Intercellular contact is sufficient to drive fibroblast-to-myofibroblast transitions

Introduction: Wound healing is a critical function of the human body facilitated by fibroblasts. Upon injury, fibroblasts transition to myofibroblasts, a phenotype that is significantly more contractile, enabling wound closure and healing by secreting a collagen rich extracellular matrix (ECM) [1]. Once tissue repair is complete, myofibroblasts are deactivated or cleared through apoptosis. However, in disease, the myofibroblast phenotype persists, causing reorganization of collagen fibrils and excess collagen deposition resulting in stiffening of the ECM leading to fibrosis, organ dysfunction, and ultimately, death [2]. Despite therapies to modulate matrix stiffness with collagen-degrading enzymes, and drugs to target collagen deposition, diseases like idiopathic pulmonary fibrosis continue to progress [3]. Clearly, there are alternative mechanisms that are yet unknown which operate independently of recognized biochemical (ex: TGF-β1) and biomechanical (ex: matrix stiffening) signals from the ECM, contributing to fibrosis. Our work investigates why myofibroblast phenotype persists even in the absence of biochemical and mechanical cues from the ECM. Here, we show that fibroblast-to-myofibroblast transitions can be triggered solely by physical contact between fibroblasts and myofibroblasts, and that such phenotype transitions are regulated by GqGPCR signaling.

Materials and

Methods: 1.Fabrication of an optically clear substrate: NuSil is an optically clear, biologically inert PDMS substrate with a tunable Young’s modulus (0.3 to 70 kPa) [4]. Equal parts of gel-8100 A and B (Carpinteria, CA, USA) were mixed to obtain a substrate with Young’s modulus E=300Pa. The substrate was then spin-coated onto 30-mm-diameter, #1.5 glass coverslips for 50s to produce a 100-μm-thick layer. They were settled on a level surface at room temperature for 1-hour and cured overnight at 60°C. These were then secured in sterile 40-mm Bioptech dishes (Biological Optical Technologies, Butler, PA, USA) and protein coated with 0.1% gelatin solution for use in cell culture. 2.Generating a pure fibroblast cell population expressing Td-tomato and a pure myofibroblast population: NIH-3T3 mouse fibroblasts (ATCC) were transduced with a lentiviral vector encoding td-tomato, followed by antibiotic selection to obtain a pure td-tomato cell population. To overcome any confounding effect of mechanical memory, cells were cultured for up to 4 additional passages on the 300Pa substrate before being used in experiments. Subsequently, non-transduced NIH-3T3 mouse fibroblasts were cultured for a prolonged period on tissue culture plastic (>100kPa) to obtain a pure myofibroblast population. 3. Platform to study fibroblast-to-myofibroblast transition: Fibroblasts and myofibroblasts were co-cultured (with or without a physical barrier to separate cell types while allowing diffusion) on the 300Pa soft substrate, cultured under standard conditions for 3 days in the absence of any external chemical or mechanical cues and confirmed for fibroblast-to-myofibroblast transitions using αSMA+ stress fiber staining and traction force microscopy.

Results, Conclusions, and Discussions: We demonstrate that fibroblasts cultured on a soft substrate (E=300 Pa) can transition to a myofibroblast phenotype even in the absence of external chemical or mechanical cues from a stiff matrix (Fig 1A). We show that cell-cell contact between myofibroblasts, and fibroblasts is sufficient to drive this phenotype transition and myofibroblast activation (Fig 1B). Our experimental data shows that the contact of myofibroblasts with fibroblasts results in increased cytoskeletal tension in the fibroblasts, and that these cell-cell contacts trigger GqGPCR activation in fibroblasts prior to the myofibroblast transition.

Our findings demonstrate that fibroblast to myofibroblast transition can occur even in the absence of external biochemical and mechanical cues from the ECM. Instead, this transition is solely driven by direct contact between fibroblasts and myofibroblasts. Current approaches to target fibrotic diseases like Idiopathic pulmonary fibrosis (IPF) focus on reducing matrix stiffness by removing the excess collagen deposition in fibrotic tissue. Our results provide an additional mechanism for targeting fibrosis and the persistence of myofibroblasts in diseases like IPF. We are currently working on providing experimental data to understand the underlying mechanobiological mechanisms.

Acknowledgements (Optional): Support for this work was provided by the National Science Foundation Career Grant (#2047207)

References: [1] Darby IA, Laverdet B, Bonté F, Desmoulière A. Fibroblasts and myofibroblasts in wound healing. Clin Cosmet Investig Dermatol.2014;7:301-311. Published 2014 Nov 6. doi:10.2147/CCID.S50046 [2] Zhou Y, Huang X, Hecker L, et al. Inhibition of mechanosensitive signaling in myofibroblasts ameliorates experimental pulmonary fibrosis. J Clin Invest. 2013;123(3):1096-1108. doi:10.1172/JCI66700 [3] Mei Q, Liu Z, Zuo H, Yang Z, Qu J. Idiopathic Pulmonary Fibrosis: An Update on Pathogenesis. Front Pharmacol. 2022;12:797292. Published 2022 Jan 19. doi:10.3389/fphar.2021.797292 [4] Yoshie H, Koushki N, Kaviani R, et al. Traction Force Screening Enabled by Compliant PDMS Elastomers. Biophys J. 2018;114(9):2194-2199. doi:10.1016/j.bpj.2018.02.045