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Interrogating the cellular and molecular mechanisms of tissue fibrosis

Primary Supervisor: Dr Jill Johnson, Life & Health Sciences

Secondary supervisor: Dr Patricia Perez-Esteban

PhD project title: Interrogating the cellular and molecular mechanisms of tissue fibrosis

University of Registration: Aston University

Project outline:

Background: Scar formation is a vital mechanism of tissue repair following injury. However, healthy tissue repair can develop into pathological fibrosis, which ultimately leads to tissue destruction and organ failure. Fibrosis is associated with chronic inflammation, oxidative stress, and ageing. However, there are currently no treatment options for organ fibrosis, and these diseases impose a significant burden on public health care systems and have detrimental impacts on patient quality of life. Importantly, little is known about the factors that initiate fibrosis. This studentship will build on previous work in Dr Jill Johnson’s research group that has identified pericytes as the primary driver of fibrosis. Pericytes provide support to capillaries throughout the body1 and are particularly important in maintaining healthy tissue structure. Importantly, pericytes are strongly associated with tissue fibrosis in the lung, liver, and kidney.2-4 Recent studies have shown that pericytes contribute to fibrosis by uncoupling from local blood vessels, followed by migration to the site of inflammation and differentiation into scar-forming myofibroblasts5 (Figure 1). However, the mechanisms by which pericytes transform into scar-forming cells (myofibroblasts) are currently unknown.

Furthermore, the mechanical microenvironment of cells has a significant effect on their activity and proliferation, driving the progression of fibrosis.6 The stiffness (Young’s elastic modulus) of human tissue ranges from 0.1-10 kPa,7 which significantly deviates from traditional plastic culture platforms (Young’s elastic modulus in the order of GPa). In this study, we will mimic the mechanical microenvironment of healthy and fibrotic tissue using innovative cell culture techniques.

Aims: To investigate the mechanisms responsible for myofibroblast transformation. using in vitro methods to assess the impact of fibrotic mediators on pericyte function.

Hypothesis: Pro-fibrotic growth factors will lead to pericyte-myofibroblast transition and contribute to fibrosis.

Methods: 1. Using in vitro two-dimensional pericyte culture, we will determine the dose and duration of fibrosis-associated growth factor treatment (EGF, bFGF, and TGF-β) resulting in pericyte transition into myofibroblasts. The readouts will include procollagen I and α-smooth muscle actin expression, cell migration using scratch assays, and cell contractility using collagen gel contraction assays.

  1. The student will establish microvascular organoids using the n3D NanoShuttle-PL magenetic system (already established in the Johnson lab). These organoids are composed of human pericytes and endothelial cells to mimic the microvascular environment and to establish the impact of growth factor treatment on pericyte/endothelial cell connectivity and vascular stability. Readouts will include the analysis of confocal images of immunostained organoids following dose-response and time-response studies.
  2. In collaboration with Dr Patricia Perez-Esteban, we will mimic the mechanical microenvironment of healthy and fibrotic tissue using chemically tailored hydrogels that incorporate biocompatible materials such as collagen, agarose, or gellan gum via mechanical methods or chemical crosslinking. By modifying the chemical composition, gelling temperature, and stirring speeds, stiffness (Young’s elastic modulus) values that are representative of human tissue can be achieved (from hundreds of Pascals (Pa) to tens of kPa), as opposed to traditional plastic culture platforms (on the order of GPa).

Expected outcomes. The induction of fibrosis in pericytes will lead to:

  • increased expression of procollagen I and α-smooth muscle actin, known to be associated with fibrosis
  • decreased microvascular organoid stability
  • increased expression of pro-fibrotic markers by pericytes grown in a stiff matrix

It is expected that these outcomes will contribute to describing the molecular mechanisms by which pericytes initiate fibrosis.

References:

1. Fuxe J et al. (2011) Am J Pathol 178:2897

2. Johnson JR et al. (2015) Am J Physiol Lung Cell Mol Physiol 308(7):L658

3. Mederacke I et al. (2013) Nat Commun 4:2823

4. Wu CF et al. (2013) Am J Pathol 182:118

5. Hosaka K et al. (2016) Proc Natl Acad Sci USA 113:E5618

6. Wells et al. (2013) Biochim Biophys Acta 1832:884.

7. Achterberg et al. (2014) J Invest Dermatol 134:1862.

BBSRC Strategic Research Priority: Understanding the Rules of Life: Immunology, Stem Cells & Structural Biology Integrated Understanding of Health: Ageing & Regenerative Biology

      Techniques that will be undertaken during the project:

      • Two-dimensional and three-dimensional cell culture
      • Immunostaining
      • Fow cytometry
      • Cell migration assays
      • Cell contraction assays
      • Confocal imaging
      • Digital image analysis.

      Contact: Dr Jill Johnson, Aston University