Inflammatory remodeling of the HDL proteome impairs cholesterol efflux capacity
2015; Elsevier BV; Volume: 56; Issue: 8 Linguagem: Inglês
10.1194/jlr.m059089
ISSN1539-7262
AutoresTomáš Vaisar, Chongren Tang, Ilona Babenko, Patrick M. Hutchins, Jake Wimberger, Anthony F. Suffredini, Jay W. Heinecke,
Tópico(s)Diabetes, Cardiovascular Risks, and Lipoproteins
ResumoRecent studies demonstrate that HDL's ability to promote cholesterol efflux from macrophages associates strongly with cardioprotection in humans independently of HDL-cholesterol (HDL-C) and apoA-I, HDL's major protein. However, the mechanisms that impair cholesterol efflux capacity during vascular disease are unclear. Inflammation, a well-established risk factor for cardiovascular disease, has been shown to impair HDL's cholesterol efflux capacity. We therefore tested the hypothesis that HDL's impaired efflux capacity is mediated by specific changes of its protein cargo. Humans with acute inflammation induced by low-level endotoxin had unchanged HDL-C levels, but their HDL-C efflux capacity was significantly impaired. Proteomic analyses demonstrated that HDL's cholesterol efflux capacity correlated inversely with HDL content of serum amyloid A (SAA)1 and SAA2. In mice, acute inflammation caused a marked impairment of HDL-C efflux capacity that correlated with a large increase in HDL SAA. In striking contrast, the efflux capacity of mouse inflammatory HDL was preserved with genetic ablation of SAA1 and SAA2. Our observations indicate that the inflammatory impairment of HDL-C efflux capacity is due in part to SAA-mediated remodeling of HDL's protein cargo. Recent studies demonstrate that HDL's ability to promote cholesterol efflux from macrophages associates strongly with cardioprotection in humans independently of HDL-cholesterol (HDL-C) and apoA-I, HDL's major protein. However, the mechanisms that impair cholesterol efflux capacity during vascular disease are unclear. Inflammation, a well-established risk factor for cardiovascular disease, has been shown to impair HDL's cholesterol efflux capacity. We therefore tested the hypothesis that HDL's impaired efflux capacity is mediated by specific changes of its protein cargo. Humans with acute inflammation induced by low-level endotoxin had unchanged HDL-C levels, but their HDL-C efflux capacity was significantly impaired. Proteomic analyses demonstrated that HDL's cholesterol efflux capacity correlated inversely with HDL content of serum amyloid A (SAA)1 and SAA2. In mice, acute inflammation caused a marked impairment of HDL-C efflux capacity that correlated with a large increase in HDL SAA. In striking contrast, the efflux capacity of mouse inflammatory HDL was preserved with genetic ablation of SAA1 and SAA2. Our observations indicate that the inflammatory impairment of HDL-C efflux capacity is due in part to SAA-mediated remodeling of HDL's protein cargo. Epidemiological and clinical studies have reported a robust inverse association of HDL-cholesterol (HDL-C) levels with risk of coronary artery disease (CAD) (1.Wilson P.W. D'Agostino R.B. Levy D. Belanger A.M. Silbershatz H. Kannel W.B. Prediction of coronary heart disease using risk factor categories.Circulation. 1998; 97: 1837-1847Crossref PubMed Scopus (7437) Google Scholar). Moreover, mice with genetically engineered deficiencies in proteins involved in HDL metabolism have atherosclerotic phenotypes (2.Tall A.R. Yvan-Charvet L. Terasaka N. Pagler T. Wang N. HDL, ABC transporters, and cholesterol efflux: implications for the treatment of atherosclerosis.Cell Metab. 2008; 7: 365-375Abstract Full Text Full Text PDF PubMed Scopus (434) Google Scholar). These observations provide strong evidence that HDL plays a causal role in vascular disease and have triggered intense interest in targeting HDL for therapeutic intervention. Several recent observations have cast doubt on the hypotheses that HDL-C levels relate to CAD risk in humans and that elevating HDL-C is therapeutic (3.Rader D.J. Tall A.R. The not-so-simple HDL story: is it time to revise the HDL cholesterol hypothesis?.Nat. 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Rader D.J. et al.Cholesterol efflux and atheroprotection: advancing the concept of reverse cholesterol transport.Circulation. 2012; 125: 1905-1919Crossref PubMed Scopus (684) Google Scholar). The ability of HDL (or serum HDL, serum depleted of apoB-containing lipoproteins) to promote sterol efflux from cultured macrophages incubated with radiolabeled cholesterol can vary markedly, despite similar levels of HDL-C and apoA-I (8.de la Llera-Moya M. Drazul-Schrader D. Asztalos B.F. Cuchel M. Rader D.J. Rothblat G.H. The ability to promote efflux via ABCA1 determines the capacity of serum specimens with similar high-density lipoprotein cholesterol to remove cholesterol from macrophages.Arterioscler. Thromb. Vasc. Biol. 2010; 30: 796-801Crossref PubMed Scopus (330) Google Scholar). Therefore, HDL-C is not necessarily the major determinant of HDL's macrophage sterol efflux capacity in this system. Importantly, the efflux capacity of serum HDL is lower in individuals with prevalent CAD (9.Khera A.V. Cuchel M. de la Llera-Moya M. Rodrigues A. Burke M.F. Jafri K. French B.C. Phillips J.A. Mucksavage M.L. Wilensky R.L. et al.Cholesterol efflux capacity, high-density lipoprotein function, and atherosclerosis.N. Engl. J. Med. 2011; 364: 127-135Crossref PubMed Scopus (1540) Google Scholar, 10.Shao B. Tang C. Sinha A. Mayer P.S. Davenport G.D. Brot N. Oda M.N. Zhao X.Q. Heinecke J.W. Humans with atherosclerosis have impaired ABCA1 cholesterol efflux and enhanced high-density lipoprotein oxidation by myeloperoxidase.Circ. Res. 2014; 114: 1733-1742Crossref PubMed Scopus (149) Google Scholar, 11.Li X.M. Tang W.H. Mosior M.K. Huang Y. Wu Y. Matter W. Gao V. Schmitt D. Didonato J.A. Fisher E.A. et al.Paradoxical association of enhanced cholesterol efflux with increased incident cardiovascular risks.Arterioscler. Thromb. Vasc. Biol. 2013; 33: 1696-1705Crossref PubMed Scopus (256) Google Scholar). A recent study of a large cohort, initially free of CAD, demonstrated that sterol efflux associates strongly and negatively with the risk of future cardiac events (12.Rohatgi A. Khera A. Berry J.D. Givens E.G. Ayers C.R. Wedin K.E. Neeland I.J. Yuhanna I.S. Rader D.R. de Lemos J.A. et al.HDL cholesterol efflux capacity and incident cardiovascular events.N. Engl. J. Med. 2014; 371: 2383-2393Crossref PubMed Scopus (978) Google Scholar). This association was strengthened by multivariate adjustment, suggesting that impaired HDL function affects incident cardiovascular risk by processes distinct from those involving HDL-C, LDL-cholesterol (LDL-C), and other traditional lipid risk factors. Together, these observations indicate that the sterol efflux capacity of HDL might be a marker, and perhaps a mediator, of atherosclerotic burden that is independent of HDL-C and apoA-I levels. However, the molecular factors controlling the sterol efflux capacity of serum HDL are poorly understood (8.de la Llera-Moya M. Drazul-Schrader D. Asztalos B.F. Cuchel M. Rader D.J. Rothblat G.H. The ability to promote efflux via ABCA1 determines the capacity of serum specimens with similar high-density lipoprotein cholesterol to remove cholesterol from macrophages.Arterioscler. Thromb. Vasc. Biol. 2010; 30: 796-801Crossref PubMed Scopus (330) Google Scholar, 13.Lewis G.F. Rader D.J. New insights into the regulation of HDL metabolism and reverse cholesterol transport.Circ. Res. 2005; 96: 1221-1232Crossref PubMed Scopus (826) Google Scholar). For example, a recent study found that the majority of radiolabeled cholesterol released from macrophages did not reside in HDL, suggesting that impaired sterol efflux capacity does not necessarily reflect alterations in HDL itself (11.Li X.M. Tang W.H. Mosior M.K. Huang Y. Wu Y. Matter W. Gao V. Schmitt D. Didonato J.A. Fisher E.A. et al.Paradoxical association of enhanced cholesterol efflux with increased incident cardiovascular risks.Arterioscler. Thromb. Vasc. Biol. 2013; 33: 1696-1705Crossref PubMed Scopus (256) Google Scholar). Inflammation and metabolic disorders have been proposed to convert HDL to a dysfunctional form lacking anti-atherogenic properties (14.Van Lenten B.J. Hama S.Y. de Beer F.C. Stafforini D.M. McIntyre T.M. Prescott S.M. La Du B.N. Fogelman A.M. Navab M. Anti-inflammatory HDL becomes pro-inflammatory during the acute phase response. Loss of protective effect of HDL against LDL oxidation in aortic wall cell cocultures.J. Clin. Invest. 1995; 96: 2758-2767Crossref PubMed Scopus (705) Google Scholar, 15.Barter 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 (1059) Google Scholar, 16.Vaisar T. Shao B. Green P.S. Oda M.N. Oram J.F. Heinecke J.W. 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Serum amyloid A (SAA): influence on HDL-mediated cellular cholesterol efflux.J. Lipid Res. 1995; 36: 1058-1065Abstract Full Text PDF PubMed Google Scholar, 21.McGillicuddy F.C. de la Llera Moya M. Hinkle C.C. Joshi M.R. Chiquoine E.H. Billheimer J.T. Rothblat G.H. Reilly M.P. Inflammation impairs reverse cholesterol transport in vivo.Circulation. 2009; 119: 1135-1145Crossref PubMed Scopus (288) Google Scholar, 22.Annema W. Nijstad N. Tolle M. de Boer J.F. Buijs R.V. Heeringa P. van der Giet M. Tietge U.J. Myeloperoxidase and serum amyloid A contribute to impaired in vivo reverse cholesterol transport during the acute phase response but not group IIA secretory phospholipase A(2).J. Lipid Res. 2010; 51: 743-754Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 23.Artl A. Marsche G. Lestavel S. Sattler W. Malle E. Role of serum amyloid A during metabolism of acute-phase HDL by macrophages.Arterioscler. Thromb. Vasc. 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Rader D.J. et al.Cholesterol efflux and atheroprotection: advancing the concept of reverse cholesterol transport.Circulation. 2012; 125: 1905-1919Crossref PubMed Scopus (684) Google Scholar). In the current study, we investigated the protein cargo and function of HDL isolated from humans and mice with inflammation. Our observations indicate that inflammatory remodeling mediated by serum amyloid A (SAA) is one mechanism for generating HDL whose capacity to promote sterol efflux is impaired. The study was approved by the institutional review board of the Clinical Research Center, New Orleans, and reviewed by the institutional review board of the National Institute of Allergy and Infectious Diseases (28.Suffredini A.F. Hochstein H.D. McMahon F.G. Dose-related inflammatory effects of intravenous endotoxin in humans: evaluation of a new clinical lot of Escherichia coli O:113 endotoxin.J. Infect. Dis. 1999; 179: 1278-1282Crossref PubMed Scopus (130) Google Scholar). Twelve healthy male volunteers aged 22–49 years underwent a complete history and physical examination prior to entering the study. All subjects had normal physical exams and routine blood and urine chemistries, and none were taking medications or had known medical conditions. The 12 subjects were randomly assigned to the endotoxin injection groups. The subjects were injected iv with 1 ng/kg or 2 ng/kg National Institutes of Health (NIH) "equivalent" endotoxin (n = 4; Clinical Center Reference Endotoxin; E. coli O:113, Pharmacy Development Service, Clinical Center, NIH, Bethesda, MD) or 4 ng/kg (n = 4) of Food and Drug Administration (FDA) endotoxin (E. coli O:113, Lot EC-5; H. D. Hochstein, Center for Biologics Evaluation and Research, FDA, Bethesda, MD) (28.Suffredini A.F. Hochstein H.D. McMahon F.G. Dose-related inflammatory effects of intravenous endotoxin in humans: evaluation of a new clinical lot of Escherichia coli O:113 endotoxin.J. Infect. Dis. 1999; 179: 1278-1282Crossref PubMed Scopus (130) Google Scholar). Consistent with previous studies with these endotoxin preparations, we found that high-sensitivity C-reactive protein levels in the group that received the 4 ng/kg dose of FDA endotoxin were comparable to those in the group that received the 1 ng/kg dose of NIH endotoxin. Because SAA and CRP levels were similar in the 1 ng/kg NIH endotoxin and 4 ng/kg FDA endotoxin groups, we combined the subjects into a 1 ng/kg endotoxin group in subsequent analyses. Experiments with mice lacking SAA1.1 and SAA2.1 (termed here as Saa1/2−/− mice) were performed at the University of Kentucky. Saa1/2−/− mice and littermate WT mice were in a 129SvEv/C57BL/6 background (29.de Beer M.C. Webb N.R. Wroblewski J.M. Noffsinger V.P. Rateri D.L. Ji A. van der Westhuyzen D.R. de Beer F.C. Impact of serum amyloid A on high density lipoprotein composition and levels.J. 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Control mice were injected with 0.5 ml of sterile normal saline. Blood anticoagulated with EDTA was collected 24 h after the injection, and plasma was prepared by centrifugation. Plasma samples from two mice were combined for HDL isolation. HDL was isolated using sequential ultracentrifugation (d = 1.063–1.21 g/ml), as described for mouse (29.de Beer M.C. Webb N.R. Wroblewski J.M. Noffsinger V.P. Rateri D.L. Ji A. van der Westhuyzen D.R. de Beer F.C. Impact of serum amyloid A on high density lipoprotein composition and levels.J. Lipid Res. 2010; 51: 3117-3125Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 32.Coetzee G.A. Strachan A.F. van der Westhuyzen D.R. Hoppe H.C. Jeenah M.S. de Beer F.C. Serum amyloid A-containing human high density lipoprotein 3. Density, size, and apolipoprotein composition.J. Biol. Chem. 1986; 261: 9644-9651Abstract Full Text PDF PubMed Google Scholar) and human HDL (33.Vaisar T. Pennathur S. Green P.S. Gharib S.A. Hoofnagle A.N. 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Efflux of [3H]cholesterol was measured after a 6 h incubation with DMEM supplemented with 0.1% BSA without or with 30 μg/ml of HDL protein. Cholesterol efflux mediated by HDL was calculated as the percentage of total [3H]cholesterol (medium plus cell) released into the medium after the value obtained with DMEM/BSA alone was subtracted. Tryptic digests of mouse HDL (2 μg protein) were injected onto a C18 trap column (Paradigm Platinum Peptide Nanotrap, 0.15 × 50 mm; Michrom Bioresources, Inc.), desalted (50 μl/min) for 5 min with 1% acetonitrile/0.1% formic acid, eluted onto an analytical reverse-phase column (0.15 × 150 mm, Magic C18AQ, 5 μm, 200 Å Michrom Bioresources, Inc.), and separated at a flow rate of 1 μl/min over 180 min, using a linear gradient of 5–35% buffer B (90% acetonitrile, 0.1% formic acid) in buffer A (5% acetonitrile, 0.1% formic acid) on a Paradigm M4B HPLC (Michrom Bioresources, Inc.). Positive ion mass spectra were acquired with ESI in a linear ion trap mass spectrometer (LTQ; Thermo Electron Corp., San Jose, CA) with data-dependent acquisition (one MS survey scan followed by MS/MS scans of the eight most abundant ions in the survey scan). An exclusion window of 45 s was used after two acquisitions of the same precursor ion. Alternatively, the HDL from WT mice, with and without inflammation, were analyzed on Thermo QE+ mass spectrometer using data-independent acquisition (m/z 20 window with m/z 10 overlap, 17,500 resolution, normalized collision energy 25) (38.Egertson J.D. Kuehn A. Merrihew G.E. Bateman N.W. MacLean B.X. Ting Y.S. Canterbury J.D. Marsh D.M. Kellmann M. Zabrouskov V. et al.Multiplexed MS/MS for improved data-independent acquisition.Nat. Methods. 2013; 10: 744-746Crossref PubMed Scopus (209) Google Scholar) and data for apoA-I native and oxidized methionine- and tryptophan-containing peptides were analyzed and quantified using Skyline (39.MacLean B. 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Positive ion mass spectra were acquired with ESI in a hybrid linear ion trap-Orbitrap mass spectrometer (LTQ Orbitrap XL; Thermo Fisher, San Jose, CA) with data-dependent acquisition of MS/MS scans (linear ion trap) on the eight most abundant ions in the survey scan (Orbitrap, resolution 60,000). An exclusion window of 45 s was used after two repeated acquisitions of the same precursor ion. HDL protein was digested and analyzed essentially as previously described (40.Hoofnagle A.N. Becker J.O. Oda M.N. Cavigiolio G. Mayer P. Vaisar T. Multiple-reaction monitoring-mass spectrometric assays can accurately measure the relative protein abundance in complex mixtures.Clin. Chem. 2012; 58: 777-781Crossref PubMed Scopus (117) Google Scholar). MS/MS spectra were matched against the human International Protein Index (IPI) database (mouse v.3.54, January 2009; human v.3.72, April 2010), using the SEQUEST (v2.7) search engine with fixed Cys carbamidomethylation and variable Met oxidation modifications. SEQUEST results were further validated with Trans-Proteomic Pipeline tools, using an adjusted probability of ≥0.90 for peptides and ≥0.95 for proteins. Proteins considered for analysis had to be detected in ≥4 analyses with ≥2 unique peptides. Relative protein quantification was performed using spectral counting (41.Liu H. Sadygov R.G. Yates III, J.R. A model for random sampling and estimation of relative protein abundance in shotgun proteomics.Anal. Chem. 2004; 76: 4193-4201Crossref PubMed Scopus (2069) Google Scholar). Significant differences in spectral counts were identified using the combination of G-test and t-test together with permutation analysis to estimate false discovery rate (FDR) (42.Becker L. Gharib S.A. Irwin A.D. Wijsman E. Vaisar T. Oram J.F. Heinecke J.W. A macrophage sterol-responsive network linked to atherogenesis.Cell Metab. 2010; 11: 125-135Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). The abundance of SAA relative to apoA-I was estimated using extracted ion chromatograms and sum of peak areas for the three most abundant peptides from each protein according to the approach of Silva et al. (43.Silva J.C. Gorenstein M.V. Li G-z. Vissers J.P.C. Geromanos S.J. Absolute quantification of proteins by LCMSE: a virtue of parallel MS acquisition.Molecular & cellular proteomics. 2006; 5: 144-156Abstract Full Text Full Text PDF PubMed Scopus (1144) Google Scholar). HDL protein levels were quantified by Bradford assay and corrected to set of standards quantified by Lowry assay. HDL and plasma lipids were quantified biochemically [cholesterol and cholesteryl ester (Amplex Red, Invitrogen), triglycerides (Cayman Chemical, Ann Arbor, MI), phospholipids (Wako Chemical, Richmond, VA)]. CRP (Invitrogen, Carlsbad, CA) and SAA were measured by ELISA (Invitrogen, Camarillo, CA). HDL particle concentration was measured by calibrated ion mobility analysis (44.Hutchins P.M. Ronsein G.E. Monette J.S. Pamir N. Wimberger J. He Y. Anantharamaiah G.M. Kim D.S. Ranchalis J.E. Jarvik G.P. et al.Quantification of HDL particle concentration by calibrated ion mobility analysis.Clin. Chem. 2014; 60: 1393-1401Crossref PubMed Scopus (62) Google Scholar). Analyses were performed with the Statistical Package for the Social Sciences (SPSS v.19) and R statistical package (v.2.14). Results represent means and SEMs. The statistical significance of differences between groups was evaluated by the two-tailed Student's t-test or ANOVA with Tukey's honest significant difference (HSD) post hoc test. The human study involved 12 male subjects (40 ± 7 years old; fasting plasma levels of lipids and inflammatory proteins: HDL-C, 39.9 ± 3.8 mg/dl; LDL-C, 107 ± 4.9 mg/dl; CRP, 2.0 ± 0.6 mg/l; SAA, 14.3 ± 5.3 μg/ml). All subjects were apparently healthy, normolipidemic, not using medications, and had no symptoms or signs of inflammation. To induce inflammation, subjects were injected with the equivalent of 1 ng/kg (n = 8) or 2 ng/kg (n = 4) of reference endotoxin (28.Suffredini A.F. Hochstein H.D. McMahon F.G. Dose-related inflammatory effects of intravenous endotoxin in humans: evaluation of a new clinical lot of Escherichia coli O:113 endotoxin.J. Infect. Dis. 1999; 179: 1278-1282Crossref PubMed Scopus (130) Google Scholar). All of the subjects developed the hallmarks of acute inflammation, as indicated by: i) symptoms (chills, headache) and signs (elevated body temperature); ii) a rapid transient elevation of inflammatory cytokines; and iii) a dose-dependent increase in blood levels of CRP and SAA after the injection (28.Suffredini A.F. Hochstein H.D. McMahon F.G. Dose-related inflammatory effects of intravenous endotoxin in humans: evaluation of a new clinical lot of Escherichia coli O:113 endotoxin.J. Infect. Dis. 1999; 179: 1278-1282Crossref PubMed Scopus (130) Google Scholar). To investigate the ability of HDL and inflammatory HDL to promote sterol efflux from macrophages, we used ultracentrifugation (d = 1.063–1.21 g/ml) to isolate HDL from blood collected from each subject 30 min prior to (control HDL) and 24 h after (inflammatory HDL) the injection of saline or low doses of endotoxin. At these doses, endotoxin induced only a mild inflammation. The efflux capacity of inflammatory HDL was reduced (Fig. 1A) by ∼10% at 1 ng/kg of endotoxin and by ∼20% at 2 ng/kg (P = 0.009, ANOVA with Tukey's HSD). These observations are consistent with previous reports that the inflammatory response impairs HDL's ability to remove sterol from macrophages in mice and rabbits, as well as in humans (20.Banka C.L. Yuan T. de Beer M.C. Kindy M. Curtiss L.K. de Beer F.C. Serum amyloid A (SAA): influence on HDL-mediated cellular cholesterol efflux.J. Lipid Res. 1995; 36: 1058-1065Abstract Full Text PDF PubMed Google Scholar, 21.McGillicuddy F.C. de la Llera Moya M. Hinkle C.C. Joshi M.R. Chiquoine E.H. Billheimer J.T. Rothblat G.H. Reilly M.P. Inflammation impairs reverse cholesterol transport in vivo.Circulation. 2009; 119: 1135-1145Crossref PubMed Scopus (288) Google Scholar, 22.Annema W. Nijstad N. Tolle M. de Boer J.F. Buijs R.V. Heeringa P. van der Giet M. Tietge U.J. Myeloperoxidase and serum amyloid A contribute to impaired in vivo reverse cholesterol transport during the acute phase response but not group IIA secretory phospholipase A(2).J. Lipid Res. 2010; 51: 743-754Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 23.Artl A. Marsche G. Lestavel S. Sattler W. Malle E. Role of serum amyloid A during metabolism of ac
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