Visualization of endothelial cell cycle dynamics in mouse using the Flt-1/eGFP-anillin system

Abstract

Endothelial cell proliferation is a key process during vascular growth but its kinetics could only be assessed in vitro or ex vivo so far. To enable the monitoring and quantification of cell cycle kinetics in vivo, we have generated transgenic mice expressing an eGFP-anillin construct under control of the endothelial-specific Flt-1 promoter. This construct labels the nuclei of endothelial cells in late G1, S and G2 phase and changes its localization during the different stages of M phase, thereby enabling the monitoring of EC proliferation and cytokinesis. In Flt-1/eGFP-anillin mice, we found eGFP+ signals specifically in Ki67+/PECAM+ endothelial cells during vascular development. Quantification using this cell cycle reporter in embryos revealed a decline in endothelial cell proliferation between E9.5 to E12.5. By time-lapse microscopy, we determined the length of different cell cycle phases in embryonic endothelial cells in vivo and found a M phase duration of about 80 min with 2/3 covering karyokinesis and 1/3 cytokinesis. Thus, we have generated a versatile transgenic system for the accurate assessment of endothelial cell cycle dynamics in vitro and in vivo.

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References

  1. 1.

    Adams RH, Alitalo K (2007) Molecular regulation of angiogenesis and lymphangiogenesis. Nat Rev Mol Cell Biol 8(6):464–478. https://doi.org/10.1038/nrm2183

    CAS  Article  PubMed  Google Scholar 

  2. 2.

    Geudens I, Gerhardt H (2011) Coordinating cell behaviour during blood vessel formation. Development 138(21):4569–4583. https://doi.org/10.1242/dev.062323

    CAS  Article  PubMed  Google Scholar 

  3. 3.

    Herbert SP, Stainier DY (2011) Molecular control of endothelial cell behaviour during blood vessel morphogenesis. Nat Rev Mol Cell Biol 12(9):551–564. https://doi.org/10.1038/nrm3176

    CAS  Article  PubMed Central  PubMed  Google Scholar 

  4. 4.

    Noseda M, Chang L, McLean G, Grim JE, Clurman BE, Smith LL, Karsan A (2004) Notch activation induces endothelial cell cycle arrest and participates in contact inhibition: role of p21Cip1 repression. Mol Cell Biol 24(20):8813–8822. https://doi.org/10.1128/MCB.24.20.8813-8822.2004

    CAS  Article  PubMed Central  PubMed  Google Scholar 

  5. 5.

    Suzuki E, Nagata D, Yoshizumi M, Kakoki M, Goto A, Omata M, Hirata Y (2000) Reentry into the cell cycle of contact-inhibited vascular endothelial cells by a phosphatase inhibitor. Possible involvement of extracellular signal-regulated kinase and phosphatidylinositol 3-kinase. J Biol Chem 275(5):3637–3644

    CAS  Article  PubMed  Google Scholar 

  6. 6.

    Lerchenmuller C, Heissenberg J, Damilano F, Bezzeridis VJ, Kramer I, Bochaton-Piallat ML, Hirschberg K, Busch M, Katus HA, Peppel K, Rosenzweig A, Busch H, Boerries M, Most P (2016) S100A6 regulates endothelial cell cycle progression by attenuating antiproliferative signal transducers and activators of transcription 1 signaling. Arterioscler Thromb Vasc Biol 36(9):1854–1867. https://doi.org/10.1161/ATVBAHA.115.306415

    Article  PubMed Central  PubMed  Google Scholar 

  7. 7.

    He Z, Campolmi N, Ha Thi BM, Dumollard JM, Peoc’h M, Garraud O, Piselli S, Gain P, Thuret G (2011) Optimization of immunolocalization of cell cycle proteins in human corneal endothelial cells. Mol Vis 17:3494–3511

    CAS  PubMed Central  PubMed  Google Scholar 

  8. 8.

    Park EJ, Grabinska KA, Guan Z, Sessa WC (2016) NgBR is essential for endothelial cell glycosylation and vascular development. EMBO Rep 17(2):167–177. https://doi.org/10.15252/embr.201540789

    CAS  Article  PubMed Central  PubMed  Google Scholar 

  9. 9.

    Ehmann UK, Williams JR, Nagle WA, Brown JA, Belli JA, Lett JT (1975) Perturbations in cell cycle progression from radioactive DNA precursors. Nature 258(5536):633–636

    CAS  Article  PubMed  Google Scholar 

  10. 10.

    Kolb B, Pedersen B, Ballermann M, Gibb R, Whishaw IQ (1999) Embryonic and postnatal injections of bromodeoxyuridine produce age-dependent morphological and behavioral abnormalities. J Neurosci 19(6):2337–2346

    CAS  PubMed  Google Scholar 

  11. 11.

    Fukuhara S, Zhang J, Yuge S, Ando K, Wakayama Y, Sakaue-Sawano A, Miyawaki A, Mochizuki N (2014) Visualizing the cell-cycle progression of endothelial cells in zebrafish. Dev Biol 393(1):10–23. https://doi.org/10.1016/j.ydbio.2014.06.015

    CAS  Article  PubMed  Google Scholar 

  12. 12.

    Field CM, Alberts BM (1995) Anillin, a contractile ring protein that cycles from the nucleus to the cell cortex. J Cell Biol 131(1):165–178

    CAS  Article  PubMed  Google Scholar 

  13. 13.

    Hesse M, Raulf A, Pilz GA, Haberlandt C, Klein AM, Jabs R, Zaehres H, Fugemann CJ, Zimmermann K, Trebicka J, Welz A, Pfeifer A, Roll W, Kotlikoff MI, Steinhauser C, Gotz M, Scholer HR, Fleischmann BK (2012) Direct visualization of cell division using high-resolution imaging of M-phase of the cell cycle. Nat Commun 3:1076. https://doi.org/10.1038/ncomms2089

    Article  PubMed Central  PubMed  Google Scholar 

  14. 14.

    Herz K, Heinemann JC, Hesse M, Ottersbach A, Geisen C, Fuegemann CJ, Roll W, Fleischmann BK, Wenzel D (2012) Live monitoring of small vessels during development and disease using the Flt-1 promoter element. Basic Res Cardiol 107(2):257. https://doi.org/10.1007/s00395-012-0257-5

    Article  PubMed  Google Scholar 

  15. 15.

    Morishita K, Johnson DE, Williams LT (1995) A novel promoter for vascular endothelial growth factor receptor (Flt-1) that confers endothelial-specific gene expression. J Biol Chem 270(46):27948–27953

    CAS  Article  PubMed  Google Scholar 

  16. 16.

    Quinn G, Ochiya T, Terada M, Yoshida T (2000) Mouse Flt-1 promoter directs endothelial-specific expression in the embyroid body model of embryogenesis. Biochem Biophys Res Commun 276(3):1089–1099. https://doi.org/10.1006/bbrc.2000.3602

    CAS  Article  PubMed  Google Scholar 

  17. 17.

    Shibuya M, Ito N, Claesson-Welsh L (1999) Structure and function of vascular endothelial growth factor receptor-1 and -2. Curr Top Microbiol Immunol 237:59–83

    CAS  PubMed  Google Scholar 

  18. 18.

    Kazemi S, Wenzel D, Kolossov E, Lenka N, Raible A, Sasse P, Hescheler J, Addicks K, Fleischmann BK, Bloch W (2002) Differential role of bFGF and VEGF for vasculogenesis. Cell Physiol Biochem 12(2–3):55–62. https://doi.org/10.1159/000063781

    CAS  Article  PubMed  Google Scholar 

  19. 19.

    Malan D, Wenzel D, Schmidt A, Geisen C, Raible A, Bolck B, Fleischmann BK, Bloch W (2010) Endothelial beta1 integrins regulate sprouting and network formation during vascular development. Development 137(6):993–1002. https://doi.org/10.1242/dev.045377

    CAS  Article  PubMed  Google Scholar 

  20. 20.

    Schmidt A, Wenzel D, Thorey I, Sasaki T, Hescheler J, Timpl R, Addicks K, Werner S, Fleischmann BK, Bloch W (2006) Endostatin influences endothelial morphology via the activated ERK1/2-kinase endothelial morphology and signal transduction. Microvasc Res 71(3):152–162. https://doi.org/10.1016/j.mvr.2006.01.001

    CAS  Article  PubMed  Google Scholar 

  21. 21.

    Vosen S, Rieck S, Heidsieck A, Mykhaylyk O, Zimmermann K, Bloch W, Eberbeck D, Plank C, Gleich B, Pfeifer A, Fleischmann BK, Wenzel D (2016) Vascular repair by circumferential cell therapy using magnetic nanoparticles and tailored magnets. ACS Nano 10(1):369–376. https://doi.org/10.1021/acsnano.5b04996

    CAS  Article  PubMed  Google Scholar 

  22. 22.

    Wenzel D, Rieck S, Vosen S, Mykhaylyk O, Trueck C, Eberbeck D, Trahms L, Zimmermann K, Pfeifer A, Fleischmann BK (2012) Identification of magnetic nanoparticles for combined positioning and lentiviral transduction of endothelial cells. Pharm Res 29(5):1242–1254. https://doi.org/10.1007/s11095-011-0657-5

    CAS  Article  PubMed  Google Scholar 

  23. 23.

    Wilhelm K, Happel K, Eelen G, Schoors S, Oellerich MF, Lim R, Zimmermann B, Aspalter IM, Franco CA, Boettger T, Braun T, Fruttiger M, Rajewsky K, Keller C, Bruning JC, Gerhardt H, Carmeliet P, Potente M (2016) FOXO1 couples metabolic activity and growth state in the vascular endothelium. Nature 529(7585):216–220. https://doi.org/10.1038/nature16498

    CAS  Article  PubMed Central  PubMed  Google Scholar 

  24. 24.

    Matsumoto K, Azami T, Otsu A, Takase H, Ishitobi H, Tanaka J, Miwa Y, Takahashi S, Ema M (2012) Study of normal and pathological blood vessel morphogenesis in Flt1-tdsRed BAC Tg mice. Genesis 50(7):561–571. https://doi.org/10.1002/dvg.22031

    CAS  Article  PubMed  Google Scholar 

  25. 25.

    Klagsbrun M, D’Amore PA (1991) Regulators of angiogenesis. Annu Rev Physiol 53:217–239. https://doi.org/10.1146/annurev.ph.53.030191.001245

    CAS  Article  PubMed  Google Scholar 

  26. 26.

    Kniewallner KM, Wenzel D, Humpel C (2016) Thiazine red(+) platelet inclusions in cerebral blood vessels are first signs in an Alzheimer’s disease mouse model. Sci Rep 6:28447. https://doi.org/10.1038/srep28447

    CAS  Article  PubMed Central  PubMed  Google Scholar 

  27. 27.

    Plein A, Ruhrberg C, Fantin A (2015) The mouse hindbrain: an in vivo model to analyze developmental angiogenesis. In: Ribatti D (ed) Vascular morphogenesis: methods and protocols, methods in molecular biology, vol 1214. Springer, New York

    Google Scholar 

  28. 28.

    Pitulescu ME, Schmidt I, Benedito R, Adams RH (2010) Inducible gene targeting in the neonatal vasculature and analysis of retinal angiogenesis in mice. Nat Protoc 5(9):1518–1534. https://doi.org/10.1038/nprot.2010.113

    CAS  Article  PubMed  Google Scholar 

  29. 29.

    Ziegler N, Plate KH, Liebner S (2014) Analysis of angiogenesis in the developing mouse central nervous system. Methods Mol Biol 1135:55–68. https://doi.org/10.1007/978-1-4939-0320-7_5

    Article  PubMed  Google Scholar 

  30. 30.

    Meduri G, Bausero P, Perrot-Applanat M (2000) Expression of vascular endothelial growth factor receptors in the human endometrium: modulation during the menstrual cycle. Biol Reprod 62(2):439–447

    CAS  Article  PubMed  Google Scholar 

  31. 31.

    Ziegler T, Nerem RM (1994) Effect of flow on the process of endothelial cell division. Arterioscler Thromb 14(4):636–643

    CAS  Article  PubMed  Google Scholar 

  32. 32.

    Sikora-Polaczek M, Hupalowska A, Polanski Z, Kubiak JZ, Ciemerych MA (2006) The first mitosis of the mouse embryo is prolonged by transitional metaphase arrest. Biol Reprod 74(4):734–743. https://doi.org/10.1095/biolreprod.105.047092

    CAS  Article  PubMed  Google Scholar 

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Acknowledgements

We thank A. Nagy (Toronto, Canada) for providing G4 mouse ES cells. Moreover, we would like to acknowledge P. Freitag (University of Bonn, Germany) for excellent technical assistance and D. Korzus (University of Bonn) for help with determination of estrus cycle.

Funding

M.P. is supported by the Max Planck Society, the European Research Council (ERC) Starting Grant ANGIOMET (311546), the Deutsche Forschungsgemeinschaft (SFB 834), the Excellence Cluster Cardiopulmonary System (EXC 147/1), the LOEWE Grant Ub-Net, the DZHK (German Center for Cardiovascular Research), the Stiftung Charité, and the European Molecular Biology Organization Young Investigator Programme.

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KH has generated Flt-1/eGFP-anillin mice, performed expression analysis of eGFP-anillin at different stages and performed live monitoring including quantitative analyses, AR has acquired pictures of sections from embryonic and adult tissue and established live monitoring of the embryos, CS has acquired data from hindbrains and retinas, ST and ME have generated Flt-1/tdsred mice and contributed to the writing of the manuscript, MP has supervised hindbrain and retina analysis and contributed to the writing of the manuscript, MH has generated Flt-1/eGFP-anillin mice by complementation of ES cells with diploid mouse embryos, BF has contributed to the design of the study and the writing of the manuscript, DW has designed the study, supervised analysis and wrote the manuscript.

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Correspondence to Daniela Wenzel.

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Herz, K., Becker, A., Shi, C. et al. Visualization of endothelial cell cycle dynamics in mouse using the Flt-1/eGFP-anillin system. Angiogenesis 21, 349–361 (2018). https://doi.org/10.1007/s10456-018-9601-1

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Keywords

  • Endothelial cell
  • Proliferation
  • Angiogenesis
  • Anillin
  • Cell cycle