Fibroblast growth factor-2 binding to the thrombospondin-1 type III repeats, a novel antiangiogenic domain

Abstract

Thrombospondin-1, an antiangiogenic matricellular protein, binds with high affinity to the angiogenic fibroblast growth factor-2, affecting its bioavailability and activity. The present work aimed at further locating the fibroblast growth factor-2 binding site of thrombospondin-1 and investigating its activity, using recombinant thrombospondin-1 proteins. Only recombinant constructs containing the thrombospondin-1 type III repeats bound fibroblast growth factor-2, whereas other domains, including the known anti-angiogenic type I repeats, were inactive. Binding was specific and inhibited by the anti thrombospondin-1 monoclonal antibody B5.2. Surface plasmon resonance analysis on BIAcore revealed a binding affinity (K(d)) of 310nM for the type III repeats and 11nM for intact thrombospondin-1. Since the type III repeats bind calcium, the effect of calcium on thrombospondin-1 binding to fibroblast growth factor-2 was investigated. Binding was modulated by calcium, as thrombospondin-1 or the type III repeats bound to fibroblast growth factor-2 only in calcium concentrations <0.3mM. The type III repeats inhibited binding of fibroblast growth factor-2 to endothelial cells, fibroblast growth factor-2-induced endothelial cell proliferation in vitro and angiogenesis in the chorioallantoic membrane assay in vivo, thus indicating the antiangiogenic activity of the domain. In conclusion, this study demonstrates that the fibroblast growth factor-2 binding site of thrombospondin-1 is located in the type III repeats. The finding that this domain is active in inhibiting angiogenesis indicates that the type III repeats represent a novel antiangiogenic domain of thrombospondin-1.

Antiangiogenic activity of trabectedin in myxoid liposarcoma: Involvement of host TIMP‐1 and TIMP‐2 and tumor thrombospondin‐1

“…It acts through multiple mechanisms, including direct interaction with endothelial cell receptors and sequestration of angiogenic factors and proteases. [26][27][28][29][30] TSP-1 is a critical determinant of angiogenesis, dormancy and growth in different types of sarcomas. [18][19][20]31 In agreement with this suppressive activity, TSP-1 expression is downregulated in highly vascularized sarcomas, including myxoid liposarcoma, [17][18][19] and its induced expression impairs the in vivo growth of Ewing's sarcoma models.…”

Thrombospondin-based antiangiogenic therapy

“…46 Another region of TSP-1, designated the type 3 repeats, inhibits angiogenesis by directly binding and sequestering fibroblast growth factor-2. 47 TSP-1, through its C-terminal domain, binds CD47; this leads to various events that alter angiogenesis, which include the disruption of constitutive association between CD47 and VEGFR2 48 as well as the inhibition of VEGF-dependent NO signaling. 49 There are 3 TSRs in TSP-1 and TSP-2.…”

Targeting tumor angiogenesis with TSP-1-based compounds: rational design of antiangiogenic mimetics of endogenous inhibitors

“…Thrombin-generated, proteolytic fragments of TSP-1 were first used, and showed that the FGF-2-binding ability was retained by the 140 kDa carboxy-terminal domain [29,30]. Using recombinant portions of TSP-1, we identified a previously undescribed antiangiogenic site in the type III repeats of TSP-1, and demonstrated that binding of the angiogenic factor FGF-2 to this site inhibits angiogenesis by sequestration of FGF-2 [31]. Finally, the peptide array technology allowed us to identify three potentially active linear sequences involved in the interaction of FGF-2 with the type III repeats of TSP-1 [68].…”

“…TSP-1 binds FGF-2, with high affinity (in the nanomolar range), similar to the affinity of the growth factor for heparin [29,31]. Consequently to this interaction, TSP-1 prevents FGF-2 binding to HSPG in the extracellular matrix and on the surface of endothelial cells [30], inhibiting FGF-2-mediated pro-angiogenic activation of endothelial cells, and depleting the extracellular matrix of stored FGF-2, an important component in the process of neovascularization [29,30].…”

“…The interaction of TSP-1 with the angiogenic factors FGF-2, VEGF, HGF, PDGF and the viral protein Tat [28][29][30][31][32], might be viewed as a general mechanism of angiogenesis regulation and thus a paradigm for designing therapeutic intervention.…”