Effects of fatty acids on endothelial cells: inflammation and monocyte adhesion

Abstract

Background: Diet is known to have an important impact on cardiovascular health. n-3 Fatty acids (FAs), found in high quantity in fish oil, have demonstrated beneficial effects in patients with coronary artery disease. The role of n-6 FAs remains more controversial. The objective of this study was to examine the effect of arachidonic acid (AA), an n-6 FA, and eicosapentanoic acid (EPA), an n-3 FA, on the interaction between monocytes and endothelial cells (ECs).

Design: We used a cellular model of ECs (EA.hy.926) and monocytes (human leukemic myelomonocytic U937). Confluent ECs were treated with AA or EPA, in the presence of tumor necrosis factor-alpha (TNF-α) or vehicle alone for either 4 or 24h. Adhesion of monocytes to the endothelial monolayer was performed. For gene expression, reverse transcription, followed by real-time quantitative polymerase chain reaction, was performed.

Results: There was a significant increase in adhesion of monocytes to the endothelial monolayer in the presence of n-6 FAs, both in the presence and in the absence of TNF-α at 4 and 24h. The adhesion of monocytes to the endothelial monolayer was decreased with n-3 FAs at 24h. Intercellular adhesion molecule 1, vascular cell adhesion molecule 1, E-Selectin, Interleukin 6, and TNF-α were significantly increased in ECs treated with n-6 FAs.

Conclusions: We conclude that AA increases inflammation and enhances the ability of ECs to bind monocytes in vitro. EPA leads to a decrease in the ability of EA.hy.926 to bind monocytes, although the effect appears more modest. Taken together, these data indicate that the n-6 FA AA could potentiate inflammation and early events of atherosclerosis.

Published by Elsevier Inc.

Figures

Figure 1. Monocyte Adhesion After Treatment of Endothelial cells with n-3 and n-6 Fatty Acids for 4 hours (A) and 24 hours (B)

To assess the effects of FA on endothelial cells’ ability to bind monocytes, ECs were grown to confluency and treated with n-3 and n-6 FA (5ug/ml) with or without TNF-α (100ng/ml) for 4 and 24 hours. At 4 hours, in the absence or presence of TNF-α, adhesion of monocytes to the endothelial monolayer is higher in the n-6 FA treated cells than in the non-treated or n-3 FA treated cells. This remains true at 24 hours. Means ± SD (n=8). *= P ≤ 0.05 for n-3 or n-6 treatment compared to the control without FA. † ≤ P ≤ 0.001 between n-3 and n-6 FA treatment groups.

Figure 2. Monocyte Adhesion to Endothelial Cells at Fluorescence Microscopy after Treatment with n-6 Fatty Acids

Confluent ECs were incubated with 5 μg/ml AA (n-6 FA) or vehicle alone for 5 hr prior to a 30-minute incubation with calcein-AM labeled U937 cells. The coverslips were gently dipped in PBS to remove unbound U937 cells then imaged by fluorescence microscopy. ECs were labeled with Hoescht stain (Blue) and U937 cells were labeled with Calcein-Am (Green). Fluorescence microscopy demonstrates an increase in monocytes binding to ECs after treatment with n-6 FA.

Figure 3. Effect of Pre-Treatment of Endothelial cells with Indomethacin, a COX-2 inhibitor on Monocyte Adhesion

To evaluate if the cellular effects of n-6 FA are mediated through the cyclooxygenase (COX) pathway, ECs were exposed to indomethacin, a COX-2 inhibitor, at the dose of 50 uM in the presence and absence of the n-6 FA AA. Indomethacin decreased the adhesion of monocytes to the n-6 treated endothelial monolayer. Indomethacin alone did not affect adhesion. Means ± SD (n=8). *=P ≤ 0.001 n-6 treated endothelial monolayer compared to no-treatment. † = P = 0.001 for n-6 treated endothelial monolayer compared to n-6 treated endothelial monolayer following pre-treatment with indomethacin.

Figure 4. Gene Expression in Endothelial Cells After Treatment of n-3 and n-6 Fatty Acids using qRTPCR analysis

To assess molecular mechanisms involved in the alterations in monocyte binding to treatment with FAs (4 hours), qRTPCR analysis of gene expression was done. Treatment of endothelial cells with FA in both the activated (with TNFα) and non-activated states shows a marked increase in expression of adhesion molecules and inflammatory mediators in n-6 compared to n-3 FA. Means ± SD (n=4). *= P ≤ 0.05 n-3 or n-6 treatment compared to the control without FA. †= P ≤ 0.05 between n-3 and n-6 FA treatment groups.