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

输血是最常见的医学疗法之一，影响着世界上数百万的患者。 美国每年。接受红细胞（RBC）输注的患者通常病情危重，通常需要 多个单位。红细胞可以储存长达六周，并且在输血后仍然具有功能;然而，储存 红细胞经历各种代谢和结构变化，统称为红细胞储存 损伤。在过去的十年中，储存对RBC功能和活力的不利影响已经出现， 受到审查。红细胞储存损伤现在已经得到很好的表征，并被归咎于不良的临床 RBC输注后的结果，特别是接受多单位储存RBC的患者。 尽管对红细胞贮存损伤有了更深入的了解，但其临床后果仍不确定 因为临床试验数据一直不明确。该试点项目的目标是研究一个重要的 RBC储存病变的组成部分，RBC微粒（RMP）的产生，并表征非- 已经充分研究了RMP被摄取并将其货物转移到内皮细胞（EC）的能力，内皮细胞（EC）排列在血管内皮细胞的周围。 血管壁。我们假设从储存的红细胞中释放的RMP可以改变细胞中的基因表达， EC通过转移微小RNA（miRNA）和血红素，从而改变EC基因表达、表型， 和功能我们估计每单位储存的红细胞的细胞外液可以有多达1000万个 因此，RBC输注后RMP介导的细胞间通讯的可能性很高。在 初步研究，我们发现，从储存的红细胞中释放的RMP相对不均匀， 大小，形状，以及它们切割染料钙黄绿素-AM的程度。RMPs中miRNAs的丰度较高 也存在于RBC中，并且RMP容易被培养的EC吸收。此外，RMP处理的EC 单核细胞粘附减少，活性氧减少，小管形成增加 （血管生成）。这些发现与传统观点相反，传统观点认为RMP对免疫系统有毒， 血管系统我们认为，RMPs介导红细胞储存损伤的抗炎作用， 红细胞储存损伤的其他成分促进促炎性变化，这是一种新的范式， 解释储存红细胞临床研究中相互矛盾的数据。在这里，我们将进一步描述 RBC储存对RMP miRNA和血红素含量的影响以及RMP将miRNA和血红素转移到RBC的能力。 培养的EC和切除的小鼠睾丸。我们还将评估转移的miRNA抑制 其靶基因在EC中的表达，并随后改变EC表型和功能。最后我们将 确定静脉输注的RMP是否可以被吸收并改变小鼠的血管炎症 做过腹主动脉缩窄手术的病人总之，我们预计，拟议的研究 将为储存的红细胞对血管功能的影响提供新的见解， 对RMP介导的EC表型和功能调节机制的进一步研究。

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.