Abstract Body (Do not enter title and authors here): Background: The Src family of non-receptor protein tyrosine kinases participate in numerous cell-signaling pathways, including cell-cell and cell-extracellular matrix interactions. In endothelial cells (ECs), Src kinases, including c-Src, participate in angiogenesis. The omega-3 fatty acid eicosapentaenoic acid (EPA) delivered as icosapent ethyl (IPE) and certain GLP-1 receptor agonists (GLP1-RA) independently reduce cardiovascular (CV) risk through potentially multifactorial mechanisms. Yet, the combined effects of these agents on CV risk remain poorly understood.

Hypothesis: We suspected the combination of the GLP-1RA liraglutide (lira) and EPA would modulate protein expression, including c-Src, in ECs during inflammation.

Methods: Human umbilical vein ECs (HUVECs) were challenged with Ang II (100 nM) for 2 h, then treated with lira (50 nM) and/or EPA (40 µM) for 24 h. Global proteomic analysis performed by mass spectroscopy measured the relative expression levels simultaneously. Significant changes in expression between treatment groups >1-fold (based on log₂ scale) were included in gene set enrichment analyses (GSEA). Previous studies identified increased heme oxygenase-1 (HO-1) levels with EPA and lira separately, so we measured HO-1 immunochemically as an indicator of drug activity.

Results: EPA, lira, and the combination all increased HO-1 levels >200% versus Ang II alone (p<0.01), demonstrating the activity of these agents in ECs. Compared with Ang II, there were over 40 significantly modulated proteins unique to the combination including c-Src, which increased 1.1-fold relative to Ang II alone (p = 7.59 × 10^-5). GSEA revealed numerous pathways significantly affected by this increase, including “focal adhesion” (GO:0005925). Within this pathway, the combination treatment significantly modulated 10 proteins (including c-Src) of which 6 were similarly modulated by EPA alone while lira alone did not affect these proteins. Either EPA or the combination also increased levels of endoglin and several integrin family members (B1, B3, A2).

Conclusion: In ECs, EPA and lira enhanced expression of Src kinase and related signaling pathways and proteins compared to their separate effects. These pathways indicate the activation of EC migration and junctions in response to this combination. These potentially beneficial effects of EPA and a GLP-1RA combination on protein expression during inflammation may enhance CV protection compared to their separate actions.