Angiostatin_4.5-mediated apoptosis of vascular endothelial cells

Angiostatin, a proteolytic cleavage product of plasminogen, acts via a selective, yet poorly understood mechanism to potently inhibit angiogenesis (M. S. O'Reilly et al., Cell, 79: 315-328, 1994). Vascular endothelial cell proliferation assays revealed that angiostatin_4.5, a naturally occurring human isoform consisting of plasminogen kringle domains 1-4 and most of kringle domain 5 (G. A. Soff, Cancer Metastasis Rev., 19: 97-107, 2000), dose dependently reduces cell number despite the presence of a potent stimulus of proliferation. Flow cytometry using the vital dyes Hoechst 33342 and Pyronin Y revealed that ∼40% of both control and antgiostatin_4.5-treated cells were in the proliferative phase, indicating that cell cycle progression is not impaired by exposure to angiostatin_4.5. Both bovine aortic endothelial cells and human umbilical endothelial cells were shown to undergo apoptosis in response to angiostatin_4.5. Caspases-3, -8, and -9 activation, specified by cleavage of fluorophore-conjugated specific peptide substrates, revealed a cascade of caspase activation that peaks at 36 h of angiostatin_4.5 treatment. Angiostatin_4.5 exposure induced release of cytochrome c from mitochondria in a caspase-dependent manner, but a pan-caspase inhibitor, zVAD-fmk, blocked cytochrome c release. Overall, these data indicate that human angiostatin_4.5 may function in vivo to block blood vessel formation by specifically inducing vascular endothelial cells to apoptose in a process likely involving both the intrinsic and extrinsic apoptosis pathways.