Angiogenic sprouting and capillary lumen formation modeled by human umbilical vein endothelial cells (HUVEC) in fibrin gels: the role of fibroblasts and Angiopoietin-1.

Angiogenesis is a multistep process of critical importance both in development and in physiological and pathophysiological processes in the adult. It involves endothelial cell (EC) sprouting from the parent vessel, followed by migration, proliferation, alignment, tube formation, and anastomosis to other vessels. Several in vitro models have attempted to recreate this complex sequence of events with varying degrees of success. We report an optimized protocol for human umbilical vein EC in which EC sprout from the surface of beads embedded in fibrin gels. Fibroblast-derived factors, other than Angiopoietin-1, promote sprouting, lumen formation, and long-term stability of neovessels. Analysis by time-lapse and still photomicroscopy demonstrates dynamic vessels guided by a "tip cell" that extends numerous processes into the gel. Behind this cell a lumen forms, surrounded by a single layer of polarized EC. The growing sprouts express notch 1, notch 4, and delta 4, as well as the downstream notch effector HESR-1. Importantly, cells can be infected with adenovirus to high efficiency without compromising sprout formation, thus allowing for manipulation of gene expression. This improved model recapitulates all the major steps of angiogenesis seen in vivo and provides a powerful model for analysis of this complex phenomenon.