Aberrant RBC SNO transport and endothelial adhesion in sepsis

脓毒症中红细胞 SNO 转运异常和内皮粘附

• 批准号: 10377331
• 负责人: TIMOTHY J MCMAHON
• 金额: --
• 依托单位: DURHAM VA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-01-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10377331
• 关键词: Acute Lung Injury; Address; Adhesions; Adhesives; Adoptive Transfer; Amino Acid Transporter; Anemia; Animal Model; Antibiotics; Autologous; Basement membrane; Biochemical; Blood; Blood Cells; Blood Vessels; Blood capillaries; Cell Adhesion; Cells; Characteristics; Competence; Critical Illness; Cryopreservation; Data; Depressed mood; Endothelial Cells; Endothelium; Engineering; Erythrocytes; Event; Extravasation; Failure; Human; Hypoxia; Immune response; Impairment; In Vitro; Infection; Injury; Innate Immune Response; Laminin; Lung; Measures; Mediating; Methods; Modeling; Mus; Nitric Oxide; Organ; Outcome; Oxygen; Pathway interactions; Patients; Physiological; Population; Process; Randomized Clinical Trials; Rest; Risk Factors; Role; S-Nitrosothiols; SKIL gene; Secondary to; Sepsis; Signal Transduction; Signaling Molecule; Specimen; System; Testing; Transfusion; Veterans; Wild Type Mouse; adhesion receptor; cecal ligation puncture; clinically significant; comorbidity; experimental study; extracellular; in vivo; indexing; inhibitor; innovation; model design; mouse model; neutrophil; novel; organ injury; paracrine; patient population; response; response to injury; septic; septic patients

In critically ill patients, anemia is a well-established risk factor for poor outcomes but, paradoxically, liberal RBC transfusion in these patients does not improve outcomes as compared with a restrictive strategy in which patients remain moderately anemic. Changes taking place very early in the storage process may contribute by impairing RBC function. We have identified functional and biochemical changes in the first day of RBC storage that contribute to a proadhesive effect of blood banking. Most recently, we identified a novel mechanism whereby healthy human RBCs export the nitric oxide (NO) derivative, S-nitrosothiol (SNO), to oppose endothelial adhesion of RBCs in vitro and in vivo in mice. This RBC SNO export is depressed after RBC storage. To study the physiological significance of this pathway, we engineered a mouse in which the responsible SNO transporter is inducibly deleted from endothelial cells. ECs from this mouse import SNO poorly as predicted, and RBC adhesion is increased in the lungs of these mice after transfusion. In mice with sepsis (injurious response to serious infection), we find that RBC export of SNO is decreased even though RBC SNO content is elevated. We hypothesize that in sepsis, intercellular SNO transport is deficient, promoting the adhesivity of RBCs (native or transfused) and neutrophils. We will test this hypothesis by accomplishing these Specific Aims: 1. Determine the influence of sepsis on antiadhesive SNO export from RBCs. We find that after cecal ligation and puncture (CLP), the ability of murine RBCs to export antiadhesive SNO is deficient. We will test whether the SNO deficiency and proadhesive effect in this sepsis model are related, such as by testing rescue using extracellular CSNO (S-nitrosocysteine). We will test whether SNO export by one RBC population can influence the adhesivity of other RBCs in paracrine fashion. The relevance to human sepsis in Veterans will be tested by studying the LAT1-dependent ability of patient RBCs to export SNO basally and in hypoxia. 2. Determine the influence of LAT1-mediated SNO export on neutrophil adhesion. Neutrophil adhesion to the endothelium is a key early event in immune responses to infection. Antiadhesive NO/SNO is abundant in sepsis and may limit organ damage. We will determine the role of LAT1 and SNO export in modulating the adhesivity of neutrophils. We will determine the role of RBC SNO export in modulating the adhesion of neutrophils. We will identify the adhesion receptors and counterreceptors mediating LAT1/SNO-sensitive neutrophil adhesion. Finally, we will determine the role of LAT1-mediated SNO export (and EC SNO import) in the adhesivity of adoptively transferred neutrophils. 3. Determine the role of SNO export by RBCs and neutrophils in adhesion and extravasation in a mouse model of CLP-induced sepsis. We will measure the adhesion of both RBCs and neutrophils, and indices of organ injury, in sepsis secondary to CLP in our mice conditionally deficient in EC LAT1. We will determine the influence in septic (post-CLP) mice of autologous or heterologous RBC transfusion as a function of SNO export competence (LAT1 deletion or inhibitors). Given our demonstration that the proadhesive effect of RBC storage can be mitigated by RBC renitrosylation, we will investigate the influence of RBC SNO augmentation in a mouse model of sepsis (CLP)-induced cellular adhesion and organ injury. Numerous supportive and other therapies have failed in sepsis, including RBC transfusion for anemia, a clinically significant comorbidity in sepsis. Our innovative mechanistic focus on altered intercellular SNO transport, facilitates the identification of translatable solutions made possible in the form of vehicle RBC transfusates.

在重症患者中，贫血是差效果不佳的危险因素 与患者的限制性策略相比，这些患者的输血不能改善结果 保持中等贫血。在存储过程的早期发生的变化可能会通过损害而造成 RBC功能。我们已经确定了RBC存储第一天的功能和生化变化 有助于血液库的原始作用。最近，我们确定了一种新型机制 健康的人RBC导出一氧化氮（NO）衍生物S-硝基硫醇（SNO），以反对内皮 小鼠的体外和体内RBC的粘附。 RBC存储后，此RBC SNO导出会降低。学习 该途径的生理意义，我们设计了一只小鼠，负责的SNO转运蛋白 从内皮细胞中诱导地诱导。如预期的那样，该小鼠的EC进口很差，RBC 输血后，这些小鼠的肺中的粘附增加。在患有败血症的小鼠中（对 严重的感染），我们发现，即使RBC SNO含量升高，RBC的SNO出口也有所减少。我们 假设在败血症中，细胞间的SNO转运不足，促进了RBC的粘附性（天然或 输血）和中性粒细胞。我们将通过实现这些特定目标来检验这一假设： 1。确定败血症对RBC的抗污染SNO导出的影响。我们发现盲肠之后 连接和穿刺（CLP），鼠RBC导出抗粘附SNO的能力不足。我们将测试 该败血症模型中的SNO缺乏症和原始效应是否相关，例如通过测试救援 使用细胞外CSNO（S-亚硝基细胞半胱氨酸）。我们将测试一个RBC人口的SNO出口是否可以 以旁分泌方式影响其他RBC的粘附性。在退伍军人中与人类败血症的相关性将是 通过研究患者RBC的LAT1依赖性能力的测试，基本和缺氧。 2。确定LAT1介导的SNO输出对中性粒细胞粘附的影响。中性粒细胞粘附 内皮是免疫反应感染的关键早期事件。抗凝聚力NO/SNO很丰富 败血症并可能限制器官损伤。我们将确定LAT1和SNO导出在调制该的作用 中性粒细胞的粘附性。我们将确定RBC SNO导出在调节粘附方面的作用 中性粒细胞。我们将确定介导LAT1/SNO敏感的粘附受体和受体受体 中性粒细胞粘附。最后，我们将确定LAT1介导的SNO导出（和EC SNO进口）在 收养中性粒细胞的粘附性。 3。确定rbcs和中性粒细胞在小鼠粘附和渗出中的SNO导出的作用 CLP诱导的败血症模型。我们将测量RBC和中性粒细胞的粘附，以及 器官损伤，在我们的小鼠中有条件缺乏EC LAT1的败血症中。我们将确定 自体或异源RBC输血的化粪池（CLP后）小鼠的影响 能力（LAT1缺失或抑制剂）。考虑到我们的演示RBC存储的原始作用 可以通过RBC果仁糖基化来缓解，我们将研究RBC SNO增强的影响。 败血症（CLP）诱导的细胞粘附和器官损伤的模型。 败血症中有许多支持性和其他疗法失败，包括贫血的RBC输血，A 败血症的临床合并症。我们的创新机械重点是改变细胞间SNO 运输，促进以车辆RBC的形式识别可翻译解决方案 输血。