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• W2160840655 abstract "LCAT, a plasma enzyme that esterifies cholesterol, has been proposed to play an antiatherogenic role, but animal and epidemiologic studies have yielded conflicting results. To gain insight into LCAT and the role of free cholesterol (FC) in atherosclerosis, we examined the effect of LCAT over- and underexpression in diet-induced atherosclerosis in scavenger receptor class B member I-deficient [Scarab(−/−)] mice, which have a secondary defect in cholesterol esterification. Scarab(−/−)×LCAT-null [Lcat(−/−)] mice had a decrease in HDL-cholesterol and a high plasma ratio of FC/total cholesterol (TC) (0.88 ± 0.033) and a marked increase in VLDL-cholesterol (VLDL-C) on a high-fat diet. Scarab(−/−)×LCAT-transgenic (Tg) mice had lower levels of VLDL-C and a normal plasma FC/TC ratio (0.28 ± 0.005). Plasma from Scarab(−/−)×LCAT-Tg mice also showed an increase in cholesterol esterification during in vitro cholesterol efflux, but increased esterification did not appear to affect the overall rate of cholesterol efflux or hepatic uptake of cholesterol. Scarab(−/−)×LCAT-Tg mice also displayed a 51% decrease in aortic sinus atherosclerosis compared with Scarab(−/−) mice (P < 0.05). In summary, we demonstrate that increased cholesterol esterification by LCAT is atheroprotective, most likely through its ability to increase HDL levels and decrease pro-atherogenic apoB-containing lipoprotein particles. LCAT, a plasma enzyme that esterifies cholesterol, has been proposed to play an antiatherogenic role, but animal and epidemiologic studies have yielded conflicting results. To gain insight into LCAT and the role of free cholesterol (FC) in atherosclerosis, we examined the effect of LCAT over- and underexpression in diet-induced atherosclerosis in scavenger receptor class B member I-deficient [Scarab(−/−)] mice, which have a secondary defect in cholesterol esterification. Scarab(−/−)×LCAT-null [Lcat(−/−)] mice had a decrease in HDL-cholesterol and a high plasma ratio of FC/total cholesterol (TC) (0.88 ± 0.033) and a marked increase in VLDL-cholesterol (VLDL-C) on a high-fat diet. Scarab(−/−)×LCAT-transgenic (Tg) mice had lower levels of VLDL-C and a normal plasma FC/TC ratio (0.28 ± 0.005). Plasma from Scarab(−/−)×LCAT-Tg mice also showed an increase in cholesterol esterification during in vitro cholesterol efflux, but increased esterification did not appear to affect the overall rate of cholesterol efflux or hepatic uptake of cholesterol. Scarab(−/−)×LCAT-Tg mice also displayed a 51% decrease in aortic sinus atherosclerosis compared with Scarab(−/−) mice (P < 0.05). In summary, we demonstrate that increased cholesterol esterification by LCAT is atheroprotective, most likely through its ability to increase HDL levels and decrease pro-atherogenic apoB-containing lipoprotein particles. The reverse cholesterol transport (RCT) pathway plays a key role in protecting against atherosclerosis. During RCT, excess cholesterol is effluxed from peripheral cells, such as aortic macrophages, and is transported back to the liver where it can be excreted or converted to a bile salt. LCAT plays a vital role in this process (1Kunnen S. Van Eck M. Lecithin:cholesterol acyltransferase: old friend or foe in atherosclerosis?.J. Lipid Res. 2012; 53: 1783-1799Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar), as it is the only known plasma enzyme that esterifies free cholesterol (FC). The esterification of cholesterol promotes net cholesterol efflux by trapping it in the hydrophobic core of lipoproteins, thus preventing its back exchange (2Schwartz C.C. VandenBroek J.M. Cooper P.S. Lipoprotein cholesteryl ester production, transfer, and output in vivo in humans.J. Lipid Res. 2004; 45: 1594-1607Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). Cholesterol esterification also promotes cholesterol efflux by increasing the concentration gradient of FC between cells and extracellular lipoprotein acceptors (2Schwartz C.C. VandenBroek J.M. Cooper P.S. Lipoprotein cholesteryl ester production, transfer, and output in vivo in humans.J. Lipid Res. 2004; 45: 1594-1607Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar, 3Czarnecka H. Yokoyama S. Regulation of cellular cholesterol efflux by lecithin:cholesterol acyltransferase reaction through nonspecific lipid exchange.J. Biol. Chem. 1996; 271: 2023-2028Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Radiolabeled cholesterol tracer studies in humans indicate that once esterified by LCAT, cholesteryl esters are preferentially returned to the liver (2Schwartz C.C. VandenBroek J.M. Cooper P.S. Lipoprotein cholesteryl ester production, transfer, and output in vivo in humans.J. Lipid Res. 2004; 45: 1594-1607Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar), in part by scavenger receptor class B member I (SR-BI) (4Schwartz C.C. Zech L.A. VandenBroek J.M. Cooper P.S. Cholesterol kinetics in subjects with bile fistula. Positive relationship between size of the bile acid precursor pool and bile acid synthetic rate.J. Clin. Invest. 1993; 91: 923-938Crossref PubMed Scopus (47) Google Scholar, 5Shamburek R.D. Zech L.A. Cooper P.S. Vandenbroek J.M. Schwartz C.C. Disappearance of two major phosphatidylcholines from plasma is predominantly via LCAT and hepatic lipase.Am. J. Physiol. 1996; 271: E1073-E1082Crossref PubMed Google Scholar). As a consequence of its proposed anti-atherogenic effects, LCAT is now being investigated as a possible therapeutic target (6Chen Z. Wang S.P. Krsmanovic M.L. Castro-Perez J. Gagen K. Mendoza V. Rosa R. Shah V. He T. Stout S.J. et al.Small molecule activation of lecithin cholesterol acyltransferase modulates lipoprotein metabolism in mice and hamsters.Metabolism. 2012; 61: 470-481Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 7Rousset X. Shamburek R. Vaisman B. Amar M. Remaley A.T. Lecithin cholesterol acyltransferase: an anti- or pro-atherogenic factor?.Curr. Atheroscler. Rep. 2011; 13: 249-256Crossref PubMed Scopus (71) Google Scholar, 8Rousset X. Vaisman B. Amar M. Sethi A.A. Remaley A.T. Lecithin: cholesterol acyltransferase–from biochemistry to role in cardiovascular disease.Curr. Opin. Endocrinol. Diabetes Obes. 2009; 16: 163-171Crossref PubMed Scopus (138) Google Scholar, 9Shamburek R.D. Freeman L. Sampson M. Bakker-Arkema R. Krause B. Auerbach B. Homan R. Shamburek A. Schwartz C. Amar M. et al.Human enzyme replacement therapy in a patient with familial lecithin cholesterol acyltransferase deficiency: rapid appearance of normal appearing HDL.Circulation. 2013; 128: A18673Google Scholar). In addition to promoting RCT, the esterification of cholesterol may have other benefits in preventing the development of atherosclerosis. Excess FC in plasma membranes can adversely affect the function of numerous plasma membrane proteins, and high levels of FC are cytotoxic (10Kellner-Weibel G. Jerome W.G. Small D.M. Warner G.J. Stoltenborg J.K. Kearney M.A. Corjay M.H. Phillips M.C. Rothblat G.H. Effects of intracellular free cholesterol accumulation on macrophage viability: a model for foam cell death.Arterioscler. Thromb. Vasc. Biol. 1998; 18: 423-431Crossref PubMed Scopus (138) Google Scholar, 11Warner G.J. Stoudt G. Bamberger M. Johnson W.J. Rothblat G.H. Cell toxicity induced by inhibition of acyl coenzyme A:cholesterol acyltransferase and accumulation of unesterified cholesterol.J. Biol. Chem. 1995; 270: 5772-5778Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar). Inhibition of the intracellular esterification of cholesterol by ACAT inhibitors has been shown to accelerate atherosclerosis and increase cardiovascular events in clinical trials (12Nicholls S.J. Sipahi I. Andrews J. Wolski K. Schoenhagen P. Crowe T. Desai M.Y. Tuzcu E.M. Nissen S.E. Proatherogenic impact of the ACAT inhibitor pactimibe in patients with coronary artery disease and multiple risk factors: insights from the ACTIVATE study.Circulation. 2006; 114: II-224Google Scholar). Recently, the enrichment of FC in red blood cell (RBC) plasma membranes has also been implicated in the development of atherosclerosis (13Hung K.T. Berisha S.Z. Ritchey B.M. Santore J. Smith J.D. Red blood cells play a role in reverse cholesterol transport.Arterioscler. Thromb. Vasc. Biol. 2012; 32: 1460-1465Crossref PubMed Scopus (41) Google Scholar, 14Lin H.L. Xu X.S. Lu H.X. Zhang L. Li C.J. Tang M.X. Sun H.W. Liu Y. Zhang Y. Pathological mechanisms and dose dependency of erythrocyte-induced vulnerability of atherosclerotic plaques.J. Mol. Cell. Cardiol. 2007; 43: 272-280Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 15Tziakas D. Chalikias G. Grapsa A. Gioka T. Tentes I. Konstantinides S. Red blood cell distribution width: a strong prognostic marker in cardiovascular disease: is associated with cholesterol content of erythrocyte membrane.Clin. Hemorheol. Microcirc. 2012; 51: 243-254Crossref PubMed Scopus (52) Google Scholar), and RBC cholesterol content has been proposed as a possible cardiovascular biomarker (15Tziakas D. Chalikias G. Grapsa A. Gioka T. Tentes I. Konstantinides S. Red blood cell distribution width: a strong prognostic marker in cardiovascular disease: is associated with cholesterol content of erythrocyte membrane.Clin. Hemorheol. Microcirc. 2012; 51: 243-254Crossref PubMed Scopus (52) Google Scholar, 16Tziakas D.N. Kaski J.C. Chalikias G.K. Romero C. Fredericks S. Tentes I.K. Kortsaris A.X. Hatseras D.I. Holt D.W. Total cholesterol content of erythrocyte membranes is increased in patients with acute coronary syndrome: a new marker of clinical instability?.J. Am. Coll. Cardiol. 2007; 49: 2081-2089Crossref PubMed Scopus (90) Google Scholar). Patients with a wide variety of primary dyslipidemias, such as abetalipoproteinemia, for example, are known to have elevated levels of FC in RBC membranes due to an altered exchange of excess FC on lipoproteins with circulating RBCs (2Schwartz C.C. VandenBroek J.M. Cooper P.S. Lipoprotein cholesteryl ester production, transfer, and output in vivo in humans.J. Lipid Res. 2004; 45: 1594-1607Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar, 17Vayá A. Martínez Triguero M. Réganon E. Vila V. Martínez Sales V. Solá E. Hernández Mijares A. Ricart A. Erythrocyte membrane composition in patients with primary hypercholesterolemia.Clin. Hemorheol. Microcirc. 2008; 40 ([Erratum. 2009. 41: 149.]): 289-294Crossref PubMed Scopus (23) Google Scholar). The enrichment of FC in RBC membranes can lead to altered cell shape and function (18Cooper R.A. Durocher J.R. Leslie M.H. Decreased fluidity of red cell membrane lipids in abetalipoproteinemia.J. Clin. Invest. 1977; 60: 115-121Crossref PubMed Scopus (112) Google Scholar, 19Holm T.M. Braun A. Trigatti B.L. Brugnara C. Sakamoto M. Krieger M. Andrews N.C. Failure of red blood cell maturation in mice with defects in the high-density lipoprotein receptor SR-BI.Blood. 2002; 99: 1817-1824Crossref PubMed Scopus (106) Google Scholar). FC on lipoproteins is also known to exchange with the plasma membrane of platelets, and cholesterol enrichment has been demonstrated to promote platelet activation (20Shattil S.J. Bennett J.S. Colman R.W. Cooper R.A. Abnormalities of cholesterol-phospholipid composition in platelets and low-density lipoproteins of human hyperbetalipoproteinemia.J. Lab. Clin. Med. 1977; 89: 341-353PubMed Google Scholar, 21Knöfler R. Urano T. Taminato T. Yoshimi T. Nakano T. Nakajima K. Takada Y. Takada A. Daily variation of serum lipids in relation to the circadian rhythm of platelet aggregation in healthy male persons.Clin. Chim. Acta. 1995; 239: 109-119Crossref PubMed Scopus (7) Google Scholar, 22Shattil S.J. Anaya-Galindo R. Bennett J. Colman R.W. Cooper R. Platelet hypersensitivity induced by cholesterol incorporation.J. Clin. Invest. 1975; 55: 636-643Crossref PubMed Scopus (342) Google Scholar). To better understand the role of LCAT, particularly in the context of increased FC, we describe here the development of two novel transgenic (Tg) mice, namely SR-BI-deficient [Scarab(−/−)]×LCAT-null [Lcat(−/−)] mice (S−L−), which lack LCAT, and Scarab(−/−)×LCAT-Tg mice (S−L++), which overexpress human LCAT. RBCs and platelets from Scarab(−/−)×Lcat(+/+) (S−L+) mice are known to have elevated levels of FC (19Holm T.M. Braun A. Trigatti B.L. Brugnara C. Sakamoto M. Krieger M. Andrews N.C. Failure of red blood cell maturation in mice with defects in the high-density lipoprotein receptor SR-BI.Blood. 2002; 99: 1817-1824Crossref PubMed Scopus (106) Google Scholar), and HDL from these mice is increased in size and displays an impairment in cholesterol esterification, as only 40–50% of the cholesterol is esterified compared with approximately 75% observed in WT mice (23Trigatti B. Rayburn H. Viñals M. Braun A. Miettinen H. Penman M. Hertz M. Schrenzel M. Amigo L. Rigotti A. et al.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 (440) Google Scholar, 24Arai T. Rinninger F. Varban L. Fairchild-Huntress V. Liang C.P. Chen W. Seo T. Deckelbaum R. Huszar D. Tall A.R. Decreased selective uptake of high density lipoprotein cholesteryl esters in apolipoprotein E knock-out mice.Proc. Natl. Acad. Sci. USA. 1999; 96: 12050-12055Crossref PubMed Scopus (73) Google Scholar, 25Braun A. Trigatti B.L. Post M.J. Sato K. Simons M. Edelberg J.M. Rosenberg R.D. Schrenzel M. Krieger M. Loss of SR-BI expression leads to the early onset of occlusive atherosclerotic coronary artery disease, spontaneous myocardial infarctions, severe cardiac dysfunction, and premature death in apolipoprotein E-deficient mice.Circ. Res. 2002; 90: 270-276Crossref PubMed Scopus (426) Google Scholar, 26Kozarsky K.F. Donahee M.H. Glick J.M. Krieger M. Rader D.J. Gene transfer and hepatic overexpression of the HDL receptor SR-BI reduces atherosclerosis in the cholesterol-fed LDL receptor-deficient mouse.Arterioscler. Thromb. Vasc. Biol. 2000; 20: 721-727Crossref PubMed Scopus (306) Google Scholar, 27Van Eck M. Twisk J. Hoekstra M. Van Rij B.T. Van der Lans C.A.C. Bos I.S.T. Kruijt J.K. Kuipers F. Van Berkel T.J.C. Differential effects of scavenger receptor BI deficiency on lipid metabolism in cells of the arterial wall and in the liver.J. Biol. Chem. 2003; 278: 23699-23705Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, 28Yu H. Zhang W. Yancey P.G. Koury M.J. Zhang Y. Fazio S. Linton M.F. Macrophage apolipoprotein E reduces atherosclerosis and prevents premature death in apolipoprotein E and scavenger receptor-class BI double-knockout mice.Arterioscler. Thromb. Vasc. Biol. 2006; 26: 150-156Crossref PubMed Scopus (35) Google Scholar). The decrease in cholesterol esterification in S−L+ mice is presumably because of the decreased efficiency of large HDL particles to activate LCAT (29Ma K. Forte T. Otvos J.D. Chan L. Differential additive effects of endothelial lipase and scavenger receptor-class B type I on high-density lipoprotein metabolism in knockout mouse models.Arterioscler. Thromb. Vasc. Biol. 2005; 25: 149-154Crossref PubMed Scopus (29) Google Scholar, 30Barter P.J. Hopkins G.J. Gorjatschko L. Lipoprotein substrates for plasma cholesterol esterification: influence of particle size and composition of the high density lipoprotein subfraction 3.Atherosclerosis. 1985; 58: 97-107Abstract Full Text PDF PubMed Scopus (51) Google Scholar). Additionally, S−L+ mice have been described as having an increased propensity to diet-induced atherosclerosis despite their increased HDL-cholesterol (HDL-C) levels, possibly because of decreased hepatic delivery of cholesterol (23Trigatti B. Rayburn H. Viñals M. Braun A. Miettinen H. Penman M. Hertz M. Schrenzel M. Amigo L. Rigotti A. et al.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 (440) Google Scholar, 24Arai T. Rinninger F. Varban L. Fairchild-Huntress V. Liang C.P. Chen W. Seo T. Deckelbaum R. Huszar D. Tall A.R. Decreased selective uptake of high density lipoprotein cholesteryl esters in apolipoprotein E knock-out mice.Proc. Natl. Acad. Sci. USA. 1999; 96: 12050-12055Crossref PubMed Scopus (73) Google Scholar, 25Braun A. Trigatti B.L. Post M.J. Sato K. Simons M. Edelberg J.M. Rosenberg R.D. Schrenzel M. Krieger M. Loss of SR-BI expression leads to the early onset of occlusive atherosclerotic coronary artery disease, spontaneous myocardial infarctions, severe cardiac dysfunction, and premature death in apolipoprotein E-deficient mice.Circ. Res. 2002; 90: 270-276Crossref PubMed Scopus (426) Google Scholar, 26Kozarsky K.F. Donahee M.H. Glick J.M. Krieger M. Rader D.J. Gene transfer and hepatic overexpression of the HDL receptor SR-BI reduces atherosclerosis in the cholesterol-fed LDL receptor-deficient mouse.Arterioscler. Thromb. Vasc. Biol. 2000; 20: 721-727Crossref PubMed Scopus (306) Google Scholar, 27Van Eck M. Twisk J. Hoekstra M. Van Rij B.T. Van der Lans C.A.C. Bos I.S.T. Kruijt J.K. Kuipers F. Van Berkel T.J.C. Differential effects of scavenger receptor BI deficiency on lipid metabolism in cells of the arterial wall and in the liver.J. Biol. Chem. 2003; 278: 23699-23705Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, 28Yu H. Zhang W. Yancey P.G. Koury M.J. Zhang Y. Fazio S. Linton M.F. Macrophage apolipoprotein E reduces atherosclerosis and prevents premature death in apolipoprotein E and scavenger receptor-class BI double-knockout mice.Arterioscler. Thromb. Vasc. Biol. 2006; 26: 150-156Crossref PubMed Scopus (35) Google Scholar). In this study, we show that the alteration of LCAT expression in Scarab(−/−) mice results in profound changes in the percentage of cholesterol esterified and in the distribution of cholesterol on lipoproteins, RBCs, and platelets. Furthermore, we demonstrate that decreased cholesterol esterification caused by the loss of LCAT in S−L− mice further accentuates the development of atherosclerosis when compared with S−L+ mice. LCAT-Tg mice, containing 240 copies of the human LCAT gene, but with only a 2-fold increase in the rate of plasma cholesterol esterification (31Vaisman B.L. Klein H.G. Rouis M. Berard A.M. Kindt M.R. Talley G.D. Meyn S.M. Hoyt Jr, R.F. Marcovina S.M. Albers J.J. et al.Overexpression of human lecithin cholesterol acyltransferase leads to hyperalphalipoproteinemia in transgenic mice.J. Biol. Chem. 1995; 270: 12269-12275Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar), and Lcat(−/−) mice (32Lambert G. Sakai N. Vaisman B.L. Neufeld E.B. Marteyn B. Analysis of glomerulosclerosis and atherosclerosis in lecithin cholesterol acyltransferase-deficient mice.J. Biol. Chem. 2001; 276: 15090-15098Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar), both on a C57BL/6N background, were crossed with S−L+ mice (Jackson Laboratory, stock number 3379) which were backcrossed three times onto the C57BL/6N background. The resultant new strains were designated as either S−L++ or S−L− mice (31Vaisman B.L. Klein H.G. Rouis M. Berard A.M. Kindt M.R. Talley G.D. Meyn S.M. Hoyt Jr, R.F. Marcovina S.M. Albers J.J. et al.Overexpression of human lecithin cholesterol acyltransferase leads to hyperalphalipoproteinemia in transgenic mice.J. Biol. Chem. 1995; 270: 12269-12275Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 32Lambert G. Sakai N. Vaisman B.L. Neufeld E.B. Marteyn B. Analysis of glomerulosclerosis and atherosclerosis in lecithin cholesterol acyltransferase-deficient mice.J. Biol. Chem. 2001; 276: 15090-15098Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). Probucol was added to the chow of S−L+ mice to facilitate breeding (33Miettinen H.E. Rayburn H. Krieger M. Abnormal lipoprotein metabolism and reversible female infertility in HDL receptor (SR-BI)-deficient mice.J. Clin. Invest. 2001; 108: 1717-1722Crossref PubMed Scopus (145) Google Scholar). C57BL/6N mice (WT) were purchased from Taconic (Albany, NY). Mice were housed under controlled conditions, with a 12/12 h light/dark cycle, and fed either a standard rodent autoclaved chow diet containing 4.0% fat (NIH31 chow diet; Zeigler Brothers Inc., Gardners, PA) or a Western diet (TD.88137 Adjusted Calories Diet; Harlan Teklad, Madison, WI). Only female mice were used in this study and, starting at 2 months of age, were fed ad libitum with a Western diet or chow diet for 7 months. All animal procedures were approved by a National Institutes of Health Institutional Animal Care and Use Committee (protocol H-0050R2). Unless otherwise noted, all chemicals used were obtained from Sigma-Aldrich (St. Louis, MO). Blood samples were collected from the periorbital sinus of eyes, with a heparinized capillary tube, and EDTA-plasma was obtained by centrifugation for 10 min at 3,000 g at 4°C. EDTA-whole blood (500 μl) was used to determine blood cell populations, using a Cell-Dyn 3700 (Abbott, Abbott Park, IL). For lipid extraction, blood cells were washed with normal saline and centrifuged at 4°C, 3,000 g for 10 min. Lipids were extracted by lysing the RBCs with an equal volume of distilled water followed by extraction of lipids by an overnight incubation with 20 vol of chloroform/methanol (2:1). Total cholesterol (TC), FC, and phospholipids were analyzed by enzymatic assays from Wako Chemicals (Richmond, VA). Triglyceride levels were measured by an enzymatic assay from Roche Diagnostics (Indianapolis, IN). Lipoprotein profiles in plasma were obtained by fast-protein LC (FPLC) (Akta FPLC; GE Healthcare) on two Superose-6 columns in series. For each group, 400 μl of pooled plasma from at least four mice was used for FPLC and lipids were measured enzymatically. To analyze the protein content of FPLC fractions, 50 μl from four fractions corresponding to 2 ml of elution volume were pooled together and 2 mg of lipid removal agent (Sigma) were added to concentrate lipoprotein samples. After washing, lipoproteins were eluted from the lipid removal agent with SDS loading buffer (Life Technologies, Grand Island, NY). For immunoblotting, equal volumes of sample were loaded onto a 4–12% Bis-Tris gel (Life Technologies) and electrophoresed at 200 V in MOPS buffer. Proteins were transferred to polyvinylidene difluoride membrane (Life Technologies), using a Pierce G2 Fast Blotter (Thermo Scientific, Rockford, IL) for 15 min at 25 V and 3.2 A. Following the protein transfer, membranes were blocked with 5% milk and 3% BSA. Membranes were stained sequentially with the following antibodies: mouse anti-mouse apoB-100/48 (5 μg/ml, 1:500) (kindly provided by Dr. Steve Young), rabbit anti-mouse apoA-I serum (1:2,000), and apoE serum (1:1,000) (Meridian Life Science, Inc., Memphis, TN). Primary antibodies were detected with the appropriate secondary antibody conjugated to HRP (Abcam, Cambridge, MA). Staining was visualized on an Omega Lum C (Aplegen, San Francisco, CA) using the WesternBright Quantum detection kit (Advansta Inc., Menlo Park, CA). FPLC fractions were pooled into three peaks corresponding to VLDL, LDL, and HDL and labeled 1, 2, and 3, respectively (Fig. 2). Lipoproteins were concentrated using lipid removal agent (Sigma). Samples were digested with trypsin at 37°C overnight. The digest was separated with a nanoLC system and detected in data-dependent analysis mode on an LTQ Orbitrap Elite or Fusion (Thermo Fisher Scientific, San Jose, CA). Relative abundance of each protein of interest was estimated based on its total spectrum counts across the fractions/samples. The VLDL triglyceride production study was carried out as previously described (34Maugeais C. Tietge U.J. Tsukamoto K. Glick J.M. Rader D.J. Hepatic apolipoprotein E expression promotes very low density lipoprotein-apolipoprotein B production in vivo in mice.J. Lipid Res. 2000; 41: 1673-1679Abstract Full Text Full Text PDF PubMed Google Scholar). Plasma from fasting mice was collected following injection with Triton WR-1339 at 0, 30, 60, 120, and 180 min postinjection. All reagents used in real time PCR experiments were obtained from Life Technologies unless otherwise noted. RNA was isolated from frozen liver tissue using RNAlater®-ICE. RNA was extracted from thawed tissue, using Trizol, and reverse transcription was carried out using Moloney murine leukemia virus reverse transcriptase oligo(dT) primers, and 1 μg of total RNA. Gene expression analysis was performed with TaqMan® Universal PCR Master Mix and commercially available primers for murine Apob, Hmgcr, Ldlr, and Pcsk9 on an ABI 7900. Values were normalized to murine Actb and then normalized to expression of each gene in the WT mice. Values are expressed as log(2−(ΔΔCT)). apoB-depleted serum was prepared by precipitation with polyethylene glycol (20%, v/v, in glycine buffer, pH 7.4). Global cholesterol efflux capacity of serum HDL samples was determined as described in detail elsewhere (35Mweva S. Paul J.L. Cambillau M. Goudouneche D. Beaune P. Simon A. Fournier N. Comparison of different cellular models measuring in vitro the whole human serum cholesterol efflux capacity.Eur. J. Clin. Invest. 2006; 36: 552-559Crossref PubMed Scopus (24) Google Scholar, 36de 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 (329) Google Scholar, 37Kempen H.J. Gomaraschi M. Bellibas S.E. Plassmann S. Zerler B. Collins H.L. Adelman S.J. Calabresi L. Wijngaard P.L. Effect of repeated apoA-IMilano/POPC infusion on lipids, (apo)lipoproteins, and serum cholesterol efflux capacity in cynomolgus monkeys.J. Lipid Res. 2013; 54: 2341-2353Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). In brief, global cholesterol efflux was measured using J774 mouse macrophage cells in the presence of cAMP. Cells were preincubated with [3H]cholesterol and ACAT inhibitor Sandoz 58-035 (but not preloaded with mass cholesterol) overnight. Cells were then incubated overnight in 0.2% BSA with cAMP. After washing, cells were incubated for 4 h with the serum HDL samples (apoB-depleted serum) added at 2.8% (v/v). [3H]cholesterol released to serum after 4 h was measured by liquid scintillation counting. Cholesterol efflux is expressed as the radiolabel released as a percentage of [3H]cholesterol within cells before addition of serum. All efflux values were corrected by subtracting the small amount of radioactive cholesterol released from cells incubated with serum-free medium. When indicated, J774 cells for some experiments were first loaded with Ac-LDL (180 μg/ml) for 72 h. Following cholesterol loading, these cells were incubated with 2.8% apoB-depleted serum overnight. TC was determined in cell layers and standardized to total protein as described below. For determination of ex vivo LCAT activity, lipid was extracted from an aliquot of the efflux medium, and thin-layer chromatography was performed to quantitate the proportion of [3H]FC and [3H]cholesterol ester in the sample. Ex vivo LCAT activity is expressed as the proportion of [3H]cholesterol ester formed as a percentage of the total [3H]cholesterol released during the 4 h efflux period. For mass cholesterol uptake assay, 1.5 × 106 RAW cells (ATCC, Manassas, VA) were added to a 6-well tissue culture treated plate (Corning Inc., Corning, NY). Opti-MEM (Invitrogen) plus 3% mouse serum was added to the cells and incubated overnight. Cells were washed three times in PBS (Invitrogen), then lipids were extracted with 2:1 hexane:isopropanol solution. After removal of the organic solvent, protein from cells was extracted with 0.1 N NaOH/0.1% SDS. Protein concentration from each well was determined by BCA assay (Pierce, Rockford, IL). Lipids were resuspended in 100% ethanol, and levels of cholesterol were measured using the Amplex Red assay (Invitrogen). Hepatic cell uptake was performed in Fu5AH rat hepatoma cells, as previously described (38Alexander E.T. Vedhachalam C. Sankaranarayanan S. de la Llera-Moya M. Rothblat G.H. Rader D.J. Phillips M.C. Influence of apolipoprotein A-I domain structure on macrophage reverse cholesterol transport in mice.Arterioscler. Thromb. Vasc. Biol. 2011; 31: 320-327Crossref PubMed Scopus (24) Google Scholar), with the following modifications. Briefly, labeled media from the efflux assay were pooled and then applied to Fu5AH hepatoma cells. [3H]cholesterol uptake from serum, after incubation for 2 h, was measured by liquid scintillation counting. Cholesterol uptake is expressed as the radiolabel within cells as a percentage of [3H]cholesterol that was added to cells. To detect the level of cholesterol in erythrocytes and platelets, 15 μl of whole blood was washed once with ice-cold PBS. Cells were resuspended in 80 μl of antibody solution and incubated at room temperature for 20 min. After incubation, cells were fixed with 4% paraformaldehyde for 1 h at 4°C. Following fixation, cells were washed one time with PBS and resuspended in 500 μl of a 120 μg/ml filipin solution (Polysciences, Inc., Warrington, PA) supplemented with 2% dextran. Antibodies for erythrocytes and platelets, TER-119 and CD41 (BD Biosciences, San Jose, CA), respectively, were used at a 1/100 dilution. Cells were kept on ice until they could be analyzed by flow cytometry, using an LSRFortessa or FACsARIA (BD Biosciences). To assess the level of reticulated platelets, cells were stained as previously described (39Matic G.B. Rothe G. Schmitz G. Flow cytometric analysis of reticulated platelets.Curr. Protoc. Cytom. 2001; 17: 7.10.1-7.10.6Crossref Google Scholar, 40Dole V.S. Matuskova J. Vasile E" @default.
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• W2160840655 date "2015-07-01" @default.
• W2160840655 modified "2023-10-17" @default.
• W2160840655 title "Increased plasma cholesterol esterification by LCAT reduces diet-induced atherosclerosis in SR-BI knockout mice" @default.
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• W2160840655 doi "https://doi.org/10.1194/jlr.m048629" @default.
• W2160840655 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/4479333" @default.

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