Exploring mechanotransduction in substrates mimicking healthy and fibrotic microenvironment

Lead Research Organisation: Imperial College London
Department Name: Dept of Bioengineering

Abstract

Chronic liver fibrosis is the main cause of organ failure in liver diseases of any aetiology1. Chronic fibrosis, if untreated leads to cirrhosis and ultimately organ failure, these patients are also more susceptible to developing liver cancer, particularly hepatocellular carcinoma(HCC)2. Liver cancer can arise from virulent factors like hepatitis B/C, excessive alcohol consumption and non-alcoholic fatty liver disease(NASH). These factors introduce damage to liver tissue, as a result activate the wound healing response with an upregulation and secretion of extracellular matrix (ECM) proteins and cytokines1. With limited therapies currently treating this disease, the most effective one being liver transplant, alternative solutions need to be sought out in order to target the progression and reversal of fibrosis.
Hepatic stellate cells (HSCs) have been identified as main effectors of the fibrotic response3. In healthy liver, HSCs are found in a quiescent state, where their main role is homeostasis of the liver and retinoid storage4. Upon tissue insult, they become 'activated', highly migratory and secrete aberrant ECM proteins, resulting in ECM remodelling and stiffness(fibrosis)5. This response results in a new dynamic environment with increased rigidity of the matrix which leads to enhancement of contractile forces in cells to maintain a tensional homeostasis and activated phenotype. This further results in a pro fibrotic feedback loop of increased activation of cells and increased secretion of ECM proteins producing a stiffer matrix.
The aim of this study is to elucidate the role of hepatic stellate cells in the progression of fibrosis, understand the mechanisms behind this response and target the activation of these cells to revert them back to a quiescent state to halt the progression and the possible reversal of chronic liver injury. We plan to demonstrate: (i) how HSC activation depends on substrate stiffness, (ii) how these cells mechanosense their microenvironment, (iv) become activated and adopt a myofibroblastic-like phenotype, and (v) become migratory and secrete factors to further contribute to substrate stiffness. By designing polyacrylamide gels with varying rigidities, and seeding cells on these hydrogels, we can manipulate the stiffness of the substrate and monitor the changes that emerge intracellularly. By using polyacrylamide gels, as opposed to tissue culture plastic/glass we will be able to provide more realistic in-vitro results which better depict the liver microenvironment by mimicking the stiffness of a healthy and fibrotic liver to monitor how these cells behave in response to mechanical cues.

Publications

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Studentship Projects

Project Reference Relationship Related To Start End Student Name
EP/N509486/1 01/10/2016 30/09/2021
1975895 Studentship EP/N509486/1 30/09/2017 29/09/2021 Gulcen Yeldag
 
Title polyacrylamide gel preparation for durotaxis 
Description A method used to study durotaxis is producing hydrogels with a stiffness gradient which can be obtained through photo-polymerisation, To prepare gels using photoactivation and a sharp boundary between rigidities, hydrophobic slides and hydrophilic coverslips were prepared. The preparation of polyacrylamide (PAA) solution was prepared adapting the protocol described by Sheth et al. Using 40% acrylamide (Sigma Aldrich) and 2% Bis solution (BioRad) mixed with PBS to give a final concentration of 8% acrylamide to 0.2% Bis with 0.02mg of photoinitiator Irgacure (2-Hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone) (Sigma Aldrich). This solution was then vortexed and 15 µl drops were added onto the hydrophobic slides, a coverslip was added on to each of the drops and the slides were placed under UV light for 2.5 minutes. This would create a uniform soft gel. After photoactivation the slides were then removed and a black opaque tape was added to half of the coverslip, covering one side of the gel and leaving the other half exposed (Figure 2). The slides were then photoactivated for a further 5 minutes under UV ensuring only the exposed hydrogel would be further photoactivated. The resulting hydrogel was a uniform gel with a very distinct boundary i.e no gradient allowing cells to be seeded across the boundary where single cells could bridge both rigidities. 
Type Of Material Improvements to research infrastructure 
Year Produced 2019 
Provided To Others? No  
Impact this method was created in order to obtain a uniform hydrogel with 2 distinct rigidities within the same same gel, eliminating the presence of a gradient. the idea was to seed cells exactly on the boundary where a single cell could bridge two different rigidities i.e soft and stiff. This method could then be used to analyse the changes in focal adhesion dynamics within the same cell.