Hepcidin Deficiency Protects Against Atherosclerosis

Abstract

Objective- Inflammatory stimuli enhance the progression of atherosclerotic disease. Inflammation also increases the expression of hepcidin, a hormonal regulator of iron homeostasis, which decreases intestinal iron absorption, reduces serum iron levels and traps iron within macrophages. The role of macrophage iron in the development of atherosclerosis remains incompletely understood. The objective of this study was to investigate the effects of hepcidin deficiency and decreased macrophage iron on the development of atherosclerosis. Approach and Results- Hepcidin- and LDL (low-density lipoprotein) receptor-deficient ( Hamp^-/-/ Ldlr^-/-) mice and Hamp^+/+/ Ldlr^-/- control mice were fed a high-fat diet for 21 weeks. Compared with control mice, Hamp^-/-/ Ldlr^-/- mice had decreased aortic macrophage activity and atherosclerosis. Because hepcidin deficiency is associated with both increased serum iron and decreased macrophage iron, the possibility that increased serum iron was responsible for decreased atherosclerosis in Hamp^-/-/ Ldlr^-/- mice was considered. Hamp^+/+/ Ldlr^-/- mice were treated with iron dextran so as to produce a 2-fold increase in serum iron. Increased serum iron did not decrease atherosclerosis in Hamp^+/+/ Ldlr^-/- mice. Aortic macrophages from Hamp^-/-/ Ldlr^-/- mice had less labile free iron and exhibited a reduced proinflammatory (M1) phenotype compared with macrophages from Hamp^+/+/ Ldlr^-/- mice. THP1 human macrophages treated with an iron chelator were used to model hepcidin deficiency in vitro. Treatment with an iron chelator reduced LPS (lipopolysaccharide)-induced M1 phenotypic expression and decreased uptake of oxidized LDL. Conclusions- In summary, in a hyperlipidemic mouse model, hepcidin deficiency was associated with decreased macrophage iron, a reduced aortic macrophage inflammatory phenotype and protection from atherosclerosis. The results indicate that decreasing hepcidin activity, with the resulting decrease in macrophage iron, may prove to be a novel strategy for the treatment of atherosclerosis.

Keywords: atherosclerosis; hepcidins; inflammation; iron; macrophages.

Figure 1:

Hepcidin deficiency in Ldlr^−/− mice was associated with decreased atherosclerosis. (A) Macrophage activity was decreased in the aortas of Hamp^−/−/ Ldlr^−/− mice compared to the aortas of Hamp^+/+/ Ldlr^−/− mice as determined using ProSense near-infrared fluorescence. Results from three representative pairs of mice are shown. (B) Quantification of ProSense signal from Hamp^−/−/ Ldlr^−/− mice revealed a 45% reduction in macrophage activity compared to Hamp^+/+/ Ldlr^−/− mice (p<0.0001, n=23 and 27, respectively). (C) There was decreased lipid accumulation in the aortas of Hamp^−/−/ Ldlr^−/− mice compared to the aortas of Hamp^+/+/ Ldlr^−/− mice as determined using Oil Red O stain. Aortas from the same three pairs of mice as in A are shown. (D) Quantification of the area and intensity of Oil Red O stain revealed a 74% decrease in aortic lipid in Hamp^−/−/ Ldlr^−/− mice compared to Hamp⁺/ Ldlr^−/− mice (p=0.005, n=6 for each). (E) Consistent with the observed decrease in aortic macrophage activity, there was a 67% decrease in the mRNA levels of CD68 in the aortas of Hamp^−/−/ Ldlr^−/− mice compared to controls (p=0.002, n=12 and 16, respectively). The final weight (F) and fasting serum LDL (G) were decreased in Hamp^−/−/ Ldlr^−/− mice compared to control mice (p=0.01 and 0.003, respectively). In (F) and (G), n=23 for Hamp^−/−/ Ldlr^−/− mice and n=27 for Hamp^+/+/ Ldlr^−/− mice.

Figure 2.

Iron-loading in Ldlr^−/− mice did not decrease atherosclerosis. (A) Compared to mice treated with dextran alone, intraperitoneal injection of iron dextran into Ldlr^−/− mice produced a greater than two-fold increase in the level of serum iron (p<0.0001, n=6 and 7 respectively). (B) There was a 19% reduction in final body weight (p=0.0003) and (C) a 37% decrease in fasting LDL levels in iron-treated Ldlr^−/− mice compared to controls (p=0.013). (D) There was no difference in macrophage activity in the aortas of iron-treated, compared to control-treated, Ldlr^−/− mice as determined using ProSense near-infrared fluorescence (three representative pairs of aortas are shown). (E) Quantification of ProSense signal revealed no difference in macrophage activity (p=0.12). (F) There was no difference in lipid accumulation in the aortas of iron-treated Ldlr^−/− mice compared to control mice as determined using Oil Red O stain. Aortas from the same three pairs of mice as in (D) are shown. (G) There was also no difference in the area and intensity of aortic staining with Oil Red O between iron-injected and control mice (p=0.16).

Figure 3.

Aortic and peritoneal macrophages from hepcidin-deficient mice as well as human macrophages treated with an iron chelator exhibit a reduced M1 phenotype and reduced uptake of oxidized LDL. (A) Pooled aortic CD11b-positive leukocytes from Hamp^−/−/ Ldlr^−/− mice have higher median calcein fluorescence (and therefore a lower labile intracellular iron concentration) than CD11b-positive leukocytes from Hamp^+/+/ Ldlr^−/− mice. (B) CD11b-positive macrophages isolated from the aortas of Hamp^−/−/ Ldlr^−/− mice fed a high-fat diet (n=6) exhibited lower rates of CD38 positivity (an M1 marker) on flow cytometric analysis and (C) similar rates of CD206 positivity (an M2 marker) compared to CD11b-positive macrophages isolated from the aortas of Hamp^+/+/ Ldlr^−/− mice (n=5). (D) Peritoneal macrophages isolated from Hamp^−/−/ Ldlr^−/− mice 4 days after intraperitoneal injection of thioglycollate had lower protein levels of iNOS (an M1 marker) relative to arginase-1 (an M2 marker) compared to peritoneal macrophages isolated from Hamp^+/+/ Ldlr^−/− mice. No difference in CD11b protein levels was observed. (E) Compared to peritoneal macrophages from Hamp^+/+/ Ldlr^−/− mice, macrophages from Hamp^−/−/ Ldlr^−/− mice had a decreased ratio of iNOS to Arg1. (F) THP1 cells (a human monocyte cell line) treated with PMA for 24 hours were incubated in the presence or absence of LPS or DFP (an iron chelator). LPS-treated cells exhibited increased iNOS mRNA levels; however, iNOS mRNA levels were not increased in cells treated with LPS and DFP (n=5 in each group). (G) THP1 cells incubated in culture media containing 100 μM ferric ammonium citrate and labeled oxidized LDL (oxLDL) were treated with either DFP (100 μM) or vehicle. Treatment with DFP resulted in reduced oxLDL uptake by macrophages as determined by flow cytometry. A representative pair of histograms is shown. (H) Comparison of mean fluorescence intensity (MFI) after uptake of labeled oxLDL by Fe-treated or Fe and DFP-treated THP1 cells (n=6 experiments each). (I) Treatment of THP1 cells with oxLDL increased ABCA1 mRNA levels, but this increase was blunted by treatment with ferric ammonium citrate.