Abstract
In sprouting angiogenesis, specialized endothelial tip cells lead the outgrowth of blood-vessel sprouts towards gradients of vascular endothelial growth factor (VEGF)-A1,2. VEGF-A is also essential for the induction of endothelial tip cells2, but it is not known how single tip cells are selected to lead each vessel sprout, and how tip-cell numbers are determined. Here we present evidence that delta-like 4 (Dll4)–Notch1 signalling regulates the formation of appropriate numbers of tip cells to control vessel sprouting and branching in the mouse retina. We show that inhibition of Notch signalling using γ-secretase inhibitors, genetic inactivation of one allele of the endothelial Notch ligand Dll4, or endothelial-specific genetic deletion of Notch1, all promote increased numbers of tip cells. Conversely, activation of Notch by a soluble jagged1 peptide leads to fewer tip cells and vessel branches. Dll4 and reporters of Notch signalling are distributed in a mosaic pattern among endothelial cells of actively sprouting retinal vessels. At this location, Notch1-deleted endothelial cells preferentially assume tip-cell characteristics. Together, our results suggest that Dll4–Notch1 signalling between the endothelial cells within the angiogenic sprout serves to restrict tip-cell formation in response to VEGF, thereby establishing the adequate ratio between tip and stalk cells required for correct sprouting and branching patterns. This model offers an explanation for the dose-dependency and haploinsufficiency of the Dll4 gene3,4,5, and indicates that modulators of Dll4 or Notch signalling, such as γ-secretase inhibitors developed for Alzheimer’s disease, might find usage as pharmacological regulators of angiogenesis.
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Acknowledgements
We thank F. Radke for providing Notch1floxed/floxed mice. Support from the following foundations and granting agencies is acknowledged: Swedish Cancer Society, Association for International Cancer Research, European Union, the Novo Nordisk, Strategic Research, Söderberg, Hedlund, Wallenberg and Inga-Britt and Arne Lundberg Foundations (to C.B.); National Institutes of Health (US, NIH) and JH (USPHS National Research Service Award) (to L.I.-A.). H.G., L.-K.P. and P.L. are supported by Cancer Research UK. We acknowledge the Swegene Centre for Cellular Imaging at Gothenburg University for the use of imaging equipment, and the Light Microscopy Service and Peptide Synthesis Laboratory, London Research Institute (Cancer Research UK) for technical assistance.
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[Competing Interests Statement: M.H. and M.K. are employed by AngioGenetics Sweden AB. C.B. receives funding from and is a consultant of AngioGenetics Sweden AB.]
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This file contains Supplementary Methods, Supplementary Figures S1-S11 with Legends and additional references. (PDF 7910 kb)
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Hellström, M., Phng, LK., Hofmann, J. et al. Dll4 signalling through Notch1 regulates formation of tip cells during angiogenesis. Nature 445, 776–780 (2007). https://doi.org/10.1038/nature05571
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DOI: https://doi.org/10.1038/nature05571
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