Influence of angiogenesis inhibitors on endothelial cell morphology in vitro

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Abstract
Human umbilical vein endothelial cells (HUVEC) propagated in co‐culture with fibroblasts form capillary‐like networks of tubes. Here we characterize the morphology and ultrastructure of HUVEC in such co‐cultures and investigate the influence of different angiogenesis inhibitors on endothelial cell morphology. Addition of angiogenesis inhibitors to the co‐culture disrupted endothelial network formation and influenced endothelial cell morphology in two distinct ways. Instead of characteristic capillary‐like networks, the endothelial cell morphology appeared as either short cords or compact cell clusters of variable size. Electron microscopy (EM) showed that in co‐culture untreated HUVEC formed capillary‐like tubes with lumina and retained important ultrastructural and physiological properties of endothelial cells in functional vessels as they contained both Weibel‐Palade bodies and transport vesicles. Immuno‐EM showed that the endothelial cell marker CD 31 stained endothelial membranes at cell‐cell contacts, and at the luminal and abluminal side of the capillary‐like tubes, although most abundantly at the luminal membranes. No ultrastructural signs of apoptosis were seen in HUVEC in inhibitor‐treated co‐cultures. Our results demonstrate that treatment with levamisole or anti‐VEGF inhibits endothelial cell differentiation into tubes or instead induces formation of compact endothelial cell clusters. Treatment with platelet factor 4, suramin and TNP‐470 results in formation of short endothelial cell cords. We discuss the implications of these findings.