Tat-APE1/ref-1 protein inhibits TNF-a-induced endothelial cell activation Yun Jeong Song, Ji Young Lee, Hee Kyoung Joo, Hyo Shin Kim, Sang Ki Lee, Kwon Ho Lee, Chung-Hyun Cho, Jin Bong Park, Byeong Hwa Jeon * Research Institute of Medical Sciences, Department of Physiology, College of Medicine, Chungnam National University, 6 Munhwa-dong, Jung-gu, Daejeon 301-131, Republic of Korea Received 8 January 2008 Available online 17 January 2008 Abstract Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/ref-1) is a multifunctional protein involved both in DNA base excision repair and redox regulation. In this study we evaluated the protective role of Tat-mediated APE1/ref-1 transduction on the tumor necro- sis factor (TNF)-a-activated endothelial activation in cultured human umbilical vein endothelial cells. To construct Tat-APE1/ref-1 fusion protein, human full length of APE1/ref-1 was fused with Tat-protein transduction domain. Purified Tat-APE1/ref-1 fusion protein efficiently transduced cultured endothelial cells in a dose-dependent manner and reached maximum expression at 1 h after incubation. Transduced Tat-APE1/ref-1 showed inhibitory activity on the TNF-a-induced monocyte adhesion and vascular cell adhesion mole- cule-1 expression in cultured endothelial cells. These results suggest Tat-APE1/ref-1 might be useful to reduce vascular endothelial acti- vation or vascular inflammatory disorders. Ó 2008 Elsevier Inc. All rights reserved. Keywords: Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/ref-1); Tat-mediated transduction; Monocyte adhesion; Vascular cell adhesion molecule-1; Endothelial cells; Vascular endothelial activation The direct intracellular delivery of proteins has been dif- ficult to achieve primarily due to the bioavailability barrier of the plasma membrane, which effectively prevents the uptake of macromolecules by limiting their passive entry. One approach to circumvent these problems is the use of HIV-TAT-mediated protein transduction [1]. HIV-Tat protein contained 9–11 amino acids for its transduction function [2]. Protein transduction domains offer an exciting therapeutic opportunity for the treatment of many dis- eases, such as airway inflammation, stroke and diabetes [3–5]. Atherosclerosis is an inflammatory disease. Monocyte rolling and adhesion to the vascular endothelial lining and subsequent diapedesis are not only the first steps, but also seem to be crucial events in the pathological process [6]. As the phenotype of endothelial cells is subject to change by oxidative stress or inflammatory cytokine, such as tumor necrosis factor (TNF)-a, endothelial activation is implicated in the pathogenesis of cardiovascular disorders [7]. Apurinic/apyrimidinic endonuclease/redox factor-1 (APE1/ref-1) is an essential endonuclease in the base exci- sion repair pathway of oxidatively damaged DNA, as well as having reducing properties that promote the binding of redox-sensitive transcription factors such as activator pro- tein-1 to their cognate DNA sequences [8,9]. In addition to a nuclear role of APE1/ref-1, an extra-nuclear role of APE1/ref-1 in the regulation of endothelial oxidative stress has been uncovered. In particular, APE1/ref-1 suppresses oxidative stress through modulation of cytoplasmic rac1- regulated ROS generation [10,11] and increases nitric oxide bioavailability through phosphorylation of endothelial nitric oxide synthase [12]. Our aim was to evaluate the potential usefulness of a Tat-APE1/ref-1 fusion protein to inhibit vascular endothe- 0006-291X/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2008.01.037 * Corresponding author. Fax: +82 42 585 8440. E-mail address: bhjeon@cnu.ac.kr (B.H. Jeon). www.elsevier.com/locate/ybbrc Available online at www.sciencedirect.com Biochemical and Biophysical Research Communications 368 (2008) 68–73