Amyloid-B peptide on endothelial adhesion and its related cellular pathways

描述（由申请人提供）：该项目的总体目标是测试假设淀粉样β肽（A¿）在不同的自组装形式，单体，低聚物和原纤维中，直接影响脑内皮功能，因为它改变了1)粘附分子（例如P-和e -选择素）和肌动蛋白聚合的表达；2)细胞力学性能；以及粘附力；3)这些变化是由A¿与晚期糖基化终产物受体（RAGE）结合诱导NADPH氧化酶和MAP激酶激活的信号通路引起的。脑微血管内皮的改变与阿尔茨海默病（AD）密切相关。在AD大脑中经常观察到脑血管中A¿沉积增加，血管壁中单核细胞和活化的小胶质细胞的存在增加。最近的研究表明，外周单核细胞可以跨越血脑屏障（BBB）并在脑实质内分化为小胶质细胞。体外研究表明，A¿沉积在内皮细胞层增强单核细胞的迁移。因此，单核细胞向大脑的迁移增加被认为是促使疾病发展向AD大脑特征的氧化和炎症条件恶化的原因。与我们的假设一致，我们的初步数据表明，A¿寡聚物增加了内皮细胞表面p -选择素的免疫反应性和内皮细胞内肌动蛋白的聚合。由于单核细胞在血脑屏障上的迁移既是一个机械过程，也是一个生化过程，因此内皮细胞粘附分子的表达和机械特性是需要研究的关键因素。在这方面，A¿对内皮细胞粘附分子表达和力学性能的直接影响尚未完全阐明。因此，我们对A¿如何改变单核细胞与内皮表面之间粘附因子的研究将为AD的进展机制和干预策略的制定提供高度相关的新生物物理信息。为了验证我们的中心假设，我们提出了三个具体目标：目标1：确定A¿单体，低聚物和原纤维对永生化脑内皮细胞（CECs）中P-和E-选择素表达和肌动蛋白聚合的影响。目的2：测量A¿单体、低聚物和原纤维对CECs细胞力学和粘附性能的影响。目的3：确定RAGE、NADPH氧化酶和MAP激酶（即p38、ERK和JNK）在a2诱导的内皮粘附改变中的作用。这些目标将通过各种生物物理和生化技术来实现，包括定量免疫荧光显微镜、原子力显微镜和各种生化方法。

DESCRIPTION (provided by applicant): The overall goal of this project is to test the hypothesis that amyloid beta-peptide (A¿) in different self-assembled forms, monomers, oligomers and fibrils, directly impacts cerebral endothelial functions because it alters 1) the expression of adhesion molecules (e.g. P- and E-selectins) and actin polymerization; 2) cellular mechanical properties; and the forces involved in adhesion; and 3) these alterations are resulted from the signaling pathway involving the binding of A¿ to receptor for advanced glycation end products (RAGE) to induce activations of NADPH oxidase and MAP kinases. Alteration of the cerebral microvascular endothelium is intimately associated with Alzheimer's Disease (AD). Increased deposition of A¿ in cerebral vasculature and the increased presence of monocytes in the vessel wall and of activated microglia are frequently observed in AD brains. Recent studies show that peripheral monocytes can migrate across the blood-brain barrier (BBB) and differentiate into microglia within the brain parenchyma. In vitro studies demonstrated that A¿ deposition at the endothelial cell layer enhances the transmigration of monocytes. Thus, increased transmigration of monocytes into brains is thought to drive the disease development towards exacerbation of the oxidative and inflammatory conditions characteristic of the AD brain. Consistent with our hypothesis, our preliminary data show that A¿ oligomer increases the immunoreactivity of P-selectin at the endothelial surface and actin polymerization within endothelial cells. Since the transmigration of monocytes across the BBB is both a mechanical and a biochemical process, the expression of adhesion molecules and mechanical properties of endothelial cells are the critical factors that require investigation. In this regard, the direct effects of A¿ on the expression of adhesion molecules and the mechanical properties of endothelial cell have yet to be fully elucidated. Therefore, our study on how A¿ alters the factors governing the adhesion between monocytes and endothelial surfaces will provide highly relevant and novel biophysical information about the mechanism of progression of AD and development of intervention strategy. To test our central hypothesis, we propose three specific aims: Aim 1: Determine the effects of A¿ monomers, oligomers and fibrils on the expression of P- and E- selectins and actin polymerization in immortalized cerebral endothelial cells (CECs). Aim 2: Measure the effects of A¿ monomers, oligomers and fibrils on the cell mechanical and adhesion properties of CECs. Aim 3: Determine the roles of RAGE, NADPH oxidase and MAP kinases (i.e. p38, ERK and JNK) in A2-induced alterations in endothelial adhesion. These aims will be accomplished by various biophysical and biochemical techniques including quantitative immunofluorescent microscopy, atomic force microscopy and various biochemical approaches. PUBLIC HEALTH RELEVANCE: Alteration of the cerebral microvascular endothelium is intimately associated with Alzheimer's Disease. We use atomic force microscopy to investigate the mechanism underlying the altered adhesion properties of endothelium induced by amyloid beta peptide. The mechanism is important in part of Alzheimer's disease development.