Regulation of Angiogenic Functions by Angiopoietins through Calcium-Dependent Signaling Pathways

Figure 6

2-APB and 8Br-cADPR impair Angs-dependent cell migration on HUVECs. Ang-1 and Ang-2 induce Ca²⁺-dependent FAK phosphorylation. Confluent HUVECs were pretreated or not with 20 μM BAPTA-AM for 1 h and stimulated with 100 ng/mL Ang-1 or 200 ng/mL Ang-2 for 30 min. Blots of total cell lysate were probed for the phosphorylation of downstream target FAK. To ensure equal loading membranes were reprobed for the total amount of the indicated protein and for β-actin (a). Cells pretreated or not with 75 μM 2-APB or 30 μM 8Br-cADPR and stimulated with either Ang-1 or Ang-2 (b1, b2, c1, and c2). Representative blots are shown from three to five independent experiments. Ang-1-induced EC migration is affected by treatment with the second messengers inhibitor 2-APB while Ang-2-induced cell migration appears to be both IP₃- and cADPR-dependent. Scratch assay to evaluate the cell migration capability in the indicated experimental conditions. Wounded monolayers at the time of manual damage ( h upper panel) and after 24 h treatment (lower panel) with Ang-1 (d), Ang-2 (e), and inhibitors as indicated. Pictures are representative of three independent experiments.

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