A recombinant NH(2)-terminal heparin-binding domain of the adhesive glycoprotein, thrombospondin-1, promotes endothelial tube formation and cell survival: a possible role for syndecan-4 proteoglycan.

Thrombospondin-1 (TSP-1) is a multifunctional protein known to modulate angiogenesis, endothelial cell adhesion and apoptosis. In this study, we have demonstrated that TSP18, a recombinant 18 kDa protein encompassing the N-terminal residues 1-174 of human TSP-1, accelerated the process of tube-like structures formation by human umbilical vein endothelial cells (HUVECs) when included in fibrin matrices at 0.55-2.2 microM concentrations, for times ranging from 24 to 72 h. This effect was specifically inhibited by V58A4, a Mab raised against TSP18. Whole TSP-1 showed a dual effect, weakly enhancing tube formation at 22 nM (10 microg/ml), but causing inhibition at 45 and 90 nM (20 and 40 microg/ml, respectively). In order to investigate the possible effects of TSP18 on cell adhesion and viability, we performed adhesion assays on different protein supports. HUVECs adhered more weakly on TSP-1-coated surfaces, remaining round-shaped, as compared to the well-spread phenotype displayed on fibronectin and gelatin. Cells adhering on TSP18-coated surfaces displayed a well spread phenotype, with this adhesion strongly inhibited by heparin. The binding of TSP18 to endothelial membrane extracts was blocked by a monoclonal IgG directed against the cell surface proteoglycan syndecan-4. The DNA fragmentation patterns and the nuclear morphology were comparable for HUVECs adhering on all proteins, including TSP18, showing minimal cell apoptosis. Our results indicate that the N-terminal region of TSP-1 constitutes a suitable adhesive support for HUVECs, protecting them from apoptosis, possibly mediated by syndecan-4 proteoglycan.