Methylglyoxal-derived hemoglobin advanced glycation end products induce apoptosis and oxidative stress in human umbilical vein endothelial cells.

The presence of excess glucose promotes hemoglobin glycation via the biochemical modification of hemoglobin by dicarbonyl products. However, the precise effects of Hb-AGEs in human umbilical vein endothelial cells (HUVECs) are not known to date. Therefore, we investigated the tentative effects of Hb-AGEs in HUVECs. Initially, we used the AGE formation assay to examine the selectivity of MGO toward various proteins. Among all proteins, MGO-Hb-AGEs formation was higher compared to the formation of other dicarbonyl-mediated AGEs. Our next data demonstrated that treatment with 0.5 mg/mL of Hb-AGEs-4w significantly reduced cell viability in HUVECs. Further, we evaluated the role of MGO in conformational and structural changes in Hb. The results showed that Hb demonstrated a highly altered conformation upon incubation with MGO. Moreover, Hb-AGEs-4w treatment strongly increased ROS production, and decreased mitochondrial membrane potential in HUVECs, and moderately reduced the expression of phosphorylated forms of p-38 and JNK. We observed that Hb-AGEs-4w treatment increased the number of apoptotic cells and the Bax/Bcl-2 ratio and cleaved the nuclear enzyme PARP in HUVECs. Finally, Hb-AGEs also inhibited migration and proliferation of HUVECs, thus be physiologically significant in endothelial dysfunction. Taken together, our data suggest that Hb-AGEs may play a critical role in inducing vascular endothelial cell damage. Therefore, this study may provide a plausible explanation for the potential Hb-AGEs in human endothelial cell dysfunction of diabetic patients.

Haptoglobin Therapeutics and Compartmentalization of Cell-Free Hemoglobin Toxicity.

Hemolysis and accumulation of cell-free hemoglobin (Hb) in the circulation or in confined tissue compartments such as the subarachnoid space is an important driver of disease. Haptoglobin is the Hb binding and clearance protein in human plasma and an efficient antagonist of Hb toxicity resulting from physiological red blood cell turnover. However, endogenous concentrations of haptoglobin are insufficient to provide protection against Hb-driven disease processes in conditions such as sickle cell anemia, sepsis, transfusion reactions, medical-device associated hemolysis, or after a subarachnoid hemorrhage. As a result, there is increasing interest in developing haptoglobin therapeutics to target 'toxic' cell-free Hb exposures. Here, we discuss key concepts of Hb toxicity and provide a perspective on the use of haptoglobin as a therapeutic protein.

A wealth of genotype-specific proteoforms fine-tunes hemoglobin scavenging by haptoglobin.

The serum haptoglobin protein (Hp) scavenges toxic hemoglobin (Hb) leaked into the bloodstream from erythrocytes. In humans, there are two frequently occurring allelic forms of Hp, resulting in three genotypes: Homozygous Hp 1-1 and Hp 2-2, and heterozygous Hp 2-1. The Hp genetic polymorphism has an intriguing effect on the quaternary structure of Hp. The simplest form, Hp 1-1, forms dimers consisting of two α1ß units, connected by disulfide bridges. Hp 2-1 forms mixtures of linear (α1)2(α2)n-2(ß)n oligomers (n > 1) while Hp 2-2 occurs in cyclic (α2)n(ß)n oligomers (n > 2). Different Hp genotypes bind Hb with different affinities, with Hp 2-2 being the weakest binder. This behavior has a significant influence on Hp's antioxidant capacity, with potentially distinctive personalized clinical consequences. Although Hp has been studied extensively in the past, the finest molecular details of the observed differences in interactions between Hp and Hb are not yet fully understood. Here, we determined the full proteoform profiles and proteoform assemblies of all three most common genetic Hp variants. We combined several state-of-the-art analytical methods, including various forms of chromatography, mass photometry, and different tiers of mass spectrometry, to reveal how the tens to hundreds distinct proteoforms and their assemblies influence Hp's capacity for Hb binding. We extend the current knowledge by showing that Hb binding does not just depend on the donor's genotype, but is also affected by variations in Hp oligomerization, glycosylation, and proteolytic processing of the Hp α-chain.

Cell-free hemoglobin augments acute kidney injury during experimental sepsis.

Acute kidney injury is a common complication of severe sepsis and contributes to high mortality. The molecular mechanisms of acute kidney injury during sepsis are not fully understood. Because hemoproteins, including myoglobin and hemoglobin, are known to mediate kidney injury during rhabdomyolysis, we hypothesized that cell-free hemoglobin (CFH) would exacerbate acute kidney injury during sepsis. Sepsis was induced in mice by intraperitoneal injection of cecal slurry (CS). To mimic elevated levels of CFH observed during human sepsis, mice also received a retroorbital injection of CFH or dextrose control. Four groups of mice were analyzed: sham treated (sham), CFH alone, CS alone, and CS + CFH. The addition of CFH to CS reduced 48-h survival compared with CS alone (67% vs. 97%, P = 0.001) and increased the severity of illness. After 24 and 48 h, CS + CFH mice had a reduced glomerular filtration rate from baseline, whereas sham, CFH, and CS mice maintained baseline glomerular filtration rate. Biomarkers of acute kidney injury, neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule-1 (KIM-1), were markedly elevated in CS+CFH compared with CS (8-fold for NGAL and 2.4-fold for KIM-1, P < 0.002 for each) after 48 h. Histological examination showed a trend toward increased tubular injury in CS + CFH-exposed kidneys compared with CS-exposed kidneys. However, there were similar levels of renal oxidative injury and apoptosis in the CS + CFH group compared with the CS group. Kidney levels of multiple proinflammatory cytokines were similar between CS and CS + CFH groups. Human renal tubule cells (HK-2) exposed to CFH demonstrated increased cytotoxicity. Together, these results show that CFH exacerbates acute kidney injury in a mouse model of experimental sepsis, potentially through increased renal tubular injury.

Development of novel oxygen carriers by coupling hemoglobin to functionalized multiwall carbon nanotubes.

Preparation of stable and effective artificial oxygen carriers (AOCs) is a promising strategy to temporarily replace transfused blood and solve tissue hypoxia. Developing hemoglobin (Hb) loaded particles is one of the main ways to prepare suitable AOCs. Particles with a hierarchical micro/nanostructure can be loaded with plenty of proteins and have attracted great attention. Therefore, multiwall carbon nanotubes (MWCNTs) were chosen to fabricate AOCs. To improve the Hb-loading capacity of MWCNTs, functionalized MWCNTs, including carboxyl-functionalized MWCNTs (MWCNT-COOH), amino-functionalized MWCNTs (MWCNT-NH2), and heparin-conjugated MWCNTs (MWCNT-Hep), were prepared. Then, in this study, Hb was coupled to the functionalized MWCNTs to fabricate the AOCs. The functionalized MWCNTs and the AOCs were characterized by FTIR, SEM, TEM, and zeta potential analysis. The oxygen/Hb-loading capacity of the AOCs was also measured. The adverse effects of the AOCs on human umbilical vein endothelial cells (HUVECs) and human red blood cells (RBCs) were evaluated. The results showed that (1) the functional groups were grafted on the surface of the MWCNTs, and Hb was bound to the functionalized MWCNTs, thus the AOCs were successfully prepared; (2) MWCNT-Hep-Hb had the most stable dispersibility (i.e., the most negative zeta potential) in 0.9 wt% NaCl solution (MWCNT-Hep-Hb < MWCNT-COOH-Hb < MWCNT-Hb < MWCNT-NH2-Hb < 0); (3) MWCNT-Hep had the best Hb-loading capability, which was three times that of purified MWCNTs; (4) with concentrations increased up to 400 µg mL-1, MWCNT-Hep-Hb still had the highest cell viability (97.63% > 80%, ISO 10993-5:2009) and excellent blood biocompatibility. Therefore, MWCNT-Hep-Hb might be a satisfactory candidate as a blood substitute.

Renal Toxicodynamic Effects of Extracellular Hemoglobin After Acute Exposure.

Plasma hemoglobin (Hb) is elevated in some hematologic disease states, during exposures to certain toxicants, and with the use of some medical devices. Exposure to free Hb can precipitate oxidative reactions within tissues and alter the normal physiological function of critical organ systems. As kidney structures can be highly sensitive to Hb exposures, we evaluated the acute dose dependent renal toxicologic response to purified Hb isolated from RBCs. Male Hartley guinea pigs (n = 5 per group) were dosed with 0.9% saline (2 ml), 15, 75, 150, or 300 mg of purified Hb, infused over a 2-h period. The primary endpoints of this study were to define toxicokinetic parameters after increasing doses of purified Hb, identify clinically recognized and experimental markers of acute kidney injury (AKI), and determine relevant toxicological parameters and potential causes of renal toxicity in this model. Experimental findings demonstrated a dose dependent increase in Cmax after a 2-h infusion, which correlated with an elevation in serum creatinine, renal Kim-1 mRNA expression and increased urinary Kim-1. Renal NGAL mRNA expression and urinary NGAL excretion were also increased in several groups, but these parameters did not correlate with exposure. Iron increased in the renal cortex as Hb exposure increased and its deposition colocalized with 4-hydroxy-nonenal and 8-Oxo-2-deoxyguanosine immune reactivity, suggesting oxidative stressors may contribute to AKI in animals exposed to Hb. The results presented here suggest that Cmax may effectively predict the risk of AKI in normal healthy animals exposed to Hb.

Protective effect of vitreous against hemoglobin neurotoxicity.

Hemorrhage into the brain parenchyma or subarachnoid space is associated with edema and vascular injury that is likely mediated at least in part by the toxicity of hemoglobin. In contrast, extravascular blood appears to be less neurotoxic when localized to the retina or adjacent vitreous, the gel filling the posterior segment of the eye. In this study, the hypothesis that vitreous protects neurons from hemoglobin toxicity was investigated in a primary cortical cell culture model. Consistent with prior observations, hemoglobin exposure for 24 h resulted in death of most neurons without injury to co-cultured glia. Neuronal loss was reduced in a concentration-dependent fashion by bovine vitreous, with complete protection produced by 3% vitreous solutions. This effect was associated with a reduction in malondialdehyde but an increase in cell iron. At low vitreous concentrations, its ascorbate content was sufficient to account for most neuroprotection, as equivalent concentrations of ascorbate alone had a similar effect. However, other vitreous antioxidants provided significant protection when applied at concentrations present in undiluted vitreous, and prevented all neuronal loss when combined in the absence of ascorbate. These results indicate that vitreous is an antioxidant cocktail that robustly protects neurons from hemoglobin toxicity, and may contribute to the relative resistance of retinal neurons to hemorrhagic injury.

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.

Hemopexin increases the neurotoxicity of hemoglobin when haptoglobin is absent.

Hemopexin (Hpx) binds heme with extraordinary affinity, and after haptoglobin may provide a second line of defense against the toxicity of extracellular hemoglobin (Hb). In this series of experiments, the hypothesis that Hpx protects neurons from Hb neurotoxicity was evaluated in murine primary cultures containing neurons and glial cells. Contrary to hypothesis, Hpx increased neuronal loss due to micromolar concentrations of Hb by 4- to 12-fold, as measured by LDH release assay; conversely, the neurotoxicity of hemin was completely prevented. The endogenous fluorescence of Hpx was quenched by Hb, consistent with transfer of Hb-bound heme to Hpx. This was associated with precipitation of globin chains, as detected by immunostaining and fluorescent Hb labeling. A portion of this precipitate attached firmly to cells and could not be removed by multiple washes. Concomitant treatment with haptoglobin (Hp) prevented globin precipitation and most of the increase in neuronal loss. Hpx weakly attenuated the increase in culture non-heme iron produced by Hb treatment, quantified by ferrozine assay. However, Hb-Hpx toxicity was iron-dependent, and was blocked by deferoxamine and ferrostatin-1. Up-regulation of cell ferritin expression, a primary cell defense against Hb toxicity, was not observed on western blots of culture lysates that had been concomitantly treated with Hpx. These results suggest that Hpx destabilizes Hb in the absence of haptoglobin, leading to globin precipitation and exacerbation of iron-dependent oxidative cell injury. Combined therapy with hemopexin plus haptoglobin may be preferable to hemopexin alone after CNS hemorrhage.

The Use of an Acellular Oxygen Carrier in a Human Liver Model of Normothermic Machine Perfusion.

BACKGROUND: Normothermic machine perfusion of the liver (NMP-L) is a novel technique that preserves liver grafts under near-physiological conditions while maintaining their normal metabolic activity. This process requires an adequate oxygen supply, typically delivered by packed red blood cells (RBC). We present the first experience using an acellular hemoglobin-based oxygen carrier (HBOC) Hemopure in a human model of NMP-L. METHODS: Five discarded high-risk human livers were perfused with HBOC-based perfusion fluid and matched to 5 RBC-perfused livers. Perfusion parameters, oxygen extraction, metabolic activity, and histological features were compared during 6 hours of NMP-L. The cytotoxicity of Hemopure was also tested on human hepatic primary cell line cultures using an in vitro model of ischemia reperfusion injury. RESULTS: The vascular flow parameters and the perfusate lactate clearance were similar in both groups. The HBOC-perfused livers extracted more oxygen than those perfused with RBCs (O2 extraction ratio 13.75 vs 9.43 % ×10 per gram of tissue, P = 0.001). In vitro exposure to Hemopure did not alter intracellular levels of reactive oxygen species, and there was no increase in apoptosis or necrosis observed in any of the tested cell lines. Histological findings were comparable between groups. There was no evidence of histological damage caused by Hemopure. CONCLUSIONS: Hemopure can be used as an alternative oxygen carrier to packed red cells in NMP-L perfusion fluid.

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.

Propofol Enhances Hemoglobin-Induced Cytotoxicity in Neurons.

BACKGROUND: It has been increasingly suggested that propofol protects against hypoxic-/ischemic-induced neuronal injury. As evidenced by hemorrhage-induced stroke, hemorrhage into the brain may also cause brain damage. Whether propofol protects against hemorrhage-induced brain damage remains unknown. Therefore, in this study, we investigated the effects of propofol on hemoglobin-induced cytotoxicity in cultured mouse cortical neurons. METHODS: Neurons were prepared from the cortex of embryonic 15-day-old mice. Hemoglobin was used to induce cytotoxicity in the neurons. The neurons were then treated with propofol for 4 hours. Cytotoxicity was determined by lactate dehydrogenase release assay. Caspase-3 activation was examined by Western blot analysis. Finally, the free radical scavenger U83836E was used to examine the potential involvement of oxidative stress in propofol's effects on hemoglobin-induced cytotoxicity. RESULTS: We found that treatment with hemoglobin induced cytotoxicity in the neurons. Propofol enhanced hemoglobin-induced cytotoxicity. Specifically, there was a significant difference in the amount of lactate dehydrogenase release between hemoglobin plus saline (19.84% ± 5.38%) and hemoglobin plus propofol (35.79% ± 4.41%) in mouse cortical neurons (P = 0.00058, Wilcoxon Mann-Whitney U test, n = 8 in the control group or the treatment group). U83836E did not attenuate the enhancing effects of propofol on hemoglobin-induced cytotoxicity in the neurons, and propofol did not significantly affect caspase-3 activation induced by hemoglobin. These data suggested that caspase-3 activation and oxidative stress might not be the underlying mechanisms by which propofol enhanced hemoglobin-induced cytotoxicity. Moreover, these data suggested that the neuroprotective effects of propofol would be dependent on the condition of the brain injury, which will need to be confirmed in future studies. CONCLUSIONS: These results from our current proof-of-concept study should promote more research in vitro and in vivo to develop better anesthesia care for patients with hemorrhagic stroke.

Hemoglobin-induced neuronal degeneration in the hippocampus after neonatal intraventricular hemorrhage.

Neuronal degeneration following neonatal intraventricular hemorrhage (IVH) is incompletely understood. Understanding the mechanisms of degeneration and cell loss may point toward specific treatments to limit injury. We evaluated the role of hemoglobin (Hb) in cell death after intraventricular injection in neonatal rats. Hb was injected into the right lateral ventricle of post-natal day 7 rats. Rats exposed to anesthesia were used for controls. The CA-1 region of the hippocampus was analyzed via immunohistochemistry, hematoxylin and eosin (H&E) staining, Fluoro-Jade C staining, Western blots, and double-labeling stains. Compared to controls, intraventricular injection of Hb decreased hippocampal volume (27% decrease; p<0.05), induced neuronal loss (31% loss; p<0.01), and increased neuronal degeneration (2.7 fold increase; p<0.01), which were all significantly reduced with the iron chelator, deferoxamine. Hb upregulated p-JNK (1.8 fold increase; p<0.05) and increased expression of the Hb/haptoglobin endocytotic receptor CD163 in neurons in vivo and in vitro (cultured cortical neurons). Hb induced expression of the CD163 receptor, which co-localized with p-JNK in hippocampal neurons, suggesting a potential pathway by which Hb enters the neuron to result in cell death. There were no differences in neuronal loss or degenerating neurons in Hb-injected animals that developed hydrocephalus versus those that did not. Intraventricular injection of Hb causes hippocampal neuronal degeneration and cell loss and increases brain p-JNK levels. p-JNK co-localized with the Hb/haptoglobin receptor CD163, suggesting a novel pathway by which Hb enters the neuron after IVH to result in cell death.

Contenido de plomo y peso de la placenta y su relación con la edad gestacional, peso, longitud y hemoglobina al nacimiento en una zona metalúrgica del Perú / Lead content and placental weight and its association with gestational age, weight, length and hemoglobin in newborns of metallurgical region - Peru

En el estudio transversal relacional se estimó la relación cuantitativa entre la concentración de plomo en la placenta y el peso de la misma; a su vez la relación entre el peso de la placenta con la edad gestacional, peso, longitud y concentración de hemoglobina de recién nacidos en una región metalúrgica de Perú. Se evaluaron 40 productos del parto colectados en un periodo de tres meses, cuando la fundición funcionaba con normalidad, pues actualmente ha suspendido su actividad. El muestreo biológico siguió protocolos ya establecidos, y el plomo se cuantificó por absorción atómica con horno de grafito en el Instituto Peruano de Energía Nuclear. Se realizaron análisis bivariados de regresión y correlación lineal de Pearson. Los promedios y desviación estándar de la edad gestacional, contenido de plomo en la placenta, peso de la placenta y el peso, longitud y hemoglobia de los neonatos fueron: 39,20+1,18 semanas; 319+215,86 ng/g; 504,25+83,53 g; 3191,75+310,61 g; 49,72+1,26 cm y 16,76+1,88 g/dL, respectivamente. El modelo de regresión lineal fue el que se ajustó mejor a las variables estudiadas. Los niveles altos de plomo en la placenta se correlacionaron negativamente con su propio peso; a su vez, los mayores pesos de la placenta se correlacionaron positivamente con el peso, longitud y contenido de hemoglobina de los recién nacidos (AU)

This cross-sectional and correlational study has estimated the quantitative relationship between placental weight and the concentration of lead in the placenta; has also estimated the association between placental weight with gestational age, weight, length and concentration of hemoglobin in newborns in a metallurgical region of Peru, when the Oroya smelter was working normally, because at present it has ceased its activity. Forty birth products, collected over a period of three months in II Hospital EsSalud La Oroya were valued. Biological sampling responded to preset protocols. Lead quantified by atomic absorption spectrometry with graphite furnace in the Peruvian Institute of Nuclear Energy. Bivariate regression analysis and Pearson linear correlation were performed to establish associations between assessed variables. The averages and standard deviation of gestational age, lead content in the placenta, placental weight; birth weight, length and hemoglobin level of neonates were 39.20+1.18 weeks, 319+215.86 ng/g, 504.25+83.53 g, 3191.75+310.61 g; 49.72+1.26 cm and 16.76+1.88 g/dL, respectively. The regression model best adapted to the assessed variables was the linear model. Higher levels of lead in the placenta were negatively correlated with the placental weight. The greatest placental weight was positively correlated to birth weight, length and hemoglobin concentrations of newborns (AU)

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.

Peroxynitrite decomposition catalyst prevents matrix metalloproteinase-9 activation and neurovascular injury after hemoglobin injection into the caudate nucleus of rats.

Hemoglobin (Hb) is a major constituent of blood and a potent mediator of oxidative or nitrative stress after intracerebral hemorrhage (ICH). Our previous study demonstrated that Hb could induce abundant peroxynitrite (ONOO(-)) formation in vivo, which may be involved in the blood-brain barrier (BBB) disruption, however, the drug intervention is absent and also the underlying mechanism. Using an experimental stroke model by injecting Hb into the caudate nucleus of male Sprague-Dawley rats, we assessed the role of ONOO(-) decomposition catalyst, 5,10,15,20-tetrakis (4-sulfonatophenyl) porphyrinato iron(III) [FeTPPS] in the activation of MMP-9 and Hb-induced neurovascular injuries. 3-Nitrotyrosine (3-NT, as an index of ONOO(-) formation) and NF-κB expression was measured by western blot (WB) and immunohistochemistry (IHC)/immunofluorescence (IF). Activity of MMP was evaluated by in situ zymography. Neurovascular injury was assessed using zonula occludens-1 (ZO-1) by WB and IF, fibronectin (FN) and neuron-specific nuclear protein (NeuN) IHC. Perihematomal cell death was determined by TUNEL assay. Behavioral outcome was measured by modified neurological severity score (mNSS) test. At the injured striata, profuse 3-NT was produced and mainly expressed in neutrophils and microglia/macrophages. 3-NT formation significantly colocalized with nuclear factor-κB (NF-κB) expression. In situ zymography showed that gelatinase activity was mostly co-localized with neurons and blood vessel walls and partly with neutrophils and microglia/macrophages. Enhanced 3-NT production, NF-κB induction and MMP-9 activation were obviously reduced after FeTPPS treatment. Hb-induced injury to tight junction protein (ZO-1), basal lamina of FN-immunopositive microvasculature and neural cells was evidently ameliorated by FeTPPS. In addition, apoptotic cell numbers as well as behavioral deficits were also improved. The present study shows that the administration of the ONOO(-) decomposition catalyst FeTPPS protects against Hb-induced neurovascular injuries and improves neurological function, which possibly in part by suppressing MMP-9 activation.

Absence of developmental toxicity in a canine model after infusion of a hemoglobin-based oxygen carrier: Implications for risk assessment.

Bovine-derived hemoglobin-based oxygen carriers (HBOCs) have been investigated for use in humans (HBOC-201) and approved for veterinary medicine (HBOC-301). We infused pregnant beagles with HBOC-201 to test whether HBOC-induced developmental toxicity previously observed in rats would occur in a species devoid of an inverted visceral yolk sac (invVYS). Phase 1 assessed developmental toxicity of 6g/kg HBOC-201 on gestational day (GD) 21. Phase 2 investigated single infusions of 6g/kg HBOC-201 on one of GDs 21, 25, 29 or 33. Phase 3 studied multiple sequential infusions on GDs 21, 23,25,27,29, 31, and 33 at 0.52g/kg/day (3.6g/kg total dose). Mild to moderate maternal toxicity occurred in all phases. There was an unequivocal absence of developmental toxicity in all phases. Overall, our hypothesis that HBOC, which interferes with the function of the invVYS, would not affect the offspring in dogs was supported. The implications relative to human risk are discussed.

Developmental toxicity in rats of a hemoglobin-based oxygen carrier results from impeded function of the inverted visceral yolk sac.

HBOC-201 is a bovine-derived, cross-linked, and stabilized hemoglobin (250kDa) in physiological saline. Daily intravenous infusions of HBOC (1.95, 3.90, or 5.85g/kg/day) during gestational days (GDs) 6-18 in Sprague-Dawley rats caused fetal mortality, reduced birth weight, and malformations. Subsequent single-day infusions (5.85g/kg/day) showed that developmental toxicity was limited to GDs 7-9 when histiotrophic nutrition via the inverted visceral yolk sac (invVYS) is essential. Histiotrophic nutrition is receptor-mediated endocytosis of bulk maternal proteins and subsequent lysosomal degradation providing amino acids and other nutrients for embryonic growth. Controls for protein content, oncotic properties, and hemoglobin content indicated that toxicity was due to hemoglobin. Rat whole embryo cultures verified HBOC interference with invVYS transport capacity and resultant deficient embryonic nutrition. These mechanisms of action are not expected to impact human development based on differences in VYS morphology and function, although a complete understanding of early human embryonic nutrition is lacking.

Interaction of recombinant octameric hemoglobin with endothelial cells.

Hemoglobin-based oxygen carriers (HBOCs) may generate oxidative stress, vasoconstriction and inflammation. To reduce these undesirable vasoactive properties, we increased hemoglobin (Hb) molecular size by genetic engineering with octameric Hb, recombinant (r) HbßG83C. We investigate the potential side effects of rHbßG83C on endothelial cells. The rHbßG83C has no impact on cell viability, and induces a huge repression of endothelial nitric oxide synthase gene transcription, a marker of vasomotion. No induction of Intermolecular-Adhesion Molecule 1 and E-selectin (inflammatory markers) transcription was seen. In the presence of rHbßG83C, the transcription of heme oxygenase-1 (oxidative stress marker) is weakly increased compared to the two other HBOCs (references) or Voluven (control). This genetically engineered octameric Hb, based on a human Hb ßG83C mutant, leads to little impact at the level of endothelial cell inflammatory response and thus appears as an interesting molecule for HBOC development.

Role of hemoglobin and iron in hydrocephalus after neonatal intraventricular hemorrhage.

BACKGROUND: Neonatal germinal matrix hemorrhage/intraventricular hemorrhage is common and often results in hydrocephalus. The pathogenesis of posthemorrhagic hydrocephalus is not fully understood. OBJECTIVE: To explore the potential role of hemoglobin and iron released after hemorrhage. METHODS: Artificial cerebrospinal fluid (aCSF), hemoglobin, or iron was injected into the right lateral ventricle of postnatal day-7 Sprague Dawley rats. Ventricle size, heme oxygenase-1 (HO-1) expression, and the presence of iron were evaluated 24 and 72 hours after injection. A subset of animals was treated with an iron chelator (deferoxamine) or vehicle for 24 hours after hemoglobin injection, and ventricle size and cell death were evaluated. RESULTS: Intraventricular injection of hemoglobin and iron resulted in ventricular enlargement at 24 hours compared with the injection of aCSF. Protoporphyrin IX, the iron-deficient immediate heme precursor, did not result in ventricular enlargement after injection into the ventricle. HO-1, the enzyme that releases iron from heme, was increased in the hippocampus and cortex of hemoglobin-injected animals at 24 hours compared with aCSF-injected controls. Treatment with an iron chelator, deferoxamine, decreased hemoglobin-induced ventricular enlargement and cell death. CONCLUSION: Intraventricular injection of hemoglobin and iron can induce hydrocephalus. Treatment with an iron chelator reduced hemoglobin-induced ventricular enlargement. This has implications for the pathogenesis and treatment of posthemorrhagic hydrocephalus. ABBREVIATIONS: aCSF, artificial cerebrospinal fluidDAB, 3,3'-diaminobenzidine-4HClGMH-IVH, germinal matrix hemorrhage/intraventricular hemorrhageHO-1, heme oxygenase-1ICH, intracerebral hemorrhagePBS, phosphate-buffered salineSVZ, subventricular zoneTBST, tris-buffered saline with Tween 20.