Fluorescence and Bioluminescence Imaging of Angiogenesis in Flk1-Nano-lantern Transgenic Mice

Angiogenesis is important for normal development as well as for tumour growth. However, the molecular and cellular mechanisms underlying angiogenesis are not fully understood, partly because of the lack of a good animal model for imaging. Here, we report the generation of a novel transgenic (Tg) mouse that expresses a bioluminescent reporter protein, Nano-lantern, under the control of Fetal liver kinase 1 (Flk1). Flk1-Nano-lantern BAC Tg mice recapitulated endogenous Flk1 expression in endothelial cells and lymphatic endothelial cells during development and tumour growth. Importantly, bioluminescence imaging of endothelial cells from the aortic rings of Flk1-Nano-lantern BAC Tg mice enabled us to observe endothelial sprouting for 18 hr without any detectable phototoxicity. Furthermore, Flk1-Nano-lantern BAC Tg mice achieved time-lapse luminescence imaging of tumour angiogenesis in freely moving mice with implanted tumours. Thus, this transgenic mouse line contributes a unique model to study angiogenesis within both physiological and pathological contexts.

[1]  Takeshi Miyamoto,et al.  Neurons Limit Angiogenesis by Titrating VEGF in Retina , 2014, Cell.

[2]  S. Nishikawa,et al.  Molecular basis for Flk1 expression in hemato-cardiovascular progenitors in the mouse , 2011, Development.

[3]  Satoru Takahashi,et al.  Flk1-GFP BAC Tg mice: an animal model for the study of blood vessel development. , 2010, Experimental animals.

[4]  R. Jain.  Normalization of Tumor Vasculature: An Emerging Concept in Antiangiogenic Therapy , 2005, Science.

[5]  Takeharu Nagai,et al.  Luminescent proteins for high-speed single-cell and whole-body imaging , 2012, Nature Communications.

[6]  Lei Xu,et al.  Normalization of the vasculature for treatment of cancer and other diseases. , 2011, Physiological reviews.

[7]  Janet Rossant,et al.  Endothelial cells and VEGF in vascular development , 2005, Nature.

[8]  Holger Gerhardt,et al.  VEGF and Notch in tip and stalk cell selection. , 2013, Cold Spring Harbor perspectives in medicine.

[9]  Satoru Takahashi,et al.  Study of normal and pathological blood vessel morphogenesis in Flt1‐tdsRed BAC Tg mice , 2012, Genesis.

[10]  T. Veikkola,et al.  Signalling via vascular endothelial growth factor receptor‐3 is sufficient for lymphangiogenesis in transgenic mice , 2001, The EMBO journal.

[11]  P. Carmeliet,et al.  Gene-targeting of Phd2 improves tumor response to chemotherapy and prevents side-toxicity. , 2012, Cancer cell.

[12]  E. Raymond,et al.  Safety, pharmacokinetic, and antitumor activity of SU11248, a novel oral multitarget tyrosine kinase inhibitor, in patients with cancer. , 2006, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[13]  Identification of Vascular Endothelial Growth Factor (VEGF) Receptor-2 (Flk-1) Promoter/Enhancer Sequences Sufficient for Angioblast and Endothelial Cell-Specific Transcription in Transgenic Mice , 1999 .

[14]  M. Shibuya.  Structure and dual function of vascular endothelial growth factor receptor-1 (Flt-1). , 2001, The international journal of biochemistry & cell biology.

[15]  M Intaglietta,et al.  Tissue perfusion inhomogeneity during early tumor growth in rats. , 1979, Journal of the National Cancer Institute.

[16]  Mina J. Bissell,et al.  The perivascular niche regulates breast tumor dormancy , 2013, Nature Cell Biology.

[17]  B. Vojnovic,et al.  Use of the mouse aortic ring assay to study angiogenesis , 2011, Nature Protocols.

[18]  Frederik De Smet,et al.  Heterozygous Deficiency of PHD2 Restores Tumor Oxygenation and Inhibits Metastasis via Endothelial Normalization , 2009, Cell.

[19]  K. Alitalo,et al.  VEGF guides angiogenic sprouting utilizing endothelial tip cell filopodia , 2003, The Journal of cell biology.

[20]  J. Rossant,et al.  Deletion of the selection cassette, but not cis-acting elements, in targeted Flk1-lacZ allele reveals Flk1 expression in multipotent mesodermal progenitors. , 2006, Blood.

[21]  Apurva A Desai,et al.  Sorafenib in advanced clear-cell renal-cell carcinoma. , 2007, The New England journal of medicine.

[22]  J. Ambati,et al.  Alternatively spliced VEGF receptor-2 is an essential endogenous inhibitor of lymphatic vessels , 2009, Nature Medicine.

[23]  Y. Mukouyama,et al.  TGFβ signaling is required for sprouting lymphangiogenesis during lymphatic network development in the skin , 2013, Development.

[24]  Robert S. Kerbel,et al.  Antiangiogenic therapy: impact on invasion, disease progression, and metastasis , 2011, Nature Reviews Clinical Oncology.

[25]  J. Berlin,et al.  Bevacizumab plus irinotecan, fluorouracil, and leucovorin for metastatic colorectal cancer. , 2004, The New England journal of medicine.

[26]  Dieter Häussinger,et al.  Sorafenib in advanced hepatocellular carcinoma. , 2008, The New England journal of medicine.

[27]  Napoleone Ferrara,et al.  Angiogenesis as a therapeutic target , 2005, Nature.

[28]  Satoru Takahashi,et al.  Primitive erythropoiesis from mesodermal precursors expressing VE-cadherin, PECAM-1, Tie2, endoglin, and CD34 in the mouse embryo. , 2006, Blood.

[29]  H. Kidoya,et al.  Identification and characterization of a resident vascular stem/progenitor cell population in preexisting blood vessels , 2012, The EMBO journal.

[30]  Sonja Loges,et al.  Recent molecular discoveries in angiogenesis and antiangiogenic therapies in cancer. , 2013, The Journal of clinical investigation.

[31]  H. Okano,et al.  Subventricular Zone‐Derived Neural Progenitor Cells Migrate Along a Blood Vessel Scaffold Toward The Post‐stroke Striatum , 2010, Stem cells.

[32]  E. Perez,et al.  Paclitaxel plus bevacizumab versus paclitaxel alone for metastatic breast cancer. , 2007, The New England journal of medicine.

[33]  Lena Claesson-Welsh,et al.  Signal transduction by VEGF receptors in regulation of angiogenesis and lymphangiogenesis. , 2006, Experimental cell research.

[34]  J. Folkman.  Tumor angiogenesis: therapeutic implications. , 1971, The New England journal of medicine.

[35]  N. Ferrara,et al.  The biology of VEGF and its receptors , 2003, Nature Medicine.

[36]  M. Shibuya,et al.  Isolation and function of mouse tissue resident vascular precursors marked by myelin protein zero , 2011, The Journal of experimental medicine.

[37]  Rakesh K. Jain,et al.  Principles and mechanisms of vessel normalization for cancer and other angiogenic diseases , 2011, Nature Reviews Drug Discovery.

[38]  J Folkman,et al.  Transplacental carcinogenesis by stilbestrol. , 1971, The New England journal of medicine.

[39]  Janet Rossant,et al.  Failure of blood-island formation and vasculogenesis in Flk-1-deficient mice , 1995, Nature.