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。可以预防急性冠脉综合征的治疗方法是非常有必要的，但由于缺乏对引发急性冠脉综合征的事件的了解，这种治疗方法的开发一直停滞不前。感染和血管闭塞都被认为是导致急性冠脉综合征的原因，然而，急性冠脉综合征的细胞和分子机制还不完全清楚。我们建议使用一种综合生理学方法来检验这一假说，即急性冠脉综合征是由于中性粒细胞和血小板聚集物堵塞肺小动脉而引起的，这一聚集过程需要P-选择素和GPIba在中性粒细胞上分别与P-选择素-糖蛋白-配体-1(PSGL)-1和Mac-1结合的膜延伸上表达P-选择素和GPIba。在目标1中，我们将使用活体多光子激发(MPE)对活体转基因SCD小鼠肺血管的成像来证明，血管内(IV)给药导致中性粒细胞在肺小动脉聚集、血小板在聚集的中性粒细胞上成核、微血栓形成、血流减少和肺损伤。PI之前已经表明，中性粒细胞形成吊索，这是装饰着PSGL-1和CD18的长膜延伸。在目标2中，我们将从SCD患者和种族匹配的对照组中提取血液，通过体外培养人肺内皮细胞的微流体通道，并使用荧光显微镜来证明，通过阻断血小板上的P-选择素或中性粒细胞上的Mac-1或阻止中性粒细胞和血小板形成吊索，可以防止血小板在中性粒细胞上的成核。在目标3中，我们将展示缺乏血小板P-选择素和中性粒细胞Mac-1的嵌合SCD小鼠不会发生肺小动脉闭塞，并在内毒素静脉攻击后保护其免受ACS的影响。这些目标的成功实现将证明，可以通过抑制血小板P-选择素和中性粒细胞Mac-1来预防ACS。

 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.