Cdc42 regulates branching in angiogenic sprouting in vitro

Abstract

Objectives The morphogenetic events that occur during angiogenic sprouting involve several members of the Rho family of GTPases, including Cdc42. However, the precise roles of Cdc42 in angiogenic sprouting have been difficult to elucidate owing to the lack of models to study these events in vitro. Here, we aim to identify the roles of Cdc42 in branching morphogenesis in angiogenesis. Methods Using a 3D biomimetic model of angiogenesis in vitro, where endothelial cells were seeded inside a cylindrical channel within collagen gel and sprouted from the channel in response to a defined biochemical gradient of angiogenic factors, we inhibited Cdc42 activity with a small molecule inhibitor ML141 and examined the effects of Cdc42 on the morphogenetic processes of angiogenic sprouting. Results We find that partial inhibition of Cdc42 had minimal effects on directional migration of endothelial cells, but led to fewer branching events without affecting the length of these branches. We also observed that antagonizing Cdc42 reduced collective migration in favor of single cell migration. Additionally, Cdc42 also regulated the initiation of filopodial extensions in endothelial tip cells. Conclusions Our findings suggest that Cdc42 can affect multiple morphogenetic processes during angiogenic sprouting and ultimately impact the architecture of the vasculature.

Consensus guidelines for the use and interpretation of angiogenesis assays

“…The use of aortic rings (see below) and that of specific microfluidic devices represent a further tool to describe this process in a 3D architecture [45]. For instance, wound healing assay exploited by single-cell analysis and by using chimeric EC sheets obtained by infecting cells with different fluorescent proteins [34, 45, 46] was instrumental to describe the following steps of EC collective migration: (1) In resting state, ECs undergo random cell motility in the monolayer with a regulated dynamics of homotypic cell junctions; (2) the presence of cell-free space (i.e., the wound) and a chemotactic gradient results in the appearance at the sheet margin of leader cells, which is characterized by an aggressive phenotype with prominent stress fibers, ruffling lamellipodia and enlarged focal adhesions, formation of peripheral actin cables, and discontinuous adherens junctions, which indicate mechanical coupling between leader and follower cells in the migrating cluster [47]; (3) as leaders start to migrate in the free space, a follower phenotype appears within cells of the monolayer.…”

Dynamin Binding Protein Is Required for Xenopus laevis Kidney Development

“…Cdc42 is a Rho family small GTPase that was first discovered in Saccharomyces cerevisiae (Johnson and Pringle, 1990). It plays a role in cell migration, polarity, differentiation and proliferation, as well as branching of blood vessels and regulation of actin dynamics (Melendez et al, 2013; Schulz et al, 2015; Mizukawa et al, 2017; Nguyen et al, 2017; Lavina et al, 2018). Cdc42 is a molecular switch that cycles between active (GTP-bound) and inactive (GDP-bound) states through its interaction with guanine exchange factors (GEFs) and GTPase activating proteins (GAPs) (Bishop and Hall, 2000; Schmidt and Hall, 2002).…”

Dynamin binding protein is required forXenopus laeviskidney development

“…Cdc42 is a Rho family small GTPase that was first discovered in Saccharomyces cerevisiae (Johnson and Pringle, 1990). It plays a role in cell migration, polarity, differentiation and proliferation, as well as branching of blood vessels and regulation of actin dynamics (Lavina et al, 2018;Melendez et al, 2013;Mizukawa et al, 2017;Nguyen et al, 2017;Schulz et al, 2015). Cdc42 is a molecular switch that cycles between active (GTP-bound) and inactive (GDPbound) states through its interaction with guanine exchange factors (GEFs) and GTPase activating proteins (GAPs) (Bishop and Hall, 2000;Schmidt and Hall, 2002).…”