A monoclonal antibody to assess oxidized cholesteryl esters associated with apoAI and apoB-100 lipoproteins in human plasma

Atherosclerosis is associated with increased lipid peroxidation, leading to generation of multiple oxidation-specific epitopes (OSEs), contributing to the pathogenesis of atherosclerosis and its clinical manifestation. Oxidized cholesteryl esters (OxCEs) are a major class of OSEs found in human plasma and atherosclerotic tissue. To evaluate OxCEs as a candidate biomarker, we generated a novel mouse monoclonal Ab (mAb) specific to an OxCE modification of proteins. The mAb AG23 (IgG1) was raised in C57BL6 mice immunized with OxCE-modified keyhole limpet hemocyanin, and hybridomas were screened against OxCE-modified BSA. This method ensures mAb specificity to the OxCE modification, independent of a carrier protein. AG23 specifically stained human carotid artery atherosclerotic lesions. An ELISA method, with AG23 as a capture and either anti-apoAI or anti-apoB-100 as the detection Abs, was developed to assay apoAI and apoB-100 lipoproteins that have one or more OxCE epitopes. OxCE-apoA or OxCE-apoB did not correlate with the well-established oxidized phospholipid-apoB biomarker. In a cohort of subjects treated with atorvastatin, OxCE-apoA was significantly lower than in the placebo group, independent of the apoAI levels. These results suggest the potential diagnostic utility of a new biomarker assay to measure OxCE-modified lipoproteins in patients with CVD. Atherosclerosis is associated with increased lipid peroxidation, leading to generation of multiple oxidation-specific epitopes (OSEs), contributing to the pathogenesis of atherosclerosis and its clinical manifestation. Oxidized cholesteryl esters (OxCEs) are a major class of OSEs found in human plasma and atherosclerotic tissue. To evaluate OxCEs as a candidate biomarker, we generated a novel mouse monoclonal Ab (mAb) specific to an OxCE modification of proteins. The mAb AG23 (IgG1) was raised in C57BL6 mice immunized with OxCE-modified keyhole limpet hemocyanin, and hybridomas were screened against OxCE-modified BSA. This method ensures mAb specificity to the OxCE modification, independent of a carrier protein. AG23 specifically stained human carotid artery atherosclerotic lesions. An ELISA method, with AG23 as a capture and either anti-apoAI or anti-apoB-100 as the detection Abs, was developed to assay apoAI and apoB-100 lipoproteins that have one or more OxCE epitopes. OxCE-apoA or OxCE-apoB did not correlate with the well-established oxidized phospholipid-apoB biomarker. In a cohort of subjects treated with atorvastatin, OxCE-apoA was significantly lower than in the placebo group, independent of the apoAI levels. These results suggest the potential diagnostic utility of a new biomarker assay to measure OxCE-modified lipoproteins in patients with CVD. The concept of sterile inflammation induced by host-derived damage-associated molecular patterns (DAMPs) helps to explain the inflammatory mechanisms of many chronic diseases. It is now well-documented that DAMPs share structural motifs with microbial pathogen-associated molecular patterns and activate innate immune pattern-recognition receptors (1Miller Y.I. Choi S-H. Wiesner P. Fang L. Harkewicz R. Hartvigsen K. Boullier A. Gonen A. Diehl C.J. Que X. et al.Oxidation-specific epitopes are danger associated molecular patterns recognized by pattern recognition receptors of innate immunity.Circ. Res. 2011; 108: 235-248Crossref PubMed Scopus (457) Google Scholar). While these responses may provide initial benefits, the ensuing inflammatory responses may be harmful if unconstrained and prolonged, such as occurs in atherosclerosis, a life-long chronic inflammation of medium/large arteries leading to CVD. Oxidized cholesteryl esters (OxCEs) are the DAMPs arising during the development of atherosclerosis (1Miller Y.I. Choi S-H. Wiesner P. Fang L. Harkewicz R. Hartvigsen K. Boullier A. Gonen A. Diehl C.J. Que X. et al.Oxidation-specific epitopes are danger associated molecular patterns recognized by pattern recognition receptors of innate immunity.Circ. Res. 2011; 108: 235-248Crossref PubMed Scopus (457) Google Scholar, 2Choi S-H. Yin H. Ravandi A. Armando A. Dumlao D. Kim J. Almazan F. Taylor A.M. McNamara C.A. Tsimikas S. et al.Polyoxygenated cholesterol ester hydroperoxide activates TLR4 and SYK dependent signaling in macrophages.PLoS One. 2013; 8: e83145Crossref PubMed Scopus (39) Google Scholar, 3Ravandi A. Leibundgut G. Hung M.Y. Patel M. Hutchins P.M. Murphy R.C. Prasad A. Mahmud E. Miller Y.I. Dennis E.A. et al.Release and capture of bioactive oxidized phospholipids and oxidized cholesteryl esters during percutaneous coronary and peripheral arterial interventions in humans.J. Am. Coll. Cardiol. 2014; 63: 1961-1971Crossref PubMed Scopus (80) Google Scholar, 4Choi S.H. Sviridov D. Miller Y.I. Oxidized cholesteryl esters and inflammation.Biochim. Biophys. Acta Mol. Cell Biol. Lipids. 2017; 1862: 393-397Crossref PubMed Scopus (50) Google Scholar). We have demonstrated that cholesterol binds to MD-2 (5Choi S.H. Kim J. Gonen A. Viriyakosol S. Miller Y.I. MD-2 binds cholesterol.Biochem. Biophys. Res. Commun. 2016; 470: 877-880Crossref PubMed Scopus (12) Google Scholar), a ligand-binding coreceptor for the prototypic pattern-recognition receptor, toll-like receptor-4 (TLR4). MD-2 has a hydrophobic pocket and, remarkably, shares a significant structural homology with Niemann-Pick disease type C2, a lysosomal cholesterol-binding protein (6Gruber A. Mancek M. Wagner H. Kirschning C.J. Jerala R. Structural model of MD-2 and functional role of its basic amino acid clusters involved in cellular lipopolysaccharide recognition.J. Biol. Chem. 2004; 279: 28475-28482Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). Further, cholesterol esterified to an oxidized polyunsaturated fatty acyl chain, i.e., OxCE, when bound to TLR4/MD-2, induces TLR4 dimerization and activates inflammatory responses in macrophages (2Choi S-H. Yin H. Ravandi A. Armando A. Dumlao D. Kim J. Almazan F. Taylor A.M. McNamara C.A. Tsimikas S. et al.Polyoxygenated cholesterol ester hydroperoxide activates TLR4 and SYK dependent signaling in macrophages.PLoS One. 2013; 8: e83145Crossref PubMed Scopus (39) Google Scholar). Importantly, specific OxCE molecules first identified in test-tube oxidation reactions are also found in cellular systems, in vascular lesions of experimental animals, and in plasma and atherosclerotic plaques isolated from human CVD patients (3Ravandi A. Leibundgut G. Hung M.Y. Patel M. Hutchins P.M. Murphy R.C. Prasad A. Mahmud E. Miller Y.I. Dennis E.A. et al.Release and capture of bioactive oxidized phospholipids and oxidized cholesteryl esters during percutaneous coronary and peripheral arterial interventions in humans.J. Am. Coll. Cardiol. 2014; 63: 1961-1971Crossref PubMed Scopus (80) Google Scholar, 7Folcik V.A. Cathcart M.K. Predominance of esterified hydroperoxy-linoleic acid in human monocyte-oxidized LDL.J. Lipid Res. 1994; 35: 1570-1582Abstract Full Text PDF PubMed Google Scholar, 8Hoppe G. Ravandi A. Herrera D. Kuksis A. Hoff H.F. Oxidation products of cholesteryl linoleate are resistant to hydrolysis in macrophages, form complexes with proteins, and are present in human atherosclerotic lesions.J. Lipid Res. 1997; 38: 1347-1360Abstract Full Text PDF PubMed Google Scholar, 9Suarna C. Dean R.T. May J. Stocker R. Human atherosclerotic plaque contains both oxidized lipids and relatively large amounts of α-tocopherol and ascorbate.Arterioscler. Thromb. Vasc. Biol. 1995; 15: 1616-1624Crossref PubMed Scopus (312) Google Scholar, 10Upston J.M. Niu X. Brown A.J. Mashima R. Wang H. Senthilmohan R. Kettle A.J. Dean R.T. Stocker R. Disease stage-dependent accumulation of lipid and protein oxidation products in human atherosclerosis.Am. J. Pathol. 2002; 160: 701-710Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 11Hutchins P.M. Moore E.E. Murphy R.C. Electrospray tandem mass spectrometry reveals extensive and non-specific oxidation of cholesterol esters in human peripheral vascular lesions.J. Lipid Res. 2011; 52: 2070-2083Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). For example, cholesteryl arachidonate and cholesteryl linoleate oxidation products are detected in human plasma from CVD patients and in human atherosclerotic lesions (2Choi S-H. Yin H. Ravandi A. Armando A. Dumlao D. Kim J. Almazan F. Taylor A.M. McNamara C.A. Tsimikas S. et al.Polyoxygenated cholesterol ester hydroperoxide activates TLR4 and SYK dependent signaling in macrophages.PLoS One. 2013; 8: e83145Crossref PubMed Scopus (39) Google Scholar, 3Ravandi A. Leibundgut G. Hung M.Y. Patel M. Hutchins P.M. Murphy R.C. Prasad A. Mahmud E. Miller Y.I. Dennis E.A. et al.Release and capture of bioactive oxidized phospholipids and oxidized cholesteryl esters during percutaneous coronary and peripheral arterial interventions in humans.J. Am. Coll. Cardiol. 2014; 63: 1961-1971Crossref PubMed Scopus (80) Google Scholar, 11Hutchins P.M. Moore E.E. Murphy R.C. Electrospray tandem mass spectrometry reveals extensive and non-specific oxidation of cholesterol esters in human peripheral vascular lesions.J. Lipid Res. 2011; 52: 2070-2083Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar), as well as in experimental studies with hypercholesterolemic mice and zebrafish (12Harkewicz R. Hartvigsen K. Almazan F. Dennis E.A. Witztum J.L. Miller Y.I. Cholesteryl ester hydroperoxides are biologically active components of minimally oxidized LDL.J. Biol. Chem. 2008; 283: 10241-10251Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 13Fang L. Harkewicz R. Hartvigsen K. Wiesner P. Choi S.H. Almazan F. Pattison J. Deer E. Sayaphupha T. Dennis E.A. et al.Oxidized cholesteryl esters and phospholipids in zebrafish larvae fed a high-cholesterol diet: macrophage binding and activation.J. Biol. Chem. 2010; 285: 32343-32351Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Human atherosclerotic lesions contain OxCE not only in a free lipid form but also as covalent adducts to proteins (14Kawai Y. Saito A. Shibata N. Kobayashi M. S. T. K. binding of oxidized cholesteryl esters to for modification of and Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). these we suggest that OxCE is a major arising in hypercholesterolemic and human to vascular inflammation and S.H. Sviridov D. Miller Y.I. Oxidized cholesteryl esters and inflammation.Biochim. Biophys. Acta Mol. Cell Biol. Lipids. 2017; 1862: 393-397Crossref PubMed Scopus (50) Google Scholar, Y.I. role of oxidized lipids and lipoproteins in 2017; Full Text Full Text PDF PubMed Scopus Google Scholar). In we detection of OxCE epitopes in plasma may have diagnostic and as a new biomarker. We developed the monoclonal Ab (mAb) AG23 that OxCE covalent adducts of the protein. AG23 specifically stained atherosclerotic lesions in human carotid AG23 was in with apoAI or apoB-100 to measure OxCE associated with or lipoproteins in human This OxCE assay demonstrated in a cohort of subjects treated with atorvastatin, was a significant in apoAI not associated with in apoAI levels. Human for was from to a by the of Human Human were also from the (2Choi S-H. Yin H. Ravandi A. Armando A. Dumlao D. Kim J. Almazan F. Taylor A.M. McNamara C.A. Tsimikas S. et al.Polyoxygenated cholesterol ester hydroperoxide activates TLR4 and SYK dependent signaling in macrophages.PLoS One. 2013; 8: e83145Crossref PubMed Scopus (39) Google and the A. Tsimikas S. Witztum J.L. The of and of in hypercholesterolemic humans.J. Am. Coll. Cardiol. 2008; PubMed Scopus Google Scholar). Human carotid were from patients carotid with a by the of Human were to by the and of the of were to and a with and were mice were from and The for OxCE-modified keyhole limpet and were To the cholesteryl arachidonate was oxidized as (2Choi S-H. Yin H. Ravandi A. Armando A. Dumlao D. Kim J. Almazan F. Taylor A.M. McNamara C.A. Tsimikas S. et al.Polyoxygenated cholesterol ester hydroperoxide activates TLR4 and SYK dependent signaling in macrophages.PLoS One. 2013; 8: e83145Crossref PubMed Scopus (39) Google to of OxCE in was in a with of in and for with was to an of oxidized cholesteryl arachidonate in a and the was one more This modification of the with OxCE was to protein with a large of the of the of in were to the and The was a against to and sterile The was and The from was with OxCE a the as for The in the was with as a were as in A. Que X. A. M.Y. Wiesner P. D. M. et with is specific and dependent for development of an Lipid Res. 2014; Full Text Full Text PDF PubMed Scopus Google Scholar). The for was from an in S. Miller E. Witztum J.L. of monoclonal to epitopes of oxidized lipoproteins from of epitopes of oxidized in human PubMed Scopus Google Scholar). mice were immunized with in for a and as in the mice were and were by ELISA to binding to the and as in The mice with the an with the in the mice were and were and with a The were in were to and To but not from were screened by ELISA against an detection Ab were by in and by ELISA for binding to to The was by in in a with with and with The AG23 and were by mice were from and for mice D. of in of the and oxidation of Biol. Chem. Full Text Full Text PDF PubMed Scopus Google were a from and were with were fed a high-cholesterol cholesterol for cholesterol was Atherosclerosis was as S. A. Hartvigsen K. E. M.Y. Pattison J. M. J. T. et oxidation-specific and atherosclerosis Am. Coll. Cardiol. 2011; PubMed Scopus Google Scholar). atherosclerosis was by from the the were stained with a and was or was to and as S.H. Wiesner P. Almazan F. Kim J. Miller Y.I. dependent to minimally oxidized One. PubMed Scopus Google Scholar). were and a were with for or to a The were with in by with with for and by detection with a and an mAb binding to an of epitopes was by ELISA as A. Que X. A. M.Y. Wiesner P. D. M. et with is specific and dependent for development of an Lipid Res. 2014; Full Text Full Text PDF PubMed Scopus Google Scholar). In were with in with or mAb was and for were detected an a and a Ab binding was as detected or with were to in with AG23 was in and for The ELISA was as and of AG23 was in the or of of complexes were by a and were in an ELISA as of was were as as A. Que X. A. M.Y. Wiesner P. D. M. et with is specific and dependent for development of an Lipid Res. 2014; Full Text Full Text PDF PubMed Scopus Google Scholar). An ELISA was with in cholesterol was in and for The was from the and for free cholesterol a from The were and in the AG23 binding assay as was and The were with and with for AG23 was in the and of or of complexes were as and the was to in a were with an with for and with were with for with and of in the of was as a were with with a To OxCE in human ELISA were In were with of a mouse monoclonal specific for human apoB-100 Witztum J.L. S. of the of by a new monoclonal PubMed Google Scholar), or with a apoAI Ab by with for were and for by AG23 detection and In were with with plasma by apoB-100 Ab or apoAI Ab from and In were with AG23 and detected with We assay as in OxCE of the or protein but only if more than one OxCE is as in were or more than as in and the with were mice were immunized with and screened for Ab by binding to were generated from the of the mice with the and screened for binding to was for its binding to but not To the of the was to a and were for binding The was and by as in the and The AG23 Ab stained but not a a stained AG23 binding to oxidation-specific epitopes (OSEs), and oxidized in was but not with for AG23 binding the specificity of the Ab to the OxCE of the carrier protein. To the specificity of AG23 to the OxCE covalent modification of a we the with OxCE, the for the and and a AG23 binding to but not The OxCE modification for the in the was not to in a covalent OxCE and the and AG23 did not to the In a both and with for AG23 binding in a of with cholesterol free hydrolysis of the ester in the OxCE and AG23 binding to the from cholesteryl was This that the cholesterol is of the recognized by AG23 or that was for the of the AG23 to be OxCE products were detected in human atherosclerotic lesions lipid and mass spectrometry (3Ravandi A. Leibundgut G. Hung M.Y. Patel M. Hutchins P.M. Murphy R.C. Prasad A. Mahmud E. Miller Y.I. Dennis E.A. et al.Release and capture of bioactive oxidized phospholipids and oxidized cholesteryl esters during percutaneous coronary and peripheral arterial interventions in humans.J. Am. Coll. Cardiol. 2014; 63: 1961-1971Crossref PubMed Scopus (80) Google Scholar, 8Hoppe G. Ravandi A. Herrera D. Kuksis A. Hoff H.F. Oxidation products of cholesteryl linoleate are resistant to hydrolysis in macrophages, form complexes with proteins, and are present in human atherosclerotic lesions.J. Lipid Res. 1997; 38: 1347-1360Abstract Full Text PDF PubMed Google Scholar, 9Suarna C. Dean R.T. May J. Stocker R. Human atherosclerotic plaque contains both oxidized lipids and relatively large amounts of α-tocopherol and ascorbate.Arterioscler. Thromb. Vasc. Biol. 1995; 15: 1616-1624Crossref PubMed Scopus (312) Google Scholar, 10Upston J.M. Niu X. Brown A.J. Mashima R. Wang H. Senthilmohan R. Kettle A.J. Dean R.T. Stocker R. Disease stage-dependent accumulation of lipid and protein oxidation products in human atherosclerosis.Am. J. Pathol. 2002; 160: 701-710Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 11Hutchins P.M. Moore E.E. Murphy R.C. Electrospray tandem mass spectrometry reveals extensive and non-specific oxidation of cholesterol esters in human peripheral vascular lesions.J. Lipid Res. 2011; 52: 2070-2083Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar), we to AG23 OxCE in human lesions AG23 specifically stained of human carotid artery atherosclerotic and with AG23 with the in a These results suggest that AG23 OxCE epitopes arising during the development of human In an we OxCE in plasma from and mice fed a high-cholesterol The has as a major in oxidation in T. modification of and PubMed Scopus Google Scholar, L. in 2004; PubMed Scopus Google Scholar), with a for the J. H. H. The cholesterol and not Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, P.M. Murphy R.C. Cholesteryl ester acyl oxidation and in of oxidized Lipid Res. Full Text Full Text PDF PubMed Scopus Google Scholar). the of high-cholesterol of were not but the of OxCE were significantly lower in the plasma of mice with that of mice The lower plasma of OxCE with of atherosclerotic lesions in mice the in with J. A. A. H. N. T. L. E. D. in PubMed Scopus Google Scholar). We to we OxCE epitopes in human the was from human plasma and AG23 was to OxCE epitopes the detection were for of the the to and of we the and AG23 as a capture Ab and a anti-apoB-100 as the detection Ab In was with a of detected in human plasma in amounts of be well the may have one or more OxCE epitopes. To OxCE associated with apoAI we the AG23 capture but the apoB-100 Ab with a anti-apoAI In we detected for i.e., of apoAI lipoproteins with one or more OxCE epitopes in an assay of capture with AG23 for a detection of OxCE epitopes. In a human the of is the assay was and the apoAI lipoproteins were first the and AG23 the binding of AG23 was the detection in the of We did not OxCE associated with we an assay in AG23 was as a capture Ab and AG23 was for detection This only if lipoproteins or proteins or more OxCE epitopes to The that of binding were detected in only a plasma and were in that the of lipoproteins only one or a OxCE epitopes to For we the results of and in that in these be detection the OxCE-apoB and OxCE-apoA levels. To of and in the cohort (2Choi S-H. Yin H. Ravandi A. Armando A. Dumlao D. Kim J. Almazan F. Taylor A.M. McNamara C.A. Tsimikas S. et al.Polyoxygenated cholesterol ester hydroperoxide activates TLR4 and SYK dependent signaling in macrophages.PLoS One. 2013; 8: e83145Crossref PubMed Scopus (39) Google Scholar), we in OxCE-apoB and OxCE-apoA OxCE-apoA did not correlate with cholesterol was the assay A. Witztum J.L. Tsimikas S. Oxidized phospholipids a biomarker disease and 2011; PubMed Scopus Google and OxCE-apoB or for OxCE-apoB and OxCE-apoA and in in a new we OxCE-apoB and OxCE-apoA in plasma from a in human subjects atorvastatin, atorvastatin, or was and of A. Tsimikas S. Witztum J.L. The of and of in hypercholesterolemic humans.J. Am. Coll. Cardiol. 2008; PubMed Scopus Google Scholar). The was not in was in OxCE-apoB or OxCE-apoA placebo and of OxCE-apoB were significantly in the and with the placebo with the in apoB-100 In to not significantly apoAI levels. and in OxCE-apoA were not by in apoAI Further, we in OxCE for and both OxCE-apoB and apoB-100 in the of subjects in the of apoAI OxCE-apoA was significantly in the group, but not the placebo of OxCE in in demonstrated with and, remarkably, was and OxCE-apoB or OxCE-apoA for OxCE-apoB and OxCE-apoA and in and were by in a new and were by In we have developed the that a covalent modification of proteins and with The Ab specifically human atherosclerotic lesions and detection of OxCE epitopes in human The ELISA assay that we have developed of lipoproteins that have one or more OxCE but not measure the of OxCE results of a with AG23 as both capture and detection but in only a that lipoproteins and components of plasma have more than one OxCE This is in to the multiple human A. Witztum J.L. Tsimikas S. Oxidized phospholipids a biomarker disease and 2011; PubMed Scopus Google Scholar, X. D. R. Witztum J.L. Tsimikas S. of oxidized phospholipids to in patients treated with the Am. Coll. Cardiol. PubMed Scopus (50) Google Scholar, S. P. J. P. M. A. Witztum J.L. S. and and of Am. Coll. Cardiol. PubMed Scopus Google Scholar, X. D. R. Witztum J.L. Tsimikas S. Oxidized phospholipids and or Am. Coll. Cardiol. 2017; PubMed Scopus Google Scholar). is found in and the of and correlate C. A. A. E. Miller C.J. S. et novel of as a carrier of oxidized phospholipids in human Lipid Res. 2008; Full Text Full Text PDF PubMed Scopus Google Scholar). and OxCE is that the of associated with apoAI lipoproteins are K. A. A. Witztum J.L. E. Tsimikas S. of oxidized phospholipids in patients with Lipid Res. Full Text Full Text PDF PubMed Scopus Google Scholar), OxCE was detected in apoAI lipoproteins in the human plasma that we These results suggest that OxCE-apoA be a independent biomarker, the with was OxCE-apoA and or apoAI or levels. OxCE-apoA and OxCE-apoA were independent of the of studies OxCE-apoA OxCE-apoB have Oxidation of cholesteryl arachidonate with a free the in of OxCE, results in of the acyl (2Choi S-H. Yin H. Ravandi A. Armando A. Dumlao D. Kim J. Almazan F. Taylor A.M. McNamara C.A. Tsimikas S. et al.Polyoxygenated cholesterol ester hydroperoxide activates TLR4 and SYK dependent signaling in macrophages.PLoS One. 2013; 8: e83145Crossref PubMed Scopus (39) Google Scholar, R. Hartvigsen K. Almazan F. Dennis E.A. Witztum J.L. Miller Y.I. Cholesteryl ester hydroperoxides are biologically active components of minimally oxidized LDL.J. Biol. 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J. of lipid and in PubMed Scopus Google and, as we in have lower plasma of OxCE epitopes. the of and hydroperoxide products the acyl of was not to of epitopes in during to and but not in in extensive lipid oxidation is by of fatty acyl and OxCE contains (2Choi S-H. Yin H. Ravandi A. Armando A. Dumlao D. Kim J. Almazan F. Taylor A.M. McNamara C.A. Tsimikas S. et al.Polyoxygenated cholesterol ester hydroperoxide activates TLR4 and SYK dependent signaling in macrophages.PLoS One. 2013; 8: e83145Crossref PubMed Scopus (39) Google Scholar), to such as when to protein to form as form covalent adducts with proteins K. E. adducts in oxidized and human a with PubMed Scopus Google Scholar, and N. Y. PubMed Scopus Google Scholar, J.M. L. S. R. in detection and of Biol. PubMed Scopus Google Scholar). the of is in an of the OxCE recognized by We an of cholesteryl arachidonate oxidation with a free to and The that cholesterol AG23 binding to the of cholesteryl its in the OxCE recognized by or that is for the of the AG23 to be more of the is by in the mass spectrometry to proteins and We also not oxidized cholesteryl linoleate covalent modification of a protein be recognized by An Ab to the oxidized cholesteryl has to epitopes in atherosclerotic lesions (14Kawai Y. Saito A. Shibata N. Kobayashi M. S. T. K. binding of oxidized cholesteryl esters to for modification of and Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), and oxidized cholesteryl linoleate is a OxCE found in human lesions (3Ravandi A. Leibundgut G. Hung M.Y. Patel M. Hutchins P.M. Murphy R.C. Prasad A. Mahmud E. Miller Y.I. Dennis E.A. et al.Release and capture of bioactive oxidized phospholipids and oxidized cholesteryl esters during percutaneous coronary and peripheral arterial interventions in humans.J. Am. Coll. Cardiol. 2014; 63: 1961-1971Crossref PubMed Scopus (80) Google Scholar, 11Hutchins P.M. Moore E.E. Murphy R.C. Electrospray tandem mass spectrometry reveals extensive and non-specific oxidation of cholesterol esters in human peripheral vascular lesions.J. Lipid Res. 2011; 52: 2070-2083Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). AG23 is from to as the of were by with cholesterol or Y. Y. M. M. S. M. and as structural for cholesterol 2010; PubMed Scopus Google Scholar). In to a of the cholesterol in or in the lipid from that of of cholesterol was in large by the Y. Y. M. M. S. M. and as structural for cholesterol 2010; PubMed Scopus Google Scholar), in that cholesteryl arachidonate hydroperoxides activate TLR4 and inflammatory responses in macrophages (2Choi S-H. Yin H. Ravandi A. Armando A. Dumlao D. Kim J. Almazan F. Taylor A.M. McNamara C.A. Tsimikas S. et al.Polyoxygenated cholesterol ester hydroperoxide activates TLR4 and SYK dependent signaling in macrophages.PLoS One. 2013; 8: e83145Crossref PubMed Scopus (39) Google to an Ab to biologically active adducts of oxidized cholesteryl arachidonate to proteins. In we developed a new OxCE epitopes and potential new biomarker for OxCE in apoB-100 and apoAI studies CVD and provide in to clinical with damage-associated molecular pattern cholesterol keyhole limpet cholesterol monoclonal Ab oxidation-specific oxidized cholesteryl ester oxidized cholesterol toll-like receptor-4