Thrombospondin promotes endothelial cell apoptosis

Thrombospondin (TSP) levels are elevated in sickle cell anemia and cancer patients and high levels can be accompanied by acute vaso-occlusive episodes. Earlier work in our laboratory has shown that TSP inhibits endothelial cell specific, vascular endothelial growth factor (VEGF) induced microvascular endothelial proliferation, tube formation, and angiogenesis. VEGF acts as a pro-angiogenic as well as anti-apoptotic factor via the MAPK/ERK signaling pathway in EC. We examined if TSP acts as an anti-angiogenic factor by inhibiting the anti-apoptotic effect of VEGF in human dermal microvascular endothelial cells (HDMEC). Cells from two different key steps of angiogenesis: 1) proliferation and 2) tube formation, were assayed for apoptosis. After 48 hours of culture, TSP (0.22 nM) induced 30% more apoptosis in VEGF (2.2 nM) induced proliferating HDMEC as observed by propidium iodide staining, followed by FACS analysis (p<0.05). Cell cycle analysis showed a significantly higher percentage of cells in GO/G1 phase in VEGF induced cultures compared to those with both VEGF and TSP (p<0.01). Similarly, TSP (0.22 nM) also inhibited VEGF (2.2 nM) induced endothelial tube formation on Matrigel. Cultures replete with both TSP and VEGF were accompanied by 10 % more dead cells and 28% more apoptotic cells as compared to those with only VEGF (p<0.02 and 0.002, respectively). Therefore, the anti-angiogenic activity of TSP may in part be due to its preventing the anti-apoptotic activity of VEGF. We observed that these responses of TSP are dependent on specific intracellular signaling pathways. TSP activated the release of mitochondrial cytochrome c in VEGF induced cultures and activated caspase-3, followed by cleavage of poly (ADP-ribose) polymerase (PARP). TSP induced apoptosis in VEGF cultures could be prevented completely by caspase inhibitor z-VAD-FMK. TSP did not have any effect on VEGF induced MAPK phosphorylation, thus suggesting that caspase activation either directly or indirectly via cytochrome c release leads to TSP induced apoptosis. We speculate that blood vessel wall damage in sickle disease could be a result of high blood plasma levels of TSP during vaso-occlusive crises.