Formation of capillary-like tubes by vascular endothelial cells cocultivated with keratocytes

This report describes an in vitro system that could be useful for investigating corneal neovascularization. When isolated bovine capillary endothelial cells (CEC) were cocultivated with rabbit corneal keratocytes, capillary-like cords extended actively from the endothelial cells in the multilayers of the keratocytes. The developing cords continued to elongate, branch out, and anastomose with each other, forming a capillary-like network by the 12th day of coculturing. Electron microscopic observation revealed that the cords were tubular structures composed of three to seven endothelial cells in the multilayers of keratocytes, and that a basal lamina-like matrix was situated at the abluminal face of the endothelial cells. The growth of cellular cords from the cloned CEC also occurred when CEC were seeded onto a cell-free extracellular matrix that had been synthesized by keratocytes. Keratocyte-conditioned medium alone did not stimulate proliferation of CEC. These observations clearly indicate that the formation of capillary-like structures by CEC depended upon the presence of an extracellular matrix produced by keratocytes, rather than upon the keratocytes themselves or any other keratocyte byproduct. This simple in vitro experimental system is proposed as a useful tool for studying corneal neovascularization.