Hemoglobin as a source of iron overload in multiple sclerosis: does multiple sclerosis share risk factors with vascular disorders?

Abstract

Although iron is known to be essential for the normal development and health of the central nervous system, abnormal iron deposits are found in and around multiple sclerosis (MS) lesions that themselves are closely associated with the cerebral vasculature. However, the origin of this excess iron is unknown, and it is not clear whether this is one of the primary causative events in the pathogenesis of MS, or simply another consequence of the long-lasting inflammatory conditions. Here, applying a systems biology approach, we propose an additional way for understanding the neurodegenerative component of the disease caused by chronic subclinical extravasation of hemoglobin, in combination with multiple other factors including, but not limited to, dysfunction of different cellular protective mechanisms against extracellular hemoglobin reactivity and oxidative stress. Moreover, such considerations could also shed light on and explain the higher susceptibility of MS patients to a wide range of cardiovascular disorders.

Introduction

“When partners can’t agree

Their dealings come to naught

And trouble is their labor’s only fruit”.

Ivan Krylov’s fable “Swan, Pike, and Crawfish”.

Multiple sclerosis (MS) is a human disease characterized by active degradation of the myelin sheath of the central nervous system, and northern-latitude countries such as Canada are affected disproportionately [1, 2]. The disease was first formally framed by Jean-Martin Charcot about 150 years ago, and then a unifying review was expounded by Russell Brain in the 1930s [3–6]. Yet the early cellular events remain unknown, and the causes of the disease are agreed to be multifactorial. About 7 years ago, Prof. Anat Achiron, in the editorial to an issue of Autoimmunity Reviews, compared MS with the mythological Daedalus’s labyrinth that has never been conquered [7]. The disease has many different facets, some of which tend to lead our views of it (cf. [6]).

The commonest conception of MS is derived from its initial animal model (experimental autoimmune encephalomyelitis, EAE), and defines it to be a demyelinating autoimmune disease mediated by T cell response against myelin proteins [8–11]. In this “outside-in” mechanism of disease, autoantibodies and T cells directed to myelin may be responsible for demyelination and subsequent damage to neuropil [12, 13]. Prolonged demyelination results, eventually, in complete axonal degeneration [14]. The inflammatory face of MS, together with use of anti-inflammatory drugs to relieve the symptoms, strongly supports the autoimmune character of the disease.

However, there is histopathological evidence that MS could alternatively or additionally be considered a degenerative disorder rather than strictly an autoimmune disease [15–18]. Thus, a compelling “inside-out” model of disease progression has arisen, which suggests that MS is caused by a cytodegenerative process aimed at the oligodendrocyte-myelin complex (Fig. 1) [19, 20]. Gradual demyelination can then lead to an autoimmune response and a cycle of further degeneration, characteristic of the most common relapsing-remitting manifestation of MS. This model suggests that the autoimmune and/or inflammatory response is a secondary reaction to the primary process that causes death of oligodendrocytes and demyelination of axons in the central nervous system [13, 20–23].

Fig. 1.

Model illustrating how MS may be primarily caused by a cytodegenerative process aimed at the oligodendrocyte/myelin complex (adapted from Dr. Stys, Calgary [20]). Gradual demyelination and disruption can then lead to an autoimmune response and a cycle of further degeneration, characteristic of the most common relapsing-remitting manifestation of MS. Chronic demyelination results eventually in complete axonal degeneration

The etiology of MS remains unsolved despite the many different methodologies that have been used to study it, including but not limited to immunological, genetic, clinical, epidemiological, environmental, and psychological (e.g., [24–31]). The difficulty arises in trying to integrate the knowledge acquired by diverse approaches on varied populations, or even from the studies of completely different diseases that may help to understand the pathogenesis of MS [26, 28]. Moreover, it is agreed that there is no single factor that causes the disease, but instead one must look for a group of factors such as genetic, nutritional, environmental, etc., that only in unfortunate combination could lead to illness (e.g., [32, 33]). Such a holistic, non-reductionist line of reasoning (generally called “systems biology”) could enable us to interpret the same results in a slightly different way, and suggest additional new mechanisms by which MS can arise and progress [26, 34, 35]. Systems biology is also being concurrently applied to other complex neurological diseases, such as Parkinson’s disease [36–39].

Iron in MS

Since Charcot’s first histological characterization of multiple sclerosis [3], a relationship has been observed between cerebral vasculature and MS lesions [40–42]. Moreover, an abnormal iron accumulation has been shown histologically and biochemically in MS lesions, particularly in the walls of the dilated veins in cerebral MS plaques [41, 43–55]. Studies of MS patients show evidence of iron accumulation even in grey matter structures such as the thalamus, globus pallidus, red nucleus, substantia nigra, putamen, caudate nucleus, and in the hippocampus, which go beyond any age-related effects [48, 56]. Indeed, the brains of patients with MS show iron staining in macrophages and microglia, around sites of inflammation, and near demyelinated plaques in grey and white matter [53]. Iron is normally present in the brain in high concentrations, since it is essential for normal oligodendroglial and myelin development [57–60]. The deleterious effects of abnormal iron accumulations on MS metabolism in the later stages of the disease have recently been reviewed [61–63].

There is no unequivocal answer to the question about the potential origin of this excess iron, and to whether this is one of the primary causative events in the pathogenesis of MS, or simply another consequence of the long-lasting inflammatory conditions. On the one hand, the accumulation of iron could be attributed to the early preclinical phase of MS that later will induce inflammatory changes, which in turn will result in increased permeability of the blood–brain barrier (BBB). On the other hand, an abnormal permeability of the BBB by itself could result in slow, subclinical infiltration of the peripheral blood constituents into the central nervous system. Therefore, sometimes MS is considered to be a disease of the BBB [64, 65], but in general, the extent of iron accumulation in grey matter structures and lesions has been shown to be a good predictor of disability progression in MS, as well as the extent of lesion accumulation and level of cell death [53, 54, 62, 66]. Of note is the recent identification of polymorphisms in ferroportin, a transmembrane iron transporter: the FPN1-8GG homozygous genotype has been shown to be associated with an increased MS susceptibility risk of more than fourfold [67].

Iron deposition has been observed in other demyelinating diseases, such as Hurst acute hemorrhagic leukoencephalitis [52], and in neurodegenerative diseases such as Alzheimer’s and Parkinson’s [37, 68–70], as well as in cerebrovascular disease, and during normal aging (references in [71, 72]). It has been suggested that normal age-related iron accumulation may further exacerbate the disease process in patients with Parkinson’s and Alzheimer’s diseases [68, 71], as well as with progressive forms of MS [17]. The potential effect of iron chelators has been studied for different non-demyelinating neurodegenerative diseases with abnormal iron accumulation (e.g., as reviewed in [63, 73]). These studies showed mixed effects that probably depended on the different metabolic routes and the mechanism of action of the iron chelator employed in each case. However, the general pattern was that an agent capable of penetrating the BBB had a more beneficial effect.

Here, we propose that the scenario described in Fig. 2 could be involved in the pathogenesis of MS. In this model, local iron overload in MS lesions is related to the very early stages of disease, and is not just a result of long-lasting inflammation caused by autoimmune attack [47, 48, 53, 74]. Several studies support the idea that iron overload in MS occurs already in the initial stages. For example, susceptibility-weighted magnetic-resonance imaging (MRI) is a powerful method of analyzing iron and vasculature in MS lesions [47, 48, 51, 53, 66, 69, 75–77], and has recently demonstrated an abnormal iron accumulation in adolescent MS [78] and in clinically isolated syndrome [79]. Furthermore, although the EAE model is not fully representative of MS, it is noteworthy that an iron-deficient diet is the most effective way (at least more effective than pharmacological compounds) in inhibiting or preventing EAE [80], and that susceptibility-weighted MRI has demonstrated the presence of iron deposits and deoxyhemoglobin in lesions in afflicted mice [81]. The importance of iron is emphasized even more in the studies that have tested different iron chelators (deferoxamine, deferiprone, dexrazoxane, and clioquinol) in EAE models induced by different antigen administration protocols ([82–86], reviewed in [63]). All four agents showed an effect on disease course, but the mechanism of their action remains elusive. It is unknown whether iron chelators act directly by inactivating free iron and lowering the amount of iron deposits, thus preventing/inhibiting oxidative stress, or if they suppress the immunological response.

Fig. 2.

Myelin damage by extravasation of erythrocytes and release of hemoglobin and heme: oxidative damage to lipids and proteins; membrane damage and cell death; inflammation and release of immunogenic peptides; protective mechanisms. BBB blood–brain barrier, RBC red blood cells, Hb hemoglobin, Hpt haptoglobin, Hpx hemopexin, HO heme-oxygenase, ROS reactive oxygen species. Hemin (ferric protoporphyrin IX) is the initial breakdown product of extracellular hemoglobin, and biliverdin, CO, and Fe²⁺ are the products of enzymatic (HO-1) heme catabolism

Hemoglobin as a source of iron overload in MS lesions

We suggest next that the major source of that iron can be hemoglobin (Hb), which is the major protein in erythrocytes. Subclinical capillary and venous hemorrhages leading to blood extravasation have been previously recorded, and potentially could result in the release of extracellular Hb [40, 42, 44]. Hemoglobin is a major iron-containing protein, and we posit here that its long-term release and degradation may potentially be an additional source for the iron accumulation and neurodegeneration seen in MS (cf. [70]). In the later, clinical phases of the disease, none-heme iron from intracellular stores in dying oligodendrocytes is released [62]. Here, our argument pertains to the pre-clinical stages before symptoms are manifested.

Any type of seemingly minor yet chronic cerebrovascular abnormality and/or damage to the blood–brain barrier, whether one that resulted from the preexisting inflammatory processes or another from yet unknown factors, can potentially lead to intravascular hemolysis, or to extravasation of erythrocytes and extravascular hemolysis. This situation resembles several pathologies that are completely different from MS: atherosclerotic lesion formation (intravascular hemolysis), chronic venous disorders, and chronic hemorrhagic micro-stroke (intra- or extravascular hemolysis), all resulting in extracellular hemoglobin. Under these conditions, as a result of hemolysis, hemoglobin will appear in its ferrous (Fe²⁺) form that can readily be oxidized to the ferric (Fe³⁺) or ferryl (Fe⁴⁺) forms [87]. Extracellular hemoglobin is a reactive molecule due to three major properties: (1) the oxidative activity of intact hemoglobin (and/or its chains) as a classic peroxidase (as horse radish peroxidase—HRP) or via formation of a ferryl radical; (2) the oxidative activity of heme released from the hemoprotein (and/or its chains); and (3) the oxidative reactivity of free iron released from heme.

Each or all three mechanisms could be involved also in MS progression. For instance, intact hemoglobin can act as peroxidase under oxidative stress or inflammatory conditions [88]. Also, heme can be released from hemoglobin and, being a hydrophobic molecule, can readily enter hydrophobic domains of biological membranes [89], or be complexed by essential structural molecules such as myelin basic protein [90, 91]. In other words, free heme can enter the myelin sheath and cause oxidative damage directly to the lipids and proteins [92]. Those oxidatively damaged proteins could initiate the secondary autoimmune response. Lastly, uncomplexed heme is not a stable molecule, and deteriorates or can be enzymatically oxidized to produce biliverdin (which is quickly converted to bilirubin), ferrous iron (Fe²⁺), and carbon monoxide (CO). Under this scenario, free iron can give rise to oxidative stress and generation of free radicals [93]. Unconjugated bilirubin has been shown to affect oligodendrocyte differentiation and axonal migration in culture [94].

Although mechanistically these three ways of hemoglobin-induced damage are different, the outcome could be the same, namely local oxidative stress, inflammation, and tissue damage. Thus, considering the fact that the hemoglobin extravasation is a minor but chronic process, over a certain period of time the trauma can progress to a clinical stage when severe demyelination and inflammation become noticeable. There is no evidence for any major abnormalities in cerebrospinal vasculature that are unique to MS patients [56, 95–97]. If there were, then this direct hemoglobin-induced damage could explain the pathogenesis of MS and be considered the cause of the disease. The true situation is definitely more complicated and involves further risk factors or pathological processes, e.g., the expression of protective proteins such as haptoglobin (Hpt), hemopexin (Hpx), and heme-oxygenase-1 (HO-1), as depicted in Fig. 2.

Further risk factors that can contribute to MS onset and progression

The order of events in the cascade of the pathological processes that leads to MS is tremendously important. However, even without any necessity for primacy, iron or its potential precursor hemoglobin definitely are not inert byproducts in the system, and could be responsible for an exacerbation of inflammation, oxidative stress, and neurodegeneration. Then, the pertinent question is: do dysfunctions in inherent mechanisms involved in protection from extracellular hemoglobin-induced damage represent additional risk factors that shift the balance towards MS?

There are several known physiological mechanisms to cope with extracellular hemoglobin and to prevent damage associated with it. The first defense line will be haptoglobin (Hpt), one of the few acute-phase reactive proteins conserved in all vertebrates [98]. The strong association of Hpt with Hb, and the high conservation of the hpt gene across species, indicate that recognition and binding affinity are key physiological roles of Hpt. It appears that, by binding extracellular hemoglobin, Hpt can serve as a vascular antioxidant [99, 100]. Haptoglobin has been shown to completely inhibit the oxidative activity of Hb toward lipids [99] as well as the ApoB protein [101], and can neutralize Hb toxicity in primary neuronal culture [102]. Humans have two different Hpt alleles, designated as Hpt1 and Hpt2 [98, 103]. Homozygotes for Hpt1 and Hpt2 alleles form Hpt1-1 and Hpt2-2 phenotypes, respectively, and individuals heterozygous for the alleles display a mixed Hpt1-2 phenotype.

In the past decade, a direct link has been found between haptoglobin phenotype and susceptibility to cardiovascular diseases. The Hpt1-1 phenotype has been associated with less frequent and less severe complications than the Hpt2-2 phenotype [103]. These differences are well expressed under oxidative conditions existing in diabetic patients [104]. Altogether, it seems established that individuals with the Hpt2-2 phenotype have a higher risk to develop iron-related vascular pathologies. Regarding MS, it has been documented that Hpt levels are altered in the plasma of patients [104, 105] and, using the EAE model in Hpt-deficient mice, Hpt has been shown to be involved in reducing the severity of an autoimmune inflammatory process [106]. Therefore, it will be of interest to study further the correlation between Hpt alleles or polymorphism and MS, particularly since epidemiological studies show that the frequency of the Hpt1 allele is lower in those regions with a high incidence of MS [103].

Hemopexin (Hpx) is the second defense line against Hb-induced oxidative damage following intravascular hemolysis [72, 107–109]. It has the highest binding affinity to heme amongst plasma proteins. Its neuroprotective effect has been shown in animal models of intra-cranial hemorrhage and ischemia [108–110]. It has been shown that hpx knock-out mice have underdeveloped myelin, and that oligodendrocytes were the most susceptible cells for oxidative damage due to iron overload [111]. More recently, Hpx has been shown to be a new differentiation factor for oligodendrocytes, and to be essential for myelination [112]. Moreover, hemopexin-deficient mice have been found to develop EAE more severely [113].

Another important player in the protection from extracellular hemoglobin and oxidative stress-related damage is the cellular-inducible enzyme heme oxygenase-1 (HO-1), also known as heat-shock protein 32 (HSP32) [92] (the HO-2 isoform is constitutively expressed). This enzyme is one of the key proteins in mediation of cell survival and resistance to oxidative stress. HO-1 catalyzes the rate-limiting step of heme catabolism, leading to the formation of CO along with bilirubin and Fe²⁺ in vivo; the last is sequestered rapidly by ferritin, which is co-induced with HO-1 [114]. HO-1 is induced in cells by several stress factors such as heme and H₂O₂ [115], and can provide protection against different forms of cellular stress [116]. Mice that are deficient in HO-1 develop a chronic inflammatory state [117], and the only human reported to lack HO-1 activity died of an inflammatory syndrome [118].

It has been speculated that the protective functions provided by HO-1 in vivo could be mediated through the products of heme enzymatic catabolism, i.e., CO-related signaling and bilirubin being an antioxidant. However, the anti-oxidative effect could be also achieved by elimination of oxidatively active heme. Again, the levels of this enzyme have been shown to be increased in MS, though it is not known whether as a response to oxidative stress or to extracellular hemoglobin [119–123]. Thus, since changes in expression level or in activity of HO-1 in MS patients could lead to ineffective scavenging of an active heme, or to overproduction of CO that can directly inhibit activity of different heme-containing proteins, this protective pathway requires more research to understand if this could be another risk factor in the concert of unfortunate events that results in demyelinating disease.

Vascular comorbidities in MS: shared risk factors?

In the light of the above, it is not surprising that MS patients have a significantly higher frequency of cardiovascular diseases [124, 125], and a much higher risk of death from cardiovascular diseases compared to an age-matched cohort [126, 127]. Another recent study has evaluated the association between the rate of disability progression in MS patients and five different conditions associated with cardiovascular ailments (diabetes, hypertension, heart disease, hypercholesterolemia, and peripheral vascular disease) [128]. The results unequivocally indicated that participants reporting one or more vascular comorbidities at the time of MS diagnosis, or at any time during the disease course, exhibited an increased risk of faster progression to ambulatory disability. Furthermore, cholesterol and lipoprotein metabolism is linked to MS progression. High total cholesterol levels in MS patients, for instance, are associated with increased disability progression [129] and a higher number of MS lesions [130]. Moreover, in other autoimmune diseases, such as systemic lupus erythematosus and rheumatoid arthritis, changes in lipid metabolism and increased risk for cardiovascular disorders were reported [131, 132]. All in all, it is widely accepted now that the risk of arterial and venous vascular diseases is much higher in patients with MS [133]. However, the biological mechanisms to describe this relationship have not yet been elucidated and require further study.

The vascular role of hemoglobin may be a source of shared risk factors between MS and vascular diseases, as well as the increased risk of the latter (including myocardial infarction, deep venous thrombosis and pulmonary embolism) after the first years of MS diagnosis [124, 133–135]. For example, it is known that endothelial dysfunction and lipoprotein oxidative modifications are key players in the pathogenesis of atherosclerosis, and extracellular hemoglobin has a central role in both processes [136]. Cerebral endothelial dysfunction is also involved in the pathology of MS [137] and, inflammation, oxidative stress, and immunological components are evident in both MS and atherosclerosis. The presence of hemoglobin exacerbates the inflammatory response and activates the innate immune system through Toll-like receptor 4, which is present on microglia and other innate immune cells [138, 139]. However, it is still unclear whether the pathogenesis of MS and other vascular disorders is similar and influenced by the same risk factors, or if these conditions could increase the risk for each other.

Concluding remarks

In summary, we hypothesize that chronic extravasation of hemoglobin, in combination with multiple other factors including, but not limited to, dysfunction of different cellular protective mechanisms against extracellular hemoglobin reactivity and oxidative stress, could represent an additional facet of MS pathology. Together with different dietary habits and other environmental variables such as sun exposure, these factors can play their synergistic role in causing MS and could explain the higher susceptibility of MS patients to a wide range of cardiovascular disorders. Finally, such a more comprehensive picture of the complexity of MS with more attention to iron dysregulation in the central nervous system will allow one to re-postulate questions about its origin and discover new potential avenues for medical intervention (cf. [6]).

Abbreviations

BBB

Blood–brain barrier

EAE

Experimental autoimmune/allergic encephalomyelitis

Hb

Hemoglobin

HO

Heme oxygenase

Hpt

Haptoglobin

Hpx

Hemopexin

MRI

Magnetic resonance imaging

MS

Multiple sclerosis