Effect of 18-carbon cis and trans fatty acids on inflammatory responses of cultured human endothelial cells

Abstract

The endothelium plays a key role in maintaining vascular homeostasis by regulating vascular tone and permeability. Endothelial dysfunction is a pro-inflammatory state characterized by chronic activation of the endothelium, which leads to atherosclerosis and cardiovascular disease (CVD). Intake of trans fatty acids (TFAs) is associated with an increased risk of CVD and other chronic diseases and this risk is usually associated with industrial TFAs rather than ruminant TFAs; however the effect of TFAs on inflammation remains unclear. The aim of this project was to compare the inflammatory response in cultured EA.hy926 endothelial cells (ECs) exposed to different 18-carbon trans fatty acids (trans vaccenic acid (TVA), elaidic acid (EA), cis vaccenic acid, conjugated cis-9,trans-11 and trans-10, cis-12 linoleic acids (CLAs)) of ruminant and industrial origin and to two 18-carbon cis fatty acids (oleic and linoleic).

EA.hy926 cells remained viable at the different FA concentrations used, with the exception of CLA10,12 at 50 μM. In addition, the FAs tested were incorporated into ECs in culture in a dose dependent manner, and this incorporation was accompanied by the appearance of some metabolic products. EA increased production of most of the cytokines and adhesion molecules measured after inflammatory stimulation, while TVA caused their reduction in non-stimulated cells and showed a neutral effect in tumour necrosis factor (TNF)-α stimulated ECs. EA also showed some anti-inflammatory effects under basal conditions when used at low concentrations. In relation to the effect of the FAs on expression of inflammatory pathway genes, EA (50 µM) induced a significant upregulation of toll-like receptor (TLR)-4 and cyclooxygenase (COX)-2 gene expression in TNF-α stimulated cells. In contrast, TVA (1 µM) induced a significant downregulation of nuclear factor kappa B subunit 1 (NFκB1) in TNF-α stimulated cells. TVA (10 µM) also decreased the relative gene expression of COX-2 under basal conditions. EA (50 µM) induced a significant increase of THP-1 monocyte cell adhesion to ECs, both with and without TNF-α stimulation. In contrast, TVA (1 µM) reduced THP-1 monocyte adhesion, under basal conditions and after TNF-α stimulation, and decreased the level of surface expression of ICAM-1 when used at 50 µM.

In relation to the effects of the CLAs, CLA9,11 had neutral or anti-inflammatory effects in non-stimulated ECs, reducing the levels of monocyte chemoattractant protein 1, regulated on activation normal T expressed and secreted, interleukin (IL)-8 and IL-6. CLA10,12 exposure reduced the same cytokines in basal conditions, which the exception of IL-6, which was increased. In contrast, CLA10,12 increased the levels of most of the inflammatory mediators after TNF-α stimulation. CLA9,11 had null effects after inflammatory stimulation. Both CLAs increased NFκB gene expression under basal conditions, together with a reduction of peroxisome proliferator activated receptor α, COX-2 and IL-6 gene expression. Instead, both CLAs (1 μM) decreased the NFκB mRNA appearance after TNF-α stimulation, but only CLA9,11 maintained this downregulation at 10 μM. CLA10,12 had no effect on THP-1 adhesion while decreasing significantly the % of cells expressing ICAM-1 and also levels of ICAM-1 expression per cell when used at 10 µM. Although CLA9,11 did not have any effect on ICAM-1 cell surface expression, it reduced the monocyte adhesion to the EA.hy926 cell monolayer.

The results suggest that TVA has some anti-inflammatory properties, while EA enhances the response to an inflammatory stimulus. In addition, both CLAs showed similar effects under basal conditions, acting generally in a neutral or modest anti-inflammatory way. In contrast, after TNF-α stimulation, CLA10,12 showed some pro-inflammatory effects. This suggests that the EC responses to some FAs may change depending on the presence of inflammation. These findings suggest differential effects induced by the TFAs tested, in general fitting with the idea that industrial TFAs and ruminant TFAs can have different and perhaps opposing biological actions.

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Identifiers

ORCID for Philip Calder: orcid.org/0000-0002-6038-710X

ORCID for Elizabeth Miles: orcid.org/0000-0002-8643-0655

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