Specific Interaction of Angiostatin with Integrin αvβ3 in Endothelial Cells

Abstract

Angiostatin, the N-terminal four kringles (K1-4) of plasminogen, blocks tumor-mediated angiogenesis and has great therapeutic potential. However, angiostatin's mechanism of anti-angiogenic action is unclear. We found that bovine arterial endothelial (BAE) cells adhere to angiostatin in an integrin-dependent manner and that integrins ␣ v ␤ 3 , ␣ 9 ␤ 1 , and to a lesser extent ␣ 4 ␤ 1 , specifically bind to angiostatin. ␣ v ␤ 3 is a predominant receptor for angiostatin on BAE cells, since a functionblocking antibody to ␣ v ␤ 3 effectively blocks adhesion of BAE cells to angiostatin, but an antibody to ␣ 9 ␤ 1 does not. ⑀-Aminocaproic acid, a Lys analogue, effectively blocks angiostatin binding to BAE cells, indicating that an unoccupied Lys-binding site of the kringles may be required for integrin binding. It is known that other plasminogen fragments containing three or five kringles (K1-3 or K1-5) have an anti-angiogenic effect, but plasminogen itself does not. We found that K1-3 and K1-5 bind to ␣ v ␤ 3 , but plasminogen does not. These results suggest that the anti-angiogenic action of angiostatin may be mediated via interaction with ␣ v ␤ 3 . Angiostatin binding to ␣ v ␤ 3 does not strongly induce stress-fiber formation, suggesting that angiostatin may prevent angiogenesis by perturbing the ␣ v ␤ 3 -mediated signal transduction that may be necessary for angiogenesis.Angiogenesis is a complex multistep process that includes endothelial cell proliferation, migration, and differentiation, degradation of extracellular matrices, tube formation, and sprouting of new capillary branches. Several recent studies show that tumor growth and metastasis are angiogenesis-dependent (1-6). Tumors often overexpress several pro-angiogenic molecules, including members of the fibroblast growth factor (7) and the vascular endothelial growth factor/vascular permeability factor families (8, 9). Excessive angiogenesis is a part of the pathology of cancer, and preventing this process is a promising therapeutic strategy.A number of fragments or cryptic domains of large protein molecules have been identified as angiogenesis inhibitors. Angiostatin, a proteolytic fragment of plasminogen (1), inhibits proliferation (10), blocks migration (11), and increases apoptosis (12) of endothelial cells. It has been reported that angiostatin binds to ATP synthase on the cell surface (13) and that the binding of angiostatin to ATP synthase is related to angiostatin's anti-angiogenic effect (e.g. the down-regulation of endothelial cell proliferation) (13). Likewise, endostatin (a proteolytic fragment of collagen XVIII) (14), the 16-kDa N-terminal fragment of prolactin (15), an N-terminally truncated platelet factor 4 (16), and a C-terminal fragment of matrix metalloprotease 2 named PEX (17) are potent angiogenesis inhibitors. Recently, several non-collagenous domains from collagen type IV (canstatin, arrestin, and tumstatin) have been shown to have anti-angiogenic activities (18 -21). These endogenous angiogenesis inhibitors have a lot of therapeutic p...

Correlation between the tumoral expression of β3-integrin and outcome in cervical cancer patients who had undergone radiotherapy

“…This has been demonstrated for endostatin (Rehn et al, 2001), angiostatin (Tarui et al, 2001), thrombospondin-1 (for a review, see Bornstein, 2001) and tumstatin (Maeshima et al, 2000). Indeed, when molecules such as angiostatin Mauceri et al, 1998), endostatin (Hanna et al, 2000) or neutralising anti-human VEGF 165 antibodies (Gorski et al, 1999;Lee et al, 2000) are administered together with ionising radiation, the cytotoxic effects of the latter upon endothelial cells are potentiated in vivo, resulting in an enhanced antitumoral effect.…”

Angiostatin's molecular mechanism: Aspects of specificity and regulation elucidated

“…Takada's group investigated bovine arterial endothelial (BAE) cell binding to a variety of proteins, including angiostatin, coated onto plastic microtiter plates [Tarui et al, 2001]. They found that BAE cells bound to angiostatin in a dose-and magnesium-dependent manner, and that binding was saturable at approximately 200 nM angiostatin.…”

“…A potential criticism of the Tarui et al [2001] study is that the molecular basis of angiostatinintegrin interactions remains completely uncharacterized. We note that there are no RGD motifs found in either human or mouse plasminogen/angiostatin sequences, although other sequences could be involved.…”

“…Moreover, whole cell binding experiments do not reveal whether the mode of action is direct or indirect, via, for example, a binding site that is distinct from but influenced by an integrin. We further note that Tarui et al [2001] investigated the heterologous binding of human angiostatin to bovine EC. We are not aware of any homologous human-human angiostatin binding studies as on the date of submission of this manuscript.…”

A multiprong approach to cancer gene therapy by coencapsulated cells

“…This may be related to angiostatin's ability to alter neutrophil trafficking 34 or adhesion molecule expression on endothelial cells. Angiostatin, when bound to integrins, 35 may alter trafficking of lymphocytes, while the expression of hypoxic-induced proangiogenic factors in tumors can alter the expression of cell adhesion molecules involved in systemic cell trafficking. 36,37 Hence, this alteration by angiostatin in the trafficking of cells that have receptors for IL-2 may affect how IL-2 is distributed throughout the host.…”