived FFAs. As shown in the left part of Figure 7, snpPLA₂ immobilized in the C6S matrix (A) exhibited 7-fold greater activity on LDL phospholipids than the heparin matrix (B). The remnant LDL particles and snpPLA₂ were eluted with an NaCl gradient shown in the right part of Figure 7. LDL elutes at lower NaCl concentrations than does snpPLA₂, indicating a lower affinity for GAGs versus snpPLA₂. These experiments were run in the presence of albumin in the eluant (10 mg/mL) that should take up all FFAs and lysophospholipids formed. As expected, there was no difference in the electrophoretic mobility between the remnant LDL particles and native LDL evaluated by agarose gel electrophoresis (data not shown). Electron microscopy with negative staining³⁹ revealed a heterogeneous size population of LDL particles eluted from the C6S column. There were small LDL particles, "native"-like LDL particles, and larger particles, suggesting fusion or aggregation of LDL particles after snpPLA₂ hydrolysis in the C6S matrix (data not shown). The snpPLA₂ activity was also determined in the collected fractions. It was clear that the affinity for heparin was higher than that for C6S, because the enzyme eluted at higher concentrations of NaCl from the heparin matrix. The recovery of snpPLA₂ from the 2 GAG columns was similar (9% more active snpPLA2 was eluted from the C6S column compared with the heparin column). The data presented in Figure 7 are representative of experiments performed 3 times. However, the efficiency of binding of snpPLA2 and LDL to C6S or heparin varied slightly between experiments. Therefore, it was not possible to keep the concentrations of substrate and enzyme constant for repetitive experiments. These variables taken together made it difficult to present, in one figure, the mean and SD of several experiments with any accuracy.

View larger version (27K): [in this window] [in a new window]   Figure 7. Activity of snpPLA2 immobilized in GAG matrixes (A, C6S matrix; B, heparin matrix). Equal amounts of LDL, containing 10 µmol/L BHT, were bound to the GAG columns containing (+) or lacking (-) 50 µg snpPLA2 in buffer: 10 mmol/L HEPES, 20 mmol/L NaCl, 10 mmol/L CaCl2, 2 mmol/L MgCl2, and 10 mg/mL FFA-free albumin, pH 8.0. The flow was stopped and the columns were incubated for 20 hours at 37°C. The FFAs were eluted with the same buffer. The bar graphs in the left part of the figure show the amount of LDL-derived FFAs determined with the NEFA-C kit from each GAG column containing (+) or lacking (-) snpPLA2. The right part of the figure shows the elution profile of LDL and snpPLA2 from the respective GAG matrix by use of a linear NaCl gradient (dashed line). The cholesterol content in each fraction was measured using the CHOD-PAP method (absorbance determined at 490 nm). The elution profile of snpPLA2 was monitored by measuring the enzyme activity in each fraction with the NEFA-C kit using L--phosphatidylcholine–containing mixed micelles as the substrate (absorbance determined at 550 nm). LDL eluted from the column without snpPLA2 is indicated by open squares; LDL eluted from the column also containing snpPLA2, open triangles; and elution of snpPLA2, filled circles. The figure shows a representative experiment performed 3 times.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
PGs and GAGs are functional modulators of an extensive collection of molecules of biological importance that include water, ions, and proteins such as growth factors, cytokines, enzymes, and lipoproteins.^{1 8 19} Most of the proteins interacting with sulfated PGs, as snpPLA₂, share positively charged lysine- and arginine-rich segments that associate ionically with the sulfate and carboxyl groups of the GAG moiety.⁴⁰ We recently described the extracellular location of snpPLA₂ in the human intima. The enzyme is abundant in atherosclerotic lesions, and the isolated arterial enzyme can act on LDL phospholipids.^{23 24} Electron microscopy studies using immunogold techniques revealed that the majority of snpPLA₂ is localized in the intimal ECM in atherosclerotic lesions from human coronary arteries.²⁷ The intimal ECM is also where LDL appears to be accumulated and modified during atherogenesis.^{19 20} Therefore, the above results suggest that snpPLA₂ and its potential substrate, the phospholipids on the LDL, may be colocalized by their association to PGs of the intimal ECM. The snpPLA₂ activity on LDL may be a focal source of lysophospholipids and FFAs that are postulated as proinflammatory, and potentially atherogenic, substances.^{41 42 43 44 45}

Vascular SMCs appear to be the main source of snpPLA₂ in the human intima.²⁴ Therefore, it is interesting to characterize the molecular interactions that could locate and control the enzyme in this environment. SMCs in culture provide a tool for such studies but certainly cannot reflect the entire structural complexity of the arterial intima. PGs produced by SMCs seems to exist as soluble components, extractable with mild solutions, and as components of organized structures that need high-ionic-strength solutions, chaotropic agents, and enzymes to be dissociated. In previous experiments, we characterized the associations of snpPLA₂ with the PGs secreted by human SMCs in culture as soluble components of the medium.²³ These may represent the soluble forms of intimal PGs containing mostly versican. In the present work, we report the association of the enzyme with biglycan and perlecan, which are structural elements of the SMC basement membrane.¹ The results suggest that there may be a functional difference between snpPLA₂ associated with PGs rich in CS, as biglycan, and those rich in HS, like perlecan. We have previously demonstrated the potential of soluble GAGs to modulate snpPLA₂ activity.²³ Here we used another model consisting of immobilized GAGs that may mimic the more structurally rigid conditions in the ECM. The results suggest that snpPLA₂ bound to CSPGs in vivo may be active toward colocalized LDL also bound to CSPGs.

During atherogenesis moderate cell proliferation is accompanied by extensive ECM production with an increased content of pericellular and extracellular PGs.⁸ Arterial wall CS- and dermatan sulfate–rich PG contents increase while the amount of HSPG decreases during atherogenesis.^{46 47} The displacement experiments reported in Figure 2 suggest that in in vivo situations like this, the extracellular CSPGs (eg, versican and biglycan) may be quantitatively more important retainers of active snpPLA₂ than are HSPGs (eg, perlecan). Biglycan belongs to the family of small, leucine-rich PGs and is secreted in the ECM environment of many tissues. It has a core protein of 45 kDa to which 2 GAG chains are attached (dermatan sulfate, C6S, or C4S type), and it can interact with many collagens, but especially type I.⁴⁸ The degree of interspersed dermatan sulfate units may vary, and this may lead to altered affinity for its ligands, eg, snpPLA₂.

In the experimental model of the nonsoluble ECM used in this study, heparin and HS were not able to displace matrix-bound snpPLA₂. These data suggest that perlecan might not be important in the retention of snpPLA₂ in this model, but instead, biglycan is the key player. One possibility is that only small amounts of HS are present in the ECM. GAG analysis of ECM from SMCs shows that it mainly contains CS-type GAGs. Furthermore, other proteins could be present bound to the HS chains in the ECM. This situation may prevent the binding of snpPLA₂ to perlecan. However, the isolation of PGs from the ECM is done using 8 mol/L urea. This procedure allows the isolation of PGs by dissociating any material bound to the PGs. The isolated HSPGs are concentrated in solution, are susceptible to degradation by HS I, and are able to interact with snpPLA₂. Taken together, this may explain the inability of heparin/HS to release ECM-bound snpPLA₂ despite the ability of snpPLA₂ to interact with isolated HSPGs.

Recently, Collins-Tozer and Carew⁴⁹ found that early in atherogenesis there is a remarkable increase in the retention of LDL by the ECM. Therefore, the colocalization of snpPLA₂ and its potential substrate may occur early during lesion development, especially if, as suggested by Nakano and coworkers,⁵⁰ the production of snpPLA₂ by vascular SMCs can be increased by proinflammatory cytokines that can be associated with atherogenesis (E.H.-C. et al, unpublished data, 1998). The contribution of snpPLA₂ to atherogenesis remains speculative. However, our results are compatible with a role for snpPLA₂ as an agent that, by its location, could form proinflammatory lipids from LDL entrapped in the CSPGs of the arterial intima.

	Acknowledgments

 
This work was supported by grants from the Medical Research Council (project No. 4531 to G.B.), the Swedish Heart and Lung Foundation (project No. 63503 to G.B. and No. 61538 to E.H.-C.), the Royal Society of Arts and Sciences in Göteborg, Sweden (to E.H.-C.), the Adlerbertska Research Foundation (to E.H.-C.), and Astra Hässle AB, Mölndal, Sweden (to G.B.). Prof Germán Camejo is acknowledged for his valuable help in preparing this manuscript and Birgitta Rosengren for skillful technical assistance.

Received January 28, 1998; accepted June 1, 1998.

	References

Top Abstract Introduction Methods Results Discussion References

 

1. Iozzo RV, Murdoch AD. Proteoglycans of the extracellular environment: clues from the gene and protein side offer novel perspectives in molecular diversity and function. FASEB J. 1996;10:598–614.[Abstract]
2. Fager G, Camejo G, Olsson U, Östergren-Lundén G, Lustig F, Bondjers G. Binding of platelet-derived growth factor and low density lipoproteins to glycosaminoglycan species produced by human arterial smooth muscle cells. J Cell Physiol. 1995;163:380–392.[Medline] [Order article via Infotrieve]
3. Ross CR, Kubinak S, Hale CC. Purification of a basic fibroblast growth factor-binding proteoglycan from bovine cardiac plasma membrane. Biochim Biophys Acta. 1993;1145:219–226.[Medline] [Order article via Infotrieve]
4. Suzu S, Kimura F, Yamada M, Yanai N, Kawashima T, Nagata N, Motoyoshi K. Direct interaction of proteoglycan macrophage colony-stimulating factor and basic fibroblast growth factor. Blood. 1994;83:3113–3119.[Abstract/Free Full Text]
5. Hildebrand A, Romaris M, Rasmussen LM, Heinegard D, Twardzik DR, Border WA, Ruoslahti E. Interaction of the small interstitial proteoglycans biglycan, decorin and fibromodulin with transforming growth factor ß. Biochem J. 1994;302:527–534.
6. Lortat-Jacob H, Kleinman HK, Grimaud JA. High-affinity binding of interferon- to a basement membrane complex (Matrigel). J Clin Invest. 1991;87:878–883.
7. Hurt-Camejo E, Rosengren B, Camejo G, Sartipy P, Fager G, Bondjers G. Interferon - binds to extracellular matrix chondroitin-sulfate proteoglycans, thus enhancing its cellular response. Arterioscler Thromb Vasc Biol. 1995;15:1456–1465.[Abstract/Free Full Text]
8. Wight TN. The extracellular matrix. In: Fuster V, Ross R, Topol EJ, eds. Atherosclerosis and Coronary Artery Disease. Philadelphia, Pa: Lippincott-Raven; 1996:421–440.
9. Cardoso LEM, Mourão PAS. Glycosaminoglycan fractions from human arteries presenting diverse susceptibilities to atherosclerosis have different binding affinities to plasma LDL. Arterioscler Thromb. 1994;1:115–124.
10. Stary HC, Chandler AB, Dinsmore RE, Fuster V, Glagov S, Insull W Jr, Rosenfeld ME, Schwartz CJ, Wagner WD, Wissler RW. A definition of advanced types of atherosclerotic lesions and a histological classification of atherosclerosis: a report from the Committee on Vascular Lesions of the Council on Arteriosclerosis, American Heart Association. Circulation. 1995;92:1355–1374.[Abstract/Free Full Text]
11. Srinivasan SR, Yost C, Bhandaru RR, Radahakrishnamurthy B, Berenson G. Lipoprotein-glycosaminoglycan interactions in aortas of rabbits fed atherogenic diets containing different fats. Atherosclerosis. 1982;43:289–301.[Medline] [Order article via Infotrieve]
12. Steele RH, Wagner WD. Lipoprotein interaction with artery wall derived proteoglycan: comparison between atherosclerosis-susceptible WC-2 and resistant Show Racer pigeons. Atherosclerosis. 1987;65:63–73.[Medline] [Order article via Infotrieve]
13. Camejo G, Olofsson SO, Lopez F, Carlsson P, Bondjers G. Identification of apoB-100 segments mediating the interaction of low density lipoproteins with arterial proteoglycans. Arteriosclerosis. 1988;8:368–377.[Abstract/Free Full Text]
14. Hollmann JA, Schmidt A, von Bassewitz DB, Buddecke E. Relationship of sulfated glycosaminoglycans and cholesterol content in normal and atherosclerotic human aortas. Arteriosclerosis. 1989;9:154–158.[Abstract/Free Full Text]
15. Nievelstein PF, Fogelman AM, Mottino G, Frank JS. Lipid accumulation in rabbit aortic intima 2 hours after bolus infusion of low density lipoprotein: a deep-etch and immunolocalization study of ultrarapidly frozen tissue. Arterioscler Thromb. 1991;11:1795–1805.[Abstract/Free Full Text]
16. Pentikäinen MO, Öörni K, Lassila R, Kovanen PT. The proteoglycan decorin links low density lipoproteins with collagen type I. J Biol Chem. 1997;272:7633–7638.[Abstract/Free Full Text]
17. Galis ZS, Alavi MZ, Moore S. Co-localization of aortic apolipoprotein B and chondroitin sulfate in an injury model of atherosclerosis. Am J Pathol. 1993;142:1432–1438.[Abstract]
18. Vijayagopal P, Figueroa JE, Fontenot JD, Glancy DL. Isolation and characterization of a proteoglycan variant from human aorta exhibiting a marked affinity for low density lipoprotein and demonstration of its enhanced expression in atherosclerotic plaque. Atherosclerosis. 1996;127:192–203.
19. Hurt-Camejo E, Olsson U, Wiklund O, Bondjers G, Camejo G. Cellular consequences of the association of apoB lipoproteins with proteoglycans: potential contribution to atherogenesis. Arterioscler Thromb Vasc Biol. 1997;17:1011–1017.[Free Full Text]
20. Williams KJ, Tabas I. The response-to-retention hypothesis of early atherogenesis. Arterioscler Thromb Vasc Biol. 1995;15:551–561.[Free Full Text]
21. Hurt-Camejo E, Camejo G. Potential involvement of type II phospholipase A2 in atherosclerosis. Atherosclerosis. 1997;132:1–8.[Medline] [Order article via Infotrieve]
22. Kleinman Y, Krul ES, Burnes M, Aronson W, Pfleger B, Schonfeld G. Lipolysis of LDL with phospholipase A2 alters the expression of selected apoB-100 epitopes and the interaction of LDL with cells. J Lipid Res. 1988;29:729–743.[Abstract]
23. Sartipy P, Johansen B, Camejo G, Rosengren B, Bondjers G, Hurt-Camejo E. Binding of human phospholipase A2 type II to proteoglycans: differential effect of glycosaminoglycans on enzyme activity. J Biol Chem. 1996;271:26307–26314.[Abstract/Free Full Text]
24. Hurt-Camejo E, Andersen S, Standal R, Rosengren B, Sartipy P, Stadberg E, Johansen B. Localization of nonpancreatic secretory phospholipase A2 in normal and atherosclerotic arteries: activity of the isolated enzyme on low-density lipoproteins. Arterioscler Thromb Vasc Biol. 1997;17:300–309.[Abstract/Free Full Text]
25. Dua R, Cho W. Inhibition of human secretory class II phospholipase A2 by heparin. Eur J Biochem. 1994;221:481–490.[Medline] [Order article via Infotrieve]
26. Murakami M, Nakatani Y, Kudo I. Type II secretory phospholipase A2 associated with cell surfaces via C-terminal heparin-binding lysine residues augments stimulus-initiated delayed prostaglandin generation. J Biol Chem. 1996;271:30041–30051.[Abstract/Free Full Text]
27. Romano M, Romano E, Björkerud S, Hurt-Camejo, E. Ultrastructural localization of secretory type II phospholipase A₂ in atherosclerotic and nonatherosclerotic regions of human arteries. Arterioscler Thromb Vasc Biol. 1997;18:519–525.[Abstract/Free Full Text]
28. Camejo G, Fager G, Rosengren B, Hurt-Camejo E, Bondjers G. Binding of low density lipoproteins by proteoglycans synthesized by proliferating and quiescent human arterial smooth muscle cells. J Biol Chem. 1993;268:14131–14137.[Abstract/Free Full Text]
29. Knudsen BS, Harpel PC, Nachman RL. Plasminogen activator inhibitor is associated with the extracellular matrix of cultured bovine smooth muscle cells. J Clin Invest. 1987;80:1082–1089.
30. Johansen B, Kramer RM, Hession C, McGray P, Pepinsky BR. Expression, purification and biochemical comparison of natural and recombinant human non-pancreatic phospholipase A2. Biochem Biophys Res Commun. 1992;187:544–551.[Medline] [Order article via Infotrieve]
31. Faassen AE, Achrager JA, Klein DJ, Oegema TR, Couchman JR, McCarthy JB. A cell surface chondroitin sulfate proteoglycan, immunologically related to CD44, is involved in type I collagen-mediated melanoma cell motility and invasion. J Cell Biol. 1992;116:521–531.[Abstract/Free Full Text]
32. Whitelock JM, Murdoch AD, Iozzo RV, Underwood AP. The degradation of human endothelial cell-derived perlecan and release of bound basic fibroblast growth factor by stromelysin, collagenase, plasmin, and heparanases. J Biol Chem. 1996;271:10079–10086.[Abstract/Free Full Text]
33. Schägger H, von Jagow G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem. 1987;166:368–379.[Medline] [Order article via Infotrieve]
34. Nelimarkka L, Kainulainen V, Schönherr E, Moisander S, Jortikka M, Lammi M, Elenius K, Jalkanen M, Järveläinen H. Expression of small extracellular chondroitin/dermatan sulfate proteoglycans is differentially regulated in human endothelial cells. J Biol Chem. 1997;272:12730–12737.[Abstract/Free Full Text]
35. Fisher LW, Stubbs JT III, Young MF. Antisera and cDNA probes to human and certain animal model bone matrix noncollagenous proteins. Acta Orthop Scand Suppl.. 1995;66:61–65.
36. Siedel J, Hagele EO, Ziegenhorn J, Wahlefeld AW. Reagent for the enzymatic determination of serum total cholesterol with improved lipolytic efficiency. Clin Chem. 1983;29:1075–1080.[Abstract/Free Full Text]
37. Noble RP. Electrophoretic separation of plasma lipoproteins in agarose gel. J Lipid Res. 1968;9:693–700.[Abstract]
38. Dennis EA. Diversity of group types, regulation, and function of phospholipase A2. J Biol Chem. 1994;269:13057–13060.[Free Full Text]
39. Forte TM, Nordhausen RW. Electron microscopy of negatively stained lipoproteins. Methods Enzymol. 1986;128:442–457.[Medline] [Order article via Infotrieve]
40. Jackson RL, Busch SJ, Cardin AD. Glycosaminoglycans: molecular properties, protein interactions, and role in physiological processes. Physiol Rev. 1991;71:481–539.[Free Full Text]
41. Kume N, Gimbrone MA Jr. Lysophosphatidylcholine transcriptionally induces growth factor gene expression in cultured human endothelial cells. J Clin Invest. 1994;93:907–911.
42. Kume N, Cybulsky MI, Gimbrone MA Jr. Lysophosphatidylcholine, a component of atherogenic lipoproteins, induces mononuclear leukocyte adhesion molecules in cultured human and rabbit arterial endothelial cells. J Clin Invest. 1992;90:1138–1144.
43. Chai YC, Howe PH, DiCorleto PE, Chisolm GM. Oxidized low density lipoprotein and lysophosphatidylcholine stimulated cell cycle entry in vascular smooth muscle cells: evidence for release of fibroblast growth factor-2. J Biol Chem. 1996;271:17791–17797.[Abstract/Free Full Text]
44. Amri EZ, Ailhaud G, Grimaldi PA. Fatty acids as signal transducing molecules: involvement in the differentiation of preadipose to adipose cells. J Lipid Res. 1994;35:930–937.[Abstract]
45. Quinn MT, Parthasarathy S, Steinberg D. Lysophosphatidylcholine: a chemotactic factor for human monocytes and its potential role in atherosclerosis. Proc Natl Acad Sci U S A. 1988;85:2805–2809.[Abstract/Free Full Text]
46. Völker W, Schmidt A, Oortmann W, Broszey T, Faber V, Buddecke E. Mapping of proteoglycans in atherosclerotic lesions. Eur Heart J. 1990;11:29–40.[Abstract/Free Full Text]
47. Yla-Herttuala S, Sumuvuori H, Karkola K, Mottonen M, Nikkari T. Glycosaminoglycans in normal and atherosclerotic human coronary arteries. Lab Invest. 1986;54:402–407.[Medline] [Order article via Infotrieve]
48. Schönherr E, Witsch-Prehm P, Harrach B, Robenek H, Rauterberg J, Kresse H. Interaction of biglycan with type I collagen. J Biol Chem. 1995;270:2776–2783.[Abstract/Free Full Text]
49. Collins-Tozer E, Carew TE. Residence time of low-density lipoprotein in the normal and atherosclerotic rabbit aorta. Circ Res. 1997;80:208–218.[Abstract/Free Full Text]
50. Nakano T, Ohara O, Teraoka H, Arita H. Group II phospholipase A2 mRNA synthesis is stimulated by two distinct mechanisms in rat vascular smooth muscle cells. FEBS Lett. 1990;1:171–174.

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