Aberrant RBC SNO transport and endothelial adhesion in sepsis

在危重病人中，贫血是预后不良的一个公认的危险因素，但矛盾的是，自由红细胞

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.