? DESCRIPTION (provided by applicant): Remodeling of the extracellular matrix (ECM) is central to the pathogenesis of chronic respiratory diseases, including pulmonary fibrosis, pulmonary hypertension, asthma and COPD. While much emphasis has been placed on studying individual cell types and how they interact with the ECM, comparatively little attention has focused on how homeostatic maintenance of the ECM is coordinated amongst multiple lung resident cell types, and how cell interactions in the setting of injury or disease either restore homeostasis or generate pathological matrix remodeling. A major limitation preventing investigation of these crucial cell-cell and cell-matrix interactions is the absence of experimentally tractable multicellular culture systems which incorporate 3D matrices capable of long-term remodeling. Another major limitation stems from the limited number, availability, proliferative capacity, and phenotypic stability of primary human cells, which are a critical resource if we are to elucidate the normal homeostatic and pathological matrix remodeling processes that underlie human health and disease. Our goal in this proposal is to develop a cell culture model system that (1) facilitates long-term culture study of homeostatic and pathologic matrix remodeling under the control of multiple interacting primary lung cell types; (2) enables repeated non-destructive imaging and sampling to identify cellular and soluble cues that correlate with, and ultimately predict, the cell-cell and cell-matrix interactions underlying matri remodeling; and (3) provides a platform for studying primary human cells in small quantities as a step toward enhanced phenotypic fidelity and personalized medicine. These design goals will be met through two interrelated specific aims. In Aim 1 we will develop and optimize a microfluidic system and culture conditions permitting stable, long-term co-culture of lung epi/endothelium and extracellular matrix-embedded fibroblasts. We will evaluate multiple physiologic metrics of ECM and tissue remodeling in co-culture, and evaluate their sensitivity and signal to noise ratio using culture conditions known to promote matrix/tissue remodeling in vivo (e.g. TGF-beta stimulation). In Aim 2 we will incorporate primary human cells into the microfluidic co-culture system as a step toward personalized medicine, therapeutic prioritization, and biologic discovery. We will use approved and failed anti-fibrosis therapies to assess the predictive capacity of this system as proof of concept. To capitalize on the discovery capabilities of the system, we will collect matrix compartment fluid samples for proteomic analysis to identify novel candidate mediators differentially expressed during matrix remodeling. This project will generate a new experimental platform that enables repeated real-time analysis of cell-cell and cell-matrix interactions during matrix remodeling, a process central to the pathogenesis of multiple respiratory diseases. Incorporation of primary human cells with low or no passaging into the platform will enhance the biofidelity of experiments and offer a unique resource for personalized medicine, therapeutic evaluation and biologic discovery in the realm of lung matrix remodeling.
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