Cohesive and anisotropic vascular endothelial cell motility driving angiogenic morphogenesis

Abstract

Vascular endothelial cells (ECs) in angiogenesis exhibit inhomogeneous collective migration called "cell mixing", in which cells change their relative positions by overtaking each other. However, how such complex EC dynamics lead to the formation of highly ordered branching structures remains largely unknown. To uncover hidden laws of integration driving angiogenic morphogenesis, we analyzed EC behaviors in an in vitro angiogenic sprouting assay using mouse aortic explants in combination with mathematical modeling. Time-lapse imaging of sprouts extended from EC sheets around tissue explants showed directional cohesive EC movements with frequent U-turns, which often coupled with tip cell overtaking. Imaging of isolated branches deprived of basal cell sheets revealed a requirement of a constant supply of immigrating cells for ECs to branch forward. Anisotropic attractive forces between neighboring cells passing each other were likely to underlie these EC motility patterns, as evidenced by an experimentally validated mathematical model. These results suggest that cohesive movements with anisotropic cell-to-cell interactions characterize the EC motility, which may drive branch elongation depending on a constant cell supply. The present findings provide novel insights into a cell motility-based understanding of angiogenic morphogenesis.

Figure 1

(a) Fluorescence (green) and phase-contrast fusion image of branch structures that sprout from a mouse aortic explant at day 7. (b) Fluorescence image and tracking of individual EC nuclei in an elongating branch using the tracking system. Dotted line shows base line (x = 0). Position and trajectory of each EC are depicted by red point and colored line, respectively.

Figure 2

Time evolution of individual EC positions along each elongating branch (x-axis) #1 (a) and #2 (b). Each line with different color represents the trajectory of individual EC. Replotted data with regard to U-turn cells are shown in the insets. (c) Time-lapse images of ECs including a U-turn cell in elongating branch observed for 36 hours. Each image is a superimposition of phase-contrast and fluorescence microscopy (green) ones. Red and yellow arrows indicate tip and following cells, respectively.

Figure 3

Time-lapse images of cell nuclei in branches #1 (a) and #2 (b) obtained by fluorescence microscopy. An arrow on a cell nucleus shows a velocity vector. Time evolution of tip cell position in branches #1 (c) and #2 (d). Time evolution of number of cells in branches #1 (e) and #2 (f).

Figure 4

Aortic sheet removal assay. (a) Phase-contrast image of aortic sheet from which branches are radially sprouting. (b) Fluorescence (green) and phase-contrast fusion image of branch structures three days after removal of aortic sheet removal, as shown in Fig. 4(a). Note that new branches are sprouting within the cutting line toward opposite direction from the former sprouting before removal of aortic sheet. (c) Fluorescence and phase-contrast fusion image of isolated branch at 12 hours. Dotted line shows original boundary between an aortic sheet and new branches. (d) Tracking of individual EC nuclei on fluorescence image of isolated branch at 12 hours. (e) Time evolution of cell positions in the isolated branch for 12 hours. Each line with different color represents trajectory of individual EC.

Figure 5

(a) Estimated force F(x) of branch #2 with R_d = 50, N = 25. (b)$-{\int }_0^{x}F(\mu )d\mu$, potential of (a).

Figure 6

Estimated forces (a) F₁(x), (b) F₂(x), and (c) F₃(x). These functions were obtained in accordance with experimental data of branch #2. (R_d = 50, N = 25). Potentials of each forces, (d) $-{\int }_0^x{F}_1(\mu )d\mu$, (e) $-{\int }_0^x{F}_2(\mu )d\mu$, (f) $-{\int }_0^x{F}_3\left(\mu \right)d\mu$, respectively.