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• W1995304881 abstract "We have observed that molecular constructs based on multiple apoA-I mimetic peptides attached to a branched scaffold display promising anti-atherosclerosis functions in vitro. Building on these promising results, we now describe chronic in vivo studies to assess anti-atherosclerotic efficacy of HDL-like nanoparticles assembled from a trimeric construct, administered over 10 weeks either ip or orally to LDL receptor-null mice. When dosed ip, the trimer-based nanolipids markedly reduced plasma LDL-cholesterol levels by 40%, unlike many other apoA-I mimetic peptides, and were substantially atheroprotective. Surprisingly, these nanoparticles were also effective when administered orally at a dose of 75 mg/kg, despite the peptide construct being composed of l-amino acids and being undetectable in the plasma. The orally administered nanoparticles reduced whole aorta lesion areas by 55% and aortic sinus lesion volumes by 71%. Reductions in plasma cholesterol were due to the loss of non-HDL lipoproteins, while plasma HDL-cholesterol levels were increased. At a 10-fold lower oral dose, the nanoparticles were marginally effective in reducing atherosclerotic lesions. Intriguingly, analogous results were obtained with nanolipids of the corresponding monomeric peptide. These nanolipid formulations provide an avenue for developing orally efficacious therapeutic agents to manage atherosclerosis. We have observed that molecular constructs based on multiple apoA-I mimetic peptides attached to a branched scaffold display promising anti-atherosclerosis functions in vitro. Building on these promising results, we now describe chronic in vivo studies to assess anti-atherosclerotic efficacy of HDL-like nanoparticles assembled from a trimeric construct, administered over 10 weeks either ip or orally to LDL receptor-null mice. When dosed ip, the trimer-based nanolipids markedly reduced plasma LDL-cholesterol levels by 40%, unlike many other apoA-I mimetic peptides, and were substantially atheroprotective. Surprisingly, these nanoparticles were also effective when administered orally at a dose of 75 mg/kg, despite the peptide construct being composed of l-amino acids and being undetectable in the plasma. The orally administered nanoparticles reduced whole aorta lesion areas by 55% and aortic sinus lesion volumes by 71%. Reductions in plasma cholesterol were due to the loss of non-HDL lipoproteins, while plasma HDL-cholesterol levels were increased. At a 10-fold lower oral dose, the nanoparticles were marginally effective in reducing atherosclerotic lesions. Intriguingly, analogous results were obtained with nanolipids of the corresponding monomeric peptide. These nanolipid formulations provide an avenue for developing orally efficacious therapeutic agents to manage atherosclerosis. Despite the widespread use of lipid-lowering drugs and changes in lifestyle, cardiovascular disease remains the major cause of death in developed countries. As a consequence, there has been intense interest in new therapeutic approaches, such as those that increase plasma HDL levels and/or improve the function of HDL (1Spillmann F. Schultheiss H-P. Tschoepe C. Van Linthout S. High-density lipoprotein-raising strategies: update 2010.Curr. Pharm. Des. 2010; 16: 1517-1530Crossref PubMed Scopus (14) Google Scholar, 2Murphy A.J. Remaley A.T. Sviridov D. HDL therapy: two kinds of right?.Curr. Pharm. Des. 2010; 16: 4134-4147Crossref PubMed Scopus (11) Google Scholar, 3Tardif J.C. Emerging high-density lipoprotein infusion therapies: fulfilling the promise of epidemiology?.J. Clin. Lipidol. 2010; 4: 399-404Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). HDL is crucial in protecting against the buildup of fatty plaques in the arteries, which is a major contributor to cardiovascular disease (1Spillmann F. Schultheiss H-P. Tschoepe C. Van Linthout S. High-density lipoprotein-raising strategies: update 2010.Curr. Pharm. Des. 2010; 16: 1517-1530Crossref PubMed Scopus (14) Google Scholar, 2Murphy A.J. Remaley A.T. Sviridov D. HDL therapy: two kinds of right?.Curr. Pharm. Des. 2010; 16: 4134-4147Crossref PubMed Scopus (11) Google Scholar, 3Tardif J.C. Emerging high-density lipoprotein infusion therapies: fulfilling the promise of epidemiology?.J. Clin. Lipidol. 2010; 4: 399-404Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). Treatments for atherosclerosis involving iv infusions of reconstituted HDL (rHDL) particles or apoA-I, the major protein component (∼70%) of HDL, have provided compelling evidence for protective effects in animal models (4Patel S. Di Bartolo B.A. Nakhla S. Heather A.K. Mitchell T.W. Jessup W. Celermajer D.S. Barter P.J. Rye K-A. Anti-inflammatory effects of apolipoprotein A-I in the rabbit.Atherosclerosis. 2010; 212: 392-397Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 5Valenta D.T. Bulgrien J.J. Banka C.L. Curtiss L.K. Overexpression of human apoA-I transgene provides long-term atheroprotection in LDL receptor-deficient mice.Atherosclerosis. 2006; 189: 255-263Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 6Shah P.K. Yano J. Reyes O. Chyu K-Y. Kaul S. Bisgaier C.L. Drake S. Cercek B. High-dose recombinant apolipoprotein A-I Milano mobilizes tissue cholesterol and rapidly reduces plaque lipid and macrophage content in apolipoprotein E-deficient mice. Potential implications for acute plaque stabilization.Circulation. 2001; 103: 3047-3050Crossref PubMed Scopus (345) Google Scholar, 7Miyazaki A. Sakuma S. Morikawa W. Takiue T. Miake F. Terano T. Sakai M. Hakamata H. Sakamoto Y. Natio M. et al.Intravenous injection of rabbit apolipoprotein A-I inhibits the progression of atherosclerosis in cholesterol-fed rabbits.Arterioscler. Thromb. Vasc. Biol. 1995; 15: 1882-1888Crossref PubMed Scopus (167) Google Scholar, 8Rubin E.M. Krauss R.M. Spangler E.A. Verstuyft J.G. Clift S.M. Inhibition of early atherogenesis in transgenic mice by human apolipoprotein A-I.Nature. 1991; 353: 265-267Crossref PubMed Scopus (860) Google Scholar), albeit with more mixed results in humans (3Tardif J.C. Emerging high-density lipoprotein infusion therapies: fulfilling the promise of epidemiology?.J. Clin. Lipidol. 2010; 4: 399-404Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 9Smits L.P. Kootte R.S. Stroes E.S. Reversal of atherosclerosis with apolipoprotein A1: Back to basics.Atherosclerosis. 2014; 232: 217-219Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar, 10Tardif J.C. Ballantyne C.M. Barter P. Dasseux J.L. Fayad Z.A. Guertin M.C. Kastelein J.J.P. Keyserling C. Klepp H. Koenig W. et al.Effects of the high-density lipoprotein mimetic agent CER-001 on coronary atherosclerosis in patients with acute coronary syndromes: a randomized trial epub ahead of print..Eur. Heart J. 2014; (doi:10.1093/eurheartj/ehu171.)Crossref Scopus (191) Google Scholar). Indeed, while a promising opportunity exists for medical advances in combatting atherosclerosis through the modulation of HDL, recent findings engendered some controversy about this approach. For example, in clinical trials with niacin or cholesterol ester transfer protein (CETP) inhibitors (dalcetrapib and torcetrapib) there was a lack of cardiovascular benefit, although total plasma HDL levels were increased (11Nicholls S.J. Is niacin ineffective? Or did AIM-HIGH miss its target?.Cleve. Clin. J. Med. 2012; 79: 38-43Crossref PubMed Scopus (36) Google Scholar, 12Joy T.R. Hegele R.A. The failure of torcetrapib: what have we learned?.Br. J. Pharmacol. 2008; 154: 1379-1381Crossref PubMed Scopus (42) Google Scholar, 13Rader D.J. Tall A.R. The not-so-simple HDL story: Is it time to revise the HDL cholesterol hypothesis?.Nat. Med. 2012; 18: 1344-1346Crossref PubMed Scopus (215) Google Scholar). In addition, the viewpoint from epidemiological data that higher total plasma HDL levels lower the risks for cardiovascular disease was undermined by a recent meta-analysis (14Voight B.F. Peloso G.M. Orho-Melander M. Frikke-Schmidt R. Barbalic M. Jensen M.K. Hindy G. Hólm H. Ding E.L. Johnson T. et al.Plasma HDL cholesterol and risk of myocardial infarction: a mendelian randomisation study.Lancet. 2012; 380: 572-580Abstract Full Text Full Text PDF PubMed Scopus (1648) Google Scholar). Thus, a mounting collection of information (15Soran H. Hama S. Yadav R. Durrington P.N. HDL functionality.Curr. Opin. Lipidol. 2012; 23: 353-366Crossref PubMed Scopus (99) Google Scholar, 16Heinecke J.W. A new era for quantifying HDL and cardiovascular risk?.Nat. Med. 2012; 18: 1346-1347Crossref PubMed Scopus (52) Google Scholar, 17Besler C. Luscher T.F. Landmesser U. Molecular mechanisms of vascular effects of high-density lipoprotein: alterations in cardiovascular disease.EMBO Mol. Med. 2012; 4: 251-268Crossref PubMed Scopus (161) Google Scholar) suggests that elevating HDL levels is insufficient in protecting against atherosclerosis. This issue becomes more complex in that one must consider the importance of HDL functional properties (13Rader D.J. Tall A.R. The not-so-simple HDL story: Is it time to revise the HDL cholesterol hypothesis?.Nat. Med. 2012; 18: 1344-1346Crossref PubMed Scopus (215) Google Scholar, 15Soran H. Hama S. Yadav R. Durrington P.N. HDL functionality.Curr. Opin. Lipidol. 2012; 23: 353-366Crossref PubMed Scopus (99) Google Scholar, 16Heinecke J.W. A new era for quantifying HDL and cardiovascular risk?.Nat. Med. 2012; 18: 1346-1347Crossref PubMed Scopus (52) Google Scholar, 17Besler C. Luscher T.F. Landmesser U. Molecular mechanisms of vascular effects of high-density lipoprotein: alterations in cardiovascular disease.EMBO Mol. Med. 2012; 4: 251-268Crossref PubMed Scopus (161) Google Scholar), including specific subtypes of HDL particles or specific HDL functional properties (18Rothblat G.H. Phillips M.C. High-density lipoprotein heterogeneity and function in reverse cholesterol transport.Curr. Opin. Lipidol. 2010; 21: 229-238Crossref PubMed Scopus (270) Google Scholar, 19Camont L. Chapman M.J. Kontush A. Biological activities of HDL subpopulations and their relevance to cardiovascular disease.Trends Mol. Med. 2011; 17: 594-603Abstract Full Text Full Text PDF PubMed Scopus (317) Google Scholar, 20Redondo S. Martinez-Gonzalez J. Urraca C. Tejerina T. Emerging therapeutic strategies to enhance HDL function.Lipids Health Dis. 2011; 10: 175Crossref PubMed Scopus (29) Google Scholar). The use of rHDL or apoA-I as therapeutic agents may be impeded by high cost, complex manufacturing processes, and lack of oral administration. Further, their compositions are not conducive to traditional medicinal chemistry manipulations to optimize HDL-like function. Thus, there has been considerable interest in employing a design approach that involves shorter peptides that mimic apoA-I (21Katsuura G. Shinohara S. Shintaku H. Eigyo M. Matsushita A. Protective effect of CCK-8 and ceruletide on glutamate-induced neuronal cell death in rat neuron cultures: possible involvement of CCK-B receptors.Neurosci. Lett. 1991; 132: 159-162Crossref PubMed Scopus (29) Google Scholar). From numerous studies on apoA-I mimetic peptides based on one or two amphiphilic α-helical segments (22Getz G.S. Wool G.D. Reardon C.A. Biological properties of apolipoprotein A-I mimetic peptides.Curr. Atheroscler. Rep. 2010; 12: 96-104Crossref PubMed Scopus (29) Google Scholar, 23Osei-Hwedieh D.O. Amar M. Sviridov D. Remaley A.T. Apolipoprotein mimetic peptides: mechanisms of action as anti-atherogenic agents.Pharmacol. Ther. 2011; 130: 83-91Crossref PubMed Scopus (54) Google Scholar, 24Van Lenten B.J. Wagner A.C. Anantharamaiah G.M. Navab M. Reddy S.T. Buga G.M. Fogelman A.M. Apolipoprotein A-I mimetic peptides.Curr. Atheroscler. Rep. 2009; 11: 52-57Crossref PubMed Scopus (76) Google Scholar, 25Gordon S.M. Davidson W.S. Apolipoprotein A-I mimetics and high-density lipoprotein function.Curr. Opin. Endocrinol. Diabetes Obes. 2012; 19: 109-114Crossref PubMed Scopus (29) Google Scholar, 26Hovingh G.K. Bochem A.E. Kastelein J.J. Apolipoprotein A-I mimetic peptides.Curr. Opin. Lipidol. 2010; 21: 481-486Crossref PubMed Scopus (23) Google Scholar), a wide range of peptide sequences have been found that are more-or-less effective in various aspects of apoA-I mimicry. Many of these peptides have no sequence homology to apoA-I, as they capitalize on an amphiphilic α-helical structural motif, and some are composed of all d-amino acids (21Katsuura G. Shinohara S. Shintaku H. Eigyo M. Matsushita A. Protective effect of CCK-8 and ceruletide on glutamate-induced neuronal cell death in rat neuron cultures: possible involvement of CCK-B receptors.Neurosci. Lett. 1991; 132: 159-162Crossref PubMed Scopus (29) Google Scholar). Although in vivo efficacy has been demonstrated for certain peptides in animal models of atherosclerosis, their mechanisms of action remain the subject of active research (27Joy T.R. Novel HDL-based therapeutic agents.Pharmacol. Ther. 2012; 135: 18-30Crossref PubMed Scopus (24) Google Scholar, 28D'Souza W. Stonik J.A. Murphy A. Demosky S.J. Sethi A.A. Moore X.L. Chin-Dusting J. Remaley A.T. Sviridov D. Structure/function relationships of apolipoprotein A-I mimetic peptides: implications for antiatherogenic activities of high-density lipoprotein.Circ. Res. 2010; 107: 217-227Crossref PubMed Scopus (66) Google Scholar, 29Wool G.D. Vaisar T. Reardon C.A. Getz G.S. An apoA-I mimetic peptide containing a proline residue has greater in vivo HDL binding and anti-inflammatory ability than the 4F peptide.J. Lipid Res. 2009; 50: 1889-1900Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 30Lu S.C. Atangan L. Won Kim K. Chen M.M. Komorowski R. Chu C. Han J. Hu S. Gu W. Veniant M. et al.An apoA-I mimetic peptibody generates HDL-like particles and increases alpha-1 HDL subfraction in mice.J. Lipid Res. 2012; 53: 643-652Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar, 31Zhao Y. Imura T. Leman L.J. Curtiss L.K. Maryanoff B.E. Ghadiri M.R. Mimicry of high-density lipoprotein: functional peptide-lipid nanoparticles based on multivalent peptide constructs.J. Am. Chem. Soc. 2013; 135: 13414-13424Crossref PubMed Scopus (60) Google Scholar, 32Ou J. Wang J. Xu H. Ou Z. Sorci-Thomas M.G. Jones D.W. Signorino P. Densmore J.C. Kaul S. Oldham K.T. et al.Effects of D-4F on vasodilation and vessel wall thickness in hypercholesterolemic LDL receptor-null and LDL receptor/apolipoprotein A-I double-knockout mice on Western diet.Circ. Res. 2005; 97: 1190-1197Crossref PubMed Scopus (108) Google Scholar, 33Navab M. Anantharamaiah G.M. Fogelman A.M. An apolipoprotein A-I mimetic works best in the presence of apolipoprotein A-I.Circ. Res. 2005; 97: 1085-1086Crossref PubMed Scopus (14) Google Scholar). A leading hypothesis is that the peptides stimulate reverse cholesterol transport (RCT), in a manner similar to apoA-I, by promoting cholesterol efflux from macrophage cells. Available evidence suggests that the peptides bind to lipoproteins (especially HDLs) in vivo (29Wool G.D. Vaisar T. Reardon C.A. Getz G.S. An apoA-I mimetic peptide containing a proline residue has greater in vivo HDL binding and anti-inflammatory ability than the 4F peptide.J. Lipid Res. 2009; 50: 1889-1900Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 30Lu S.C. Atangan L. Won Kim K. Chen M.M. Komorowski R. Chu C. Han J. Hu S. Gu W. Veniant M. et al.An apoA-I mimetic peptibody generates HDL-like particles and increases alpha-1 HDL subfraction in mice.J. Lipid Res. 2012; 53: 643-652Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar, 31Zhao Y. Imura T. Leman L.J. Curtiss L.K. Maryanoff B.E. Ghadiri M.R. Mimicry of high-density lipoprotein: functional peptide-lipid nanoparticles based on multivalent peptide constructs.J. Am. Chem. Soc. 2013; 135: 13414-13424Crossref PubMed Scopus (60) Google Scholar), and work in concert with endogenous apoA-I to improve the function of HDL (32Ou J. Wang J. Xu H. Ou Z. Sorci-Thomas M.G. Jones D.W. Signorino P. Densmore J.C. Kaul S. Oldham K.T. et al.Effects of D-4F on vasodilation and vessel wall thickness in hypercholesterolemic LDL receptor-null and LDL receptor/apolipoprotein A-I double-knockout mice on Western diet.Circ. Res. 2005; 97: 1190-1197Crossref PubMed Scopus (108) Google Scholar, 33Navab M. Anantharamaiah G.M. Fogelman A.M. An apolipoprotein A-I mimetic works best in the presence of apolipoprotein A-I.Circ. Res. 2005; 97: 1085-1086Crossref PubMed Scopus (14) Google Scholar). Plasma total cholesterol lowering does not appear to be a general or primary mechanism, because some mimetic peptides that were shown to be atheroprotective in animals did not significantly affect plasma lipid levels (34Navab M. Oral administration of an apo A-I mimetic peptide synthesized from D-amino acids dramatically reduces atherosclerosis in mice independent of plasma cholesterol.Circulation. 2002; 105: 290-292Crossref PubMed Scopus (369) Google Scholar, 35Garber D.W. Datta G. Chaddha M. Palgunachari M.N. Hama S.Y. Navab M. Fogelman A.M. Segrest J.P. Anantharamaiah G.M. A new synthetic class A amphipathic peptide analogue protects mice from diet-induced atherosclerosis.J. Lipid Res. 2001; 42: 545-552Abstract Full Text Full Text PDF PubMed Google Scholar); further, two mimetic peptides with similar cholesterol-reducing properties had different atheroprotective effects (36Handattu S.P. Nayyar G. Garber D.W. Palgunachari M.N. Monroe C.E. Keenum T.D. Mishra V.K. Datta G. Anantharamaiah G.M. Two apolipoprotein E mimetic peptides with similar cholesterol reducing properties exhibit differential atheroprotective effects in LDL-R null mice.Atherosclerosis. 2013; 227: 58-64Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Other proposed mechanisms for these peptides include anti-inflammatory and anti-oxidant effects, as well as the binding of oxidized or pro-atherogenic lipids, such as lysophosphatidic acid (LPA), in the plasma or intestine (37Navab M. Reddy S.T. Van Lenten B.J. Buga G.M. Hough G. Wagner A.C. Fogelman A.M. High-density lipoprotein and 4F peptide reduce systemic inflammation by modulating intestinal oxidized lipid metabolism: novel hypotheses and review of literature.Arterioscler. Thromb. Vasc. Biol. 2012; 32: 2553-2560Crossref PubMed Scopus (54) Google Scholar, 38Navab M. Reddy S.T. Anantharamaiah G.M. Imaizumi S. Hough G. Hama S. Fogelman A.M. Intestine may be a major site of action for the apoA-I mimetic peptide 4F whether administered subcutaneously or orally.J. Lipid Res. 2011; 52: 1200-1210Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 39Van Lenten B.J. Wagner A.C. Jung C-L. Ruchala P. Waring A.J. Lehrer R.I. Watson A.D. Hama S. Navab M. Anantharamaiah G.M. et al.Anti-inflammatory apoA-I-mimetic peptides bind oxidized lipids with much higher affinity than human apoA-I.J. Lipid Res. 2008; 49: 2302-2311Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). We recently reported on the development of branched multivalent apoA-I mimetic constructs, in which multiple copies of a modified α-helical segment derived from apoA-I were appended to a scaffold (31Zhao Y. Imura T. Leman L.J. Curtiss L.K. Maryanoff B.E. Ghadiri M.R. Mimicry of high-density lipoprotein: functional peptide-lipid nanoparticles based on multivalent peptide constructs.J. Am. Chem. Soc. 2013; 135: 13414-13424Crossref PubMed Scopus (60) Google Scholar). In spite of their abiotic structures and distinctly different molecular topology from the linear arrangement of helical segments in the native protein, these branched, multivalent constructs bound lipids to generate HDL-like nanoparticles, exchanged into native HDLs in human plasma, promoted cellular cholesterol efflux, and induced remodeling of large mature HDLs to smaller lipid-poor (pre-β) particles. The lipid nanoparticles derived from the multivalent constructs were superior to those from either the corresponding monomeric parent peptide or a monomeric 4F reference peptide in all functional aspects tested (31Zhao Y. Imura T. Leman L.J. Curtiss L.K. Maryanoff B.E. Ghadiri M.R. Mimicry of high-density lipoprotein: functional peptide-lipid nanoparticles based on multivalent peptide constructs.J. Am. Chem. Soc. 2013; 135: 13414-13424Crossref PubMed Scopus (60) Google Scholar). The multivalent peptides showed impressive stability toward proteolytic digestion and had long plasma half-lives in mice. As such, we hypothesized that our multivalent apoA-I mimetics would exhibit improved atheroprotection compared with the parent monomeric peptide. In this report, we describe results of chronic in vivo efficacy studies in LDL receptor-null (LDLr−/−) mice, a standard mouse model of atherosclerosis, for nanoparticle formulations of the trimeric peptide construct and the parent monomeric peptide. Administration of a nanolipid formulation of the trimeric construct, either ip or po, reduced plasma cholesterol and atherosclerotic lesions in the mice to a degree comparable with the most effective apoA-I mimetics previously reported (and administered parenterally) (21Katsuura G. Shinohara S. Shintaku H. Eigyo M. Matsushita A. Protective effect of CCK-8 and ceruletide on glutamate-induced neuronal cell death in rat neuron cultures: possible involvement of CCK-B receptors.Neurosci. Lett. 1991; 132: 159-162Crossref PubMed Scopus (29) Google Scholar). Oral efficacy was observed in spite of the peptide segments being comprised of l-amino acids, which would make them vulnerable to rapid proteolytic degradation. This nanolipid material, containing a multivalent peptide, provides a new approach for the development of orally efficacious agents to manage atherosclerosis. All peptide native-ligation reactions involved a 1.5-fold excess of purified peptide relative to the number of thioesters in the scaffold. Ligations were performed in 200 mM MOPS buffer containing 7 M guanidine hydrochloride (Gdn·HCl), 100 mM tris(2-carboxyethyl)phosphine (TCEP), pH 7.5, at room temperature for 6–12 h. At completion of the reaction, iodoacetamide (∼50-fold excess relative to Cys) was added to cap the free thiol moieties on Cys residues. After 5 min, CF3CO2H was added to quench the reaction, and the product was purified by reverse-phase HPLC. For detailed synthetic procedures for the peptide constructs, see our prior report (31Zhao Y. Imura T. Leman L.J. Curtiss L.K. Maryanoff B.E. Ghadiri M.R. Mimicry of high-density lipoprotein: functional peptide-lipid nanoparticles based on multivalent peptide constructs.J. Am. Chem. Soc. 2013; 135: 13414-13424Crossref PubMed Scopus (60) Google Scholar). (R)-(+)-1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) was dissolved in 0.5 ml of CHCl3 in a test tube and dried into a thin film by blowing N2 gas into the tube while vortexing. The lipid film was further dried under reduced pressure overnight in a desiccator. Multilamellar vesicles (MLVs) were obtained by suspending the dried lipids into PBS [10 mM phosphate (pH 7.4), 136 mM NaCl] with vortexing and/or sonication. MLVs were typically prepared at concentrations of 10 mM. To prepare peptide-lipid nanoparticles, a stock solution of peptide in PBS was added to 10 mM MLVs at a 1:10 (helix:lipid) molar ratio, and the solutions were vigorously stirred for 24 h at 22°C. The DMPC unilamellar vesicles (ULVs) (18 mM) were made by extrusion DMPC MLVs through 0.2 μm Nucleopore track-etched membranes (Whatman) in the Avanti mini extruder, and sterile filtered before ip administration to mice. All procedures involving live animals were approved by the Scripps Research Institute Institutional Animal Care and Use Committee. LDLr−/− mice were fed a chow diet until they were 10 weeks old, when they were switched to a high-fat diet (HFD) (Harlan Teklad 94059). At the time that the HFD was started, peptide/DMPC nanoparticles were administered by daily ip injection or by the oral route ad libitum in the drinking water for 10 weeks in the continued presence of the HFD. Mice receiving ip injections of PBS or DMPC ULVs served as controls for the ip groups. Mice receiving drinking water containing 1% sucrose/PBS or DMPC MLVs served as controls for the oral groups. The mice were bled after an overnight fast (∼15 h) after 2 weeks of treatment and at the time of harvest (10 weeks); the plasma was used to determine lipoprotein profiles, biomarkers including total cholesterol levels, triglycerides, plasma serum amyloid A (SAA), plasma 15(S)-HETE levels, and plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) concentrations. Atherosclerosis in the aorta from the proximal ascending aorta to the bifurcation of the iliac artery was assessed as described previously (40Mullick A.E. Tobias P.S. Curtiss L.K. Modulation of atherosclerosis in mice by Toll-like receptor 2.J. Clin. Invest. 2005; 115: 3149-3156Crossref PubMed Scopus (464) Google Scholar). Briefly, the dissected aorta was pinned flat on black wax, stained with Sudan IV, and digitally photographed at a fixed magnification. Total aortic areas and atherosclerotic lesion areas were calculated by using Adobe Photoshop CS4, Chromatica V, and National Institutes of Health Scion Image software. Results are reported as lesion area as a percentage of total en face aortic area. As a second assessment of atherosclerosis, lesions of the aortic root (heart sinus) were analyzed. Briefly, hearts were fixed, frozen, and sectioned on a Leica cryostat. For each aortic sinus cusp, sections were collected from the beginning of the sinus for a distance of 500 μm into the sinus. Sections (10 μm thick) were stained with oil red O and counterstained with Gill hematoxylin 1 (Fisher). Stained sections were photographed and digitized. Lesion volume in the first 500 μm of each cusp was estimated from four sections spaced at 140 μm. Lesion volume was calculated from an integration of the measured cross-sectional areas. Female LDLr−/− mice were administered peptide/DMPC for 10 weeks as described above. On weeks 2 and 6, the monomer and trimer concentration in the plasma of randomly selected mice was measured 2 h and 4 h after ip administration, respectively, by LC-MS in the selected ion monitoring (SIM) mode. These times correspond to the respective tmax values for the peptide agents, as determined previously (31Zhao Y. Imura T. Leman L.J. Curtiss L.K. Maryanoff B.E. Ghadiri M.R. Mimicry of high-density lipoprotein: functional peptide-lipid nanoparticles based on multivalent peptide constructs.J. Am. Chem. Soc. 2013; 135: 13414-13424Crossref PubMed Scopus (60) Google Scholar). Details of LC-MS SIM quantification methods are described in our previously published study (31Zhao Y. Imura T. Leman L.J. Curtiss L.K. Maryanoff B.E. Ghadiri M.R. Mimicry of high-density lipoprotein: functional peptide-lipid nanoparticles based on multivalent peptide constructs.J. Am. Chem. Soc. 2013; 135: 13414-13424Crossref PubMed Scopus (60) Google Scholar). Plasma total cholesterol and triglyceride were determined using a cholesterol Amplex assay kit (Invitrogen) and a triglyceride quantification kit (BioVision), respectively. Plasma SAA and 15-HETE levels were measured by using a SAA Mouse ELISA kit (Life Technologies) and a 15(S)-HETE EIA kit (Cayman), respectively, following the manufacturer's instructions. At the time of harvest (10 weeks), blood (∼0.5 ml) was collected by cardiac puncture into EDTA anti-coagulant-coated tubes, and centrifuged at 4°C for 10 min at 5,000 rpm. Plasma samples were stored at −80°C until analysis was conducted. Plasma ALT and AST concentrations were measured using Infinity ALT (GPT) and AST (GOT) liquid stable reagents, respectively, which is a colorimetric kinetic assay (Thermo Scientific). Assays were performed in accordance with the manufacturer's recommendations, adjusting the reagent volumes (20 μl plasma + 200 μl reagent, 0.69 cm light pathlength of the solution in the well) for analysis of samples in 96-well flat bottom microplate format. Lipid extracts of liver tissue were assayed for cholesterol and triglyceride according to the manufacturer's protocols using the cholesterol Amplex assay kit (Invitrogen) and the triglyceride quantification kit (BioVision), respectively. Briefly, liver tissue was homogenized in 5% NP-40 in water (1:15 w/v). Samples were slowly heated to 80°C for 10 min. Insoluble materials were removed by centrifugation (13,000 rpm, 10 min). Cholesterol and triglyceride concentrations in the supernatant were determined by the enzyme based fluorometric assays. Peptides and peptide-DMPC nanoparticles were tested for interference on cholesterol solubility in artificially prepared micelles. Peptide-DMPC nanoparticles were prepared as described above, and purified by size-exclusion chromatography (SEC) using disposable Sephadex G-25 columns (illustra NAP-25 columns, GE Healthcare). Solutions of peptides and purified peptide-DMPC nanoparticles were further concentrated using Amicon centrifugal filters (Millipore) with 3 K and 10 K molecular weight cut-offs, respectively. Neomycin and cholestyramine were used as positive control compounds, as these compounds are known to inhibit intestinal cholesterol absorption by precipitating micellar lipids and sequestrating bile acids, respectively (41Thompson G.R. MacMahon M. Claes P. Precipitation by neomycin compounds of fatty acid and cholesterol from mixed micellar solutions.Eur. J. Clin. Invest. 1970; 1: 40-47Crossref PubMed Scopus (29) Google Scholar, 42Miettinen T.A. Effects of neomycin alone and in combination with cholestyramine on serum methyl sterols and conversion of acetate and mevalonate to cholesterol.Scand. J. Clin. Lab. Invest. 1982; 42: 189-196Crossref PubMed Google Scholar, 43Bergen S.S. Van Itallie T.B. Tennent D.M. Sebrell W.H. Effect of an anion exchange resin on serum cholesterol in man.Proc. Soc. Exp. Biol. Med. 1959; 102: 676-679Crossref" @default.
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• W1995304881 date "2014-10-01" @default.
• W1995304881 modified "2023-10-14" @default.
• W1995304881 title "In vivo efficacy of HDL-like nanolipid particles containing multivalent peptide mimetics of apolipoprotein A-I" @default.
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