Lymphatic EC response to VEGFC

Series:	IN_VITRO DIFFERENTIATION SERIES
Species:	Human (Homo sapiens)
Genomic View:	Zenbu
Expression table:	FILE
Link to TET:	TET
Sample providers :	Michael Detmar
Germ layer:	mesoderm
Primary cells or cell line:	primary cells
Time span:	8 hours
Number of time points:	16

Overview

The lymphatic vasculature plays a critical role in the maintenance of tissue fluid balance, the uptake of dietary fats and the immune response. Lymphatic vessels are also actively involved in pathological conditions, in particular in promoting cancer metastasis to lymph nodes and in controlling chronic inflammatory diseases (1, 2, 3, 4). The growth of new lymphatic vessels from pre-existing vasculature is called lymphangiogenesis. The main pathway regulating lymphangiogenesis is VEGF-C signaling via its receptor VEGFR-3 on lymphatic endothelial cells. Fully mature VEGF-C has affinity for VEGFR-2 which is also expressed on lymphatic endothelial cells (LEC) (5). A mutated version of VEGF-C, VEGF-C156S, in which cysteine 156 is replaced by a serine, specifically activates VEGFR-3 but not VEGFR-2 (6). Specific activation of VEGFR-3 by VEGF-C156S is sufficient to induce lymphangiogenesis, as demonstrated in K14-VEGF-C156S mice. Upon stimulation, the receptor dimerizes and is phosphorylated at several tyrosine residues. These phosphorylation sites activate several adaptor molecules along with further downstream mediators including JNK, ERK1/2, PI3K and PKB/AKT, ultimately leading phenotypic changes of LECs. However, the exact transcriptional mediators are not fully investigated yet. These transcription factors might be essential in mediating the effect of VEGF-C and could serve as potential therapeutic targets of the lymphatic endothelium. References: (1) Skobe et al. (2001) Nat Med, 7(2):192-8 (2) Hirakawa et al. (2007) Blood, 109(3):1010-7 (3) Hirakawa et al. (2005) J Exp Med, 201(7):1089-99 (4) Huggenberger et al. (2010) J Exp Med, 207(10):2255-69 (5) Joukov et al. (1997) EMBO J, 16:3898-3911 (6) Joukov et al. (1998) J Biol Chem, 273:65599-6602

Sample description

We have used primary lymphatic endothelial cells, which have previously been isolated from human foreskin (7). Cells were cultured on collagen-coated culture dishes in EBM medium (Lonza) supplemented with 20% FBS (Life Technologies), 1x penicillin/streptomycin (Life Technologies), 2mM L-Glutamine (Life Technologies), 25 µg/ml cAMP (Sigma-Aldrich), and 10 µg/ml hydrocortisone (Sigma-Aldrich). For this study, we used cells isolated from 3 individual donors, at low passage numbers (<=6). Cells were seeded at 70% confluency and were starved in EBM + 0.2% BSA over night before stimulation with 1.5 µg/ml recombinant human VEGF-C156S (Kari Alitalo, University of Helsinki, Finland) for 0 min, 15 min, 30 min, 45 min, 60 min, 80 min, 100 min, 2 h, 2.5h, 3 h, 3.5 h, 4 h, 5 h, 6 h, 7 h, or 8 h (16 different time points). The 0 min time point of treatment served as a control for the other time points (Figure 1). Figure 1: Schematic overview of the experimental procedure for the transcriptome analysis. Starved lymphatic endothelial cells (LECs) were treated with VEGF-C156S (1.5μg/ml) for various time periods from 0 min to 480 min. Treatment was terminated by adding TRIzol and isolating the RNA for CAGE sequencing. References: (7) Hirakawa et al. (2003) Am J Pathol, 162(2):575-86

Quality control

Marker gene expression Marker gene expression Key genes of interest behaved the same way in all three isolates. As expected, there was a rapid induction of the known VEGF-C target gene DLL4 (8), whereas SOX18, a master transcription factor of lymphatic cell identity, was robustly expressed over the whole time course, and was only mildly induced by VEGF-C156S (Figure 2). Figure 2: TPM expression profiles of individual replicates for the VEGF-C target genes Dll4 and SOX18. An early up-regulation of SOX18 and Dll4 can be observed after VEGF-C156S stimulation. References: (8) Zheng et al. (2011) Blood, 118(4):1154-62

Profiled time course samples

Only samples that passed quality controls (Arner et al. 2015) are shown here. The entire set of samples are downloadable from FANTOM5 human / mouse samples

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12260-130A1	Lymphatic Endothelial cells response to VEGFC	00hr00min	biol_rep1 (MM XIX - 1)
12261-130A2	Lymphatic Endothelial cells response to VEGFC	00hr15min	biol_rep1 (MM XIX - 2)
12262-130A3	Lymphatic Endothelial cells response to VEGFC	00hr30min	biol_rep1 (MM XIX - 3)
12263-130A4	Lymphatic Endothelial cells response to VEGFC	00hr45min	biol_rep1 (MM XIX - 4)
12264-130A5	Lymphatic Endothelial cells response to VEGFC	01hr00min	biol_rep1 (MM XIX - 5)
12265-130A6	Lymphatic Endothelial cells response to VEGFC	01hr20min	biol_rep1 (MM XIX - 6)
12266-130A7	Lymphatic Endothelial cells response to VEGFC	01hr40min	biol_rep1 (MM XIX - 7)
12267-130A8	Lymphatic Endothelial cells response to VEGFC	02hr00min	biol_rep1 (MM XIX - 8)
12268-130A9	Lymphatic Endothelial cells response to VEGFC	02hr30min	biol_rep1 (MM XIX - 9)
12269-130B1	Lymphatic Endothelial cells response to VEGFC	03hr00min	biol_rep1 (MM XIX - 10)
12270-130B2	Lymphatic Endothelial cells response to VEGFC	03hr30min	biol_rep1 (MM XIX - 11)
12271-130B3	Lymphatic Endothelial cells response to VEGFC	04hr	biol_rep1 (MM XIX - 12)
12272-130B4	Lymphatic Endothelial cells response to VEGFC	05hr	biol_rep1 (MM XIX - 13)
12273-130B5	Lymphatic Endothelial cells response to VEGFC	06hr	biol_rep1 (MM XIX - 14)
12274-130B6	Lymphatic Endothelial cells response to VEGFC	07hr	biol_rep1 (MM XIX - 15)
12275-130B7	Lymphatic Endothelial cells response to VEGFC	08hr	biol_rep1 (MM XIX - 16)
12382-131E6	Lymphatic Endothelial cells response to VEGFC	00hr00min	biol_rep2 (MM XIV - 1)
12383-131E7	Lymphatic Endothelial cells response to VEGFC	00hr15min	biol_rep2 (MM XIV - 2)
12384-131E8	Lymphatic Endothelial cells response to VEGFC	00hr30min	biol_rep2 (MM XIV - 3)
12385-131E9	Lymphatic Endothelial cells response to VEGFC	00hr45min	biol_rep2 (MM XIV - 4)
12386-131F1	Lymphatic Endothelial cells response to VEGFC	01hr00min	biol_rep2 (MM XIV - 5)
12387-131F2	Lymphatic Endothelial cells response to VEGFC	01hr20min	biol_rep2 (MM XIV - 6)
12388-131F3	Lymphatic Endothelial cells response to VEGFC	01hr40min	biol_rep2 (MM XIV - 7)
12389-131F4	Lymphatic Endothelial cells response to VEGFC	02hr00min	biol_rep2 (MM XIV - 8)
12390-131F5	Lymphatic Endothelial cells response to VEGFC	02hr30min	biol_rep2 (MM XIV - 9)
12391-131F6	Lymphatic Endothelial cells response to VEGFC	03hr00min	biol_rep2 (MM XIV - 10)
12392-131F7	Lymphatic Endothelial cells response to VEGFC	03hr30min	biol_rep2 (MM XIV - 11)
12393-131F8	Lymphatic Endothelial cells response to VEGFC	04hr	biol_rep2 (MM XIV - 12)
12394-131F9	Lymphatic Endothelial cells response to VEGFC	05hr	biol_rep2 (MM XIV - 13)
12395-131G1	Lymphatic Endothelial cells response to VEGFC	06hr	biol_rep2 (MM XIV - 14)
12396-131G2	Lymphatic Endothelial cells response to VEGFC	07hr	biol_rep2 (MM XIV - 15)
12397-131G3	Lymphatic Endothelial cells response to VEGFC	08hr	biol_rep2 (MM XIV - 16)
12504-133A2	Lymphatic Endothelial cells response to VEGFC	00hr00min	biol_rep3 (MM XXII - 1 )
12505-133A3	Lymphatic Endothelial cells response to VEGFC	00hr15min	biol_rep3 (MM XXII - 2)
12506-133A4	Lymphatic Endothelial cells response to VEGFC	00hr30min	biol_rep3 (MM XXII - 3)
12507-133A5	Lymphatic Endothelial cells response to VEGFC	00hr45min	biol_rep3 (MM XXII - 4)
12508-133A6	Lymphatic Endothelial cells response to VEGFC	01hr00min	biol_rep3 (MM XXII - 5)
12509-133A7	Lymphatic Endothelial cells response to VEGFC	01hr20min	biol_rep3 (MM XXII - 6)
12510-133A8	Lymphatic Endothelial cells response to VEGFC	01hr40min	biol_rep3 (MM XXII - 7)
12511-133A9	Lymphatic Endothelial cells response to VEGFC	02hr00min	biol_rep3 (MM XXII - 8)
12512-133B1	Lymphatic Endothelial cells response to VEGFC	02hr30min	biol_rep3 (MM XXII - 9)
12513-133B2	Lymphatic Endothelial cells response to VEGFC	03hr00min	biol_rep3 (MM XXII - 10)
12514-133B3	Lymphatic Endothelial cells response to VEGFC	03hr30min	biol_rep3 (MM XXII - 11)
12515-133B4	Lymphatic Endothelial cells response to VEGFC	04hr	biol_rep3 (MM XXII - 12)
12516-133B5	Lymphatic Endothelial cells response to VEGFC	05hr	biol_rep3 (MM XXII - 13)
12517-133B6	Lymphatic Endothelial cells response to VEGFC	06hr	biol_rep3 (MM XXII - 14)
12518-133B7	Lymphatic Endothelial cells response to VEGFC	07hr	biol_rep3 (MM XXII - 15)
12519-133B8	Lymphatic Endothelial cells response to VEGFC	08hr	biol_rep3 (MM XXII - 16)