Cellular Mechanisms for the Antiangiogenic Activity of Angiostatin

Angiostatin inhibits proliferation, migration, and tube formation by endothelial cells. Angiostatin induces the apoptosis of endothelial cells in culture and in mouse models. Although most studies have focused on the endothelial effects of angiostatin, other cells may also be targets for angiostatin. Angiostatin may have a more potent effect on circulating, bone marrow-derived endothelial progenitor cells than it has on endothelial cells. We have shown that smooth muscle cell (SMC) migration and proliferation are inhibited by angiostatin. Furthermore, hepatocyte growth factor (HGF) signaling pathways were inhibited by angiostatin in SMCs. Benelli et al. [1] have demonstrated that angiostatin inhibits monocyte migration to MCP-1 and fMLP. An even more potent effect of angiostatin was exhibited in inhibiting neutrophil migration to CXCR1 and CXCR2 agonists (IL-8 and MlP-2). These nonendothelial cellular effects of angiostatin may be important because leukocytes contribute to angiogenesis and smooth muscle cells are essential for arteriogenesis.

Proposed Molecular Targets of Angiostatin

One of the first known binding sites for angiostatin on endothelial cells was the F1-F0 ATP synthase (Figure 1). The ATP synthase has historically been considered to be an exclusively mitochondrial enzyme. Moser et al. not only localized ATP synthase on the endothelial cell (EC) surface, but demonstrated that it is active in extracellular ATP synthesis. Antibodies to ATP synthase prevented the antiproliferative effect of angiostatin on endothelial cells. The proton pumping function of ATP synthase is also blocked by angio-statin, endangering the ability of ECs to survive in hypoxic, acidic microenvironments.

Tarui and colleagues reported that bovine aortic endothe-lial cells adhered to angiostatin via the integrin receptor avb3. Since angiostatin did not induce stress fiber formation upon binding to the integrin, these investigators hypothesized that angiostatin competes with the natural ligands for avb3, producing an antiangiogenic effect. Blockade of avb3 by angiostatin could inhibit EC migration. It is also possible that this effect reduces MMP-2 activation, which is enhanced by the ligation of pro-MMP-2 with avb3.

The competitive inhibition of avb3 by angiostatin could alter focal adhesion kinase (FAK) activity. Integrins do not possess intrinsic kinase activity, but instead form complexes with signaling molecules at focal adhesion contact sites. FAK is a tyrosine kinase that is activated by integrin clustering, although there are also other mechanisms for activating FAK independent of cell-ECM interactions. FAK is also activated by ligation of some growth factor receptors. Claesson-Welch and colleagues [2] have shown that angio-statin upregulates FAK activity in endothelial cells in an RGD-independent and therefore probably integrin-independent fashion. These investigations suggested that the aberrant upregulation of FAK could contribute to inhibition of EC migration and to the induction of apoptosis.

Figure 1 Reported molecular targets of angiostatin. A section of a cell membrane is diagrammed showing the molecular targets of angiostatin. Angiostatin binds to the a/b subunits of ATP synthase (A), limiting extracellular ATP synthesis and increasing the intracellular [H+] (B). Angiostatin also binds to annexin II (C), displacing lys-plasminogen, thereby preventing plasmin generation. Plasmin has several important pericellular activities, including activation of TGF-b, degrading ECM proteins, and activating MMPs. Integrin receptors may also contribute to MMP activation. Angiostatin has also been reported to bind to the vitronectin receptor (avb3), probably inhibiting the binding of ECM protein ligands (D). Focal adhesion kinase (FAK) is coupled to the intracellular domain of integrin receptors and also to some growth factor receptors (E). Angiostatin has been reported to activate FAK phosphorylation. Angiostatin also prevents signaling through the hepatocyte growth factor (HGF) c-Met pathway (F), by binding to c-Met. One of the actions of HGF c-Met that is blocked by Angiostatin is phosphorylation and activation of Akt (protein kinase B). Angiostatin inhibits migration and tube formation that is promoted by the cell-surface associated protein angiomotin (G). Angiostatin uncouples eNOS from hsp90 (H), leading to reduced production of NO, with increased production of O2. Finally, angiostatin promotes Ca2+ influx by an unknown mechanism (I). Several molecular targets of angiostatin, including ATP synthase, eNOS, integrins, and annexin II, may associate with caveolae, representing a potential mechanism by which an inhibitory effect on a variety of targets could be enhanced.

Figure 1 Reported molecular targets of angiostatin. A section of a cell membrane is diagrammed showing the molecular targets of angiostatin. Angiostatin binds to the a/b subunits of ATP synthase (A), limiting extracellular ATP synthesis and increasing the intracellular [H+] (B). Angiostatin also binds to annexin II (C), displacing lys-plasminogen, thereby preventing plasmin generation. Plasmin has several important pericellular activities, including activation of TGF-b, degrading ECM proteins, and activating MMPs. Integrin receptors may also contribute to MMP activation. Angiostatin has also been reported to bind to the vitronectin receptor (avb3), probably inhibiting the binding of ECM protein ligands (D). Focal adhesion kinase (FAK) is coupled to the intracellular domain of integrin receptors and also to some growth factor receptors (E). Angiostatin has been reported to activate FAK phosphorylation. Angiostatin also prevents signaling through the hepatocyte growth factor (HGF) c-Met pathway (F), by binding to c-Met. One of the actions of HGF c-Met that is blocked by Angiostatin is phosphorylation and activation of Akt (protein kinase B). Angiostatin inhibits migration and tube formation that is promoted by the cell-surface associated protein angiomotin (G). Angiostatin uncouples eNOS from hsp90 (H), leading to reduced production of NO, with increased production of O2. Finally, angiostatin promotes Ca2+ influx by an unknown mechanism (I). Several molecular targets of angiostatin, including ATP synthase, eNOS, integrins, and annexin II, may associate with caveolae, representing a potential mechanism by which an inhibitory effect on a variety of targets could be enhanced.

We have demonstrated that angiostatin inhibits HGF-induced signaling in endothelial and smooth muscle cells. We hypothesized that angiostatin, which has significant homology to HGF, would competitively inhibit HGF from binding to its receptor (c-Met). Angiostatin inhibited the HGF-induced tyrosine phosphorylation of c-Met, as well as the phosphorylation of Akt and ERK1/2.

Angiostatin-mediated EC apoptosis has been reported to involve calcium signaling. Jiang and colleagues [3] have shown that angiostatin induces an acute rise in [Ca2+] of human and bovine endothelial cells. Treatment with endostatin evoked a similar response and attenuated the acute elevation in [Ca2+] with subsequent administration of VEGF and FGF-2. Angiostatin is able to deplete thapsigargin-sensitive intracellular Ca2+ stores that are normally released by muscarinic agonists. The cellular target responsible for the effects on [Ca2+] in response to angiostatin are currently unknown. Kringle 5 of plasminogen, also an inhibitor of angiogenesis, has been reported to induce a rise in [Ca2+] in HUVEC by binding to a cell-surface voltage-dependent anion channel [4]. It is unknown whether angiostatin affects the same or similar ion channels.

Angiostatin may inhibit pericellular plasmin generation, limiting the ability of the EC to dissolve its basement membrane, and preventing migration in the early phases of angiogenesis. There are at least two mechanisms for this effect. Stack and colleagues [5] demonstrated that angio-statin binds to t-pA and inhibits plasminogen activation. Another mechanism could be through the interaction of angiostatin with annexin II. Annexin II is an endothelial cell surface protein that binds to both lys-plasminogen and t-pA, enhancing plasmin generation. Tuszynski et al. reported that angiostatin binds to annexin II on the EC surface, providing another potential mechanism by which angiostatin could inhibit plasmin production at the EC surface. Plasmin has important roles in angiogenesis by activating MMps and TGF-ß1 and promoting cell migration.

Angiomotin is a 72-kDa cell surface associated protein localized to the lamellipodia of the leading edge of migrating capillary endothelial cells and is colocalized with FAK. Angiomotin-transfected cells also have increased FAK activity in response to angiostatin. When angiomotin-transfected cells are treated with angiostatin, migration, and tube formation are significantly inhibited. Although a detailed mechanism of action of angiomotin has not been determined, a PDZ-binding domain appears to have a critical role.

Essentials of Human Physiology

Essentials of Human Physiology

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