PLASMINOGEN ACTIVATORS AUGMENT ENDOTHELIAL-CELL ORGANIZATION IN-VITROBY 2 DISTINCT PATHWAYS

Endothelial cell differentiation into capillary structures is a comple x process that requires the concerted effects of several extracellular matrix proteases, including plasminogen activators. Here, the role of tissue-type plasminogen activator (tPA) and urokinase-type plasminoge n activator (uPA) was evaluated in an in vitro model of endothelial mo rphogenesis involving organization of human umbilical vein endothelial cells into tubular structures when they are cultured on the basement membrane preparation, Matrigel. Both uPA and tPA were detected in HUVE C cultures on Matrigel, and inhibitors of plasminogen activators or of serine proteases decreased the extent of the tube network formed by t he cells. The decrease resulting from serine protease inhibitors was a dditive to that from matrix metalloproteinase inhibitors which have pr eviously been shown to decrease tube formation in this model, suggesti ng that the two classes of proteases modulate tube formation by distin ct mechanisms. Plasminogen activator inhibitor (PAI)-1 decreased tube formation by 50% when added up to 4.5 h after the initiation of an 18 h assay and caused 25% inhibition when added 9.5 h after culture initi ation, indicating that the effects of plasminogen activators are not l imited to an early event in the differentiation process. Steady-state expression of mRNA for uPA increased during the first several hours of culture on Matrigel, further supporting a role for PA activity throug hout the process of tube formation. These findings suggested that PAs may affect multiple events during tube-forming activity. A fucosylated peptide comprising the amino-terminal domain of uPA that binds to the uPA receptor (uPAR) but lacking proteolytic activity enhanced tube fo rmation. In contrast, a defucosylated form of the same peptide had no effect. Since fucosylation of this fragment has been shown to be essen tial in other models of cell stimulation by uPA-uPAR interaction, thes e data support the hypothesis that uPA enhances endothelial morphogene sis both through proteolytic activity and via uPAR occupancy. Plasmino gen activators could facilitate angiogenesis in vivo. (C) 1995 Wiley-L iss, Inc.