, Volume 21, Issue 2, pp 203–214 | Cite as

Tip-cell behavior is regulated by transcription factor FoxO1 under hypoxic conditions in developing mouse retinas

  • Moe Fukumoto
  • Kanako Kondo
  • Kazumasa Uni
  • Tomoko Ishiguro
  • Mikiko Hayashi
  • Shinnosuke Ueda
  • Itsuki Mori
  • Kenta Niimi
  • Fumi Tashiro
  • Satsuki Miyazaki
  • Jun-Ichi Miyazaki
  • Shinobu InagakiEmail author
  • Tatsuo FuruyamaEmail author
Original Paper


Forkhead box protein O1 (FoxO1) is a transcription factor and a critical regulator of angiogenesis. Various environmental stimuli, including growth factors, nutrients, shear stress, oxidative stress and hypoxia, affect FoxO1 subcellular localization and strongly influence its transcriptional activity; however, FoxO1-localization patterns in endothelial cells (ECs) during development have not been clarified in vivo. Here, we reported that FoxO1 expression was observed in three layers of angiogenic vessels in developing mouse retinas and that among these layers, the front layer showed high levels of FoxO1 expression in the nuclei of most tip ECs. Because tip ECs migrate toward the avascular hypoxic area, we focused on hypoxia as a major stimulus regulating FoxO1 subcellular localization in tip cells. In cultured ECs, FoxO1 accumulated into the nucleus under hypoxic conditions, with hypoxia also inducing expression of tip-cell-specific genes, including endothelial-specific molecule 1 (ESM1), which was suppressed by FoxO1 knockdown. Additionally, in murine models, EC-specific FoxO1 deletion resulted in reduced ESM1 expression and suppressed tip-cell migration during angiogenesis. These findings indicated roles for FoxO1 in tip-cell migration and that its transcriptional activity is regulated by hypoxia.


Tip-cell Migration Hypoxia Forkhead transcription factor ESM1 Mouse retina 



We thank Dr. Bernd Arnold for Tie2CreER T 2mice and Manabu Sakai for helpful discussion.


This work was supported by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (24590258 and 15K08142 to TF; 16K08442 to SI).

Supplementary material

10456_2017_9588_MOESM1_ESM.docx (15 kb)
Supplementary material 1 (DOCX 15 kb)
10456_2017_9588_MOESM2_ESM.jpg (160 kb)
Supplementary material 2 (JPEG 159 kb)


  1. 1.
    Gerhardt H, Golding M, Fruttiger M, Ruhrberg C, Lundkvist A, Abramsson A et al (2003) VEGF guides angiogenic sprouting utilizing endothelial tip cell filopodia. J Cell Biol 161:1163–1177. CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Phng LK, Gerhardt H (2009) Angiogenesis: a team effort coordinated by Notch. Dev Cell 16:196–208. CrossRefPubMedGoogle Scholar
  3. 3.
    Benedito R, Roca C, Sorensen I, Adams S, Gossler A, Fruttiger M et al (2009) The Notch ligands Dll4 and Jagged1 have opposing effects on angiogenesis. Cell 137:1124–1135. CrossRefPubMedGoogle Scholar
  4. 4.
    del Toro R, Prahst C, Mathivet T, Siegfried G, Kaminker JS, Larrivee B et al (2010) Identification and functional analysis of endothelial tip cell-enriched genes. Blood 116:4025–4033. CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Arima S, Nishiyama K, Ko T, Arima Y, Hakozaki Y, Sugihara K et al (2011) Angiogenic morphogenesis driven by dynamic and heterogeneous collective endothelial cell movement. Development 138:4763–4776. CrossRefPubMedGoogle Scholar
  6. 6.
    Blanco R, Gerhardt H (2013) VEGF and Notch in tip and stalk cell selection. Cold Spring Harb Perspect Med. PubMedPubMedCentralGoogle Scholar
  7. 7.
    Leveen P, Pekny M, Gebremedhin S, Swolin B, Larsson E, Betsholtz C (1994) Mice deficient for PDGF-B show renal, cardiovascular, and hematological abnormalities. Genes Dev 8:1875–1887. CrossRefPubMedGoogle Scholar
  8. 8.
    Strasser GA, Kaminker JS, Tessier-Lavigne M (2010) Microarray analysis of retinal endothelial tip cells identifies CXCR4 as a mediator of tip cell morphology and branching. Blood 115(24):5102–5110. CrossRefPubMedGoogle Scholar
  9. 9.
    Suchting S, Freitas C, le Noble F, Benedito R, Breant C, Duarte A et al (2007) The Notch ligand Delta-like 4 negatively regulates endothelial tip cell formation and vessel branching. Proc Natl Acad Sci USA 104:3225–3230. CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Ogg S, Paradis S, Gottlieb S, Patterson GI, Lee L, Tissenbaum HA et al (1997) The Fork head transcription factor DAF-16 transduces insulin-like metabolic and longevity signals in C-elegans. Nature 389:994–999CrossRefPubMedGoogle Scholar
  11. 11.
    Furuyama T, Kitayama K, Shimoda Y, Ogawa M, Sone K, Yoshida-Araki K et al (2004) Abnormal angiogenesis in Foxo1 (Fkhr)-deficient mice. J Biol Chem 279:34741–34749. CrossRefPubMedGoogle Scholar
  12. 12.
    Wilhelm K, Happel K, Eelen G, Schoors S, Oellerich MF, Lim R et al (2016) FOXO1 couples metabolic activity and growth state in the vascular endothelium. Nature 529:216–226. CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Biggs WH, Meisenhelder J, Hunter T, Cavenee WK, Arden KC (1999) Protein kinase B/Akt-mediated phosphorylation promotes nuclear exclusion of the winged helix transcription factor FKHR1. Proc Natl Acad Sci USA 96:7421–7426. CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Essers MAG, Weijzen S, de Vries-Smits AMM, Saarloos I, de Ruiter ND, Bos JL et al (2004) FOXO transcription factor activation by oxidative stress mediated by the small GTPase Ral and JNK. EMBO J 23:4802–4812. CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Forde A, Constien R, Grone HJ, Hammerling G, Arnold B (2002) Temporal Cre-mediated recombination exclusively in endothelial cells using Tie2 regulatory elements. Genesis 33:191–197. CrossRefPubMedGoogle Scholar
  16. 16.
    Miyazaki S, Minamida R, Furuyama T, Tashiro F, Yamato E, Inagaki S et al (2012) Analysis of Foxo1-regulated genes using Foxo1-deficient pancreatic beta cells. Genes Cells 17:758–767. CrossRefPubMedGoogle Scholar
  17. 17.
    Abid R, Guo SD, Minami T, Spokes KC, Ueki K, Skurk C et al (2004) Vascular endothelial growth factor activates PI3K/Akt/forkhead signaling in endothelial cells. Arterioscler Thromb Vasc Biol 24:294–300. CrossRefPubMedGoogle Scholar
  18. 18.
    Asada S, Daitoku H, Matsuzaki H, Saito T, Sudo T, Mukai H et al (2007) Mitogen-activated protein kinases, Erk and p38, phosphorylate and regulate Foxo1. Cell Signal 19:519–527. CrossRefPubMedGoogle Scholar
  19. 19.
    van den Berg MCW, Burgering BMT (2011) Integrating opposing signals toward Forkhead Box O. Antiox Redox Signal 14:607–621. CrossRefGoogle Scholar
  20. 20.
    Awad H, Nolette N, Hinton M, Dakshinamurti S (2014) AMPK and FoxO1 regulate catalase expression in hypoxic pulmonary arterial smooth muscle. Ped Pulmonol 49:885–897. CrossRefGoogle Scholar
  21. 21.
    Chow KT, Timblin GA, McWhirter SM, Schlissel MS (2013) MK5 activates Rag transcription via Foxo1 in developing B cells. J Exp Med 210:1621–1634. CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Lobov IB, Renard RA, Papadopoulos N, Gale NW, Thurston G, Yancopoulos GD et al (2007) Delta-like ligand 4 (DII4) is induced by VEGF as a negative regulator of angiogenic sprouting. Proc Natl Acad Sci USA 104:3219–3224. CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Shin JW, Huggenberger R, Detmar M (2008) Transcriptional profiling of VEGF-A and VEGF-C target genes in lymphatic endothelium reveals endothelial-specific molecule-1 as a novel mediator of lymphangiogenesis. Blood 112:2318–2326. CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Rocha SF, Schiller M, Jing D, Li H, Butz S, Vestweber D et al (2014) Esm1 modulates endothelial tip cell behavior and vascular permeability by enhancing VEGF bioavailability. Circ Res 115:581. CrossRefPubMedGoogle Scholar
  25. 25.
    Huang HJ, Tindall DJ (2007) Dynamic FoxO transcription factors. J Cell Sci 120:2479–2487. CrossRefPubMedGoogle Scholar
  26. 26.
    Park DY, Lee J, Kim J, Kim K, Hong S, Han S et al (2017) Plastic roles of pericytes in the blood-retinal barrier. Nat Commun 8:16. CrossRefGoogle Scholar
  27. 27.
    Sawamiphak S, Seidel S, Essmann CL, Wilkinson GA, Pitulescu ME, Acker T et al (2010) Ephrin-B2 regulates VEGFR2 function in developmental and tumour angiogenesis. Nature 465:487-U115. CrossRefGoogle Scholar
  28. 28.
    Jeltsch M, Leppanen VM, Saharinen P, Alitalo K (2013) Receptor tyrosine kinase-mediated angiogenesis. Cold Spring Harb Perspect Biol. PubMedPubMedCentralGoogle Scholar
  29. 29.
    Manalo DJ, Rowan A, Lavoie T, Natarajan L, Kelly BD, Ye SQ et al (2005) Transcriptional regulation of vascular endothelial cell responses to hypoxia by HIF-1. Blood 105:659–669. CrossRefPubMedGoogle Scholar
  30. 30.
    Bechard D, Gentina T, Delehedde M, Scherpereel A, Lyon M, Aumercier M et al (2001) Endocan is a novel chondroitin sulfate/dermatan sulfate proteoglycan that promotes hepatocyte growth factor/scatter factor mitogenic activity. J Biol Chem 276:48341–48349. CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media B.V., part of Springer Nature 2017

Authors and Affiliations

  1. 1.Group of Neurobiology, Division of Health Science, Graduate School of MedicineOsaka UniversitySuitaJapan
  2. 2.Department of Stem Cell Regulation Research, Graduate School of MedicineOsaka UniversitySuitaJapan
  3. 3.Kagawa Prefectural University of Health SciencesTakamatsuJapan

Personalised recommendations