Visualization of endothelial cell cycle dynamics in mouse using the Flt-1/eGFP-anillin system
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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.
KeywordsEndothelial cell Proliferation Angiogenesis Anillin Cell cycle
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.
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.
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.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
- 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 CrossRefPubMedCentralPubMedGoogle Scholar
- 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):3637–3644CrossRefPubMedGoogle Scholar
- 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 CrossRefPubMedCentralPubMedGoogle Scholar
- 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 CrossRefPubMedCentralPubMedGoogle Scholar
- 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 CrossRefPubMedGoogle Scholar
- 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 CrossRefPubMedGoogle Scholar
- 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 CrossRefPubMedGoogle Scholar
- 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 CrossRefPubMedGoogle Scholar
- 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 CrossRefPubMedCentralPubMedGoogle Scholar
- 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 CrossRefPubMedGoogle Scholar
- 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 YorkGoogle Scholar