Heme is an iron-containing porphyrin of vital importance since it constitutes the prosthetic moiety of several hemoproteins. It can interact with different inactive apo-heme proteins giving rise to active hemoproteins, whose function is ultimately determined by the properties of the polypeptide bound to it (Dawson, 1988). Thanks to its ability to coordinate an iron atom inside its structure, heme is engaged in controlled oxido-reducing reactions that allow hemoproteins to work properly. For this reason, heme is involved in a multitude of crucial biological functions. In hemoglobin and myoglobin, it is used for oxygen transport and storage, respectively, whereas in cytochromes it is involved in electron transport, energy generation, and chemical transformation. In catalases and peroxidases, heme functions in hydrogen peroxide inactivation or activation, respectively, and in tryptophan pyrrolase, it catalyzes the oxidation of tryptophan (Kumar and Bandyopadhyay, 2005). Furthermore, heme is indispensable for a wide array of other important enzyme systems, such as cyclooxygenase and nitric-oxide synthase (Seed and Willoughby, 1997).

Other than acting as a prostetic group in hemoproteins, heme itself may influence the expression of many genes. In non erythroid cells, heme regulates its own production by down-regulating heme biosynthesis at the level of the rate-limiting enzyme 5-aminolevulinic acid (ALA) synthase 1 (ALAS1) and by up-regulating its metabolism (Yamamoto et al., 1982). Conversely, in erythroid cells, heme acts as a positive feedback regulator for its synthesis and inhibits its degradation (Sassa, 1976; Rutherford and Harrison, 1979). Heme may control gene expression at almost all levels by regulating transcription, mRNA stability, splicing, protein synthesis, and post-translational modification (Ponka, 1999; Zhu et al., 1999). Genes coding for globins, heme biosynthetic enzymes, heme-oxygenase (HO)-1, ferroportin, cytochromes, myeloperoxidase, and transferrin receptor are all regulated by heme. Most of these genes are regulated via heme response elements (HREs) and the mammalian transcription repressor, Bach1 (Ogawa et al., 2001). Heme responsive elements (HREs) are located in enhancer regions of genes induced by heme itself. The minimal heme response element was identified as an extended activator protein 1 (AP-1, c-Fos/c-Jun family) binding site, TGCTGAGTCAT/C. In addition to the AP-1 heptad (TGAGTCA), this element also contains an interdigitated antioxidant response element (ARE), GCnnnGTCA. This motif resembles the binding site of the transcription factors of the sMaf family (Maf recognition element, MARE; consensus sequence: TGCTGAC(G)TCAGCA), which contains an ARE core for transcription factors of the NF-E2 family (NF-E2, Nrf1, Nrf2) (Inamdar et al., 1996). The transcription factor Bach1 interacts with proteins of the Maf-related family and the resulting heterodimer binds HRE elements, thus repressing gene transcription. Under conditions of intracellular heme accumulation, heme binds to Bach1, thus leading to a conformational change, a decrease in DNA binding activity and finally its removal from HREs (Marro et al., 2010). This allows Maf-Maf, Nrf2-Maf, and other activating heterodimers (Nrf2-AP-1) to occupy the HRE sites in gene promoters and leads to increased transcription of target genes.

In addition, heme regulates differentiation and proliferation of various cell types. It stimulates neuronal differentiation of mouse neuroblastoma cells (Ishii and Maniatis, 1978), erythroid differentiation of erythroleukemia cells (Granick and Sassa, 1978), formation of erythroid colonies in mouse as well as in human bone marrow cultures (Partanen et al., 1988; Abraham et al., 1991), differentiation of 3T3 fibroblasts into adipocytes (Chen and London, 1981), and it stimulates cell growth of cultured fibroblasts (Verger et al., 1983).

All together, these functions can be regarded as the “good face” of heme, without which many critical biological processes would not occur.

In contrast to the positive functions of heme, free heme excess can cause cell damage and tissue injury since heme catalyzes the formation of reactive oxygen species (ROS), resulting in oxidative stress. Heme that is not bound to proteins is considered the labile heme pool; this portion of heme is derived from newly synthesized heme that has not yet been incorporated into hemoproteins, or heme that has been released from hemoproteins under oxidative conditions. “Free heme” is an abundant source of redox-active iron that can participate in the Fenton reaction to produce toxic free hydroxyl radicals. ROS damage lipid membranes, proteins and nucleic acids, activate cell signaling pathways and oxidant-sensitive, proinflammatory transcription factors, alter protein expression, and perturb membrane channels (Vercellotti et al., 1994; Jeney et al., 2002). Heme toxicity is further exacerbated by its ability to intercalate into lipid membranes. Due to its lipophilic nature, heme may initially lodge within the hydrophobic phospholipid bilayer. Within this highly oxidizable matrix, iron catalyzes the oxidation of cell membrane and promotes the formation of cytotoxic lipid peroxide, which enhances membrane permeability, thus promoting cell lysis and death (Balla et al., 1991; Ryter and Tyrrell, 2000; Kumar and Bandyopadhyay, 2005; Tolosano et al., 2010). Additionally, heme is a potent hemolytic agent. It affects erythrocyte membrane stability as a result of ROS formation and oxidative membrane damage. Finally, heme is strongly pro-inflammatory since it induces the recruitment of leukocytes, platelets and red blood cells to the vascular endothelium, it oxidizes low-density lipoproteins and it consumes nitric oxide, thus impairing vascular function (Figure 1).

When intracellular heme accumulation occurs, heme is able to exert its pro-oxidant and cytotoxic action. The cellular free heme pool may increase after extracellular heme overload, increased heme synthesis, accelerated hemoprotein breakdown, impaired incorporation into apo-hemoproteins, or impaired HO activity, resulting in ROS formation, oxidative damage and cell injury. Several pathological conditions are associated with hemolysis or myolysis, and tissues can subsequently be exposed to large amounts of free heme (sickle cell anemia, thalassemia, malaria, paroxysomal nocturnal hemoglobinuria, etc.) (Gozzelino et al., 2010).

In summary, heme is a double-faced molecule: physiological amounts of heme act in gene regulation or as the functional group of hemoproteins, providing essential cellular functions, whereas excessive free heme levels result in oxidative stress and tissue injury. Therefore, the amount of free heme must be tightly controlled to maintain cellular homeostasis and avoid pathological conditions. To this purpose, mammals evolved several defense mechanisms to specifically counteract free heme-mediated oxidative stress and inflammation (Wagener et al., 2003; Gozzelino et al., 2010).

Here the mechanisms involved in the maintenance of heme homeostasis and in the control of heme levels are reviewed: the regulation of both extracellular and intracellular heme content is described, with particular emphasis on the emerging role of heme transporters.

The first level of regulation of cellular heme content occurs at the level of heme synthesis control. Heme is synthesized through a series of eight enzymatic reactions (Figure 2). Work in the past decade has shown that heme synthesis is an almost ubiquitous process. The biosynthesis of heme starts in the mitochondrial matrix with the condensation of succinyl-CoA and glycine to form ALA, a reaction catalyzed by the enzyme ALAS. There are two isoforms of ALAS: Alas1 gene is located on chromosome 3 in humans and codes for an ubiquitously expressed protein whereas Alas2 gene is on the X chromosome and codes for an erythroid-specific protein (Bishop et al., 1990). The two isoforms of ALAS mainly differ for their mode of regulation, as discussed later in this section.

ALA is exported in the cytosol soon after its synthesis. The precise molecular mechanism by which ALA is transported through the two mitochondrial membranes is not completely understood. Two mitochondrial inner membrane proteins, SLC25A38 (solute carrier family 25, member 38) and the ATP-binding cassette transporter ABCB10 have been proposed to play this role.

Yeast lacking YDL119c, the ortholog of SLC25A38, shows a defect in the biosynthesis of ALA (Guernsey et al., 2009). Thus, it has been suggested that SLC25A38 could facilitate the production of ALA by importing glycine into mitochondria or by exchanging glycine for ALA across the mitochondrial inner membrane. Recently, Bayeva et al. reported that the silencing of ABCB10 causes a decrease of cellular and mitochondrial heme levels. The administration of ALA fully restores heme level in ABCB10-downregulated cells whereas Alas2 overexpression fails to do this. Thus, it has been proposed that ABCB10 could facilitate mitochondrial ALA synthesis or its export from mitochondria (Bayeva et al., 2013).

Both proteins are located on the inner mitochondrial membrane; the ALA transporter on the outer mitochondrial membrane still remains to be identified.

In the cytosol, two molecules of ALA are condensed to form the monopyrrole porphobilinogen, a reaction catalyzed by aminolevulinate dehydratase (ALAD). Then, the enzyme hydroxymethylbilane synthase (HMBS) catalyzes the head-to-tail synthesis of four porphobilinogen molecules to form the linear tetrapyrrole hydroxymethylbilane which is converted to uroporphyrinogen III by uroporphyrinogen synthase (UROS). The last cytoplasmic step, the synthesis of coproporphyrinogen III (CPgenIII), is catalyzed by uroporphyrinogen decarboxylase (UROD) (Ajioka et al., 2006). All the remaining steps of heme biosynthesis take place inside mitochondria, thus CPgenIII needs to be transported in the mitochondrial intermembrane space. It has been initially proposed that the ATP-binding cassette transporter ABCB6 could play this role (Krishnamurthy et al., 2006). However, data concerning the localization and function of ABCB6 in mitochondria are controversial. ABCB6 was also found to be expressed on the plasma membrane, in the Golgi compartment and in lysosomes. Some works even fail to detect ABCB6 in mitochondria (Paterson et al., 2007; Tsuchida et al., 2008; Kiss et al., 2012). In addition, ABCB6 has been associated to other functions unrelated to porphyrin homeostasis: ABCB6 contributes to anticancer drug resistance (Kelter et al., 2007); it was identified as the genetic basis of the Lan blood group antigen expressed on red blood cells (Helias et al., 2012); defects in Abcb6 cause an inherited developmental defect of the eye, with no known relationship with porphyrins accumulation (Wang et al., 2012). Recently, it has been reported that Abcb6^−/− mice completely lack mitochondrial ATP-driven import of CPgenIII and shows the up-regulation of compensatory porphyrin and iron pathways. Abcb6^−/− mice are phenotypically normal; increased mortality and reduced heme synthesis were observed following phenylhydrazine administration thus suggesting that Abcb6 is essential during conditions of high porphyrin demand (Ulrich et al., 2012).

In the mitochondrial intermembrane space, CPgenIII is converted to protoporphyrinogen IX by the enzyme coproporphyrinogen III oxidase (CPOX), a homodimer weakly associated with the outside of the inner mitochondrial membrane. The following oxidation of protoporphyrinogen IX to protoporphyrin IX (PPIX) is catalyzed by protoporphyrinogen oxidase (PPOX), located on the outer surface of the inner mitochondrial membrane. Finally, ferrous iron is incorporated into PPIX to form heme in the mitochondrial matrix, a reaction catalyzed by the enzyme ferrochelatase (FECH) (Ajioka et al., 2006). It has been reported that FECH is part of a multi-enzyme complex composed by the mitochondrial iron importer MITOFERRIN1 (MFRN1) and the ATP-binding cassette transporter ABCB10. The association of FECH with MFRN1 allows the coupling of mitochondrial iron import and the integration of iron into PPIX ring while ABCB10 stabilizes MFRN1 expression (Chen et al., 2009, 2010).

Once synthesized, heme must be exported through the two mitochondrial membranes for incorporation into hemoproteins. The only mitochondrial heme exporter identified to date is the mitochondrial isoform of Flvcr1 (Feline Leukemia Virus subgroup C Receptor 1) gene. Two different isoforms of FLVCR1 exist, FLVCR1a and FLVCR1b, expressed on the plasma membrane and mitochondria respectively. FLVCR1b derives from an alternative transcription start site located in the first intron of the Flvcr1 gene thus resulting in the production of a shorter protein (Chiabrando et al., 2012). Flvcr1a transcript codes for a protein with 12 transmembrane domains (Tailor et al., 1999; Quigley et al., 2000) while Flvcr1b mRNA codes for a protein with just six transmembrane domains (Chiabrando et al., 2012). The role of FLVCR1b as a mitochondrial heme exporter is suggested by in vitro data indicating that its overexpression promotes heme synthesis whereas its silencing causes detrimental heme accumulation in mitochondria. Moreover, FLVCR1b expression is upregulated following the stimulation of heme synthesis in vitro (Chiabrando et al., 2012). According to its role as a mitochondrial heme exporter, FLVCR1b is essential for erythroid differentiation both in vitro and in vivo (as discussed in section Control of Heme Export). The submitochondrial localization of FLVCR1b is still unknown as well as its ability to interact with other mitochondrial transporters. Further work is needed to definitively understand how heme is transported across the two mitochondrial membranes.

The importance of controlling the rate of heme synthesis is highlighted by the fact that mutations in many genes coding for enzymes involved in this pathway cause specific pathological conditions characterized by the accumulation of toxic heme precursors (as discussed in section Pathological Conditions Associated with Alterations of Heme Synthesis). The regulation of heme synthesis mainly occurs at the level of ALAS, the first and rate-limiting enzyme of the heme biosynthetic pathway. It has been demonstrated that heme plays an essential role in the regulation of its own synthesis by regulating the expression of ALAS.

In non-erythroid cells, heme synthesis is dependent on the activity of ALAS1. It has been reported that ALAS1 is directly regulated by heme levels through several mechanisms: heme negatively controls the transcription (Yamamoto et al., 1982), translation (Sassa and Granick, 1970; Yamamoto et al., 1983) and stability (Hamilton et al., 1991) of Alas1 mRNA. In addition, it has been shown that ALAS1 contains three heme regulatory motifs (HRMs). Heme binding to two of these HRMs, located in the mitochondrial targeting sequence of ALAS1, inhibits the transport of ALAS1 precursor protein in mitochondria (Yamauchi et al., 1980). The regulation of ALAS1 by heme represents a crucial negative feedback mechanism to maintain appropriate intracellular heme levels in non-erythroid cells, thus avoiding heme-induced oxidative damage.

In erythroid cells, heme synthesis is exclusively dependent on the activity of ALAS2. Contrary to ALAS1, heme does not inhibit the expression of ALAS2, as high amount of heme are required for the differentiation of erythroid progenitors. The expression of ALAS2 is controlled at multiple levels. The transcription of Alas2 is regulated by erythroid-specific transcription factors, like GATA1 (Surinya et al., 1998; Kaneko et al., 2013). At the post-transcriptional level, the expression of ALAS2 is regulated by iron availability. The 5' untranslated region of Alas2 mRNA contains an iron responsive element (IRE) which interacts with iron regulatory protein (IRP) 1 and 2. The IRE binding activity of IRPs is regulated by cellular iron. In iron-deficient cells, the binding of IRPs to the 5'IRE inhibits the translation of Alas2 mRNA. In iron-repleted cells, IRPs are degraded thus stimulating the translation of Alas2 mRNA. This mechanism ensures the coordination of heme synthesis to the availability of iron thus avoiding the production of potentially toxic heme precursors when iron concentrations are limiting. The regulation of the IRE-binding activity of IRPs by cellular iron occurs through several mechanisms. The IRE binding activity of IRP1 is controlled by iron-sulfur clusters assembly while that of IRP2 by its oxidation, ubiquitination and degradation by the proteasome (Hentze et al., 2010). This latter process is dependent on iron but also on heme availability. It has been reported that IRP2 contains a HRM; heme binding to the HRM mediates the oxidation of IRP2 that triggers its ubiquitination and degradation (Yamanaka et al., 2003; Ishikawa et al., 2005). Thus, during the differentiation of erythroid progenitors, increased cellular iron level stimulates the translation of Alas2 mRNA by inducing the degradation of IRPs. The following accumulation of heme contributes to the oxidation and degradation of IRP2, further enhancing heme synthesis. This positive feedback mechanism allows a sustained production of heme for hemoglobin synthesis in differentiating erythroid progenitors.

The rate of de novo heme synthesis has to be proportionate to its rate of incorporation into newly synthesized apo-hemoproteins. This is obtained at different levels via the control of heme synthesis as well as apo-hemoproteins synthesis. As reported in section Heme as a Modulator of Gene Expression and Cell Proliferation/Differentiation, heme itself can induce the transcription and the expression of several apo-hemoproteins, such as hemoglobin, myoglobin, and neuroglobin (Bruns and London, 1965; Tahara et al., 1978; Zhu et al., 1999, 2002; Correia et al., 2011), as well as cytochromes and many other heme-containing proteins. This evolutionary conserved strategy prevents intracellular heme accumulation, presumably limiting heme cytotoxicity (Figure 2). Additionally, in non-erythroid cells, heme is able to regulate its own synthesis and degradation via inhibition of ALAS1 expression/activity and induction of HO-1 expression/activity, thus properly balancing the amount of synthesized heme with that one incorporated into hemoproteins or catabolized (Zheng et al., 2008; Correia et al., 2011).

HO is the primary enzyme involved in heme degradation and plays an important role in the protection of cells from heme-induced oxidative stress. It is a 32 kDa protein, mainly located on membranes of the smooth endoplasmic reticulum, able to break down the pro-oxidant heme into the antioxidant biliverdin, the vasodilator carbon monoxide (CO) and iron (Fe²⁺) (Figure 2) (Gozzelino et al., 2010; Tolosano et al., 2010). Biliverdin is then reduced to bilirubin by the enzyme biliverdin reductase. To date, three isoforms of HO have been identified: HO-1, HO-2, and HO-3.

HO-1 is highly inducible by a variety of stimuli including oxidative stress, heat shock, hypoxia, ischemia-reperfusion, lipopolysaccharide, heavy metals, cytokines and its substrate heme. Heme is the most potent physiologic inducer of HO-1. The enzyme activity was shown to increase in many tissues, including the liver, kidney, adrenals, ovaries, lung, skin, intestine, heart, and peritoneal macrophages. HO-2 is ubiquitously expressed and participates in the normal heme capturing and metabolism. The isoenzymes HO-1 and HO-2 are products of two different genes. The two forms show only 45% aminoacid homology but they share a region with 100% secondary structure homology, that corresponds to the catalytic site (Maines, 1988). HO-3 has poor heme-degrading capacity (Wagener et al., 2003).

Herein, we focus on the inducible isoform HO-1.

HO-1 plays a vital function in heme degradation and protects against heme-mediated oxidative injury. Overexpression of HO-1 is associated to the resolution of inflammation through the generation of beneficial molecules like CO, bilirubin, and ferritin resulting from catabolism of toxic heme (Wagener et al., 2001, 2003; Kapturczak et al., 2004). Bilirubin efficiently scavenges peroxyl radicals, thereby inhibiting lipid peroxidation, attenuating heme-induced oxidative stress, cell activation and death (Dore et al., 1999; Soares et al., 2004; Kawamura et al., 2005). CO controls the activity of several heme proteins and causes vasodilation. It also exerts anti-inflammatory effects by inhibiting the expression of pro-inflammatory cytokines (Ndisang et al., 2003; Beckman et al., 2009). Finally, ferritin, by sequestering toxic free iron, limits microrganism growth and ROS production.

HO-1 confers cytoprotection against different forms of programmed cell death, including apoptosis driven by heme and tumor necrosis factor (Gozzelino and Soares, 2011). This cytoprotective effect is driven by heme degradation per se as well as by its end products, CO and biliverdin/bilirubin (Yamashita et al., 2004; Gozzelino et al., 2010). CO can exert cytoprotective effects via the modulation of cellular signal pathways, including the p38 mitogen activating protein kinase (Ndisang et al., 2003). In addition, CO can bind Fe in the heme pockets of hemoproteins, inhibiting heme release and preventing its cytotoxic effects (Seixas et al., 2009; Ferreira et al., 2011). On the other hand, biliverdin has been described to participate in an antioxidant redox cycle in which, once produced by HO, biliverdin is reduced to bilirubin by biliverdin reductase. This is followed by the subsequent oxidation of bilirubin by ROS back to biliverdin, forming a catalytic antioxidant cycle that is driven by NADPH, the reducing cofactor of biliverdin reductase. This cycle has the ability to strongly suppress the oxidizing and toxic potential of hydrogen peroxide and other ROS, thus acting as one of the most powerful anti-oxidant physiological system.

Another detoxifying system is represented by ferritin, an evolutionarily conserved Fe sequestering protein that acts as the major intracellular depot of non-metabolic iron (Balla et al., 1992a,b; Berberat et al., 2003; Cozzi et al., 2004; Pham et al., 2004). Ferritin is a multimeric protein composed of 24 subunits of two types, the heavy chain (H-Ft) and the light chain (L-Ft) and has a very high capacity for storing iron (up to 4500 mol of iron per mol of ferritin). H-Ft manifests ferroxidase activity that catalyses the oxidation of ferrous iron to ferric iron, thus favoring its storage in L-Ft (Hentze et al., 2004) and limiting its participation in the production of free radicals via Fenton reaction (Pham et al., 2004). Together, HO and ferritin allow rapid shifting of iron from heme into ferritin core where it is less available to catalyze deleterious reactions. By increasing the expression of HO-1 and ferritin, cells can survive to lethal heme-induced oxidative stress (Balla et al., 2005; Gozzelino and Soares, 2013).

Recent evidence demonstrated that also heme export out of the cell significantly contributes to the regulation of intracellular heme levels. Two heme exporters located at the plasma membrane have been identified to date: FLVCR1a and ABCG2 (ATP-Binding Cassette, subfamily G, member 2) (Figure 2).

A further level of control of intracellular heme content is represented by the modulation of heme import inside the cells (Figure 2). To date the only known proteins with a well-established function as heme importers are HRGs (Rajagopal et al., 2008; White et al., 2013), reviewed by Hamza et al. in the present Research Topic. Two other putative heme importers have been described, the Feline leukemia virus subgroup C receptor 2 (FLVCR2) and the Heme carrier protein 1/Proton-coupled folate transporter (HCP1/PCFT).

Many pathological conditions are associated with hemolysis and extracellular heme release. Among them, sickle cell anemia, β-thalassemia, malaria, paroxysomal nocturnal hemoglobinuria are the most paradigmatic (Gozzelino et al., 2010). In all these conditions, the pool of circulating Hx is diminished and, consequently, the plasma heme scavenging capacity is strongly reduced.

In the last decade, the use of a knockout mouse model for Hx was valuable to elucidate its function as well as its potential use as a therapeutic molecule in the prevention of heme adverse effects (Table 1). Additionally, other functions of Hx not clearly related to its role as an heme scavenger, have also been described thanks to the use of knock-out mice (Fagoonee et al., 2008; Spiller et al., 2011; Rolla et al., 2013).

Works on Hx^−/− mice have demonstrated that these animals have alterations in heme/iron homeostasis in the duodenum (Fiorito et al., 2013) and brain (Morello et al., 2009, 2011), are highly sensitive to acute hemolysis (Vinchi et al., 2008) and show a defective recovery after intravascular hemolysis, suffering from severe renal damage associated with iron loading and lipid peroxidation (Tolosano et al., 1999, 2002). After acute heme overload, Hx^−/− mice develop severe hepatic red blood cell congestion associated to cell iron redistribution, lipid peroxidation and inflammation. This is likely due to the increased endothelial activation and vascular permeability occurring in Hx^−/− mice compared to wild-type controls (Vinchi et al., 2008). Endothelial activation is a proinflammatory and procoagulant state of the endothelial cells lining the lumen of blood vessels. This state is mainly characterized by an increased expression of adhesion molecules on endothelial cell surface, which promotes the adherence of leukocytes as well as platelets and red blood cells, thus favoring inflammation, clot and eventually thrombus formation. According to its function as a free heme scavenger, Hx is expected to counteract heme toxicity on the vascular endothelium and increasing experimental evidence is supporting this concept.

Similarly, sickle cell disease and β-thalassemia mice show heme-driven endothelial activation, vaso-occlusion and cardiovascular dysfunction that could be efficiently recovered through the administration of an Hx-based therapy (Vinchi et al., 2013). This recent observation further strengthens the concept that heme triggers vascular inflammation and damage, and emphasizes the importance of Hx in counteracting heme-driven cardiovascular dysfunction associated with hemolytic conditions. This could have relevance, in the future, for therapeutic interventions against cardiovascular and endothelial dysfunctions in hemolytic patients (Vinchi and Tolosano, 2013).

Malaria is another well-known hemolytic condition, associated with the accumulation of high concentrations of free heme in plasma. The appearance of hemoglobin and heme in plasma has been linked to the development of cerebral malaria, which remains the most severe and difficult to treat complication of the infection (Ferreira et al., 2008; Seixas et al., 2009). The potential protective effect of both Hp and Hx in this pathology still needs to be elucidated.

Recently, hemolysis has been observed to occur after red blood cell transfusion, one of the most common therapeutic interventions in medicine. Over the last two decades, however, transfusion practices have been restricted, limiting unnecessary transfusions (Barr and Bailie, 2011). The adverse effects of transfusions seem to be mainly related to the storage period between blood donation and transfusion (Wang et al., 2012). Transfusions of old blood in animal models result in both intravascular and extravascular hemolysis and cause hypertension, acute renal failure, hemoglobinuria, and vascular injury. Conversely, old blood transfusion together with the hemoglobin scavenger Hp attenuated most of the transfusion-related adverse effects (Baek et al., 2012; Schaer et al., 2013b). Whether a similar protection, alone or in association with Hp, could be afforded by Hx still needs to be explored.

Not long ago, severe sepsis was found accompanied by hemolysis and Hx exhaustion in mice (Larsen et al., 2012). Larsen and coworkers showed that the administration of exogenous Hx was protective against organ injury and prevents the lethal outcome of severe sepsis in mice (Larsen et al., 2010). The protective effect in this model is related to the ability of Hx to counteract heme proinflammatory effects upon pathogen infection.

Furthermore, a neuroprotective effect for Hx was also described. Hx was found expressed by cortical neurons and present in mouse cerebellum, cortex, hippocampus, and striatum. Upon experimental ischemia, neurologic deficits as well as infarct volumes in the brain were increased in Hx deficient mice, indicating that Hx regulates extracellular free heme levels and the heme-Hx complexes protect primary neurons against the heme-induced toxicity (Li et al., 2009).

The rationale for the use of Hx as a therapeutic is based on the idea that it acts by scavenging circulating free heme, the ultimate mediator of hemoglobin toxicity. In particular, the crucial role of Hx in the protection from heme toxicity has relevance to pathological conditions associated with hemolysis that are often characterized by partial/total exhaustion of hemoglobin/heme scavengers. Therefore, replenishing the circulating stores of heme scavengers, thereby compensating for the loss of heme scavenging capacity in plasma, may be used as a therapeutic approach to target circulating free heme and prevent its deleterious effects (Schaer and Buehler, 2013; Schaer et al., 2013b; Vinchi and Tolosano, 2013). The recent demonstration of the effectiveness of an Hx therapy in mouse models of sickle cell anemia, β-thalassemia and sepsis opens new perspectives concerning the use of this molecule as a new therapeutic drug in hemolytic diseases.

To date, we are still far from the use of heme scavengers as therapeutics and no clinical trials are ongoing. Anyway, the possible use of these molecules for therapeutic purposes has elicited the interest of the research community in the field as well as of several pharmaceutical companies. Pre-clinical studies are ongoing with the aim of translating the protective effects of heme scavengers into clinical practice in the near future.

Humans differ quantitatively in their ability to build up an HO-1 response and variations in HO-1 expression dictate the pathologic outcome of a broad range of diseases. This differential response seems to be modulated by two polymorphisms in the HO-1 gene promoter region. Several studies demonstrated that the ability of a patient to respond strongly in terms of upregulating HO-1 may be an important endogenous protective factor.

A well described (GT)n microsatellite polymorphism in the HO-1 promoter is thought to regulate the extension of HO-1 induction as well as its response to many stimuli. Individuals with a lower number of (GT)n repeats retain the ability to more efficiently induce HO-1 than individuals with a higher number of repeats. Multiple studies demonstrate that individuals with fewer repeats are less prone to certain pathologies. It is interesting to observe that most of the pathologies affected by this polymorphism in humans overlap with those in which the outcome is exacerbated by HO-1 deletion (e.g., endotoxin shock, severe sepsis, atherosclerosis, myocardial infarction, ischemia reperfusion injury). Additionally, one study correlated the presence of the shorter (GT)n polymorphism with a longer life of the individual. Individuals who lived longer were more likely to have the “high transcriptional induction” genotype, associated with the short HO polymorphism (Kimpara et al., 1997; Hirai et al., 2003; Denschlag et al., 2004; Exner et al., 2004).

Studies comparing the outcome of several experimental pathologic conditions in HO-1^−/− or wild-type mice show that when HO-1 is lacking, the pathologic conditions studied are exacerbated and result in high incidence of mortality. Pharmacological inhibition of HO activity mimics the phenotypes associated with HO-1 deletion, while, on the other hand, induction of HO-1 or administration of its reaction products, CO and biliverdin, is protective and usually ameliorates the pathologic conditions. The cytoprotective effects of HO-1 and/or of its final products, CO and biliverdin/bilirubin have been demonstrated in a variety of diseases, including immune-mediated inflammatory diseases (rejection of transplanted organs, autoimmune diseases, asthma, arthritis, colitis, pancreatitis, recurrent abortions), infectious diseases (severe malaria, sepsis, endotoxin shock), cardiovascular diseases (atherosclerosis, myocardial infarction, endothelial dysfunction, vaso-occlusion) and ischemia-reperfusion injury (Gozzelino et al., 2010).

Several molecules that exert a beneficial effect against different diseases are known to act via HO-1 induction. Some of these synthetic compounds include sialic acid, statins and rapamycin. In addition, several molecules produced under physiologic conditions might act in a similar manner, including the anti-inflammatory cytokine interleukin (IL)-10, some prostaglandins, VEGF (vascular endothelial growth factor), stromal cell-derived factor 1, nitric oxide (NO) and NGF (nerve growth factor) (Gozzelino et al., 2010). The mechanisms via which these molecules could induce the expression of HO-1 is the inhibition of Bach1 activity and the activation of transcription factors that promote HO-1 transcription, such as Nrf2. Alternatively, therapeutic induction of endogenous HO-1 might be obtained via the delivery of non-cytotoxic amount of heme or heme-containing proteins, the so-called preconditioning. In this manner, the administration of hemoglobin or heme was shown to improve survival in response to endotoxic shock (Otterbein et al., 1995), to reduce liver and kidney injury (Nath et al., 1992) and decrease vascular stasis (Belcher et al., 2006, 2010). Similarly, the preconditioning of Hx^−/− mice with a small dose of hemin strongly decreases their susceptibility to acute heme overload, thus limiting heme-mediated tissue damage (Vinchi et al., 2008). Interestingly, a physiological paradigm of heme preconditioning is represented by sickle cell trait, in the heterozygous form. This mutation confers protection against severe forms of malaria due to the low concentrations of heme in the circulation that lead to induction of endogenous HO-1 expression and protection against Plasmodium infection (Ferreira et al., 2011). In this condition, chronic hemolysis associated to sickle cell disease induces a state of malaria tolerance by inducing HO-1 up-regulation, which directly supports heme degradation and drives the generation of anti-inflammatory and anti-oxidant metabolites. Additionally, the ability of CO to directly bind hemoglobin and inhibit its oxidation, thus avoiding heme release from oxidized hemoglobin, has been recently shown to prevent experimental cerebral malaria, thus highlighting a new and key mechanism of HO-mediated protection in pathologies associated with hemoglobin/heme release (Pamplona et al., 2007; Ferreira et al., 2008; Seixas et al., 2009).

Clinical trials based on the use of CO are currently ongoing to evaluate the potential of CO as a therapeutic agent in humans. CO is administered by inhalation for the treatment of pathologies such as pulmonary arterial hypertension, post-operative ileus and idiopathic pulmonary fibrosis (see http://www.clinicaltrials.gov website). Alternatively, in the last decade a great effort was made to design and study molecules able to bind and deliver CO in biological systems, known as CO-releasing molecules (CO-RMs) (Motterlini et al., 2005). In the next years the evaluation of the therapeutic potential of CO via inhalation or CO-RM administration will reveal whether this way may be pursued as a therapeutic strategy to counteract pathological outcome due to heme-driven toxicity.

Recently, FLVCR1 has been reported to be the causative gene for PCARP (OMIM: #609033) (Rajadhyaksha et al., 2010; Ishiura et al., 2011) (Table 2). PCARP is a childhood-onset, autosomal-recessive, neurodegenerative syndrome with the clinical features of sensory ataxia and retinitis pigmentosa. PCARP begins in infancy with areflexia and retinitis pigmentosa. During infancy, night blindness, peripheral visual loss with subsequent progressive constriction of the visual field and loss of central retinal function became apparent. The sensory ataxia caused by the degeneration of the posterior column of the spinal cord results in a loss of proprioceptive sensation. Scoliosis, camptodactyly, achalasia, gastrointestinal dysmothility and a sensory peripheral neuropathy are variable features of the disease. PCARP is considered a sensory ganglionopathy causing a degeneration of central and peripheral axons without evidence of primary demyelination (Higgins et al., 1997).

Four different homozygous mutations in FLVCR1 gene have been reported to date. Interestingly, three of these mutations (c.361A>G, c.574T>C, and c.721G>A) (Rajadhyaksha et al., 2010) occur in the first exon of FLVCR1 gene, thus probably affecting only FLVCR1a. However, we cannot exclude the possibility that these mutations could interfere with any still unknown regulatory sequences important for FLVCR1b expression. The other mutation (c1477G>C) (Ishiura et al., 2011) is in the tenth exon of FLVCR1 gene, common to both Flvcr1a and Flvcr1b transcripts (Chiabrando et al., 2012), thus probably affecting both transporters. Each mutation corresponds to a specific aminoacid substitution (Asn121Asp, Cys192Arg, Ala241Thr, and Gly493Arg) within putative transmembrane domains of FLVCR1: 1, 3, 5, and 12 transmembrane domains, respectively. Yanatori et al. (2012) investigated the consequences of the reported FLVCR1 mutations on the plasma membrane heme exporter FLVCR1a. In vitro studies indicate that all the four identified mutations affect the subcellular localization, half-life and heme-export function of FLVCR1a (Yanatori et al., 2012). However, the consequences of all these mutations on the expression, localization and activity of FLVCR1b have not been investigated yet. It has been proposed that the mutant FLVCR1a proteins fail to fold properly in the endoplasmic reticulum and are rapidly degraded in the lysosomes; loss of FLVCR1a could lead to heme overload, oxidative stress and apoptosis of photoreceptors in the retina and sensory neurons in the posterior column of the spinal cord of PCARP patients (Yanatori et al., 2012). Actually, this is merely a hypothesis and evidences of heme overload, heme-induced toxicity, and apoptosis in PCARP patients or mouse models of the disease are still lacking. Of note, apoptosis is the final and common cause of photoreceptors degeneration in several forms of retinitis pigmentosa (Marigo, 2007).

The reason why the loss of FLVCR1a could affect two specific sensory modalities, vision and proprioception, is unknown. The expression levels of Flvcr1 mRNA have been confirmed in the mouse brain (neocortex, striatum, hippocampus, and cerebellum), posterior column of the spinal cord, retina and retinal pigment epithelium. The highest levels of Flvcr1 mRNA have been found in the retina and spinal cord thus suggesting a correlation between the regional specificity of Flvcr1 mRNA expression and the selective degenerative pathology of PCARP (Rajadhyaksha et al., 2010; Gnana-Prakasam et al., 2011). However, FLVCR1 is ubiquitously expressed (Quigley et al., 2004; Chiabrando et al., 2012) and the observation that mutations in the FLVCR1 gene cause sensory ataxia and retinitis pigmentosa was completely unexpected. Two different mouse models of FLVCR1 deficiency have been generated; in both cases, the loss of specific FLVCR1 isoforms severely affects embryonic development (Keel et al., 2008; Chiabrando et al., 2012), a stronger phenotype compared to that of PCARP patients. Furthermore, the mouse models of Flvcr1 deficiency suggest an essential role of FLVCR1 isoforms in erythroid differentiation and in the maintenance of endothelial integrity (Keel et al., 2008; Chiabrando et al., 2012). PCARP patients did not suffer from anemia (Rajadhyaksha et al., 2010; Yanatori et al., 2012). On the other hand, neurological defects have not been reported in mouse models of Flvcr1 deficiency. The reasons of the discrepancies between the human disease and the mouse models need to be further investigated.

Further work is needed to understand how the loss of a ubiquitously expressed heme exporter could lead to the selective pathological features of PCARP.

A role for FLVCR1 in the pathogenesis of Diamond Blackfan Anemia (DBA; OMIM: #105650) has been proposed due to the observation that the feline leukemia virus subgroup C causes a pure cell aplasia in cats by interfering with the expression of FLVCR1 and that mice lacking both FLVCR1 isoforms phenocopy the human disease (Tailor et al., 1999; Quigley et al., 2000; Keel et al., 2008).

DBA is an autosomal dominant disorder with incomplete penetrance due to mutations in several genes coding for ribosomal proteins; the most common mutation is in ribosomal protein S19 gene (RPS19) (Campagnoli et al., 2008). DBA patients are characterized by a severe block of erythroid differentiation at the proerythroblast stage associated to congenital malformations and cancer predisposition (Flygare and Karlsson, 2007; Chiabrando and Tolosano, 2010; Narla and Ebert, 2010). Accordingly, FLVCR1 deficient mice are characterized by a block of erythroid differentiation as well as growth defects (Keel et al., 2008).

Mutations in FLVCR1 gene have not been identified in DBA patients (Quigley et al., 2005). Rey et al. (2008) identified several alternatively spliced Flvcr1 transcripts, encoding proteins with aberrant expression and function. Due to the experimental strategy, only Flvcr1a transcript was evaluated in this work. Interestingly, the aberrant alternative splicing of Flvcr1a is increased in immature erythroid cells of some DBA patients negative for RPS19 mutations, while the expression of the wild-type protein is decreased (Rey et al., 2008). The molecular mechanism leading to the aberrant alternative splicing of Flvcr1a in DBA is unknown. These data suggest that decreased FLVCR1a expression and alteration of heme metabolism could contribute to the pathogenesis of DBA.

The identification of FLVCR1b essential role in heme synthesis and erythroid differentiation, leads to the hypothesis that alteration of FLVCR1b expression could also concur to the pathogenesis of DBA (Chiabrando et al., 2012). Whether aberrant alternative splicing of Flvcr1b transcript also occurs in DBA immature erythroid cells, still needs to be addressed.

Mouse models of Flvcr1 deficiency are also characterized by defective growth (Keel et al., 2008) and skeletal malformations (Chiabrando et al., 2012). Thus, FLVCR1a could also have a role in the development of the congenital malformations observed in DBA patients.

Although FLVCR1 is far to be the causative gene for DBA, we could speculate that the aberrant expression of FLVCR1 isoforms could contribute to different pathological features of DBA.

Considering the essential role of FLVCR1 isoforms during erythroid differentiation, it will be interesting to analyze their expression also in other erythroid diseases. An alteration of the expression of FLVCR1 isoforms may play a role in the pathogenesis of disorders characterized by an imbalance between heme and globin synthesis too.

The recent finding that liver conditional Flvcr1a^−/− mice show a reduced expression and activity of cytochromes P450 suggests that heme export may have an impact on cytochrome function. About 50% of hepatic heme is used for synthesis of P450 enzymes, which metabolize exogenous and endogenous compounds, including hormones, xenobiotics, drugs, and carcinogens. It is well-known that a reduction in heme availability due to an enhanced heme degradation leads to the impairment of cytochrome function (Correia et al., 2011). On the other hand, the lack of FLVCR1a-mediated heme export in hepatocytes, although leading to an increase in the cytosolic heme pool, similarly causes a reduction in cytochrome activity. In hepatocytes, heme is likely formed in excess over its metabolic needs and heme homeostasis is ensured by a combination of synthetic, degradative, and export mechanisms. FLVCR1a, by exporting heme excess out of the cell, controls the size of the cytosolic heme pool, thus allowing proper heme synthesis and new cytochrome formation. FLVCR1a loss causes heme synthesis inhibition, due to the expansion of the cytosolic heme pool, ultimately leading to a decrease in cytochrome function (Vinchi et al., 2014). These observations have potential implications for hepatic metabolism of xenobiotics and drugs. In particular, deletion or mutations in Flvcr1a and other pathologic conditions that reduce its expression potentially cause a reduction in cytochrome activity, thus altering drug metabolism. Therefore, individuals that routinely assume drugs for therapeutic purposes may show a reduced ability to induce cytochromes P450 and to metabolize pharmaceuticals, thus being more susceptible to drug intoxication. In the near future, the mouse model of FLVCR1a deletion in hepatocytes could be applied for metabolic studies to address the importance of hepatic heme export upon drug administration.

As stated above, ABCG2 was initially shown to transport a wide range of substrates including drugs, xenobiotics, food metabolites and heme. Only recently, genetic studies in humans have identified urate as a physiological substrate of ABCG2, perhaps the most relevant. Indeed, a genome-wide association study identified common alleles in ABCG2 as associated with serum urate level and risk of gout (OMIM: #138900) (Table 2) (Dehghan et al., 2008). ABCG2 expression in the apical membrane of human proximal tubule cells (Doyle and Ross, 2003), the main site of urate handling in the kidney, is consistent with a role for ABCG2 in urate excretion. Gout is a common form of inflammatory arthritis and occurs when uric acid crystallizes in the form of monosodium urate, precipitating in joints, on tendons, and in the surrounding tissues. These crystals then trigger a local immune-mediated inflammatory reaction. Genetic studies identified a SNP in the exon 5 of ABCG2 gene that leads to a glutamine-to-lysine amino acid substitution (Q141K) (Dehghan et al., 2008). The Q141K variant has been shown to have a significant reduced capacity to transport urate and, when expressed in mammalian cells, Q141K ABCG2 expression is significantly lower than that of wild-type protein (Imai et al., 2002; Mizuarai et al., 2004; Morisaki et al., 2005; Woodward et al., 2009). Another study on the Japanese population identified a different variant as associated to hyperuricemia and gout (Yamagishi et al., 2010).

Many additional SNPs in ABCG2 gene have been identified and associated to altered drug resistance (Woodward et al., 2011). Future studies will have to address whether additional SNPs also affect serum urate concentration in human. Finally, how these polymorphisms affect heme transport remains to be determined.

Mutations in the FLVCR2 gene have been associated to the Fowler syndrome (OMIM: #225790) (Table 2), a rare lethal autosomal-recessive disorder of brain angiogenesis, first described in 1972 (Fowler et al., 1972). The Fowler syndrome is a cerebral proliferative glomeruloid vasculopathy resulting in abnormally thickened and aberrant perforating vessels, forming glomeruloid structures throughout the central nervous system. These defects lead to hydranencephaly and hydrocephaly and are restricted to the central nervous system parenchyme. In addition to cerebral proliferative glomeruloid vasculopathy, fetuses affected by the Fowler syndrome also show limb deformities.

Several kinds of mutations were identified in cases of Fowler syndrome (Meyer et al., 2010; Thomas et al., 2010), but it is not clear how these mutations could affect FLVCR2 expression, functions or localization.

The mechanism by which FLVCR2 mutations could lead to abnormal angiogenesis is also unknown. Some hypotheses have been put forward.

First, it has been postulated that the role of FLVCR2 in calcium trafficking, proposed by Brasier et al. (2004), could explain its involvement in Fowler syndrome. Indeed, the altered angiogenesis in Fowler syndrome has been supposed to be due to a deficit in pericytes, cells essential for capillary stabilization and remodeling during brain angiogenesis (Thomas et al., 2010). These cells interact with endothelial cells, whose proliferation and motility are calcium-dependent events. Moreover, the Fowler syndrome has been associated with varying degrees of calcification throughout the central nervous system (Harding et al., 1995).

Although possible, this hypothesis seems to be inadequate because the involvement of FLVCR2 in calcium transport has not been demonstrated. Moreover, it is not clear whether the effects on pericytes observed in fetuses affected by Fowler syndrome is the primary cause or an effect of the disease.

A second hypothesis to explain FLVCR2 implication in the Fowler syndrome is based on its heme import activity. It has been observed in some cases of Fowler syndrome that there is a deficiency in the heme-dependent complexes IV of the mitochondrial electron transport chain, essential for the production of adenosine triphosphate by oxidative phosphorylation. Since young neurons that migrate to the neocortex are dependent on oxidative phosphorylation, it has been speculated that Flvcr2 disruption could lead to dysfunction of the mitochondrial electron transport chain, thus causing the neurodegeneration and the developmental abnormalities found in Fowler syndrome (Duffy et al., 2010).

Finally, a third hypothesis is that alteration on FLVCR2 heme-import activity could account for cellular iron overload, a condition known to be associated to neurodegeneration and bone abnormalities (Duffy et al., 2010).

Although all conceivable, the three hypotheses need experimental verification and it remains unclear why the damaged areas in Fowler syndrome are restricted to the central nervous system and whether the proliferative vasculopathy is primary or secondary to the neurodegeneration.

The only known disorder associated to mutations in the HCP1/PCFT gene relates to its role in the transport of folates, while the effects of the loss of its heme-import functions seem to be negligible, suggesting the instauration of alternative compensatory mechanisms to support cellular heme-iron uptake in the absence of HCP1/PCFT.

Mutations in the HCP1/PCFT gene have been associated to the hereditary folate malabsorption syndrome (HFM; OMIM: #229050), a rare autosomal recessive disorder first described in 1961 (Luhby et al., 1961) (Table 2).

Folates are essential cofactors required for key epigenetic and biosynthetic processes, such as de novo synthesis of purines and pyrimidines, methionine and deoxythymidylate monophosphate. Mammals do not synthesize folates, and body needs are met by duodenal and jejunal absorption of folates contained in the diet.

The basis for HFM is the impaired intestinal folate absorption, resulting in severe systemic and cerebrospinal fluid folate deficiency in a few months after birth.

Patients affected by HFM show anemia, diarrhea, pancytopenia, hypoimmunoglobulinemia, and frequent neurological dysfunctions like developmental delays, cognitive impairment, and epilepsy. Many kinds of mutations have been reported (Qiu et al., 2006; Zhao et al., 2007; Shin et al., 2011; Diop-Bove et al., 2013), occurring in different sites of HCP1/PCFT gene and with different effects on the protein encoded by the mutated alleles.

If untreated, the disease is fatal and, if treatment is delayed, the neurologic deficits can become permanent. Normalization of blood, and rarely cerebrospinal fluid, folate level could be achieved by administration of low doses of parenteral folates or of pharmacological doses of oral folates. In the latter case, folates are probably absorbed by the bidirectional Reduced Folate Carrier 1 (RFC1), a folate importer alternative to HCP1/PCFT, which is expressed in the upper small intestine. RFC1 shows a lower affinity for folates as compared with HCP1/PCFT, but can compensate for HCP1/PCFT loss in HFM patients treated with high doses of folates (Qiu et al., 2006; Zhao et al., 2007).

The Hcp1/Pcft^−/− mouse represents the murine model for this HFM syndrome (Salojin et al., 2011) (Table 1). Hcp1/Pcft^−/− mice die by 10–12 weeks of age and show a haematological profile which closely resembles that observed in human HFM patients.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.