LDL particle core enrichment in cholesteryl oleate increases proteoglycan binding and promotes atherosclerosis
2013; Elsevier BV; Volume: 54; Issue: 9 Linguagem: Inglês
10.1194/jlr.m039644
ISSN1539-7262
AutoresJohn Melchior, Janet K. Sawyer, Kathryn L. Kelley, Ramesh Shah, Martha D. Wilson, Roy R. Hantgan, Lawrence L. Rudel,
Tópico(s)Advanced Glycation End Products research
ResumoSeveral studies in humans and animals suggest that LDL particle core enrichment in cholesteryl oleate (CO) is associated with increased atherosclerosis. Diet enrichment with MUFAs enhances LDL CO content. Steroyl O-acyltransferase 2 (SOAT2) is the enzyme that catalyzes the synthesis of much of the CO found in LDL, and gene deletion of SOAT2 minimizes CO in LDL and protects against atherosclerosis. The purpose of this study was to test the hypothesis that the increased atherosclerosis associated with LDL core enrichment in CO results from an increased affinity of the LDL particle for arterial proteoglycans. ApoB-100-only Ldlr−/− mice with and without Soat2 gene deletions were fed diets enriched in either cis-MUFA or n-3 PUFA, and LDL particles were isolated. LDL:proteogylcan binding was measured using surface plasmon resonance. Particles with higher CO content consistently bound with higher affinity to human biglycan and the amount of binding was shown to be proportional to the extent of atherosclerosis of the LDL donor mice. The data strongly support the thesis that atherosclerosis was induced through enhanced proteoglycan binding of LDL resulting from LDL core CO enrichment. Several studies in humans and animals suggest that LDL particle core enrichment in cholesteryl oleate (CO) is associated with increased atherosclerosis. Diet enrichment with MUFAs enhances LDL CO content. Steroyl O-acyltransferase 2 (SOAT2) is the enzyme that catalyzes the synthesis of much of the CO found in LDL, and gene deletion of SOAT2 minimizes CO in LDL and protects against atherosclerosis. The purpose of this study was to test the hypothesis that the increased atherosclerosis associated with LDL core enrichment in CO results from an increased affinity of the LDL particle for arterial proteoglycans. ApoB-100-only Ldlr−/− mice with and without Soat2 gene deletions were fed diets enriched in either cis-MUFA or n-3 PUFA, and LDL particles were isolated. LDL:proteogylcan binding was measured using surface plasmon resonance. Particles with higher CO content consistently bound with higher affinity to human biglycan and the amount of binding was shown to be proportional to the extent of atherosclerosis of the LDL donor mice. The data strongly support the thesis that atherosclerosis was induced through enhanced proteoglycan binding of LDL resulting from LDL core CO enrichment. Atherosclerosis is the disease process in the artery wall leading to clinical complications of coronary heart disease, a disease that has cost more lives in the United States over the past century than the next four diseases combined (1Roger V.L. Go A.S. Lloyd-Jones D.M. Benjamin E.J. Berry J.D. Borden W.B. Bravata D.M. Dai S. Ford E.S. Fox C.S. et al.Heart disease and stroke statistics–2012 update: a report from the American Heart Association.Circulation. 2012; 125: e1002Google Scholar). The subendothelial retention and accumulation of LDL in the artery wall is now recognized as an initiating event in the pathophysiology of atherosclerosis (2Williams K.J. Tabas I. The response-to-retention hypothesis of early atherogenesis.Arterioscler. Thromb. Vasc. Biol. 1995; 15: 551-561Crossref PubMed Google Scholar). After LDL particles enter the intima of the artery wall, they can interact with proteoglycans, which are structural proteins residing in the extracellular matrix that consist of one or more glycosaminoglycan (GAG) side-chains attached to core proteins. The LDL-proteoglycan complex is generally regarded as an electrostatic interaction (3Camejo G. The interaction of lipids and lipoproteins with the intercellular matrix of arterial tissue: its possible role in atherogenesis.Adv. Lipid Res. 1982; 19: 1-53Crossref PubMed Google Scholar). The sulfation pattern of the acidic sugar groups that form the GAG chains on the proteoglycans results in an overall negative charge that can interact with clusters of positively charged amino acid residues on apoB-100, the primary apolipoprotein present on the LDL particle (4Skålén K. Gustafsson M. Rydberg E.K. Hulten L.M. Wiklund O. Innerarity T.L. Boren J. Subendothelial retention of atherogenic lipoproteins in early atherosclerosis.Nature. 2002; 417: 750-754Crossref PubMed Scopus (728) Google Scholar). A proteoglycan frequently deposited in atherosclerotic plaques of humans (5O'Brien K.D. Olin K.L. Alpers C.E. Chiu W. Ferguson M. Hudkins K. Wight T.N. Chait A. Comparison of apolipoprotein and proteoglycan deposits in human coronary atherosclerotic plaques: colocalization of biglycan with apolipoproteins.Circulation. 1998; 98: 519-527Crossref PubMed Scopus (246) Google Scholar, 6Zeng X. Chen J. Miller Y.I. Javaherian K. 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Wilson M.D. Sawyer J.K. Rudel L.L. Dietary fat-induced alterations in atherosclerosis are abolished by ACAT2-deficiency in ApoB100 only, LDLr−/− mice.Arterioscler. Thromb. Vasc. Biol. 2007; 27: 1396-1402Crossref PubMed Scopus (44) Google Scholar, 9Rudel L.L. Bond M.G. Bullock B.C. LDL heterogeneity and atherosclerosis in nonhuman primates.Ann. N. Y. Acad. Sci. 1985; 454: 248-253Crossref PubMed Scopus (48) Google Scholar, 10Rudel L.L. Johnson F.L. Sawyer J.K. Wilson M.S. Parks J.S. Dietary polyunsaturated fat modifies low-density lipoproteins and reduces atherosclerosis of nonhuman primates with high and low diet responsiveness.Am. J. Clin. Nutr. 1995; 62: 463S-470SCrossref PubMed Scopus (57) Google Scholar–11Rudel L.L. Parks J.S. Sawyer J.K. Compared with dietary monounsaturated and saturated fat, polyunsaturated fat protects African green monkeys from coronary artery atherosclerosis.Arterioscler. Thromb. Vasc. Biol. 1995; 15: 2101-2110Crossref PubMed Scopus (178) Google Scholar). In nonhuman primates, consumption of dietary MUFAs resulted in LDL particles containing a CE core enriched in cholesterol oleate (CO), which were positively associated with coronary artery atherosclerosis (r= 0.8) (10Rudel L.L. Johnson F.L. Sawyer J.K. Wilson M.S. Parks J.S. Dietary polyunsaturated fat modifies low-density lipoproteins and reduces atherosclerosis of nonhuman primates with high and low diet responsiveness.Am. J. Clin. Nutr. 1995; 62: 463S-470SCrossref PubMed Scopus (57) Google Scholar, 11Rudel L.L. Parks J.S. Sawyer J.K. Compared with dietary monounsaturated and saturated fat, polyunsaturated fat protects African green monkeys from coronary artery atherosclerosis.Arterioscler. Thromb. Vasc. Biol. 1995; 15: 2101-2110Crossref PubMed Scopus (178) Google Scholar). Importantly, a similar diet-related shift in the CE FA composition of LDL has been observed in humans in which consumption of oleate-enriched diets resulted in elevations in the percentage of CO in the LDL CE (12Reaven P. Parthasarathy S. Grasse B.J. Miller E. Steinberg D. Witztum J.L. Effects of oleate-rich and linoleate-rich diets on the susceptibility of low density lipoprotein to oxidative modification in mildly hypercholesterolemic subjects.J. Clin. Invest. 1993; 91: 668-676Crossref PubMed Scopus (327) Google Scholar). Additionally, the large Atherosclerosis Risk in Communities Study revealed a significant positive association between the percentage of plasma CE as CO and carotid artery intima-media thickness in patients with preclinical atherosclerosis (13Ma J. Folsom A.R. Lewis L. Eckfeldt J.H. Relation of plasma phospholipid and cholesterol ester fatty acid composition to carotid artery intima-media thickness: the Atherosclerosis Risk in Communities (ARIC) Study.Am. J. Clin. Nutr. 1997; 65: 551-559Crossref PubMed Scopus (76) Google Scholar). Steroyl O-acyltransferase 2 (SOAT2) is a membrane-bound enzyme localized to the endoplasmic reticulum in hepatocytes and enterocytes that is at least partially responsible for the incorporation of CO into apoB-containing lipoproteins (14Parini P. Davis M. Lada A.T. Erickson S.K. Wright T.L. Gustafsson U. Sahlin S. Einarsson C. Eriksson M. Angelin B. et al.ACAT2 is localized to hepatocytes and is the major cholesterol-esterifying enzyme in human liver.Circulation. 2004; 110: 2017-2023Crossref PubMed Scopus (168) Google Scholar). Gene deletion of Soat2 in mouse models has been shown to protect against LDL CO enrichment and the development of atherosclerosis, supporting the claim that inhibition of CO production is a viable therapeutic target in the treatment of disease (8Bell III, T.A. Kelley K. Wilson M.D. Sawyer J.K. Rudel L.L. Dietary fat-induced alterations in atherosclerosis are abolished by ACAT2-deficiency in ApoB100 only, LDLr−/− mice.Arterioscler. Thromb. Vasc. Biol. 2007; 27: 1396-1402Crossref PubMed Scopus (44) Google Scholar, 15Lee R.G. Kelley K.L. Sawyer J.K. Farese Jr, R.V. Parks J.S. Rudel L.L. Plasma cholesteryl esters provided by lecithin:cholesterol acyltransferase and acyl-coenzyme a:cholesterol acyltransferase 2 have opposite atherosclerotic potential.Circ. Res. 2004; 95: 998-1004Crossref PubMed Scopus (100) Google Scholar). Although evidence consistently implicates CO as an important component in the development of atherosclerosis, the mechanism by which it promotes accelerated disease development has remained elusive. The primary goal of this study was to establish whether LDL particle core enrichment with CO enhances interactions between the LDL particle and resident arterial proteoglycans, which could accelerate the development of atherosclerosis via increased LDL particle retention. To accomplish this goal, we developed a novel assay using surface plasmon resonance (SPR) technology to quantify the interaction of LDL particles with arterial proteoglycans. The apoB-100-only Ldlr−/− mouse (75% C57Bl/6, 25% 129S/Sv) (16Veniant M.M. Zlot C.H. Walzem R.L. Pierotti V. Driscoll R. Dichek D. Herz J. Young S.G. Lipoprotein clearance mechanisms in LDL receptor-deficient "Apo-B48-only" and "Apo-B100-only" mice.J. Clin. Invest. 1998; 102: 1559-1568Crossref PubMed Scopus (119) Google Scholar) used in this study was a gift from Dr. Steven Young. Two mouse lines on the apoB-100-only LDL receptor-deficient background were compared for these studies; apoB-100-only Ldlr−/−, Soat2+/+ and apoB-100-only Ldlr−/−, Soat2−/−, which were generated as previously described (8Bell III, T.A. Kelley K. Wilson M.D. Sawyer J.K. Rudel L.L. Dietary fat-induced alterations in atherosclerosis are abolished by ACAT2-deficiency in ApoB100 only, LDLr−/− mice.Arterioscler. Thromb. Vasc. Biol. 2007; 27: 1396-1402Crossref PubMed Scopus (44) Google Scholar). Littermate controls were used to account for genetic heterogeneity. At about 6 weeks of age, mice were switched from a rodent chow diet to one of two semipurified diets consisting of 10% energy as fat with 0.02% cholesterol (wet weight) enriched in either cis-MUFAs (55% of total FAs) or long-chain n-3 PUFAs (20% of total FAs). Details of the dietary ingredients and FA compositions have been previously described (8Bell III, T.A. Kelley K. Wilson M.D. Sawyer J.K. Rudel L.L. Dietary fat-induced alterations in atherosclerosis are abolished by ACAT2-deficiency in ApoB100 only, LDLr−/− mice.Arterioscler. Thromb. Vasc. Biol. 2007; 27: 1396-1402Crossref PubMed Scopus (44) Google Scholar). All experimental animals were euthanized between 10 and 16 weeks of diet treatment. Various endpoints were assessed during this period at the time points indicated. While the plasma cholesterol concentrations and atherosclerosis extent both tend to rise with time of diet exposure, the mice used for LDL binding studies were also evaluated for atherosclerosis extent so that direct comparisons could be made. Mice were maintained in an American Association for Accreditation of Laboratory Animal Care-approved pathogen-free animal facility, and the institutional Animal Care and Use Committee at Wake Forest University Health Sciences approved all experimental protocols. Blood was taken from each mouse at the terminal time point via heart puncture and transferred to tubes containing 10 μl of protease inhibitor cocktail (Sigma P2714) in 5% Azide and 5% EDTA, and lipids and lipoproteins were measured. Plasma was separated from red blood cells by centrifugation at 7,500 g for 15 min at 4°C. Total plasma cholesterol (TPC) concentrations were determined by enzymatic assay as described previously (17Carr T.P. Andresen C.J. Rudel L.L. Enzymatic determination of triglyceride, free cholesterol, and total cholesterol in tissue lipid extracts.Clin. Biochem. 1993; 26: 39-42Crossref PubMed Scopus (483) Google Scholar, 18Temel R.E. Lee R.G. Kelley K.L. Davis M.A. Shah R. Sawyer J.K. Wilson M.D. Rudel L.L. Intestinal cholesterol absorption is substantially reduced in mice deficient in both ABCA1 and ACAT2.J. Lipid Res. 2005; 46: 2423-2431Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). Cholesterol distribution among lipoprotein classes was determined after an aliquot of whole plasma was fractionated by gel filtration chromatography using a Superose-6 10/30 column (GE Healthcare) run at a flow rate of 0.5 ml/min as previously described (15Lee R.G. Kelley K.L. Sawyer J.K. Farese Jr, R.V. Parks J.S. Rudel L.L. Plasma cholesteryl esters provided by lecithin:cholesterol acyltransferase and acyl-coenzyme a:cholesterol acyltransferase 2 have opposite atherosclerotic potential.Circ. Res. 2004; 95: 998-1004Crossref PubMed Scopus (100) Google Scholar). It has previously been reported that isolation of LDL particles by density gradient ultracentrifugation (UC) can dissociate minor apolipoproteins from the particle surface, which could play a role in binding to arterial proteoglycans (19Stahlman M. Davidsson P. Kanmert I. Rosengren B. Boren J. Fagerberg B. Camejo G. Proteomics and lipids of lipoproteins isolated at low salt concentrations in D2O/sucrose or in KBr.J. Lipid Res. 2008; 49: 481-490Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). Thus, a majority of the binding experiments used LDL particles isolated chromatographically as previously described (8Bell III, T.A. Kelley K. Wilson M.D. Sawyer J.K. Rudel L.L. Dietary fat-induced alterations in atherosclerosis are abolished by ACAT2-deficiency in ApoB100 only, LDLr−/− mice.Arterioscler. Thromb. Vasc. Biol. 2007; 27: 1396-1402Crossref PubMed Scopus (44) Google Scholar). A Superose 6 10/30 column was equilibrated with sample buffer (100 mM HEPES, 20 mM NaCl, 2 mM MgCl2, 2 mM CaCl2, pH 7.4) containing 50 μl of protease inhibitor (Sigma P8340) per liter of buffer. For LDL isolation, 350 μl of plasma diluted 1:1 with PBS was injected onto the column, and fractions were eluted at a flow rate of 0.4 ml/min. For LDL binding assays, a narrow center of the peak window (23–28 min) was collected and pooled. For LDL compositional analysis, the entire window corresponding to the LDL peak was collected and pooled. After collection, LDLs were maintained at 4°C until analysis. For comparison, subsets of LDL were also isolated between densities of 1.019 and 1.063 g/ml by ultracentrifugation from pooled plasma (n = 3 mice per group) of all four experimental groups, as previously described (20Lee R.G. Shah R. Sawyer J.K. Hamilton R.L. Parks J.S. Rudel L.L. ACAT2 contributes cholesteryl esters to newly secreted VLDL, whereas LCAT adds cholesteryl ester to LDL in mice.J. Lipid Res. 2005; 46: 1205-1212Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). All centrifugation steps were performed at room temperature (RT) and the floated LDL was stored at either RT or 4°C until experimental use. Recombinant BGN was produced using a stable transfected 293-EBNA cell line at LifeCell Corporation. The cell line was created by transferring a BGN polyhistidine fusion construct, originally created for expression using a vaccinia virus expression system (21Hocking A.M. Strugnell R.A. Ramamurthy P. McQuillan D.J. Eukaryotic expression of recombinant biglycan. Post-translational processing and the importance of secondary structure for biological activity.J. Biol. Chem. 1996; 271: 19571-19577Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar), into the pCEP4 (Invitrogen Corporation; Carlsbad, CA) expression vector. The resulting plasmid was transfected into 293-EBNA cells and stable-expressing cells selected by culturing in the presence of hygromycin B. Following amplification, cells were seeded into a Celligen Plus bioreactor (New Brunswick Scientific; Edison, NJ) containing 100 g Fibra-Cel disks and grown to saturation. Protein production was initiated by replacing the culture media with serum-free DMEM. Conditioned media was collected every 48 h, with fresh media added back to the bioreactor. Following concentration of the conditioned media using a Pellicon 2 tangential flow system (Millipore Corporation; Bedford, MA), recombinant biglycan was purified using nickel chelating chromatography and elution with a gradient of 0–250 mM imidazole in 20 mM Tris-HCl, 500 mM NaCl, 0.2% CHAPS, pH 8.0. Protein core that had not been well glycosylated was subsequently separated from proteoglycan following anion exchange chromatography on Q-Sepharose and elution with a linear gradient of 0.15–2 M NaCl in PBS, 0.2% CHAPS. Using Coomassie staining, there was no detectable unglycosylated core protein remaining in the proteoglycan preparation as seen after polyacrylamide gel electrophoresis. A Biacore T100 SPR platform (22Hantgan R.R. Stahle M.C. Integrin priming dynamics: mechanisms of integrin antagonist-promoted alphaIIbbeta3:PAC-1 molecular recognition.Biochemistry. 2009; 48: 8355-8365Crossref PubMed Scopus (35) Google Scholar, 23Hantgan R.R. Stahle M.C. Horita D.A. Entropy drives integrin alphaIIbbeta3:echistatin binding–evidence from surface plasmon resonance spectroscopy.Biochemistry. 2008; 47: 2884-2892Crossref PubMed Scopus (12) Google Scholar) was used to quantify the interaction of LDL with immobilized BGN. In the first step, the dextran surfaces of a CM5 biosensor chip (Biacore, Inc.; Piscataway, NJ), for both reference and sample channels, were activated for amine coupling (22Hantgan R.R. Stahle M.C. Integrin priming dynamics: mechanisms of integrin antagonist-promoted alphaIIbbeta3:PAC-1 molecular recognition.Biochemistry. 2009; 48: 8355-8365Crossref PubMed Scopus (35) Google Scholar). Following surface activation, an antigen affinity-purified polyclonal goat IgG, specific to BGN core protein (R and D Systems; AF2667), was covalently coupled to the dextran surface of the chip through its lysine residues, reproducibly yielding a sparse IgG monolayer in both channels (sample channel, 14,464 ± 1,606 response unit (RU); reference channel, 11,643 ± 2,720 RU; n = 5). Ethanolamine treatment was then used to block any unreacted sites on the chip surface. Following mAb immobilization, the system was equilibrated with sample buffer. Each binding cycle began with the delivery of recombinant human BGN (24Berendsen A.D. Fisher L.W. Kilts T.M. Owens R.T. Robey P.G. Gutkind J.S. Young M.F. Modulation of canonical Wnt signaling by the extracellular matrix component biglycan.Proc. Natl. Acad. Sci. USA. 2011; 108: 17022-17027Crossref PubMed Scopus (115) Google Scholar, 25Groeneveld T.W. Oroszlan M. Owens R.T. Faber-Krol M.C. Bakker A.C. Arlaud G.J. McQuillan D.J. Kishore U. Daha M.R. Roos A. Interactions of the extracellular matrix proteoglycans decorin and biglycan with C1q and collectins.J. Immunol. 2005; 175: 4715-4723Crossref PubMed Scopus (102) Google Scholar, 26Mercado M.L. Amenta A.R. Hagiwara H. Rafii M.S. Lechner B.E. Owens R.T. McQuillan D.J. Froehner S.C. Fallon J.R. Biglycan regulates the expression and sarcolemmal localization of dystrobrevin, syntrophin, and nNOS.FASEB J. 2006; 20: 1724-1726Crossref PubMed Scopus (49) Google Scholar–27Rafii M.S. Hagiwara H. Mercado M.L. Seo N.S. Xu T. Dugan T. Owens R.T. Hook M. McQuillan D.J. Young M.F. et al.Biglycan binds to alpha- and gamma-sarcoglycan and regulates their expression during development.J. Cell. Physiol. 2006; 209: 439-447Crossref PubMed Scopus (46) Google Scholar), which had been purified by ion exchange chromatography as described above and kindly provided by Dr. Rick Owens (LifeCell Corporation; Branchburg, NJ). The BGN was added to the sample channel at a concentration of 34 μg/ml protein at a flow rate of 20 μl/min for 180 s followed by a 300 s stabilization period to achieve ∼10% saturation of the mAb sample channel surface (1,239 ± 18 RU, n = 30) (see supplementary Fig. IA). BGN capture was quite stable, with an average RU loss of only 3.6 ± 0.9% over 1,700 s. Immunocapturing BGN through its protein core increases the exposure of its GAG chains to enable LDL binding. After BGN immobilization, various LDL particles were delivered in sample buffer at a flow rate of 30 μl/min for 700 s (binding) to both the reference (mAb only) and sample channels (mAb-BGN), followed by sample buffer at the same rate for 1,000 s (dissociation) (see supplementary Fig. IB). After the binding interaction, 3 M sodium thiocynate was delivered at a flow rate of 30 μl/min to remove the LDL:BGN complex, yielding a fresh mAb surface (Residual RU after each wash =−1 ±−1, n = 6). The biosensor surface was then equilibrated for 300 s with sample buffer prior to the next cycle of BGN capture, LDL binding, and regeneration. A majority of the binding assays were performed using LDL isolated from whole plasma by HPLC. LDLs were maintained at 10°C in the SPR sample compartment, and data were collected at 25°C. Importantly, binding experiments were performed with fresh LDL (within 24 h of preparation of plasma) to avoid any potential loss in binding signal over time (see supplementary Fig. II). Binding kinetics and concentration curves (0–40 μg/ml LDL cholesterol) for LDL isolated from pooled plasma samples (n = 3 mice/pool) can be found in Fig. 2. All remaining binding experiments were performed on LDL from individual mice at a concentration of 30 μg/ml of LDL cholesterol. To ensure that CE composition-related differences in binding were not influenced by extraneous proteins coeluting with the LDL particles isolated by HPLC, comparator binding assays were also performed on LDL isolated by UC. Because particle storage temperature and binding temperature also could affect LDL binding, particles isolated and stored at RT were maintained at 25°C in the sample compartment, and data were collected at either 37°C or 25°C (see supplementary Fig. IIIA, B). Particles were also stored at 4°C and maintained in the sample compartment at 10°C, and binding data were collected at 25°C (see supplementary Fig. IIIC for comparison). LDL particle size was determined using size exclusion chromatography with concomitant dynamic light scattering (DLS). After LDL particle isolation, 150 μl of the LDL was injected onto the Superose 6 column and eluted with sample buffer at a flow rate of 0.4 ml/min. Detection was performed at a wavelength of 661 nm by refractive index detector model Optilab rEX (Wyatt Technology) and MALS detector model DAWN HELEOS II (Wyatt Technology). Signals from the detectors were processed using ASTRA software, version 6.0.1.10 (Wyatt Technology), and the hydrodynamic radius was evaluated at the peak of the size distribution curve, generated as a function of particle retention time. Chemical compositions of the isolated LDLs were determined with enzymatic assays for total cholesterol (TC), free cholesterol (FC), triglyceride (TG), and phospholipid (PL) (TC by Pointe Scientific, Inc.; FC, TG, and PL by Wako Chemicals USA). Total CE mass was calculated for individual LDLs by subtracting the FC from the TC and then multiplying by 1.67 to account for the mass of the acyl chain. LDL CEs were detected and quantified by mass spectrometry as previously described (28Miller C.D. Thomas M.J. Hiestand B. Samuel M.P. Wilson M.D. Sawyer J.K. Rudel L.L. Cholesteryl esters associated with ACAT2 predict coronary artery disease in patients with symptoms of acute coronary syndrome.Acad. Emerg. Med. 2012; 19: 673-682Crossref PubMed Scopus (15) Google Scholar). For CE composition analyses, 10 μl of the LDL isolated by HPLC was stored at −80°C under argon to prevent oxidation prior to analysis. At the time of analysis, samples were thawed and diluted in 1 ml of methanol solution containing 500 pg/μl of 17:0 CE as the internal standard and 1 ng/μl of sodium formate. CEs were measured using a Quattro II mass spectrometer equipped with a Z-spray interface (capillary voltage = 3.2 kV; cone voltage = 50 V; source temperature =−80°C; desolvation temperature = 200°C). Samples were maintained at 15°C in a temperature-controlled Spark Holland Reliance autosampler/stacker until analysis. Twenty-five microliters of each sample was infused into the mass spectrometer at 10 μl/min. CEs were quantified in the positive-ion mode by monitoring the common neutral loss of 368.5 Da. The CE molar concentrations were calculated from individual profiles (see supplementary Fig. IV) and the internal standard. Individual CE measurements are reported as percentages of total CE mass. Protein was measured by the method of Lowry et al. (29Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. Protein measurement with the Folin phenol reagent.J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar) on the ultracentrifuge-isolated LDL. Estimates of the protein for the column-isolated LDL were extrapolated by using the TC:PRO ratio of the centrifugally isolated LDL preparations. Data for the LDL chemical compositions are expressed as a percentage of the total lipid plus protein mass. Aortic atherosclerosis was evaluated using both en face and chemical techniques. At the time of necropsy, aortas were removed from mice and preserved in 10% neutral buffered formalin. After a minimum of 24 h fixation, all extraneous tissue and arterial branches were removed from the aorta. In a subset of three mice per experimental group, the aorta was opened longitudinally, pinned flat, and photographed with an mm marker on the photo. After the image was grabbed using Image J software, the entire surface of the aorta and each lesion area were digitized using a digitizing tablet interfaced to a computer using the mm marker as a correction factor. Lesion areas were defined as raised opaque areas on the aortic surface. The percent surface area with lesions was calculated as a simple ratio of aortic lesion area to aorta total surface area. In aortae from all animals, including those used for surface measurements, lipids were extracted in 2:1 chloroform-methanol in a test tube containing a known amount of 5α-cholestane as an internal standard. TC and FC (the latter after saponification) were determined by gas-liquid chromatography, and aortic CE concentration was calculated. The correlation between aortic CE concentrations and surface area occupied by lesion was consistently r≥ 0.9 in this and other experiments (see supplementary Fig. V). All statistical analyses were conducted using JMP Software (version 5.0.1.2; Cary, NC). Two-way analysis of variance was used for analyses in which two independent variables were compared, and statistical significance is indicated where observed. Relationships between binding and disease parameters were analyzed by regression analysis and least-squares best-fit regression lines; regression coefficients are included in those figures. The data shown in Fig. 1 are from mice of each diet and genotype group as measured after 11 weeks of diet exposure. Two-way ANOVA was used to identify statistically significant differences. In apoB-100-only Ldlr−/− mice, consumption of a diet containing n-3 PUFA limited diet-induced hypercholesterolemia compared with mice fed the cis-MUFA diet (Fig. 1A). No significant difference in TPC between Soat2+/+ versus Soat2−/− mice was seen. Cholesterol distributions among the lipoprotein classes showed that the increase in TPC levels observed in the cis-MUFA-fed mice was a result of significant elevations in cholesterol of both VLDL and LDL, with LDLc comprising about 80% of the total cholesterol pool in both Soat2 +/+ and Soat2 −/− mice (Fig. 1B,C). No significant difference between Soat2 genotypes was seen for either VLDL or LDL cholesterol concentrations by two-way ANOVA. For HDL cholesterol, both dietary FA (P= 0.0008) and Soat2 genotype (P= 0.03) effects were found to be significant by two-way ANOVA. Percentage compositions of LDL particles isolated by HPLC from individual mice are shown in Table 1. The primary differences in lipid components were limited to the core lipids, i.e., CE and TG. Compared with LDL of Soat2+/+ mice, LDL particles produced by the Soat2−/− mice contained lower percentages of CE and higher percentages of TG, and these differences appeared to be independent of diet. The combined proportions of surface constituents (FC, PL, plus PRO) versus core lipids (CE plus TG) were similar for all groups, and particle diameters, obtained by DLS, showed only a small range of sizes among the particles (23–26 nm). Soat2+/+ mice consuming the cis-MUFA diet had slightly larger LDL particles
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