Elucidating the morphogenic and signaling roles of defined growth factors controlling human endothelial cell lumen formation versus sprouting behavior.

We have previously shown that five growth factors (Factors) [i.e. insulin, fibroblast growth factor-2 (FGF-2), stem cell factor (SCF), interleukin-3 (IL-3), and stromal-derived factor-1 alpha (SDF-1α)] in combination are necessary for human ECs to undergo tube morphogenesis, a process requiring both lumen formation and sprouting behavior. Here, we investigate why they are required by subdividing the Factors into 4 separate groups: Insulin only, Insulin + FGF-2, no FGF-2 (all Factors but without FGF-2), and all Factors. We demonstrate that the Insulin only condition fails to support EC morphogenesis or survival, the Insulin + FGF-2 condition supports primarily EC lumen formation, and the no FGF-2 condition supports EC sprouting behavior. By comparison, the all Factors condition more strongly stimulates both EC lumen formation and sprouting behavior and signaling analysis revealed prolonged stimulation of multiple pro-morphogenic signals coupled with inhibition of pro-regressive signals. Pharmacological inhibition of Jak kinases more selectively blocks EC sprouting behavior while inhibition of Raf, PI3-kinase, and Akt kinases shows selective blockade of lumen formation. Inhibition of Src family kinases and Notch leads to increased sprouting coupled to decreased lumen formation, while inhibition of Pak, Mek and mTor kinases block both sprouting and lumen formation. These findings reveal novel downstream biological and signaling activities of defined Factors that are required for the assembly of human EC-lined capillary tube networks.