Haptoglobin and Hemopexin Redirect Heme-Driven Oxidative Stress and Neurotoxicity in Organotypic Brain Slices
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE299269 | GEO | 2025/12/29
REPOSITORIES: GEO
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Project description:Severe presentations of malaria emerge as parasites from Plasmodium spp. proliferate and lyse red blood cells (RBC), producing extracellular hemoglobin (HB). The heme prosthetic groups are released upon HB oxidation generating circulating labile heme. Here we asked whether scavenging of extracellular HB and/or labile heme, by haptoglobin (HP) and/or hemopexin (HPX), respectively, is protective against severe presentations of malaria. We found that circulating labile heme is an independent risk factor for cerebral and non-cerebral severe presentations of P. falciparum malaria. Labile heme was negatively correlated with HP and HPX, which were however, not risk factors for severe P. falciparum malaria. Genetic HP and/or HPX deletion in mice led to accumulation of labile heme in plasma and kidneys in response to Plasmodium (chabaudi chabaudi) infection. This was associated with increased mortality and acute kidney injury (AKI) in ageing but not adult mice, corroborated in P. falciparum malaria by a an inverse correlation between HPX and heme with serological markers of AKI. In conclusion, HP and HPX exert an age-dependent protective effect against malaria that counters the pathogenesis of AKI in mice and presumably in humans.
2024-01-30 | GSE253390 | GEO
Project description:Maintaining hemopexin (HPX) plasma levels protects against heme-activated inflammation and the toxicity of heme and its iron during hemolysis. Plasma heme regulates HPX turnover in hepatocytes, thus changing plasma HPX. However, the cell biology of hepatic HPX-mediated heme delivery is not fully elucidated. The low density-lipoprotein related protein 1 (LRP1) binds heme-HPX and targets it for lysosomal degradation. Nevertheless, heme from heme-HPX is delivered to the liver, and apo-HPX recycles without degradation. Heme-HPX endocytosis also occurs in mouse embryonic fibroblast LRP1-/-PEA 13 cells. Here, we characterize HPX receptors and HPX “interactomes” in a human neutrophil model, promyelocytic HL-60 cells, hepatoma HepG2 cells and primary human hepatocytes. Methods and Results: Immunoblots revealed that HL-60 cells lack LRP1; and immunocytochemistry data established that in HL60 cells, HPX trafficked with transferrin and transferrin receptor 1(TfR1) in Rab5-positive early endosomes supporting following a clathrin-mediated endocytotic pathway. Heme-HPX affinity chromatography of whole cell extracts isolated TfR1 and LRP1 as heme-HPX binding proteins and other proteins of novel HPX interactomes identified by their peptide sequences. TfR1 down-regulation in HL-60 cells decreased surface binding and intracellular HPX, implicating TfRs in heme-HPX endocytosis. In LRP+/+ HepG2 cells, HPX trafficked in endosomes with LRP1 and TfR1, or with TfR1 alone, and with TfR2, supporting that TfR2 potentially provides liver targeting of heme-HPX; Tfr1 and 2 could both account for apo-HPX recycling. LRP1 and TfR1 were identified in heme-HPX Affi-isolates from primary human hepatocytes, and this HPX interactome also included proteins associated with hemostasis, inflammation control, coagulation regulation, iron transport and body fluid regulation. Discussion: After heme-HPX uptake by human hepatocytes, HPX is recycled or degraded depending upon the relative expression of surface TfR1, TfR2 and LRP1. The overlapping and distinct roles of these receptors are discussed. TfR1 is a scavenger receptor like LRP1; nevertheless, specific hepatic HPX receptors may exist. Increasing knowledge of HPX biology will elucidate the causes that change plasma HPX, thus improving care in the clinic and in veterinary medicine, with increased understanding of the hematological adaptations to weightlessness that lead to clotting or anemia from hemolysis in astronauts and, potentially, space tourists.
2025-12-22 | PXD060789 | Pride
Project description:Hemoglobin (Hb) released from red blood cells during hemolysis is a trigger of oxidative vascular damage with endothelial cells being a primary target. The Hb and heme scavenger proteins haptoglobin (Hp) and hemopexin (Hx) have been characterized as a sequential defense system with Hp as the primary protector and Hx as a backup when all Hp is depleted during more severe or prolonged hemolysis. The paradigm of sequential protection by the two scavengers is based on observational patient data proposed by Mueller-Eberhardt. In this study we present a mechanistic rationale for these clinical observations using a novel in vitro model of Hb triggered endothelial damage. We identified oxyHb(Fe2+) transformation to ferric Hb(Fe3+), free heme transfer from ferric Hb(Fe3+) to lipoprotein and subsequent oxidative reactions in the lipophilic phase. The accumulation of toxic lipid peroxidation products liberated during oxidation reactions lead to endothelial damage characterized by a specific gene expression pattern, reduced cellular ATP and monolayer disintegration. Quantitative analysis of key biochemical and functional parameters allowed us to precisely define the mechanisms and concentrations required for Hp and Hx to prevent this toxicity. In the case of Hp we defined an exponential relationship between Hp availability relative to oxyHb(Fe2+) and related protective activity. This exponential relationship demonstrates that large Hp quantities are required to prevent Hb toxicity. In contrast, the linear relationship between Hx concentration and protective activity allows for significant protection by the backup scavenger during conditions of large excess of free oxyHb(Fe2+) that occurs when all Hp is consumed. The diverse protective function of Hp and Hx in this model can be explained by the different target specificities of the two proteins.
2018-02-25 | GSE66283 | GEO
Project description:Haptoglobin and Hemopexin are plasma acute phase proteins that bind with high affinity hemoglobin and heme, respectively. Several studies have demonstrated that they have a key role in the protection against oxidative stress and inflammation. However, little is known about the functional modules in which they are involved. To gain insights into this issue, we integrated bioinformatic and experimental approaches to identify genes coexpressed with Haptoglobin or Hemopexin and modulated in Haptoglobin and/or Hemopexin knock-out mice. These genes have a high probability to be functionally related to Haptoglobin and/or Hemopexin. We performed a gene expression analysis of the livers of Hp- and/or Hx-null mice compared to wild-type controls. The mice used in the following experiments, i.e. wild-type (Hp+/Hx+/), Hp-null (Hp-/-Hx+/), Hx-null (Hp+/Hx-/-), and HpHx-null (Hp-/-Hx-/-), were littermates derived by breeding F1 double heterozygous Hp+/-Hx+/- mice in the mixed genetic background C57/BLJ6 X 129Sv. We have three experimental points: Hx-null mice, Hp-null mice and HpHx-null mice. At least 5 adult mice per genotype were perfused via aorta with PBS, sacrificed and their liver pooled. Thirty microgram of total RNA were subjected to direct labeling reaction by incorporation of cyanin 3 (Cy3) or cyanin 5 (Cy5) fluorescent dyes into the cDNA by priming with oligo(dT). Four replicates were set up for each experimental point. In order to exclude artifacts resulting from different dye usage, we employed the dye-swap approach.
2006-10-31 | E-GEOD-3656 | biostudies-arrayexpress
Project description:Hemolysis of senescent erythrocytes occurs in the spleen, resulting in hemoglobin (Hb) release. Several diseases are associated with an increased risk of erythrocyte rupture. Iron recycling from Hb and Hb detoxification have been attributed to the sequestration of Hb-haptoglobin complexes by macrophages, however, other routes of Hb clearance might exist. We identified liver sinusoidal endothelial cells (LSECs) as the major cell type that sequesters Hb. Hb uptake involves macropinocytosis and is independent of haptoglobin and the Hb-haptoglobin receptor CD163. LSECs expressed heme oxygenase 1 and hepcidin-controlled ferroportin and were the most efficient cellular scavengers of Hb at doses below the haptoglobin binding capacity. Erythrocyte transfusion assays demonstrated that while splenic red pulp macrophages are efficient in erytrophagocytosis, liver Kupffer cells and LSECs are primarily involved in the clearance of erythrocyte ghosts and Hb, respectively. Using time-course injections of high doses of Hb in mice, we detected transient hepatic iron retention, early induction of Hmox1 specifically in LSECs, and elevation of ferroportin levels. This was associated with transcriptional activation of LSEC Bmp6, followed by hepcidin-mediated transient hypoferremia. Injection of Hb and iron-citrate elicited distinct transcriptional signatures in LSECs and the early Bmp6 induction was phenocopied by erythrocyte lysis upon phenylhydrazine. Collectively, we propose that LSECs provide a key mechanism for Hb clearance, a function that establishes the spleen-liver axis for physiological iron recycling from Hb and contributes to heme detoxification during hemolysis, coupled with the induction of the BMP6-hepcidin axis that restores homeostasis.
2025-08-01 | GSE240270 | GEO
Project description:After intracranial hemorrhages, heme is released from cell-free hemoglobin. This red blood cell toxin may drive secondary brain injury at the hematoma-brain interface. This study aimed to generate a spatially resolved map of the transcriptome-wide gene expression changes in the heme-exposed brain and define the potential therapeutic activity of the heme-binding protein hemopexin.
2021-11-25 | GSE182127 | GEO
Project description:Extracellular hemoglobin (Hb) has been recognized as a disease trigger in hemolytic conditions such as sickle cell disease, malaria and blood transfusion. In vivo, many of the adverse effects of free Hb can be attenuated by the Hb scavenger acute phase protein haptoglobin (Hp). The primary physiologic disturbances that can be caused be free Hb are found within the cardiovascular system and Hb triggered oxidative toxicity towards the endothelium has been promoted as a potential mechanism. The molecular mechanisms of this toxicity as well as of the protective activities of Hp are not yet clear. Within this study we systematically investigated the structural, biochemical and cell biologic nature of Hb toxicity in an endothelial cell system under peroxidative stress. We identified two principal mechanisms of oxidative Hb toxicity that are mediated by globin degradation products and by modified lipoprotein species, respectively. The two damage pathways trigger diverse and discriminative inflammatory and cytotoxic responses. Hp provides structural stabilization of Hb and shields Hb’s oxidative reactions with lipoproteins providing dramatic protection against both pathways of toxicity. By these mechanisms Hp shifts Hb’s destructive pseudo-peroxidative reaction into a potential anti-oxidative function during peroxidative stress.
2015-04-30 | GSE46471 | GEO
Project description:Methods of structural mass spectrometry have become more popular to study protein structure and dynamics. Among them, fast photochemical oxidation of proteins (FPOP) has several advantages such as irreversibility of modifications and more facile determination of the site of modification with single residue resolution. In the present study, FPOP analysis was applied to study the hemoglobin (Hb) – haptoglobin (Hp) complex allowing identification of respective regions altered upon the complex formation. The footprinting using a timsTOF Pro mass spectrometer allowed to obtain structural information for 84 and 76 residues in Hp and Hb, respectively, including statistically significant differences in the modification extent below 0.3 %. The most affected residues upon the complex formation were Met76 and Tyr140 in Hbα, and Tyr280 and Trp284 in Hpβ. The data allowed determination of amino acids directly involved in Hb – Hp interactions and those located outside of the interaction interface yet affected by the complex formation. Also, previously modeled interaction between Hb βTrp37 and Hp βPhe292 was not confirmed by our data.
2021-12-30 | PXD021621 | Pride
Project description:Hemoglobin (Hb) is released from red blood cells (RBC) during intravascular hemolysis. Cell free Hb has been implicated to play a pathogenic role in hemolytic diseases such as sickle cell anemia or malaria. Hydrogen peroxide (H2O2) is the most prevalent reactive oxygen species which is produced in excess during inflammation or tissue injury and has been included as a potential mediator of oxidative stress related cell and tissue damage. The biologic significance of Hbs peroxidase activity as an anti-oxidant is controversial as the radicals and higher oxidation iron species which are potentially released during these reactions could be a source of exaggerated oxidative damage. Using a cell culture model of low-flux continuous H2O2 generation by glucose-oxidase (GOx) Hb revealed the potential to protect vascular smooth muscle cells and macrophages from H2O2 mediated glutathione depletion and cell death. As revealed by gene array analysis, Hb effectively abolished H2O2 induced oxidative stress response in these cells. Through electron microscopy and quantitative mass spectrometry extensive oxidation of specific Hb amino acid residues, polymerization and precipitation was defined. This may be a consequence of potential protective mechanism within Hb. The net biologic effect of Hb, when present in oxidative rich environments, is likely a result of the balance of H2O2 consumption (protection) on one hand and the production of free radicals (damage) on the other hand. Further the extensive structural changes occurring during the reaction of ferrous Hb with H2O2 could 'absorb' a significant amount of oxidative impact and thereby further protect the environment from heme derived free radical damage. The intrinsic peroxidase activity of hemoglobin seems to be a constitutive 'enzymatic' function of Hb which adds a novel facet to the multivalent role played by the abundant oxygen carrier protein.
2009-09-15 | E-MEXP-2263 | biostudies-arrayexpress
Project description:Extracellular hemoglobin (Hb) has been recognized as a disease trigger in hemolytic conditions such as sickle cell disease, malaria and blood transfusion. In vivo, many of the adverse effects of free Hb can be attenuated by the Hb scavenger acute phase protein haptoglobin (Hp). The primary physiologic disturbances that can be caused be free Hb are found within the cardiovascular system and Hb triggered oxidative toxicity towards the endothelium has been promoted as a potential mechanism. The molecular mechanisms of this toxicity as well as of the protective activities of Hp are not yet clear. Within this study we systematically investigated the structural, biochemical and cell biologic nature of Hb toxicity in an endothelial cell system under peroxidative stress. We identified two principal mechanisms of oxidative Hb toxicity that are mediated by globin degradation products and by modified lipoprotein species, respectively. The two damage pathways trigger diverse and discriminative inflammatory and cytotoxic responses. Hp provides structural stabilization of Hb and shields Hb’s oxidative reactions with lipoproteins providing dramatic protection against both pathways of toxicity. By these mechanisms Hp shifts Hb’s destructive pseudo-peroxidative reaction into a potential anti-oxidative function during peroxidative stress. HPAEC: A two color common reference design was chosen with 4-8 independent biological replicates of each condition. Each experimental sample (Cy5 labeled) was hybridized against a non-treated reference sample (Cy3 labeled). HUVEC: A two color common reference design was chosen with 3-4 independent biological replicates of each condition. Each experimental sample (Cy5 labeled) was hybridized against a non-treated reference sample (Cy3 labeled).
2015-04-30 | E-GEOD-46471 | biostudies-arrayexpress