Figure 3.

Disorganized cell junctions and impaired function in 3D models of angiogenesis upon CCM3 gene inactivation. (A) Scheme of endothelial tight and adherens junctions. ICS = intracellular space. ECS = extracellular space. ZO-1 = zonula occludens protein 1. JAM = junctional adhesion molecule. (B) In contrast to wild-type CI-huVECs, ${\mathit{CCM}\mathit{3}}^{-\mathrm{/}-}$ CI-huVECs demonstrated numerous small gaps (white arrowheads) and a less homogeneous pattern in VE-cadherin staining (red). Scale bar $\stackrel{\wedge }{=}$ 20 µm. They also displayed significant actin stress fiber formation (green, phalloidin staining). Scale bar $\stackrel{\wedge }{=}$ 25 µm. DAPI (blue) was used to stain cell nuclei. (C) ${\mathit{CCM}\mathit{3}}^{-\mathrm{/}-}$ CI-huVECs demonstrated impaired spheroid formation and VEGF-induced sprouting. The number and length of sprouts formed by ${\mathit{CCM}\mathit{3}}^{-\mathrm{/}-}$ spheroids upon stimulation with 25 ng/ml VEGF-A were significantly reduced. Scale bar $\stackrel{\wedge }{=}$ 100 µm. (D) CCM3 gene inactivation had cell type-specific effects on endothelial tube formation on Matrigel. While tubes formed by ${\mathit{CCM}\mathit{3}}^{-\mathrm{/}-}$ CI-huVECs were unstable and had fallen apart 17 h after seeding on Matrigel, ${\mathit{CCM}\mathit{3}}^{-\mathrm{/}-}$ hCMEC/D3 cells formed more stable meshes than hCMEC/D3 wild-type controls. Scale bar $\stackrel{\wedge }{=}$ 500 µm.