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.
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Key Points
One of the most important functions of PUFA is related to their enzymatic conversion into eicosanoids. PUFA are released from membrane phospholipids by the action of various phospholipases and metabolized to different eicosanoids. Arachidonic acid (20:4ω-6, ARA) is the substrate for two classes of enzymes, cyclooxygenases (COX) which produce the 2-series of prostaglandins, prostacyclins and thromboxanes, and lipoxygenases (LOX) which catalyze the biosynthesis of hydroxyeicosatetraenoic acids (HETE) and the 4-series leukotrienes. EPA exhibits a similar metabolism to ARA, but it is metabolized to the 3-series prostaglandins, prostacyclins and thromboxanes through the action of the COX enzymes and 5-series leukotrienes and hydroxyeicosapentaenoic acids (HEPE) from LOX. DHA is mainly converted to D-series resolvins by LOX.
Human umbilical vein endothelial cells (HUVEC) can be regarded as a valuable tool for studying different biological aspects of endothelial cells. Their availability, easy to isolate in a pure form, low cost, rapid culturing and proliferation in simple laboratory settings (e.g., medium containing fetal bovine serum) made them a very attractive system for modelling the influence of ω-3 and ω-6 PUFA on the production of their associated pro- and anti-inflammatory biomarkers through the cyclic (COX enzyme) and linear (LOX enzyme) pathways.
The present research aims at studying the production of cyclooxygenase and lipoxygenase metabolites by HUVEC exposed to different combinations of PUFA. The research assesses specifically the levels of prostaglandins (PGE2, PGE3), prostacyclins (6-keto-PGF1α, Δ17-6-keto-PGF1α), resolvins (RvD1, RvD2, RvD3, RvD4 and 17-epi-RvD1) and leukotriene (LTB4) released into endothelial cell growth basal medium-2 (EBM-2) by HUVEC exposed to ARA, EPA, DHA and ALA, 18:3n-3. The study showed the inhibitory effect of DHA and ALA on the production of pro-inflammatory COX metabolites, the lack of correlation between EPA and production of anti-inflammatory prostaglandin or prostacyclin, the association between ARA and increased production of all pro-inflammatory metabolites, and the enhanced production of LOX metabolites by DHA. This research assesses the influence of single and combined ω-3 and ω-6 PUFA on the simultaneous production of eicosanoids and resolvins’ metabolites by HUVEC.
The present research has examined the association between different ω-3 and ω-6 PUFA and the release of ten different COX and LOX metabolites in cell culture medium by HUVEC. The present research confirmed earlier published results on the anti-inflammatory role of ω-3 PUFA (DHA and ALA), and the lack of effectivity of EPA towards the production of PGE3 in a HUVEC model. However, the non-significant production of PGE3 by EPA seems to be controversial and it has been reported previously in mammalian cell models, including HUVEC. In addition, exposing the cells to exogenous ARA resulted in a high production of COX metabolites. The high levels of COX metabolites were suppressed when ARA was combined with DHA, ALA or DHA×ALA. DHA enhanced all the LOX metabolites (LTB4 and resolvins), while ALA enhanced only the levels of the LTB4 but had no effect on resolvins.