SNO transport regulates endothelial adhesion of RBCs

项目总结 输注红细胞(RBC)费用昂贵，是VA和VERT患者最常进行的程序之一 其他医疗保健设置。然而，越来越多的证据表明，许多贫血患者可能不会受益。 从红细胞输注，血液功能下降，即使在很短的储存期。我们已经确定了一个 红细胞通常将一氧化氮(NO)衍生信号输出到血管系统的新机制， 促进血液本身的流动。S-亚硝硫醇(SNO)是由NO前体在红细胞中形成的硫醇加合物。 与血红蛋白中与氧化有关的变构转变相一致。红细胞输出这些血管调节因子 SNO“按需”，以微调局部血流并防止RBC与内皮细胞黏附 (ECS)。初步研究首次表明，小载体SNO的细胞间运输在 防止红细胞与内皮细胞的黏附。这些新发现证明了血液流动调节的作用， RBC通过细胞间SNO信号传导，并在储存后损伤这一功能。我们将测试 假设SNO转运对红细胞的血管调节功能至关重要，其损失是可以补救的 通过实现以下特定目标，促进输血后肺部发病率的“储存性损伤”：目标1： 确定RBC LAT1在调节人红细胞输出SNO中的作用。特定的管道，通过它 RBC输出SNO组仍未确定，但我们的初步研究表明RBC具有重要作用 SNO导出中的LAT1。我们将使用新的工具和药理学方法来确定LAT1在 RBC SNO出口，以及加强这一活动的措施。我们还将鉴定细胞内的RBC分子 有益SNO恢复的目标。目的2：确定通过LAT1导入SNO在内皮细胞中的作用 细胞在限制健康人红细胞粘附性中的作用。我们证明了SNO可以在银行中恢复 RBC在储存诱导的丢失后，SNO的恢复限制了RBC-EC的黏附。我们将决定 LAT1介导的内皮细胞SNO输入是否是RBC SNO抗黏附效应所必需的 使用遗传和药理学方法，并测试替代机制的作用。目标3： 在人的小鼠模型中确定LAT1在RBC SNOS存储敏感效应中的作用 红细胞输注。我们开发了一种创新的人-RBC输血小鼠模型，概括了关键 临床表型，如肺功能障碍。我们将使用活体显微镜，一种新的有条件的小鼠 EC LAT1缺陷和输液标记以确定RBC SNO在限制输血后的作用 发病率，特别是红细胞粘附性和血液氧合功能低下以及组织氧气输送。既天真又幼稚 将采用二次打击(脂多糖后输注)、急性和慢性模型。确定企业的角色 在健康和疾病中，从红细胞到内皮细胞的信号转导中的SNO运输是迈向我们长期- 术语科学目标：了解RBCs输出调解人以调节其自身的机制 顺应组织需求的流动。这些研究有望为输血实践中的变化提供信息 改善退役军人患者的风险与利益平衡。

PROJECT SUMMARY Red blood cell (RBC) transfusion is costly, and is among the procedures most frequently performed in VA and other health care settings. Growing evidence, however, suggests that many anemic patients may not benefit from RBC transfusion, and that blood function declines even during short storage periods. We have identified a novel mechanism whereby RBCs normally export nitric oxide (NO)-derived signals to the vasculature, facilitating the flow of blood itself. S-nitrosothiols (SNOs) are thiol adducts formed in RBCs from precursor NO in concert with the oxygenation-linked allosteric transition in hemoglobin. RBCs export these vasoregulatory SNOs “on demand” in order to fine-tune regional blood flow and prevent RBC adhesion to the endothelium (ECs). Preliminary Studies show for the first time that intercellular transport of small carrier SNOs is critical in the prevention of RBC adhesion to ECs. These novel findings demonstrate the role of blood flow-regulating, intercellular SNO signaling by the RBC, and impairment in this function after storage. We will test the hypothesis that SNO transport is critical to the vasoregulatory function of RBCs, and its loss a remediable “storage lesion” promoting post-transfusion lung morbidity, by accomplishing these Specific Aims: Aim 1: Determine the role of RBC LAT1 in mediating SNO export by human RBCs. The specific conduit by which RBCs export SNO groups remains undetermined, but our Preliminary Studies indicate a significant role of RBC LAT1 in SNO export. We will use new tools and pharmacological approaches to determine the role of LAT1 in RBC SNO export, and measures to augment this activity. We will also identify intracellular RBC molecular targets of beneficial SNO restoration. Aim 2: Determine the role of SNO import via LAT1 in endothelial cells in limiting the adhesivity of healthy human RBCs. We showed that SNO can be restored in banked RBCs after storage-induced loss, and that SNO restoration limits RBC-EC adhesion. We will determine whether LAT1-mediated SNO import by ECs is necessary for the RBC SNO-induced antiadhesive effect by using genetic and pharmacological approaches, and test the role of alternative mechanisms. Aim 3: Determine the role of LAT1 in the storage-sensitive effects of RBC SNOs in mouse models of human RBC transfusion. We developed an innovative mouse model of human-RBC transfusion, recapitulating key clinical phenotypes such as lung dysfunction. We will use intravital microscopy, a novel mouse conditionally deficient in EC LAT1, and transfusate labeling to determine the role of RBC SNO in limiting post-transfusion morbidity, specifically RBC adhesion and depressed blood oxygenation and tissue O2 delivery. Both naïve and two-hit (transfusion after lipopolysaccharide), acute and chronic models will be used. Determining the role of SNO transport in signaling from RBCs to ECs in health and disease is the next logical step toward our long- term scientific objective: to understand the mechanisms whereby RBCs export mediators to regulate their own flow in response to tissue needs. These studies are expected to inform changes in transfusion practice to improve the risk-benefit balance for Veteran patients.