Coadministration of PEGylated apohemoglobin and haptoglobin can limit vascular dysfunction in the microcirculation and prevent acute inflammation.

Unfortunately, during pathological conditions resulting in chronic hemolysis cell-free hemoglobin (Hb) is released into the circulation that releases free heme, resulting in several complications. One approach to prevent these toxicities is the administration of supplemental scavenger proteins, haptoglobin (Hp) and hemopexin (Hpx). The goal of this body of work is to objectively measure the levels of vascular reactivity and inflammatory profiles after an infusion of acellular hemoglobin in animals that were given a coadministration of PEGylated human apohemoglobin (PEG-apoHb), a hemopexin (Hpx)-mimetic that can scavenge free heme from hemoglobin, together with human plasma-derived Hp that can scavenge dimerized Hb. Using intravital microscopy, Golden Syrian hamsters instrumented with a dorsal window chamber were used to evaluate the in vivo effects of four experimental groups that were then challenged with a hypovolemic injection (10% of the animal's blood volume) of human Hb (hHb, 5 g/dL). The four experimental groups consisted of: 1) lactated Ringer's solution (control), 2) PEG-apoHb only, 3) Hp only, and 4) PEG-apoHb + Hp. The microvascular hemodynamics (diameter and flow) in arterioles and venules were recorded at baseline, 20 min after treatment, and 20 min after hHb challenge. Systemic parameters (blood pressure and heart rate), blood gases (pH, Pco2, and Po2), blood parameters (Hb concentration and hematocrit), and multiorgan functionality/inflammation were also measured. Our results suggest that coadministration of PEG-apoHb + Hp as a booster before the infusion of acellular hemoglobin significantly prevented vasoconstriction in the microcirculation, significantly increased the number of functional capillaries, and significantly reduced inflammation.NEW & NOTEWORTHY Coadministration of PEGylated human apohemoglobin (PEG-apoHb)-a hemopexin (Hpx) mimetic that can scavenge free heme-and human plasma-derived haptoglobin (Hp) that can scavenge hemoglobin (Hb), reduces microcirculatory dysfunction and cardiac and kidney inflammation in a Hb-challenge model.

Hemopexin alleviates cognitive dysfunction after focal cerebral ischemia-reperfusion injury in rats.

BACKGROUND: Ischemia-reperfusion (I/R) is a critical pathophysiological basis of cognitive dysfunction caused by ischemia stroke. Heme-oxygenase-1 (HO-1) is the rate-limiting enzyme for the elimination of excessive free heme by combining with hemopexin (HPX), a plasma protein that contributes to eliminating excessive free heme during ischemia stroke. This study aimed to elucidate whether HPX could alleviate cognitive dysfunction in rats subjected to cerebral I/R. METHODS: Rats were randomly divided into five groups: sham, MCAO, Vehicle, HPX and HPX + protoporphyrin IX (ZnPPIX). Cerebral I/R was induced by MCAO. Saline, vehicle, HPX and HPX + ZnPPIX were injected intracerebroventricularly at the moment after reperfusion. Morris water maze (MWM) test was used to detect the learning and cognitive function. Western blot was used to detect the expression of HO-1 in ischemic penumbra. CD31/vWF double labeling immunofluorescence was used to detect the neovascularization in the penumbra hippocampus. The structure and function of blood-brain barrier (BBB) was detected by the permeability of Evans Blue (EB), water content of the brain tissue, the Ang1/Ang2 and VE-cadherin expression. RESULTS: Our study verified that HPX improved the learning and memory capacity. Hemopexin up-regulated HO-1 protein expression, the average vessel density in the penumbra hippocampus and the VE- cadherin expression but decreased the permeability of EB, the water content of brain tissue and the ratio of Ang1/Ang2. The effects were reversed by ZnPPIX, an inhibitor of HO-1. CONCLUSION: HPX can maintain the integrity of the blood-brain barrier and alleviate cognitive dysfunction after cerebral I/R through the HO-1 pathway.

When might transferrin, hemopexin or haptoglobin administration be of benefit following the transfusion of red blood cells?

PURPOSE OF REVIEW: After transfusion, a percentage of red blood cells undergo hemolysis within macrophages. Intravascular exposures to hemin and hemoglobin (Hb) can occur after storage bag hemolysis, some transfusion reactions, during use of medical assist devices and in response to bacterial hemolysins. Proteins that regulate iron, hemin and Hb either become saturated after iron excess (transferrin, Tf) or depleted after hemin (hemopexin, Hpx) and Hb (haptoglobin, Hp) excess. Protein saturation or stoichiometric imbalance created by transfusion increases exposure to non-Tf bound iron, hemin and Hb. Tf, Hpx and Hp are being developed for hematological disorders where iron, hemin and Hb contribute to pathophysiology. However, complexed to their ligands, each represents a potential iron source for pathogens, which may complicate the use of these proteins. RECENT FINDINGS: Erythrophagocytosis by macrophages and processes of cell death that lead to reactive iron exposure are increasingly described. In addition, the effects of transfusion introduced circulatory hemin and Hb are described in the literature, particularly following large volume transfusion, infection and during concomitant medical device use. SUMMARY: Supplementation with Tf, Hpx and Hp suggests therapeutic potential in conditions of extravascular/intravascular hemolysis. However, their administration following transfusion may require careful assessment of concomitant disease.

Hemopexin promotes angiogenesis via up-regulating HO-1 in rats after cerebral ischemia-reperfusion injury.

BACKGROUND: Ischemia-reperfusion (I/R) is a critical pathophysiological change of ischemic stroke. Heme-oxygenase-1 (HO-1) is a rate-limiting enzyme of eliminating excessive free heme by combining with hemopexin (HPX), a plasma protein contributing to alleviating infarct size due to ischemia stroke. This study was to investigate whether HPX could improve angiogenesis after cerebral ischemia-reperfusion via up-regulating HO-1. METHODS: Rats were randomly divided into five groups: sham, MCAO, MCAO + Vehicle, MCAO + HPX and MCAO + HPX + protoporphyrin IX (ZnPPIX, an HO-1 inhibitor). Cerebral I/R was induced by MCAO. Saline, vehicle, HPX and HPX + ZnPPIX were respectively given to MCAO group, MCAO + Vehicle group, MCAO + HPX group and MCAO + HPX + ZnPPIX group at the moment after reperfusion by intracerebroventricular injection. Neurological behavioral scores(NBS) was assessed at 24 h and 7d after I/R. Real-time polymerase chain reaction (RT-PCR) was used to analyze the mRNA level of HO-1. Angiogenesis in penumbra area was assessed by immunofluorescence detection at 7d after I/R. Serum endothelial nitric oxide synthase (eNOS) was assessed by enzyme linked immunosorbent assay (ELISA) at 24 h and 7d after I/R. RESULTS: Compared with sham group, the NBS and the mRNA levels of HO-1 at 24 h and 7d after I/R in MCAO group decreased notably (P < 0.05), the new vessel density in ischemia penumbra increased notably at 7d after I/R (P < 0.05), the serum eNOS level increased at 24 h and 7d after I/R (P < 0.05). MCAO group and MCAO + Vehicle group showed no significant differences (P > 0.05). In the MCAO + HPX group, compared with MCAO + Vehicle group, the NBS and the mRNA levels of HO-1 increased drastically at 24 h and 7d after I/R (P < 0.05), the new vessel density in ischemia penumbra increased significantly at 7d after I/R (P < 0.05), the serum eNOS level at 24 h and 7d after I/R ascended notably (P < 0.05). Compared with MCAO + HPX group, the NBS assessment, new vessel density and serum eNOS level decreased at corresponding time points after I/R in MCAO + HPX+ ZnPPIX group (P < 0.05). CONCLUSION: HPX can promote angiogenesis after cerebral ischemia-reperfusion injury in rats via up-regulating HO-1.

Endothelial dysfunction inhibits the ability of haptoglobin to prevent hemoglobin-induced hypertension.

Intravascular hemolysis produces injury in a variety of human diseases including hemoglobinopathies, malaria, and sepsis. The adverse effects of increased plasma hemoglobin are partly mediated by depletion of nitric oxide (NO) and result in vasoconstriction. Circulating plasma proteins haptoglobin and hemopexin scavenge extracellular hemoglobin and cell-free heme, respectively. The ability of human haptoglobin or hemopexin to inhibit the adverse effects of NO scavenging by circulating murine hemoglobin was tested in C57Bl/6 mice. In healthy awake mice, the systemic hemodynamic effects of intravenous coinfusion of cell-free hemoglobin and exogenous haptoglobin or of cell-free hemoglobin and hemopexin were compared with the hemodynamic effects of infusion of cell-free hemoglobin or control protein (albumin) alone. We also studied the hemodynamic effects of infusing hemoglobin and haptoglobin as well as injecting either hemoglobin or albumin alone in mice fed a high-fat diet (HFD) and in diabetic (db/db) mice. Coinfusion of a 1:1 weight ratio of haptoglobin but not hemopexin with cell-free hemoglobin prevented hemoglobin-induced systemic hypertension in healthy awake mice. In mice fed a HFD and in diabetic mice, coinfusion of haptoglobin mixed with an equal mass of cell-free hemoglobin did not reverse hemoglobin-induced hypertension. Haptoglobin retained cell-free hemoglobin in plasma, but neither haptoglobin nor hemopexin affected the ability of hemoglobin to scavenge NO ex vivo. In conclusion, in healthy C57Bl/6 mice with normal endothelium, coadministration of haptoglobin but not hemopexin with cell-free hemoglobin prevents acute hemoglobin-induced systemic hypertension by compartmentalizing cell-free hemoglobin in plasma. In murine diseases associated with endothelial dysfunction, haptoglobin therapy appears to be insufficient to prevent hemoglobin-induced vasoconstriction.NEW & NOTEWORTHY Coadministraton of haptoglobin but not hemopexin with cell-free hemoglobin prevents hemoglobin-induced systemic hypertension in mice with a normal endothelium. In contrast, treatment with the same amount of haptoglobin is unable to prevent hemoglobin-induced vasoconstriction in mice with hyperlipidemia or diabetes mellitus, disorders that are associated with endothelial dysfunction.

Haptoglobin or Hemopexin Therapy Prevents Acute Adverse Effects of Resuscitation After Prolonged Storage of Red Cells.

BACKGROUND: Extracellular hemoglobin and cell-free heme are toxic breakdown products of hemolyzed erythrocytes. Mammals synthesize the scavenger proteins haptoglobin and hemopexin, which bind extracellular hemoglobin and heme, respectively. Transfusion of packed red blood cells is a lifesaving therapy for patients with hemorrhagic shock. Because erythrocytes undergo progressive deleterious morphological and biochemical changes during storage, transfusion of packed red blood cells that have been stored for prolonged intervals (SRBCs; stored for 35-40 days in humans or 14 days in mice) increases plasma levels of cell-free hemoglobin and heme. Therefore, in patients with hemorrhagic shock, perfusion-sensitive organs such as the kidneys are challenged not only by hypoperfusion but also by the high concentrations of plasma hemoglobin and heme that are associated with the transfusion of SRBCs. METHODS: To test whether treatment with exogenous human haptoglobin or hemopexin can ameliorate adverse effects of resuscitation with SRBCs after 2 hours of hemorrhagic shock, mice that received SRBCs were given a coinfusion of haptoglobin, hemopexin, or albumin. RESULTS: Treatment with haptoglobin or hemopexin but not albumin improved the survival rate and attenuated SRBC-induced inflammation. Treatment with haptoglobin retained free hemoglobin in the plasma and prevented SRBC-induced hemoglobinuria and kidney injury. In mice resuscitated with fresh packed red blood cells, treatment with haptoglobin, hemopexin, or albumin did not cause harmful effects. CONCLUSIONS: In mice, the adverse effects of transfusion with SRBCs after hemorrhagic shock are ameliorated by treatment with either haptoglobin or hemopexin. Haptoglobin infusion prevents kidney injury associated with high plasma hemoglobin concentrations after resuscitation with SRBCs. Treatment with the naturally occurring human plasma proteins haptoglobin or hemopexin may have beneficial effects in conditions of severe hemolysis after prolonged hypotension.

Apolipoprotein E-/- Mice Lacking Hemopexin Develop Increased Atherosclerosis via Mechanisms That Include Oxidative Stress and Altered Macrophage Function.

OBJECTIVE: We previously reported that hemopexin (Hx), a heme scavenger, is significantly increased and associated with proinflammatory high-density lipoprotein under atherogenic conditions. Although it is established that Hx together with macrophages plays a role in mitigating oxidative damage, the role of Hx in the development of atherosclerosis is unknown. APPROACH AND RESULTS: We used Hx and apoE double-knockout mice (HxE(-/-)) to determine the role of Hx in the development of atherosclerosis. HxE(-/-) mice had significantly more free heme, reactive oxygen species, and proinflammatory high-density lipoprotein in their circulation, when compared with control apoE(-/-) mice. Atherosclerotic plaque area (apoE(-/-)=9.72±2.5×10(4) µm(2) and HxE(-/-)=27.23±3.6×10(4) µm(2)) and macrophage infiltration (apoE(-/-)=38.8±5.8×10(3) µm(2) and HxE(-/-)=103.4±17.8×10(3) µm(2)) in the aortic sinus were significantly higher in the HxE(-/-) mice. Atherosclerotic lesions in the aortas were significantly higher in the HxE(-/-) mice compared with apoE(-/-) mice. Analysis of polarization revealed that macrophages from HxE(-/-) mice were more M1-like. Ex vivo studies demonstrated that HxE(-/-) macrophage cholesterol efflux capacity was significantly reduced when compared with apoE(-/-) mice. Injection of human Hx into HxE(-/-) mice reduced circulating heme levels and human Hx pretreatment of naive bone marrow cells ex vivo resulted in a shift from M1- to M2-like macrophages. CONCLUSIONS: We conclude that Hx plays a novel protective role in alleviating heme-induced oxidative stress, improving inflammatory properties of high-density lipoprotein, macrophage phenotype and function, and inhibiting the development of atherosclerosis in apoE(-/-) mice.

Expression of hemopexin in acute rejection of rat liver allograft identified by serum proteomic analysis.

Acute rejection (AR) and acceptance of allograft after liver transplantation (LTx) remain critical issues that need addressing to improve prognosis. We therefore performed rat orthotopic LTx and proteomic analyses to screen for immune response-related biomarkers in sera. Markers identified were validated at the mRNA and/or protein levels, and the molecules of interest were functionally explored. Compared with syngeneic controls, signs of AR as well as spontaneous acceptance were observed in hematoxylin and eosin-stained sections of liver allografts. In accordance with the severity of AR, 30 protein spots displaying significant changes in abundance were identified using two-dimensional differential gel electrophoresis. Ultimately, 14 serum proteins were sequenced and five spots of interest were identified as hemopexin (HPX). Expression of HPX was significantly and inversely associated with the severity of AR at both the mRNA and protein levels. In vitro, Mt-1, Ho-1, Fth, Ifn-γ, and Il-17 transcripts were significantly upregulated in lysates of lymphocytes stimulated with HPX, whereas Il-10 markedly was remarkably downregulated. Interferon-γ, IL-10, and IL-17 proteins in the supernatant of HPX-stimulated lymphocytes were significantly altered in keeping with the mRNA level. Our data facilitated the generation of a proteomic profile to enhance the understanding of rat liver AR. In view of finding that the HPX serum level is negatively associated with the severity of AR of rat liver allograft, we propose that in vitro treatment with HPX regulates cytokine expression in rat lymphocytes.

Local intracerebral delivery of endogenous inhibitors by osmotic minipumps effectively suppresses glioma growth in vivo.

The systemic administration of endogenous inhibitors significantly reduced the growth of human glioma in vivo, but required the production of a large amount of biologically active protein. In this study we reduced the amount of protein needed and optimized the therapeutical response by delivering the endogenous inhibitors locally into the brain by osmotic minipumps. Human hemopexin fragment of MMP-2 or COOH-terminal fragment of platelet factor-4 were delivered locally and continuously into the brain of mice implanted intracranially with glioma cells, by osmotic minipumps connected to an intracranial catheter. Local delivery of human hemopexin fragment of MMP-2 and COOH-terminal fragment of platelet factor-4 significantly inhibited the growth of well-established malignant glioma in nude and BALB/C mice. When the inhibitors were given at the same concentration, the efficacy of the local delivery was much higher than that reached with the systemic administration, both when the inhibitor was administered daily or continuously by s.c. minipumps. Moreover, the local delivery reduced the amount of protein needed to reach a significant therapeutic response. Intracerebral delivery maintained a long-term control of glioma growth and inhibited glioma recurrence in a surgical resection model. Treatment showed no side effects. Histochemical analysis of tumors showed that the tumor growth inhibition was the result of a decrease in tumor vasculature and a change in tumor vessel morphology. Our data demonstrate that local intracerebral delivery of endogenous inhibitors effectively inhibits malignant glioma growth and reduces the amount of protein needed to reach a therapeutical response.