Thrombospondin Inhibits VEGF-Induced Endothelial Survival and Cell Cycle Progression

Abstract

Thrombospondin (TSP) is an anti-angiogenic protein that inhibits vascular endothelial growth factor (VEGF)- induced endothelial cell growth and survival. We examined the intracellular mechanism(s) of the inhibition of VEGF- induced endothelial survival by TSP. We show that antibodies against the type I-, type II-, type-III repeats, carboxy terminal domain and N-terminal region of TSP blocked TSP-induced endothelial apoptosis. TSP promotes apoptosis by stimulating the release of cytochrome c from mitochondria and activating caspase-3 activity and cleavage of poly-ADP- ribose-polymerase (PARP). In addition, TSP inhibits VEGF-induced cell cycle progression in the G0/G1, S and G2/M phases of the cell cycle. The inhibitory effect of TSP on the cell cycle is accompanied by inhibition of cyclins A- and E- dependent kinase activity and prevention of the translocation of cell cycle regulatory proteins cyclins A and E to the nucleus. Furthermore, TSP upregulates the cell cycle regulatory phosphatases p21 CIP/WAF-1 and p27 KIP-1 . These results suggest that TSP stimulates apoptosis and cell cycle arrest by stimulating cytochrome c release and activation of caspase-3 activity; and inhibition of cell cycle regulatory checkpoints involving cyclin A and E dependent kinases that are in turn controlled by upregulation of p21 CIP/WAF-1 and p27 KIP-1 . Our data suggest the possibility that different domains of TSP are associated with the anti-angiogenic activity of TSP utilizing both endothelial apoptosis and cell cycle arrest.