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Hemopexin is a vital glycoprotein for processing excessive iron in blood and functions as an iron scavenger in mammals. Teleosts however, unlike mammals, have two known hemopexin paralogs called warm temperature acclimation-related 65 kDa protein (Wap65-1 and Wap65-2, collectively termed Wap65s). Although Wap65s in rainbow trout have been considered notable biomarkers with significantly higher and/or lower expression under conditions of stress or disease, the individual roles, similarities and differences between the two paralogs are not well known. The aim of this study was to gain an understanding of the characteristics and functions of trout Wap65s from the perspective of iron-metabolism, physiological roles, and relevant immunological responses. The expression of Wap65-1 and -2 in this study was determined in the face of challenges by Aeromonas salmonicida, infectious hematopoietic necrosis virus (IHNV), and iron-dextran. Immuno-histochemistry (IHC) was employed to localize the major cell types for Wap65-2 expression, and trout leukocytes were isolated and incubated with LPS and OxLDL for comprehending the immunological characteristics of Wap65-2. We demonstrate that Wap65-1 is expressed only in the liver but Wap65-2 is systemically expressed in most organs and tissues. Interestingly, Wap65-1 expression was not significantly changed under A. salmonicida and iron-dextran administration, but was significantly decreased under IHNV. In contrast, Wap65-2 was up-regulated in all challenged groups, however with different expression patterns in the blood and liver. These results suggested that the two paralogs may participate in different biological roles. IHC showed that Wap65-2 antibody had high affinity for leukocyte-like cells, and macrophages but not lymphocytes significantly increased expression under LPS and OxLDL stimulation. These results support the conclusion that trout Wap65-2, not Wap65-1 may have conventional hemopexin functions such as reported in mammals including effects on iron metabolism, inflammation, and acute-phase protein., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

After intracranial hemorrhage, heme is released from cell-free hemoglobin. This red blood cell component may drive secondary brain injury at the hematoma‒brain interface. This study aimed to generate a spatially resolved map of transcriptome-wide gene expression changes in the heme-exposed brain and to define the potential therapeutic activity of the heme-binding protein, hemopexin. We stereotactically injected saline, heme, or heme‒hemopexin into the striatum of C57BL/6J mice. After 24 h, we elucidated the two-dimensional spatial transcriptome by sequencing 21760 tissue-covered features, at a mean transcript coverage of 3849 genes per feature. In parallel, we studied the extravasation of systemically administered fluorescein isothiocyanate labeled (FITC)-dextran, magnetic resonance imaging features indicative of focal edema and perfusion, and neurological functions as translational correlates of heme toxicity. We defined a cerebral heme-response signature by performing bidimensional differential gene expression analysis, based on unsupervised clustering and manual segmentation of sequenced features. Heme exerted a consistent and dose-dependent proinflammatory activity in the brain, which occurred at minimal exposures, below the toxicity threshold for the induction of vascular leakage. We found dose-dependent regional divergence of proinflammatory heme signaling pathways, consistent with reactive astrocytosis and microglial activation. Co-injection of heme with hemopexin attenuated heme-induced gene expression changes and preserved the homeostatic microglia signature. Hemopexin also prevented heme-induced disruption of the blood‒brain barrier and radiological and functional signals of heme injury in the brain. In conclusion, we defined heme as a potent inflammatoxin that may drive secondary brain injury after intracerebral hemorrhage. Co-administration of hemopexin attenuated the heme-derived toxic effects on a molecular, cellular, and functional level, suggesting a translational therapeutic strategy., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

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 Ca 2+ transient amplitudes and fractional shortening in primary cardiomyocytes exposed to free heme. In keeping with these Ca 2+ 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 Ca 2+ 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., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Conventional animal tests such as leukocytosis promoting tests have been used for decades to evaluate toxicity of pertussis vaccine. Here, we examined gene expression in relation to the vaccine toxicity using a DNA microarray. Comparison of conventional animal test data with the DNA microarray-based gene expression data revealed a gene expression pattern highly correlated with leukocytosis in animals. Of 10,490 rat genes analyzed, two genes, alpha1-acid-glycoprotein (Agp) and hemopexin (Hpx), were found up-regulated by the toxin administration in a dose-dependent manner (assayed by a quantitative PCR based on the microarray). Variation of the gene expression was very small amongst the test animals, and the results were highly reproducible. These findings suggest that gene expression analysis of vaccine-treated animals can be used as an accurate and simple method of pertussis vaccine safety assessment.

In order to make clear whether the expression of biological activities and antitumor polysaccharides are under genetic control, the responses of mice to lentinan, a beta-1,6;1,3-glucan, in the induction of several acute phase proteins (APPs) and T-cell-mediated vascular dilation and hemorrhage (VDH) were investigated. Twenty inbred strains of mice were divided into two groups according to their phenotypes in the induction of APPs when they were administered lentinan i.p. at a dose of 10 mg/kg; sensitive strains showed a marked increase in levels of APPs and resistant strains showed as low a level of APPs as non-treated control mice. No sex-related differences and no relation with H-2 halotypes were found in the responses. Only low-level responses were observed in F1 hybrid mice obtained by crosses between a sensitive and a resistant strain, indicating that the low APP response to lentinan is dominant. The N2 progeny between the F1 and a high responder segregated into high and low responders at a ratio of almost 1:1. These results suggest that a single major gene on an autosome is responsible for the induction of APPs. The induction of VDH also depended on the strains of mice. However, the strain distribution pattern of the VDH phenotype was distinct from that of the APP phenotype, indicating that the VDH-controlling gene was different from the APP-controlling gene. Further analyses with F1 hybrid and backcross progeny mice suggested that the high VDH response was dominant, and that the phenotype was determined by a single major gene.

1. Of ten protein systems studied in mouflon (Ovis musimon), five were polymorphic (Tf, Hpx, EsA, X-protein, Cat). Electrophoretic mobilities of mouflon proteins did not differ from those of sheep. 2. Mouflon haemoglobin B and sheep haemoglobin B differed in isoelectric focusing. 3. Haemopexin levels in mouflon were determined by rocket immunoelectrophoresis. A trimodal distribution was apparent, with no haemopexin, low and high levels of the protein. The results are indicative of genetic control of haemopexin levels, one of the alleles being inactive (Hpx0).