OC 45.4 - Transcriptomic Analysis Of Plasma And Thrombus Extracellular Vesicles (Evs) For The Molecular Diagnosis Of Ischemic Stroke Etiology

Background: Ischemic stroke (IS) results from an acute thrombus obstruction of a cerebral artery that causes high morbidity and mortality worldwide. Almost 45% of all IS are from unknown origin complicating the precise secondary treatment. Extracellular vesicles (EVs) are membrane nanoparticles that contain nucleic acids from the cell of origin. The study of their content might help to define a personalized molecular pattern to guide clinicians in the diagnosis and the therapeutic strategy for each patient.

Aims: To analyse the transcriptomic content of circulating and thrombus derived EVs according to IS etiology to assess their molecular signature.

Methods: We isolate EVs from IS patients with large vessels occlusion by high-speed centrifugation (2x20.000g, 70min) from citrated platelet-free plasma and Accutase-digested thrombus. To characterize EVs size and cellular origin, nanoparticle tracking analysis (NTA) and flow cytometry were performed. Transcriptomic profiles of medium/large EVs were generated using the MARS-Seq RNA-Seq protocol (n=5/group).

Results: EVs uptaking Carboxyfluorescein succinimidyl ester (CFSE) were mainly from platelet and erythrocyte origin. NTA shows a polydisperse heterogeneous vesicle distribution (133.5-300 nm). By RNAseq we identified 95 differentially expressed genes (DEG) in plasma EVs (p < 0.01) and 21 in thrombus EVs (p < 0.01), which could discriminate IS patients by etiology. Among these DEG, plasma EVs from cardioembolic patients were enriched in circulatory system development, regulation of neuron death and T cell activation pathways, whereas, thrombi EVs presented differences in transcripts related to hematopoiesis and catabolic process. In atherothrombotic patients, lipid catabolic process, and regulation of MAPK were the enriched pathways in plasma while ion and transmembrane transport were in thrombi EVs.

Conclusion(s): Transcriptomic analysis of circulating and thrombus-derived EVs in IS patients might be useful to identify molecular signatures of IS etiologies.

Funding: ISCIII (PI19/00065"), co-funded by ERDF, "A way to make Europe", RICORs-ictus (RD21/0006/0008), CIBERCV (CB16/11/00371), and Virto Group (Navarra, Spain).