Transcriptomic profiling of thrombus-derived extracellular vesicles reveals PECAM-1 as a potential inflammatory marker for cardioembolic stroke patients.

Accurate etiological diagnosis ensures appropriate secondary prevention of ischemic stroke (IS) patients. However, about 25–40% of IS cases remain of undetermined origin (referred to as ESUS if an unknown embolic source is suspected), highlighting the need to improve diagnostic precision. To identify potential biomarkers of IS etiologies, we analyzed the transcriptomic content of extracellular vesicles (EVs) from thrombi obtained after thrombectomy, including patients with cardioembolic (CE, n = 10), atherothrombotic (AT, n = 10) and ESUS (n = 10) IS (discovery cohort). mRNA levels of those differentially expressed genes in thrombus EVs were determined in microvascular brain endothelial cells after stimulation, revealing PECAM-1, as the most promising candidate. Next, PECAM-1 levels were determined in thrombi (n = 65) and serum samples (n = 95) of an independent validation cohort of IS patients, using immunohistochemistry and ELISA respectively. The transcriptome profiling and bioinformatics analysis revealed 1,514 gene transcripts in thrombus EVs, of which 114 were differentially expressed in AT vs CE etiologies. The genes upregulated in EVs from CE thrombi were enriched in pathways related to cell surface interactions at the vascular wall, leukocyte migration and neutrophil degranulation. Consistent with the increased PECAM-1 expression in thrombus EVs from CE vs AT etiologies, the endothelial expression of PECAM-1 increased after thrombin exposure and diminished upon atherogenic stimulation (oxLDL and TNFα). Moreover, thrombus expression of PECAM-1 was localized predominantly in platelet rich areas containing or surrounded by inflammatory cells and fibrin, and was higher in CE stroke thrombi. Serum levels of PECAM-1 were associated [OR (95%CI) 1.52 (1.14–2.03), p = 0.004] with the severity of stroke at Hospital admission. These findings collectively suggest that the transcriptional study of thrombus EVs may be useful to unravel the molecular pathways behind thrombus formation, and to explore local biological differences between IS etiologies. Specifically, PECAM-1, an adhesion molecule implicated in immuno-inflammatory processes, was increased in thrombi EVs and thrombi of patients with CE IS. Regarding the use of PECAM-1 as systemic biomarker, it did not discriminate IS etiologies, but was positively correlated with worse neurological outcome, suggesting a possible role of PECAM-1 in processes leading to brain injury post-IS.