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Title: Studies on a disintegrin and metalloproteinase with thrombospondin motifs-1, -4 and -5 and the regulation of their gene expression in macrophages
Author: Ashlin, Timothy
ISNI:       0000 0004 2734 8349
Awarding Body: Cardiff University
Current Institution: Cardiff University
Date of Award: 2012
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A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) are a family of proteins that are closely related to the matrix metalloproteinases (MMPs). It has been suggested that the proteins have a critical role in the breakdown of articular cartilage during osteoarthritis (OA). More recently it has been suggested that their actions could potentially regulate atherosclerotic plaque stability. Atherosclerosis is a chronic, inflammatory disorder characterised by lipid and cholesterol accumulation and the development of fibrotic plaques within the walls of large and medium arteries. The stability of the plaques is very important because clinical symptoms are only presented after rupture of the unstable plaques, leading to thrombosis and ischemia. During the current study, immunohistochemical analysis confirmed that ADAMTS-­1, -­4 and -­5 were being expressed within human carotid atherosclerotic lesions; macrophages were identified as major contributors to their expressions. Following on from this THP-­1 macrophages were stimulated with transforming growth factor-­β (TGF-­β), interferon-­γ (IFN-­γ), TNF-­like protein 1A (TL1A), interleukin (IL)-­17A and IL-­33. The regulation of ADAMTS-­1, -­4 and -­5 expressions were analysed using quantitative polymerase chain reactions (QPCR) and western blots. It was shown that TGF-­β increased the expressions of ADAMTS-­1 and -­5 and decreased the expression of ADAMTS-­4. IL-­33 decreased the expressions of ADAMTS-­1, -­4 and -­5 and IFN-­γ also decreased the expression of ADAMTS-­1. TL1A and IL-­17A stimulation of macrophages had no regulatory actions over ADAMTS-­1, -­4 or -­5 expressions. Looking at evidence from previous studies, TL1A and IL-­17A were identified as agents that could potentially act in synergy to amplify pro­inflammatory cytokine responses. To investigate this further, THP-­1 macrophages were stimulated with TL1A and IL-­17A, TL1A and IFN-­γ and also IL-­17A combined with IFN-­γ. TL1A and IL-­17A were shown to act in synergy to increase the expressions of ADAMTS-­1, -­4 and -­5 in macrophages. The regulation of ADAMTS-­1, -­4 and -­5 expressions in macrophages by IL-­33 was studied further. The mechanism of signal transduction was studied using RNA interference (RNAi) targeting extracellular signal-­‐regulated kinases (ERK)-­1, ERK-­2, p38, c-­Jun N-­terminal kinases(JNK)-­1/2, c-­Jun, phosphoinositide 3-­kinase(PI3K)-­γ, PI3K-­δ, p50, p65 and Janus kinase(JAK)-­1/2. It was determined that the attenuation of ADAMTS-­1, -­4 and -­5 expressions occurred through transcriptional regulation that was dependent on the ST2 receptor. ERK-­1, ERK-­2, JNK-­1/2, c-­Jun, PI3K-­γ and PI3K-­δ were also involved in the signal transduction of the response. The cellular roles of ADAMTS activity within atherosclerotic disease progression remain poorly understood. During the current study adenoviral vectors were created that delivered shRNA-­targeting ADAMTS-­1, -­4 and -­5. The adenoviral vectors were utilised in studies designed to investigate the roles of ADAMTS-­1, -­4 and -­5 during macrophage migration and foam cell formation. The studies showed that knockdown of ADAMTS-­1, -­4 and -­5 had no effect on macrophage migration or foam cell formation. More research is required into the cellular roles that ADAMTS proteases play during atherosclerotic disease progression. The field of research is now growing and could potentially provide some exciting opportunities for novel therapeutics of the future.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: QH Natural history ; QH301 Biology ; QH426 Genetics