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We studied the formation of capillary tubes by endothelial cells which were sandwiched between two fibrin gels under serum-free conditions. After formation of the overlying fibrin gel, the endothelial cell monolayer rearranged into an extensive net of capillary tubes. Tube formation was apparent at 5 h and was fully developed by 24 h. The capillary tubes were vacuolated, and both intracellular and intercellular lumina were present. Maximal tube formation was observed with fibrin II (which lacks both fibrinopeptide A and B), minimal tube formation with fibrin I (which lacks only fibrinopeptide A), and complete absence of tube formation with fibrin 325 (which lacks the NH2- terminal beta 15-42 sequence, in addition to fibrinopeptides A and B). The inability of fibrin 325 to stimulate capillary tube formation supports the idea that beta 15-42 plays an important role in this process, and its importance was confirmed by the finding that exogenous soluble beta 15-42 inhibited fibrin II-induced capillary tube formation. This effect was specific for fibrin, since beta 15-42 did not inhibit tube formation by endothelial cells sandwiched between collagen gels. The interaction of the apical surface of the endothelial cell with the overlying fibrin II gel, as opposed to the underlying fibrin gel upon which the cells were seeded, was necessary for capillary tube formation. These studies suggest that the beta 15-42 sequence of fibrin interacts with a component of the apical cell surface and that this interaction plays a fundamental role in the induction of endothelial capillary tube formation.

We studied the interaction of [125I]fibronectin with human umbilical vein endothelial cells. Endothelial cell monolayers cross-linked [125I]fibronectin which had been preadsorbed to gelatin-coated dishes. The cross-linking of the substrate-immobilized [125I]fibronectin was mediated by cell-associated tissue transglutaminase and occurred more rapidly during the first 30 min after endothelial cell seeding but also continued for several hours after the cells were fully spread. The processing of the [125I]fibronectin was associated with the basolateral surface of the endothelial cell, as demonstrated by the finding that cross-linking did not occur when [125I]fibronectin was presented to the apical surface of confluent monolayers. Transglutaminase activity was not necessary for attachment and spreading of HUVEC on a fibronectin/gelatin matrix. The presence of a nonpeptidyl transglutaminase inactivator rendered the cells more susceptible to detachment by trypsin and destabilized the association of fibronectin with the subendothelial extracellular matrix. Thus, endothelial cells process fibronectin into cross-linked multimers due to the expression of tissue transglutaminase at the basal surface of the cell. This process may serve to stabilize the extracellular matrix and to firmly anchor the cells to the basement membrane.

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