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Novel Function of Ascorbic Acid as an Angiostatic Factor

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Abstract

Endothelial permeability is increased by vascular endothelial cell growth factor and decreased by antioxidants. Whether or not l-ascorbic acid (Asc), which decreases endothelial permeability by stimulating the endothelial barrier function, is anti-angiogenic (angiostatic) remains unknown. We examined the role of Asc on angiogenesis using two assay systems. At first, the potential role of Asc on four steps of angiogenesis was investigated in cultured bovine microvascular endothelial cells. Asc inhibited the formation of vessel-like tubular structures of endothelial cells cultured on Matrigel; however, it did not decrease the activity of plasminogen activator (PA), which creates the space into which vascular vessels extend. Furthermore, even at high concentrations, Asc did not inhibit either the proliferation or migration of endothelial cell cultures. Secondly, whether Asc inhibited in vivo angiogenesis or not was studied on chick chorioallantoic membrane (CAM) during the 4–6 days of embryogenesis when neovascularization is rapid. It also revealed that angiogenesis was dose-dependently inhibited by Asc from 0.5 μmol/CAM with half-maximal inhibition at 2.5 μmol/CAM. Because it was previously reported that the endothelial barrier function decreases permeability via the stimulation of collagen synthesis induced by Asc, we treated CAM with the inhibitor of collagen synthesis, l-azetidine 2-carboxylic acid (AzC). This compound partially attenuated the angiostatic function of Asc on CAM. To understand the involvement of an antioxidant activity in the angiostatic function of Asc, we further examined the effect of glutathione (GSH), which is an endogenous antioxidant, on angiogenesis in CAM and endothelial cells. GSH inhibited CAM angiogenesis, as well as the formation of vessel-like tubular structures of endothelial cell cultures on Matrigel. Both Asc and GSH inhibited hydrogen peroxide (H2O2) induced tubular morphogenesis. These findings suggest that Asc affects angiogenesis through both its antioxidant properties and the stimulation of collagen synthesis. As the angiostatic activity of Asc may be one of the many effects involved in host resistance to the growth or invasiveness of solid cancer, it may be useful as a supplementary therapy in various angiogenic diseases.

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References

  1. Folkman J. Tumor angiogenesis: Therapeutic implications. N Engl J Med 1971; 285: 1182–6.

    Article  PubMed  CAS  Google Scholar 

  2. Malhotra R, Stenn KS, Fernandez LA, Braverman IM. Angiogenic properties of normal and psoriatic skin associated with epidermis, not dermis. Lab Invest 1989; 61: 162–5.

    PubMed  CAS  Google Scholar 

  3. Fava RA, Olsen NJ, Spencer-Green G et al. Vascular permeability factor/endothelial growth factor(VPF/VEGF): Accumulation and expression in human synovial fluids and rheumatoid synovial tissue. J Exp Med 1994; 180: 341–6.

    Article  PubMed  CAS  Google Scholar 

  4. Adamis AP, Miller JW, Bernal MT et al. Increased vascular endothelial growth factor levels in the vitreous of eyes with proliferative diabetic retinopathy. Am J Ophthalmol 1994; 118: 445–50.

    PubMed  CAS  Google Scholar 

  5. O'Brien ER, Garvin MR, Dev R et al. Angiogenesis in human coronary atherosclerotic plaques. Am J Pathol 1994; 145: 883–94.

    PubMed  Google Scholar 

  6. Lind J. A Treatise of the Scurvy. Edinburgh, UK: Edinburgh University Press 1953.

    Google Scholar 

  7. Bendich A, Langseth L. The health effects of vitamin C supplementation: A review. J Am Coll Nutr 1995; 14: 124–36.

    PubMed  CAS  Google Scholar 

  8. Lynch SM, Gaziano JM, Frei B. Ascorbic acid and atherosclerotic cardiovascular disease. Subcell Biochem 1996; 25: 331–67.

    PubMed  CAS  Google Scholar 

  9. King CG, Burns JJ. Second Conference on Vitamin C. Ann N Y Acad Sci 1975; 258: 253–306.

    Google Scholar 

  10. Murad S, Tajima S, Johnson GR et al. Collagen synthesis in cultured human skin fibroblast: Effect of ascorbic acid and its analog. J Invest Dermatol 1983; 81: 158–62.

    Article  PubMed  CAS  Google Scholar 

  11. Ivanov VO, Ivanova SV, Niedzwiecki A. Ascorbate affects proliferation of guinea-pig vascular smooth muscle cells by direct and extracellular matrix-mediated effects. J Mol Cell Cardiol 1997; 29: 3293–303.

    Article  PubMed  CAS  Google Scholar 

  12. Utoguchi N, Ikeda K, Saeki K et al. Ascorbic acid stimulates barrier function of cultured endothelial cell monolayer. J Cell Physiol 1995; 163: 393–9.

    Article  PubMed  CAS  Google Scholar 

  13. Connolly DT, Heuvelman DM, Nelson R et al. Tumor vascular permeability factor stimulates endothelial cell growth and angiogenesis. J Clin Invest 1989; 84: 1470–8.

    PubMed  CAS  Google Scholar 

  14. Ames BN, Shigenaga M, Hagen TM. Oxidants, antioxidants and the degenerative diseases of aging. Proc Natl Acad Sci USA 1993; 90: 7915–22.

    Article  PubMed  CAS  Google Scholar 

  15. Sandoval M, Zhang XJ, Liu X et al. Peroxynitrite-induced apoptosis in T84 and RAW 264.7 cells: Attenuation by L-ascorbic acid. Free Radic Biol Med 1997; 22: 489–95.

    Article  PubMed  CAS  Google Scholar 

  16. Ali MH, Schlidt SA, Chandel NS et al. Endothelial permeability and IL-6 production during hypoxia: Role of ROS in signal transduction. Am J Physiol 1999; 277: L1057–65.

    PubMed  CAS  Google Scholar 

  17. Anderson MT, Staal FJT, Gitler C et al. Separation of oxidantinitiated and redox-regulated steps in the NF-jB signal transduction pathway. Proc Natl Acad Sci USA 1994; 91: 11527–31.

    Article  PubMed  CAS  Google Scholar 

  18. Brauchle M, Funk JO, Kind P, Werner S. Ultraviolet B and H2O2 are potent inducer of vascular endothelial growth factor expression in cultured keratinocytes. J Biol Chem 1996; 271: 21793–7.

    Article  PubMed  CAS  Google Scholar 

  19. Kuroki M, Voest EE, Amano S et al. Reactive oxygen intermediates increase vascular endothelial growth factor expression in vitro and in vivo. J Clin Invest 1996; 98: 1667–75.

    Article  PubMed  CAS  Google Scholar 

  20. Wu J, Akaike T, Hayashida K et al. Enhanced vascular permeability in solid tumor involving peroxynitrite and matrix metalloproteionases. Jpn J Cancer Res 2001; 92: 439–51.

    PubMed  CAS  Google Scholar 

  21. Jiang C, Agarwal R, Lu J. Anti-angiogenic potential of a cancer chemopreventive flavonoid antioxidant, silymarin: Inhibition of key attributes of vascular endothelial cells and angiogenic cytokine secretion by cancer epithelial cells. Biochem Biophys Res Commun 2000; 276: 371–8.

    Article  PubMed  CAS  Google Scholar 

  22. Hirano F, Tanaka H, Miura T et al. Inhibition of NF-jBdependent transcription of human immunodeficiency virus 1 promoter by a phosphodiester compound of vitamin C and vitamin E, EPC-K1. Immunopharmacology 1998; 39: 31–8.

    Article  PubMed  CAS  Google Scholar 

  23. Nagao N, Etoh T, Yamaoka S et al. Enhanced invasion of Taxexpressing fibroblasts into the basement membrane is repressed by phosphorylated ascorbate with simultaneous decreases in intracellular oxidative stress and NF-kappa B activation. Antioxid Redox Signal 2000; 2: 727–38.

    Article  PubMed  CAS  Google Scholar 

  24. Bowie AG, O'Neill LA. Vitamin C inhibits NF-kappa B activation by TNF via the activation of p38 mitogen-activated protein kinase. J Immunol 2000; 165: 7180–8.

    PubMed  CAS  Google Scholar 

  25. Rodriguez-Porcel M, Lerman LO, Holmes DRJ et al. Chronic antioxidant supplementation attenuates nuclear factor-kappa B activation and preserves endothelial function in hypercholesterolemic pigs. Cardiovasc Res 2002; 53: 1010–8.

    Article  PubMed  CAS  Google Scholar 

  26. Liu JW, Nagao N, Kageyama K, Miwa N. Antimetastatic and anti-invasive ability of phospho-ascorbyl palmitate through intracellular ascorbate enrichment and the resultant antioxidant action. Oncol Res 1999; 11: 479–87.

    PubMed  CAS  Google Scholar 

  27. Gross J, Azizkhan RG, Biswas C et al. Inhibition of tumor growth, vascularization, and collagenolysis in the rabbit cornea by medroxyprogesterone. Proc Natl Acad Sci USA 1981; 78: 1176–1180.

    Article  PubMed  CAS  Google Scholar 

  28. Berman M, Winthrop S, Ausprunk D et al. Plasminogen activator (urokinase) causes vascularization of the cornea. Invest Ophthalmol Vis Sci 1982; 22: 191–9.

    PubMed  CAS  Google Scholar 

  29. Gross JL, Moscatelli D, Rifkin DB. Increased capillary endothelial cell protease activity in response to angiogenic stimuli in vitro. Proc Natl Acad Sci USA 1983; 80: 2623–7.

    Article  PubMed  CAS  Google Scholar 

  30. Mignatti P, Rifkin DB. Biology and biochemistry of proteinases in tumor invasion. Physiol Rev 1993; 73: 161–95.

    PubMed  CAS  Google Scholar 

  31. Ashino H, Shimamura M, Oikawa T et al. Inhibition of angiogenesis by diisopropyl fluorophosphate. Washington, DC: American Society for Microbiology 1994.

    Google Scholar 

  32. Shimamura M, Ashino-Fuse H, Oikawa T, Iwaguchi T. Actinonin, a proteinase inhibitor, potently inhibits angiogenesis. J Cancer Res Clin Oncol 1990; 116(Suppl I): 14.

    Google Scholar 

  33. Taraboletti G, Garofalo A, Belotti D et al. Inhibition of angiogenesis and murine hemangioma growth by batimastat, a synthetic inhibitor of matrix metalloproteinases. J Natl Cancer Inst 1995; 87: 293–8.

    PubMed  CAS  Google Scholar 

  34. Ashino-Fuse H, Takano Y, Oikawa T et al. Medroxyprogesterone acetate, an anti-cancer and anti-angiogenic steroid, inhibits the plasminogen activator in bovine endothelial cells. Int J Cancer 1989; 44: 859–64.

    PubMed  CAS  Google Scholar 

  35. Montesano R, Orci L. Tumor-promoting phorbol esters induce angiogenesis in vitro. Cell 1985; 42: 469–77.

    Article  PubMed  CAS  Google Scholar 

  36. Ingber D, Folkman J. Inhibition of angiogenesis through modulation of collagen metabolism. Lab Invest 1988; 59: 44–51.

    PubMed  CAS  Google Scholar 

  37. Nicosia RF, Belser P, Bonanno E, Diven J. Regulation of angiogenesis in vitro by collagen metabolism. In Vitro Cell Dev Biol 1991; 27A: 961–6.

    PubMed  CAS  Google Scholar 

  38. Mosmann T. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J Immunol Meth 1983; 65: 55–63.

    Article  CAS  Google Scholar 

  39. Shimada H, Mori T, Takada A et al. Use of chromogenic substrate S-2251 for determination of plasminogen activator in rat ovaries. Thromb Haemost 1981; 46: 507–10.

    PubMed  CAS  Google Scholar 

  40. Ashton AW, Yokota R, John G et al. Inhibition of endothelial cell migration, intercellular communication, and vascular tube formation by thromboxane A2. J Biol Chem 1999; 274: 35562–70.

    Article  PubMed  CAS  Google Scholar 

  41. Tanaka NG, Sakamoto N, Tohgo A et al. Inhibitory effects of anti-angiogenic agents on neovascularization and growth of the chorioallantoic membrane (CAM): The possibility of a new CAM assay for angiogenesis inhibition. Exp Pathol 1986; 30: 143–50.

    PubMed  CAS  Google Scholar 

  42. Inada Y, Hagiwara H, Kojima S et al. Synergism of vitamins A and C on fibrinolysis. Biochem Biophys Res Commun 1985; 130: 182–7.

    Article  PubMed  CAS  Google Scholar 

  43. Castronovo V, Belotti D. TNP-470 (AGM-1470): Mechanism of action and early clinical development. Eur J Cancer 1996; 32A: 2520–7.

    Article  PubMed  CAS  Google Scholar 

  44. O'Reilly MS, Holmgren L, Shing Y et al. Angiostatin: A novel angiogenesis inhibitor that mediates the suppression of metastases by a Lewis lung carcinoma. Cell 1994; 79: 315–28.

    Article  PubMed  Google Scholar 

  45. Cai T, Fassina G, Morini M et al. N-Acetylcysteine inhibits endothelial cell invasion and angiogenesis. Lab Invest 1999; 79: 1151–9.

    PubMed  CAS  Google Scholar 

  46. Shono T, Ono M, Izumi H et al. Involvement of the transcription factor NF-κB in tubular morphogenesis of human microvascular endothelial cells by oxidative stress. Mol Cell Biol 1996; 16: 4231–9.

    PubMed  CAS  Google Scholar 

  47. Carcamo JM, Pedraza A, Borquez-Ojeda O, Golde DW. Vitamin C suppresses TNFα-induced NF-κB activation by inhibiting I κB α phosphorylation. Biochemistry 2002; 41: 12995–13002.

    Article  PubMed  CAS  Google Scholar 

  48. Cameron E, Pauling L, Leibovitz B. Ascorbic acid and cancer: A review. Cancer Res 1979; 39: 663–81.

    PubMed  CAS  Google Scholar 

  49. Geesin JC, Darr D, Kaufman R, Murad S. Ascorbic acid specifically increases type I and type III procollagen messenger RNA levels in human skin fibroblast. J Invest Dermatol 1988; 90: 420–4.

    Article  PubMed  CAS  Google Scholar 

  50. Jimenez S, Harsch M, Rosenbloom J. Hydroxyproline stabilizes the triple helix of chick tendon collagen. Biochem Biophys Res Commun 1973; 52: 106–14.

    Article  PubMed  CAS  Google Scholar 

  51. Berg RA, Prockop DJ. The thermal transition of a non-hydroxylated form of collagen: Evidence for a role for hydroxyproline in stabilizing the triple-helix of collagen. Biochem Biophys Res Commun 1973; 52: 115–20.

    Article  PubMed  CAS  Google Scholar 

  52. Blanck TJ, Peterkofsky B. The stimulation of collagen secretion by ascorbate as a result of increased proline hydroxylation in chick embryo fibroblasts. Arch Biochem Biophys 1975; 171: 259–67.

    Article  PubMed  CAS  Google Scholar 

  53. Schwartz E, Bienkowski RS, Coltoff-Schiller B et al. Changes in the components of extracellular matrix and in growth properties of cultured aortic smooth muscle cells upon ascorbate feeding. J Cell Biol 1982; 92: 462–70.

    Article  PubMed  CAS  Google Scholar 

  54. Hata R, Sunada H, Arai K et al. Regulation of collagen metabolism and cell growth by epidermal growth factor and ascorbate in cultured human skin fibroblasts. Eur J Biochem 1988; 173: 261–7.

    Article  PubMed  CAS  Google Scholar 

  55. Knowles HJ, Raval RR, Harris AL, Ratcliffe PJ. Effect of ascorbate on the activity of hypoxia-inducible factor in cancer cells. Cancer Res 2003; 63: 1764–8.

    PubMed  CAS  Google Scholar 

  56. Grant DS, Lelkes PI, Fukuda K, Kleinman HK. Intracellular mechanisms involved in basement membrane induced blood vessel differentiation in vitro. In Vitro Cell Dev Biol 1991; 27A: 327–36.

    PubMed  CAS  Google Scholar 

  57. Schnaper HW, Grant DS, Stetler-Stevenson WG et al. Type IV collagenase(S) and TIMPs modulate endothelial cell morphogenesis in vitro. J Cell Physiol 1993; 156: 235–46.

    Article  PubMed  CAS  Google Scholar 

  58. Vincent L, Varet J, Pille JY et al. Efficacy of dendrimer-mediated angiostatin and TIMP-2 gene delivery on inhibition of tumor growth and angiogenesis: In vitro and in vivo studies. Int J Cancer 2003; 105: 419–29.

    Article  PubMed  CAS  Google Scholar 

  59. O'Reilly MS, Boehm T, Shing Y et al. Endostatin: An endogenous inhibitor of angiogenesis and tumor growth. Cell 1997; 88: 277–85.

    Article  PubMed  Google Scholar 

  60. Norrby K. The proline analog L-azetidine-2–carboxylic acid modifies the neovascularization pattern by inhibiting branching or tortuosity and stimulating spatial expansion in the rat mesentery. Int J Microcirc Clin Exp 1993; 12: 119–29.

    PubMed  CAS  Google Scholar 

  61. Yasuda M, Ohzeki Y, Shimizu S et al. Stimulation of in vitro angiogenesis by hydrogen peroxide and the relation with ETS-1 in endothelial cells. Life Sci 1999; 64: 249–58.

    Article  PubMed  CAS  Google Scholar 

  62. Cameron E, Rotman D. Ascorbic acid, cell proliferation, and cancer. Lancet 1972; 1: 542.

    Article  PubMed  CAS  Google Scholar 

  63. Cameron E, Pauling L. Ascorbic acid and the glycosaminoglycans: An orthomolecular approach to cancer and other diseases. Oncology 1973; 27: 181–92.

    Article  PubMed  CAS  Google Scholar 

  64. Kuribayashi N, Sakagami H, Sakagami T et al. Induction of DNA fragmentation in human myelogenous leukemic cell lines by sodium 5,6–Benzylidene-L-Ascorbate and its related compounds. Anticancer Res 1994; 14: 969–76.

    PubMed  CAS  Google Scholar 

  65. Oikawa T, Okayasu I, Ashino H et al. Three novel synthetic retinoids, Re 80, Am 580 and Am 80, all exhibit anti-angiogenic activity in vivo. Eur J Pharmacol 1993; 249: 113–6.

    Article  PubMed  CAS  Google Scholar 

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Authors and Affiliations

  1. Department of Molecular Biology, The Tokyo Metropolitan Institute of Medical Science, Tokyo Metropolitan Organization for Medical Research, Tokyo, Japan

    Hiromi Ashino & Seiichi Kawashima

  2. Medical R&D Center, The Tokyo Metropolitan Institute of Medical Science, Tokyo Metropolitan Organization for Medical Research, Tokyo, Japan

    Mariko Shimamura, Tsutomu Oikawa & Takao Iwaguchi

  3. UNITIKA Ltd. R&D Institute, Kyoto, Japan

    Hiroshi Nakajima, Munehiko Dombou & Satoshi Kawanaka

  4. Department of Technical Development, AKOKASEI Co., Ltd., Hyogo, Japan

    Hiroshi Nakajima

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  1. Hiromi Ashino
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Ashino, H., Shimamura, M., Nakajima, H. et al. Novel Function of Ascorbic Acid as an Angiostatic Factor. Angiogenesis 6, 259–269 (2003). https://doi.org/10.1023/B:AGEN.0000029390.09354.f8

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