A new HDL mimetic peptide that stimulates cellular cholesterol efflux with high efficiency greatly reduces atherosclerosis in mice
2010; Elsevier BV; Volume: 51; Issue: 6 Linguagem: Inglês
10.1194/jlr.m003665
ISSN1539-7262
AutoresJohn K. Bielicki, Haiyan Zhang, Yuan Cortez, Ying Zheng, Vasanthy Narayanaswami, Arti Patel, Jan Johansson, Salman Azhar,
Tópico(s)Peroxisome Proliferator-Activated Receptors
ResumoHere, we report the creation of a single-helix peptide (ATI-5261) that stimulates cellular cholesterol efflux with Km molar efficiency approximating native apolipoproteins. Anti-atherosclerosis activity of ATI-5261 was evaluated in LDLR−/− and apolipoprotein (apo)E−/− mice ∼5–7 months of age, following 13–18 weeks on a high-fat Western diet (HFWD). Treatment of fat-fed LDLR−/− mice with daily intraperitoneal injections of ATI-5261 (30 mg/kg) for 6 weeks reduced atherosclerosis by 30%, as judged by lesion area covering the aorta (7.9 ± 2 vs.11.3 ± 2.5% control, P = 0.011) and lipid-content of aortic sinus plaque (25 ± 5.8 vs. 33 ± 4.9% control, P = 0.014). In apoE−/− mice, the peptide administered 30 mg/kg ip on alternate days for 6 weeks reduced atherosclerosis by ∼45% (lesion area = 15 ± 7 vs. 25 ± 8% control, P = 0.00016; plaque lipid-content = 20 ± 6 vs. 32 ± 8% control, P < 0.0001). Similar reductions in atherosclerosis were achieved using ATI-5261:POPC complexes. Single intraperitoneal injection of ATI-5261 increased reverse cholesterol transport from macrophage foam-cells to feces over 24–48 h. In summary, relatively short-term treatment of mice with the potent cholesterol efflux peptide ATI-5261 reduced substantial atherosclerosis. This was achieved using an L-amino acid peptide, in the presence of severe hypercholesterolemia/HFWD, and did not require daily injections or formulation with phospholipids when administered via intraperitoneal injection. Here, we report the creation of a single-helix peptide (ATI-5261) that stimulates cellular cholesterol efflux with Km molar efficiency approximating native apolipoproteins. Anti-atherosclerosis activity of ATI-5261 was evaluated in LDLR−/− and apolipoprotein (apo)E−/− mice ∼5–7 months of age, following 13–18 weeks on a high-fat Western diet (HFWD). Treatment of fat-fed LDLR−/− mice with daily intraperitoneal injections of ATI-5261 (30 mg/kg) for 6 weeks reduced atherosclerosis by 30%, as judged by lesion area covering the aorta (7.9 ± 2 vs.11.3 ± 2.5% control, P = 0.011) and lipid-content of aortic sinus plaque (25 ± 5.8 vs. 33 ± 4.9% control, P = 0.014). In apoE−/− mice, the peptide administered 30 mg/kg ip on alternate days for 6 weeks reduced atherosclerosis by ∼45% (lesion area = 15 ± 7 vs. 25 ± 8% control, P = 0.00016; plaque lipid-content = 20 ± 6 vs. 32 ± 8% control, P < 0.0001). Similar reductions in atherosclerosis were achieved using ATI-5261:POPC complexes. Single intraperitoneal injection of ATI-5261 increased reverse cholesterol transport from macrophage foam-cells to feces over 24–48 h. In summary, relatively short-term treatment of mice with the potent cholesterol efflux peptide ATI-5261 reduced substantial atherosclerosis. This was achieved using an L-amino acid peptide, in the presence of severe hypercholesterolemia/HFWD, and did not require daily injections or formulation with phospholipids when administered via intraperitoneal injection. apolipoprotein alanine aminotransferase aspartate aminotransferase circular dichroism C-terminal high-fat Western diet low-density lipoprotein receptor red blood cells reverse cholesterol transport Plasma HDLs are thought to protect against atherosclerosis via mechanisms related, in part, to reverse cholesterol transport (RCT) (1Gordon T. Castelli W.P. Hjortland M.C. Kannel W.B. Dawber T.R. High density lipoprotein as a protective factor against coronary heart disease: the Framingham Study.Am. J. Med. 1977; 62: 707-714Abstract Full Text PDF PubMed Scopus (4037) Google Scholar, 2Johansson J. Carlson L.A. Landou C. Hamsten A. 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ApolipoproteinA-I mimetic peptides: possible new agents for the treatment of atherosclerosis.Curr. Opin. Investig. Drugs. 2007; 8: 201-212PubMed Google Scholar, 14Tall A.R. Yvan-Charvet L. Terasaka N. Pagler T. Nan W. HDL, ABC transporters and cholesterol efflux: implications for the treatment of atherosclerosis.Cell Metab. 2008; 7: 365-375Abstract Full Text Full Text PDF PubMed Scopus (419) Google Scholar). The mechanisms by which apolipoprotein mimetic peptides reduce atherosclerosis are not fully understood. The most widely studied 4F peptide reduces atherosclerosis in animal models via mechanisms related to binding of oxidized lipid (12Navab M. Anantharamaiah G.M. Reddy S.T. Hama S. Hough G. Grijalva V.R. Yu N. Ansell B.J. Datta G. Garber D.W. et al.ApolipoproteinA-I mimetic peptides.Arterioscler. Thromb. Vasc. Biol. 2005; 25: 1325-1331Crossref PubMed Scopus (225) Google Scholar, 15Navab M. Anantharamaiah G.M. Hama S. Garber D.W. Chaddha M. Hough G. Lallone R. Fogelman A.M. 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Efflux of cellular cholesterol and phospholipids to lipid-free apolipoproteins and class A amphipathic peptides.Biochemistry. 1995; 34: 7955-7965Crossref PubMed Scopus (192) Google Scholar, 18Remaley A.T. Thomas F. Stonik J.A. Demosky S.J. Bark S.E. Neufeld E.B. Bocharoc A.V. Vishnyakova T.G. Patterson A.P. Eggerman T.L. et al.Synthetic amphipathic helical peptides promote lipid efflux from cells by an ABCA1-dependent and an ABCA1-independent pathway.J. Lipid Res. 2003; 44: 828-836Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 19Sethi A.A. Stonik J.A. Thomas F. Demosky S.J. Amar M. Neufeld E. Brewer H.B. Davidson W.S. D'Souza W. Sviridov D. et al.Asymmetry in the lipid affinity of bi-helical amphipathic peptides: a structural determinant for the specificity of ABCA1-dependent cholesterol efflux by peptides.J. Biol. Chem. 2008; 283: 32273-32282Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 20Tang C. Vaughan A.M. Anantharamaiah G.M. Oram J.F. Janus kinas 2 modulates the lipid-removing but not protein-stabilizing interactions of amphipathic helices with ABCA1.J. Lipid Res. 2006; 47: 107-114Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Other peptides that have been described target the macrophage cholesteryl ester cycle and LCAT activation to modulate HDL cholesterol efflux and RCT (21Tam S.P. Ancsin J.B. Tan R. Kisilevsky R. Peptides derived from serum amyloid A prevent, and reverse, aortic lipid lesion in apoE−/− mice.J. Lipid Res. 2005; 46: 2091-2101Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 22Datta G. Chaddha M. Hama S. Navab M. Fogelman A.M. Garber D.W. Mishra V.K. Epand R.M. Epand R.F. Lund-Katz S. et al.Effects of increasing hydrophobicity on the physical-chemical and biological properties of a class A amphipathic helical peptide.J. Lipid Res. 2001; 42: 1096-1104Abstract Full Text Full Text PDF PubMed Google Scholar, 23Anantharamaiah G.M. Mishra V.K. Garber D.W. Datta G. 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Of particular interest is stimulating cholesterol efflux via the membrane ABCA1, which enhances RCT and protects against macrophage foam-cell formation and atherosclerosis (24Wang X. Collins H.L. Ranalletta M. Fuki I.V. Billheimer J.T. Rothblat G.H. Tall A.R. Rader D.J. Macrophage ABCA1 and ABCG1, but not SR-B1, promote macrophage reverse cholesterol transport in vivo.J. Clin. Invest. 2007; 117: 2216-2224Crossref PubMed Scopus (460) Google Scholar, 25Singaraja R.R. Fievet C. Castro G. James E.R. Hennuyer N. Clee S.M. Bissada N. Choy J.C. Fruchart J.C. McManus B.M. et al.Increased ABCA1 activity protects against atherosclerosis.J. Clin. Invest. 2002; 110: 35-42Crossref PubMed Scopus (247) Google Scholar). Many apolipoproteins stimulate ABCA1 cholesterol efflux, suggesting common determinants may govern the efflux process (26Remaley A.T. Stonik J.A. Demosky S.J. Neufeld E.B. Bocharov A.V. Vishnyakova T.G. Eggerman T.L. Patterson A.P. Duverger N.J. Santamarina-Fojo S. et al.Apolipoprotein specificity for lipid efflux by the human ABCA1 transporter.Biochem. Biophys. Res. Commun. 2001; 280: 818-823Crossref PubMed Scopus (275) Google Scholar). The nature of these determinants is not known with certainty, but class A amphipathic α-helices are thought to be involved (17Yancey P.G. Bielicki J.K. Johnson W.J. Lund-Katz S. Palgunachari M.N. Anantharamaiah G.M. Segrest J.P. Phillips M.C. Rothblat G.H. Efflux of cellular cholesterol and phospholipids to lipid-free apolipoproteins and class A amphipathic peptides.Biochemistry. 1995; 34: 7955-7965Crossref PubMed Scopus (192) Google Scholar, 27Natarajan P. Forte T.M. Chu B. Phillips M.C. Oram J.F. Bielicki J.K. Identification of an apolipoproteinA-I structural element that mediates cellular cholesterol efflux and stabilizes ATP binding cassette transporter A1.J. Biol. Chem. 2004; 279: 24044-24052Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 28Vedhachalam C. Narayanaswami V. Neto N. Forte T.M. Phillips M.C. Lund-Katz S. Bielicki J.K. The C-terminal lipid-binding domain of apolipoproteinE is a highly efficient mediator of ABCA1-dependent cholesterol efflux that promotes the assembly of high density lipoproteins.Biochemistry. 2007; 46: 2583-2593Crossref PubMed Scopus (91) Google Scholar). Apolipoproteins are largely composed of various types of amphipathic α-helices having polar and nonpolar surfaces (29Segrest J.P. Jones M.K. De Loof H. Brouillette C.G. Venkatachalapathi Y.V. Anantharamaiah G.M. The amphipathic helix in the exchangeable apolipoproteins: a review of secondary structure and function.J. Lipid Res. 1992; 33: 141-165Abstract Full Text PDF PubMed Google Scholar). It is generally believed that two (or more) of these α-helices linked via proline are required to mediate cholesterol efflux effectively (17Yancey P.G. Bielicki J.K. Johnson W.J. Lund-Katz S. Palgunachari M.N. Anantharamaiah G.M. Segrest J.P. Phillips M.C. Rothblat G.H. Efflux of cellular cholesterol and phospholipids to lipid-free apolipoproteins and class A amphipathic peptides.Biochemistry. 1995; 34: 7955-7965Crossref PubMed Scopus (192) Google Scholar, 28Vedhachalam C. Narayanaswami V. Neto N. Forte T.M. Phillips M.C. Lund-Katz S. Bielicki J.K. The C-terminal lipid-binding domain of apolipoproteinE is a highly efficient mediator of ABCA1-dependent cholesterol efflux that promotes the assembly of high density lipoproteins.Biochemistry. 2007; 46: 2583-2593Crossref PubMed Scopus (91) Google Scholar, 30Wool G.D. Reardon C.A. Getz G.S. Apolipoprotein A-I mimetic peptide helix number and helix linker influence potentially anti-atherogenic properties.J. Lipid Res. 2008; 49: 1268-1283Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Consequently, there is a deficiency of small peptides for stimulating cholesterol efflux with high potency. Difficulties designing peptides with secondary structure also limits scientific and clinical pursuits (31Walensky L.D. Kung A.L. Escher I. Malia T.J. Barbuto S. Wright R.D. Wagner G. Verdine G.L. Korsmeyer S.J. Activation of apoptosis in vivo by a hydrocarbon-stapled BH3 helix.Science. 2004; 305: 1466-1470Crossref PubMed Scopus (1087) Google Scholar). Here we report the design of a single-helix peptide that stimulates cholesterol efflux with high efficiency. This was achieved by engineering a class A α-helix from a short segment derived from the C-terminal domain of apoE. The synthetic peptide displayed exceptional α-helicity and solubility characteristics, stimulated cholesterol efflux similar to native proteins on a molar basis, and reduced atherosclerosis in hyperlipidemic mice. ATI-5261 was synthesized from L-amino acids and capped with N-terminal acetyl and C-terminal amide groups (Biosynthesis Inc., TX). Lyophilized peptide (∼95% pure) was routinely dissolved to ∼3–4 mg/ml PBS (pH = 7.4), but stock solutions of >140 mg/ml could easily be prepared. The latter represents ∼100- to 1000-fold greater solubility than current HDL mimetic peptides (22Datta G. Chaddha M. Hama S. Navab M. Fogelman A.M. Garber D.W. Mishra V.K. Epand R.M. Epand R.F. Lund-Katz S. et al.Effects of increasing hydrophobicity on the physical-chemical and biological properties of a class A amphipathic helical peptide.J. Lipid Res. 2001; 42: 1096-1104Abstract Full Text Full Text PDF PubMed Google Scholar). Complexes of ATI-5261 and POPC were prepared by cholate dialysis (32Nichols A.V. Gong E.L. Blanche P.J. Forte T.M. Characterization of discoidal complexes of phosphatidylcholine, apolipoproteinA-I, and cholesterol by gradient gel electrophoresis.Biochim. Biophys. Acta. 1983; 750: 353-364Crossref PubMed Scopus (76) Google Scholar). Peptide concentrations were determined by absorbance at 280 nm. Mean hydrophobicity and amphiphilicity were calculated as described (27Natarajan P. Forte T.M. Chu B. Phillips M.C. Oram J.F. Bielicki J.K. Identification of an apolipoproteinA-I structural element that mediates cellular cholesterol efflux and stabilizes ATP binding cassette transporter A1.J. Biol. Chem. 2004; 279: 24044-24052Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Circular dichroism (CD) spectroscopy was carried out on a Jasco 810 spectropolarimeter at 25°C, using lipid-free peptide in 10 mM phosphate buffer (pH = 7.4) and an average of four scans (20 nm/min/scan) (33Choy N. Raussens V. Narayanaswami V. Inter-molecular coiled-coil formation in human apolipoprotein E C-terminal domain.J. Mol. Biol. 2003; 334: 527-539Crossref PubMed Scopus (100) Google Scholar). Cholesterol efflux activities were determined as before (27Natarajan P. Forte T.M. Chu B. Phillips M.C. Oram J.F. Bielicki J.K. Identification of an apolipoproteinA-I structural element that mediates cellular cholesterol efflux and stabilizes ATP binding cassette transporter A1.J. Biol. Chem. 2004; 279: 24044-24052Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 28Vedhachalam C. Narayanaswami V. Neto N. Forte T.M. Phillips M.C. Lund-Katz S. Bielicki J.K. The C-terminal lipid-binding domain of apolipoproteinE is a highly efficient mediator of ABCA1-dependent cholesterol efflux that promotes the assembly of high density lipoproteins.Biochemistry. 2007; 46: 2583-2593Crossref PubMed Scopus (91) Google Scholar). Efflux efficiency (Km) was calculated using the Michaelis-Menten equation (Graph-Pad Prism4) and 4 h data with 0.1 to 10 μg ATI-5261/ml and 1 to 20 μg apoA-I/ml. Activity of lipid-free ATI-5261 to lyse human RBCs was evaluated as described (34Giunta S. Galeazzi R. Valli M.B. Corder E.H. Galeazzi L. Transferrin neutralization of amyloid β 25–35 cytotoxicity.Clin. Chim. Acta. 2004; 350: 129-136Crossref PubMed Scopus (26) Google Scholar). Percent hemolytic activity was calculated from absorbance readings at 540 nm, using maximum values obtained in the presence of 1% triton X-100. ATI-5261 was selected over other peptides because of superior efflux efficiency and high aqueous solubility (27Natarajan P. Forte T.M. Chu B. Phillips M.C. Oram J.F. Bielicki J.K. Identification of an apolipoproteinA-I structural element that mediates cellular cholesterol efflux and stabilizes ATP binding cassette transporter A1.J. Biol. Chem. 2004; 279: 24044-24052Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). For the present studies, plasma half-life of 125I-ATI-5261 in apoE−/− mice and rats (4.2 and 9.5 ± 1.4 h, respectively, n = 4) and uptake into plasma (rats, ∼30%) following intraperitoneal injection indicated the peptide was suitable for further study. All procedures were approved by institutional Animal Welfare Research Committee. Seven- or eight-week-old LDLR−/− and apoE−/− mice (male, C57BL/6) from Jackson Laboratories (Bar Harbor, ME) were housed in a pathogen-free environment (12-h light/dark cycle) and given free access to food and drinking water. Mice were acclimated 1 week on chow diet before receiving high-fat Western diet (HFWD) (Harlan TekLad TD.88137) to promote atherosclerosis. LDLR−/− mice were fed HFWD for 7 weeks, then injected (intraperitoneally) with peptide (30 mg/kg body weight) daily for 6 weeks in the continued presence of HFWD. ApoE−/− mice were fed the HFWD for 18 weeks before injections (intraperitoneal) of ATI-5261 or ATI-5261:POPC complexes for 6 weeks. ApoE−/− mice were fed chow diet during peptide intervention. After anesthesia with isoflurane, mice were euthanized and their hearts and whole aortas were perfused with saline. Hearts were embedded in OCT compound (Tissue-Teck), frozen on dry ice, and stored until sectioning. Serial 10 μm sections (every fifth from the middle of the ventricle until the appearance of the aortic valve and every second section from the appearance to the disappearance of the aortic leaflets) were collected on poly-D-lysine coated slides, stained with Oil Red O and hematoxylin, and counterstained with Fast Green (10Shah P.K. Nilsson J. Kaul S. Fishbein M.C. Ageland H. Hamsten A. Johansson J. Karpe F. Cercek B. Effects of recombinant apolipoproteinA-IMilano on aortic atherosclerosis in apolipoproteinE-deficient mice.Circulation. 1998; 97: 780-785Crossref PubMed Scopus (226) Google Scholar). Quantification of atheromatous lesions were performed by computerized analysis (Image Pro Plus, Version 6.0; Media Cybernetics, Inc.), and expressed as average % lesion area occupied with lipid from six sections/mouse. The descending thoracic- and abdominal-aorta (up to bifurcation of common iliac arteries) were stored overnight in Histochoice fixation, split open longitudinally, and stained with Oil Red O. The percentage of aortic surface covered by atheroma was determined by computer-assisted planimetry (10Shah P.K. Nilsson J. Kaul S. Fishbein M.C. Ageland H. Hamsten A. Johansson J. Karpe F. Cercek B. Effects of recombinant apolipoproteinA-IMilano on aortic atherosclerosis in apolipoproteinE-deficient mice.Circulation. 1998; 97: 780-785Crossref PubMed Scopus (226) Google Scholar). Aspartate- and alanine-aminotransferase activities (AST and ALT, respectively) in plasma were quantified by IDEXX Laboratories, Inc. (Maine) to assess liver toxicity/necrosis. Plasma total-cholesterol levels were determined using commercial kits. For aminotransferase and cholesterol measurements, blood was drawn from the retro-orbital plexus at termination of atherosclerosis studies, i.e., 2 h after final injection of ATI-5261 and ATI-5261:POPC complexes (30 mg/kg dose). Ability of ATI-5261 to stimulate RCT in vivo was assessed as described (4Zhang Y. Zanotti I. Reilly M.P. Glick J.M. Rothblat G.H. Rader D.J. Overexpression of apolipoproteinA-I promotes reverse transport of cholesterol from macrophages to feces in vivo.Circulation. 2003; 108: 661-663Crossref PubMed Scopus (370) Google Scholar). The outcome of this assay was used to identify the peptide injection schedule (daily or alternate days) for atherosclerosis studies; thus RCT was evaluated in atherosclerotic mice (i.e., not exposed to 6 weeks peptide intervention). Data were analyzed by ANOVA and Student's t-test, using unequal variance and two-tail test. Members of our group recently found that the C-terminal (CT) domain of apoE was a potent mediator of ABCA1 cholesterol efflux and that the entire CT domain (aa216-299) was required for activity (28Vedhachalam C. Narayanaswami V. Neto N. Forte T.M. Phillips M.C. Lund-Katz S. Bielicki J.K. The C-terminal lipid-binding domain of apolipoproteinE is a highly efficient mediator of ABCA1-dependent cholesterol efflux that promotes the assembly of high density lipoproteins.Biochemistry. 2007; 46: 2583-2593Crossref PubMed Scopus (91) Google Scholar). This previous study also revealed that relatively hydrophobic segments (aa260-299) containing a class G α-helix conferred efflux efficiency, whereas the early portion (aa216-237) of the first helical segment of the CT domain possessed acidic residues endowing class A structure. Presently, we tested whether these features of hydrophobic character and class A acidic residues could be compressed into a single relatively small α-helix to create a peptide that stimulates cholesterol efflux with high efficiency. A segment corresponding to the central (aa238-266) CT region was used as a template to design the efflux peptide (Fig. 1). The engineered peptide (designated ATI-5261) possessed an α-helical content of ∼70–80% in the absence of lipid, which was ∼2-fold higher than the original aa238-266 segment (Fig. 1). The mean hydrophobicity and hydrophobic moment of ATI-5261 were −0.22 and 0.24, respectively, versus values of −0.34 and 0.14 for the original aa238-266 sequence. Therefore, ATI-5261 displayed greater hydrophobicity and amphiphilicity characteristics compared with the original sequence from which it was designed. The aa238-266 peptide failed to stimulate cholesterol efflux from cAMP treated macrophages (Fig. 2A), consistent with previous results using peptides based on the apoE CT domain (28Vedhachalam C. Narayanaswami V. Neto N. Forte T.M. Phillips M.C. Lund-Katz S. Bielicki J.K. The C-terminal lipid-binding domain of apolipoproteinE is a highly efficient mediator of ABCA1-dependent cholesterol efflux that promotes the assembly of high density lipoproteins.Biochemistry. 2007; 46: 2583-2593Crossref PubMed Scopus (91) Google Scholar). In contrast, ATI-5261 stimulated high levels of cholesterol efflux from macrophages treated with cAMP and low levels of efflux in the absence of ABCA1 induction (Fig. 2A, B). This behavior was similar to native apolipoproteins (Fig. 2C). The peptide stimulated cholesterol efflux at concentrations where apoA-I was largely ineffective (Fig. 2D), reaching maximal efflux at 3 μg peptide/ml (Fig. 2D). As a result, ATI-5261 stimulated cholesterol efflux with a Km molar efficiency approximating apoA-I (Fig. 2D). These Km values were similar to those previously reported for apoA-I, E, and the CT- domain of apoE (28Vedhachalam C. Narayanaswami V. Neto N. Forte T.M. Phillips M.C. Lund-Katz S. Bielicki J.K. The C-terminal lipid-binding domain of apolipoproteinE is a highly efficient mediator of ABCA1-dependent cholesterol efflux that promotes the assembly of high density lipoproteins.Biochemistry. 2007; 46: 2583-2593Crossref PubMed Scopus (91) Google Scholar). Complexes of ATI-5261:POPC (7–8 nm) possessed ∼4-fold greater cholesterol efflux capacity than the lipid-free peptide (Fig. 2E, F). This was associated with an increased Vmax for activity obtained with complexes versus the lipid-free peptide (Fig. 2G). The former was likely attributed to phospholipid and ABCA1-independent mechanisms. However, cholesterol efflux to complexes increased substantially with cAMP treatment of cells, suggesting a portion of efflux was attributed to ABCA1. This was verified using ABCA1 expressing HeLa cells (16 ± 0.4 vs. 22 ± 3% efflux/24 h from nonexpressing and ABCA1-expressing cells, respectively, 50 μg complexes/ml, n = 3). The ABCA1 component of efflux was not attributed to contamination of the complexes with lipid-free peptide (Fig. 2E, right). Therefore, ATI-5261 formulated with POPC was not prevented from mediating ABCA1 efflux, as expected for apoA-I on HDL (36Wang N. Silver D.L. Costet P. Tall A.R. Specific binding of apoA-I, enhanced cholesterol efflux, and altered plasma membrane morphology in cells expressing ABC1.J. Biol. Chem. 2000; 275: 33053-33058Abstract Full Text Full Text PDF PubMed Scopus (496) Google Scholar). The peptide-POPC formulation, however, stimulated cholesterol efflux with lower efficiency versus lipid-free ATI-5261, as judged by a higher Km for activity (Fig. 2G). Daily intraperitoneal treatments of atherosclerotic LDLR−/− mice with lipid-free ATI-5261 for 6 weeks reduced atherosclerosis by ∼30% (Fig. 3). Total-cholesterol concentrations in plasma were similar in control and peptide treated mice at termination (2552 ± 586 and 2809 ± 436 mg/dl, respectively), indicating that ATI-5261 reduced substantial atherosclerosis in the presence of severe and persistent hypercholesterolemia. ATI-5261 stimulated macrophage RCT in apoE−/− mice over 24–48 h, as judged by an increase in fecal [3H]sterol secretion (Fig. 4A). The latter suggested ATI-5261 may reduce atherosclerosis if provided at 48 h intervals, as opposed to daily injections. This was tested by treating apoE−/− mice with either daily ip injections of ATI-5261 (15 mg/kg) or injections every other day (30 mg/kg) for 6 weeks. Both protocols significantly reduced plaque lesions (Fig. 4B), producing 20 and 47% reductions in atherosclerosis with daily and alternative-day injection schedules, respectively. The latter was verified in a second apoE−/− mouse study (18 weeks HFWD), where 10 mg/kg injected ip on alternate days (6 weeks) reduced the lipid content of aortic -sinus plaque (26 ± 5 vs. 36 ± 4 control, respectively, n = 8/group, P = 0.00088) in continued presence of HFWD. ATI-5261:POPC complexes administered on alternate days also reduced (∼40–45%) atherosclerosis in apoE−/− mice (Fig. 5). This effect was similar to the lipid-free peptide. The complex
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