BACKGROUND: Peripheral artery disease (PAD) is a major cause of chronic vascular pathologies in developed countries. PAD patients are at high risk of thrombotic events -myocardial infarction, stroke- and death, urging to find new prognostic tools. Extracellular vesicles (EVs) contain nucleic acids from the cell of origin that might represent their molecular imprint. We hypothesize that transcriptomic study of circulating EVs might be useful in search for new long-term prognostic markers in PAD patients.
METHODS: EVs were isolated by centrifugation (2x20.000, 70min) from citrated platelet-free plasma (PFP) of PAD patients with intermittent claudication (IC, grade IIa), critic limb ischemia (CLI, grade IV), and age and sex matched controls (n=12/group). Poly-A transcripts were isolated from EVs samples and sequencing libraries were prepared using a low input scRNA-seq protocol. After sequencing (Illumina NextSeq500), data was processed and differentially expressed genes were obtained by means of a parametric (Limma, R/Bioconductor) and non-parametric (Kruskal-Wallis) approach. Protein levels of a selected gene, S100A9, were measured in serum (Human MRP8/14, BioLegend).
RESULTS: The bioinformatic analysis yielded 15 differentially expressed genes, which could cluster PAD patients by disease severity (IC, CLI and controls). Serum levels of S100A9 were higher in PAD patients (n=317) compared to healthy controls (n=100, p<0.05), and were associated to amputation (p<0.01). 
CONCLUSIONS: We have established a workflow for studying the transcriptome of circulating EVs in PAD patients and identified S100A9 as possible biomarker. For future studies we propose to analyse EVs by cell origin, enabling the identification of new prognostic markers in PAD.

04 - TRANSCRIPTOMIC ANALYSIS OF EXTRACELLULAR VESICLES FOR BIOMARKER DISCOVERY IN PERIPHERAL ARTERY DISEASE (PAD)