Hepcidin Destabilizes Atherosclerotic Plaque Via Overactivating Macrophages After Erythrophagocytosis
Objective—To explore a direct and causal relationship between vascular hepcidin and atherosclerotic plaque stability.
Methods and Results—Accelerated atherosclerotic lesions were established by perivascular collar placement in apolipoprotein E–deficient (ApoE−/−) mice. Adenoviral overexpression of hepcidin in the carotid artery during plaque formation enhanced intraplaque macrophage infiltration and suppressed the contents of collagen and vascular smooth muscle cells, whereas hepcidin shRNA treatment exerts opposite effects. The overexpression or knockdown of hepcidin did not affect plaque lipid deposition but increased or decreased oxidized low-density lipoprotein (ox-LDL) levels within intraplaque macrophages. In cultured macrophages, ox-LDL not only increased reactive oxygen species formation, inflammatory cytokine production, and apoptosis but also upregulated hepcidin expression. However, hepcidin did not exaggerate the ox-LDL–induced activation of macrophages until an onset of erythrophagocytosis. Whereas hepcidin was critical for the upregulation of L-ferritin and H-ferritin in both ox-LDL–treated erythrophagocytosed macrophages and atherosclerotic plaques, the adding of iron chelators suppressed the intracellular lipid accumulation, reactive oxygen species formation, inflammatory cytokine expression, and apoptosis in erythrophagocytosed macrophages.
Conclusion—Hepcidin promotes plaque destabilization partly by exaggerating inflammatory cytokine release, intracellular lipid accumulation, oxidative stress, and apoptosis in the macrophages with iron retention.
It has been documented for decades that a state of sustained iron depletion or mild iron deficiency protects against atherosclerosis.1 Although a few risk factors for cardiovascular disease including overactivation of the renin-angiotensin system and polymorphisms of haptoglobin or heme oxygenase promoter are associated with increased atherosclerotic plaque iron,1 an approach of iron depletion either delays the onset of atherogenesis or stabilizes plaque.2 Importantly, the storage and processing of iron from erythrophagocytosis by macrophages within plaque appear to play a key role in plaque progression. Accordingly, we have demonstrated that erythrocytes induce plaque vulnerability in a dose-dependent manner in a rabbit model of intraplaque hemorrhage.3 However, a plausible link between the retention of iron in macrophages and atherosclerotic lesion formation and development remains unknown.
Recently, hepcidin has been demonstrated to be a key peptide in the regulation of iron homeostasis.4,5 Hepcidin binds to the iron transporter Ferroportin 1 (FPN1) on the cell surface and induces FPN1 internalization and degradation.6 As a result, the intracellular iron level is elevated. Hepcidin is produced by a wide variety of cells including macrophages,5 and the expression of hepcidin is regulated by a number of factors. For instance, hepcidin expression is increased in response to iron supplement and inflammation and decreased in response to iron insufficiency.6 Notably, hepcidin is a major determinant of the amount of iron retained in macrophages.7 Therefore, it has been suggested that hepcidin promotes atherosclerosis progression by slowing or preventing the mobilization of iron from macrophages within the atherosclerotic lesion.8 Valenti et al9 found that the serum hepcidin and macrophage iron levels correlated with MCP-1 release and vascular damage in patients with metabolic syndrome. More recently, Saeed et al10 showed that reduction of the iron levels of macrophages via systemic pharmacological suppression of hepcidin increased expression of cholesterol efflux transporters and attenuated atherosclerosis. Nevertheless, a direct and causal relationship between vascular hepcidin and atherosclerotic plaque stability and the potential mechanisms have not yet been explored.
In the present study, we studied the potential role of vascular hepcidin in atherosclerotic plaque stability by local hepcidin gain- and loss-of-function approaches in a mouse model of accelerated atherosclerosis and explored the underlying mechanisms in macrophages under a proatherogenic microenvironment.
Detailed material and methods are described in the online-only Data Supplement.
Preparation of Adenoviral Vectors
Recombinant adenoviruses (Ad) carrying the murine hepcidin (Ad-hepcidin) and its shRNA or a control transgene EGFP (Ad-EGFP) were prepared.
Animal Model and Gene Transfer
In the first part of the in vivo study, 40 male apolipoprotein E (apoE)−/− mice were randomly divided into a control group and a model group (n=20 each). Mice in the model group underwent constrictive collar placement around the left common carotid artery near its bifurcation as previously described.11 In the second part of the in vivo study, 75 male apoE−/− mice underwent constrictive collar placement and were randomly divided into 3 groups (n=25 each) for adenoviral gene delivery of Ad-EGFP, Ad-hepcidin, and Ad-hepcidin shRNA, respectively.
Serum Lipid, Glucose, and Iron Measurement
At the end of the second part of the in vivo study, serum total cholesterol (TC), triglycerides (TG), low-density lipoprotein (LDL-C) cholesterol, and high-density lipoprotein cholesterol (HDL) and glucose concentrations were measured.
Tissue Preparation and Histological Analysis
Antihepcidin monoclonal antibody (1:150, Abcam, Cambridge, United Kingdom) was used for hepcidin staining in vivo. Positive staining areas of macrophages, smooth muscle cells (SMCs), lipids, collagen, IL-6, MCP-1, TNFα, MMP-2, hepcidin, H-ferritin, and L-ferritin were quantified. The vulnerable index was calculated as described in previous studies.12
Tissue sections of the carotid arteries were incubated with double primary antibodies, including those against macrophages and hepcidin, SMCs and hepcidin, macrophages, and oxidized LDL (ox-LDL) as well as SMCs and ox-LDL. Fluorescent images were obtained by a laser scanning confocal microscopy.
Cell Culture and Treatment
J774 macrophages were chosen for erythrophagocytosis as previously described.13 Monolayers of J774 macrophages with or without erythrophagocytosis were treated with ox-LDL, synthetic human hepcidin, desferrioxamine (DFO), and ferrous chelator 2,2′-bipyridyl (BPDL).14,15 Monolayers of J774 macrophages transfected with control or hepcidin siRNAs were subjected to erythrophagocytosis and given different treatments.
Quantitative Real-Time PCR
The mRNA expression levels of hepcidin, IL-6, MCP-1, TNFα, MMP-2, FPN1, H-ferritin, and L-ferritin were quantified.
Western Blot Analysis
The protein expression levels of IL-6, MCP-1, TNFα, H-ferritin, and L-ferritin were quantitatively analyzed.
Antihepcidin monoclonal antibodies (1:100, Abcam) were applied for immunofluorescent staining of hepcidin. Expression and localization of hepcidin and FPN1 in J774 macrophages were examined.
Quantification of Reactive Oxygen Species Production
Fluorescence measurement of reactive oxygen species (ROS) was performed with Flow Cytometer, and the data were analyzed with Cell Quest Pro.
Detection of Apoptosis
Apoptosis was assessed by terminal deoxynucleotidyl transferase end-labeling staining (TUNEL).
The concentrations of hepcidin in serum and supernatant were determined by ELISA.
Quantification of Intracellular Lipids
The lipids of macrophages with different treatments were extracted with the Folch method, and the intracellular TC, TG, and LDL-C were measured by enzymatic assay.
Measurement of Nonheme Iron by Atomic Absorption Spectrometry
The levels of nonheme iron in atherosclerotic plaques were measured by flame atomic absorption spectrometry.
The data are expressed as mean±SEM. An independent-samples t test was used to compare continuous data for between-group differences, and comparisons among groups involved the use of ANOVA with least-squares difference post hoc test used for multiple comparisons. P<0.05 was considered statistically significant.
Upregulation of Hepcidin Expression in Atherosclerotic Plaques
To clarify the role of hepcidin in atherogenesis, we first examined hepcidin expression in the carotid plaques in ApoE−/− mice. Relative to the homolateral carotid arteries in the control group without atherosclerotic lesions, both mRNA and protein expression levels of hepcidin were upregulated in the carotid plaques (Figure IA– IC in the online-only Data Supplement). These findings indicated a potential role of vascular hepcidin in the pathogenesis of atherosclerosis.9
Critical Role of Hepcidin in Plaque Instability and Inflammation
Second, we applied local hepcidin gain- and loss-of-function approaches by adenoviral delivery of hepcidin and its shRNA into the atherosclerotic carotid arteries to address a precise role of vascular hepcidin in plaque stability. Because GFP provides a convenient monitor for checking the efficiency of adenovirus infection, the GFP fluorescence in the carotid plaques infected with Ad-EGFP, Ad-EGFP-hepcidin (Ad-hepcidin), and Ad-EGFP-hepcidin shRNA (Ad-hepcidin shRNA) was examined. Elevated and comparable levels of fluorescent densities were observed in plaques of these infected carotid arteries, appearing at 1 week after infection and sustaining for additional 2 weeks. In our preliminary study, we measured the ratio of the GFP-positive staining area to the plaque area in the 3 treatment groups of mice 2 weeks after the infection, and the ratio was 0.67±0.15, 0.68±0.08, and 0.64±0.17 in the Ad-EGFP, Ad-hepcidin, and Ad-hepcidin shRNA groups, respectively (P>0.05), which demonstrates a similar infection efficiency among these 3 groups. At the end of the experiment, hepcidin expression in the carotid arteries was upregulated in the Ad-hepcidine group but downregulated in the Ad-hepcidin shRNA group (Figure 1A through 1C). To further characterize the intraplaque cell types of hepcidin overexpression, we double-stained hepcidin and macrophages or SMCs of atherosclerotic carotid plaques transfected with Ad-hepcidin and found that the local hepcidin gain-of-function approach led to an increase of hepcidin expression in intraplaque macrophages and SMCs (Figure 1D). In contrast, the manipulation of hepcidin expression hardly affected the serum lipid and glucose levels (Table in the online-only Data Supplement). There was no significant difference of serum hepcidin and hepatic hepcidin mRNA levels among the 3 groups of mice (Figure 1E and 1F).
It is worth noting that neither the overexpression nor knockdown of hepcidin altered the plaque size, relative to the control (Figure 2B). However, the plaque composition including macrophages, SMCs, and collagen was significantly affected by the hepcidin overexpression or knockdown. The hepcidin overexpression increased intraplaque macrophages and decreased intraplaque SMCs, whereas hepcidin knockdown exerted opposite effects (Figure 2A and 2D). There was no significant difference of total lipid levels in the plaques among the Ad-EGFP, Ad-hepcidin, and Ad-hepcidin shRNA groups (Figure 2A and 2D). It is interesting that the expression level of ox-LDL in intraplaque macrophages was substantially enhanced by hepcidin overexpression but was dramatically suppressed by hepcidin knockdown (Figure 2G and 2H). Accordingly, the plaque vulnerability index was elevated by the hepcidin overexpression but reduced by the hepcidin knockdown, respectively (Figure 2C), which suggests that hepcidin plays a critical role in plaque destabilization.
Considering a crucial role of inflammation in the pathogenesis of atherosclerosis,16 we further examined whether hepcidin overexpression or knockdown regulates vascular inflammatory responses in the atherosclerotic lesions. The results showed that the expression levels of IL-6, MCP-1, TNFα, and MMP-2 were substantially enhanced by hepcidin overexpression but were dramatically suppressed by hepcidin knockdown (Figure 2A, 2E, and 2F), which suggests that hepcidin destabilizes atherosclerotic plaques at least partly by exaggerating inflammatory responses in atherosclerotic lesions.
Essential Role of Hepcidin in ox-LDL–mediated Phenotypic Modulation of Macrophages After Erythrophagocytosis
Because of the potential link of hepcidin, macrophage iron, vascular inflammatory responses, atherosclerotic lesion progression, and plaque stability aforementioned, we further explored the molecular mechanisms by which hepcidin destabilizes plaques in macrophages. Compared with the control, the expression of hepcidin was time- and dose-dependently upregulated in macrophages treated with ox-LDL (Figure 3A). The ox-LDL–induced upregulation of hepcidin was transient, peaked at 2 hours after the stimulation, and thereafter declined to the basal level within 24 hours, with the maximum effective dose of 50 μg/mL (Figure 3A through 3C), suggesting a regulatory role of hepcidin in the ox-LDL–mediated proatherogenic effects such as oxidative stress, inflammatory cytokine secretion, and apoptosis in macrophages. Because hepcidin expression was augmented in intraplaque macrophages (Figure 1D) and the proatherogenic phenotypic switching of macrophages is closely linked with the cellular LDL oxidation and iron loading,17 the effect of ox-LDL–mediated proatherogenic activation of macrophages was determined in the setting of iron loading by erythrophagocytosis. As postulated, the ox-LDL–induced accumulation of intracellular lipids, upregulation of IL-6, MCP-1, and TNFα expression, augmentations of ROS formation, and apoptosis were further enhanced in the macrophages after erythrophagocytosis (Figure IIA–IIG in the online-only Data Supplement). Interestingly, hepcidin hardly enhanced the ox-LDL–induced proatherogenic activation of macrophages (Figure IIIA–IIIG in the online-only Data Supplement), but the enhancement became quite obvious in the setting of erythrophagocytosis (Figure 3D through 3J), indicating a unique role of hepcidin in ox-LDL–mediated phenotypic modulation of the iron-loaded macrophages. To further study the role of hepcidin in the proatherogenic activation of macrophages after erythrophagocytosis, we applied hepcidin RNA interference approach by using hepcidin siRNA in macrophages. The hepcidin knockdown efficacy was between 70–80% (Materials and Methods in the online-only Data Supplement). The silencing of hepcidin decreased the ox-LDL–induced intracellular lipids accumulation (Figure 4A) and inhibited both basal and ox-LDL–induced inflammatory cytokine expression, oxidative stress, and apoptosis in erythrophagocytosed macrophages (Figure 4B through 4G). Taken together, these results demonstrate that hepcidin is a novel positive regulator of ox-LDL–mediated proatherogenic activation of macrophages in the setting of erythrophagocytosis, contributing to the plaque instability.
Hepcidin-Upregulated Expression of Ferritin in Erythrophagocytosed Macrophages and Atherosclerotic Plaques
Finally, we explored the interplay of iron loading, ox-LDL, and hepcidin in macrophages, which might result in plaque destabilization in vitro and in vivo. We observed a time-dependent upregulation of L-ferritin and H-ferritin, which are iron-storage proteins, and FPN1, an iron-export protein. The expression of FPN1 reached a peak at 4 hours, declined thereafter, and returned to the basal level by 24 hours after erythrophagocytosis, whereas the expression of H-ferritin and L-ferritin reached a peak at 4 hours and 6 hours, respectively, and sustained at least 24 hours after erythrophagocytosis (Figure VA in the online-only Data Supplement). Hepcidin further upregulated the H-ferritin and L-ferritin expression but downregulated the FPN1 expression in the erythrophagocytosed macrophages (Figure 5G and 5H and Figure VB and VC in the online-only Data Supplement). These results demonstrated the critical role of hepcidin in regulating iron levels in the iron-loaded macrophages after erythrophagocytosis as previously described.18 Importantly, both basal expression and ox-LDL–mediated upregulation of ferritin protein levels were dramatically inhibited by the silencing of hepcidin in erythrophagocytosed macrophages (Figure 5I and 5J). Although there was no significant difference of serum iron levels in the Ad-GFP, Ad-hepcidin, and Ad-hepcidin shRNA groups (Figure 5F), the nonheme iron level and the expression of Ferritin mRNA and protein in the atherosclerotic plaque was upregulated or downregulated by the local adenoviral overexpression of hepcidin or by its shRNA, respectively (Figure 5A through 5E). These results demonstrate that hepcidin controls iron trapping in erythrophagocytosed macrophages and atherosclerotic lesions.
Considering a key role of hepcidin in elevating ox-LDL levels in intraplaque macrophages (Figure 2G and 2H) and the suppression of hepcidin inhibitor on lipid accumulation, iron retention, and ROS formation,10 we further determined the potential interaction of hepcidin, iron retention, and ox-LDL loading on proatherosclerotic activation of macrophages. Macrophage intracellular iron was scavenged by iron chelators including DFO and ferrous chelator BPDL as described elsewhere (Figure IVA and IVB in the online-only Data Supplement),14,15 The increased lipids accumulation, proinflammatory cytokine production, ROS formation, and apoptosis in erythrophagocytosed macrophages were inhibited by adding of DFO and BPDL (Figure IVC–IVI in the online-only Data Supplement), indicating that the trapped iron in erythrophagocytosed macrophages is redox active. Moreover, erythrophagocytosis per se had a minor effect on the intracellular levels of lipids including TC, TG, and LDL-C in the macrophages without ox-LDL loading (Figure IIA in the online-only Data Supplement), and the adding of DFO and BPDL hardly affected the lipid levels in the phagocytosed macrophages without ox-LDL treatment (Figure IVC in the online-only Data Supplement). However, the elevated intracellular lipid levels in macrophages due to ox-LDL loading were further augmented by erythrophagocytosis (Figure IIA in the online-only Data Supplement). These results suggest that trapped iron promotes lipid accumulation in macrophages at a setting of proatherosclerotic dysregulation of lipid metabolism. Whereas the synergistic effect of ox-LDL loading and erythrophagocytosis on intracellular lipid accumulation in macrophages was inhibited by hepcidin silencing (Figure 4A) and the iron chelators (Figure IVC in the online-only Data Supplement), it was further enhanced by hepcidin (Figure 3D). Taken together, these findings support a notion that the interaction of hepcidin, trapped iron, and accumulated lipids is critical for proatherosclerotic activation of macrophages contributing to destabilization.
In this study, we provided several novel findings regarding the hepcidin-mediated atherosclerosis as follows: (1) Hepcidin is a positive regulator of atherosclerotic plaque instability; (2) hepcidin is essential for ox-LDL–mediated phenotypic switching of iron-loaded macrophages leading to atherosclerotic plaque destabilization; (3) hepcidin upregulates ferritin trapping iron in macrophages, whereas iron loading in turn facilitates the hepcidin-mediated phenotypic switch of macrophages; and (4) the interaction of hepcidin, trapped iron, and accumulated lipids is critical for proatherosclerotic activation of macrophages contributing to destabilization. To our knowledge, these results demonstrate for the first time that hepcidin is a positive regulator of atherosclerotic plaque destabilization via regulating iron homeostasis in macrophages.
Although iron retention as a key mechanism of atherosclerosis has been proposed for decades,19 only recently did some authors find that the storage and processing of iron from erythrophagocytosis by macrophages within plaque is an important source of iron retention in atherosclerotic lesions.20 Loading of iron in macrophages promotes lipid accumulation and induces oxidative stress.17 Oxidative reactions associated with the overloaded iron and lipids facilitate macrophage apoptosis with the release of cellular contents into the lesion, which further enhances inflammatory responses. In contrast, administration of desferrioxamine, an iron chelator, attenuated inflammation and macrophage-specific gene expression in atherosclerotic lesions of ApoE−/− mice.14 A recent study found that pathological iron metabolism in macrophages contributes to vulnerability of human carotid plaque.21 However, the critical regulator of the phenotypic switching of the iron loaded macrophages remains to be verified. In this context, our results demonstrated that there is the endogenous expression in the atherosclerotic plaque and hepcidin is a critical mediator of plaque destabilization and ox-LDL–exaggerated lipids accumulation, oxidative stress, inflammation, and apoptosis in macrophages after erythrophagocytosis, thus providing a novel insight into the understanding of “iron hypothesis” in the pathogenesis of atherosclerosis.19 In addition, we observed that hepcidin downregulated FPN1 expression and upregulated L-ferritin and H-ferritin expression in macrophages after erythrophagocytes. Since hepcidin has been demonstrated to suppress iron release from macrophages after erythrophagocytosis via downregulating the expression of iron-export FPN1,6 and ferritin is an iron-storage protein,22 it is most likely that hepcidin suppresses iron release from proatherogenically activated macrophages via both inhibiting iron export and increasing iron storage. With the consideration of the fact that hepcidin did not facilitate ox-LDL–induced proatherogenic activation of macrophages until an onset of erythrophagocytosis, it is conceivable that loading iron and upregulating hepcidin might form a positive feedback loop in the phenotypic modulation of macrophages leading to the plaque instability. This notion is actually supported by a recent study demonstrating that hepcidin and macrophage iron correlate with vascular damage in high-risk individuals with metabolic alterations.9 The “iron hypothesis” that iron deficiency may play a protective role against atherosclerosis has been criticized by the lack of an increased risk for arterial structural lesions in genetic hemochromatosis.23 However, the very low level of hepcidin concentration with decreased retention of iron in macrophages in hemochromatosis may explain this paradox, though the precise mechanism remains to be explored.24 This study not only revealed a unique role of hepcidin in mediating plaque instability but also provided direct evidence to clarify the paradoxical issues observed in hemochromatosis.
As observed, the expression of IL-6, MCP-1, TNFα, and MMP-2 was enhanced by hepcidin in atherosclerotic lesions; meanwhile, several studies revealed that hepcidin could be induced by inflammation cytokines.25 Therefore, at certain stages of plaque progression, inflamed atherosclerotic tissue may upregulate the production of hepcidin, which in turn augments macrophage iron retention and iron-associated inflammation. Moreover, we observed that trapped iron was not able to augment intracellular lipid accumulation in macrophages until an ox-LDL loading, and hepcidin increased intracellular lipid level only in macrophages with trapped iron as well as ox-LDL loading. Collectively, these findings indicate that the interaction of hepcidin, trapped iron, and accumulated lipids is critical for proatherosclerotic activation of macrophages leading to plaque destabilization. Our results were consistent with a recent report that pharmacological suppression of hepcidin decreased lipid accumulation, intracellular iron, and ROS formation in macrophages.10 However, in contrast to their finding that systemic suppression of hepcidin inhibited early aortic lesion formation, our study found that local knockdown of hepcidin did not affect the size of advanced carotid plaques in mice. It is likely that short-term manipulation of hepcidin expression in an already established plaque may not be adequate to assess the role of hepcidin in plaque formation, and further studies are warranted to address this important issue.
It has been recently documented that intraplaque hemorrhage not only stimulates the progression of atherosclerosis but also promotes the transition of plaques from a stable to an unstable lesion.26 Moreover, plaques with this intraplaque hemorrhage are vulnerable to new plaque hemorrhage.27 In a rabbit model of intraplaque hemorrhage, we found that erythrocytes may induce plaque destabilization in a dose-dependent fashion.3 Many substances including iron were released from hemoglobin and taken by macrophages via CD163 receptor.28 Meanwhile, the expression of hepcidin is promoted by inflammation, which leads to the more iron retention in macrophages. Thus, whether hepcidin regulates CD163 receptor function to participate in the intraplaque hemorrhage-induced atherosclerosis progression deserves further investigation.
In summary, we showed that in a mouse model of atherosclerosis, the expression of hepcidin was upregulated in atherosclerotic plaque and that hepcidin is a positive regulator of plaque instability and inflammation. Only in the setting of erythrophagocytosis did hepcidin preferentially enhance the ox-LDL–induced proatherogenic activation of macrophages leading to plaque destabilization. Hepcidin upregulated the expression of ferritin in erythrophagocytosed macrophages and atherosclerotic plaques, and the iron trapping might be critical for the hepcidin-mediated phenotypic switching of macrophages leading to the plaque instability (Figure 6).
We thank Prof Guo Qing Liu at Beijing University, Prof Zhong Ming Qian at Hongkong Polytechnic University, and Prof Ming Xiang Zhang, Prof Fan Jiang, Prof Fan Yi, Dr Chun Xi Liu, Dr Xu Ping Wang, and Dr Hong Jiang at Shandong University for their excellent technical assistance.
Sources of Funding
This work was supported by the National 973 Basic Research Program of China (No. 2012CB518603, 2011CB503906), the National High-tech Research and Development Program of China (No. 2012AA02A510), the Program of Introducing Talents of Discipline to Universities (No.B07035), the State Program of National Natural Science Foundation of China for Innovative Research Group (No. 81021001), the State Key Program of National Natural Science of China (No. 60831003), grants of the National Natural Science Foundation of China (No. 30900607, 30971096, 30972809, 81100206, 81173251, 81170207, 81000126), grants of the Natural Science Foundation of Shandong Province, P. R. China (No. ZR2011HQ039), and the Foundation for Excellent Young Scientists of Shandong Province, P. R. China (No. BS2009SW026).
The online-only Data Supplement is available with this article at http://atvb.ahajournals.org/lookup/suppl/doi:10.1161/ATVBAHA.112.246108/-/DC1.
- Received August 26, 2011.
- Accepted February 13, 2012.
- © 2012 American Heart Association, Inc.
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