Elsevier

Atherosclerosis

Volume 223, Issue 2, August 2012, Pages 306-313
Atherosclerosis

Copper chelation by tetrathiomolybdate inhibits vascular inflammation and atherosclerotic lesion development in apolipoprotein E-deficient mice

https://doi.org/10.1016/j.atherosclerosis.2012.06.013Get rights and content

Abstract

Endothelial activation, which is characterized by upregulation of cellular adhesion molecules and pro-inflammatory chemokines and cytokines, and consequent monocyte recruitment to the arterial intima are etiologic factors in atherosclerosis. Redox-active transition metal ions, such as copper and iron, may play an important role in endothelial activation by stimulating redox-sensitive cell signaling pathways. We have shown previously that copper chelation by tetrathiomolybdate (TTM) inhibits LPS-induced acute inflammatory responses in vivo. Here, we investigated whether TTM can inhibit atherosclerotic lesion development in apolipoprotein E-deficient (apoE−/−) mice. We found that 10-week treatment of apoE−/− mice with TTM (33–66 ppm in the diet) reduced serum levels of the copper-containing protein, ceruloplasmin, by 47%, and serum iron by 26%. Tissue levels of “bioavailable” copper, assessed by the copper-to-molybdenum ratio, decreased by 80% in aorta and heart, whereas iron levels of these tissues were not affected by TTM treatment. Furthermore, TTM significantly attenuated atherosclerotic lesion development in whole aorta by 25% and descending aorta by 45% compared to non-TTM treated apoE−/− mice. This anti-atherogenic effect of TTM was accompanied by several anti-inflammatory effects, i.e., significantly decreased serum levels of soluble vascular cell and intercellular adhesion molecules (VCAM-1 and ICAM-1); reduced aortic gene expression of VCAM-1, ICAM-1, monocyte chemotactic protein-1, and pro-inflammatory cytokines; and significantly less aortic accumulation of M1 type macrophages. In contrast, serum levels of oxidized LDL were not reduced by TTM. These data indicate that TTM inhibits atherosclerosis in apoE−/− mice by reducing bioavailable copper and vascular inflammation, not by altering iron homeostasis or reducing oxidative stress.

Highlights

► The copper chelator, tetrathiomolybdate, reduced bioavailable copper in apoE−/− mice. ► No severe anemia or altered iron homeostasis in aorta and heart were observed. ► Atherosclerosis in the descending aorta was attenuated by 45%. ► Inflammatory mediators, but not LDL oxidation, were significantly reduced. ► Copper appears to play a critical role in vascular inflammation and atherosclerosis.

Introduction

As the leading cause of mortality in developed countries, atherosclerosis is a systemic multifactorial disease characterized by lipid deposition and hardening of the vascular wall of large and medium-sized arteries. In recent years, the role of inflammation in atherosclerosis has been increasingly recognized, with evidence showing that the initiation and progression of atherosclerotic lesion development is accompanied by persistent vascular inflammation [1]. Hence, the recruitment of inflammatory leukocytes from the circulation to the arterial wall is a significant feature of atherogenesis. After rolling along the vascular endothelium, monocytes transmigrate into the intima of the arterial wall, where they differentiate into macrophages and ingest modified lipoproteins. As a consequence, these macrophages become lipid-laden “foam cells”, which are the main structural component of the atherosclerotic fatty streak.

The mechanisms of arterial monocyte recruitment and retention are characterized by the expression of cellular adhesion molecules and chemokines, such as vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), and monocyte chemotactic protein-1 (MCP-1), by vascular endothelial cells [2]. Several studies have shown that animal models of atherosclerosis were protected from vascular lesion development when either MCP-1 or ICAM-1 was genetically ablated [3], [4]. The expression of adhesion molecules, chemokines, and pro-inflammatory cytokines by endothelial cells is orchestrated primarily by the transcription factor, nuclear factor-κB (NF-κB) [5]. The expression of cellular adhesion molecules and chemokines can be induced by pro-inflammatory cytokines, such as tumor necrosis factor α (TNFα), via activation of NF-κB.

Inflammatory processes in atherosclerosis may be driven, in part, by reactive oxygen species. There is increasing evidence that superoxide radicals and hydrogen peroxide generated by isoforms of NADPH oxidase in vascular cells, such as NOX 1, 2, 4, and 5, play an important role in inflammatory endothelial activation [6]. Redox-active transition metals, such as copper and iron, also have been implicated in atherogenesis through mechanisms involving redox-sensitive cell signaling pathways and activation of NF-κB [7], [8]. Treatment with desferrioxamine has been shown to reduce iron levels in atherosclerotic lesions and suppress lesion development in cholesterol-fed rabbits [9] and apolipoprotein E-deficient (apoE−/−) mice [10]. Copper has been shown to stimulate migration and proliferation of human endothelial cells [11]. Copper deficiency was found to be associated with down-regulation of inflammatory responses and angiogenesis in mice [12]. Our laboratory has previously demonstrated that activation of human endothelial cells by pro-inflammatory cytokines, e.g., TNFα, can be suppressed by desferrioxamine or the copper chelating agent, neocuproine, supporting the notion that redox-active transition metals play a role in the inflammatory responses in these cells [8].

In the present study, we used tetrathiomolybdate (TTM) to investigate the role of copper in atherosclerosis. TTM is a small hydrophilic compound that chelates copper with high specificity. Currently under development as a drug to treat Wilson's disease, an autosomal recessive genetic disease characterized by excessive copper accumulation in the liver, TTM has demonstrated a good safety index [13]. The copper-TTM complex is metabolized in the liver and then cleared through bile [14]. Accumulating evidence indicates that TTM treatment inhibits expression and lowers circulating levels of a number of angiogenic, growth-promoting, and inflammatory mediators, such as vascular endothelial growth factor, TNFα, interleukin(IL)-1α, IL-1β, and IL-6 [12], [15]. It has also been shown that TTM at a physiologically relevant dose inhibits vascular endothelial cell proliferation [16]. Moreover, recent studies suggest that TTM inhibits expression of inflammatory mediators through attenuation of NF-κB activation [12], [17]. We have recently shown that TTM significantly inhibits lipopolysaccharide (LPS)-induced acute inflammatory responses in mice [18]. In the present study, we investigated whether TTM can inhibit vascular inflammation and atherosclerotic lesion development in apoE−/− mice.

Section snippets

Animals and experimental procedures

Female C57BL/6N and apoE−/− mice on a C57BL/6 background at 4–5 weeks of age and weighing about 12–14 g were purchased from Jackson Laboratory (Bar Harbor, ME). The animals were housed under pathogen-free conditions in a temperature and humidity-controlled environment (12-h light/dark cycle). All animal procedures were reviewed and approved by the Institutional Animal Care and Use Committee of Oregon State University.

The apoE−/− mice were fed a high-fat, high-cholesterol (HFHC) Western-type

Tetrathiomolybdate effectively reduces bioavailable copper without adverse side effects

Serum ceruloplasmin, as assessed by its ferroxidase activity, is an established marker of bioavailable copper status [13]. Dietary treatment of apoE−/− mice with TTM for 10 weeks (33 ppm TTM for 2 weeks followed by 66 ppm TTM for 8 weeks) significantly reduced the serum level of ceruloplasmin by 47% compared to non-TTM treated control apoE−/− mice (Table 1). In contrast, no statistically significant differences were observed between TTM-treated and control mice in either final body weight

Discussion

We have previously reported that tetrathiomolybdate inhibits LPS-induced acute inflammatory responses in mice, likely by inhibiting activation of the redox-sensitive transcription factors, NF-κB and AP-1 [18]. The current study confirms the anti-inflammatory effects of dietary TTM treatment and demonstrates that TTM also inhibits atherosclerotic lesion development in a well-established murine model of human atherosclerosis.

Administration of TTM for 10 weeks significantly reduced bioavailable

Acknowledgment

The work described in this paper was supported by National Center for Complementary and Alternative Medicine (NCCAM) Grant P01 AT002034. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the National Institutes of Health. The authors thank Dr. Robert Tanguay from the Department of Environmental & Molecular Toxicology at Oregon State University for his assistance with image analysis of atherosclerotic lesions. We also acknowledge the

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