Discovery of New Pathways and Mechanisms of Notch Signalling in Angiogenesis


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Esther Bridges1,Anassuya Ramachandran1,Jie Zuo1,Tessa Peterkin1,Martin Taylor2,Harukazu Suzuki3,Philip Pinheiro1,Ji-Liang Li1,Roger Patient1,Adrian Harris1
1Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, United Kingdom,2Human Genetics Unit, University of Edinburgh, Edinburgh, United Kingdom,3RIKEN Omics Science Center, Yokohama Institute, Yokohama, Japan

Background

The Notch ligand Delta-like 4 (Dll4) functions as a negative regulator of tumour angiogenesis (formation of new blood vessels). However, little is still known about the mechanisms/genes involved in the downstream responses following Dll4/Notch signalling, their subsequent role in tumour vascular biology and how they are coupled to other angiogenic signalling pathways. Our aim is to identify and validate these genes and the mechanisms.

Method

Deep sequence analysis (CAGE array) of Human Umbilical Vein Endothelial Cells (HUVEC) stimulated with human recombinant tethered Dll4 (rhDll4) for 16h was validated by real time QPCR in HUVEC and a glioblastoma cell line U87. Selected candidates and their knockout phenotype was screened in various models of angiogenesis, including the in vitro-in vivo hanging drop assay and the zebrafish model.

Results

The CAGE array was successfully validated by examining the expression profile over time of selected candidates (e.g. SULF1, RHOQ) in HUVEC and U87 cells, following stimulation with rhDll4. All genes examined were significantly induced in HUVEC compared to U87 cells; RHOQ was observed to be up-regulated in HUVEC cells only. Loss of expression of RHOQ in HUVEC reduced sprouting in the hanging drop assay, conversely loss of SULF1 increased sprouting. In situ expression profiles suggest these genes are affiliated with blood vessels in zebrafish.

CAGE analysis revealed induction of several FGFR signalling components (e.g Flrt2, FGF2) in HUVEC cells, following stimulation with rhDll4. Interestingly, we observed that FGF signalling up-regulated Dll4 expression in HUVEC cells via a gamma secretase sensitive pathway.

Conclusion

New mechanisms and genes are currently being identified which are coupled to the notch signalling pathway.  Deep sequence analysis of HUVEC cells stimulated with rhDll4 has identified many candidates for further characterisation; for example SULF1 and RHOQ. Evidence of cross talk between the FGF signalling pathway and Notch signalling was also observed.