Abstract Body: Background: Atherosclerotic cardiovascular disease is characterized, in part, by endothelial dysfunction and impaired efferocytosis (clearing of dead cells). Endothelial cells (ECs) are poised to regulate efferocytosis within atherogenic vessels due to their strategic location, ability to respond to insults, and release of soluble mediators. The role of EC-macrophage communication in efferocytosis regulation and disease progression has yet to be fully understood. We hypothesize that ECs, in response to atheroprone stimuli, regulate macrophage efferocytosis through extracellular vesicle (EV) mediated cell-cell communication.

Methods: Human Umbilical Vein Endothelial Cells (HUVECs) were activated with TNFα or left untreated (quiescent) and cocultured with THP-1 macrophages (MΦ) using transwells for 24h. RT-PCR was employed to measure the abundance of inflammatory and efferocytosis-related transcripts (e.g. ADAM17, SIRPα, MerTK, and LRP-1) in cocultured THP-1 MΦ. An in vitro efferocytosis assay was performed using the cocultured THP-1 MΦ, where THP-1 MΦ were incubated with pHrodo-stained apoptotic cells at a 3:1 ratio for 2h; efferocytosis capacity was measured using confocal microscopy. EVs were isolated from the secretome of quiescent and TNFα-stimulated ECs using ultracentrifugation and characterized per MISEV2023 guidelines. EC-derived EVs were added to THP-1 MΦ, at a physiological concentration, for downstream efferocytosis assay and transcriptomic analysis.

Results: THP-1 MΦ cocultured with activated ECs showed an increase in inflammatory markers and ADAM17, while decreasing MerTK and LRP-1, compared to naïve THP-1 MΦ (n=6, p<0.05). Efferocytic capacity was reduced in these MΦ (n=5, p≤0.0001) compared to naïve and THP-1 MΦ coculture with quiescent ECs. Activated ECs released more EVs compared to quiescent ECs (n=5, p=0.0063). THP-1 MΦ treated with activated EC-EVs showed a downregulation in efferocytic capacity (n=3, p<0.05), compared to naïve, activated EV-depleted secretome, and quiescent EC-EVs treatment. THP-1 MΦ treated with activated EC-EVs showed an upregulation in SIRPα and downregulation in MerTK and LRP-1, compared to quiescent EC-EV treated MΦ (n=6, p<0.05).

Conclusion: Our data reveal a critical role of activated EC-EVs in impairing macrophage efferocytosis and promoting a proinflammatory phenotype. Understanding EC-EV-mediated regulation of efferocytosis would provide insight into novel therapeutic strategies for atherosclerosis treatment.