Cell Surface ATP Synthase: A Potential Target for Anti-Angiogenic Therapy - Extracellular ATP and Adenosine as Regulators of Endothelial Cell Function - page 144

disruption cell signaling through ATP binding to cell surface purinoreceptors. The
relative contributions of these two actions have not yet been elucidated, however
most attention has focused on pH homeostasis due to the differential effects of
angiostatin and anti-ATP synthase antibodies at neutral and low pH e . It has been
reported that angiostatin disrupts endothelial cell tube formation under acidic con-
ditions (pH e 6.7), but not at a normal physiologic pH of 7.3 [42]. Additionally,
angiostatin treatment of endothelial cells under conditions of low pH e results
in increased toxicity of angiostatin to the cells [44]. A pH-dependent effect on
angiostatin-induced cell death has also been shown in tumor cells expressing cell
surface ATP synthase [13].
In both endothelial cells and ATP synthase-expressing tumor cells, exposure
to acidic conditions causes a greater decrease in intracellular pH (pH i ) in cells
treated with angiostatin than untreated cells. These results prompted our laboratory
to hypothesize that angiostatin induces cell death by disrupting pH i homeostasis.
ATP synthase is more active in generating ATP at pH e 6.7 than at 7.2, and this enzy-
matic activity likely tempers the pH i decrease that occurs in acidic conditions by
transporting protons out of the cell [13, 14]. This activity is puzzling, given that
it involves the energetically unfavorable synthesis of ATP under conditions when
protons are being moved against their concentration gradient in order to regulate
pH i . This confounding activity could be explained by the localization of cell surface
ATP synthase in caveolae, where pH can potentially differ from other areas along
the plasma membrane due to the distinct composition of proteins present in these
invaginations [14]. Regardless of the mechanism, the experimental evidence dis-
cussed above indicates increased ATP synthesis by cell surface ATP synthase under
acidic conditions.
Like angiostatin, the first inhibitory monoclonal antibody against the subunit

of ATP synthase, MAb3D5AB1, reduces extracellular ATP synthesis under low pH e
conditions (Fig. 9.6a). Exposure of HUVEC to a drop in pH e of 7.3 to 6.7 resulted
inapH i decrease of 0.41 when challenged in the presence of 67 nM MAb3D5AB1,
relative to a decrease of only 0.25 in the absence of the antibody (Fig. 9.6b) [15].
This effect by the monoclonal antibody is more potent (

β

∼

15-fold) than that of angio-

∼ μ

statin, which must be present at a concentration of M to achieve this degree

of pH i decrease [3]. Again mimicking the properties of angiostatin, MAb3D5AB1
inhibits endothelial cell tube formation by 60% under acidic conditions (pH e 6.7),
but not at the normal physiologic pH e of 7.4. In vivostudies also demonstrate anti-
angiogenic activity in the acidic microenvironment of the chicken CAM model, but
not in the rat corneal neovascularization assay, where the pH is

1

∼ 7.4 as would be

expected in this normal tissue [15]. The two other anti- -ATP synthase antibodies

(McAb178-5G10 and mAb6F2C4) recently reported in the literature also inhibit
extracellular ATP synthesis, HUVEC proliferation, and endothelial cell tube forma-
tion preferentially in an acidic environment [45, 54]. These results have garnered
particular interest, given that this pH-dependent effect would likely target the anti-
angiogenic effects of antibodies against the

β

β subunit to acidic microenvironments

such as tumors, while sparing normal tissues.