Thrombospondin Inhibits VEGF-Induced Endothelial Survival and Cell Cycle Progression

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 VEGFinduced 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-ADPribose-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 Aand Edependent 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.