Treatment of vocal folds fibrosis by targeting the mechanobiological MRTF-A/SRF axis

Introduction: Vocal folds (VF) are connective tissues for voice production. The VFs are highly mechanically active, oscillating up to 300 Hz during normal phonation and thus subjecting resident vocal fold fibroblasts to significant mechanical loading. Upon injury due to prolonged intubation, trauma, or iatrogenic complications, VF fibroblasts differentiate into contractile, α-SMA+ myofibroblasts which secrete an excess of collagen-rich extracellular matrix (ECM). This leads to vocal fold scarring (VFS), characterized by a loss in tissue pliability and overall ECM stiffening. A positive mechanobiological feedback loop is established to further promote myofibroblast differentiation and scarring. Myofibroblast differentiation can be induced through biochemical cues, such as TGF-β1, or biophysical interactions, such as matrix stiffness. Currently, steroid injection laryngoplasty is the predominant treatment modality for VFS, but this approach leads to unwanted side effects such decrease in vocal fold mass and muscle atrophy. There exists an unmet need to develop a VFS therapy that specifically targets pro-fibrotic signaling cascades which drive airway scarring. Herein, by analyzing the transcriptome of human vocal fold fibroblasts and myofibroblasts, we identified the MRTF-A/SRF axis as important for VF myofibroblast differentiation. We then validate the role of MRTF-A/SRF in regulating airway fibroblast phenotype using a small molecule MRTF-A/SRF pathway inhibitor, CCG-257081. Finally, we show that targeted inhibition of the MRTF-A/SRF axis may be a potential therapeutic for VFS using our novel murine model of VFS.

Materials and

Methods: Five donors of human VF fibroblasts were cultured for in vitro studies with or without TGF-β1 and CCG-257081 (MRTF-A inhibitor). Immunocytochemistry (ICC) was performed for α-SMA, MRTF-A, SRF, and CCN2 protein expression. Quantitative reverse transcription polymerase chain reaction (RT-qPCR) provided quantitative data for fibrotic gene expression (COL1A1, COL1A2, ACTA-2, ITGB1, FN1, and CCN2). To investigate stiffness-induced myofibroblast differentiation, we seeded soft (1kPa) or stiff (20 kPa) collagen-coated polyacrylamide gels with VF fibroblasts and stimulated cells with or without CCG-257081. ICC was used to measure α-SMA, MRTF-A, and SRF expression. We established a novel murine model for VFS to determine the efficacy of MRTF-A inhibition via CCG-257081 to abrogate VFS in vivo. We induced VFS through abrasion injury with a wire brush. Mice injected with CCG-257081 or a vehicle control (VC) daily by intraperitoneal administration. After 7 days, VFs were analyzed via histology and immunostaining.

Results, Conclusions, and Discussions:

Results:
In both TGF-β1-induced and stiffness-induced myofibroblast differentiation, we observed nuclear trans-localization of MRTF-A and SRF, suggesting increased transcriptional activity. CCG-257081 significantly reduced levels of fibrotic genes COL1A1, COL1A2, ACTA-2, ITGB1, FN1, and CCN2 and decreased α-SMA expression. Further, VF fibroblasts treated with CCG-257081 showed decreased cell contractility in collagen hydrogels. These data indicate that MRTF-A inhibition prevents myofibroblast differentiations.
In our murine model of VFS, we observed characteristic epithelial thickening, increased collagen deposition, and decreased glycosaminoglycan content after VF injury. However, when mice were given daily CCG-257081 injections, we observed reduced epithelial thickness and collagen secretion, but increased glycosaminoglycan abundance, similar to sham controls. Treated mice also exhibited reduced MRTF-A and SRF protein expression and a lower percentage of α-SMA+ myofibroblasts.

Conclusion:
CCG-257081 decreased fibrotic expression for biochemically and biophysically induced myofibroblasts in vitro. CCG-257081 reduced VFS in mice. Inhibition of the MRTF-A/SRF pathway is a promising strategy in resolving vocal fold fibrosis.

Discussions:
Given their high mechanical activity, vocal folds depend on a regulated extracellular matrix (ECM) to function effectively, enabling operation at frequencies of up to 300 Hertz. Inhibition of the MRTF-A/SRF axis has shown preclinical efficacy in preventing scar tissue formation and fibrosis in other tissues such as lung, dermis, and eye. In our work, the small molecule MRTF-A/SRF pathway inhibitor, CCG-257081, demonstrated successful suppression of the MRTF-A/SRF axis both in vitro and in vivo. Significant differences in myofibroblast phenotype markers were observed following treatment with CCG-257081, both in cells and tissue samples. This discovery holds promise for mechanobiological therapeutic strategies for VFS.

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