Angiostatin inhibits endothelial MMP-2 and MMP-14 expression: a hypoxia specific mechanism of action

Abstract

Angiostatin is an angiogenesis inhibitor in part generated by and released from platelets. Since platelets upon thrombus formation can give rise to areas of hypoxia, we investigated the effects of angiostatin on endothelial cell migration and apoptosis during hypoxia. Human microvascular endothelial cells (HMVEC-L) were exposed to angiostatin under normoxic or hypoxic conditions. Apoptosis was measured by flow-cytometry. HMVEC-L migration was studied using a modified Boyden Chamber assay, in which migration is MMP-dependent. MMP-2, MMP-14, and VEGF levels were measured using immunoblot, Q-PCR and ELISA. During hypoxia HMVEC-L were protected from angiostatin-induced apoptosis due to increased hypoxia-induced VEGF expression. However, MMP-dependent migration of HMVEC-L was inhibited by angiostatin under hypoxic but not normoxic conditions. Angiostatin decreased MMP-2 at the gene and protein levels only in HMVEC-L exposed to hypoxia. A similar result was obtained for MMP-14. Higher angiostatin concentrations, as would be seen during thrombosis, induced HMVEC-L apoptosis, which was not rescued by VEGF. Under hypoxic conditions angiostatin's primary anti-angiogenic mechanism is likely inhibition of endothelial cell MMP-dependent endothelial cell migration. Only at higher concentrations does angiostatin induce endothelial cell death. This study identifies a novel angiostatin anti-angiogenesis mechanism that is only triggered under pathological-like conditions.