Principal Supervisor: Dr Jill JohnsonLink opens in a new window

Co-supervisor: Dr Emma Shepherd

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

University of Registration: University of Aston

Project outline:

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 are a type of mesenchymal progenitor cell that support 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 myofibroblasts^5,6 (Figure 1). However, the mechanism by which chronic inflammation affects the differentiation capacity of pericytes is currently unknown.

Mesenchymal stem cells (MSCs) are multipotent adult stem cells with self-renewal and differentiation capacity that can be readily isolated from bone marrow, but all tissues harbor MSC-like cells as part of the microvasculature, i.e. pericytes. MSCs can differentiate into multiple cell lineages under specific differentiation conditions. Intriguingly, recent studies have shown that inflammatory mediators affect the differentiation capacity of MSCs, i.e. low levels of TNF-α promote osteogenic differentiation in bone marrow-derived MSCs.⁷

Aims: To investigate the mechanisms regulating the differentiation capacity of pericytes in health and disease.

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

Objectives:

1. Using in vitro two-dimensional pericyte culture, we will investigate the mechanisms of pericyte-to-myofibroblast differentiation by varying the dose and duration of treatment with inflammatory mediators known to promote myofibroblast differentiation (IL-13, EGF, bFGF, and TGF-β). Readouts will include qPCR array analysis of fibrosis-associated genes (Qiagen RT² Profiler™ PCR Array Human Fibrosis), FACS analysis of myofibroblast surface markers (CD105 and CD49e), immunostaining for procollagen I and α-smooth muscle actin expression, cell migration assays, and collagen gel contraction assays.
2. Differentiation assays will be performed on healthy and inflammatory-mediator treated human pericytes. Readouts will include qPCR array analysis (Human Mesenchymal Stem Cell qPCR Array, Stemcell Technologies) and MSC differentiation assays to assess the capacity of pericytes to differentiate into adipocytes, chondrocytes, and osteocytes using MesenCult™ differentiation kits (Stemcell Technologies).
3. Experiments will be performed to validate the findings from the first two aims in an in vivo setting. To achieve this, a mouse model of chronic inflammation and fibrosis⁸ will be used to query pericyte phenotype using FACS (CD105 and CD49e, CXCR4). Additionally, magnetic cell separation techniques will be used to isolate pericytes from healthy and inflamed mice to analyse gene expression and differentiation capacity using the methods described in Aim 2 (Mouse Mesenchymal Stem Cell qPCR Array, MesenCult™).

Expected outcomes. Pericytes will respond to inflammatory mediators by transforming into myofibroblasts and demonstrating more robust differentiation capacity.

This project will contribute to describing the molecular mechanisms by which pericytes initiate fibrosis. The student will gain experience in key biosciences laboratory and bioinformatics data analysis techniques and gain a solid grounding in fibrosis research.

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. Bignold R et al. (2021) Front Allergy 2:786034.6. Bignold R et al. (2022) Respir Res. 2022 23(1):183. 7. Russell T et al. (2021) Cells 10(2):341.8. Johnson JR (2004) Am J Respir Crit Care Med 169(3):378-85.

 

BBSRC Strategic Research Priority: Understanding the rules of life - Immunology, Stem Cells, Structural Biology and Integrated Understanding of health - Ageing and Regenerative Biology.

Techniques that will be undertaken during the project:
Two-dimensional and three-dimensional cell culture, immunostaining, flow cytometry, cell migration assays, cell contraction assays, confocal imaging, digital image analysis.

Contact: Dr Jill JohnsonLink opens in a new window