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

用于筛选免疫细胞-血管相互作用的基于 nanoIEA 的高通量测定法

• 批准号: 10592897
• 负责人: Deok-Ho Kim
• 金额: $ 21.17万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-19 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10592897
• 关键词: Address; Adherens Junction; Animal Genetics; Animal Model; Animals; Autoimmune Diseases; Basement membrane; Biological Assay; Biology; Blood; Blood - brain barrier anatomy; Blood Vessels; COVID-19; Categories; Cell Adhesion; Cell Communication; Cell-Cell Adhesion; Cells; Cellular Morphology; Chronic; Coagulation Process; Coculture Techniques; Communicable Diseases; Complement; Deposition; Dermal; Disease; Drug Screening; Electrical Resistance; Electrodes; Endothelial Cells; Endothelium; Epithelium; Experimental Models; Extravasation; Functional disorder; Gene Expression; Goals; Human; Immune; Immune System Diseases; Immunity; Immunology; Impairment; In Vitro; Infection; Inflammation; Inflammatory; Intercellular Junctions; Investigation; Lesion; Lung; Lupus; Lymphatic; Measurement; Measures; Mediating; Methods; Modeling; Nature; Pattern; Pericytes; Permeability; Pharmaceutical Preparations; Phenotype; Physiological; Play; Pluripotent Stem Cells; Process; Rapid screening; Reading; Role; Screening procedure; Sepsis; Signal Transduction; Site; Skin; System; Systems Analysis; Technology; Therapeutic; Time; Vascular Diseases; Vascular Permeabilities; cell behavior; cell motility; clinically relevant; cytokine; drug candidate; electric impedance; endothelial dysfunction; experience; glomerular filtration; high throughput screening; improved; in vitro Model; in vivo; interstitial; knock-down; lung microvascular endothelial cells; migration; nanopattern; novel; pathogen; personalized medicine; polarized cell; prevent; screening; trafficking

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.

项目摘要 血管通过将免疫细胞运送到感染部位，在维持宿主免疫力方面发挥核心作用。 在此过程中，血管经历内皮连接重塑以控制血管通透性 和免疫细胞外渗。在感染下，血管变得可渗透，并允许免疫细胞 渗出并杀死病原体。一旦感染得到解决，可渗透的血管变得更少， 渗透性和限制间质免疫细胞的数量。然而，有时在炎症中， 受到干扰，导致血管延长，渗透性过高。这种血管功能障碍有助于 免疫性疾病，如慢性炎症、狼疮和自身免疫性疾病。已知内皮细胞 细胞排列对于维持完整的细胞-细胞粘附和促进连接成熟是至关重要的。尽管 细胞排列在功能性内皮中的重要性，目前可用的高通量方法， 作为实时细胞分析（RTCA）和跨上皮/跨内皮电阻（TEER）系统， 随机接种的细胞没有成功地测量通过该孔的细胞阻抗或电阻。 活体样控制内皮细胞形态、排列和成熟的细胞-细胞连接。而且 目前的技术缺乏周细胞与内皮细胞的共培养。在这份提案中，我们将制定一项高- 能够更快地进行药物筛选和机制研究的通量、高含量功能筛选测定 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 基于纳米图案化IEA的功能测定用于周细胞覆盖的 内皮细胞为了建立基于纳米图案化的IEA的测定，我们将确定用于连接的条件。 有或没有周细胞的人真皮和肺微血管内皮细胞的成熟，集中于（i） 细胞排列的程度;（ii）粘附连接、极化和基底膜标记物的表达; (iii)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， 稳定和免疫细胞粘附。我们接下来将评估免疫细胞通过内皮外渗 在非炎症条件下，以确定稳态血管中的免疫细胞行为（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。实现这一 目的，我们将检查五种不同类型的血管内皮细胞屏障功能和免疫细胞外渗。 炎症细胞因子和各种水平的底物硬度，考虑皮肤和肺微环境 (Aim 2.1）。最后，我们将确定潜在的目标和药物，以扭转血管功能障碍，重点是 单独或组合地消除细胞因子效应和僵硬效应（目标2.2）。总之，我们的 系统将构成对现有技术的重大改进，因为它代表了一种新的高性能， 用于功能成熟的血液内皮及其与免疫细胞的相互作用的通量筛选工具。