Advertisement

Molecular and Cellular Biochemistry

, Volume 291, Issue 1–2, pp 119–126 | Cite as

Comparative Study on in Vitro Effects of Homocysteine Thiolactone and Homocysteine on HUVEC Cells: Evidence for a Stronger Proapoptotic and Proinflammative Homocysteine Thiolactone

  • Mohsen Kerkeni
  • Mehdi Tnani
  • Laurence Chuniaud
  • Abdelhedi Miled
  • Khira Maaroufi
  • François Trivin
Article

Abstract

Hyperhomocysteinemia is an independent risk factor for the development of atherosclerosis. However the underlying mechanisms responsible for endothelial cell injury with increased plasma concentration of homocysteine or homocysteine derivatives remains still incompletely elucidated. In this study, we investigated the ability of homocysteine (Hcy) and homocysteine thiolactone (HcyT) to induce cell death and IL-8 secretion in primary human umbilical vein endothelial cells (HUVEC). Hcy and HcyT were both cytotoxic and capable of promoting cell death, as measured by caspase-3 activation and DNA fragmentation. ELISA assays clearly demonstrated that Hcy and HcyT strongly activated IL-8 release. Furthermore, our results showed that HcyT was much more efficient than Hcy in activating caspase-3 or in inducing IL-8 secretion. The use of antioxidants such as vitamin C and vitamin E strongly but not completely reduced programmed cell death and chemokine release suggesting that other pathways different than reactive oxygen species are also involved. This study suggests that Homocysteine derivatives like HcyT might possess stronger cytotoxicity and pro-inflammatory properties and that Hcy derivatives levels should therefore be more taken into account during diagnostics.

Keywords

homocysteine thiolactone homocysteine apoptosis proinflammation atherosclerosis antioxidants 

Abbreviations:

Hcy

homocysteine

HcyT

homocysteine thiolactone

HUVEC

human umbilical vein endothelial cells

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. 1.
    Wilcken DEL, Dudman NPB: Homocystinuria and atherosclerosis In: AJ Lusis, JI Rotter, RS Sparkes (eds). Molecular Genetics of Coronary Artery Disease; Candidate Genes and Process in Atherosclerosis. Monograms in Human Genetics, Karger, New York, NY, 1992Google Scholar
  2. 2.
    Mudd SH, Havlik R, Levy HL, McKusicK VA, Feinleib M: Cardiovascular risk in Heterozygotes for homocystinuria. Am J Hum Genet 34: 1018–1021, 1982PubMedGoogle Scholar
  3. 3.
    Frosst P, Blom HJ, Milos R, Goyette P, Sheppard CA, Matthews RJ, Boers GJ, den Heijer M, Kluijtmans LA, van den Heuvel LP, Rozen R: A candidate genetic risk factor for vascular disease: a common mutation in methylenetetrahydrofolate reductase: isolation of cDNA, mapping and mutation identification. Nat Genet 7: 195–200, 1995Google Scholar
  4. 4.
    Selhub J, Jacques PF, Wilson PWF, Rush D, Rosenberg IH: Vitamin status and intake as primary determinants of homocysteinemia in an elderly population. J Am Med Assoc 270: 2693–2698, 2001CrossRefGoogle Scholar
  5. 5.
    Skurk S, Walsh K: A new mechanism of Homocysteine-Mediated Endothelial Cell Cytotoxicity. Hypertension 43: 1168–1170, 2004CrossRefPubMedGoogle Scholar
  6. 6.
    Clarke R, Daly L, Robinson K, Naughten E, Cahalane S, Fowler B and Graham I: Hyperhomocysteinemia: an independent risk factor for vascular disease. N Engl J Med 324: 1149–1155, 1991PubMedCrossRefGoogle Scholar
  7. 7.
    Audelin MC, Genest J Jr: Homocysteine and cardiovascular disease in diabetes mellitus. Atherosclerosis 159: 497–511, 2001CrossRefPubMedGoogle Scholar
  8. 8.
    Herrmann W, Knapp JP: Hyperhomocysteinemia: a new risk factor for degenerative diseases. Clin Lab 48: 471–481, 2002PubMedGoogle Scholar
  9. 9.
    Polidori MC, Marvardi M, Cheribini A, Senin U, Mecocci P: Heart disease and cardiovascular risk factors in the cognitively impaired elderly: implication for Alzheimer's dementia. Aging 13: 231–239, 2001PubMedGoogle Scholar
  10. 10.
    Schroecksnadel K, Frick B, Wirleitner B, Winkler C, Schennach H, Fuchs D: Moderate Hyperhomocysteinemia and immune activation. Curr Pharm Biotechnol 5: 107–118, 2004CrossRefPubMedGoogle Scholar
  11. 11.
    Den Heijer M, Koster T, Blom HJ, Bos GM, Breit E, Reitsma PH, et al.: Hyperhomocysteinemia as a risk factor for deep-vein thrombosis. N Engl J Med 344: 759–762, 1996CrossRefGoogle Scholar
  12. 12.
    McCully KS: Homocysteine and vascular disease. Nat Med 2: 386–389, 1996CrossRefPubMedGoogle Scholar
  13. 13.
    Lawrence de Koning AB Werstuck GH, Zhou J, Austin RC: Hyperhomocysteinemia and its role in the development of atherosclerosis. Clin Biochem 36: 431–441, 2003CrossRefPubMedGoogle Scholar
  14. 14.
    Bessede G, Miguet C, Gambert P, Neel D, Lizard G: Efficiency of Homocysteine plus Copper in inducing apoptosis is inversely proportional to γ glutamyl transpeptidase activity. FASEB J 15: 1927–1940, 2001CrossRefPubMedGoogle Scholar
  15. 15.
    Di Simone N, Maggiano N, Caliandro D, Riccardi P, Evangelista A, Carducci B, Caruso A: Homocysteine induces trophoblasts cell death with apoptotic features. Biol Reprod 69: 1129–1134, 2003CrossRefPubMedGoogle Scholar
  16. 16.
    Poddar R, Sivasubramania N, Dibello PM, Robinson K, Jacobsen D: Homocysteine induces expression and secretion of monocyte chemoattractant protein-1 and interleukine-8 in human aortic endothelial cells: implications for vascular disease. Circulation 103: 2717–2723, 2001PubMedGoogle Scholar
  17. 17.
    Jakubowski H: Molecular basis of homocysteine toxicity in humans. Cell Mol Life Sci 61: 470–487, 2004CrossRefPubMedGoogle Scholar
  18. 18.
    McCully KS: Chemical pathology of homocysteine. II. Carcinogenesis and homocysteine thiolactone metabolism. Ann Clin Labo Sci 24: 27–59, 1994Google Scholar
  19. 19.
    Huang RF, Huang SM, Lin BS, Wei JS, and Liu TZ: Homocysteine thiolactone induces apoptotic DNA damage mediated by increased intracellular hydrogen peroxide and caspase-3 activation in HL-60 cells. Life Sci 68: 2799–2811, 2001CrossRefPubMedGoogle Scholar
  20. 20.
    Mercie P, Garnier O, Lascoste L, Renard M, Closse C, Durrieu F, et al.: Homocysteine thiolactone induces caspase-independent vascular endothelial cell death apoptosic features. Apoptosis 5: 403–411, 2000CrossRefPubMedGoogle Scholar
  21. 21.
    Renvoize C, Biola A, Pallardy M, Breard J: Apoptosis: identification of dying cells. Cell Biol toxicol 14: 111–120, 1998CrossRefPubMedGoogle Scholar
  22. 22.
    Loscalzo J: The oxidant stress of hyperhomocysteinemia. J Clin Invest 98: 5–7, 1996PubMedGoogle Scholar
  23. 23.
    Lang D, Kredan MB, Moat SJ, Hussain SA, Powell CA, Bellamy MF, Powers HJ, Lewis MJ: Homocysteine-induced inhibition of endothelium relaxation in rabbit aorta: role for superoxide anions. Arterioscler Thromb Vasc Biol 20: 422–427, 2000PubMedGoogle Scholar
  24. 24.
    Hermann C, Zeiher AM, Dmmeler S: Shear stress inhibits H2O2-induced apoptosis in human endothelial cells by modulation of the glutathione redox cycle and nitric oxide synthase. Arterioscler Thromb Vasc Biol 17: 3588–3592, 1997PubMedGoogle Scholar
  25. 25.
    Dipietrantonio AM, Hsieh TC, Wu JM: Activation of caspase-3 in HL-60 cells exposed to hydrogen peroxide. Biochem Biophys Res Commun 255: 477–482, 1999CrossRefPubMedGoogle Scholar
  26. 26.
    Barroso MP, Diaz CG, Lluch GL, Malagon MM, Crane FL, Navas P: Ascorbate and alpha-tocopherol prevent apoptosis induced by serum removal independent of Bcl-2. Arch. Biochem Biophys 343: 243–248, 1997CrossRefPubMedGoogle Scholar
  27. 27.
    Haendeler J, Zeiher AM, Dimmeter S: Vitamin C and Vitamin E prevent lipopolysaccharide-induced apoptosis in humans endothelial cells by modulation of Bcl-2 and Bax. Eur J Pharmacol 317: 407–411, 1996CrossRefPubMedGoogle Scholar
  28. 28.
    Wu J, Karlsson K, Danielsson A: Effects of vitamins E, C and catalase on bromobenzene- and hydrogen peroxide-induced intracellular oxidation and DNA single-strand breakage in HepG2 cells. J Hepatol 26: 669–677, 1997CrossRefPubMedGoogle Scholar
  29. 29.
    Upchurch GR, Welch GN, Fabian AJ, Freedman JE, Jhonson JL, Keaney JF, Loscalzo J: Homocysteine decrease bioavailable nitric oxide by a mechanism involving glutathione peroxidase. J Biol Chem 272: 17012–1701, 1997CrossRefPubMedGoogle Scholar
  30. 30.
    Heinecke JW, Rosen H, Suzuki LA, Chait A: The role of sulfur containing amino acids in superoxide production and modification of low density lipoprotein arterial smooth muscle cell. J Biol Chem 262: 10098–10103, 1987PubMedGoogle Scholar
  31. 31.
    Outinen PA, Sood SK, Pfeifer SI, Pamidi S, Podor TJ, Li J, Weitz JL, Austin RC: Homocysteine induced endoplasmic reticulum stress and growth arrest leads to specific changes in gene expression in human vascular endothelial cell. Blood 94: 959–967, 1999PubMedGoogle Scholar
  32. 32.
    Outinen PA, Sood SK, Liaw PC, Sarge KD, Maeda N, Hirsh J, Podor TJ, Weitz JL, Austin RC: Characterization of the stress inducing effects of homocysteine. Biochem J 332: 213–221, 1998PubMedGoogle Scholar
  33. 33.
    Ron D: Hyperhomocysteinemia and function of the endoplasmic reticulum. J Clin Invest 107: 1221–1222, 2001PubMedGoogle Scholar
  34. 34.
    Kokame K, Kato H, Miyata T: Homocysteine-respondent genes in vascular endothelial cells identified by differential display analysis. GRP78//BIP and novel genes. J Biol Chem 271: 29659–29665, 1996CrossRefPubMedGoogle Scholar
  35. 35.
    Cai Y, Zhang C, Nawa T, Aso T, Tanaka M, Oshiro S, Ichijo H, Kitajima S: Homocysteine responsive ATF3 gene expression in human vascular endothelial cells: Activation of c-jun NH(2) terminal Kinase and promoter responsive element. Blood 96: 2140–2148, 2000PubMedGoogle Scholar
  36. 36.
    Zhang C, Cai Y, Adachi MT, Oshiro S, Aso T, Kaufman RJ, Kitajima S: Homocysteine induces programmed cell death in human vascular endothelial cells through activation of the unfolded protein response. J Biol Chem 276: 35867–35874, 2001CrossRefPubMedGoogle Scholar
  37. 37.
    Hultberg B, Andersson A, Isaksson A: Metabolism of homocysteine, its relation to the other cellular thiols and its mechanism of cell damage in a cell culture line (human histiocytic cell line U937). Biochim Biophys Acta 1269: 6–12, 1995CrossRefPubMedGoogle Scholar
  38. 38.
    Huang RF, Huang SM, Lin BS, Hung CW, Lu HT: N-Acetylcystein, Vitamin C and vitamin E diminish homocysteine thiolactone induced apoptosis in human promyeloid HL-60 cells. J Nutr 132: 2151–2156, 2002PubMedGoogle Scholar
  39. 39.
    Jaffe EA, Nachmann RL, Becker CG, Minick CR: Culture of human endothelial cells derived from unbilical veins: Identification by morphologic and immunologic criteria. J Clin Invest 52: 2745, 1973PubMedGoogle Scholar
  40. 40.
    Denizot F, Lang R: Rapid colorimetric assay for cell growth and survival: Modifications to the tetrazolium dye procedure giving improved sensitivity and reliability. J Immunol Methods 89: 271–277, 1986CrossRefPubMedGoogle Scholar
  41. 41.
    Slater TF, Sawyer B, Strauli U: Studies on succinate-tetrazolium reductase systems. III. Points of coupling of four different tetrazolium salts. Biochem Biophys Acta 77: 383–393, 1963CrossRefPubMedGoogle Scholar
  42. 42.
    Foster V: Mechanisms leading to myocardial infarction: insight from studies of vascular biology. Circulation 90: 2126–2146, 1994PubMedGoogle Scholar
  43. 43.
    Harker LA, Ross R, Slichter SJ, Scott CR: Homocysteine induced atherosclerosis: The role of endothelial cell injury and platelet response in its genesis. J Clin Invest 58: 731–741, 1976PubMedGoogle Scholar
  44. 44.
    Ross R: Atherosclerosis-an inflammatory disease. N Engl J Med 340: 115–126, 1999CrossRefPubMedGoogle Scholar
  45. 45.
    McCully KS: Homocysteine and vascular disease. Nat Med 2: 386–389, 1996CrossRefPubMedGoogle Scholar
  46. 46.
    Eberhardt RT, Forgione MA, Cao A, Leopold JA, Rudd MA, Trolliet M, Heydrick S, Stark R, Klings ES, Moldovan NI, Yaghoubi M, Glodshmidt-Clermont PJ, Farber HW, Cohen R, Loscalzo J: Endothelial dysfunction in a murine model of mild hyperhomocysteinemia. J Clin Invest 106: 483–491, 2000PubMedGoogle Scholar
  47. 47.
    Tsai JC, Perella MA, Yoshizumi M, Hsiech CM, Haber E, Shlegel R, Lee ME: Promotion of vascular smooth muscle cell growth by homocysteine: a link to atherosclerosis. Proc Natl Acad Sci USA 91: 6369–6373, 1994PubMedCrossRefGoogle Scholar
  48. 48.
    Stamler JS, Osborne JA, Jaraki O, Rabbani LE, Mullins M, Singel D: Adverse vascular effects of homocysteine are modulated by endothelium-derived relaxing factors and related oxides of nitrogen. J Clin Invest 91: 308–318, 1993PubMedGoogle Scholar
  49. 49.
    Stamler JS, Loscalzo J: Endothelium derived relaxing factor modulates the atherothrombogenic effects of homocysteine. J Cardiovasc Pharmacol 20: 202–204, 1992Google Scholar
  50. 50.
    Grow AJ, Cobb F, and Stamler JS: Homocysteine, nitric oxide and nitrosothiols. In: Homocysteine in health and disease, D Jacobsen, R Carmel (eds). Cambridge University Press, Cambridge, 2001 pp. 39–45Google Scholar
  51. 51.
    Jakubowski H: Calcium-dependent human serum homocysteine thiolactone hydrolase: A protective mechanism against protein N homocysteinylation. J Biol Chem 275: 3957–3962, 2000CrossRefPubMedGoogle Scholar
  52. 52.
    Suhara T, Fukuo K, Yasuda O, Tsubakimoto M, Takemura Y, Kawamoto H, et al.: Homocysteine enhances endothelial apoptosis via upregulation of Fas mediated pathways. Hypertention 43: 1208–1213, 2004CrossRefGoogle Scholar
  53. 53.
    Jakubowski H: Metabolism of homocysteine thiolactone in human cell culture. J Biol Chem 272: 1935–1942, 1997PubMedGoogle Scholar
  54. 54.
    Jakubowski H: Protein homocysteinylation possible mechanism underlying pathological consequences of elevated homocysteine levels. FASEB J 13: 2277–2283, 1999PubMedGoogle Scholar
  55. 55.
    Jakubowski H, Zhang L, Bardeguez A, Aviv A: Homocysteine-thiolactone and protein homocysteinylation in human endothelial cells: implications for atherosclerosis. Cir Res 87: 45–51, 2000Google Scholar
  56. 56.
    Wang G, Siow YL, O K: Homocysteine induces monocyte chemoattractant protein-1 expression by activation NF-kappa B in THP-1 macrophages. Am J Physiol Heart Circ Physiol 280: 2840–2847, 2001Google Scholar
  57. 57.
    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, 2001CrossRefPubMedGoogle Scholar
  58. 58.
    Collins T, Cybulsky MI: NF-kappaB: pivotal mediator or innocent bystander in atherogenesis? J Clin Invest 107: 255–264, 2001PubMedCrossRefGoogle Scholar
  59. 59.
    Au-Yeung KKW, Woo CWH, Sung FL, Yip JCW, Siow YL, Karmin O: Hyperhomocysteinemia activates Nuclear Factor-kB in endothelial cells via oxidative stress. Cir Res 94: 28–36, 2004CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, Inc. 2006

Authors and Affiliations

  • Mohsen Kerkeni
    • 1
    • 2
    • 5
  • Mehdi Tnani
    • 1
  • Laurence Chuniaud
    • 2
  • Abdelhedi Miled
    • 3
  • Khira Maaroufi
    • 1
  • François Trivin
    • 2
    • 4
  1. 1.Research Unit 03/UR/08-14, Faculty of PharmacyMonastirTunisia
  2. 2.Department of BiochemistryHospital Saint-JosephParisFrance
  3. 3.Department of Biochemistry and Toxicology CHU HachedSousseTunisia
  4. 4.Department of Clinical BiochemistryFrançois Rabelais UniversityToursFrance
  5. 5.Department of BiochemistryHospital Saint-JosephParis cedex 14France

Personalised recommendations