Systemic downregulation of EV-associated MiRNAs following remote ischemic preconditioning.

Remote ischemic preconditioning (RIPC) is a non-invasive strategy to protect tissues from ischemia-reperfusion injury (IRI), yet its systemic molecular mediators remain incompletely understood. Extracellular vesicles (EVs), particularly their microRNA (miRNA) cargo, have emerged as potential conveyors of RIPC-induced protection. In this translational study, we performed paired EV-miRNA profiling in five human subjects undergoing free flap surgery, with plasma collected before and 30 min after a standardized RIPC protocol. EVs were isolated, RNA was extracted and profiled using a 384-plex qPCR array. Differential expression was assessed using paired statistics and false discovery correction. Functional impact was inferred via validated target mapping, gene set enrichment analysis (GSEA), and miRNA-gene-pathway network modeling. RIPC induced a rapid, systemic shift in the circulating EV-miRNA profile. Principal component analysis revealed consistent directional changes post-RIPC. Four miRNAs, hsa-miR-505-3p (log2FC = -3.30, p = 0.025), hsa-miR-374a-5p (log2FC = -4.52, p = 0.028), hsa-miR-200a-3p (log2FC = -7.27, p = 0.044), and hsa-miR-181b-5p (log2FC = -3.94, p = 0.045), were significantly downregulated across the cohort. No miRNAs were consistently upregulated. GSEA of validated targets showed positive enrichment of stress-responsive and pro-survival pathways, including TNF-alpha signaling, TGF-beta, Notch, hypoxia, angiogenesis, and apoptosis. Network analysis identified miR-181b-5p and miR-374a-5p as central regulators affecting multiple interconnected pathways. These results suggest that RIPC elicits systemic protection not by introducing new molecular signals but by selectively removing suppressive EV-bound miRNAs. This may reflect a subtractive regulatory mechanism and enable rapid derepression of protective gene programs, offering a plausible explanation for the early benefits of RIPC. These findings should be validated in larger, multi-timepoint cohorts.