Targeting cerebrovascular endothelial cells as a therapeutic approach for amyloid pathogenesis

以脑血管内皮细胞为靶点治疗淀粉样变 项目摘要 淀粉样蛋白-(A-β)在脑血管中的积聚可导致脑组织发育 淀粉样血管病(CAA)。CAA是伴随阿尔茨海默病(AD)出现的一种病理特征 在高频率，突出了血管Aβ在阿尔茨海默病等痴呆中的潜在重要作用。而当 β介导血脑屏障有害作用的基础可能是多因素的， 大量研究表明，A-β参与了内皮细胞通透性的增加。确切的原因是 对于慢性再生障碍性贫血的血脑屏障功能障碍还不是很清楚，但Aβ从大脑的清除受到损害。 血脑屏障以及降低A血管周围引流的有效性被提出以增强 老年人脑血管和脑实质淀粉样蛋白沉积。尽管我们理解 在导致血脑屏障功能障碍和清除Aβ的途径中，治疗Aβ的药物的可用性 与发病机制相关的疾病，如CAA和AD，仍然缺乏。这个项目的长期目标是发展 以血脑屏障为靶点的治疗方法，以恢复其功能并最大限度地清除大脑中的Aβ，这是 对预防或延缓CAA和AD的发病很重要。这个项目的总体目标是充分描述一个 实验性内皮细胞血脑屏障模型作为一种有效的高通量筛选(HTS)格式来识别 治疗血管性Aβ病因性疾病(AD、CAA和血管性痴呆)。中心假说 是利用高度新颖的脑血管内皮细胞血脑屏障模型的高通量筛查(HTS)可以 用于识别有益地调节A清除和减少A介导的增加的小分子 血脑屏障渗透性。我们将通过追求以下具体目标来验证这一假设：1)细胞的利用率 基于直线的血脑屏障模型筛选A介导的内皮细胞功能紊乱的调节物。这 目标将通过调查以下子目标来实现：1)在现场筛选化合物 42寡聚体对脑血管内皮细胞通透性的影响 黄色(LY)作为总渗透性标志。降低介导的渗透率的化合物将是 高级对子目标1B；1B)使用GOLD确定血脑屏障模型中A清除的刺激物 作为A清除终点的碘化A的标准测量。Hit化合物将在#年进行进一步研究。 Aim 2.2)验证和研究Aim1中HIT化合物的调节能力 紧密连接和A清除蛋白的表达(转运和降解)。这一目标将是 通过测试以下子目标来完成：2a)进行二次确认并建立配置文件 目标1、2B中确定的HITS动力学比较改善42低聚物诱导的通透性 与亚目的2A、2C相比，原代脑血管内皮细胞的A清除量降低) HIT化合物改善血脑屏障密封性、完整性和清除的机理研究 从2B开始。3)测试目标2中的前两个命中在体内调节血管和实质A的能力 在CAA小鼠模型中的蓄积和血脑屏障完整性。要使用的方法和技术来实现 上述目标包括体外细胞培养、高通量筛选、转运、渗透和清除。 研究，A动力学，从野生型小鼠脑中分离微血管，以及在慢性再生障碍性贫血模型中的体内研究。 产生的数据将为未来的临床研究提供候选治疗分子进行测试。

Targeting cerebrovascular endothelial cells as a therapeutic approach for amyloid pathogenesis Project Summary The accumulation of amyloid- (Aβ) in the brain blood vessels can result in the development of cerebral amyloid angiopathy (CAA). CAA is a pathological feature present concomitantly with Alzheimer’s disease (AD) at a high frequency, highlighting a potentially important role for vascular Aβ in dementias such as AD. While the basis by which Aβ mediates deleterious effect on the blood-brain barrier (BBB) is likely multifactorial, numerous studies indicate a role for Aβ mediated increases in endothelial cell permeability. The exact causes for BBB dysfunction in CAA are not well known, however impaired clearance of Aβ from the brain across the BBB as well as a reduction in the efficacy of the perivascular drainage of A have been proposed to enhance accumulation of cerebrovascular and parenchymal amyloid deposits in the elderly. Despite our understanding of the pathways responsible for BBB dysfunction and clearance of Aβ, the availability of drugs to treat Aβ pathogenesis related disorders, CAA and AD, remains lacking. The long-term goal of this project is to develop therapeutics that target the BBB to restore its function and maximize clearance of Aβ from the brain, which is important to prevent or delay onset of CAA and AD. The overall goal of this project is to fully characterize an experimental endothelial BBB model as an effective high-throughput screening (HTS) format to identify therapeutics for vascular Aβ pathogenesis disorders (AD, CAA and vascular dementia). The central hypothesis is that high-throughput screening (HTS) utilizing a highly novel cerebrovascular endothelial BBB model can be used to identify small molecules which beneficially regulate A clearance and reduce A mediated increases in BBB permeability. We will test this hypothesis by pursuing the following specific aims: 1) Utilization of a cell line-based BBB model to screen for modulators of A mediated disturbances of endothelial cell function. This aim will be accomplished by investigating the following sub-aims: 1A) screen for compounds, in the presence of A42 oligomers, for their effect on the gross permeability of cerebrovascular endothelial cells using Lucifer Yellow (LY) as a gross permeability marker. Compounds which reduce A-mediated permeability will be advanced to Sub-Aim 1B; 1B) identify stimulators of A clearance across the BBB model using the gold standard measure of iodinated A as the endpoint for A clearance. Hit compounds will be further examined in Aim 2. 2) Validation and mechanistic investigation of hit compounds from Aim1 for their ability to modulate expression of tight junction and A clearance proteins (transport and degradation). This aim will be accomplished by testing the following sub-aims: 2A) conduct secondary confirmation and establish profiles for hits identified in Aim 1, 2B) comparison of hits kinetics for amelioration of A42 oligomers induced permeability and reduced A clearance in primary cerebrovascular endothelial cells with those of Sub-Aim 2A, 2C) mechanistic investigation for improved BBB tightness, integrity and A clearance by hit compounds selected from 2B. 3) Test ability of the top 2 hits from Aim 2 to in vivo modulate vascular and parenchymal A accumulation, and BBB integrity in a mouse model of CAA. Methods and technique to be used to accomplish the above aims include in vitro cell culture, high-throughput screening, transport, permeability and clearance studies, A kinetics, microvessels isolation from brains of wild type mice, and in vivo studies in CAA model. The data produced will provide candidate therapeutic molecules to test in future clinical studies.