In 3D settings, endothelial cells have been shown to depend on actomyosin-based contractile forces to invade their surrounding matrix and maintain sprout structures after invasion ( Kniazeva and Putnam, 2009). For instance, studies using natural and fibrillar matrices have demonstrated that actomyosin-based cell contractility is necessary to support the formation of multicellular networks through processes mimicking vasculogenesis ( Lyle et al., 2012 Davidson et al., 2019). In endothelial cells, in particular, myosin-mediated cell contractility has been investigated in the context of multicellular organization. Similarly, in 3D and in vivo assays, studies have demonstrated that cell contractility is needed to enable multicellular 3D invasion and proper morphogenesis of epithelial ductal structures ( Ewald et al., 2008 Gjorevski et al., 2015). For example, in epithelial cells, two-dimensional (2D) assays have been utilized to study the impact of external factors such as substrate stiffness and the role of actomyosin-based contractility in inducing leader cell formation and maintaining coordinated movements of cell cohorts during planar migration ( Ng et al., 2012 Rausch et al., 2013). The important role of cell-mediated forces in multicellular migration and morphogenesis has been highlighted in various cell types and through a variety of in vitro approaches.
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Although the molecular drivers of vascular sprouting and tip and stalk cell specification, via the VEGFR2 and Notch1-DLL4 pathways for example, have been extensively described, much less is known about how cellular mechanics and force regulation influence sprout morphogenesis ( Gerhardt et al., 2003 Hellström et al., 2007 Benedito et al., 2009).
As they advance, tip cells retain physical cell–cell adhesions with a trailing cohort of stalk cells that form the lumenized trunk of the sprout ( Adams and Alitalo, 2007 Carmeliet et al., 2009). The three-dimensional (3D), multicellular structures that form during sprouting are classically composed of leader endothelial cells, or tip cells, that proteolytically degrade the surrounding extracellular matrix to migrate toward sources of angiogenic factors. This cooperative movement of cells is of particular importance during sprouting morphogenesis because the endothelial cells that make up new vessels must form patent, nonleaky structures capable of supporting blood flow.
This is a highly regulated process that is critical in a variety of pathological and developmental morphogenic events such as tumor cell invasion and sprouting angiogenesis ( Friedl and Gilmour, 2009 Scarpa and Mayor, 2016). Together, these studies reveal a critical role for NMIIA-mediated contractile forces in maintaining multicellularity during sprouting and highlight the central role of forces in regulating cell–cell adhesions during collective motility.Ĭollective migration is a process in which cohorts of cells move in a coordinated manner so that cell–cell contacts are maintained. Using CRISPR/Cas9-mediated gene editing, we further identified NMIIA as the major isoform responsible for regulating multicellularity and cell contractility during sprouting. Closer examination of cell–cell junctions revealed that blebbistatin impaired adherens-junction organization, particularly between tip and stalk cells. Surprisingly, inhibiting cellular contractility with blebbistatin did not affect the extent of cellular invasion but resulted in cell–cell dissociation primarily between tip and stalk cells. Three-dimensional maps of mechanical deformations generated by sprouts revealed that mainly leader cells, not stalk cells, exert contractile forces on the surrounding matrix. To address this question, we investigated the role of cellular contractility in sprout morphogenesis, using a biomimetic model of angiogenesis.
Although several signaling pathways have been identified as controlling sprouting, it remains unclear to what extent this process is mechanoregulated. During sprouting, tip cells and ensuing stalk cells migrate collectively into the extracellular matrix while preserving cell–cell junctions, forming patent structures that support blood flow. Angiogenic sprouting is a critical process involved in vascular network formation within tissues.