Safety and feasibility of the gene transfer of hemopexin for conditions with increased free heme.

Heme is a fundamental molecule for several biological processes, but when released in the extracellular space such as in hemolytic diseases, it can be toxic to cells and tissues. Hemopexin (HPX) is a circulating protein responsible for removing free heme from the circulation, whose levels can be severely depleted in conditions such as sickle cell diseases. Accordingly, increasing HPX levels represents an attractive strategy to mitigate the deleterious effects of heme in these conditions. Gene transfer of liver-produced proteins with adeno-associated virus (AAV) has been shown to be an effective and safety strategy in animal and human studies mainly in hemophilia. Here, we report the feasibility of increasing HPX levels using an AAV8 vector expressing human HPX (hHPX). C57Bl mice were injected with escalating doses of our vector, and expression was assessed by enzyme immunoassay (ELISA), Western blot, and quantitative polymerase chain reaction (qPCR). In addition, the biological activity of transgenic hHPX was confirmed using two different models of heme challenge consisting of serial heme injections or phenylhydrazine-induced hemolysis. Sustained expression of hHPX was confirmed for up to 26 weeks in plasma. Expression was dose-dependent and not associated with clinical signs of toxicity. hHPX levels were significantly reduced by heme infusions and phenylhydrazine-induced hemolysis. No clinical toxicity or laboratory signs of liver damage were observed in preliminary short-term heme challenge studies. Our results confirm that long-term expression of hHPX is feasible and safe in mice, even in the presence of heme overload. Additional studies are needed to explore the effect of transgenic HPX protein in animal models of chronic hemolysis.

Genomic organization and gene evolution of two warm temperature acclimation proteins (Wap65s) of Micropterus salmoides and their responses to temperature and bacterial/viral infections.

Warm temperature acclimation-related 65-kDa proteins (Wap65s) are fish plasma acute-phase glycoproteins homologous to hemopexin with high affinity and clearance for heme. The study characterized Mswap65-1 and Mswap65-2 genes in Micropterus salmoides. Structural analysis showed MsWap65s contained conserved heme-binding sites. MsWap65-1 had a chloride-binding site similar to hemopexin, while MsWap65-2 had an additional calcium-binding site. Phylogenetic and Ka/Ks analysis showed that fish Wap65s were evolutionarily conserved and underwent strong purifying selection. Functional divergence analysis indicated that fish Wap65-2 retained the putative function of ancestral Wap65, while Wap65-1 underwent neofunctional differentiation. QPCR showed Mswap65s were predominantly expressed in liver, but prolonged hyperthermy inhibited Mswap65-2 expression. Mswap65-2 expression was up-regulated in liver and spleen after Nocardia seriolae infection, while Mswap65-1 was down-regulated. MsWap65-2 may be associated with pathogenesis and play potential role in pathogen resistance. LMBV infection resulted in both significant downregulation of Mswap65s were both significantly down-regulated, with differences observed between sexes. We speculated the immune system might suppress expression after viral infection. Exogenous rMsWap65s were prepared, and injection of rMsWap65s alleviated phenylhydrazine-induced hemolysis and inhibited increases in heme, complement C3 and inflammatory symptoms. Our results contribute to an advanced understanding of the functions and mechanisms of MsWap65s in stress resistance.

Identification and characterization of warm temperature acclimation proteins (Wap65s) in rainbow trout (Oncorhynchus mykiss).

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.

Heme oxygenase-1 and hemopexin gene polymorphisms and the risk of anti-tuberculosis drug-induced hepatotoxicity in China.

Objective: To assess whether the risk of anti-tuberculosis drug-induced hepatotoxicity (ATDH) might be influenced by heme oxygenase-1 (HMOX1) and hemopexin (HPX) gene polymorphisms. Methods: A dynamic anti-tuberculosis treatment cohort was constructed, and the 1:4 matched nested case-control study was analysed. Eight single-nucleotide polymorphisms (SNPs) of the two genes were selected for genotyping and Bonferroni correction was performed to correct for multiple comparison. Results: Overall, 7.8% of patients developed ATDH. SNP rs1807714 in the HMOX1 gene had decreased effects on the risk of moderate and severe hepatotoxicity under the dominant and additive models, and hepatocellular injury under the additive model. SNP rs2682099 in the HPX gene had increased effects on the risk of moderate and severe hepatotoxicity under the recessive model. However, these associations disappeared after Bonferroni correction. Conclusion:HMOX1 and HPX gene polymorphisms might not be associated with susceptibility to ATDH in the Chinese population.

Analysis of hemopexin plasma levels in patients with age-related macular degeneration.

Purpose: A protein quantitative trait locus (pQTL) analysis recently revealed a strong association between hemopexin (HPX) levels and genetic variants at the complement factor H (CFH) locus. In this study, we aimed to determine HPX plasma levels in patients with age-related macular degeneration (AMD) and to compare them with those in controls. We also investigated whether genetic variants at the CFH locus are associated with HPX plasma levels. Methods: HPX levels were quantified in 200 advanced AMD cases and 200 controls using an enzyme-linked immunosorbent assay and compared between the two groups. Furthermore, HPX levels were analyzed per genotype group of three HPX-associated variants (rs61818956, rs10494745, and rs10801582) and four AMD-associated variants (rs794362 [proxy for rs187328863], rs570618, rs10922109, and rs61818924 [proxy for rs61818925]) at the CFH locus. Results: HPX levels were similar in the control group compared with the AMD group. The three variants at the CFH locus, which were previously associated with the HPX levels, showed no association with the HPX levels in our data set. No significant differences in HPX levels were detected between the different genotype groups of AMD-associated variants at the CFH locus. Conclusions: In this study, HPX levels were not associated with AMD or AMD-associated variants at the CFH locus. The finding of a previous pQTL study that variants at the CFH locus were associated with HPX levels was also not confirmed in this study.

Spatial transcriptome analysis defines heme as a hemopexin-targetable inflammatoxin in the brain.

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.

Hemopexin Modulates Expression of Complement Regulatory Proteins in Rat Glomeruli.

In systemic hemolysis and in hematuric forms of kidney injury, the major heme scavenging protein, hemopexin (HPX), becomes depleted, and the glomerular microvasculature (glomeruli) is exposed to high concentrations of unbound heme, which, in addition to causing oxidative injury, can activate complement cascades; thus, compounding extent of injury. It is unknown whether unbound heme can also activate specific complement regulatory proteins that could defend against complement-dependent injury. Isolated rat glomeruli were incubated in media supplemented with HPX-deficient (HPX-) or HPX-containing (HPX+) sera as a means of achieving different degrees of heme partitioning between incubation media and glomerular cells. Expression of heme oxygenase (HO)-1 and of the complement activation inhibitors, decay-accelerating factor (DAF), CD59, and complement receptor-related gene Y (Crry), was assessed by western blot analysis. Expression of HO-1 and of the GPI-anchored DAF and CD59 proteins increased in isolated glomeruli incubated with HPX- sera with no effect on Crry expression. Exogenous heme (hemin) did not further induce DAF but increased Crry expression. HPX modulates heme-mediated induction of complement activation controllers in glomeruli. This effect could be of translational relevance in glomerular injury associated with hematuria.

Modular Platform for the Development of Recombinant Hemoglobin Scavenger Biotherapeutics.

Cell-free hemoglobin (Hb) is a driver of disease progression in conditions with intravascular or localized hemolysis. Genetic and acquired anemias or emergency medical conditions such as aneurysmal subarachnoid hemorrhage involve tissue Hb exposure. Haptoglobin (Hp) captures Hb in an irreversible protein complex and prevents its pathophysiological contributions to vascular nitric oxide depletion and tissue oxidation. Preclinical proof-of-concept studies suggest that human plasma-derived Hp is a promising therapeutic candidate for several Hb-driven diseases. Optimizing the efficacy and safety of Hb-targeting biotherapeutics may require structural and functional modifications for specific indications. Improved Hp variants could be designed to achieve the desired tissue distribution, metabolism, and elimination to target hemolytic disease states effectively. However, it is critical to ensure that these modifications maintain the function of Hp. Using transient mammalian gene expression of Hp combined with co-transfection of the pro-haptoglobin processing protease C1r-LP, we established a platform for generating recombinant Hp-variants. We designed an Hpß-scaffold, which was expressed in this system at high levels as a monomeric unit (mini-Hp) while maintaining the key protective functions of Hp. We then used this Hpß-scaffold as the basis to develop an initial proof-of-concept Hp fusion protein using human serum albumin as the fusion partner. Next, a hemopexin-Hp fusion protein with bispecific heme and Hb detoxification capacity was generated. Further, we developed a Hb scavenger devoid of CD163 scavenger receptor binding. The functions of these proteins were then characterized for Hb and heme-binding, binding of the Hp-Hb complexes with the clearance receptor CD163, antioxidant properties, and vascular nitric oxide sparing capacity. Our platform is designed to support the generation of innovative Hb scavenger biotherapeutics with novel modes of action and potentially improved formulation characteristics, function, and pharmacokinetics.

Adenoviral transfer of hemopexin gene attenuates oxidative stress and apoptosis in cultured primary cortical neuron cell exposed to blood clot.

BACKGROUND: A growing body of experimental evidence suggests that hemin released from heme is a potent oxidant and accumulates in intracranial hematomas. Hemopexin (Hpx) decreases hemin accumulation and catabolism by nerve cells. In previous study, we observed that Hpx gene knockout aggravated striatal injury and worsened behavioral deficits of mice subjected to intracerebral hemorrhage. AIM: To examine the effect of Hpx on oxidative damage and apoptosis in cultured nerve cells with blood clot. METHODS: Neuron and glial cells were transfected with adenoviral Hpx gene. Transfected primary neuron-glial cells were co-cultured with 50 µl of arterial blood clot using insert transwells. The sham group was co-coulture with 50 µl of DMEM/F12, which contained 28 µl of serum; the control group was transfected with adenoviral vector. At 12 and 24 h, the level of malonaldehyde (MDA), surperoxide dismutase (SOD) concentration, glutathione (GSH), apoptosis, expression of HO-1 and caspase-3 were detected. RESULTS: MDA level was decreased (P < 0.01) whereas SOD and GSH concentration were increased in the Hpx group (P < 0.05 and P < 0.01, respectively). Results of flow cytometry revealed no significant difference in apoptosis between the Hpx group and model group at 12 h. However, the percentage of cells undergoing apoptosis in the Hpx group was decreased at 24 h compared with the model group (P < 0.01). HO-1 expression decreased in the Hpx group at 24 h (P < 0.01) while caspase-3 expression decreased at both 12 and 24 h (P < 0.011 and P < 0.05, respectively) compared with the model group. CONCLUSION: Hpx protected nerve cells exposed to blood from injury by anti-oxidation and a decrease in the expression of HO-1 and caspase-3.

Proteomic Profiling of Exosomes Derived from Plasma of HIV-Infected Alcohol Drinkers and Cigarette Smokers.

Abuse of alcohol and tobacco could exacerbate HIV pathogenesis by transferring materials through exosomes (small nanovesicles). Exosomes present a stable and accessible source of information concerning the health and/or disease status of patients, which can provide diagnostic and prognostic biomarkers for myriad conditions. Therefore, we aimed to study the specific exosomal proteins that are altered in both HIV-infected subjects and alcohol/tobacco users. Exosomes were isolated from plasma of the following subjects: a) HIV-negative subjects (healthy), b) HIV-positive subjects (HIV), c) HIV-negative alcohol drinkers (drinkers), d) HIV-negative tobacco smokers (smokers), e) HIV-positive drinkers (HIV + drinkers), and f) HIV-positive smokers (HIV + smokers). Quantitative proteomic profiling was then performed from these exosomes. Sixteen proteins were significantly altered in the HIV group, ten in drinkers, four in HIV + drinkers, and fifteen in smokers compared to healthy subjects. Only one protein, fibulin-1 (FBLN1), was significantly altered in HIV + smokers. Interestingly, hemopexin was not significantly altered in drinkers or HIV patients but was significantly altered in HIV + drinkers. Further, our study is the first to show properdin expression in plasma exosomes, which was decreased in HIV + smokers and HIV + drinkers compared to HIV patients. The present findings suggest that hemopexin and properdin show potential as markers for physiological effects that may arise in HIV-infected individuals who abuse alcohol and tobacco. Graphical abstract This study presents a proteomic analysis of plasma-derived exosomes from HIV-infected alcohol drinkers and smokers. Among the proteins altered due to drug-abuse, hemopexin and properdin were of highest significance. These proteins can be potential biomarkers for co-morbid conditions associated with drug abuse in HIV-patients.

Omega-3 Fatty Acids Responsive Proteins and Reduction in Breast Density in Obese Postmenopausal Women.

We reported that breast density (BD) was inversely correlated with the plasma level of DHA in postmenopausal obese, but not in nonobese, women given Lovaza (n-3FA). To identify protein biomarkers for the possible differential effect of n-3FA on BD between obese and nonobese women, an iTRAQ method was performed to analyze plasma from obese and lean women at each time point (baseline, 12 and 24-months, n = 10 per group); 173 proteins with >95% confidence (Unuses Score >1.3 and local false discovery rate estimation <5%) were identified. Comparative analysis between various groups identified several differentially expressed proteins (hemopexin precursor, vitamin D binding protein isoform 1 precursor [VDBP], fibronectin isoform 10 precursor [FN], and α-2 macroglobulin precursor [A2M]). Western blot analysis was performed to verify the differential expression of proteins in the iTRAQ study, and those found to be altered in a tumor protective fashion by an n-3FA rich diet in our previous preclinical study; gelsolin, VDBP, and FN were altered by n-3FA in a manner consistent with reduction in inflammation in obese women. To test the impact of our findings on breast cancer risk reduction by n-3FA, a posthoc analysis revealed that n-3FA administration reduced BD selectively in obese postmenopausal women.

A tale of two genes: divergent evolutionary fate of haptoglobin and hemopexin in hemoglobinless Antarctic icefishes.

The evolution of Antarctic notothenioid fishes in the isolated freezing Southern Ocean has led to remarkable trait gains and losses. One of the most extraordinary was the loss of the major oxygen carrier hemoglobin (Hb) in the icefishes (family Channichthyidae). Although the mechanisms of this loss and the resulting compensatory changes have been well studied, the impact of Hb loss on the network of genes that once supported its recycling and disposal has remained unexplored. Here, we report the functional fate and underlying molecular changes of two such key Hb-supporting proteins across the icefish family - haptoglobin (Hp) and hemopexin (Hx), crucial in removing cytotoxic free Hb and heme, respectively. Hp plays a critical role in binding free Hb for intracellular recycling and absent its primary client, icefish Hp transcription is now vanishingly little, and translation into a functional protein is nearly silenced. Hp genotype degeneration has manifested in separate lineages of the icefish phylogeny with three distinct nonsense mutations and a deletion frame shift, as well as mutated polyadenylation signal sequences. Thus, Hb loss appears to have diminished selective constraint on Hp maintenance, resulting in its stochastic, co-evolutionary drift towards extinction. Hx binds free heme for iron recycling in hepatocytes. In contrast to Hp, Hx genotype integrity is preserved in the icefishes and transcription occurs at levels comparable to those in the red-blooded notothenioids. The persistence of Hx likely owes to continued selective pressure for its function from mitochondrial and non-Hb cellular hemoproteins.

Proteomic Characterization of the Heart and Skeletal Muscle Reveals Widespread Arginine ADP-Ribosylation by the ARTC1 Ectoenzyme.

The clostridium-like ecto-ADP-ribosyltransferase ARTC1 is expressed in a highly restricted manner in skeletal muscle and heart tissue. Although ARTC1 is well studied, the identification of ARTC1 targets in vivo and subsequent characterization of ARTC1-regulated cellular processes on the proteome level have been challenging and only a few ARTC1-ADP-ribosylated targets are known. Applying our recently developed mass spectrometry-based workflow to C2C12 myotubes and to skeletal muscle and heart tissues from wild-type mice, we identify hundreds of ARTC1-ADP-ribosylated proteins whose modifications are absent in the ADP-ribosylome of ARTC1-deficient mice. These proteins are ADP-ribosylated on arginine residues and mainly located on the cell surface or in the extracellular space. They are associated with signal transduction, transmembrane transport, and muscle function. Validation of hemopexin (HPX) as a ARTC1-target protein confirmed the functional importance of ARTC1-mediated extracellular arginine ADP-ribosylation at the systems level.

Hemopexin is required for adult neurogenesis in the subventricular zone/olfactory bulb pathway.

The neural stem cells (NSCs) of the subventricular zone (SVZ) reside within a specialized niche critical for neurogenesis. Hemopexin, a plasma glycoprotein, has been extensively studied as a heme scavenger at the systemic level. However, little is known about its function in the central nervous system, especially in neurogenesis. In the present study, we demonstrate that deletion of hemopexin leads to neurogenic abnormalities in the SVZ/olfactory bulb (OB) pathway. The lateral ventricle is enlarged in hemopexin-deficient mice, and more apoptosis was observed in Dcx+ cells. Lineage differentiation of NSCs was also inhibited in the SVZ of hemopexin-deficient mice, with more stem cells stayed in an undifferentiated, GFAP+ radial glia-like cell stage. Moreover, hemopexin deletion resulted in impaired neuroblast migration in the rostral migratory stream. Furthermore, exogenous hemopexin protein inhibited apoptosis and promoted the migration and differentiation of cultured NSCs. Finally, immunohistochemical analysis demonstrated that deletion of hemopexin reduced the number of interneurons in the OB. Together, these results suggest a new molecular mechanism for the NSC niche that regulates adult neurogenesis in the SVZ/OB pathway. Our findings may benefit the understanding for olfactory system development.

Co-precipitation molecules hemopexin and transferrin may be key molecules for fibrillogenesis in TTR V30M amyloidogenesis.

The disease model of familial amyloidotic polyneuropathy-7.2-hMet30 mice-manifests amyloid deposition that consists of a human amyloidogenic mutant transthyretin (TTR) (TTR V30M). Our previous study found amyloid deposits in 14 of 27 7.2-hMet30 mice at 21-24 months of age. In addition, non-fibrillar TTR deposits were found in amyloid-negative 7.2hMet30 mice. These results suggested that TTR amyloidogenesis required not only mutant TTR but also an additional factor (or factors) as an etiologic molecule. To determine the differences in serum proteome in amyloid-positive and amyloid-negative mice in the 7.2-hMet30 model, we used proteomic analyses and studied serum samples obtained from these mice. Hemopexin (HPX) and transferrin (Tf) were detected in the serum samples from amyloid-positive mice and were also found in amyloid deposits via immunohistochemistry, but serum samples from amyloid-negative mice did not contain HPX and Tf. These two proteins were also not detected in non-fibrillar TTR deposits. In addition, in silico analyses suggested that HPX and Tf facilitate destabilization of TTR secondary structures and misfolding of TTR. These results suggest that HPX and Tf may be associated with TTR amyloidogenesis after fibrillogenesis in vivo.

Hypothermal stress induced differential expression profiles of the immune response gene, warm-temperature-acclimation associated 65-kDa protein (Wap65), in the liver of fresh water and seawater milkfish, Chanos chanos.

The milkfish (Chanos chanos), an important aquaculture species, is intolerant to cold environments. Temperature fluctuations in the environment affect the physiological response, behavior, and survival rate of the fish. The warm-temperature-acclimation associated 65-kDa protein (Wap65) of teleosts was identified after heat shock treatment and has two isoforms. Both the isoforms were involved in the induction of immune responses in fish. They showed high degree of sequence conservation with the mammalian hemopexin and had high affinity for heme, which helped in the neutralization of free-heme and its transport to the liver. In this study, we isolated and characterized the two isoforms of wap65 genes (Ccwap65-1 and Ccwap65-2) from the liver of milkfish. The Ccwap65-1 and Ccwap65-2 are mainly expressed in livers of milkfish. In hypothermal treatment, the expression levels of Ccwap65-2 in the livers of SW and FW milkfish were up-regulated after exposure to low temperature (18 °C) for 12 h and 96 h compared to those in the normal temperature (28 °C) group, respectively. After intraperitoneal injection of lipopolysaccharide (LPS), the expression of Ccwap65-2 was elevated in both SW and FW milkfish, whereas that of Ccwap65-1 was not affected in both the groups. Thus, Ccwap65-2 expressed in the milkfish liver under hypothermal stress was identified as a novel immune biomarker. In addition, according to the transcriptome database, up-regulation of the other immune-response genes indicated increased pathogen infection status under hypothermal stress. Acute increase in the expression of hepatic Ccwap65-2 in response to pathogen infection might lead to better cold tolerance of SW milkfish compared to that of the FW individuals upon cold challenge.

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.

Placental Proteomics Provides Insights into Pathophysiology of Pre-Eclampsia and Predicts Possible Markers in Plasma.

Pre-eclampsia is a hypertensive disorder characterized by the new onset of hypertension >140/90 mmHg and proteinuria after the 20th week of gestation. The disorder is multifactorial and originates with abnormal placentation. Comparison of the placental proteome of normotensive (n = 25) and pre-eclamptic (n = 25) patients by gel-free proteomic techniques identified a total of 2145 proteins in the placenta of which 180 were differentially expressed (>1.3 fold, p < 0.05). Gene ontology enrichment analysis of biological process suggested that the differentially expressed proteins belonged to various physiological processes such as angiogenesis, apoptosis, oxidative stress, hypoxia, and placental development, which are implicated in the pathophysiology of pre-eclampsia. Some of the differentially expressed proteins were monitored in the plasma by multiple reaction monitoring analysis, which showed an increase in apolipoproteins A-I and A-II in gestational weeks 26-30 (2-fold, p < 0.01), while haptoglobin and hemopexin decreased in gestational weeks 26-30 and week 40/at delivery (1.8 fold, p < 0.01) in pre-eclamptic patients. This study provides a proteomic insight into the pathophysiology of pre-eclampsia. Identified candidate proteins can be evaluated further for the development of potential biomarkers associated with pre-eclampsia pathogenesis.

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.

Deletion of the hemopexin or heme oxygenase-2 gene aggravates brain injury following stroma-free hemoglobin-induced intracerebral hemorrhage.

BACKGROUND: Following intracerebral hemorrhage (ICH), red blood cells release massive amounts of toxic heme that causes local brain injury. Hemopexin (Hpx) has the highest binding affinity to heme and participates in its transport, while heme oxygenase 2 (HO2) is the rate-limiting enzyme for the degradation of heme. Microglia are the resident macrophages in the brain; however, the significance and role of HO2 and Hpx on microglial clearance of the toxic heme (iron-protoporphyrin IX) after ICH still remain understudied. Accordingly, we postulated that global deletion of constitutive HO2 or Hpx would lead to worsening of ICH outcomes. METHODS: Intracerebral injection of stroma-free hemoglobin (SFHb) was used in our study to induce ICH. Hpx knockout (Hpx(-/-)) or HO2 knockout (HO2(-/-)) mice were injected with 10 µL of SFHb in the striatum. After injection, behavioral/functional tests were performed, along with anatomical analyses. Iron deposition and neuronal degeneration were depicted by Perls' and Fluoro-Jade B staining, respectively. Immunohistochemistry with anti-ionized calcium-binding adapter protein 1 (Iba1) was used to estimate activated microglial cells around the injured site. RESULTS: This study shows that deleting Hpx or HO2 aggravated SFHb-induced brain injury. Compared to wild-type littermates, larger lesion volumes were observed in Hpx(-/-) and HO2(-/-) mice, which also bear more degenerating neurons in the peri-lesion area 24 h postinjection. Fewer Iba1-positive microglial cells were detected at the peri-lesion area in Hpx(-/-) and HO2(-/-) mice, interestingly, which is associated with markedly increased iron-positive microglial cells. Moreover, the Iba1-positive microglial cells increased from 24 to 72 h postinjection and were accompanied with improved neurologic deficits in Hpx(-/-) and HO2(-/-) mice. These results suggest that Iba1-positive microglial cells could engulf the extracellular SFHb and provide protective effects after ICH. We then treated cultured primary microglial cells with SFHb at low and high concentrations. The results show that microglial cells actively take up the extracellular SFHb. Of interest, we also found that iron overload in microglia significantly reduces the Iba1 expression level and resultantly inhibits microglial phagocytosis. CONCLUSIONS: This study suggests that microglial cells contribute to hemoglobin-heme clearance after ICH; however, the resultant iron overloads in microglia appear to decrease Iba1 expression and to further inhibit microglial phagocytosis.