VEGFC
Primary lymphatic endothelial cell response to vascular endothelial growth factor C (VEGFC)
Time course name: human_Lymphatic_Endothelial_cells_response_to_VEGFC
Sample provider: Michael Detmar
Introduction
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.
Samples
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.
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
(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
(7) Hirakawa et al. (2003) Am J Pathol, 162(2):575-86
(8) Zheng et al. (2011) Blood, 118(4):1154-62
Beginning of non-public section
hCAGE sequencing
The quality analysis was done in collaboration with Dr. Anthony Mathelier and Prof. Wyeth Wasserman and revealed the following: The number of expressed genes as well as the TPM distribution show a highly consistent level in all three replicates. Additionally, it was identified that the fold changes are rather small in all replicates (Figure 3).
Figure 3: TPM distribution in the three LEC replicates treated with VEGF-C156S. A violin plot of the distribution of TPM values per time point is shown for each replicate that was treated with VEGF-C156S. The distribution is similar with some higher TPM values in replicate 2.
Bioinformatics collaborator
Dr. Anthony Mathelier and Prof. Wyeth Wasserman
Principal Component Analysis
An important point that we would like to stress is that we used three different isolates from three different humans for this experimental set-up rather than performing the same experiment three times with the same primary cell isolate. Thus, when analyzing the data and performing the PCA analysis this needs to be taken into consideration. Dr. Kim-Anh Le Cao (University of Queensland, Australia) filtered the data (discarded low standard deviations across CAGE clusters) and log2 transformed the data. Then a multilevel transformation was applied for this data set (Figure 4).
Figure 4: Principal component analysis of the three replicates.
Related samples
We have a large collection of microarray data from different tissues. Especially, we do have human lymphatic and blood vascular endothelial cell comparisons but also data ex vivo isolated lymphatic and blood vascular endothelial cells from mice that were analyzed for their gene expression profile under basal conditions and also under different stimulatory conditions like inflammation or VEGF-C over-expression.
Results
Zenbu configurations and status
- Lymphatic Endothelial cells response to VEGFC(COMPLETED)
- Lymphatic Endothelial cells response to VEGFC timecourse, October 26th 2012
Expression profiles
- Gene and cluster expression for the VEGFC response series
MARA based network results
Self-organising maps
Quality control by RIKEN
Short RNA expression
ISMARA analysis results
All samples: http://ismara.unibas.ch/timecourses/VEGFC/ismara_report/index.html
Replicate averaged: http://ismara.unibas.ch/timecourses/VEGFC-avgd/averaged_report/index.html
For more information, see ISMARA.
Paper Outline
This section is work-in-progress.
Tentative title: HOXD10 is a major mediator of VEGF-C induced lymphangiogenesis
Figure 1: Prominent up-regulation of transcription factors (TFs) after stimulation of VEGFR-3 in LEC
• Gene ontology analysis
• Number of all genes and especially transcription factors over-expressed over the time course
Figure 2: VEGF-C156S leads to up-regulation of immediate early response transcription factors
• Validation of transcription factor up-regulation by RT-PCR analysis
• data analysis and RT-PCR confirmation revealed that TFs are differentially regulated between 30-80min
• most highly regulated TFs are e.g.: EGR1, EGR3, EGR2, ATF3, SNAI1, HES1, FOSL1
Figure 3: HOXD10 is a down-stream mediator of VEGFR-3 induced gene expression in LEC
• oPOSSUM analysis
• expression levels in lymphatic endothelial cells of HOXD10 in vitro (human) and ex vivo (mouse)
Figure 4: Regulation of HOXD10 target genes
• validation of viral transduction (over-expression and knock-down)
• target gene regulation
Figure 5: HOXD10 is necessary for the induction of a VEGF-C156S target gene
Figure 6: HOXD10’s involvement in regulating functional aspects of lymphatic endothelial cells
• results of functional assays
