This project is based upon the hypothesis that intracellular antioxidants can prevent endothelial dysfunction related to the initiation of atherosclerosis. Oxidative stress (an imbalance between oxidants and antioxidants in favor of the former) has been implicated as an etiologic factor in human atherosclerosis, both through the oxidative modification of low-density lipoprotein (LDL) and the stimulation of monocyte-endothelial interactions. While our and other investigators' previous studies have evaluated the effects of LDL-associated and extracellular antioxidants on these pro-atherogenic, redox-sensitive processes, little is known about the role of intracellular antioxidants. Vitamin C and glutathione are accumulated and synthesized by human cells, respectively, and play a central role in cellular antioxidant defenses, and thus cellular integrity and survival. Therefore, the overall objective of this proposal is to identify the role of intracellular vitamin C and glutathione in endothelial cell-mediated LDL oxidation and endothelial dysfunction. The experimental model will be cultured human aortic endothelial cells (HAECs).
The first aim i s to characterize athe transport kinetics and intracellular metabolism of vitamin C by these cells. The interrelationships of intracellular vitamin C and glutathione will be studied by examining the effects of cellular vitamin C loading on the levels and redox status of intracellular glutathione, and conversely the effects of manipulating cellular glutathione status on dehydroascorbic acid reduction, an important step in vitamin C metabolism. Next, we will identify the role of intracellular vitamin C and glutathione in HAEC- mediated LDL oxidation. Cellular superoxide generation, metal ion reduction, and thiol production will be studied as possible mechanisms for any observed effects. Finally, in the third aim we will determine the role of cellular vitamin C and glutathione status in HAEC dysfunction as manifested by increased monocyte adhesion. Possible mechanisms will be explored by studying expression of vascular cell adhesion molecule-1 (VCAM- 1) and intercellular adhesion molecule-1 (ICAM-1), and activation and nuclear translocation of the redox-sensitive transcription factor nuclear factor kappaB (NFkappaB). This information will provide a better understanding of the mechanisms by which antioxidants modify the initial events of atherogenesis.
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