Matrix Stiffness and Architecture Drive Fibro-adipogenic Progenitors’ Activation into Myofibroblasts

Abstract

BackgroundFibro-adipogenic progenitors (FAPs) are essential in supporting regeneration in skeletal muscle but pathologically become the main source of excess extracellular matrix (ECM) deposition in fibrosis. Fibrosis can create a progressively degenerative feedback loop with fibrotic tissue leading to an increase in the fibrotic FAP phenotype. Fibrotic ECM has altered mechanical and architectural properties, but the interactions between FAPs and the ECM are not well understood.MethodsIn this study, we aimed to examine FAPs’ sensitivity to their environment and its effect on cell fate. Polyacrylamide and collagen hydrogels were utilized to investigate substrate stiffness and ECM architecture. Collagen hydrogels were manipulated to mimic aspects of healthy and fibrotic ECM including density, crosslinking, fibril size, and alignment. Collagen-coated polyacrylamide gels were utilized to analyze substrate stiffnesses that are physiologically relevant. YAP nuclear localization, proliferation, and myofibroblast activation were measured using immunofluorescence and custom FIJI: ImageJ scripts. Results Substrates of fibrotic stiffnesses had increased myofibroblast activation compared to substrates mimicking healthy muscle. The increase in myofibroblast activation correlated strongly with in Yes-associated protein (YAP) nuclear localization. Collagen crosslinking and increased fibril size inhibited myofibroblast activation, independent of YAP localization. Alignment of collagen did not affect FAP behavior.ConclusionsThis study shows that FAPs possess the ability to sense their ECM environment and that influences cell fate. Therefore, ECM mechanics and architecture are key targets to reduce the progression of fibrosis.