Targeting cerebrovascular endothelial cells as a therapeutic approach for amyloid pathogenesis

 描述(申请人提供)：以脑血管内皮细胞为靶点作为淀粉样蛋白发病的治疗方法。淀粉样蛋白β在脑血管中的积聚可导致脑淀粉样血管病的发生。CAA是阿尔茨海默病(AD)伴发的一种高频率病理特征，突出了血管A？在痴呆(如AD)中的潜在重要作用。虽然Aü介导血脑屏障(BBB)有害效应的基础可能是多因素的，但大量研究表明，Aü介导的内皮细胞通透性增加也起到了作用。慢性再生障碍性贫血患者血脑屏障功能障碍的确切原因尚不清楚，但已有研究认为，血脑屏障清除功能受损以及A-β血管周围引流的效果降低是促进老年人脑血管和实质淀粉样蛋白沉积的原因之一。尽管我们了解了导致血脑屏障功能障碍和清除A？的途径，但治疗A？病程相关疾病CAA和AD的药物仍然缺乏。该项目的长期目标是开发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A？，这对预防或推迟CAA和AD的发病非常重要。该项目的总体目标是将实验性内皮BBB模型完全表征为一种有效的高通量筛选(HTS)形式，以确定治疗血管A？发病机制障碍(AD、CAA和血管性痴呆)的药物。中心假设是，利用新型脑血管内皮细胞血脑屏障模型的高通量筛选可用于识别有利于调节Aβ清除和降低Aβ介导的血脑屏障通透性增加的小分子。我们将通过追求以下特定目标来验证这一假设：1)利用基于细胞系的血脑屏障模型来筛选Aβ介导的内皮细胞功能紊乱的调节器。这一目标将通过调查以下子目标来实现：1)在Aβ42低聚体存在的情况下，使用荧光黄(LY)作为总渗透性标记筛选化合物，以了解它们对脑血管内皮细胞总渗透性的影响。降低Aβ介导的通透性的化合物将被推进到子目标1B；1B)使用碘化Aβ作为Aβ清除的终点的黄金标准来确定整个血脑屏障模型中Aβ清除的刺激物。来自Aim1的HIT化合物的有效性和机制研究将在Aim 2)验证和机制研究中进一步检验其调节紧密连接表达的能力 和Aβ清除蛋白(运输和降解)。这一目标将通过测试以下子目标来实现：2A)对目标1、2B中确定的HITS进行二次确认并建立概况)比较目标1、2B中用于改善Aβ42寡聚体诱导的原代脑血管内皮细胞通透性和减少Aβ清除的HITS动力学与子目标2A、2C中的那些)通过从2B中选择的HIT化合物改善血脑屏障密封性、完整性和Aβ清除的机理研究。3)在慢性再生障碍性贫血小鼠模型中，测试目标2中的前两个HITS在体内调节血管和实质Aβ积聚和血脑屏障完整性的能力。实现上述目标的方法和技术包括体外细胞培养、高通量筛选、转运、通透性和清除性研究、Aβ动力学、野生型小鼠脑微血管分离和体内研究。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。

 DESCRIPTION (provided by applicant): Targeting cerebrovascular endothelial cells as a therapeutic approach for amyloid pathogenesis. The accumulation of amyloid-β (Aß) 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 Aß in dementias such as AD. While the basis by which Aß mediates deleterious effect on the blood-brain barrier (BBB) is likely multifactorial, numerous studies indicate a role for Aß mediated increases in endothelial cell permeability. The exact causes for BBB dysfunction in CAA are not well known, however impaired clearance of Aß from the brain across the BBB as well as a reduction in the efficacy of the perivascular drainage of Aβ 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 Aß, the availability of drugs to treat Aß 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 Aß 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 Aß 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 Aβ clearance and reduce Aβ 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 Aβ 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 Aβ42 oligomers, for their effect on the gross permeability of cerebrovascular endothelial cells using Lucifer Yellow (LY) as a gross permeability marker. Compounds which reduce Aβ-mediated permeability will be advanced to Sub-Aim 1B; 1B) identify stimulators of Aβ clearance across the BBB model using the gold standard measure of iodinated Aβ as the endpoint for Aβ 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 Aβ 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 Aβ42 oligomers induced permeability and reduced Aβ clearance in primary cerebrovascular endothelial cells with those of Sub-Aim 2A, 2C) mechanistic investigation for improved BBB tightness, integrity and Aβ 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 Aβ accumulation, and BBB integrity in a mouse model of CAA. Methods and techniques to be used to accomplish the above aims include in vitro cell culture, high-throughput screening, transport, permeability and clearance studies, Aβ 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.