Angiogenic morphogenesis driven by dynamic and heterogeneous collective endothelial cell movement

Abstract

Recently, roles of Delta-like 4 (Dll4)-Notch signaling in angiogenesis have been demonstrated by a series of reports (Ridgway et al., 2006;Hellstrom et al., 2007;Siekmann and Lawson, 2007;Suchting et al., 2007). Murine retina heterozygous for a null mutation of the Dll4 gene showed excessive branching and this was recapitulated by administering a -secretase inhibitor, Development 138, 4763-4776 (2011Development 138, 4763-4776 ( ) doi:10.1242 SUMMARYAngiogenesis is a complex process, which is accomplished by reiteration of modules such as sprouting, elongation and bifurcation, that configures branching vascular networks. However, details of the individual and collective behaviors of vascular endothelial cells (ECs) during angiogenic morphogenesis remain largely unknown. Herein, we established a time-lapse imaging and computer-assisted analysis system that quantitatively characterizes behaviors in sprouting angiogenesis. Surprisingly, ECs moved backwards and forwards, overtaking each other even at the tip, showing an unknown mode of collective cell movement with dynamic 'cell-mixing'. Mosaic analysis, which enabled us to monitor the behavior of individual cells in a multicellular structure, confirmed the 'cell-mixing' phenomenon of ECs that occurs at the whole-cell level. Furthermore, an in vivo EC-tracking analysis revealed evidence of cell-mixing and overtaking at the tip in developing murine retinal vessels. In parametrical analysis, VEGF enhanced tip cell behavior and directed EC migration at the stalk during branch elongation. These movements were counter-regulated by EC-EC interplay via -secretase-dependent Dll4-Notch signaling, and might be promoted by EC-mural cell interplay. Finally, multiple regression analysis showed that these molecule-mediated tip cell behaviors and directed EC migration contributed to effective branch elongation. Taken together, our findings provide new insights into the individual and collective EC movements driving angiogenic morphogenesis. The methodology used for this analysis might serve to bridge the gap in our understanding between individual cell behavior and branching morphogenesis.

Blood vessel anastomosis is spatially regulated by Flt1 during angiogenesis

“…Thus, cells with reduced Flt1, which are likely to have higher levels of VEGF signaling, are favored as sites of stable connection, and this bias depends on the expression of the membrane-bound decoy VEGF receptor, mFlt1. Heterogeneous Flt1 expression and Flk1 activity influence sprout initiation and extension (Chappell et al, 2009;Arima et al, 2011;Jakobsson et al, 2010;Chappell et al, 2013), and our data extend the regulatory impact of heterogeneous VEGF signaling in the patterning of blood vessels to anastomosis. Membrane-bound Flt1 appears essential for the anastomosis selectivity, which is consistent with its cellautonomous role in endothelial cells, relative to sFlt1 that is secreted and has potential to affect aspects of sprouting non-cellautonomously (Chappell et al, 2009;.…”

A cellular Potts model analyzing differentiated cell behavior during in vivo vascularization of a hypoxic tissue

“…First of all, we have assumed that tip cells maintain their fate for the duration of the entire vascular elongation or until anastomosis events. However, it is useful to underline that few recent in vivo and in vitro studies have shown that, at least in some situations, a dynamic and continuous phenotypic interchange between stalk and tip individuals may also occur [3,5,28]. The hypotheses underlying the role played by the two VEGF isoforms are of course a simplified setting of the "real" physiological picture, although they are commonly accepted and used in the computational literature.…”

Endothelin-2 signaling in the neural retina promotes the endothelial tip cell state and inhibits angiogenesis

“…Additionally, Vegfa, acting through Vegfr2, directly promotes the tip cell fate, including filopodia formation. Recent time lapse imaging studies of vascular development in zebrafish and mammalian EC dynamics in explant culture show that the tip cell and stalk cell states are highly plastic, with frequent exchanges between the two cell states (8,9).…”