Polyunsaturated Fatty Acid (PUFA) Composition of Growth Medium Changes the Atherogenic Potential of Human Aortic Endothelial Cells (HAECs) Following Endotoxin Stimulation

<b>Background/Objectives:</b> Endothelial activation by lipopolysaccharides (LPS) contributes to inflammation and the development of cardiovascular disease, making <i>n-3</i> polyunsaturated fatty acids (PUFAs) potential modulators capable of mitigating endothelial dysfunction. The current study examines the effects of long-chain eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), along with their precursor, α-linolenic acid (ALA), on oxidative stress, adhesion molecule expression, and cytokine milieu in LPS-stimulated human aortic endothelial cells (HAECs). <b>Methods:</b> HAECs (fifth passage) were cultured in control medium under standard conditions: ~37 °C, 5% CO₂, ≥80% humidity. Cells were incubated in control basal cell medium or medium supplemented with ALA, EPA, DHA, and their combination (50 µM; <i>n</i> = 5 per group). After 48 h, cells were treated overnight (~16 h) with LPS from <i>E. coli</i> (0.75 and 1 µg/mL). HAECs and supernatants were collected for flow cytometry, Luminex, and ELISA assays. Significance was assessed using two-way analysis of variance ANOVA, followed by post hoc analyses (<i>p</i> < 0.05). Spearman's correlation analysis was performed between markers, and <i>p</i>-values were adjusted using the Benjamini-Hochberg (BH) correction. <b>Results:</b> PUFA supplementation, particularly with DHA and ALA, significantly reduced intracellular reactive oxygen species (ROS) production and the expression of adhesion molecules (ICAM-1, E-selectin) in HAECs under both basal and LPS-stimulated inflammatory conditions. All PUFAs reduced pro-inflammatory cytokine levels (IFNγ, TNFα, IL-6), while ALA increased IL-1α and endoglin expression, indicating differential immunomodulatory effects. EPA exhibited antioxidant and anti-inflammatory effects primarily at higher LPS concentrations. Correlation analysis demonstrated strong interdependence between oxidative stress, inflammatory markers, and vascular activation, further confirming PUFA-mediated endothelial protection. <b>Conclusions:</b> PUFA supplementation produced molecule-specific effects on endothelial inflammation. DHA and ALA consistently showed anti-inflammatory and antioxidative effects, while EPA's beneficial effect was more pronounced under inflammatory conditions, emphasising the importance of PUFA type and context in managing vascular inflammation.