Thiol isomerases and ERO1 alpha in sickle cell vaso-occlusion

项目概要 临床前和临床研究提供了令人信服的证据，表明活化的中性粒细胞粘附 血管内皮有助于镰状细胞病 (SCD) 中的细胞聚集和血管闭塞 (VOC)。 VOC 与严重疼痛危象和急性胸部综合征（常见并发症和原因）呈正相关 SCD 患者的死亡。目前，VOC 的有效处理方案仍然有限。因此，更好的 了解中性粒细胞粘附功能的调节机制将有助于开发新疗法 用于处理 SCD 中的 VOC。我们之前发现中性粒细胞表面结合蛋白二硫键异构酶 (PDI) 对于 αMβ2 整合素的配体结合活性和中性粒细胞-内皮细胞相互作用至关重要 血管炎症。这些发现提出了几个基本问题： 细胞表面结合的 PDI 受到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。我们的初步数据表明，内质网蛋白 57（ERp57，一种硫醇 与 PDI 结构相似的异构酶）和 ER 氧化还原素 1α（ERO1α，硫醇异构酶的关键氧化酶） 在 ER 中）在活化的中性粒细胞表面检测到，并在以下过程中发挥重叠和不同的作用： 在炎症条件下调节中性粒细胞粘附功能。使用生物化学、细胞和动物 使用新型小鼠模型、阻断抗体和细胞不可渗透的肽进行研究，我们将检验该假设 细胞外 ERp57 和 PDI 与 ERO1α 一起靶向不同的表面受体并调节 配体结合功能，有助于血管内细胞间相互作用和 SCD 中的 VOC。在目标 1 中，使用 通过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 ERO1α 调节中性粒细胞粘附功能。在目标 2 中，使用体内成像技术， 我们将测试细胞外 ERp57、PDI 和 ERO1α 的抑制是否会减弱血管内细胞间的作用 SCD 小鼠外周血管和肺血管中的聚集和 VOC。在目标 3 中，使用来自 SCD患者在不同VOC条件下，我们会检测细胞外硫醇异构酶和ERO1α是否存在 有助于细胞间聚集并与 SCD 患者的急性 VOC 状况相关。 我们的研究将为介导血管内细胞间的新分子机制提供见解 相互作用，可用于设计安全有效的策略来治疗 VOC 介导的 SCD 病症。

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.