Effects of Native and Oxidized Low Density Lipoproteins on Formation and Inactivation of EDRF and Vascular Smooth Muscle

  • E. Bassenge
  • J. Galle


Native and oxidized low density lipoproteins (LDL) were investigated for their direct influence on endothelium-derived relaxing factor (EDRF)-formation, EDRF-activity, and vascular smooth muscle tone. Native (n) LDL was isolated from fresh human plasma via sequential ultracentrifugation, and oxidized by Cu2+-incubation. EDRF released from cultured endothelial cells was inactivated by both n-LDL and ox-LDL (1 mg/ml), as detected in a bioassay system. n-LDL reduced the EDRF-mediated vasodilations of the detector segments by 38.5 ± 5.3%, and ox-LDL by 55.5 ± 4.6%. The effects of lipoproteins on EDRF-formation were studied on cultured endothelial cells, preincubated with either n-LDL or ox-LDL (lmg/ml for 1 hour) and stimulated for EDRF-release with bradykinin after washout of the lipoproteins. EDRF was assessed by measuring its stimulatory effect on the activity of a purified soluble guanylate cyclase. Preincubation with both n-LDL and ox-LDL did not reduce the bradykinin-induced EDRF-formation. Accordingly, acetylcholine-induced, EDRF-mediated dilations of intact rabbit femoral artery segments were not impaired by luminal exposure to n-LDL or ox-LDL (lmg/ml for 1 hour).

Effects of n-LDL and ox-LDL on vascular smooth muscle tone were investigated in isolated perfused rabbit femoral arteries. Perfusion of endothelium-intact and endothe-lium-denuded segments with ox-LDL (80–500 ug protein/ml) caused no, or only weak vasoconstriction in the absence of contractile agonists. However, in the presence of ox-LDL, vasoconstrictions to threshold concentrations of norepinephrine (NE), serotonin (5-HT), phenylephrine (PE) and potassium were significantly enhanced. Native LDL (80–1000µg/ml) had no effect on vascular tone, neither in the presence nor absence of contractile agonists. Incubation with verapamil, diltiazem and nitrendipine inhibited vasoconstrictions evoked by ox-LDL. The contractile responses to ox-LDL were significantly greater in endothelium-denuded segments than in endothelium-intact segments.

In conclusion, neither n-LDL nor ox-LDL acutely impair the formation of EDRF, but inactivate EDRF after its release from endothelial cells. n-LDL has no direct influence on vascular smooth muscle tone, but ox-LDL greatly enhances vasoconstrictions to various contractile agonists by direct interaction with vascular smooth muscle. Thus, in regions of lipoprotein accumulation in the arterial wall, the inactivation of EDRF and the potentiation of agonist-induced vasoconstrictions may favor inappropriate vasoconstrictions.


Contractile Response Guanylate Cyclase Organ Bath Soluble Guanylate Cyclase Detector Segment 
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  1. 1.
    Steinberg D, Parthasarathy S, Carew TE, Khoo JC, Witztum JL (1989) Beyond cholesterol: Modifications of low-density lipoprotein that increase its atherogenicity. N Engl J Med 320: 915–924PubMedCrossRefGoogle Scholar
  2. 2.
    Bossaller C, Habib GB, Yamamoto H, Williams C, Wells S, Henry PD (1987) Impaired muscarinic endothelium-dependent relaxation and cyclic guanosine 5’-monophosphate formation in atherosclerotic human coronary artery and rabbit aorta. J Clin Invest 79: 170–174PubMedCrossRefGoogle Scholar
  3. 3.
    Osborne JA, Lento PH, Siegfried MR, Stahl GL, Fusman B, Lefer AM (1989) Cardiovascular effects of acute hypercholesterolemia in rabbits. J Clin Invest 83: 465–473PubMedCrossRefGoogle Scholar
  4. 4.
    Hof RP, Hof A (1988) Vasoconstrictor and vasodilator effects in normal and atherosclerotic conscious rabbits. Br J Pharmacol 95: 1075–1080PubMedGoogle Scholar
  5. 5.
    Verbeuren T, Jordaens F, Zonnekeyn L, Van Hove C, Coene M, Herman A (1986) 1. Endothelium-dependent and endothelium-independent contractions and relaxations in isolated arteries of control and hypercholesterolemic rabbits. Circ Res 58: 552–564PubMedGoogle Scholar
  6. 6.
    Wines PA, Schmitz JM, Pfister SL, Clubb FJ, Buja LM, Willerson JT, Campbell WB (1989) Augmented vasoconstrictor responses to serotonin precede development of atherosclerosis in aorta of WHHL rabbit. Arteriosclerosis 9: 195–202PubMedCrossRefGoogle Scholar
  7. 7.
    Rosendorf C, Hoffman JIE, Verrier ED, Rouleau J, Boerboom LE (1981) Cholesterol potentiates the coronary artery response to norepinephrine in anesthetized and conscious dogs. Circ Res 48: 320–329Google Scholar
  8. 8.
    Tomoike H, Egashira K, Yamamoto Y, Nakamura M (1989) Enhanced responsiveness of smooth muscle, impaired endothelium-dependent relaxation and the genesis of coronary spasm. Am J Cardiol 63: 33E–39EPubMedCrossRefGoogle Scholar
  9. 9.
    Henry PD, Yokoyama M (1980) Supersensitivity of atherosclerotic rabbit aorta to ergonovine. Mediation by a serotonergic mechanism. J Clin Invest 66: 306–313Google Scholar
  10. 10.
    Heistad DD, Armstrong ML, Marcus ML, Piegors DJ, Mark AL (1984) Augmented responses to vasoconstrictor stimuli in hypercholesterolemic and atherosclerotic monkeys. Circ Res 54: 711–718PubMedGoogle Scholar
  11. 11.
    Holland JA, Pritchard KA, Rogers NJ, Stemerman MB. Perturbation of cultured human endothelial cells by atherogenic levels of low density lipoprotein (1988) Am J Phathol 132: 474–478Google Scholar
  12. 12.
    Cathcart MK, Morel DW, Chisolm GM. Monocyte and neutrophils oxidize low density lipoprotein making it cytotoxic (1985) J Leukocyte Biol 38: 341–350PubMedGoogle Scholar
  13. 13.
    Hennig B, Chow CK (1988) Lipid peroxidation and endothelial cell injury: implications in atherosclerosis. Free Radical Biol Med 4: 99–106CrossRefGoogle Scholar
  14. 14.
    Andrews HE, Bruckdorfer KR, Dunn RC, Jacobs M (1987) Low-density lipoproteins inhibit endothelium-dependent relaxation in rabbit aorta. Nature 327: 237–239PubMedCrossRefGoogle Scholar
  15. 15.
    Hoff HF, Morton RE (1985) Lipoproteins containing apoB extracted from human aortas. Ann NY Acad Sci 454: 183–194PubMedCrossRefGoogle Scholar
  16. 16.
    Daugherty A, Zweifel BS, Sobel BE, Schonfeld G (1988) Isolation of low density lipoprotein from atherosclerotic vascular tissue of Watanabe heritable hyperlipidemic rabbits. Arteriosclerosis 8: 768–777PubMedCrossRefGoogle Scholar
  17. 17.
    Palinski W, Rosenfeld ME, Ylä-Herttuala S, Gurtner GC, Socher SS, Butler SW, Parthasarathy S, Carew TE, Steinberg D, Witzum JL (1989) Low density lipoprotein undergoes oxidative modification in vivo. Proc Natl Acad Sci USA 86: 1372–1376PubMedCrossRefGoogle Scholar
  18. 18.
    Ylä-Herttuala S, Palinsky W, Rosenfeld ME, Parthasarathy S, Carew TE, Butler S, Witzum JL, Steinberg D (1989) Evidence for the presence of oxidatively modified low density lipoprotein in atherosclerotic lesions of rabbit and man. J Clin Invest 84: 1086–1095PubMedCrossRefGoogle Scholar
  19. 19.
    Bassenge E, Busse R (1988) Endothelial modulation of coronary tone. Prog Cardiovasc Dis 30: 349–380PubMedCrossRefGoogle Scholar
  20. 20.
    Edelstein C, Scanu AM (1986) Precautionary measures for collecting blood destinated for lipoprotein isolation. In: Segrest JP, Albers JJ (eds) Methods in enzymology. Academic Press, New York, pp 151–155Google Scholar
  21. 21.
    Havel RJ, Eder HA, Bragdon JH (1955) The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum. J Clin Invest 34: 1345–1353PubMedCrossRefGoogle Scholar
  22. 22.
    Bradford M (1975) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal Biochem 72: 248–254CrossRefGoogle Scholar
  23. 23.
    Esterbauer H, Striegel G, Puhl H, Rotheneder M (1989) Continuous monitoring of in vitro oxidation of human low density lipoprotein. Free Radical Res Comm 6: 67–75CrossRefGoogle Scholar
  24. 24.
    Steinbrecher UP, Witztum JL, Parthasarathy S, Steinberg D (1987) Decrease in reactive amino groups during oxidation or endothelial cell modification of LDL. Correlation with changes in receptor-mediated catabolism. Arteriosclerosis 7: 135–143PubMedCrossRefGoogle Scholar
  25. 25.
    Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685PubMedCrossRefGoogle Scholar
  26. 26.
    Lückhoff A, Busse R, Winter I, Bassenge E (1987) Characterization of vascular relaxant factor released from cultured endothelial cells. Hypertension 9: 295–303PubMedGoogle Scholar
  27. 27.
    Förstermann U, Goppelt-Strübe M, Frolich JC, Busse R (1986) Inhibitors of acyl-coenzyme A: Lysolecithin acyltranferase activates the production of endothelium-derived vascular relaxing factor. J Pharmacol Exp Ther 238: 352–359PubMedGoogle Scholar
  28. 28.
    Mülsch A, Böhme E, Busse R (1987) Stimulation of soluble guanylate cyclase by endothelium-derived relaxing factor from cultured endothelial cells. Eur J Pharmacol 135: 247–250PubMedCrossRefGoogle Scholar
  29. 30.
    Busse R, Pohl U, Kellner C, Klemm U (1983) Endothelial cells are involved in the vasodilatory response to hypoxia. Pflügers Arch 397: 78–80PubMedCrossRefGoogle Scholar
  30. 31.
    Galle J, Bassenge E, Busse R (1990) Oxidized low density lipoproteins potentiate vasoconstrictions to various contractile agonists by direct interaction with vascular smooth muscle. Circ Res 66: 1287–1293PubMedGoogle Scholar
  31. 32.
    Palmer RMJ, Ferrige AG, Moncada S (1987) Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature 327:524–526PubMedCrossRefGoogle Scholar
  32. 33.
    Cox DA, Vita JA, Treasure CB, Fish RD, Alexander RW, Ganz P, Selwyn AP (1989) Atherosclerosis impairs flow-mediated dilation of coronary arteries in humans. Circulation 80: 458–465PubMedCrossRefGoogle Scholar
  33. 34.
    Verbeuren TJ, Jordaens FH, VanHove CE, VanHoydonck AE, Herman AG (to be published) Release and vascular activity of the endothelium-derived relaxant factor in atherosclerotic rabbit aorta. Eur J PharmacolGoogle Scholar
  34. 35.
    Tomita T, Ezaki M, Miwa M, Nakamura K, Inoue Y (1990) Rapid and reversible inhibition by low density lipoprotein of the endothelium-dependent relaxation to hemostatic substances in porcine coronary arteries. Heat and acid labile factors in low density lipoprotein mediate the inhibition. Circ Res 66: 18–27Google Scholar
  35. 36.
    Kugiyama K, Bucay M, Morrisett JD, Roberts R, Henry PD (1989) Oxidized LDL impairs endothelium-dependent arterial relaxation. Circulation 80 (Suppl 2): 279Google Scholar
  36. 37.
    Vedernikov Y, Lankin V, Tikhaze A, Vikhert A (1988) Lipoproteins as factors in vessel tone and reactivity modulation. Basic Res Cardiol 83: 590–596PubMedCrossRefGoogle Scholar
  37. 38.
    Esterbauer H, Jürgens G, Quehenberger O, Koller E (1987) Autoxidation of human low density lipoprotein: Loss of polyunsaturated fatty acids and vitamin E and generation of aldehydes. J Lipid Res 28: 495–509PubMedGoogle Scholar
  38. 39.
    Morel DW, Hessler JR, Chisolm GM (1983) Low density cytotoxicity induced by free radical peroxidation of lipid. J Lipid Res 24: 1070–1076PubMedGoogle Scholar
  39. 40.
    Hessler JR, Robertson AL, Chisolm JR, Chisolm GM (1979) LDL induced cytotoxicity and its inhibition by HDL in human vascular smooth muscle and endothelial cells in culture. Ateriosclerosis 32: 213–229CrossRefGoogle Scholar
  40. 41.
    Esterbauer H, Rotheneder M, Striegel G, Waeg G, Ashy A, Sattler W, Jürgens G (1989) Vitamin E and other lipophiiic antioxidants protect LDL against oxidation. Fat Sci Technol 8: 316–324Google Scholar
  41. 42.
    Link WF (1965) Solubilities of inorganic and metal-organic compounds vol 2, 4th edn. American Chemical Society, Wasington DC, p 792Google Scholar
  42. 43.
    Griffith TM, Henderson AH, Edwards DH, Lewis MJ (1984) Isolated perfused rabbit coronary artery and aortic strip preparations: the role of endothelium-derived relaxant factor. J Physiol (Lond) 351: 13–24Google Scholar
  43. 44.
    Cohen RA, Zitnay KM, Weisbrod RM, Tesfamariam B (1988) Influence of the endothelium on tone and the response of isolated pig coronary artery to norepinephrine. J Pharmacol Exp Ther 244: 550–555PubMedGoogle Scholar
  44. 45.
    Chiba S, Tsukada M (1984) Potentiation of KCl-induced vasoconstriction by saponin treatment in isolated canine mesenteric arteries. Jpn J Pharmacol 36: 535–537PubMedCrossRefGoogle Scholar
  45. 46.
    Pohl U, Busse R (1987) Endothelium-derived relaxant factor inhibits the effect of nitrocompounds in isolated arteries. Am J Physiol 252: H307–H313PubMedGoogle Scholar
  46. 47.
    Shirasaki Y, Su C (1985) Endothelium removal augments vasodilation by sodium nitroprusside and sodium nitrite. Eur J Pharmacol 114: 93–96PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Tokyo 1991

Authors and Affiliations

  • E. Bassenge
  • J. Galle
    • 1
  1. 1.Department of Applied PhysiologyUniversity of FreiburgFreiburgFederal Republic of Germany

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