The role of APOE genotype in modulating the transcriptomics profile of brain endothelial cells in vitro. The role of APOE genotype in modulating the transcriptomics profile of brain endothelial cells in vitro

Human APOE genotype is intricately involved in the homeostasis of the central nervous system, which is particularly evident from its association with neurodegenerative disease. Compared to APOE3, APOE4 is associated with greater cognitive decline in aging, poorer outcomes following stroke and traumatic brain injury, and is a major genetic risk factor for Alzheimer’s disease. Human APOE genotypes can differentially modulate neuronal function via both direct and indirect pathways. Of the indirect pathways, increasing evidence supports that APOE4-associated neurovascular dysfunction plays a significant role in neurodegeneration. For example, deficits in cerebral blood flow, neuronal-vascular coupling and blood-brain barrier integrity are observed with APOE4 both during aging and in Alzheimer’s disease patients; data that has been recapitulated in mouse models. Therefore, the identification of cellular mechanistic pathways that contribute to APOE-modulated vascular function is important for fundamental and disease-focused research. Highly specialized brain endothelial cells (BECs) are essential for virtually all neurovascular functions and, therefore, the influence of apoE on the cerebrovasculature likely involves alterations in brain endothelial cells function, directly or via supporting cells. ApoE synthesis has been demonstrated in multiple cell types in both the brain (e.g. astrocytes, pericytes and neurons) and periphery (e.g. hepatocytes and macrophages). Current research has largely focused on the effects of apoE produced by supporting cell types, particularly astrocytes and pericytes, on BEC function. However, less explored is whether BECs produce apoE to modulate their function. Data from limited studies on apoE production by BECs and APOE genotype-specific functional differences are conflicting. Evaluation of whether the APOE genotype of BECs modulates their function is important, as this could represent a novel mechanisms that contributes to altered cerebrovascular function in aging and AD. The goal of this study was to evaluate the role of human APOE genotype in modulating the phenotype and/or function of brain endothelial cells in vitro. To this end, we utilized primary brain endothelial cells isolated from APOE-targeted replacement mice that express human APOE3 or APOE4 under the endogenous mouse APOE promoter. BECs are highly specialized and functionally complex to maintain homeostasis of the central nervous system and APOE genotype could theoretically alter levels of any number of molecules and associated functions. Thus, without a priori knowledge we initially evaluated the extent that APOE modulated the transcriptomic profile of brain endothelial cells. Overall design: Two genotypes of brain endothelial cells (APOE3, APOE4) with 8 biological replicates. A biological replicate correspnds to an isoaltion from a different animal.

人类载脂蛋白E（apolipoprotein E，APOE）基因型与中枢神经系统内稳态密切相关，这一点从其与神经退行性疾病的关联中便可清晰窥见。相较于APOE3，APOE4与衰老过程中更为显著的认知衰退、脑卒中及创伤性脑损伤后的不良预后相关，同时也是阿尔茨海默病（Alzheimer’s disease，AD）的主要遗传风险因子。人类APOE基因型可通过直接与间接两条通路差异化调控神经元功能。在间接通路中，越来越多的证据表明，APOE4相关的神经血管功能障碍在神经退行性病变中发挥关键作用。例如，无论在衰老过程中还是阿尔茨海默病患者体内，APOE4均会导致脑血流量、神经血管耦合及血脑屏障完整性受损；相关实验结果已在小鼠模型中得到重现。因此，阐明介导APOE调控血管功能的细胞机制通路，对于基础研究及疾病相关研究均具有重要价值。高度特化的脑内皮细胞（brain endothelial cells，BECs）几乎介导所有神经血管功能，因此apoE对脑血管系统的影响很可能通过直接或间接改变脑内皮细胞功能实现。现有研究证实，大脑内（如星形胶质细胞、周细胞及神经元）与外周组织（如肝细胞、巨噬细胞）中的多种细胞类型均可合成apoE。当前研究大多聚焦于支持细胞（尤其是星形胶质细胞与周细胞）产生的apoE对BEC功能的调控作用，而关于BEC自身是否产生apoE以调控其功能的探索则相对匮乏。目前针对BEC产生apoE的有限研究数据，以及APOE基因型特异性功能差异的相关结果尚存争议。评估BEC的APOE基因型是否调控其自身功能具有重要意义，因为这或许能揭示衰老及阿尔茨海默病患者脑血管功能异常的全新机制。本研究旨在探究人类APOE基因型在体外环境下调控脑内皮细胞表型和/或功能中的作用。为此，我们利用了APOE靶向替换小鼠来源的原代脑内皮细胞——这些小鼠在内源性小鼠APOE启动子的调控下表达人类APOE3或APOE4。BECs具有高度特化的功能特性，维持中枢神经系统内稳态的过程极为复杂，理论上APOE基因型可改变多种分子的表达水平及相关功能。因此，在缺乏先验知识的前提下，我们首先评估了APOE对脑内皮细胞转录组谱的调控程度。整体实验设计：两组基因型的脑内皮细胞（APOE3、APOE4），各设置8个生物学重复。每个生物学重复对应自不同个体动物的一次细胞分离操作。