GBT021601 improves red blood cell health and the pathophysiology of sickle cell disease in a murine model.

The pathophysiologic mechanism of sickle cell disease (SCD) involves polymerization of deoxygenated haemoglobin S (HbS), leading to red blood cell (RBC) sickling, decreased RBC deformability, microvascular obstruction, haemolysis, anaemia and downstream clinical complications. Pharmacological increase in the concentration of oxygenated HbS in RBCs has been shown to be a novel approach to inhibit HbS polymerization and reduce RBC sickling and haemolysis. We report that GBT021601, a small molecule that increases HbS-oxygen affinity, inhibits HbS polymerization and prevents RBC sickling in blood from patients with SCD. Moreover, in a murine model of SCD (SS mice), GBT021601 reduces RBC sickling, improves RBC deformability, prolongs RBC half-life and restores haemoglobin levels to the normal range, while improving oxygen delivery and increasing tolerance to severe hypoxia. Notably, oral dosing of GBT021601 in animals results in higher levels of Hb occupancy than voxelotor and suggests the feasibility of once-daily dosing in humans. In summary, GBT021601 improves RBC health and normalizes haemoglobin in SS mice, suggesting that it may be useful for the treatment of SCD. These data are being used as a foundation for clinical research and development of GBT021601.

The Molecular Size of Bioengineered Oxygen Carriers Determines Tissue Oxygenation in a Hypercholesterolemia Guinea Pig Model of Hemorrhagic Shock and Resuscitation.

Polymerized human hemoglobin (PolyhHb) has shown promise in preclinical hemorrhagic shock settings. Different synthetic and purification schemes can control the size of PolyhHbs, yet research is lacking on the impact of polymerized hemoglobin size on tissue oxygenation following hemorrhage and resuscitation in specialized animal models that challenge their resuscitative capabilities. Pre-existing conditions that compromise the vasculature and end organs, such as the liver, may limit the effectiveness of resuscitation and exacerbate the toxicity of these molecules, which is an important but minimally explored therapeutic dimension. In this study, we compared the effective oxygen delivery of intermediate molecular weight PolyhHb (PolyhHb-B3; 500-750 kDa) to high molecular weight PolyhHb (PolyhHb-B4; 750 kDa-0.2 µm) for resuscitative effectiveness in guinea pig models subjected to hemorrhagic shock. We evaluated how the size of PolyhHb impacts hemodynamics and tissue oxygenation in normal guinea pigs and guinea pigs on an atherogenic diet. We observed that while PolyhHb-B3 and -B4 equivalently restore hemodynamic parameters of normal-dieted guinea pigs, high-fat-dieted guinea pigs resuscitated with PolyhHb-B4 have lower mean arterial pressures, impaired tissue oxygenation, and higher plasma lactate levels than those receiving PolyhHb-B3. We characterized the plasma of these animals following resuscitation and found that despite similar oxygen delivery kinetics, circulating PolyhHb-B3 and -B4 demonstrated a size-dependent increase in the plasma viscosity, consistent with impaired perfusion in the PolyhHb-B4 transfusion group. We conclude that intermediate-sized PolyhHbs (such as -B3) are ideal for further research given the effective resuscitation of hemorrhagic shock based on tissue oxygenation in hypercholesterolemic guinea pigs.

Changes in oxygen delivery during experimental models of cerebral malaria.

Cerebral malaria (CM) is a severe manifestation of malaria that commonly occurs in children and is hallmarked by neurologic symptoms and significant Plasmodium falciparum parasitemia. It is currently hypothesized that cerebral hypoperfusion from impaired microvascular oxygen transport secondary to parasitic occlusion of the microvasculature is responsible for cerebral ischemia and thus disease severity. Animal models to study CM, are known as experimental cerebral malaria (ECM), and include the C57BL/6J infected with Plasmodium berghei ANKA (PbA), which is ECM-susceptible, and BALB/c infected with PbA, which is ECM-resistant. Here we sought to investigate whether changes in oxygen (O2) delivery, O2 flux, and O2 utilization are altered in both these models of ECM using phosphorescence quenching microscopy (PQM) and direct measurement of microvascular hemodynamics using the cranial window preparation. Animal groups used for investigation consisted of ECM-susceptible C57BL/6 (Infected, n = 14) and ECM-resistant BALB/c (Infected, n = 9) mice. Uninfected C57BL/6 (n = 6) and BALB/c (n = 6) mice were included as uninfected controls. Control animals were manipulated in the exact same way as the infected mice (except for the infection itself). C57BL/6 ECM animals at day 6 of infection were divided into two cohorts: Early-stage ECM, presenting mild to moderate drops in body temperature (>34 < 36 °C) and Late-stage ECM, showing marked drops in body temperature (<33 °C). Data taken from new experiments conducted with these animal models were analyzed using a general linear mixed model. We constructed three general linear mixed models, one for total O2 content, another for total O2 delivery, and the third for total O2 content as a function of convective flow. We found that in both the ECM-susceptible C57BL/6J model and ECM-resistant BALB/c model of CM, convective and diffusive O2 flux along with pial hemodynamics are impaired. We further show that concomitant changes in p50 (oxygen partial pressure for 50% hemoglobin saturation), only 5 mmHg in the case of late-stage CM C57BL/6J mice, and O2 diffusion result in insufficient O2 transport by the pial microcirculation, and that both these changes are required for late-stage disease. In summary, we found impaired O2 transport and O2 affinity in late-stage ECM, but only the former in either early-stage ECM and ECM-resistant strains.

The Double-Edged Sword of Erythrocytes in Health and Disease via Their Adhesiveness.

Their widespread presence throughout the vasculature, coupled with their reactivity, and thereby to their potential to release reactive oxidative species, or to utilize their anti-oxidative capacities, has promoted much discussion of the role(s) of red blood cells (RBCs) in the progression of health or, alternatively, a wide range of disease states. Moreover, these role(s) have been linked to the development of adhesiveness and, in fact, thereby to the essential pathway to their eventual clearance, e.g., by macrophages in the spleen. These disparate roles coupled with the mechanisms involved are reviewed and given. Following an analysis, novel perspectives are provided; these perspectives can lead to novel assays for identifying the potential for RBC adhesiveness as suggested herein. We describe this paradigm, that involves RBC adhesiveness, hemolysis, and ghost formation, with examples including, inter alia, the progression of atherosclerosis and the suppression of tumor growth along with other disease states.

Hyperspectral wide-field-of-view imaging to study dynamic microcirculatory changes during hypoxia.

Hyperspectral imaging (HSI) provides a fast, reliable, and noninvasive way for the study of vascular microcirculation in animal models. Rapid hyperspectral imaging of large portions of the microcirculatory preparation is critical for understanding the function and regulation of vascular microcirculatory networks. This report presents the application of an off-the-shelf benchtop HSI linear scanning system to acquire larger field-of-view images of microcirculatory preparations. The HSI line detector was displaced perpendicular to the scanning direction to map larger areas, with a rate of displacement determined by the scanning rate and the exposure time. Analysis of the collected image was used to assess dynamic changes in the microcirculation. The system records dynamic changes in microvascular hemoglobin oxygen (HbO2) saturation and vascular morphology during hypoxia and reoxygenation and has similar acquisition speeds to commonly referenced spectral-scanning HSI systems. In addition, the HbO2 saturations collected via HSI closely correlate with those collected by phosphorescence quenching microscopy. The reported system enables dynamic functional imaging of the microcirculation for broad experimental and clinical applications.NEW & NOTEWORTHY This study presents a novel bench setup and algorithm to measure intravascular hemoglobin oxygen saturation in microcirculation. Wide-field hyperspectral imaging allows for rapid quantification of intravascular changes in hemoglobin saturation. The method described in this manuscript can expand the understanding of oxygen delivery to tissues in vivo.

Increased hemoglobin affinity for oxygen with GBT1118 improves hypoxia tolerance in sickle cell mice.

Therapeutic agents that increase the Hb affinity for oxygen (O2) could, in theory, lead to decreased O2 release from Hb and impose a hypoxic risk to tissues. In this study, GBT1118, an allosteric modifier of Hb affinity for O2, was used to assess the impact of increasing Hb affinity for O2 on brain tissue oxygenation, blood pressure, heart rate, O2 delivery, and tolerance to hypoxia in Townes transgenic sickle cell disease (SCD) mice. Brain oxygenation and O2 delivery were studied during normoxia and severe hypoxic challenges. Chronic treatment with GBT1118 increased Hb affinity for O2, reducing the Po2 for 50% HbO2 saturation (P50) in SCD mice from 31 mmHg to 18 mmHg. This treatment significantly reduced anemia, increasing hematocrit by 33%, improved cardiac output (CO), and O2 delivery and extraction. Chronically increasing Hb affinity for O2 with GBT1118 preserved cortical O2 tension during normoxia, improved cortical O2 tension during hypoxia, and increased tolerance to severe hypoxia in SCD mice. Independent of hematological changes induced by chronic treatment, a single dose of GBT1118 significantly improved tolerance to hypoxia, highlighting the benefits of increasing Hb affinity for O2 and consequently reducing sickling of RBCs in blood during hypoxia in SCD.NEW & NOTEWORTHY Chronic pharmacologically increased hemoglobin affinity for oxygen in sickle cell disease mice alleviated hematological consequences of sickle cell disease, increasing RBC half-life, hematocrit, and hemoglobin concentration, while also decreasing reticulocyte count. Additionally, chronically increased hemoglobin affinity for oxygen significantly improved survival as well as cortical tissue oxygenation in sickle cell disease mice during hypoxia, suggesting that oxygen delivery and utilization is improved by increased hemoglobin affinity for oxygen.

Negative pressure increases microvascular perfusion during severe hemorrhagic shock.

Hemorrhagic shock (HS) is a severe life-threatening condition characterized by loss of blood volume and a lack of oxygen (O2) delivery to tissues. The objective of this study was to examine the impact of manipulating Starling forces in the microcirculation during HS to increase microvascular perfusion without restoring blood volume or increasing O2 carrying capacity. To decrease interstitial tissue pressure, we developed a non-contact system to locally apply negative pressure and manipulate the pressure balance in capillaries, while allowing for visualization of the microcirculation. Golden Syrian hamsters were instrumented with dorsal window chambers and subjected to a controlled hemorrhaged of 50% of the animal's blood volume without any fluid resuscitation. A negative pressure chamber was attached to the dorsal window chamber and a constant negative pressure was applied. Hemodynamic parameters (including microvascular diameter, blood flow, and functional capillary density [FCD]) were measured before and during the four hours following the hemorrhage, with and without applied negative pressure. Blood flow significantly increased in arterioles during negative pressure. The increase in flow through arterioles also improved microvascular perfusion as reflected by increased FCD. These results indicate that negative pressure increases flow in the microcirculation when fluid resuscitation is not available, thus restoring blood flow, oxygen delivery, and preventing the accumulation of metabolic waste. Applying negative pressure might allow for control of microvascular blood flow and oxygen delivery to specific tissue areas.

Purification and analysis of a protein cocktail capable of scavenging cell-free hemoglobin, heme, and iron.

BACKGROUND: Hemolysis releases toxic cell-free hemoglobin (Hb), heme, and iron, which overwhelm their natural scavenging mechanisms during acute or chronic hemolytic conditions. This study describes a novel strategy to purify a protein cocktail containing a comprehensive set of scavenger proteins for potential treatment of hemolysis byproducts. STUDY DESIGN AND METHODS: Tangential flow filtration was used to purify a protein cocktail from Human Cohn Fraction IV (FIV). A series of in vitro assays were performed to characterize composition and biocompatibility. The in vivo potential for hemolysis byproduct mitigation was assessed in a hamster exchange transfusion model using mechanically hemolyzed blood plasma mixed with the protein cocktail or a control colloid (dextran 70 kDa). RESULTS: A basis of 500 g of FIV yielded 62 ± 9 g of a protein mixture at 170 g/L, which bound to approximately 0.6 mM Hb, 1.2 mM heme, and 1.2 mM iron. This protein cocktail was shown to be biocompatible in vitro with red blood cells and platelets and exhibits nonlinear concentration dependence with respect to viscosity and colloidal osmotic pressure. In vivo assessment of the protein cocktail demonstrated higher iron transport to the liver and spleen and less to the kidney and heart with significantly reduced renal and cardiac inflammation markers and lower kidney and hepatic damage compared to a control colloid. DISCUSSION: Taken together, this study provides an effective method for large-scale production of a protein cocktail suitable for comprehensive reduction of hemolysis-induced toxicity.

Safety profile of high molecular weight polymerized hemoglobins.

BACKGROUND: Hemoglobin (Hb)-based oxygen (O2 ) carriers (HBOCs) are being developed as alternatives to red blood cells and blood when these products are unavailable. Clinical trials of previous HBOC generations revealed side effects, including hypertension and vasoconstriction, that were not observed in preclinical studies. Large molecular weight (MW) polymerized bovine Hb (PolybHb) represents a new class of HBOC with promising results. We evaluated the safety profile of PolybHb after an exchange transfusion (ET) in guinea pigs (GPs). This study compares changes in indices of cardiac, inflammatory, and organ function after ET with high (R-state) and low (T-state) O2 affinity PolybHb with high MW. STUDY DESIGN AND METHODS: Guinea pigs underwent a 20% ET with PolybHb. To assess the implication of PolybHb ET on the microcirculation, hamsters instrumented with a dorsal window chamber were subjected to a similar volume ET. RESULTS: T and R-state PolybHb did not induce significant alterations in cardiac function. T-state PolybHb induced mild vasoconstriction shortly after transfusion, while R-state did not have acute effects on microvascular tone. CONCLUSION: Large MW PolybHbs were found to be safe and efficacious in increasing O2 carrying capacity and the O2 affinity of the PolybHb did not affect O2 delivery or extraction by tissues in relevant preclinical models. In conclusion, these results suggest that both T-state and R-state PolybHb are safe and do not impair O2 delivery. The results are encouraging and support further evaluation of high MW PolybHbs and their future feasibility compared to allogenic blood in a trauma model.

Tumor vascular status controls oxygen delivery facilitated by infused polymerized hemoglobins with varying oxygen affinity.

Oxygen (O2) delivery facilitated by hemoglobin (Hb)-based O2 carriers (HBOCs) is a promising strategy to increase the effectiveness of chemotherapeutics for treatment of solid tumors. However, the heterogeneous vascular structures present within tumors complicates evaluating the oxygenation potential of HBOCs within the tumor microenvironment. To account for spatial variations in the vasculature and tumor tissue that occur during tumor growth, we used a computational model to develop artificial tumor constructs. With these simulated tumors, we performed a polymerized human hemoglobin (hHb) (PolyhHb) enhanced oxygenation simulation accounting for differences in the physiologic characteristics of human and mouse blood. The results from this model were used to determine the potential effectiveness of different treatment options including a top load (low volume) and exchange (large volume) infusion of a tense quaternary state (T-State) PolyhHb, relaxed quaternary state (R-State) PolyhHb, and a non O2 carrying control. Principal component analysis (PCA) revealed correlations between the different regimes of effectiveness within the different simulated dosage options. In general, we found that infusion of T-State PolyhHb is more likely to decrease tissue hypoxia and modulate the metabolic rate of O2 consumption. Though the developed models are not a definitive descriptor of O2 carrier interaction in tumor capillary networks, we accounted for factors such as non-uniform vascular density and permeability that limit the applicability of O2 carriers during infusion. Finally, we have used these validated computational models to establish potential benchmarks to guide tumor treatment during translation of PolyhHb mediated therapies into clinical applications.

RRx-001 Increases Erythrocyte Preferential Adhesion to the Tumor Vasculature.

Red blood cells (RBCs) serve a variety of functions beyond mere oxygen transport both in health and pathology. Notably, RRx-001, a minimally toxic pleiotropic anticancer agent with macrophage activating and vascular normalization properties currently in Phase III trials, induces modification to RBCs which could promote vascular adhesion similar to sickle cells. This study assessed whether RBCs exposed to RRx-001 adhere to the tumor microvasculature and whether this adhesion alters tumor viability. We next investigated the biomechanics of RBC adhesion in the context of local inflammatory cytokines after treatment with RRx-001 as a potential mechanism for preferential tumor aggregation. Human HEP-G2 and HT-29 tumor cells were subcutaneously implanted into nu/nu mice and were infused with RRx-001-treated and Technetium-99m (99mTc)-labeled blood. RBC adhesion was quantified in an in vitro human umbilical vein endothelial cell (HUVEC) assay under both normoxic and hypoxic conditions with administration of either lipopolysaccharide (LPS) or Tumor necrosis alpha (TNFα) to mimic the known inflammation in the tumor microenvironment. One hour following administration of 99mTc labeled RBCs treated with 10 mg/kg RRx-001, we observed an approximate 2.0-fold and 1.5-fold increase in 99mTc-labeled RBCs compared to vehicle control in HEPG2 and HT-29 tumor models, respectively. Furthermore, we observed an approximate 40% and 36% decrease in HEP-G2 and HT-29 tumor weight, respectively, following treatment with RRx-001. To quantify RBC adhesive potential, we determined τ50, or the shear stress required for 50% disassociation of RBCs from HUVECs. After administration of TNF-α under normoxia, τ50 was determined to be 4.5 dynes/cm2 (95% CI: 4.3-4.7 dynes/cm2) for RBCs treated with 10 µM RRx-001, which was significantly different (p < 0.05) from τ50 in the absence of treatment. Under hypoxic conditions, the difference of τ50 with (4.8 dynes/cm2; 95% CI: 4.6-5.1 dynes/cm2) and without (2.6 dynes/cm2; 95% CI: 2.4-2.8 dynes/cm2) 10 µM RRx-001 treatment was exacerbated (p = 0.05). In conclusion, we demonstrated that RBCs treated with RRx-001 preferentially aggregate in HEP-G2 and HT-29 tumors, likely due to interactions between RRx-001 and cysteine residues within RBCs. Furthermore, RRx-001 treated RBCs demonstrated increased adhesive potential to endothelial cells upon introduction of TNF-α and hypoxia suggesting that RRx-001 may induce preferential adhesion in the tumor but not in other tissues with endothelial dysfunction due to conditions prevalent in older cancer patients such as heart disease or diabetic vasculopathy.

Hemoglobin ß93 Cysteine Is Not Required for Export of Nitric Oxide Bioactivity From the Red Blood Cell.

BACKGROUND: Nitrosation of a conserved cysteine residue at position 93 in the hemoglobin ß chain (ß93C) to form S-nitroso (SNO) hemoglobin (Hb) is claimed to be essential for export of nitric oxide (NO) bioactivity by the red blood cell (RBC) to mediate hypoxic vasodilation and cardioprotection. METHODS: To test this hypothesis, we used RBCs from mice in which the ß93 cysteine had been replaced with alanine (ß93A) in a number of ex vivo and in vivo models suitable for studying export of NO bioactivity. RESULTS: In an ex vivo model of cardiac ischemia/reperfusion injury, perfusion of a mouse heart with control RBCs (ß93C) pretreated with an arginase inhibitor to facilitate export of RBC NO bioactivity improved cardiac recovery after ischemia/reperfusion injury, and the response was similar with ß93A RBCs. Next, when human platelets were coincubated with RBCs and then deoxygenated in the presence of nitrite, export of NO bioactivity was detected as inhibition of ADP-induced platelet activation. This effect was the same in ß93C and ß93A RBCs. Moreover, vascular reactivity was tested in rodent aortas in the presence of RBCs pretreated with S-nitrosocysteine or with hemolysates or purified Hb treated with authentic NO to form nitrosyl(FeII)-Hb, the proposed precursor of SNO-Hb. SNO-RBCs or NO-treated Hb induced vasorelaxation, with no differences between ß93C and ß93A RBCs. Finally, hypoxic microvascular vasodilation was studied in vivo with a murine dorsal skin-fold window model. Exposure to acute systemic hypoxia caused vasodilatation, and the response was similar in ß93C and ß93A mice. CONCLUSIONS: RBCs clearly have the fascinating ability to export NO bioactivity, but this occurs independently of SNO formation at the ß93 cysteine of Hb.

Apohemoglobin-haptoglobin complex attenuates the pathobiology of circulating acellular hemoglobin and heme.

Haptoglobin (Hp) is the plasma protein that binds and clears cell-free hemoglobin (Hb), whereas apohemoglobin (apoHb, i.e., Hb devoid of heme) can bind heme. Therefore, the apoHb-Hp protein complex should facilitate holoHb-apoHb αß-dimer exchange and apoHb-heme intercalation. Thus, we hypothesized that apoHb-Hp could facilitate both Hb and heme clearance, which, if not alleviated, could have severe microcirculatory consequences. In this study, we characterized apoHb-Hp and Hb/heme ligand interactions and assessed their in vivo consequences. Hb exchange and heme binding with the apoHb-Hp complex was studied with transfer assays using size-exclusion high-performance liquid chromatography coupled with UV-visible spectrophotometry. Exchange/transfer experiments were conducted in guinea pigs dosed with Hb or heme-albumin followed by a challenge with equimolar amounts of apoHb-Hp. Finally, systemic and microcirculatory parameters were studied in hamsters instrumented with a dorsal window chamber via intravital microscopy. In vitro and in vivo Hb exchange and heme transfer experiments demonstrated proof-of-concept Hb/heme ligand transfer to apoHb-Hp. Dosing with the apoHb-Hp complex reversed Hb- and heme-induced systemic hypertension and microvascular vasoconstriction, reduced microvascular blood flow, and diminished functional capillary density. Therefore, this study highlights the apoHb-Hp complex as a novel therapeutic strategy to attenuate the adverse systemic and microvascular responses to intravascular Hb and heme exposure.NEW & NOTEWORTHY This study highlights the apoHb-Hp complex as a novel therapeutic strategy to attenuate the adverse systemic and microvascular responses to intravascular Hb and heme exposure. In vitro and in vivo Hb exchange and heme transfer experiments demonstrated proof-of-concept Hb/heme ligand transfer to apoHb-Hp. The apoHb-Hp complex reverses Hb- and heme-induced systemic hypertension and microvascular vasoconstriction, preserves microvascular blood flow, and functional capillary density. In summary, the unique properties of the apoHb-Hp complex prevent adverse systemic and microvascular responses to Hb and heme-albumin exposure and introduce a novel therapeutic approach to facilitate simultaneous removal of extracellular Hb and heme.

Enzyme promiscuity drives branched-chain fatty acid synthesis in adipose tissues.

Fatty acid synthase (FASN) predominantly generates straight-chain fatty acids using acetyl-CoA as the initiating substrate. However, monomethyl branched-chain fatty acids (mmBCFAs) are also present in mammals but are thought to be primarily diet derived. Here we demonstrate that mmBCFAs are de novo synthesized via mitochondrial BCAA catabolism, exported to the cytosol by adipose-specific expression of carnitine acetyltransferase (CrAT), and elongated by FASN. Brown fat exhibits the highest BCAA catabolic and mmBCFA synthesis fluxes, whereas these lipids are largely absent from liver and brain. mmBCFA synthesis is also sustained in the absence of microbiota. We identify hypoxia as a potent suppressor of BCAA catabolism that decreases mmBCFA synthesis in obese adipose tissue, such that mmBCFAs are significantly decreased in obese animals. These results identify adipose tissue mmBCFA synthesis as a novel link between BCAA metabolism and lipogenesis, highlighting roles for CrAT and FASN promiscuity influencing acyl-chain diversity in the lipidome.

Red Blood Cell Metabolic Responses to Torpor and Arousal in the Hibernator Arctic Ground Squirrel.

Arctic ground squirrels provide a unique model to investigate metabolic responses to hibernation in mammals. During winter months these rodents are exposed to severe hypothermia, prolonged fasting, and hypoxemia. In the light of their role in oxygen transport/off-loading and owing to the absence of nuclei and organelles (and thus de novo protein synthesis capacity), mature red blood cells have evolved metabolic programs to counteract physiological or pathological hypoxemia. However, red blood cell metabolism in hibernation has not yet been investigated. Here we employed targeted and untargeted metabolomics approaches to investigate erythrocyte metabolism during entrance to torpor to arousal, with a high resolution of the intermediate time points. We report that torpor and arousal promote metabolism through glycolysis and pentose phosphate pathway, respectively, consistent with previous models of oxygen-dependent metabolic modulation in mature erythrocytes. Erythrocytes from hibernating squirrels showed up to 100-fold lower levels of biomarkers of reperfusion injury, such as the pro-inflammatory dicarboxylate succinate. Altered tryptophan metabolism during torpor was here correlated to the accumulation of potentially neurotoxic catabolites kynurenine, quinolinate, and picolinate. Arousal was accompanied by alterations of sulfur metabolism, including sudden spikes in a metabolite putatively identified as thiorphan (level 1 confidence)-a potent inhibitor of several metalloproteases that play a crucial role in nociception and inflammatory complication to reperfusion secondary to ischemia or hemorrhage. Preliminary studies in rats showed that intravenous injection of thiorphan prior to resuscitation mitigates metabolic and cytokine markers of reperfusion injury, etiological contributors to inflammatory complications after shock.

Cardioprotective Effect of Phase 3 Clinical Anticancer Agent, RRx-001, in Doxorubicin-Induced Acute Cardiotoxicity in Mice.

Anthracycline chemotherapy (e.g., doxorubicin or DOX) is associated with a cumulative dose-dependent cardiac dysfunction that may lead to congestive heart failure, which limits both its use and usefulness in the clinic. The cardiotoxicity may manifest acutely and/or months or years after treatment with doxorubicin has ended. Experimental and human data have demonstrated that angiotensin-converting enzyme/angiotensin-receptor antagonists mediate a cardioprotective effect against anthracycline toxicity. In this study, with the angiotensin receptor blocker, candesartan, as a positive control, we evaluated whether pretreatment with the hypoxic nitric oxide generating anticancer agent, RRx-001, could reduce acute DOX-induced cardiotoxicity. A total of 24 BALB/c mice were randomized for prophylactic treatment with vehicle, RRx-001, candesartan, or no-intervention control. Within each of the three intervention arms, mice received treatment with DOX. Murine pressure-volume analysis was performed with microconductance catheters to characterize the degree of cardiovascular dysfunction within each group. The following hemodynamic parameters were monitored: left ventricular systolic pressure (LVSP), heart rate, and maximal rate of increase of left ventricular pressure (±d P/d tmax). Five days after doxorubicin injection, untreated (with RRx-001) mice displayed significantly impaired systolic (LVSP, -27%; d P/d tmax, -25%; left ventricular developed pressure (LVDP), +33%; P < 0.05) and global (stroke volume (SV), -52%; ejection fraction (EF), -20%; stroke work (SW), -62.5%; heart rate (HR), -18%; cardiac output (CO), -57%; mean blood arterial pressure (MAP), -30%; systemic vascular resistance (SVR), +20%; P < 0.05) LV functions when compared with the untreated (with RRx-001) group. In contrast, RRx-001-treated mice showed improved variables of systolic (LVSP, +27%; d P/d tmax, +25%; LVDP, -33%; P < 0.05) and global (SV, +52%; EF, +20%; SW, +62.5%; HR, +18%; CO, +57%; MAP, +30%; SVR, -20%; P < 0.05) LV functions compared with untreated doxorubicin mice. Similar to the positive control, candesartan, the cardiotoxic effects of DOX in mice were partially attenuated by the prophylactic administration of RRx-001. These results suggest that RRx-001 as a multifunctional anticancer agent, which sensitizes cancer cells to the cytotoxic effects of chemotherapy and radiation, may also have beneficial cardioprotective effects.

Do Skeletal Dynamics Mediate Sugar Uptake and Transport in Human Erythrocytes?

We explore, herein, the hypothesis that transport of molecules or ions into erythrocytes may be affected and directly stimulated by the dynamics of the spectrin/actin skeleton. Skeleton/actin motions are driven by thermal fluctuations that may be influenced by ATP hydrolysis as well as by structural alterations of the junctional complexes that connect the skeleton to the cell's lipid membrane. Specifically, we focus on the uptake of glucose into erythrocytes via glucose transporter 1 and on the kinetics of glucose disassociation at the endofacial side of glucose transporter 1. We argue that glucose disassociation is affected by both hydrodynamic forces induced by the actin/spectrin skeleton and by probable contact of the swinging 37-nm-long F-actin protofilament with glucose, an effect we dub the "stickball effect." Our hypothesis and results are interpreted within the framework of the kinetic measurements and compartmental kinetic models of Carruthers and co-workers; these experimental results and models describe glucose disassociation as the "slow step" (i.e., rate-limiting step) in the uptake process. Our hypothesis is further supported by direct simulations of skeleton-enhanced transport using our molecular-based models for the actin/spectrin skeleton as well as by experimental measurements of glucose uptake into cells subject to shear deformations, which demonstrate the hydrodynamic effects of advection. Our simulations have, in fact, previously demonstrated enhanced skeletal dynamics in cells in shear deformations, as they occur naturally within the skeleton, which is an effect also supported by experimental observations.

Platelet inhibitory effects of the Phase 3 anticancer and normal tissue cytoprotective agent, RRx-001.

The platelet inhibitory effects of the Phase 3 anticancer agent and nitric oxide (NO) donor, RRx-001, (1-bromoacetyl-3,3-dinitroazetidine) were examined ex vivo and compared with the diazeniumdiolate NO donor, diethylenetriamine NONOate (DETA-NONOate), which spontaneously releases nitric oxide in aqueous solution. In the absence of red blood cells and in a dose-dependent manner, DETA-NONOate strongly inhibited platelet aggregation induced by several stimuli (ADP, epinephrine and collagen) whereas RRx-001 only slightly inhibited platelet aggregation under the same conditions in a dose-dependent manner; these antiaggregant effects were blocked when both DETA-NONOate and RRx-001 were co-incubated with carboxy-PTIO (CPTIO 0.01-100 micromol), a widely accepted NO scavenger. However, in the presence of red blood cells from healthy human donors, RRx-001, which binds covalently to haemoglobin (Hb) and catalyses the production of NO from endogenous nitrite, more strongly inhibited the aggregation of platelets than DETA-NONOate in a dose-dependent manner likely because haemoglobin avidly scavenges nitric oxide and reduces its half-life; the RRx-001-mediated platelet inhibitory effect was increased in the presence of nitrite. The results of this study suggest that RRx-001-bound Hb (within RBCs) plays an important role in the bioconversion of NO2- to NO. , which makes RRx-001 a more physiologically relevant inhibitor of platelet aggregation than other nitric oxide donors, whose effects are attenuated in the presence of red blood cells. Therefore, RRx-001-mediated platelet inhibition is a potentially useful therapeutic property, especially in hypercoagulable cancer patients that are at an increased risk of thrombotic complications.

Endothelin receptor B, a candidate gene from human studies at high altitude, improves cardiac tolerance to hypoxia in genetically engineered heterozygote mice.

To better understand human adaptation to stress, and in particular to hypoxia, we took advantage of one of nature's experiments at high altitude (HA) and studied Ethiopians, a population that is well-adapted to HA hypoxic stress. Using whole-genome sequencing, we discovered that EDNRB (Endothelin receptor type B) is a candidate gene involved in HA adaptation. To test whether EDNRB plays a critical role in hypoxia tolerance and adaptation, we generated EdnrB knockout mice and found that when EdnrB (-/+) heterozygote mice are treated with lower levels of oxygen (O2), they tolerate various levels of hypoxia (even extreme hypoxia, e.g., 5% O2) very well. For example, they maintain ejection fraction, cardiac contractility, and cardiac output in severe hypoxia. Furthermore, O2 delivery to vital organs was significantly higher and blood lactate was lower in EdnrB (-/+) compared with wild type in hypoxia. Tissue hypoxia in brain, heart, and kidney was lower in EdnrB (-/+) mice as well. These data demonstrate that a lower level of EDNRB significantly improves cardiac performance and tissue perfusion under various levels of hypoxia. Transcriptomic profiling of left ventricles revealed three specific genes [natriuretic peptide type A (Nppa), sarcolipin (Sln), and myosin light polypeptide 4 (Myl4)] that were oppositely expressed (q < 0.05) between EdnrB (-/+) and wild type. Functions related to these gene networks were consistent with a better cardiac contractility and performance. We conclude that EDNRB plays a key role in hypoxia tolerance and that a lower level of EDNRB contributes, at least in part, to HA adaptation in humans.