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.
Ferrari do Outeiro-Bernstein
Marianna A
MA
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.
Nunes
Sara Santana
SS
Andrade
Ana Carolina Magalhães
AC
Alves
Tercia Rodrigues
TR
Legrand
Chantal
C
Morandi
Verônica
V
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