Poster 144 - Studying Contractile Forces of TGF-β Induced Myofibrotic Activity by Jayden Wong

Saturday, October 26, 2024

10:00 AM – 12:00 PM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Jayden Wong

High School Student Researcher
University of California Irvine
Irvine, California, United States

Primary Investigator(s)

Quinton Smith, PhD

Assistant Professor
University of California, Irvine, United States

Co-Author(s)

Christopher T. Clark, MS

PhD Candidate
University of California Irvine
Irvine, California, United States

Introduction:

Introduction: Driving most chronic liver diseases, liver fibrosis is the overaccumulation of extracellular matrix proteins following wound-healing processes, replacing liver parenchyma as connective scar tissue (Batteler R, 2005). Early stressors in the liver advance into liver cirrhosis, a disease state responsible for 14.6% percent of all deaths in the US (Chen C, 2022). While recent advancements have enabled novel drugs to join liver transplantation as treatment methods, fibrosis models remain in high demand to inform drug discovery and understand disease progression conditions. As the master regulator of fibrosis, transforming growth factor (TGF)-β is often focused on for its induction of myofibroblasts from fibroblast populations in vitro (Meng Xm, 2016). In this state, cells have increased extracellular matrix (ECM) deposition and heightened cellular contractile force, a characteristic attributed to increased alpha-smooth muscle actin (αSMA) expression in stress fibers (Iwaisako K, 2012). The increase in contractile strength of myofibroblasts can be measured through traction force microscopy (TFM), where cells, seeded atop bead-laden gels, pull on both the gel and the beads, enabling cell force quantification through bead displacement analysis (Zancla A, 2022). By utilizing TFM to verify the effectiveness of modeling fibrotic response, we can investigate the advancement of fibrotic progression in relation to contractile cell force.
Hypothesis: Can an in vitro model of fibrotic response be induced in human dermal fibroblasts through TGF-β exposure and quantified through traction force microscopy?

Materials and

Methods: Materials and

Methods: Human dermal fibroblasts (HDFs; PromoCell) were cultured with Fibroblast Growth Medium (FGM-2; PromoCell) before being passaged onto 10.61 kPa polyacrylamide (PAA) gels containing 1% 0.5 μm fluorescent microbeads for TFM trials. For staining trials, HDFs were passaged onto 15 mm glass coverslips. All samples were coated with 0.05mg/mL of collagen1 in 6-well plates at a uniform density of 10,000 cells/cm2. Cells were exposed to either a vehicle control or TGF-β at a concentration of 5ng/mL, with media replaced each day for two days. In TFM trials, bead-laden PAA gels were first imaged to access cell attachment, then cells were released after a 5-minute trypsin exposure and imaged again afterward. Obtained images were quantified in FIJI for bead displacement and traction force using Particle Image Velocimetry (PIV) and Fourier Transform Traction Cytometry (FTTC), respectively. For staining trials, cells were then fixed with 4% paraformaldehyde (PFA) and stained for DAPI, phalloidin (F-actin), fibronectin (FN1), and alpha smooth muscle actin (αSMA). The level of αSMA and FN1 expression was imaged and quantified using FIJI to measure the mean fluorescence intensity.

Results, Conclusions, and Discussions: Results and

Discussion: HDFs expressed significantly higher FN1 expression in 5ng/mL TGF-β wells compared to control conditions. Quantification of staining exhibited a likewise increase in αSMA expression, with a corresponding increase in stress fibers. Quantified cell contractility was obtained using aligned bead images to construct traction force measurements from displacement fields. Collectively, TFM enabled the observation of standard fibroblast contractility and cell forces, aiding the observation that HDFs exhibit increased contractile forces in the presence of TGF-β, a driver of fibrotic processes.

Conclusions: Adhesion and maintenance of bead-laden PAA cultured cells confirmed a baseline cellular force reading for a non-activated state. This cellular contraction was quantified by tracked bead movement and then used with known characteristics of the hydrogel to determine the force exerted by cells on their substrate. Following 5ng/mL TGF-β exposure, HDFs demonstrated higher FN1 and αSMA expression than the control, exhibiting heightened ECM deposition and contractility of fibrotic cells. Thus, TGF-β exposed cells can serve as a model of fibrotic response in vitro, with contractile force as a viable benchmark for the shift between fibroblast and myofibroblast states. Future studies include adapting this model to liver-specific stellate cells for developing interventions to abrogate fibrosis.

Acknowledgements (Optional): I would like to thank my Ph.D. student advisor Christopher T. Clark for his mentorship and Dr. Quinton Smith from the Department of Chemical and Biomolecular Engineering at the University of California, Irvine for allowing me to work in his lab. The research was funded by the Howard Hughes Medical Institute (HHMI).

References (Optional): 1. Bataller, R. “Liver Fibrosis.” J Clin Invest 115(2), 209–218 (2005).
2. Chen, C. “Surveillance Report #118: LIVER CIRRHOSIS MORTALITY IN THE UNITED STATES: NATIONAL, STATE, AND REGIONAL TRENDS, 2000–2019”. NIAAA (2022)
3. Meng, Xm. “TGF-β: the master regulator of fibrosis.” Nat Rev Nephrol 12, 325–338 (2016).
4. Iwaisako, K. “What’s new in liver fibrosis? The origin of myofibroblasts in liver fibrosis.” J Gastroenterol Hepatol 27, 65-68 (2012).
5. Zancla, A. “A primer to traction force microscopy.” J Biol Chem, 298(5), 101867 (2022).