A8404 Anti-inflammatory activity of secreted APE1/Ref-1 in vascular endothelial cells

Objectives: Apurinic apyrimidinic endonuclease 1/Redox factor-1 (APE1/Ref-1) is a multifunctional protein that plays a DNA repair and redox regulation with secretory activity. The aim of this study was to evaluate the biological role of extracellular APE1/Ref-1 in human cultured endothelial cells. Methods: Intracellular acetylation is induced by the incubation of trichostatin A, secreted APE1/Ref-1 is quantified with sandwich ELISA. Results: During treatment of trichostatin A in culture, a time-dependent increase in secreted APE1/Ref-1 was confirmed. Trichostatin A significantly suppressed vascular cell adhesion molecule-1 expression in response to TNF-α. The acetyl moiety of acetylated-APE1/Ref-1 was rapidly removed based on the removal kinetics. Additionally, recombinant human APE1/Ref-1 with reducing activity induced a conformational change in rhTNF-α receptor 1 by thiol-disulfide exchange. Following treatment with the neutralizing anti-APE1/Ref-1 antibody, inflammatory signals via the binding of TNF-α to TNFR1 were remarkably recovered, leading to up-regulation of reactive oxygen species generation and VCAM-1. Conclusion: Furthermore, rhAPE1/Ref-1 inhibited VCAM-1 expression in interleukin-1β-stimulated endothelial cells, and inhibited inducible nitric oxide synthase. These results strongly indicate that anti-inflammatory effects in TNF-α-stimulated endothelial cells by acetylation are tightly linked to secreted APE1/Ref-1, which inhibits inflammatory cytokines binding to its receptors.