Metal Chelators and Thiols in Endothelial Function

该项目的前提是，细胞氧化还原环境的失衡会导致内皮细胞活化，黏附分子和趋化因子的表达增加。血管内皮细胞活化是动脉粥样硬化和心血管疾病(CVD)的关键启动和传播事件。在初步研究中，我们发现铁络合剂去铁胺(DFO)和铜络合剂新铜氨酸(NC)，以及二硫醇化合物(R)-α-亚碘酸(LA)和吡咯烷二硫代氨基甲酸酯(PDTC)可抑制TNFα诱导的 激活人主动脉内皮细胞(HAEC)，主要通过抑制血管内皮细胞的激活 转录因子NFkappaB和SP-1。因此，本项目将研究金属螯合剂和二硫醇化合物通过阻止氧化还原敏感细胞信号转导的氧化失活和随后氧化还原敏感转录因子诱导基因转录来抑制内皮细胞激活，从而抑制动脉粥样硬化的假设。目的1研究EDTA、上述金属螯合剂和二硫醇对肿瘤坏死因子α-过氧化氢或过氧亚硝酸根刺激的血管内皮细胞黏附分子和趋化因子表达的影响。通过评估氧化还原敏感的信号转导分子硫氧还蛋白和PTEN，以及氧化还原敏感的转录因子NFkappaB、SP-1和AP-1的状态和活性，将探索潜在的机制。目的2采用C57BL/6J小鼠炎症模型，确定金属螯合剂和二硫醇在体内抑制内皮细胞活化的效果。将在相关的血管床中评估内皮激活、信号转导的氧化还原状态和转录因子，以及金属状态。目的3研究金属螯合剂和二硫醇对载脂蛋白E基因敲除小鼠局部和全身动脉粥样硬化病变发展的影响。小鼠将被喂以西式饮食，并用成功的金属螯合剂和AIMS 1和2中确定的二硫醇治疗。动脉粥样硬化病变区域将在相关的血管床中进行量化。血管内皮细胞活化的上述标志物和 氧化还原环境将被确定，并与动脉粥样硬化病变区域和成分、金属状态和血浆脂类进行比较。为了与CERCAT的总体目标保持一致，该项目将在体外和体内研究CAM金属螯合剂和抗氧化剂的机制和有效性，确定相关的生物靶点，并为支持或反对CAM疗法在CVD中的人体研究提供基本的理论基础。

This project is based upon the premise that an imbalance in cellular redox environment causes endothelial activation with increased expression of adhesion molecules and chemokines. Endothelial activation is a pivotal initiating and propagating event in atherosclerosis and cardiovascular diseases (CVD). In preliminary studies we found that the iron-chelator desferrioxamine (DFO) and the copper-chelator neocuproine (NC), and the dithiol compounds (R)-alpha-Iipoic acid (LA) and pyrrolidine dithiocarbamate (PDTC) inhibit TNFalpha-induced activation of human aortic endothelial cells (HAEC), mainly by inhibiting activation of the transcription factors NFkappaB and SP-1. Therefore, this project will investigate the hypothesis that metal chelators and dithiol compounds inhibit endothelial activation and, thus, atherosclerosis by preventing oxidative inactivation of redox-sensitive cellular signal transducers and subsequent induction of gene transcription by redox-sensitive transcription factors. Aim 1 will determine the effects of EDTA and the above metal chelators and dithiols on adhesion molecule and chemokine expression in HAEC stimulated with TNFalpha H2O2, or peroxynitrite. Underlying mechanisms will be explored by assessing the redox state and activity of the redox-sensitive signal transducers thioredoxin and PTEN, and the redox-sensitive transcription factors NFkappaB, SP-1, and AP-1. Aim 2 will determine the efficacy of metal chelators and dithiols in inhibiting endothelial activation in vivo, using inflammatory models in C57BL/6J mice. Endothelial activation, redox state of signal transducers, and transcription factors will be assessed in relevant vascular beds, as well as metal status. Aim 3 will determine the effects of metal chelators and dithiols on local and systemic atherosclerotic lesion development in apolipoprotein E knockout mice. Mice will be fed a Western-type diet and treated with successful metal chelators and dithiols identified in Aims 1 and 2. Atherosclerotic lesion areas will be quantified in relevant vascular beds. The above markers of vascular endothelial activation and redox environment will be determined and compared to atherosclerotic lesion areas and composition, metal status, and plasma lipids. In keeping with the overall goals of CERCAT, this project will study the mechanisms and efficacy of CAM metal chelators and antioxidants in vitro and in vivo, identify relevant biological targets, and provide the essential rationale for or against human studies of CAM therapies in CVD.