Orientation of endothelial cell division is regulated by VEGF signaling during blood vessel formation

Abstract

New blood vessel formation requires the coordination of endothelial cell division and the morphogenetic movements of vessel expansion, but it is not known how this integration occurs. Here, we show that endothelial cells regulate division orientation during the earliest stages of blood vessel formation, in response to morphogenetic cues. In embryonic stem (ES) cell-derived vessels that do not experience flow, the plane of endothelial cytokinesis was oriented perpendicular to the vessel long axis. We also demonstrated regulated cleavage orientation in vivo, in flow-exposed forming retinal vessels. Daughter nuclei moved away from the cleavage plane after division, suggesting that regulation of endothelial division orientation effectively extends vessel length in these developing vascular beds. A gainof-function mutation in VEGF signaling increased randomization of endothelial division orientation, and this effect was rescued by a transgene, indicating that regulation of division orientation is a novel mechanism whereby VEGF signaling affects vessel morphogenesis. Thus, our findings show that endothelial cell division and morphogenesis are integrated in developing vessels by flow-independent mechanisms that involve VEGF signaling, and this cross talk is likely to be critical to proper vessel morphogenesis. IntroductionBlood vessels form and expand in both development and disease, via processes that include vasculogenesis, angiogenesis, and intussusception (reviewed in Risau, 1 Eichmann et al, 2 Coultas et al 3 ). Sprouting angiogenesis is the coordinated migration of groups of endothelial cells from vessels and their subsequent fusion to form new interconnections. In this way, simple vascular tubes are ramified and extended to form a primitive vascular plexus. This vessel plexus forms at numerous sites in the embryo, including the yolk sac, the head mesenchyme, and surrounding the neural tube. The primitive vascular plexus is then remodeled under the influence of blood flow and interactions with mural cells. Thus, the initial pattern of vessels serves as a template for remodeling that leads to a mature vasculature.During formation of the primitive vascular plexus, several cellular processes must be regulated and integrated. Specifically, endothelial cells respond to some morphogenetic cues by sprouting, while actively dividing to expand the pool of endothelial cells. One level of integration occurs via the signaling pathways that promote angiogenesis, because many affect both endothelial cell division and morphogenesis. The VEGF signaling pathway is an example of this mode of integration, because it regulates both cell division and branching morphogenesis (reviewed in Rousseau et al, 4 Kliche and Waltenberger, 5 Ferrara et al,6 Nagy and Senger, 7 and Shibuya and Claesson-Welsh 8 ). VEGF-A (VEGF) binds 2 highaffinity receptors on endothelial cells, flk-1 (VEGFR-2) and flt-1 (VEGFR-1), and perturbation of VEGF signaling by genetic deletion of either receptor affects both endothelial cell division and morphogenesis. ...

p38α MAP kinase‐deficient mouse embryonic stem cells can differentiate to endothelial cells, smooth muscle cells, and neurons

“…They were apparently formed from multiple cells and likely represented the primitive vessels (supplemental data). Similar structures derived from ESCs were observed and defined as vessels with lumens in a recent study by Zeng et al (2007). Quantitative analysis of the vessel structures developed from wild type and p38␣ Ϫ/Ϫ ESCs was difficult due to heterogeneity in length, diameter, and morphology.…”

“…For instance, the roles of VEGF signaling pathways in vasculogenesis and angiogenesis have been elegantly illustrated in recent studies using in vitro ESC differentiation systems (Schuh et al, 1999;Zeng et al, 2007;Vittet et al, 1996), which support and complement the findings from in vivo studies (Drake et al, 1998(Drake et al, , 2000. The objective of this study was to assess an overall role of p38␣ in ESC differentiation.…”

VEGF and endothelial guidance in angiogenic sprouting

“…Monitoring endothelial cell division in mouse embryonic stem cell-derived embryoid bodies, they found that stalk cells normally divided with their plane of cytokinesis perpendicular to the vessel long axis. 28 This polarized cell division promoted vessel elongation during sprouting. Interestingly, stalk cell polarization occured independently of flow, but was instead regulated by heparin-binding VEGF isoforms.…”

VEGF-Directed Blood Vessel Patterning: From Cells to Organism

“…Endothelial cells subsequently orient in the plane of the vessel ( planar cell polarity) in response to shear stress provided by blood flow, at least in large conduit vessels (for review, see McCue et al 2004). Finally, they also orient their spindles as they divide, and in developing vessel networks this orientation puts the cleavage plane perpendicular to the vessel long axis and extends the length of the vessel (Zeng et al 2007). …”

“…However, oriented endothelial divisions appear to be flow independent and VEGF dependent at early developmental stages. Embryonic stem cell -derived vessels that do not experience flow orient endothelial cell divisions with the cleavage plane perpendicular to the vessel long axis, and retinal vessels near the vascular front that likely have low shear stress also orient endothelial divisions, perhaps in order to extend vessel length in expanding vascular networks (Zeng et al 2007). Elevated VEGF signaling due to loss of the negative modulator VEGFR-1 (Flt-1) results in randomization of endothelial division orientation, although expression of only the VEGF120 isoform does not significantly randomize endothelial division orientation in developing retinas (Zeng et al 2007;SM Taylor, H Park, G Zeng, et al, unpubl.…”