Original Contribution Long-term exposure to oxidized low-density lipoprotein enhances tumor necrosis factor-a-stimulated endothelial adhesiveness of monocytes by activating superoxide generation and redox-sensitive pathways Jaw-Wen Chen * , Yung-Hsiang Chen, Shing-Jong Lin National Yang-Ming University School of Medicine, Cardiovascular Research Center, and Division of Cardiology, Department of Medicine, Taipei Veterans General Hospital, 201, Sec. 2, Shih-Pai Road, Taipei, Taiwan, Republic of China Received 11 May 2005; revised 4 October 2005; accepted 6 October 2005 Available online 2 November 2005 Abstract This study was conducted to investigate the role of oxidized low-density lipoprotein (LDL) in monocyte/mononuclear cell (MNC) activation during atherogenesis. First, the activity of MNCs was studied in patients with coronary artery disease (CAD). Compared to normal subjects, phorbol 12-myristate 13-acetate (PMA)-stimulated reactive oxygen species (ROS) production and the adhesiveness to endothelial cells were increased in MNCs from CAD patients. After 24-h coculture with oxidized LDL, ROS elaboration of MNCs was significantly increased in CAD patients. It was further correlated to the endothelial adhesiveness of MNCs (r = 0.561, P < 0.05). Secondly, in an in vitro model for long-term, direct effects of oxidized LDL on murine monocytoid cells (MMCs), oxidized LDL, but not native LDL, induced ROS production of MMCs in a time-dependent manner up to a 4-day coincubation (261% elevation, P < 0.05). Four-day coincubation with ox-LDL enhanced cytoplasmic InB phosphorylation and nuclear factor kappa B (NF-nB) translocation and increased endothelial adhesiveness of MMCs. The long-term exposure to oxidized LDL also significantly enhanced tumor necrosis factor-a (TNF-a)-stimulated ROS production and endothelial adhesiveness of MMCs, which could be completely abolished by the short-term existence of pyrrolidine dithiocarbamate (PDTC), an antioxidant and NF-nB blocker and by long-term coincubation with N-acetylcysteine, a nonspecific antioxidant. Accordingly, circulating MNCs were activated with increased endothelial adhesiveness in CAD patients. Long-term exposure to oxidized LDL could directly activate MNCs ex vivo and MMCs in vitro and enhance TNF-a-stimulated endothelial adhesiveness through the redox-dependent NF-nB transcriptional pathway. The findings suggest the pivotal role of oxidized LDL-induced oxidative stress in monocyte activation during atherogenesis. D 2005 Elsevier Inc. All rights reserved. Keywords: Antioxidant; Atherogenesis; Cell adhesion; Endothelial cells; Mononuclear cells; Nuclear factor kappa B; Oxidized low-density lipoprotein; Reactive oxygen species; Tumor necrosis factor-a; Free radical Introduction Enhanced monocyte adherence to vascular endothelium is one of the earliest signs of atherogenesis observed at the cellular level [1,2]. It has been demonstrated that risk factors including hypertension, hyperglycemia, tobacco smoke, and lipoproteins especially low-density lipoprotein (LDL) with various degrees of oxidization, so-called oxidized LDL, can impair nitric oxide production of endothelial cells and increase their generation of superoxide, resulting in an increase of intra- as well as extracellular oxidative stress [3–7]. The increased superoxide generation in endothelial cells may then activate redox-sensitive transcriptional pathways such as nuclear factor kappa B (NF-nB), resulting in enhanced expression of 0891-5849/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.freeradbiomed.2005.10.037 Abbreviations: CAD, coronary artery disease; D-MEM, Dulbecco’s modified Eagle’s medium; EMSA, electrophoretic mobility-shift assay; HAECs, human aortic endothelial cells; IL-1h, interleukin-1h; LDH, lactate dehydrogenase; LDL, low-density lipoprotein; MMCs, murine monocytoid cells; MNCs, mononuclear cells; NAC, N- acetylcysteine; NF-nB, nuclear factor kappa B; ox-LDL, oxidized low-density lipoprotein; PBS, phosphate- buffered saline; PDTC, pyrrolidine dithiocarbamate; PMA, phorbol 12- myristate 13-acetate; ROS, reactive oxygen species; TNF-a, tumor necrosis factor-a; VCAM-1, vascular cell adhesion molecule-1. * Corresponding author. Fax: +886 2 2871 1601. E-mail address: jwchen@vghtpe.gov.tw (J.-W. Chen). Free Radical Biology & Medicine 40 (2006) 817 – 826 www.elsevier.com/locate/freeradbiomed