VEGFR-3 controls tip to stalk conversion at vessel fusion sites by reinforcing Notch signalling

Abstract

Angiogenesis, the growth of new blood vessels, involves specification of endothelial cells to tip cells and stalk cells, which is controlled by Notch signalling, whereas vascular endothelial growth factor receptor (VEGFR)-2 and VEGFR-3 have been implicated in angiogenic sprouting. Surprisingly, we found that endothelial deletion of Vegfr3, but not VEGFR-3-blocking antibodies, postnatally led to excessive angiogenic sprouting and branching, and decreased the level of Notch signalling, indicating that VEGFR-3 possesses passive and active signalling modalities. Furthermore, macrophages expressing the VEGFR-3 and VEGFR-2 ligand VEGF-C localized to vessel branch points, and Vegfc heterozygous mice exhibited inefficient angiogenesis characterized by decreased vascular branching. FoxC2 is a known regulator of Notch ligand and target gene expression, and Foxc2+/−; Vegfr3+/− compound heterozygosity recapitulated homozygous loss of Vegfr3. These results indicate that macrophage-derived VEGF-C activates VEGFR-3 in tip cells to reinforce Notch signalling, which contributes to the phenotypic conversion of endothelial cells at fusion points of vessel sprouts.

In vivo imaging of lymphatic vessels in development, wound healing, inflammation, and tumor metastasis

“…EGFP fluorescence is also detected in growing blood capillaries of the postnatal retina (Fig. 1C, III and IV) where VEGFR3 regulates angiogenic sprouting (21).…”

“…We have generated a reporter mouse line for in vivo imaging of lymphatic vessels in which a dual reporter for fluorescence and luminescence (EGFPLuc) is expressed under the endogenous transcriptional control of the Vegfr3 (vascular endothelial growth factor receptor 3) gene, the first lymphatic marker discovered (20). Although Vegfr3 is also expressed, to some extent, in the tip cells of the newly formed blood capillaries (21)(22)(23) and in fenestrated endothelium (24), it is still one of the best markers for all lymphatic endothelial cells. Outside the vascular system Vegfr3 expression has been described in subpopulations of monocytes and macrophages (25) and in embryonic osteoblasts and neural progenitors (26,27).…”

Interaction of tumor cells and lymphatic vessels in cancer progression

“…31 VEGFR-3 may function to limit excessive angiogenic signaling through VEGFR-2 on blood vascular endothelial cells. 32 This effect of VEGFR-3 seems to be independent of the intrinsic kinase activity of the receptor. Upon cell attachment to the extracellular matrix component collagen I and activation of integrin b1 expressed on the cell membrane, VEGFR-3 can be phosphorylated by Src kinase independently of VEGF ligand binding or activity of the VEGFR-3 kinase domain.…”

“…Upon cell attachment to the extracellular matrix component collagen I and activation of integrin b1 expressed on the cell membrane, VEGFR-3 can be phosphorylated by Src kinase independently of VEGF ligand binding or activity of the VEGFR-3 kinase domain. 32,33 Recent studies have also shown that both VEGFR-2 and VEGFR-3 are expressed by the blood vascular endothelial cells, where VEGF-C may induce VEGFR-2/VEGFR-3 heterodimerization and downstream signaling in part explaining the redundancy of VEGFR-3 ligands in blood vascular development. 34 VEGF-C and VEGF-D may also function in monocyte and macrophage recruitment as VEGFR-3 expression has been found on some of these cells.…”

Inhibition of Notch signaling induces extensive intussusceptive neo-angiogenesis by recruitment of mononuclear cells

“…It leads to splitting of the receptor by TACE and c-secretase and subsequent translocation of its intracellular portion (NICD) into the nucleus. The subsequent interaction with RBP-Jk/CBF-1 protein complex results in expression of genes normally silent in absence of Notch signal such as basic helix-loophelix genes Hes 1, 5, 7 and Hesr/Hey 1, 2, L. In mice, loss and gain of function studies for Notch receptors and Notch ligands including Dll4/Jag1 and Dll1 has shown that Notch signaling is essential for normal arterial-venous differentiation and remodeling and for angiogenic sprouting [11][12][13][14]. In angiogenic sprouts, Dll4/Notch signaling acts as a negative regulator of endothelial tip cell differentiation.…”