Native low density lipoprotein increases the production of both nitric oxide and reactive oxygen species in the human umbilical vein endothelial cells

  • Hyun Joong Yoon
  • Kee Oh Chay
  • Sung Yeul YangEmail author
Research Article



Nitric oxide synthases (NOSs) are a unique family of enzymes that catalyze the production of nitric oxide (NO) from l-arginine. Atherogenic action of oxidized low-density lipoproteins (oxLDL) may be mediated partly by the formation of NO in endothelial cells.


The objective of this study was to identify sources of reactive oxygen species (ROS) causing native LDL (nLDL)-induced senescence of cultured human umbilical vein endothelial cells (HUVECs).


HUVECs were treated with nLDL and NO production was assessed using Griess reagent as substrate and spectrophotometry in the absence or presence of specific inhibitors of endothelial NOS (eNOS) and inducible NOS (iNOS). In addition, expression levels of eNOS and iNOS were measured with ELISA and western blotting, and ROS was evaluated using 2′,7′-dichlorofluorescin diacetate (DCF-DA) and a fluorescence microplate reader.


NO formation in nLDL-treated HUVECs was significantly increased. Long-term treatment with nLDL up-regulated both eNOS and iNOS proteins. Such increase of NO production in HUVECs induced by nLDL was significantly suppressed by treatment with iNOS-selective inhibitor 1400 W, but not by the eNOS-selective inhibitor L-NIO. Native LDL treatment uncoupled Hsp90, the regulatory binding protein of eNOS, from the enzyme in HUVECs. Native LDL also significantly increased ROS production in HUVECs.


These findings suggest that oxidative stress originated from induction of iNOS and eNOS could be a causative factor for nLDL-induced senescence of HUVECs.


Native low density lipoprotein Human umbilical vein endothelial cell Nitric oxide Reactive oxygen species Inducible nitric oxide synthase 


Compliance with ethical standards

Conflict of interest

Yoon, H. J., Chay, K.O., and Yang, S.Y. declares that they have no conflicts of interest.


  1. Bredt DS, Snyder SH (1990) Isolation of nitric oxide synthase, a calmodulin-requiring enzyme. Proc Natl Acad Sci USA 87:682–685CrossRefGoogle Scholar
  2. Bredt DS, Hwang PM, Glatt CE, Lowenstein C, Reed RR, Snyder SH (1991) Cloned and expressed nitric oxide synthase structurally resembles cytochrome P-450 reductase. Nature 351:714–718CrossRefGoogle Scholar
  3. Dimmeler S, Haendeler J, Galle J, Zeiher AM (1997) Oxidized low-density lipoprotein induces apoptosis of human endothelial cells by activation of CPP32-like proteases. A mechanistic clue to the ‘response to injury’ hypothesis. Circulation 95(7):1760–1763CrossRefGoogle Scholar
  4. Ehara S, Ueda M, Naruko T, Haze K, Itoh A, Otsuka M, Komatsu R, Matsuo T, Itabe H, Takano T, Tsukamoto Y, Yoshiyama M, Takeuchi K, Yoshikawa J, Becker AE (2001) Elevated levels of oxidized low density lipoprotein show a positive relationship with the severity of acute coronary syndromes. Circulation 103:1955–1960CrossRefGoogle Scholar
  5. Galle J, Bengen J, Schollmeyer P, Wanner C (1995) Impairment of endothelium-dependent dilation in rabbit renal arteries by oxidized lipoprotein(a). Role of oxygen-derived radicals. Circulation 92(6):1582–1589CrossRefGoogle Scholar
  6. Gleissner CA, Leitinger N, Ley K (2007) Effects of native and modified low-density lipoproteins on monocyte recruitment in atherosclerosis. Hypertension 50(2):276–283CrossRefGoogle Scholar
  7. Guzik TJ, Korbut R, Adamek-Guzik T (2003) Nitric oxide and superoxide in inflammation and immune regulation. J Physiol Pharmacol 54(4):469–487Google Scholar
  8. Honjo T, Otsui K, Shiraki R, Kawashima S, Sawamura T, Yokoyama M, Inoue N (2008) Essential role of NOXA1 in generation of reactive oxygen species induced by oxidized low-density lipoprotein in human vascular endothelial cells. Endothelium 15:137–141CrossRefGoogle Scholar
  9. Hulthe J, Fagerberg B (2002) Circulating oxidized LDL is associated with subclinical atherosclerosis development and inflammatory cytokines (AIR Study). Arterioscler Thromb Vasc Biol 22:1162–1167CrossRefGoogle Scholar
  10. Ignarro LJ, Cirino G, Casini A, Napoli C (1999) Nitric oxide as signaling molecule in the vascular system: an overview. J Cardiovasc Pharmacol 34:879–886CrossRefGoogle Scholar
  11. Imanishi T, Hano T, Sawamura T, Nishio I (2004) Oxidized low-density lipoprotein induces endothelial progenitor cell senescence, leading to cellular dysfunction. Clin Exp Pharmacol Physiol 31(7):407–413CrossRefGoogle Scholar
  12. Khan BV, Parthasarathy SS, Alexander RW, Medford RM (1995) Modified low density lipoproteins and its constituents augment cytokineactivated vascular cell adhesion molecule-1 gene expression in human vascular endothelial cells. J Clin Invest 95:1262–1270CrossRefGoogle Scholar
  13. Kugiyama K, Kerns SA, Morrisett JD, Roberts R, Henry PD (1990) Impairment of endothelium-dependent arterial relaxation by lysolecithin in modified low-density lipoproteins. Nature 344:160–162CrossRefGoogle Scholar
  14. Lamas S, Marsden PA, Li GK, Tempst P, Michel T (1992) Endothelial nitric oxide synthase: molecular cloning and characterization of a distinct constitutive enzyme isoform. Proc Natl Acad Sci USA 89:6348–6352CrossRefGoogle Scholar
  15. Lin JH, Zhu Y, Liao HL, Kobari Y, Groszek L, Stemerman MB (1996) Induction of vascular cell adhesion molecule-1 by low-density lipoprotein. Atherosclerosis 127:185–194CrossRefGoogle Scholar
  16. Lind M, Hayes A, Caprnda M, Petrovic D, Rodrigo L, Kruzliak P, Zulli A (2017) Inducible nitric oxide synthase Good or bad? Biomed Pharmacother 93:370–375CrossRefGoogle Scholar
  17. Ling W, Lougheed M, Suzuki H (1997) Oxidized or acetylated low density lipoproteins are rapidly cleared by the liver in mice with disruption of the scavenger receptor class A type I/II gene. J Clin Invest 100:244–252CrossRefGoogle Scholar
  18. Moncada S, Palmer RM, Higgs EA (1991) Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacol Rev 43:109–142Google Scholar
  19. Nishi K, Itabe H, Uno M, Kitazato KT, Horiguchi H, Shinno K, Nagahiro S (2002) Oxidized LDL in carotid plaques and plasma associates with plaque instability. Arterioscler Thromb Vasc Biol 22:1649–1654CrossRefGoogle Scholar
  20. Oh ST, Park H, Yoon HJ, Yang SY (2017) Long-term treatment of native LDL induces senescence of cultured human endothelial cells. Oxid Med Cell Longev 2017:6487825Google Scholar
  21. Pritchard KA Jr, Schwarz SM, Medow MS, Stemerman MB (1991) Effect of low-density lipoprotein on endothelial cell membrane fluidity and mononuclear cell attachment. Am J Physiol 260:C43–C49CrossRefGoogle Scholar
  22. Pritchard KA Jr, Groszek L, Smalley DM, Sessa WC, Wu M, Villalon P, Wolin MS, Stemerman MB (1995) Native low-density lipoprotein increases endothelial cell nitric oxide synthase generation of superoxide anion. Circ Res 77:510–518CrossRefGoogle Scholar
  23. Robbesyn F, Salvayre R, Negre-Salvayre A (2004) Dual role of oxidized LDL on the NF-kappaB signaling pathway. Free Radic Res 38:541–551CrossRefGoogle Scholar
  24. Royall JA, Ischiropoulos H (1993) Evaluation of 2′,7′-dichlorofluorescin and dihydrorhodamine 123 as fluorescent probes for intracellular H2O2 in cultured endothelial cells. Arch Biochem Biophys 302:348–355CrossRefGoogle Scholar
  25. Schmidt HH, Murad F (1991) Purification and characterization of a human NO synthase. Biochem Biophys Res Commun 181:1372–1377CrossRefGoogle Scholar
  26. Schmidt HH, Seifert R, Bohme E (1989) Formation and release of nitric oxide from human neutrophils and HL-60 cells induced by a chemotactic peptide, platelet activating factor and leukotriene B4. FEBS Lett 244:357–360CrossRefGoogle Scholar
  27. Sessa WC, Harrison JK, Barber CM et al (1992) Molecular cloning and expression of a cDNA encoding endothelial cell nitric oxide synthase. J Biol Chem 267:15274–15276Google Scholar
  28. Shi Y, Lüscher TF, Camici GG (2014) Dual role of endothelial nitric oxide synthase in oxidized LDL-induced, p66Shc-mediated oxidative stress in cultured human endothelial cells. PLoS ONE. Google Scholar
  29. Singal PK, Khaper N, Palace V et al (1998) The role of oxidative stress in the genesis of heart disease. Cardiovasc Res 40(3):426–432CrossRefGoogle Scholar
  30. Smalley DM, Lin JH, Curtis ML, Kobari Y, Stemerman MB, Pritchard KA Jr (1996) Native LDL increases endothelial cell adhesiveness by inducing intercellular adhesion molecule-1. Arterioscler Thromb Vasc Biol 16:585–590CrossRefGoogle Scholar
  31. Sohn HY, Krotz F, Gloe T, Keller M, Theisen K, Klauss V, Pohl U (2003) Differential regulation of xanthine and NAD(P)H oxidase by hypoxia in human umbilical vein endothelial cells. Role of nitric oxide and adenosine. Cardiovasc Res 58:638–646CrossRefGoogle Scholar
  32. Steinberg D (1997) Low density lipoprotein oxidation and its pathological significance. J Biol Chem 272:20963–20966CrossRefGoogle Scholar
  33. Van Berkel TJ, De Rijke YB, Kruijt JK (1991) Different fate in vivo of oxidatively modified low density lipoprotein and acetylated low density lipoprotein in rats. Recognition by various scavenger receptors on Kupffer and endothelial liver cells. J Biol Chem 266:2282–2289Google Scholar
  34. Vannini F, Kashfi K, Nath N (2015) The dual role of iNOS in cancer. Redox Biol 6:334–343CrossRefGoogle Scholar
  35. Varadharaj S, Porter K, Pleister A, Wannemacher J, Sow A, Jarjoura D, Zweier JL, Khayat RN (2015) Endothelial nitric oxide synthase uncoupling: a novel pathway in OSA induced vascular endothelial dysfunction. Respir Physiol Neurobiol 207:40–47CrossRefGoogle Scholar
  36. Verhoye E, Langlois MR (2009) Circulating oxidized low-density lipoprotein: a biomarker of atherosclerosis and cardiovascular risk? Clin Chem Lab Med 47:128–137Google Scholar
  37. Xie QW, Cho HJ, Calaycay J, Mumford RA, Swiderek KM, Lee TD, Ding A, Troso T, Nathan C (1992) Cloning and characterization of inducible nitric oxide synthase from mouse macrophages. Science 256:225–228CrossRefGoogle Scholar
  38. Xia Y, Tsai AL, Berka V, Zweier JL (1998) Superoxide generation from endothelial nitric-oxide synthase. A Ca2+/calmodulin-dependent and tetrahydrobiopterin regulatory process. J Biol Chem 273:25804–25808CrossRefGoogle Scholar
  39. Zhao R, Ma X, Xie X, Shen GX (2009) Involvement of NADPH oxidase in oxidized LDL-induced upregulation of heat shock factor-1 and plasminogen activator inhibitor-1 in vascular endothelial cells. Am J Physiol Endocrinol Metab 297:E104–E111CrossRefGoogle Scholar
  40. Zhao K, Huang Z, Lu H, Zhou J, Wei T (2010) Induction of inducible nitric oxide synthase increases the production of reactive oxygen species in RAW264.7 macrophages. Biosci Rep 30(4):233–241CrossRefGoogle Scholar

Copyright information

© The Genetics Society of Korea 2019

Authors and Affiliations

  1. 1.Department of Biochemistry, Medical School, The Research Institute of Medical ScienceChonnam National UniversityHwasun-gunSouth Korea

Personalised recommendations