Virchows Archiv

, Volume 426, Issue 5, pp 479–486 | Cite as

Hypoxia-induced expression of vascular endothelial growth factor by retinal glial cells promotes in vitro angiogenesis

  • Y. Hata
  • K. Nakagawa
  • K. Sueishi
  • T. Ishibashi
  • H. Inomata
  • H. Ueno
Original Article


To determine whether retinal glial cells (RGCs) participate in the paracrine regulation of retinal neovascularization, we investigated whether cultured RGCs synthesize and release vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) under normoxic or hypoxic conditions. Northern blot analysis demonstrated that cultured RGCs transcribed both VEGF mRNA with two molecular bands approximately 3.9 and 4.3 kilobases (kb), and bFGF mRNA with approximately 3.7 and 6.0 kb. The expression of VEGF mRNA was greatly enhanced by hypoxic cultivation (2% oxygen) when compared with normoxic cultivation (20% oxygen), while the expression of bFGF mRNA by RGCs was not significantly affected by hypoxia. The effects of RGCs-conditioned media (CM) on tritiated-thymidine incorporation and in vitro angiogenesis by retinal capillary endothelial cells (RECs) in producing the formation of capillary-like tubes in type I collagen gels, were evident in the observation that RGCs-CM harvested after hypoxic cultivation significantly enhanced tritiated-thymidine incorporation (1.9 times, P<0.01) and in vitro angiogenesis (2.4 times, P<0.01) compared with the normoxic RGCs-CM. These enhancing effects of RGCs-CM at hypoxia were suppressed by anti-VEGF neutralizing antibody. Furthermore, RECs were shown to express mRNA encoding the VEGF receptor flt-1 by northern blot analysis. These results suggest that VEGF expressed by RGCs under hypoxic conditions plays an integral role in the initiation and progression of retinal neovascularization in a paracrine manner.

Key words

Vascular endothelial growth factor Retinal glial cells Hypoxia Angiogenesis Retinal capillary endothelial cells 


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  1. 1.
    Adamis AP, Shima DT, Yeo K-T, Yeo T-K, Brown LF, Berse B, D'Amore PA, Folkman J (1993) Synthesis and secretion of vascular permeability factor/vascular endothelial growth factor by human retinal pigment epithelial cells. Biochem Biophys Res Commun 193:631–638Google Scholar
  2. 2.
    Aiello LP, Ferrara N, King GL (1994) Hypoxic regulation of vascular endothelial growth factor: Characterization in retinal microvascular pericytes and pigment epithelial cells (abstract). Invest Ophthalmol Vis Sci 5:1868Google Scholar
  3. 3.
    Alvarez JA, Baird A, Tatum A, Daucher J, Chorsky R, Gonzalez AM, Stopa EG (1992) Localization of basic fibroblast growth factor and vascular endothelial growth factor in human glial neoplasms. Mod Pathol 5:303–307Google Scholar
  4. 4.
    Blanchard KL, Acquaviva AM, Galson DL, Bunn HF (1992) Hypoxic induction of the human erythropoietin gene: Cooperation between the promoter and enhancer, each of which contains steroid receptor response elements. Mol Cell Biol 12:5373–5385Google Scholar
  5. 5.
    Brown LF, Berse B, Jackman RW, Tognazzi K, Manseau EJ, Senger DR, Dvorak HF (1993) Expression of vascular permeability factor (vascular endothelial growth factor) and its receptors in adenocarcinomas of the gastrointestinal tract. Cancer Res 53:4727–4735Google Scholar
  6. 6.
    Carter BZ, Malter JS (1991) Biology of disease. Regulation of mRNA stability and its relevance to desease. Lab Invest 65:610–621Google Scholar
  7. 7.
    Chomczynski P, Sacchi N (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159CrossRefPubMedGoogle Scholar
  8. 8.
    Connolly DT (1991) Vascular permeability factor: A unique regulator of blood vessel function. J Cell Biochem 47:219–223PubMedGoogle Scholar
  9. 9.
    Connolly DT, Olander JV, Heuvelman D, Nelson R, Monsell R, Siegel N, Haymore BL, Leimgruber R, Feder J (1989) Human vascular permeability factor: Isolation from U937 cells. J Biol Chem 264:20017–20024Google Scholar
  10. 10.
    Feinberg AP, Vogelstein B (1983) A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132:6–13PubMedGoogle Scholar
  11. 11.
    Ferrara N, Henzel WJ (1989) Pituitary follicular cells secrete a novel heparin-binding growth factor specific for vascular endothelial cells. Biochem Biophys Res Commun 161:851–858PubMedGoogle Scholar
  12. 12.
    Ferrara N, Houck KA, Jakeman LB, Winer J, Leung DW (1991) The vascular endothelial growth factor family of polypeptides. J Cell Biochem 47:211–218Google Scholar
  13. 13.
    Ferrara N, Houck K, Jakeman L, Leung DW (1992) Molecular and biological properties of the vascular endothelial growth factor family of proteins. Endocr Rev 13:18–32Google Scholar
  14. 14.
    Finkenzeller G, Marme D, Weich HA, Hug H (1992) Platelet-derived growth factor-induced transcription of vascular endothelial growth factor gene is mediated by protein kinase C. Cancer Res 52:4821–4823Google Scholar
  15. 15.
    Folkman J, Shing Y (1992) Angiogenesis. J Biol Chem 267:10931–10934PubMedGoogle Scholar
  16. 16.
    Goto F, Goto K, Weindel K, Folkman J (1993) Synergistic effects of vascular endothelial growth factor and basic fibroblast growth factor on the proliferation and cord formation on bovine capillary endothelial cells within collagen gels. Lab Invest 69:508–517Google Scholar
  17. 17.
    Hosoda Y, Okada M, Matsumura M, Ogino N, Honda Y, Nagai Y (1993) Epiretinal membrane of proliferative diabetic retinopathy: An immunohistochemical study. Ophthalmic Res 25:289–294Google Scholar
  18. 18.
    Houck KA, Ferrara N, Winer J, Cachianes G, Li B, Leung DW (1991) The vascular endothelial growth factor family: identification of a fourth molecular species and characterization of alternative splicing of RNA. Mol Endocrinol 5:1806–1814Google Scholar
  19. 19.
    Ishibashi T, Tanaka K, Taniguchi Y (1980) Disruption of blood-retinal barrier in experimental diabetic rats: An electron microscopic study. Exp Eye Res 30:401–410Google Scholar
  20. 20.
    Jakeman LB, Winer J, Bennett GL, Altar A, Ferrara N (1992) Binding sites for vascular endothelial growth factor are localized on endothelial cells in adult rat tissues. J Clin Invest 89:244–253Google Scholar
  21. 21.
    Karakurum M, Shreeniwas R, Chen J, Pinsky D, Yan S-D, Anderson M, Sunouchi K, Major J, Hamilton T, Kuwabara K, Rot A, Nowygrod R, Stern D (1994) Hypoxic induction of inter-leukin-8 gene expression in human endothelial cells. J Clin Invest 93:1564–1570Google Scholar
  22. 22.
    Ladoux A, Frelin C (1993) Hypoxia is a strong inducer of vascular endothelial growth factor mRNA expression in the heart. Biochem Biophys Res Commun 195:1005–1010Google Scholar
  23. 23.
    Laterra J, Guerin C, Goldstein GW (1990) Astrocytes induce neural microvascular endothelial cells to form capillary-like structures in vitro. J Cell Physiol 144:204–215Google Scholar
  24. 24.
    Leung DW, Cachianes G, Kuang W-J, Goeddel DV, Ferrara N (1989) Vascular endothelial growth factor is a secreted angiogenic mitogen. Science 246:1306–1309PubMedGoogle Scholar
  25. 25.
    Miyazono K, Okabe T, Ishibashi S, Urabe A, Takaku F (1985) A platelet factor stimulating the proliferation of vascular endothelial cells: Partial purification and characterization. Exp Cell Res 159:487–494Google Scholar
  26. 26.
    Morii K, Tanaka R, Washiyama K, Kumanishi T, Kuwano R (1993) Expression of vascular endothelial growth factor in capillary hemangioblastoma. Biochem Biophys Res Commun 194:749–755Google Scholar
  27. 27.
    Murata T, Ishibashi T, Inomata H, Sueishi K (1994) Media conditioned by coculture of pericytes and endothelial cells under a hypoxic state stimulate in vitro angiogenesis. Ophthalmic Res 26:23–31Google Scholar
  28. 28.
    Nork TM, Wallow IHL, Sramek SJ, Anderson G (1987) Müller's cell involvement in proliferative diabetic retinopathy. Arch Ophthalmol 105:1424–1429Google Scholar
  29. 29.
    Penfold PL, Provis JM, Madigan MC, Driel D van, Billson FA (1990) Angiogenesis in normal human retinal development: the involvement of astrocytes and macrophages. Graefes Arch Clin Exp Ophthalmol 228:255–263Google Scholar
  30. 30.
    Pepper MS, Ferrara N, Orci L, Montesano R (1992) Potent synergism between vascular endothelial growth factor and basic fibroblast growth factor in the induction of angiogenesis in vitro. Biochem Biophys Res Commun 189:824–831Google Scholar
  31. 31.
    Plate KH, Breier G, Weich HA, Risau W (1992) Vascular endothelial growth factor is a potential tumour angiogenesis factor in human gliomas in vivo. Nature 359:845–848Google Scholar
  32. 32.
    Plate K, Wizigmann-Voos S, Breier G, Risau W (1994) Evidence for paracrine growth regulatory mechanism in human hemangioblastomas (abstract). Clin Neuropathol 13:264Google Scholar
  33. 33.
    Sakuda H, Nakashima Y, Kuriyama S, Sueishi K (1992) Media conditioned by smooth muscle cells cultured in a variety of hypoxic environments stimulates in vitro angiogenesis: A relationship to transforming growth factor-β1. Am J Pathol 141:1507–1516Google Scholar
  34. 34.
    Sato Y, Rifkin DB (1989) Inhibition of endothelial cell movement by pericytes and smooth muscle cells: Activation of a latent transforming growth factor-β1-like molecule by plasmin during co-culture. J Cell Biol 109:309–315Google Scholar
  35. 35.
    Sato Y, Tsuboi R, Lyons R, Moses H, Rifkin DB (1990) Characterization of the activation of latent TGF-β by co-cultures of endothelial cells and pericytes or smooth muscle cells: A self-regulating system. J Cell Biol 111:757–763Google Scholar
  36. 36.
    Schultz GS, Grant MB (1991) Neovascular growth factors. Eye 170–180Google Scholar
  37. 37.
    Shweiki D, Itin A, Soffer D, Keshet E (1992) Vascular endothelial growth factor induced by hypoxia may mediate hypoxia-initiated angiogenesis. Nature 359:843–845CrossRefPubMedGoogle Scholar
  38. 38.
    Simorre-Pinatel V, Guerrin M, Chollet P, Penary M, Clamens S, Malecaze F, Plouet J (1994) Vasculotropin-VEGF stimulates retinal capillary endothelial cells through an autocrine pathways. Invest Ophthalmol Vis Sci 35:3393–3400Google Scholar
  39. 39.
    Su T, Gillies MC (1992) A simple method for the in vitro culture of human retinal capillary endothelial cells. Invest Ophthalmol Vis Sci 33:2809–2813Google Scholar
  40. 40.
    Tagami M, Yamagata K, Fujino H, Kubota A, Nara Y, Yamori Y (1992) Morphological differentiation of endothelial cells co-cultured with astrocytes on type-I or type-IV collagen. Cell Tissue Res 268:225–232Google Scholar
  41. 41.
    Terada Y, Tomita K, Nonoguchi H, Marumo F (1992) Different localization of two types of endothelin receptor mRNA in microdissected rat nephron segments using reverse transcription and polymerase chain reaction assay. J Clin Invest 90:107–112Google Scholar
  42. 42.
    Vries C de, Escobedo JA, Ueno H, Houck K, Ferrara N, Williams LT (1992) The fms-like tyrosine kinase, a receptor for vascular endothelial growth factor. Science 255:989–991Google Scholar
  43. 43.
    Yanoff M (1966) Diabetic retinopathy. New Engl J Med 274:1344–1349Google Scholar
  44. 44.
    Yasunaga C, Naskashima Y, Sueishi K (1989) A role of fibrinolytic activity in angiogenesis. Quantitative assay using in vitro method. Lab Invest 61:698–704Google Scholar

Copyright information

© Springer-Verlag 1995

Authors and Affiliations

  • Y. Hata
    • 2
  • K. Nakagawa
    • 1
  • K. Sueishi
    • 1
  • T. Ishibashi
    • 2
  • H. Inomata
    • 2
  • H. Ueno
    • 3
  1. 1.Department of Pathology 1, Faculty of MedicineKyushu University 60FukuokaJapan
  2. 2.Department of Ophthalmology, Faculty of MedicineKyushu UniversityFukuokaJapan
  3. 3.Department of Cardiology, Faculty of MedicineKyushu UniversityFukuokaJapan

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