Session: 16-01-01: Government Agency Student Poster Competition

Paper Number: 150507

150507 - Mechanotransduction of Cellular Pre-Stress 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 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 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 (IPF) continue to progress [3]. Clearly, there are alternative mechanisms that are yet unknown, operating 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. We show that fibroblast-to-myofibroblast transitions can be triggered solely by physical contact between fibroblasts and myofibroblasts through mechanotransduction of intrinsic cellular pre-stress. Our findings underscore the critical role of direct mechanical interactions between cells in fibrosis, suggesting that these interactions could modulate cytoskeletal pre-stress and thereby represent novel targets for therapeutic intervention in fibrotic diseases. This shift toward intracellular and intercellular mechanical forces opens new avenues for understanding and possibly mitigating fibrosis more effectively. Moreover, these insights extend to broader pathological contexts, including cancer, where cell-cell interactions are pivotal in driving disease progression. 

Methodology: Fabrication of an optically clear substrate: NuSil is an optically clear, biologically inert PDMS substrate with Young’s modulus tunable in the range from 0.3 to 70 kPa [4]. Equal parts of NuSil gel-8100 parts A and B (NuSil, CA, USA) were mixed to obtain a substrate with Young’s modulus E=300Pa and spin-coated onto 30-mm-diameter, glass coverslips to produce a 100μm-thick layer. They were cured overnight at 60°C and secured in sterile 40-mm Bioptech dishes (Biological Optical Technologies, PA, USA). Traction Force Microscopy: NuSil substrates were coated with a layer of 0.2µm-diameter red fluorescent carboxylate-modified microspheres (Invitrogen, CA, USA) and left to adhere at room temperature for 1-hour. Substrates were washed 3x with PBS, and protein coated with 0.1% gelatin solution for use in cell culture. Generating a pure fibroblast and myofibroblast population: To differentiate fibroblasts from myofibroblasts, NIH 3T3-mouse fibroblasts (ATCC) were transduced with a lentiviral vector encoding td-tomato (red-fluorescence), followed by antibiotic selection to obtain a pure td-tomato population. To overcome any confounding effect of mechanical memory, cells were cultured for up to 4 additional passages on the 300Pa soft substrate before being used in experiments. Subsequently, non-transduced 3T3-mouse fibroblasts were cultured for a prolonged period on tissue culture plastic (>100kPa) to obtain a pure myofibroblast population. Co-culture platform to study fibroblast-to-myofibroblast transition: Equal number of fibroblasts and myofibroblasts were plated together (or with a physical barrier to separate the cell types while allowing diffusion) on the soft substrate, cultured for 3-days in the absence of any external chemical/ mechanical cues and analyzed for fibroblast to myofibroblast transitions using fluorescent staining for myofibroblast markers.

Preliminary results and conclusions: Our findings demonstrate that when fibroblasts are co-cultured (1:1) with myofibroblasts on the 300Pa substrate, fibroblasts transition to a myofibroblast phenotype even in the absence of external chemical or mechanical cues. We show that this transition is solely driven by cell-cell contact between myofibroblasts and fibroblasts and is sufficient for myofibroblast activation. Our traction-force data indicates that the interaction between fibroblasts and myofibroblasts leads to increased cytoskeletal tension in fibroblasts as early as the first day of culture. This increase in cytoskeletal tension precedes phenotypic transitions (observed only on the third day). Our ongoing work is focused on understanding the underlying mechanobiological mechanisms (ie-GqGPCR activation) in fibroblasts prior to myofibroblast transition. Our results provide a new mechanism for persistence of myofibroblasts and targeting fibrosis in diseases like IPF.

References-[1]Darby-IA,et-al-Fibroblasts-and-myofibroblasts-in-wound-healing-doi-10-2147/CCID-S50046-[2]-Zhou-Y-et-al-Inhibition-of-mechanosensitive-signaling-in-myofibroblasts-ameliorates-experimental-pulmonary-fibrosis-doi-10-1172/JCI66700-[3]-Mei-Q-et-al.-Idiopathic-Pulmonary-Fibrosis-An-Update-on-Pathogenesis-doi-10-3389/fphar-2021-797292-[4]-Yoshie-H-et-al-Traction-Force-Screening-Enabled-by-Compliant-PDMS-Elastomers-doi-10-1016/j.bpj.2018.02.045 

Presenting Author: Vasuretha Chandar Northeastern University

Presenting Author Biography: Vasuretha Chandar is a second-year PhD candidate in the Department of Bioengineering at Northeastern University, working under the supervision of Dr. Harikrishnan Parameswaran. Her primary research focuses on understanding the continued progression of diseases like Idiopathic Pulmonary Fibrosis despite current treatments, such as cytokine and growth factor inhibitors to prevent collagen deposition, and collagen-degrading enzymes to soften the stiff extracellular matrix. She is currently investigating the mechanisms that drive the transition of fibroblasts to myofibroblasts even in the absence of external chemical and mechanical cues, aiming to identify new mechanobiological pathways for treating fibrosis.

Authors:

Vasuretha Chandar Northeastern University
Benjamin Goykadosh Northeastern University
Harikrishnan Parameswaran Northeastern University