Using axolotls to define innate mechanisms for combatting fibrosis

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Soft tissue fibrosis and cutaneous scarring represent huge clinical burdens to 100 million patients per year, and therapeutic options are currently quite limited. Novel approaches to combat fibrosis and scarring are necessary. Efficient wound closure is a crucial part of wound healing. Without it, injuries would be far more susceptible to fluid loss and infection. It has been hypothesized that scarring evolved as a solution to maximize healing speed. This hypothesis, however, does not explain why regenerative animals, with arguably the most remarkable healing abilities, are capable of scar-free healing. Here, I propose that understanding how axolotl salamanders heal wounds scarlessly and antagonize fibrosis during regeneration will provide critical therapeutic approaches. To overcome the impressive ability of these animals to combat fibrosis, I will create both genetic- and chemical-based fibrotic models in axolotls. I have already successfully managed to develop a chemical-based model using the drug bleomycin, which limits the animals' regenerative ability, and a genetic- based model using tsp-1 loss-of-function mutants, which reduces regenerative rate and blastema size. In Aim 1, I will perform hypothesis-driven, as well as discovery-based, studies to interrogate the balance between fibrosis and regeneration. I will use genome editing to create tsp-1/tsp-2 and tsp-1/tsp-4 double-mutant axolotls, which are predicted to have exacerbated fibrotic phenotypes. In parallel, I will use established Thrombospondin-inhibiting peptides (shown to promote fibrotic phenotypes in cultured human dermal fibroblasts). For both models, I will identify candidate molecular targets that antagonize fibrosis in axolotls using RNAseq and differential gene expression analysis. In collaboration with tissue engineers, I will then use these findings to develop micro-patterned, silk-based hydrogels loaded with biologicals designed to inhibit fibrosis that could be later developed toward human treatments. In Aim 2, I will identify mechanisms whereby axolotls might maintain scar-free tissue in the presence of silicone implants that routinely lead to fibrosis in human patients, necessitating their replacement. These mechanisms represent novel approaches for future therapies that might be preventively employed in human patients necessitating medical implants. They might also be active in remediating existing fibrosis around implants. In parallel, I will test the axolotl extracellular matrix's capability to remodel transplanted human fibrotic tissues. Together, these approaches are extremely novel. They leverage both the explosion of molecular genetic tools now available in these remarkable animals, and they capitalize on natural solutions to fibrotic insults that have yet to be applied to humans. These strategies could provide powerful new approaches to improving fibrosis outcomes in human patients.