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People living with sickle cell disease (SCD) face intermittent acute pain episodes due to vaso-occlusion primarily treated palliatively with opioids. Hemolysis of sickle erythrocytes promotes release of heme, which activates inflammatory cell adhesion proteins on endothelial cells and circulating cells, promoting vaso-occlusion. In this study, plasma-derived hemopexin inhibited heme-mediated cellular externalization of P-selectin and von Willebrand factor, and expression of IL-8, VCAM-1, and heme oxygenase-1 in cultured endothelial cells in a dose-responsive manner. In the Townes SCD mouse model, intravenous injection of free hemoglobin induced vascular stasis (vaso-occlusion) in nearly 40% of subcutaneous blood vessels visualized in a dorsal skin-fold chamber. Hemopexin administered intravenously prevented or relieved stasis in a dose-dependent manner. Hemopexin showed parallel activity in relieving vascular stasis induced by hypoxia-reoxygenation. Repeated IV administration of hemopexin was well tolerated in rats and non-human primates with no adverse findings that could be attributed to human hemopexin. Hemopexin had a half-life in wild-type mice, rats, and non-human primates of 80–102 h, whereas a reduced half-life of hemopexin in Townes SCD mice was observed due to ongoing hemolysis. These data have led to a Phase 1 clinical trial of hemopexin in adults with SCD, which is currently ongoing.

Background Germinal matrix intraventricular hemorrhage (GM-IVH) is associated with deposition of redox active cell-free hemoglobin (Hb), derived from hemorrhagic cerebrospinal fluid (CSF), in the cerebrum and cerebellum. In a recent study, using a preterm rabbit pup model of IVH, intraventricularly administered haptoglobin (Hp), a cell-free Hb scavenger, partially reversed the damaging effects observed following IVH. Together, this suggests that cell-free Hb is central in the pathophysiology of the injury to the immature brain following GM-IVH. An increased understanding of the causal pathways and metabolites involved in eliciting the damaging response following hemorrhage is essential for the continued development and implementation of neuroprotective treatments of GM-IVH in preterm infant. Methods We exposed immature primary rat mixed glial cells to hemorrhagic CSF obtained from preterm human infants with IVH (containing a mixture of Hb-metabolites) or to a range of pure Hb-metabolites, incl. oxidized Hb (mainly metHb with iron in Fe3+), oxyHb (mainly Fe2+), or low equivalents of heme, with or without co-administration with human Hp (a mixture of isotype 2-2/2-1). Following exposure, cellular response, reactive oxygen species (ROS) generation, secretion and expression of pro-inflammatory cytokines and oxidative markers were evaluated. Results Exposure of the glial cells to hemorrhagic CSF as well as oxidized Hb, but not oxyHb, resulted in a significantly increased rate of ROS production that positively correlated with the rate of production of pro-inflammatory and oxidative markers. Congruently, exposure to oxidized Hb caused a disintegration of the polygonal cytoskeletal structure of the glial cells in addition to upregulation of F-actin proteins in microglial cells. Co-administration of Hp partially reversed the damaging response of hemorrhagic CSF and oxidized Hb. Conclusion Exposure of mixed glial cells to oxidized Hb initiates a pro-inflammatory and oxidative response with cytoskeletal disintegration. Early administration of Hp, aiming to minimize the spontaneous autoxidation of cell-free oxyHb and liberation of heme, may provide a therapeutic benefit in preterm infant with GM-IVH.

Hemoglobin (Hb) is one of the most abundant proteins in the human body. When red blood cells rupture, cell-free Hb may initiate adverse pathophysiological reactions. Pathophysiology triggered by cell-free Hb plays an important role in modifying the phenotype of sickle cell disease (SCD). SCD is caused by a single nucleotide mutation of the β-globin gene resulting in Hemoglobin-S (HbS) instead of the normal HbA found in healthy individuals. Polymerization of HbS shortens the lifespan of sickle red blood cells and promotes intra- and extravascular hemolysis. In cell-free Hb ferrous Hb (Fe2+) is oxidized into ferric Hb (Fe3+) promoting the dissociation and transfer of heme into lipid compartments where it triggers lipid peroxidation and generation of cytotoxic and pro-inflammatory reaction products. These processes promote endothelial cell activation and damage. The endogenous plasma protein hemopexin exhibits the highest binding affinity for heme and binds heme in a 1:1 binding ratio. Heme bound to hemopexin is rendered relatively non-reactive and is delivered safely to hepatocytes for endocytosis and degradation. To investigate the endothelial-protective function of hemopexin based on its ability to scavenge heme, we exposed human umbilical vein endothelial cells (HUVEC) in vitro to heme(NaOH) in the presence or absence of different hemopexin doses. As a read-out, different markers for endothelial cell activation were analyzed by either flow cytometry or multiplexed particle-based flow cytometry (Luminex). Briefly, confluent HUVEC were preincubated with hemopexin at different concentrations for 5 min before stimulation with heme(NaOH) for 25 min. Following stimulation cells were analyzed by flow cytometry for expression of membrane bound P-Selectin, a robust marker of endothelial cell activation. Alternatively, heme(NaOH) stimulation of hemopexin-preincubated HUVEC was conducted for 16 h and cell culture supernatants were analyzed by Luminex for three additional well-characterized plasma markers of endothelial cell activation: pro-inflammatory cytokine IL-8, cell adhesion molecule VCAM-1 and glycoprotein Von Willebrand factor (vWF). In the absence of hemopexin, heme(NaOH) consistently induced robust cell surface expression of P-Selectin and elevated levels of soluble IL-8, VCAM-1 and vWF. However, hemopexin completely blocked the stimulatory potential of heme as HUVEC exposed to pre-formed heme:hemopexin complexes showed unchanged P-Selectin expression levels compared to negative control samples. We found that hemopexin reduced heme(NaOH)-mediated P-selectin expression on HUVEC in a dose-dependent fashion. Once an equimolar ratio between heme and hemopexin was reached, P-selectin expression was abolished as shown in figure 1. In addition to P-Selectin, hemopexin also had a strong effect to reduce the heme-induced expression of IL-8, VCAM-1 and vWF to background levels. Thus, the presented data underlines on the one hand the stimulatory capacity of heme(NaOH) on endothelial cells and demonstrates on the other hand the potential of hemopexin to efficiently neutralize free heme. In a stoichiometric fashion, hemopexin potently prevents the pro-inflammatory effect of heme on endothelial cells. Hence, our study suggests a protective role of hemopexin for endothelial cells exposed to elevated levels of cell-free heme due to intravascular hemolysis. Additional experiments are required to elucidate the effect of hemopexin on the endothelium in more detail. Combined with our other lines of data, our results further support the investigation of hemopexin as a potential therapeutic agent in the treatment of sickle cell disease. Disclosures Adam: CSL Behring AG: Current Employment. Gentinetta:CSL Behring: Current Employment. Diditchenko:CSL Behring AG: Current Employment. Schaub:CSL Behring AG: Current Employment. Kato:CSL Behring AG: Current Employment. Brinkman:CSL Behring: Current Employment. Zuercher:CSL Behring AG: Current Employment.

Polymerization of hemoglobin-S (HbS) in the deoxy conformation shortens the lifespan of sickle red blood cells and promotes intravascular and extravascular hemolysis. When sickle red blood cells are lysed intravascularly, HbS is released into the vascular space where it can consume nitric oxide and be oxidized to higher oxidative forms. During these reactions, ferric (Fe3+) HbS is formed, which readily releases heme. The released heme can activate the innate immune pattern recognition receptor toll-like receptor 4 (TLR4) on endothelium, leading to P-selectin expression, NF-ĸB activation, and microvascular stasis in sickle cell disease (SCD) mice with implanted dorsal skin-fold chambers (DSFCs). Heme-induced TLR4 signalling and stasis are blocked by administering hemopexin with the heme. Plasma hemopexin has the highest binding affinity for free heme, rendering it relatively nonreactive and delivering it safely to the liver for endocytosis and degradation by heme oxygenase-1 (HO-1). Due to chronic hemolysis, hemopexin levels are depleted in SCD patients and mice. We previously reported that intravenous hemopexin induces hepatic HO-1 activity and inhibits ongoing, spontaneous stasis in the venules of SCD mice for up to 48 hours. Inhibition of HO-1 activity with tin protoporphyrin (SnPP) and the administration of carbon monoxide (CO) to SnPP-treated mice demonstrated that HO-1 and its reaction product CO play important roles in the protection against stasis. In the current studies, we asked the question how much of the hemopexin-mediated protection is due to preventing heme activation of TLR4 signalling versus induction of HO-1 and CO production. We wondered if hemopexin complexed with heme would be as effective as hemopexin alone in treating a vaso-occlusive crisis (VOC) induced with hemoglobin heme. First, in a stasis prevention model, we intravenously administered different doses of hemopexin in Townes SCD mice with a DSFC one hour prior to a hemoglobin challenge. Briefly, anesthetized mice were placed on an intravital microscopy stage, and 20-24 flowing subcutaneous venules were selected and mapped. After baseline selection of flowing venules and one hour after administration of different doses of hemopexin (0 - 160 mg/kg body weight), microvascular stasis was triggered by infusing hemoglobin (1 µmol/kg). The same vessels were re-examined for stasis (no flow) and percent stasis was calculated. Our results show that hemopexin effectively reduced stasis in a dose responsive manner when delivered one hour prior to the hemoglobin challenge (Figure 1A). Second, we evaluated hemopexin in a VOC acute treatment model by infusing various doses of hemopexin 30 minutes after the intravenous hemoglobin challenge. We found a dose-dependent effect of hemopexin to reduce and resolve microvascular stasis during an acute VOC one hour after hemopexin infusion (Figure 1B). We obtained similar results after inducing stasis with lipopolysaccharide (LPS) or hypoxia-reoxygenation in place of hemoglobin challenge. Third, we evaluated equimolar hemopexin-heme complexes in a VOC acute treatment model by infusing the same doses of hemopexin-heme 40 minutes after the intravenous hemoglobin challenge (1 µmol/kg) to further elucidate the effects of heme scavenging versus HO-1 induction. Our results showed a striking, dose-dependent reduction in stasis, albeit to a detectably lower degree than hemopexin free of heme (Figure 1C). In summary, hemopexin has a dose-dependent effect to prevent and treat vaso-occlusion induced in a mouse model of SCD by hemoglobin, LPS, and hypoxia-reoxygenation. This effect is partially replicated by hemopexin pre-complexed with heme, suggesting that the beneficial effect is not limited to clearance of circulating heme and prevention of TLR4 signalling. The partial effectiveness of hemopexin-heme complex is consistent with our prior published data implicating CO generation by HO-1 as playing a role in the relief of vaso-occlusion by hemopexin. We conclude that hemopexin shows promise for the treatment of VOC. Additional experiments are needed to clarify the activity of hemopexin in clearance-dependent and -independent mechanisms of resolution of vaso-occlusion. Clinical trials of hemopexin are warranted to evaluate its safety, tolerability and potential efficacy in relieving vaso-occlusive pain crisis in patients with SCD. Disclosures Gentinetta: CSL Behring: Current Employment. Vercellotti:CSL Behring: Research Funding. Kato:CSL Behring AG: Current Employment. Brinkman:CSL Behring: Current Employment. Zuercher:CSL Behring AG: Current Employment. Belcher:Mitobridge-Astellas: Consultancy, Research Funding; CSL Behring: Consultancy, Research Funding; Hillhurst Biopharmaceuticals: Consultancy.

During hemolysis, hemoglobin and heme released from red blood cells promote oxidative stress, inflammation and thrombosis. Plasma haptoglobin and hemopexin scavenge free hemoglobin and heme, respectively, but can be depleted in hemolytic states. Haptoglobin and hemopexin supplementation protect tissues, including the vasculature, liver and kidneys. It is widely assumed that these protective effects are due primarily to hemoglobin and heme clearance from the vasculature. However, this simple assumption does not account for the consequent cytoprotective adaptation seen in cells and organs. To further address the mechanism, we used a hyperhemolytic murine model (Townes-SS) of sickle cell disease to examine cellular responses to haptoglobin and hemopexin supplementation. A single infusion of haptoglobin or hemopexin (± equimolar hemoglobin) in SS-mice increased heme oxygenase-1 (HO-1) in the liver, kidney and skin several fold within 1 hour and decreased nuclear NF-ĸB phospho-p65, and vaso-occlusion for 48 hours after infusion. Plasma hemoglobin and heme levels were not significantly changed 1 hour after infusion of haptoglobin or hemopexin. Haptoglobin and hemopexin also inhibited hypoxia/reoxygenation and lipopolysaccharide-induced vaso-occlusion in SS-mice. Inhibition of HO-1 activity with tin protoporphyrin blocked the protections afforded by haptoglobin and hemopexin in SS-mice. The HO-1 reaction product carbon monoxide, fully restored the protection, in part by inhibiting Weibel-Palade body mobilization of P-selectin and von Willebrand factor to endothelial cell surfaces. Thus, the mechanism by which haptoglobin and hemopexin supplementation in hyperhemolytic SS-mice induces cytoprotective cellular responses is linked to increased HO-1 activity.

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.

Free hemoglobin and hemin, released by red blood cells during intravascular hemolysis, promote vasculopathy, inflammation, thrombosis, and renal injury. Plasma haptoglobin and hemopexin tightly bind free hemoglobin and hemin, respectively, thwarting these clinical sequelae. In sickle cell disease (SCD), chronic hemolysis can deplete plasma haptoglobin and hemopexin in humans and mice. To explore mechanisms mediating this protection and provide a basis for supplementation in SCD patients, dorsal skin fold chambers were implanted onto Townes-SS mice and microvascular stasis (% non-flowing venules) was measured in response to a hemoglobin challenge. Human haptoglobin, hemopexin, or albumin was co-infused with hemoglobin or 1 hour after hemoglobin at equimolar concentrations. Sickle mice co-infused with hemoglobin/haptoglobin, hemoglobin/hemopexin or hemoglobin/haptoglobin/hemopexin had less stasis 1 to 4 hours after infusion, compared to albumin- and saline-treated mice (*p Figure Figure. Disclosures Belcher: CSL-Behring: Research Funding; Imara: Research Funding. Chen:Imara: Research Funding. Brinkman:CSL-Behring: Employment. Vercellotti:CSL-Behring: Research Funding; Imara: Research Funding.

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 as well as chronic inflammation. Here we demonstrate that in addition to activating the vascular endothelium, hemoglobin and heme alter the macrophage phenotype in sickle cell disease. We show that exposure of cultured macrophages to hemolytic aged red blood cells, heme or iron causes their functional phenotypic change towards a pro-inflammatory phenotype, with increased expression levels of inflammatory markers such as IL-6 (P We further demonstrate that the heme scavenger hemopexin protects reticulo-endothelial macrophages from heme overload in heme-loaded Hx-null mice (P Taken together, our data suggest that therapeutical administration of hemopexin is beneficial to counteract heme-driven macrophage-mediated inflammation in sickle cell disease. Disclosures Brinkman: CSL Behring: Employment. Zuercher:CSL Behring: Employment.