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S. Becerra, J. Amaral, L. Notari; Interactions Between Pigment Epithelium-Derived Factor (PEDF) and F1 ATP Synthase Provide Insights into Its Antiangiogenic Mechanism. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2969.
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© ARVO (1962-2015); The Authors (2016-present)
PEDF is a potent blocker of angiogenesis. Angiostatin and piceatannol bind and inhibit the F1 catalytic domain of H+-ATP synthase on endothelial cell-surfaces leading to inhibition of migration and proliferation. The purpose of this study is to examine the interactions between PEDF and F1-ATP synthase.
PEDF was purified from media of stably transfected BHK cells with full-length human PEDF cDNA expression vectors. F1 was purified from yeast cells bearing His-tagged F1-ß subunit expression vectors. Complex formation was assayed by size-ultrafiltration. Protein-protein interactions were examined by surface plasmon resonance with immobilized PEDF or angiostatin. Human microvascular endothelial cells (HMVECs) were cultured and membrane fractions were prepared. ATP synthase protein was followed by immunoreactions with anti-F1-ß subunit on western blots and by surface plasmon resonance. ATP production was assayed using CellTiter Glow® Luminescent reagent.
HMVECs membrane protein fractions contained both F1-ß subunit-immunoreacting proteins and PEDF-binding components. F1-ß subunit antibodies recognized PEDF-binding components in the HMVEC membrane fractions. Direct binding to purified yeast F1 showed PEDF binding with high affinity, and angiostatin binding with lower affinity. PEDF efficiently competed with angiostatin for F1 binding. To determine whether the mechanism by which PEDF inhibits angiogenesis involves ATP synthesis inhibition, we examined the extracellular ATP synthesis activity of HMVECs in the presence of PEDF. HMVECs produced ATP extracellularly, and PEDF, angiostatin and piceatannol significantly inhibited this activity.
These results demonstrate a direct interaction of PEDF with ATP synthase. They imply that ATP synthase inhibition might mediate the antiangiogenic activity of PEDF via mechanisms similar to those used by angiostatin.
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