This F31 fellowship application outlines the training and research plan that will prepare Jessica Ackerman for a future career as an independent investigator with expertise in sophisticated imaging of fibrotic pathologies. Tendon injuries represent a large clinical burden, and healing often results in impaired restoration of mechanical properties. Healing of the tendon occurs through a scar-mediated process that is not well understood, leading to a lack of therapeutic targets. Currently no biological therapy is in use clinically to attenuate scarring following tendon injury. Myofibroblasts play a key role in pathological fibrosis, primarily through excessive deposition of matrix at the site of injury. Although there is evidence of myofibroblast involvement in tendon healing, their contribution to the process has yet to be investigated in detail. We have shown previously that resident tendon cells (tenocytes) differentiate to myofibroblasts during healing. Additionally, we have observed a decrease in myofibroblast content in a model of regenerative tendon healing, as well as alterations in macrophage presence and polarization. Macrophage-myofibroblast cross talk has been implicated in fibrosis in a number of other tissues, and M?-polarization state may affect the eventual outcome of healing. Thus, our central hypothesis is that M?-myofibroblast/tenocyte crosstalk is critical for tenocyte differentiation to myofibroblasts, and that inducible depletion of myofibroblasts following tendon injury will attenuate scar formation and improve tendon healing. To assess the presence and persistence of myofibroblasts and the effects of myofibroblast depletion, we will use Postn-CreER to drive recombination of the Ai9 fluorescent reporter or ROSA-DTA construct, respectively in activated myofibroblasts (Aim 1). Mice will undergo our well-established murine model of tendon injury, involving complete transection and repair of the flexor digitorum longus (FDL) tendon in the hindpaw. The temporal and molecular profile of the phenotypic shift of tenocytes to myofibroblasts will be defined in vitro (Aim 2A). Further, we will explore crosstalk between tenocytes/myofibroblasts and macrophages. Data suggests that paracrine signaling via macrophages, specifically the M1 phenotype, is sufficient to drive myofibroblast differentiation, which we will test with tenocytes in vitro (Aim 2B). Lastly, we plan to examine the effect of differentiated myofibroblasts on macrophage polarization and function in vitro to establish how each cell type may impact the other during the healing process (Aim 2C). Overall we plan to use the data from performing these studies to inform our approach towards the goal of establishing a viable biological therapy to treat scarring following tendon injury.
Acute tendon injuries often lead to unsatisfactory outcomes for patients, due in large part to an excessive scar tissue response. Limited understanding of the cellular and molecular mechanisms guiding tendon healing has prevented development of an effective biological therapy. The proposed research plan will address this gap in knowledge in the tendon field through investigation of the contribution of myofibroblasts, a major matrix- producing cell, to the process of scar-mediated tendon healing.
