Transcriptomic analysis of circulating extracellular vesicles in peripheral arterial diseasegaining insight into the pathophysiology of the disease

Resumen

BACKGROUND: Peripheral arterial 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 genes with prognostic potential, as well as with a functional role in the pathophysiology of PAD. 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 sequenced using a low input RNA-seq protocol. Protein levels of two selected genes, calprotectin (S100A8/A9) and LCN2, were measured in serum of the PAD cohort (n=331 patients). Using a model of hind limb ischemia (HLI) in wild type and knockout mice, and in vitro in cell culture, we characterized the functional role and molecular regulation of candidate genes in PAD pathophysiology. RESULTS: The transcriptomic study of circulating EVs identified 15 differentially expressed genes, which could cluster PAD patients by disease severity (IC, CLI and controls). Combination of serum calprotectin and LCN2 significantly improved risk prediction assessment of amputation and mortality of PAD subjects. Lcn2-/- mice subjected to HLI presented impaired inflammatory response and muscle regeneration. Muscle expression of the miR-138 was significantly and inversely correlated to Lcn2 mRNA levels, suggesting that it might modulate LCN2 expression in the skeletal regeneration after HLI. CONCLUSIONS: These results reinforce the value of studying the content of circulating EVs as a source of biomarkers and novel therapeutic targets in cardiovascular diseases, particularly, in PAD. Furthermore, LCN2 in combination with calprotectin emerges as a potential prognostic biomarker for assessing the risk of mortality or amputation. Finally, LCN2 has shown to participate in the inflammatory response after ischemia, being necessary for the effective muscle regeneration process.