This project is based upon the hypothesis that the cellular redox status plays a critical role in preventing endothelial cell dysfunction related to atherogenesis. Oxidative stress (an imbalance of oxidants and anti- oxidants in favor of the former) has been implicated as an etiologic factor in atherogenesis, both through the oxidation of low-density lipoprotein (LDL) and its sequelae and the stimulation of monocyte-endothelial interactions. While previous studies have evaluated the effects of LDL- associated and extracellular anti-oxidants on these pro-atherogenic, redox- sensitive processes, little is known about the role of intracellular antioxidants. Vitamin C and glutathione are accumulated and synthesized, respectively, in millimoral concentrations by human cells, and play a control role in the cellular antioxidant defense system, and thus in cellular integrity and function in the face of an oxidant challenge. Therefore, the overall objective of this project is to identify the role of cellular vitamin C and glutathione status in endothelial activation in vitro and in vivo. The in vitro system will be cultured human aortic endothelial cells (HAEC), and the in vivo system guinea pigs, which, like humans, cannot synthesize ascorbic acid.
The first aim i s to characterize and manipulate the vitamin and glutathione status of HAEC. The effects of vitamin C loading on cellular glutathione status, and conversely the effects of manipulating cellular glutathione status on vitamin C status will be studied.
Aim 2 will identify the role of cellular redox status in HAEC- mediated LDL oxidation. Underlying mechanisms will be explored by measuring cellular superoxide and thiol production, and by inhibiting nitric oxide synthesis. In the third aim we will determine the role of cellular vitamin C and glutathione status in HAEC activation, i.e. expression of cellular adhesion molecules (CAMs) and monocyte chemotactic protein-1 (MCP-1) and monocyte chemotactic protein-1 (MCP-1) and monocyte adhesion. Underlying mechanisms will be explored by studying the nuclear translocation of the redox-sensitive transcription factor NfkappaB and the involvement of nitric oxide.
In aim 4, the in vivo relevance of the HAEC studies will be investigated. The vitamin C and/or glutathione status of guinea pigs fed a standard or 0.3% cholesterol-containing diet will be manipulated, and circulating levels of MCP-1, CAMs and autoantibodies to oxidized LDL will be measured, as well as aortic CAM and MCP-1 expression, NfkappaB activation, monocyte adhesion, and the extent of atherosclerotic lesion formation. This information will provide a better understanding of the mechanisms by which antioxidants modify atherogenesis.
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