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. 2012;7(7):e40497.
doi: 10.1371/journal.pone.0040497. Epub 2012 Jul 6.

In vitro assays using primary embryonic mouse lymphatic endothelial cells uncover key roles for FGFR1 signalling in lymphangiogenesis

Affiliations

In vitro assays using primary embryonic mouse lymphatic endothelial cells uncover key roles for FGFR1 signalling in lymphangiogenesis

Jan Kazenwadel et al. PLoS One. 2012.

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.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Isolation and purity of primary mouse embryonic dermal lymphatic (LEC) and blood vascular (BEC) endothelial cells.
(a) Schematic representation of mouse embryonic dermal endothelial cell isolation. Skin of E16.5 embryos was removed and digested to generate a single cell suspension. Macrophages and hematopoietic cells were depleted using anti-F4/80 and anti-CD45 antibodies, in combination with anti-rat magnetic beads. LEC were captured using anti-LYVE-1 antibody and anti-rabbit magnetic beads, prior to isolation of BEC using anti-CD31 antibody and anti-rat magnetic beads. (b) Analysis of mRNA levels of established markers of LEC identity in LEC and BEC isolated from E16.5 dermis. (c) Analysis of mRNA levels of known markers of BEC (Flt1, Nrp1, Cd34), macrophage (Emr1), vascular smooth muscle (Acta2) and keratinocyte (Krt14) identity in LEC and BEC isolated from E16.5 dermis. Data were normalised to Actb and show mean ± s.d. of triplicate samples. Data are representative of at least three independent cell isolations, each prepared from multiple litters of embryos.
Figure 2
Figure 2. Purity of isolated embryonic dermal LEC and BEC populations assessed by immunostaining.
Analysis of markers indicative of LEC and BEC identity on cells isolated from E16.5 dermis and cultured in EGM-2MV for 72 h. LEC are uniformly Prox1 and CD31 positive and depleted of hematopoietic cells. BEC are uniformly CD31, CD34, VEGFR2 and Nrp1 positive and devoid of LEC and hematopoietic cells. Scale bars represent 40 µm.
Figure 3
Figure 3. FGF2 stimulates primary mouse LEC proliferation.
(a) Primary LEC were cultured in EBM-2+0.5 mg ml−1 Albumax (Control) or EBM-2+0.5 mg ml−1 Albumax containing FGF2 or VEGFC at the indicated concentrations for 48 h. LEC proliferation was measured using the CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega). Data shown represent mean ± s.e.m. and are derived from 3 independent cell isolations, each prepared from multiple litters of embryos, and 5 replicates of each treatment (n = 15). (b) FGF2 stimulated LEC proliferation is inhibited by an FGFR tyrosine kinase inhibitor but not by VEGFR inhibitors. Primary LEC were cultured in EBM-2+0.5 mg ml−1 Albumax (Control), or EBM-2+0.5 mg ml−1 Albumax and FGF2 (10 ng ml−1), together with the tyrosine kinase inhibitors SU5402 (10 µM, FGFR inhibitor), SU5416 (5 µM, VEGFR-2 inhibitor) or MAZ51 (5 µM, VEGFR-3 inhibitor). LEC proliferation was measured using the CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega). Data shown represent mean ± s.e.m. and are derived from 3 independent cell isolations prepared from multiple litters of embryos and 5 replicates of each treatment (n = 15). **P<0.01 ***P<0.001.
Figure 4
Figure 4. FGF2 and VEGF-C promote migration of primary mouse LEC.
(a) Confluent monolayers of primary LEC were scratched and cultured in EBM-2+0.5% FBS (Control), or EBM-2+0.5% FBS containing FGF2 (10 ng ml−1) ± SU5402 (10 µM) or VEGF-C (200 ng ml−1) for 8 h. Dotted white lines mark the boundaries of the wound at 0 h. Scale bars represent 125 µm. (b) Quantification of area migrated in 8 h. Data represent mean ± s.e.m. and are derived from 3 independent cell isolations, each prepared from multiple litters of embryos, and 5 replicates of each treatment (n = 15). *P<0.05, **P<0.01, ***P<0.001.
Figure 5
Figure 5. FGF2 and VEGF-C promote tube formation of primary mouse LEC.
(a) Primary LEC were cultured for 24 h and imaged immediately following the addition of Matrigel. (b) Primary LEC were cultured for 24 h followed by addition of Matrigel alone or Matrigel containing FGF2 (10 ng ml−1), VEGF-C (200 ng ml−1) or a combination of FGF2 and VEGF-C. Images were captured after a further 48 hours. (c) Primary LEC were cultured for 24 h followed by addition of Matrigel containing FGF2 (10 ng ml−1) or a combination of FGF2 (10 ng ml−1) and VEGF-C (200 ng ml−1) and tyrosine kinase inhibitors SU5402 (10 µM, FGFR1), SU5416 (5 µM, VEGFR-2) or MAZ51 (5 µM, VEGFR-3). Three replicates of each treatment were performed and images are representative of at least three independent cell isolations. Inset panels in (c) illustrate magnified views of boxed regions. Scale bars represent 250 µm. Quantification of average vessel diameter (d) using Lymphatic Vessel Analysis Protocol (LVAP) and ImageJ software and branch points per well (e) using AngioTool software , for each treatment indicated. Data show mean ± s.e.m. and are derived from 2 independent cell isolations, each prepared from multiple litters of embryos, and 3 replicates of each treatment (n = 6). *P<0.05, **P<0.01, ***P<0.001.
Figure 6
Figure 6. FGFR1 is important for LEC proliferation.
(a) FGF receptor profile in primary embryonic mouse dermal LEC and BEC. Real-time RT-PCR analysis of Fgfr1-4 mRNA levels in freshly isolated E16.5 LEC and BEC. Data are normalised to Actb and show mean ± s.d. of triplicate samples. Data are representative of at least three independent cell isolations from multiple litters of embryos. (b) FGFR1 is localized to the nucleus of LEC following stimulation with FGF2. Immunostaining of primary LEC cultured for 24 h in either EBM-2+0.5 mg ml−1 Albumax (serum starved) or EGM-2MV containing FGF2 (complete media). Scale bars represent 40 µm. (c) siRNA mediated knockdown of FGFR1 in primary embryonic LEC. Primary LEC were cultured for 24 h prior to transfection with control or Fgfr1 siRNA. Fgfr1 mRNA levels were analysed 72 h post-transfection. Data are normalised to Actb and represent mean ± s.e.m. Data are derived from 3 independent cell isolations, each prepared from multiple litters of embryos, and 3 transfections per isolation (n = 9). ***P<0.001. (d) FGFR1 protein levels were assessed by Western blot 72 h post-transfection and quantified relative to β-actin. (e) Immunostaining of primary LEC cultured in complete medium for 72 h after transfection with control or Fgfr1 siRNA revealed efficient reduction in FGFR1 protein levels. Scale bars represent 100 µm. (f) FGFR1 is important for LEC proliferation. Primary LEC were cultured for 24 h prior to treatment with control or Fgfr1 siRNA. LEC proliferation was measured by counting cells 72 h post-transfection. Data show mean ± s.e.m. Data are derived from 2 independent cell isolations, each prepared from multiple litters of embryos and multiple transfections per isolation (n = 11). ***P<0.001. (g) Primary LEC were cultured in EGM-2MV (complete media, CM) for 24 h prior to treatment with SU5402 (10 µM) for 72 h. LEC proliferation was measured using the CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega). Data shown represent mean ± s.e.m. and are derived from 3 independent cell isolations prepared from multiple litters of embryos and multiple replicates of each treatment (n = 14). *P<0.05.

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