On the Possible Role of Reactive Oxygen Species in Angiogenesis

  • Peter I. LelkesEmail author
  • Kenneth L. Hahn
  • Drew A. Sukovich
  • Soverin Karmiol
  • Donald H. Schmidt
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 454)


Human microvascular endothelial cells grown on a 3-D reconstituted extracellular matrix (Matrigel) spontaneously and rapidly form a capillary network of tubular structures, thus modeling part of the angiogenic cascade. Exposure of the cells at the time of plating onto Matrigel to a brief episode of hypoxia (40–60) min and subsequent reoxygenation, significantly accelerated (up to 3-fold) the rate of tubular morphogenesis, as determined by computer-aided morphometry. This effect was not dependent on activation of PKC or upregulation/release of angiogenic growth factors. Rather, hypoxia/reoxygenation (H/R), but not hypoxia alone, caused the formation of reactive oxygen species (ROS) and the activation of the nuclear transcription factor NFκB, both of which were inhibited by ROS-scavengers, such as pyrollidine dithiocarbamate. Tube formation was inhibited, also under normoxic conditions, by diverse ROS antagonists in a dose-dependent fashion. Our results indicate that angiogenesis is accompanied by and/or requires generation of ROS. We hypothesize that in the clinical setting of hypoxia/reoxygenation during ischemic preconditioning, enhanced activation of ROS-dependent intracellular signaling may accelerate the rate of neovascularization also in vivo, thus contributing to the alleviation of certain ischemic lesions.


Tube Formation Normoxic Condition Vascular Endothelial Cell Growth Factor Angiogenic Growth Factor Endogenous Reactive Oxygen Species 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Andrews, N.C. and Faller, D.V. (1991) A rapid micropreparation technique for extraction of DNA-binding proteins from limiting numbers of mammalian cells. Nucleic Acids Res. 19: 2499PubMedCentralPubMedCrossRefGoogle Scholar
  2. Auerbach, R., Auerbach, W., and Polakowski, I. (1991) Assays for angiogenesis: a review. Pharmac. Ther. 51: 1–11.CrossRefGoogle Scholar
  3. Baatout, S. (1997) Endothelial differentiation using Matrigel. Anticancer Res. 17: 451–455.PubMedGoogle Scholar
  4. Beit-Yannai E., Zhang R., Trembovler V., Samuni A., and Shohami E. (1996) Cerebroprotective effect of stable nitroxide radicals in closed head injury in the rat. Brain Res 717: 22–28.PubMedCrossRefGoogle Scholar
  5. Brogi, E., Schatteman, G., Wu, T., Kim, E.A., Varticovski, L., Keyt, B., and Isner, J.M. (1996) Hypoxia-induced paracrine regulation of vascular endothelial growth factor receptor expression. J. Clin. Invest. 97: 469–476.PubMedCentralPubMedCrossRefGoogle Scholar
  6. Collins, T. (1993) Biology of disease: Endothelial nuclear factor-κB and the initiation of the atherosclerotic lesion. Lab. Invest. 68: 499–508.PubMedGoogle Scholar
  7. Collins, T., Read, M.A., Neish, A.S., Whitley, M.Z., Thanos, D., and Maniatis, T. ( 1995) Transcriptional regulation of endothelial cell adhesion molecules: NF-κB and cytokine-inducible enhancers. FASEB J. 9: 899–909.PubMedGoogle Scholar
  8. Davis, C.M., Danehower, S.C., Laurenza, A., and Molony, J.L. (1993) Identification of a role of the vitronectin receptor and protein kinase C in the induction of endothelial cell vascular formation. J. Cell. Biochem. 51: 206–218.PubMedCrossRefGoogle Scholar
  9. Foresti, R., Clark, J.E., Green, C.J., and Motterlini, R. (1997) Thiol compounds interact with nitric oxide in regulating heme oxygenase-1 induction in endothelial cells — Involvement of Superoxide and peroxynitrite anions. J. Biol. Chem. 272: 18411–18417.PubMedCrossRefGoogle Scholar
  10. Grant, D.S., Lelkes, P.I., Fukuda, K., and Kleinman, H.K. (1991) Intracellular mechanisms involved in basement membrane induced blood vessel differentiation in vitro. In Vitro Cell. Dev. Biol. 27A: 327–336.PubMedCrossRefGoogle Scholar
  11. Gupta, M.R, Evanoff, V., and Hart, C.M. (1997) Nitric oxide attenuates hydrogen peroxide-mediated injury to porcine pulmonary artery endothelial cells. Am. J. Physiol. 272: L1133–L1141.PubMedGoogle Scholar
  12. Hahn, K.A., Schmidt, D.H., and Lelkes, P.I. (1996) Hypoxia enhances angiogenesis of human microvascularendo-thelial cells cultured on Matrigel. In: Molecular, Cellular and Clinical Aspects of Angiogenesis (M.E. Maragoudakis, ed.), pp 260–261, Plenum Press, New York and London.Google Scholar
  13. Haralabopoulos, G.C., Grant, D.S., Kleinman, H.K., Lelkes, P.I, Papaioannou, S.P., and Maragoudakis, M.E. (1994) Inhibitors of basement membrane collagen synthesis prevent endothelial cell alignment in Matrigel in vitro and angiogenesis in vivo. Lab. Invest. 71: 575–582.PubMedGoogle Scholar
  14. Kanda, K., Hayman, G.T., Silverman, M.D., and Lelkes, P.I. (1998) Differential expression of ICAM-1 and VCAM-1 in human endothelial cell and smooth muscle cells derived from distinct vascular beds. Endothelium(in press).Google Scholar
  15. Khachigian, L.M., Lindner, V., Williams, A.J., and Collins, T. (1996) Egr-1-induced endothelial gene expression: a common theme in vascular injury. Science 271: 1427–1431.PubMedCrossRefGoogle Scholar
  16. Kinsella, J.L., Grant, D.S., Weeks, B.S., and Kleinman, H.K. (1992) Protein kinase C regulates endothelial cell tube formation on basement membrane matrix, Matrigel. Exp. Cell Res. 199: 56–62.PubMedCrossRefGoogle Scholar
  17. Klein, C.L., Köhler, H., Bittinger, F., Otto, M., Hermanns, L, and Kirkpatrick, C.J. (1995) Comparative studies on vascular endothelium in vitro. 2. Hypoxia: Its influences on endothelial cell proliferation and expression of cell adhesion molecules. Pathobiology 63: 1–8.PubMedCrossRefGoogle Scholar
  18. Kubota, Y., Kleinman, H.K., Martin, G.R., and Lawley, T.J. (1988) Role of laminin and basement membrane in the morphological differentiation of human endothelial cells into capillary-like structures. J. Cell Biol. 107: 1589–1598.PubMedCrossRefGoogle Scholar
  19. Lander, H.M. (1997) An essential role for free radicals and derived species in signal transduction. FASEB J. 11 Google Scholar
  20. Laniado-Schwartzman, M., Lavrovsky, Y, Stoltz, R.A., Conners, M.S., Falck, J.R., Chauhan, K., and Abraham, N.G. (1994) Activation of nuclear factor KB and oncogene expression by 12(R)-hydroxy-eicosatrienoic acid, an angiogenic factor in microvessel endothelial cells. J. Biol. Chem. 269: 24321–24327.PubMedGoogle Scholar
  21. Lefer, A.M., Tsao, P.S., Lefer, D.J., and Ma, X.-L. (1991) Role of endothelial dysfunction in the pathogenesis of reperfusion injury after myocardial ischemia. FASEB J. 5: 2029–2034.PubMedGoogle Scholar
  22. Lelkes, P.I., Hahn, K.A., Karmiol, S., and Schmidt, D.H. (1998), Hypoxia/Reoxygenation enhances tube formation of cultured human microvascular endothelial cells: The role of reactive oxygen species. In: Angiogenesis (M.E. Maragoudakis, ed.), Plenum Press, New York and London, in press.Google Scholar
  23. Lelkes, P.I., Manolopoulos, V.G., Silverman, M., Zhang, S., Karmiol, S., and Unsworth, B.R. (1996) On the possible role of endothelial cell heterogeneity in angiogenesis. In: Molecular, cellular, and clinical aspects of angiogenesis, 1–18. Edited by Maragoudakis, M.E. New York, Plenum Press.Google Scholar
  24. Manolopoulos, V.G., Samet, M.M., and Lelkes, P.I. (1995) Regulation of the adenylyl cyclase signaling system in various types of cultured endothelial cells. J. Cell. Biochem. 57: 590–598.PubMedCrossRefGoogle Scholar
  25. Maragoudakis, M.E., Tsopanoglou, N.E., and Haralabopoulos, G. (1993) Regulation of angiogenesis via protein kinase C. In: Vascular Endothelium, pp. 81–85. (J.D. Catravas, ed.), New York, Plenum PressCrossRefGoogle Scholar
  26. Maulik, N., Watanabe, M., Zu, Y.L., Huang, C.K., Cordis, G.A., Schley, J.A., and Das, D.K. (1996) Ischemic preconditioning triggers the activation of MAP kinases and MAPKAP kinase 2 in rat hearts. FEBS Lett. 396:233–237.PubMedCrossRefGoogle Scholar
  27. Maxwell S.R. (1995) Prospects for the use of antioxidant therapies. Drugs 49:345–361.PubMedCrossRefGoogle Scholar
  28. Millauer, B., Wizigmann-Voos, S., Schnürch, H., Martinez, R., Moller, N.P.H., Risau, W., and Ullrich, A (1993) High affinity VEGF binding and developmental expression suggest Flk-1 as a major regulator of vasculogenesis and angiogenesis. Cell 72: 835–846.PubMedCrossRefGoogle Scholar
  29. Montesano, R. and Orci, L. (1985) Tumor-promoting phorbol esters induce angiogenesis in vitro. Cell 42:469–477.Google Scholar
  30. Morbidelli, L., Chang, C.H., Douglas, J.G., Granger, H.J., Ledda, F., and Ziehe, M. (1996) Nitric oxide mediates mitogemc effect of VEGF on coronary venular endothelium. Am. J. Physiol. 270: H411–H415.PubMedGoogle Scholar
  31. Papadimitriou, E., Manolopoulos, V.G., Maragoudakis, M.E., Unsworth, B.R., and Lelkes, P.I. (1997) Thrombin modulates vectorial secretion of extracellular matrix proteins in cultured endothelial cells Am J Phvsiol 272: C1112–C1122.Google Scholar
  32. Pepper, M.S. (1997) Manipulating angiogenesis — From basic science to the bedside. Arterioscler Thromb. Vasc Biol. 17: 605–619.PubMedCrossRefGoogle Scholar
  33. Phillips, P.G., Birnby, L.M., and Narendran, A. (1995) Hypoxia induces capillary network formation in cultured bovine pulmonary microvessel endothelial cells. Am. J. Physiol. Lung Cell. Mol. Physiol. 268: L789–L800.Google Scholar
  34. Pipili-Synetos, E., Papageorgiou, A., Sakkoula, E., Sotiropoulou, G., Fotsis, T., Karakiulakis, G., and Maragoudakis, M.E. (1995) Inhibition of angiogenesis, tumour growth and metastasis by the NO-releasing vasodilators, isosorbide mononitrate and dinitrate. Br. J. Pharmacol. 116: 1829–1834.PubMedCentralPubMedCrossRefGoogle Scholar
  35. Pipili-Synetos, E., Sakkoula, E., Haralabopoulos, G., Andriopoulou, P., Peristeris, P., and Maragoudakis, M.E. (1994) Evidence that nitric oxide is an endogenous anti-angiogenic mediator. Br. J. Pharmacol. 111: 894–902.PubMedCentralPubMedCrossRefGoogle Scholar
  36. Pipili-Synetos, E., Sakkoula, E., and Maragoudakis, M.E. (1993) Nitric oxide is involved in the regulation of angiogenesis. Br. J. Pharmacol. 108: 855–857.PubMedCentralPubMedCrossRefGoogle Scholar
  37. Przyklenk, K. and Kloner, R.A. (1996) Role of protein kinase C in ischemic preconditioning: in search of the “pure and simple truth”. Basic Res. Cardiol. 91: 41–43.PubMedCrossRefGoogle Scholar
  38. Read, M.A., Whitley, M.Z., Williams, A.J., and Collins, T. (1994) NF-κB and IκBα: an inducible regulatory system in endothelial activation. J. Exp. Med. 179: 503–512.PubMedCrossRefGoogle Scholar
  39. Royall, J.A. and 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: 348–355.PubMedCrossRefGoogle Scholar
  40. Sahai, A., Patel, M.S., Zavosh, A.S., and Tannen, R.L. (1994) Chronic hypoxia impairs the differentiation of 3T3Ll fibroblast in culture: role of sustained protein kinase C activation. J. Cell. Physiol. 160: 107–112.PubMedCrossRefGoogle Scholar
  41. Samet, M.M. and Lelkes, P.I. (1993) Flow patterns and endothelial cell morphology in a simplified model of an artificial ventricle. Cell Biophys. 23: 139–163.PubMedCrossRefGoogle Scholar
  42. Seetharam, L., Gotoh, N., Maru, Y., Neufeld, G., Yamaguchi, S., and Shibuya, M. (1995) a unique signal transduction from FLT tyrosine kinase, a receptor for vascular endothelial growth factor VEGF. Oncogene 10: 135–147.PubMedGoogle Scholar
  43. Shono, T., Ono, M., Izumi, H., Jimi, S., Matsushima, K., Okamoto, T., Kohno, K., and Kuwano, M. (1996) Involvement of the transcription factor NF-kappaB in tubular morphogenesis of human microvascular endothelial cells by oxidative stress. Mol. Cell. Biol. 16: 4231–4239.PubMedCentralPubMedGoogle Scholar
  44. Shweiki, D., Itin, A., Soffer, D., and Keshet, E. (1992) Vascular endothelial growth factor induced by hypoxia may mediate hypoxia-initiated angiogenesis. Nature 359: 843–845.PubMedCrossRefGoogle Scholar
  45. Silverman, M.D., Manolopoulos, V.G., Unsworth, B.R., and Lelkes, P.I. (1996) Tissue factor expression is differentially modulated by cyclic mechanical strain in various human endothelial cells. Blood Coagul. Fibrinolysis 7: 281–288.PubMedCrossRefGoogle Scholar
  46. Stoltz, R.A., Abraham, N.G., and Laniado-Schwartzman, M.L. (1996) The role of NFKB in the angiogenic response of coronary microvessel endothelial cells. Proc. Natl. Acad. Sci. USA 93: 2832–2837.PubMedCentralPubMedCrossRefGoogle Scholar
  47. Strasser, R., Htun, P., and Schaper, W. (1996) Salvage of jeopardized myocardium by ischemie preconditioning: Is the quest over? Mol. Cell. Biochem. 161: 209–215.CrossRefGoogle Scholar
  48. Takagi, H., King, G.L., Ferrara, N., and Aiello, L.P. (1996) Hypoxia regulates vascular endothelial growth factor receptor KDR/Flkgene expression through adenosine A2 receptors in retinal capillary endothelial cells. Invest. Ophthalmol. Vis. Sci. 37: 1311–1321.PubMedGoogle Scholar
  49. Tsopanoglou, N.E., Haralabopoulos, G.C., and Maragoudakis, M.E. (1995) Opposing effects on modulation of angiogenesis by protein kinase C and cAMP-mediated pathways. J. Vasc. Res. 270: 8367–8372.Google Scholar
  50. Tsopanoglou, N.E., Pipili-Synetos, E., and Maragoudakis, M.E. (1993) Protein kinase C involvement in the regulation of angiogenesis. J. Vasc. Res. 30: 202–208.PubMedCrossRefGoogle Scholar
  51. Tucci, M., Hammerman, S.I., Furfaro, S., Saukonnen, J.J., Conca, T.J., and Farber, H.W. (1997) Distinct effect of hypoxia on endothelial cell proliferation and cycling. Am. J. Physiol. 272: C1700–C1708.PubMedGoogle Scholar
  52. Ware, J.A. and Simons, M. (1997) Angiogenesis in ischemie heart disease. Nature Medicine 3: 158–164.PubMedCrossRefGoogle Scholar
  53. White, B.C., Grossman, L.I., and Krause, G.S. (1993) Brain injury by global ischemia and reperfusion. Neurology 43: 1656–1665.PubMedCrossRefGoogle Scholar
  54. Wojta, J., Jones, R.L., Binder, B.R., and Hoover, R.L. (1988) Reduction in pO2 decreases the fibrinolytic potential of cultured bovine endothelial cells derived from pulmonary arteries and lung microvasculature. Blood 71: 1703–1706.PubMedGoogle Scholar
  55. Ziehe, M., Morbidelli, L., Choudhuri, R., Zhang, H.-T, Donnini, S., and Granger, H.T. (1997) Nitric oxide synthase lies downstream from vascular endothelial growth factor-induced but not basic fibroblast growth factor-induced angiogenesis. J. Clin. Invest. 99: 2625–2634.CrossRefGoogle Scholar
  56. Ziehe, M., Morbidelli, L., Masini, E., Amerini, S., Granger, H.J., Maggi, C.A., Geppetti, P., and Ledda, F. (1994) Nitric oxide mediates angiogenesis in vivo and endothelial cell growth and migration in vitro promoted by substance P. J. Clin. Invest. 94: 2036–2044.CrossRefGoogle Scholar
  57. Ziehe, M., Morbidelli, L., Parenti, A., and Ledda, F. (1995) Nitric oxide modulates angiogenesis elicited by prostaglandin El in rabbit cornea. Adv. Prostaglandin Thromboxane Leukocyte Res. 23: 495–497.Google Scholar
  58. Ziehe, M., Parenti, A., Ledda, F., Dell’Era, P., Granger, H.J., Maggi, C.A., and Presta, M. (1997) Nitric oxide promotes proliferation and plasminogen activator production by coronary venular endothelium through endogenous bFGF. Circ. Res. 80: 845–852.CrossRefGoogle Scholar
  59. Zulueta, J.J., Sawhney, R., Yu, F.S., Cote, C.C., and Hassoun, P.M. (1997) Intracellular generation of reactive oxygen species in endothelial cells exposed to anoxia-reoxygenation. Am. J. Physiol. 272: L897–L902.PubMedGoogle Scholar
  60. Zünd, G., Nelson, D.P., Neufeld, E.J., Dzus, A.L., Bischoff, J., Mayer, J.E., and Colgan, S.P. (1996) Hypoxia enhances stimulus-dependent induction of E-selectin on aortic endothelial cells. Proceedings of the National Academy of Sciences of the United States of America 93: 7075–7080.PubMedCentralPubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 1998

Authors and Affiliations

  • Peter I. Lelkes
    • 1
    Email author
  • Kenneth L. Hahn
    • 2
  • Drew A. Sukovich
    • 3
  • Soverin Karmiol
    • 4
  • Donald H. Schmidt
    • 2
  1. 1.Laboratory of Cell Biology, Department of MedicineUniversity of Wisconsin Medical SchoolMilwaukeeUSA
  2. 2.Section of Cardiology Department of MedicineUniversity of Wisconsin Medical SchoolUSA
  3. 3.Cardiovascular ResearchBerlex BiosciencesRichmondUSA
  4. 4.BioWhittaker, Inc.WalkersvilleUSA

Personalised recommendations