Figure 1.
FXIa induces VE-cadherin shedding and EC permeability. (A) HUVECs were grown on gelatin-coated glass coverslips and incubated with FXIa (30 nM) for 2 to 6 hours. Cells were fixed and stained for VE-cadherin (green), actin (red), and nuclei (blue). Bar, 50 μm. (B) HUVECs were incubated with FXIa for 1 to 6 hours. Cells were lysed and analyzed by western blotting using an anti–C-terminal VE-cadherin antibody. Results are representative of 3 experiments. Significance (∗P < .05) determined by Kruskal-Wallis testing with Dunns correction for multiple comparisons. Data are mean ± standard error of the mean (SEM; n = 3). (C) HUVECs were incubated with FXIa (5 or 30 nM), thrombin (10 nM), kallikrein (30 nM), or FXIIa (30 nM) for 6 hours. Cells were lysed and analyzed by western blotting using an anti–C-terminal anti–VE-cadherin antibody. Results are representative of 3 experiments. Significance (∗P < .05) determined by Kruskal-Wallis testing with Dunns correction for multiple comparisons. Data are mean ± SEM (n = 3). (D) HUVECs were incubated with FXIa (30 nM), or FXa (10 or 30 nM), or FIXa (10 or 30 nM) for 6 hours. Cells were lysed and analyzed by western blotting using an anti–C-terminal VE-cadherin antibody. Results are representative of 3 experiments. Significance (∗P < .05) determined by Kruskal-Wallis testing with Dunns correction for multiple comparisons. Data are mean ± SEM (n = 3). (E) HUVECs were grown to confluence on gelatin-coated Transwell filters and incubated with thrombin, FXa, or FXIa (5 nM), for 2 or 6 hours. Permeability of Evans Blue-BSA was measured after 60 minutes of incubation. Data are mean ± SEM (n = 3). Significance (∗P < .05) was determined by Kruskal-Wallis testing with Dunns correction for multiple comparisons. (F) HUVECs were incubated with FXIa (1, 5, or 30 nM), TNFα (10 ng/mL), or VEGF (100 ng/mL) for 6 hours. Cells were lysed and analyzed by western blotting using an anti–C-terminal VE-cadherin, anti–ICAM-1, or anti–VCAM-1 antibodies. Results are representative of 3 experiments. Data are mean ± SEM. Significance (∗P < .05) was determined by Kruskal-Wallis testing with Dunns correction for multiple comparisons. (G) HUVECs were grown to confluence on gelatin-coated Transwell filters and incubated with FXIa (30 nM), VEGF (100 ng/mL), or TNFα (10 ng/mL) for 6 hours. Permeability of Evans Blue-BSA was measured after 60 minutes of incubation. Data are mean ± SEM (n = 3). Significance (∗P < .05) was determined by Kruskal-Wallis testing with Dunns correction for multiple comparisons.

FXIa induces VE-cadherin shedding and EC permeability. (A) HUVECs were grown on gelatin-coated glass coverslips and incubated with FXIa (30 nM) for 2 to 6 hours. Cells were fixed and stained for VE-cadherin (green), actin (red), and nuclei (blue). Bar, 50 μm. (B) HUVECs were incubated with FXIa for 1 to 6 hours. Cells were lysed and analyzed by western blotting using an anti–C-terminal VE-cadherin antibody. Results are representative of 3 experiments. Significance (∗P < .05) determined by Kruskal-Wallis testing with Dunns correction for multiple comparisons. Data are mean ± standard error of the mean (SEM; n = 3). (C) HUVECs were incubated with FXIa (5 or 30 nM), thrombin (10 nM), kallikrein (30 nM), or FXIIa (30 nM) for 6 hours. Cells were lysed and analyzed by western blotting using an anti–C-terminal anti–VE-cadherin antibody. Results are representative of 3 experiments. Significance (∗P < .05) determined by Kruskal-Wallis testing with Dunns correction for multiple comparisons. Data are mean ± SEM (n = 3). (D) HUVECs were incubated with FXIa (30 nM), or FXa (10 or 30 nM), or FIXa (10 or 30 nM) for 6 hours. Cells were lysed and analyzed by western blotting using an anti–C-terminal VE-cadherin antibody. Results are representative of 3 experiments. Significance (∗P < .05) determined by Kruskal-Wallis testing with Dunns correction for multiple comparisons. Data are mean ± SEM (n = 3). (E) HUVECs were grown to confluence on gelatin-coated Transwell filters and incubated with thrombin, FXa, or FXIa (5 nM), for 2 or 6 hours. Permeability of Evans Blue-BSA was measured after 60 minutes of incubation. Data are mean ± SEM (n = 3). Significance (∗P < .05) was determined by Kruskal-Wallis testing with Dunns correction for multiple comparisons. (F) HUVECs were incubated with FXIa (1, 5, or 30 nM), TNFα (10 ng/mL), or VEGF (100 ng/mL) for 6 hours. Cells were lysed and analyzed by western blotting using an anti–C-terminal VE-cadherin, anti–ICAM-1, or anti–VCAM-1 antibodies. Results are representative of 3 experiments. Data are mean ± SEM. Significance (∗P < .05) was determined by Kruskal-Wallis testing with Dunns correction for multiple comparisons. (G) HUVECs were grown to confluence on gelatin-coated Transwell filters and incubated with FXIa (30 nM), VEGF (100 ng/mL), or TNFα (10 ng/mL) for 6 hours. Permeability of Evans Blue-BSA was measured after 60 minutes of incubation. Data are mean ± SEM (n = 3). Significance (∗P < .05) was determined by Kruskal-Wallis testing with Dunns correction for multiple comparisons.

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