Mechanoregulatory Role of Extra Domain A Fibronectin Matrices During Wound Repair

Introduction: Fibronectin (Fn) is a ~500kDa extracellular matrix dimeric glycoprotein with variable structure and conformation sensitive to cell traction and microenvironmental forces. An alternatively spliced Fn isoform with an Extra Doman A (EDA) inserted is known to be synthesized in adult wounds and pathologies, including fibrosis.^1,2 Regeneration, or recapitulation of the original tissue structure function, remains the holy grail of wound healing; however, it is reliably observed in humans only in fetal wounds whereas wound repair generates a fibrotic (scar) response, which is an increased deposition of ECM proteins.¹ When cells attach to Fn, Yes-associated protein (YAP) is activated and translocated into the nucleus; YAP is known to be essential for wound healing.^3,4 However, the mechanoregulatory role of Fn’s EDA region during wound closure (migration) remains unclear. We aim to characterize EDA Fn microarchitecture and investigate if the EDA region on Fn activates YAP to regulate migration during wound repair.


Materials and Methods: This wound assay comprises human dermal fibroblasts cultured on plasma Fn-coated polydimethysiloxane (PDMS) substrates with stiffnesses mimicking wounded (18kPa), normal (55kPa), and fibrotic (146kPa) tissue (Fig1A). PDMS mask removal simulated wounding. Test conditions include Irigenin (inhibits binding to EDA domain of Fn⁵), CA3 (partial YAP inhibitor⁶), with dimethylsiloxane and media controls. Paraformaldehyde fixed samples were immunostained for EDA-Fn, f-actin, YAP, and nuclei; wound sites were imaged with an Olympus Fluoview FV1200 confocal microscope, 30x objective, and the resulting images, 10 z-slices, 2 µm apart, were max intensity projected into a single image for analysis. Live imaging was taken once every 10 minutes for 24-72h post mask removal with a Keyence BZ-X810 microscope, 10x objective. Migration, nuclear/cytoplasmic YAP ratio, and matrix microarchitecture were characterized in ImageJ: TWOMBLI,⁷ and TrackMate.⁸ GraphPad-Prism 9 calculated one-way ANOVAs and Tukey’s multiple comparisons post-hoc analyses.


Results, Conclusions, and Discussions:

Initial EDA Fn assembled in the baseline wound assay (Fig1B-C) shows stiffness-dependent alignment and density trends, and significant branching to stiffness correlation (Fig1D-E). Blocking cell-binding to the EDA region of Fn (Irigenin) reverses the initial stiffness driven matrix alignment profile and increases overall branching and generates EDA Fn matrices with non-significantly variable density within the first 24h (Fig1M-O) compared to control (Fig1G,H). Cells treated with Irigenin assembled EDA Fn matrices in wound sites with thicker fibrils, smaller pores, and increases in branching, density (Fig1I,J). YAP inhibitor CA3 generates similar EDA Fn matrix effects (Fig1K,L) and trends as blocking the EDA Fn-cell binding region (Irigenin): smaller porosity, increased branching and density, and similar 24h alignment trends (Fig1M-O). In the initial 24h, before contact inhibition decreases nuclear translocation of YAP, substrate-driven YAP activation was no longer present in the inhibitor conditions. Specifically, cells on the fibrotic substrate with the most aligned EDA fibronectin fibers showed the most YAP activation; but interestingly, when blocking adhesion to EDA or abrogating YAP signaling in the fibrotic condition, there was a significant increase in YAP activation with corresponding decrease in cell population (Fig1P-R). When evaluating cell migration, inhibiting binding to EDA or abrogating YAP signaling within wound and normal stiffness conditions (with dysregulated EDA Fn matrix and less YAP signaling) led to a significant decrease in migration speed compared to respective controls. Meanwhile, within the fibrotic condition, when blocking adhesion to EDA or abrogating YAP signaling, the increased YAP signaling rescued cell migration (Fig1S-U).



Substrate stiffness alters matrix microarchitecture. Inhibition of the EDA Fn-cell binding region (Irigenin) or abrogating YAP signaling (CA3) can similarly alter initial substrate-driven matrix properties, decrease YAP signaling compared to control and decrease migration, except the inhibited fibrotic conditions, which contrarily shows rescued migration speed. This data suggests tissue stiffness regulates wound-site EDA Fn remodeling through the EDA Fn specific cell-binding region with potential downstream effector YAP to modulate cell-matrix remodeling, migration during wound closure. Our work investigates how tissue stiffness modulates Fn mechanotransduction with the end goal of tuning Fn matrices to alter wound healing from fibrotic repair to regenerative repair.




Acknowledgements (Optional):

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