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Type: Journal article
Title: In Vitro assays using primary embryonic mouse lymphatic endothelial cells uncover key roles for FGFR1 signalling in lymphangiogenesis
Author: Kazenwadel, J.
Secker, G.
Betterman, K.
Harvey, N.
Citation: PLoS One, 2012; 7(7):1-12
Publisher: Public Library of Science
Issue Date: 2012
ISSN: 1932-6203
Statement of
Jan Kazenwadel, Genevieve A. Secker, Kelly L. Betterman and Natasha L. Harvey
Abstract: Despite the importance of blood vessels and lymphatic vessels during development and disease, the signalling pathways underpinning vessel construction remain poorly characterised. Primary mouse endothelial cells have traditionally proven difficult to culture and as a consequence, few assays have been developed to dissect gene function and signal transduction pathways in these cells ex vivo. Having established methodology for the purification, short-term culture and transfection of primary blood (BEC) and lymphatic (LEC) vascular endothelial cells isolated from embryonic mouse skin, we sought to optimise robust assays able to measure embryonic LEC proliferation, migration and three-dimensional tube forming ability in vitro. In the course of developing these assays using the pro-lymphangiogenic growth factors FGF2 and VEGF-C, we identified previously unrecognised roles for FGFR1 signalling in lymphangiogenesis. The small molecule FGF receptor tyrosine kinase inhibitor SU5402, but not inhibitors of VEGFR-2 (SU5416) or VEGFR-3 (MAZ51), inhibited FGF2 mediated LEC proliferation, demonstrating that FGF2 promotes proliferation directly via FGF receptors and independently of VEGF receptors in primary embryonic LEC. Further investigation revealed that FGFR1 was by far the predominant FGF receptor expressed by primary embryonic LEC and correspondingly, siRNA-mediated FGFR1 knockdown abrogated FGF2 mediated LEC proliferation. While FGF2 potently promoted LEC proliferation and migration, three dimensional tube formation assays revealed that VEGF-C primarily promoted LEC sprouting and elongation, illustrating that FGF2 and VEGF-C play distinct, cooperative roles in lymphatic vascular morphogenesis. These assays therefore provide useful tools able to dissect gene function in cellular events important for lymphangiogenesis and implicate FGFR1 as a key player in developmental lymphangiogenesis in vivo.
Keywords: Endothelial Cells; Animals; Mice, Inbred C57BL; Mice; Fibroblast Growth Factor 2; Vascular Endothelial Growth Factor C; Cell Separation; Signal Transduction; Cell Proliferation; Cell Movement; Lymphangiogenesis; Female; Receptor, Fibroblast Growth Factor, Type 1; Primary Cell Culture
Rights: Copyright: © 2012 Kazenwadel et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
RMID: 0020120706
DOI: 10.1371/journal.pone.0040497
Appears in Collections:Medicine publications

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