Adhesion mechanisms mediating sickle cell vasoocclusion in vivo

描述（由申请人提供）：本提案的长期目标是表征镰状细胞病中介导血管闭塞（VOC）的粘附机制。我们之前在镰状细胞小鼠中使用活体显微镜显示，镰状红细胞（rbc）与体内炎症小静脉中的粘附白细胞（wbc）相互作用。红细胞和白细胞之间的相互作用似乎具有重要的生物学意义，因为它们在野生型和镰状细胞小鼠中都存在，并且它们在镰状细胞小鼠小静脉中的数量与微血管血流速度降低和活体显微镜下存活时间缩短相关。此外，抑制红细胞-白细胞相互作用可以保护镰状细胞小鼠免受VOC的侵害。最近的研究使用一种新型的数字多通道宽视场荧光显微镜系统已经确定了多形核中性粒细胞（pmn）作为捕获循环红细胞的主要白细胞亚群。e -选择素介导的信号在激活pmn捕获红细胞中起关键作用。PI实验室的其他研究表明，PMN上的所有e -选择素配体（ESL）活性都是由三种糖蛋白PSGL-1、CD44和ESL-1赋予的。在此，我们拟进一步研究挥发性有机化合物在镰状细胞小鼠体内的细胞和分子机制。在特异性目标1中，我们将确定哪种ESL诱导白细胞激活和循环红细胞的捕获。我们将使用遗传缺失（Cd44 /, Selplg /，或缺乏两者的小鼠）和RNA干扰（针对ESL-1的短发夹RNAi），通过慢病毒转导引入造血干细胞（hsc）。在特异性目标2中，我们将使用高速数字视频显微镜定义介导循环镰状红细胞捕获的白细胞微结构域和分子决定因子。在C57BL/6小鼠中进行的初步实验表明，聚集在粘附白细胞前沿的受体介导了红细胞的捕获，b2整合素Mac-1的表达至关重要。在Specific Aim 3中，我们将研究合成所有选择素配体所需的白细胞聚焦转移酶（Fut7）作为镰状细胞VOC的治疗靶点的作用，使用RNAi下调hsc中Fut7的表达，然后生成辐射嵌合体，并使用缺乏Fut7的镰状细胞小鼠。在最后一篇Specific Aim中，我们将使用我们已经生成的白细胞粘附缺陷动物模型来评估白细胞粘附缺陷对慢性溶血的长期影响，以及对靶器官功能和病理的影响。提出的研究将为白细胞在镰状细胞血管闭塞中的作用提供新的机制见解，并可能导致新的治疗选择，以预防或治疗这种使人衰弱的疾病。

DESCRIPTION (provided by applicant): The long-term objective of this proposal is to characterize the adhesion mechanisms mediating vaso-occlusion (VOC) in sickle cell disease. We have previously shown using intravital microscopy in sickle cell mice that sickle red blood cells (RBCs) interact with adherent leukocytes (WBCs) in inflamed venules in vivo. The interactions between RBCs and WBCs appear to be biologically important since they occur in both wild-type and sickle cell mice, and their numbers in sickle cell mouse venules correlate with reduced microvascular blood flow velocities and shorter survival during intravital microscopy. In addition, inhibition of RBC-WBC interactions protects sickle cell mice from VOC. Recent studies using a novel digital multichannel widefield fluorescence microscopy system has identified polymorphonuclear neutrophils (PMNs) as the major leukocyte subset capturing circulating RBCs. E-selectin-mediated signaling plays a critical role in enabling activated PMNs to capture RBCs. Other studies from the PI's laboratory have revealed that all E-selectin ligand (ESL) activity on PMN is conferred by three glycoproteins, PSGL-1, CD44 and ESL-1. Here, we propose to study further the cellular and molecular mechanisms mediating VOC in sickle cell mice. In Specific Aim 1, we will identify which ESL induces leukocyte activation and the capture of circulating RBCs. We will use genetic deletion (Cd44 / , Selplg / , or mice lacking both) and RNA interference (short hairpin RNAi targeting ESL-1) introduced by lentiviral transduction of hematopoietic stem cells (HSCs). In Specific Aim 2, we will define the microdomains and molecular determinants on leukocytes that mediate the capture of circulating sickle RBCs using high-speed digital videomicroscopy. Preliminary experiments in C57BL/6 mice suggest that receptors that cluster to the leading edge of adherent leukocytes mediate the capture of RBCs, and that expression of the b2 integrin Mac-1 is critical. In Specific Aim 3, we will investigate the role of the leukocyte fucosyltransferase (Fut7), required for the synthesis of all selectin ligands, as therapeutic target for sickle cell VOC using RNAi downregulation of Fut7 expression in HSCs followed by generation of radiation chimeras, and using sickle cell mice deficient in Fut7. In the last Specific Aim, we will use the animal models of leukocyte adhesion defects that we have generated to evaluate the long-term impact of leukocyte adhesion deficits on chronic hemolysis, and on the function and pathology of target organs. The proposed studies will provide new mechanistic insights into the role of leukocytes in sickle cell vascular occlusion and may lead to new therapeutic options to prevent or treat this debilitating illness.