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.
Departamento de Biologia Celular e Genética, Instituto de Biologia Roberto Alcantara Gomes, Universidade do Estado do Rio de Janeiro (UERJ), Rua São Francisco Xavier, 524 PHLC sala 205, Maracanã, Rio de Janeiro, RJ, Brazil.
Marianna A
MA
Ferrari do Outeiro-Bernstein
Sara Santana
SS
Nunes
Ana Carolina Magalhães
AC
Andrade
Tercia Rodrigues
TR
Alves
Chantal
C
Legrand
Verônica
V
Morandi
D001665:Binding Sites; D001792:Blood Platelets / Q000378:metabolism; D002448:Cell Adhesion; D015815:Cell Adhesion Molecules / Q000494:pharmacology; D002454:Cell Differentiation; D002470:Cell Survival / Q000187:drug effects; D004727:Endothelium / Q000166:cytology* / Q000187:drug effects* / Q000378:metabolism; D005337:Fibrin / Q000378:metabolism; D006493:Heparin / Q000378:metabolism*; D006801:Humans; D008562:Membrane Glycoproteins / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011509:Proteoglycans / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D053671:Syndecan-4; D019700:Thrombospondin 1 / Q000737:chemistry* / Q000378:metabolism* / Q000494:pharmacology; D014471:Umbilical Veins / Q000166:cytology
D015815:Cell Adhesion Molecules; D008562:Membrane Glycoproteins; D011509:Proteoglycans; D011994:Recombinant Proteins; C508304:SDC4 protein, human; D053671:Syndecan-4; D019700:Thrombospondin 1; D005337:Fibrin; D006493:Heparin