Editors' ChoiceAngiogenesis

VEGFR Internalization Promotes Sprouting

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Science Signaling  12 Mar 2013:
Vol. 6, Issue 266, pp. ec63
DOI: 10.1126/scisignal.2004133

Angiogenesis, or the building of blood vessels, involves the sprouting of highly mobile endothelial tip cells that extend processes called filopodia. Mature vessels have quiescent endothelial cells that form tight junctions, creating the endothelial barrier. Vascular endothelial growth factor receptors (VEGFRs) and their ligands are critical to angiogenesis, and binding of VEGF to its receptor stimulates endocytosis. Nakamaya et al. investigated the role of VEGFR endocytosis in the postnatal vascularization of the mouse retina. Unexpectedly, VEGFR2 immunostaining was highest in the established vasculature of the center retina, and it was barely detectable in the angiogenic sprouts. The abundance of VEGFR2 and VEGFR3 in the sprouts was increased by intraocular injection of a proteasome inhibitor, a lysosome inhibitor, or inhibitors of clathrin-mediated endocytosis. Perinuclear accumulation of dye-labeled VEGF was greatest in the cells at the angiogenic front. The transmembrane protein EphrinB2 is implicated in VEGFR internalization, and proteins affinity-purified with the cytoplasmic region of Ephrin B2 contained components of the clathrin-mediated endocytosis machinery (the adaptor AP2, clathrin heavy chain, dynamin, and the clathrin-associated sorting protein Dab2). The interaction of EphrinB2 with Dab2 was mediated by PAR-3, which acted as a molecular adaptor for VEGFR2, VEGFR3, EphrinB2, Dab2, and atypical protein kinase C (aPKC). Coimmmunoprecipitation of VEGFR3, Dab2, and PAR-3 was detected in lung lysates, and endothelial-cell–specific loss of PAR-3 reduced the coimmunoprecipitation of Dab2 and VEGFR2. Knockdown of Dab2 or PAR-3 decreased the uptake and perinuclear accumulation of exogenously added VEGF to cultured mouse endothelial cells and compromised downstream VEGFR signaling to the guanosine triphosphatase Rac1 and the mitogen-activated protein kinases ERK1 and -2. Inducible endothelial-cell–specific genetic knockout of Dab2 or PAR-3 reduced the size and complexity of the endothelial network in the retina and was associated with decreased tip cell number, sprout length, and number of filopodia. Furthermore, uptake of dye-labeled VEGF was reduced at the angiogenic front. In contrast, inducible endothelial-cell–specific genetic knockout of the aPKC PKCλ resulted in increased accumulation of dye-labeled VEGF in the central vasculature of the retina, reduced abundance of VEGFRs in the central region, and aberrant sprouting of the vasculature of the central retina. In vitro assays indicated that PKCλ phosphorylated a Dab2 fragment, which reduced the interaction between the Dab2 fragment and VEGFRs. Inhibition of aPKC enhanced VEGFR internalization and downstream signaling in cultured endothelial cells. Phosphorylated (and activated) aPKC was predominantly detected in the central retinal vasculature, suggesting that in mature vessels of the central retina, spatial regulation of aPKC activity contributed to the stabilization of VEGFR but reduced its endocytosis and signaling.

M. Nakayama, A. Nakayama, M. van Lessen, H. Yamamoto, S. Hoffmann, H. C. A. Drexler, N. Itoh, T. Hirose, G. Breier, D. Vestweber, J. A. Cooper, S. Ohno, K. Kaibuchi, R. H. Adams, Spatial regulation of VEGF receptor endocytosis in angiogenesis. Nat. Cell Biol. 15, 249-260 (2013). [PubMed]

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