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...