Cholesterol deposition in macrophages: foam cell formation mediated by cholesterol-enriched oxidized low density lipoprotein.

Open AccessPublished:January 01, 1997DOI:
      This paper is only available as a PDF. To read, Please Download here.
      Oxidized low density lipoprotein (LDL) is thought to mediate the transformation of macrophages to cholesterol-rich foam cells. Yet convincing evidence for this process is lacking in vitro. We suggest that oxidized LDL-mediated foam cell formation is not seen in vitro because the cholesteryl ester content of LDL particles (oxidized in the presence of transition metals) is dramatically reduced. Thus, if oxidized LDL could be cholesterol-enriched prior to its addition to macrophages, this lipoprotein would be made more capable of inducing the cellular deposition of cholesteryl esters. When we enriched cupric sulfate-oxidized LDL with cholesterol by incubation of this lipoprotein with unesterified cholesterol/phosphatidylcholine liposomes and added it to mouse peritoneal macrophage cultures, we found that: a) the enrichment of oxidized LDL with cholesterol did not alter the extent of oxidized LDL degradation; b) the cells accumulated massive amounts of cholesteryl ester (148 microg/mg cell protein) and unesterified cholesterol (260 microg/mg cell protein) after 24 h of incubation; and c) Sephacryl S-1000 chromatography of the cholesterol-enriched oxidized LDL verified the formation of large oxidized LDL-unesterified cholesterol/phosphatidylcholine complexes. These results demonstrate that oxidized LDL, when cholesterol-enriched, can mediate the formation of macrophage foam cells in culture


        • Fowler S.D.
        • Mayer E.P.
        • Greenspan P.
        Foam cells and atherogenesis.
        Ann. NY Acad. Sci. 1985; 454: 79-90
        • Steinberg D.
        • Parthasarathy S.
        • Carew T.E.
        • Khoo J.C.
        • Witztum J.L.
        Beyond cholesterol: modifications of low density lipoprotein that increase its athero-genicity.
        N. Engl. J. Med. 1989; 320: 915-924
        • Palinski W.
        • Rosenfeld M.E.
        • Yl-Herttuala S.
        • Gurtner G.C.
        • Socher S.S.
        • Butler S.W.
        • Parthasarathy S.
        • Carew T.E.
        • Steinberg D.
        • Witztum J.L.
        Low density lipoprotein undergoes oxidative modification in vivo.
        Proc. Natl. Acad. Sci. USA. 1989; 86: 1372-1376
        • Henriksen T.
        • Mahoney E.M.
        • Steinberg D.
        Enhanced macrophage degradation of biologically modified low density lipoprotein.
        Arteriosclerosis. 1983; 3: 149-159
        • Roma P.
        • Catapano A.L.
        • Bertulli S.M.
        • Varesi L.
        • Fu-magalli R.
        • Bernini F.
        Oxidized LDL increase free cholesterol and fail to stimulate cholesterol esterification in murine macrophages.
        Biochem. Biophys. Res. Commun. 1990; 171: 123-131
        • Musanti R.
        • Ghiselli G.
        Interaction of oxidized HDLs with J774-A1 macrophages causes intracellular accumulation of unesterified cholesterol.
        Arterioscler. Thromb. 1993; 13: 1334-1345
        • Ryu B-H.
        • Mao F.W.
        • Lou P.
        • Gutman R.L.
        • Greenspan P.
        Cholesteryl ester accumulation in macrophages treated with oxidized low density lipoprotein.
        Biosci. Biotech. Biochem. 1995; 59: 1619-1622
        • Klinkner A.M.
        • Waites C.R.
        • Kerns W.D.
        • Bugelski P.J.
        Evidence of foam cell and cholesterol crystal formation in macrophages incubated with oxidized LDL by fluorescence and electron microscopy.
        J. Histochem. Cy-tochem. 1995; 43: 1071-1078
        • McCloskey H.M.
        • Rothblat G.H.
        • Glick J.M.
        Incubation of acetylated low density lipoprotein with cholesterol-rich dispersions enhances cholesterol uptake by macrophages.
        Biochim. Biophys. Acta. 1987; 921: 320-332
        • Havel R.J.
        • Eder H.A.
        • Bragdon J.H.
        The distribution and chemical composition of ultracentrifu-gally separated lipoproteins in human serum.
        j. Clin. Invest. 1955; 34: 1345-1353
        • St. Clair R.W.
        • Mitschelen J.J.
        • Leight M.
        Metabolism by cells in culture of low-density lipoproteins of abnormal composition from non-human primates with diet-induced hypercholesterolemia.
        Biochim. Biophys. Acta. 1980; 618: 63-79
        • Yagi K.
        A simple fluorometric assay for lipoperox-ide in blood plasma.
        Biochem. Med. 1976; 15: 212-216
        • Glick J.M.
        • Adelman S.J.
        • Phillips M.C.
        • Rothblat G.H.
        Cellular cholesteryl ester clearance.
        J. Biol. Chem. 1983; 258: 13425-13430
        • Bartlett G.R.
        Phosphorus assay in column chromatography.
        J. Biol. Chem. 1959; 234: 466-468
        • Greenspan M.D.
        • Lo C-Y. L.
        • Hanf D.P.
        • Yudko-vitz J.B.
        Separation and identification of triglycerides, cholesteryl esters, cholesterol, 7-dehydrocholesterol, doli-chol, ubiquinone, tocopherol and retinol by high performance liquid chromatography with a diode array detector.
        J. Lipid Res. 1988; 29: 971-976
        • Lowry' O.H.
        • Rosebrough N.J.
        • Farr A.L.
        • Randall R.J.
        Protein mesurement with the Folin phenol reagent.
        J. Biol. Chem. 1951; 193: 265-275
        • Bligh E.G.
        • Dyer W.J.
        Rapid method of total lipid extraction and purification.
        Can.]. Biochem. Physiol. 1959; 37: 911-917
        • Goldstein J.I..
        • Brown M.S.
        Binding and degradation of low density lipoproteins by cultured human fibroblasts.
        J. Biol. Chem. 1974; 249: 5153-5162
        • Vercaemst R.
        • Union A.
        • Rosseneu M.
        Separation and quantitation of free cholesterol and cholesteryl esters in a macrophage cell line by high performance liquid chromatography.
        J. Chromatogr. 1989; 494: 43-52
        • Goldstein J.I.
        • Dana S.E.
        • Brown M.S.
        Esterification of low density lipoprotein cholesterol in human fibroblasts and its absence in homozygous familial hypercholesterolemia.
        Proc. Natl. Acad. Sci. USA. 1974; 71: 4288-4292
        • Goldstein J.L.
        • Hoff H.F.
        • Ho Y.K.
        • Basil S.K.
        • Brown M.S.
        Stimulation of cholesteryl ester synthesis in macrophages by extracts of atherosclerotic human aortas and complexes of albuminJcholesteryl esters.
        Arteriosclerosis. 1981; 1: 210-226
        • Mattson L.
        • Johansson H.
        • Ottosson M.
        • Bondjers G.
        • Wiklund O.
        Expression of lipoprotein lipase mRNA and secretion in macrophages isolated from human atherosclerotic aorta.
        J. Clin. Invest. 1993; 92: 1759-1765
        • Yui S.
        • Sasaki T.
        • Miyazaki A.
        • Horiuchi S.
        • Yama-zaki M.
        Induction of murine macrophage growth by modified LDLs.
        Arlerioscler. Thromb. 1993; 13: 331-337
        • Greenspan P.
        • Mayer E.P.
        • Fowler S.D.
        Nile red: a selective fluorescent stain for intracellular lipid droplets.
        j. Cell Biol. 1985; 100: 965-973
        • Brown A.J.
        • Dean R.T.
        • Jessup W.
        Free and esterified oxysterol: formation during copper-oxidation of low density lipoprotein and uptake by macrophages.
        J. lipid Res. 1996; 37: 320-335
        • Greenspan P.
        Phosphatidylserine-mediated delivery of cholesterol to macrophages: a novel experimental method for the generation of foam cells.
        Biochim. Biophys. Acta. 1990; 1045: 94-96
        • Stocker R.
        • Bowry V.W.
        • Frei B.
        Ubiquinol-10 protects human low density lipoprotein more efficiently against lipid peroxidation than does tocopherol.
        Proc. Natl. Acad. Sci. USA. 1991; 88: 1646-1650
        • Kritharides L.
        • Jessup W.
        • Gifford J.
        • Dean R.T.
        A method for defining the stages of low density lipoprotein oxidation by the separation of cholesterol- and cholesteryl ester oxidation products using HPLC.
        Anal. Biochem. 1993; 213: 79-89
        • Steinbrecher U.P.
        • Witztum J.L.
        • Parthasarathy S.
        • Steinberg D.
        Decrease in the reactive ami no groups during oxidation or endothelial cell modification of LDL: correlation with changes in receptor-mediated catabolism.
        Arteriosclerosis. 1987; 7: 135-143
        • Reaven 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 hypercho-lesterolemic subjects.
        J. Clin. Invest. 1993; 91: 668-676
        • Steinbrecher U.P.
        • Lougheed M.
        Scavenger receptor-independent stimulation of cholesterol esterification in macrophages by low density lipoprotein extracted from human aortic intima.
        Arteriosckr. Thromb. 1992; 12: 608-625
        • Tertov V.V.
        • Orekhov A.N.
        • Sobenin I.A.
        • Gabbasov Z.A.
        • Popov E.G.
        • Yaroslavov A.A.
        • Smirnov V.N.
        Three types of naturally occurring modified lipoproteins induce intracellular lipid accumulation due to lipoprotein aggregation.
        Circ. Res. 1992; 71: 218-228
        • Khoo J.C.
        • Miller E.
        • McLoughlin P.
        • Steinberg D.
        Enhanced macrophage uptake of low density lipoprotein after self-aggregation.
        Arteriosclerosis. 1988; 8: 348-358
        • Suits A.G.
        • Chait A.
        • Aviram M.
        • Heinecke J.W.
        Phagocytosis of aggregated lipoprotein by macrophages: low density lipoprotein receptor-dependent foam-cell formation.
        Proc. Natl. Acad. Sci. USA. 1989; 86: 2713-2717
        • Vijayagopal P.
        • Srinivasan S.R.
        • Radhakrishnamurthy B.
        • Berenson G.S.
        Human monocyte-derived macrophages bind low-density-lipoprotein-proteoglycan complexes by a receptor different from the low-density-lipo-protein receptor.
        Biochem. J. 1993; 289: 837-844
        • Greenspan P.
        • Ryu B-H.
        • Mao F.W.
        • Gutman R.L.
        Association of negatively charged phospholipids with low-density lipoprotein (LDL) increases its uptake and the deposition of cholesteryl esters by macrophages.
        Biochim. Biophys. Acta. 1995; 1257: 257-264
        • HofF H.F.
        • Whitaker T.E.
        • O̕Neil J.
        Oxidation of low density lipoprotein leads to particle aggregation and altered macrophage recognition.
        J. Biol. Chem. 1992; 267: 602-609
        • Brown M.S.
        • Basu S.K.
        • Falck J.R.
        • Ho Y.K.
        • Goldstein J.L.
        The scavenger cell pathway for lipoprotein degradation: specificity of the binding site that mediates the uptake of negatively charged LDL by macrophages.
        J. Supramol. Struct. 1980; 13: 67-81
        • Zhang H.
        • Basra H.J.K.
        • Steinbrecher U.P.
        Effects of oxidatively modified LDL on cholesterol esterification in cultured macrophages.
        J. Lipid Res. 1990; 31: 13611369
        • Maor I.
        • Aviram M.
        Oxidized low density lipoprotein leads to macrophage accumulation of unesterified cholesterol as a result of lysosomal trapping of the lipoprotein hydrolyzed cholesteryl ester.
        J. Lipid Res. 1994; 35: 803-819
        • Haberland M.E.
        • Fong D.
        • Cheng L.
        Malondi-aldehyde altered protein occurs in atheroma of Watanabe heritable hyperlipidemic rabbits.
        Science. 1988; 241: 215-218
        • Yla-Herttuala S.
        • Palinski W.
        • Rosenfeld M.E.
        • Parthasarathy S.
        • Carew T.E.
        • Butler S.
        • Witztum J.L.
        • Steinberg D.
        Evidence for the presence of oxidatively modified low density lipoprotein in atherosclerotic lesions of rabbit and man.
        J. Clin. Invest. 1989; 84: 1086-1095
        • Chao F-F.
        • Amende L.M.
        • Blanchette-Mackie E.J.
        • Skarlatos S.
        • Gamble W.
        • Resau J.H.
        • Mergner W.T.
        • Kruth H.S.
        Unesterified cholesterol-rich particles in atherosclerotic lesions of human and rabbit aortas.
        Am. J. Pathol. 1988; 131: 73-83
        • Chao F-F.
        • Blanchette-Mackie E.J.
        • Dickens B.F.
        • Gamble W.
        • Kruth H.S.
        Development of unesterified cholesterol-rich lipid particles in atherosclerotic lesions of WHHL and cholesterol-fed NZW rabbits.
        J. Lipid Res. 1994; 35: 71-83
        • Mora R.
        • Simionescu M.
        • Simionescu N.
        Purification and partial characterization of extracellular liposomes isolated from the hyperlipidemic rabbit aorta.
        J. Lipid Res. 1990; 31: 1793-1807
        • Kruth H.S.
        Filipin-positive, Oil red O-negative particles in atherosclerotic lesions induced by cholesterol feeding.
        Lab. Invest. 1983; 50: 87-93
        • Kruth H.S.
        Localization of unesterified cholesterol in human atherosclerotic lesions.
        Am. J. Pathol. 1984; 114: 201-208
        • Kruth H.S.
        Subendothelial accumulation of unesterified cholesterol.
        Atherosclerosis. 1985; 57: 337-341
        • Simionescu N.
        • Vasile E.
        • Lupu F.
        • Popescu G.
        • Simionescu M.
        Prelesional events in atherosclerosis. Accumulation of extracellular cholesterol-rich liposomes in the arterial intima and cardiac valves of the hyperlipidemic rabbit.
        Am. J. Pathol. 1986; 123: 109-125
        • Chao F-F.
        • Blanchette-Mackie E.J.
        • Tertov V.V.
        • Skarlatos S.I.
        • Chen Y-J.
        • Kruth H.S.
        Hydrolysis of cholesteryl ester in low density lipoprotein converts this lipoprotein to a liposome.
        J. Biol. Chem. 1992; 267: 4992-4998
        • Chung B.H.
        • Tallis G.
        • Yalamoori V.
        • Ananthara-maiah G.M.
        • Segrest J.P.
        Lipid-like particles isolated from human atherosclerotic plaques are structurally and compositionally similar to surface remnants of triglyceride-rich lipoproteins.
        Arterioscler. Thromb. 1994; 14: 622-635