Binding and displacement of vascular endothelial growth factor (VEGF) by thrombospondin: Effect on human microvascular endothelial cell proliferation and angiogenesis

Vascular endothelial growth factor (VEGF) is a specific angiogenic factor, and thrombospondin (TSP), is a potent inhibitor of angiogenesis. To better understand the role of TSP as an anti-angiogenic agent, we have identified its specific domains that participate in its anti-angiogenic activity and examined the mechanism of its inhibitory effect on VEGF₁₆₅ induced angiogenesis. Exogenously added TSP inhibited VEGF₁₆₅ induced angiogenesis (proliferation and tube formation of human dermal microvascular endothelial cells {HDMEC} and neovascular outgrowth from human arterial rings). Although both VEGF₁₆₅ and TSP are heparin binding proteins, TSP had a higher affinity for ¹²⁵I-heparin than VEGF₁₆₅ (K_d1 4 nM and K_d2 14 nM for TSP; K_d 91 nM for VEGF₁₆₅). TSP displaced 36% of ¹²⁵I-VEGF₁₆₅ from HDMEC and this was comparable to the 27% reduction in ¹²⁵I-VEGF₁₆₅ binding to HDMEC upon cleavage of cell surface heparan sulfate (HS). About 35% of the mitogenic activity of VEGF₁₆₅ was attributable to its heparin binding region. These results indicate that a proportion of the mitogenic activity of VEGF₁₆₅ is inhibited by TSP via competition for cell surface HS. Further, ¹²⁵I-VEGF₁₆₅ bound directly to TSP in a saturable, concentration dependent manner, and heparin modulated this binding. The mAbs to the heparin binding domain to the type 1 and type 3 repeats of TSP inhibited the binding of VEGF₁₆₅ to TSP, and also blocked the inhibitory effect of TSP on VEGF₁₆₅ induced HDMEC proliferation. We conclude that (i) the anti-angiogenic activity of TSP is localized in its heparin binding domain and type 1 and type 3 repeats (ii) TSP inhibits angiogenesis by at least two separate mechanisms, (a) displacement of VEGF₁₆₅ from endothelial cell HS and (b) direct binding to VEGF₁₆₅.