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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.

Human apolipoprotein A-I preparations reconstituted with phospholipids (reconstituted high-density lipoprotein [HDL]) have been used in a large number of animal and human studies to investigate the physiological role of apolipoprotein A-I. Several of these studies observed that intravenous infusion of reconstituted HDL might cause transient elevations in plasma levels of hepatic enzymes. Here we describe the mechanism of this enzyme release. Observations from several animal models and in vitro studies suggest that the extent of hepatic transaminase release (alanine aminotransferase [ALT]) correlates with the movement of hepatic cholesterol into the blood after infusion. Both the amount of ALT release and cholesterol movement were dependent on the amount and type of phospholipid present in the reconstituted HDL. As cholesterol is known to dissolve readily in phospholipid, an HDL preparation was loaded with cholesterol before infusion into rats to assess the role of diffusion of cholesterol out of the liver and into the reconstituted HDL. Cholesterol-loaded HDL failed to withdraw cholesterol from tissues and subsequently failed to cause ALT release. To investigate further the role of cholesterol diffusion, we employed mice deficient in SR-BI, a transporter that facilitates spontaneous movement of cholesterol between cell membranes and HDL. These mice showed substantially lower movement of cholesterol into the blood and markedly lower ALT release. We conclude that initial depletion of hepatic cholesterol initiates transient ALT release in response to infusion of reconstituted HDL. This effect may be controlled by appropriate choice of the type and amount of phospholipid in reconstituted HDL. Copyright © 2015 John Wiley & Sons, Ltd.

Aim: We have recently demonstrated that reconstituted high-density lipoprotein (rHDL) delivered immediately after myocardial infarction reduces infarct size and improves heart function in mice ( Heywood SE, Sci Transl Med, 2017 ). We now examine potential immunomodulatory actions of HDL that may underlie these effects. Methods: In male C57BL/6 mice, a single intravenous bolus of rHDL (CSL-111, 80mg/kg of human apoA-I, or saline) was delivered at the time of reperfusion following 30mins of surgically-induced ischemia. Effects on the inflammatory response were studied throughout the 5 days post ischemia-reperfusion. Results: Twenty-four hours after ischemia-reperfusion, rHDL reduced the number of circulating leukocytes (versus saline phigh ) than the anti-inflammatory subtype (Ly6C low ; pHigh monocytes 5 days post ischemia in the LV of mice treated with rHDL (p Conclusion: rHDL limits the myocardial post-ischemic inflammatory response by effects in both the LV and by directly modulating inflammatory cells. These finding suggest a novel treatment modality for acute coronary syndromes.

Human apolipoprotein A-I preparations reconstituted with phospholipids (reconstituted high-density lipoprotein [HDL]) have been used in a large number of animal and human studies to investigate the physiological role of apolipoprotein A-I. Several of these studies observed that intravenous infusion of reconstituted HDL might cause transient elevations in plasma levels of hepatic enzymes. Here we describe the mechanism of this enzyme release. Observations from several animal models and in vitro studies suggest that the extent of hepatic transaminase release (alanine aminotransferase [ALT]) correlates with the movement of hepatic cholesterol into the blood after infusion. Both the amount of ALT release and cholesterol movement were dependent on the amount and type of phospholipid present in the reconstituted HDL. As cholesterol is known to dissolve readily in phospholipid, an HDL preparation was loaded with cholesterol before infusion into rats to assess the role of diffusion of cholesterol out of the liver and into the reconstituted HDL. Cholesterol-loaded HDL failed to withdraw cholesterol from tissues and subsequently failed to cause ALT release. To investigate further the role of cholesterol diffusion, we employed mice deficient in SR-BI, a transporter that facilitates spontaneous movement of cholesterol between cell membranes and HDL. These mice showed substantially lower movement of cholesterol into the blood and markedly lower ALT release. We conclude that initial depletion of hepatic cholesterol initiates transient ALT release in response to infusion of reconstituted HDL. This effect may be controlled by appropriate choice of the type and amount of phospholipid in reconstituted HDL. Copyright © 2015 John WileySons, Ltd.

Objective— The ability of high-density lipoprotein (HDL) to remove cholesterol from atherosclerotic plaque is thought to underlie its inverse correlation with cardiovascular risk. Our objective was to produce and characterize a human apolipoprotein AI (apoA-I) product optimized to treat clinical atherosclerotic disease. Approach and Results— A new formulation of full length, plasma-derived human apoA-I termed CSL112 was designed to maximize the cholesterol efflux from cells and exhibit favorable pharmacological properties. CSL112 is a disc-shaped particle that strongly elevates cholesterol esterification and shows good pharmacokinetics in rabbits. Infusion of CSL112 into rabbits caused a strong and immediate increase in the ATP binding cassette transporter A1 (ABCA1)-dependent efflux capacity of plasma, an increase in plasma unesterified cholesterol and rapid subsequent cholesterol esterification. In the presence of human plasma, CSL112 was significantly more potent than native HDL at enhancing cholesterol efflux from macrophages, and the efflux elevation was predominantly via the ABCA1 transporter. Consistent with this observation, addition of CSL112 to plasma led to generation of high levels of HDL-VS, a favorable substrate for ABCA1. The lipid profile of plasma did not affect these behaviors. In studies with whole human blood, CSL112 reduced expression of intercellular adhesion molecule 1 and cytokine secretion, and as with cholesterol efflux, these activities were substantially greater than those of native HDL assayed in parallel. Conclusions— CSL112 has favorable pharmacological properties and strongly elevates the ability of plasma to withdraw cholesterol from cells. Preferential elevation of ABCA1-dependent efflux may target atherosclerotic plaque for cholesterol removal and this property makes CSL112 a promising candidate therapy for acute coronary syndrome.

The ability of apoA-I to remove cholesterol from atherosclerotic plaque is thought to underlie its inverse correlation with cardiovascular risk. CSL112 was designed as an infusible formulation of human apoA-I to rapidly remove plaque cholesterol and reduce risk after an ACS. We have recently shown that infusion of CSL112 into rabbits caused a strong and immediate increase in the ABCA1-dependent efflux capacity of plasma, an increase in plasma unesterified cholesterol and rapid subsequent esterification of the cholesterol. In the presence of human plasma, CSL112 was significantly more potent than human HDL 3 at enhancing cholesterol efflux, and the efflux elevation was predominantly via the ABCA1 transporter. Consistent with this observation, addition of CSL112 to plasma led to generation of high levels of PreBeta1-HDL, a favorable substrate for ABCA1. Phase 1 studies of CSL112 have confirmed the elevation of efflux capacity in humans and the rapid generation of PreBeta1. Here we examine the steps leading to formation of PreBeta1-HDL. Plasma spiked with 0.8 mg/mL CSL112 and held at 0[[Unable to Display Character: ⁰]]C did not increase PreBeta1-HDL levels. However, incubation at 37[[Unable to Display Character: ⁰]]C caused the generation of PreBeta1 that reached half-maximal levels at 15-20 min and maximal levels at 2 h (7-fold over baseline). By contrast, addition of HDL 3 to plasma caused minimal generation of PreBeta1-HDL at any time point. Native gradient gel electrophoresis showed that formation of PreBeta1 was accompanied by elevation in the size and protein content of HDL 3 and HDL 2 . These data suggest that the remarkable ability of CSL112 to elevate efflux and PreBeta1 derives from engaging plasma proteins in a process of lipoprotein remodeling.

The ability of HDL to remove cholesterol from macrophages in atherosclerotic plaque is thought to underlie its inverse correlation with cardiovascular risk. Indeed, cholesterol efflux from macrophages is a functional serum marker that has an even stronger inverse association with coronary heart disease than HDL cholesterol (Khera et al. 2011, NEJM 364;2;127). We recently described CSL112, a novel formulation of full length human apoA-I and phospholipids designed to resemble nascent HDL and to maximize cholesterol efflux from macrophages. Here we studied the effects of CSL112 vs HDL3 on ex vivo cholesterol efflux in human plasma and serum. Addition of HDL3 to pooled plasma significantly increased ABCA1-independent efflux but had little or no effect on ABCA1-dependent efflux. In contrast, the addition of CSL112 increased not only ABCA1-independent efflux but also strongly increased ABCA1-dependent efflux in a concentration dependent fashion. Through a range of concentrations, CSL112 was significantly more efficacious in global cholesterol efflux than was HDL3. For example, at 5 μg/mL, CSL112 increased global efflux by 152% ± 5.6% vs 66% ± 7.4% for HDL3. Additional studies examined serum samples from five subjects with a range of lipid phenotypes including low, normal and high levels of HDL. When spiked into these sera, CSL112 increased both ABCA1-dependent and independent efflux in all subjects, regardless of lipid profile. In all subjects, the relative increase in ABCA1-dependent efflux was larger than that observed with ABCA1-independent efflux indicating selectivity of the interaction of CSL112 with the ABCA1 transporter. We speculate that the disproportionate rise in ABCA1-dependent efflux with CSL112 may be explained by the observation that PreBeta1-HDL levels measured by ELISA increase dramatically upon incubation of CSL112 with human plasma. We conclude that CSL112 robustly enhances the capacity of human serum to promote cholesterol efflux; the enhancement appears independent of lipid phenotype, is greater than that observed with HDL3 and that the efflux derives disproportionately from the ABCA1 pathway.

The efflux of cellular cholesterol mediated by apoA-I and ATP-binding cassette transporter A1 (ABCA1) is thought to underlie many of the cardiovascular benefits of HDL. Several groups have manufactured apoA-I preparations with the aim of making an infused HDL therapy for the acute treatment of