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Effects of oxidative modification of cholesterol in isolated low density lipoproteins on cultured smooth muscle cells

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It has been proposed that low density lipoprotein (LDL) must undergo oxidative modification before it can participate in atherosclerosis. The present paper studied the effect of cholesterol oxidation in LDL on cultured vascular smooth muscle cells. LDL was oxidized by cholesterol oxidase (3-β-hydroxy-steroid oxidase) which catalyzes the oxidation of cholesterol to 4-cholesten-3 one and other oxidized cholesterol derivatives. Cholesterol oxidase treatment of LDL did not result in lipid peroxidation. Cultured rabbit aortic smooth muscle cells were morphologically changed following exposure to cholesterol oxidized LDL. Nile red, a hydrophobic probe which can selectively stain intracellular lipid droplets, was applied to detect the cellular lipid content after treatment with oxidized or non-oxidized LDL cholesterol. LDL which did not undergo oxidation of its cholesterol had no effect on the cells. However, cellular nile red fluorescence intensity was increased as the pre-incubation time of cholesterol oxidase with LDL increased. This was supported by HPLC analysis which revealed that the oxidized cholesterol content of treated cells increased. These findings suggest that cholesterol oxidation of LDL can alter lipid deposition in the cells and change cell morphology. The oxidation of cholesterol in vivo may play an important role in the modification of LDL which could contribute to the generation of the lipid-laden foam cells.

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References

  1. Palinski W, Rosenfeld ME, Yla-Herttuala S, Gartner GC, Socher SS, Butler SW, Parthasarathy S, Carew TE, Steinberg D, Witztum JL: Low density lipoprotein undergoes oxidative modification in vivo. Proc Natl Acad Sci USA 86: 1372–1376, 1989

    Google Scholar 

  2. Hegele RA: Lipoprotein (a): an emerging risk factor for atherosclerosis. Can J Cardiol 5: 263–265, 1989

    Google Scholar 

  3. Goldstein JL, Brown MS: The low-density lipoprotein pathway and its relation to atherosclerosis. Annu Rev Biochem 46: 897–930, 1977

    Google Scholar 

  4. Steinberg D: Lipoproteins and atherosclerosis: a look back and a look ahead. Arteriosclerosis 3: 283–301, 1983

    Google Scholar 

  5. Brown MS, Goldstein JL: Lipoprotein metabolism in the macrophage: implications for cholesterol deposition in atherosclerosis. Anna Rev Biochem 52: 223–261, 1983

    Google Scholar 

  6. Goldstein JL, Ho YK, Basu SK, Brown MS: Binding site on macrophages that mediates uptake and degradation of acetylated low density lipoprotein, producing massive cholesterol deposition. Proc Natl Acad Sci USA 76: 333–337, 1979

    Google Scholar 

  7. Buja LM, Kita T, Goldstein JL, Watanabe Y, Brown MS: Cellular pathology of progressive atherosclerosis in the WHHL rabbit: an animal model of familial hypercholesterolemia. Arteriosclerosis 3: 87–101, 1983

    Google Scholar 

  8. Rosenfeld ME, Tsukada T, Gown AM, Ross R: Fatty streak initiation in Watanabe Heritable Hyperlipemic and comparably hypercholesterolemic fat-fed rabbits. Arteriosclerosis 1: 9–23, 1987

    Google Scholar 

  9. Kuzuya M, Naito M, Funaki C, Hayashi T, Asai K, Kuzuya F: Protective role of intracellular glutathione against oxidized low density lipoprotein in cultured endothelial cells. Biochem Biophys Res Commun 163: 1466–1472, 1989

    Google Scholar 

  10. Steinberg D, Parthasarathy S, Carew TE: In vivo inhibition of foam cell development by probucol in Watanabe rabbits. Am J Cardiol 62: 6B-12B, 1988

    Google Scholar 

  11. Steinberg D, Parthasarathy S, Carew TE, Khoo JC, Witztum JL: Beyond cholesterol: Modifications of low-density lipoprotein that increase its atherogenicity. N Engl J Med 320: 915–924, 1989

    Google Scholar 

  12. Kodama T, Freeman M, Rohrer L, Zabrecky J, Matsudaira P, Krieger M: Type I macrophage scavenger receptor contains α-helical and collagen-like coiled coils. Nature 343: 531–535, 1990

    Google Scholar 

  13. Brown MS, Goldstein JL: Scavenging for receptors. Atherosclerosis 343: 508–509, 1990

    Google Scholar 

  14. Esterbauer H, Jurgens G, Quehenberger O, Koller E: Autooxidation of human low density lipoprotein: loss of polyunsaturated fatty acids and vitamin E and generation of aldehydes. J Lipid Res 28: 495–509, 1987

    Google Scholar 

  15. Steinbrecher UP, Witztum JL, Parthasarathy S, Steinberg D: Decrease in reactive amino groups during oxidation or endothelial cell modification of LDL: correlation with changes in receptor-mediated catabolism. Arteriosclerosis 1: 135–143, 1987

    Google Scholar 

  16. Steinbrecher UP: Oxidation of human low density lipoprotein results in derivatization of lysine residues of apolipoprotein B by lipid peroxide decomposition products. J Biol Chem 262: 3603–3608, 1987

    Google Scholar 

  17. Parthasarathy S, Fong LG, Otero D, Steinberg D: Recognition of solubilized apoproteins from delipidated, oxidized low density lipoprotein (LDL) by the acetyl-LDL receptor. Proc Natl Acad Sci USA 84: 537–540, 1987

    Google Scholar 

  18. Steinbrecher UP, Lougheed M, Kwan WC, Dirks M: Recognition of oxidized low density lipoprotein by the scavenger receptor of macrophages results from derivatization of apolipoprotein B by products of fatty acid peroxidation. J Biol Chem 264: 15216–15223, 1989

    Google Scholar 

  19. Pierce GN, Langer GA, Wright GB, Kutryk MJB: Calcium is rapidly exchangeable in cultured vascular smooth muscle cells from rabbit aorta. J Mol Cell Cardiol 21: 889–899, 1989

    Google Scholar 

  20. Chapman MJ, Goldstein S, Lagrange D, Lapland PM: A density gradient ultracentrifugal procedure for the isolation of the major lipoprotein classes from human serum. J Lipid Res 22: 339–358, 1981

    Google Scholar 

  21. Radding CM, Steinberg D: Studies on the synthesis and secretion of serum lipoproteins by rat liver slices. J Clin Invest 39: 1560–1569, 1960

    Google Scholar 

  22. Lowry OH, Rosebrough NJ, Farr AL, Randall AJ: Protein measurement with the Folin phenol reagent. J Biol Chem 193: 265–275, 1951

    CAS  PubMed  Google Scholar 

  23. Sale FO, Marchesini S, Fishman PH, Berra B: A sensitive enzymatic assay for determination of cholesterol in lipid extracts. Anal Biochem 142: 347–350, 1984

    Google Scholar 

  24. Greenspan P, Fowler SD: Spectrofluorometric studies of the lipid probe, nile red. J Lipid Res 26: 781–789, 1985

    Google Scholar 

  25. Greenspan P, Mayer EP, Fowler SD: Nile red: A selective fluorescent stain for intracellular lipid droplets. J Cell Biol 100: 965–973, 1985

    Google Scholar 

  26. Liu KZ, Maddaford TG, Ramjiawan B, Kutryk MJB, Pierce GN: Effects of cholesterol oxidase on cultured vascular smooth muscle cells. Mol Cell Biochem 108: 39–48, 1991

    Google Scholar 

  27. Sevanian A, McLeod LL: Cholesterol autoxidation in phospholipid membrane bilayers. Lipids 22: 627–636, 1987

    Google Scholar 

  28. Brown WJ, Warfel J, Greenspan P: Use of vile red stain in the detection of cholesteryl ester accumulation in acid lipase-deficient fibroblasts. Arch Pathol Lab Med 112: 295–297, 1988

    Google Scholar 

  29. Yla-Herttuala S, Palinski W, Rosenfeld ME, Parthasarathy S, Carew TE, Butler S, Witztum JL, Steinberg D: Evidence for the presence of oxidatively modified low density lipoprotein in atherosclerotic lesions of rabbit and man. J Clin Invest 84: 1086–1095, 1989

    Google Scholar 

  30. Thurnhofer H, Gains N, Mutsch B, Hauser H: Cholesterol oxidase as a structural probe of biological membranes: its application to brush-border membrane. Biochim Biophys Acta 856: 174–181, 1986

    Google Scholar 

  31. Peng SK, Tham P, Taylor CB, Mikkelson B: Cytotoxicity of oxidation derivatives of cholesterol on cultured aortic smooth muscle cells and their effect on cholesterol biosynthesis. Am J Clin Nutr 32: 1033–1042, 1979

    Google Scholar 

  32. Ogiso T, Iwaki M, Kurata A, Saito H: Effect of phospholipase A2 and cholesterol oxidase on perazine and promethazine penetration into human erythrocytes and on fluidity of the membrane. Chem Pharm Bull 36: 2168–2175, 1988

    Google Scholar 

  33. Kutryk MJB, Maddaford TG, Ramjiawan B, Pierce GN: Oxidation of membrane cholesterol alters active and passive transsarcolemmal calcium movement. Circ Res 68: 18–26, 1991

    Google Scholar 

  34. Salen G, Shefer S, Berginer VM: In the metabolic basis of inherited disease. In: JB Stanbury et al. (eds), Ed. 5. McGraw-Hill, New York, 1983, pp 713–730

  35. Kritchevsky D: Cholesterol. John Wiley and Sons, Inc., New York, 1958, p 132

    Google Scholar 

  36. Linder R: Alteration of mammalian membranes by the cooperative and antagonistic actions of bacterial proteins. Biochem Biophys Acta 779: 423–435, 1984

    Google Scholar 

  37. Hubbard RW, Ono Y, Sanchez A: Atherogenic effect of oxidized products of cholesterol. Prog Food Nutr Sci 13: 17–44, 1989

    Google Scholar 

  38. Redwell S, Dean RT, Jessup W: The action of defined oxygen-centred free radicals on human low-density lipoprotein. Biochem J 262: 707–712, 1989

    Google Scholar 

  39. Morel DW, Hessler JR, Chisolm GM: Low density lipoprotein cytotoxicity induced by free radical peroxidation of lipid. J Lipid Res 24: 1070–1076, 1983

    Google Scholar 

  40. Terao J, Sugino K, Matsushita S: Fe2+ and ascorbic acid induced oxidation of cholesterol in phosphatidylcholine liposomes and its inhibition by α-tocopherol. J Nutr Sci Vitaminol 31: 499–508, 1985

    Google Scholar 

  41. McCord JM: Free radicals and heart disease. In: JC Somogyi, HR Muller (eds) Nutritional Impact of Food Processing. Bibl Nutr Dicta, Karger, 1989, 43: 327–337

  42. Fowler SD, Mayer EP, Greenspan P: Foam cells and atherogenesis. Ann NY Acad Sci 454: 79–90, 1985

    Google Scholar 

  43. Fogelman AM, Shechter I, Seager J, Hokon M, Child JS, Edwards PA: Malondialdehyde alteration of low density lipoproteins leads to cholesteryl ester accumulation in hu man monocyte-macrophages. Proc Natl Acad Sci USA 77: 2214–2218, 1980

    Google Scholar 

  44. Lee DM: Malondialdehyde formation in stored plasma. Biochem Biophys Res Commun 95: 1663–1672, 1980

    Google Scholar 

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Liu, K., Ramjiawan, B., Kutryk, M.J.B. et al. Effects of oxidative modification of cholesterol in isolated low density lipoproteins on cultured smooth muscle cells. Mol Cell Biochem 108, 49–56 (1991). https://doi.org/10.1007/BF00239541

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  • DOI: https://doi.org/10.1007/BF00239541

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