Altered TGFB1 regulated pathways promote accelerated tendon healing in the superhealer MRL/MpJ mouse

Abstract

To better understand the molecular mechanisms of tendon healing, we investigated the Murphy Roth’s Large (MRL) mouse, which is considered a model of mammalian tissue regeneration. We show that compared to C57Bl/6J (C57) mice, injured MRL tendons have reduced fibrotic adhesions and cellular proliferation, with accelerated improvements in biomechanical properties. RNA-seq analysis revealed that differentially expressed genes in the C57 healing tendon at 7 days post injury were functionally linked to fibrosis, immune system signaling and extracellular matrix (ECM) organization, while the differentially expressed genes in the MRL injured tendon were dominated by cell cycle pathways. These gene expression changes were associated with increased α-SMA+ myofibroblast and F4/80+ macrophage activation and abundant BCL-2 expression in the C57 injured tendons. Transcriptional analysis of upstream regulators using Ingenuity Pathway Analysis showed positive enrichment of TGFB1 in both C57 and MRL healing tendons, but with different downstream transcriptional effects. MRL tendons exhibited of cell cycle regulatory genes, with negative enrichment of the cell senescence-related regulators, compared to the positively-enriched inflammatory and fibrotic (ECM organization) pathways in the C57 tendons. Serum cytokine analysis revealed decreased levels of circulating senescence-associated circulatory proteins in response to injury in the MRL mice compared to the C57 mice. These data collectively demonstrate altered TGFB1 regulated inflammatory, fibrosis, and cell cycle pathways in flexor tendon repair in MRL mice, and could give cues to improved tendon healing.

Earlier proteoglycan turnover promotes higher efficiency matrix remodeling in MRL/MpJ tendons

“…Despite the increased ECM turnover, MRL/MpJ explants experienced the same diminished biomechanical function as B6 explants by 14 days in culture. While previous studies have shown that MRL/MpJ flexor tendons at this age have a lower baseline modulus compared with B6 tendons, 26 we found only a difference in maximum stress at baseline (Supporting Information: Figure ), which could be due to differences in testing protocols. We may need to consider different modes of mechanical evaluation for our studies.…”

“…In other forms of mechanical injury, such as overuse and overload injuries, inflammatory genes including IL-6, TNF-α, IL-1β, and COX-2 were identified to increase in vivo and in vitro. 41,42 26 we found only a difference in maximum stress at baseline (Supporting Information: Figure S7), which could be due to differences in testing protocols. We may need to consider different modes of mechanical evaluation for our studies.…”

“…While foundational work studying MRL/MpJ mice focused on in vivo tendon healing, [14][15][16][17]26,27 our study investigated innate ECM remodeling through the use of a stress-deprived explant model. We found a more robust response to a loss of mechanical tension in the F I G U R E 3 (A) Total protein synthesis, (B) total collagen content, (C) sGAG synthesis, (D) total glycosaminoglycan content, and (E) MMP activity for B6 (blue, circles) and MRL (orange, squares) throughout culture.…”

“…While foundational work studying MRL/MpJ mice focused on in vivo tendon healing, 14–17,26,27 our study investigated innate ECM remodeling through the use of a stress‐deprived explant model. We found a more robust response to a loss of mechanical tension in the MRL/MpJ tendon explants, characterized by an altered profile and timing of matrix turnover.…”

“…Significance is noted at *p < 0.05 and a trend is noted at # p < 0.1. MRL/MpJ, Murphy Roths Large.extend to the FDL,26 allowing it to serve as an effective model for exploring the innate cellular mechanisms of MRL/MpJ tendon injury response. Finally, it must be considered that our measures of synthesis and degradation are general, measuring all sulfated GAGs, total protein, and general MMP activity.…”