The Production of Microparticles During RBC Storage and Their Impact on Endothelial Phenotype In-vitro and In-vivo

Emory University, Atlanta GA

Investigators

Abstract

Transfusion of blood represents one of the most common medical therapies, impacting millions of patients in the U.S. each year. Recipients of red blood cell (RBC) transfusions are often critically ill and usually require multiple units. RBCs can be stored up to six weeks and still be functional after transfusion; however, stored RBCs undergo a variety of metabolic and structural changes, collectively referred to as the RBC storage lesion. Over the last decade, the detrimental effects of storage on RBC functionality and viability have come under scrutiny. The RBC storage lesion has now been well characterized and blamed for adverse clinical outcomes after RBC transfusion, particularly those in patients who receive multiple units of stored RBCs. Despite a greater understanding of the RBC storage lesion, its clinical consequences remain uncertain because clinical trial data have been equivocal. The goal of this pilot project is to examine one important component of the RBC storage lesion, the production of RBC microparticles (RMPs), and characterize the not- well-studied ability of RMPs to be taken up and transfer their cargo to the endothelial cells (ECs), which line the walls of blood vessels. We hypothesize that RMPs released from stored RBCs can alter gene expression in ECs through the transfer of microRNA (miRNA) and heme, thereby altering EC gene expression, phenotype, and function. We estimate that the extracellular fluid of each unit of stored RBCs can have up to 10 million RMPs, so the potential for RMP-mediated cell-to-cell communication after RBC transfusion is high. In preliminary studies, we have found that RMPs released from stored RBCs were relatively heterogeneous in size, shape, and the degree to which they cleave the dye calcein-AM. RMPs had high abundance of miRNAs that are also found in RBCs, and RMPs were readily taken up by cultured ECs. Furthermore, RMP-treated ECs had reduced monocyte adhesion, decreased reactive oxygen species, and increased tube formation (angiogenesis). These findings are counter to the traditional view of RMPs as being toxic to the immune and vascular systems. We propose that RMPs mediate anti-inflammatory effects of the RBC storage lesion while other components of the RBC storage lesion promote pro-inflammatory changes, a novel paradigm that might explain conflicting data from clinical studies of stored RBCs. Here, we will further characterize the effect of RBC storage on RMP miRNA and heme content as well as the ability of RMPs to transfer miRNA and heme to cultured ECs and excised mouse aortas. We will also assess the ability of transferred miRNA to suppress expression of their target genes in ECs and subsequently alter EC phenotype and function. Finally, we will determine whether RMPs transfused intravenously can be taken up and alter vascular inflammation in mice that have undergone abdominal aorta coarctation surgery. Together, we anticipate that the proposed studies will provide novel insights into the effects of stored RBCs on vascular function that will serve as the basis for future studies of the mechanisms responsible for RMP-mediated modulation of EC phenotype and function.