Skip to main content
SpringerLink
Log in

Possible involvement of NADPH oxidase and JNK in homocysteine-induced oxidative stress and apoptosis in human umbilical vein endothelial cells

  • Published:
Cardiovascular Toxicology Aims and scope Submit manuscript

Abstract

Hyperhomocysteinemia is an independent risk factor for cardiovascular diseases, although the mechanism leading to vascular dysfunction is not clear. The aim of this study was to examine the effect of homocysteine (Hcy) on oxidative stress and apoptosis in human umbilical vein endothelial cells (HUVECs). HUVECs were challenged for 24 h with Hcy (10 μM-3 mM) in the presence of various stress signaling inhibitors, including the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor apocynin (100 μM), the p38 mitogen-activated protein kinase inhibitor SB203580 (2.5 μM), the extracellular signal-regulated kinase inhibitor U0126 (2.5 μM), the stress-activated protein kinase (SAPK)/c-Jun NH2-terminal kinase (JNK) inhibitor JNK inhibitor II (10 μM), and antioxidants α-tocopherol (5 μg/mL) and N-acetyl cysteine (NAC, 2 mM). Reactive oxygen species (ROS) were detected using 5-(6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate. Apoptosis was evaluated by 4′,6′-diamidino-2′-phenylindoladihydrochloride staining, annexin-V phosphatidyl-serine/propidium iodide, and caspase-3 assay. NADPH oxidase and SAPK/JNK signal were evaluated with immunoblotting. Hcy significantly enhanced ROS generation and apoptosis after 24-h incubation. Apocynin prevented Hcy-induced ROS generation but only partially restored Hcy-induced apoptosis. JNK inhibitor II, α-tocopherol, and NAC partially reduced Hcy-induced apoptosis, although SB203580 and U0126 had no effect. Immunoblotting analysis confirmed upregulation of NADPH oxidase and SAPK/JNK signaling. Collectively, our results suggested that Hcy may induce oxidative stress and apoptosis through an NADPH oxidase and/or JNK-dependent mechanisms(s).

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Domagala, T.B., Undas, A., Libura, M., and Szczeklik, A. (1998). Pathogenesis of vascular disease in hyperhomocysteinaemia. J. Cardiovasc. Risk 5:239–247.

    Article  PubMed  CAS  Google Scholar 

  2. Fallon, U.B., Ben-Shlomo, Y., Elwood, P., Ubbink, J.B., and Smith, G.D. (2001). Homocysteine and coronary heart disease in the Caerphilly cohort: a 10 year follow up. Heart 85:153–158.

    Article  PubMed  CAS  Google Scholar 

  3. Mujumdar, V.S., Aru, G.M., and Tyagi, S.C. (2001) Induction of oxidative stress by homocyst(e)ine impairs endothelial function. J. Cell. Biochem. 82:491–500.

    Article  PubMed  CAS  Google Scholar 

  4. Li, N., Yi, F.X., Rute, E., Zhang, D.X., Slocum, G.R., and Zou, A.P. (2002). Effects of homocysteine on intracellular nitric oxide and superoxide levels in the renal arterial endothelium. Am. J. Physiol. Heart Circ. Physiol. 283:H1237-H1243.

    PubMed  CAS  Google Scholar 

  5. Weiss, N., Keller, C., Hoffmann, U., and Loscalzo, J. (2002). Endothelial dysfunction and atherothrombosis in mild hyperhomocysteinemia. Vasc. Med. 7:227–239.

    Article  PubMed  Google Scholar 

  6. Jacobsen, D.W. (1998). Homocysteine and vitamins in cardiovascular disease. Clin. Chem. 44:1833–1843.

    PubMed  CAS  Google Scholar 

  7. Majors, A., Ehrhart, L.A., and Pezacka, E.H. (1997). Homocysteine as a risk factor for vascular disease: enhanced collagen production and accumulation by smooth muscle cells. Arterioscler. Thromb Vasc. Biol. 17:2074–2081.

    PubMed  CAS  Google Scholar 

  8. Nygard, O., Vollset, S.E., Refsum, H., Brattstrom, L., and Ueland, P.M. (1999). Total homocysteine and cardiovascular disease. J. Intern. Med. 246:425–454.

    Article  PubMed  CAS  Google Scholar 

  9. Privratsky, J.R., Wold, L.E., Sowers, J.R., Quinn, M.T., and Ren, J. (2003). AT1 blockade prevents glucose-induced cardiac dysfunction in ventricular myocytes: role of the AT1 receptor and NADPH oxidate. Hypertension 42:206–212.

    Article  PubMed  CAS  Google Scholar 

  10. Fuentes, L., Perez, R., Nieto, M.L., Balsinde, J., and Balboa, M.A. (2003). Bromoenol lactone promotes cell death by a mechanism involving phosphatidate phosphohydrolase-1 rather than calcium-independent phospholipase A2. J. Biol. Chem. 278:44683–44690.

    Article  PubMed  CAS  Google Scholar 

  11. Jamali, R., Bao, M., and Arnqvist, H.J. (2003). IGF-I but not insulin inhibits apoptosis at a low concentratin in vascular smooth muscle cells. J. Endocrinol. 179:267–274.

    Article  PubMed  CAS  Google Scholar 

  12. Wang, X., Martindale, J.L., and Holbrook, N.J. (2000). Requirement for ERK activation in cisplatin-induced apoptosis. J. Biol. Chem. 275:39435–39443.

    Article  PubMed  CAS  Google Scholar 

  13. Shi, X., and Nuttall, A.L. (2003): Upregulated iNOS and oxidative damage to the cochlear stria vascularis due to noise stress. Brain Res. 967:1–10.

    Article  PubMed  CAS  Google Scholar 

  14. Yuan, Q, Ray, R.M., and Johnson, L.R. (2002). Polyamine depletion prevents camptothecin-induced apoptosis by inhibiting the release of cytochrome c. Am. J. Physiol. Cell Physiol. 282:C1290-C1297.

    PubMed  CAS  Google Scholar 

  15. Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248–254.

    Article  PubMed  CAS  Google Scholar 

  16. Zhang, X., Klein, A.L., Alberle, N.S. II, Norby, F.L., Ren, B.H., Duan, J., et al. Cardiac-specific overexpression of catalase rescues ventricular myocytes from ethanol-induced cardiac contractile defect. J. Mol. Cell. Cardiol. 35:645–652.

  17. Huang, R.F., Huang, S.M., Lin, B.S., Wei, J.S., and Liu, T.Z. (2001). 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.

    Article  PubMed  CAS  Google Scholar 

  18. Rodrigo, R., Passalacqua, W., Araya, J., Orellana, M., and Rivera, G. (2003). Implications of oxidative stress and homocysteine in the pathophysiology of essential hypertension. J. Cardiovasc. Pharmacol. 42:453–461.

    Article  PubMed  CAS  Google Scholar 

  19. Zhu, B.T. (2002). On the mechanism of homocysteine pathophysiology and pathogenesis: a unifying hypothesis. Histol. Histopathol. 17:1283–1291.

    PubMed  CAS  Google Scholar 

  20. Assanelli, D., Bonanome, A., Pezzini, A., Albertini, F., Maccalli, P., Grassi, M., et al. (2004). Folic acid and Vitamin E supplementation effects on homocysteinemia, endothelial function and plasma antioxidant capacity in young myocardial-infarction patients. Pharmacol. Res. 49:79–84.

    Article  PubMed  CAS  Google Scholar 

  21. Eikelboom, J.W., Lonn, E., Genest, J. Jr., Hankey, G., and Yusuf, S. (1999). Homocyst(e) ine and cardiovascular disease: a critical review of the epideniologic evidence. Ann. Intern. Med. 131:363–375.

    PubMed  CAS  Google Scholar 

  22. Ford, E.S., Smith, S.J., Stroup, D.F., Steinberg, K.K., Mueller, P.M., and Thacker, S.B. (2002). Homocyst(e) ine and cardiovascular disease: a systematic review of the evidence with special emphasis on case-control studies and nested case-control studies. Int. J. Epidemiol. 31:59–70.

    Article  PubMed  Google Scholar 

  23. Cai, H., Griendling, K.K., and Harrison, D.G. (2003). The vascular NAD(P)H oxidases as therapeutic targets in cardiovascular diseases. Trends Pharmacol. Sci. 24:471–478.

    Article  PubMed  CAS  Google Scholar 

  24. Martindale, J.L., and Holbrook, N.J. (2002). Cellular response to oxidative stress: signaling for suicide and survival. J. Cell. Physiol. 192:1–15.

    Article  PubMed  CAS  Google Scholar 

  25. Torres, M., and Forman, H.J. (2003). Redox signaling and the MAP kinase pathways. Biofactors 17:287–296.

    PubMed  CAS  Google Scholar 

  26. Cai, Y., Zhang, C., Nawa, T., Aso, T., Tanaka, M., Oshiro, S., et al. (2000). Homocysteine-responsive ATF3 gene expression in human vascular endothelial cells: activation of c-Jun NH(2)-terminal kinase and promoter response element. Blood 96:2140–2148.

    PubMed  CAS  Google Scholar 

  27. Zhang, C., Kawauchi, J., Adachi, M.T., Hashimoto, Y., Oshiro, S., Aso, T., et al. (2001). Activation of JNK and transcriptional repressor ATF3/LRF1 through the IRE1/TRAF2 pathway is implicated in human vascular endothelial cell death by homocysteine. Biochem. Biophys. Res. Commun. 289:718–724.

    Article  PubMed  CAS  Google Scholar 

  28. Benhar, M., Engelberg, D., and Levitzki, A. (2002). ROS, stress-activated kinases and stress signaling in cancer. EMBO Rep. 3:420–425.

    Article  PubMed  CAS  Google Scholar 

  29. Kuranaga, E., and Miura, M. (2002). Molecular genetic control of caspases and JNK-mediated neural cell death. Arch. Histol. Cytol. 65:291–300.

    Article  PubMed  CAS  Google Scholar 

  30. Kyriakis, J.M., and Avruch, J. (2001). Mammalian mitogen-activated protein kinase signal transduction pathways activated by stress and inflammation. Physiol. Rev. 81: 807–869.

    PubMed  CAS  Google Scholar 

  31. Lin, A., and Dibling, B. (2002). The true face of JNK activation in apoptosis. Aging Cell 1:112–116.

    Article  PubMed  CAS  Google Scholar 

  32. Luo, J., Sun, Y., Lin, H., Qian, Y., Li, Z., Leonard, S.S., et al. (2003). Activation of JNK by vanadate induces a Fas-associated death domain (FADD)-dependent death of cerebellar granule progenitors in vitro. J. Biol. Chem. 278: 4542–4551.

    Article  PubMed  CAS  Google Scholar 

  33. Lin, A. (2003). Activation of the JNK signaling pathway: breaking the brake on apoptosis. Bioessays 25:17–24.

    Article  PubMed  Google Scholar 

  34. Crott, J., and Fenech, M. (2001). Preliminary study of the genotoxic potential of homocysteine in human lymphocytes in vitro. Mutagenesis 16:213–217.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Zoology and Physiology, University of Wyoming, Laramie, WY

    Zhaojie Zhang & Qun Ren

  2. Division of Pharmaceutical Sciences and Center for Cardiovascular Research and Alternative Medicine, University of Wyoming, 82071-3375, Laramie, WY

    Feng Dong, Xiaochun Zhang, Shi-Yan Li, Bruce Culver &  Jun Ren

Authors
  1. Feng Dong
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Xiaochun Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Shi-Yan Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Zhaojie Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Qun Ren
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Bruce Culver
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Jun Ren
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Jun Ren.

Additional information

The first two authors contributed equally to this work.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Dong, F., Zhang, X., Li, SY. et al. Possible involvement of NADPH oxidase and JNK in homocysteine-induced oxidative stress and apoptosis in human umbilical vein endothelial cells. Cardiovasc Toxicol 5, 9–20 (2005). https://doi.org/10.1385/CT:5:1:009

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1385/CT:5:1:009

Key Words

Search

Navigation