Browsing by Author "Giembycz, Mark A."

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    Modulation of TGFβ1-induced Fibroblast-to-Myofibroblast Transition in response to Prostaglandin E2 Production by Human Rhinovirus-Infected Airway Epithelial Cells
    (2020-05-08) Pham, Diana Minh; Leigh, Richard A.; Proud, David G.; Kelly, Margaret Mary; Giembycz, Mark A.
    One of the cardinal features of asthma is the presence of airway remodeling, the structural changes that contribute to exaggerated narrowing of the airway. Fibroblast-to-myofibroblast transition (FMT) is an airway remodeling phenomenon whereby fibroblasts develop phenotypic characteristics of myofibroblasts. This increase in myofibroblasts leads to the excessive deposition of extracellular matrix proteins, leading to the thickening of the airway walls. Transforming growth factor beta-1 (TGFβ1) is highly expressed in asthmatics and is known to upregulate fibroblast alpha-smooth muscle actin (α-SMA) expression, a characteristic marker of myofibroblasts. Given that human rhinovirus (HRV) infections are postulated to be involved in the pathogenesis of airway remodeling in asthma, we originally hypothesized that human airway epithelial cells promoted TGFβ1-induced FMT based on α-SMA expression by secreting mediator(s) upon HRV infection. Interestingly, our data consistently demonstrated that supernatants from HRV-infected epithelial cells inhibited TGFβ1-induced α-SMA in fibroblasts compared to supernatants from non-infected epithelial cells. This led us to hypothesize that HRV-infected airway epithelial cells released PGE2 which acts via EP1-4 receptors on fibroblasts to suppress TGFβ1-induced α-SMA expression. Using an in vitro cell culture model, we confirmed HRV-infection of epithelial cells result in the upregulation of PGE2 and validated that PGE2 inhibits TGFβ1-induced α-SMA protein in fibroblasts. Furthermore, supernatants from epithelial cells treated with diclofenac, a non-selective cyclooxygenase inhibitor, prior to HRV infection had reduced ability to inhibit TGFβ1-induced α-SMA expression in fibroblasts. Finally, we demonstrated that PGE2 acts on the fibroblast EP2 receptor to downregulate TGFβ1-induced FMT. In conclusion, our research findings may account for why healthy, non-asthmatic individuals are prevented from developing TGFβ1-induced FMT following repeated rhinovirus infections. This thesis instigates future investigation on whether well-characterized asthmatic cells behave differently to render it vulnerable to TGFβ1-induced FMT following rhinovirus infections.
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    Regulation of cAMP-Dependent Gene Expression in Airway Epithelial Cells: A Transcriptional and Pharmacodynamic Analysis
    (2019-08-02) Yan, Dong; Giembycz, Mark A.; Slater, Donna M.; Yipp, Bryan G.; Hollenberg, Morley Donald
    Asthma and chronic obstructive pulmonary disease (COPD) are leading causes of death worldwide and their prevalence is predicted to increase over the next 20 years. Current treatments focus on providing symptomatic relief, with bronchodilators, and gaining disease control by suppressing inflammation. Long-acting β2-adrenoceptor agonists (LABAs) are effective bronchodilators that work by elevating cAMP in airway smooth muscle (ASM). Less appreciated and understood is the ability of cAMP to regulate gene expression. For example, previous studies have shown LABAs to protect ASM against pro-contractile stimuli by up-regulating RGS2, which attenuates signalling mediated through Gq-linked receptors. Based on these historical data, the first objective of the work reported here was to “mine” the LABA-regulated transcriptome for additional genes with disease-relevant functions. In the BEAS-2B airway epithelial cell line and in human primary bronchial epithelial cells (HBEC), the LABAs, indacaterol and salmeterol, promoted robust transcriptional responses. Functional annotation and gene ontology analyses identified genes with both anti-inflammatory and adverse effect potential. Moreover, there was significant overlap in gene expression changes between BEAS-2B and HBEC, and between the two LABAs. These observations suggested that pro- inflammatory gene expression changes could explain the known toxicity of chronic, β2- adrenoceptor agonist monotherapy initially reported in patients with asthma in the 1960s. Conversely, the expression of anti-inflammatory genes could help prevent exacerbations. Roflumilast is a phosphodiesterase (PDE) 4 inhibitor approved by the United States Food and Drug Administration as an ‘add-on’ therapy for patients with severe, bronchitic COPD with a history of frequent exacerbations. The second objective was to determine if inhibiting cAMP degradation could augment the expression of the LABA-regulated transcriptome and assess whether this could help explain the clinical activity of roflumilast. In BEAS-2B cells, roflumilast N-oxide (the active metabolite of roflumilast) augmented gene expression changes induced by a submaximal concentration of salmeterol, although the magnitude of the effect was dependent on the gene of interest. Further analysis established that this was because the sensitivity of the salmeterol-regulated transcriptome varied by a factor of 7.5-fold, which could be consistent with gene- dependent differences in regulation downstream of cAMP-dependent protein kinase. The final objective was to examine the pharmacology of GS-5759. This is a novel, bifunctional ligand composed a b2-adrenoceptor agonist linked to an analogue of the PDE4 inhibitor, GSK 256066, by a pent-1-yn-1ylbenzene spacer. Pharmacodynamic studies established that the PDE4 inhibitor pharmacophore produced a 35.5-fold increase in affinity for the b2-adrenoceptor relative to the monofunctional parent compound in the absence of any change in efficacy. However, this was not related to inhibition of PDE4 but to non-allosteric, ‘forced proximity’ binding, which predicts an increased in retention time of a compound near the orthosteric site of the receptor. In the context of obstructive lung diseases LABAs are established bronchodilators. The data presented in this thesis demonstrate that these drugs alone and in combination with a PDE4 inhibitor also have significant genomic actions. The possibility that gene expression changes occur in patients taking inhaled b2-adrenoceptor agonists and have functional consequences should be considered.
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    The Spatial-temporal Contribution of Prostaglandin E2 Pathway to Rat Myometrial Contractile Function during Pregnancy and Labour
    (2020-09-24) Liwa, Anthony Cuthbert; Slater, Donna M.; Cole, William C.; Davidge, Sandra Thomas; Macdonald, Justin Anthony; Giembycz, Mark A.; Roth, Sheldon H.
    A key function of the uterus during pregnancy is to accommodate the growing fetus in a relatively quiescent environment. Towards the end of pregnancy, the uterus transforms to an active organ capable of generating forceful contractions during labour. Prostaglandins are thought to play a key role in the parturition process, with prostaglandin E2 (PGE2) being produced by uterine tissues, and having possible roles including stimulation of cervical ripening and uterine contractions. PGE2 exhibits diverse physiological actions, most probably depending on expression levels of selective PGE2 (EP) receptor subtypes; in the uterus EP1 and EP3 receptors may stimulate myometrial contraction, whereas EP2 and EP4 receptors may evoke relaxation. There is no consensus concerning their relative importance in regulating uterine function, and whether there are spatial- and/or time-dependent alterations in how PGE2 regulates uterine contractility via changes in PGE2 synthesis and/or EP receptor expression. We have hypothesised that, in myometrial tissue, expression of enzymes involved in PGE2 synthesis and/or the four EP receptors are regulated to provide for region- and time-dependent variations in the contribution of PGE2 to uterine quiescence in pregnancy and contractility in labour. An in vitro tissue bath was utilised to examine myometrial contractility responses to PGE2 and EP antagonists. Real-time polymerase chain reaction and western blotting experiments were used to determine expression of key enzymes involved in PGE2 synthesis plus the EP receptors. Uterine tissues from upper and lower uterine tissues at days 15-21 of gestation and during labour were used for the experiments. Our findings demonstrate the presence of spontaneous contractions in ex vivo uterine tissues at all time points studied. No regional difference in the magnitude of uterine contraction was observed with the exception of upper day 21, in which contractility was greater than in the upper compared to the lower uterus. The addition of PGE2 increased the spontaneous contractions in non-pregnant and day 15-21 pregnant tissues, but not those from rats in active labour. EP3 antagonist (L-798106) did not alter the actions of PGE2, whereas PGE2-evoked contractions of day 18 pregnant, but not in labour myometrium, were enhanced in the presence of EP4 antagonist (ONO-AE3-208). No significant differences in the expression of PGE2 isoenzyme and EP receptor mRNA and protein expression were detected in upper or lower uterine tissues during pregnancy and labour, with the exception of increased COX1 and decreased EP3 mRNA levels in tissues from rats in labour. These results suggest that rat uterine tissues can synthesize PGE2 throughout pregnancy and parturition but based on assessment of its effect on contractility in vitro, PGE2 only stimulates myometrial contractility during gestation and not in labour. PGE2 may therefore play a role in enhancing contractility of non-labour myometrium, but perhaps not in the regulation of myometrial contractility during labour. Alterations in the expression of PGE2 synthesizing enzymes and EP receptors would not appear to be the primary determinant of uterine tissue responsiveness. Furthermore, our results do not support the concept of functional regionality in terms of response to PGE2 of the rat uterus. The exact mechanisms of PGE2 regulation of uterine contractility await further investigation.
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    Transcriptional regulation by glucocorticoids: a comparative transcriptome analysis preceding mechanistic and functional assessments of KLF9 induction
    (2020-09-15) Mostafa, Mahmoud; Newton, Robert; Giembycz, Mark A.; Cobb, Jennifer A.
    Regulation of gene expression by glucocorticoid receptor (GR) is not only central to numerous endocrine processes, but also critical for the control of inflammation by glucocorticoids administered for the treatment of inflammatory diseases, including asthma. Advancing our knowledge of glucocorticoid biology as well as developing GR ligands with improved therapeutic profile are hindered by an inadequate understanding of the mechanisms and impacts of gene regulation by glucocorticoids. Furthermore, owing to their pleiotropic effects on many different target cells, transcriptomic responses to glucocorticoids vary widely between cell types. This confounds the identification of key glucocorticoid-regulated genes. In this thesis, transcriptomic responses to glucocorticoid were compared between A549, BEAS-2B, and primary HBE cells. These are models for airway epithelial cells, which represent a key player in asthma pathogenesis and response to inhaled glucocorticoid therapies. While the variability in glucocorticoid-modulated gene expression between epithelial cell variants was surprisingly high, the genes regulated in common may represent key players in eliciting glucocorticoid effects. Among the genes that were induced in common, transcriptional regulators, such as KLF9, were highly enriched and hypothesized to contribute to later gene expression changes, including repression of inflammatory genes. Mechanistically, ligand-activated GR binds and activates multiple conserved enhancer regions upstream of the KLF9 gene. These include a promoter proximal region that was appeared to be essential for the constitutive expression and glucocorticoid-mediated induction of KLF9. In fact, GR recruitment and transcriptional activation at KLF9 upstream enhancers were highly conserved among cell lines and primary cells, demonstrating reliability of cell lines in mechanistic interrogations of conserved genes. Assessment of KLF9 functions was attempted via transcriptome analysis of CRISPR-edited KLF9 KO lines. While procedural artifacts hindered the identification of transcriptomic impacts of KLF9 induction, candidate gene investigations following overexpression or knockdown of KLF9 suggested a role for the constitutively expressed KLF9 in limiting the expression of AKAP12 and RGS2. Yet, the functional impacts of glucocorticoid-induced KLF9 in pulmonary epithelial cells remain undetermined. Collectively, these results establish a platform for identifying key glucocorticoid-regulated genes in the airways, and guiding the selection of cell line models for in vitro investigations of such genes.