Vitamin C, glutathione and endothelium derived NO

本申请基于细胞内 抗氧化状态是内皮细胞的重要组成部分， 功能障碍，并表征动脉粥样硬化和衰老， 有助于血管疾病的临床表现。的 内皮细胞通常维持血管内稳态，部分是通过 内皮源性一氧化氮（EDNO）的作用。引人注目 有证据表明，EDNO的作用对血管 氧化应激（氧化剂和抗氧化剂之间的不平衡， 前者）。早期研究检查受损的机制 EDNO的作用涉及抗氧化酶，如SOD或脂溶性 只是现在正在探索中。本提案的目标是定义角色 维生素C和谷胱甘肽（GSH），两种主要的细胞内水- 体内可溶性抗氧化剂种类对内皮功能的影响。 我们将使用人主动脉内皮细胞（HAECs）作为实验材料， 模型和我们的指标内皮功能将是生物活性 EDNO。在追求这一项目的目标时，我们将考虑双重 维生素C和GSH在细胞内的作用。两种化合物均提供 减少关键细胞功能的当量， 细胞内抗氧化保护。我们将首先测试维生素C的作用 和GSH在EDNO作用和产生中的作用。我们 将决定EDNO作用对细胞内容物的依赖性 以及维生素C和GSH的氧化还原状态。一旦确定，我们将 探讨eNOS活性、辅因子 可用性，eNOS，自动失活和NO与超氧化物的相互作用。 维生素C和GSH的作用也将在HAEC中进行测试， 生理学相关的氧化应激源如超氧化物， 过氧化物或氧化LDL。最后，维生素C和GSH对 将在豚鼠模型中测试EDNO的体内作用。豚鼠 将使维生素C和/或GSH轻微缺乏， 饮食药理学手段和EDNO作用的影响 使用生物测定法测定并与氧化应激的标志物相关。 胆固醇喂养将作为一种生理来源， 氧化应激和维生素C和GSH的叠加作用 在这个模型中也有探索。这些研究将提供更多 深入了解维生素C和GSH在维持血管 稳态，并可能建议治疗策略， 动脉粥样硬化性血管疾病

This application is based upon the hypothesis that intracellular antioxidant status is an important component of the endothelial cell dysfunction and characterizes atherosclerosis and aging and that contributes to the clinical manifestations of vascular disease. The endothelium normally maintains vascular homeostasis, in part, through the action of endothelium-derived nitric oxide (EDNO). Compelling evidence indicates that EDNO action is particularly sensitive to vascular oxidative stress (an imbalance between oxidants and antioxidants in favor of the former). Early research examining the mechanism(s) of impaired EDNO action involved antioxidant enzymes such as SOD or lipid-soluble only now being explored. The goal of this proposal is to define to role(s) of vitamin C and glutathione (GSH), the two principal intracellular water- soluble antioxidant species in vivo, on endothelial function. We will use human aortic endothelial cells (HAECs) as out experimental model and our index endothelial function will be the bioactivity of EDNO. In pursuing the goal of this project, we will consider the dual roles out of vitamin C and GSH within the cell. Both compounds provide reducing equivalents for critical cellular functions and are important for intracellular antioxidant protection. We will first test the role of vitamin C and GSH in EDNO action and production using unstressed HAECs. We will determine the dependence of EDNO action on the cellular content and redox state of vitamin C and GSH. once this is established, we will investigate operative mechanisms such as eNOS activity, cofactor availability, eNOS, auto-inactivation and NO interaction with superoxide. The role of vitamin C and GSH will also be tested in HAECs exposed to physiologically relevant sources of oxidative stress such as superoxide, peroxides, or oxidized LDL. Finally, the roles of vitamin C and GSH on EDNO action in vivo will be tested in guinea pig models. Guinea pigs will be rendered marginally vitamin C and/or GSH deficient through dietary of pharmacologic means and the implications for EDNO action determined using bioassays and related to markers of oxidative stress. Cholesterol -feeding will be employed as a physiologic source of oxidative stress and the superimposed effects of vitamin C and GSH explored in this model as well. These studies should provide additional insight into the role(s) of vitamin C and GSH in maintaining vascular homeostasis and may suggest treatment strategies for patients with atherosclerotic vascular disease.