The EV–Oxylipin Axis in Atherosclerosis: Characterization of oxylipin cargo in plasma-derived extracellular vesicles from Ldlr^-/- mice

Atherosclerosis is a chronic inflammatory disease driven by dysregulated lipid metabolism and immune activation. Extracellular vesicles (EVs) have emerged as important mediators of intercellular communication in cardiovascular disease, capable of transferring bioactive cargo including lipids. Oxylipins are bioactive oxidized metabolites of polyunsaturated fatty acids regulate inflammation, vascular tone, and immune activation. Despite growing interest in both EVs and oxylipins, the oxylipin cargo of circulating EVs and how it is remodeled under atherogenic conditions remain poorly understood. To characterize the oxylipin composition of plasma-derived EV subpopulations from Ldlr^-/- mice and determine how atherogenic dietary conditions alter the EV–oxylipin profile.

Plasma from Ldlr^-/- mice fed a Western diet (WD) or control diet (CD) was fractionated by size exclusion chromatography into an LDL-enriched fraction (F0) and two EV-enriched fractions (F1, F2). EV isolation was confirmed by nanoparticle tracking analysis (NTA). Oxylipins were quantified by targeted mass spectrometry and normalized to total protein.

NTA showed WD mice produced EVs with higher concentrations and larger particle sizes, particularly in F1 (~30 × 10⁶ vs ~12 × 10⁶ particles/mL; ~141 vs ~117 nm). We detected 72 oxylipin species across eight fatty acid precursors, dominated by arachidonic acid (AA, n=33), followed by LA (n=11), EPA (n=11), DHA (n=9), ALA, DHGLA, GLA, and mead acid. AA-derived pro-inflammatory mediators including HETEs, KETEs, DiHETrEs, and prostanoids (PGD2, PGE2, PGF2α, 6-keto-PGF1α) were broadly upregulated in WD fractions. CD fractions showed enrichment of EPA-derived HEPEs and DHA-derived HDoHEs, including the pro-resolving mediator RvD2. In the EV1 fraction, 16 oxylipins were detected exclusively under WD nearly three times the 6 oxylipins unique to CD suggesting broader activation of AA-dependent enzymatic pathways under atherogenic stress.

These findings reveal a diet-driven remodeling of the EV–oxylipin axis in Ldlr^-/- mice, shifting from a pro-resolving signature under CD toward a pro-inflammatory eicosanoid-enriched profile under WD, implicating EV-associated oxylipins in vascular inflammation during atherogenesis.