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 copper, as indicated by a 47% decrease of serum ceruloplasmin, without adversely affecting liver function. Hematological data showed that hematocrit, MCV, and RBC, WBC, lymphocyte, monocyte, neutrophil, eosinophil, and platelet counts also were unaffected by TTM treatment. In contrast, hemoglobin, MCH, and MCHC decreased in TTM-treated animals; nevertheless, the values remained well within the normal range of hematological parameters [21
]. Together with the lack of behavioral changes, these data indicate that the TTMtreated mice did not suffer from severe anemia. Histopathological analysis of liver and kidney further confirmed no signs of deleterious effects of TTM treatment. Consistent with studies using higher doses of TTM [12
], these observations suggest that TTM is a safe drug to lower copper status without causing adverse side effects.
Tetrathiomolybdate chelates bioavailable copper by forming a tripartite TTM-copper-protein complex. A recent study on TTM’s impact on copper physiology revealed that TTM specifically complexes with copper and its intracellular chaperon, Atx1, through formation of a sulfur-bridged copper-molybdenum cluster [24
]. The formation of this stable TTM-copper-Atx1 complex primarily contributes to the inhibition of copper delivery to the trans
Golgi network and downstream incorporation into cuproproteins. Due to the slow clearance of TTM-copper-protein complexes, total serum or tissue copper concentrations may not be immediately decreased by TTM treatment, although bioavailable copper is strongly reduced. Therefore, serum ceruloplasmin, the biosynthesis of which depends on liver copper status, is commonly measured as a surrogate marker of bioavailable copper in studies investigating the effect of TTM [13
]. In tissues, TTM chelates copper intracellularly [24
] and renders it non-available for biological functions; hence, the copper-to-molybdenum ratio indicates bioavailable copper status [18
]. In agreement with the declined ceruloplasmin level, the copper-to-molybdenum ratio in tissues decreased greatly in our animals.
The ferroxidase activity of ceruloplasmin is required for iron transport from the liver into the bloodstream. As expected, together with the TTM-induced decrease of serum ceruloplasmin, iron levels were significantly increased in liver and decreased in serum. Given the established role of tissue iron in atherosclerotic lesion development [9
], it is possible that a systemic change in iron homeostasis secondary to copper chelation may have played a role in modulating the atherosclerotic phenotype of our mice. However, we observed no changes of iron levels in heart and aorta of TTM-treated mice. Further analysis of mRNA and protein levels of TfR, a sensitive marker of intracellular iron status, also showed no TTM-induced changes in aorta and heart. In contrast, as discussed above, bioavailable copper levels in these tissues were dramatically reduced by TTM. Our data, therefore, indicate that TTM treatment of apoE−/− mice did not alter vascular iron homeostasis or cause severe anemia, but instead inhibited atherosclerosis primarily by reducing bioavailable copper.
The levels of soluble adhesion molecules in serum are closely correlated with overt atherosclerosis in humans [22
]. Serum levels of sVCAM-1 and sICAM-1, and mRNA levels of these adhesion molecules, MCP-1, and pro-inflammatory cytokines in aorta and heart, decreased substantially in TTM-treated animals compared to controls. This was paralleled by a decrease of aortic macrophage markers, especially M1 type macrophages. Taken together, these data suggest that TTM down-regulated expression of inflammatory mediators in vascular cells, thereby reducing monocyte recruitment to the arterial wall and—consequently—vascular inflammation and atherosclerotic lesion development.
Copper is a functional component of the innate immune system, as copper deficiency is associated with reduced neutrophil and macrophage functions and attenuated expression of pro-inflammatory cytokines in animals and cultured cells [25
]. Copper-lowering therapy with TTM has been shown to cause anti-inflammatory, anti-fibrotic, and anti-cancer effects through multiple mechanisms. Accumulating evidence suggests that NF-κB may be the primary target through which TTM suppresses expression of a wide spectrum of inflammation- and proliferation-related genes [12
]. We have previously shown that NF-κB is the main target mediating the anti-inflammatory effects of TTM in animals challenged with LPS [18
]. In the present study, it is likely that TTM prevented transcription of inflammatory mediators, including adhesion molecules and pro-inflammatory chemokines and cytokines in aorta and heart by attenuating NF-κB activation.
In addition to vascular inflammation, hypercholesterolemia and hypertriglyceridemia are major risk factors for atherosclerosis. ApoE−/− mice exhibit substantially higher levels of serum cholesterol and triglycerides compared to wild-type mice. Our data indicate that TTM moderately decreased serum VLDL cholesterol in apoE−/− mice, although the underlying mechanism is unknown. Both copper overload and severe deficiency have been found to cause hypercholesterolemia in experimental animals by inducing 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase [27
]. Surprisingly, we also observed a moderate, TTM-induced increase of serum triglycerides, suggesting a complex role of copper in lipid metabolism.
As an essential trace element, copper is required as a co-factor for several enzymes, e.g.
, cytochrome c
oxidase, copper-zinc superoxide dismutase, ceruloplasmin, and tyrosinase. However, copper is implicated in atherosclerosis due to its redox-activity [29
]. It has been shown that implanting a copper ion-releasing cuff around rat carotid arteries resulted in formation of arteriosclerosis-like lesions, suggesting that copper directly induces vascular inflammation and atherogenesis [30
]. Oxidative modification of LDL is closely associated with atherosclerosis, and ceruloplasmin has been suggested as a relevant pathophysiological source of redox-active copper that can oxidatively modify LDL [31
]. However, our results indicate that the substantial reduction of serum ceruloplasmin did not affect circulating levels of OxLDL. Therefore, TTM likely inhibited atherosclerosis by reducing vascular inflammation, not by an antioxidant effect.
Morphometric analysis of aortic lesions revealed that dietary TTM supplementation resulted in moderate but statistically significant inhibition of atherosclerotic lesion formation. The relatively small decrease of lesion development in the aortic arch compared to the descending aorta suggests that TTM primarily inhibits the initiation of atherosclerosis, as the nascent lesions formed in the descending aorta were reduced by nearly 50%. The inhibition of lesion development was accompanied by suppressed expression of inflammatory mediators in the aorta. This decrease in vascular inflammation is likely the primary cause for the decrease in atherosclerotic lesion formation, although the lower serum cholesterol levels may also have contributed somewhat to TTM’s anti-atherogenic effect.
In conclusion, the present study demonstrates that copper chelation with TTM effectively inhibits atherosclerotic lesion development in apoE−/− mice and ameliorates inflammation in the cardiovascular system. While these data may provide the proof-of-concept that copper plays a critical role in vascular inflammation and atherosclerosis, the potential therapeutic implications of using TTM as an adjunct in the prevention or treatment of cardiovascular diseases and inflammatory conditions in humans remain to be investigated.