Toll-Like Receptor 4 Mediates Endothelial Cell Activation Through NF-kB but Is Not Associated with Endothelial Dysfunction in Patients with Rheumatoid Arthritis Rossella Menghini 1 , Umberto Campia 2 *, Manfredi Tesauro 1 , Arianna Marino 1 , Valentina Rovella 1 , Giuseppe Rodia 1 , Francesca Schinzari 3 , Barbara Tolusso 4 , Nicola di Daniele 1 , Massimo Federici 1,5 , Angelo Zoli 4 , Gianfranco Ferraccioli 4 , Carmine Cardillo 3 1 Department of System Medicine, University of Tor Vergata, Rome, Italy, 2 Division of Cardiology, MedStar Heart Institute, Washington, DC, United States of America, 3 Department of Internal Medicine, Catholic University Medical School, Rome, Italy, 4 Department of Rheumatology, Catholic University Medical School, Rome, Italy, 5 Center for Atherosclerosis, Policlinico Tor Vergata, Rome, Italy Abstract Objective: To investigate the effects of TLR4 antagonism on human endothelial cells activation and cytokine expression, and whether the Asp299Gly TLR4 polymorphism is associated with better endothelial function in patients with rheumatoid arthritis (RA). Methods: Human aortic endothelial cells (HAECs) were treated with lipopolysaccharide (LPS), OxPAPC, and free fatty acids (FFA) at baseline and after incubation with the TLR4 antagonist eritoran (E5564). Cytokine expression was assessed by quantitative real-time PCR. In vivo endothelial function was assessed as brachial artery flow-mediated dilation (FMD) in RA patients with the wild type gene (aa) and with the Asp299Gly TLR4 polymorphic variant (ag). Results: In HAEC, TLR4 antagonism with eritoran inhibited LPS-induced mRNA expression of IL-6, IL-8, TNFa, CCL-2, VCAM and ICAM (P,0.05 for all) and inhibited Ox-PAPC-induced mRNA expression of IL-8 (P,0.05) and IL-6, albeit not to a statistically significant level (p = 0.07). In contrast, eritoran did not affect FFA-induced mRNA expression of IL-6 (P.0.05). In 30 patients with RA (15 with the ag allele) undergoing measurement of FMD, no differences in FMD and plasma levels of IL- 6, IL-8, VCAM, and ICAM were found between the aa and the ag phenotype (P.0.05 for all). Conclusions: TLR4 signaling in endothelial cells may be triggered by LPS and oxidized phospholipids, leading to endothelial activation and inflammation, which are inhibited by eritoran. Our in vivo investigation, however, does not support an association between the Asp299Gly TLR4 polymorphism and improved endothelium-dependent vasodilator function in patients with RA. Further study is needed to better understand the potential role of TLR4 on endothelial dysfunction in this and other patient populations. Citation: Menghini R, Campia U, Tesauro M, Marino A, Rovella V, et al. (2014) Toll-Like Receptor 4 Mediates Endothelial Cell Activation Through NF-kB but Is Not Associated with Endothelial Dysfunction in Patients with Rheumatoid Arthritis. PLoS ONE 9(6): e99053. doi:10.1371/journal.pone.0099053 Editor: Angelo Scuteri, INRCA, Italy Received January 28, 2014; Accepted May 9, 2014; Published June 11, 2014 Copyright: ß 2014 Menghini et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work as supported by an intramural grant of Tor Vergata University to Dr. Menghini. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: umberto.campia@medstar.net Introduction Chronic inflammation represents a pivotal mechanism in the pathogenesis of atherosclerosis [1]. Interestingly, recent evidence suggests that innate immunity may also contribute to the development of vascular damage by interacting with inflammatory pathways [2]. In particular, toll-like receptors (TLRs) are increasingly being recognized as a link between the innate immune system, inflammation, and atherogenesis. This family of innate immune receptors is expressed by endothelial cells, in which they trigger various signaling pathways and lead to cell activation, increased expression of inflammatory cytokines and adhesion molecules, and endothelial dysfunction [3,4]. While initially identified as sensors of microbial invasion, TLRs are now known to be activated also by endogenous ligands produced in inflamed tissues, potentially leading to further inflammation and perpetu- ating an inflammatory milieu [3]. Among them, TLR4, a receptor for lipopolysaccharide (LPS) from Gram negative bacterial cell walls, also exhibits affinity for fatty acids [5], extracellular matrix components, fibrinogen, and various heat shock proteins [6]. Of note, TRL4 signaling leads to activation of NF-kB [4], a pathway associated with andothelial injury [7], and TRL4 expression is increased in human atherosclerotic plaques [8]. Additionally, lack of TLR4 reduces atherosclerosis and alters plaque phenotype in apoE-deficient mice fed a high-cholesterol diet [9]. In agreement with these data, clinical evidence indicates that the Asp299Gly TLR4 polymorphism, a functional variant in the TLR4 gene (896ARG) that attenuates receptor signaling and diminishes the PLOS ONE | www.plosone.org 1 June 2014 | Volume 9 | Issue 6 | e99053