Abstract 19424: Red Blood Cell Derived Microparticles Transfer Hemoglobin to Vascular Endothelial Cells, Activate the Heme Oxygenase System, and Mitigate Inflammatory Response
Introduction: Cell-derived microparticles (MPs) can shuttle cellular contents such as RNA or protein between cells, representing a novel mechanism of intercellular communication. Red blood cell-derived microparticles (RMPs) are among the most abundant MPs in human circulation and accumulate in stored human red blood cell transfusion units. Little is known regarding the effects of RMPs on vascular endothelial cells.
Hypothesis: RMPs would be internalized by human aortic endothelial cells (HAECs), transfer RNA and protein cargo, and alter the response of cells to inflammatory stimuli.
Methods: RMPs were isolated from stored human red blood cell transfusion units by centrifugation, labeled with a fluorescent membrane dye (PKH-26), and incubated with HAECs for 24 hours at a concentration of 6 x 106 MPs/mL, after which the cells were washed extensively. Internalization of RMPs by HAECs was assessed by confocal microscopy and flow cytometry. Monocyte adhesion to HAECs +/- LPS was assessed with a static monocyte adhesion assay, and levels of intracellular reactive oxygen species (ROS) +/- TBHP were measured with ROSstar. Changes in RNA and protein were assessed with RT-qPCR and Western blot, respectively.
Results: Confocal microscopy and flow cytometry studies confirmed cellular internalization of RMPs by HAECs. RMPs significantly reduced monocyte adhesion to HAECs in the presence of LPS (0.53 fold, p<0.05) and reduced intracellular levels of ROS in the presence of TBHP (0.78 fold, p<0.05). The presence of hemoglobin was detected in RMP-treated cells but not in untreated controls, and heme oxygenase-1 was upregulated at both the mRNA (2.2-fold, p<0.05) and protein level (3-fold, p<0.05). Although levels of miR-451 were dramatically increased in cells incubated with RMPs (~500-fold, p<0.05) we did not observe changes in expression of miR-451 target genes.
Conclusions: RMPs transfer hemoglobin to HAECs in vitro, thereby activating the heme oxygenase pathway, which may account for the observed anti-inflammatory and antioxidative effects of RMPs on HAECs. These studies raise questions regarding the possibility of RMP mediated communication between red blood cells and the vascular endothelium, as well as the potential effects of RMPs in transfused blood.
Author Disclosures: A. Mitchell: None. W. Gray: None. I. Mohamed: None. S. Thomas: None. K. Rooney: None. J.D. Roback: None. C.D. Searles: None.
- © 2016 by American Heart Association, Inc.