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

红细胞储存过程中微粒的产生及其对体内外内皮表型的影响

• 批准号: 9167980
• 负责人: CHARLES D SEARLES
• 金额: $ 19.23万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-08-01 至 2018-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9167980
• 关键词: Address; Adhesions; Adult; Adverse effects; Affect; Age; Anti-Inflammatory Agents; Anti-inflammatory; Aorta; Aortic coarctation; Apoptosis; Apoptotic; Binding; Biology; Blood; Blood Banks; Blood Transfusion; Blood Vessels; Blood Volume; Bulla; Cell Communication; Cell physiology; Cells; Child; Cleaved cell; Clinical; Clinical Research; Clinical Trials; Conflict (Psychology); Critical Illness; DNA; Data; Development; Disease; Dyes; Encapsulated; Endothelial Cells; Erythrocyte Transfusion; Erythrocytes; Extracellular Fluid; Extracellular Protein; Future; Gene Expression; Gene Targeting; Goals; Health; Heme; Immune system; In Vitro; Inflammatory; Lesion; Life; Link; Mediating; Medical; Membrane; Mesentery; Messenger RNA; Metabolic; MicroRNAs; Modality; Mus; Operative Surgical Procedures; Outcome; Patient-Focused Outcomes; Patients; Phenotype; Pilot Projects; Plasma; Production; Reactive Oxygen Species; Risk; Role; Safety; Shapes; Sickle Cell Anemia; Signal Transduction; Source; Techniques; Therapeutic; Tissues; Transfusion; Tube; Untranslated RNA; Vascular System; Vasodilation; Vesicle; Work; abdominal aorta; angiogenesis; base; calcein AM; common treatment; exosome; experience; extracellular; improved; in vivo; insight; migration; monocyte; novel; novel marker; novel therapeutics; pediatric patients; vascular inflammation; vector; vesicular release

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.

血液输血代表了最常见的医疗疗法之一，影响了数百万患者 美国每年。红细胞（RBC）输血的接受者通常病重病，通常需要 多个单元。 RBC可以存储长达六个星期，并且在输血后仍能发挥作用。但是，存储了 RBC经历了各种代谢和结构变化，共同称为RBC存储 病变。在过去的十年中，存储对RBC功能和生存能力的有害影响已经出现 在审查下。 RBC储存病变现已被很好地表征和指责 RBC输血后的结果，尤其是那些接受多个单位存储的RBC的患者。 尽管对RBC储存病变有更深入的了解，但其临床后果仍然不确定 因为临床试验数据是模棱两可的。该试点项目的目标是检查一个重要的 RBC存储病变的组成部分，RBC微粒（RMP）的产生，并表征非 - RMP被占用并将其货物转移到内皮细胞（EC）的能力充分的能力， 血管墙。我们假设从存储的RBC释放的RMP可以改变基因表达 通过microRNA（miRNA）和血红素的转移EC，从而改变了EC基因表达，表型， 和功能。我们估计每个存储的RBC的每个单元的细胞外流体最多可以拥有1000万 RMP，因此RBC输血后RMP介导的细胞对细胞通信的潜力很高。在 初步研究，我们发现从存储的RBC释放的RMPS在 大小，形状以及它们裂开染料钙软件剂的程度。 RMP的miRNA丰富 在RBC中也可以找到，RMP很容易被培养的EC占据。此外，经过RMP处理的ECS 单核细胞粘附降低，活性氧降低并增加了管的形成 （血管生成）。这些发现与RMP的传统观点背道而驰，因为 血管系统。我们建议RMP介导RBC储存病变的抗炎作用，而 RBC储存病变的其他组成部分促进促炎性变化，这是一种新型范式，可能 从存储的RBC的临床研究中解释矛盾的数据。在这里，我们将进一步描述 RBC在RMP miRNA和血红素含量上存储以及RMP将miRNA和血红素传递到的能力 培养的EC和切除的小鼠主动脉。我们还将评估转移miRNA抑制的能力 其靶基因在EC中的表达，随后改变EC的表型和功能。最后，我们会的 确定RMP是否可以静脉输血并改变小鼠的血管炎症 经过腹主动脉缩窄手术。一起，我们预计拟议的研究 将提供有关存储的RBC对血管功能的影响的新颖见解，这将作为血管功能的基础 对负责RMP介导的EC表型和功能调节机制的未来研究。