Eicosapentaenoic acid (EPA) reduced expression of neutrophil activation proteins in endothelial cells during challenge with angiotensin II (Ang II) and cytokines

Abstract

Abstract Funding Acknowledgements Type of funding sources: Private company. Main funding source(s): Amarin Pharmaceutical, Inc. Elucida Research LLC. Background Neutrophil degranulation contributes to atherogenesis and tissue injury in response to cytokine release and activation of the renin-angiotensin system (RAS), along with its effector angiotensin II (Ang II). Mixed omega-3 fatty acid (n3-FA) formulations of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have not shown reduction in CV events compared to EPA-only formulations (REDUCE-IT), but the mechanisms of atheroprotection with EPA are not well understood. Purpose We measured the effects of EPA on expression of proteins related to neutrophil activation in vascular endothelial cells (ECs) under various inflammatory challenges, specifically exposure to Ang II and the cytokine IL-6 in vitro. Methods Human umbilical vein ECs (HUVECs) were challenged with the cytokine IL-6 at (12 ng/mL) and Ang II (100 nM) for 2 h, then treated with EPA (40 µM) for 24 h. Global proteomic analysis was performed using LC/MS to measure relative expression levels of over 2,500 proteins simultaneously. Only significant (p<0.05) changes in expression between treatment groups >1-fold (100%) were analyzed by differential enrichment analysis of proteomics data (DEP) and included in gene set enrichment analyses (GSEA). Results EPA significantly down/up-regulated expression of 507/505 and 544/472 proteins, respectively, compared with Ang II and IL-6 alone. The GSEA revealed EPA treatment significantly modulated expression of 88 and 80 proteins within the neutrophil degranulation pathway (Gene Ontology ID: 0043312), compared to Ang II and IL-6, respectively. Of these proteins, 20 were similarly modulated by EPA relative to each stimulus (13 decreased, 7 increased expression). This included a 110% decrease in expression of heat shock protein 90 (110%) and HSP 86 (110%) and a 140% and 110% increase in junction plakoglobin relative to Ang II and IL-6, respectively, which is also involved in endothelial cell-cell adhesion. Conclusion EPA modulated expression of multiple proteins linked to neutrophil degranulation following challenge with either Ang II or IL-6. The effects of EPA on neutrophil protein expression may lead to reduced inflammation and risk for patients with CV disease as demonstrated in outcome trials.