Elsevier

Atherosclerosis

Volume 207, Issue 1, November 2009, Pages 38-44
Atherosclerosis

LXR-activating oxysterols induce the expression of inflammatory markers in endothelial cells through LXR-independent mechanisms

https://doi.org/10.1016/j.atherosclerosis.2009.04.001Get rights and content

Abstract

Aims

Liver X receptors α and β (LXRα, LXRβ) are key regulators of cholesterol homeostasis. The effects of LXR ligands on endothelial cells are largely unknown. While oxysterol LXR agonists can increase the endothelial–leukocyte interaction, synthetic LXR agonists are anti-atherogenic and anti-inflammatory. Mechanistic differences may underlie such findings.

Methods and results

LXRα and LXRβ were found to be expressed in human endothelial cells. While synthetic LXR agonists could blunt the LPS-induced up-regulation of adhesion molecules (ICAM-1, VCAM-1, E-Selectin), 22-hydroxycholesterol and 24,25-epoxycholesterol enhanced such response. Microarray profiling further showed that the endothelial gene expression fingerprints of 22-hydroxycholesterol and T0901317 largely differed and unexpectedly shared only a restricted number of genes. Indeed, 22-hydroxycholesterol down-regulated eNOS and up-regulated a vast cohort of inflammatory mediators such as adhesion molecules, cytokines, enzymes and transcription factors. Other LXR-activating oxysterols such as 24,25-epoxycholesterol, 25-hydroxycholesterol and 27-hydroxycholesterol could also stimulate the endothelial expression of inflammatory markers, although significant differences were observed. These effects persisted in LXR-silenced cells, confirming the mechanistic dissociation of oxysterol and LXR pathways. Furthermore, the oxysterol-induced expression of inflammatory markers was not secondary to cell apoptosis and may relate to oxidative stress.

Conclusions

LXR-activating oxysterols comprehensively activate the expression of endothelial inflammation markers independently from LXRs. At proper dosage, synthetic LXR agonists are safe on endothelial cells and may even transrepress inflammatory reactions.

Introduction

Nuclear receptors LXRα and LXRβ (Liver X receptors, NR1H3 and NR1H2) are master regulators of body cholesterol and control its intestinal absorption, reverse transport and liver metabolism [1]. Endogenous LXR agonists include 22-hydroxy, 24,25-epoxy, 25-hydroxy and 27-hydroxycholesterol (22OH, 24,25E, 25OH, 27OH). These oxysterol compounds derive from cholesterol via enzymatic and non-enzymatic reactions. The latter depend on oxidative radicals, while the enzymatic production of LXR-activating oxysterols parallels cholesterol loading [2], [3]. Recently, several synthetic LXR agonists have been developed, such as T0901317 and GW3965. The administration of these compounds to apoE- and LDR-deficient mice reduces the development and progression of atherosclerosis, owing to a more efficient reverse cholesterol transport and to a net inhibition of plaque inflammation [4], [5], [6], [7], [8], [9], [10]. LXR agonists have thus emerged as promising anti-atherosclerotic drugs.

In vivo, endothelial cells can be exposed to LXR-activating oxysterols released from atherosclerotic plaques, oxidized lipoproteins and lipid-loaded cells [3], [11]. Endothelial cells express functional LXRs and are able to up-regulate ABC transporters upon treatment with LXR agonists [12], [13]. Moreover, shear stress has been shown to activate the expression and function of endothelial LXRs [14]. However, it was also previously shown that 25OH can also augment eicosanoid release from cultured endothelial cells and increase endothelial–leukocyte interaction through the up-regulation of VCAM1 [15], [16]. Furthermore, 25OH can increase cytokine production in non-endothelial cells [17], [18]. Contradicting the anti-atherosclerotic actions of synthetic LXR agonists, LXR-activating oxysterols have thus been proposed as pro-inflammatory and atherogenic mediators. Such paradox prompts to underlying differences in mechanism and awaits further characterization. The objective of the present study was therefore to specifically and comprehensively examine the effects of LXR agonists on human endothelial cells. In particular, our specific aims were: (1) to clarify if LXR agonists can indeed modulate the expression of endothelial inflammatory markers and (2) to uncover any mechanistic dissociation between oxysterol and synthetic LXR agonists in endothelial cells.

Section snippets

Materials and methods

For detailed procedures, see Supplementary Materials and Methods.

LXRs are expressed and functional in human endothelial cells

LXRα and LXRβ mRNA and protein were detected in both HUVEC and HUAEC and showed a preferential nuclear localization (Fig. 1A and B). Treatment of HUVEC with 22OH, 24,25E, T0901317 and GW3965 lead to a significant up-regulation of LXR target gene ABCA1, indicating that endothelial LXRs can regularly transactivate and recruit the transcriptional machinery (Fig. 1C). Accordingly, the transcriptional inhibitor actinomycin D could blunt the up-regulation of ABCA1 by LXRs.

LXR agonists modulate endothelial responses to inflammatory stimuli

Opposing effects have been

Discussion

In the present work, we have shown that oxysterol LXR agonists comprehensively stimulate inflammatory pathways in endothelial cells, leading to the up-regulation of adhesion molecules (ICAM1, VCAM1, SELE), chemokines (IL8, IL1α, CCLs and CXCLs), transcription factors (EGR1, FOS) and enzymes (COX2), and to the down-regulation of eNOS. These findings are in line with previous studies, which have reported that 25OH can promote endothelial–leukocyte interaction, augment eicosanoid release from

Acknowledgments

The study was supported by grants from M.U.R. (ex 60%), Regione Piemonte (Ric. Fin. 2006 to FV and 2008 to FM), University of Torino and Compagnia di San Paolo di Torino.

References (30)

  • N. Zelcer et al.

    Liver X receptors as integrators of metabolic and inflammatory signaling

    J Clin Invest

    (2006)
  • S.B. Joseph et al.

    Synthetic LXR ligand inhibits the development of atherosclerosis in mice

    Proc Natl Acad Sci USA

    (2002)
  • B. Hu et al.

    Discovery of phenyl acetic acid substituted quinolines as novel liver X receptor agonists for the treatment of atherosclerosis

    J Med Chem

    (2006)
  • M.N. Bradley et al.

    Ligand activation of LXR{beta} reverses atherosclerosis and cellular cholesterol overload in mice lacking LXR{alpha} and apoE

    J Clin Invest

    (2007)
  • D. Peng et al.

    Differential anti-atherosclerotic effects in the innominate artery and aortic sinus by the liver X receptor agonist T0901317

    Atherosclerosis

    (2008)
  • Cited by (59)

    • Liver X receptor activation mitigates oxysterol-induced dysfunction in fetoplacental endothelial cells

      2024, Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids
    • Oxysterols and nuclear receptors

      2019, Molecular and Cellular Endocrinology
    • Up-regulation of COX-2 and mPGES-1 by 27-hydroxycholesterol and 4-hydroxynonenal: A crucial role in atherosclerotic plaque instability

      2018, Free Radical Biology and Medicine
      Citation Excerpt :

      Of note, LXR activation induces the expression of genes different from those stimulated by endogenous oxysterols. Therefore, although LXR-activating oxysterols might reduce inflammation, they can also act by activating opposing pathways and inducing expression of inflammation markers independently of LXRs [73]. Another beneficial effect of 27-OH is due to its ability to protect human macrophages from cholesterol overload [75] since oxysterols, by binding to LXR, might act as “cholesterol sensors”, increasing the expression of target genes associated with reverse cholesterol transport [74].

    View all citing articles on Scopus
    View full text