Elsevier

Atherosclerosis

Volume 179, Issue 2, April 2005, Pages 247-254
Atherosclerosis

Very low density lipoprotein potentiates tumor necrosis factor-α expression in macrophages

https://doi.org/10.1016/j.atherosclerosis.2004.12.002Get rights and content

Abstract

High levels of the triacylglycerol-rich lipoproteins, very low density lipoprotein (VLDL) and intermediate density lipoprotein (IDL) have been identified as independent risk factors for coronary heart disease, and inflammation is thought to contribute to atherosclerosis and its complications. To understand how dyslipidemia promotes inflammation, we have characterised the effects of VLDL treatment on production of tumor necrosis factor-α (TNF) by human monocyte-derived macrophages. VLDL strongly potentiated lipopolysaccharide (LPS)-induced expression of TNF mRNA and secretion of TNF protein. VLDL activated mitogen-activated protein kinase-ERK kinase 1/2 (MEK1/2), and potentiated LPS-induced MEK1/2 activation. The MEK1/2 inhibitor U0126 strongly diminished TNF expression, indicating that MEK1/2 plays a central role in the regulation of TNF expression. VLDL did not activate transcription factors NF-κB and PPAR-γ, but it activated AP-1 at least as potently as LPS, and potentiated LPS-induced activation of AP-1. The inhibitor U0126 completely prevented this potentiation. Inhibition of AP-1 by decoy oligonucleotides abolished potentiation of TNF secretion by VLDL. In conclusion, VLDL treatment potentiates TNF expression in macrophages by activation of MEK1/2 and AP-1. These findings suggest that triacylglycerol-rich lipoproteins are involved in inflammatory processes associated with atherosclerosis.

Introduction

Even without prior oxidative modification, chylomicron remnants, VLDL and IDL can be taken up in large amounts by macrophages. Lipid accumulation in macrophages leads to formation of foam cells, a distinct cell type found in fatty streaks and more advanced atherosclerotic lesions. Apart from effects on the endothelial expression of cell adhesion molecules [1], the effects of intracellular lipid accumulation on inflammatory gene expression have not been extensively studied.

VLDL (Sf 20 to 60) and IDL (Sf 12 to 20) have been shown to be independently associated with the presence, severity, and progression of atherosclerosis [2], [3]. VLDL and VLDL remnants have been found in atherosclerotic plaques [4].

Increased levels of TNF have been detected in young survivors of myocardial infarction [5]. TNF expression in humans is also elevated in atheroma [6] and in macrophages from atherosclerotic lesions [7]. Arbustini et al. [8] observed that TNF expression was highest in lipid-rich regions of atherosclerotic lesions.

It was recently reported that, compared to normolipidemic controls, patients with familial hypercholesterolemia exhibit significantly lower TNF production in blood stimulated ex vivo by LPS [9]. In contrast, the same investigators observed that LPS treatment of blood from hypertriglyceridemic patients led to production of more TNF than did the same treatment of blood from control subjects, and they also found that lowering of serum triacylglycerol decreased the TNF levels [9]. These findings suggest that VLDL and other triacylglycerol-rich lipoproteins (but not LDL) augment production of TNF, and this conclusion is supported by results obtained in hypercholesterolemic rabbits [10]. We have previously shown that treatment of LDL has a suppressive effect on TNF expression in macrophages, likely mediated by downregulation of transcription factor AP-1 [11]. In the present study, we examined how VLDL influences TNF expression. Mitogen-activated protein kinase (MAPK) pathways are known to regulate TNF expression in macrophages [12]. Using U0126, a specific inhibitor of MEK1/2 activity, we studied MAPK involvement in TNF expression induced by LPS and VLDL.

Section snippets

Materials

[γ-32P]ATP (185 TBq/mmol/L) was purchased from Amersham Biosciences (Buckinghamshire, UK). The oligonucleotides for mobility shift assays of AP-1 and NF-κB were obtained from Promega (Madison, WI) and Santa Cruz Biotechnology (Santa Cruz, CA). TNF was from R&D Systems (Minneapolis, MN). Tris–buffered saline (pH 7.2) was from Pierce (Rockford, IL) and Ficoll-Paque was purchased from Amersham Biosciences and was used according to the instructions of the manufacturer. Macrophage-SFM culture medium

Dose response study

Cells were treated with 0, 5, 10, 25, 50, 75 and 100 μg/mL VLDL for 8 h, and thereafter, medium was replaced with VLDL-free medium, and the cells were further incubated for 12 h in the absence or presence of 1 μg/mL LPS. The culture medium was collected, and TNF secretion was analyzed by ELISA. The VLDL concentration of 75 μg/mL gave the strongest, statistically significant potentiation of TNF secretion (Fig. 1A). This concentration was therefore used in subsequent experiments.

Effect of VLDL on LPS-induced TNF synthesis

In our preliminary

Discussion

Our results show that VLDL potentiates LPS-induced TNF expression in macrophages, as determined by Northern blotting and analysis of TNF in culture medium by ELISA. Activation of MEK1/2 and AP-1 was observed in cells treated with VLDL alone, but was much stronger in cells treated with LPS and VLDL. TNF expression was strongly inhibited by U0126, a specific inhibitor of MEK1/2 activity. MEK1/2 has been shown to be involved in AP-1 activation [20]. Accordingly, U0126 inhibited the VLDL-induced

Acknowledgements

This study was supported by grants from the Swedish Research Council (no. 8311), the Swedish Heart and Lung Foundation, the Wallenberg Foundation, the Swedish Medical Society, the Crafoord Foundation, Ernhold Lundström Foundation, Malmö University Hospital, The Royal Physiographic Society, and Lars Hierta Foundation. We thank Anna Larsson and Linda Andersson for expert technical assistance.

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