The Effect of Omega-3 and Omega-6 Polyunsaturated Fatty Acids on the Production of Cyclooxygenase and Lipoxygenase Metabolites by Human Umbilical Vein Endothelial Cells

Abstract

Although the correlation between polyunsaturated fatty acids (PUFA) and the production of pro- and anti-inflammatory metabolites is well documented, little is known about the simultaneous effect of different PUFA on the production of cyclooxygenase and lipoxygenase metabolites. The present research examines the association between different omega-3 (ω-3) and omega-6 (ω-6) PUFA and the release of four cyclooxygenase and six lipoxygenase metabolites in cell medium by human umbilical vein endothelial cells (HUVEC). The different combinations of ω-3 and ω-6 PUFA were prepared according to a full 2⁴ factorial design that enables studying not only the main effects but also the different interactions between fatty acids. In addition, interactions diagrams and principal component analysis were useful tools for interpreting higher order interactions. To the best of our knowledge, this is the first report addressing the combined effect of ω-3 and ω-6 PUFA on the signaling of prostaglandins, prostacyclins, leukotrienes and resolvins by HUVEC.

Keywords: arachidonic acid; docosahexaenoic acid; eicosanoids; eicosapentaenoic acid; human umbilical vein endothelial cells; leukotrienes; polyunsaturated fatty acids; prostacyclins; resolvins; α-linolenic acid.

Figure 1

Metabolism of different ω-3 and ω-6 PUFA. EPA and ARA compete for the same cyclooxygenase (COX) and lipoxygenase (LOX) enzymes and they are converted into prostaglandins, prostacyclins and thromboxanes by COX enzymes and into leukotrienes, lipoxins, hydroxyeicosatetraenoic acids by different LOX enzymes. DHA produces the anti-inflammatory resolvins D (RvD) through the action of LOX enzymes (5-LOX and 15-LOX). The studied PUFA and metabolites in the present research are indicated by blue and red stars, respectively.

Figure 2

Average production of prostaglandins and prostacyclins by HUVEC exposed to different PUFA combinations. The asterisks indicate significant values at p < 0.05 (*), p < 0.001 (**) and p < 0.000 (***). The actual numerical p-values are described in Table S2.

Figure 3

Average production of leukotriene and resolvins by HUVEC exposed to different PUFA combinations. The asterisks indicate significant values at p < 0.05 (*), p < 0.001 (**) and p < 0.000 (***).The actual numerical p-values are described in Table S2.

Figure 4

Average production of resolvins by HUVEC exposed to different PUFA combinations. The asterisks indicate significant values at p < 0.05 (*), p < 0.001 (**) and p < 0.000 (***).The actual numerical p-values are described in Table S2.

Figure 5

Interaction diagrams for elucidating the effect between two different PUFA on the production of cyclooxygenase metabolites by HUVEC. Explanation about how the diagrams were calculated is given in Section 3.2 and supporting Figure S1. Values in picograms (pg) are expressed as average ± standard error.

Figure 6

Interaction diagrams for elucidating the effect between two different PUFA on the production of lipoxygenase metabolites by HUVEC. Explanation about how the diagrams were calculated is given in Section 3.2 and supporting Figure S1. Values in picograms (pg) are expressed as average ± standard error.

Figure 7

PCA (a) score and (b) loading plots showing the relationship between the different combinations of ω-3 and ω-6 PUFA according to the proposed 2⁴ factorial design described in Table 1 and the relationship between the different COX and LOX metabolites, respectively. The plots show that DHA and ARA are the main substrates behind the production of LOX and COX metabolites, respectively. DHA is associated with a high production of resolvins, which is counter regulated by ARA through a high production of prostaglandins. The loading plot confirms that 78.12% of the total data variability is explained by the production of resolvins and prostaglandins. The numbers inside the nine different clusters correspond to the different PUFA combinations. 1=ALL; 2=ALL-ARA; 3=ALL-ALA; 4=DHA+EPA; 5=ALL-EPA; 6=DHA+ALA; 7=DHA+ARA; 8=DHA; 9=ALL-DHA; 10=EPA+ALA; 11=EPA+ARA; 12=EPA; 13=ALA+ARA; 14=ALA; 15=ARA; 16=+EtOH; 17=-EtOH.