The aim of this thesis was to investigate the effects of antioxidant supplementation and/or exercise training on parameters that influence endothelial cell protection. It is unclear whether injury to the endothelium is a cause or product of atherosclerosis plaque formation. The initial cause of endothelial damage in vivo is unclear and may be caused by oxidative stress as increased levels are seen in atherosclerotic plaques and reactive oxygen species stimulate apoptosis of endothelial cells in vitro. However, large clinical trials using various antioxidant supplements and protocols have failed to provide conclusive evidence for a causal role of oxidative stress. Regular exercise prevents cardiovascular disease (CVD) and improves endothelial function, yet acute exercise also causes oxidative stress. Therefore, the intention of this thesis was to further elucidate the role of oxidative stress in endothelial cell protection by examining the effects of antioxidants and/or exercise on various cytoprotective mechanisms using both in vitro and in vivo models. The two antioxidants used in this thesis, α-lipoic acid and α-tocopherol, were chosen as they are both potent antioxidants and function in complementary roles when administered together. This thesis is divided into seven chapters. The first and last provide a general introduction and summary respectively while the second chapter is a review of the relevant literature. The other four chapters are a series of separate scientific studies designed to address the various aspects of the aim of the thesis.
Chapter 2 is a review of the literature in this field. The first section describes the role of the endothelium in atherosclerosis and the importance of maintaining the structural integrity of this layer to prevent plaque formation. The second section provides an overview of the apoptotic pathway, including anti- and pro-apoptotic proteins and the effects of altered expressions of these proteins on cell viability. The third section describes the actions of the two antioxidants used in this thesis and provides a justification for the use of these compounds. The fourth section examines the effects of increased levels of oxidative stress on endothelial cells. Finally, the fifth section reviews the effects of exercise on endothelial cell protection and is an abbreviated version of the review currently in press in the International Journal of Cardiology
The first experimental study, presented in Chapter 3, investigates the effects of antioxidant supplementation and/or exercise training on apoptotic proteins in endothelial cells. The justification for this study was that atherosclerotic plaques contain apoptotic endothelial cells in addition to increased levels of oxidative stress. While acute exercise induces oxidative stress, regular exercise prevents CVD whereas antioxidant supplementation elicits little or no benefit. To address these issues, male rats received a control or antioxidant-supplemented diet (α-lipoic acid and α-tocopherol1) and were assigned to sedentary or exercise-trained groups for 14 weeks. Expressions of the anti-apoptotic protein Bcl-2 and the pro-apoptotic protein Bax were measured in endothelial cells. Antioxidant supplementation increased Bcl-2 but not Bax compared to non-supplemented animals. Exercise training had no effect on either protein when undertaken alone or combined with antioxidant supplementation. These results are novel and indicate that α-lipoic acid and α-tocopherol supplementation may provide greater protection against apoptosis than exercise training. These data have been accepted for publication in the Journal of Molecular and Cellular Cardiology.
The second experimental study, presented in Chapter 4, examines the effects of α-lipoic acid and α-tocopherol on protective pathways in endothelial cells exposed to oxidative and non-oxidative stresses in vitro. This study was designed to investigate the mechanisms responsible for the findings in the previous study and to elucidate whether the effects found in vivo were a result of the antioxidant or non-antioxidant properties of each compound. To achieve this, endothelial cells were incubated with α-lipoic acid and/or α-tocopherol prior to incubation with oxidative or non-oxidative stress. Expressions of various cytoprotective proteins were measured in addition to activity of the proteolytic enzyme caspase-3. α-lipoic acid increased caspase-3 activity in unstressed cells and decreased the expression of an activated cell cycle protein in cells exposed to the non-oxidative stress in a dose-dependent manner, α-tocopherol had no effect on the parameters measured. These findings provide an insight into the use of novel antioxidant combinations to enhance endothelial cell survival and may indicate a pro-apoptotic effect of α-lipoic acid at high concentrations. These data have been accepted for publication in Apoptosis.
The third experimental study, outlined in Chapter 5, contains a sub-study from the major intervention study described in Chapter 3 and examines the effects of α-lipoic acid and α-tocopherol supplementation and/or endurance training on the antioxidant defences of erythrocytes. The rationale for performing this study was that both intense exercise and oxidative stress cause injury to erythrocytes, which in turn, creates a procoagulant environment and may promote endothelial cell damage. Young male rats received a control or antioxidant-supplemented diet (α-lipoic acid and α-tocopherol) and were assigned to sedentary or exercise-trained groups for 14 weeks. Erythrocyte activities of endogenous antioxidant enzymes were measured in addition to plasma oxidative stress. Antioxidant supplementation and exercise training alone caused no change in antioxidant enzyme activities. However, when combined, the activities of some but not all enzymes were increased. There were no changes in plasma oxidative stress. These data suggest that the combination of antioxidant supplementation and exercise training is more effective in altering erythrocyte antioxidant enzyme defences than either intervention alone and have been submitted to Redox Report.
The last experimental study, presented in Chapter 6, investigates the effects of combined supplementation of α-lipoic acid and α-tocopherol on bleeding tendency as a procoagulant environment causes increased endothelial cell damage. Male rats received a control or antioxidant-supplemented diet (α-lipoic acid and α-tocopherol) for 14 weeks. Antioxidant supplemented animals exhibited a significantly prolonged bleeding time compared to those that consumed the control diet. There was no change in plasma oxidative stress. The findings of this study suggest that α-lipoic acid and α-tocopherol supplementation may benefit individuals with CVD who typically exhibit elevated levels of blood coagulation. These data have been accepted for publication in Clinical and Applied Thrombosis - Hemostasis.
Chapter 7 summarises the main findings of this thesis and discusses the implications of these findings in the context of known and potential interventions for the prevention of CVD.
In summary, the data presented in this thesis suggest a beneficial role for the combination of α-lipoic acid and α-tocopherol in various pathways associated with endothelial cell protection in vivo but a potentially apoptotic effect of α-lipoic acid at high concentrations. In addition, exercise training alone did not increase any of the protective parameters measured, which suggests that the beneficial effects of regular exercise are mediated by pathways other than those examined in this thesis.