Specific binding of angiogenin (ANG) to calf pulmonary artery endothelial cells was demonstrated. Cellular binding at 4TC of 125-Ilabeled human recombinant ANG was time and concentration dependent, reversible, and saturable in the presence of increasing amounts of the unlabeled molecules. The interaction was shown to be specific since a large excess of unlabeled ANG reduced labeled ANG binding by >80%, whereas similar doses of RNase A, a structurally related protein, had no effect. Scatchard analyses of binding data revealed two apparent components. High-affinity sites with an apparent dissociation constant of 5 x 10-9 M were shown to represent cell-specific interactions. The second component, comprising low-affinity/high-capacity sites with an apparent dissociation constant of 0.2 x 10-6 M, was essentially associated with pericellular components. High-affinity ANG binding sites varied with cell density and were found on other endothelial cells from bovine aorta, cornea, and adrenal cortex capillary but not on Chinese hamster lung fibroblasts. Divalent copper, a modulator of angiogenesis, was found to induce a severalfold increase in specific cell-bound radioactivity. Placental ribonuclease inhibitor, a tight-binding inhibitor of both ribonucleolytic and angiogenic activities of ANG, abolished 1251-labeled human recombinant ANG binding only in the absence of copper.

Angiogenin is a potent inducer of blood-vessel formation with ribonucleolytic activity. Angiogenin binds to high affinity endothelial cell receptors and with lower affinity to extracellular matrix components. Here we report the effect of copper and zinc on these interactions. There was a 4.3-fold increase in angiogenin binding to calf pulmonary artery endothelial cells in the presence of Cu2+ in vitro. A 3.8-fold increase was observed with Zn2+, whereas Ni2+, Co2+, or Li+ had no effect. Specific angiogenin binding to the lower affinity matrix sites was increased by 2.7- and 1.9-fold in the presence of Cu2+ and Zn2+ respectively. Metal ion affinity chromatography and atomic absorption spectrometry were used to show the direct interaction of angiogenin with copper and zinc ions. Angiogenin bound 2.4 mol of copper per mole of protein. We suggest that copper, a modulator of angiogenesis in vivo, may be involved in the regulation of the biological activity of angiogenin.

The binding of urokinase (u-PA) to its cell surface receptor (u-PAR) is critical for tumor cell invasion. Here, we report that the disruption of this binding by an u-PAR antagonist ATF-HSA inhibits in vitro the motility of endothelial cells in a dose-dependent manner. This inhibition was also observed when the cells were first stimulated with potent angiogenic factors, including bFGF or VEGF.[3H]thymidine incorporation assay demonstrated that ATF-HSA did not affect the cell proliferation. ATF-HSA was more potent than plasmin inhibitors, suggesting that it exerts its effects not solely by inhibiting the remodeling of the extracellular matrix. In fact, analysis of the cell shape change during migration revealed for the first time that its effect is related to a decrease in cell deformability. These results suggest that u-PAR antagonist may be a new approach to control angiogenesis.

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