(271e) Cdc42 Regulates Formation of Branches during Chemotactic Invasion of 3D Angiogenic Sprouting

Angiogenesis is a process where new blood vessels form from existing vasculature. Endothelial cells from capillaries respond to chemotactic angiogenic cues by extending filopodial protrusions, digesting the vascular basement membrane, and invading into the interstitial matrix as tip cells with following stalk cells. These multicellular sprouts eventually develop into mature vessels with branched architectures. During the process of forming branched vascular network, the cytoskeleton of the endothelial cells undergoes dynamic changes. Rho GTPase proteins are known to regulate actin cytoskeletal dynamics in cell migration during organ development and tissue morphogenesis. Among Rho GTPase proteins, Cdc42 has a conserved role in regulating actin cytoskeleton dynamics, filopodial extensions, cell polarity, and migration. However, its role in angiogenic sprouting, especially in the context of chemotactic migration in angiogenesis, remains elusive due to the lack of existing in vitro platforms to truly capture angiogenic invasion in 3D microenvironment.

Recently, we have developed an AngioChip, an in vitro model wherein endothelial cells lining a perfusable lumen can be induced with angiogenic factors to sprout and invade into the surrounding extracellular matrix [1]. In this model, many of the morphogenic features of sprouting angiogenesis are recapitulated, including tip cells, stalk cells, and multicellular branched networks. Here, using this system, we sought to investigate the effects of Cdc42 on the morphogenic processes of angiogenesis by using a small molecule inhibitor of Cdc42. Unlike other studies of tubulogenesis and Matrigelâ„¢ assays, where endothelial cell network formation occurred in a uniform distribution of biochemical stimuli, our system employed a biochemical gradient not only to stimulate the formation of multicellular sprout structures, but also to unveil the effects of Cdc42 in the context of chemotactic migration in angiogenic sprouting. We observed that Cdc42 mediates several aspects of the morphogenetic processes of angiogenesis. Partial abrogation of Cdc42 activity reduces formation of new branches but not on the length of branches. Disruption of Cdc42 activity also reduces the extent of collective cell migration and unexpectedly results in many more filopodial protrusions [2]. Together our 3D biomimetic model provides a unique opportunity to study the role of Cdc42 in branching angiogenesis. A concrete understanding of vessel branching and how it is regulated by Cdc42 will provide valuable insight into designing and engineering vascular networks to support functional tissue implants.

[1] Nguyen, D.H., Stapleton, S.C., Yang, M.T., Cha, S.S., Choi, C.K., Galie, P.A., Chen, C.S. (2013). Biomimetic model to reconstitute angiogenic sprouting morphogenesis in vitro. Proceedings of the National Academy of Sciences. 110(17): 6712-6717.

[2] Nguyen, D.H., Gao, L., Wong, A., Chen, C.S. (2017). Cdc42 regulates branching in angiogenic sprouting in vitro. Microcirculation (Epub ahead of print).