PIEZO1-mediated mechanotransduction regulates collagen synthesis on nanostructured 2D and 3D models of fibrosis.

Progressive fibrosis can lead to tissue malfunction and organ failure due to the pathologic accumulation of a collagen-rich extracellular matrix. In vitro models provide useful tools for deconstructing the roles of specific biomechanical or biological mechanisms, such as substrate micro- and nanoscale architecture, in these processes for identifying potential therapeutic targets. Here, we investigated how the mechanosensitive ion channel PIEZO1 influences fibrotic gene and protein expression in adipose-derived stem cells (hASCs). Specifically, we examined the role of PIEZO1 and the mechanosensitive transcription factors YAP/TAZ in sensing aligned or non-aligned substrate architecture to regulate collagen formation. We utilized both 2D microphotopatterned substrates and 3D electrospun polycaprolactone (PCL) substrates to study the role of culture dimensionality. We found that PIEZO1 regulates collagen synthesis in hASCs in a manner that is sensitive to substrate architecture. Activation of PIEZO1 induced significant morphological changes in hASCs, particularly when cultured on aligned substrates, leading to a 30-40 % reduction in cell spreading area and increased cell elongation, in 3D-aligned cultures. Picrosirius Red staining and immunoblotting revealed that PIEZO1 activation reduced collagen accumulation in 3D culture. While YAP translocated to the cytoplasm following PIEZO1 activation, depleting YAP and TAZ did not change collagen expression significantly downstream of PIEZO1 activation, implying that YAP/TAZ translocation from the nucleus and decreased collagen synthesis may be independent consequences of PIEZO1 activation. Our studies demonstrate a role for PIEZO1 in cellular mechanosensing of substrate architecture and provide targetable pathways for treating fibrosis and for enhancing tissue-engineered and regenerative approaches for fibrous tissue repair. STATEMENT OF SIGNIFICANCE: This study examines how cells sense and respond to their physical environment via PIEZO1 mechanotransduction. We discovered that cells use PIEZO1 to detect the alignment of surrounding structures, influencing the production of collagen - a key component in fibrosis. Our study used both 2D and 3D models to mimic different tissue environments, providing new insights into how cellular responses change in more complex settings. Importantly, we found that activating PIEZO1 alters cell shape and collagen production, especially on aligned surfaces. Interestingly, while PIEZO1 activation caused YAP translocation to the cytoplasm, this translocation did not directly affect collagen production. This work advances our understanding of fibrosis development and identifies PIEZO1 as a potential target for new therapies.