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.
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