Fig 1.
Tie-2 is required for the morphological responses of endothelial cells triggered by shear stress.
(A–C) HUVECs transfected with control or Tie-2 siRNA were exposed to15 dyn/cm2 of shear stress for 24 hours. The resulting cells were stained for VE-cadherin (green) (A). Percentage of aligned cell to shear stress direction within 30° (B) and elongation index (C) were quantified using ImageJ/Fiji. Data presented as mean ± SEM. P values were calculated with two-way ANOVA followed by Tukey’s multiple test. Scale bar: 50 μm.
Fig 2.
Tie-2 is required for nuclear exclusion of FOXO1 induced by shear stress.
(A, B) HUVECs transfected with control or FOXO1 siRNA were exposed to 15 dyn/cm2 of shear stress for 24 hours. The resulting cells were stained for FOXO1 (red) and VE-cadherin (green)(A). Quantification of nuclear FOXO1 intensity with ImageJ/Fiji (B). Data presented as mean ± SEM. P values were calculated with two-way ANOVA followed by Tukey’s multiple test. Scale bar indicates 25 μm.
Fig 3.
FOXO1 is required for cell morphological responses induced by shear stress.
(A-D) HUVECs transfected with control or FOXO1 siRNA were exposed to 15 dyn/cm2 shear stress for 24 hours. The resulting cells were stained for FOXO1 (red) and VE-cadherin (green) (A). Percentage of aligned cell to shear stress direction within 30° (B) and elongation index (C) were quantified with ImageJ/Fiji. Data presented as mean ± SEM. P values were calculated with two-way ANOVA followed by Tukey’s multiple test. Scale bar indicates 50 μm.
Fig 4.
Shear stress-stimulated Tie-2-FOXO1 signaling regulates autophagosome formation.
(A-B) HUVECs transfected with control, Tie-2 or FOXO1 siRNA were exposed to shear stress at 15 dyn/cm2 for 24 hours. The resulting cells were stained for LC3B (red) and VE-cadherin (green) (A). Areas of LC3B puncta in each cell were quantified with ImageJ/Fiji (B) (C–D) HUVECs were treated with 600 ng/ml Angpt1 for 24 hrs followed by staining for LC3B (red) and VE-cadherin (green) (C). Areas of LC3B puncta in each cell were quantified with ImageJ/Fiji (D). (E–F) HUVECs transfected with control or Tie-2 siRNA were treated with 600 ng/ml Angpt1 for 30 minutes, followed by staining for FOXO1 (red) and VE-cadherin (green) (E). Nuclear FOXO1 levels were quantified with ImageJ/Fiji (F). Data presented as mean ± SEM. P values were calculated with two-way ANOVA followed by Tukey’s multiple test (B and F) or Mann–Whitney U test (D). Scale bars indicate 20 μm (A and C) or 50 μm (E).
Fig 5.
Autophagy is required for cell alignment under laminar shear stress.
(A) To test siRNA efficiency, lysates of control or Atg5 siRNA treated HUVEC were immunoblotted for Atg5 and α-tubulin as a loading control 48 hours later. (B) To test the effect of Atg5 silencing on LC3B activity, lysates of control or Atg5 siRNA-treated HUVEC were immunoblotted for LC3B and α-tubulin as a loading control 48 hours later. The inactive and active forms of LC3B were distinguished by their electrophoretic mobility. Quantification is shown on the right. (C-E) HUVECs transfected with control or Atg5 siRNA were exposed to 15 dyn/cm2 shear stress for 24 hours followed by staining for VE-cadherin (green) (C). Percentage of aligned cell to shear stress direction within 30° (D) and elongation index (E) were quantified with ImageJ/Fiji. Data are presented as mean ± SEM. P values were calculated with Mann–Whitney U test (B) and two-way ANOVA followed by Tukey’s multiple test (D and E). Scale bars indicate 50 μm.
Fig 6.
Proposed mechanism by which Tie-2 regulates the morphological responses of endothelial cells to shear stress via FOXO1.
Laminar shear stress stimulates the activation of Tie-2, which leads to the translocation of FOXO1 from the nucleus to the cytosol where it triggers autophagosome formation which supports cell alignment.