High-throughput nanoIEA-based Assay for Screening Immune Cell-Vascular Interactions

项目总结 血管通过将免疫细胞运送到感染部位，在维持宿主免疫方面发挥着核心作用。 在这个过程中，血管经历内皮连接重塑来控制血管的通透性。 和免疫细胞渗出。在感染下，血管变得可渗透，并允许免疫细胞 渗出并杀死间质中的病原体。一旦感染消除，通透性血管就会变得更少。 并限制间质免疫细胞的数量。然而，有时在炎症中，重塑 会受到干扰，导致血管长时间、高通透性。这种血管功能障碍导致 免疫性疾病，如慢性炎症、狼疮和自身免疫性疾病。众所周知，血管内皮细胞 细胞排列是维持完整的细胞间黏附和促进连接成熟的关键。尽管 细胞排列在功能性内皮中的意义，目前可用的高通量方法，如 作为实时细胞分析(RTCA)和跨上皮/跨内皮电阻(TEER)系统 随机播种的细胞没有成功地通过输入测量单元阻抗或电阻 活体样控制内皮细胞形态、排列和成熟的细胞-细胞连接。此外， 目前的技术缺乏周细胞与内皮细胞的共培养。在这项建议中，我们将发展一种高度- 能够更快地进行药物筛选和机制研究的吞吐量、高含量功能筛选试验 对血管屏障功能和免疫细胞外渗的影响。为了实现我们的目标，我们将建立一个 基于纳米IEA的功能分析高通量筛选周细胞表型 内皮细胞。为了建立基于纳米粒子的IEA检测，我们将确定连接条件 人真皮和肺微血管内皮细胞在有或无周细胞作用下的成熟，关注(I) 细胞排列程度；(Ii)黏附连接、极化和基底膜标记物的表达； (3)血管屏障功能(目标1.1)。然后我们将评估与血管相关的血管基因表达谱 稳定和免疫细胞黏附。接下来，我们将评估免疫细胞通过内皮细胞的外渗。 在非炎症条件下测定稳态血管中免疫细胞的行为(目标1.2)。 接下来，我们将验证该系统对炎症诱导的血管功能障碍的实用性。要做到这一点 目的：我们将检测五种不同类型的血管内皮细胞屏障功能和免疫细胞外渗。 考虑皮肤和肺微环境的炎性细胞因子和不同水平的基质硬度 (目标2.1)。最后，我们将通过关注以下方面来确定逆转血管功能障碍的潜在靶点和药物 单独或合并消除细胞因子效应和僵硬效应(目标2.2)。总而言之，我们的 系统将构成对现有技术的重大改进，因为它代表着一种新的高度的 功能成熟的血液内皮细胞及其与免疫细胞相互作用的吞吐量筛选工具。

PROJECT SUMMARY Blood vessels play a central role in maintaining host immunity by transporting immune cells to sites of infection. During the process, blood vessels experience endothelial junction remodeling to control vascular permeability and immune cell extravasation. Under infection, blood vessels become permeable and allow immune cells to extravasate and kill pathogens in the interstitium. Once the infection is resolved, permeable vessels become less permeable and limit the number of interstitial immune cells. However, sometimes in inflammation, the remodeling is perturbed, resulting in prolonged, hyper-permeable blood vessels. This vascular dysfunction contributes to immune diseases, such as chronic inflammation, lupus, and autoimmune disease. It is known that endothelial cell alignment is crucial to maintain intact cell-cell adhesion and promote junction maturation. Despite the significance of the cell alignment in functional endothelium, currently available high-throughput methods, such as real-time cell analysis (RTCA) and trans-epithelial/trans-endothelial electrical resistance (TEER) systems with randomly seeded cells have not successfully measured cell impedance or electrical resistance through the in vivo-like controlled endothelial cell morphology, alignment, and matured cell-cell junctions. Furthermore, the current technologies lack pericyte co-culture with endothelial cells. In this proposal, we will develop a high- throughput, high-content functional screening assay capable of faster drug screening and mechanistic studies on blood vessel barrier function and immune cell extravasation. To achieve our goals, we will establish a nanopatterned IEA-based functional assay for high-throughput phenotype screening of pericyte-covered endothelium. To establish the nanopatterned IEA-based assay, we will determine conditions for junctional maturation of human dermal and lung microvascular endothelial cells with or without pericytes, focusing on (i) degree of cell alignment; (ii) expression of adherens junctions, polarization, and basement membrane markers; (iii) vascular barrier function (Aim 1.1). We will then assess vascular gene expression profiles related to vessel stabilization and immune cell adhesion. We will next evaluate immune cell extravasation through the endothelium in the non-inflammatory condition to determine immune cell behaviors in steady-state blood vessels (Aim 1.2). Next, we will validate the utility of the system for inflammation-induced blood vessel dysfunction. To achieve this aim, we will examine the endothelial barrier function and immune cell extravasation in five different categories of inflammatory cytokines and various levels of substrate stiffness considering skin and lung microenvironments (Aim 2.1). Lastly, we will identify potential targets and drugs to reverse vessel dysfunction by focusing on abrogation of the cytokine effect and the stiffness effect, separately or in combination (Aim 2.2). In summary, our system will constitute a significant improvement over existing technologies as it represents a novel high- throughput screening tool for functionally matured blood endothelium and their interactions with immune cells.