Circulating Extracellular Vesicle Cargo as Bioinformants of 'at-risk'Carotid Artery Stenosis

Extracellular Vesicles

Raju, Sneha, Dakota Gustafson, Kamalben Prajapati, Natalie J. Galant, Steven R. Botts, Giuseppe Papia, Jason E. Fish, and Kathryn L. Howe. "Circulating Extracellular Vesicle Cargo as Bioinformants of ‘at-risk’Carotid Artery Stenosis." Journal of Vascular Surgery 74, no. 3 (2021): e237-e238.

Objectives Carotid artery atherosclerosis is a major cause of ischemic stroke. Managing patients with asymptomatic disease remains challenging, given the lack of reliable tests to identify the subgroup of patients prone to plaque progression and stroke. Given the functional and diagnostic roles of extracellular vesicle (EV) contents, we hypothesized that plasma EV-derived microRNA (miRNA) differs between symptomatic and asymptomatic patients. Methods EVs were isolated via serial centrifugation followed by enrichment using size exclusion chromatography (SEC) (qEVoriginal columns 70 nm; Izon Science Ltd). EV isolation was confirmed according to MISEV 2018 guidelines: Western blot analysis of common EV markers (CD63, CD81, Alix), nanoparticle tracking analysis (NTA), and cryogenic transmission electron microscopy (Cryo-TEM). Lipoprotein contamination was assessed via enzyme-linked immunosorbent assay of individual SEC fractions (R&D Systems; DAPA10, DAPB00). Next-generation sequencing was performed on EVs (HTG Molecular Diagnostics, Inc.), and differential miRNA expression evaluated using Partek Genomics Suite software (version 8.0). Results Twelve patient plasma samples were collected (n = 6 symptomatic; n = 6 asymptomatic). The average age of the cohort was 70.0 ± 5.7 years (asymptomatic, 67.0 ± 5.5 vs symptomatic, 72.5 ± 5.5 years). All patients had severe stenoses with similar peak systolic velocity (asymptomatic 403.2 ± 84.43 vs symptomatic 371.6 ± 175.25; P = .50) and internal carotid artery (ICA):common carotid artery (CCA) ratios (asymptomatic, 5.36 ± 1.07 vs symptomatic, 7.3 ± 5.00; P = .50). CD63 expression confirmed EV enrichment in fractions 7 to 10, with minimal lipoprotein contamination. EV isolation was further confirmed by CD81 and Alix expression (n = 3 patient samples per group). Cryo-TEM identified EVs as bi-layered nanoparticles with electron dense cores (Fig 1). NTA revealed no significant differences in EV concentration or size between groups (n = 3; P > .05). Principal component and heatmap analysis of miRNA sequencing data revealed symptomatic carotid plasma samples clustered together, whereas asymptomatic samples were either starkly different (n = 5) or approximated the symptomatic profiles (n = 1), suggesting a disease gradient (Fig 2). When symptomatic carotid plasma EV-miRNA profiles were compared with asymptomatic specimens, 190 miRNAs were differentially expressed, with miRNA-654-5p and miRNA-127-3p being the most upregulated, and downregulated, respectively (P < .05, fold-change −2< or >2, excluding miRNA with counts <100). Gene set enrichment identified regulation of protein metabolic processes, and negative regulation of cell communication, signaling, and signal transduction as predicted targets of differentially expressed EV-miRNA (P-value < .05). Conclusions Plasma EV-miRNA profiles may differentiate symptomatic vs asymptomatic carotid stenosis and, together with clinical characteristics, may be used in risk stratification of asymptomatic patients.

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