Fibroblast Extracellular Matrix Remodeling: Differences in Idiopathic and Normal Pulmonary Fibrosis
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Date
2022-11
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Abstract
Pulmonary fibrosis is a chronic lung disease that occurs when lung tissue becomes damaged as a result of injury or inflammation. The main effector cell in fibrosis is the fibroblasts, which causes remodeling of extracellular matrix (ECM) leading to a scar-like tissue, increased lung stiffness, reduced lung elasticity, and decreased gas exchange. A severe form of lung fibrosis is Idiopathic Pulmonary Fibrosis (IPF), which is characterized by progressive, non-resolving accumulation of ECM and has a survival of only 2-5 years after diagnosis. While the cause of IPF is unknown, it is thought to be the result of repetitive injuries to the alveolar epithelium that cause fibroblast differentiation and activation into a myofibroblast phenotype. Recent studies using scRNA sequencing have transcriptomically identified a variety of different fibroblast subpopulations; however, understanding the role of these various fibroblast subsets in remodeling ECM has not been determined. In order to measure fibroblast functions, we used confocal reflectance microscopy to quantitatively characterize ECM structure and remolding by fibroblasts isolated from normal and IPF patients.
Primary human IPF and normal fibroblasts were seeded on 45 uL bovine collagen gels at a density of 10,000 cells/mL and allowed to compact and reorganize collagen fibers. The collagen gels were comprised of two layers. The first layer was 20 uL of bovine collagen that was placed in a 37 degrees C incubator for 2 hours. The second layer was then added which contained a cell and collagen mix with a volume of 25 uL. Gels were imaged 24 hours post seeding using a Leica Stellaris 5 confocal laser scanning microscope configured to collect images in reflectance mode using a 63x oil-immersion objective and a 488-nm argon laser. Images were collected with a step size of 0.3 um for 50 steps to create a z stack of images. Outlines of cells were used to calculate cell area and circularity in each slice and histograms of pixel intensity in each slice were analyzed for standard deviation as a measure of fiber bundling (i.e. lower standard deviation indicates more bundling).
Results: After 24 hours, normal fibroblasts were circular in shape and small collagen fibers were uniformly distributed throughout the gel with some bundling around cells and moderate alignment of fibers in between cells. In contract, IPF fibroblasts had a highly elongated morphology with highly aligned fibers in between cells. Measurements of cell area and circularity confirm elongated morphology in IPF fibroblasts and measurements of collagen distribution confirm increased bundling (lower standard deviation) in IPF seeded gels. Interestingly, reflectance intensity was lower in IPF seed gels indicating possible uptake and degradation.
Conclusions: These results show that IPF fibroblasts overall exhibit more remodeling of the ECM than normal fibroblasts. The larger cell area and circularity of the IPF fibroblasts shows that they are more active because of their elongated shape. The histograms demonstrate that the normal fibroblasts have a more even distribution of collagen than the IPF fibroblasts, which is further evidence that the IPF cells are bundling up or degrading the collagen at a higher rate than the normal cells. Future studies will use this system to investigate how genomic changes in fibroblasts sub-population alter their collagen remodeling capabilities. The significance of this work is a quantitative platform that can assess fibroblast remodeling that can be used in future studies to better understand how different sub-populations of fibroblasts isolated from pulmonary fibrosis patients influence the ECM remodeling that leads to disease.
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Keywords
fibroblast, ECM, Pulmonary fibrosis, simulation