Evaluation and Characterization of Endothelial Cell Invasion and Sprouting Behavior.

Here, we describe highly reproducible methods to investigate human EC invasion and sprouting behavior in 3D collagen matrices. Two assay models are presented whereby ECs are induced to sprout from a monolayer surface or from aggregated ECs suspended within a collagen gel matrix. In each case, the assays are performed using serum-free defined media containing a combination of five growth factors (Factors): FGF-2, SCF, IL-3, SDF-1α, and insulin. In both models, marked EC sprouting occurs with leading EC tip cells over a 12-24 h period. To illustrate their utility, we present data showing the influence of various pharmacologic inhibitors directed to membrane-type matrix metalloproteinases (MT-MMPs), protein kinase C alpha (PKCα), Src family kinases, and Notch-dependent signaling. Marked inhibition of sprouting is observed after blockade of MT-MMPs and PKCα, while strong increases in sprouting and EC tip cell number is observed following blockade of Src kinases, Notch signaling or both. Interestingly, the increased sprouting behavior observed following Src or Notch blockade directly correlates with a loss in the ability of ECs to form lumens. These defined in vitro assay models allow for a genetic and signaling dissection of EC tip cells vs. lumen forming ECs, which are both necessary for the formation of branching networks of tubes during vascular morphogenic events.