Stability of Liposomal Membrane of Hemoglobin-Vesicles (Artificial Red Cells) for Over Years of Storage Evaluated Using Liquid Chromatography-Mass Spectrometry.

Hemoglobin-Vesicles (Hb-V) are artificial oxygen carriers encapsulating a purified and concentrated Hb solution in liposomes composed of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), cholesterol, 1,5-O-dihexadecyl-N-succinyl-l-glutamate (DHSG), and 1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine-N-poly(ethylene glycol) (PEG5000) (DSPE-PEG). The safety and efficacy of Hb-V have been studied extensively by both preclinical and clinical test methods. Deoxygenation of Hb-V prevents autoxidation of Hb and can extend its shelf life to 2 years at room temperature. However, the lipid components raise concerns about hydrolysis because Hb-V is dispersed in saline. For this study, we attempted to estimate the lipid degradation of long-term stored Hb-V using liquid chromatography-mass spectrometry. Analyses of lipid components extracted from the stored Hb-V showed that the degradation increased depending on the storage temperature. The calculated % remaining of intact lipids of Hb-V were 98.1% after 4 years and 90.4% after 7.2 years at 4 °C, 95.8% after 1 year and 86.7% after 2 years at 25 °C, and 85.6% after 6 months at 40 °C. The main degradation products were lyso-PC and palmitic acid which are hydrolyzed at the ester bond of DPPC. A few hydrolyzed products of DHSG and DSPE-PEG were also detected in Hb-V, but almost no degradation or oxidation products derived from cholesterol could be identified. A shear test of Hb-V at 1500 s-1 showed no significant increase in Hb leakage after storage of 2 years at 25 °C and 6 months at 40 °C. Lipid degradation products including free fatty acids would decrease the pH of the Hb-V dispersion and synergistically facilitate degradation, but it maintained pH 6.5 during 6 years at 4 °C, 2 years at 25 °C, and 3 months at 40 °C because of its high buffering capacity. These results indicate that the storage conditions for Hb-V are appropriate to minimize lipid degradation in the long term.

Carbon monoxide-loaded cell therapy as an exercise mimetic for sarcopenia treatment.

Sarcopenia is characterized by loss of muscle strength and muscle mass with aging. The growing number of sarcopenia patients as a result of the aging population has no viable treatment. Exercise maintains muscle strength and mass by increasing peroxisome growth factor activating receptor γ-conjugating factor-1α (PGC-1α) and Akt signaling in skeletal muscle. The present study focused on the carbon monoxide (CO), endogenous activator of PGC-1α and Akt, and investigated the therapeutic potential of CO-loaded red blood cells (CO-RBCs), which is bioinspired from in vivo CO delivery system, as an exercise mimetic for the treatment of sarcopenia. Treatment of C2C12 myoblasts with the CO-donor increased the protein levels of PGC-1α which enhanced mitochondrial biogenesis and energy production. The CO-donor treatment also activated Akt, indicating that CO promotes muscle synthesis. CO levels were significantly elevated in the skeletal muscle of normal mice after intravenous administration of CO-RBCs. Furthermore, CO-RBCs restored the mRNA expression levels of PGC-1α in the skeletal muscle of two experimental sarcopenia mouse models, denervated (Den) and hindlimb unloading (HU) models. CO-RBCs also restored muscle mass in Den mice by activating Akt signaling and suppressing the muscle atrophy factors myostatin and atrogin-1, and oxidative stress. Treadmill tests further showed that the reduced running distance in HU mice was significantly restored by CO-RBC administration. These findings suggest that CO-RBCs have potential as an exercise mimetic for sarcopenia treatment.

Using hemoglobin vesicles to treat operative hemorrhagic shock after pneu- monectomy in dog models: an experimental study.

Hemoglobin vesicles (HbVs), considered as red blood cell substitutes, are liposomes encapsulating purified hemoglobin, with a phospholipid bilayer membrane (diameter: 250 nm; P50, 28 Torr). In this study, we aimed to investigate HbV function during hemorrhagic shock in lung resection and analyze the details of oxygen delivery. Left pneumonectomy was performed in dogs under mechanical ventilation, followed by rapid exsanguination of approximately 30% of the total circulating blood volume, which led to shock, reducing the mean arterial pressure (MAP) by approximately 60% of baseline. Subsequently, either 5% human serum albumin (HSA) or HbVs suspended in 5% HSA were infused for resuscitation. The MAP only recovered to 75% of baseline after HSA administration, but fully recovered (100%) after HbV administration, with significant differences between the groups (P < 0.005). Oxygen delivery was restored in the HbV group and was significantly higher than that in the HSA group (P < 0.0001). The infusion of HbVs dispersed in a 5% HSA solution compensated for the rapid loss of approximately 30% of the total circulating blood volume in a dog pneumonectomy model, even with impaired lung function. Thus, HbVs can be used for resuscitation from hemorrhagic shock during thoracic surgery.

Absence of Anaphylactic Reactions to Injection of Hemoglobin Vesicles (Artificial Red Cells) to Rodents.

The safety and efficacy of hemoglobin vesicles (HbVs) as artificial oxygen carriers encapsulating a purified and concentrated Hb solution in liposomes have been studied extensively. The HbV surface, modified with PEG by incorporating a PEG-conjugated phospholipid, is beneficial for storage and biocompatibility. However, it might be possible that interaction of PEG and the pre-existing anti-PEG antibody in the bloodstream causes acute adverse reaction. This study used two sets of experiments with rats and guinea pigs to ascertain whether the anti-PEG antibody generated by the PEG-modified HbV injection can induce anaphylactic reactions. SD rats received repeated intravenous injection of HbV at a dose rate of 16 or 32 mL/kg three times. Not anti-PEG IgG but anti-PEG IgM was detected. Nevertheless, no anaphylactic reaction occurred. Guinea pigs were used to study the presence of active systemic anaphylaxis further after injections of the PEG-modified liposomes used for HbV. The animals were sensitized by three repeated subcutaneous injections of PEG-modified liposomes (PEG-liposome) along with adjuvant at 1 week intervals. For comparison, unmodified liposomes (liposome) and 10 times excessively PEG-modified liposomes with ionizable lipid (10PEG-DODAP-liposome) were used. Inclusion of PEG modification induced not only anti-PEG IgM but also anti-PEG IgG. Three weeks after the final injection, intravenous injection of both PEG-liposome and liposome (1 mL/kg) induced no anaphylactic reaction. However, the injection of 10PEG-DODAP-liposome showed one lethal anaphylaxis case and one mild anaphylaxis case. Antisera obtained from the animal sensitized as described above were inoculated (0.05 mL) intradermally into fresh guinea pigs. The presence of passive cutaneous anaphylaxis was evaluated after intravenous injections (1 mL/kg) of three liposomes with Evans blue. No dye leakage was detected at any inoculated skin point for PEG-liposome or liposome, but a slight leakage was detected in one inoculated skin point for 10PEG-DODAP-liposome. These results indicate the absence of acute allergic reactions at repeated injections of HbVs despite the anti-PEG antibody induction. Not all the PEG-modified liposomes show anaphylaxis, and it may depend on the amount of PEGylated phospholipid and lipid composition of PEG-modified liposomes.

Elucidating the binding properties of methemoglobin in red blood cell to cyanide, hydrosulfide, and azide ions using artificial red blood cell.

Methemoglobin (metHb), the oxidized form of hemoglobin, lacks the ability of reversible oxygen binding; however, it has a high binding affinity to toxic substances such as cyanide, hydrosulfide, and azide. This innate property of metHb offers the clinical option to treat patients poisoned with these toxins, by oxidizing the endogenous hemoglobin in the red blood cells (RBCs). The binding properties of naked metHb (isolated from RBC) with these toxins has been studied; however, the binding behaviors of metHb under the intracellular conditions of RBC are unclear because of the difficulty in detecting metHb status changes in RBC. This study aimed to elucidate the binding properties of metHb in RBC under physiological and poisoned conditions using artificial RBC, which was hemoglobin encapsulated in a liposome. The mimic-circumstances of metHb in RBC (metHb-V) was prepared by oxidizing the hemoglobin in artificial RBC. Spectroscopic analysis indicated that the metHb in metHb-V exhibited a binding behavior different from that of naked metHb, depending on the toxic substance: When the pH decreased, (i) the cyanide binding affinity of metHb-V remained unchanged, but that of naked metHb decreased (ii) the hydrosulfide binding affinity was increased in metHb-V but was decreased in naked metHb. (iii) Azide binding was increased in metHb-V, which was similar to that in naked metHb, irrespective of the pH change. Thus, the binding behavior of intracellular metHb in the RBC with cyanide, hydrosulfide, and azide under physiological and pathological conditions were partly elucidated using the oxidized artificial RBC.

Machine perfusion preservation with hemoglobin based oxygen vesicles alleviate ultrastructural damages in porcine liver donated after cardiac death.

Midthermic machine perfusion (MMP) of post-circulatory arrest donor liver grafts has the advantage of preserving the functional ultrastructure of hepatocytes in donor grafts. It was reported that oxygenation during MMP reduces portal venous resistance and increases bile production. The MMP with hemoglobin-based oxygen vesicles (HbV) keeps the lower aspartate aminotransferase level (an indicator of liver injury) and maintains the functional ultrastructure of mitochondria in the hepatocytes. To evaluated differences of ultrastructural damages in donor livers between the MMP with and without HbV, porcine liver grafts after 60 min of warm ischemia were perfused at 22°C for 4 h with or without HbV, and a part of liver grafts were analyzed by transmission electron microscopy (TEM) and osmium-maceration scanning electron microscopy (OM-SEM). The remaining grafts were perfused with autologous blood at 38°C for 2 h in an isolated liver reperfusion model (IRM) that mimics the inside of the body after transplantation, and then analyzed by TEM and OM-SEM. Hepatocytes after MMP had small round mitochondria with rod-shaped cristae and reticulovesicular rough endoplasmic reticulum (rER) in both HbV(+) and HbV(-) livers. After IRM of HbV(+) livers, the well-developed lamellar rER was often found in hepatocytes. Liver sinusoidal endothelial cells (LSECs) after MMP contained some large vacuolar structures containing amorphous garbage in the cytoplasm, and their size along with appearance frequency were smaller and lower, respectively, in HbV(+) livers than HbV(-). Oxygenation during the MMP by using HbV suppressed the ultrastructural damages in donor livers, in particular for the LSECs. RESEARCH HIGHLIGHTS: Liver sinusoidal endothelial cells after midthermic machine perfusion had large vacuolar organelles with amorphous garbage. Oxygenation during the perfusion made them less and smaller, ultrastructurally supporting its utility.

Carbon Monoxide-Loaded Red Blood Cell Prevents the Onset of Cisplatin-Induced Acute Kidney Injury.

Cisplatin-induced acute kidney injury (AKI) is an important factor that limits the clinical use of this drug for the treatment of malignancies. Oxidative stress and inflammation are considered to be the main causes of not only cisplatin-induced death of cancer cells but also cisplatin-induced AKI. Therefore, developing agents that exert antioxidant and anti-inflammatory effects without weakening the anti-tumor effects of cisplatin is highly desirable. Carbon monoxide (CO) has recently attracted interest due to its antioxidant, anti-inflammatory, and anti-tumor properties. Herein, we report that CO-loaded red blood cell (CO-RBC) exerts renoprotective effects on cisplatin-induced AKI. Cisplatin treatment was found to reduce cell viability in proximal tubular cells via oxidative stress and inflammation. Cisplatin-induced cytotoxicity, however, was suppressed by the CO-RBC treatment. The intraperitoneal administration of cisplatin caused an elevation in the blood urea nitrogen and serum creatinine levels. The administration of CO-RBC significantly suppressed these elevations. Furthermore, the administration of CO-RBC also reduced the deterioration of renal histology and tubular cell injury through its antioxidant and anti-inflammatory effects in cisplatin-induced AKI mice. Thus, our data suggest that CO-RBC has the potential to substantially prevent the onset of cisplatin-induced AKI, which, in turn, may improve the usefulness of cisplatin-based chemotherapy.

Development of liposomal contrast agent with high iodine concentration and minimal effect on renal function.

Purpose: The use of contrast media is essential to achieve high accuracy in diagnostic imaging. Iodine contrast media, one of these contrast media, has nephrotoxicity as a side effect. Therefore, the development of iodine contrast media that can reduce nephrotoxicity is expected. Since liposomes are generally adjustable in size (100-300 nm) and are not filtered by the renal glomerulus, we hypothesized that iodine contrast media could be encapsulated in liposomes and administered to avoid the nephrotoxicity of iodine contrast media. The aim of this study is to develop an iomeprol-containing liposome (IPL) agent with high iodine concentration and to investigate the effect of intravenous administration of IPL on renal function in a rat model with chronic kidney injury. Materials and methods: IPLs were prepared by encapsulating an iomeprol (400mgI/mL) solution in liposomes by a kneading method using a rotation-revolution mixer. Radiodensities of iomeprol and IPL were measured. IPL or iopamidol at normal dose (0.74 g I/kg) or high dose (3.7 g I/kg) was administered to healthy and 5/6-nephrectomized rats (n = 3-6). Serum creatinine (sCr) and histopathological change of tubular epithelial cells were evaluated after injection. Results: The iodine concentration of IPL was 220.7 mgI/mL, equivalent to 55.2% of the iodine concentration of iomeprol. The CT values of IPL was 4731.6 ± 53.2 HU, 59.04% that of iomeprol. The ratios of change in sCr in 5/6-nephrectomized rats that received high-dose iopamidol were 0.73, which were significantly higher than that in 5/6-nephrectomized rats that received high-dose IPL (-0.03) (p = 0.006). Change in foamy degeneration of tubular epithelial cells was confirmed in 5/6-nephrectomized rats that received high-dose iopamidol than that in the sham control group and healthy rats that received normal dose iopamiron (p = 0.016, p = 0.032, respectively). Foamy degeneration of tubular epitherial cells was rarely observed in the IPL injection group. Conclusions: We developed new liposomal contrast agents that have high iodine concentration and minimal effect on renal function.

Liposome-Encapsulated Hemoglobin Vesicle Improves Persistent Anti-arrhythmogenesis through Improving Myocardial Electrical Remodeling and Modulating Cardiac Autonomic Activity in a Hemorrhagic Shock-Induced Rat Heart Model.

OBJECTIVE: Shock heart syndrome (SHS) is associated with lethal arrhythmias (ventricular tachycardia/ventricular fibrillation, VT/VF). We investigated whether liposome-encapsulated human hemoglobin vesicles (HbVs) has comparable persistent efficacy to washed red blood cells (wRBCs) for improving arrhythmogenesis in the subacute to chronic phase of SHS. METHODS: Optical mapping analysis (OMP), electrophysiological study (EPS), and pathological examinations were performed on blood samples from Sprague-Dawley rats following induction of hemorrhagic shock. After hemorrhagic shock, the rats were immediately resuscitated by transfusing 5% albumin (ALB), HbV, or wRBCs. All rats survived for 1 week. OMP and EPS were performed on Langendorff-perfused hearts. Spontaneous arrhythmias and heart rate variability (HRV) were evaluated using awake 24-h telemetry, cardiac function by echocardiography, and pathological examination of Connexin43. RESULTS: OMP showed significantly impaired action potential duration dispersion (APDd) in the left ventricle (LV) in the ALB group whereas APDd was substantially preserved in the HbV and wRBCs groups. Sustained VT/VF was easily provoked by EPS in the ALB group. No VT/VF was induced in the HbV and wRBCs groups. HRV, spontaneous arrhythmias, and cardiac function were preserved in the HbV and wRBCs groups. Pathology showed myocardial cell damage and Connexin43 degradation in the ALB group, all of which were attenuated in the HbV and wRBCs groups. CONCLUSION: LV remodeling after hemorrhagic shock caused VT/VF in the presence of impaired APDd. Similar to wRBCs, HbV persistently prevented VT/VF by inhibiting persistent electrical remodeling, preserving myocardial structures, and ameliorating arrhythmogenic modifying factors in the subacute to chronic phase of hemorrhagic shock-induced SHS.

Carbon monoxide combined with artificial blood cells acts as an antioxidant for tissues thermally-damaged by dye laser irradiation.

Artificial red blood cells [i.e., hemoglobin vesicles (HbVs)] can be used as photosensitizers in pulsed-dye laser (PDL) treatment for port wine stains in animal models. Small HbVs are distributed in the vicinity of the endothelial cells of the blood vessels. In our previous in vivo experiments, both HbVs and red blood cells absorbed photons of the laser and generated heat, contributing to removal of very small blood vessels and large deeper subcutaneous blood vessels with PDL irradiation. Herein, we tested carbon monoxide-bound HbVs (CO-HbVs) that would produce heat energy while releasing CO in vessels after dye laser irradiation in a rabbit auricle model. We conducted this experiment to confirm secondary progression of thermal injury being reduced with the antioxidative property of CO. We histopathologically evaluated the damages to the large vessels and surrounding dermal tissue following PDL irradiation alone or subsequent to the intravenous injection of the qualified HbVs. The soft tissue damages were graded on a five-point scale and compared statistically. Intravenous CO-HbVs significantly reduced damage to the surrounding tissue after subsequent PDL irradiation; however, the degree of damage to the larger vessel wall resulted in a variety of changes, including a slight increase in our histopathological grades. This beneficial effect in dye laser treatment for port wine stains may be the result of the antioxidative property of CO against free radicals in the zone of stasis that may still be theoretically viable in burns. This effect of CO protecting tissues from thermal damage is consistent with previous reports of CO as a reducing agent. If the reducing agent can be delivered directly to the affected area immediately after the burn injury, even in a small amount, the complex inflammatory cascade may be reduced and unnecessary inflammation after laser treatment that lowers the patient's quality of life can be avoided.

Examination of central nervous system by functional observation battery after massive intravenous infusion of carbon monoxide-bound and oxygen-bound hemoglobin vesicles in rats.

Carbon monoxide (CO) is known as a toxic gas inducing "CO poisoning", which acutely affects the central nervous system (CNS) and which persistently affects brain functions depending on the exposure time and CO concentration. By contrast, in pathological rodent models, intravenous infusion of CO-bound hemoglobin vesicles (CO-HbV) has shown various beneficial effects such as anti-oxidative and anti-inflammatory reactions. This study assessed effects of CO-HbV infusion on CNS using a functional observation battery, sensory reflexes, grip strength, and landing foot splay measurements. The test fluids were CO-HbV and O2-bound HbV (O2-HbV) suspended in saline ([Hb] â€‹= â€‹10 â€‹g/dL), and saline alone for comparison. The rats received either 16 or 32 â€‹mL/kg of fluid intravenously at 1.5 â€‹mL/min/kg. Observations were made before infusion, and at 5 â€‹min, 4, 8, 24, 48 and 72 â€‹h after infusion. Massive doses of 16 and 32 â€‹mL/kg respectively corresponded to about 29 and 57% of the whole circulating blood volume (56 â€‹mL/kg). No toxicological effect was observed in any measurement item for any group in comparison to the control saline infusion group. Histopathological examination of hippocampal tissue at 14 days after infusion showed the number of necrotic cells to be minimal. Results obtained from rats in this experiment suggest that the massive intravenous infusion of CO-HbV yields beneficial anti-oxidative and anti-inflammatory effects without showing CO-poisoning-related symptoms of CNS damage.

Structural insights into the substrate specificity of IMP-6 and IMP-1 metallo-ß-lactamases.

IMP-type metallo-ß-lactamases confer resistance to carbapenems and a broad spectrum of ß-lactam antibiotics. IMP-6 and IMP-1 differ by only a point mutation: Ser262 in IMP-1 and Gly262 in IMP-6. The kcat/Km values of IMP-1 for imipenem and meropenem are nearly identical; however, for IMP-6, the kcat/Km for meropenem is 7-fold that for imipenem. In clinical practice, this may result in an ineffective therapeutic regimen and, consequently, in treatment failure. Here, we report the crystal structures of IMP-6 and IMP-1 with the same space group and similar cell constants at resolutions of 1.70 and 1.94 Å, respectively. The overall structures of IMP-6 and IMP-1 are similar. However, the loop region (residues 60-66), which participates in substrate binding, is more flexible in IMP-6 than in IMP-1. This difference in flexibility determines the substrate specificity of IMP-type metallo-ß-lactamases for imipenem and meropenem. The amino acid at position 262 alters the mobility of His263; this affects the flexibility of the loop via a hydrogen bond with Pro68, which plays the role of a hinge in IMP-type metallo-ß-lactamases. The substitution of Pro68 with a glycine elicited an increase in the Km of IMP-6 for imipenem, whereas the affinity for meropenem remained unchanged.

Early reperfusion with hemoglobin vesicles into tracheal subepithelial capillaries in a mouse tracheal transplant model.

Hemoglobin vesicles (HbVs), liposomes containing concentrated hemoglobin extracted from outdated human red blood cells (RBC), are artificial oxygen carriers with a small particle size. To evaluate the reperfusion of capillaries with HbVs in a tracheal transplant model and compare it with that of RBC. Isogenic mice were used as donors and recipients in a parallel trachea transplant model. Both ends of the donor trachea were anastomosed end-laterally to the recipient trachea to form in parallel. After transplantation, 0.3 mL of HbV solution (Hb concentration, 10 g/dL) was administered via the tail vein. The recipients were euthanized 1, 4, 6, and 8 h after surgery (n = 5 in each group). The tracheas were harvested, and tracheal subepithelial capillaries (SEC) reperfusion was histologically evaluated. A significant number of particles defined as HbV by electron microscopy were observed in the SEC of the grafted tracheas 4 h after the transplant surgery and HbV administration when no RBC were found in the SECs. The number increased 6 and 8 h later. Our findings suggest that HbVs, which are smaller than RBC, can reperfuse the capillaries of grafts earlier than RBCs after transplantation and contribute to the oxygenation of transplanted tissues.

Exploring how plasma- and muscle-related parameters affect trout hemolysis as a route to prevent hemoglobin-mediated lipid oxidation of fish muscle.

Hemoglobin (Hb) is a powerful promoter of lipid oxidation, particularly in muscle of small pelagic fish species and fish by-products, both having high Hb-levels and highly unsaturated lipids. As Hb is located within the red blood cells (RBCs) it is here hypothesized that the perishable polyunsaturated fatty acids (PUFAs) can be protected from oxidation by limiting hemolysis during early fish processing. Using a model system consisting of washed-resuspended trout (Oncorhynchus mykiss) RBCs (wr-RBCs), the aim of this study was to evaluate how RBC lysis under cold storage was affected by selected parameters linked to blood or muscle: bacterial growth, energy status, pH, RBC membrane lipid oxidation and colloidal osmotic pressure (COP). The results indicated that bacterial growth had a modest effect on hemolysis while pH-values typical for post mortem fish muscle (6.4-6.8), and absence of glucose or albumin stimulated hemolysis. The rapid hemolysis observed at pH 6.4-6.8 correlated with lipid oxidation of the RBC membrane, while the lower hemolysis at pH 7.2-8.0 occurred with low, or without any RBC membrane lipid oxidation. When hemin was added to the RBCs at pH 6.8 hemolysis was induced without parallel RBC membrane oxidation, pointing at Hb-autoxidation and hemin-release per se as important events triggering lysis in fish muscle. Altogether, the study provided valuable findings which ultimately can aid development of new tools to combat lipid oxidation in post mortem fish muscle by limiting hemolysis.

Liposomal methemoglobin as a potent antidote for hydrogen sulfide poisoning.

Hydrogen sulfide (H2S) induces acute and lethal toxicity at high concentrations. However, no specific antidotes for H2S poisoning have been approved. Liposomal methemoglobin (metHb@Lipo) was developed as an antidote for cyanide poisoning. As the toxic mechanism of H2S poisoning is the same as that of cyanide poisoning, metHb@Lipo could potentially be used as an antidote for H2S poisoning. In this study, we evaluated the antidotal efficacy of metHb@Lipo against H2S poisoning. Stopped-flow rapid-scan spectrophotometry clearly showed that metHb@Lipo scavenged H2S rapidly. Additionally, metHb@Lipo showed cytoprotective effects against H2S exposure in H9c2 cells by maintaining mitochondrial function. MetHb@Lipo treatment also improved the survival rate after H2S exposure in vivo, with the maintenance of cytochrome c oxidase activity and suppression of metabolic acidosis. Moreover, metHb@Lipo therapy maintained significant antidotal efficacy even after 1-year-storage at 4-37 °C. In conclusion, metHb@Lipo is a candidate antidote for H2S poisoning.

A bioinspired carbon monoxide delivery system prevents acute kidney injury and the progression to chronic kidney disease.

Renal ischemia-reperfusion (IR)-induced tissue hypoxia causes impaired energy metabolism and oxidative stress. These conditions lead to tubular cell damage, which is a cause of acute kidney injury (AKI) and AKI to chronic kidney disease (CKD). Three key molecules, i.e., hypoxia-inducible factor-1α (HIF-1α), AMP-activated protein kinase (AMPK), and nuclear factor E2-related factor 2 (Nrf2), have the potential to protect tubular cells from these disorders. Although carbon monoxide (CO) can comprehensively induce these three molecules via the action of mitochondrial reactive oxygen species (mtROS), the issue of whether CO induces these molecules in tubular cells remains unclear. Herein, we report that CO-enriched red blood cells (CO-RBC) cell therapy, the inspiration for which is the in vivo CO delivery system, exerts a renoprotective effect on hypoxia-induced tubular cell damage via the upregulation of the above molecules. Experiments using a mitochondria-specific antioxidant provide evidence to show that CO-driven mtROS partially contributes to the upregulation of the aforementioned molecules in tubular cells. CO-RBC ameliorates the pathological conditions of IR-induced AKI model mice via activation of these molecules. CO-RBC also prevents renal fibrosis via the suppression of epithelial mesenchymal transition and transforming growth factor-ß1 secretion in an IR-induced AKI to CKD model mice. In conclusion, our results confirm that the bioinspired CO delivery system prevents the pathological conditions of both AKI and AKI to CKD via the amelioration of hypoxia inducible tubular cell damage, thereby making it an effective cell therapy for treating the progression to CKD.

Intraosseous infusion of liposome-encapsulated hemoglobin (HbV) acutely prevents hemorrhagic anemia-induced lethal arrhythmias, and its efficacy persists with preventing proarrhythmic side effects in the subacute phase of severe hemodilution model.

BACKGROUND: Artificial oxygen carriers (HbV) can treat hemorrhagic shock with lethal arrhythmias (VT/VF). No reports exist on subacute HbV's effects. METHODS: Acute and subacute resuscitation effects with anti-arrhythmogenesis of HbV were studied in 85% blood exchange rat model (85%-Model). Lethal 85%-Model was created by bone marrow transfusion and femoral artery bleeding in 80 SD rats in HbV-administered group (HbV-group), washed erythrocyte-administered group (wRBC-group), and 5% albumin-administered group (ALB-group). Survival rates, anti-arrhythmic efficacy by optical mapping system (OMP) with electrophysiological study (EPS) in Langendorff heart, cardiac autonomic activity by heart rate variability (HRV) and ventricular arrhythmias by 24-h electrocardiogram telemetry monitoring (24 h-ECG) in awake, and left ventricular function by echocardiography (left ventricular ejection fraction [LVEF]) were measured. RESULTS: All rats in HbV- and wRBC-groups survived for 4 weeks, whereas no rats in ALB-group. HbV and wRBC acutely suppressed VT/VF in Langendorff heart through ameliorating action potential duration dispersion (APDd) analyzed by OMP with EPS. For subacute analysis, 50% blood exchange by 5% albumin was used (ALB-group 50). Subacute salutary effect on APDd and VT/VF inducibility was confirmed in HbV- and wRBC-groups. 24 h-ECG showed that HbV and wRBC suppressed none-sustained ventricular tachycardia (NSVT) and sympathetic component of HRV (LF/HF) with preserved LVEF (HbV-group, wRBC-group vs. ALB-group 50; NSVT numbers/days, 0.5 ± 0.3, 0.4 ± 0.3 vs. 3.9 ± 1.2*; LF/HF, 1.1 ± 0.2, 0.8 ± 0.2 vs. 3.5 ± 1.0*; LVEF, 84 ± 5, 83 ± 4, vs. 77 ± 4%*; *p < 0.05). CONCLUSIONS: Collectively, HbV has sustained antiarrhythmic effect in subacute 85%-Model by ameliorating electrical remodeling and improving arrhythmogenic modifying factors (HRV and LVEF). These findings are useful in now continuing clinical trials of HbV.

Effect of carbon monoxide administration using haemoglobin-vesicles on the hippocampal tissue.

Carbon monoxide (CO) is a toxic gas that causes neuropathy. However, CO is endogenously produced in small amounts showing various beneficial effects. We hypothesized that CO-bound haemoglobin-vesicle (HbV) administration would reduce cerebral ischaemia-reperfusion injury without causing neuropathy. Three experiments were conducted. First, rats were exposed to CO inhalation to create a CO-poisoning group, and they were sacrificed on 0, 7, 14, and 21 days after CO exposure. Histopathologically, hippocampal damage was prominent at 14 days. Second, the rats were administered with CO-HbV equivalent to 50 or 25% of circulating blood volume (CO-HbV50 or CO-HbV25 group). Rats were sacrificed 14 days after administration. Third, rats put into haemorrhagic shock by 50% of circulating blood withdrawal were resuscitated using saline, autologous blood, and CO-HbV. They were sacrificed 14 days after resuscitation. Hippocampal damage assessment clarified that almost no necrotic cells were observed in the CO-HbV50 group. Necrotic cells in the CO-HbV25 group were comparable to those found for the control group. In rats resuscitated from haemorrhagic shock, the hippocampal damage in the group using CO-HbV was the mildest. Administration of CO-HbV did not lead to marked hippocampal damage. Furthermore, CO-HbV was effective at preventing cerebral ischaemia-reperfusion injury after haemorrhagic shock.

Bioinspired carbon monoxide delivery using artificial blood attenuates the progression of obliterative bronchiolitis via suppression of macrophage activation by IL-17A.

Carbon monoxide (CO) is expected to attenuate the progression of obliterative bronchiolitis (OB), which is a serious complication after lung transplantation. However, issues in terms of feasible exogenous CO supply, such as continuousness and safety, remain unsolved. Here, we applied nano red blood cells, namely hemoglobin vesicles (Hb-V), as a CO cargo based on the biomimetic concept and investigated the therapeutic potential of CO-loaded Hb-V on OB in orthotopic tracheal transplant model mice. The CO-loaded Hb-V was comprised of negatively charged liposomes encapsulating carbonylhemoglobin with a size of ca. 220 nm. The results of histological evaluation showed that allograft luminal occlusion and fibrosis were significantly ameliorated by treatment with CO-loaded Hb-V compared to treatment with saline, cyclosporine, and Hb-V. The therapeutic effects of CO-loaded Hb-V on OB were due to the suppression of M1 macrophage activation in tracheal allografts, resulting from decreased IL-17A production. Furthermore, the expression of TNF-α and TGF-ß in tracheal allografts was decreased by CO-loaded Hb-V treatment but not saline and Hb-V treatment, indicating that CO liberated from CO-loaded Hb-V inhibits epithelial-mesenchymal transition. These findings suggest that CO-loaded Hb-V exerts strong therapeutic efficacy against OB via the regulation of macrophage activation by IL-17A and TGF-ß-driven epithelial-mesenchymal transition.