Thiol isomerases and ERO1 alpha in sickle cell vaso-occlusion

项目摘要 临床前和临床研究提供了令人信服的证据，表明中性粒细胞被激活 在镰状细胞病(SCD)中，血管内皮细胞参与细胞间的聚集和血管闭塞(VOC)。 VOC与严重疼痛危象、急性胸腔综合征、常见并发症及病因呈正相关 SCD患者的死亡率。目前，VOC的有效治疗选择仍然有限。因此，一个更好的 了解中性粒细胞黏附功能的调节机制将有助于开发新的治疗方法 用于治疗慢性阻塞性肺疾病的VOC。我们先前发现中性粒细胞表面结合蛋白二硫键异构酶 (PDi)对αMβ2整合素的配体结合活性和中性粒细胞与内皮细胞的相互作用至关重要 血管发炎。这些发现提出了几个基本的问题，即人类的活动 细胞表面结合的PDI被控制以及其他硫醇异构酶是否也调节中性粒细胞的功能 表面受体。我们的初步数据显示内质网蛋白57(ERp57，一种硫醇 与PdI结构相似的异构酶)和硫醇异构酶的关键氧化酶ER氧化还原蛋白1α(ERO 1α 在内质网中)在激活的中性粒细胞表面被检测到，并在 调节炎症条件下中性粒细胞的黏附功能。使用生化、细胞和动物 用新的小鼠模型、封闭抗体和细胞不通透性多肽进行研究，我们将检验这一假说 细胞外ERP57和PDI与ERO1α共同作用于不同的表面受体，调节 配体结合功能，有助于SCD的血管内细胞-细胞相互作用和VOC。在目标1中，使用 一系列的生化和细胞生物学研究，我们将确定中性粒细胞硫醇的分子基础 异构酶和ERO_1α在调节中性粒细胞黏附功能中的作用在目标2中，使用活体成像技术， 我们将测试抑制细胞外ERP57、PDI和ERO1α是否减弱血管内细胞-细胞 SCD小鼠外周血管和肺血管的聚集和挥发性有机化合物。在目标3中，使用来自 SCD患者在不同的VOC条件下，我们将检测胞外硫醇异构酶和ERO_1α 促进细胞-细胞聚集，并与SCD患者的急性VOC病情相关。 我们的研究将为调节血管内细胞-细胞的新的分子机制提供见解 交互作用，可用于设计安全有效的策略来治疗SCD中VOC介导的疾病。

Project Summary Preclinical and clinical studies have provided compelling evidence that adherent neutrophils on activated vascular endothelium contribute to cell-cell aggregation and vaso-occlusion (VOC) in sickle cell disease (SCD). VOC positively correlates with severe pain crisis and acute chest syndrome, the common complication and cause of death in SCD patients. Currently, the effective treatment options for VOC remain limited. Thus, a better understanding of the regulatory mechanisms of neutrophil adhesive function will help develop novel therapies for treating VOC in SCD. We previously discovered that neutrophil surface-bound protein disulfide isomerase (PDI) is crucial for the ligand-binding activity of αMβ2 integrin and neutrophil-endothelial cell interactions during vascular inflammation. These findings have raised several fundamental questions regarding how the activity of cell surface-bound PDI is controlled and whether other thiol isomerases also regulate the function of neutrophil surface receptors. Our preliminary data demonstrate that endoplasmic reticulum protein 57 (ERp57, a thiol isomerase with structural similarity with PDI) and ER oxidoreductin 1α (ERO1α, a key oxidase of thiol isomerases in the ER) are detected on the surface of activated neutrophils and play overlapping and distinct roles in regulating neutrophil adhesive function under inflammatory conditions. Using biochemical, cellular, and animal studies with novel mouse models, blocking antibodies and cell-impermeable peptides, we will test the hypothesis that extracellular ERp57 and PDI in concert with ERO1α target different surface receptors and regulate the ligand-binding function, contributing to intravascular cell-cell interactions and VOC in SCD. In Aim 1, using a series of biochemical and cell biological studies, we will determine the molecular basis of neutrophil thiol isomerases and ERO1α in regulating neutrophil adhesive function. In Aim 2, using in vivo imaging techniques, we will test whether inhibition of extracellular ERp57, PDI and ERO1α attenuates intravascular cell-cell aggregation and VOC in peripheral and pulmonary vessels of SCD mice. In Aim 3, using blood samples from SCD patients under different VOC conditions, we will test whether extracellular thiol isomerases and ERO1α contribute to cell-cell aggregation and correlate with acute VOC conditions in SCD patients. Our studies will provide insights into novel molecular mechanisms mediating intravascular cell-cell interactions, which can be used to design safe and effective strategies to treat VOC-mediated conditions in SCD.