Pulmonary arteriole occlusion by platelet-neutrophil micro-emboli in Acute Chest Syndrome

 描述（由申请人提供）：急性胸部综合征（ACS）是镰状细胞病（SCD）患者发病的主要原因。可以预防ACS的治疗是非常必要的，但由于缺乏对引发ACS的事件的了解，这种治疗的发展已经停滞。感染和血管闭塞被认为是导致ACS的原因;然而，ACS的细胞和分子机制尚未完全了解。我们建议使用一个综合的生理学方法来测试的假设，ACS是由肺小动脉阻塞的中性粒细胞和血小板的聚集体，这个过程中的聚集需要P-选择素和GPIba上的膜延伸被称为“吊索”的血小板结合P-选择素-糖蛋白-配体-1（PSGL）-1和Mac-1，分别在中性粒细胞吊索。在目的1中，我们将使用活的转基因SCD小鼠中的肺脉管系统的体内多光子激发（MPE）成像来证明细菌脂多糖（LPS）的血管内（IV）施用导致肺小动脉中的中性粒细胞积聚、血小板在积聚的中性粒细胞上成核、微血栓的形成、血流的消除和肺损伤的连续步骤。PI先前已经表明，中性粒细胞形成吊索，其是用PSGL-1和CD 18装饰的长膜延伸。在目标2中，我们将使从SCD患者和种族匹配的对照受试者抽取的血液流过与人肺内皮细胞一起培养的体外微流体通道，并使用荧光显微镜来证明可以通过阻断血小板上的P-选择素或中性粒细胞上的Mac-1或防止中性粒细胞和血小板形成吊带来防止中性粒细胞上的血小板成核。在目的3中，我们将证明血小板P-选择素和中性粒细胞Mac-1缺陷的嵌合SCD小鼠不会发生肺小动脉闭塞，并且在用LPS IV攻击后免受ACS。这些目标的成功完成将证明ACS可以通过抑制血小板P-选择素和中性粒细胞Mac-1来预防。

 DESCRIPTION (provided by applicant): Acute chest syndrome (ACS) is a leading cause of morbidity among Sickle Cell Disease (SCD) patients. Therapies that can prevent ACS are highly warranted but the development of such treatments has been stalled by the lack of knowledge of the events that initiate ACS. Both infection and vaso-occlusion are believed to cause ACS; however, the cellular and molecular mechanism of ACS is not completely understood. We propose to use an integrative physiologic approach to test the hypothesis that ACS is caused by occlusion of the pulmonary arterioles by aggregates of neutrophils and platelets and this process of aggregation requires P-selectin and GPIba expressed on membrane extensions known as "slings" on platelets binding to P-selectin- glycoprotein-ligand-1 (PSGL)-1 and Mac-1, respectively on neutrophil slings. In Aim 1, we will use in vivo multi- photon excitation (MPE) imaging of the lung vasculature in live transgenic SCD mice to prove that intravascular (IV) administration of bacterial lipopolysaccharide (LPS) results in sequential steps of neutrophil accumulation in the pulmonary arteriole, platelet nucleation on accumulated neutrophils, formation of micro-thrombus, abrogation of blood flow and lung injury. The PI has shown previously that neutrophils form slings, which are long membrane extensions decorated with PSGL-1 and CD18. In Aim 2, we will flow blood drawn from SCD patients and race-matched control subjects through in vitro microfluidic channels cultured with human lung endothelial cells and use fluorescence microscopy to prove that platelet nucleation on neutrophils can be prevented by either blocking P-selectin on platelets or Mac-1 on neutrophils or preventing sling formation by neutrophils and platelets. In the Aim 3, we will show that chimeric SCD mice deficient in platelet P-selectin and neutrophil Mac-1 do not develop pulmonary arteriole occlusion and are protected from ACS following IV challenge with LPS. The successful completion of these Aims will demonstrate that ACS can be prevented by inhibiting platelet P-selectin and neutrophil Mac-1.