Pflügers Archiv - European Journal of Physiology

, Volume 455, Issue 5, pp 811–818 | Cite as

Endothelial function in aorta segments of apolipoprotein E-deficient mice before development of atherosclerotic lesions

  • Paul FransenEmail author
  • Tim Van Assche
  • Pieter-Jan Guns
  • Cor E. Van Hove
  • Gilles W. De Keulenaer
  • Arnold G. Herman
  • Hidde Bult
Cardiovascular System


Acetylcholine (ACh)-induced relaxation declines in apolipoprotein E-deficient (apoE−/−) mouse aortas, but only after atherosclerotic plaque formation. This study investigated intracellular calcium concentrations [Ca2+]i and changes in phenylephrine-induced contractions as index of baseline nitric oxide (NO) bioavailability before plaque development. Isometric contractions of thoracic aorta rings of young (4 months) apoE−/− and C57BL/6J (WT) mice were evoked by phenylephrine (3 × 10−9–3 × 10−5 M) in the presence and absence of endothelial cells (ECs) or NO synthase (NOS) inhibitors. [Ca2+]i (Fura-2 AM) and endothelium-dependent relaxation were measured at baseline and after ACh stimulation. Segments of apoE−/− mice were significantly more sensitive and developed more tension than WT segments in response to phenylephrine. The differences disappeared after NOS inhibition or EC removal or upon increasing [Ca2+]i in apoE−/− strips with 10−6 M cyclopiazonic acid or 10−7 M Ca2+-ionophore A23187. Expression of endothelial NOS (eNOS) mRNA was similar in apoE−/− and WT aorta segments. Basal [Ca2+]i was significantly lower in apoE−/− than in WT strips. Relaxation by ACh (3 × 10−9–10−5 M) was time- and dose-dependently related to [Ca2+]i, but neither ACh-induced relaxation nor Ca2+ mobilization were diminished in apoE−/− strips. In conclusion, basal, but not ACh-induced NO bioavailability, was compromised in lesion-free aorta of apoE−/− mice. Decreased basal NO bioavailability was not related to lower eNOS expression, but most likely related to lower basal [Ca2+]i. These findings further point to important differences between basal and stimulated eNOS activity.


Atherosclerosis Apolipoprotein E-deficient mouse Endothelial cell Intracellular calcium [Ca2+]i Nitric oxide synthase Thoracic aorta Basal nitric oxide 





apolipoprotein E-deficient


intracellular calcium concentration


cyclopiazonic acid


endothelial cell


\( N^{\omega } \)-nitro-l-arginine methyl ester


\( N^{\omega } \)-nitro-l-arginine


nitric oxide


nitric oxide synthase


endothelial nitric oxide synthase


wild type



The technical assistance of Valerie Croons and Ludo Zonnekeyn and the secretarial help of Liliane Van den Eynde are greatly appreciated. This work was supported by grants from Bijzonder Onderzoek Fonds of the University of Antwerp to Paul Fransen (project 1083, BOF KP 2005) and Fonds voor Wetenschappelijk Onderzoek (FWO, Vlaamse Gemeenschap, project G.0174.06).


  1. 1.
    Barton M, Haudenschild CC, d’Uscio LV, Shaw S, Munter K, Lüscher TF (1998) Endothelin ETA receptor blockade restores NO-mediated endothelial function and inhibits atherosclerosis in apolipoprotein E-deficient mice. Proc Natl Acad Sci USA 95:14367–14372PubMedCrossRefGoogle Scholar
  2. 2.
    Bult H (1996) Nitric oxide and atherosclerosis: possible implications for therapy. Mol Med Today 2:510–518PubMedCrossRefGoogle Scholar
  3. 3.
    Burdyga T, Shmygol A, Eisner DA, Wray S (2003) A new technique for simultaneous and in situ measurements of Ca2+ signals in arteriolar smooth muscle and endothelial cells. Cell Calcium 34:27–33PubMedCrossRefGoogle Scholar
  4. 4.
    Cohen RA, Plane F, Najibi S, Huk I, Malinski T, Garland CJ (1997) Nitric oxide is the mediator of both endothelium-dependent relaxation and hyperpolarization of the rabbit carotid artery. Proc Natl Acad Sci USA 94:4193–4198PubMedCrossRefGoogle Scholar
  5. 5.
    Cohen RA, Weisbrod RM, Gericke M, Yaghoubi M, Bierl C, Bolotina VM (1999) Mechanism of nitric oxide-induced vasodilatation: refilling of intracellular stores by sarcoplasmic reticulum Ca2+ ATPase and inhibition of store-operated Ca2+ influx. Circ Res 84:210–219PubMedGoogle Scholar
  6. 6.
    Crauwels HM, Van Hove CE, Holvoet P, Herman AG, Bult H (2003) Plaque-associated endothelial dysfunction in apolipoprotein E-deficient mice on a regular diet. Effect of human apolipoprotein AI. Cardiovasc Res 59:189–199PubMedCrossRefGoogle Scholar
  7. 7.
    Dowell FJ, Martin W, Dominiczak AF, Hamilton CA (1999) Decreased basal despite enhanced agonist-stimulated effects of nitric oxide in 12-week-old stroke-prone spontaneously hypertensive rat. Eur J Pharmacol 379:175–182PubMedCrossRefGoogle Scholar
  8. 8.
    Feron O, Dessy C, Moniotte S, Desager JP, Balligand JL (1999) Hypercholesterolemia decreases nitric oxide production by promoting the interaction of caveolin and endothelial nitric oxide synthase. J Clin Invest 103:897–905PubMedCrossRefGoogle Scholar
  9. 9.
    Feron O, Michel JB, Sase K, Michel T (1998) Dynamic regulation of endothelial nitric oxide synthase: complementary roles of dual acylation and caveolin interactions. Biochemistry 37:193–200PubMedCrossRefGoogle Scholar
  10. 10.
    Fleischhacker E, Esenabhalu VE, Holzmann S, Skrabal F, Koidl B, Kostner GM, Graier WF (2000) In human hypercholesterolemia increased reactivity of vascular smooth muscle cells is due to altered subcellular Ca(2+) distribution. Atherosclerosis 149:33–42PubMedCrossRefGoogle Scholar
  11. 11.
    Fleming I, Busse R (1999) Signal transduction of eNOS activation. Cardiovasc Res 43:532–541PubMedCrossRefGoogle Scholar
  12. 12.
    García-Cardeña G, Martasek P, Masters BS, Skidd PM, Couet J, Li S, Lisanti MP, Sessa WC (1997) Dissecting the interaction between nitric oxide synthase (NOS) and caveolin. Functional significance of the NOS caveolin binding domain in vivo. J Biol Chem 272:25437–25440PubMedCrossRefGoogle Scholar
  13. 13.
    Hill CE, Rummery N, Hickey H, Sandow SL (2002) Heterogeneity in the distribution of vascular gap junctions and connexins: implications for function. Clin Exp Pharmacol Physiol 29:620–625PubMedCrossRefGoogle Scholar
  14. 14.
    Jen CJ, Chan HP, Chen HI (2002) Chronic exercise improves endothelial calcium signaling and vasodilatation in hypercholesterolemic rabbit femoral artery. Arterioscler Thromb Vasc Biol 22:1219–1224PubMedCrossRefGoogle Scholar
  15. 15.
    Kamata K, Nakajima M (1998) Ca2+ mobilization in the aortic endothelium in streptozotocin-induced diabetic and cholesterol-fed mice. Br J Pharmacol 123:1509–1516PubMedCrossRefGoogle Scholar
  16. 16.
    Kauser K, da Cunha V, Fitch R, Mallari C, Rubanyi GM (2000) Role of endogenous nitric oxide in progression of atherosclerosis in apolipoprotein E-deficient mice. Am J Physiol Heart Circ Physiol 278:H1679–H1685PubMedGoogle Scholar
  17. 17.
    Kawashima S (2004) Malfunction of vascular control in lifestyle-related diseases: endothelial nitric oxide (NO) synthase/NO system in atherosclerosis. J Pharmacol Sci 96:411–419PubMedCrossRefGoogle Scholar
  18. 18.
    Kuhlencordt PJ, Gyurko R, Han F, Scherrer-Crosbie M, Aretz TH, Hajjar R, Picard MH, Huang PL (2001) Accelerated atherosclerosis, aortic aneurysm formation, and ischemic heart disease in apolipoprotein E/endothelial nitric oxide synthase double-knockout mice. Circulation 104:448–454PubMedCrossRefGoogle Scholar
  19. 19.
    Landmesser U, Hornig B, Drexler H (2004) Endothelial function: a critical determinant in atherosclerosis? Circulation 109:II27–II33PubMedCrossRefGoogle Scholar
  20. 20.
    Liu J, García-Cardeña G, Sessa WC (1996) Palmitoylation of endothelial nitric oxide synthase is necessary for optimal stimulated release of nitric oxide: implications for caveolae localization. Biochemistry 35:13277–13281PubMedCrossRefGoogle Scholar
  21. 21.
    Millanvoye-Van Brussel E, Topal G, Brunet A, Do Pham T, Deckert V, Rendu F, David-Dufilho M (2004) Lysophosphatidylcholine and 7-oxocholesterol modulate Ca2+ signals and inhibit the phosphorylation of endothelial NO synthase and cytosolic phospholipase A2. Biochem J 380:533–539PubMedCrossRefGoogle Scholar
  22. 22.
    Mizuno O, Kobayashi S, Hirano K, Nishimura J, Kubo C, Kanaide H (2000) Stimulus-specific alteration of the relationship between cytosolic Ca(2+) transients and nitric oxide production in endothelial cells ex vivo. Br J Pharmacol 130:1140–1146PubMedCrossRefGoogle Scholar
  23. 23.
    Nakashima Y, Raines EW, Plump AS, Breslow JL, Ross R (1998) Upregulation of VCAM-1 and ICAM-1 at atherosclerosis-prone sites on the endothelium in the ApoE-deficient mouse. Arterioscler Thromb Vasc Biol 18:842–851PubMedGoogle Scholar
  24. 24.
    Oemar BS, Tschudi MR, Godoy N, Brovkovich V, Malinski T, Lüscher TF (1998) Reduced endothelial nitric oxide synthase expression and production in human atherosclerosis. Circulation 97:2494–2498PubMedGoogle Scholar
  25. 25.
    Ohi Y, Takai N, Muraki K, Watanabe M, Imaizumi Y (2001) Ca2+-images of smooth muscle cells and endothelial cells in one confocal plane in femoral artery segments of the rat. Jpn J Pharmacol 86:106–113PubMedCrossRefGoogle Scholar
  26. 26.
    Oishi H, Budel S, Schuster A, Stergiopulos N, Meister JJ, Beny JL (2001) Cytosolic-free calcium in smooth-muscle and endothelial cells in an intact arterial wall from rat mesenteric artery in vitro. Cell Calcium 30:261–267PubMedCrossRefGoogle Scholar
  27. 27.
    Ozaki M, Kawashima S, Yamashita T, Hirase T, Namiki M, Inoue N, Hirata K, Yasui H, Sakurai H, Yoshida Y, Masada M, Yokoyama M (2002) Overexpression of endothelial nitric oxide synthase accelerates atherosclerotic lesion formation in apoE-deficient mice. J Clin Invest 110:331–340PubMedCrossRefGoogle Scholar
  28. 28.
    Pelat M, Dessy C, Massion P, Desager JP, Feron O, Balligand JL (2003) Rosuvastatin decreases caveolin-1 and improves nitric oxide-dependent heart rate and blood pressure variability in apolipoprotein E−/− mice in vivo. Circulation 107:2480–2486PubMedCrossRefGoogle Scholar
  29. 29.
    Pohl U, de Wit C (1999) A unique role of NO in the control of blood flow. News Physiol Sci 14:74–80PubMedGoogle Scholar
  30. 30.
    ‘t Hoen PA, Van der Lans CA, Van Eck M, Bijsterbosch MK, Van Berkel TJ, Twisk J (2003) Aorta of ApoE-deficient mice responds to atherogenic stimuli by a prelesional increase and subsequent decrease in the expression of antioxidant enzymes. Circ Res 93:262–269PubMedCrossRefGoogle Scholar
  31. 31.
    Van Assche T, Fransen P, Guns PJ, Herman AG, Bult H (2007) Altered Ca2+ handling of smooth muscle cells in aorta of apolipoprotein E-deficient mice before development of atherosclerotic lesions. Cell Calcium 41:295–302PubMedCrossRefGoogle Scholar
  32. 32.
    van Haperen R, de Waard M, van Deel E, Mees B, Kutryk M, van Aken T, Hamming J, Grosveld F, Duncker DJ, de Crom R (2002) Reduction of blood pressure, plasma cholesterol, and atherosclerosis by elevated endothelial nitric oxide. J Biol Chem 277:48803–48807PubMedCrossRefGoogle Scholar
  33. 33.
    Vanhoutte PM (1988) Aging and vascular responsiveness. J Cardiovasc Pharmacol 12(Suppl 8):S11–S19PubMedGoogle Scholar
  34. 34.
    Weisbrod RM, Griswold MC, Du Y, Bolotina VM, Cohen RA (1997) Reduced responsiveness of hypercholesterolemic rabbit aortic smooth muscle cells to nitric oxide. Arterioscler Thromb Vasc Biol 17:394–402PubMedGoogle Scholar

Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  • Paul Fransen
    • 1
    Email author
  • Tim Van Assche
    • 1
  • Pieter-Jan Guns
    • 1
  • Cor E. Van Hove
    • 1
  • Gilles W. De Keulenaer
    • 1
  • Arnold G. Herman
    • 1
  • Hidde Bult
    • 1
  1. 1.Division of Pharmacology, Faculties of Medicine and Pharmaceutical SciencesUniversity of AntwerpWilrijkBelgium

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