Mechanical regulation of angiogenesis in a biomimetic model

Collective cell migration is required for numerous developmental and pathological processes including angiogenesis, branching morphogenesis, and cancer progression. Dynamic regulation of cell-cell adhesions, transmission of long-range contractile forces across cells, and remodeling of the extracellular matrix (ECM) are all required for successful multicellular invasion. While actomyosin contractility is well studied in single cells on flat surfaces, less is understood about its regulation of collective cell migration, including during angiogenesis whereby endothelial cells from existing vessels invade as multicellular sprouts to form new vessels. Here, we have engineered a novel organotypic model of angiogenic sprouting and neovessel formation that originates from pre-formed artificial vessels fully encapsulated within a 3D ECM. Using this model, we screened the effects of angiogenic factors and identified two distinct cocktails that promoted robust multicellular endothelial sprouting. The angiogenic sprouts in our system exhibited hallmark structural features of in vivo angiogenesis, including directed invasion of leading cells that developed filopodia-like protrusions characteristic of tip cells and following polarized stalk cells that line lumens connecting back to parent vessels. Ultimately, sprouts bridged between pre-formed channels and formed perfusable neovessels. Using this model, we investigated the effects of angiogenic inhibitors on sprouting morphogenesis using quantitative evaluation metrics. Together, these results demonstrate an in vitro 3D biomimetic model that reconstitutes the morphogenetic steps of angiogenic sprouting. We used this biomimetic model to characterize the role of actomyosin contractility during multicellular sprout extension. We also described differences in tip cell-stalk cell and stalk cell-stalk cell adhesions by evaluating vascular endothelial (VE)-cadherin organization. Inhibition of actomyosin contractility through non-muscle myosin II caused a decrease in VE-cadherin organization at cell-cell adhesions, and a loss of cell-cell contact between leading tip cell and the following stalk during sprout extension. This effect is rescued when cells express a form of VE-cadherin that stabilizes its interactions with the actin cytoskeleton. Our findings reveal contractility is required for multicellular invasion during angiogenic sprout extension, and are validated using an in silico model developed by Bentley et al. 2014 to simulate cell dynamics during sprouting, and recapitulated in an in vivo model of mouse retinal angiogenesis