Fig. 7: Humanized TAAB activates Tie2 and its downstream signaling in HUVECs.

a, b Immunoblot analysis for relative phosphorylation ratios of Tie2 and Akt after treatment of HUVECs with different concentrations (1 and 10 μg/ml) of humanized TAABs (hzTAAB-H1L1, H1L2, H2L1, H2L2) (a). Densitometric analyses (b) of p-Tie2/Tie2 and p-Akt/Akt ratios are shown. Data from three independent experiments (n = 3) were analyzed and expressed as mean ± SD. P values by two-tailed unpaired t test. ns, not significant. c, d Biological effects of hzTAAB-H2L2 in HUVECs. Effect of hzTAAB-H2L2 on serum deprivation-induced apoptosis in HUVECs (Top). HUVECs were incubated with serum-free medium containing 10 μg/ml of hzTAAB-H2L2, hTAAB, or 1 μg/ml of COMP-Angpt1. Migration activity was measured using a modified Boyden chamber assay (Middle). HUVECs were seeded in the upper layer of 8-μm pore membranes. Serum-free medium containing 10 μg/ml of hzTAAB-H2L2, hTAAB, or 1 μg/ml of COMP-Angpt1, was added to the bottom chamber. Migrated cells were fixed and stained after 9-h incubation. HUVECs were plated on Matrigel-coated wells and incubated with serum-free medium containing 10 μg/ml of hzTAAB-H2L2, hTAAB, or 1 μg/ml of COMP-Angpt1. Phase-contrast microscopy images were acquired 7 h after treatment. Tube-formation activity was measured as total tube length (Bottom). Scale bars, 100 μm. Quantification (d) of TUNEL-positive cell, migrated HUVEC, and sprouts for (c). Data from three independent experiments (n = 3) were analyzed and expressed as mean ± SEM (***P < 0.001 vs. control). P values by one-way ANOVA test followed by Sidak’s multiple comparisons test. e Representative images of HUVECs showing hzTAAB-H2L2-induced Tie2 translocation to cell–cell contacts and hzTAAB-H2L2-induced nuclear clearance of FOXO1. Serum-starved HUVECs were treated with hTAAB-H2L2 (10 μg/ml) or COMP-Angpt1 (1 μg/ml) for 30 min. No treatment was used as a negative control. Goat anti-human Tie2 and Alexa Fluor 488-conjugated donkey anti-goat antibodies were used for Tie2 visualization, and rabbit anti-FOXO1 and Alexa Fluor 594-conjugated donkey anti-rabbit antibodies were used for FOXO1 visualization. Scale bars, 20 μm. DAPI, 4’, 6-diamidino-2-phenylindole. Similar results were observed in three independent experiments. f Representative images of HUVEC spheroids 3D culture after stimulation with COMP-Angpt1 (1 μg/ml), hTAAB (10 μg/ml), or hzTAAB (10 μg/ml) with or without VEGF-A for 48 h (left). Scale bars, 100 μm. Comparison of the number and width of sprouts is shown in the right panel and each dot indicates a value from n = 4–6 spheroids/group (mean ± SD). P values by two-tailed Mann–Whitney U test. g Immunoblot analysis of the relative phosphorylation ratios of Tie2 and Akt after treatment of HUVECs with ABTAA or hzTAAB-H2L2 (10 μg/ml) with or without Angpt2 (1 μg/ml). Densitometric analyses of p-Tie2/Tie2 and p-Akt/Akt ratios are shown in the right panel. Data from three independent experiments (n = 3) were analyzed and expressed as mean ± SD (***P < 0.001 vs. control; ^##P = 0.0051 ABTAA vs. Angpt2+ABTAA; ^###P < 0.001 ABTAA vs. Angpt2+ABTAA). P values by one-way ANOVA test followed by Sidak’s multiple comparisons test. ns, not significant.