Cell Surface ATP Synthase: A Potential Target for Anti-Angiogenic Therapy - Extracellular ATP and Adenosine as Regulators of Endothelial Cell Function - page 139

and subunits are localized to caveolae [46, 47]. Furthermore, several other research

groups have confirmed the presence of enzymatically active F 1 F 0 ATP synthase on
the surface of endothelial cells [2, 47, 54]. It is worth noting that the orientation
of ATP synthase in the plasma membrane of eukaryotic cells is opposite to the ori-
entation in bacteria. Thus, ATP hydrolysis and synthesis occurs in the extracellular
milieu of endothelial cells, potentially influencing extracellular ADP/ATP levels and
affecting signaling via purinergic receptors (Fig. 9.1b).

β

9.2 ATP Synthase and Angiogenesis

9.2.1 Receptor for Angiostatin

Angiostatin is an endogenous anti-angiogenic molecule formed by the proteolytic
cleavage of plasminogen. Its structure consists of the first three or four disulfide-
linked loops (kringles 1-3 or 1-4) of plasminogen. In 1994, Judah Folkman and his
group reported the isolation of this circulating 38 kDa proteolytic fragment from the
serum and urine of mice bearing Lewis lung carcinoma tumors [28]. They demon-
strated that angiostatin specifically inhibits endothelial cell proliferation in vitro
and prevents angiogenesis inthe chicken chorioallantoic membrane (CAM) model.
Additionally, they found that angiostatin inhibits growth of tumor metastases. Later
studies also showed that systemic treatment of immunodeficient mice with angio-
statin induces a dramatic size reduction in primary human tumors, followed by the
onset of dormancy [27]. These findings generated interest in the use of angiostatin
as a cancer therapeutic; however, the encouraging effects of angiostatin in animal
tumor models did not translate into success in human clinical trials. The lack of effi-
cacy in these trials was largely due to the short serum half-life of angiostatin ( 20

minutes), which necessitates the use of high doses and frequent administration to
achieve a therapeutic effect [4, 43].
The discovery of ATP synthase as one of the receptors of angiostatin shed light
on at least one component of the mechanism of action of angiostatin. Our labo-
ratory utilized affinity chromatography to identify proteins located in the plasma
membrane of human umbilical vein endothelial cells (HUVEC) that bound to angio-
statin. A single

∼

∼

55-kDa protein was eluted from the angiostatin-Sepharose column.
This protein was identified as either the subunit of ATP synthase based on

amino-terminal sequencing, mass spectrometry, and peptide mass fingerprinting.
Incubation of HUVEC with a polyclonal antibody against the

α or β

α subunit inhibited

binding of angiostatin to the cells by 56% [26]. The lack of complete binding

inhibition could in part be due to the presence of other receptors for angiostatin
on endothelial cells. Additionally, angiostatin also binds to the

∼

β subunit of ATP

synthase, which is 23% homologous and 57% similar to the α subunit [26]. The

activity of angiostatin actually depends upon this β subunit interaction, as the use

of a polyclonal antibody against the subunit inhibits endothelial proliferation like

angiostatin, whereas an antibody against the

β

α subunit does not [25, 26]. Similarly,