Deciphering the myofibroblast life cycle in skin wound healing

PROJECT SUMMARY/ABSTRACT Myofibroblasts generate scar tissue in wound healing and most forms of organ fibrosis. Aberrant wound healing produces a large clinical burden, including deficient wound healing (dehiscence and chronic non-healing wounds) and excessive wound healing (keloids and hypertrophic scars). During wound healing, quiescent fibroblasts are activated to proliferate, migrate, and differentiate into myofibroblasts, which are critical for proper wound healing. After the wound is closed, SMApositive myofibroblasts disappear from the skin. Where do they go? Recent studies show that many myofibroblasts revert to deactivated myofibroblasts or post-contractile fibroblasts. This project begins with preliminary data showing that many PostnMerCreMer labeled skin myofibroblasts revert to a deactivated SMAnegative phenotype and persist in the lower scar long after wound closure, suggesting that survival is regulated rather than stochastic. Myofibroblasts in the lower scar express higher levels of the Hippo-regulated transcriptional cofactors YAP and TAZ compared to myofibroblasts in the upper scar. This project will create a molecular map of the myofibroblast life cycle and will test the hypothesis that myofibroblasts convert to a deactivated phenotype in late wound healing through YAP/TAZ-dependent survival. To generate a molecular map of the myofibroblast life cycle (Aim 1), the NuTRAP system will be used to isolate nuclei and translating mRNAs in vivo, specifically from cells of the fibroblast-myofibroblast lineage, at each stage of the life-cycle. Then, ATAC-seq and RNA-seq will be performed and data sets integrated to create a map of how chromatin and gene expression coordinately change as fibroblasts differentiate into myofibroblasts and then convert into deactivated myofibroblasts. scRNA-seq will also be used to characterize deactivated myofibroblast heterogeneity and to map the lineage of PostnMerCreMer labeled cells. Second, this proposal will use genetic approaches to investigate the role of Hippo-YAP/TAZ signaling in myofibroblast deactivation and survival (Aim 2). The results of this project will move the field forward by characterizing the myofibroblast life cycle in the skin and exploring the latent fibrogenic potential of deactivated myofibroblasts. This will be significant to human health because it may open the way to precisely modulate these signals as a treatment for skin fibrosis and regenerative medicine.