Ashworth_Briggs_whole_thesis.pdf (3.43 MB)
Aqueous humour protein imbalances in primary open-angle glaucoma and their effects on trabecular meshwork cell function
thesisposted on 2023-05-28, 09:16 authored by Ashworth Briggs, EL
Purpose Glaucoma is the leading cause for irreversible blindness worldwide. The term encompasses several ocular disease subtypes with the same end result, namely damage to the optic nerve head, of which primary open-angle glaucoma (POAG) is the most common. Despite extensive research, the aetiology and progression of POAG remain largely elusive. The development of early diagnostic strategies and more effective treatment approaches requires a better understanding of the cellular mechanisms involved in POAG. Problem Elevated intraocular pressure (IOP) is a key risk factor for POAG and the focus of all current treatment strategies. IOP homeostasis is regulated by the trabecular meshwork (TM), a specialised tissue within the anterior chamber of the eye that determines the rate of aqueous humour outflow. In POAG, a dysfunctional TM leads to elevated IOP and consequently damage to the optic nerve head. To date, the processes involved in TM dysfunction remain unclear. Methodology Two studies investigated imbalances in human aqueous humour protein concentrations using multiplex immunoassays. Proteins measured included matrix metalloproteinases (MMPs) and their endogenous tissue inhibitors (TIMPs), various cytokines, and growth factors. Samples from POAG patients were compared to non-glaucomatous cataract patients and results were correlated to disease descriptors including age, IOP, cup-disk-ratio (CDR), Humphrey's visual field pattern standard deviation (PSD), and disease duration since diagnosis. Based on the results from the second study, the anti-inflammatory cytokine uteroglobin was selected for in vitro investigation using a human TM cell model. TM cells were first characterised using a panel of TM marker genes and two functional assays: Dexamethasone-stimulated myocilin upregulation and collagen gel contraction. For the latter, contractile (human fibroblast) and non-contractile (HeLa) control cell lines were included, and ROCK inhibitor Y-27632 was used to inhibit contraction. Subsequently, the collagen gel contraction assay was used to investigate the effect of 1 and 2 ˜í¬¿g/ml uteroglobin on TM cell contraction over a period of 96 hours. Results The first study found significantly elevated levels of TIMP1, TIMP2, TIMP4, and MMP2 concentrations in aqueous humour samples from POAG compared to cataract control. Imbalances in MMP/TIMP molar ratios were also determined in POAG, several of which correlated with IOP and PSD, but not with other disease descriptors. In the second study, several cytokines and growth factors, including uteroglobin, HGF, and FLRG, were detected in aqueous humour samples from POAG and cataract control patients. Uteroglobin and FLRG concentrations correlated significantly with age in POAG but not cataract samples. Furthermore, HGF concentrations resulted in a negative correlation with disease duration. Gene expression and immunofluorescence experiments using primary TM cells determined expression of the uteroglobin receptor LMBR1L but not uteroglobin itself. Treatment of primary TM cells with recombinant uteroglobin did not significantly affect cell contraction at the concentrations tested. Conclusions The differences identified in MMPs, TIMPs, and various cytokine concentrations in aqueous humour from POAG patients add to our knowledge on aqueous humour imbalances in POAG. The analysis of MMP-to-TIMP ratios showed a shift towards increased TIMP levels, suggesting a potential increase in MMP inhibition, which may affect extracellular matrix composition and thus contribute to elevated aqueous humour outflow resistance. Several of the cytokines reported in the second study have not previously been quantified in aqueous humour. Correlations of age with uteroglobin and FLRG in POAG may indicate an increased need for anti-inflammatory (uteroglobin) or anti-calcification (FLRG) activity in the aging glaucomatous TM. While no effect of uteroglobin on TM cell contraction was found in the final study, further experiments may elucidate the effects this protein has on TM cell function. Specifically, phospholipase A2 activity, tissue transglutaminase activity, and phagocytosis assays are required to determine the potential effect of uteroglobin on TM cells. Determination of the effects of the aqueous humour proteins quantified in this thesis on TM cell behaviour may lead to new insights with regards to TM dysfunction in POAG and possibly provide new targets for pharmaceutical intervention or result in diagnostic biomarkers for POAG.
Rights statementCopyright 2018 the author Chapter 2 appears to be the equivalent of a post-print version of an article published as: Ashworth Briggs, E. L., Toh, T. Y., Eri, R., Hewitt, A. W., Cook, A. L., 2015. TIMP1, TIMP2, and TIMP4 are increased in aqueous humor from primary open angle glaucoma patients, Molecular vision, 21, 1162-1172. Copyright 2015 Molecular Vision. The article and all supplementary materials are made freely available online immediately with a Creative Commons Attribution-NonCommercial-NoDerivatives License 3.0, or CC BY-NC-ND 3.0 (see http://creativecommons.org/licenses/by-nc-nd/3.0/ for license terms). The authors retain copyright and grant Molecular Vision an irrevocable, royalty-free, perpetual license to publish and distribute the article, in all formats now known or later developed, and to identify Molecular Vision as the original publisher. Chapter 3 appears to be the equivalent of a post-print version of an article published as: Ashworth Briggs, E. L., Toh, T. Y., Eri, R., Hewitt, A. W., Cook, A. L., 2018. Uteroglobin and FLRG concentrations in aqueous humor are associated with age in primary open angle glaucoma patients, BMC ophthalmology, 18, 57. Copyright The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.