Endothelial cell rearrangements during vascular patterning require PI3-kinase-mediated inhibition of actomyosin contractility

Abstract

Angiogenesis is a dynamic process relying on endothelial cell rearrangements within vascular tubes, yet the underlying mechanisms and functional relevance are poorly understood. Here we show that PI3Kα regulates endothelial cell rearrangements using a combination of a PI3Kα-selective inhibitor and endothelial-specific genetic deletion to abrogate PI3Kα activity during vessel development. Quantitative phosphoproteomics together with detailed cell biology analyses in vivo and in vitro reveal that PI3K signalling prevents NUAK1-dependent phosphorylation of the myosin phosphatase targeting-1 (MYPT1) protein, thereby allowing myosin light chain phosphatase (MLCP) activity and ultimately downregulating actomyosin contractility. Decreased PI3K activity enhances actomyosin contractility and impairs junctional remodelling and stabilization. This leads to overstretched endothelial cells that fail to anastomose properly and form aberrant superimposed layers within the vasculature. Our findings define the PI3K/NUAK1/MYPT1/MLCP axis as a critical pathway to regulate actomyosin contractility in endothelial cells, supporting vascular patterning and expansion through the control of cell rearrangement.

Distinct Vegfa isoforms control endothelial cell proliferation through PI3 kinase signalling mediated regulation of cdkn1a/p21

“…Another study showed that Y1212 in VEGFR2 was important to control PI3K signaling pathway activation upstream of myc-dependent EC proliferation (Testini et al, 2019). In line with our observations in the mouse retina, blocking PI3K signaling in this setting reduced the number of phospho-histone 3 positive ECs (Ola et al, 2016) and led to a reduction in EdU incorporation (Angulo-Urarte et al, 2018). Importantly, activating mutations in PIK3CA can lead to venous malformations that are characterized by increased EC proliferation (Castel et al, 2016; Castillo et al, 2019; Castillo et al, 2016).…”

“…For example, the PI3K pathway controls EC survival in response to VEGFA in cultured cells (Gerber et al, 1998; Nakatsu et al, 2003), while in vivo studies in the developing mouse retina (Graupera et al, 2008) and in zebrafish embryos (Nicoli et al, 2012) suggested that PI3K signalling predominantly regulates EC migration. More recent studies, however, also suggested a function of PI3K signalling in EC proliferation during retinal development (Angulo-Urarte et al, 2018; Ola et al, 2016) and in vascular malformations (Castel et al, 2016; Castillo et al, 2019; Castillo et al, 2016). Further work in mouse and in cell culture has shown that ERK signalling downstream of VEGFA stimulates EC proliferation (Koch and Claesson-Welsh, 2012), vessel integrity (Ricard et al, 2019) and artery formation (Simons and Eichmann, 2015).…”

ECM concentration and cell‐mediated traction forces play a role in vascular network assembly in 3D bioprinted tissue

“…The action of actin‐mediated contractile forces in the hydrogel constructs may result in changes in ECM mechanics that affect vessel‐like network formation. Regulation of actomyosin‐mediated contractility is believed to be essential for endothelial cell organization during vascular network formation (Angulo‐Urarte et al, ). Treatment with the inhibitors, Y27632 and blebbistatin, resulted in reduction of total length of vessel‐like structures to different extents in the hydrogel constructs.…”

A multiscale model of complex endothelial cell dynamics in early angiogenesis

“…Angiogenesis, the process by which new blood vessels are generated from a pre-existing vascular network, has been extensively investigated in recent years [1][2][3][4][5][6][7][8][9][10]. However, understanding of the complex behaviour of endothelial cells (ECs) during angiogenesis remains incomplete.…”

“…As a consequence, a dynamic coupling between cell phenotypes and rearrangements is established. The functional role of cell mixing, and how it is affected by variations in the gene expression patterns of ECs, are unclear, although it is acknowledged that cell rearrangements greatly influence the pattern of the vascular network and its functionality [1,7,14,15].…”