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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.

项目总结