Single Molecule Analysis of Erythrocyte Adhesion in Sickle Cell Disease

镰状细胞病中红细胞粘附的单分子分析

• 批准号: 7066633
• 负责人: DAVID E. GOLAN
• 金额: $ 21.19万
• 依托单位: BOSTON MEDICAL CENTER
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7066633
• 关键词: CD2 molecule; T lymphocyte; blood vessel occlusion; cell adhesion; cell cell interaction; clinical research; confocal scanning microscopy; cooperative study; erythrocytes; fluorescence microscopy; fluorescence resonance energy transfer; human subject; integrins; intravital microscopy; leukocyte activation /transformation; leukocyte adhesion molecules; membrane proteins; shear stress; sickle cell anemia; thrombospondins; vascular cell adhesion molecule; vascular endothelium

Adhesive interactions involving sickle erythrocytes are critically important in the pathophysiology of vaso-occlusive crisis, hemolytic anemia, and other clinical manifestations of sickle cell disease. These interactions appear to be mediated by the abnormal expression and/or function of adhesion molecules on the surface of sickle erythrocytes. Recent studies in model membranes and intact biological systems have begun to elucidate the biochemical and biophysical properties of adhesion molecules that are necessary for stable adhesion; these properties include adhesion molecule expression, size, lateral mobility, surface density, surface distribution, and affinity for the molecule's cognate ligand. Although a number of molecular interactions involved in sickle lerythrocyte adhesion to vascular endothelial cells and T lymphocytes have been identified, the properties of these molecular interactions that are important for stable adhesion remain to be characterized. We have developed a unique set of biophysical and imaging techniques to study, at the level of individual adhesion molecules, the molecular interactions involved in cell-cell adhesion. The methods include fluorescence photobleaching recovery, polarized fluorescence depletion, single particle tracking, laser optical tweezers, glass-supported planar bilayer membranes, fluorescence resonance energy transfer, and dynamic in vitro and in vivo (intravital) adhesion assays. Here we propose to apply these methods to the study of (1) membrane protein iand lipid dynamics in sickle erythrocytes, (2) adhesive interactions between sickle erythrocytes and activated vascular endotheliai cells, and (3) adhesive interactions between sickle erythrocytes and activated T lymphocytes. We shall use these methods to study adhesive interactions involving the adhesion molecules VLA-4 (alpha4beta1integrin), CD36, and CD2 on sickle erythrocytes, VCAM-1 and alpha-v, beta3 integrin on activated vascular endothelial cells, CD58 on activated T lymphocytes, and the adhesive plasma protein thrombospondin. Results from these studies are expected to lead to a quantitative understanding of important molecular and cellular events in the pathophysiology of sickle cell disease, and, ideally, to point the way to targeted therapies that interrupt the most critical aspects of these molecular and cellular events.

涉及镰状红细胞的粘合剂相互作用在血管钙化危机，溶血性贫血和其他镰状细胞疾病的其他临床表现的病理生理学中至关重要。这些相互作用似乎是由镰状红细胞表面粘附分子的异常表达和/或功能介导的。在模型膜和完整生物系统中的最新研究已开始阐明稳定粘附所必需的粘附分子的生化和生物物理特性。这些 特性包括粘附分子表达，大小，横向迁移率，表面密度，表面分布以及对分子的同源配体的亲和力。尽管已经鉴定出与血管内皮细胞和T淋巴细胞相对于镰状LeryThrocyte粘附的许多分子相互作用，但这些分子相互作用的特性对于稳定粘附很重要。我们开发了一组独特的生物物理和成像技术，以在单个粘附分子的水平上研究与细胞细胞粘附有关的分子相互作用。这些方法包括荧光 光漂白恢复，极化荧光消耗，单个颗粒跟踪，激光光学镊，玻璃支持的平面双层膜，荧光共振能传递以及体外和体内动态（玻璃体）粘附力。在这里，我们建议将这些方法应用于（1）镰状红细胞中的膜蛋白Iand脂质动力学，（2）（2）（2）镰状红细胞和活化的血管内皮细胞之间的粘合性相互作用，以及（3）（3）镰状ERHES的粘膜相互作用，镰状erythrocythrocythocythrocytes tlymphythcyte t lymphyperpated tlymphyphypypypypytessessessessestessestessestessestessestessestessestessestessestes。 We shall use these methods to study adhesive interactions involving the adhesion molecules VLA-4 (alpha4beta1integrin), CD36, and CD2 on sickle erythrocytes, VCAM-1 and alpha-v, beta3 integrin on activated vascular endothelial cells, CD58 on activated T lymphocytes, and the adhesive plasma protein血小板传播。这些研究的结果预计将导致对镰状细胞病的病理生理学中重要的分子和细胞事件的定量理解，理想情况下，可以为中断这些分子和细胞事件最关键方面的靶向疗法指向。