Skip to main content

Suppression of vascular network formation by chronic hypoxia and prolyl-hydroxylase 2 (phd2) deficiency during vertebrate development

Angiogenesis volume 19, pages 119–131 (2016)Cite this article

Abstract

In the adult, new vessels and red blood cells form in response to hypoxia. Here, the oxygen-sensing system (PHD–HIF) has recently been put into focus, since the prolyl-hydroxylase domain proteins (PHD) and hypoxia-inducible factors (HIF) are considered as potential therapeutic targets to treat ischemia, cancers or age-related macula degeneration. While the oxygen-sensing system (PHD–HIF) has been studied intensively in this respect, only little is known from developing vertebrate embryos since mutations within this pathway led to an early decease of embryos due to placental defects. During vertebrate embryogenesis, a progenitor cell called hemangioblast is assumed to give rise to blood cells and blood vessels in a process called hematopoiesis and vasculogenesis, respectively. Xenopus provides an ideal experimental system to address these processes in vivo, as its development does not depend on a functional placenta and thus allows analyzing the role of oxygen directly. To this end, we adopted a computer-controlled four-channel system, which allowed us to culture Xenopus embryos under defined oxygen concentrations. Our data show that the development of vascular structures and blood cells is strongly impaired under hypoxia, while general development is less compromised. Interestingly, suppression of Phd2 function using specific antisense morpholinos or a chemical inhibitor resulted in mostly overlapping vascular defects; nevertheless, blood cell was formed almost normally. Our results provide the first evidence that oxygen via Phd2 has a decisive influence on the formation of the vascular network during vertebrate embryogenesis. These findings may be considered in certain potential treatment concepts.

This is a preview of subscription content, access via your institution.

Access options

Buy single article

Instant access to the full article PDF.

USD 39.95

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

References

  1. Ny A, Autiero M, Carmeliet P (2006) Zebrafish and Xenopus tadpoles: small animal models to study angiogenesis and lymphangiogenesis. Exp Cell Res 312(5):684–693. doi:10.1016/j.yexcr.2005.10.018

    Article  CAS  PubMed  Google Scholar 

  2. Warkman AS, Krieg PA (2007) Xenopus as a model system for vertebrate heart development. Semin Cell Dev Biol 18(1):46–53. doi:10.1016/j.semcdb.2006.11.010

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Coffin JD, Harrison J, Schwartz S, Heimark R (1991) Angioblast differentiation and morphogenesis of the vascular endothelium in the mouse embryo. Dev Biol 148(1):51–62

    Article  CAS  PubMed  Google Scholar 

  4. Risau W, Flamme I (1995) Vasculogenesis. Annu Rev Cell Dev Biol 11:73–91. doi:10.1146/annurev.cb.11.110195.000445

    Article  CAS  PubMed  Google Scholar 

  5. Klagsbrun M, Moses MA (1999) Molecular angiogenesis. Chem Biol 6(8):R217–R224. doi:10.1016/S1074-5521(99)80081-7

    Article  CAS  PubMed  Google Scholar 

  6. Ray PS, Estrada-Hernandez T, Sasaki H, Zhu L, Maulik N (2000) Early effects of hypoxia/reoxygenation on VEGF, ang-1, ang-2 and their receptors in the rat myocardium: implications for myocardial angiogenesis. Mol Cell Biochem 213(1–2):145–153

    Article  CAS  PubMed  Google Scholar 

  7. Epstein AC, Gleadle JM, McNeill LA, Hewitson KS, O’Rourke J, Mole DR, Mukherji M, Metzen E, Wilson MI, Dhanda A, Tian YM, Masson N, Hamilton DL, Jaakkola P, Barstead R, Hodgkin J, Maxwell PH, Pugh CW, Schofield CJ, Ratcliffe PJ (2001) C. elegans EGL-9 and mammalian homologs define a family of dioxygenases that regulate HIF by prolyl hydroxylation. Cell 107(1):43–54

    Article  CAS  PubMed  Google Scholar 

  8. Yu F, White SB, Zhao Q, Lee FS (2001) HIF-1alpha binding to VHL is regulated by stimulus-sensitive proline hydroxylation. Proc Natl Acad Sci USA 98(17):9630–9635. doi:10.1073/pnas.181341498

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Iwai K, Yamanaka K, Kamura T, Minato N, Conaway RC, Conaway JW, Klausner RD, Pause A (1999) Identification of the von Hippel–lindau tumor-suppressor protein as part of an active E3 ubiquitin ligase complex. Proc Natl Acad Sci USA 96(22):12436–12441

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Semenza GL (2001) HIF-1, O(2), and the 3 PHDs: how animal cells signal hypoxia to the nucleus. Cell 107(1):1–3

    Article  CAS  PubMed  Google Scholar 

  11. Takahata S, Sogawa K, Kobayashi A, Ema M, Mimura J, Ozaki N, Fujii-Kuriyama Y (1998) Transcriptionally active heterodimer formation of an Arnt-like PAS protein, Arnt3, with HIF-1a, HLF, and clock. Biochem Biophys Res Commun 248(3):789–794. doi:10.1006/bbrc.1998.9012

    Article  CAS  PubMed  Google Scholar 

  12. Aragones J, Schneider M, Van Geyte K, Fraisl P, Dresselaers T, Mazzone M, Dirkx R, Zacchigna S, Lemieux H, Jeoung NH, Lambrechts D, Bishop T, Lafuste P, Diez-Juan A, Harten SK, Van Noten P, De Bock K, Willam C, Tjwa M, Grosfeld A, Navet R, Moons L, Vandendriessche T, Deroose C, Wijeyekoon B, Nuyts J, Jordan B, Silasi-Mansat R, Lupu F, Dewerchin M, Pugh C, Salmon P, Mortelmans L, Gallez B, Gorus F, Buyse J, Sluse F, Harris RA, Gnaiger E, Hespel P, Van Hecke P, Schuit F, Van Veldhoven P, Ratcliffe P, Baes M, Maxwell P, Carmeliet P (2008) Deficiency or inhibition of oxygen sensor Phd1 induces hypoxia tolerance by reprogramming basal metabolism. Nat Genet 40(2):170–180. doi:10.1038/ng.2007.62

    Article  CAS  PubMed  Google Scholar 

  13. Wright G, Higgin JJ, Raines RT, Steenbergen C, Murphy E (2003) Activation of the prolyl hydroxylase oxygen-sensor results in induction of GLUT1, heme oxygenase-1, and nitric-oxide synthase proteins and confers protection from metabolic inhibition to cardiomyocytes. J Biol Chem 278(22):20235–20239. doi:10.1074/jbc.M301391200

    Article  CAS  PubMed  Google Scholar 

  14. Toescu EC (2004) Hypoxia response elements. Cell Calcium 36(3–4):181–185. doi:10.1016/j.ceca.2004.02.020

    Article  CAS  PubMed  Google Scholar 

  15. Kotch LE, Iyer NV, Laughner E, Semenza GL (1999) Defective vascularization of HIF-1alpha-null embryos is not associated with VEGF deficiency but with mesenchymal cell death. Dev Biol 209(2):254–267. doi:10.1006/dbio.1999.9253

    Article  CAS  PubMed  Google Scholar 

  16. Ramirez-Bergeron DL, Runge A, Adelman DM, Gohil M, Simon MC (2006) HIF-dependent hematopoietic factors regulate the development of the embryonic vasculature. Dev Cell 11(1):81–92. doi:10.1016/j.devcel.2006.04.018

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. van Rooijen E, Voest EE, Logister I, Bussmann J, Korving J, van Eeden FJ, Giles RH, Schulte-Merker S (2010) von Hippel–Lindau tumor suppressor mutants faithfully model pathological hypoxia-driven angiogenesis and vascular retinopathies in zebrafish. Dis Models Mech 3(5–6):343–353. doi:10.1242/dmm.004036

    Article  Google Scholar 

  18. Gnarra JR, Ward JM, Porter FD, Wagner JR, Devor DE, Grinberg A, Emmert-Buck MR, Westphal H, Klausner RD, Linehan WM (1997) Defective placental vasculogenesis causes embryonic lethality in VHL-deficient mice. Proc Natl Acad Sci USA 94(17):9102–9107

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Takeda K, Ho VC, Takeda H, Duan LJ, Nagy A, Fong GH (2006) Placental but not heart defects are associated with elevated hypoxia-inducible factor alpha levels in mice lacking prolyl hydroxylase domain protein 2. Mol Cell Biol 26(22):8336–8346. doi:10.1128/MCB.00425-06

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Jiang H, Guo R, Powell-Coffman JA (2001) The Caenorhabditis elegans hif-1 gene encodes a bHLH-PAS protein that is required for adaptation to hypoxia. Proc Natl Acad Sci USA 98(14):7916–7921. doi:10.1073/pnas.141234698

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Anderson LL, Mao X, Scott BA, Crowder CM (2009) Survival from hypoxia in C. elegans by inactivation of aminoacyl-tRNA synthetases. Science 323(5914):630–633. doi:10.1126/science.1166175

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Zhou D, Xue J, Lai JC, Schork NJ, White KP, Haddad GG (2008) Mechanisms underlying hypoxia tolerance in Drosophila melanogaster: hairy as a metabolic switch. Plos Genet 4(10):e1000221. doi:10.1371/journal.pgen.1000221

    Article  PubMed  PubMed Central  Google Scholar 

  23. Jaspers RT, Testerink J, Della Gaspera B, Chanoine C, Bagowski CP, van der Laarse WJ (2014) Increased oxidative metabolism and myoglobin expression in zebrafish muscle during chronic hypoxia. Biol Open 3(8):718–727. doi:10.1242/bio.20149167

    Article  PubMed  PubMed Central  Google Scholar 

  24. Gracey AY, Lee TH, Higashi RM, Fan T (2011) Hypoxia-induced mobilization of stored triglycerides in the euryoxic goby Gillichthys mirabilis. J Exp Biol 214(Pt 18):3005–3012. doi:10.1242/jeb.059907

    Article  CAS  PubMed  Google Scholar 

  25. Rattner BA, Michael SD, Brinkley HJ (1978) Embryonic implantation, dietary intake, and plasma GH concentration in pregnant mice exposed to hypoxia. Aviat Space Environ Med 49(5):687–691

    CAS  PubMed  Google Scholar 

  26. Golan H, Huleihel M (2006) The effect of prenatal hypoxia on brain development: short- and long-term consequences demonstrated in rodent models. Dev Sci 9(4):338–349. doi:10.1111/j.1467-7687.2006.00498.x

    Article  PubMed  Google Scholar 

  27. Wikenheiser J, Doughman YQ, Fisher SA, Watanabe M (2006) Differential levels of tissue hypoxia in the developing chicken heart. Dev Dyn 235(1):115–123. doi:10.1002/dvdy.20499

    Article  CAS  PubMed  Google Scholar 

  28. Hollemann T, Panitz F, Pieler T (1998) In situ hybridization: an improved whole-mount method for Xenopus embryos. In: Richter DJ (ed) A comparative methods approach to the study of oocytes and embryos. Oxford University Press, New York, pp 279–290

    Google Scholar 

  29. Nieuwkoop PD, Faber J (1967) Normal table of Xenopus laevis (Daudin). North Holland, Amsterdam

    Google Scholar 

  30. Saka Y, Smith JC (2001) Spatial and temporal patterns of cell division during early Xenopus embryogenesis. Dev Biol 229(2):307–318. doi:10.1006/dbio.2000.0101

    Article  CAS  PubMed  Google Scholar 

  31. Hensey C, Gautier J (1997) A developmental timer that regulates apoptosis at the onset of gastrulation. Mech Dev 69(1–2):183–195

    Article  CAS  PubMed  Google Scholar 

  32. Pfirrmann T, Lokapally A, Andreasson C, Ljungdahl P, Hollemann T (2013) SOMA: a single oligonucleotide mutagenesis and cloning approach. Plos One 8(6):e64870. doi:10.1371/journal.pone.0064870

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Evans JP, Kay BK (1992) Biochemical fractionation of oocytes: freon extraction to remove yolk proteins from Oocyte Lysates. In: Kay KK, Peng HB (eds) Xenopus laevis: practical uses in cell and molecular biology. Methods in cell biology, vol 36. Academic Press, pp 133–147

  34. McGrath KE, Palis J (2005) Hematopoiesis in the yolk sac: more than meets the eye. Exp Hematol 33(9):1021–1028. doi:10.1016/j.exphem.2005.06.012

    Article  PubMed  Google Scholar 

  35. Walmsley M, Cleaver D, Patient R (2008) Fibroblast growth factor controls the timing of Scl, Lmo2, and Runx1 expression during embryonic blood development. Blood 111(3):1157–1166. doi:10.1182/blood-2007-03-081323

    Article  CAS  PubMed  Google Scholar 

  36. Hartenstein V (1989) Early neurogenesis in Xenopus: the spatio-temporal pattern of proliferation and cell lineages in the embryonic spinal cord. Neuron 3(4):399–411

    Article  CAS  PubMed  Google Scholar 

  37. Kim SG, Buel GR, Blenis J (2013) Nutrient regulation of the mTOR complex 1 signaling pathway. Mol Cells 35(6):463–473. doi:10.1007/s10059-013-0138-2

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Brugarolas J, Lei K, Hurley RL, Manning BD, Reiling JH, Hafen E, Witters LA, Ellisen LW, Kaelin WG Jr (2004) Regulation of mTOR function in response to hypoxia by REDD1 and the TSC1/TSC2 tumor suppressor complex. Genes Dev 18(23):2893–2904. doi:10.1101/gad.1256804

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Reiling JH, Hafen E (2004) The hypoxia-induced paralogs Scylla and Charybdis inhibit growth by down-regulating S6 K activity upstream of TSC in Drosophila. Genes Dev 18(23):2879–2892. doi:10.1101/gad.322704

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Dunwoodie SL (2009) The role of hypoxia in development of the Mammalian embryo. Dev Cell 17(6):755–773. doi:10.1016/j.devcel.2009.11.008

    Article  CAS  PubMed  Google Scholar 

  41. Fraisl P, Mazzone M, Schmidt T, Carmeliet P (2009) Regulation of angiogenesis by oxygen and metabolism. Dev Cell 16(2):167–179. doi:10.1016/j.devcel.2009.01.003

    Article  CAS  PubMed  Google Scholar 

  42. Han D, Wen L, Chen Y (2012) Molecular cloning of phd1 and comparative analysis of phd1, 2, and 3 expression in Xenopus laevis. Sci World J 2012:689287. doi:10.1100/2012/689287

    Google Scholar 

  43. Neuhaus H, Muller F, Hollemann T (2010) Xenopus er71 is involved in vascular development. Dev Dyn 239(12):3436–3445. doi:10.1002/dvdy.22487

    Article  CAS  PubMed  Google Scholar 

  44. Salanga MC, Meadows SM, Myers CT, Krieg PA (2010) ETS family protein ETV2 is required for initiation of the endothelial lineage but not the hematopoietic lineage in the Xenopus embryo. Dev Dyn 239(4):1178–1187. doi:10.1002/dvdy.22277

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Kaelin WG Jr, Ratcliffe PJ (2008) Oxygen sensing by metazoans: the central role of the HIF hydroxylase pathway. Mol Cell 30(4):393–402. doi:10.1016/j.molcel.2008.04.009

    Article  CAS  PubMed  Google Scholar 

  46. Majmundar AJ, Wong WJ, Simon MC (2010) Hypoxia-inducible factors and the response to hypoxic stress. Mol Cell 40(2):294–309. doi:10.1016/j.molcel.2010.09.022

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Huang ST, Vo KC, Lyell DJ, Faessen GH, Tulac S, Tibshirani R, Giaccia AJ, Giudice LC (2004) Developmental response to hypoxia. FASEB J 18(12):1348–1365. doi:10.1096/fj.03-1377com

    Article  CAS  PubMed  Google Scholar 

  48. Moore LG (2003) Fetal growth restriction and maternal oxygen transport during high altitude pregnancy. High Alt Med Biol 4(2):141–156. doi:10.1089/152702903322022767

    Article  PubMed  Google Scholar 

  49. Ream M, Ray AM, Chandra R, Chikaraishi DM (2008) Early fetal hypoxia leads to growth restriction and myocardial thinning. Am J Physiol Regul Integr Comp Physiol 295(2):R583–R595. doi:10.1152/ajpregu.00771.2007

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Abbott BD, Buckalew AR (2000) Placental defects in ARNT-knockout conceptus correlate with localized decreases in VEGF-R2, Ang-1, and Tie-2. Dev Dyn 219(4):526–538. doi:10.1002/1097-0177(2000)9999:9999<:AID-DVDY1080>3.0.CO;2-N

    Article  CAS  PubMed  Google Scholar 

  51. Cowden Dahl KD, Fryer BH, Mack FA, Compernolle V, Maltepe E, Adelman DM, Carmeliet P, Simon MC (2005) Hypoxia-inducible factors 1alpha and 2alpha regulate trophoblast differentiation. Mol Cell Biol 25(23):10479–10491. doi:10.1128/MCB.25.23.10479-10491.2005

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Laplante M, Sabatini DM (2012) mTOR signaling in growth control and disease. Cell 149(2):274–293. doi:10.1016/j.cell.2012.03.017

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgments

We thank M. Inui for the ami-SK plasmid, W. Knöchel for the beta-globin probe, Aldo Ciau-Uitz for the lmo2 plasmid, Paul Krieg for cardiac troponin and the NIBB for flk1 and flt1 probes. We like to thank Juliane Herfurth for her excellent technical help.

Funding

The work was supported by a grant from the “Roux-Forschungsförderprogramm” MLU Halle-Wittenberg [Grant Number FKZ 26/21].

Author information

Authors and Affiliations

  1. Department of Medical Molecular Biology, Medical Faculty, Institute for Physiological Chemistry, Martin-Luther-University Halle-Wittenberg, Hollystrasse 1, 06114, Halle (Saale), Germany

    Sanjeeva Metikala, Herbert Neuhaus & Thomas Hollemann

Authors
  1. Sanjeeva Metikala
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Herbert Neuhaus
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Thomas Hollemann
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Thomas Hollemann.

Ethics declarations

Conflict of interest

The authors have no conflict of interest to declare.

Electronic supplementary material

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Metikala, S., Neuhaus, H. & Hollemann, T. Suppression of vascular network formation by chronic hypoxia and prolyl-hydroxylase 2 (phd2) deficiency during vertebrate development. Angiogenesis 19, 119–131 (2016). https://doi.org/10.1007/s10456-015-9492-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10456-015-9492-3

Keywords

  • Xenopus
  • Angiogenesis
  • Vasculogenesis
  • Hypoxia
  • Prolyl-hydroxylase domain
  • phd2