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miRNAs are short, noncoding RNAs that circulate in plasma within membrane-bound vesicles, such as microparticles (MPs), or bound to proteins or lipoproteins. Circulating miRNAs are putative biomarkers for a wide range of diseases, including cardiovascular disease. Our group and others have evidence to suggest MP-encapsulated miRNAs, rather than total plasma miRNAs, may harbor more specific and functionally relevant biomarkers. We tested the hypothesis that MP-encapsulated miRNA levels would differ between patients with mild or severe peripheral artery disease (PAD) and healthy controls matched for age, gender, and race. Banked plasma stored at -80°C was obtained for 5 healthy subjects, 6 patients with mild PAD (ABI 0.8-1.0), and 10 patients with severe PAD (ABI < 0.4). MPs were isolated using a standard differential centrifugation protocol and quantified by flow cytometry. Four miRNAs implicated in vascular disease (miR-126-3p, miR-126-5p, miR-21, and miR-222) were quantified with qRT-PCR. Relative expression for each miRNA (-2ΔCt) was calculated and divided the total number of MPs to determine miRNA expression per MP. There was a non-significant trend towards increased levels of MPs in patients with mild (19,462 MPs/μl ± 5,213) and severe PAD (21,688 MPs/μl ± 7,175) compared to healthy controls (7244 MPs/μl ± 919). MP levels of miR-126-5p were increased among healthy subjects compared to patients with mild and severe PAD (p = 0.003), while MP miR-126-3p levels were significantly lower in healthy subjects (p = 0.002). miR-21 and miR-222 did not differ significantly between groups. None of the four miRNAs distinguished mild versus severe PAD. In conclusion, our data suggests that MP-mediated transport of miR-126-5p and miR-126-3p is dysregulated in the setting of mild and severe vascular disease, and supports the concept that the plasma MP fraction may harbor disease-associated miRNA profiles.

Microparticles (MPs) circulate in human blood and can transfer functional miRNA to recipient cells. Red blood cell derived MPs (RMPs) accumulate during storage of human erythrocyte transfusion units, but it is not known whether RMPs can deliver functional miRNAs to endothelial cells. We hypothesized that the RMPs could transfer functional miR-451 to human arterial endothelial cells (HAECs), and that RMP-mediated miRNA transfer could affect clinical outcomes after stored RBC transfusion. miR-451, an erythrocyte specific miRNA, was confirmed to be abundant in RMPs and absent in HAECs, making it a useful miRNA to assess for transfer. RMPs were isolated from a 42 day-old transfusion unit and confirmed by both flow cytometry and electron microscopy. HAECs were incubated for 2 hours with calcein-labeled RMPs derived from 2 ml of blood product. After incubation cells were washed with phosphate buffered saline (PBS) and fixed for microscopy or harvested for RNA. Confocal microscopy confirmed the uptake of RMPs by treated cells. miRNA-451 levels increased ~900-fold in HAECs after treatment with RMPs (fold change = 927 ± 245, p < 1.0 x 10-4). To assess whether transferred miR-451 was functional, we measured mRNA levels of two targets of miR-451 in recipient HAECs, macrophage migration inhibitory factor (MIF) and sphingosine-1-phosphate receptor 2 (S1PR2). RMP treatment decreased expression of MIF (–2.8 fold ± 0.08, p = 0.047) and S1PR2 (–2.9 fold ± 0.06, p = 0.007) as compared to untreated controls, suggesting the transferred miR-451 is functional. Further studies aimed at confirming miR-451 dependent changes in MIF and S1PR2 are underway. This data supports the concept that RMPs, in particular those that accumulate during RBC storage, can function as signaling molecules via MP-mediated miRNA transfer. MP-mediated miRNA transfer represents a novel mechanism by which stored blood products may exert previously unrecognized effects upon transfusion.