Aligned Fibronectin Microenvironment Temporally Facilitates Profibrotic Fibroblast Activation via Integrin α5β1

ABSTRACT Excessive, aligned nanofibrous extracellular matrix deposition is a main feature of fibrosis, a pathology indicative of poor prognosis in various chronic diseases. Fibronectin is one of the major nanofibrous extracellular matrix proteins that contribute to tissue homeostasis and repair via its cell surface receptors, integrins. Although mechanosensing of aligned fibronectin by fibroblasts is also implicated in the initiation and progression of fibrogenesis, the mechanisms involved remain uncertain. To investigate how profibrotic cell responses and fibrotic tissue development are triggered through these interactions in vitro, we utilized fibroblasts expressing fibronectin‐binding integrins (αV‐class and α5β1 integrins) cultured on fibronectin‐coated electrospun nanofibers with random or aligned networks. During early adhesion, fibroblasts employ a specific integrin, α5β1 integrin, to respond to the aligned nanofibrous fibronectin microenvironment by strengthening cell and F‐actin alignments, nascent focal adhesion, and alpha‐smooth muscle actin expression that are signatures of early pro‐fibrotic activities. Interestingly, α5β1 integrin‐mediated fibronectin alignment sensing continues to form fibrotic tissues at a later stage associated with higher alignment, greater alpha‐smooth muscle actin expression, and extensive extracellular matrix deposition. This mechanistic insight paves the way to better understand the role of fibronectin and its properties in pathophysiology, representing a new target for potential applications in drug discovery.