Saudi Cultural Missions Theses & Dissertations
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Item Embargo Investigating the ability of statin-mediated ERK5 activation to protect cardiac microvascular endothelial cells and cardiomyocytes against the adverse effects of doxorubicin.(Saudi Digital Library, 0025-04-10) Almaghrabi, Shrouq; Cross, MichaelDoxorubicin is a widely used chemotherapeutic agent known for its efficacy against various cancers; however, its clinical utility is limited by cardiotoxic side effects that involve both endothelial dysfunction and direct cardiomyocyte damage. This doctoral research investigates the protective role of statins—specifically simvastatin and atorvastatin—in mitigating doxorubicin-induced toxicity through activation of the ERK5 signaling pathway. Using human and mouse cardiac microvascular endothelial cells (HCMECs and MCMECs), as well as human cardiomyocyte-like AC16 cells, the study demonstrates that statins restore ERK5 phosphorylation and upregulate vasoprotective transcription factors KLF2 and KLF4. These molecular changes were associated with decreased inflammatory marker expression, preserved tight junction integrity, and improved cellular viability. The findings highlight a novel therapeutic potential for statins in preserving cardiovascular health during chemotherapy and underscore the significance of ERK5 as a molecular target for cardioprotection.29 0Item Restricted Therapeutic Interventions to Target Fetoplacental Vascular Dysregulation in Fetal Growth Restriction(The University of Manchester, 2024-06-21) Almohammadi, Lujain; Brownbill, PaulFetal growth restriction (FGR) affects 3-7% of pregnancies and is a condition where the fetus fails to reach its genetic growth potential, often due to placental dysfunction. This can lead to stillbirth and in survivors increased risk of morbidity and ill health in later life. FGR is associated with reduced fetoplacental blood flow and elevated placental oxidative stress. Nitric oxide (NO) plays a crucial role in increasing blood flow, but in FGR, NO bioavailability and endothelial nitric oxide synthase (eNOS)-dependent vasodilation are impaired. Shear stress, which is the frictional force between the flowing blood and the endothelium, is a powerful stimulator of NO synthesis and vasodilation in several vascular beds. Flow mediated vasodilation (FMVD) is dysregulated in FGR. NOs is mainly produced by eNOS, using L-arginine as a substrate and various cofactors including tetrahydrobiopterin (BH4). Arginase-2 (ARG-2) competes with eNOS for L-arginine, thereby inhibiting NOS-dependent relaxation. BH4 is an essential cofactor for eNOS enzyme activity; without BH4, eNOS creates superoxide instead of NO. In this thesis, the focus was on investigating the effectiveness of (S-(2-boronoethyl)-L-cysteine) (BEC) as a potent and specific arginase inhibitor, along with BH4 both alone and in combination. Here two hypotheses were tested (a) Arginase inhibition by BEC, alone and with BH4, would augment NO production by the fetoplacental endothelium. (b) Arginase inhibition by BEC will reduce vascular tone and improve FMVD in normal pregnancy (NP) and FGR. The aim was to identify potential therapeutic strategies that could increase NO bioavailability, lower vascular resistance and improve FMVD in FGR. Two placental preparations were used to test the hypotheses (a) primary cultures of human placental arterial endothelial cells (HPAECs) (b) dual perfusion of the human placental cotyledon ex vivo. Experiments were performed on placentas from normal pregnancy (defined as the delivery of a singleton infant at term with an individualised birthweight ratio (IBR) between the 20th and 80th centiles) and FGR (defined as an infant with an IBR below the 3rd centile). Using HPAECs maintained in static culture conditions, the effects of BEC, BH4 and BEC+BH4 were investigated on the expression of cell stress related proteins (Proteome Profiler Human Cell Stress Array), the phosphorylation of protein kinases in the eNOS pathway (Human Phosphokinase Array) and on arginase activity (MAK384, Sigma-Aldrich kit). The effects of BEC and BH4 on the ratio of phospho-eNOS (S1177) to total eNOS, and on NO production (nitrite measured in culture medium after 48hr treatment using the Greiss reaction) were assessed in static conditions and in response to shear stress of 20dyn.cm2 (cells maintained under flow: Ibidi fluidic units). The effect of BEC on the fetoplacental vasculature of FGR was studied using the dual placental perfusion preparation. Baseline fetal inflow hydrostatic pressure (FIHP; a measure of fetoplacental vascular resistance), and the reduction in FIHP in response to incremental increases in flow rate (FMVD), were measured in NP and FGR. Initial studies to assess potential concentration-dependent effects of BEC and BH4 on HPAEC viability, showed that metabolism (MTT assay) was unaffected by BEC (0.125 µM -1250µM) but reduced by BH4 (at 0.2µM -20µM) (n=6 placentas: NP). BEC (12.5µM) reduced the expression of 15 pro-survival proteins, necessary for the regulation of cellular oxidative stress (expression <40% of control; pooled cell lysate from 3 placentas; NP); this effect was not reversed by BH4 (20 µM). BH4 alone increased the expression of proteins involved in cellular growth, angiogenesis, proliferation, activation of eNOS, regulation of inflammatory responses, and protection from reactive oxygen species (ROS). In general, neither BEC nor BH4 increased the expression of phosphorylated proteins involved in eNOS signalling. The ratio of active eNOS to total eNOS was also unaffected by BEC and BH4 in static culture conditions or under flow in NP (n=3-6) or FGR (n=2). Neither BEC nor BH4 increased NO production by HPAECs of NP (n=3-6) or FGR (n=2). However, BEC and BH4 failed to inhibit arginase enzyme activity in NP (n=6) or FGR (n=2). There was no evidence that BEC could lower fetoplacental vascular resistance or increase FMVD upon ex vivo placental perfusion in normal pregnancy (n=5). In the current study, reduced FMVD was not evident in FGR and BEC did not affect FIHP or FMVD (n=3) The limited number of samples used in this study precludes statistical analyses of some of the data and definitive conclusions cannot yet be made. The indication from the preliminary findings, that BEC failed to activate eNOS or stimulate NO production by HPAECs, could be related to its failure to inhibit arginase activity and/or its effect to lower the expression of anti-oxidant proteins. BH4 reduced cell metabolism but in general, increased the expression of proteins that have a cell protective effect. BH4 but did not elevate NO production alone or in concert with BEC. There was no evidence of the ability of the fetoplacental circulation to elicit a vasodilatory response upon perfusion with BEC arginase inhibitor in NP or FGR, arginase activity was not assessed in this preparation. Based on the preliminary findings, it seems that BEC might not be an effective therapeutic intervention for improving fetoplacental blood flow in cases of FGR. It is possible that BEC could increase oxidative stress through off-target actions, which could explain its lack of effect. Further studies are needed to determine whether BH4 could be a viable strategy for enhancing the ability of placental endothelial cells (ECs) to generate NO in FGR and reduce vascular resistance to improve blood flow.19 0Item Restricted The Development of the Lung Metastatic Niche in Murine Breast Cancer(University of Oxford, 2024-05-15) Lin, Salwa; Harris, Adrian L.; Banham, Alison H.; De Val, SarahCancer metastasis causes 90% of cancer deaths. Patients with secondary lung metastases often show only modest responses to chemotherapy, immunotherapy, and anti-angiogenic therapy, and can develop resistance. Remodelling of the metastatic niche and the use of alternative, non-angiogenic, vascularisation pathways such as vessel co-option are thought to contribute to resistance to therapies. However, there is only limited knowledge about the molecular mechanism underlying metastatic niche formation, and the expression profile of genes driving tumour vessel co-option is poorly understood. In part, this is due to the paucity of in vitro and in vivo models that accurately recapitulate the metastatic niche in human cancers. Here, I developed a preclinical E0771 breast cancer (BC) lung metastasis mouse model with which I investigated tumour vessel development and the metastatic niche. First, MRI imaging and histological assessments were used to study vessel growth patterns, suggesting the predominant use of vessel co-option by metastases at an early stage of development, while the induction of angiogenesis was seen during the progression to subpleural metastases. Next, I combined metastatic niche labelling with single-cell RNA sequencing (scRNA-seq) to study the transcriptome of the stroma during metastatic progression. This analysis detected a shift in endothelial cell (EC) subtypes between early and late metastatic timepoints. Interestingly, co-opted capillaries were found to enhance their OXPHOS-related genes and downregulate genes linked to tight junctions, suggesting a hyperpermeability response that may facilitate tumour cell extravasation. Further analysis of this scRNA-seq data also identified cancer-associated mesenchymal cells (CAMCs) in the late metastatic niche, which acted as the primary source of the pro-angiogenic factor VEGFA. Lineage tracing analysis using Wt1-CreERT2;tdTomato reporter mice also examined whether pleural mesothelial cells exhibited pro-metastatic capacity, but suggested that they had little capacity, migrating only underneath the subpleura and few co-expressed CAMC markers. This finding was supported by the scRNA-seq data, which suggested that pleural mesothelial cells partially contribute to the growth of the subpleural metastases but were not the primary origin of the CAMC subtypes. In conclusion, analysis of this preclinical lung metastasis model has enabled me to demonstrate the temporal and spatial heterogeneity present within the metastatic niche and distal cells, and to interrogate the gene expression patterns involved in vessel co-opting and angiogenic metastases. This data could provide important information when selecting vascular drugs to target early vs late metastasis, whilst this extensive transcriptome dataset of lung stromal cells in the metastatic niche and distal regions can provide a basis for further research into the mechanisms involved.21 0Item Restricted Investigation of the role of nicotinic acetylcholine receptors in the control of vascular tone(Saudi Digital Library, 2023) Alkhammash, Abdullah; Lever, RebeccaThe endothelium is a thin monolayer of cells that lines the interior surface of all blood vessels and plays several important physiological roles, including in vascular tone modulation via endothelium-dependent relaxation mechanisms. Cholinergic agonists mediate endothelium-dependent relaxation by activating muscarinic acetylcholine receptors (mAChRs) on endothelial cells (ECs). Activating mAChRs releases nitric oxide (NO) and prostacyclin (PGI2 ) and facilitates endothelium-dependent hyperpolarisation (EDH); subsequently, relaxation of adjacent smooth muscle cells (SMCs) occurs. ECs express mAChRs and nicotinic acetylcholine receptors (nAChRs). While it has been established that M 3 mAChRs mediate endothelium-dependent relaxation, the functional roles and activities of nAChRs in regulating vascular tone are still unclear. Alpha 7 nAChRs (α7 nAChRs), which are expressed more widely in non-neuronal cells, have been shown to play an essential role in several physiological and pathophysiological conditions. The present work investigated the role that α7 nAChRs play in resistance arteries, particularly in endothelium-dependent relaxation. Wire (tension) myography was used to study pharmacological responses in rat mesenteric arteries to investigate the relative effects of cholinergic receptors in mediating functional responses to acetylcholine (ACh). Phenylephrine (PE) or U46619 was applied to precontracted arterial segments, which were relaxed using cholinergic agonists in the presence or absence of α7 nAChR antagonists. Nitrite (NO2 - ) and PGI2 were also added to conditioned media to investigate the response of cultured ECs to cholinergic receptor agonists and antagonists. The results show that selective α7 nAChR antagonists enhance ACh-induced endothelium-dependent relaxation in rat mesenteric arteries. In summary, α7 nAChRs act in opposition to mAChRs in response to agonists such as ACh, and these receptors may therefore play a functional role in controlling vascular tone.123 0