Effect of selenomethionine supplementation on the inflammatory response associated with atherosclerosis development

Abstract

Atherosclerosis is the major cause of cardiovascular disease and features the deposition of fatty material and cholesterol within the arterial wall. The fatty steaks progressively develop into atheroma and vulnerable plaques, increasing the risk of clinical manifestations, such as stroke and heart attack. Atherosclerosis is a chronic inflammatory disease of the vasculature characterised by the ongoing infiltration of a variety of immune cells, in particular neutrophils and macrophages, at sites of damage within the vessel wall. The activation of these inflammatory cells function to both eliminate cell debris and promote beneficial vessel structural remodelling, but also contribute to lesion formation and plaque progression. As a part of their inflammatory repertoire, neutrophils release extracellular traps, termed “NETs”, aimed to confine infection, signal to and promote macrophage infiltration and activation. Besides their immune defence role, NETs have been implicated in atherosclerosis, with elevated NET levels shown to be associated with plaque formation and vascular damage during the disease progression. It has been proven that activated macrophages can also release extracellular traps (METs) in response to inflammatory stimulation, which also participates in atherogenesis. Activation of infiltrated neutrophils within vessel intima produces excess reactive oxidant species (ROS) and results in the release of the enzyme myeloperoxidase (MPO). This event exacerbates oxidant generation and contributes to lesion development by accelerating the formation of lipid-filled foam cells, which increases overall oxidative stress. Monocyte-derived macrophages are recruited by a series of inflammatory events to the sites of damages within the vessel wall and atherosclerotic lesions. These cells further differentiate into two macrophage phenotypes: pro-inflammatory M1 macrophages and alternatively-activated M2 macrophages, each with distinct effects on atherosclerosis development. How the balance between populations of M1 and M2 macrophages relates to atherosclerotic disease development is complex and remains unclear and complicated. Chapter 3 of this thesis examines the ability of M1 and M2 differentiated human monocyte derived macrophages (HMDM) to produce METs in response to a variety of inflammatory stimuli as well as the oxidant HOCl. The polarisation of the macrophages prior to HOCl exposure revealed a greater propensity for inflammatory M1 macrophages to produce METs, whereas M2 macrophages were less susceptible to HOCl insult. Similarly, M1 macrophages also produced ETs on exposure to phorbol myristate acetate (PMA), interleukin- 8 (IL-8) and tumour necrosis factor α (TNFα). Importantly, this Chapter highlighted M1 macrophages elicit MET formation independent of histone citrullination and is not affected by PAD activation. The results from this Chapter suggest that the common NET inhibitors, such as Cl-amidine, may not be a good candidate in MET prevention. Given that the critical role of inflammatory oxidative stress in MET formation and atherosclerosis development, antioxidants are considered as a potential therapeutic avenue. The dietary supplement selenomethionine (SeMet) as a form of antioxidant therapy was used in this thesis to investigate the therapeutic effect of selenium supplements. Selenium is an essential trace element that functions in the regulation of the immune response by both bolstering the endogenous thioredoxin and glutathione antioxidant defence systems and by directly scavenging damaging oxidant species. SeMet is an organic form of selenium, which has better bioavailability compared to other selenium sources. Chapter 4 investigates the efficacy of SeMet supplementation within an in vitro cell culture system in alternating the macrophage differentiation and modulating ET generation from pro-inflammatory stimulated macrophages and neutrophils. Although SeMet showed the potential effect on the modulation of inflammatory cytokine expression within HMDM during the initial stages of pro-inflammatory stimulated polarisation of macrophages, it had no subsequent effect on the extent of MET formation from M1 polarised HMDM. In contrast, SeMet had inhibitory effects on the NET formation from neutrophils isolated from patients with acute coronary symptom (ACS). These latter data indicate that SeMet supplementation has high potential in the modulation of the immune response during chronic disease pathogenesis. In order to further explore the role of SeMet supplementation in the prevention of atherosclerosis, studies were designed in Chapter 5 to examine its capacity in reduction of lesion burden development and the modulation of inflammatory responses during pathogenesis of the disease through enhancing antioxidant ability within an apolipoprotein E deficient (ApoE-/-) mouse model. The dietary supplementation in SeMet (2 mg/kg) increased the tissue concentration of Se, and the expression and activity of glutathione peroxidase, compared to non-supplemented controls. Additionally, SeMet significantly reduced atherosclerotic plaque formation in mouse aorta, resulted in a more stable lesion phenotype and improved vessel function. Most importantly, SeMet supplemented mice showed a slight decrease in pro-inflammatory response accompany with modulated enhanced antioxidant capacity and improved arterial endothelial function. In summary, the results of this thesis contribute to the knowledge regarding macrophages and neutrophils in mediating extracellular trap formation, with the results suggesting this may be relevant in pathological conditions characterised by chronic inflammation. Furthermore, this thesis broadened knowledge regarding the therapeutic efficacy of selenium supplements, particularly in the form of SeMet in the prevention of atherosclerosis. Taken together, this work highlights the potential beneficial effect of SeMet supplementation as a therapeutic strategy for atherosclerosis.