Multiple forms of angiostatin induce apoptosis in endothelial cells

Angiostatin is a circulating inhibitor of angiogenesis generated by proteol ytic cleavage of plasminogen. In this study we have used recombinant human and murine angiostatins (kringles 1-4) as well as native human angiostatin (prepared by elastase digestion of plasminogen [kringles 1-3] or by plasmin autocatalysis in the presence of a free sulfhydryl donor [kringles 1-4]). We report that angiostatin reduces endothelial cell number in a 4-day proli feration assay without affecting cell cycle progression into S-phase (as de termined by bromodeoxyuridine labeling). This suggested that the reduction in cell number in the proliferation assay might in part be due to cytotoxic ity. This was confirmed by the observation that ethidium homodimer incorpor ation (a measure of plasma membrane integrity) into endothelial cells was i ncreased by angiostatin in a manner similar to that seen with tumor necrosi s factor-alpha (TNF-alpha) and transforming growth factor-beta 1 (TGF-beta 1), both of which induce apoptosis in endothelial cells. In contrast to TNF -alpha and TGF-beta 1, angiostatin did not induce cytotoxicity in human MRC -B fibroblast, rat smooth muscle, canine MDCK epithelial, or murine B16-F10 melanoma cell lines. Angiostatin-induced apoptosis was confirmed by endoth elial cell nuclear acridine orange incorporation as well as by annexin V an d TUNEL staining. These in vitro findings point to endothelial cell apoptos is as a mechanism for the antiangiogenic effect of angiostatin in vivo. (C) 1998 by The American Society of Hematology.