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.

在危重病人中，贫血是一个公认的不良结局的危险因素，但矛盾的是，自由红细胞 与限制性策略相比，这些患者的输血并不能改善结果 仍处于中度贫血状态。在存储过程的早期发生的更改可能会损害 红细胞功能。我们已经确定了RBC储存第一天的功能和生化变化 有助于血库的前黏附效应。最近，我们发现了一种新的机制， 健康人红细胞输出一氧化氮(NO)衍生物S亚硝硫醇(SNO)以对抗内皮细胞 小鼠红细胞体内外黏附的实验研究。该RBC SNO输出在RBC存储后被抑制。学习 这一途径的生理意义，我们设计了一种小鼠，在其中负责SNO转运体 可诱导性地从内皮细胞中删除。这只小鼠的ECS导入SNO效果不佳，RBC 输血后，这些小鼠的肺部粘附性增加。在脓毒症小鼠中(对 严重感染)，我们发现，即使RBC SNO含量增加，RBC对SNO的输出也减少。我们 假设在脓毒症中，细胞间SNO转运不足，从而促进红细胞(天然或 输血)和中性粒细胞。我们将通过实现以下具体目标来检验这一假设： 1.确定脓毒症对红细胞输出抗黏附SNO的影响。我们发现盲肠后 结扎和穿孔(CLP)，小鼠红细胞输出抗黏附SNO的能力不足。我们将测试 在这个脓毒症模型中，SNO缺乏和前黏附效应是否相关，例如通过测试救援 使用胞外CSNO(S-亚硝半胱氨酸)。我们将测试一个RBC人口的SNO输出是否可以 以旁分泌方式影响其他红细胞的粘附性。退伍军人与人类败血症的相关性将是 通过研究患者红细胞在碱性和低氧条件下依赖LAT1输出SNO的能力进行测试。 2.确定LAT1介导的SNO输出对中性粒细胞黏附的影响。中性粒细胞与 内皮细胞是感染免疫反应中的一个关键早期事件。防粘剂NO/SNO在 败血症，并可能限制器官损害。我们将确定LAT1和SNO导出在调节 中性粒细胞的粘附性。我们将确定RBC SNO输出在调节细胞黏附中的作用 中性粒细胞。我们将鉴定介导LAT1/SNO敏感性的黏附受体和拮抗受体 中性粒细胞黏附。最后，我们将确定LAT1介导的SNO导出(和EC SNO导入)在 过继转移的中性粒细胞的粘附性。 3.确定红细胞和中性粒细胞输出SNO在小鼠黏附和渗出中的作用 CLP致脓毒症模型的建立。我们将测量红细胞和中性粒细胞的粘附性，以及 器官损伤，在我们的小鼠继发于CLP的脓毒症中EC LAT1条件缺陷。我们将确定 SNO输出对脓毒症(CLP后)小鼠自体或异体RBC输注的影响 能力(LAT1缺失或抑制物)。鉴于我们的论证，红细胞储存的前黏附效应 RBC-SNO增加对小鼠的影响。 脓毒症(CLP)诱导的细胞黏附和器官损伤模型。 许多支持疗法和其他疗法在败血症中都失败了，包括针对贫血的红细胞输注， 败血症的临床显著合并症。我们的创新机制专注于改变细胞间SNO 运输，便于识别以车辆RBC形式实现的可翻译解决方案 输血。