Dermal Wounding Reveals Focal Adhesion Kinase Dependent Tissue-Resident Fibroblast Progenitors
Malini Chinta, BA, Deshka Foster, MD, MA, Alan Nguyen, Ankit Salhotra, Gunsagar Gulati, R. Chase Ransom, R. Ellen Jones, MD, Ashley L. Titan, MD, Clement D. Marshall, MD, Shamik Mascharak, Michael Hu, MD, Michael Januszyk, MD, PhD, Geoffrey C. Gurtner, MD, Derrick C. Wan, MD, Jeffrey A. Norton, MD, Howard Y. Chang, MD, PhD, Gerlinde Wernig, MD, Michael T. Longaker, MD, MBA.
Stanford University, Palo Alto, CA, USA.
Purpose: Wound healing is a complex process that involves the extensive coordination of different cell types, most significant of which is the fibroblast. Fibroblasts play a key role in wound healing by assisting in wound closure and tissue remodeling. Significant research efforts have gone into identifying and characterizing specific subsets of fibroblasts involved in wound healing. However, unanswered questions in wound healing remain including where these cells originate from and what their progenitor phenotypes are. Our lab has previously shown that scar fibrosis is dependent on focal adhesion kinase (FAK) signaling and mechanotransduction pathways. The aim of our research was to characterize fibroblast progenitor-type phenotypes in the setting of dermal injury and to understand the role of FAK-signaling in fibroblast proliferation during wound healing. Methods: We used the Rainbow mouse model (Rosa26VT2/GK3), which has a four-color reporter construct at the Rosa-26 locus. Following the induction of Cre-recombinase, cells express one of four fluorescent proteins, and all daughter cells are labeled with the same color. We created wounds in the dorsal dermis of Rainbow mice using a stented wound-healing model which mimics human wound healing kinetics. We used local application of tamoxifen liposomes to induce Cre recombinase in tissue-resident cells at the time of injury (Fig. 1A-B). Confocal imaging was conducted on whole mount (Fig. 1B) and sectioned (Fig. 1C) wound specimens after tissue clearing. Using an unbiased FACS strategy, we isolated wound-healing fibroblasts based on their rainbow color and their position (outer edge vs. center of wound) for RNA-seq at various timepoints post-operatively (POD 2, 7 and 14) (Fig 1D). Topical FAK-inhibitor versus vehicle control was applied to Rainbow mouse dorsal dermal wounds and imaged with confocal microscopy. Results: Confocal imaging shows distinct radial proliferation of tissue-resident, progenitor-type fibroblasts that are activated along the wound edge (area of greatest tension) and expand towards the center of the wound, using the Rainbow mouse model with an activated fibroblast (aSMA-CreERT2) driver (Fig. 1B). These linearly-expanding clones can be further appreciated in the dermis on wound cross section using a ubiquitous (Actin-CreERT2) driver with our Rainbow mouse model, compared with unwounded, control skin (Fig. 1C). Bulk RNA-seq of wound healing fibroblasts shows significant differences in gene expression patterns between fibroblasts isolated from the outer versus inner portions of the wound, and upregulation of FAK-pathway genes. Fibroblast heterogeneity is observed on single-cell RNA-seq of fibroblasts isolated based on their rainbow color (Fig. 1D). Application of an FAK-inhibitor shows disruption of fibroblast clonal proliferation compared with control wounds on confocal imaging. Conclusions: Dermal fibroblasts undergo clonal expansion in a distinct radial pattern in response to wounding, suggesting the presence of tissue-resident progenitor-type fibroblasts that are activated with injury. Differences in “outer” and “inner” fibroblasts are observed on RNA-seq, with significant heterogeneity amongst the fibroblasts isolated at various timepoints. The clonal proliferation of wound-healing fibroblasts is dependent on FAK-pathway signaling.
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