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Molecular Medicine

, Volume 21, Issue 1, pp 709–716 | Cite as

Low Oxygen Tension Primes Aortic Endothelial Cells to the Reparative Effect of Tissue-Protective Cytokines

  • Lamia Heikal
  • Pietro Ghezzi
  • Manuela Mengozzi
  • Gordon Ferns
Research Article

Abstract

Erythropoietin (EPO) has both erythropoietic and tissue-protective properties. The EPO analogues carbamylated EPO (CEPO) and pyroglutamate helix B surface peptide (pHBSP) lack the erythropoietic activity of EPO but retain the tissue-protective properties that are mediated by a heterocomplex of EPO receptor (EPOR) and the β common receptor (βCR). We studied the action of EPO and its analogues in a model of wound healing where a bovine aortic endothelial cells (BAECs) monolayer was scratched and the scratch closure was assessed over 24 h under different oxygen concentrations. We related the effects of EPO and its analogues on repair to their effect on BAECs proliferation and migration (evaluated using a micro-Boyden chamber). EPO, CEPO and pHBSP enhanced scratch closure only at lower oxygen (5%), while their effect at atmospheric oxygen (21%) was not significant. The mRNA expression of EPOR was doubled in 5% compared with 21% oxygen, and this was associated with increased EPOR assessed by immunofluorescence and Western blot. By contrast, βCR mRNA levels were similar in 5% and 21% oxygen. EPO and its analogues increased both BAECs proliferation and migration, suggesting that both may be involved in the reparative process. The priming effect of low oxygen tension on the action of tissue-protective cytokines may be of relevance to vascular disease, including atherogenesis and restenosis.

Notes

Acknowledgments

Supported by European Regional Development Fund, Project “Peptide Research Network of Excellence,” to P Ghezzi.

Supplementary material

10020_2015_2101709_MOESM1_ESM.pdf (1.3 mb)
Supplementary material, approximately 1.32 MB.

References

  1. 1.
    Jelkmann W. (1992) Erythropoietin: structure, control of production, and function. Physiol. Rev. 72:449–89.CrossRefGoogle Scholar
  2. 2.
    Watowich SS, Hilton DJ, Lodish HF. (1994) Activation and inhibition of erythropoietin receptor function- Role of receptor dimerization. Mol. Cell. Biol. 14:3535–49.CrossRefGoogle Scholar
  3. 3.
    Semenza GL. (2009) Regulation of oxygen homeostasis by hypoxia-inducible factor 1. Physiology (Bethesda). 24:97–106.Google Scholar
  4. 4.
    Brines M, Cerami A. (2005) Emerging biological roles for erythropoietin in the nervous system. Nat. Rev. Neurosci. 6:484–94.CrossRefGoogle Scholar
  5. 5.
    Mengozzi M, Ermilov P, Annenkov A, Ghezzi P, Pearl F. (2014) Definition of a family of tissue-protective cytokines using functional cluster analysis: a proof-of-concept study. Front. Immunol. 5:115.CrossRefGoogle Scholar
  6. 6.
    Grasso G, Sfacteria A, Cerami A, Brines M. (2004) Erythropoietin as a tissue-protective cytokine in brain injury: What do we know and where do we go? Neuroscientist. 10:93–8.CrossRefGoogle Scholar
  7. 7.
    Brines M, et al. (2004) Erythropoietin mediates tissue protection through an erythropoietin and common beta-subunit heteroreceptor. Proc. Natl. Acad. Sci. U. S. A. 101:14907–12.CrossRefGoogle Scholar
  8. 8.
    Marzo F, et al. (2008) Erythropoietin in heart and vessels: focus on transcription and signalling pathways. J. Thromb. Thrombolysis. 26:183–7.CrossRefGoogle Scholar
  9. 9.
    Brines M, Cerami A. (2012) The receptor that tames the innate immune response. Mol. Med. 18:486–96.CrossRefGoogle Scholar
  10. 10.
    Congote LF, Sadvakassova G, Dobocan MC, DiFalco MR, Li Q. (2010) Erythropoietin-dependent endothelial proteins: Potential use against erythropoietin resistance. Cytokine. 51:113–8.CrossRefGoogle Scholar
  11. 11.
    Varani J, Ward PA. (1994) Mechanisms of endothelial cell injury in acute inflammation. Shock. 2:311–9.CrossRefGoogle Scholar
  12. 12.
    Sanchis-Gomar F, Perez-Quilis C, Lippi G. (2013) Erythropoietin receptor (EpoR) agonism is used to treat a wide range of disease. Mol. Med. 19:62–4.CrossRefGoogle Scholar
  13. 13.
    Martinez-Estrada OM, Rodriguez-Millan E, Gonzalez-de Vicente E, Reina M, Vilaro S, Fabre M. (2003) Erythropoietin protects the in vitro blood-brain barrier against VEGF-induced permeability. Eur. J. Neurosci. 18:2538–44.CrossRefGoogle Scholar
  14. 14.
    Chong ZZ, Kang JQ, Maiese K. (2002) Angiogenesis and plasticity: Role of erythropoietin in vascular systems. J. Hematother. Stem Cell Res. 11:863–71.CrossRefGoogle Scholar
  15. 15.
    Anagnostou A, et al. (1994) Erythropoietin receptor messenger-RNA expression in human endothelial cells. Proc. Natl. Acad. Sci. U. S. A. 91:3974–8.CrossRefGoogle Scholar
  16. 16.
    Noguchi CT, Wang L, Rogers HM, Teng R, Jia Y. (2008) Survival and proliferative roles of erythropoietin beyond the erythroid lineage. Expert Rev. Mol. Med. 10:1–23.CrossRefGoogle Scholar
  17. 17.
    Bahlmann FH, et al. (2004) Erythropoietin regulates endothelial progenitor cells. Blood. 103:921–6.CrossRefGoogle Scholar
  18. 18.
    Sautina L, et al. (2010) Induction of nitric oxide by erythropoietin is mediated by the beta common receptor and requires interaction with VEGF receptor 2. Blood. 115:896–905.CrossRefGoogle Scholar
  19. 19.
    Nangaku M. (2013) Tissue protection by erythropoietin: new findings in a moving field. Kidney Int. 84:427–9.CrossRefGoogle Scholar
  20. 20.
    Dumont F, Bischoff P. (2010) Non-erythropoietic tissue-protective peptides derived from erythropoietin: WO2009094172. Expert Opin. Ther. Pat. 20:715–23.CrossRefGoogle Scholar
  21. 21.
    Broxmeyer HE. (2013) Erythropoietin: multiple targets, actions, and modifying influences for biological and clinical consideration. J. Exp. Med. 210:205–8.CrossRefGoogle Scholar
  22. 22.
    Phrommintikul A, Haas SJ, Elsik M, Krum H. (2007) Mortality and target haemoglobin concentrations in anaemic patients with chronic kidney disease treated with erythropoietin: a meta-analysis. Lancet. 369:381–8.CrossRefGoogle Scholar
  23. 23.
    Wun T, Law L, Harvey D, Sieracki B, Scudder SA, Ryu JK. (2003) Increased incidence of symptomatic venous thrombosis in patients with cervical carcinoma treated with concurrent chemotherapy, radiation, and erythropoietin. Cancer. 98:1514–20.CrossRefGoogle Scholar
  24. 24.
    Coleman TR, et al. (2006) Cytoprotective doses of erythropoietin or carbamylated erythropoietin have markedly different procoagulant and vasoactive activities. Proc. Natl. Acad. Sci. U. S. A. 103:5965–70.CrossRefGoogle Scholar
  25. 25.
    Bohr S, et al. (2015) Modulation of cellular stress response via the erythropoietin/CD131 heteroreceptor complex in mouse mesenchymal-derived cells. J. Mol. Med. (Berl). 93:199–210.CrossRefGoogle Scholar
  26. 26.
    Leist M, et al. (2004) Derivatives of erythropoietin that are tissue protective but not erythropoietic. Science. 305:239–42.CrossRefGoogle Scholar
  27. 27.
    Brines M, et al. (2008) Nonerythropoietic, tissue-protective peptides derived from the tertiary structure of erythropoietin. Proc. Natl. Acad. Sci. U. S. A. 105:10925–30.CrossRefGoogle Scholar
  28. 28.
    Erbayraktar Z, Erbayraktar S, Yilmaz O, Cerami A, Coleman T, Brines M. (2009) Nonerythropoietic tissue protective compounds are highly effective facilitators of wound healing. Mol. Med. 15:235–41.CrossRefGoogle Scholar
  29. 29.
    Fiordaliso F, et al. (2005) A nonerythropoietic derivative of erythropoietin protects the myocardium from ischemia-reperfusion injury. Proc. Natl. Acad. Sci. U. S. A. 102:2046–51.CrossRefGoogle Scholar
  30. 30.
    Ueba H, et al. (2013) Suppression of coronary atherosclerosis by helix B surface peptide, a nonerythropoietic, tissue-protective compound derived from erythropoietin. Mol. Med. 19:195–202.CrossRefGoogle Scholar
  31. 31.
    Mengozzi M, et al. (2012) Erythropoietin-induced changes in brain gene expression reveal induction of synaptic plasticity genes in experimental stroke. Proc. Natl. Acad. Sci. U. S. A. 109:9617–22.CrossRefGoogle Scholar
  32. 32.
    Cervellini I, Annenkov A, Brenton T, Chernajovsky Y, Ghezzi P, Mengozzi M. (2013) Erythropoietin (EPO) increases myelin gene expression in CG4 oligodendrocyte cells through the classical EPO receptor. Mol. Med. 19:223–9.CrossRefGoogle Scholar
  33. 33.
    Zwezdaryk KJ, et al. (2007) Erythropoietin, a hypoxia-regulated factor, elicits a pro-angiogenic program in human mesenchymal stem cells. Exp. Hematol. 35:640–52.CrossRefGoogle Scholar
  34. 34.
    Bohr S, et al. (2013) Alternative erythropoietin-mediated signaling prevents secondary microvascular thrombosis and inflammation within cutaneous burns. Proc. Natl. Acad. Sci. U. S. A. 110:3513–8.CrossRefGoogle Scholar
  35. 35.
    Padgett DA, Marucha PT, Sheridan JF. (1998) Restraint stress slows cutaneous wound healing in mice. Brain Behav. Immun. 12:64–73.CrossRefGoogle Scholar
  36. 36.
    Ferri C, et al. (2010) Recombinant human erythropoietin stimulates vasculogenesis and wound healing in a patient with systemic sclerosis complicated by severe skin ulcers. Clin. Exp. Dermatol. 35:885–7.CrossRefGoogle Scholar
  37. 37.
    Guenter CI, et al. (2013) A multi-center study on the regenerative effects of erythropoietin in burn and scalding injuries: study protocol for a randomized controlled trial. Trials. 14:124.CrossRefGoogle Scholar
  38. 38.
    Bernaudin M, et al. (1999) A potential role for erythropoietin in focal permanent cerebral ischemia in mice. J. Cereb. Blood Flow Metab. 19:643–51.CrossRefGoogle Scholar
  39. 39.
    Moore E, Bellomo R. (2011) Erythropoietin (EPO) in acute kidney injury. Ann. Intensive Care. 1:3.CrossRefGoogle Scholar
  40. 40.
    Wang L, et al. (2011) Tumor necrosis factor alpha primes cerebral endothelial cells for erythropoietin-induced angiogenesis. J. Cereb. Blood Flow Metab. 31:640–7.CrossRefGoogle Scholar
  41. 41.
    Beleslin-Cokic BB, Cokic VP, Yu XB, Weksler BB, Schechter AN, Noguchi CT. (2004) Erythropoietin and hypoxia stimulate erythropoietin receptor and nitric oxide production by endothelial cells. Blood. 104:2073–80.CrossRefGoogle Scholar
  42. 42.
    Beleslin-Cokic BB, et al. (2011) Erythropoietin and hypoxia increase erythropoietin receptor and nitric oxide levels in lung microvascular endothelial cells. Cytokine. 54:129–35.CrossRefGoogle Scholar
  43. 43.
    Trincavelli ML, et al. (2013) Regulation of erythropoietin receptor activity in endothelial cells by different erythropoietin (EPO) derivatives: an in vitro study. Int. J. Mol. Sci. 14:2258–81.CrossRefGoogle Scholar
  44. 44.
    Su K-H, et al. (2011) β common receptor integrates the erythropoietin signaling in activation of endothelial nitric oxide synthase. J. Cell. Physiol. 226:3330–9.CrossRefGoogle Scholar
  45. 45.
    Cokic BBB, Cokic VP, Suresh S, Wirt S, Noguchi CT. (2014) Nitric oxide and hypoxia stimulate erythropoietin receptor via MAPK kinase in endothelial cells. Microvasc. Res. 92:34–40.CrossRefGoogle Scholar
  46. 46.
    Velly L, Pellegrini L, Guillet B, Bruder N, Pisano P. (2010) Erythropoietin 2nd cerebral protection after acute injuries: A double-edged sword? Pharmacol. Ther. 128:445–59.CrossRefGoogle Scholar
  47. 47.
    Xu B, Dong G-H, Liu H, Wang Y-Q, Wu H-W, Jing H. (2005) Recombinant human erythropoietin pretreatment attenuates myocardial infarct size: a possible mechanism involves heat shock Protein 70 and attenuation of nuclear factor-kappaB. Ann. Clin. Lab. Sci. 35:161–8.PubMedGoogle Scholar
  48. 48.
    Nakao T, Shiota M, Tatemoto Y, Izumi Y, Iwao H. (2007) Pravastatin induces rat aortic endothelial cell proliferation and migration via activation of PI3K/Akt/mTOR/p70 S6 kinase signaling. J. Pharmacol. Sci. 105:334–41.CrossRefGoogle Scholar
  49. 49.
    Cooke JP. (2003) NO and angiogenesis. Atheroscler. Suppl. 4:53–60.CrossRefGoogle Scholar
  50. 50.
    Gammella E, Leuenberger C, Gassmann M, Ostergaard L. (2013) Evidence of synergistic/additive effects of sildenafil and erythropoietin in enhancing survival and migration of hypoxic endothelial cells. Am. J. Physiol. Lung Cell. Mol. Physiol. 304:L230–9.CrossRefGoogle Scholar
  51. 51.
    Liu N, Tian J, Cheng J, Zhang J. (2013) Effect of erythropoietin on the migration of bone marrow-derived mesenchymal stem cells to the acute kidney injury microenvironment. Exp. Cell Res. 319:2019–27.CrossRefGoogle Scholar
  52. 52.
    Lester RD, Jo M, Campana WM, Gonias SL. (2005) Erythropoietin promotes MCF-7 breast cancer cell migration by an ERK/mitogenactivated protein kinase-dependent pathway and is primarily responsible for the increase in migration observed in hypoxia. J. Biol. Chem. 280:39273–7.CrossRefGoogle Scholar
  53. 53.
    Clowes AW, Reidy MA, Clowes MM. (1983) Kinetics of cellular proliferation after arterial injury. I. Smooth muscle growth in absence of endothelium. Lab. Invest. 49:327–33.PubMedGoogle Scholar
  54. 54.
    Bennett CL, et al. (2008) Venous thromboembolism and mortality associated with recombinant erythropoietin and darbepoetin administration for the treatment of cancer-associated anemia. JAMA. 299:914–24.CrossRefGoogle Scholar

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Authors and Affiliations

  • Lamia Heikal
    • 1
  • Pietro Ghezzi
    • 1
  • Manuela Mengozzi
    • 1
  • Gordon Ferns
    • 1
  1. 1.Brighton and Sussex Medical School, Division of Medical EducationMayfield HouseFalmerUK

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