Cell Surface ATP Synthase: A Potential Target for Anti-Angiogenic Therapy - Extracellular ATP and Adenosine as Regulators of Endothelial Cell Function - page 140

a monoclonal antibody against the subunit inhibits endothelial cell tube forma-

tion, while six monoclonal antibodies against the

β

subunit tested by our laboratory
do not induce inhibition in this in vitro assay predictive of anti-angiogenic activity
[15]. Thus, use of the anti-

α

subunit polyclonal antibody may have only partially
inhibited the interaction of angiostatin with the adjacent

α

subunit via steric hin-
drance and/or binding of the polyclonal antibody to epitope(s) on the

β

β subunit that

are shared with the subunit.

Use of an antibody against ATP synthase to inhibit interaction of angiostatin with
the receptor largely inhibits the anti-proliferative effect of angiostatin on endothelial
cells (

α

∼

80%) [26]. Another group demonstrated that kringles 1-5 from plasmino-
gen also bind to cell surface ATP synthase, activating caspases-8, -9, and -3 and
leading to endothelial cell apoptosis [41]. These results indicate that cell surface
ATP synthase is involved in the anti-angiogenic effect of angiostatin. A therapeutic
mimetic able to bind this receptor could potentially overcome the pharmacokinetic
disadvantages associated with recombinant angiostatin.
Further studies of angiostatin revealed that it blocks the enzymatic activity of
ATP synthase [25]. In the absence of angiostatin, HUVEC synthesize approximately
40 pmol of ATP per million cells. Treatment with 1 M angiostatin reduces this

extracellular ATP production by 81%, while treatment with polyclonal antibodies
against the

μ

subunits of ATP synthase inhibits ATP generation by 65 and
57%, respectively. Oligomycin, a known inhibitor of ATP synthase enzymatic activ-
ity, decreases ATP synthesis by 84% (Fig. 9.3). Purified bovine F 1 ATP synthase was
also utilized to test the effect on ATP hydrolysis, as the F 1 subunit in the absence of
the F 0 proton channel can only catalyze ATP hydrolysis. Angiostatin and polyclonal

α and β

Fig. 9.3 Inhibition of HUVEC extracellular ATP synthesis by polyclonal antibodies against ATP
synthase. Extracellular ATP generation by HUVEC in the presence of 50 μ M ADP was measured
by bioluminescent luciferase assay. Cells were treated with 1 μ M angiostatin ( 1 ), 1 mg/ml poly-
clonal antibody against the α subunit of ATP synthase ( 2 ), 0.5 mg/ml polyclonal antibody against
the β subunit of ATP synthase ( 3 ), 1 mg/ml pre-immune serum ( 4 ), or 50 μ g/ml oligomycin ( 5 ).
Inhibition was calculated relative to cells incubated in media only. Antibodies directed against ATP
synthase inhibited extracellular ATP generation, although to a lesser extent than angiostatin and
oligomycin, a known F 0 inhibitor. (Adapted from [25], reprinted by permission of Proceedings
of the National Academy of Sciences, Copyright 2001 National Academy of Sciences, USA
[25])