HIF-1α-Dependent Metabolic Reprogramming, Oxidative Stress, and Bioenergetic Dysfunction in SARS-CoV-2-Infected Hamsters.

The mechanistic interplay between SARS-CoV-2 infection, inflammation, and oxygen homeostasis is not well defined. Here, we show that the hypoxia-inducible factor (HIF-1α) transcriptional pathway is activated, perhaps due to a lack of oxygen or an accumulation of mitochondrial reactive oxygen species (ROS) in the lungs of adult Syrian hamsters infected with SARS-CoV-2. Prominent nuclear localization of HIF-1α and increased expression of HIF-1α target proteins, including glucose transporter 1 (Glut1), lactate dehydrogenase (LDH), and pyruvate dehydrogenase kinase-1 (PDK1), were observed in areas of lung consolidation filled with infiltrating monocytes/macrophages. Upregulation of these HIF-1α target proteins was accompanied by a rise in glycolysis as measured by extracellular acidification rate (ECAR) in lung homogenates. A concomitant reduction in mitochondrial respiration was also observed as indicated by a partial loss of oxygen consumption rates (OCR) in isolated mitochondrial fractions of SARS-CoV-2-infected hamster lungs. Proteomic analysis further revealed specific deficits in the mitochondrial ATP synthase (Atp5a1) within complex V and in the ATP/ADP translocase (Slc25a4). The activation of HIF-1α in inflammatory macrophages may also drive proinflammatory cytokine production and complement activation and oxidative stress in infected lungs. Together, these findings support a role for HIF-1α as a central mediator of the metabolic reprogramming, inflammation, and bioenergetic dysfunction associated with SARS-CoV-2 infection.

Structural Integrity of the Alveolar-Capillary Barrier in Cynomolgus Monkeys Challenged with Fully Virulent and Toxin-Deficient Strains of Bacillus anthracis.

Inhalational anthrax, a disease caused by inhaling Bacillus anthracis spores, leads to respiratory distress, vascular leakage, high-level bacteremia, and often death within days. Anthrax lethal toxin and edema toxin, which are composed of protective antigen (PA) plus either lethal factor (LF) or edema factor (EF), respectively, play an important yet incompletely defined role in the pulmonary pathophysiology. To better understand their contribution, we examined the structural integrity of the alveolar-capillary barrier in archival formalin-fixed lungs of cynomolgus monkeys challenged with the fully virulent B. anthracis Ames wild-type strain or the isogenic toxin-deficient mutants ΔEF, ΔLF, and ΔPA. Pulmonary spore challenge with the wild-type strain caused high mortality, intra-alveolar hemorrhages, extensive alveolar septal sequestration of bacteria and neutrophils, diffuse destabilization of epithelial and endothelial junctions, increased markers of coagulation and complement activation (including tissue factor and C5a), and multifocal intra-alveolar fibrin deposition. ΔEF challenge was lethal and showed similar alveolar-capillary alterations; however, intra-alveolar hemorrhages, bacterial deposition, and markers of coagulation or complement were absent or markedly lower. In contrast, ΔLF or ΔPA challenges were nonlethal and showed no signs of alveolar bacterial deposition or alveolar-capillary changes. These findings provide evidence that lethal toxin plays a determinative role in bacterial dissemination and alveolar-capillary barrier dysfunction, and edema toxin may significantly exacerbate pulmonary pathologies in a systemic infection.

Comprehensive Biochemical and Biophysical Characterization of Hemoglobin-Based Oxygen Carrier Therapeutics: All HBOCs Are Not Created Equally.

The development of hemoglobin (Hb)-based oxygen carriers (HBOCs) has been hampered because of safety concerns in humans. Chemical and/or genetic modifications of the Hb introduce varied structural and conformational constraint on the molecule that resulted in proteins with diverse allosteric responses, nitrosative and oxidative side reactions. Here, we present for the first time a comprehensive biochemical and biophysical comparison of human, bovine, and genetically engineered HBOCs that have been tested in humans. We evaluate oxygen equilibrium and ligand binding kinetics under different experimental conditions as well as their autoxidation kinetics, redox reactions, and heme release. We determined the effects of HBOCs on cellular redox states and mitochondrial respiration. Taken together, these experiments provide a better understanding of the relationship between the structure-function and oxidative reactivity of these proteins. One can therefore select independently among these diverse properties to engineer a safe and effective HBOC with improved biochemical/biophysical characteristics.

1918 pandemic influenza virus and Streptococcus pneumoniae co-infection results in activation of coagulation and widespread pulmonary thrombosis in mice and humans.

To study bacterial co-infection following 1918 H1N1 influenza virus infection, mice were inoculated with the 1918 influenza virus, followed by Streptococcus pneumoniae (SP) 72 h later. Co-infected mice exhibited markedly more severe disease, shortened survival time and more severe lung pathology, including widespread thrombi. Transcriptional profiling revealed activation of coagulation only in co-infected mice, consistent with the extensive thrombogenesis observed. Immunohistochemistry showed extensive expression of tissue factor (F3) and prominent deposition of neutrophil elastase on endothelial and epithelial cells in co-infected mice. Lung sections of SP-positive 1918 autopsy cases showed extensive thrombi and prominent staining for F3 in alveolar macrophages, monocytes, neutrophils, endothelial and epithelial cells, in contrast to co-infection-positive 2009 pandemic H1N1 autopsy cases. This study reveals that a distinctive feature of 1918 influenza virus and SP co-infection in mice and humans is extensive expression of tissue factor and activation of the extrinsic coagulation pathway leading to widespread pulmonary thrombosis.

Ultraviolet light and riboflavin accelerates red blood cell dysfunction in vitro and in a guinea pig transfusion model.

BACKGROUND: Quality assessment of modified or processed red blood cell (RBC) components, such as pathogen-reduced RBCs, using only in vitro testing may not always be predictive of in vivo performance. Mouse or rat in vivo models are limited by a lack of applicability to certain aspects of human RBC biology. Here, we used a guinea pig model to study the effects of riboflavin combined with UV light on the integrity of RBCs in vitro and following transfusion in vivo. MATERIALS AND METHODS: Guinea pig RBCs were collected from whole blood (WB) treated with varying UV doses (10, 20, 40 or 80 J/mL) in the presence of riboflavin (UVR-RBCs). In vitro tests for UVR-RBCs included hemolysis, osmotic fragility, and cellular morphology by scanning electron microscopy. Guinea pigs transfused with one-day post-treatment UVR-RBCs were evaluated for plasma hemoglobin (Hb), non-transferrin bound iron (NTBI), total iron and Perls-detectable hemosiderin deposition in the spleen and kidney, and renal uptake of Hb. RESULTS: Acute RBC injury was dose dependently accelerated after treatment with UV light in the presence of riboflavin. Aberrant RBC morphology was evident at 20, 40, and 80 J/mL, and membrane lysis with Hb release was prominent at 80 J/mL. Guinea pigs transfused with 40 and 80 J/mL UVR-RBCs showed increased plasma Hb levels, and plasma NTBI was elevated in all UVR-RBC groups (10-80 J/mL). Total iron levels and Perls-hemosiderin staining in spleen and kidney as well as Hb uptake in renal proximal tubules were increased 8 hours post-transfusion with 40 and 80 J/mL UVR-RBCs. DISCUSSION: UVR-RBCs administered to guinea pigs increased markers of intravascular and extravascular hemolysis in a UV dose-dependent manner. This model may allow for the discrimination of RBC injury during testing of extensively processed RBCs intended for transfusion.

Renal glomerular and tubular responses to glutaraldehyde- polymerized human hemoglobin.

Hemoglobin-based oxygen carriers (HBOCs) are being developed as oxygen and volume replacement therapeutics, however, their molecular and cellular effects on the vasculature and different organ systems are not fully defined. Using a guinea pig transfusion model, we examined the renal glomerular and tubular responses to PolyHeme, a highly characterized glutaraldehyde-polymerized human hemoglobin with low tetrameric hemoglobin content. PolyHeme-infused animals showed no major changes in glomerular histology or loss of specific markers of glomerular podocytes (Wilms tumor 1 protein, podocin, and podocalyxin) or endothelial cells (ETS-related gene and claudin-5) after 4, 24, and 72 h. Relative to sham controls, PolyHeme-infused animals also showed similar expression and subcellular distribution of N-cadherin and E-cadherin, two key epithelial junctional proteins of proximal and distal tubules, respectively. In terms of heme catabolism and iron-handling responses, PolyHeme induced a moderate but transient expression of heme oxygenase-1 in proximal tubular epithelium and tubulointerstitial macrophages that was accompanied by increased iron deposition in tubular epithelium. Contrary to previous findings with other modified or acellular hemoglobins, the present data show that PolyHeme does not disrupt the junctional integrity of the renal glomerulus and tubular epithelium, and triggers moderate activation of heme catabolic and iron sequestration systems likely as part of a renal adaptive response.

Intranasal or airborne transmission-mediated delivery of an attenuated SARS-CoV-2 protects Syrian hamsters against new variants.

Detection of secretory antibodies in the airway is highly desirable when evaluating mucosal protection by vaccines against a respiratory virus, such as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We show that intranasal delivery of an attenuated SARS-CoV-2 (Nsp1-K164A/H165A) induces both mucosal and systemic IgA and IgG in male Syrian hamsters. Interestingly, either direct intranasal immunization or airborne transmission-mediated delivery of Nsp1-K164A/H165A in Syrian hamsters offers protection against heterologous challenge with variants of concern (VOCs) including Delta, Omicron BA.1, BA.2.12.1 and BA.5. Vaccinated animals show significant reduction in both tissue viral loads and lung inflammation. Similarly attenuated viruses bearing BA.1 and BA.5 spike boost variant-specific neutralizing antibodies in male mice that were first vaccinated with modified vaccinia virus Ankara vectors (MVA) expressing full-length WA1/2020 Spike protein. Together, these results demonstrate that our attenuated virus may be a promising nasal vaccine candidate for boosting mucosal immunity against future SARS-CoV-2 VOCs.

Blood-brain barrier disruption and oxidative stress in guinea pig after systemic exposure to modified cell-free hemoglobin.

Systemic exposure to cell-free hemoglobin (Hb) or its breakdown products after hemolysis or with the use of Hb-based oxygen therapeutics may alter the function and integrity of the blood-brain barrier. Using a guinea pig exchange transfusion model, we investigated the effect of a polymerized cell-free Hb (HbG) on the expression of endothelial tight junction proteins (zonula occludens 1, claudin-5, and occludin), astrocyte activation, IgG extravasation, heme oxygenase (HO), iron deposition, oxidative end products (4-hydroxynonenal adducts and 8-hydroxydeoxyguanosine), and apoptosis (cleaved caspase 3). Reduced zonula occludens 1 expression was observed after HbG transfusion as evidenced by Western blot and confocal microscopy. Claudin-5 distribution was altered in small- to medium-sized vessels. However, total expression of claudin-5 and occludin remained unchanged except for a notable increase in occludin 72 hours after HbG transfusion. HbG-transfused animals also showed increased astrocytic glial fibrillary acidic protein expression and IgG extravasation after 72 hours. Increased HO activity and HO-1 expression with prominent enhancement of HO-1 immunoreactivity in CD163-expressing perivascular cells and infiltrating monocytes/macrophages were also observed. Consistent with oxidative stress, HbG increased iron deposition, 4-hydroxynonenal and 8-hydroxydeoxyguanosine immunoreactivity, and cleaved caspase-3 expression. Systemic exposure to an extracellular Hb triggers blood-brain barrier disruption and oxidative stress, which may have important implications for the use of Hb-based therapeutics and may provide indirect insight on the central nervous system vasculopathies associated with excessive hemolysis.

Active and Passive Immunization of Syrian Hamsters with An Attenuated SARS-CoV-2 Protects against New Variants of Concern.

Detection of secretory antibodies in the airway is highly desirable when evaluating mucosal protection by a vaccine against a respiratory virus like the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We show that a single intranasal delivery of an attenuated SARS-CoV-2 (Nsp1-K164A/H165A) induced both mucosal and systemic IgA and IgG in Syrian hamsters. Interestingly, either active or passive immunization of hamsters with Nsp1-K164A/H165A offered protection against heterologous challenge with variants of concern (VOCs) including Delta, Omicron BA.1, and Omicron BA.2.12.1. Among challenged animals, Nsp1-K164A/H165A vaccination specifically reduced viral loads in the respiratory tract and suppressed infection-induced macrophage accumulation and MX1 upregulation in the lung. The absence of variant-specific mucosal and systemic antibodies was associated with breakthrough infections, particularly of the nasal cavity following challenges with Omicron isolates. Together, our study demonstrates that an attenuated nasal vaccine may be developed to boost mucosal immunity against future SARS-CoV-2 VOCs.

Intranasal delivery of a rationally attenuated SARS-CoV-2 is immunogenic and protective in Syrian hamsters.

Few live attenuated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines are in pre-clinical or clinical development. We seek to attenuate SARS-CoV-2 (isolate WA1/2020) by removing the polybasic insert within the spike protein and the open reading frames (ORFs) 6-8, and by introducing mutations that abolish non-structural protein 1 (Nsp1)-mediated toxicity. The derived virus (WA1-ΔPRRA-ΔORF6-8-Nsp1K164A/H165A) replicates to 100- to 1000-fold-lower titers than the ancestral virus and induces little lung pathology in both K18-human ACE2 (hACE2) transgenic mice and Syrian hamsters. Immunofluorescence and transcriptomic analyses of infected hamsters confirm that three-pronged genetic modifications attenuate the proinflammatory pathways more than the removal of the polybasic cleavage site alone. Finally, intranasal administration of just 100 PFU of the WA1-ΔPRRA-ΔORF6-8-Nsp1K164A/H165A elicits robust antibody responses in Syrian hamsters and protects against SARS-CoV-2-induced weight loss and pneumonia. As a proof-of-concept study, we demonstrate that live but sufficiently attenuated SARS-CoV-2 vaccines may be attainable by rational design.

Sodium nitrite induces acute central nervous system toxicity in guinea pigs exposed to systemic cell-free hemoglobin.

Systemic cell-free hemoglobin (Hb) released via hemolysis disrupts vascular homeostasis, in part, through the scavenging of nitric oxide (NO). Sodium nitrite (NaNO(2)) therapy can attenuate the hypertensive effects of Hb. However, the chemical reactivity of NaNO(2) with Hb may enhance heme- or iron-mediated toxicities. Here, we investigate the effect of NaNO(2) on the central nervous system (CNS) in guinea pigs exposed to systemic cell-free Hb. Intravascular infusion of NaNO(2), at doses sufficient to alleviate Hb-mediated blood pressure changes, reduced the expression of occludin, but not zona occludens-1 (ZO-1) or claudin-5, in cerebral tight junctions 4h after Hb infusion. This was accompanied by increased perivascular heme oxygenase-1 expression, neuronal iron deposition, increased astrocyte and microglial activation, and reduced expression of neuron-specific nuclear protein (NeuN). These CNS changes were not observed in animals treated with Hb or NaNO(2) alone. Taken together, these findings suggest that the use of nitrite salts to treat systemic Hb exposure may promote acute CNS toxicity.

Lung epithelial and endothelial damage, loss of tissue repair, inhibition of fibrinolysis, and cellular senescence in fatal COVID-19.

Coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is characterized by respiratory distress, multiorgan dysfunction, and, in some cases, death. The pathological mechanisms underlying COVID-19 respiratory distress and the interplay with aggravating risk factors have not been fully defined. Lung autopsy samples from 18 patients with fatal COVID-19, with symptom onset-to-death times ranging from 3 to 47 days, and antemortem plasma samples from 6 of these cases were evaluated using deep sequencing of SARS-CoV-2 RNA, multiplex plasma protein measurements, and pulmonary gene expression and imaging analyses. Prominent histopathological features in this case series included progressive diffuse alveolar damage with excessive thrombosis and late-onset pulmonary tissue and vascular remodeling. Acute damage at the alveolar-capillary barrier was characterized by the loss of surfactant protein expression with injury to alveolar epithelial cells, endothelial cells, respiratory epithelial basal cells, and defective tissue repair processes. Other key findings included impaired clot fibrinolysis with increased concentrations of plasma and lung plasminogen activator inhibitor-1 and modulation of cellular senescence markers, including p21 and sirtuin-1, in both lung epithelial and endothelial cells. Together, these findings further define the molecular pathological features underlying the pulmonary response to SARS-CoV-2 infection and provide important insights into signaling pathways that may be amenable to therapeutic intervention.

Anthrax lethal toxin enhances IkappaB kinase activation and differentially regulates pro-inflammatory genes in human endothelium.

Anthrax lethal toxin (LT) was previously shown to enhance transcriptional activity of NF-kappaB in tumor necrosis factor-alpha-activated primary human endothelial cells. Here we show that this LT-mediated increase in NF-kappaB activation is associated with the enhanced degradation of the inhibitory proteins IkappaBalpha and IkappaBbeta but not IkappaBepsilon. Moreover, this was accompanied by enhanced activation of the IkappaB kinase complex (IKK), which is responsible for targeting IkappaB proteins for degradation. Importantly, LT enhancement of IkappaBalpha degradation was completely blocked by a selective IKKbeta inhibitor, whereas IkappaBbeta degradation was attenuated, suggesting a mechanistic link. Consistent with the above data, LT-cotreated cells show elevated phosphorylation of two IKK substrates, IkappaBalpha and p65, both of which were blocked by incubation with the IKKbeta inhibitor. Consistent with NF-kappaB activation, LT increased transcription of the NF-kappaB regulated gene CD40. Conversely, LT inhibited transcription of another NF-kappaB-regulated gene, CCL2. This inhibition was linked to the LT-mediated suppression of another CCL2-regulating transcription factor, AP-1 (activator protein-1). These data suggest that LT-mediated enhancement of NF-kappaB is IKK-dependent, but importantly, the net effect of LT on the transcription of proinflammatory genes is driven by the cumulative effect of LT on the particular set of transcription factors that regulate a given promoter. Together, these findings provide new mechanistic insight on how LT may disrupt the host response to anthrax.

Acellular haemoglobin attenuates hypoxia-inducible factor-1alpha (HIF-1alpha) and its target genes in haemodiluted rats.

Hb (haemoglobin)-based blood substitutes represent a class of therapeutics designed to correct oxygen deficit under conditions of anaemia and traumatic blood loss. The influences of these agents on HIF-1alpha (hypoxia-inducible factor-1alpha) target genes involved in adaptation to hypoxia have so far not been studied. In the study presented here, rats underwent 80% ET (exchange transfusion) with either HS (hetastarch) or a polymerized Hb OG (Oxyglobin). HS induced dramatic EPO (erythropoietin) gene transcription, reaching a maximum at 4 h post-ET. In contrast, OG suppressed EPO transcription until approx. 24 h post-ET. Large plasma EPO levels that were observed post-ET with HS were significantly blunted in animals transfused with OG. OG, unlike HS, induced a sharp increase in HO-1 (haem oxygenase-1) transcription at 4 h, which declined rapidly within 24 h, whereas modest increases in iNOS [inducible (nitric oxide synthase)] and constitutive NOS [eNOS (endothelial NOS)] were detected over the control. Our results demonstrate for the first time that severe haemodilution-induced erythropoietic responses in kidneys were attenuated by a low-oxygen-affinity cell-free Hb and suggest that tissue-specific oxygen-sensing pathways can be influenced by allosterically modified Hbs.

Reversible renal glomerular dysfunction in guinea pigs exposed to glutaraldehyde-polymerized cell-free hemoglobin.

Chemically modified hemoglobin (Hb)-based oxygen carriers are promising oxygen replacement therapeutics however their potential renal effects are not fully understood. Using a guinea pig exchange transfusion model, we examined the effects of glutaraldehyde-polymerized bovine hemoglobin (HbG) on the permeability and integrity of the glomerular filtration barrier (GFB), which is comprised of podocytes, fenestrated endothelium, and the glomerular basement membrane. HbG induced marked proteinuria characterized in part by the loss of high molecular weight proteins, including albumin, immunoglobulin, and transferrin, at 4 and 12 h post-infusion that resolved by 72 h. This correlated with HbG-induced GFB alterations based on the reduced expression of specific markers of podocytes (podocin, nephrin, podocalyxin, and Wilms Tumor 1 protein) and endothelial cells (ETS-related gene and claudin-5). Lectin binding studies also demonstrated marked but reversible alterations to the GFB glycocalyx accompanied by increased intraglomerular HbG deposition and 4-HNE protein adduct expression indicative of oxidative damage. Together, these findings indicate that HbG induces reversible glomerular barrier dysfunction in conjunction with transient GFB changes providing new insight into the renal response to chemically modified Hb therapeutics.

Poly(I:C) Induces Human Lung Endothelial Barrier Dysfunction by Disrupting Tight Junction Expression of Claudin-5.

Viral infections are often accompanied by pulmonary microvascular leakage and vascular endothelial dysfunction via mechanisms that are not completely defined. Here, we investigated the effect of the Toll-like receptor 3 (TLR3) ligand polyinosinic-polycytidylic acid [Poly(I:C)], a synthetic analog of viral double-stranded RNA (dsRNA) commonly used to simulate viral infections, on the barrier function and tight junction integrity of primary human lung microvascular endothelial cells. Poly(I:C) stimulated IL-6, IL-8, TNFα, and IFNß production in conjunction with the activation of NF-κB and IRF3 confirming the Poly(I:C)-responsiveness of these cells. Poly(I:C) increased endothelial monolayer permeability with a corresponding dose- and time-dependent decrease in the expression of claudin-5, a transmembrane tight junction protein and reduction of CLDN5 mRNA levels. Immunofluorescence experiments revealed disappearance of membrane-associated claudin-5 and co-localization of cytoplasmic claudin-5 with lysosomal-associated membrane protein 1. Chloroquine and Bay11-7082, inhibitors of TLR3 and NF-κB signaling, respectively, protected against the loss of claudin-5. Together, these findings provide new insight on how dsRNA-activated signaling pathways may disrupt vascular endothelial function and contribute to vascular leakage pathologies.

Transcriptional Suppression of Renal Antioxidant Enzyme Systems in Guinea Pigs Exposed to Polymerized Cell-Free Hemoglobin.

Hemoglobin-based oxygen carriers (HBOCs) are being developed as oxygen and plasma volume-expanding therapeutics though their potential to promote oxidative tissue injury has raised safety concerns. Using a guinea pig exchange transfusion model, we examined the effects of polymerized bovine hemoglobin (HbG) on the transcriptional regulation, activity, and expression of the renal antioxidant enzymes; superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx). HbG infusion downregulated the mRNA levels for genes encoding SOD isoforms 1-3, GPx1, GPx3, GPx4, and CAT. This transcriptional suppression correlated with decreased enzymatic activities for SOD, CAT, and GPx. Immunostaining revealed decreased protein expression of SOD1, CAT, and GPx1 primarily in renal cortical tubules. DNA methylation analyses identified CpG hypermethylation in the gene promoters for SOD1-3, GPx1, GPx3, and GPx4, suggesting an epigenetic-based mechanism underlying the observed gene repression. HbG also induced oxidative stress as evidenced by increased renal lipid peroxidation end-products and 4-HNE immunostaining, which could be the result of the depleted antioxidant defenses and/or serve as a trigger for increased DNA methylation. Together, these findings provide evidence that the renal exposure to HbG suppresses the function of major antioxidant defense systems which may have relevant implications for understanding the safety of hemoglobin-based products.

Sodium nitrite potentiates renal oxidative stress and injury in hemoglobin exposed guinea pigs.

Methemoglobin-forming drugs, such as sodium nitrite (NaNO2), may exacerbate oxidative toxicity under certain chronic or acute hemolytic settings. In this study, we evaluated markers of renal oxidative stress and injury in guinea pigs exposed to extracellular hemoglobin (Hb) followed by NaNO2 at doses sufficient to simulate clinically relevant acute methemoglobinemia. NaNO2 induced rapid and extensive oxidation of plasma Hb in this model. This was accompanied by increased renal expression of the oxidative response effectors nuclear factor erythroid 2-derived-factor 2 (Nrf-2) and heme oxygenase-1 (HO-1), elevated non-heme iron deposition, lipid peroxidation, interstitial inflammatory cell activation, increased expression of tubular injury markers kidney injury-1 marker (KIM-1) and liver-fatty acid binding protein (L-FABP), podocyte injury, and cell death. Importantly, these indicators of renal oxidative stress and injury were minimal or absent following infusion of Hb or NaNO2 alone. Together, these results suggest that the exposure to NaNO2 in settings associated with increased extracellular Hb may potentiate acute renal toxicity via processes that are independent of NaNO2 induced erythrocyte methemoglobinemia.

A role for the myoglobin redox cycle in the induction of endothelial cell apoptosis.

This study investigates the potential role of the ferric/ferryl redox cycle of myoglobin (Mb) in the development of endothelial cell injury. Bovine aortic endothelial cells were incubated with ferric Mb (0.5-100 micro M) in the presence or absence of low steady states of H(2)O(2) (3-4 micro M) generated by glucose oxidase (GOX). The reaction of ferric Mb with H(2)O(2) generated ferryl Mb as monitored spectrophotometrically. Ferryl Mb formation correlated with the induction of apoptosis as indicated by morphological criteria, caspase 3 activation, phosphatidylserine (PS) externalization, and nuclear condensation by Hoechst 33342 staining. The addition of ascorbate or catalase inhibited the formation of ferryl Mb and the onset of apoptosis, whereas apoptosis was enhanced in cells depleted of intracellular glutathione by pretreatment with buthionine sulfoximine. Mb and Mb/GOX suppressed cell cycle progression, but only Mb/GOX produced significant cell loss revealed by the accumulation of sub G1 events. These results suggest a role for the Mb redox cycle in the induction of endothelial cell apoptosis, which may be relevant in the pathophysiology of diseases characterized by the release of Mb from damaged muscle.

Extracellular Hb enhances cardiac toxicity in endotoxemic guinea pigs: protective role of haptoglobin.

Endotoxemia plays a major causative role in the myocardial injury and dysfunction associated with sepsis. Extracellular hemoglobin (Hb) has been shown to enhance the pathophysiology of endotoxemia. In the present study, we examined the myocardial pathophysiology in guinea pigs infused with lipopolysaccharide (LPS), a Gram-negative bacterial endotoxin, and purified Hb. We also examined whether the administration of the Hb scavenger haptoglobin (Hp) could protect against the effects observed. Here, we show that Hb infusion following LPS administration, but not either insult alone, increased myocardial iron deposition, heme oxygenase-1 expression, phagocyte activation and infiltration, as well as oxidative DNA damage and apoptosis assessed by 8-hydroxy-2'-deoxyguanosine (8-OHdG) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) immunostaining, respectively. Co-administration of Hp significantly attenuated the myocardial events induced by the combination of LPS and Hb. These findings may have relevant therapeutic implications for the management of sepsis during concomitant disease or clinical interventions associated with the increased co-exposures to LPS and Hb, such as trauma, surgery or massive blood transfusions.