VEGFC induced cell cycle arrest mediates sprouting and differentiation of venous and lymphatic endothelial cells

Abstract

The emergence and growth of new vessels requires a tight synchronization between proliferation, differentiation and sprouting, traditionally thought to be controlled by mitogenic signals, especially of the VEGF family. However, how these cues are differentially transduced, by sometimes even neighboring endothelial cells (ECs), remains unclear. Here we identify cell-cycle progression as a new regulator of EC sprouting and differentiation. Using transgenic zebrafish illuminating cell-cycle stages, we show that venous and lymphatic precursors sprout from the Cardinal Vein (CV) exclusively in G0/G1, and reveal that cell-cycle arrest is induced in these ECs by p53 and the CDK-inhibitors p27 and p21. Moreover, we demonstrate that in vivo, chemical and genetic cell-cycle inhibition, results in massive vascular growth. Mechanistically, we identify the mitogenic VEGFC/VEGFR3/ERK axis as direct inducer of cell-cycle arrest in angiogenic ECs and characterize the cascade of events governing venous vs. lymphatic segregation and sprouting. Overall, our results uncover an unexpected mechanism whereby mitogen-controlled cell-cycle arrest boosts sprouting, raising important questions about the use of cell-cycle inhibitors in pathological angiogenesis and lymphangiogenesis.

Zebrafish mutants provide insights into Apolipoprotein B functions during embryonic development and pathological conditions

“…Study approval. Zebrafish were raised by standard methods and handled according to, and following approval by, the Weizmann Institute Animal Care and Use Committee (68).…”

Fluid flow to mimic organ function in 3D in vitro models

“…The activation of this signaling pathway potentiates LEC proliferation and expansion of the network. 86 Additionally, laminar shear stress induced by intraluminal flow inhibits NOTCH1 and induces Ca2+ signaling and the transcription factors KLF2/4 that promote LEC proliferation and sprouting 87–89 till a more mature network consisting in capillaries and collecting lymphatic vessels (LVs) is formed. Indeed, for the formation of collecting LVs and valves, lymph flow sensing by LECs is needed in areas of flow recirculation.…”

Mechanisms and cell lineages in lymphatic vascular development

“…These progenitors undergo asymmetric division and translocate to the dorsal wall of the vein [ 87 , 88 ]. Live imaging of a fucci reporter, which identifies cells in specific stages of the cell cycle, showed that lymphatic progenitors undergo cell cycle arrest prior to emigrating from the posterior cardinal vein [ 89 ]. Upon their exit from the vein, LECs migrate along intersomitic arterial vessels, in a manner dependent on Vegfc [ 47 , 48 , 87 , 90 ] and Cxcl12a and Cxcl12b gradients [ 91 ], and form the lymphatics of the zebrafish trunk via lymphangiogenesis [ 47 , 48 , 50 ].…”