Saudi Cultural Missions Theses & Dissertations
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Item Restricted Elucidation of the Role of Methylarginine Metabolism in Regulation of Nitric Oxide Production and Inflammation(University of Glasgow, 2024) Alshuwayer, Noha Ali S; Leiper, James; Mercer, JohnAtherosclerosis is a major global health issue, and inflammation is important in its pathogenesis. Many atherosclerosis risk factors lead to reduced nitric oxide (NO) bioavailability. Asymmetric dimethylarginine (ADMA), an independent cardiovascular disease risk factor and NO synthase inhibitor, is metabolised by dimethylarginine dimethylaminohydrolase (DDAH). DDAH2 is the isoform present in the immune system. A deeper understanding of ADMA metabolism could help reveal new therapies for atherosclerosis. However, it is debated if DDAH2 hydrolyses ADMA. There is evidence that DDAH2 has NO-independent cellular functions, and research in our group showed that DDAH2 regulates macrophage functions. This thesis initially aimed to investigate the role of DDAH2 in regulating inflammation in atherosclerosis models. However, this was derailed by limitations imposed by the Covid-19 pandemic. Therefore, models of inflammation were used. Genes and mechanisms associated with inflammation and atherosclerosis were investigated. RAW 264.7 murine macrophage cell line and bone marrow-derived macrophages (BMDM) were validated for suitability to study the DDAH-ADMA-NOS pathway. To better understand the functions of DDAH2, a macrophage-specific Ddah2 null mouse model was re-derived and validated. RNA sequencing data previously generated by our group from peritoneal macrophages of the same model was re-analysed and revealed almost 5,000 genes to be DDAH-dependent and required for normal immune response. More than 200 Reactome pathways appeared enriched, with apoptosis being the most enriched. The in silico data was validated in vitro in DDAH2-knockout peritoneal macrophages from the macrophage-specific Ddah2 null mouse model. Inferred hypotheses were investigated in DDAH2-Knockout BMDMs from the macrophage-specific Ddah2 null mouse model with confirmatory studies on C57BL/6J BMDMs using ADMA. The in vitro analysis in the BMDMs showed no conclusive evidence supporting the in silico data that DDAH2 regulates the investigated genes (except Il17a), nor did ADMA alter the gene response to LPS. Il17a was shown by the in silico analysis to be regulated by DDAH2 and was validated in vitro in peritoneal macrophages by both RT-qPCR and ELISA. Given the significant role of IL17A in inflammation and its existing use in treating systemic inflammatory conditions such as psoriasis, this thesis proposes DDAH2 as a potential therapeutic target for inflammatory diseases in general and atherosclerosis in particular.15 0Item Restricted Systemic Inflammation, Arterial Stiffness, and Vascular Endothelial Dysfunction in Patients with Chronic Lung Disease(University of Dundee, 2024-05-22) Arafah, Abdullah M.; Khan, FaiselChronic lung disease (CLD) is considered a heterogeneous, complex, and multicomponent condition. Types of CLD include bronchiectasis, chronic obstructive pulmonary disease (COPD), and asthma. Cardiovascular events and peripheral vascular disease are highly prevalent among patients who are known to have CLD. It is increasingly acknowledged that cardiovascular comorbidities contribute to the disease’s severity. The underlying mechanisms that link CLD and cardiovascular disease (CVD) are inadequately understood. Systemic inflammation is a key component that could describe the link between CLD and CVD. Changes in vascular endothelial function accompany the increased cardiovascular events in CLD. Atherosclerosis and calcification of macrovascular and microvascular lead to further decrease vascular compliance. These structural changes in the vascular wall contribute to increased arterial stiffness observed in patients with CLD. Endothelial dysfunction and arterial stiffness are early signs of vascular disease and the development of cardiovascular events. Chronic systemic inflammation plays a vital role in linking CLD to the development of endothelial dysfunction and arterial stiffness. Therefore, this study aims to investigate the association between CLD and CVD. To successfully achieve the aims of this project, four work packages were employed, including a systematic review, a retrospective study, a Mendelian randomisation study, and a cross-section study involving the BRIDGE study. The systematic review study related to arterial stiffness in patients with CLD using various pulse wave velocity (PWV) methods, which assessed and summarised the outcomes of all relevant studies regarding the link between CLD and CVD. The retrospective study analysed anonymous data from the SUMMIT study to assess the vascular function in patients with CLD in the presence of CVD and type 2 diabetes mellitus, and shows a significantly greater PWV; p-value = 0.015 and carotid intima-media thickness (CIMT) in the CLD patients; p-value = 0.001. The Mendelian randomisation study investigated potential genetic causal links between CLD and arterial stiffness, which shows a significant association; p-value = 0.021. The cross-section study and BRIDGE study utilised biomarkers to determine if there are shared pathways that contribute to the development and progression of CLD and CVD, and shows significant differences in PWV, and microvascular function; p-value = 0.001, blood biomarkers include adiponectin, VCAM-1, GDF-15, coagulation factor III, syndecan-1, and matrix metalloproteinase (MMP-10); p-value = 0.001. In conclusion, this study revealed a significant association between CLD and CVD. Therefore, monitoring CVD risk, including assessment of endothelial dysfunction and arterial stiffness, in patients with CLD might be helpful for risk stratification and for identifying future CVD pathologies and disease progression. This emphasises the need to identify and manage comorbid CLD and CVD to target new or existing therapeutic approaches to control systemic inflammation and improve overall lung and cardiovascular health.9 0Item Restricted Anti-atherogenic actions of hydroxytyrosol(Saudi Digital Library, 2023-11-02) Alotibi, Reem Mansour; Ramji, DipakIntroduction: Atherosclerosis is a chronic inflammatory disorder characterised by the accumulation of lipids in the arterial wall, and is considered to be a major contributor to cardiovascular disease (CVD). Worldwide, CVD is responsible for a third of all deaths. Current pharmacological therapeutic agents for CVD, such as statin therapy, are not fully effective and have considerable residual risk for the disease. Alternative agents for the prevention and treatment of the disease are therefore required. Hydroxytyrosol (HT) is a polyphenol compound found mainly in olive oil. HT has been reported to prevent CVD predominantly due to its antioxidant effects. HT is the only polyphenol recognised by the European Food Safety Authority as a protector against low-density lipoprotein-mediated oxidative damage. Previous investigations carried out in the host laboratory showed that HT altered multiple atherosclerosis-associated risk factors in wild-type mice fed a high-fat diet (HFD) for 3 weeks, as well as various anti-inflammatory and anti-atherogenic actions on human monocytes/macrophages in vitro. Unfortunately, the actions of HT in atherosclerosis in vivo are poorly understood. The aim of this study therefore was to elucidate its effects on atherosclerosis progression and regression in a mouse model system. Methods: In order to study the development of atherosclerosis in vivo, 8-week-old male or female low-density lipoprotein receptor-deficient (ldlr-/-) mice were given HFD alone or in combination with 10 mg/kg/day of HT for 12 weeks. For regression studies, the mice were fed a HFD for 12 weeks to promote the formation of established lesions, and then switched to normal chow diet (NCD) alone or in combination with HT. The two procedures were then followed up by an in-depth investigations of atherosclerosis- associated risk factors and the plaques that formed in the aortic root. RNA-sequencing and bioinformatic analyses was used to assess changes in gene expression and associated pathways in the thoracic aorta. The liver was also analysed in relation to non-alcoholic fatty liver disease (NAFLD) that is often associated with atherosclerosis and extended via the use of an in vitro hepatoma HepG2 cell culture model system. Results: As part of the progression study, female ldlr-/- mice that had received HT supplemented HFD for 12 weeks had attenuated weight gain, plaque size in the aortic root and neutrophil content in the peripheral blood, while male ldlr-/- mice had attenuated occlusion and T cells in the peripheral blood. In both cases, there was reduced plaque inflammation and improved plasma lipid profile and plaque stability. As part of the regression studies on male ldlr-/- mice, intervention with HT combined with NCD reversed hepatic injury and enhanced plaque stability (to a greater extent than NCD intervention alone). Conclusions: These findings provide support for the anti-atherogenic actions of HT as well as its possible use as an alternative nutraceutical agent for preventing the development of atherosclerosis. This is potentially possible due to the lack of adverse effects as well as the relatively low cost in comparison to that of typical pharmacological treatments. Additional research is necessary in order to determine the mechanisms that are responsible for these favourable anti-atherogenic and other beneficial changes.16 0Item Restricted Molecular simulation of discoidal HDL lipoprotein particles(Saudi Digital Library, 2023-05-20) Siddiq, Hind Ahmed Ali; Anwar, JamshedHigh-density lipoprotein (HDL) has been determined to play an essential role as an atheroprotective in atherosclerosis – the thickening and hardening of arteries. HDL particles are important delivery vehicles in the reverse cholesterol transport (RCT) pathway wherein excess cholesterol is removed from peripheral tissues and transported to the liver for redistribution or removal from the body. The particles undergo a sequential development from being lipid-free comprising only the protein Apolipoprotein A-I (ApoA-I), to discoidal HDL, and finally forming spherical HDL. ApoA-I appears to stabilise the particles as well as influencing the activity of the enzymes ABCA and LCAT, and recognition of the HDL particles by the scavenger receptors in the liver. Apolipoprotein Zaragoza (ApoA-I Z) and Apolipoprotein Milano (ApoA-I M) are natural mutations of Apolipoprotein A-I. Carriers of these mutations have a lower level of HDL-cholesterol and yet a low risk of cardiovascular disease. Discoidal HDL (dHDL) particles take the form of a lipid disk stabilised by two ApoA-I molecules surrounding the edges. Here we have investigated the structure and physical characterisation of dHDL using molecular dynamics simulations. Being soft matter, the structure of dHDL and its self-assembly is still not entirely resolved. We have employed molecular dynamics simulation (using both atomistic and coarse-grained models), including enhanced sampling methods (temperature replica exchange, Hamiltonian replica exchange, Jarzynski’s non-equilibrium, metadynamic, and umbrella sampling approach) to explore the interactions of ApoA-I proteins in isolation, self-assembly of dHDL particles, and the free energy surface for the chain-chain interaction of ApoA-I proteins within the dHDL complex. Simulations have been carried out on the wild type and the ApoA-I mutants ApoA-I Z and ApoA-I M. The simulations of ApoA-I monomers and dimers in water solutions indicate that wild-type ApoA-I is more stable and more rapid in changing conformation than ApoA-I Z and ApoA-I M mutants. With respect to the self-assembly of dHDL, the standard MD simulations do not converge to equilibrium as the emergent structure becomes kinetically locked. The use of thermal replica exchange is also ineffective, being inefficient for large systems. Hamiltonian exchange wherein the ApoA-I protein chains are gradually transformed to generate a soft-core potential was observed to be more effective and indeed generated the double-belt structure with the experimentally known helix 5/2 registration. The free energy surface for the ApoA-I – ApoA-I pair interaction/registry in dHDL was inaccessible by Jarzynsky’s non-equilibrium approach and metadynamics but could be characterised using umbrella sampling. The surface suggests that H5/2 is the most stable form in both ApoA-1 and ApoA-I Z in dHDL.14 0Item Restricted Investigation of the actions of Resveratrol on atherosclerosis development using in vitro and in vivo model systems(2023-07-05) Alahmadi, Alaa; Ramji, DipakBackground: Atherosclerosis continues to be a major contributor to cardiovascular disease (CVD), which is one of the leading causes of morbidity and mortality globally. The current pharmacological strategies targeting hyperlipidaemia, such as statins, have shown limited effectiveness in combating cardiovascular risk and have other issues. Considering the limitations associated with statins and other pharmacotherapies, alternative avenues need to be sought. Nutraceuticals, such as resveratrol (RSV), have been highlighted as potential candidates for atherosclerosis prevention and treatment due to its demonstrated ability to modify several atherogenic risk factors and its excellent safety profile. Unfortunately, its effects on the full range of atherosclerotic processes along with the underlying molecular mechanisms are not fully understood. Therefore, the main aims of this study were to investigate the effects of RSV on key cellular processes associated with atherosclerosis development in vitro and to elucidate its effects on atherosclerosis progression in a mouse model system. Methods: Various in vitro assays were carried out using different cell lines and primary cell cultures to investigate the effect of RSV treatment on a range of key cellular processes associated with atherosclerosis development. Furthermore, to investigate the effect of RSV on atherosclerotic plaque progression in vivo, 8-week-old male low-density lipoprotein receptor-deficient (LDLR-/-) mice were fed either a high-fat diet (HFD) or HFD-supplemented with RSV for 12 weeks. This was followed by a comprehensive analysis of risk factors associated with disease initiation and progression, such as plasma lipid profile and staining of resident cells (e.g. macrophages, T-cells and smooth muscle cells (SMCs)) in the plaque. Results: RSV attenuated several key atherosclerosis-associated processes in vitro, such as monocyte migration towards monocyte chemoattractant protein-1(MCP-1), reactive oxygen species (ROS) production in all investigated cell types, and foam cell formation. Furthermore, RSV reduced human aortic smooth muscle cells (HASMCs) invasion and enhanced their proliferation, and exhibited anti-inflammatory actions. Regarding in vivo progression study, mice that received RSV-supplemented HFD for 12 weeks showed an improvement in plasma lipid profile, attenuation of plaque inflammation and enhancement markers of plaque stability. Furthermore, additional investigation on liver samples showed that RSV has the ability to reduce steatosis. Conclusion: The findings from this study provide valuable insights into the anti-atherogenic actions of RSV and implicate it as a potential nutraceutical candidate that could be used globally as a part of ongoing atherosclerotic CVD prevention and management strategies due to the lack of undesirable side effects and the comparatively low cost compared to standard pharmacological medications. The potential of RSV should be investigated however in large clinical trials.48 0