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

基本信息

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
项目类别：
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

项目摘要

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.

输血是最常见的医疗疗法之一，影响着数百万患者每年美国。红细胞 (RBC) 输血者通常病情危重，通常需要多个单位。红细胞可以保存长达六周，并且在输血后仍然具有功能；然而，存储红细胞经历各种代谢和结构变化，统称为红细胞储存病变。在过去的十年中，存储对红细胞功能和活力的不利影响已经显现正在接受审查。红细胞贮积病变现已得到充分表征，并被归咎于临床不良反应红细胞输注后的结果，特别是接受多个单位储存红细胞的患者。尽管对红细胞贮积病变有了更深入的了解，但其临床后果仍不确定因为临床试验数据模棱两可。该试点项目的目标是检查一项重要的红细胞储存损伤的组成部分，红细胞微粒（RMP）的产生，并表征非- 经过充分研究的 RMP 被吸收并将其货物转移至内皮细胞 (EC) 的能力，内皮细胞排列在血管壁。我们假设从储存的红细胞中释放的 RMP 可以改变 ECs通过转移microRNA（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最多可达1000万个 RMP，因此红细胞输注后 RMP 介导的细胞间通讯的潜力很高。在初步研究，我们发现从储存的红细胞中释放的 RMP 具有相对异质性。大小、形状以及它们裂解染料钙黄绿素-AM 的程度。 RMP 具有高丰度 miRNA RBC 中也存在这些物质，并且 RMP 很容易被培养的 EC 吸收。此外，经 RMP 处理的 EC 单核细胞粘附减少，活性氧减少，管形成增加（血管生成）。这些发现与 RMP 对免疫和免疫系统有毒的传统观点相反。血管系统。我们认为 RMP 介导红细胞储存病变的抗炎作用，而红细胞储存损伤的其他成分促进促炎性变化，这是一种可能的新范例解释储存红细胞临床研究中相互矛盾的数据。在这里，我们将进一步表征 RBC 对 RMP miRNA 和血红素含量的储存以及 RMP 将 miRNA 和血红素转移到培养的 EC 和切除的小鼠主动脉。我们还将评估转移的 miRNA 抑制的能力其靶基因在 EC 中表达，随后改变 EC 表型和功能。最后，我们将确定静脉输注的 RMP 是否可以被小鼠吸收并改变血管炎症接受过腹主动脉缩窄手术的人。我们共同预计拟议的研究将为储存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。