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

描述(申请人提供)：本项目的总体目标是测试假说，即不同的自组装形式，单体，低聚物和纤维，直接影响脑血管内皮细胞功能，因为它改变1)黏附分子的表达(例如，P-和E-选择素)和肌动蛋白聚合；2)细胞机械特性；以及3)这些改变是由涉及Aβ与晚期糖基化终产物受体(RAGE)结合的信号通路引起的，以诱导NADPH氧化酶和MAP激酶的激活。脑微血管内皮细胞的改变与阿尔茨海默病(AD)密切相关。阿尔茨海默病患者脑血管内Aβ沉积增多，血管壁单核细胞和激活的小胶质细胞增多。最近的研究表明，外周血单核细胞可以通过血脑屏障(BBB)在脑实质内分化为小胶质细胞。体外研究表明，在血管内皮细胞层沉积A‘可促进单核细胞的迁移。因此，单核细胞向大脑中迁移的增加被认为是导致AD大脑特有的氧化和炎症条件恶化的原因。与我们的假设一致，我们的初步数据显示，A？寡聚体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。由于单核细胞跨血脑屏障的迁移既是一个机械的过程，也是一个生化的过程，因此内皮细胞的黏附分子的表达和力学性质是需要研究的关键因素。在这方面，Aβ对内皮细胞黏附分子表达和力学性质的直接影响尚未完全阐明。因此，我们对Aβ如何改变单核细胞与内皮细胞表面黏附因子的研究将为AD的进展机制和干预策略的发展提供高度相关的新的生物物理信息。为了验证我们的中心假设，我们提出了三个具体的目标：目标1：确定A单体、寡聚体和纤维对永生化脑内皮细胞(CECs)P-和E-选择素表达以及肌动蛋白聚合的影响。目的2：检测A单体、低聚物和纤维对CECs细胞力学和黏附性能的影响。目的：探讨RAGE、NADPH氧化酶和MAP激酶(即p38、ERK和JNK)在A2诱导的内皮细胞黏附改变中的作用。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。 公共卫生相关性：脑微血管内皮细胞的改变与阿尔茨海默病密切相关。我们使用原子力显微镜来研究淀粉样β蛋白诱导的内皮细胞黏附特性改变的机制。这一机制在阿尔茨海默病的部分发展过程中起着重要作用。

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.