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Peptides derived from serum amyloid A prevent, and reverse, aortic lipid lesions in apoE−/− mice

2005; Elsevier BV; Volume: 46; Issue: 10 Linguagem: Inglês

10.1194/jlr.m500191-jlr200

1539-7262

S P Tam, John B. Ancsin, Ruth Tan, Robert Kisilevsky,

Pancreatitis Pathology and Treatment

Macrophages (Mϕ) at sites of acute tissue injury accumulate and export cholesterol quickly. This metabolic activity is likely dependent on the physiological function of a major acute-phase protein, serum amyloid A 2.1 (SAA2.1), that is synthesized by hepatocytes as part of a systemic response to acute injury. Our previous studies using cholesterol-laden J774 mouse Mϕ showed that an N-terminal domain of SAA2.1 inhibits acyl-CoA:cholesterol acyltransferase activity, and a C-terminal domain enhances cholesteryl ester hydrolase activity. The net effect of this enzymatic regulation is to drive intracellular cholesterol to its unesterified state, the form readily exportable to an extracellular acceptor such as HDL. Here, we demonstrate that these domains from mouse SAA2.1, when delivered in liposomal formulation, are effective at preventing and reversing aortic lipid lesions in apolipoprotein E-deficient mice maintained on high-fat diets. Furthermore, mouse SAA peptides, in liposomal formulation, are effective at regulating cholesterol efflux in THP-1 human Mϕ, and homologous domains from human SAA are effective in mouse J774 cells. These peptides operate at the level of the foam cell in the reverse cholesterol pathway and therefore may be used in conjunction with other agents that act more distally in this process.Such human peptides, or small molecule mimics of their structure, may prove to be potent antiatherogenic agents in humans. Macrophages (Mϕ) at sites of acute tissue injury accumulate and export cholesterol quickly. This metabolic activity is likely dependent on the physiological function of a major acute-phase protein, serum amyloid A 2.1 (SAA2.1), that is synthesized by hepatocytes as part of a systemic response to acute injury. Our previous studies using cholesterol-laden J774 mouse Mϕ showed that an N-terminal domain of SAA2.1 inhibits acyl-CoA:cholesterol acyltransferase activity, and a C-terminal domain enhances cholesteryl ester hydrolase activity. The net effect of this enzymatic regulation is to drive intracellular cholesterol to its unesterified state, the form readily exportable to an extracellular acceptor such as HDL. Here, we demonstrate that these domains from mouse SAA2.1, when delivered in liposomal formulation, are effective at preventing and reversing aortic lipid lesions in apolipoprotein E-deficient mice maintained on high-fat diets. Furthermore, mouse SAA peptides, in liposomal formulation, are effective at regulating cholesterol efflux in THP-1 human Mϕ, and homologous domains from human SAA are effective in mouse J774 cells. These peptides operate at the level of the foam cell in the reverse cholesterol pathway and therefore may be used in conjunction with other agents that act more distally in this process. Such human peptides, or small molecule mimics of their structure, may prove to be potent antiatherogenic agents in humans. Acute tissue injury commonly results in local cell death and the generation of large quantities of cell membrane fragments rich in cholesterol. As part of the reactive acute inflammatory process, macrophages (Mϕ) are mobilized to such sites of injury, ingest these fragments, and acquire a considerable cholesterol load. A removal mechanism is required to mobilize this cholesterol either for reuse or excretion. Our past results suggested that a physiological role of one isoform of a major acute-phase protein synthesized by the liver in response to tissue injury, mouse serum amyloid A 2.1 (mSAA2.1), is the regulation of Mϕ cholesterol export (1Ely S. Bonatesta R. Ancsin J.B. Kindy M. Kisilevsky R. The in-vitro influence of serum amyloid A isoforms on enzymes that regulate the balance between esterified and un-esterified cholesterol.Amyloid. 2001; 8: 169-181Google Scholar, 2Tam S.P. Flexman A. Hulme J. Kisilevsky R. Promoting export of macrophage cholesterol: the physiological role of a major acute-phase protein, serum amyloid A 2.1.J. Lipid Res. 2002; 43: 1410-1420Google Scholar, 3Kisilevsky R. Tam S.P. Macrophage cholesterol efflux and the active domains of serum amyloid A2.1.J. Lipid Res. 2003; 44: 2257-2269Google Scholar). Fragmentation of this protein into peptides that span its entire length has revealed that the N-terminal region, mSAA2.11–20, is a potent in vitro and in vivo inhibitor of Mϕ ACAT (3Kisilevsky R. Tam S.P. Macrophage cholesterol efflux and the active domains of serum amyloid A2.1.J. Lipid Res. 2003; 44: 2257-2269Google Scholar). A separate region at the C terminus, mSAA2.174–103, contains a domain that enhances the in vitro and in vivo activity of neutral cholesteryl ester hydrolase (CEH) (3Kisilevsky R. Tam S.P. Macrophage cholesterol efflux and the active domains of serum amyloid A2.1.J. Lipid Res. 2003; 44: 2257-2269Google Scholar). In combination, these two peptides drive stored cholesteryl esters into their unesterified form, which, in the presence of a functional cholesterol transporter and an extracellular cholesterol acceptor such as HDL, is rapidly exported from the Mϕ (2Tam S.P. Flexman A. Hulme J. Kisilevsky R. Promoting export of macrophage cholesterol: the physiological role of a major acute-phase protein, serum amyloid A 2.1.J. Lipid Res. 2002; 43: 1410-1420Google Scholar). These results suggested that such peptides may be useful in mobilizing cholesterol from Mϕ at sites of atherogenesis. To examine this possibility, we prepared liposomal formulations of the active SAA2.1 peptides and tested their ability to prevent, or cause the regression of, aortic lipid lesions in apolipoprotein E-deficient (apoE−/−) mice maintained on a high-fat atherogenic diet. To determine whether these peptides, as liposomal formulations, are effective only in mouse Mϕ, we examined the influence of these peptides on 1) ACAT, CEH, and cholesterol efflux activities in cholesterol-laden THP-1 human Mϕ, and 2) the effect of homologous human peptides on cholesterol-laden J774 mouse Mϕ. All chemicals were reagent grade and purchased from Fisher Scientific (Nepean, Ontario, Canada), Sigma (St. Louis, MO), ICN (Aurora, OH), or Bio-Rad (Hercules, CA). DMEM, RPMI-1640 medium, and FBS were purchased from Life Technologies (Burlington, Ontario, Canada). Radiolabeled [1-14C]oleic acid (52 mCi/mmol), [1,2,6,7-3H(N)]cholesterol (82 Ci/mmol), and cholesteryl-1,2,6,7-3H(N)]oleate (84 Ci/mmol) were obtained from DuPont-New England Nuclear (Boston, MA). Swiss-white CD1 6–8 week old female mice were obtained from Charles River Laboratories (Montreal, Quebec, Canada). Mice were kept in a temperature-controlled room on a 12 h light/dark cycle. They were fed Purina Lab Chow pellets and water ad libitum. Female homozygous apoE−/− mice (C57BL/6J-Apoe tm1Unc) were purchased from Jackson Laboratories (Bar Harbor, ME) at 8 weeks of age and fed Purina Lab Chow ad libitum until 12 weeks of age. For "prevention" experiments, apoE−/− mice were divided into six groups of animals. One group remained on lab chow and the other five were fed a high-fat diet containing 7.5% cocoa butter, 1.25% cholesterol, and 0.5% cholic acid (C13002; Research Diets, Inc.). When the animals were placed on the high-fat diet, they were given either no treatment or liposomes containing 15 μg of mSAA2.11–20, 15 μg of mSAA2.174–103, or a combination of both peptides (7.5 μg of each). Each liposome regimen was given every 4th day as a 100 μl intravenous injection via the tail vein. In addition, as a control, one group was given four injections of protein-free liposomes. For the "regression" experiment, mice were divided into five groups of animals. One group received the standard diet and the other four groups were fed the high-fat diet until they were euthanized. At 16 weeks of age, among the groups of mice receiving the high-fat diet for 4 weeks, one group received no treatment; the other three groups were given four injections of liposomes containing 15 μg of mSAA2.11–20, 15 μg of mSAA2.174–103, or a combination of these two peptides (7.5 μg of each). All animals were euthanized 4 days after the final injection of liposomes. Each of these experiments was performed twice. Animals were euthanized by CO2 nacrosis and exsanguinated by cardiac puncture into heparin-coated syringes for plasma lipid analyses. Total plasma, LDL, and HDL cholesterol and triglyceride levels were determined with Roche modular automated instruments in the clinical laboratories of Kingston General Hospital. The aortas were perfused with 10 ml of PBS via the left ventricle and teased free from the body but left attached to the heart. The adventitial adipose tissue was removed, and the aortas were opened longitudinally, pinned out as described (4Tangirala R.K. Rubin E.M. Palinski W. Quantitation of atherosclerosis in murine models: correlation between lesions in the aortic origin and in the entire aorta, and differences in the extent of lesions between sexes in LDL receptor-deficient and apolipoprotein E-deficient mice.J. Lipid Res. 1995; 36: 2320-2328Google Scholar), washed with 60% isopropanol for 3 min, stained with Oil Red O (0.4% in 60% isopropanol) for 3 min, rinsed in 60% isopropanol for 3 min, and then fixed in 10% formalin for 2 min. Once fixed, the aortas were stored in 10% formalin until the lipid lesions were quantified. Quantification of the percentage of the aortic surface occupied by Oil Red O-positive lesions was performed with a program and apparatus from MCID M2 Imaging Research, Inc. (St. Catherines, Ontario, Canada) as described previously (5Kisilevsky R. Lemieux L.J. Fraser P.E. Kong X.Q. Hultin P.G. Szarek W.A. Arresting amyloidosis in vivo using small-molecule anionic sulphonates or sulphates: implications for Alzheimer's disease.Nat. Med. 1995; 1: 143-148Google Scholar). Peptides corresponding to mSAA2.11–20, mSAA2.121–50, mSAA2.151–80, mSAA2.174–103, and human (h)SAA1.1/2.11–23 (note that the sequence of the human isoforms is identical over the first 50 residues) were prepared as described previously (3Kisilevsky R. Tam S.P. Macrophage cholesterol efflux and the active domains of serum amyloid A2.1.J. Lipid Res. 2003; 44: 2257-2269Google Scholar). In addition, an R linked to the N terminus of mSAA1.11–20 was synthesized. These peptides were obtained from the Protein Synthesis Laboratory at Queen's University or The Hospital for Sick Children (Toronto, Ontario, Canada). The purity of the synthetic peptides was established by analytical HPLC and ion-spray mass spectrometry. To mimic the ingestion of cell membrane fragments by Mϕ at sites of tissue injury, red blood cells (RBCs) were used as a source of cholesterol, as described previously (2Tam S.P. Flexman A. Hulme J. Kisilevsky R. Promoting export of macrophage cholesterol: the physiological role of a major acute-phase protein, serum amyloid A 2.1.J. Lipid Res. 2002; 43: 1410-1420Google Scholar). Similar quantities of cholesterol, 175 μg, were used in all experiments. The concentration of cholesterol in the membrane preparations was determined as described previously (2Tam S.P. Flexman A. Hulme J. Kisilevsky R. Promoting export of macrophage cholesterol: the physiological role of a major acute-phase protein, serum amyloid A 2.1.J. Lipid Res. 2002; 43: 1410-1420Google Scholar). HDL and acute-phase high density lipoprotein (AP-HDL) were isolated from normal and inflamed mice, respectively, using sequential density flotation as described previously (6Ancsin J.B. Kisilevsky R. The heparin/heparan sulfate-binding site on apo-serum amyloid A: implications for the therapeutic intervention of amyloidosis.J. Biol. Chem. 1999; 274: 7172-7181Google Scholar, 7Ancsin J.B. Kisilevsky R. Laminin interactions with the apoproteins of acute-phase HDL: preliminary mapping of the laminin binding site on serum amyloid A.Amyloid. 1999; 6: 37-47Google Scholar). The isolation, separation, and purification of apoA-I, SAA1.1, and SAA2.1 from acute-phase mouse plasma was performed as described previously (6Ancsin J.B. Kisilevsky R. The heparin/heparan sulfate-binding site on apo-serum amyloid A: implications for the therapeutic intervention of amyloidosis.J. Biol. Chem. 1999; 274: 7172-7181Google Scholar, 7Ancsin J.B. Kisilevsky R. Laminin interactions with the apoproteins of acute-phase HDL: preliminary mapping of the laminin binding site on serum amyloid A.Amyloid. 1999; 6: 37-47Google Scholar). The purity of the isolated proteins was established by mass spectrometry and N-terminal sequencing. Each of the intact proteins (apoA-I, SAA1.1, and SAA2.1) or the various synthetic peptides listed above were reconstituted with lipids to form liposomes using the procedure described by Jonas, Kezdy, and Wald (8Jonas A. Kezdy E. Wald J.H. Defined apolipoprotein A-I conformations in reconstituted high density lipoprotein discs.J. Biol. Chem. 1989; 264: 4818-4825Google Scholar), as detailed previously (2Tam S.P. Flexman A. Hulme J. Kisilevsky R. Promoting export of macrophage cholesterol: the physiological role of a major acute-phase protein, serum amyloid A 2.1.J. Lipid Res. 2002; 43: 1410-1420Google Scholar, 3Kisilevsky R. Tam S.P. Macrophage cholesterol efflux and the active domains of serum amyloid A2.1.J. Lipid Res. 2003; 44: 2257-2269Google Scholar). When assessing the effects of the various apolipoproteins or peptides, these were always used as liposomes. Free peptides have no effect in culture or in vivo. Mouse J774 Mϕ (TIB-67) and human THP-1 monocytes (TIB-202) were obtained from the American Type Culture Collection (Manassas, VA). J774 cells were cultured on six-well tissue culture plates at 106 cells/well and grown to 90% confluence in 2 ml of DMEM supplemented with 10% FBS. The medium was changed three times per week. THP-1 cells were maintained in RPMI-1640 medium containing 10% FBS according to the instructions supplied by the American Type Culture Collection. These monocytes were differentiated into Mϕ with 100 nM phorbol myristate acetate. The cells were seeded onto six-well tissue culture dishes at 106 cells per well and maintained in medium containing phorbol myristate acetate (100 nM). Media were replaced every 2 days, and experiments were started after 7 days in culture, when the cells morphologically were Mϕ. THP-1 or J774 cells were loaded with cholesterol using RBC membrane fragments as described previously (2Tam S.P. Flexman A. Hulme J. Kisilevsky R. Promoting export of macrophage cholesterol: the physiological role of a major acute-phase protein, serum amyloid A 2.1.J. Lipid Res. 2002; 43: 1410-1420Google Scholar, 3Kisilevsky R. Tam S.P. Macrophage cholesterol efflux and the active domains of serum amyloid A2.1.J. Lipid Res. 2003; 44: 2257-2269Google Scholar). ACAT activity was determined by measuring the incorporation of [1-14C] oleic acid into cholesteryl esters as described previously (2Tam S.P. Flexman A. Hulme J. Kisilevsky R. Promoting export of macrophage cholesterol: the physiological role of a major acute-phase protein, serum amyloid A 2.1.J. Lipid Res. 2002; 43: 1410-1420Google Scholar, 3Kisilevsky R. Tam S.P. Macrophage cholesterol efflux and the active domains of serum amyloid A2.1.J. Lipid Res. 2003; 44: 2257-2269Google Scholar). ACAT activity was determined in phorbol myristate acetate-treated THP-1 cells without cholesterol loading, in cholesterol-laden cells, and in cholesterol-laden cells that were then cultured in medium supplemented with 50 μg/ml native HDL, AP-HDL, protein-free liposomes, or liposomes containing 2 μM (final culture concentration) murine apoA-I, mSAA1.1, or mSAA2.1. To map the domains in mSAA2.1 that decreased ACAT activity, THP-1 Mϕ were loaded with cholesterol and labeled as described above, then incubated with liposomes containing 7.5 μg of the peptide of interest. These included mSAA2.11–20, mSAA2.121–50, mSAA2.151–80, and mSAA2.174–103. To determine whether homologous human peptides had an effect on ACAT activity similar to mSAA2.11–20 in J774 cells, these cells were loaded with cholesterol and labeled as described above, then incubated with liposomes containing hSAA1.1/2.11–23, or mSAA1.11–20 as a negative control, or mSAA1.11–20 with R added at the N terminus. After 3 h incubations in the media described above, the cells were incubated for 3 h with [14C]oleate (9Oram J.F. Mendez A.J. Slotte J.P. Johnson T.F. High density lipoprotein apolipoproteins mediate removal of sterol from intracellular pools but not from plasma membranes of cholesterol-loaded fibroblasts.Arterioscler. Thromb. 1991; 11: 403-414Google Scholar, 10Mendez A.J. Anantharamaiah G.M. Segrest J.P. Oram J.F. Synthetic amphipathic helical peptides that mimic apolipoprotein A-I in clearing cellular cholesterol.J. Clin. Invest. 1994; 94: 1698-1705Google Scholar), chilled on ice, and washed twice with PBS-BSA and twice with PBS and [3H]cholesteryl oleate (6,000 dpm/well) added as an internal standard to monitor extraction efficiency. The lipids were analyzed by thin-layer chromatography as described previously (9Oram J.F. Mendez A.J. Slotte J.P. Johnson T.F. High density lipoprotein apolipoproteins mediate removal of sterol from intracellular pools but not from plasma membranes of cholesterol-loaded fibroblasts.Arterioscler. Thromb. 1991; 11: 403-414Google Scholar, 10Mendez A.J. Anantharamaiah G.M. Segrest J.P. Oram J.F. Synthetic amphipathic helical peptides that mimic apolipoprotein A-I in clearing cellular cholesterol.J. Clin. Invest. 1994; 94: 1698-1705Google Scholar). The radioactivity in cholesteryl ester spots was corrected for the extraction efficiency and used as a measure of ACAT activity. Rates of hydrolysis of radiolabeled cholesteryl ester in THP-1 cells were determined exactly as described previously with J774 cells in the presence of 2 μg/ml Sandoz 58-035 (an ACAT inhibitor) to prevent the reesterification of liberated [14C]oleate and free cholesterol (2Tam S.P. Flexman A. Hulme J. Kisilevsky R. Promoting export of macrophage cholesterol: the physiological role of a major acute-phase protein, serum amyloid A 2.1.J. Lipid Res. 2002; 43: 1410-1420Google Scholar, 3Kisilevsky R. Tam S.P. Macrophage cholesterol efflux and the active domains of serum amyloid A2.1.J. Lipid Res. 2003; 44: 2257-2269Google Scholar). At various times under the different culture conditions, cellular lipids were extracted and analyzed for cholesteryl ester radioactivity as described above. THP-1 and J774 cells were laden with cholesterol by incubating with RBC membrane fragments that had been equilibrated previously with 0.5 μCi/ml [3H]cholesterol (2Tam S.P. Flexman A. Hulme J. Kisilevsky R. Promoting export of macrophage cholesterol: the physiological role of a major acute-phase protein, serum amyloid A 2.1.J. Lipid Res. 2002; 43: 1410-1420Google Scholar, 3Kisilevsky R. Tam S.P. Macrophage cholesterol efflux and the active domains of serum amyloid A2.1.J. Lipid Res. 2003; 44: 2257-2269Google Scholar). Cholesterol pools were allowed to equilibrate for 18 h in culture, and efflux was examined after treatment with the different isoforms of SAA, or their peptides, as described previously (2Tam S.P. Flexman A. Hulme J. Kisilevsky R. Promoting export of macrophage cholesterol: the physiological role of a major acute-phase protein, serum amyloid A 2.1.J. Lipid Res. 2002; 43: 1410-1420Google Scholar, 3Kisilevsky R. Tam S.P. Macrophage cholesterol efflux and the active domains of serum amyloid A2.1.J. Lipid Res. 2003; 44: 2257-2269Google Scholar). To determine cholesterol export in vivo, experiments were conducted as described and validated previously (2Tam S.P. Flexman A. Hulme J. Kisilevsky R. Promoting export of macrophage cholesterol: the physiological role of a major acute-phase protein, serum amyloid A 2.1.J. Lipid Res. 2002; 43: 1410-1420Google Scholar). Briefly, J774 Mϕ were laden with RBC membranes and [3H]cholesterol as described above, washed with PBS/BSA, and then detached from the culture dishes. Five million cells in 200 μl of DMEM were injected into mice via the tail vein. At various times thereafter, ∼25 μl of blood was collected from the tail vein of each animal into heparinized capillary tubes, then centrifuged for 5 min in an Adams Autocrit Centrifuge to separate RBCs from plasma. Cholesterol efflux was determined by measuring the appearance of [3H]cholesterol in plasma by scintillation spectrometry. To study whether the export of cholesterol from these injected J774 cells to plasma is influenced by mSAA2.11–20, mSAA1.11–20, or hSAA1.1/2.11–23, 100 μl of liposomes containing 15 μg of one of these peptides was injected intravenously, and at various times after this injection, ∼25 μl of blood was collected from the tail vein of each animal and the plasma was analyzed by scintillation spectrometry. Protein concentration was determined by the method of Lowry and coworkers (11Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. Protein measurement with the Folin phenol reagent.J. Biol. Chem. 1951; 193: 265-275Google Scholar), with the aid of a Bio-Rad protein assay kit. Previous results with mSAA2.11–20 and mSAA2.174–103 acting on mouse Mϕ ACAT and CEH activities, respectively, and on Mϕ cholesterol export in culture and in vivo suggested that these peptides may have antiatherogenic activity (2Tam S.P. Flexman A. Hulme J. Kisilevsky R. Promoting export of macrophage cholesterol: the physiological role of a major acute-phase protein, serum amyloid A 2.1.J. Lipid Res. 2002; 43: 1410-1420Google Scholar, 3Kisilevsky R. Tam S.P. Macrophage cholesterol efflux and the active domains of serum amyloid A2.1.J. Lipid Res. 2003; 44: 2257-2269Google Scholar). To assess this possibility, the effects of mSAA2.11–20 and mSAA2.174–103, individually and in combination, were examined in apoE−/− mice placed on a high-fat diet. Two protocols were used, a prevention mode, wherein the treatment with the requisite peptides began as the mice were placed on the high-fat diet, and a regression mode, wherein the mice were on a high-fat diet for 28 days before treatment with the requisite peptides commenced. In the prevention mode, six groups of mice were examined, those on 1) the high-fat diet; 2) standard lab chow (low-fat diet); 3) the high-fat diet given mSAA2.11–20; 4) the high-fat diet given mSAA2.174–103; 5) the high-fat diet given mSAA2.11–20 + mSAA2.174–103; and 6) the high-fat diet given peptide-free liposomes. In each case, the peptides were given as liposomes once every 4 days (four doses), at the termination of which the animals were euthanized and the aortas were stained with Oil Red O. The endothelial surface was examined en face, and the aortic area positive for lipid was expressed as a percentage of the total aortic area. These data were then normalized relative to the mean lipid-positive area in the untreated group fed the high-fat diet. These data are shown in Fig. 1. Mice on the low-fat diet had 67 ± 7% less area occupied by lipid lesions relative to mice on the high-fat diet. Among the groups on the high-fat diet, those treated with protein-free liposomes or liposomes containing mSAA2.11–20, mSAA2.174–103, or mSAA2.11–20 + mSAA2.174–103 had, respectively, 14 ± 19%, 45 ± 8%, 41 ± 13%, and 73 ± 5% less area occupied by lipid lesions relative to mice on the high-fat diet. The value with protein-free liposomes is not significantly different from that of the mice on the high-fat diet itself. The value with mSAA2.11–20 + mSAA2.174–103 is equivalent to that of mice on the low-fat diet. The P values for the groups of mice treated with the various peptides are <0.05 and are indicated in the legend to Fig. 1. These data indicate that peptides mSAA2.174–103, mSAA2.11–20, and particularly mSAA2.11–20 + mSAA2.174–103 are effective at inhibiting aortic lipid accumulation in apoE−/− mice. In the regression mode, apoE−/− mice were first placed on the high-fat diet for a period of 4 weeks. They were then divided into four groups, all of which continued on the diet for an additional 16 days; group 1 received no treatment, group 2 was treated with mSAA2.11–20, group 3 was treated with mSAA2.174–103, and group 4 was treated with mSAA2.11–20 + mSAA2.174–103. In each case, the peptides were administered as liposomes once every 4 days (four doses). A fifth group consisted of apoE−/− mice on standard lab chow for 44 days. The results are illustrated in Fig. 2. After 44 days, mice on the low-fat diet had 86 ± 7% less aortic area occupied by lipid lesions relative to those on the high-fat diet. Among the groups on the high-fat diet, those treated with mSAA2.11–20, mSAA2.174–103, or mSAA2.11–20 + mSAA2.174–103 had, respectively, 34 ± 13%, 48 ± 8%, and 71 ± 4% less area occupied by lipid lesions relative to those on the high-fat diet. The P values are indicated in the legend to Fig. 2. These data indicate that peptides mSAA2.174–103, mSAA2.11–20, and particularly mSAA2.11–20 + mSAA2.174–103 are effective at reducing aortic lipid accumulation in apoE−/− mice. A visual comparison of the aortic lesions in an apoE−/− mouse on a high-fat diet for 44 days and one treated with peptides mSAA2.11–20 + mSAA2.174–103 over the final 16 days is given in Fig. 3. The effect of the high-fat diet, and treatments, on plasma lipid parameters (triglycerides and total HDL and LDL cholesterol concentrations) are indicated in Table 1. There was a 3- to 5-fold increase in plasma cholesterol parameters when the mice were placed on the high-fat diet; however, there was no apparent effect of the peptides singly, or in combination, on these parameters, despite the fact that these peptides clearly affected the degree of the aortic lipid lesions. A comparison of the plasma lipid parameters between mice that were on the high-lipid diet for 16 days versus 44 days suggests a modest increase in HDL cholesterol in the latter group but a reduction in the other plasma lipid values in the groups fed this diet for longer periods of time. The reasons for these changes are not obvious but may relate to a reduction in the intake of diet over time (there was no difference in body weight between the mice in the various groups; data not shown), perhaps an increase in HDL production, or adaptations to a high-fat diet that are not immediately apparent. Nevertheless, it is important to note that the increase in HDL cholesterol and the decrease in LDL/HDL cholesterol ratio did not protect against the development of lipid lesions unless liposomes containing SAA peptides were also added.TABLE 1Plasma lipid parameters of apolipoprotein E-deficient mice on standard lab chow, high-fat diet, and high-fat diet and treatment with mSAA2.174–103, mSAA2.11–20, or mSAA2.11–20 + mSAA2.174–103 in the prevention and regression protocolsTreatmentNTGTCHDL-CLDL-CTC/HDL-CLDL-C/HDL-CLFD51.00 ± 0.1119.7 ± 2.308.90 ± 0.7310.3 ± 1.602.16 ± 0.101.16 ± 0.10HFD93.01 ± 0.4075.8 ± 6.4120.8 ± 0.6353.6 ± 6.003.62 ± 0.272.58 ± 0.26mSAA2.11–20103.07 ± 0.5698.6 ± 4.8025.6 ± 1.3871.8 ± 4.653.97 ± 0.322.92 ± 0.31mSAA2.174–103103.49 ± 0.7691.0 ± 6.3023.4 ± 1.1066.3 ± 5.303.92 ± 0.182.82 ± 0.17Both peptides103.61 ± 0.6876.8 ± 7.5019.9 ± 1.6055.4 ± 6.803.93 ± 0.392.92 ± 0.38PFL52.00 ± 0.2784.3 ± 3.4023.0 ± 0.9360.4 ± 2.803.54 ± 0.102.58 ± 0.12*HFD91.88 ± 0.2774.1 ± 4.3926.2 ± 2.2047.1 ± 4.163.02 ± 0.331.97 ± 0.32*mSAA2.11–2091.69 ± 0.2066.5 ± 4.5525.0 ± 1.7440.9 ± 3.222.71 ± 0.111.66 ± 0.09*mSAA2.174–10381.89 ± 0.2372.5 ± 4.9830.1 ± 3.6641.6 ± 3.282.54 ± 0.201.53 ± 0.20*Both peptides61.12 ± 0.1864.0 ± 2.1126.9 ± 3.6336.6 ± 1.922.40 ± 0.821.38 ± 0.07Values, except for ratios, are in mmol/l ± SEM. N, number of mice per group; TG, total triglyceride; TC, total cholesterol; HDL-C, high density lipoprotein cholesterol; LDL-C, low density lipoprotein cholesterol; LFD, standard lab chow; HFD, high-fat diet; PFL, protein-free liposomes; mSAA, mouse serum amyloid A. Treatment groups with asterisks are from the regression protocol, as described in Materials and Methods. Open table in a new tab Values, except for ratios, are in mmol/l ± SEM. N, number of mice per group; TG, total triglyceride; TC, total cholesterol; HDL-C, high density lipoprotein cholesterol; LDL-C, low density lipoprotein cholesterol; LFD, standard lab chow; HFD, high-fat diet; PFL, protein-free liposomes; mSAA, mouse serum amyloid A. Treatment groups with asterisks are from the regression protocol, as described in Materials and Methods. Our previous studies with J774 Mϕ demonstrated that mSAA2.1 had an ACAT-inhibitory domain at its N terminus and a CEH-enhancing domain at its C terminus (2Tam S.P. Flexman A. Hulme J. Kisilevsky R. Promoting export of macrophage cholesterol: the physiological role of a major acute-phase protein, serum amyloid A 2.1.J. Lipid Res. 2002; 43: 1410-1420Google Scholar, 3Kisilevsky R. Tam S.P. Macrophage cholesterol efflux and the active domains of serum amyloid A2.1.J. Lipid Res. 2003; 44: 2257-2269Google Scholar). Operating individually or in concert, these peptides have proven remarkably effective in culture and in vivo at promoting the rapid efflux of cholesterol from cholesterol-laden cells (2Tam S.P. Flexman A. Hulme J. Kisilevsky R. Promoting export of macrophage cholesterol: the physiological role of a major acute-phase protein, serum amyloid A 2.1.J. Lipid Res. 2002; 43: 1410-1420Google Scholar, 3Kisilevsky R. Tam S.P. Macrophage cholesterol efflux and the active domains of serum amyloid A2.1.J. Lipid Res. 2003; 44: 2257-2269Google Scholar). To determine whether these results are peculiar to mouse cells, we examined the effect of mouse AP-HDL, mSAA2.1, and mSAA2.1 peptides (each as liposomes) on ACAT and CEH activity and cholesterol export with a human Mϕ cell line, THP-1. Figure 4illustrates the baseline ACAT activity of THP-1 cells, the effect of feeding these cells mouse erythrocyte membrane fragments (as a source of cholesterol), and the subsequent influence of HDL or AP-HDL on such activity. As shown previously with J774 cells (2Tam S.P. Flexman A. Hulme J. Kisil