Wound Healing Myofibroblasts Proliferate Clonally and in a Mechanoresponsive Manner

Abstract

INTRODUCTION: Fibroblasts are integral in regulating tissue homeostasis. Notably, activated fibroblasts (myofibroblasts) play critical roles in wound healing including producing and depositing extracellular matrix and driving wound closure. Much research has been aimed at characterizing subpopulations of fibroblasts with specific activities in wound healing; however, information regarding the origins and heterogeneity of myofibroblasts remains incompletely explored. Our lab has previously shown that fibroblast activity in wound healing is dependent on tissue mechanics (specifically focal-adhesion kinase signaling). The aim of our research was to determine the origins of myofibroblasts that respond to dermal injury and to understand their proliferation, mechanoresponsiveness, and heterogeneity. METHODS: In order to study the characteristics of wound healing myofibroblasts, we used the rainbow reporter mouse (Rosa26VT2/GK3). The rainbow system has a 4-color reporter construct, which after induction and recombination, cells express 1 of 4 fluorescent proteins and all progeny cells are then marked with the same color as their parent cell permitting precise clonal analysis and lineage tracing. We used a wounding model that mimics human wound healing kinetics in which full-thickness wounds were created on the dorsal dermis of Rainbow mice and stented with silicone rings. Wound tissues were examined using confocal microscopy. In order to investigate the influence of local tissue mechanics in our wound healing model, a small molecule FAK-inhibitor (or vehicle control) was applied to the mouse wounds. Imaris software was used for imaging analysis. RESULTS: Imaging analysis suggests the presence of progenitor-type fibroblasts that proliferate clonally and radially during wound healing. Clonal expansion can be appreciated when comparing cross-sectional images of uninjured control dermis to wounded dermis. Using an activated fibroblast driver (aSMA-CreERT2) with the rainbow mouse model, radial proliferation of fibroblast clones is observed. Bulk RNA-seq data shows that the clonal proliferation of wound healing fibroblasts demonstrates an upregulation in mechanoresponsive gene pathways (ie, those involving expression of FAK). Imaging analysis reveals that FAK-inhibition results in disordered clonal proliferation when compared with normal wound controls. CONCLUSIONS: Fibroblast proliferation in response to dermal injury is clonal, suggesting the presence of progenitor-type fibroblasts. This clonal proliferation is FAK-dependent suggesting that this phenomenon is highly reliant on mechanical signaling pathways.