Laminin Modulates Morphogenic Properties of the Collagen XVIII Endostatin Domain

Abstract

We have shown previously that the oligomeric endostatin domain of collagen XVIII (NC1) functioned as a motility-inducing factor regulating the extracellular matrix-dependent morphogenesis of endothelial cells. This motogenic activity gave rise to structures resembling filipodia and lamellipodia and was dependent on Rac, Cdc42, and mitogen-activated protein kinase. Here, we demonstrate that these properties of endostatin are primarily mediated by laminin in the basement membrane and heparan sulfates on the cell surface. The sites of interaction between laminin and oligomeric endostain include the N-terminal regions of all three laminin chains (amino acids 204 -1243 of the ␣ chain, 932-1161 of the ␤ chain, and 150 -965 of the ␥ chain). A monoclonal antibody that blocks the interactions between endostatin and laminin was utilized to inhibit the motogenic activity of endostatin. In parallel, we have engineered selective point mutations and produced recombinant forms that lack binding to heparan sulfates on the cell surface. Our data are consistent with a model of endostatin with two binding sites: one mainly to laminin in the basement membrane and the other to heparan sulfates on the cell surface. The two binding domains on endostatin appear to be separate with the possibility of some overlap between the two sites.

Endostatin therapy reveals a U-shaped curve for antitumor activity

“…A detailed description of this procedure was given previously. 39 Serum samples (100 ml) of AAV-1/cFcESand AAV-1/GFP-infected mice were compared with different concentrations of recombinant cFcES. The immunoblot was probed with rabbit anti-canine endostatin antibody (1:2000).…”

“…39 Serum samples (250 ml) of AAV-1/cFcES-and AAV-1/GFP-infected mice were used and compared to recombinant cFcES (10 mg/ml).…”

Endostatin Has ATPase Activity, Which Mediates Its Antiangiogenic and Antitumor Activities

“…It can initiate endothelial cell apoptosis through the induction of VDAC1 phosphorylation, which facilitates the mitochondrial permeability transition pore (mPTP) opening (10). Moreover, it also binds to cell surface receptors such as integrins (11), glypicans (12), laminin (13) and nucleolin (14), and thereby regulates a myriad of signaling cascades. For example, endostatin binding to integrin a5b1 results in the inhibition of the FAK-c-Raf-MEK1/2-p38-ERK1 MAPK pathway (15).…”

Targeted Delivery of an Antibody–Mutant Human Endostatin Fusion Protein Results in Enhanced Antitumor Efficacy

“…Inhibition of tube assembly has been previously reported with the NC1 domain of collagen XVIII and oligomeric forms of endostatin (17,18). The increased in vitro antiangiogenic effect of aHER2-huEndo fusion proteins relative to native endostatin may be due to presentation of endostatin as a dimer as previously reported (17,18).…”

“…It has been suggested that initial preparations of endostatin had suboptimal biologic activity and a modified formulations (e.g., EndoStar) may be more potent and have been approved for use in China (14)(15)(16). Endostatin may be more potent when delivered as a dimer or trimer than when delivered in monomeric form (17,18). Finally, a mutant form of endostatin (P125A) has been reported to have enhanced antiangiogenic activity (19)(20)(21).…”