VEGF induces signalling and angiogenesis by directing VEGFR2 internalisation through macropinocytosis

Abstract

Endocytosis plays a crucial role in receptor signalling. VEGFR2 (also known as KDR) and its ligand VEGFA are fundamental in neovascularisation. However, our understanding of the role of endocytosis in VEGFR2 signalling remains limited. Despite the existence of diverse internalisation routes, the only known endocytic pathway for VEGFR2 is the clathrin-mediated pathway. Here, we show that this pathway is the predominant internalisation route for VEGFR2 only in the absence of ligand. Intriguingly, VEGFA induces a new internalisation itinerary for VEGFR2, the pathway of macropinocytosis, which becomes the prevalent endocytic route for the receptor in the presence of ligand, whereas the contribution of the clathrin-mediated route becomes minor. Macropinocytic internalisation of VEGFR2, which mechanistically is mediated through the small GTPase CDC42, takes place through macropinosomes generated at ruffling areas of the membrane. Interestingly, macropinocytosis plays a crucial role in VEGFAinduced signalling, endothelial cell functions in vitro and angiogenesis in vivo, whereas clathrin-mediated endocytosis is not essential for VEGFA signalling. These findings expand our knowledge on the endocytic pathways of VEGFR2 and suggest that VEGFA-driven internalisation of VEGFR2 through macropinocytosis is essential for endothelial cell signalling and angiogenesis.

Dynasore impairs VEGFR2 signalling in an endocytosis-independent manner

“…Given that both tools (siRNA of CHC and dynasore) inhibited effectively the uptake of transferrin (a typical cargo-marker of CME) (Fig. 1a,b), it is concluded that partial inhibition of VEGFR2 internalisation (by the above tools) is not due to insufficient blockage of CME, but rather due to a partial contribution of this pathway in VEGFR2 internalisation, which is in line with previous reports1819242628. Furthermore, since CHC knockdown and dynasore inhibited internalisation of VEGFR2 to the same extent (up to 20–30%), we conclude that these tools are consistent in blocking CME of VEGFR2.…”

“…To assess the amount of internalised VEGFR2, we followed an anti-VEGFR2 antibody uptake protocol, followed by analysis by confocal microscopy1821232637, in primary human umbilical vein endothelial cells (HUVECs). This method reveals the molecules of newly internalised VEGFR2, while it excludes the non-internalised and intracellular pools of the receptor.…”

“…5) to prevent dynamin-dependent endocytosis (DDE). At this step (step 1), dynasore inhibits both DDE and the off-targets (illustrated in the scheme as “DDE blocked” and “off-targets are inhibited by dynasore”, respectively), while the receptor can still internalise through dynamin-independent endocytosis18 (noted in the scheme as “DIE”). In step 2, the cells are washed, to remove the drug.…”

“…Given the pivotal role of VEGFR2 signalling in vascular homeostasis, as well as in cancer progression and other angiogenesis-related diseases, unraveling the underlying mechanisms that govern VEGFR2 endocytosis has been imperative for the comprehension of vascular pathogenesis and for targeted therapy1617. Although the main VEGF-induced endocytic route of VEGR2 is macropinocytosis18, which plays critical role in VEGF functions18, a part of the receptor is also internalised via clathrin- and dynamin-dependent endocytosis181920212223242526272829303132. Intriguingly, the role of this route in the regulation of VEGFR2 signalling remains controversial.…”

“…Intriguingly, the role of this route in the regulation of VEGFR2 signalling remains controversial. Thus, on one hand, knockdown of clathrin or of other molecules of the clathrin machinery have no effect or they augment VEGF-induced activation of ERK1/2182122242829, yet, on the other hand, dynasore attenuates VEGFR2 signalling23263334. Intriguingly, given that small molecule inhibitors may have off-target effects1335, it is unclear whether the inhibitory effect of dynasore in VEGF signalling is due to interference with endocytosis itself or due to concomitant modulation of other molecules that participate in the signalling process (i.e.…”