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Canadian Journal of Cardiology

INCREASED EICOSAPENTAENOIC ACID TO ARACHIDONIC ACID (EPA/AA) RATIO IN BRAIN ENDOTHELIAL CELLS ASSOCIATED WITH REDUCED EXPRESSION OF NEUTROPHIL DEGRANULATION PROTEINS DURING INFLAMMATION

      Background

      Endothelial cell (EC) dysfunction results in inflammation and increased atherothrombotic risk, including stroke. The omega-3 fatty acid (FA) eicosapentaenoic acid (EPA) improves arterial compliance in a manner predicted by circulating EPA/AA ratios, an indicator of cardiovascular (CV) risk. Treatment with icosapent ethyl (IPE), a formulation of highly purified EPA, reduced composite CV events, including stroke, in REDUCE-IT that correlated with on-treatment EPA levels. We measured the effects of EPA on the EPA/AA ratio and protein expression in endothelial cells from brain ECs (BECs) following cytokine challenge.

      Methods and Results

      BECs were challenged with IL-6 (12 ng/mL) for 2 hours and then treated with EPA (40 μM) or equivolume vehicle and incubated for 24 hours. Samples were allocated for FA analysis or proteomic analysis. Total cellular FAs were extracted, derivatized to FA methyl esters (FAME), and analyzed using gas chromatography. Total protein content was also measured and used to normalize fatty acid content. Global proteomic analysis was performed using LC/MS to measure relative expression levels of proteins between treatment groups. Significant (p < 0.05) changes in expression between treatment groups (>1-fold) were analyzed using differential enrichment analysis of proteomics data (DEP). Biological pathways were analyzed using proteins that survived the cutoff criteria via gene set enrichment analysis (GSEA). EPA treatment significantly modulated proteins in the “neutrophil degranulation” pathway (GO:0043312) with pathway adjusted-p values of 3.68 × 10-13. EPA significantly altered expression of 66 proteins in this pathway, including increasing peroxiredoxin-6 and heat shock protein 90, relative to IL-6. These changes in protein expression in BECs correlated with large, significant increases in the EPA/AA ratio from 0.025 ± 0.002 to 1.72 ± 0.20 (>67-fold increase, p< 0.001).

      Conclusion

      EPA favorably modulated proteins in brain ECs related to neutrophil degranulation in a manner that correlated with increases in the EPA/AA ratio. The anti-inflammatory benefits may contribute to reduced stroke risk with IPE as demonstrated in outcome trials.