Angiopoietin-like 4-Induced 3D Capillary Morphogenesis Correlates to Stabilization of Endothelial Adherens Junctions and Restriction of VEGF-Induced Sprouting

Figure 1. Regulation of 3D capillary morphogenesis by ANGPTL4. (A) 3D capillary formation was assessed after 4 days of culture in absence (control) or presence of ANGPTL4 (2.5 µg/mL) and/or VEGF (2.5 ng/mL). Capillaries sprouting from fluorescent bead (red) were stained for F-actin (green) and nuclei (blue). Scale bar: 100 µm. (B) Median values of capillary number/bead (+interquartile). (C) Median values of branch-point number/bead (+interquartile). (D) Median values of total capillary length/bead (+interquartile). (E) Capillaries stained for F-actin (green) and nuclei (blue). Scale bar: 100 µm. (F) Mean values of capillary diameter/bead (+SEM). Values were measured in three independent experiments of at least triplicate wells. ns: p > 0.05, ** p ≤ 0.01, *** p ≤ 0.001 ((B–D): Kruskal–Wallis, (F): one-way ANOVA).

Figure 2. Regulation of VE-cadherin pattern in sprouting capillaries by ANGPTL4. (A) Z-stack projection of sprouting capillaries after 4 days of culture with ANGPTL4 (2.5 µg/mL) and/or VEGF (2.5 ng/mL). Capillaries were stained for VE-cadherin (green) and nuclei (blue). Scale bar: 20 µm. White boxed areas are presented in (B). Scale bar: 10 µm. (C) 3D reconstruction of capillaries presented in red boxed images. These images are representative of three independent experiments.

Figure 3. Regulation of migratory-related endothelial behavior by ANGPTL4 and VEGF in the early step of angiogenesis. (A) Endothelial cells seeded on beads and incubated without or with ANGPTL4 (2.5 µg/mL) and/or VEGF (50 ng/mL) for 5 h. Cells were fixed and labelled for VE-cadherin (green), F-actin (red), and nuclei (blue). The number of visible nuclei per bead is indicated below the panels. Scale bar: 50 µm. (B) Median values of cell perimeter (+interquartile). (C) Median values of cell circularity index (+interquartile). Values were measured on 62 to 83 cells per conditions in at least three independent experiments. ns: p > 0.05, ** p ≤ 0.01, *** p ≤ 0.001 (Kruskal–Wallis).

Figure 4. Regulation of VE-cadherin and endothelial spheroid integrity by ANGPTL4 and VEGF. (A) Endothelial spheroids treated without or with ANGPTL4 (2.5 µg/mL) and/or VEGF (50 ng/mL) for 24 h. Spheroids were fixed and labelled for VE-cadherin (green) and nuclei (blue). White boxed areas are presented in top right insert. Scale bar: 50 µm. (B) Mean values of spheroid circularity index (+SEM). Values were measured in three independent experiments. ns: p > 0.05, *** p ≤ 0.001 (one-way ANOVA).

Figure 5. Regulation of 3D endothelial cell migration by ANGPTL4 and VEGF. (A) Cell responses were assessed by incubating endothelial cells seeded on beads with endothelial growth medium without or with ANGPTL4 (2.5 µg/mL) and/or VEGF (2.5 ng/mL) for 48 h. Cells were fixed and labelled for F-actin (green) and nuclei (blue). Scale bar: 100 µm. (B) Median values of normalized maximal distance covered by cell (nucleus) from the bead for each condition (+interquartile). (C) Median values of normalized number of nuclei emerging from a bead (+interquartile). Nuclei closely surrounding the bead are not considered. Individual values are normalized to the mean value of VEGF condition for each experiment. Values were measured in three independent experiments. ns: p > 0.05, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 (Kruskal-Wallis).

Figure 6. Short- and long-term regulation of VEGFR2 signaling. Endothelial cells were stimulated without or with ANGPTL4 (2.5 or 5 µg/mL) and/or VEGF (50 ng/mL). Phosphorylation of VEGFR2 at tyrosine sites 951 and 1175 was analyzed after 2 min (A–C) or 5 h (D,E) stimulation. Phosphorylation of ERK was analyzed after 2 min (F,G) or 5 h (H,I) treatment by ANGPTL4 (2.5 µg/mL) and/or VEGF (50 ng/mL). Immunoblots are representative of several independent experiments (3 for (B,E,G,I); 5 for (C)). Blot image cuts are indicated by dotted white lines. Actin immunoblot was used as loading control. Graphs represent mean values (+SEM) of the ratio of phosphorylated protein over total protein. Individual values are normalized to the mean value of VEGF condition for each experiment. ns: p > 0.05, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 (one-way ANOVA).