The Histochemical Journal

, Volume 32, Issue 1, pp 41–51

Histochemical Evidence for Inducible Nitric Oxide Synthase in Advanced but Non-ruptured Human Atherosclerotic Carotid Arteries

  • Delphine Behr-Roussel
  • Alain Rupin
  • Patricia Sansilvestri-Morel
  • Jean-Noël Fabiani
  • Tony J. Verbeuren


In response to cytokine stimulation, the inducible isoform of the nitric oxide synthase (iNOS) produces large amounts of nitric oxide with potential consequences in the pathophysiology of atherosclerosis. Previous investigations have demonstrated the presence of iNOS in human atherosclerotic lesions. The goal of this study was to evaluate the occurrence of the expression of iNOS in ruptured versus non-ruptured human carotid atherosclerotic plaques. Using plastic-embedded sections, we performed in situ hybridization and immunohistochemistry on very advanced atherosclerotic lesions type V (non-ruptured) and type VI (ruptured) from 12 atheromatous carotid arteries from endarterectomy and six non-atherosclerotic internal mammary arteries from aorto-coronary bypass. Only one internal mammary artery expressed iNOS in the endothelium. In contrast, iNOS mRNA and protein were repeatedly expressed in advanced lesions type V in 5/7 cases, particularly in inflammatory regions. Specific cell markers identified iNOS-positive cells as macrophages and T-lymphocytes but also as smooth muscle cells and endothelial cells adjacent to these inflammatory regions. Nitration of protein tyrosines was not always associated to iNOS expression but more likely to the presence of inflammatory cells. In complicated lesions type VI, the occurrence of iNOS mRNA and protein expression diminished drastically (1/5 cases). Combined expression of iNOS mRNA and protein is frequently found in advanced but non-ruptured human atherosclerotic carotid lesions while it becomes rare after the plaque has ruptured. These findings suggest that iNOS could be an active participant in the plaque rupture event.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References cited

  1. Abe J-I, Berk B (1998) Reactive oxygen species as mediators of signal transduction in cardiovascular disease. Trends Cardiovasc Med 8: 59-64.Google Scholar
  2. Barath P, Fisherlein M, Cao J, Berenson J, Helfant R, Forrester J (1990) Detection and localization of tumor necrosis factor in human atheroma. Am J Cardiol 65: 297-302.Google Scholar
  3. Beckmann J, Ye Y, Anderson P, Chen J, Accavitti M, Tarpey M, White C (1994) Extensive nitration of protein tyrosines in human atherosclerosis detected by immunohistochemistry. Biol Chem Hoppe-Seyler 375: 81-88.Google Scholar
  4. Behr D, Rupin A, Fabiani J-N, Verbeuren TJ (1999) Distribution and prevalence of inducible nitric oxide synthase in atherosclerotic vessels from long-term cholesterol-fed rabbits. Atherosclerosis 142: 335-344.Google Scholar
  5. Brüne B, Knethen AV, Sandau KB (1998) Nitric oxide and its role in apoptosis. Eur J Pharmacol 351: 261-272.Google Scholar
  6. Buttery L, Springall D, Chester A, Evans T, Stanfield N, Parums D, Yacoub M, Polak J (1996) Inducible nitric oxide synthase is present within human atherosclerotic lesions and promotes the formation and activity of peroxynitrite. Lab Invest 75: 77-85.Google Scholar
  7. Cox K, De Leon D, Angerer L, Angerer R (1984) Detection of mRNAs in sea urchin embryos by in situ hybridization using asymmetric RNA probes. Dev Biol 101: 485-502.Google Scholar
  8. Esaki T, Hayashi T, Muto E, Yamada K, Kuzuya M, Iguchi A (1997) Expression of inducible nitric oxide synthase in T lymphocytes and macrophages of cholesterol-fed rabbits. Atherosclerosis 128: 39-46.Google Scholar
  9. Fujisawa H, Ogura T, Kurashima Y, Yokoyama T, Yamashita J, Esumi H (1994) Expression of two types of nitric oxide synthase mRNA in human neuroblastoma cell lines. J Neurochem 63: 140-145.Google Scholar
  10. Kishikawa H, Shimokawa T, Watanabe T (1993) Localization of T lymphocytes and macrophages expressing IL-1, IL-2 receptor, IL-6 and TNF in human aortic intima. Role of cell-mediated immunity in human atherogenesis. Virchows Arch 423: 433-442.Google Scholar
  11. Luoma JS, Stralin P, Marklund SL, Hiltunen TP, Särkioja T, Ylä-Herttuala S (1998) Expression of extracellular SOD and INOS in macrophages and smooth muscle cells in human and rabbit atherosclerotic lesions. Colocalization with epitopes characteristic of oxidized LDL and peroxynitrite-modified proteins. Arterioscler Thromb Vasc Biol 18: 157-167.Google Scholar
  12. Mallat Z, Heymes C, Ohan J, Faggin E, Leseche G, Tedgui A (1999) Expression of interleukin-10 in advanced human atherosclerotic plaques. Relation to inducible nitric oxide synthase expression and cell death. Arterioscler Thromb Vasc Biol 19: 611-616.Google Scholar
  13. Ogura T, Yokoyama T, Fujisawa H, Kurashima Y, Esumi H (1993) Structural diversity of neuronal nitric oxide synthase mRNA in the nervous system. Biochem Biophys Res Com 193: 1014-1022.Google Scholar
  14. Okamoto T, Akaike T, Nagano T, Miyajima S, Suga M, Ando M, Ichimori K, Maeda H (1997) Activation of human neutrophil procollagenase by nitrogen dioxide and peroxynitrite: a novel mechanism for procollagenase activation involving nitric oxide. Arch Biochem Biophys 342: 261-274.Google Scholar
  15. Padgett EL, Pruett SB (1992) Evaluation of nitric oxide production by human monocyte-derived macrophages. Biochem Biophys Res Com 186: 775-787.Google Scholar
  16. Rajagopalan S, Meng XP, Ramasamy S, Harrison DG, Galis ZS (1996) Reactive oxygen species produced by macrophage-derived foam cells regulate the activity of vascular matrix metalloproteinases in vitro. Implications for atherosclerotic plaque stability. J Clin Invest 98: 2572-2579.Google Scholar
  17. Ravalli S, Albala A, Ming M, Szabolcs M, Barbone A, Michler RE, Cannon PJ (1998) Inducible nitric oxide synthase expression in smooth muscle cells and macrophages of human transplant coronary artery disease. Circulation 97: 2338-2345.Google Scholar
  18. Rupin A, Behr D, Verbeuren TJ (1996) Increased activity of guanylate cyclase in the atherosclerotic rabbit aorta: role of non-endothelial nitric oxide synthases. Br J Pharmacol 119: 1233-1238.Google Scholar
  19. Sasaki K, Hattori T, Fujisawa T, Takahashi K, Inouhe H, Takigawa M (1998) Nitric oxide mediates interleukin-1-induced gene expression of matrix metalloproteinases and basic fibroblast growth factor in cultured rabbit articular chondrocytes. J Biochem (Tokyo) 123: 431-439.Google Scholar
  20. Schroeder A, Falk E (1996) Pathophysiology and inflammatory aspects of plaque rupture. Cardiol Clin 14: 211-220.Google Scholar
  21. Simonet S, Porro De Bailliencourt J, Descombes JJ, Mennecier P, Laubie M, Verbeuren TJ (1993) Hypoxia causes an abnormal contractile response in the atherosclerotic rabbit aorta: implication of reduced nitric oxide and cGMP production. Circ Res 72: 616-630.Google Scholar
  22. Stary HC, Chandler AB, Dinsmore RE, Fuster V, Glagov S, Insull Jr, W, Rosenfeld ME, Schwartz CJ, Wagner WD, Wissler RW (1995) A definition of advanced types of atherosclerotic lesions and a histological classification of atherosclerosis. A report from the committee on vascular lesions of the council on arteriosclerosis, AHA. Arterioscler Thromb Vasc Biol 15: 1512-1531.Google Scholar
  23. Trachtman H, Futterweit S, Singhal P (1995) Nitric oxide modulates the synthesis of extracellular matrix proteins in cultured rat mesengial cells. Biochem Biophys Res Commun 207: 120-125.Google Scholar
  24. Van Der Wal AC, Becker AE, Van Der Loos CM, Das PK (1994) Site of intimal rupture or erosion of thrombosed coronary atherosclerotic plaques is characterized by an inflammatory process irrespective of the dominant plaque morphology. Circulation 89: 36-44.Google Scholar
  25. Van Der Wal AC, Becker AE (1999) Atherosclerotic plaque rupture-pathologic basis of plaque stability and instability. Cardiovasc Res 41: 334-344.Google Scholar
  26. Verbeuren TJ, Bonhomme E, Laubie M, Simonet S (1993) Evidence for induction of non-endothelial NO synthase in aortas of cholesterol-fed rabbits. J Cardiovasc Pharmacol 21: 841-845.Google Scholar
  27. White CR, Brock TA, Chang L-Y, Capro J, Briscoe P, Ku D, Bradley WA, Gianturco SH, Gore J, Freeman BA, Tarpey MM (1994) Superoxide and peroxynitrite in atherosclerosis. Proc Natl Acad Sci USA 91: 1044-1048.Google Scholar
  28. Wilcox JN, Subramanian RR, Sundell CL, Tracey WR, Pollock JS, Harrison DG, Marsden PA (1997) Expression of multiple isoforms of nitric oxide synthase in normal and atherosclerotic vessels. Arterioscler Thromb Vasc Biol 17: 2479-2488.Google Scholar
  29. Wu W, Chen Y, Hazen SL (1999) Eosinophil peroxidase nitrates protein tyrosyl residues. Implications for oxidative damage by nitrating intermediates in eosinophilic inflammatory disorders. J Biol Chem 274: 25933-25944.Google Scholar

Copyright information

© Kluwer Academic Publishers 2000

Authors and Affiliations

  • Delphine Behr-Roussel
    • 1
  • Alain Rupin
    • 2
  • Patricia Sansilvestri-Morel
    • 2
  • Jean-Noël Fabiani
    • 1
  • Tony J. Verbeuren
    • 2
  1. 1.Department of Cardiovascular Surgery and Laboratoire d'Etude des Greffes et Prothèses CardiaquesHôpital BroussaisParisFrance
  2. 2.Division of AngiologyServier Research InstituteSuresnesFrance

Personalised recommendations