Vascular inflammation plays a central role in the complex chain of events leading to atherosclerotic lesion development (1
). There is accumulating, although controversial, evidence that redox-active transition metal ions such as iron or copper are causative agents in the pathogenesis of atherosclerosis and its clinical sequelae (15
). Thus, intracellular metal chelation may have anti-atherogenic effects and improve cardiovascular outcome (29
). In this study, we found that the iron chelator, desferrioxamine, reduces inflammation in LPS-exposed C57BL mice and inhibits inflammation and atherosclerotic lesion development in the aorta of apoE−/− mice.
Reactive oxygen species have been implicated as key mediators of cell signaling pathways that activate redox-sensitive transcription factors, which in turn upregulate inflammatory gene expression (31
). It has been shown that ROS production and LDL oxidation by cultured vascular cells are mediated by transition metal ions (32
), and the oxidative capacity of these cells is greatly enhanced by micromolar concentrations of iron (24
). In addition, LPS and iron may enhance cellular superoxide production by increasing NADPH oxidase activity (25
). Catalytic or labile iron also has been shown to induce inflammatory responses in vascular endothelial cells, as manifested, e.g
., by increased expression of adhesion molecules and IL-6 and increased adherence of monocytes to these cells (24
Therefore, removal of intracellular labile iron may decrease ROS production and oxidative stress, thereby diminishing cell and tissue injury and inflammatory responses. In agreement with this notion, the iron chelator, DFO, has been shown to effectively inhibit iron-mediated ROS production in vitro
and in vivo
) and protect cells from H2
-induced DNA damage (38
). Furthermore, DFO can protect against ROS-induced myocardial and coronary endothelial ischemia-reperfusion injury (39
) and improve endothelium-dependent vasodilation in CVD patients (41
). Consistent with these observations, our data show that DFO inhibits LPS-induced acute inflammatory responses in vivo
, as evidenced by reduced serum levels of adhesion molecules and MCP-1 and decreased aortic NFκB and AP-1 activation.
As explained, inflammation and oxidative stress in the arterial wall are prominent features of atherosclerosis. Ross (1
) observed that feeding an atherogenic diet to experimental animals induced adhesion of monocytes and other inflammatory cells to the arterial wall in a matter of days. The antioxidant and anti-inflammatory effects of DFO most likely account for its observed anti-atherogenic effect in apoE−/− mice in the present study, which is in agreement with an earlier report that DFO decreases atherosclerotic lesion development in cholesterol-fed rabbits (20
). In our study, DFO not only inhibited aortic atherosclerosis, but also significantly decreased macrophage accumulation, as assessed by CD68 expression. Aortic gene expression of MCP-1, a chemokine that plays a critical role in vascular monocyte recruitment and atherogenesis (1
), was markedly–albeit non-significantly–reduced by DFO. In addition, DFO treatment significantly decreased gene expression of VCAM-1, ICAM-1, MCP-1, TNFα, and IL-6 in the heart, which is compatible with the notion of decreased inflammation and atherosclerotic lesion development in coronary arteries. We also found that DFO significantly increased tissue expression of TfR in heart and liver, a sensitive and reliable marker of intracellular labile iron status. While these data cannot establish cause-and-effect, they suggest that (intracellular) iron plays a critical role in inflammation and atherosclerosis, and that iron chelation by DFO inhibits atherosclerotic lesion development, in part, by suppressing vascular inflammation and monocyte-macrophage recruitment.
The finding that DFO exerted anti-inflammatory effects in apoE−/− mice is further buttressed by our observations that DFO suppressed LPS-induced acute inflammatory responses in C57BL mice and inhibited TNFα-induced expression of adhesion molecules and MCP-1 in human aortic endothelial cells (23
). These studies also revealed a common underlying mechanism for the anti-inflammatory effect of DFO, viz
., inhibition of NFκB activation, a redox-sensitive transcription factor that regulates expression of many inflammatory genes (11
). In most cell types, NFκB can be activated by a diverse range of stimuli, suggesting that several signaling pathways are involved. Lipid peroxidation has been reported to play a role in TNFα-induced NFκB activation (42
). Redox-active transition metal ions play a key role in the initiation and propagation of lipid peroxidation, leading to the generation of peroxyl and alkoxyl radicals, as well as lipid hydroperoxides and numerous reactive breakdown products (33
). DFO, which strongly inhibits iron-dependent lipid peroxidation, also strongly inhibited TNFα-induced NFκB activation in endothelial cells (42
). Consistent with these data, in the present in vivo
study we found that DFO treatment strongly inhibited LPS-induced NFκB activation in aorta.
The macrophage is a key cell type in the formation and fate of an atherosclerotic plaque. The accumulation of iron by plaque macrophages, mainly via erythrophagocytosis, promotes lipid uptake by stimulating expression of the macrophage scavenger receptor–1 (43
). Oxidative reactions associated with the acquisition of iron and lipid may facilitate macrophage apoptosis, with the release of their cellular content into the atherosclerotic lesion. The release of iron and lipids contributes to the cellular mass of the lesion and causes further monocyte recruitment to the arterial wall, thereby amplifying the atherosclerotic process. Interruption of iron acquisition and storage in plaque macrophages by iron restriction or iron chelation inhibits lesion initiation and progression (19
). In agreement with these observations, our results provide new evidence that DFO treatment inhibits macrophage accumulation in the arterial wall.
It should be noted that large doses of DFO appear to be safe in experimental animals, e.g.
, 540 mg/kg/d for 7 days in rats (44
) and 170 mg/kg/d in mice (45
). Our preliminary data also showed that DFO at a daily dose of 500 mg/kg for seven days exerted no toxic effects. In our study, treating apoE−/− mice for 10 weeks with 100 mg/kg/d DFO did not cause significant growth retardation or body weight loss. Consistent with previous reports (20
), we found that DFO did not affect serum iron, ferritin, and hemoglobin levels, as well as hematocrit. Hence, mice treated with DFO likely had sufficient iron stores to maintain normal erythropoiesis. However, we did find that DFO significantly decreased liver iron levels and increased cardiac and hepatic TfR expression, indicating that DFO effectively depleted tissue iron stores.
In conclusion, our study indicates that the iron chelator, DFO, inhibits inflammation and atherosclerotic lesion development in experimental mice, and provides the proof-of-concept that iron plays an important role in the pathogenesis of atherosclerosis. The relationship between iron stores and CVD in humans and the potential benefits of chelation of excess iron or limiting iron intake as a complementary strategy to prevent or treat atherosclerosis in humans requires further investigation.