Title: Normal high density lipoprotein inhibits three steps in the formation of mildly oxidized low density lipoprotein: steps 2 and 3
Abstract: Treatment of human artery wall cells with apolipoprotein A-I (apoA-I), but not apoA-II, with an apoA-I peptide mimetic, or with high density lipoprotein (HDL), or paraoxonase, rendered the cells unable to oxidize low density lipoprotein (LDL). Human aortic wall cells were found to contain 12-lipoxygenase (12-LO) protein. Transfection of the cells with antisense to 12-LO (but not sense) eliminated the 12-LO protein and prevented LDL-induced monocyte chemotactic activity. Addition of 13(S)-hydroperoxyoctadecadienoic acid [13(S)-HPODE] and 15(S)-hydroperoxyeicosatetraenoic acid [15(S)-HPETE] dramatically enhanced the nonenzymatic oxidation of both 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (PAPC) and cholesteryl linoleate. On a molar basis 13(S)-HPODE and 15(S)-HPETE were approximately two orders of magnitude greater in potency than hydrogen peroxide in causing the formation of biologically active oxidized phospholipids (m/z 594, 610, and 828) from PAPC. Purified paraoxonase inhibited the biologic activity of these oxidized phospholipids. HDL from 10 of 10 normolipidemic patients with coronary artery disease, who were neither diabetic nor receiving hypolipidemic medications, failed to inhibit LDL oxidation by artery wall cells and failed to inhibit the biologic activity of oxidized PAPC, whereas HDL from 10 of 10 age- and sex-matched control subjects did. We conclude that a) mildly oxidized LDL is formed in three steps, one of which involves 12-LO and each of which can be inhibited by normal HDL, and b) HDL from at least some coronary artery disease patients with normal blood lipid levels is defective both in its ability to prevent LDL oxidation by artery wall cells and in its ability to inhibit the biologic activity of oxidized PAPC. —Navab, M., S. Y. Hama, G. M. Anantharamaiah, K. Hassan, G. P. Hough, A. D. Watson, S. T. Reddy, A. Sevanian, G. C. Fonarow, and A. M. Fogelman. Normal high density lipoprotein inhibits three steps in the formation of mildly oxidized low density lipoprotein: steps 2 and 3. J. Lipid Res. 2000. 41: 1495–1508. Treatment of human artery wall cells with apolipoprotein A-I (apoA-I), but not apoA-II, with an apoA-I peptide mimetic, or with high density lipoprotein (HDL), or paraoxonase, rendered the cells unable to oxidize low density lipoprotein (LDL). Human aortic wall cells were found to contain 12-lipoxygenase (12-LO) protein. Transfection of the cells with antisense to 12-LO (but not sense) eliminated the 12-LO protein and prevented LDL-induced monocyte chemotactic activity. Addition of 13(S)-hydroperoxyoctadecadienoic acid [13(S)-HPODE] and 15(S)-hydroperoxyeicosatetraenoic acid [15(S)-HPETE] dramatically enhanced the nonenzymatic oxidation of both 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (PAPC) and cholesteryl linoleate. On a molar basis 13(S)-HPODE and 15(S)-HPETE were approximately two orders of magnitude greater in potency than hydrogen peroxide in causing the formation of biologically active oxidized phospholipids (m/z 594, 610, and 828) from PAPC. Purified paraoxonase inhibited the biologic activity of these oxidized phospholipids. HDL from 10 of 10 normolipidemic patients with coronary artery disease, who were neither diabetic nor receiving hypolipidemic medications, failed to inhibit LDL oxidation by artery wall cells and failed to inhibit the biologic activity of oxidized PAPC, whereas HDL from 10 of 10 age- and sex-matched control subjects did. We conclude that a) mildly oxidized LDL is formed in three steps, one of which involves 12-LO and each of which can be inhibited by normal HDL, and b) HDL from at least some coronary artery disease patients with normal blood lipid levels is defective both in its ability to prevent LDL oxidation by artery wall cells and in its ability to inhibit the biologic activity of oxidized PAPC. —Navab, M., S. Y. Hama, G. M. Anantharamaiah, K. Hassan, G. P. Hough, A. D. Watson, S. T. Reddy, A. Sevanian, G. C. Fonarow, and A. M. Fogelman. Normal high density lipoprotein inhibits three steps in the formation of mildly oxidized low density lipoprotein: steps 2 and 3. J. Lipid Res. 2000. 41: 1495–1508. We previous reported (1Navab M. Imes S. Hama S. Hough G. Ross L. Bork R. Valente A.J. Berliner J.A. Drinkwater D.C. Laks M. Fogelman A.M. Monocyte transmigration induced by modification of low density lipoprotein in cocultures of human aortic wall cells is due to induction of monocyte chemotactic protein-1 synthesis and is abolished by high density lipoprotein.J. Clin. Invest. 1991; 88: 2039-2046Google Scholar) that high density lipoprotein (HDL) but not apolipoprotein A-I (apoA-I), when added to human artery wall cell cocultures together with low density lipoprotein (LDL), prevented the oxidation of the LDL by the artery wall cells. In those experiments, the apoA-I was kept in the culture together with the artery wall cells and the LDL (1Navab M. Imes S. Hama S. Hough G. Ross L. Bork R. Valente A.J. Berliner J.A. Drinkwater D.C. Laks M. Fogelman A.M. Monocyte transmigration induced by modification of low density lipoprotein in cocultures of human aortic wall cells is due to induction of monocyte chemotactic protein-1 synthesis and is abolished by high density lipoprotein.J. Clin. Invest. 1991; 88: 2039-2046Google Scholar). Subsequently, in pursuing the mechanisms for the ability of HDL to protect LDL against oxidation by human artery wall cells, we observed that if the apoA-I was incubated with the cells and then removed prior to the addition of the LDL, the artery wall cells were then unable to oxidize the added LDL. This suggested to us that apoA-I might be able to remove from cells not only cholesterol and phospholipids but perhaps oxidized lipids as well. These preliminary findings, which have been reported in abstract form (2Hama S. Jin L. Navab M. Fogelman A.M. Apolipoprotein A-I can remove lipid molecules from native LDL rendering it resistant to oxidation by cultured artery wall cells.Circulation. 1997; 96 (Abstract): I-485Google Scholar, 3Hama S. Hough G. Jin L. Van Lenten B.J. Anantharamaiah G.M. Watson A.D. Faull K. Navab M. Laks H. Fogelman A.M. Apolipoprotein A-I mimic peptides inhibit oxidation of low density lipoprotein by artery wall cells and the resulting monocyte interactions.Circulation. 1998; 98 (Abstract): I-252Google Scholar), prompted us to perform the studies detailed in this article. The experiments detailed in this and the accompanying article (3Navab M. Hama S.Y. Cooke C.J. Anantharamaiah G.M. Chaddha M. Jin L. Subbanagounder G. Faull K.F. Reddy S.T. Miller N.E. Fogelman A.M. Normal high densisty lipoprotein inhibits three steps in the formation of mildly oxidized low density lipoprotein: step 1.J. Lipid Res. 2000; 41: 1481-1494Google Scholar) have led us to propose that the biologically active lipids in mildly oxidized LDL are formed in a series of three steps. The first step is the seeding of LDL with lipid oxidation products including those of the metabolism of linoleic and arachidonic acid. The evidence of the first step is presented in the accompanying article. In this article we present evidence regarding the second step in LDL oxidation by artery wall cells. We demonstrate that 12-lipoxygenase (12-LO) protein is present in human artery wall cells and is required for the production of mildly oxidized LDL by the artery wall cells. Stocker and colleagues (4Neuzil J. Upston J.M. Witting P.K. Scott K.F. Stocker R. Secretory phospholipase A2 and lipoprotein lipase enhance 15-lipoxygenase-induced enzymic and nonenzymic lipid peroxidation in low-density lipoproteins.Biochemistry. 1998; 37: 9203-9210Google Scholar, 5Upston J.M. Neuzil J. Witting P.K. Alleva R. Stocker R. Oxidation of free fatty acids in low density lipoprotein by 15-lipoxygenase stimulates nonenzymic, alpha-tocopherol-mediated peroxidation of cholesteryl esters.J. Biol. Chem. 1997; 272: 30067-30074Google Scholar) have presented indirect evidence that lipoxygenases mediate the peroxidation of cholesteryl linoleate largely by a nonenzymatic process. We demonstrate in this article that the nonenzymatic oxidation of cholesteryl linoleate is greatly enhanced by the presence of 13-hydroperoxyoctadecadienoic acid [13(S)-HPODE]. We also propose in this article that the third step in the formation of mildly oxidized LDL is the nonenzymatic oxidation of LDL phospholipids that occurs when a critical threshold of "seeding molecules" (e.g., 13(S)-HPODE and 15-hydroperoxyeicosatetraenoic acid [15(S)-HPETE]) is reached in the LDL. We present evidence in this article to indicate that when these seeding molecules reach a critical level, they cause the nonenzymatic oxidation of a major LDL phospholipid, 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (PAPC). This results in the formation of the three biologically active oxidized phospholipids: 1-palmitoyl-2-oxovaleryl-sn-glycero-3-phosphocholine (POVPC, m/z 594), 1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine (PGPC, m/z 610), and 1-palmitoyl-2-(5,6-epoxyisoprostane E2)-sn-glycero-3-phosphocholine (PEIPC, m/z 828) (6Watson A.D. Structural identification of a novel proinflammatory epoxyisoprostane phospholipid in mildly oxidized low density lipoprotein.J. Biol. Chem. 1999; 274: 24787-24798Google Scholar, 7Watson A.D. Leitinger N. Navab M. Faull K.F. Hörkkö S. Witztum J.L. Palinski W. Schwenke D. Salomon R.G. Sha W. Subbanagounder G. Fogelman A.M. Berliner J.A. Structural identification by mass spectrometry of oxidized phospholipids in minimally oxidized low density lipoprotein that induces monocyte/endothelial interactions and evidence for their presence in vivo.J. Biol. Chem. 1997; 272: 13597-13607Google Scholar). The experiments described in this article also indicate that in contrast to the case for normal HDL, HDL taken from patients with coronary artery disease, who show normal blood lipid levels, and were neither diabetic nor taking hypolipidemic medications, did not protect LDL against oxidation by human artery wall cells and failed to inhibit the biological activity of oxidized PAPC. The arachidonic acid analog 5,8,11,14-eicosatetraynoic acid (ETYA) was obtained from Biomol (Plymouth Meeting, PA). Polyclonal antiserum against recombinant human platelet-type 12-lipoxygenase was obtained from Cayman Chemical (Ann Arbor, MI). Monoclonal antibody against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was purchased from Chemicon (Temecula, CA). Phosphorothioate oligonucleotides directed against human platelet-type 12-lipoxygenase were purchased from GIBCO-BRL (Gaithersburg, MD). The antisense oligonucleotide sequence used was 5′-CTCAGGAGGGTGTAAACA-3′, the corresponding sense oligonucleotide sequence was 5′-TGTTTACAC CCTCCTGAG-3′, and the scrambled oligonucleotide sequence used was 5′-AAGATTGCGCGACGATGA-3′. SuperFect reagent was purchased from Qiagen (Chatsworth, CA). All other materials were from sources described in the accompanying article. Lipoproteins, cocultures, monocyte isolation, monocyte chemotaxis assays, and monocyte adhesion assays were prepared and/or performed as described in the accompanying article. Blood samples were collected from patients referred to the cardiac catheterization laboratory at the Center for Health Sciences at the University of California, Los Angeles (Los Angeles, CA). After signing a consent form approved by the Human Research Subject Protection Committee of the University of California, Los Angeles, each patient donated a fasting blood sample collected in a heparinized tube. LDL and/or HDL were isolated by fast protein liquid chromatography (FPLC) from the blood samples collected from patients who had angiographically documented coronary atherosclerosis but who had normal total cholesterol (<200 mg/dl), LDL-cholesterol (<130 mg/dl), HDL-cholesterol (males >45 mg/dl, females >50 mg/dl), and triglycerides (<150 mg/dl), who were not taking hypolipidemic medications, and who were not diabetic. Data from some patients and some controls previously reported by us (8Navab M. Hama-Levy S. Van Lenten B.J. Fonarow G.C. Cardinez C.J. Castellani L.W. Brennan M-L. Lusis A.J. Fogelman A.M. Mildly oxidized LDL induces an increased apolipoprotein J/paraoxonase ratio.J. Clin. Invest. 1997; 99: 2005-2019Google Scholar) have been included with additional new data. The inclusion of previously reported patients is explicitly indicated in the appropriate figure legend. HDL was isolated from each individual and paraoxonase activity was determined as previously described (8Navab M. Hama-Levy S. Van Lenten B.J. Fonarow G.C. Cardinez C.J. Castellani L.W. Brennan M-L. Lusis A.J. Fogelman A.M. Mildly oxidized LDL induces an increased apolipoprotein J/paraoxonase ratio.J. Clin. Invest. 1997; 99: 2005-2019Google Scholar). The ability of the HDL from each subject to protect LDL against oxidation by human artery wall cell cocultures was then determined by techniques previously described (1Navab M. Imes S. Hama S. Hough G. Ross L. Bork R. Valente A.J. Berliner J.A. Drinkwater D.C. Laks M. Fogelman A.M. Monocyte transmigration induced by modification of low density lipoprotein in cocultures of human aortic wall cells is due to induction of monocyte chemotactic protein-1 synthesis and is abolished by high density lipoprotein.J. Clin. Invest. 1991; 88: 2039-2046Google Scholar, 8Navab M. Hama-Levy S. Van Lenten B.J. Fonarow G.C. Cardinez C.J. Castellani L.W. Brennan M-L. Lusis A.J. Fogelman A.M. Mildly oxidized LDL induces an increased apolipoprotein J/paraoxonase ratio.J. Clin. Invest. 1997; 99: 2005-2019Google Scholar). The LDL used for testing the ability of HDL to protect LDL against oxidation by human artery wall cells was prepared from a normal donor and was aliquoted and cryopreserved in sucrose as previously described (9Rumsey S.C. Galeano N.F. Arad Y. Deckelbaum R.J. Cryopreservation with sucrose maintains normal physical and biological properties of human plasma low density lipoproteins.J. Lipid Res. 1992; 33: 1551-1561Google Scholar). To determine the capacity of HDL to inactivate oxidized phospholipids, in some cases 100 μg of oxidized PAPC (Ox-PAPC) per milliliter (9Rumsey S.C. Galeano N.F. Arad Y. Deckelbaum R.J. Cryopreservation with sucrose maintains normal physical and biological properties of human plasma low density lipoproteins.J. Lipid Res. 1992; 33: 1551-1561Google Scholar) was incubated in test tubes with HDL at 250 μg/ml in 10% lipoprotein-deficient serum (LPDS) in medium 199 (M199) at 37°C with gentle mixing. The HDL-Ox-PAPC mixture was then added to endothelial monolayers and monocyte binding was determined. Fibroblasts that were transfected with vector alone or cells that overexpressed 15-LO were a generous gift of J. Witztum and P. Reaven. In the present experiments, the fibroblasts were incubated with or without apoA-I (100 μg/ml). After 3 h of incubation at 37°C with gentle mixing, the culture supernatants were removed, apoA-I was separated by FPLC, and the level of hydroperoxides was determined in lipid extracts of the culture supernatants and in lipid extracts of apoA-I. Human artery wall cell cocultures were preincubated for 30 min with ETYA at a concentration of 10−8 mol/liter or with cinnamyl-3,4-dihydroxy-α-cyanocynamate (CDC; from Biomol) at a concentration of 10−8 mol/liter in M199 containing 10% LPDS. The cocultures were then washed and LDL was added at 250 μg/ml and incubated for 8 h. The supernatants were removed and assayed for Auerbach lipid hydroperoxide equivalents and monocyte chemotactic activity was determined as described in the accompanying article. Human aortic endothelial cells (HAEC) and artery wall cell cocultures were prepared as described previously (1Navab M. Imes S. Hama S. Hough G. Ross L. Bork R. Valente A.J. Berliner J.A. Drinkwater D.C. Laks M. Fogelman A.M. Monocyte transmigration induced by modification of low density lipoprotein in cocultures of human aortic wall cells is due to induction of monocyte chemotactic protein-1 synthesis and is abolished by high density lipoprotein.J. Clin. Invest. 1991; 88: 2039-2046Google Scholar). Cells were harvested in a buffer containing 50 mm Tris-HCl (pH 7.5), 100 mm NaCl, 5 mm sodium orthovanadate, 1% Triton X-100, aprotinin (1 μg/ml) and leupeptin (1 μg/ml). Samples were boiled for 10 min and protein concentrations were determined by the Bradford assay. Seventy-five micrograms of total protein from each sample was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; 5% stacking and 8% resolving gel). The proteins were blotted onto nitrocellulose membranes with a semidry transfer apparatus (Bio-Rad, Hercules, CA). The filters were incubated for 1 h in phosphate-buffered saline (PBS) containing 0.2% Tween 20 and 10% nonfat dried milk, washed in PBS containing 0.2% Tween 20 and 1% nonfat dried milk, and incubated with a polyclonal anti-12-LO antibody (1:1,000 dilution) for 1 h, and Western analysis was performed with 12-LO antibody at a 1:1,000 dilution. After three additional washes in PBS containing 0.2% Tween 20 and 1% nonfat dried milk, the filters were incubated with a secondary antibody (anti-rabbit IgG conjugated to horseradish peroxidase; Sigma, St. Louis, MO) at a dilution of 1:4,000. Immunodetection was per formed with ECL reagents (Pharmacia, Piscataway, NJ). The filters were exposed to Kodak XAR-5 film (Eastman Kodak, Rochester, NY). Artery wall cocultures were set up in six-well plates as described previously (1Navab M. Imes S. Hama S. Hough G. Ross L. Bork R. Valente A.J. Berliner J.A. Drinkwater D.C. Laks M. Fogelman A.M. Monocyte transmigration induced by modification of low density lipoprotein in cocultures of human aortic wall cells is due to induction of monocyte chemotactic protein-1 synthesis and is abolished by high density lipoprotein.J. Clin. Invest. 1991; 88: 2039-2046Google Scholar). Antisense, sense, or scrambled oligonucleotides were used at a final concentration of 1 μm. For each transfection, an appropriate amount of the oligonucleotide was diluted in 200 μl of serum-free M199 medium in a 0.5-ml Eppendorf tube. Three microliters of SuperFect reagent was added to each tube, vortexed for 10 sec, and allowed to incubate at room temperature for 15 min to allow SuperFect reagent -DNA complex formation. During the incubation the artery wall cocultures were washed with PBS and supplemented with 0.8 ml of complete M199 medium. The transfection complexes were added to the wells and incubated for 2 h. The cocultures were washed in PBS and supplemented with complete M199 medium. Eighteen hours later the transfection protocol was repeated, and appropriate cocultures received LDL (250 μg/ml) to be oxidatively modified by the artery wall cells. Six hours later, supernatants were collected and were transferred to fresh untransfected cocultures that served as target for induction and release into supernatant of monocyte chemotactic activity under the influence of the transferred LDL. After incubation for 5 h, the coculture medium was changed to serum-free medium and incubated for an additional 6 h to accumulate monocyte chemotactic activity, which was then determined as described in Materials and Methods. In addition, the coculture lysates obtained from the first set that was used for modification of LDL were analyzed for 12-LO protein expression as described above. 13(S)-HPODE or 15(S)-HPETE or vehicle alone was added at various concentrations to PAPC, mixed and evaporated to form a thin film, and allowed to oxidize in air. In some experiments, PAPC was evaporated, forming a thin film, and allowed to oxidize in air with 100 μl containing hydrogen peroxide at various concentrations. The samples were extracted with chloroform–methanol 2:1 (v/v) (10Folch J. Lees M. Sloane Stanley G.H. A simple method for the isolation and purification of total lipids from animal tissues.J. Biol. Chem. 1957; 226: 497-509Google Scholar) and in the case of the hydrogen peroxide experiments by addition of five parts chloroform–methanol 2:1 (v/v) to one part aqueous solution, mixing, and centrifugation. The chloroform phase was collected and analyzed by electrospray ionization mass spectrometry (ESI-MS) in the positive ion mode. The level of the remaining PAPC and the oxidized phospholipids that formed were determined and expressed in relation to the internal standard, 1,2-ditetradecanoyl-rac-glycerol-3-phosphocholine (DMPC, m/z 678.3). Fast performance liquid chromatography and reversed-phase high performance liquid chromatography (RP-HPLC) were performed as described in the accompanying article. For the detection of cholesteryl linoleate hydroperoxide an Alltech Associates (Deerfield, IL) Alltima 250 × 4.6 mm, 5-μm RP-HPLC C18 column was used to separate and detect cholesteryl linoleate hydroperoxide at 234 nm and cholesteryl linoleate at 205 nm. The mobile solvent consisted of acetonitrile–2-propanol–water 44:54:2 (v/v/v) at 1.0 ml/min. Lipids were resuspended in the mobile solvent for injection. ESI-MS in the positive or negative ion mode was per formed according to the protocol and conditions previously described (6Watson A.D. Structural identification of a novel proinflammatory epoxyisoprostane phospholipid in mildly oxidized low density lipoprotein.J. Biol. Chem. 1999; 274: 24787-24798Google Scholar, 7Watson A.D. Leitinger N. Navab M. Faull K.F. Hörkkö S. Witztum J.L. Palinski W. Schwenke D. Salomon R.G. Sha W. Subbanagounder G. Fogelman A.M. Berliner J.A. Structural identification by mass spectrometry of oxidized phospholipids in minimally oxidized low density lipoprotein that induces monocyte/endothelial interactions and evidence for their presence in vivo.J. Biol. Chem. 1997; 272: 13597-13607Google Scholar). Briefly, ESI-MS was performed with an API III triple-quadrupole biomolecular mass analyzer (Perkin-Elmer, Norwalk, CT) fitted with an articulated, pneumatically assisted nebulization probe and an atmospheric pressure ionization source (7Watson A.D. Leitinger N. Navab M. Faull K.F. Hörkkö S. Witztum J.L. Palinski W. Schwenke D. Salomon R.G. Sha W. Subbanagounder G. Fogelman A.M. Berliner J.A. Structural identification by mass spectrometry of oxidized phospholipids in minimally oxidized low density lipoprotein that induces monocyte/endothelial interactions and evidence for their presence in vivo.J. Biol. Chem. 1997; 272: 13597-13607Google Scholar). Positive ion flow injection analysis was done with acetonitrile–water–formic acid 50:50:0.1 (v/v/v) and negative ion flow injection analysis was done with methanol–water 50:50 (v/v) containing 10 mm ammonium acetate. For quantitative analysis, 1,2-ditetradecanoyl-rac-glycerol-3-phosphocholine (DMPC) or heptadecanoic acid were used as internal standards. Ions were scanned at a step size of 0.3 Da. Data were processed by software provided by PE SCIEX (PE Biosystems, Foster City, CA). Oxidized PAPC, POVPC, PGPC, or PEIPC was incubated in M199 without or with purified human paraoxonase (1 × 10−2 U/ml) for 3 h with gentle mixing at 37°C. Paraoxonase was removed from the mixture by ultrafiltration with 30-kDa cutoff spin filters and the lipids were incubated with human aortic wall cocultures in M199 with 10% LPDS for 8 h at 37°C. The cocultures were washed and incubated with fresh medium for an additional 4 h at 37°C. The supernatants were analyzed for monocyte chemotactic activity. Protein content of lipoproteins was determined by a modification (11Lorenzen A. Kennedy S.W. A fluorescent based protein assay for use with a microplate reader.Anal. Biochem. 1993; 214: 346-348Google Scholar) of the Lowry assay (12Lowry O.H. Rosebrough M.J. Farr A.L. Randall R.J. Protein measurement with the Folin phenol reagent.J. Biol. Chem. 1951; 193: 265-275Google Scholar). Lipid hydroperoxide levels were measured by the assay described by Auerbach, Kiely, and Cornicelli (13Auerbach B.J. Kiely J.S. Cornicelli J.A. A spectrophotometric microtiter-based assay for detection of hydroperoxy derivatives of linoleic acid.Anal. Biochem. 1992; 201: 375-380Google Scholar). In some experiments, where indicated, the lipid in culture supernatants containing LDL that was oxidized by the artery wall cell cocultures was extracted with chloroform–methanol and hydroperoxides determined by the Auerbach method. Peroxidation of cholesteryl linoleate was accomplished as described in the accompanying article. Paraoxonase activity was measured as previously described (14Gan K.N. Smolen A. Eckerson H.W. La Du B.N. Purification of human serum paraoxonase/arylesterase. Evidence for one esterase catalyzing both activities.Drug Metab. Dispos. 1991; 19: 100-106Google Scholar). Statistical significance was determined by model 1 analysis of variance (ANOVA). The analyses were carried out first by ANOVA in an Excel application to determine whether differences existed among the group means, followed by a paired Student's t-test to identify the significantly different means, when appropriate. Significance is defined as P < 0.05. The accompanying article demonstrated that LDL contains "seeding molecules" necessary for LDL oxidation by artery wall cells. We previously reported (1Navab M. Imes S. Hama S. Hough G. Ross L. Bork R. Valente A.J. Berliner J.A. Drinkwater D.C. Laks M. Fogelman A.M. Monocyte transmigration induced by modification of low density lipoprotein in cocultures of human aortic wall cells is due to induction of monocyte chemotactic protein-1 synthesis and is abolished by high density lipoprotein.J. Clin. Invest. 1991; 88: 2039-2046Google Scholar, 15Berliner J.A. Territo M.C. Sevanian A. Ramin S. Kim J.A. Bamshad B. Esterson M. Fogelman A.M. Minimally modified low density lipoprotein stimulates monocyte endothelial interactions.J. Clin. Invest. 1990; 85: 1260-1266Google Scholar) that freshly isolated LDL does not induce monocyte adherence to endothelial cells and does not induce monocyte chemotaxis whereas mildly oxidized LDL induces both (1Navab M. Imes S. Hama S. Hough G. Ross L. Bork R. Valente A.J. Berliner J.A. Drinkwater D.C. Laks M. Fogelman A.M. Monocyte transmigration induced by modification of low density lipoprotein in cocultures of human aortic wall cells is due to induction of monocyte chemotactic protein-1 synthesis and is abolished by high density lipoprotein.J. Clin. Invest. 1991; 88: 2039-2046Google Scholar, 15Berliner J.A. Territo M.C. Sevanian A. Ramin S. Kim J.A. Bamshad B. Esterson M. Fogelman A.M. Minimally modified low density lipoprotein stimulates monocyte endothelial interactions.J. Clin. Invest. 1990; 85: 1260-1266Google Scholar). The ability of mildly oxidized LDL to induce monocyte adherence and chemotaxis was based on the presence in the mildly oxidized LDL of three oxidized phospholipids with characteristic m/z ratios (m/z 594, 610, and 828) (6Watson A.D. Structural identification of a novel proinflammatory epoxyisoprostane phospholipid in mildly oxidized low density lipoprotein.J. Biol. Chem. 1999; 274: 24787-24798Google Scholar, 7Watson A.D. Leitinger N. Navab M. Faull K.F. Hörkkö S. Witztum J.L. Palinski W. Schwenke D. Salomon R.G. Sha W. Subbanagounder G. Fogelman A.M. Berliner J.A. Structural identification by mass spectrometry of oxidized phospholipids in minimally oxidized low density lipoprotein that induces monocyte/endothelial interactions and evidence for their presence in vivo.J. Biol. Chem. 1997; 272: 13597-13607Google Scholar). Because freshly isolated LDL did not induce monocyte adherence or monocyte chemotactic activity, we concluded that the seeding molecules in freshly isolated LDL were by themselves insufficient to generate the three biologically active oxidized phospholipids, either because the level of these seeding molecules was less than some critical threshold or because additional and different seeding molecules were required to generate the biologically active oxidized phospholipids. Thus, we concluded that at least an additional step in the formation of mildly oxidized LDL was required beyond the initial seeding. ApoA-I, but not apoA-II, renders human artery wall cells unable to oxidize LDL. We previously reported that coincubation of human artery wall cells with apoA-I and LDL did not protect the LDL against oxidation by the artery wall cells (1Navab M. Imes S. Hama S. Hough G. Ross L. Bork R. Valente A.J. Berliner J.A. Drinkwater D.C. Laks M. Fogelman A.M. Monocyte transmigration induced by modification of low density lipoprotein in cocultures of human aortic wall cells is due to induction of monocyte chemotactic protein-1 synthesis and is abolished by high density lipoprotein.J. Clin. Invest. 1991; 88: 2039-2046Google Scholar). As shown in Fig. 1, these results were confirmed (compare coincubated A-I with cultures sham treated). However, when the human artery wall cocultures were first incubated with apoA-I and the apoA-I was then removed from the cocultures prior to the addition of LDL (cultures after A-I), the artery wall cells were not able to oxidize the LDL (Fig. 1A) and monocyte chemotaxis was prevented (Fig. 1B). In contrast to the case for apoA-I, when the cultures were first incubated with apoA-II and the apoA-II was then removed, the artery wall cocultures retained their ability to oxidize LDL (Fig. 1A) and induce monocyte chemotaxis (Fig. 1B) (cultures after A-II). In other experiments, apoA-I was incubated with a first set of cocultures and then removed from the first set of cocultures and added to a second set of cocultures that had been identically treated (i.e., the second set of cocultures had been incubated with apoA-I, which was then removed). When LDL was added to this second set of cocultures, which contained apoA-I from the first set of cocultures, these reconstituted cocultures readily oxidized the LDL (Fig. 1A) and induced monocyte chemotaxis (Fig. 1B) (cultures after A-I + A-I after cultures). Similar experiments were performed with apoA-II. ApoA-II was incubated with a first set of