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Multiple forms of angiostatin induce apoptosis in endothelial cells
R Lucas1, L Holmgren, I Garcia
1Laboratory of Immunopathology of Intensive Care, Department of Anesthesiology, Geneva University Hospital, Geneva, Switzerland
Abstract:
Angiostatin is a circulating inhibitor of angiogenesis generated by proteolytic 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 proliferation assay without affecting cell cycle progression into S-phase (as determined by bromodeoxyuridine labeling). This suggested that the reduction in cell number in the proliferation assay might in part be due to cytotoxicity. This was confirmed by the observation that ethidium homodimer incorporation (a measure of plasma membrane integrity) into endothelial cells was increased by angiostatin in a manner similar to that seen with tumor necrosis factor- (TNF-) and transforming growth factor-beta1 (TGF-beta1), both of which induce apoptosis in endothelial cells. In contrast to TNF- and TGF-beta1, angiostatin did not induce cytotoxicity in human MRC-5 fibroblast, rat smooth muscle, canine MDCK epithelial, or murine B16-F10 melanoma cell lines. Angiostatin-induced apoptosis was confirmed by endothelial cell nuclear acridine orange incorporation as well as by annexin V and TUNEL staining. These in vitro findings point to endothelial cell apoptosis as a mechanism for the antiangiogenic effect of angiostatin in vivo.
Insights
Angiostatin, an angiogenesis inhibitor, reduces endothelial cell numbers by inducing apoptosis, not by affecting cell cycle progression. This specific cytotoxicity was observed only in endothelial cells, not other cell types.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Angiostatin is a naturally occurring inhibitor of angiogenesis, derived from plasminogen.
- Its mechanism of action in reducing endothelial cell proliferation requires further elucidation.
Purpose of the Study:
- To investigate the mechanism by which angiostatin reduces endothelial cell number.
- To determine if angiostatin induces endothelial cell apoptosis or affects cell cycle progression.
Main Methods:
- Utilized recombinant and native angiostatin variants (kringles 1-4 and 1-3).
- Assessed endothelial cell proliferation and cell cycle progression (bromodeoxyuridine labeling).
- Measured cytotoxicity using ethidium homodimer incorporation, acridine orange, annexin V, and TUNEL staining.
Main Results:
- Angiostatin reduced endothelial cell number without altering cell cycle progression into S-phase.
- Angiostatin increased ethidium homodimer incorporation in endothelial cells, indicating loss of membrane integrity.
- This cytotoxic effect was specific to endothelial cells and not observed in fibroblasts, smooth muscle, epithelial, or melanoma cells.
Conclusions:
- Angiostatin induces apoptosis in endothelial cells, contributing to its anti-angiogenic effects.
- The mechanism involves direct cytotoxicity to endothelial cells, distinguishing it from other growth factors.
- These in vitro findings support endothelial cell apoptosis as a key in vivo anti-angiogenic mechanism for angiostatin.