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Review
. 2021 Aug 20;35(6):387-414.
doi: 10.1089/ars.2020.8167. Epub 2021 Mar 22.

Non-Transferrin-Bound Iron in the Spotlight: Novel Mechanistic Insights into the Vasculotoxic and Atherosclerotic Effect of Iron

Francesca Vinchi  1   2
Affiliations
Review

Non-Transferrin-Bound Iron in the Spotlight: Novel Mechanistic Insights into the Vasculotoxic and Atherosclerotic Effect of Iron

Francesca Vinchi. Antioxid Redox Signal. .

Abstract

Significance: While atherosclerosis is an almost inevitable consequence of aging, food preferences, lack of exercise, and other aspects of the lifestyle in many countries, the identification of new risk factors is of increasing importance to tackle a disease, which has become a major health burden for billions of people. Iron has long been suspected to promote the development of atherosclerosis, but data have been conflicting, and the contribution of iron is still debated controversially. Recent Advances: Several experimental and clinical studies have been recently published about this longstanding controversial problem, highlighting the critical need to unravel the complexity behind this topic. Critical Issues: The aim of the current review is to provide an overview of the current knowledge about the proatherosclerotic impact of iron, and discuss the emerging role of non-transferrin-bound iron (NTBI) as driver of vasculotoxicity and atherosclerosis. Finally, I will provide detailed mechanistic insights on the cellular processes and molecular pathways underlying iron-exacerbated atherosclerosis. Overall, this review highlights a complex framework where NTBI acts at multiple levels in atherosclerosis by altering the serum and vascular microenvironment in a proatherogenic and proinflammatory manner, affecting the functionality and survival of vascular cells, promoting foam cell formation and inducing angiogenesis, calcification, and plaque destabilization. Future Directions: The use of additional iron markers (e.g., NTBI) may help adequately predict predisposition to cardiovascular disease. Clinical studies are needed in the aging population to address the atherogenic role of iron fluctuations within physiological limits and the therapeutic value of iron restriction approaches. Antioxid. Redox Signal. 35, 387-414.

Keywords: atherosclerosis; calcification; cardiovascular disease; endothelial dysfunction; inflammation; intraplaque macrophages; iron; iron-aggravated atherosclerosis; iron-loaded VSMC; nontransferrin-bound iron (NTBI); oxidized LDL; reactive oxygen species (ROS).

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Conflict of interest statement

No competing financial interests exist.

Figures

FIG. 1.
FIG. 1.
NTBI accumulates in arteries and exacerbates atherosclerosis. (A, B) Representative images of entire aorta and en-face aortic arch of ApoE-null and ApoE-null FPNC326S mice stained with the lipophilic dye Sudan IV. Plaques are visible in bright red. More and bigger plaques can be observed in ApoE-null FPNC326S mice. (C, D) Representative images of (C) innominate artery and (D) common carotid artery sections of ApoE-null and ApoE-null FPNC326S mice stained for iron with diaminobenzidine-enhanced Perls' staining. An enlarged picture shows iron deposition in brown in the smooth muscle layer of the artery in ApoE-null FPNC326S mice (C). While iron accumulates predominantly in the media layer (see arrowheads), the atherosclerotic plaque is almost completely iron spared (D). ApoE, apolipoprotein E; FPN, ferroportin; NTBI, nontransferrin-bound iron. Color images are available online.
FIG. 2.
FIG. 2.
Proatherosclerotic action of NTBI. NTBI exerts a complex multifactorial action in atherosclerosis by (i) altering the composition of the serum in terms of cholesterol amount and oxidation of lipids and proteins; (ii) causing oxidative stress, activation, and apoptosis of ECs, which trigger vascular permeability and monocyte adhesion; (iii) affecting the functional phenotype and survival of VSMCs, which induce intraplaque monocyte recruitment and calcification formation; (iv) inducing macrophage shift toward a proinflammatory phenotype with impaired cholesterol handling properties, which facilitates evolution into foam cells. EC, endothelial cell; VSMC, vascular smooth muscle cell. Color images are available online.
FIG. 3.
FIG. 3.
Molecular mechanisms underlying heme- and iron-driven EC and macrophage activation. NHBH and NTBI mediate the proinflammatory activation of ECs and macrophages by producing ROS and activating the TLR4/MyD88/MAPK/ERK/NFkB and inflammasome signaling pathways. Heme and iron induce ROS generation non-enzymatically, through the Fenton reaction and by converting organic hydroperoxides into free radicals, and enzymatically, through the activation of the p-Syk/PKC/NOX pathway. TLR4 activation by heme/iron promotes MAPKs and NFkB activation, which in turn induce the transcription of inflammation-related genes, including inflammatory cytokines and chemokines (e.g., IL-6, TNFα, IL-1β, IL-8) and adhesion molecules (e.g., E-selectin, P-selectin, ICAM-1, VCAM-1). Although heme and iron trigger ROS formation independently of TLR4 activation, ROS production is required for full MAPK activation and cytokine induction, suggesting a synergistic action of the two pathways. By inducing ROS, heme and iron also contribute to NLRP3/inflammasome activation, leading to the maturation of pro-IL1β to IL1β via caspase-1-mediated cleavage. TLR4 inhibitors and antioxidants by blocking the major heme/iron-induced pathways, and chelators (transferrin, deferoxamine, deferasirox, deferiprone, hemopexin) by scavenging heme/iron prevent the activation of ECs and macrophages in conditions where free heme and iron are elevated. ERK, extracellular-signal-regulated kinase; ICAM-1, intercellular adhesion molecule 1; IL, interleukin; MAPK, mitogen-activated protein kinase; MyD88, myeloid differentiation primary response 88; NFkB, nuclear factor kappa-light-chain-enhancer of activated B cells; NHBH, nonhemopexin-bound iron; NLRP3, NLR Family Pyrin Domain Containing 3; NOX, NADPH oxidase; PKC, protein kinase C; p-Syk = phosphorylated spleen tironase kinase; ROS, reactive oxygen species; TLR4, toll-like receptor 4; TNFα, tumor necrosis factor alpha; VCAM-1, vascular adhesion molecule 1. Color images are available online.
FIG. 4.
FIG. 4.
NTBI triggers intraplaque lipid accumulation, cell apoptosis, macrophage recruitment, VSMC phenotypic switch, and vascular calcification. (A, B) Representative images of common carotid artery of ApoE-null and ApoE-null FPNC326S mice stained with (A) Masson's trichrome, (B) Von Cossa, and (C) Tunel stains. (D, E) Representative images of innominate arteries of ApoE-null and ApoE-null FPNC326S mice stained with antibodies against (D) Mac-2 and (E) α-Sma. (A) The Masson trichrome stain shows reduced collagen deposition (blue) and increased lipid droplets (white) within the atherosclerotic plaque of ApoE-null FPNC326S compared with ApoE-null control mice. Arrowheads show lipid-filled areas. (B) The Von Cossa stain shows the presence of dark brown-stained intraplaque calcifications, which are bigger and more frequent in ApoE-null FPNC326S mice. (C) The Tunel stain reveals the presence of multiple Tunel-positive dark apoptotic cells within the plaque of the ApoE-null FPNC326S mice, which are almost absent in the ApoE-null mice. Arrowheads show Tunel-positive cells. (D) Mac-2 stains show a higher number of Mac-2-positive macrophages in the plaque of ApoE-null FPNC326S mice compared with ApoE-null mice. Arrowheads point at Mac-2-positive cells. (E) α-Sma stains show α-Sma-positive cells in the plaque of ApoE-null and ApoE-null FPNC326S mice. Arrowheads point at α-Sma-positive cells. The partial overlapping of Mac-2 and α-Sma stain suggests that VSMCs might undergo a phenotypic switching toward macrophage-like cells. αSMA, α smooth muscle actin; Mac2, macrophage 2 protein or Galectin 3. Color images are available online.
FIG. 5.
FIG. 5.
Role of macrophage iron content in atherosclerosis. (A) In early/mid-stage atherosclerotic plaques, exposure to NHBH and NTBI directs macrophages toward a proinflammatory phenotypic switching, with potential proatherosclerotic action. Iron and heme stimulate the activation of the TLR/NFkB signaling pathway, which is responsible for macrophage inflammatory activation. In a synergistic manner, oxLDL and iron activate the TLR4 pathway, which in turn triggers the autocrine release of hepcidin. This results in the exacerbation of iron accumulation, ROS production, and inflammatory response through a positive feedback loop. In addition, iron retention accelerates foam cell formation by (i) promoting LDL oxidation and (ii) inducing cholesterol mishandling through increased CD36-mediated cholesterol uptake and decreased ABC transporter ABCA1/ABCG1-mediated reverse cholesterol efflux via interference with the CYP27A1/27HC and PPARγ/LXRα signaling, respectively. Iron and lipids therefore show a synergistic action in accelerating foam cell formation. ROS and TLR4 signaling pathways play major roles in iron-driven macrophage inflammatory activation and foam cell formation, which are prevented by chelators, antioxidants, and TLR4 inhibitors. (B) In advanced hemorrhagic plaques, exposure to the Hp–Hb or Hx–heme complexes directs macrophages toward an iron-recycling phenotype characterized by increased ability to take up Hb via the CD163 receptor and export iron via FPN, which lead to reduced intracellular iron and ROS formation. These macrophages, defined as M(Hb) or Mhem macrophages, show reduced lipid retention, decreased production of inflammatory cytokines, and increased secretion of the anti-inflammatory atheroprotective cytokine IL-10. Reduced intracellular iron and ROS lower inflammatory cytokine production, and improve lipid handling by reducing cholesterol loading via CD36 downregulation and increasing reverse cholesterol efflux via ABC transporter upregulation. While these nonfoam M(Hb) macrophages are in principle atheroprotective, the progressive intracellular iron depletion leads to HIF1α stabilization and VEGF secretion. VEGF exerts proatherosclerotic effects by inducing vascular permeabilization, intraplaque neoangiogenesis, and immune cell recruitment. Whether M(Hb) macrophages play an initial protective effect, which turns into a proatherosclerotic one when severe iron depletion is achieved, remains to be determined. Whereas iron depletion and low hepcidin levels are desirable in early/mid-stage atherosclerosis to activate an atheroprotective phenotypic switching of macrophages, iron balance in late-stage atherosclerosis is preferred to prevent VEGF-related atherosclerotic effects. ABC, ATP-binding cassette; Hb, hemoglobin; HIF1α, hypoxia-inducible factor 1α; Hp, haptoglobin; Hx, hemopexin; ox-LDL, oxidized low-density lipoprotein; VEGF, vascular endothelial growth factor. Color images are available online.
FIG. 6.
FIG. 6.
Cascade of events leading to iron-aggravated atherosclerosis. NTBI accumulates in ECs and VSMCs, which eventually undergo apoptosis. Apoptotic ECs and VSMCs produce elevated levels of VEGF and MCP1, which trigger vasopermeabilization and immune cell recruitment, respectively. Increased vascular permeability induces the subendothelial infiltration of LDL, promoting plaque development. Iron-induced NO reduction contributes to proatherogenic mechanisms, including endothelial activation, vascular dysfunction, and arterial stiffness. Under these conditions, monocytes recruited to the plaque differentiate into macrophages and develop a proinflammatory phenotype due to NTBI exposure. This phenotype is associated with poor cholesterol handling ability and foam cell formation. Necrotic core and calcification appear as consequence of NTBI-induced VSMC apoptosis, foam cell development, and collagen loss. These events synergize to promote plaque instability and increased propensity to rupture, with thrombus formation. MCP1, monocyte chemoattractant protein 1; NO, nitric oxide. Color images are available online.
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