Stroma-free Hemoglobin Research Articles

Neuroprotective effects of a hemoglobin-based oxygen carrier (stroma-free hemoglobin nanoparticle) on ischemia reperfusion injury

The application of hemoglobin (Hb)-based oxygen carriers (HBOCs) to the treatment of cerebral ischemia has been investigated. A cluster of 1 Hb and 3 human serum albumins (Hb-HSA3) was found to exert neuroprotective effects on ischemia/reperfusion injury. Stroma-free hemoglobin nanoparticles (SFHbNP), a subsequently developed HBOC consisting of a spherical polymerized stroma-free Hb core with a HSA shell, contains the natural antioxidant enzyme catalase and, thus, is expected to exert additive effects. We herein investigated whether SFHbNP exerted enhanced neuroprotective effects in a rat transient middle cerebral artery occlusion (tMCAO) model. Rats were subjected to 2-hour tMCAO and divided into the following 3 groups with the intravenous administration of the respective reagents: (1) phosphate-buffered saline (PBS), as a vehicle (2) Hb-HSA3, and (3) SFHbNP. After 24-hour reperfusion, infarct and edema volumes decreased in the order of the PBS, Hb-HSA3, and SFHbNP groups, with a significant difference (p < 0.05) between the PBS and SFHbNP groups. Similar reductions were observed in oxidative stress, leukocyte recruitment, and blood–brain barrier disruption in the order of the PBS, Hb-HSA3, and SFHbNP groups. In the early phase of reperfusion within 6 h, microvascular HBOC perfusion and cerebral blood flow were maintained at high levels during the reperfusion period in the Hb-HSA3 and SFHbNP groups. However, a difference was observed in tissue oxygen partial pressure levels, which significantly decreased after 6-hour reperfusion in the Hb-HSA3 group, but remained high in the SFHbNP group. A superior oxygen transport ability appears to be related to the enhanced neuroprotective effects of SFHbNP.

The P50 value detected by the oxygenation-dissociation analyser and blood gas analyser

Oxygen tension at 50% haemoglobin saturation (P50), which reflects the degree of peripheral oxygen offloading and tissue oxygenation, plays an important role in the diagnosis and treatment of disease, as well as in transfusion research. Blood gas analysers are commonly used in clinical and obtain P50 values through complex calculations and analysis. Oxygenation-dissociation analysers are specially designed to record the oxygen dissociation curves and obtain P50 values of whole blood, red blood cells (RBCs), and stroma-free haemoglobin. However, whether the two equipment obtain comparable data is still uncertain. Herein, we used both equipment to detect P50 values of blood and stroma-free haemoglobin from human and bovine sources, venous and arterial blood of beagle and rat, and stored rat blood. For human blood, both analysers yielded similar data. P50 of the stroma-free haemoglobin and bovine blood could only be properly detected by oxygenation-dissociation analysers. Blood gas analysers showed different P50 values, while oxygenation-dissociation analysers got similar P50 values for arterial and venous samples. Oxygenation-dissociation analysers distinguished changes in P50 values during RBCs storage. Compared with the blood gas analysers, oxygenation-dissociation analysers had a stronger detection capability in P50 measurement with regard to both sample types and species.

Physicochemical properties and oxygen affinity of glutaraldehyde polymerized crocodile hemoglobin: the new alternative hemoglobin source for hemoglobin-based oxygen carriers

Hemoglobin-based oxygen carriers (HBOCs) are modified stroma-free hemoglobin molecules used in developing a blood substitute for therapeutic usage. In order to prevent hemoglobin dissociation, glutaraldehyde (GTA) was used to generate high-molecular weight heterogeneous crocodile hemoglobin (Poly-cHb). This work, Poly-cHb was created using various GTA concentrations, ranging from 0.025-0.150% (v/v). Physicochemical properties were investigated that were comparable GTA polymerized human hemoglobin (Poly-hHb). This study has revealed that GTA polymerization increases the molecular size of Native-cHbs from 14.10 nm over a range from 16.31 to 54.27 nm. Moreover, this polymerization alters the secondary structure and heme environment by decreasing the helicity ratio from 1.00 to 0.95 at the highest condition and exhibits hypochromic shift of the Soret band to be 0.88 times lower than the native. However, all Poly-cHbs still possessed higher oxygen affinity than that of Poly-hHbs with average P50 values of 13 and 21 mmHg, respectively. Although, polymerization affected the overall Poly-cHb structure slightly, but compensated by decreasing the denaturation level to lower than 10%. Thus, it is interesting to note that Poly-cHb may advantageously provide effective oxygen carriage and ability for pasteurization, which may benefit the search for new alternative hemoglobin sources for HBOC development.

Thrombin-Mediated Activation of PAR-1 Contributes to Microvascular Stasis in Mouse Models of Sickle Cell Disease Via Increased Endothelial Expression of P-Selectin and VWF

Sickle cell disease (SCD) is caused by a single nucleotide mutation in the β-globin gene, resulting in an altered red cell physiology that causes vascular complications such as hemolytic anemia, chronic inflammation, activation of coagulation, and vaso-occlusion. We have shown that infusion of hemin results in tissue factor (TF)-dependent activation of coagulation in mice. Furthermore, TF-dependent activation of coagulation contributes to inflammation in a mouse model of SCD. Interestingly, thrombin-dependent inflammation in sickle cell mice was not attenuated by deficiency of the main thrombin receptor protease activated receptor-1 (PAR-1). However, others have shown that the activation of endothelial cell PAR-1 with agonist peptide enhances interactions of these cells with sickle RBCs in a P-selectin-dependent manner. Importantly, P-selectin inhibition reduces microvascular stasis in mouse models of SCD and prevents vaso-occlusive crisis in sickle cell patients.We propose that thrombin-mediated PAR-1 activation promotes microvascular stasis in mouse models of SCD via increased expression of P-selectin and VWF on the endothelium, triggered by exocytosis of Weibel-Palade bodies.To investigate if the TF/thrombin/PAR-1 pathway contributes to microvascular stasis, dorsal skinfold chambers were implanted in NY1DD sickle mice 3 days before the experiment. Microvascular stasis was determined in 20-25 preselected micro-vessels in response to intravenous infusion of stroma-free hemoglobin (SFH, 1.6 µmol/kg) and was expressed as % non-flowing vessels (mean ± SEM). We previously demonstrated that infusion of SFH results in microvascular stasis that is inhibited by hemopexin in NY1DD mice, indicating that hemoglobin releases heme into the circulation. In NY1DD mice treated with control IgG antibody, SFH infusion caused stasis in 28.3 ± 1.7% and 36.7 ± 1.7% of preselected vessels at 1 and 4 hrs after infusion, respectively. Treatment with an inhibitory anti-TF antibody 1H1 (25 mg/kg; IP) 30 minutes before SFH infusion significantly reduced stasis to only 3.3 ± 1.5% and 2.8 ± 1.6% of vessels at 1 and 4 hrs, respectively (p<0.01). To investigate if TF contributes to SFH-induced microvascular stasis via generation of downstream coagulation proteases, NY1DD mice were fed with control chow or chow containing either Factor Xa (FXa) inhibitor rivaroxaban (0.4 mg/g chow) or thrombin inhibitor dabigatran (10 mg/g chow) ad libitum for 4 days prior to stasis experiments. We previously showed that these doses efficiently anticoagulated sickle mice without bleeding complications. FXa inhibition significantly reduced stasis at 1 hr (4.9 ± 0.1% versus 22.4 ± 3.8%, p<0.001) and 4 hrs (1.7 ± 1.6% versus 12.8 ± 1.9%, p<0.001) after SFH injection. Similarly, dabigatran also significantly attenuated SFH-induced stasis (3.1 ± 1.5% non-flowing vessels at 1 hour and 1.6 ± 1.6% non-flowing vessels at 4 hours). Next, to investigate if thrombin contributes to SFH-induced stasis through activation of PAR-1, we used bone marrow transplantation to generate sickle mice lacking PAR-1 expression in all non-hematopoietic cells. PAR-1+/+ and PAR-1-/- mice were lethally irradiated and transplanted with bone marrow from Townes sickle (SS) mice. Efficient reconstitution of bone marrow was confirmed by hemoglobin electrophoresis. We found that PAR-1-/- SS mice were significantly protected from SFH-induced stasis compared to PAR-1+/+ SS mice at both 1 hr (13.2 ± 3.4% versus 37.6 ± 3.9% stasis; P<0.001) and 4 hrs (6.6 ± 1.7% versus 18.0 ± 1.5% stasis; p<0.05) after hemoglobin challenge. We have previously shown that a monoclonal antibody to murine P-selectin or a polyclonal antibody to VWF markedly inhibit stasis in sickle mice. After the 4 hr stasis measurement, lungs were harvested from PAR-1+/+ SS and PAR-1-/- SS the mice and stained for P-selectin, VWF, and CD31 (a marker for endothelial cells). We found that intensity of P-selectin and VWF staining was markedly reduced in the lungs of PAR-1-/- SS mice compared to the staining observed in lungs of PAR-1+/+ SS mice.Our data indicates that SFH-induced microvascular stasis is significantly diminished by inhibition of TF, FXa, or thrombin activity or knockout of endothelial PAR-1, which leads to reduced expression of P-selectin and VWF on endothelium in a mouse model of SCD. We speculate that inhibiting the TF/thrombin/PAR-1 axis may reduce vaso-occlusive crises in SCD patients. DisclosuresVercellotti:CSL Behring: Research Funding. Belcher:CSL Behring: Research Funding.

Abstract TP342: Protective Effects of the Lipophilic Dadmdft Iron Chelator Against Brain Hemoglobin Toxicity

Background and Purpose: Intracerebral hemorrhage (ICH) is a devastating form of stroke, and therapies are largely non-existent. Among the deleterious processes occurring after ICH, iron toxicity is suggested to contribute to secondary injury. There is a pressing need for the continued development of new iron-chelating agents that can selectively remove released iron after ICH from brain tissues especially vulnerable to iron-induced toxicity. The DADMDFT analogue [( S )-4,5-dihydro-2-(2-hydroxyphenyl)-4-thiazolecarboxylic acid] used here is a more lipophilic iron chelator that achieves higher concentrations in the plasma and has a longer half-life than most commonly used chelators. Methods: The DADMDFT-treated mice were subjected to intrastriatal injection of stroma-free hemoglobin. Infarct volume and ferric iron distribution were assessed by using Cresyl violet and Perls’ staining respectively. Knowing that hemoglobin can activate microglial cells, thus microglial activation was monitored by Iba1 immunohistochemistry. Behavioral functions were evaluated by rotarod and neurological deficit score at 24 and 72h after hemoglobin injection. Results: We found that DADMDFT treatment at 75 and 150μmol/kg both significantly reduced the lesion volume after hemoglobin intrastriatal injection. Moreover, around the lesion site, smaller accumulation of iron was observed after DADMDFT treatment in contrast to the vehicle group. Our quantitative analyses indicated that there were more activated microglial cells at peri-lesion area with DADMDFT treatment at the dose of 75 and 150μmol/kg, as compared to the vehicle-treated group. In behavioral tests, DADMDFT treatment significantly prolonged the latency to fall during the rotarod test at 150μmol/kg at 24h after injection. In addition, DADMDFT of 75 and 150μmol/kg significantly improved the neurological deficit score at 24 and 72h after injection. Conclusions: We found that the lipophilic DADMDFT can protect brain tissue from hemoglobin toxicity. The potential mechanism of its protective effects is that DADMDFT treatment could help maintaining the cell number and phagocytosis function of activated microglia around the lesion site, which could promote the clearance of toxic ferric iron.

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

BackgroundFollowing 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.MethodsIntracerebral 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.ResultsThis 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.ConclusionsThis 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.

Abstract W MP95: Deletion of the Hemopexin or Heme Oxygenase-2 Gene Aggravates Brain Injury Following Stroma-free-hemoglobin Induced Intracerebral Hemorrhage

Introduction: The breakdown of heme-hemoglobin released after intracerebral hemorrhage (ICH) contributes to brain injury. Hemopexin (HPX) has highest binding affinity to heme as its transporter and heme oxygenase 2 (HO2) is heme rate-limiting enzyme for its degradation. However, the significance and role of HPX and HO2 on hemoglobin clearance in brain remains understudied. Methods: Intracerebral injection of stroma-free hemoglobin (SFHb) was used in our study to induced ICH. The HPX or HO2 knockout (KO) mice were injected with 10μL SFHb into the striatum. Behavioral/functional tests were performed along with anatomical changes. Iron deposition and neuronal degeneration was shown by Perl’s or Fluoro Jade B staining, respectively. Immunohistochemistry with anti-Iba1 was used to detect microglial activation around the injured site. Result: This study reveals that microglia contribute to hemoglobin clearance after SFHb injection. Deletion of HPX or HO2 aggravated the SFHb-induced brain injury. Both of HPX or HO2 KO mice showed larger lesion volume (P&lt;0.05) and more degenerating neurons (P&lt;0.01) compared with the littermate controls in peri-lesion area at 24h post-injection. However, their activated microglial cells in peri-lesion area were significantly decreased (P&lt;0.05), accompanied with markedly increased iron positive glial cells (both P&lt;0.001), which are confirmed to be microglial cells by double-staining. The data suggested that activated microglia engulfed the injected SFHb and conferred protective effects after ICH. To further address this point, primary microglial cells were treated with SFHb and the results showed that microglial cells take up extracellular SFHb and that SFHb can activate microglial cells. In addition, our in vivo work revealed that activated microglial cells were increased from 24h to 72h after injection in both HPX KO and HO2 KO mice (P&lt;0.05). Also, notable improvements on neurological deficits scoring and locomotor activities indicated that the brain damages in mice were recovering from 24h to 72h post-injection, supporting activated microglia exerted beneficial effects after ICH. Conclusions: This study suggests that HPX and HO2 contribute to hemoglobin clearance after ICH and microglia play the crucial role.

H-ferritin ferroxidase induces cytoprotective pathways and inhibits microvascular stasis in transgenic sickle mice

Hemolysis, oxidative stress, inflammation, vaso-occlusion, and organ infarction are hallmarks of sickle cell disease (SCD). We have previously shown that increases in heme oxygenase-1 (HO-1) activity detoxify heme and inhibit vaso-occlusion in transgenic mouse models of SCD. HO-1 releases Fe2+ from heme, and the ferritin heavy chain (FHC) ferroxidase oxidizes Fe2+ to catalytically inactive Fe3+ inside ferritin. FHC overexpression has been shown to be cytoprotective. In this study, we hypothesized that overexpression of FHC and its ferroxidase activity will inhibit inflammation and microvascular stasis in transgenic SCD mice in response to plasma hemoglobin. We utilized a Sleeping Beauty (SB) transposase plasmid to deliver a human wild-type-ferritin heavy chain (wt-hFHC) transposable element by hydrodynamic tail vein injections into NY1DD SCD mice. Control SCD mice were infused with the same volume of lactated Ringer’s solution (LRS) or a human triple missense FHC (ms-hFHC) plasmid with no ferroxidase activity. 8 weeks later, LRS-injected mice had ~40% microvascular stasis (% non-flowing venules) 1 h after infusion of stroma-free hemoglobin, while mice overexpressing wt-hFHC had only 5% stasis (p < 0.05), and ms-hFHC mice had 33% stasis suggesting vascular protection by ferroxidase active wt-hFHC. The wt-hFHC SCD mice had marked increases in splenic hFHC mRNA and hepatic hFHC protein, ferritin light chain (FLC), 5-aminolevulinic acid synthase (ALAS), heme content, ferroportin, nuclear factor erythroid 2-related factor 2 (Nrf2), and HO-1 activity and protein. There was also a decrease in hepatic activated nuclear factor-kappa B (NF-κB) phospho-p65 and vascular cell adhesion molecule-1 (VCAM-1). Inhibition of HO-1 activity with tin protoporphyrin demonstrated HO-1 was not essential for the protection by wt-hFHC. We conclude that wt-hFHC ferroxidase activity enhances cytoprotective Nrf2-regulated proteins including HO-1, thereby resulting in decreased NF-κB-activation, adhesion molecules, and microvascular stasis in transgenic SCD mice.

Fucosylation Inhibitor, 2-Fluorofucose, Inhibits NF-κB Activation and Vaso-Occlusion In Transgenic Sickle Mice

Polymorphonuclear cells (PMNs) roll along vessel walls by binding to E- and P-selectins on the endothelial surface. These vascular adhesion molecules are involved in the initial tethering of PMNs on endothelium rather than their firm adhesion. Endothelial cell–expressed P- and E-selectins interact with leukocyte counterreceptors P-selectin glycoprotein ligand-1 (PSGL-1) and E-selectin ligand-1 (ESL-1). These glycoprotein counterreceptors are active only when modified posttranslationally by fucosylated oligosaccharides represented by the sialyl Lewis x tetrasaccharide (sLex) or its structural variants. Because of the important role that fucose plays in protein glycosylation and cellular functions, specific inhibitors of its incorporation could have significant applications in basic research and therapy. One such inhibitor, 2-fluorofucose peracetate, was recently reported to block fucosylation of cellular proteins and to inhibit binding of PMNs to P- and E-selectin. One potential application is the inhibition of leukocyte recruitment in pro-inflammatory disease states such as sickle cell disease (SCD). P-selectin expression on endothelium has been demonstrated to play an important role in vaso-occlusion, a hallmark of SCD. Inhibition of P-selectin binding inhibits vaso-occlusion in transgenic sickle mice. Therefore, we hypothesized that 2-fluorofucose would inhibit tissue inflammation and vaso-occlusion in SCD mice. MethodsNY1DD sickle mice were given plain water or 20 or 100 mM 2-fluorofucose in their drinking water ad libitum. The first cohort of mice (n=8/group) was treated for 7 days. On day 7 the mice were sacrificed with CO2, an EDTA blood sample was collected from the heart and the liver was removed and frozen in liquid nitrogen. Total white blood cell counts and differentials were performed by manual counting using a hemocytometer and Wright-stained blood smears, respectively. Nuclear extracts were prepared from nuclei isolated from liver homogenates. Western blots of the nuclear extracts were immunostained with antibodies to NF-ĸB phospho-p65 a marker of NF-ĸB activation. The second cohort of mice (n=4/group) was also treated for 7 days. On day 4, dorsal skin fold chambers were implanted onto the mice. On day 7, flowing venules in the dorsal skin-fold chamber window were selected and mapped using intravital microscopy. Thereafter, the mice were infused via the tail vein with human stroma-free hemoglobin (0.32 µmols heme/kg), a known inducer of vascular stasis in SCD mice. At 1 and 4 hours after infusion the same venules were re-examined and the percentage of vessels that had become static (no flow) was recorded. A positive control group of NY1DD mice (n=3) with implanted dorsal skin-fold chambers was given water to drink and pretreated with hemin (40 µmols/kg X 3 days, i.p.), a known inhibitor of vascular stasis. ResultsWhite blood cells counts were 16.3±3.2 (K/µL, mean±SD) in SCD mice treated with water. The white counts increased to 22.1±5.2 in SCD mice treated with 20 mM 2-fluorofucose and 34.2±7.2 in SCD mice treated with 100 mM 2-fluorofucose, respectively (p<0.05 for all pairwise comparisons). NF-ĸB in liver and other organs is activated in SCD mice compared to normal C57BL/6 mice. Nuclear NF-ĸB phospho-p65 was partially diminished in mice treated with 20 mM 2-fluorofucose and markedly decreased in mice treated with 100 mM 2-fluorofucose or pretreated with heme (Fig. 1).Infusion of hemoglobin induced ∼30% microvascular stasis at 1 and 4 hours in SCD mice treated with water (Fig. 2). Treatment with 20 mM 2-fluorofucose partially inhibited stasis at 1 and 4 hours (p<0.05 compared to water). Treatment with 100 mM 2-fluorofucose or heme inhibited stasis to 6.7% or less at 1 and 4 hours (p<0.025 compared to water). [Display omitted] [Display omitted] ConclusionsOral administration of 2-fluorofucose has significant anti-inflammatory and anti-vaso-occlusive properties in SCD mice. Disclosures:Belcher:Seattle Genetics, Inc.: Research Funding. Chen:Seattle Genetics, Inc.: Research Funding. Nguyen:Seattle Genetics, Inc.: Research Funding. Okeley:Seattle Genetics, Inc.: Employment. Senter:Seattle Genetics, Inc.: Employment. Benjamin:Seattle Genetics, Inc.: Employment. Vercellotti:Seattle Genetics, Inc.: Research Funding.

P45: Enhanced nitrite reaction rates of MP4OX, a novel ischemic rescue therapy

Background Hemoglobin-based oxygen carriers have been associated with vasoconstriction, attributed primarily to binding of nitric oxide (NO) as a heme ligand. MP4OX, an ischemic rescue therapy, designed with high O2 affinity using poly (ethylene glycol) (PEG) conjugation chemistry, has been observed not to cause vasoconstriction and to preserve microvascular blood flow, despite reducing perivascular levels of NO [1] . Recent evidence shows that reduction of nitrite to NO by deoxyhemoglobin has the ability to vasodilate blood vessels [2] . It also has been reported that the oxidative reaction of oxyhemoglobin with nitrite may facilitate the release of NO from heme [3] . We hypothesized that MP4OX may have enhanced reaction rates with nitrite and regeneration of NO, which may contribute to its lack of systemic vasoconstriction. Materials and methods MP4OX was prepared by reacting human stroma-free hemoglobin (SFH) with 2-iminothiolane and maleimide-activated PEG to produce a PEG-hemoglobin conjugate, with approximately 8 PEGs/Hb. Rates of the deoxyHb/nitrite reaction were measured by mixing deoxygenated SFH or MP4OX anaerobically with a 10-fold excess of sodium nitrite in a sealed cuvette in the presence of sodium dithionite. The reaction was monitored spectrophotometrically, and the spectra were deconvoluted using parent spectra for deoxyhemoglobin, iron-nitrosyl-hemoglobin (HbNO), and methemoglobin. Rates of the oxyHb/nitrite reaction were measured by mixing oxygenated SFH or MP4 with a 20-fold excess of nitrite. These spectra were deconvoluted using parent spectra for oxyhemoglobin, methemoglobin, and nitrite-bound methemoglobin. Results Time courses for both deoxy-and oxyHb reactions showed autocatalytic properties that deviated from pseudo first-order kinetics. Therefore, the maximum reaction rates were used for comparison purposes. Fully deoxygenated SFH and MP4OX (i.e., MP4DX) reduced nitrite to NO with maximum rates of 0.022 and 0.206 μM/s, respectively, showing a nearly 10-fold higher rate for MP4DX compared to deoxySFH. For the oxyHb/nitrite reaction, SFH and MP4OX showed maximum rates of 0.55 and 0.92 μM/s, respectively, a nearly 2-fold enhancement for MP4OX over SFH. Conclusions Our results demonstrate that deoxygenated MP4OX is more effective at reducing nitrite to NO than unmodified Hb. In addition, oxygenated MP4OX reacts more rapidly with nitrite, which may then accelerate the release of NO from heme sites. Taken together, these enhanced nitrite reaction rates for MP4 may compensate for NO scavenging and explain, at least in part, why MP4OX does not induce vasoconstriction in vivo. The increased reaction rates of MP4OX appear to be due to R-state stabilization due to the PEG conjugation. Further studies are required to understand the extent to which MP4OX’s interactions with nitrite contribute to its ability to improve perfusion and oxygenation of ischemic tissues. Disclosure This work was funded in part by Sangart Inc. and under US Army Contract No. W81XWH1020111 to Sangart.

Lowering of elevated tissue PCO2 in a hemorrhagic shock rat model after reinfusion of a novel nanobiotechnological polyhemoglobin-superoxide dismutase-catalase-carbonic anhydrase that is an oxygen and a carbon dioxide carrier with enhanced antioxidant properties

Even though erythrocytes transport both oxygen and carbon dioxide, research on blood substitutes has concentrated on the transport of oxygen and its vasoactivity and oxidative effects. Recent study in a hemorrhagic shock animal model shows that the degree of tissue PCO2 elevation is directly related to mortality rates. We therefore prepared a novel nanobiotechnological carrier for both O2 and CO2 with enhanced antioxidant properties. This is based on the use of glutaraldehyde to crosslink stroma free hemoglobin (SFHb), superoxide dismutase (SOD), catalase (CAT) and carbonic anhydrase (CA) to form a soluble PolySFHb-SOD-CAT-CA. It was compared to blood and different resuscitation fluids on the ability to lower elevated tissue PCO2 in a 2/3 blood volume loss rat hemorrhagic shock model. Sixty minutes of sustained hemorrhagic shock at 30 mm Hg resulted in the increase of tissue PCO2 to 95 mm ± 3 mmHg from the control level of 55 mm Hg. Reinfusion of whole blood (Hb 15 g/dL with its RBC enzymes) lowered the tissue PCO2 to 72 ± 4.5 mmHg 60 minutes after reinfusion. PolySFHb-SOD-CAT-CA (SFHb 10 g/dL plus additional enzymes) was more effective than whole blood in lowering PCO2 lowering this to 66.2 ± 3.5 mmHg. Ringer's Lactated solution or polyhemoglobin lowered the elevated PCO2 only slightly to 87 ± 4.5 mmHg and 84.8 ± 1.5 mmHg, respectively. Moreover, ST-elevation for whole blood (Hb 15 g/dL) and PolySFHb-SOD-CAT-CA (Hb 10 g/dL) was respectively 12.8% ± 4% and 13.0% ± 2% of the control 60 minutes after reinfusion. Both are significantly better than those in the Ringer's lactated group and the PolyHb group. In conclusion, this novel approach for blood substitute design has resulted in a novel nanobiotechnological carrier for both O2 and CO2 with enhanced antioxidant properties.

Novel Nanodimension artificial red blood cells that act as O2 and CO2 carrier with enhanced antioxidant activity: PLA-PEG nanoencapsulated PolySFHb-superoxide dismutase-catalase-carbonic anhydrase

Poly(ethylene glycol)-Poly(lactic acid) block-copolymer (PEG-PLA) was prepared and characterized using Fourier transform infrared spectrophotometer (FTIR). Glutaraldehyde was used to crosslink stroma-free hemoglobin (SFHb), superoxide dismutase (SOD), catalase (CAT), and carbonic anhydrase (CA) into a soluble complex of PolySFHb-SOD-CAT-CA. PEG-PLA was then used to nanoencapsulated PolySFHb-SOD-CAT-CA by oil in water emulsification. This resulted in the formation of PLA-PEG-PolySFHb-SOD-CAT-CA nanocapsules that have enhanced antioxidant activity and that can transport both O2 and CO2. These are homogeneous particles with an average diameter of 100 nm with good dispersion and core shell structure, high entrapment efficiency (EE%), and nanocapsule percent recovery. A lethal hemorrhagic shock model in rats was used to evaluate the therapeutic effect of the PLA-PEG-PolySFHb-SOD-CAT-CA nanocapsules. Infusion of this preparation resulted in the lowering of the elevated tissue PCO2 and also recovery of the mean arterial pressure (MAP).

MP4CO, a Pegylated Hemoglobin Saturated with Carbon Monoxide, Inhibits Microvascular Stasis in Transgenic Sickle Mice Through Heme Oxygenase-1.

AbstractAbstract 2112Our laboratory has shown in murine models of sickle cell disease (SCD) that intravascular heme promotes oxidative stress, inflammation and microvascular stasis through toll-like receptor-4 (TLR4) signaling. Furthermore, the heme degrading enzyme, heme-oxygenase-1 (HO-1) and its by-products biliverdin and carbon monoxide (CO), inhibit these effects. CO may induce salutary effects in SCD to decrease vaso-occlusion by inhibiting hemoglobin S polymerization, vasodilation and anti-inflammatory actions, including induction of HO-1. MP4CO is a 4.3 g/dL solution of human hemoglobin conjugated with polyethylene glycol and saturated with CO. In the current studies, we tested the hypothesis that MP4CO would induce HO-1 in transgenic sickle mice and inhibit microvascular stasis in response to hypoxia/reoxygenation (H/R). Microvascular stasis (% non-flowing venules) was examined by intravital microscopy following 1hr of hypoxia (7% O2) and 1hr of reoxygenation (room air) in NY1DD transgenic sickle mice implanted 3 days earlier with a dorsal skin fold chamber window (DSFC). Five treatment groups of 3–6 mice were studied initially: 1) lactated Ringer’s solution (LRS); 2) MP4OX (oxygen saturated MP4); 3) MP4CO; 4) oxygen-saturated stroma-free hemoglobin (SFH); 5) hemin chloride, 40 nmols/g i.p. × 3 days was administered as a positive control based on the previously-demonstrated induction of HO-1. Other than hemin chloride, all solutions (LRS, MP4CO, MP4OX, SFH) were administered i.v., 0.008 mL/g. In the first study, LRS, MP4OX, MP4CO or SFH were infused 24hr prior to H/R and in the second study the same solutions were infused 30min after hypoxia, during the reoxygenation phase of the experiment.In sickle mice treated with LRS or MP4OX 24hr prior to H/R, 25% and 22% of the venules, respectively, became static in response to H/R. However, in sickle mice treated with MP4CO 24hr prior to H/R, only 9% of the venules became static (p<0.05 MP4CO vs. LRS and MP4OX). In contrast, sickle mice treated with SFH 24hr prior to H/R developed significantly more stasis (37% stasis) than sickle mice in the other treatment groups (p<0.05). As we have previously shown, pretreatment with hemin abrogated vascular stasis in sickle mice (3% stasis, p<0.05 vs. all other groups). In additional groups of sickle mice, LRS, MP4OX, MP4CO and SFH were administered 30min after hypoxia during the reoxygenation phase. After H/R, LRS-treated animals had 26% stasis, MP4OX-treated mice had 18% stasis (p<0.05 vs. LRS) and MP4CO-treated mice had11% stasis (p<0.05 vs. LRS). Infusion of SFH 30min post-hypoxia markedly worsened stasis compared to the other treatments (44% stasis, p<0.05 vs. MP4CO, LRS and MP4OX).Infusion of MP4CO, but not LRS, MP4OX or SFH, markedly induced expression of microsomal HO-1 activity and protein, suggesting HO-1 was responsible for inhibition of stasis by MP4CO. Indeed, the HO-1 inhibitor SnPP reversed the effect of MP4CO on H/R-induced stasis in sickle mice (27% stasis with SnPP + MP4CO vs. 10% with LRS + MP4CO, p<0.05). The mechanism of HO-1 induction by MP4CO was likely due to an increased expression of nuclear factor erythroid 2-related factor 2 (Nrf2), an important transcriptional regulator of HO-1. MP4CO induced strikingly more Nrf2 in liver nuclei than LRS, MP4OX or SFH. Induction of HO-1 by MP4CO decreased the inflammatory response in sickle mice as evidenced by a decrease in activated nuclear factor-kappa B (NF-kB) phospho-p65 in liver nuclei following H/R.We conclude that MP4CO enhances cytoprotective Nrf2-regulated proteins including HO-1 resulting in decreased NF-kB activation, inflammation and microvascular stasis in transgenic SCD mice. CO delivery via MP4CO may be beneficial in patients with sickle cell anemia. Disclosures:Belcher:Sangart Inc: Research Funding. Chen:Sangart Inc: Research Funding. Young:Sangart Inc: Employment. Burhop:Sangart Inc: Employment. Vercellotti:Sangart Inc: Consultancy, Research Funding.

H-Ferritin Ferroxidase Activity Induces Cytoprotective Pathways and Inhibits Microvascular Stasis in Transgenic Sickle Mice

AbstractAbstract 378Hemolysis, oxidative stress, inflammation, vaso-occlusion and organ infarction are hallmarks of sickle cell disease (SCD). We hypothesize that intravascular heme, liberated from hemoglobin S derived from hemolyzed sickle red blood cells, is fundamental to inflammation and vaso-occlusion in SCD. Heme can be degraded by heme-oxygenase-1 (HO-1), releasing Fe2+, carbon monoxide (CO), and biliverdin/bilirubin. We have previously shown that increases in HO-1 activity inhibit vascular inflammation and vaso-occlusion in transgenic mouse models of SCD. HO-1 products, CO and biliverdin/bilirubin, also are protective in SCD, but Fe2+ released from the heme ring requires further processing. The released Fe2+ from heme is oxidized by ferritin heavy chain (FHC) ferroxidase activity and safely stored as catalytically-inactive Fe3+ inside ferritin clusters. FHC overexpression has been shown to be cytoprotective in response to inflammation and oxidative stress in vivo and in vitro. In this study, we hypothesize that overexpression of FHC with its ferroxidase activity will inhibit inflammation and microvascular stasis in transgenic sickle mice in response to stroma-free hemoglobin. We utilized a Sleeping Beauty transposase plasmid to deliver a human wt-ferritin heavy chain (wt-FHC) transposable element by hydrodynamic tail vein injections to NY1DD SCD mice. Control mice were infused with the same volume of lactated Ringer’s solution LRS) or a triple missense (ms-) human FHC plasmid encoding no ferroxidase enzyme activity. Eight weeks after injection, the mice were implanted with dorsal skin-fold chambers to access microvascular blood flow. LRS-treated mice had 40% microvascular stasis (% non-flowing venules) when infused with stroma-free hemoglobin, while wt-FHC overexpressing mice had only 5% stasis (p<0.05), suggesting vascular protection by wt-FHC. Ferroxidase activity was critical in this protection as mice overexpressing ms-FHC were not protected from microvascular stasis (33%). The wt-FHC SCD mice had marked increases in FHC mRNA and protein, light chain ferritin, 5-aminolevulinic acid synthase (5-ALA-synthase), cellular heme content, ferroportin, nuclear factor erythroid 2-related factor 2 (Nrf2), nuclear FHC and microsomal HO-1 activity and protein, and a decrease in activated phosho- and total nuclear factor-kappa B (NF-κB) p65 in the nuclei of the livers. We conclude that wt-FHC ferroxidase activity enhances 5-ALA-synthase activity and cytoprotective Nrf2-regulated proteins including HO-1 resulting in decreased NF-κB-activation, inflammation and microvascular stasis in transgenic SCD mice. Therapies directed at increasing ferritin heavy chain production and decreasing reactive iron availability may be beneficial in limiting sickle cell crises and end organ damage. Disclosures:Vercellotti:Sangart Inc: Consultancy, Research Funding. Chen:Sangart Inc: Research Funding. Hebbel:Sangart Inc: Consultancy, Research Funding. Belcher:Sangart Inc: Research Funding.

Stroma-Free Hemoglobin評価法としての赤血球型物質酵素免疫測定法の確立

Stroma-Free Hemoglobin評価法としての赤血球型物質酵素免疫測定法の確立

Bis[2-(3-carboxyphenoxy)carbonylethyl]phosphinic Acid (m-BCCEP): A Novel Affinity Cross-Linking Reagent for the β-Cleft Modification of Human Hemoglobin

The design and synthesis of bis[2-(3-carboxyphenoxy)carbonylethyl]phosphinic acid (m-BCCEP, 1) as a site-directed affinity reagent for cross-linking human hemoglobin have been reported as part of our long-term goal to generate artificial blood for emergency transfusions. Molecular modeling techniques were used to design the reagent, employing crystal coordinates of human hemoglobin A(0) imported from the Protein Data Bank. It was synthesized in four steps commencing from 3-hydroxybenzoic acid. The reagent 1 was converted to its trisodium salt to allow effective cross-linking in an aqueous medium. The reagent 1, as its trisodium salt, was found to specifically cross-link stroma-free human hemoglobin A(0) in the beta-cleft under oxygenated reaction conditions at neutral pH. The SDS-PAGE analyses of the modified hemoglobin pointed to the molecular mass range of 32 kDa as anticipated. The HPLC analyses of the product suggested that the cross-link had formed between the beta(1)-beta(2) subunits. Molecular dynamics simulation studies on the reagent-HbA(0) complex suggested that the predominant amino acid residues involved in the cross-linking are N-terminus Val-1 or Lys-82 on one of the beta-subunits and Lys-144 on the other. These predictions were borne out by MALDI-TOF MS analyses data of the peptide fragments obtained from tryptic digestion of the cross-linked product. The data also suggested the presence of a minor cross-link between Val-1 and Lys-82 on the opposing subunits. The oxygen equilibrium measurements of the m-BCCEP-modified hemoglobin product at 37 degrees C showed oxygen affinity (P(50) = 25.8 Torr) comparable to that of the natural whole blood (P(50) = 27.0 Torr) and significantly lower than that of stroma-free hemoglobin (P(50) = 14.19 Torr) assayed under identical conditions. The measured Hill coefficient value of 1.91 of the m-BCCEP-modified Hb product points to the reasonable retainment of oxygen-binding cooperativity after the cross-link formation.

Use of the Labscale Tangential Flow Diafiltrator to Remove Tetrameric Hemoglobin from Polyhemoglobin, Purify Hemolysate, and Concentrate Polyhemoglobin

The Labscale tangential flow diafiltration system in conjunction with polyethersulfone Biomax filters (both purchased from Millipore Inc.) were used in the following studies: (1) A crucial step in the preparation of polyhemoglobin (polyHb) is the removal of tetrameric hemoglobin (Hb), which can cause adverse side-effects. The efficiency for this depends on the integrity of the 100kDa filters. Those with lower integrity were not effective whereas those with a higher integrity of 0.075ml/min were more effective. A filter with an integrity of 0.075ml/min can reduce the initial 11.2% tetrameric Hb concentration to 1.3% using a flow rate of 0.2ml/min. Higher flow rate of 2.2ml/min was not as effective. (2) Filtration with a 100kDa filter was quick and efficient in separating stroma-free hemoglobin solutions from cellular debris from hemolysate. (3) Both 5kDa and 100kDa filters are efficient in concentrating hemoglobin solutions.

Severe adverse events associated with hemoglobin based oxygen carriers: Role of resuscitative fluids and liquid preserved RBC

Severe adverse events have been observed following the infusion of hemoglobin based oxygen carriers in patients subjected to elective orthopedic procedures, cardiopulmonary bypass surgery, and vascular surgical procedures. Along with all three of the hemoglobin based oxygen carriers, the patients received Ringer’s d, l-lactate as the resuscitative fluid, Ringer’s d, l-lactate in the excipient medium for the stroma free hemoglobin, and liquid preserved red blood cells that had been stored at 4 °C for longer than 2 weeks. The Ringer’s d, l-lactate solution has been shown to be toxic in both animals and patients. The current formulation of Ringer’s lactate contains only the l-isomer which has been shown in animals to be less toxic than the d-isomer of lactate . In a recent publication morbidity and mortality have been reported associated with the length of storage of red blood cells at 4 °C in patients subjected to reoperative cardiac surgery. Current clinical studies to assess the safety and therapeutic effectiveness of a hemoglobin based oxygen carrier (HBOC) must consider the effects of the composition of the resuscitation solution (Ringer’s l-lactate), the composition of the excipient medium (Ringer’s l-lactate or 0.9% NaCl) for the hemoglobin based oxygen carrier, and the length of storage of the liquid preserved red blood cells infused with the hemoglobin based oxygen carrier.

Long-term effects on the histology and function of livers and spleens in rats after 33% toploading of PEG-PLA-nano artificial red blood cells.

This study is to investigate the long-term effects of nanodimension PEG-PLA artificial red blood cells containing hemoglobin and red blood cell enzymes on the liver and spleen after 1/3 blood volume top loading in rats. The experimental rats received one of the following infusions: Nano artificial red blood cells in Ringer lactate, Ringer lactate, stroma-free hemoglobin, polyhemoglobin, and autologous rat whole blood. Blood samples were taken before infusions and on days 1, 7, and 21 after infusions for analysis. Nano artificial red blood cells, polyhemoglobin, Ringer lactate and rat red blood cells did not have any significant adverse effects on alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, creatine kinase, amylase and creatine kinase. On the other hand, stroma-free hemoglobin induced significant adverse effects on liver as shown by elevation in alanine aminotransferase and aspartate aminotransferase throughout the 21 days. On day 21 after infusions rats were sacrificed and livers and spleens were excised for histological examination. Nano artificial red blood cells, polyhemoglobin, Ringer lactate and rat red blood cells did not cause any abnormalities in the microscopic histology of the livers and spleens. In the stroma-free hemoglobin group the livers showed accumulation of hemoglobin in central veins and sinusoids, and hepatic steatosis. In conclusion, injected nano artificial red blood cells can be efficiently metabolized and removed by the reticuloendothelial system, and do not have any biochemical or histological adverse effects on the livers or the spleens.

Effects of PEG-PLA-nano artificial cells containing hemoglobin on kidney function and renal histology in rats.

This study is to investigate the long-term effects of PEG-PLA nano artificial cells containing hemoglobin (NanoRBC) on renal function and renal histology after 1/3 blood volume top loading in rats. The experimental rats received one of the following infusions: NanoRBC in Ringer lactate, Ringer lactate, stroma-free hemoglobin (SFHB), polyhemoglobin (PolyHb), autologous rat whole blood (rat RBC). Blood samples were taken before infusions and on days 1, 7 and 21 after infusions for biochemistry analysis. Rats were sacrificed on day 21 after infusions and kidneys were excised for histology examination. Infusion of SFHB induced significant decrease in renal function damage evidenced by elevated serum urea, creatinine and uric acid throughout the 21 days. Kidney histology in SFHb infusion group revealed focal tubular necrosis and intraluminal cellular debris in the proximal tubules, whereas the glomeruli were not observed damaged. In all the other groups, NanoRBC, PolyHb, Ringer lactate and rat RBC, there were no abnormalities in renal biochemistry or histology. In conclusion, injection of NanoRBC did not have adverse effects on renal function nor renal histology.