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

输血是最常见的医疗方法之一，影响着全球数百万患者 美国每年都是如此。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。红细胞可储存长达六周，并在输血后仍具有功能；然而， 红细胞经历各种代谢和结构变化，统称为红细胞储存 损伤。在过去的十年里，存储对RBC功能和活性的有害影响已经到来 受到严密审查。红细胞储存性损害现在已经被很好地描述，并被认为是临床不良反应的原因。 输注红细胞后的结果，特别是在接受多单位储存红细胞的患者中。 尽管人们对红细胞储存损伤有了更多的了解，但其临床后果仍不确定。 因为临床试验数据一直模棱两可。这个试点项目的目标是检查一个重要的 RBC储存损伤的成分，RBC微粒(RMPs)的产生，并表征非- RMP被吸收并将其货物转移到内皮细胞(ECs)的能力已经得到了充分的研究，内皮细胞(ECs) 血管壁。我们假设从储存的红细胞中释放的RMPs可以改变基因的表达 ECS通过转移microRNA(MiRNA)和血红素，从而改变EC基因的表达，表型， 和功能。我们估计，每单位储存的红细胞的胞外液可以有多达1000万 RMPS，因此RBC输注后RMP介导的细胞间通讯的可能性很高。在……里面 初步研究发现，从储存的红细胞中释放的RMP在 大小，形状，以及它们切割钙黄绿素-AM的程度。RMPS具有高丰度的miRNAs 这些也存在于红细胞中，而且RMP很容易被培养的内皮细胞摄取。此外，经RMP处理的内皮细胞 减少了单核细胞的黏附，减少了活性氧，增加了管子的形成 (血管生成)。这些发现与传统观点相反，即RMPs对免疫力和 血管系统。我们认为，RMPs介导了RBC储存损伤的抗炎作用，而 红细胞储存损伤的其他成分促进促炎变化，这是一种新的范式，可能 解释储存的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 红细胞对RMP、miRNA和血红素含量的存储以及RMP将miRNA和血红素转移到 培养的内皮细胞和切除的小鼠主动脉。我们还将评估转移的miRNA抑制 其靶基因在内皮细胞中的表达，进而改变内皮细胞的表型和功能。最后，我们会 确定静脉输注的RMPs能否被摄取并改变小鼠的血管炎症 做过腹主动脉缩窄手术的人。总而言之，我们预计拟议的研究 将对储存的红细胞对血管功能的影响提供新的见解，这将作为 未来对RMP介导的EC表型和功能调节机制的研究。

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.