Saudi Cultural Missions Theses & Dissertations
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Item Restricted The influence of ECM on mesenchymal stromal/stem cell activity and their interactions with monocytes/macrophages(University of Cambridge, 2024-03-23) Alkhrayef, Mohammad Nasser M; Birch, Mark; McCaskie, AndrewOsteoarthritis is a prevalent degenerative condition affecting synovial joints, resulting in pain, stiffness, and difficulty with movement. Current treatments mainly alleviate symptoms without effectively regenerating the lost cartilage tissue. An early surgical intervention for focal cartilage damage, known as marrow stimulation or microfracture, aims to repair this damage by promoting endogenous healing. However, the resulting repair tissue often lacks the properties of native cartilage, raising questions about the long-term effectiveness of this approach. Therefore, to enhance the outcome of this osteochondral repair approach, it is necessary to understand the biological mechanisms involved in the healing process following microfracture. Osteochondral repair is a complex interplay of cellular interactions and changing extracellular matrix (ECM) environment. This thesis aimed to extend the current understanding of the biology underpinning this process, focusing on the interaction between mesenchymal stromal/stem cells (MSCs) and monocytes/macrophages within dynamically changing ECM proteins found at the injury site. We hypothesized that this interaction plays a pivotal role in determining the outcome of tissue repair. In our investigation, we first characterised the deposition and remodelling patterns of key ECM proteins, fibrin and collagen type 1, during osteochondral injury repair in C57BL/6 mice. Our findings revealed that fibrin was the predominant ECM protein in the initial seven days post-injury, which was subsequently gradually replaced by collagen type 1. At eight weeks post-injury, the tissue repair outcome at the articular surface was found to contain collagen type 1, perhaps representing an immature form of hyaline cartilage. Subsequent in vitro studies, using 3D ECM models constructed from fibrin and collagen type 1, demonstrated that these environments significantly modulate MSC morphology, proliferation, migration, and immunomodulatory activity. Notably, MSCs within these 3D environments exhibit differential responses to pro-inflammatory stimuli, with the ECM potentially acting as a reservoir for secreted cytokines and growth factors, orchestrating cellular activities over time. Three molecular mechanisms, the TNFα/NFkB pathway, TNFα/JNK/AP1 pathway, and the potential involvement of ROCK, were identified as specific effectors of MSC immunomodulatory activity within these environments. Further investigations into the crosstalk between MSCs and monocytes in controlled 3D ECM environments revealed a distinct M2 macrophage phenotype, characterized as CD206+ MerTK- CD163- CD209-. This subpopulation displayed enhanced expression of IL10, IL6, and IL8. Moreover, conditioned media from these co-cultures influenced chondrocyte migration and chondrogenesis, with TGFβ1 playing a pivotal role in these observations. In conclusion, this thesis underscores the profound influence of the ECM on cellular interactions during osteochondral repair. The insights gained pave the way for innovative therapeutic strategies, potentially enhancing tissue repair outcomes and offering improved treatments for conditions like osteoarthritis.15 0Item Restricted The Anti-inflammatory Effects of Metformin on Human Pro-inflammatory Macrophages(2023-06-23) Alqurashi, Noor; Rena, GrahamMacrophages are a type of immune cells that have a key role in atherosclerotic plaque development and as such possible target for cardiovascular disease (CVD) therapy. In mouse and human lesions, macrophages adhering to both M1 (pro-inflammatory) and M2 (anti-inflammatory) phenotypes are present, with M1 being the most common phenotype. The type 2 diabetes drug metformin is thought to reduce inflammation by reducing the polarisation process to M1 macrophages, with AMPK activation likely to be an important mediator of this effect. In this thesis, I have used state-of-the-art data-independent acquisition (DIA) proteomics to perform a systems-wide comparison of the effect of metformin and a selective AMPK agonist A769662 on the polarisation and differentiation processes in THP-1 cells, a human macrophage cell line. This work highlighted the modulation of proteins linked to inflammation, cell adhesion, migration,19 0Item Restricted ANGIOTENSIN II TYPE 2 RECEPTOR AGONIST PREVENTS THE PRO- INFLAMMATORY RESPONSE IN LPS TREATED HUMAN MACROPHAGES(ProQuest, 2020-08) Alshammari, Abdulkarim; Fagan, SusanStroke is a leading cause of long term disability and is associated with a 30% incidence of severe cognitive impairment. Sustained pro-inflammatory microglia activation contributes to, and our lab has shown that the Angiotensin II type 2 receptor (AT2R) agonist, compound 21 (C21), can prevent the development of, PSCI. We hypothesized that activation of pro-inflammatory microglia and macrophages can be prevented with C21. This was assessed using a microglial cell line (C8-B4) and THP-1 derived macrophages. The reduction in the pro- inflammatory cell markers was assessed via RT-qPCR using the following genes, IL-1b, TNFa, and NOS2. Cells were either pre-treated, prior to LPS exposure, or post-treated after LPS treatment, with C21 (100 uM). C21 effectively reduced the expression of IL-1b in a concentration-dependent manner. Both pre- and post- treatment with C21 significantly reduced the expression of pro-inflammatory markers after LPS exposure in a mouse microglial cell line and human macrophages.36 0