Abstract
Alpha-linolenic acid (ALA) and linoleic acid (LA) are precursors for longer-chain more unsaturated fatty acids and for lipid signalling molecules that may influence inflammatory processes through a variety of mechanisms. The actions of LA and ALA may be divergent and interdependent. The aim of this study was to investigate the incorporation and metabolism of ALA and LA in cultured in EA.hy926 endothelial cells and the production of inflammatory mediators (VEGF, RANTES, ICAM-1, MCP-1, IL-6 and IL-8) by these cells when exposed to different concentrations of ALA, LA and ratios of LA:ALA. Human endothelial cells were cultured with either culture medium or culture medium supplemented with ALA, LA or various ratios of LA:ALA (1:4, 1:1, 4:1, 9:1 or 19:1) followed by 24 h TNF-α stimulation; the total concentration of ALA plus LA was kept constant at 100 μM. The incorporation and metabolism of ALA and LA was measured using gas chromatography. The production of inflammatory mediators in the supernatant was assessed using a Luminex Multi-Analyte kit. Both ALA and LA were incorporated and metabolised by the endothelial cells. Cells incubated with ALA had a statistically significantly lower production of VEGF, RANTES, ICAM-1, MCP-1 and IL-6 compared to cells incubated without additional ALA. LA was not found to exert pro-inflammatory effects. Cells incubated with low LA:ALA ratios had lower production of VEGF, RANTES, MCP-1 and IL-6 when compared with a LA:ALA ratio of 19:1. These findings suggest that a low LA:ALA ratio exerts anti-inflammatory effects by lowering the production VEGF, RANTES, ICAM-1, MCP-1 and IL-6 in TNF-α stimulated endothelial cells compared to a high ratio. These effects were likely mediated by ALA, but LA may also possess some anti-inflammatory effects.
Link to Full Text
Key Points
Long chain n-3 PUFAs may exert inhibitory effects on inflammation by reducing leucocyte chemotaxis, adhesion molecule expression and leukocyte-endothelial adhesive interactions, and production of pro-inflammatory cytokines and of pro-inflammatory eicosanoids from arachidonic acid (AA). They also promote increased production of inflammation-resolving mediators. The suggested competitive mechanisms between ALA and LA imply that lower ratios of LA:ALA may be associated with a higher conversion efficiency of ALA and a lower production of inflammatory mediators when compared to higher ratios. The objectives of this study were to investigate the incorporation and metabolism of ALA and LA in cultured endothelial cells and to explore the production of inflammatory mediators by these cells when exposed to different concentrations of ALA, LA and ratios of LA:ALA. In this in vitro study, it was found that TNF-α stimulated endothelial cells preincubated with low LA: ALA ratios had a lower production of VEGF, RANTES, MCP-1 and IL-6 compared to cells preincubated with a high LA:ALA ratio. The observed anti-inflammatory effects were likely a result of increased ALA exposure because endothelial cells preincubated with ALA alone had a lower production of the same cytokines compared to DMEM controls, whereas much less of an effect was seen in cells incubated with LA alone. Both ALA and LA were incorporated into the cells in a dose-dependent pattern, but no appreciable differences were seen in the content of AA in the cells across ratios of LA:ALA. In endothelial cells incubated with ALA alone, higher contents of ETA, EPA and DPA were noted compared to DMEM control suggesting that ALA is metabolised into these longer chain PUFAs, whereas no discernible differences were seen across ratios of LA:ALA, which could indicate competition from LA on ALA metabolism. In conclusion, this study suggests that a low LA:ALA ratio exerts anti-inflammatory effects by lowering the production VEGF, RANTES, ICAM-1, MCP-1 and IL-6 in TNF-α stimulated endothelial cells compared to a high ratio, which was likely to be mediated by a higher ALA exposure rather than a lower LA exposure.