Understanding endothelial cell behaviors during sprouting angiogenesis

Abstract

Here in the first part of this Thesis, I investigated endothelial cell division in differing vascular architecture during sprouting angiogenesis in zebrafish. The vasculature of the zebrafish trunk is composed of tubes with different cellular architectures. Unicellular tubes form their lumen through membrane invagination and transcellular cell hollowing, whereas multicellular vessels become lumenized through a cord hollowing process. Endothelial cell proliferation is essential for the subsequent growth and maturation of the blood vessels. However, how cell division, lumen formation and cell rearrangement are coordinated during angiogenic sprouting has so far not been investigated at a detailed cellular level. We have analyzed and described the sequential steps of cell division (mitotic rounding, cytokinesis, actin re-distribution and adherence junction formation) in branches of differing cellular architectures during sprouting angiogenesis. In particular, we characterized mitosis and lumen dynamics within unicellular and multicellular tubes. Unicellular tubes constrict the lumen prior to mitosis and ultimately displace it from the division plane during mitosis, at which site a de-novo junction forms by the recruitment of junctional proteins at the division plane right after abscission. In contrast, the lumen of multicellular tubes remains intact throughout the cell division process and new junctions form from pre-existing junctions. Our findings illustrate that during the course of normal development, multiple tube architectures can accommodate the cell division machinery, thereby avoiding disruptions of the vascular network. In the second part of this Thesis, I investigated the lumen invagination including the aspects of endosomal trafficking, as well as the distribution of cytoskeleton and subcellular organelles (e.g. golgi) during endothelial cell behavior changes. During development, vascular networks form via vasculogenesis at early stages, followed by angiogenesis at later stages, a process in which new vessels grow from pre-existing vessels through coordinated cell division, migration, and cell rearrangements and eventually each sprout connects one with another to form vascular loops. The functionality of connected vascular networks depends on opening of luminal spaces allowing fluid flow. However, how vascular tubes establish continuous lumens within branches of endothelial cells in vivo to meet local metabolic needs remain obscure. In this study, we used a transgenic zebrafish line expressing the membrane marker CAAX-mCherry, to image apical membrane compartment with high spatial and temporal resolution. Our approach allows visualizing both the endothelial cell membrane and the apical lumen within the endothelial cells. Here we show that some vesicle-like structures, labeled with CAAX-mCherry, form and move in the cytoplasm and eventually dissolve on/fuse with the growing apical membrane. Based on these observations, our hypothesis is that the vesicular/membrane trafficking contributes to apical luminal membrane invagination. To investigate this phenomenon in more detail, we currently characterized localization and dynamics of Rab5c-early, Rab7-late, and Rab11a-recycling endosomal pathways during lumen invagination in sprouting angiogenesis. In addition, we also visualized the dynamics of the cytoskeleton (microtubules and actin) and the Golgi apparatus, which are linked to vesicle trafficking.