In Vivo Angiogenic Activity of Erythropoietin

  • Domenico Ribatti
Part of the Methods in Molecular Biology book series (MIMB, volume 982)


The role of erythropoietin (Epo) has been demonstrated in tissues outside the hematopoietic system, including the cardiovascular system, where Epo promotes various effects in endothelial cells. Here, we have demonstrated the angiogenic capacity of recombinant human Epo (rhuEpo) in vivo, by means of the chick embryo chorioallantoic membrane (CAM) assay, a well-established in vivo assay to study angiogenesis and antiangiogenesis.

Key words

Angiogenesis Chorioallantoic membrane Erythropoietin 



Supported by European Seventh Framework Programme (FTP/2007-2013) under grant agreement No 278570 to DR.


  1. 1.
    Ribatti D, Presta M, Vacca A, Ria R, Giuliani R, Dell’Era P, Nico B, Roncali L, Dammacco F (1999) Human erythropoietin induces a pro-angiogenic phenotype in cultured endothelial cells and stimulates neovascularization in vivo. Blood 93:2627–2636PubMedGoogle Scholar
  2. 2.
    Anagnostou A, Lee ES, Kessimian N, Levinson R, Steiner M (1990) Erythropoietin has a mitogenic and positive chemotactic effect on endothelial cells. Proc Natl Acad Sci U S A 87:5982–5987CrossRefGoogle Scholar
  3. 3.
    Yamaji R, Okada T, Moriya M, Naito M, Tsuruo T, Miyatake K, Nakano Y (1996) Brain capillary endothelial cells express two forms of erythropoietin receptor mRNA. Eur J Biochem 239:494–500PubMedCrossRefGoogle Scholar
  4. 4.
    Ashley RA, Dubuque SH, Dvorak B, Woodward SS, Williams SK, Kling PJ (2002) Erythropoietin stimulates vasculogenesis in neonatal rat mesenteric microvascular endothelial cells. Pediatr Res 51:472–478PubMedCrossRefGoogle Scholar
  5. 5.
    Carlini RG, Reyes AA, Rothstein M (1995) Recombinant human erythropoietin stimulates angiogenesis in vitro. Kidney Int 55:546–553CrossRefGoogle Scholar
  6. 6.
    Vogel V, Kramer HJ, Bäcker A, Meyer-Lehnert H, Jelkmann W, Fandrey J (1997) Effects of erythropoietin on endothelin-1 synthesis and cellular calcium messanger system in vascular endothelial cells. Am J Hypertens 10:289–297PubMedCrossRefGoogle Scholar
  7. 7.
    Kertesz N, Wu J, Chen TH, Sucov HM, Wu H (2004) The role of erythropoietin in regulating angiogenesis. Dev Biol 276:101–110PubMedCrossRefGoogle Scholar
  8. 8.
    Monestiroli S, Mancuso P, Burlini A, Pruneri G, Dell’Agnola C, Gobbi A, Martinelli G, Bertolini F (2001) Kinetics and viability of circulating endothelial cells as surrogate angiogenesis marker in an animal model of human lymphoma. Cancer Res 61:4341–4344PubMedGoogle Scholar
  9. 9.
    Mancuso P, Burlini A, Pruneri G, Goldhirsch A, Martinelli G, Bertolini F (2001) Resting and activated endothelial cells are increased in the peripheral blood of cancer patients. Blood 97:3658–3661PubMedCrossRefGoogle Scholar
  10. 10.
    Heeschen C, Aicher A, Lehmann R, Fichtlscherer S, Vasa M, Urbich C, Mildner-Rihm C, Martin H, Zeiher AM, Dimmeler S (2003) Erythropoietin is a potent physiologic stimulus for endothelial progenitor cell mobilization. Blood 102:1340–1346PubMedCrossRefGoogle Scholar
  11. 11.
    Yasuda Y, Fujita Y, Matsuo T, Koinuma S, Hara S, Tazaki A, Onozaki M, Hashimoto M, Musha T, Ogawa K, Fujita H, Nakamura Y, Shiozaki H, Utsumi H (2003) Erythropoietin regulates tumour growth of human malignancies. Carcinogenesis 24:1021–1029PubMedCrossRefGoogle Scholar
  12. 12.
    Kayser K, Gabius HJ (1992) Analysis of expression of erythropoietin-binding sites in human lung carcinoma by the biotinylated ligand. Zentralbl Pathol 138:266–270PubMedGoogle Scholar
  13. 13.
    Ribatti D, Vacca A, Roccaro AM, Crivellato E, Presta M (2003) Erythropoietin as an angiogenic factor. Eur J Clin Invest 33:891–896PubMedCrossRefGoogle Scholar
  14. 14.
    Perelman N, Selvaraj SK, Batra S, Luck LR, Erdreich-Epstein A, Coates TD, Kalra VK, Malik P (2003) Placenta growth factor activates monocytes and correlates with sickle cell disease severity. Blood 102:1506–1514PubMedCrossRefGoogle Scholar
  15. 15.
    Wang L, Zhang Z, Wang Y, Zhang R, Chopp M (2004) Treatment of stroke with erythropoietin enhances neurogenesis and angiogenesis and improves neurological function in rats. Stroke 35:1732–1737PubMedCrossRefGoogle Scholar
  16. 16.
    Nakano M, Satoh K, Fukumoto Y, Ito Y, Kagaya Y, Ishii N, Sugamura K, Shimokawa H (2007) Important role of erythropoietin receptor to promote VEGF expression and angiogenesis in peripheral ischemia in mice. Circ Res 100:662–669PubMedCrossRefGoogle Scholar
  17. 17.
    Okazaki T, Ebihara S, Asada M, Yamanda S, Niu K, Arai H (2008) Erythropoietin promotes the growth of tumors lacking its receptor and decreases survival of tumor-bearing mice by enhancing angiogenesis. Neoplasia 10:932–939PubMedGoogle Scholar
  18. 18.
    Hardee ME, Cao Y, Fu P, Jiang X, Zhao Y, Rabbani ZN, Vujaskovic Z, Dewhirst MW, Arcasoy MO (2007) Erythropoietin blockade inhibits the induction of tumor angiogenesis and progression. PLoS One 2:e549PubMedCrossRefGoogle Scholar
  19. 19.
    Olujohungbe A, Handa S, Holmes J (1987) Do erythropoietin accelerate malignant transformation in multiple myeloma? Postgrad Med J 73:163–164CrossRefGoogle Scholar
  20. 20.
    Bunworasate U, Arnouk H, Minderman H, O’Loughlin KL, Sait SN, Barcos M, Stewart CC, Baer MR (2001) Erythropoietin-dependent transformation of myelodysplastic syndrome to acute monoblastic leukemia. Blood 98:3492–3494PubMedCrossRefGoogle Scholar
  21. 21.
    Ribatti D (2002) A potential role of erythropoietin in angiogenesis associated with myelodysplastic syndromes. Leukemia 16:1890PubMedCrossRefGoogle Scholar
  22. 22.
    Arcasoy MO, Amin K, Karayal AF, Chou SC, Raleigh JA, Varia MA, Haroon ZA (2002) Functional significance of erythropoietin receptor expression in breast cancer. Lab Invest 82:911–918PubMedGoogle Scholar
  23. 23.
    Yasuda Y, Fujita Y, Masuda S, Musha T, Ueda K, Tanaka H, Fujita H, Matsuo T, Nagao M, Sasaki R, Nakamura Y (2002) Erythropoietin is involved in growth and angiogenesis in malignant tumours of female reproductive organs. Carcinogenesis 23:1797–1805PubMedCrossRefGoogle Scholar
  24. 24.
    Ribatti D (2010) The chick embryo chorioallantoic membrane in the study of angiogenesis and metastasis. Springer Science+Business Media, DordrechtCrossRefGoogle Scholar
  25. 25.
    Ribatti D, Marzullo A, Nico B, Crivellato E, Ria R, Vacca A (2003) Erythropoietin as an angiogenic factor in gastric carcinoma. Histopathology 42:246–250PubMedCrossRefGoogle Scholar
  26. 26.
    Ribatti D, Marzullo A, Gentile A, Longo V, Nico B, Vacca A, Dammacco F (2007) Erythropoietin/erythropoietin-receptor system is involved in angiogenesis in human hepatocellular carcinoma. Histopathology 50:591–596PubMedCrossRefGoogle Scholar
  27. 27.
    Acs G, Zhang PJ, McGrath CM, Acs P, McBroom J, Mohyeldin A, Liu S, Lu H, Verma A (2003) Hypoxia-inducible erythropoietin signaling in squamous dysplasia and squamous cell carcinoma of the uterine cervix and its potential role in cervical carcinogenesis and tumor progression. Am J Pathol 162:1789–1806PubMedCrossRefGoogle Scholar
  28. 28.
    Westenfelder C, Baranowski RL (2000) Erythropoietin stimulates proliferation of human renal carcinoma cells. Kidney Int 58:647–657PubMedCrossRefGoogle Scholar
  29. 29.
    Ribatti D, Poliani PL, Longo V, Mangieri D, Nico B, Vacca A (2007) Erythropoietin/erythropoietin receptor system is involved in angiogenesis in human neuroblastoma. Histopathology 50:636–641PubMedCrossRefGoogle Scholar
  30. 30.
    Batra S, Perelman N, Luck LR, Shimada H, Malik P (2003) Pediatric tumor cells express erythropoietin and a functional erythropoietin receptor that promotes angiogenesis and tumor cell survival. Lab Invest 83:1477–1487PubMedCrossRefGoogle Scholar
  31. 31.
    Krieg M, Marti HH, Plate KH (1998) Coexpression of erythropoietin and vascular endothelial growth factor in nervous system tumors associated with von Hippel-Lindau tumor suppressor gene loss of function. Blood 92:3388–3393PubMedGoogle Scholar
  32. 32.
    Kokhaei P, Abdalla AO, Hansson L, Mikaelsson E, Kubbies M, Haselbeck A, Jernberg-Wiklund H, Mellstedt H, Osterborg A (2007) Expression of erythropoietin receptor and in vitro functional effects of epoetins in B-cell malignancies. Clin Cancer Res 13:3536–3544PubMedCrossRefGoogle Scholar
  33. 33.
    Nico B, Annese T, Guidolin D, Finato N, Crivellato E, Ribatti D (2011) Epo is involved in angiogenesis in human glioma. J Neuroncol 102:51–58CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2013

Authors and Affiliations

  • Domenico Ribatti
    • 1
  1. 1.Department of Basic Medical Sciences, Neurosciences, and Sensory OrgansUniversity of Bari Medical SchoolBariItaly

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