Exploring the Unique Extracellular Matrix Composition of Acomys as a Potential Key to Resisting Fibrosis

Abstract

Fibrosis is a dysregulated wound healing response characterized by excessive accumulation of dense scar tissue that inhibits organ function and is estimated to contribute to up to 45% of deaths in the industrialized world. In this work, we sought to uncover new ways to address fibrosis by drawing inspiration from an animal that does not develop fibrosis. The Spiny Mouse (Acomys) has the most extensive regenerative capabilities of any known mammal and can regenerate injuries to the skin, kidney, heart, skeletal muscle, and spine with little to no fibrosis. We hypothesize that the regenerative abilities of Acomys are due, in part, to altered stiffness-mediated fibroblast-to-myofibroblast transition (FMT). In this work, we interrogated stiffness-mediated FMT in Acomys and Mus dermal fibroblasts in vitro by performing RNA Sequencing and found no differential gene expression in Acomys fibroblasts cultured on soft vs stiff substrates. We further investigated the direct impact of stiffness-mediated FMT and species differences on ECM deposition by fabricating cell-derived matrices (CDMs) from Acomys and Mus fibroblasts cultured on varying stiffnesses. After assessing the composition of these CDMs using label-free quantitative proteomics, fibrosis-associated extracellular matrix proteins including fibrillin-1, ADAMTS1, SPARC, and galectin-1 were found to be significantly reduced or absent in Acomys CDMs compared to Mus CDMs. In addition, proteins that have been connected to fibrosis resolution, including Col12a1 and clusterin, were upregulated in Acomys CDMs. When cultured on Acomys CDMs, mouse macrophages downregulated MMP9 mRNA expression and maintained increased expression of iNOS in response to IL-4, a pro-fibrotic cytokine. These results indicate a direct impact of species-specific ECM compositions on macrophage phenotype and suggest that ECM produced by Acomys fibroblasts may impede the development of a pro-fibrotic macrophage phenotype in the presence of pro-fibrotic stimuli.

Keywords: cell-derived matrices; fibroblast-to-myofibroblast transition; fibrosis; macrophages; spiny mouse (Acomys); stiffness-mediated signaling.