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S. Becerra, L. Notari, D. Mueller, T. Higuti, N. Arakaki; Pigment Epithelium–Derived Factor (PEDF) and ATP Synthase: A Mechanism of Action for Inhibition of Angiogenesis . Invest. Ophthalmol. Vis. Sci. 2006;47(13):1834.
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© ARVO (1962-2015); The Authors (2016-present)
PEDF is a potent blocker of angiogenesis in vivo and an inhibitor of endothelial tube formation. Angiostatin, another antiangiogenic factor, binds F1/F0–ATP synthase on the surface of human umbilical vein endothelial cells. These cells possess high ATP synthesis activity on the cell surface that is inhibited by angiostatin and piceatannol, an inhibitor of the F1 catalytic domain of H+–ATP synthase, without affecting intracellular ATP levels, but leading to inhibition of endothelial cell migration and proliferation. To determine whether the mechanism by which PEDF inhibits angiogenesis involves ATP synthesis inhibition, we examined the extracellular ATP synthesis activity of endothelial cells in the presence of PEDF.
Endothelial tube formation assays were performed with immortalized human microvascular endothelial cells (HMVECs). ATP production was assayed using CellTiter Glow® Luminescent reagent. Protein–protein interactions were assayed by size–ultrafiltration and surface plasmon resonance. Radioligand binding assays and PEDF–affinity column chromatography were performed with bovine retina endothelial cells (BRECs) and bovine retina. Peptide mass fingerprinting was carried out on protein extracted from excised polyacrylamide Coomassie stained bands.
HMVECs formed tubes, even without addition of proangiogenic VEGF, and both PEDF and angiostatin inhibited tube formation. HMVECs produced ATP extracellularly, and treatments with PEDF or piceatannol for 48 hours completely inhibited this activity. VEGF did not change the extracellular ATP production, but VEGF/PEDF or VEGF/piceatannol combinations partially inhibited it. Treatments of only 30 seconds with PEDF, angiostatin or piceatannol significantly inhibited the extracellular ATP synthesis activity, suggesting a direct interaction of PEDF with ATP synthase. Binding of PEDF to endothelial cell surface was assessed. BRECs exhibited saturable and high affinity PEDF–binding sites. Affinity chromatography revealed a PEDF–binding protein of 60–kDa in plasma membranes from bovine retina and BRECs. Peptide mass fingerprinting matched this protein to Bos taurus F1–ATP synthase ß–subunit. Direct PEDF binding to F1–ATP synthase was examined with highly purified yeast F1 with a His–tagged ß–subunit. PEDF bound to F1 with similar affinities to those with intact cells.
These results imply that ATP synthase inhibition might mediate the antiangiogenic activity of PEDF via mechanisms similar to those used by angiostatin.
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