VEGF-A/FGF-2 priming events are functionally separable and show distinct signaling requirements for hematopoietic cytokine-induced EC tube morphogenesis in 3D collagen matrices. (A) ECs were primed with the combination of VEGF-A/FGF-2 for the indicated times to identify the timing requirements for the priming event. After priming, ECs were placed in culture with the hematopoietic factors and fixed after 72 hours (n ≥ 10; P ≤ .01). *Denotes significance over the 0-hour time point; +significance over the 4-hour time point. (B) The indicated VEGF isoforms were tested for their ability to prime ECs for 16 hours. ECs were then placed in culture with the hematopoietic factors and fixed after 72 hours (n ≥ 10; P ≤ .01). *Denotes significance over control; ⁺denotes significance over the FGF-2 alone condition. (C) Inhibitors of p38 MAP kinase (SB203580, 10μM), protein kinase A (Rp-cAMPS, 10μM), protein kinase G (cGMPS, 10μM), Rho kinase (Y27632, 10μM) and MMPs (GM6001, 5μM) were added during the priming step or separately during the tube morphogenesis step to assess whether signaling differences exist between the 2 steps. Cultures were fixed after 72 hours, and total tube area was quantitated (n ≥ 10; P ≤ .01). *Denotes significance from control. Representative images of cultures treated with the indicated inhibitors during the priming versus morphogenesis are shown. Bar equals 100 μm.