Vitamin C, glutathione, and endothelial cell activation

这个项目是基于这样的假设：细胞氧化还原状态 在预防与以下疾病相关的内皮细胞功能障碍方面发挥关键作用 动脉粥样硬化的形成。氧化应激(氧化剂和抗氧化剂的失衡) 有利于前者的氧化剂)被认为是致病因素 在动脉粥样硬化形成中，既通过低密度脂蛋白的氧化 低密度脂蛋白及其后遗症与单核细胞-内皮细胞的刺激 互动。虽然之前的研究已经评估了低密度脂蛋白的影响- 相关的和细胞外的抗氧化剂对这些促动脉粥样硬化、氧化还原- 敏感的过程，对细胞内的作用知之甚少 抗氧化剂。维生素C和谷胱甘肽的积累和合成， 分别以毫米级浓度计算，并在人体细胞中发挥作用 在细胞抗氧化防御系统中的控制作用，从而在细胞中 在面对氧化剂挑战时的正直和功能。因此， 该项目的总体目标是确定细胞内维生素C的作用 以及谷胱甘肽在体外和体内内皮细胞激活中的状态。 体外系统将培养人主动脉内皮细胞 (HAEC)，以及体内系统的豚鼠，它和人类一样，不能 合成抗坏血酸。第一个目标是刻画和操纵 HAEC的维生素和谷胱甘肽状态。维生素C的作用 负荷对细胞谷胱甘肽状态的影响，反之 操纵细胞谷胱甘肽状态对维生素C状态的影响 学习。目标2将确定细胞氧化还原状态在HAEC中的作用- 介导低密度脂蛋白氧化。将通过以下方式探索潜在机制 测量细胞内超氧化物和硫醇的产生，并通过抑制 一氧化氮合成。在第三个目标中，我们将确定 细胞内维生素C和谷胱甘肽在HAEC激活中的状态，即 细胞黏附分子和单核细胞的表达 趋化蛋白-1和单核细胞趋化蛋白-1 (MCP-1)和单核细胞黏附。潜在的机制将是 通过研究氧化还原敏感型细胞的核转位来探索 转录因子NfkappaB与一氧化氮的参与。在AIM 4，将调查HAEC研究的体内相关性。这个 标准饲料和0.3%饲料对豚鼠维生素C和/或谷胱甘肽的影响 含胆固醇的饮食将受到操纵，循环中的 将检测氧化低密度脂蛋白的单核细胞趋化蛋白-1、细胞黏附分子和自身抗体，如 以及主动脉CAM和MCP-1的表达，NfkappaB的激活， 单核细胞黏附和动脉粥样硬化病变形成的程度。 这些信息将提供对机制的更好的理解 抗氧化剂通过其改变动脉粥样硬化的形成。

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 is 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.