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Macrophage-specific expression of group IIA sPLA2 results in accelerated atherogenesis by increasing oxidative stress

2005; Elsevier BV; Volume: 46; Issue: 8 Linguagem: Inglês

10.1194/jlr.m400469-jlr200

ISSN

1539-7262

Autores

Uwe J.F. Tietge, Domenico Praticò, Tao Ding, Colin Funk, Reeni B. Hildebrand, Theo van Berkel, Miranda Van Eck,

Tópico(s)

Lipoproteins and Cardiovascular Health

Resumo

Group IIA secretory phospholipase A2 (sPLA2) is an acute-phase protein mediating decreased plasma HDL cholesterol and increased atherosclerosis. This study investigated the impact of macrophage-specific sPLA2 overexpression on lipoprotein metabolism and atherogenesis. Macrophages from sPLA2 transgenic mice have 2.5 times increased rates of LDL oxidation (thiobarbituric acid-reactive substances formation) in vitro (59 ± 5 vs. 24 ± 4 nmol malondialdehyde/mg protein; P < 0.001) dependent on functional 12/15-lipoxygenase (12/15-LO). Low density lipoprotein receptor-deficient (LDLR−/−) mice were transplanted with bone marrow from either sPLA2 transgenic mice (sPLA2→ LDLR−/−; n = 19) or wild-type C57BL/6 littermates (C57 BL/6→LDLR−/−; n = 19) and maintained for 8 weeks on chow and then for 9 weeks on a Western-type diet. Plasma sPLA2 activity and plasma lipoprotein profiles were not significantly different between sPLA2→LDLR−/− and C57BL/6→LDLR−/− mice. Aortic root atherosclerosis was increased by 57% in sPLA2→LDLR−/− mice compared with C57BL/6→LDLR−/− controls (P < 0.05). Foam cell formation in vitro and in vivo was increased significantly. Urinary, plasma, and aortic levels of the isoprostane 8,12-iso-iPF2α-VI and aortic levels of 12/15-LO reaction products were each significantly higher (P < 0.001) in sPLA2→LDLR−/− compared with C57BL/6→LDLR−/− mice, indicating significantly increased in vivo oxidative stress in sPLA2→ LDLR−/−.These data demonstrate that macrophage-specific overexpression of human sPLA2 increases atherogenesis by directly modulating foam cell formation and in vivo oxidative stress without any effect on systemic sPLA2 activity and lipoprotein metabolism. Group IIA secretory phospholipase A2 (sPLA2) is an acute-phase protein mediating decreased plasma HDL cholesterol and increased atherosclerosis. This study investigated the impact of macrophage-specific sPLA2 overexpression on lipoprotein metabolism and atherogenesis. Macrophages from sPLA2 transgenic mice have 2.5 times increased rates of LDL oxidation (thiobarbituric acid-reactive substances formation) in vitro (59 ± 5 vs. 24 ± 4 nmol malondialdehyde/mg protein; P < 0.001) dependent on functional 12/15-lipoxygenase (12/15-LO). Low density lipoprotein receptor-deficient (LDLR−/−) mice were transplanted with bone marrow from either sPLA2 transgenic mice (sPLA2→ LDLR−/−; n = 19) or wild-type C57BL/6 littermates (C57 BL/6→LDLR−/−; n = 19) and maintained for 8 weeks on chow and then for 9 weeks on a Western-type diet. Plasma sPLA2 activity and plasma lipoprotein profiles were not significantly different between sPLA2→LDLR−/− and C57BL/6→LDLR−/− mice. Aortic root atherosclerosis was increased by 57% in sPLA2→LDLR−/− mice compared with C57BL/6→LDLR−/− controls (P < 0.05). Foam cell formation in vitro and in vivo was increased significantly. Urinary, plasma, and aortic levels of the isoprostane 8,12-iso-iPF2α-VI and aortic levels of 12/15-LO reaction products were each significantly higher (P < 0.001) in sPLA2→LDLR−/− compared with C57BL/6→LDLR−/− mice, indicating significantly increased in vivo oxidative stress in sPLA2→ LDLR−/−. These data demonstrate that macrophage-specific overexpression of human sPLA2 increases atherogenesis by directly modulating foam cell formation and in vivo oxidative stress without any effect on systemic sPLA2 activity and lipoprotein metabolism. Atherosclerosis is increasingly considered to have an inflammatory component (1Ross R. Atherosclerosis—an inflammatory disease.N. Engl. J. Med. 1999; 340: 115-126Google Scholar). While the atherosclerotic plaque displays the features of a localized inflammation within the vascular wall (2Steinberg D. Atherogenesis in perspective: hypercholesterolemia and inflammation as partners in crime.Nat. Med. 2002; 8: 1211-1217Google Scholar, 3Lee R.T. Libby P. The unstable atheroma.Arterioscler. Thromb. Vasc. Biol. 1997; 17: 1859-1867Google Scholar), markers of systemic inflammation such as C-reactive protein have been found to be predictive of future cardiovascular events (4Ridker P.M. Buring J.E. Shih J. Matias M. Hennekens C.H. Prospective study of C-reactive protein and the risk of future cardiovascular events among apparently healthy women.Circulation. 1998; 98: 731-733Google Scholar, 5Ridker P.M. Hennekens C.H. Roitman-Johnson B. Stampfer M.J. Allen J. Plasma concentration of soluble intercellular adhesion molecule 1 and risks of future myocardial infarction in apparently healthy men.Lancet. 1998; 351: 88-92Google Scholar, 6Rader D.J. Inflammatory markers of coronary risk.N. Engl. J. Med. 2000; 343: 1179-1182Google Scholar). The type IIA secretory phospholipase A2 (sPLA2) is an acute-phase protein expressed in response to a variety of proinflammatory cytokines by a number of different tissues and cell types, mainly of mesenchymal origin (7Tischfield J.A. A reassessment of the low molecular weight phospholipase A2 gene family in mammals.J. Biol. Chem. 1997; 272: 17247-17250Google Scholar, 8Kudo I. Murakami M. Phospholipase A2 enzymes.Prostaglandins Other Lipid Mediat. 2002; 68–69: 3-58Google Scholar, 9Hurt-Camejo E. Camejo G. Peilot H. Oorni K. Kovanen P. Phospholipase A2 in vascular disease.Circ. Res. 2001; 89: 298-304Google Scholar, 10Nevalainen T.J. Haapamaki M.M. Gronroos J.N. Roles of secretory phospholipases A2 in inflammatory diseases and trauma.Biochim. Biophys. Acta. 2000; 1488: 83-90Google Scholar, 11Balsinde J. Balboa M.A. Insel P.A. Dennis E.A. Regulation and inhibition of phospholipase A2.Annu. Rev. Pharmacol. Toxicol. 1999; 39: 175-189Google Scholar, 12Peilot H. Rosengren B. Bondjers G. Hurt-Camejo E. 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. 2000; 275: 22895-22904Google Scholar). Increased sPLA2 plasma levels have been reported in patients with various acute and chronic inflammatory conditions (8Kudo I. Murakami M. Phospholipase A2 enzymes.Prostaglandins Other Lipid Mediat. 2002; 68–69: 3-58Google Scholar, 10Nevalainen T.J. Haapamaki M.M. Gronroos J.N. Roles of secretory phospholipases A2 in inflammatory diseases and trauma.Biochim. Biophys. Acta. 2000; 1488: 83-90Google Scholar). Notably, patients with atherosclerotic cardiovascular disease have significantly higher circulating sPLA2 levels compared with controls (13Kugiyama K. Ota Y. Takazoe K. Moriyama Y. Kawano H. Miyao Y. Sakamoto T. Soejima H. Ogawa H. Doi H. et al.Circulating levels of secretory type II phospholipase A2 predict coronary events in patients with coronary artery disease.Circulation. 1999; 100: 1280-1284Google Scholar, 14Kugiyama K. Ota Y. Sugiyama S. Kawano H. Doi H. Soejima H. Miyamoto S. Ogawa H. Takazoe K. Yasue H. Prognostic value of plasma levels of secretory type II phospholipase A2 in patients with unstable angina pectoris.Am. J. Cardiol. 2000; 86: 718-722Google Scholar), and in one report, sPLA2 levels were even found to have a higher predictive value for future coronary events than C-reactive protein (13Kugiyama K. Ota Y. Takazoe K. Moriyama Y. Kawano H. Miyao Y. Sakamoto T. Soejima H. Ogawa H. Doi H. et al.Circulating levels of secretory type II phospholipase A2 predict coronary events in patients with coronary artery disease.Circulation. 1999; 100: 1280-1284Google Scholar). Data obtained in animal models implied that sPLA2 plays a causative role in the process of atherogenesis and indicated that local as well as systemic expression might be relevant. Transgenic mice overexpressing human sPLA2 (15Grass D.S. Felkner R.H. Chiang M-Y. Wallace R.E. Nevelainen T.J. Bennett C.F. Swanson M.E. Expression of human group II PLA2 in transgenic mice results in epidermal hyperplasia in the absence of inflammatory infiltrate.J. Clin. Invest. 1996; 97: 2233-2241Google Scholar) have decreased HDL cholesterol plasma levels attributable to increased catabolism of apolipoprotein A-I as well as HDL cholesteryl ester (16Tietge U.J.F. Maugeais C. Cain W. Grass D. Glick J.M. deBeer F.C. Rader D.J. 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. 2000; 275: 10077-10084Google Scholar, 17de Beer F.C. Connell P.M. Yu J. de Beer M.C. Webb N.R. van der Westhuyzen D.R. HDL modification by secretory phospholipase A2 promotes scavenger receptor class B type I interaction and accelerates HDL catabolism.J. Lipid Res. 2000; 41: 1849-1857Google Scholar, 18Tietge U.J.F. Maugeais C. Grass D. de Beer F.C. Rader D.J. Human secretory phospholipase A2 (sPLA2) mediates decreased plasma levels of HDL-cholesterol and apoA-I in response to inflammation independent of serum amyloid A (SAA) in human apoA-I transgenic mice.Arterioscler. Thromb. Vasc. Biol. 2002; 22: 1213-1218Google Scholar). These mice develop dramatically increased atherosclerosis when fed an atherogenic diet for 12 weeks but also even on a normal chow diet (19Ivandic B. Castellani L.W. Wang X.P. Qiao J-H. Mehrabian M. Navab M. Fogelman A.M. Grass D.S. Swanson M.E. de Beer M.C. 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. 1999; 19: 1284-1290Google Scholar). In addition, increased formation of oxidized phospholipids in sPLA2 transgenic mice has been reported (20Leitinger N. Watson A.D. Hama S.Y. Ivandic B. Qiao J-H. Huber J. Faull K.F. Grass D.S. Navab M. Fogelman A.M. et al.Role of group II secretory phospholipase A2 in atherosclerosis. 2. Potential involvement of biologically active oxidized phospholipids.Arterioscler. Thromb. Vasc. Biol. 1999; 19: 1291-1298Google Scholar). In human atherosclerotic plaques, sPLA2 is expressed mainly by vascular smooth muscle cells but also consistently by tissue macrophages within the lesion (21Hurt-Camejo E. Anderson S. Standal R. Rosengren B. Sartipy P. Stadberg E. Johanses 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: 300-309Google Scholar, 22Menschikowski M. Jaross W. Kasper M. Lattke P. Schiering A. Secretory group-II phospholipase A2 in human atherosclerotic plaques.Atherosclerosis. 1995; 118: 173-181Google Scholar, 23Elinder L.S. Dumitrescu A. Larsson P. Hedin U. Frostegard J. Claesson H.E. Expression of phospholipase A2 isoforms in human normal and atherosclerotic arterial wall.Arterioscler. Thromb. Vasc. Biol. 1997; 17: 2257-2263Google Scholar, 24Romano M. Romano E. Bjorkerud S. Hurt-Camejo E. Ultrastructural localization of secretory type II phospholipase A2 in atherosclerotic and nonatherosclerotic regions of human arteries.Arterioscler. Thromb. Vasc. Biol. 1998; 18: 519-525Google Scholar). Therefore, we hypothesized that expression of human sPLA2 exclusively in macrophages might enhance early atherogenesis after bone marrow transplantation (BMT) from sPLA2 transgenic mice into low density lipoprotein receptor-deficient (LDLR−/−) recipients. Our results demonstrate that feeding a Western-type diet for 9 weeks after BMT significantly increased atherosclerosis in the group transplanted with the sPLA2 transgenic bone marrow. These data are consistent with the results of a study that was published while our work was in progress (25Webb N.R. Bostrom M.A. Szilvassy S.J. van der Westhuyzen D.R. Daugherty A. de Beer F.C. Macrophage-expressed group IIA secretory phospholipase A2 increases atherosclerotic lesion formation in LDL receptor-deficient mice.Arterioscler. Thromb. Vasc. Biol. 2003; 23: 263-268Google Scholar). However, we provide potential mechanisms for the increased atherogenesis induced by macrophage sPLA2, namely i) by increased 12/15-lipoxygenase (12/15-LO)-dependent generation of oxidative stress, as demonstrated by enhanced LDL oxidation by sPLA2 transgenic macrophages in vitro and increased isoprostane formation in vivo, and ii) by increased foam cell formation by sPLA2-overexpressing macrophages in vitro and in vivo. The human group IIA sPLA2 transgenic mice used in this study have been described previously (15Grass D.S. Felkner R.H. Chiang M-Y. Wallace R.E. Nevelainen T.J. Bennett C.F. Swanson M.E. Expression of human group II PLA2 in transgenic mice results in epidermal hyperplasia in the absence of inflammatory infiltrate.J. Clin. Invest. 1996; 97: 2233-2241Google Scholar, 16Tietge U.J.F. Maugeais C. Cain W. Grass D. Glick J.M. deBeer F.C. Rader D.J. 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. 2000; 275: 10077-10084Google Scholar). In these mice, expression of the transgene is driven by the endogenous human group IIA sPLA2 promoter, allowing for regulatability of the transgene by inflammatory stimuli besides high-level baseline sPLA2 expression (26Tietge U.J.F. Maugeais C. Cain W. Rader D.J. Acute inflammation increases selective uptake of HDL cholesteryl esters into adrenals of mice overexpressing human sPLA2.Am. J. Physiol. Endocrinol. Metab. 2003; 285: E403-E411Google Scholar). The sPLA2 transgenic line has been backcrossed extensively to the C57BL/6 genetic background for >14 generations. The animals were caged in animal rooms with ad libitum access to water and mouse chow diet. Homozygous LDLR−/− mice were obtained from The Jackson Laboratory (Bar Harbor, ME) as mating pairs and bred at the Gorlaeus Laboratory (Leiden, The Netherlands). Mice were housed in sterilized filter-top cages and given unlimited access to food and water. Mice were maintained on sterilized regular chow, containing 4.3% (w/w) fat without added cholesterol (RM3; Special Diet Services, Witham, UK), or were fed a semisynthetic Western-type diet, containing 15% (w/w) fat and 0.25% (w/w) cholesterol (Diet W; Hope Farms, Woerden, The Netherlands). Drinking water was supplied with antibiotics (83 mg/l ciprofloxacin and 67 mg/l polymyxin B sulfate) and 6.5 g/l sucrose. Animal experiments were performed in accordance with the national laws. All experimental protocols were approved by the respective government authorities and the local ethics committees for animal experiments of Leiden University and Humboldt University. Resident peritoneal macrophages with and without thioglycollate (Sigma, Deisenhofen, Germany) stimulation for 3 days were harvested by peritoneal lavage with 10 ml of sterile PBS containing 1 mM EDTA, collected by centrifugation (1,400 rpm, 10 min, 4°C), and plated on six-well plates (BD Falcon, Franklin Lakes, NJ) at a cell density of 4 × 106 in RPMI medium (Gibco Invitrogen, Karlsruhe, Germany) supplemented with endotoxin-free 10% fetal calf serum (Specialty Media, Phillipsburg, NJ). After 4 h, plates were washed three times with PBS to remove nonadherent cells and further incubated under various experimental conditions as indicated. Resident peritoneal macrophages isolated from sPLA2 transgenic mice and C57BL/6 controls were cultured for 24 h in RPMI supplemented with 10% fetal calf serum. To assess the expression of human type IIA sPLA2, RNA was isolated using the Trizol reagent (Invitrogen), incubated with DNase I (Qiagen, Hilden, Germany), and reverse transcribed using the Omniscript RT Kit (Qiagen). PCR with specific primers to amplify the full-length human type IIA sPLA2 cDNA according to the published sequence (GenBank accession number NM_000300) was performed on aliquots of the RT-PCR product. The presence of the human type IIA sPLA2 protein in cell culture supernatants of the respective macrophage incubations was detected by Western blot as described (18Tietge U.J.F. Maugeais C. Grass D. de Beer F.C. Rader D.J. Human secretory phospholipase A2 (sPLA2) mediates decreased plasma levels of HDL-cholesterol and apoA-I in response to inflammation independent of serum amyloid A (SAA) in human apoA-I transgenic mice.Arterioscler. Thromb. Vasc. Biol. 2002; 22: 1213-1218Google Scholar) using a monoclonal antibody (Cayman Chemical, Ann Arbor, MI). The sPLA2 activity assay was performed on cell culture supernatants and mouse plasma essentially as described previously (27Tietge U.J.F. Kozarsky K.F. Donahee M.H. Rader D.J. A tetracycline-regulated adenoviral expression system for in vivo delivery of transgenes to lung and liver.J. Gene Med. 2003; 5: 567-575Google Scholar). Briefly, using a 1,2-dithio analog of diheptanoyl phosphatidylcholine (Cayman Chemical) as a substrate, free thiols were liberated by sPLA2 action and detected using DTNB. Native human LDL was isolated by sequential ultracentrifugation (1.019 < d < 1.063), dialyzed extensively against EDTA-free PBS, filter-sterilized, and used within 2 days. The protein content of the preparations was measured using the bicinchoninic acid reagents (Pierce, Rockford, IL). To study macrophage oxidation of LDL (28Tangirala R.K. Mol M.J. Palinski W. Macrophage oxidative modification of low density lipoprotein occurs independently of its binding to the low density lipoprotein receptor.J. Lipid Res. 1996; 36: 2320-2328Google Scholar), unstimulated resident peritoneal macrophages were isolated as described above and incubated in Ham's F-10 medium (Promocell GmbH, Heidelberg, Germany) containing 100 mg LDL protein/ml for the time periods indicated. At the end of the incubations, the media containing LDL were removed and centrifuged for 10 min at 1,400 rpm, and EDTA was added to the supernatants at a final concentration of 0.2 mM to stop further oxidation. The extent of LDL oxidation was assessed by measuring thiobarbituric acid-reactive substances (TBARS) formation in 100 μl aliquots as described using malondialdehyde as a standard (28Tangirala R.K. Mol M.J. Palinski W. Macrophage oxidative modification of low density lipoprotein occurs independently of its binding to the low density lipoprotein receptor.J. Lipid Res. 1996; 36: 2320-2328Google Scholar). No-cell controls were included, and the respective TBARS values were subtracted from the experimental values. Cellular protein content was assessed with the bicinchoninic acid assay after adding 0.1 M NaOH and 1% SDS to the wells as described (29Tietge U.J.F. Sun G. Czarnecki S. Yu Q.C. Lohse P. Du H. Grabowski G.A. Glick J.M. Rader D.J. Phenotypic correction of lipid storage and growth arrest in Wolman disease fibroblasts by gene transfer of lysosomal acid lipase.Hum. Gene Ther. 2001; 12: 279-289Google Scholar). Data for LDL oxidation are expressed in nanomoles of malondialdehyde produced per milligram of cellular protein. To evaluate the role of 12/15-LO in macrophage-mediated LDL oxidation, the specific inhibitor PD146176 (Sigma-Aldrich, Munich, Germany) at a concentration of 20 μM, which is nontoxic to macrophages, was added to mouse peritoneal macrophages from sPLA2 transgenic mice or wild-type C57BL/6 littermates 1 h before the addition of native LDL (30Miller Y.I. Chang M-K. Funk C.D. Feramisco J.R. Witztum J.L. 12/15-Lipoxygenase translocation enhances site-specific actin polymerization in macrophages phagocytosing apoptotic cells.J. Biol. Chem. 2001; 276: 19432-19439Google Scholar). TBARS formation was assessed after 12 h of incubation. For Western blot analysis of 12/15-LO protein, 30 μg of protein was resolved by SDS-PAGE, transferred to nitrocellulose, and probed with a polyclonal rabbit anti-mouse 12/15-LO antibody at a dilution of 1:1,000 as described previously (31Chen X.S. Kurre U. Jenkins N.A. Copeland N.G. Funk C.D. cDNA cloning, expression, mutagenesis of C-terminal isoleucine, genomic structure, and chromosomal localization of murine 12-lipoxygenases.J. Biol. Chem. 1994; 269: 13979-13987Google Scholar). To assess macrophage foam cell formation in vitro, oxidized LDL (OxLDL) was prepared by incubating native LDL with 10 μM CuSO4 in Ham's F-10 medium at 37°C for 18 h. Mouse peritoneal macrophages from sPLA2 transgenic mice or wild-type C57BL/6 littermates were incubated for 24 h with OxLDL (25 μg/ml). Then, total cholesterol and free cholesterol were measured using enzymatic colorimetric assays (Wako Chemicals, Neuss, Germany). Cholesteryl esters were determined by subtracting free cholesterol from total cholesterol values. Total RNA was extracted from thioglycollate-elicited peritoneal macrophages from control C57BL/6 mice and sPLA2 transgenic animals by the Trizol method. cDNA was synthesized from 2 μg of total RNA using RevertAid™ M-MuLV reverse transcriptase. mRNA levels were quantitatively determined on an ABI Prism® 7700 sequence detection system (Applied Biosystems, Foster City, CA) using SYBR-green technology according to the manufacturer's instructions. For detection of scavenger receptor A mRNA, 5′-GGTGGTAGTGGAGCCCATGA-3′ and 5′-CCCGTATATCCCAGCGATCA-3′ were used as forward and reverse primers, respectively. Scavenger receptor A mRNA expression levels were calculated relative to the average of the housekeeping genes HPRT (primers 5′-TTGCTCGAGATGTCATGAAGGA-3′ and 5′-AGCAGGTCAGCAAAGAACTTATAGC-3′) and 36B4 (primers 5′-GGACCCGAGAAGACCTCCTT-3′ and 5′-GCACATCACTCAGAATTTCAATGG-3′). To induce bone marrow aplasia, female LDLR−/− mice were exposed to a single dose of 9 Gray (0.19 Gray/min, 200 kV, 4 mA) total body irradiation using an Andrex Smart 225 Röntgen source (YXLON International, Copenhagen, Denmark) with a 6 mm aluminum filter 1 day before transplantation. Bone marrow was isolated by flushing the femurs and tibias from female sPLA2 transgenic mice or female wild-type C57BL/6 littermates with phosphate-buffered saline. Single-cell suspensions were prepared by passing the cells through a 30 μm nylon gauze. Irradiated recipients received 0.5 × 107 bone marrow cells by intravenous injection into the tail vein. The hematologic chimerism of the LDLR−/− mice was determined in genomic DNA from bone marrow by PCR at 17 weeks after transplantation using specific primers located in the promoter region and the first intron of the human type IIA sPLA2 transgene (15Grass D.S. Felkner R.H. Chiang M-Y. Wallace R.E. Nevelainen T.J. Bennett C.F. Swanson M.E. Expression of human group II PLA2 in transgenic mice results in epidermal hyperplasia in the absence of inflammatory infiltrate.J. Clin. Invest. 1996; 97: 2233-2241Google Scholar), generating a 280 bp product. Notably, two independent BMT experiments were performed, each involving LDLR−/− mice receiving sPLA2 transgenic bone marrow as well as mice receiving control C57BL/6 bone marrow, and the data shown represent the combination of both of these studies. After an overnight fast, ∼100 μl of blood was drawn from individual mice by tail bleeding. The concentrations of total and free cholesterol, triglycerides, and phospholipids in serum were determined using enzymatic colorimetric assays (Roche Diagnostics, Mannheim, Germany). The distribution of cholesterol and phospholipids over the different lipoprotein subclasses in serum was determined by fractionation of 30 μl of serum of each mouse using a Superose 6 column (3.2 × 30 mm, Smart-system; Pharmacia, Uppsala, Sweden) as described (32van Eck M. Bos I.S. Kaminski W.E. Orso E. Rothe G. Twisk J. Bottcher A. van Amersfoort E.S. Christiansen-Weber T.A. Fung-Leung W.P. et al.Leukocyte ABCA1 controls susceptibility to atherosclerosis and macrophage recruitment into tissues.Proc. Natl. Acad. Sci. USA. 2002; 99: 6298-6303Google Scholar). Total cholesterol and phospholipid contents in the effluent were determined using enzymatic colorimetric assays (Roche Diagnostics). To analyze the development of atherosclerosis at the aortic root, transplanted mice were killed at week 17 after transplantation after 9 weeks of feeding the high-cholesterol Western-type diet. The arterial tree was perfused in situ with phosphate-buffered saline (100 mm Hg) for 20 min via a cannula in the left ventricular apex. The aortic arch as well as the thoracic and abdominal aortas were excised and stored in 3.7% formalin (Formal-fixx; Shandon Scientific Ltd.). The total atherosclerotic lesion area in Oil Red O-stained cryostat sections of the aortic root was quantified using the Leica image-analysis system, which consisted of a Leica DMRE microscope coupled to a video camera and Leica Qwin Imaging software (Leica Ltd., Cambridge, UK). Mean lesion area (in square micrometers) was calculated from 10 Oil Red O-stained sections, starting at the appearance of the triscuspid valves. For the assessment of macrophage area, sections were immunolabeled with MOMA-2 (a generous gift of Dr. G. Kraal, Vrije Universiteit, Amsterdam, The Netherlands; dilution 1:50) for the specific detection of macrophages. The MOMA-2-positive lesion area was subsequently quantified using the Leica image-analysis system. Cellular density was determined by counting the number of nuclei per macrophage area. The amount of collagen in the lesions was determined using Masson's Trichrome Accustain according to the manufacturer's instructions (Sigma Diagnostics). For detection of the expression of 12/15-LO in atherosclerotic lesions, sections were stained using a rabbit polyclonal antibody for mouse 12/15-LO (dilution 1:100) (31Chen X.S. Kurre U. Jenkins N.A. Copeland N.G. Funk C.D. cDNA cloning, expression, mutagenesis of C-terminal isoleucine, genomic structure, and chromosomal localization of murine 12-lipoxygenases.J. Biol. Chem. 1994; 269: 13979-13987Google Scholar). Urinary, plasma, and aortic levels of the isoprostane 8,12-iso-iPF2α-VI were measured by gas chromatography-mass spectrometry as described previously (33Pratico D. Tangirala R.K. Rader D.J. Rokach J. FitzGerald G.A. Vitamin E suppresses isoprostane generation in vivo and reduces atherosclerosis in apoE-deficient mice.Nat. Med. 1998; 4: 1189-1192Google Scholar). Urine was collected for 24 h from groups of animals, blood samples collected from individual mice were immediately centrifuged at 12,000 rpm for 15 min, and plasma was separated and stored at −80°C until analysis. Samples were spiked with a known amount of internal standard, extracted and purified by thin-layer chromatography, and analyzed by negative ion chemical ionization gas chromatography-mass spectrometry (33Pratico D. Tangirala R.K. Rader D.J. Rokach J. FitzGerald G.A. Vitamin E suppresses isoprostane generation in vivo and reduces atherosclerosis in apoE-deficient mice.Nat. Med. 1998; 4: 1189-1192Google Scholar, 34Cyrus T. Yao Y. Rokach J. Tang L.X. Pratico D. Vitamin E reduces progression of atherosclerosis in low-density lipoprotein receptor deficient mice with established vascular lesions.Circulation. 2003; 107: 521-523Google Scholar). Aortas from individual mice were obtained, weighed, minced, and homogenized in PBS containing EDTA (2 mM/l) and butylated hydroxytoluene (2 mM/l), pH 7.4, and total lipid extracted using Folch solution (chloroform-methanol, 2:1, v/v). Next, base hydrolysis was performed using 15% KOH at 45°C for 1 h, and the total levels of 8,12-iso-iPF2α-VI were processed before analysis as described above. Total 12-hydroxyeicosatetraenoic acid (12-HETE) and 15-HETE levels were assayed by liquid chromatography-tandem mass spectrometry essentially as described (35Pratico D. Zhukareva V. Yao Y. Uryu K. Funk C.D. Lawson J.A. Trojanowski J.Q. Lee V. M-Y. 12/15-Lipoxygenase is increased in Alzheimer's disease: possible involvement in brain oxidative stress.Am. J. Pathol. 2004; 164: 1655-1662Google Scholar). Statistical analysis was performed using the Statistical Package for the Social Sciences (SPSS). Data are expressed as means ± SEM unless indicated otherwise. Results were analyzed using Student's t-test, and statistical significance for all comparisons was assigned at P < 0.05. First, we determined whether human sPLA2 is expressed by peritoneal macrophages from sPLA2 transgenic mice. In unelicited resident peritoneal macrophages, a weak but specific signal was reproducibly detected by RT-PCR, but protein expression could not be visualized by Western blot, indicating low-level constitutive expression of the transgene (data not shown). Consistent with our previous observation that the human sPLA2 transgene is regulatable by inflammatory stimuli in these mice (18Tietge U.J.F. Maugeais C. Grass D. de Beer F.C. Rader D.J. Human secretory phospholipase A2 (sPLA2) mediates decreased plasma levels of HDL-cholesterol and apoA-I in response to inflammation independent of serum amyloid A (SAA) in human apoA-I transgenic mice.Arterioscler. Thromb. Vasc. Biol. 2002; 22: 1213-1218Google Scholar, 26Tietge U.J.F. Maugeais C. Cain W. Rader D.J. Acute inflammation increases selective uptake of HDL cholesteryl esters into adrenals of mice overexpressing human sPLA2.Am. J. Physiol. Endocrinol. Metab. 2003; 285: E403-E411Google Scholar), a strong signal was detected for the sPLA2 mRNA by RT-PCR in elicited peritoneal macrophages from sPLA2 transgenic mice, whereas human sPLA2 mRNA was absent in control C57BL/6 mice (Fig. 1A). Western blot analysis confirmed significant sPLA2 protein expression in macrophages of sPLA2 transgenic mice (Fig. 1B). In addition, supernatants of macrophages from sPLA2 transgenic mice contained significantly higher sPLA2 activity compared with C57BL/6 controls, indicating the expression of functional sPLA2 protein in macrophages from sPLA2 transgenic mice (0.91 ± 0.15 vs. 0.27 ± 0.10 milli optical density units/min, respectively; P < 0.001) (Fig. 1C).Fig. 1Expression of functional secretory phospholipase A2 (sPLA2) enzyme by elicited mouse peritoneal macrophages from human sPLA2 transgenic mice. A: Results of RT-PCR analysis of human sPLA2 mRNA expression in macrophages from sPLA2 transgenic mice and C57BL/6 control littermates, including a water control (−) and as a positive control cloned human sPLA2 cDNA (+). B, C: Wes

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