Ferroportin inhibitor vamifeport ameliorates ineffective erythropoiesis in a mouse model of ß-thalassemia with blood transfusions.

ß-thalassemia is an inherited anemia characterized by ineffective erythropoiesis. Blood transfusions are required for survival in transfusion-dependent ß-thalassemia and are also occasionally needed in patients with non-transfusion-dependent ß-thalassemia. Patients with transfusion-dependent b-thalassemia often have elevated transferrin saturation (TSAT) and non-transferrin-bound iron (NTBI) levels, which can lead to organ iron overload, oxidative stress, and vascular damage. Vamifeport is an oral ferroportin inhibitor that was previously shown to ameliorate anemia, ineffective erythropoiesis, and dysregulated iron homeostasis in the Hbbth3/+ mouse model of ß-thalassemia, under non-transfused conditions. Our study aimed to assess the effects of oral vamifeport on iron-related parameters (including plasma NTBI levels) and ineffective erythropoiesis following blood transfusions in Hbbth3/+ mice. A single dose of vamifeport prevented the transient transfusion-mediated NTBI increase in Hbbth3/+ mice. Compared with vehicle treatment, vamifeport significantly increased hemoglobin levels and red blood cell counts in transfused mice. Vamifeport treatment also significantly improved ineffective erythropoiesis in the spleens of Hbbth3/+ mice, with additive effects observed when treatment was combined with repeated transfusions. Vamifeport corrected leukocyte counts and significantly improved iron-related parameters (serum transferrin, TSAT and erythropoietin levels) versus vehicle treatment in Hbbth3/+ mice, irrespective of transfusion status. In summary, vamifeport prevented transfusion-mediated NTBI formation in Hbbth3/+ mice. When given alone or combined with blood transfusions, vamifeport also ameliorated anemia, ineffective erythropoiesis, and dysregulated iron homeostasis. Administering vamifeport together with repeated blood transfusions additively ameliorated anemia and ineffective erythropoiesis in this mouse model, providing preclinical proof-of-concept for the efficacy of combining vamifeport with blood transfusions in ß-thalassemia.

The oral ferroportin inhibitor vamifeport improves hemodynamics in a mouse model of sickle cell disease.

Sickle cell disease (SCD) is an inherited hemolytic anemia caused by a single point mutation in the ß-globin gene of hemoglobin that leads to synthesis of sickle hemoglobin (HbS) in red blood cells (RBCs). HbS polymerizes in hypoxic conditions, leading to intravascular hemolysis, release of free hemoglobin and heme, and increased adhesion of blood cells to the endothelial vasculature, which causes painful vaso-occlusion and organ damage. HbS polymerization kinetics are strongly dependent on the intracellular HbS concentration; a relatively small reduction in cellular HbS concentration may prevent HbS polymerization and its sequelae. We hypothesized that iron restriction via blocking ferroportin, the unique iron transporter in mammals, might reduce HbS concentration in RBCs, thereby decreasing hemolysis, improving blood flow, and preventing vaso-occlusive events. Indeed, vamifeport (also known as VIT-2763), a clinical-stage oral ferroportin inhibitor, reduced hemolysis markers in the Townes model of SCD. The RBC indices of vamifeport-treated male and female Townes mice exhibited changes attributable to iron-restricted erythropoiesis: decreased corpuscular hemoglobin concentration mean and mean corpuscular volume, as well as increased hypochromic and microcytic RBC fractions. Furthermore, vamifeport reduced plasma soluble VCAM-1 concentrations, which suggests lowered vascular inflammation. Accordingly, intravital video microscopy of fluorescently labeled blood cells in the microvasculature of Townes mice treated with vamifeport revealed diminished adhesion to the endothelium and improved hemodynamics. These preclinical data provide a strong proof-of-concept for vamifeport in the Townes model of SCD and support further development of this compound as a potential novel therapy in SCD.

Acute Hemolysis and Heme Suppress Anti-CD40 Antibody-Induced Necro-Inflammatory Liver Disease.

Clearance of red blood cells and hemoproteins is a key metabolic function of macrophages during hemolytic disorders and following tissue injury. Through this archetypical phagocytic function, heme is detoxified and iron is recycled to support erythropoiesis. Reciprocal interaction of heme metabolism and inflammatory macrophage functions may modify disease outcomes in a broad range of clinical conditions. We hypothesized that acute hemolysis and heme induce acute anti-inflammatory signals in liver macrophages. Using a macrophage-driven model of sterile liver inflammation, we showed that phenylhydrazine (PHZ)-mediated acute erythrophagocytosis blocked the anti-CD40 antibody-induced pathway of macrophage activation. This process attenuated the inflammatory cytokine release syndrome and necrotizing hepatitis induced by anti-CD40 antibody treatment of mice. We further established that administration of heme-albumin complexes specifically delivered heme to liver macrophages and replicated the anti-inflammatory effect of hemolysis. The anti-inflammatory heme-signal was induced in macrophages by an increased intracellular concentration of the porphyrin independently of iron. Overall, our work suggests that induction of heme-signaling strongly suppresses inflammatory macrophage function, providing protection against sterile liver inflammation.

Agonistic Anti-CD40 Antibody Triggers an Acute Liver Crisis With Systemic Inflammation in Humanized Sickle Cell Disease Mice.

Sickle cell disease (SCD) is an inherited hemolytic disorder, defined by a point mutation in the ß-globin gene. Stress conditions such as infection, inflammation, dehydration, and hypoxia trigger erythrocyte sickling. Sickled red blood cells (RBCs) hemolyze more rapidly, show impaired deformability, and increased adhesive properties to the endothelium. In a proinflammatory, pro-coagulative environment with preexisting endothelial dysfunction, sickled RBCs promote vascular occlusion. Hepatobiliary involvement related to the sickling process, such as an acute sickle hepatic crisis, is observed in about 10% of acute sickle cell crisis incidents. In mice, ligation of CD40 with an agonistic antibody leads to a macrophage activation in the liver, triggering a sequence of systemic inflammation, endothelial cell activation, thrombosis, and focal ischemia. We found that anti-CD40 antibody injection in sickle cell mice induces a systemic inflammatory and hemodynamic response with accelerated hemolysis, extensive vaso-occlusion, and large ischemic infarctions in the liver mimicking an acute hepatic crisis. Administration of the tumor necrosis factor-α (TNF-α) blocker, etanercept, and the heme scavenger protein, hemopexin attenuated end-organ damage. These data collectively suggest that anti-CD40 administration offers a novel acute liver crisis model in humanized sickle mice, allowing for evaluation of therapeutic proof-of-concept.

Hemolysis transforms liver macrophages into antiinflammatory erythrophagocytes.

During hemolysis, macrophages in the liver phagocytose damaged erythrocytes to prevent the toxic effects of cell-free hemoglobin and heme. It remains unclear how this homeostatic process modulates phagocyte functions in inflammatory diseases. Using a genetic mouse model of spherocytosis and single-cell RNA sequencing, we found that erythrophagocytosis skewed liver macrophages into an antiinflammatory phenotype that we defined as MarcohiHmoxhiMHC class IIlo erythrophagocytes. This phenotype transformation profoundly mitigated disease expression in a model of an anti-CD40-induced hyperinflammatory syndrome with necrotic hepatitis and in a nonalcoholic steatohepatitis model, representing 2 macrophage-driven sterile inflammatory diseases. We reproduced the antiinflammatory erythrophagocyte transformation in vitro by heme exposure of mouse and human macrophages, yielding a distinctive transcriptional signature that segregated heme-polarized from M1- and M2-polarized cells. Mapping transposase-accessible chromatin in single cells by sequencing defined the transcription factor NFE2L2/NRF2 as a critical driver of erythrophagocytes, and Nfe2l2/Nrf2 deficiency restored heme-suppressed inflammation. Our findings point to a pathway that regulates macrophage functions to link erythrocyte homeostasis with innate immunity.

Line-selective macrophage activation with an anti-CD40 antibody drives a hemophagocytic syndrome in mice.

Hemophagocytic syndromes comprise a cluster of hyperinflammatory disorders, including hemophagocytic lymphohistiocytosis and macrophage activation syndrome. Overwhelming macrophage activation has long been considered a final common pathway in the pathophysiology of hemophagocytic syndromes leading to the characteristic cytokine storm, laboratory abnormalities, and organ injuries that define the clinical spectrum of the disease. So far, it is unknown whether primary macrophage activation alone can induce the disease phenotype. In this study, we established a novel mouse model of a hemophagocytic syndrome by treating mice with an agonistic anti-CD40 antibody (Ab). The response in wild-type mice is characterized by a cytokine storm, associated with hyperferritinemia, high soluble CD25, erythrophagocytosis, secondary endothelial activation with multiple organ vaso-occlusion, necrotizing hepatitis, and variable cytopenias. The disease is dependent on a tumor necrosis factor-α-interferon-γ-driven amplification loop. After macrophage depletion with clodronate liposomes or in mice with a macrophage-selective deletion of the CD40 gene (CD40flox/flox/LysMCre), the disease was abolished. These data provide a new preclinical model of a hemophagocytic syndrome and reinforce the key pathophysiological role of macrophages.

Revisiting the putative role of heme as a trigger of inflammation.

Activation of the innate immune system by free heme has been proposed as one of the principal consequences of cell-free hemoglobin (Hb) exposure. Nonetheless, in the absence of infection, heme exposures within a hematoma, during hemolysis, or upon systemic administration of Hb (eg, as a Hb-based oxygen carrier) are typically not accompanied by uncontrolled inflammation, challenging the assumption that heme is a major proinflammatory mediator in vivo. Because of its hydrophobic nature, heme liberated from oxidized hemoglobin is rapidly transferred to alternative protein-binding sites (eg, albumin) or to hydrophobic lipid compartments minimizing protein-free heme under in vivo equilibrium conditions. We demonstrate that the capacity of heme to activate human neutrophil granulocytes strictly depends on the availability of non protein-associated heme. In human endothelial cells as well as in mouse macrophage cell cultures and in mouse models of local and systemic heme exposure, protein-associated heme or Hb do not induce inflammatory gene expression over a broad range of exposure conditions. Only experiments in protein-free culture medium demonstrated a weak capacity of heme-solutions to induce toll-like receptor-(TLR4) dependent TNF-alpha expression in macrophages. Our data suggests that the equilibrium-state of free and protein-associated heme critically determines the proinflammatory capacity of the metallo-porphyrin. Based on these data it appears unlikely that inflammation-promoting equilibrium conditions could ever occur in vivo.

Heme accumulation in endothelial cells impairs angiogenesis by triggering paraptosis.

Heme is required for cell respiration and survival. Nevertheless, its intracellular levels need to be finely regulated to avoid heme excess, which may catalyze the production of reactive oxygen species (ROS) and promote cell death. Here, we show that alteration of heme homeostasis in endothelial cells due to the loss of the heme exporter FLVCR1a, results in impaired angiogenesis. In vitro, FLVCR1a silencing in endothelial cells causes defective tubulogenesis and poor viability due to intracellular heme accumulation. Consistently, endothelial-specific Flvcr1a knockout mice show aberrant angiogenesis responsible for hemorrhages and embryonic lethality. Importantly, we demonstrate that impaired heme export leads to endothelial cell death by paraptosis and provide evidence that endoplasmic reticulum (ER) stress precedes heme-induced paraptosis. These findings highlight a crucial role for the cytosolic heme pool in the control of endothelial cell survival and in the regulation of the angiogenic process. Interfering with endothelial heme export represents a valuable model for a deeper understanding of the molecular mechanisms underlying heme-triggered paraptosis and, in the future, might provide a novel tool for the modulation of angiogenesis in pathophysiologic conditions.

Data demonstrating the anti-oxidant role of hemopexin in the heart.

The data presented in this article are related to the research article entitled Hemopexin counteracts systolic dysfunction induced by heme-driven oxidative stress (G. Ingoglia, C. M. Sag, N. Rex, L. De Franceschi, F. Vinchi, J. Cimino, S. Petrillo, S. Wagner, K. Kreitmeier, L. Silengo, F. Altruda, L. S. Maier, E. Hirsch, A. Ghigo and E. Tolosano, 2017) [1]. Data show that heme induces reactive oxygen species (ROS) production in primary cardiomyocytes. H9c2 myoblastic cells treated with heme bound to human Hemopexin (Hx) are protected from heme accumulation and oxidative stress. Similarly, the heme-driven oxidative response is reduced in primary cardiomyocytes treated with Hx-heme compared to heme alone. Our in vivo data show that mouse models of hemolytic disorders, ß-thalassemic mice and phenylhydrazine-treated mice, have low serum Hx associated to enhanced expression of heme- and oxidative stress responsive genes in the heart. Hx-/- mice do not show signs of heart fibrosis or overt inflammation. For interpretation and discussion of these data, refer to the research article referenced above.

Hemopexin counteracts systolic dysfunction induced by heme-driven oxidative stress.

Heart failure is a leading cause of morbidity and mortality in patients affected by different disorders associated to intravascular hemolysis. The leading factor is the presence of pathologic amount of pro-oxidant free heme in the bloodstream, due to the exhaustion of the natural heme scavenger Hemopexin (Hx). Here, we evaluated whether free heme directly affects cardiac function, and tested the therapeutic potential of replenishing serum Hx for increasing serum heme buffering capacity. The effect of heme on cardiac function was assessed in vitro, on primary cardiomyocytes and H9c2 myoblast cell line, and in vivo, in Hx-/- mice and in genetic and acquired mouse models of intravascular hemolysis. Purified Hx or anti-oxidants N-Acetyl-L-cysteine and α-tocopherol were used to counteract heme cardiotoxicity. In mice, Hx loss/depletion resulted in heme accumulation and enhanced reactive oxygen species (ROS) production in the heart, which ultimately led to severe systolic dysfunction. Similarly, high ROS reduced systolic Ca2+ transient amplitudes and fractional shortening in primary cardiomyocytes exposed to free heme. In keeping with these Ca2+ handling alterations, oxidation and CaMKII-dependent phosphorylation of Ryanodine Receptor 2 were higher in Hx-/- hearts than in controls. Administration of anti-oxidants prevented systolic failure both in vitro and in vivo. Intriguingly, Hx rescued contraction defects of heme-treated cardiomyocytes and preserved cardiac function in hemolytic mice. We show that heme-mediated oxidative stress perturbs cardiac Ca2+ homeostasis and promotes contractile dysfunction. Scavenging heme, Hx counteracts cardiac heme toxicity and preserves left ventricular function. Our data generate the rationale to consider the therapeutic use of Hx to limit the cardiotoxicity of free heme in hemolytic disorders.

Hemopexin therapy reverts heme-induced proinflammatory phenotypic switching of macrophages in a mouse model of sickle cell disease.

Hemolytic diseases, such as sickle cell anemia and thalassemia, are characterized by enhanced release of hemoglobin and heme into the circulation, heme-iron loading of reticulo-endothelial system macrophages, and chronic inflammation. Here we show that in addition to activating the vascular endothelium, hemoglobin and heme excess alters the macrophage phenotype in sickle cell disease. We demonstrate that exposure of cultured macrophages to hemolytic aged red blood cells, heme, or iron causes their functional phenotypic change toward a proinflammatory state. In addition, hemolysis and macrophage heme/iron accumulation in a mouse model of sickle disease trigger similar proinflammatory phenotypic alterations in hepatic macrophages. On the mechanistic level, this critically depends on reactive oxygen species production and activation of the Toll-like receptor 4 signaling pathway. We further demonstrate that the heme scavenger hemopexin protects reticulo-endothelial macrophages from heme overload in heme-loaded Hx-null mice and reduces production of cytokines and reactive oxygen species. Importantly, in sickle mice, the administration of human exogenous hemopexin attenuates the inflammatory phenotype of macrophages. Taken together, our data suggest that therapeutic administration of hemopexin is beneficial to counteract heme-driven macrophage-mediated inflammation and its pathophysiologic consequences in sickle cell disease.

Haptoglobin, hemopexin, and related defense pathways-basic science, clinical perspectives, and drug development.

Hemolysis, which occurs in many disease states, can trigger a diverse pathophysiologic cascade that is related to the specific biochemical activities of free Hb and its porphyrin component heme. Normal erythropoiesis and concomitant removal of senescent red blood cells (RBC) from the circulation occurs at rates of approximately 2 × 10(6) RBCs/second. Within this physiologic range of RBC turnover, a small fraction of hemoglobin (Hb) is released into plasma as free extracellular Hb. In humans, there is an efficient multicomponent system of Hb sequestration, oxidative neutralization and clearance. Haptoglobin (Hp) is the primary Hb-binding protein in human plasma, which attenuates the adverse biochemical and physiologic effects of extracellular Hb. The cellular receptor target of Hp is the monocyte/macrophage scavenger receptor, CD163. Following Hb-Hp binding to CD163, cellular internalization of the complex leads to globin and heme metabolism, which is followed by adaptive changes in antioxidant and iron metabolism pathways and macrophage phenotype polarization. When Hb is released from RBCs within the physiologic range of Hp, the potential deleterious effects of Hb are prevented. However, during hyper-hemolytic conditions or with chronic hemolysis, Hp is depleted and Hb readily distributes to tissues where it might be exposed to oxidative conditions. In such conditions, heme can be released from ferric Hb. The free heme can then accelerate tissue damage by promoting peroxidative reactions and activation of inflammatory cascades. Hemopexin (Hx) is another plasma glycoprotein able to bind heme with high affinity. Hx sequesters heme in an inert, non-toxic form and transports it to the liver for catabolism and excretion. In the present review we discuss the components of physiologic Hb/heme detoxification and their potential therapeutic application in a wide range of hemolytic conditions.

Heme exporter FLVCR1a regulates heme synthesis and degradation and controls activity of cytochromes P450.

BACKGROUND & AIMS: The liver has one of the highest rates of heme synthesis of any organ. More than 50% of the heme synthesized in the liver is used for synthesis of P450 enzymes, which metabolize exogenous and endogenous compounds that include natural products, hormones, drugs, and carcinogens. Feline leukemia virus subgroup C cellular receptor 1a (FLVCR1a) is plasma membrane heme exporter that is ubiquitously expressed and controls intracellular heme content in hematopoietic lineages. We investigated the role of Flvcr1a in liver function in mice. METHODS: We created mice with conditional disruption of Mfsd7b, which encodes Flvcr1a, in hepatocytes (Flvcr1a(fl/fl);alb-cre mice). Mice were analyzed under basal conditions, after phenylhydrazine-induced hemolysis, and after induction of cytochromes P450 synthesis. Livers were collected and analyzed by histologic, quantitative real-time polymerase chain reaction, and immunoblot analyses. Hepatic P450 enzymatic activities were measured. RESULTS: Flvcr1a(fl/fl);alb-cre mice accumulated heme and iron in liver despite up-regulation of heme oxygenase 1, ferroportin, and ferritins. Hepatic heme export activity of Flvcr1a was closely associated with heme biosynthesis, which is required to sustain cytochrome induction. Upon cytochromes P450 stimulation, Flvcr1a(fl/fl);alb-cre mice had reduced cytochrome activity, associated with accumulation of heme in hepatocytes. The expansion of the cytosolic heme pool in these mice was likely responsible for the early inhibition of heme synthesis and increased degradation of heme, which reduced expression and activity of cytochromes P450. CONCLUSIONS: In livers of mice, Flvcr1a maintains a free heme pool that regulates heme synthesis and degradation as well as cytochromes P450 expression and activity. These findings have important implications for drug metabolism.

Acute-phase protein hemopexin is a negative regulator of Th17 response and experimental autoimmune encephalomyelitis development.

Hemopexin (Hx) is an acute-phase protein synthesized by hepatocytes in response to the proinflammatory cytokines IL-6, IL-1ß, and TNF-α. Hx is the plasma protein with the highest binding affinity to heme and controls heme-iron availability in tissues and also in T lymphocytes, where it modulates their responsiveness to IFN-γ. Recent data have questioned regarding an anti-inflammatory role of Hx, a role that may be both heme-binding dependent and independent. The aim of this study was to investigate the role of Hx in the development of a T cell-mediated inflammatory autoimmune response. During experimental autoimmune encephalomyelitis (EAE), the mouse model of multiple sclerosis, Hx content in serum increased and remained high. When EAE was induced in Hx knockout (Hx(-/-)) mice, they developed a clinically earlier and exacerbated EAE compared with wild-type mice, associated to a higher amount of CD4(+)-infiltrating T cells. The severe EAE developed by Hx(-/-) mice could be ascribed to an enhanced expansion of Th17 cells accounting for both a higher disposition of naive T cells to differentiate toward the Th17 lineage and a higher production of Th17 differentiating cytokines IL-6 and IL-23 by APCs. When purified human Hx was injected in Hx(-/-) mice before EAE induction, Th17 expansion, as well as disease severity, were comparable with those of wild-type mice. Taken together, these data indicate that Hx has a negative regulatory role in Th17-mediated inflammation and prospect its pharmacological use to limit the expansion of this cell subset in inflammatory and autoimmune disease.