Endothelial cell surface F1-F0 ATP synthase is active in ATP synthesis and is inhibited by angiostatin

Abstract

Angiostatin blocks tumor angiogenesis in vivo, almost certainly through its demonstrated ability to block endothelial cell migration and proliferation. Although the mechanism of angiostatin action remains unknown, identification of F(1)-F(O) ATP synthase as the major angiostatin-binding site on the endothelial cell surface suggests that ATP metabolism may play a role in the angiostatin response. Previous studies noting the presence of F(1) ATP synthase subunits on endothelial cells and certain cancer cells did not determine whether this enzyme was functional in ATP synthesis. We now demonstrate that all components of the F(1) ATP synthase catalytic core are present on the endothelial cell surface, where they colocalize into discrete punctate structures. The surface-associated enzyme is active in ATP synthesis as shown by dual-label TLC and bioluminescence assays. Both ATP synthase and ATPase activities of the enzyme are inhibited by angiostatin as well as by antibodies directed against the alpha- and beta-subunits of ATP synthase in cell-based and biochemical assays. Our data suggest that angiostatin inhibits vascularization by suppression of endothelial-surface ATP metabolism, which, in turn, may regulate vascular physiology by established mechanisms. We now have shown that antibodies directed against subunits of ATP synthase exhibit endothelial cell-inhibitory activities comparable to that of angiostatin, indicating that these antibodies function as angiostatin mimetics.

Figure 1

Colocalization of the α- and β-subunits of ATP synthase on the surface of HUVECs by immunostaining and confocal microscopy. (a) Nonpermeabilized HUVECs immunostained with a murine mAb specific for the α-subunit of ATP synthase. (b) The same cells immunostained with a rabbit polyclonal antiserum specific for the β-subunit of ATP synthase. (c) Composite colocalization images obtained by digital overlays of the above images. (d) Colocalization image obtained from cells permeabilized with ethanol (100%). Representative images are shown; n = 26.

Figure 2

Surface localization of the α-subunits of ATP synthase and CD31 on nonpermeabilized HUVECs by immunostaining and confocal microscopy. (A–D) Confocal optical sections were taken along the z axis every 1.5 μm. Each section is ≈0.6 μm in thickness. A series of z sections from a representative field is shown, starting with the basal surface in A and ending with the apical surface in D. (E) The same section shown in C; fluorescence from red channel only. (F) The same section shown in C; fluorescence from green channel only; n = 3.

Figure 3

Binding of angiostatin to purified bovine F₁ ATP synthase. ELISA was used to determine concentration-dependent binding of angiostatin to a constant amount of F₁ ATP synthase. Each well was coated with 1 μg of F₁ ATP synthase before addition of decreasing amounts of angiostatin. Control lane (−) shows binding of secondary antibody only (n = 6). (Inset) Apparent dissociation constants [K_d(app)] were determined from double-reciprocal plots of the binding data. Angiostatin bound to bovine F₁ ATP synthase with a K_d(app) of 14.1 nM.

Figure 4

Inhibition of purified F₁ ATP synthase by angiostatin. Purified F₁ ATP synthase activity was measured spectrophotometrically at λ = 340 nm by coupling the production of ADP to the oxidation of NADH via the pyruvate kinase and lactate dehydrogenase reaction, in which a decrease in the absorbance at λ = 340 nm indicates active protein () (28). Angiostatin (10 μM) completely inhibited purified F₁ ATP synthase activity (◊), comparable to a known F₁ ATP synthase inhibitor, NaN₃ (2%) (⧫) and an enzyme-free control (▴). Polyclonal antibodies directed against the recombinant α-subunit of ATP synthase (500 μg/ml) (▾) and β-subunit ATP synthase (700 μg/ml) (▿) abolished ATPase activity. A mAb to the α-subunit of ATP synthase (25 μg/ml) also inhibited activity (■). Control antibodies had no effect on activity (○, □). Representative data are shown; n = 3.

Figure 5

Inhibition of ATP generation by angiostatin on the surface of HUVECs as measured by bioluminescent luciferase assay. ATP generation on the surface of HUVECs was inhibited in a dose-dependent manner in the presence of increasing concentrations of angiostatin. Representative data are shown; n = 3.