Essential role of phospholipase A 2 activity in endothelial cell-induced modification of low density lipoprotein ( atherosclerosis / foam celis / macrophages / lipid peroxides )

Previous studies have established that incubation of low density lipoprotein (LDL) with cultured endotheHal cells (EC) converts it to a new form (EC-modified LDL) that is now recognized by a specific receptor on macrophages (the acetyl LDL receptor) and is taken up and degraded 3-10 times more rapidly than native LDL (biological modification). The formation of EC-modified LDL depended on generation of free radicals with consequent peroxidation of LDL lipids and was accompanied by extensive hydrolysis of LDL phosphatidylcholine at the 2-position. The present studies show that p-bromophenacyl bromide, a site-specific irreversible inhibitor of phospholipase A2 activity, blocks this hydrolysis and, at the same time, the enhanced macrophage degradation. We show further that during EC modification the apoprotein B of LDL undergoes considerable modification and that this also is prevented by the phospholipase inhibitor. Finally, as reported previously, changes similar to those observed on incubation of LDL with EC can be induced by incubation in the absence of cells but in the presence of a sufficiently high concentration of Cu2+. This also is accompanied by hydrolysis of phosphatidylcholine at the 2-position and breakdown of apoprotein B. These changes are also inhibited by p-bromophenacyl bromide, suggesting the presence of a phospholipase A2 activity associated with LDL as it is isolated. A hypothesis is presented linking lipid peroxidation, phosphatidylcholine hydrolysis, and changes in the LDL apoprotein during EC modification. Many or most of the lipid-laden foam cells of atherosclerotic lesions are derived from monocyte/macrophages (1-3). Yet, paradoxically, native low density lipoprotein (LDL) is recognized poorly by cultured macrophages and does not produce cholesterol ester accumulation in such cells in culture (4). Previously we have described a biological modification of LDL that converts it to a form recognized by macrophages and that leads to greatly enhanced cellular uptake and promotion of cholesterol ester accumulation (5-8). In part, the enhanced uptake is mediated via the "scavenger" receptorthe acetyl LDL receptor (4)-which also specifically recognizes several chemically modified forms ofLDL (9, 10). The biological modification is effected by simply incubating native LDL overnight in the presence of cultured endothelial cells (EC) (5) or smooth muscle cells (7). The product, designated EC-modified LDL (EC-LDL), shows an increase in negative charge and hydrated density and is degraded less rapidly by the native LDL receptor (7). All of these changes were recently shown to be obligatorily linked to free radicalmediated peroxidation of LDL and to be accompanied by extensive hydrolysis of LDL phosphatidylcholine (PtdCho) to lyso-PtdCho (I-PtdCho) through apparent phospholipase A2 activity (8). All of the compositional changes, as well as the biological modification (defined here as those changes that induce the increased rate of degradation of EC-LDL by macrophages), were blocked by antioxidants [vitamin E or butylated hydroxytoluene (BHT)]. They were also blocked by low concentrations of EDTA, concentrations sufficient to complex iron and copper in the medium but too low to significantly alter Ca concentrations. It was concluded that initiation of EC modification depended on peroxidative changes catalyzed by metal ions. In the present paper we show that EC modification is associated also with extensive changes in apolipoprotein B (apo B). These protein changes, the hydrolysis of PtdCho, and most of the other changes that accompany EC modification, including the biological modification, are all blocked by p-bromophenacyl bromide (pBPB), an inhibitor of phospholipase A2 activity. Thus, phospholipid breakdown is also an obligatory element in the series of changes involved in generating EC-LDL. MATERIALS AND METHODS Carrier-free Na125I and [1-14C]linoleic acid were purchased from Amersham. Ham's F-10 medium was obtained from Irvine Scientific and Dulbecco's modified Eagle's medium was from GIBCO. Fetal bovine serum was supplied by Hyclone (Logan, UT). pBPB (2,4'-dibromoacetophenone) and soybean lipoxidase were purchased from Sigma and p-bromoacetophenone and acetophenone were from Aldrich. 1Palmitoyl-2-[1-14C]linoleoyl-sn-glycero-3-phosphocholine was prepared as described (8). 1-Bromooctan-2-one was synthesized by reacting heptanoyl chloride with excess diazomethane, followed by treatment of the resulting diazoketone with 50%o (wt/vol) aqueous hydrogen bromide (28). Lipoproteins. LDL (p = 1.019-1.063) was isolated from fresh human plasma and radioiodinated as described (8). Unlabeled and labeled LDL were extensively dialyzed against phosphate-buffered saline containing 0.01% EDTA. Cells. Rabbit EC, generously provided by V. Buonassissi, were grown in Ham's F-10 medium supplemented with 15% fetal bovine serum and EC modification ofLDL was carried out as described (5). The modified LDL was used for degradation studies without reisolation. LDL was also modified in the absence of EC by incubating LDL (100 ,g/ml) in 2 ml of Ham's F-10 medium containing 5 AtM Cu2' at 370C for 24 hr (8). Abbreviations: LDL, low density lipoprotein; EC, endothelial cell(s); PtdCho, phosphatidylcholine; I-PtdCho, lyso-PtdCho; pBPB, p-bromophenacyl bromide; apo B, apolipoprotein B; B1OO, the major form of apo B with M549,000; BHT, butylated hydroxytoluene. *Present address: Division of Gastroenterology, University of British Columbia, Health Sciences Centre Hospital, 2211 Wesbrook Mall, Vancouver, BC V6T 1W5 Canada. 3000 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Proc. Nati. Acad. Sci. USA 82 (1985) 3001 Resident peritoneal macrophages were harvested from female Swiss-Webster mice by peritoneal lavage and used in experiments after 24 hr in culture as described (7). Degradation of 1251-Labeled LDL ('251-LDL). Medium containing 125I-labeled lipoprotein samples was added to macrophages at 10 ,ug of LDL protein per ml in a total volume of 1 ml in serum-free F-10 or Dulbecco's modified Eagle's medium. After 5 hr of incubation at 370C, the medium was removed and trichloroacetic acid-soluble, noniodide radioactivity was measured (8). Lipid Peroxidation. The extent of lipid peroxidation was measured as thiobarbituric acid-reactive products and expressed as malondialdehyde equivalents (11). LDL (50 ,ug of protein) in 0.5 ml was mixed with 1.5 ml of 20% trichloroacetic acid and 1.5 ml of 0.67% thiobarbituric acid in 0.05 M NaOH. After heating in a boiling water bath for 30 min, the samples were centrifuged at 2000 rpm for 10 min and the optical density was read at 532 nm. Fresh tetramethoxypropane, which produces malondialdehyde, was used as standard. Lipid extraction and analysis for lipid phosphorus were performed as described (8) and protein was determined by the method of Lowry et al. (12). Preparation of Oxidized PtdCho. One micromole of 1-palmitoyl-2-[1-_4C]linoleoyl-sn-glycero-3-phosphocholine, 5 jimol of sodium deoxycholate, and 0.2 mg of soybean lipoxidase (2 x 104 units) were incubated in 0.6 ml of 0.2 M borate buffer (pH 9.0). Fresh enzyme was added at 15-min intervals for 1 hr. The incubation mixture was extracted by the method of Bligh and Dyer (13) and the chloroform extract, containing 1-palmitoyl-2-[1-14C]linoleoylhydroperoxy-sn-glycero-3-phosphocholine, was taken to dryness. Unlabeled linoleic acid, similarly oxidized, provided oxidized fatty acid as standard for TLC. Oxidized or unoxidized PtdCho was used as the substrate for the assay of phospholipase activity in LDL. The incubation system contained 65 ,uM labeled PtdCho, 75 mM Tris (pH 7.4), 2.5 mM CaCl2, 1 mM sodium deoxycholate, and 200 ,uM BHT, with or without 100 jig of LDL protein (total volume, 0.8 ml). Incubation was carried out at 37°C for 1 hr and the products were extracted by the method of Bligh and Dyer, with 1 ml of 1 M HCl in place of water. The chloroform layer was dried and lipids were separated by TLC on silica gel G plates by using CHCl3/methanol/acetic acid/ H20, 90:10:0.5:0.5 (vol/vol), as the solvent system. Lipids were identified after brief exposure to iodine, and the bands corresponding to linoleic acid and oxidized linoleic acid were scraped, eluted, and radioassayed. Appropriate control studies showed that recoveries from both unoxidized and oxidized substrates were equal and nearly quantitative. Apparent enzyme activities are expressed in terms of nmol of labeled free fatty acid released. NaDodSO4 Gradient Gel Electrophoresis. Lipoprotein samples were extracted by the method of Bligh and Dyer, stored at -20°C, and subsequently centrifuged in a fixed-angle rotor for 10 min at 2500 rpm at room temperature. The phases were removed carefully without disturbing the protein interface. The protein was then solubilized in sample buffer (containing 2% NaDodSO4, 10% glycerol, and 5% 2-mercaptoethanol) by incubating in a boiling water bath for 3 min. Protein recoveries in experiments utilizing 125I-LDL were found to be 70-105% of that in the medium before extraction. Electrophoresis was performed using 3-15% gradient gels or precast 2.5-27% gels (Isolab, Akron, OH) at a constant current of 25 mA for 5 hr using a Pharmacia vertical gel system. Gels were stained with Coomassie blue or with silver (14).