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The HDL proteome: a marker–and perhaps mediator–of coronary artery disease

2008; Elsevier BV; Volume: 50; Linguagem: Inglês

10.1194/jlr.r800097-jlr200

1539-7262

Jay W. Heinecke,

Lipoproteins and Cardiovascular Health

One important cardioprotective function of HDL is to remove cholesterol from lipid-laden macrophages in the artery wall. HDL also exerts anti-inflammatory effects that might inhibit atherogenesis. However, HDL has been proposed to be dysfunctional in humans with established coronary artery disease (CAD), though the underlying mechanisms are unclear. Therefore, we used mass spectrometry to investigate the roles of HDL proteins in inflammation and cardiovascular disease. Shotgun proteomic analysis identified multiple complement regulatory proteins, protease inhibitors, and acute-phase response proteins in HDL, strongly implicating the lipoprotein in inflammation and the innate immune system. Moreover, mass spectrometry and biochemical analyses demonstrated that HDL3 from subjects with clinically significant CAD was selectively enriched in apolipoprotein E, suggesting that it carries a distinctive protein cargo in humans with atherosclerosis. HDL from CAD subjects also contained markedly elevated levels of chlorotyrosine and nitrotyrosine, two characteristic products of myeloperoxidase, indicating that oxidative damage might generate dysfunctional HDL. Aggressive lipid therapy with a statin and niacin remodeled the HDL proteome to resemble that of apparently healthy subjects. Collectively, our observations indicate that quantifying the HDL proteome by mass spectrometry should help identify novel anti-inflammatory and cardioprotective actions of HDL and provide insights into lipid therapy. One important cardioprotective function of HDL is to remove cholesterol from lipid-laden macrophages in the artery wall. HDL also exerts anti-inflammatory effects that might inhibit atherogenesis. However, HDL has been proposed to be dysfunctional in humans with established coronary artery disease (CAD), though the underlying mechanisms are unclear. Therefore, we used mass spectrometry to investigate the roles of HDL proteins in inflammation and cardiovascular disease. Shotgun proteomic analysis identified multiple complement regulatory proteins, protease inhibitors, and acute-phase response proteins in HDL, strongly implicating the lipoprotein in inflammation and the innate immune system. Moreover, mass spectrometry and biochemical analyses demonstrated that HDL3 from subjects with clinically significant CAD was selectively enriched in apolipoprotein E, suggesting that it carries a distinctive protein cargo in humans with atherosclerosis. HDL from CAD subjects also contained markedly elevated levels of chlorotyrosine and nitrotyrosine, two characteristic products of myeloperoxidase, indicating that oxidative damage might generate dysfunctional HDL. Aggressive lipid therapy with a statin and niacin remodeled the HDL proteome to resemble that of apparently healthy subjects. Collectively, our observations indicate that quantifying the HDL proteome by mass spectrometry should help identify novel anti-inflammatory and cardioprotective actions of HDL and provide insights into lipid therapy. HDL and LDL are the major carriers of cholesterol in human blood (1Rader D.J. Puré E. Lipoproteins, macrophage function, and atherosclerosis: beyond the foam cell?.Cell Metab. 2005; 1: 223-230Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 2Oram J.F. Heinecke J.W. ATP-binding cassette transporter A1: a cell cholesterol exporter protects against cardiovascular disease.Physiol. Rev. 2005; 85: 1343-1372Crossref PubMed Scopus (419) Google Scholar, 3Brown M.S. Goldstein J.L. A receptor-mediated pathway for cholesterol homeostasis.Science. 1986; 232: 34-47Crossref PubMed Scopus (4331) Google Scholar). Epidemiological, genetic, and clinical studies demonstrate that elevated levels of LDL or low levels of HDL are important risk factors for coronary artery disease (CAD). In striking contrast, high levels of HDL are cardioprotective. LDL promotes heart disease by delivering cholesterol to macrophages, a key early event in atherogenesis (1Rader D.J. Puré E. Lipoproteins, macrophage function, and atherosclerosis: beyond the foam cell?.Cell Metab. 2005; 1: 223-230Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 2Oram J.F. Heinecke J.W. ATP-binding cassette transporter A1: a cell cholesterol exporter protects against cardiovascular disease.Physiol. Rev. 2005; 85: 1343-1372Crossref PubMed Scopus (419) Google Scholar, 3Brown M.S. Goldstein J.L. A receptor-mediated pathway for cholesterol homeostasis.Science. 1986; 232: 34-47Crossref PubMed Scopus (4331) Google Scholar). HDL protects against atherosclerosis by removing cholesterol from artery wall macrophages by a process termed reverse cholesterol transport. HDL exhibits other biological activities that may contribute to its anti-atherogenic properties, such as the ability to inhibit inflammation (4Barter P.J. Nicholls S. Rye K.A. Anantharamaiah G.M. Navab M. Fogelman A.M. Antiinflammatory properties of HDL.Circ. Res. 2004; 95: 764-772Crossref PubMed Scopus (1044) Google Scholar). It has been proposed that its cardioprotective effects depend on the types of particles generated in vivo and that HDL in humans with established CAD is dysfunctional (4Barter P.J. Nicholls S. Rye K.A. Anantharamaiah G.M. Navab M. Fogelman A.M. Antiinflammatory properties of HDL.Circ. Res. 2004; 95: 764-772Crossref PubMed Scopus (1044) Google Scholar). Indeed, animal studies convincingly demonstrate that changes in proteins involved in HDL metabolism can promote atherosclerosis, even when plasma levels of HDL-cholesterol are elevated (5Schultz J.R. Verstuyft J.G. Gong E.L. Nichols A.V. Rubin E.M. Protein composition determines the anti-atherogenic properties of HDL in transgenic mice.Nature. 1993; 365: 762-764Crossref PubMed Scopus (246) Google Scholar, 6Trigatti B. Rayburn H. Vinals M. Braun A. Miettinen H. Penman M. Hertz M. Schrenzel M. Amigo L. Rigotti A. al et Influence of the high density lipoprotein receptor SR-BI on reproductive and cardiovascular pathophysiology.Proc. Natl. Acad. Sci. USA. 1999; 96: 9322-9327Crossref PubMed Scopus (437) Google Scholar). Moreover, in vitro studies demonstrate that oxidative damage impairs the ability of apolipoprotein A-I (apoA-I), the major HDL protein, to remove cholesterol from macrophages (7Shao B. Oda M.N. Oram J.F. Heinecke J.W. Myeloperoxidase: an inflammatory enzyme for generating dysfunctional high density lipoprotein.Curr. Opin. Cardiol. 2006; 21: 322-328Crossref PubMed Scopus (114) Google Scholar). One possible contributor to oxidative damage to HDL is myeloperoxidase (7Shao B. Oda M.N. Oram J.F. Heinecke J.W. Myeloperoxidase: an inflammatory enzyme for generating dysfunctional high density lipoprotein.Curr. Opin. Cardiol. 2006; 21: 322-328Crossref PubMed Scopus (114) Google Scholar). This heme protein, which is expressed by macrophages in human atherosclerotic lesions (8Daugherty A. Dunn J.L. Rateri D.L. Heinecke J.W. Myeloperoxidase, a catalyst for lipoprotein oxidation, is expressed in human atherosclerotic lesions.J. Clin. Invest. 1994; 94: 437-444Crossref PubMed Scopus (1122) Google Scholar), generates an array of reactive oxygen and nitrogen species in vitro. Moreover, quantification by isotope dilution gas chromatography-mass spectrometry of oxidized amino acids derived from the lipoprotein has implicated myeloperoxidase in HDL oxidation in vivo (7Shao B. Oda M.N. Oram J.F. Heinecke J.W. Myeloperoxidase: an inflammatory enzyme for generating dysfunctional high density lipoprotein.Curr. Opin. Cardiol. 2006; 21: 322-328Crossref PubMed Scopus (114) Google Scholar). For example, circulating HDL isolated from humans with established CAD contains markedly elevated levels of chlorotyrosine and nitrotyrosine, two characteristic products of myeloperoxidase, suggesting that oxidative damage by the enzyme helps generate dysfunctional HDL (7Shao B. Oda M.N. Oram J.F. Heinecke J.W. Myeloperoxidase: an inflammatory enzyme for generating dysfunctional high density lipoprotein.Curr. Opin. Cardiol. 2006; 21: 322-328Crossref PubMed Scopus (114) Google Scholar, 8Daugherty A. Dunn J.L. Rateri D.L. Heinecke J.W. Myeloperoxidase, a catalyst for lipoprotein oxidation, is expressed in human atherosclerotic lesions.J. Clin. Invest. 1994; 94: 437-444Crossref PubMed Scopus (1122) Google Scholar). During inflammation, HDL also acquires proteins that may either protect or harm the artery wall (9Van Lenten B.J. Hama S.Y. Beer F.C. de Stafforini D.M. McIntyre T.M. Prescott S.M. Du B.N. La Fogelman A.M. Navab M. Anti-inflammatory HDL becomes pro-inflammatory during acute phase response.J. Clin. Invest. 1995; 96: 2758-2767Crossref PubMed Scopus (700) Google Scholar, 10Cabana V.G. Reardon C.A. Feng N. Neath S. Lukens J. Getz G.S. Serum paraoxonase: effect of the apolipoprotein composition of HDL and the acute phase response.J. Lipid Res. 2003; 44: 780-792Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). For example, it becomes enriched in amyloid A, which can induce it to bind to proteoglycans in the artery wall, where it can be converted into an atherogenic form (11Chait A. Han C.Y. Oram J.F. Heinecke J.W. The immune system and atherogenesis. Lipoprotein-associated inflammatory proteins: markers or mediators of cardiovascular disease?.J. Lipid Res. 2005; 46: 389-403Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar). Chronic inflammation, as monitored by blood proteins such as C-reactive protein, is associated with an increased risk of CAD (11Chait A. Han C.Y. Oram J.F. Heinecke J.W. The immune system and atherogenesis. Lipoprotein-associated inflammatory proteins: markers or mediators of cardiovascular disease?.J. Lipid Res. 2005; 46: 389-403Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar). Thus, oxidative and nonoxidative pathways associated with inflammation could render HDL dysfunctional in vivo. HDL is a circulating complex of lipids and proteins that was originally defined on the basis of its density on ultracentrifugation (12Havel R.J. Eder H.A. Bragdon J.H. The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum.J. Clin. Invest. 1955; 34: 1345-1353Crossref PubMed Scopus (6475) Google Scholar). This approach tends to equate buoyant density with functional significance. By contrast, Alaupovic (13Alaupovic P. Apoliproproteins and lipoproteins.Atherosclerosis. 1971; 13: 141-146Abstract Full Text PDF PubMed Scopus (105) Google Scholar) proposed more than 30 years ago that HDL is a family of distinct particles that contain apoA-I but also vary in protein composition. This concept has been confirmed by the isolation and characterization of subspecies of HDL particles (14McVicar J.P. Kunitake S.T. Hamilton R.L. Kane J.P. Characteristics of human lipoproteins isolated by selected-affinity immunosorption of apolipoprotein A-I.Proc. Natl. Acad. Sci. USA. 1984; 81: 1356-1360Crossref PubMed Google Scholar, 15Cheung M.C. Albers J.J. Characterization of lipoprotein particles isolated by immunoaffinity chromatography. Particles containing A-I and A-II and particles containing A-I but no A-II.J. Biol. Chem. 1984; 259: 12201-12209Abstract Full Text PDF PubMed Google Scholar, 16Shiflett A.M. Bishop J.R. Pahwa A. Hajduk S.L. Human high density lipoproteins are platforms for assembly of multi-component innate immune complexes.J. Biol. Chem. 2005; 280: 32578-32585Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, 17Heinecke J.W. Mass spectrometric quantification of amino acid oxidation products in proteins: insights into pathways that promote LDL oxidation in the human artery wall.FASEB J. 1999; 13: 1113-1120Crossref PubMed Scopus (126) Google Scholar). For example, immunoaffinity isolation has identified two broad families of particles, one containing only apoA-I and the other containing both apoA-I and apoA-II (15Cheung M.C. Albers J.J. Characterization of lipoprotein particles isolated by immunoaffinity chromatography. Particles containing A-I and A-II and particles containing A-I but no A-II.J. Biol. Chem. 1984; 259: 12201-12209Abstract Full Text PDF PubMed Google Scholar). HDL subspecies are thought to be distinct metabolic entities with numerous functions, including lipid transport and promotion of cholesterol efflux from macrophages and other peripheral cells. However, HDL also has antioxidant and antiparasitic activities (4Barter P.J. Nicholls S. Rye K.A. Anantharamaiah G.M. Navab M. Fogelman A.M. Antiinflammatory properties of HDL.Circ. Res. 2004; 95: 764-772Crossref PubMed Scopus (1044) Google Scholar, 16Shiflett A.M. Bishop J.R. Pahwa A. Hajduk S.L. Human high density lipoproteins are platforms for assembly of multi-component innate immune complexes.J. Biol. Chem. 2005; 280: 32578-32585Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). The proteins it contains with potential antioxidant properties include paraoxonase-1, lecithin-cholesterol acyltransferase, and lipoprotein-phospholipase A2, and alterations to those proteins have been proposed to impair its cardioprotective function (4Barter P.J. Nicholls S. Rye K.A. Anantharamaiah G.M. Navab M. Fogelman A.M. Antiinflammatory properties of HDL.Circ. Res. 2004; 95: 764-772Crossref PubMed Scopus (1044) Google Scholar). We hypothesized that quantifying HDL's protein composition might provide insights into its antiatherogenic and anti-inflammatory properties. We therefore used shotgun proteomics, tandem mass spectrometry (MS/MS) analysis of a complex mixture of proteins (18Link A.J. Eng J. Schieltz D.M. Carmack E. Mize G.J. Morris D.R. Garvik B.M. Yates 3rd J.R. Direct analysis of protein complexes using mass spectrometry.Nat. Biotechnol. 1999; 17: 676-682Crossref PubMed Scopus (2065) Google Scholar, 19Aebersold R. Mann M. Mass spectrometry-based proteomics.Nature. 2003; 422: 198-207Crossref PubMed Scopus (5552) Google Scholar), to study the HDL proteome. In this approach, HDL is digested with trypsin, and the resulting peptides are separate by liquid chromatography and then analyzed with MS and MS/MS (20Vaisar T. Pennathur S. Green P.S. Gharib S.A. Hoofnagle A.N. Cheung M.C. Byun J. Vuletic S. Kassim S. Singh P. al et Shotgun proteomics implicates protease inhibition and complement activation in the antiinflammatory properties of HDL.J. Clin. Invest. 2007; 117: 746-756Crossref PubMed Scopus (782) Google Scholar). In the first step, the mass of intact precursor peptides is determined by MS. In the second, precursor ions are isolated from all other peptide ions and fragmented by collision-induced dissociation, which generates a set of C-terminal and N-terminal peptide ions. The masses of these product ions (i.e., the MS/MS spectrum) are searched against a protein database to identify the parent protein that contains the specific peptides that would generate the observed MS/MS spectrum (19Aebersold R. Mann M. Mass spectrometry-based proteomics.Nature. 2003; 422: 198-207Crossref PubMed Scopus (5552) Google Scholar). The MS/MS spectra can also be used to deduce peptide sequence and to identify site-specific modifications. Using gel electrophoresis with MS/MS analysis, other investigators have identified 12 proteins associated with HDL3 (the dense fraction of HDL) and 14 proteins with total HDL (21Karlsson H. Leanderson P. Tagesson C. Lindahl M. Lipoproteomics II: mapping of proteins in high-density lipoprotein using two-dimensional gel electrophoresis and mass spectrometry.Proteomics. 2005; 5: 1431-1445Crossref PubMed Scopus (151) Google Scholar). We identified all of those proteins, as well as many others (see below), suggesting that liquid chromatography MS/MS methods are more sensitive than gel-based proteomic methods for detecting HDL-associated proteins. Using shotgun proteomics, we identified 48 proteins in HDL isolated by ultracentrifugation from healthy controls and/or CAD subjects (20Vaisar T. Pennathur S. Green P.S. Gharib S.A. Hoofnagle A.N. Cheung M.C. Byun J. Vuletic S. Kassim S. Singh P. al et Shotgun proteomics implicates protease inhibition and complement activation in the antiinflammatory properties of HDL.J. Clin. Invest. 2007; 117: 746-756Crossref PubMed Scopus (782) Google Scholar). They included 22 of 23 known HDL proteins with well-characterized roles in lipid metabolism, which validates our experimental approach. Importantly, we found 13 proteins not previously known to reside in HDL. We used annotations by the Gene Ontology Consortium to connect the complex array of proteins we identified in HDL to biological processes (Fig. 1). Surprisingly, acute-phase response proteins (23 of 48), whose plasma concentrations are altered markedly by acute inflammation (20Vaisar T. Pennathur S. Green P.S. Gharib S.A. Hoofnagle A.N. Cheung M.C. Byun J. Vuletic S. Kassim S. Singh P. al et Shotgun proteomics implicates protease inhibition and complement activation in the antiinflammatory properties of HDL.J. Clin. Invest. 2007; 117: 746-756Crossref PubMed Scopus (782) Google Scholar), outnumbered proteins implicated in lipid metabolism. Several proteins not previously known to reside in HDL, including complement factors C4A/C4B and C9 as well as the complement regulatory protein vitronectin, were identified. Vitronectin is an extracellular matrix protein, which raises the possibility that certain HDL components can be derived from noncellular sources or cells distinct from those that synthesize apoA-I in the liver and intestine. The detection of multiple proteins with roles in complement activation, together with pioneering studies of HDL protein complexes that kill protozoa (16Shiflett A.M. Bishop J.R. Pahwa A. Hajduk S.L. Human high density lipoproteins are platforms for assembly of multi-component innate immune complexes.J. Biol. Chem. 2005; 280: 32578-32585Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar), is consistent with the suggestion that HDL serves as a platform for the assembly of proteins involved in the innate immune response. Given that proteolysis of structural proteins in atherosclerotic lesions is thought to play a critical role in plaque rupture, the major cause of myocardial infarction and sudden death in subjects with CAD (22Libby P. The molecular mechanisms of the thrombotic complications of atherosclerosis.J. Intern. Med. 2008; 263: 517-527Crossref PubMed Scopus (172) Google Scholar), it is noteworthy that we found a family of proteins in HDL that contain serine proteinase inhibitor domains. Serine protease inhibitors, termed serpins, are key regulators of numerous biological pathways involved in inflammation, coagulation, angiogenesis, and matrix degradation. Moreover, we also detected the thiol proteinase inhibitor kininogen-1 and haptoglobin-related protein, which contains a crippled catalytic triad residue that may allow it to act as a decoy substrate to prevent proteolysis. These observations suggest that HDL plays a previously unsuspected role in preventing plaque rupture, perhaps by protecting vascular lesions from promiscuous proteolysis. Our detection of multiple complement regulatory proteins in HDL (20Vaisar T. Pennathur S. Green P.S. Gharib S.A. Hoofnagle A.N. Cheung M.C. Byun J. Vuletic S. Kassim S. Singh P. al et Shotgun proteomics implicates protease inhibition and complement activation in the antiinflammatory properties of HDL.J. Clin. Invest. 2007; 117: 746-756Crossref PubMed Scopus (782) Google Scholar) is also noteworthy. Complement activation helps mediate tissue damage in animal models of acute myocardial infarction (23Pepys M.B. Hirschfield G.M. Tennent G.A. Gallimore J.R. Kahan M.C. Bellotti V. Hawkins P.N. Myers R.M. Smith M.D. Polara A. al et Targeting C-reactive protein for the treatment of cardiovascular disease.Nature. 2006; 440: 1217-1221Crossref PubMed Scopus (561) Google Scholar). HDL also blocks the assembly of the terminal complement attack complex on cultured endothelial cells (24Hamilton K.K. Zhao J. Sims P.J. Interaction between apolipoproteins A-I and A-II and the membrane attack complex of complement. Affinity of the apoproteins for polymeric C9.J. Biol. Chem. 1993; 268: 3632-3638Abstract Full Text PDF PubMed Google Scholar). Inhibition of complement deposition by HDL may limit injury to cardiac cells and prevent activation of the coagulant response in endothelium and platelets, two critical events in acute thrombosis. Our studies of the HDL proteome (20Vaisar T. Pennathur S. Green P.S. Gharib S.A. Hoofnagle A.N. Cheung M.C. Byun J. Vuletic S. Kassim S. Singh P. al et Shotgun proteomics implicates protease inhibition and complement activation in the antiinflammatory properties of HDL.J. Clin. Invest. 2007; 117: 746-756Crossref PubMed Scopus (782) Google Scholar), together with those of immunoaffinity-isolated HDL particles (14McVicar J.P. Kunitake S.T. Hamilton R.L. Kane J.P. Characteristics of human lipoproteins isolated by selected-affinity immunosorption of apolipoprotein A-I.Proc. Natl. Acad. Sci. USA. 1984; 81: 1356-1360Crossref PubMed Google Scholar, 15Cheung M.C. Albers J.J. Characterization of lipoprotein particles isolated by immunoaffinity chromatography. Particles containing A-I and A-II and particles containing A-I but no A-II.J. Biol. Chem. 1984; 259: 12201-12209Abstract Full Text PDF PubMed Google Scholar, 16Shiflett A.M. Bishop J.R. Pahwa A. Hajduk S.L. Human high density lipoproteins are platforms for assembly of multi-component innate immune complexes.J. Biol. Chem. 2005; 280: 32578-32585Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar), strongly support the view that HDL is a mixture of particles that carry distinct protein cargoes. Many of these proteins are involved in lipid transport and metabolism. However, others may contribute to inflammation, complement activation, and proteolysis. Some of the particles could be cardioprotective by inhibiting inflammation, removing toxic proteins and lipids from the artery wall, and delivering protective agents to arterial cells. Others could have deleterious effects, such as promoting cholesterol accumulation by arterial cells and inhibiting other cardioprotective pathways. Our observations support the proposal that alterations in HDL's protein cargo might boost the formation of inflammatory particles (4Barter P.J. Nicholls S. Rye K.A. Anantharamaiah G.M. Navab M. Fogelman A.M. Antiinflammatory properties of HDL.Circ. Res. 2004; 95: 764-772Crossref PubMed Scopus (1044) Google Scholar, 9Van Lenten B.J. Hama S.Y. Beer F.C. de Stafforini D.M. McIntyre T.M. Prescott S.M. Du B.N. La Fogelman A.M. Navab M. Anti-inflammatory HDL becomes pro-inflammatory during acute phase response.J. Clin. Invest. 1995; 96: 2758-2767Crossref PubMed Scopus (700) Google Scholar), raising the possibility that quantifying those proteins could provide insights into atherogenesis. To explore this idea, we used shotgun proteomics to compare the protein composition of HDL3 isolated from control and CAD subjects (20Vaisar T. Pennathur S. Green P.S. Gharib S.A. Hoofnagle A.N. Cheung M.C. Byun J. Vuletic S. Kassim S. Singh P. al et Shotgun proteomics implicates protease inhibition and complement activation in the antiinflammatory properties of HDL.J. Clin. Invest. 2007; 117: 746-756Crossref PubMed Scopus (782) Google Scholar). Importantly, the CAD subjects were newly diagnosed (though at least 3 months past any acute CAD event) and not on medications that alter lipid levels (see below). Using statistical approaches based on spectral counting [the number of MS/MS spectra unique to a protein (25Washburn M.P. Ulaszek R.R. Yates 3rd J.R. Reproducibility of quantitative proteomic analyses of complex biological mixtures by multidimensional protein identification technology.Anal. Chem. 2003; 75: 5054-5061Crossref PubMed Scopus (114) Google Scholar, 26Liu H. Sadygov R.G. Yates 3rd J.R. A model for random sampling and estimation of relative protein abundance in shotgun proteomics.Anal. Chem. 2004; 76: 4193-4201Crossref PubMed Scopus (2060) Google Scholar)] and random permutation analysis (20Vaisar T. Pennathur S. Green P.S. Gharib S.A. Hoofnagle A.N. Cheung M.C. Byun J. Vuletic S. Kassim S. Singh P. al et Shotgun proteomics implicates protease inhibition and complement activation in the antiinflammatory properties of HDL.J. Clin. Invest. 2007; 117: 746-756Crossref PubMed Scopus (782) Google Scholar, 27Fu X. Gharib S.A. Green P.S. Aitken M.L. Frazer D.A. Park D.R. Vaisar T. Heinecke J.W. Spectral index for assessment of differential protein expression in shotgun proteomics.J. Proteome Res. 2008; 7: 845-854Crossref PubMed Scopus (86) Google Scholar), we found five proteins that were apparently enriched in HDL isolated from the CAD subjects. These proteins related to lipid metabolism (apoE, apoC-IV, and apoA-IV), oxidative stress (paraxonase-1), and the immune system (complement factor C3). ApoA-IV, located in the apoA-I/apoC-III/apoA-IV gene cluster, inhibits atherosclerosis in mouse models of hypercholesterolemia (28Vergnes L. Baroukh N. Ostos M.A. Castro G. Duverger N. Nanjee M.N. Najib J. Fruchart J.C. Miller N.E. Zakin M.M. al et Expression of human apolipoprotein A-I/C-III/A-IV gene cluster in mice induces hyperlipidemia but reduces atherogenesis.Arterioscler. Thromb. Vasc. Biol. 2000; 20: 2267-2274Crossref PubMed Scopus (27) Google Scholar). ApoE, apoC-IV, and C3 are expressed by macrophages (1Rader D.J. Puré E. Lipoproteins, macrophage function, and atherosclerosis: beyond the foam cell?.Cell Metab. 2005; 1: 223-230Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 29Strunk R.C. Kunke K.S. Giclas P.C. Human peripheral blood monocyte-derived macrophages produce haemolytically active C3 in vitro.Immunology. 1983; 49: 169-174PubMed Google Scholar). C3 is needed to assemble the membrane attack complex of the complement system, suggesting a link between HDL, macrophages, and innate immunity. Moreover, apoE and apoC-IV are part of a gene cluster that is upregulated in LXR-stimulated macrophages (30Mak P.A. Laffitte B.A. Desrumaux C. Joseph S.B. Curtiss L.K. Mangelsdorf D.J. Tontonoz P. Edwards P.A. Regulated expression of the apolipoprotein E/C-I/C-IV/C-II gene cluster in murine and human macrophages. A critical role for nuclear liver X receptors alpha and beta.J. Biol. Chem. 2002; 277: 31900-31908Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). A number of factors related to atherosclerosis might regulate the levels of these proteins in HDL, including their production by the liver, their expression in macrophages, and remodeling of HDL particles. Our proteomic analyses suggested that apoE was enriched in HDL3 isolated from CAD subjects (20Vaisar T. Pennathur S. Green P.S. Gharib S.A. Hoofnagle A.N. Cheung M.C. Byun J. Vuletic S. Kassim S. Singh P. al et Shotgun proteomics implicates protease inhibition and complement activation in the antiinflammatory properties of HDL.J. Clin. Invest. 2007; 117: 746-756Crossref PubMed Scopus (782) Google Scholar). To confirm this observation, we measured apoE levels immunochemically in HDL3 isolated from a second set of 64 subjects enrolled in a different study: 32 with established CAD and 32 age- and sex-matched controls. To control for differences in lipid metabolism, we matched the subjects for HDL-cholesterol and triglyceride levels (20Vaisar T. Pennathur S. Green P.S. Gharib S.A. Hoofnagle A.N. Cheung M.C. Byun J. Vuletic S. Kassim S. Singh P. al et Shotgun proteomics implicates protease inhibition and complement activation in the antiinflammatory properties of HDL.J. Clin. Invest. 2007; 117: 746-756Crossref PubMed Scopus (782) Google Scholar). Levels of apoE were significantly higher in HDL3 isolated from the CAD subjects. In striking contrast, the two groups had similar levels of apoA-I and apoA-II. Our demonstration of elevated levels of apoE in HDL3 isolated from two independent groups of subjects with established CAD raises the possibility that alteration in HDL's protein content serves as a marker, and perhaps a mediator, of CAD. We used MS to test the hypothesis that aggressive lipid-lowering therapy alters the HDL proteome in humans with established CAD (31Green P.S. Vaisar T. Pennathur S. Kulstad J.J. Moore A.B. Marcovina S. Brunzell J. Knopp R.H. Zhao X.Q. Heinecke J.W. Combined statin and niacin therapy remodels the high-density lipoprotein proteome.Circulation. 2008; 118: 1259-1267Crossref PubMed Scopus (115) Google Scholar). To quantify changes in abundance, we used two complementary methods for quantifying proteins: spectral counting and extracted ion chromatograms. Spectral counting, the number of detected MS/MS spectra for a given protein, is based on the observation that tryptic peptides derived from proteins that are more abundant in a sample have a higher probability of being identified by MS/MS (25Washburn M.P. Ulaszek R.R. Yates 3rd J.R. Reproducibility of quantitative proteomic analyses of complex biological mixtures by multidimensional protein identification technology.Anal. Chem. 2003; 75: 5054-5061Crossref PubMed Scopus (114) Google Scholar, 26Liu H. Sadygov R.G. Yates 3rd J.R. A model for random sampling and estimation of relative protein abundance in shotgun proteomics.Anal. Chem. 2004; 76: 4193-4201Crossref PubMed Scopus (2060) Google Scholar). For extracted ion chromatograms, a peptide's ion current and charge state extracted from full scan mass spectra are used to construct a chromatogram (32Old W.M. Pierce K.Meyer-Arendt, L.Aveline-Wolf, K.G. Mendoza A. Sevinsky J.R. Resing K.A. Ahn N.G. Comparison of label-free methods for quantifying human proteins by shotgun proteomics.Mol. Cell. Proteomics. 2005; 4: 1487-1502Abstract Full Text Full Text PDF PubMed Scopus (1013) Google Scholar). Relative abundance is monitored as the peptide's ion current. This approach offers important technical advantages. First, both methods are label-free (i.e., do not require peptides or proteins to be labeled with mass tags or isotopes) and therefore are well suited to analyzing HDL under clinical conditions. Second, spectral counting is readily implemented with the data analysis systems used for MS/MS (25Washburn M.P. Ulaszek R.R. Yates 3rd J.R. Reproducibility of quantitative proteomic analyses of complex biological mixtures by multidimensional protein identification technology.Anal. Chem. 2003; 75: 5054-5061Crossref PubMed Scopus (114) Google Scholar, 26Liu H. Sadygov R.G. Yates 3rd J.R. A model for random sampling and estimation of relative protein abundance in shotgun proteomics.Anal. Chem. 2004; 76: 4193-4201Crossref PubMed Scopus (2060) Google Scholar). Third, extracted ion chromatograms estimate protein ratios more accurately than does spectral counting (32Old W.M. Pierce K.Meyer-Arendt, L.Aveline-Wolf, K.G. Mendoza A. Sevinsky J.R. Resing K.A. Ahn N.G. Comparison of label-free methods for quantifying human proteins by shotgun proteomics.Mol. Cell. Proteomics. 2005; 4: 1487-1502Abstract Full Text Full Text PDF PubMed Scopus (1013) Google Scholar). Finally, it is possible to compare the extracted ion chromatogram ratios of multiple peptides detected from the same protein, which should increase confidence in the results. HDL3 was isolated from six newly diagnosed CAD subjects prior to and 1 year after combination therapy with niacin (a potent HDL-cholesterol elevating drug) and atorvastatin (which markedly lowers LDL-cholesterol levels). After digesting HDL proteins with trypsin, we analyzed the resulting peptide mixture by liquid chromatography-Fourier transform-mass spectrometry (31Green P.S. Vaisar T. Pennathur S. Kulstad J.J. Moore A.B. Marcovina S. Brunzell J. Knopp R.H. Zhao X.Q. Heinecke J.W. Combined statin and niacin therapy remodels the high-density lipoprotein proteome.Circulation. 2008; 118: 1259-1267Crossref PubMed Scopus (115) Google Scholar). Spectral counting was used to initially identify proteins that appeared to be differentially expressed in HDL3 before and during therapy. To confirm these observations, we quantified extracted ion chromatograms derived from multiple peptides for each candidate protein. This approach initially identified three HDL3 proteins whose relative abundance appeared to change significantly as a result of treatment: apoE, apoF, and phospholipid transfer protein (PLTP) (31Green P.S. Vaisar T. Pennathur S. Kulstad J.J. Moore A.B. Marcovina S. Brunzell J. Knopp R.H. Zhao X.Q. Heinecke J.W. Combined statin and niacin therapy remodels the high-density lipoprotein proteome.Circulation. 2008; 118: 1259-1267Crossref PubMed Scopus (115) Google Scholar). The combination lipid therapy lowered levels of apoE; by contrast, it boosted levels of apoF and PLTP. Spectral counting also detected trends with borderline significance toward lower apoC-II and higher apoJ levels in the treated subjects. It is important to note that proteins with low spectral counts (e.g., apoF, PLTP, and apoJ) exhibit greater relative variability (27Fu X. Gharib S.A. Green P.S. Aitken M.L. Frazer D.A. Park D.R. Vaisar T. Heinecke J.W. Spectral index for assessment of differential protein expression in shotgun proteomics.J. Proteome Res. 2008; 7: 845-854Crossref PubMed Scopus (86) Google Scholar). We took advantage of the greater precision of extracted ion chromatography to confirm our observations (32Old W.M. Pierce K.Meyer-Arendt, L.Aveline-Wolf, K.G. Mendoza A. Sevinsky J.R. Resing K.A. Ahn N.G. Comparison of label-free methods for quantifying human proteins by shotgun proteomics.Mol. Cell. Proteomics. 2005; 4: 1487-1502Abstract Full Text Full Text PDF PubMed Scopus (1013) Google Scholar), finding that therapy with atorvastatin and niacin significantly lowered apoE and increased apoJ, apoF, and PLTP levels in HDL3 isolated from CAD subjects (31Green P.S. Vaisar T. Pennathur S. Kulstad J.J. Moore A.B. Marcovina S. Brunzell J. Knopp R.H. Zhao X.Q. Heinecke J.W. Combined statin and niacin therapy remodels the high-density lipoprotein proteome.Circulation. 2008; 118: 1259-1267Crossref PubMed Scopus (115) Google Scholar). Although niacin increases levels of HDL-cholesterol and apoA-I in plasma, we failed to observe a significant change in the apoA-I content of HDL3. This observation suggests that niacin increases the number of HDL particles but not the amount of apoA-I per particle. Alternatively, it may increase apoA-I levels in HDL species distinct from HDL3. To confirm that our MS techniques can quantify changes in the HDL proteome, we used two complementary approaches (31Green P.S. Vaisar T. Pennathur S. Kulstad J.J. Moore A.B. Marcovina S. Brunzell J. Knopp R.H. Zhao X.Q. Heinecke J.W. Combined statin and niacin therapy remodels the high-density lipoprotein proteome.Circulation. 2008; 118: 1259-1267Crossref PubMed Scopus (115) Google Scholar). First, we observed a strong linear correlation between apoE levels as assessed by extracted ion chromatograms and nephelometry in our initial studies. Second, we demonstrated biochemically that combination therapy with niacin and statin reduced levels of apoE in HDL3 in an independent group of 18 subjects. The validity of our approach is further supported by the observation of decreased apoE levels in HDL during statin treatment of hypertriglyceridemic subjects (33Le N.A. Li W.Innis-Whitehouse, X. Black R.Bakker-Arkema, D. Brown W.V. Lipid and apolipoprotein levels and distribution in patients with hypertriglyceridemia: effect of triglyceride reductions with atorvastatin.Metabolism. 2000; 49: 167-177Abstract Full Text PDF PubMed Scopus (67) Google Scholar) or hypertriglyceridemic subjects with type 2 diabetes mellitus (34Bach-Ngohou K. Ouguerram K. Frenais R. Maugere P. Zair B.Ripolles-Piquer, Y. Krempf M. Bard J.M. Influence of atorvastatin on apolipoprotein E and AI kinetics in patients with type 2 diabetes.J. Pharmacol. Exp. Ther. 2005; 315: 363-369Crossref PubMed Scopus (31) Google Scholar). It is noteworthy that we found elevated levels of apoE in HDL3 isolated from subjects with established CAD (20Vaisar T. Pennathur S. Green P.S. Gharib S.A. Hoofnagle A.N. Cheung M.C. Byun J. Vuletic S. Kassim S. Singh P. al et Shotgun proteomics implicates protease inhibition and complement activation in the antiinflammatory properties of HDL.J. Clin. Invest. 2007; 117: 746-756Crossref PubMed Scopus (782) Google Scholar) and that aggressive lipid therapy lowered apoE levels in HDL3 (31Green P.S. Vaisar T. Pennathur S. Kulstad J.J. Moore A.B. Marcovina S. Brunzell J. Knopp R.H. Zhao X.Q. Heinecke J.W. Combined statin and niacin therapy remodels the high-density lipoprotein proteome.Circulation. 2008; 118: 1259-1267Crossref PubMed Scopus (115) Google Scholar). We also found that aggressive lipid therapy raised levels of apoJ, apoF, and PLTP, all of which were present at lower levels in CAD subjects than control subjects (31Green P.S. Vaisar T. Pennathur S. Kulstad J.J. Moore A.B. Marcovina S. Brunzell J. Knopp R.H. Zhao X.Q. Heinecke J.W. Combined statin and niacin therapy remodels the high-density lipoprotein proteome.Circulation. 2008; 118: 1259-1267Crossref PubMed Scopus (115) Google Scholar). Thus, combination therapy remodels the HDL3 proteome to make it resemble that of apparently healthy subjects. Our observations implicate HDL in inflammation and host defense mechanisms. They further suggest that its proteome could serve as a marker, and perhaps mediator, of cardiovascular disease in humans. Monitoring the HDL proteome may provide insights into the efficacy of lipid therapy and should help identify novel cardioprotective actions of HDL.