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

基本信息

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
项目类别：
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

项目摘要

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.

项目概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着核心作用。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外渗并杀死间质中的病原体。一旦感染得到解决，通透性血管就会减少具有渗透性并限制间质免疫细胞的数量。然而，有时在炎症中，重塑受到干扰，导致血管延长、渗透性过高。这种血管功能障碍会导致免疫疾病，例如慢性炎症、狼疮和自身免疫性疾病。据了解，内皮细胞细胞排列对于维持完整的细胞间粘附和促进连接成熟至关重要。尽管功能性内皮细胞排列的重要性，目前可用的高通量方法，例如作为实时细胞分析（RTCA）和跨上皮/跨内皮电阻（TEER）系统随机接种的细胞尚未成功测量细胞阻抗或电阻类似体内的受控内皮细胞形态、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。在本提案中，我们将开发一个高高通量、高内涵的功能筛选测定，能够更快地进行药物筛选和机制研究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 IEA 的功能测定，用于周细胞覆盖的高通量表型筛选内皮细胞。为了建立基于 IEA 的纳米图案检测，我们将确定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，重点是（i）细胞排列程度； (ii) 粘附连接、极化和基底膜标记的表达； (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性和免疫细胞粘附。接下来我们将评估免疫细胞通过内皮的外渗在非炎症条件下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于炎症引起的血管功能障碍的效用。为了实现这一目标目的，我们将检查五种不同类别的内皮屏障功能和免疫细胞外渗考虑到皮肤和肺部微环境，炎症细胞因子和不同水平的基质硬度（目标 2.1）。最后，我们将重点关注逆转血管功能障碍的潜在靶点和药物单独或组合消除细胞因子效应和僵硬效应（目标 2.2）。综上所述，我们的系统将是对现有技术的重大改进，因为它代表了一种新颖的高用于功能成熟的血液内皮及其与免疫细胞相互作用的通量筛选工具。