Toll-like receptor 4 mediates endothelial cell activation through NF-κB but is not associated with endothelial dysfunction in patients with rheumatoid arthritis.

Rossella Menghini,Nicola Di Daniele,Francesca Schinzari,Manfredi Tesauro,Barbara Tolusso,Carmine Cardillo,Angelo Zoli,Gianfranco Ferraccioli,Arianna Marino,Umberto Campia,Valentina Rovella,Massimo Federici,Giuseppe Rodia,Angelo Scuteri

doi:10.1371/journal.pone.0099053

Abstract

ObjectiveTo 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).MethodsHuman 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).ResultsIn HAEC, TLR4 antagonism with eritoran inhibited LPS-induced mRNA expression of IL-6, IL-8, TNFα, 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).ConclusionsTLR4 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.

Highlights

Chronic inflammation represents a pivotal mechanism in the pathogenesis of atherosclerosis [1]
Incubation with eritoran did not lead to significant changes in mRNA levels of these cytokines compared to control
To assess whether TLR4 receptor antagonism impacts LPSinduced expression of adhesion molecules in Human aortic endothelial cells (HAECs), we measured mRNA levels of VCAM and ICAM after treatment with LPS, alone and following pretreatment with eritoran for 30 minutes. compared with control, eritoran alone did not lead to significant changes in VCAM and ICAM mRNA levels

Summary

Introduction

Chronic inflammation represents a pivotal mechanism in the pathogenesis of atherosclerosis [1]. 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]. 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 inflammatory response to LPS [10], is associated with decreased atherosclerotic risk [11]. Whether antagonism of TLR4 prevents TLR4-induced expression of inflammatory cytokines and adhesion molecules in human macrovascular endothelial cells has not been investigated in detail

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Conclusion