- Endothelial activation with increased expression of cellular adhesion molecules, chemokines and pro-inflammatory cytokines critically contributes to vascular inflammation and the initiation and progression of atherosclerosis. By affecting redox-sensitive cell signaling pathways and transcription factors, redox-active transition metal ions, such as copper and iron, may play an important role in these processes. The goals of my studies were to investigate the role of copper in systemic and vascular inflammation, endothelial activation, and atherosclerotic lesion development, using tetrathiomolybdate (TTM), a clinically-used copper chelator, as a specific means to manipulate cellular copper levels in vitro and in vivo.
Incubation of human aortic endothelial cells (HAEC) with copper induced activation of the redox-sensitive transcription factors, nuclear factor kappa B (NF-kB) and activator protein-1 (AP-1) and up-regulation of mRNA and protein levels of adhesion molecules and the chemokine, monocyte chemotactic protein-1 (MCP-1), in a dose- and time-dependent manner. Furthermore, TTM inhibited TNFa-induced increases of mRNA and protein levels of adhesion molecules and MCP-1 in a dose-dependent manner. TNFa-induced activation of NF-kB and AP-1 also was dose-dependently attenuated by TTM, and inhibition of NF-kB activity was associated with decreased phosphorylation and degradation of its cytosolic inhibitory subunit, IkBa.
In an in vivo model of systemic inflammation using of C57BL mice, oral TTM administration for three weeks significantly reduced bioavailable copper and inhibited lipopolysaccharide (LPS)-induced upregulation of inflammatory gene expression in aorta and heart. Tetrathiomolybdate also significantly inhibited LPS-induced increases of serum levels of soluble adhesion molecules, MCP-1, and pro-inflammatory cytokines. Similar inhibitory effects of TTM were observed on NF-kB and AP-1 activation in the heart and lungs.
In apolipoprotein E-deficient (apoE-/-) mice, a well established animal model of human atherosclerosis, dietary supplementation with TTM for ten weeks significantly reduced bioavailable copper and attenuated atherosclerotic lesion development, which was particularly pronounced in the descending aorta. This anti-atherogenic outcome of TTM supplementation was accompanied by several anti-inflammatory effects, such as significantly declined serum levels of soluble adhesion molecules, reduced aortic gene expression of inflammatory mediators, and less aortic macrophage accumulation. Importantly, TTM supplementation for ten weeks did not cause liver toxicity or anemia.
In conclusion, my studies provide the proof of concept that copper plays an important role in systemic and vascular inflammation and the pathogenesis of atherosclerosis, and demonstrate that tetrathiomolybdate exerts anti-inflammatory and anti-atherogenic effects in preclinical animal models. The possible implications of these findings for cardiovascular and inflammatory diseases in humans remain to be investigated.