Abstract 443: Red Blood Cell Microparticles Mediate Transfer of Functional miR-451 to Endothelial Cells in vitro

Abstract

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.