Abstract
The formation and guidance of specialized endothelial tip cells is essential for both developmental and pathological angiogenesis1. Notch-1 signalling regulates the generation of tip cells, which respond to gradients of vascular endothelial growth factor (VEGF-A)2. The molecular cues and signalling pathways that control the guidance of tip cells are poorly understood. Bidirectional signalling by Eph receptors and ephrin ligands represents one of the most important guidance cues involved in axon path finding3. Here we show that ephrin-B2 reverse signalling involving PDZ interactions regulates endothelial tip cell guidance to control angiogenic sprouting and branching in physiological and pathological angiogenesis. In vivo, ephrin-B2 PDZ-signalling-deficient mice (ephrin-B2ΔV) exhibit a reduced number of tip cells with fewer filopodial extensions at the vascular front in the mouse retina. In pathological settings, impaired PDZ signalling decreases tumour vascularization and growth. Mechanistically, we show that ephrin-B2 controls VEGF receptor (VEGFR)-2 internalization and signalling. Importantly, internalization of VEGFR2 is necessary for activation and downstream signalling of the receptor and is required for VEGF-induced tip cell filopodial extension. Together, our results suggest that ephrin-B2 at the tip cell filopodia regulates the proper spatial activation of VEGFR2 endocytosis and signalling to direct filopodial extension. Blocking ephrin-B2 reverse signalling may be an attractive alternative or combinatorial anti-angiogenic therapy strategy to disrupt VEGFR2 function in tumour angiogenesis.
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Acknowledgements
We thank R. Klein for the ephrin-B2 lox/lox mice, A. Filosa, F. Finkelmeier and H. zum Buttel for technical support, A. Filosa and B. Garvalov for comments on the manuscript and R. Adams for scientific input and reading the manuscript. We acknowledge the Max Planck Institute of Neurobiology in Martinsried (Germany) and the Edinger Institute at Frankfurt University (Germany) for support and the use of equipment and animal facilities in the early stages of this project. This work was supported by grants from the Deutsche Forschungsgemeinschaft within the SPP1190 (AC110/3-1, 3-2 to T.A. and AC180/3-1, 3-2 to A.A.-P.), the Deutsche Krebshilfe (107231 to T.A.) and the Clusters of Excellence ‘Macromolecular Complexes (CEF)’ (EXC 115) at the University Frankfurt and ‘Cardio-Pulmonary System (ECCPS)’ (EXC 147) at the Universities of Giessen and Frankfurt.
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Contributions
S.Sa. designed experiments and performed all the characterization of developmental angiogenesis of the mice, the retinal explant cultures, time lapse experiments, antibody feeding assays, part of the biochemistry and generated the astrocytomas for the tumour angiogenesis part. S.Se. performed the tumour injections and the analysis of tumour growth and vasculature. C.L.E. performed most of the biochemistry. G.A.W. and M.E.P. generated mouse mutants. T.A. designed experiments, interpreted results in the tumour angiogenesis part and wrote the manuscript. A.A.-P. designed experiments and interpreted results in the developmental part, helped with quantifications, prepared all the figures and wrote the manuscript.
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Supplementary information
Supplementary Figures
This file contains Supplementary Figures 1-12 with legends. (PDF 3538 kb)
Supplementary Movie 1
Endothelial cells overexpressing YFP-ephrinB2 extended numerous dynamic filopodia, which facilitated cell migration in the extracellular matrix. The movie is 8 h long with 96 frames at 5 min intervals. (MOV 692 kb)
Supplementary Movie 2
YFP-ephrinB2 (green) conferred a repulsive phenotype on endothelial cells. Dynamic filopodia, protruding from YFP-ephrinB2 expressing cells, led to increased cell motility. Upon contact with surrounding cells, green cells rapidly retracted which led to the exclusion of these cells from the capillary-like structures (tubes). The movie is 7 h long with 84 frames at 5 min intervals. (MOV 1671 kb)
Supplementary Movie 3
Endothelial cells lacking ephrinB2 (green) lost their contact-dependent repulsion and invasive migration and tightly assembled into capillary-like structures. The movie is 7 h long with 84 frames at 5 min intervals. (MOV 4049 kb)
Supplementary Movie 4
Deletion of the cytoplasmic tail results in the loss of function of ephrinB2. Endothelial cells expressing YFP-ephrinB2ΔC (green) lost the repulsive phenotype and integrated into the tubes, indicating the significance of reverse signaling downstream of ephrinB2 in the regulation of endothelial cell function. The movie is 8 h long with 96 frames at 5 min intervals. (MOV 1582 kb)
Supplementary Movie 5
PDZ-interactions are necessary for the repulsive activity conferred by ephrinB2. Endothelial cells expressing YFP-ephrinB2ΔV (green) integrated into the tubes. The movie is 8 h long with 96 frames at 5 min intervals. (MOV 4141 kb)
Supplementary Movie 6
Tyrosine phosphorylation is dispensable for the repulsive phenotype. Endothelial cells expressing YFP-ephrinB25Y (green) behaved like wild type cells and showed high motility and exclusion from tubular structures. The movie is 8 h long with 96 frames at 5 min intervals. (MOV 4109 kb)
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Sawamiphak, S., Seidel, S., Essmann, C. et al. Ephrin-B2 regulates VEGFR2 function in developmental and tumour angiogenesis. Nature 465, 487–491 (2010). https://doi.org/10.1038/nature08995
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DOI: https://doi.org/10.1038/nature08995
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