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Homocysteine stimulates inducible nitric oxide synthase expression in macrophages: Antagonizing effect of ginkgolides and bilobalide

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Abstract

Hyperhomocysteinemia is an independent risk factor for atherosclerotic diseases. Inducible nitric oxide synthase (iNOS) is mainly expressed in macrophages upon stimulation. Overproduction of nitric oxide (NO) by iNOS can exacerbate the development of atherosclerosis. Our previous studies demonstrated that the extract of ginkgo biloba leaves (EGb) inhibited the iNOS-mediated NO production in monocyte-derived macrophage. We also reported that homocysteine could stimulate monocyte chemoattractant protein-1 (MCP-1) expression in vascular cells causing enhanced monocyte chemotaxis. The objective of the present study was to investigate the effect of homocysteine on iNOS-mediated NO production in macrophages and the antagonizing effect of EGb. Human monocytic cell (THP-1)-derived macrophages were incubated with homocysteine for various time periods. Homocysteine at concentrations of 0.05–0.1 mM significantly stimulated NO production and iNOS activity in macrophages via increased expression of iNOS mRNA and protein. The increased iNOS expression was associated with activation of nuclear factor-kappa B (NF-κB) arising from reduced expression of inhibitor protein (IκBα) mRNA as well as increased phosphorylation of IκBα protein in homocysteine-treated cells. EGb and its terpenoids (ginkgolide A, ginkgolide B and bilobalide) could antagonize the homocysteine effect on iNOS expression in macrophages via their antioxidant effect resulting in attenuation of NF-κB activation. Taken together, our results have demonstrated that homocysteine, at pathophysiological concentrations, stimulates iNOS-mediated NO production in macrophages. EGb and its terpenoids can antagonize such stimulatory effect via antioxidation and attenuation of NF-κB activation.

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References

  1. Refsum H, Ueland PM, Nygard O, Vollset SE: Homocysteine and cardiovascular disease. Annu Rev Med 49: 31-62, 1998

    Google Scholar 

  2. Clarke R, Daly L, Robinson K, Naughten E, Cahalane S, Fowler B, Graham I: Hyperhomocysteinemia: An independent risk factor for vascular disease. N Engl J Med 324: 1149-1155, 1991

    Google Scholar 

  3. McCully KS: Homocysteine and vascular disease. Nat Med 2: 386-389, 1996

    Google Scholar 

  4. Duell PB, Malinow MR: Homocyst(e)ine: An important risk factor for atherosclerotic vascular disease. Curr Opin Lipidol 8: 28-34, 1997

    Google Scholar 

  5. Ikeda U, Ikeda M, Minota S, Shimada K: Homocysteine increases nitric oxide synthesis in cytokine-stimulated vascular smooth muscle cells. Circulation 99: 1230-1235, 1999

    Google Scholar 

  6. Harker LA, Ross R, Slichter SJ, Scott CR: Homocysteine-induced arteriosclerosis. The role of endothelial cell injury and platelet response in its genesis. J Clin Invest 58: 731-741, 1976

    Google Scholar 

  7. O K, Lynn EG, Chung YH, Siow YL, Man RYK, Choy PC: Homocysteine stimulates the production and secretion of cholesterol in hepatic cells. Biochim Biophys Acta 1393: 317-324, 1998

    Google Scholar 

  8. Sung FL, Siow YL, Wang G, Lynn EG, O K: Homocysteine stimulates the expression of monocyte chemoattractant protein-1 in endothelial cells leading to enhanced monocyte chemotaxis. Mol Cell Biochem 216: 121-128, 2001

    Google Scholar 

  9. Wang G, Siow YL, O K: Homocysteine stimulates nuclear factor kappa B activity and monocyte chemoattractant protein-1 expression in vascular smooth muscle cells: A possible role for protein kinase C. Biochem J 352: 817-826, 2000

    Google Scholar 

  10. Wang G, Siow YL, O K: Homocysteine induces monocyte chemo-attractant protein-1 expression by activating NF-kappa B in THP-1 macrophage. Am J Physiol Heart Cir Physiol 280: H2840-2847, 2001

    Google Scholar 

  11. Glass CK, Witztum JL: Atherosclerosis: The Road Ahead. Cell 104: 503-516, 2001

    Google Scholar 

  12. Gerrity RG: The role of the monocyte in atherogenesis: I. Transition of blood-borne monocytes into foam cells in fatty lesions. Am J Pathol 103: 181-190, 1981

    Google Scholar 

  13. Ross R: The pathogenesis of atherosclerosis: A perspective for the 1990s. Nature (Lond) 362: 801-809, 1993

    Google Scholar 

  14. Napoli C, Ignarro LJ: Nitric oxide and atherosclerosis. Nitric Oxide 5: 88-97, 2001

    Google Scholar 

  15. Masters BS, Martásek P, Roman LJ: Structural variations on a theme of nitric oxide production by three isoforms of nitric oxide synthase. In: G.M. Rubanyi (ed). Pathophysiology and Clinical Applications of Nitric Oxide. Harwood Academic, Amsterdam, 1999, pp 17-37

    Google Scholar 

  16. Li H, Förstermann U: Nitric oxide in the pathogenesis of vascular disease. J Pathol 190: 244-254, 2000

    Google Scholar 

  17. Cheung F, Siow YL, O K: Inhibition by ginkgolides and bilobalide of the production of nitric oxide in macrophages (THP-1) but not in endothelial cells (HUVEC). Biochem Pharmacol 61: 503-510, 2001

    Google Scholar 

  18. DeFeudis FV: Ginkgo Biloba Extract (EGb761): Pharmacological Activities and Clinical Applications. Elsevier, Paris, 1991

    Google Scholar 

  19. Ang-Lee M, Moss J, Yuen C: Herbal medicines and perioperative care. J Am Med Assoc 286: 208-216, 2001

    Google Scholar 

  20. Mckenna DJ, Jones K, Hughes K: Efficacy, safety and use of ginkgo biloba in clinical and preclinical applications. Altern Ther Health Med 7: 70-86, 88–90, 2001

    Google Scholar 

  21. Yoshikawa T, Naito Y, Kondo M: Ginkgo biloba leaf extract: Review of biological actions and clinical applications. Antioxid Redox Signal 1: 469-480, 1999

    Google Scholar 

  22. Robak J, Gryglewski RJ: Flavonoids are scavengers of superoxide anions. Biochem Pharmacol 37: 837-841, 1988

    Google Scholar 

  23. Upchurch GR Jr, Welch GN, Fabian AJ, Freedman JE, Johnson JL, Keaney JF Jr, Loscalzo J: Homocyst(e)ine decreases bioavailable nitric oxide by a mechanism involving glutathione peroxidase. J Biol Chem 272: 17012-17017, 1997

    Google Scholar 

  24. Stuhlinger MC, Tsao PS, Her JH, Kimoto M., Balint RF, Cooke JP: Homocysteine impairs the nitric oxide synthase pathway: Role of asymmetric dimethylarginine. Circ 104: 2569-2575, 2001

    Google Scholar 

  25. Welch GN, Upchurch GR Jr, Farivar RS, Pigazzi A, Vu K, Brecher P, Keaney JF Jr, Loscalzo J: Homocysteine-induced nitric oxide production in vascular smooth-muscle cells by NF-kappa B-dependent transcriptional activation of NOS2. Proc Assoc Am Physicians 110: 22-31, 1998

    Google Scholar 

  26. Kurosaka K, Watanabe N, Kobayashi Y: Production of proinflammatory cytokines by phorbol myristate acetate-treated THP-1 cells and monocyte-derived macrophages after phagocytosis of apoptotic CTLL-2 cells. J Immunol 161: 6245-6249, 1998

    Google Scholar 

  27. Tsuchiya S, Kobayashi Y, Goto Y, Okumura H, Nakae S, Konno T, Tada K: Induction of maturation in cultured human monocytic leukemia cells by a phorbol diester. Cancer Res 42: 1530-1536, 1982

    Google Scholar 

  28. Zhang YN, Zheng J, Huang GQ, Gu CY, Chen WZ: Protective effects of extract of ginkgo biloba extract (EGb) on lysophosphatidylcholine induced damages of vascular endothelial cells in vitro. Acta Pharm Sin 32: 737-739, 1997

    Google Scholar 

  29. Michelakis ED, Archer SL: The measurement of NO in biological systems using chemiluminescene. In: M. Titheradge (ed). Nitric Oxide Protocols. Humana Press, Totowa, 1998, pp 111-127

    Google Scholar 

  30. Perillo IB, Hyde RW, Olszowka AJ, Pietropaoli AP, Frasier LM, Torres A, Perkins PT, Forster RE II, Utell MJ, Frampton MW: Chemiluminescent measurements of nitric oxide pulmonary diffusing capacity and alveolar production in humans. J Appl Physiol 91: 1931-1940, 2001

    Google Scholar 

  31. Bredti DS, Shmidt H: The citrulline assay. In: M. Feelisch, J.S. Stamler (eds). Methods in Nitric Oxide Research. J. Wiley, New York, 1996, pp 249-255

    Google Scholar 

  32. Schreiber E, Matthias P, Mueller MM, Schaffner W: Rapid detection of octamer binding proteins with 'mini-extracts', prepared from a small number of cells. Nucleic Acids Res 17: 6419, 1989

    Google Scholar 

  33. Rauen U, Petrat F, Li T, De Groot H: Hypothermia injury/cold-induced apoptosis — evidence of an increase in chelatable iron causing oxidative injury in spite of low O2 −/H2O2 formation. FASEB J 14: 1953-1964, 2000

    Google Scholar 

  34. Wever R, Luscher T, Cosentino F, Rabelink T: Atherosclerosis and the two faces of endothelial nitric oxide synthase. Circulation 97: 108-112, 1998

    Google Scholar 

  35. Wilcox JN, Subramanian RR, Sundell CL, Tracey WR, Pollock JS, Harrison DG, Marsden PA: Expression of multiple isoforms of nitric oxide synthase in normal and atherosclerosis vessels. Arterioscler Thromb Vasc Biol 17: 2479-2488, 1997

    Google Scholar 

  36. Zhang X, Li H, Jin H, Ebin Z, Brodsky S, Goligorsky MS: Effects of homocysteine on endothelial nitric oxide production. Am J Physiol Renal Physiol 279: F671-F678, 2000

    Google Scholar 

  37. Chen C, Conklin BS, Ren Z, Zhong DS: Homocysteine decreases endothelial-dependent vasorelaxation in porcine arteries. J Surg Res 102: 22-30, 2002

    Google Scholar 

  38. Collins T, Cybulsky M: NF-κB: Pivotal mediator or innocent bystander in atherogenesis? J Clin Invest 107: 255-264, 2001

    Google Scholar 

  39. Valen G, Yan Z, Hansson G: Nuclear factor kappa-B and the heart. J Am Coll Cardiol 38: 307-314, 2001

    Google Scholar 

  40. Yamamoto M, Hara H, Adechi T: Effects of homocysteine on the binding of extracellular-superoxide dismutase to the endothelial cell surface. FEBS Lett 486: 159-162, 2000

    Google Scholar 

  41. Wang G, O K: Homocysteine stimulates the expression of monocyte chemoattractant protein-1 receptor (CCR2) in human monocytes: Possible involvement of oxygen free radicals. Biochem J 357: 233-240, 2001

    Google Scholar 

  42. Fukuto JM, Chaudhuri G: Inhibition of constitutive and inducible nitric oxide synthase: Potential selective inhibition. Annu Rev Pharmacol Toxicol 35: 165-194, 1995

    Google Scholar 

  43. Gravey EP, Oplinger JA, Tanoury GJ, Sherman PA, Fowler M, Marshall S, Harmon MF, Paith JE, Furfine ES: Potent and selective inhibition of human nitric oxide synthase. J Biol Chem 269: 26669-26676, 1994

    Google Scholar 

  44. Griffith MJD, Messent M, MacAllister RJ, Evans TW: Aminoguanidine selectively inhibits inducible nitric oxide synthase. Br J Pharmacol 110: 963-968, 1993

    Google Scholar 

  45. Kleijnen J, Knipschild P: Ginkgo biloba. Lancet 340: 1136-1139, 1992

    Google Scholar 

  46. Haines DD, Bak I, Ferdinandy P, Mahmoud FF, Al-Harbi SA, Blasig IE, Tosaki A: Cardioprotective effects of the calcineurin inhibitor FK506 and the PAF receptor antagonist and free radical scavenger, EGb 761, in isolated ischemic/reperfused rat hearts. J Cardiovasc Pharmacol 35: 37-44, 2000

    Google Scholar 

  47. Diamond BJ, Shiflett SC, Feiwel N, Matheis RJ, Noskin O, Richards JA, Schoenberger NE: Ginkgo biloba extract: Mechanisms and clinical indications. Arch Phys Med Rehabil 81: 668-678, 2000

    Google Scholar 

  48. Wadsworth TL, Koop DR: Effects of ginkgo biloba extract (EGb761) and quercetin on lipopolysaccharide-induced release of nitric oxide. Chem Biol Interact 137: 43-58, 2001

    Google Scholar 

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Authors and Affiliations

  1. Department of Pharmacology, Faculty of Medicine, University of Hong Kong, Hong Kong, China

    Connie W.H. Woo, Filly Cheung, Vincent W.H. Chan & Yaw L. Siow

  2. Department of Pharmacology, Faculty of Medicine, The University of Hong Kong, Faculty of Medicine Building, 2/F Laboratory Block, 21 Sassoon Road, Hong Kong, People's Republic of China

    O Karmin

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Woo, C.W., Cheung, F., Chan, V.W. et al. Homocysteine stimulates inducible nitric oxide synthase expression in macrophages: Antagonizing effect of ginkgolides and bilobalide. Mol Cell Biochem 243, 37–47 (2003). https://doi.org/10.1023/A:1021601512058

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