Fig. 6

Endothelial Tie2 activation is essential for TFMG-induced vascular normalization. a EC co-cultures, with or without siRNA-mediated silencing of endothelial Tie2 or Tie2 neutralizing antibody-mediated blockage of Tie2, were treated with 100 nM TFMG for 24 h, and the permeability (%) of FITC-dextran was measured relative to untreated control co-cultures. *P < 0.0001 vs. untreated control EC co-culture (E); ^#P < 0.01 vs. TFMG-treated EC co-culture; ^$P < 0.001 vs. TFMG-treated EC co-culture with siNC silencing (n = 3). b EA.hy926 cells were treated with 10, 100, and 1000 nM of TFMG for 24 h, and immunofluorescent images were recorded to illustrate the level of ZO-1. (right panel) Quantitation of the relative ZO-1 level was performed. *P < 0.001 vs. Ctrl; ^$P < 0.0001 vs. Ctrl (n = 3). c Western blot analyses to demonstrate the activation of Tie2 and Akt, along with the levels of ROCK-1 (Rho kinase), FoxO3a, and zonula occludens-1 (ZO-1) in endothelial cells following TFMG treatment. EA.hy926 cells were treated with 100 nM TFMG for 15, 60, and 120 min, and Western blot analysis was done to visualize the levels of phosphorylated Tie2 (p-Tie2), Tie2, p-Akt, Akt, ROCK-1, p-FoxO3a, FoxO3a, FoxO1, and ZO-1. d, e A frozen section of tumor tissues of each group was costained with anti-CD31/anti- PAR-3/anti-pTie2 antibodies (d) or anti-CD31/anti-PAR-1/anti-PAR-3 antibodies (e) and appropriate secondary fluorescent antibodies by IHC. Immunofluorescence was observed under the fluorescent microscope (× 400). Scale bar: 50 μm. f Ea.hy926 cells were transfected with negative control (NC), Gα13, Gαi, or Gαq siRNA (100 nM). Cells were harvested at 60 min after TFMG (100 nM) treatment and analyzed for activation of Tie2 signaling and Akt expression by western blot analysis. Quantitation in (b–d) were conducted using ImageJ software. g Proposed underlying mechanism of TFG and TFMG-induced vascular normalization in cancer. EPCR binding and PAR-1 activation by TFG/TFMG resulted in PAR-1/PAR-3 heterodimerization, which induced Gα13-mediated endothelial Tie2 activation, resulting finally in the induction of tight-junction proteins and vascular normalization. Data information: Data are presented as the mean ± SD. Significant enrichment: *P < 0.05; **P < 0.01; ***P < 0.001 (c, d) (Sidak’s multiple comparisons test)