Lumen formation during angiogenesis in vitro involves phagocytic activity, formation and secretion of vacuoles, cell death, and capillary tube remodelling by different populations of endothelial cells

Abstract

Background: We have utilised an in vitro model of angiogenesis to investigate the morphological changes which occur during the formation of a lumen in capillary tubes. Methods and Results: On collagen 1 gel in the presence of phorbol myristate acetate (PMA) and anti-α2β1 antibody, cell aggregation and alignment takes place within two hours of plating. The initial apparently homogeneous population of endothelial cells (EC) actually display at least three distinct phenotypes. One population, characterised by a phagocytic phenotype, migrated through the gel creating channels and defines the extent of the capillary network. These are later enveloped by a second population of cells characterised by intracellular vacuoles. The ultimate fate of these vacuoles is fusion with the plasma membrane. By 12 hours the original phagocytic cell population undergoes cell death, which morphologically appears apoptotic in nature. A consequence of the secretion of vacuoles and programmed cell death is the extensive remodelling of the capillary tubes, resulting in expansion of the intercellular space into a lumen. The remodelling results in 45% of the EC membrane contacting the lumenal surface at the expense of EC-EC and EC-matrix contact. A third population of cells implant between the EC involved in lumen formation and thus expand the size of the capillary tube. Conclusion: Thus, in the formation of a mature multicellular lumen we have identified a number of key events. First, cell-cell contact is essential in order to define the intercellular space. Second, at least three morphologically distinct subpopulations of ECs are involved. Third, vacuole formation and programmed cell death are required for expansion of the intercellular space which ultimately becomes the lumen.