Spatiotemporal Mechanoregulation of Cell Migration During Wound Healing

Introduction: Cell migration, the rate limiting element of wound healing, is sensitive to mechanoregulation by microenvironmental forces.1 Yes-associated protein (YAP) is essential for wound healing, yet conversely, also drives the strength of the fibrotic or scar tissue response; when translocated into the nucleus, YAP upregulates target genes for migration or fibrosis.2-3 However, the mechanoregulatory role and signaling of YAP during wound closure migration remains unclear in determining wound fate: regeneration or fibrosis. We aim to characterize the effect of spatiotemporal mechanoregulation of YAP signaling in the context of wound and fibrotic cell migration.

Materials and

Methods: This wound assay comprises human dermal fibroblasts cultured on plasma Fn-coated polydimethysiloxane (PDMS) substrates with stiffnesses mimicking normal, wounded (18kPa) and fibrotic (146kPa) tissue. PDMS mask removal simulated wounding. Test conditions include CA3 (partial YAP inhibitor4), with dimethylsiloxane and media controls. Cell nuclei were stained with SPY650 DNA. Live imaging of cell nuclei were taken once every 10 minutes for 24h post mask removal with a Keyence BZ-X800 microscope, 10x objective. Migration was characterized in ImageJ: TrackMate,5 Chemotaxis and Migration Tool 2.0 (ibidi, http://www.ibidi.de/applications/ap_chemo.html), and MATLAB (in-house developed leader-follower cell analysis). GraphPad-Prism 9 calculated one-way ANOVAs and Tukey’s multiple comparisons post-hoc analyses.

Results, Conclusions, and Discussions: Instantaneous velocity of cell migration on softer substrates mimicking normal wounds was significantly higher than that of cell migration on fibrotic substrates, suggesting contextual mechanotransduction drives a form of negative durotaxis.6 Partial inhibition of YAP signaling (CA3; partial inhibition prevents cell toxicity) significantly lowers migration velocity compared to DMSO controls, supporting literature that YAP is necessary to regulate wounded cell migration.2-3 Despite higher velocity, cells on normal, wound substrate stiffnesses demonstrate lower persistence over 24h, indicating more random migration. Cells treated with CA3 demonstrate the opposite trend, with partial YAP inhibition increasing persistence in normal, wound substrates and decreasing persistence in fibrotic substrates compared to DMSO controls. This suggests YAP mechanoregulates wound closure by facilitating faster, but random migration on softer, wound-like substrates while promoting slower, but persistent migration on fibrotic-like substrates. Aggregate cell data from 0-24h tracked cells throughout the wound closure process. However, spatiotemporal cues as the wound closed were identified by separating cell migration data from 0-24h into 0-12h and 12-24h segments. Velocity trends show the same trends between the aggregate and time-separated conditions; however, persistence data demonstrates temporal differences. Early (0-12h) partial YAP inhibition rescues persistence in cells on the wound substrate stiffnesses compared to controls. Continual partial YAP inhibition significantly decreases cell persistence in fibrotic substrates. This data suggests mechanoregulation of YAP over time decides a pro-migratory or a pro-fibrotic cell response. Leader (cells lining the initial wound edge) and follower (cells behind immediate wound edge) cell directionality and persistence shows macroscopically similar distribution to one another in control conditions. However, when YAP is partially inhibited, the leader and follower cells show differences in directionality and persistence, suggesting YAP regulates cell persistence. Additionally, this data supports recent literature that leader cell status is not entirely determined by location at the wound edge.7 Immediate next steps include contexualizing cell behavior during wound closure: dynamic leader-follower cell behavior, actin structure-function, (which translate environmental forces into cellular responses), and cell contractility (necessary for wound closure but also a hallmark of fibrosis) to investigate mechanisms for mechanotransduction modulation to suppress fibrotic repair while promoting regenerative healing.

Acknowledgements (Optional):

References: 1. Patten, J. Adv Drug Deliv Rev, 2021:170:353-368. 2. Lee, M. J Inv Derm, 2014:134:518-525. 3. Dupont, S. Nature, 2011:474:179-184. 4. Song, S. Mol Cancer Therapeutics, 2018:17:443-454. 5. Schindelin J., Nat. Meth, 2012:9;7;676-68. 6. Isomursu, A. Nat Materials, 2022;21, p1081–1090. 7. Yolland, L. Nat Cell Biol, 2019;21:1370–81.