Novel high molecular weight polymerized hemoglobin in a non-obese model of cardiovascular and metabolic dysfunction.

The widespread adoption of high-calorie, high-fat, high-sucrose diets (HFHSD) has become a global health concern, particularly due to their association with cardiovascular diseases and metabolic disorders. These comorbidities increase susceptibility to severe outcomes from viral infections and trauma, with trauma-related incidents significantly contributing to global mortality rates. This context underscores the critical need for a reliable blood supply. Recent research has focused on high molecular weight (MW) polymerized human hemoglobin (PolyhHb) as a promising alternative to red blood cells (RBCs), showing encouraging outcomes in previous studies. Given the overlap of metabolic disorders and trauma-related health issues, it is crucial to assess the potential toxicity of PolyhHb transfusions, particularly in models that represent these vulnerable populations. This study evaluated the effects of PolyhHb exchange transfusion in guinea pigs that had developed metabolic disorders due to a 12-week HFHSD regimen. The guinea pigs, underwent a 20 % blood volume exchange transfusion with either PolyhHb or the lower molecular weight polymerized bovine hemoglobin, Oxyglobin. Results revealed that both PolyhHb and Oxyglobin transfusions led to liver damage, with a more pronounced effect observed in HFHSD-fed animals. Additionally, markers of cardiac dysfunction indicated signs of cardiac injury in both the HFHSD and normal diet groups following the Oxyglobin transfusion. This study highlights how pre-existing metabolic disorders can exacerbate the potential side effects of hemoglobin-based oxygen carriers (HBOCs). Importantly, the newer generation of high MW PolyhHb showed lower cardiac toxicity compared to the earlier generation low MW PolyhHb, known as Oxyglobin, even in models with pre-existing endothelial and metabolic challenges.

Hypoxically stored RBC resuscitation in a rat model of traumatic brain injury and severe hemorrhagic shock.

This study aims to investigate the effects of hypoxically stored Red Blood Cells (RBCs) in a rat model of traumatic brain injury followed by severe hemorrhagic shock (HS) and resuscitation. RBCs were made hypoxic using an O2 depletion system (Hemanext Inc. Lexington, MA) and stored for 3 weeks. Experimental animals underwent craniotomy and blunt brain injury followed by severe HS. Rats were resuscitated with either fresh RBCs (FRBCs), 3-week-old hypoxically stored RBCs (HRBCs), or 3-week-old conventionally stored RBCs (CRBCs). Resuscitation was provided via RBCs transfusion equivalent to 70 % of the shed blood and animals were followed for 2 h. The control group was comprised of healthy animals that were not instrumented or injured. Post-resuscitation hemodynamics and lactate levels were improved with FRBCs and HRBCs, and markers of organ injury in the liver (Aspartate aminotransferase [AST]), lung (chemokine ligand 1 [CXCL-1] and Leukocytes count), and heart (cardiac troponin, Interleukin- 6 [IL-6] and Tumor Necrosis Factor Alpha[TNF-α]) were lower with FRBCs and HRBCs resuscitation compared to CRBCs. Following reperfusion, biomarkers for oxidative stress, lipid peroxidation, and RNA/DNA injury were assessed. Superoxide dismutase [SOD] levels in the HRBCs group were similar to the FRBCs group and levels in both groups were significantly higher than CRBCs. Catalase levels were not different than control values in the FRBCs and HRBCs groups but significantly lower with CRBCs. Thiobarbituric acid reactive substances [Tbars] levels were higher for both CRBCs and HRBCs. Hypoxically stored RBCs show few differences from fresh RBCs in resuscitation from TBI + HS and decreased organ injury and oxidative stress compared to conventionally stored RBCs.

Polymerized human hemoglobin with low and high oxygen affinity in trauma models.

The present study aimed to compare the ability of tense (T) and relaxed (R) quaternary state polymerized human hemoglobin (PolyhHb) to restore hemodynamics after severe trauma in a rat model, and to assess their relative toxicity in a guinea pigs (GPs). To assess the efficacy of these PolyhHbs in restoring hemodynamics, Wistar rats were subjected to traumatic brain injury (TBI) followed by hemorrhagic shock (HS). Animals were separated into 3 groups based on the resuscitation solution: Whole blood, T-state or R-state PolyhHb, and followed for 2 hours after resuscitation. For toxicity evaluation, GPs were subjected to HS and the hypovolemic state was maintained for 50 minutes. Then, the GPs were divided randomly into 2 groups, and reperfused with T- or R-state PolyhHb. Rats resuscitated with blood and T-state PolyhHb had a higher recovery of MAP at 30 min after resuscitation when compared to R-state PolyhHb, demonstrating the greater ability of T-state PolyhHb to restore hemodynamics compared to R-state PolyhHb. Resuscitation with R-state PolyhHb in GPs increased markers of liver damage and inflammation, kidney injury and systemic inflammation compared to the T-state PolyhHb group. Finally, increased levels of cardiac damage markers, such as troponin were observed, indicating greater cardiac injury in GPs resuscitated with R-state PolyhHb. Therefore, our results showed that T-state PolyhHb exhibited superior efficacy in a model of TBI followed by HS in rats, and presented reduced vital organ toxicity in GPs, when compared to R-state PolyhHb.

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.

Safety and efficacy of human polymerized hemoglobin on guinea pig resuscitation from hemorrhagic shock.

For the past thirty years, hemoglobin-based oxygen carriers (HBOCs) have been under development as a red blood cell substitute. Side-effects such as vasoconstriction, oxidative injury, and cardiac toxicity have prevented clinical approval of HBOCs. Recently, high molecular weight (MW) polymerized human hemoglobin (PolyhHb) has shown positive results in rats. Studies have demonstrated that high MW PolyhHb increased O2 delivery, with minimal effects on blood pressure, without vasoconstriction, and devoid of toxicity. In this study, we used guinea pigs to evaluate the efficacy and safety of high MW PolyhHb, since like humans guinea pigs cannot produce endogenous ascorbic acid, which limits the capacity of both species to deal with oxidative stress. Hence, this study evaluated the efficacy and safety of resuscitation from severe hemorrhagic shock with high MW PolyhHb, fresh blood, and blood stored for 2 weeks. Animals were randomly assigned to each experimental group, and hemorrhage was induced by the withdrawal of 40% of the blood volume (BV, estimated as 7.5% of body weight) from the carotid artery catheter. Hypovolemic shock was maintained for 50 min. Resuscitation was implemented by infusing 25% of the animal's BV with the different treatments. Hemodynamics, blood gases, total hemoglobin, and lactate were not different before hemorrhage and during shock between groups. The hematocrit was lower for the PolyhHb group compared to the fresh and stored blood groups after resuscitation. Resuscitation with stored blood had lower blood pressure compared to fresh blood at 2 h. There was no difference in mean arterial pressure between groups at 24 h. Resuscitation with PolyhHb was not different from fresh blood for most parameters. Resuscitation with PolyhHb did not show any remarkable change in liver injury, inflammation, or cardiac damage. Resuscitation with stored blood showed changes in liver function and inflammation, but no kidney injury or systemic inflammation. Resuscitation with stored blood after 24 h displayed sympathetic hyper-activation and signs of cardiac injury. These results suggest that PolyhHb is an effective resuscitation alternative to blood. The decreased toxicities in terms of cardiac injury markers, vital organ function, and inflammation following PolyhHb resuscitation in guinea pigs indicate a favorable safety profile. These results are promising and support future studies with this new generation of PolyhHb as alternative to blood when blood is unavailable.

Attenuating ischemia-reperfusion injury with polymerized albumin.

Ischemia-reperfusion injury increased vascular permeability, resulting in fluid extravasation from the intravascular compartment into the tissue space. Fluid and small protein extravasation lead to increased interstitial fluid pressure and capillary collapse, impairing capillary exchange. Polymerized human serum albumin (PolyHSA) has an increased molecular weight (MW) compared with unpolymerized human serum albumin (HSA) and can improve intravascular fluid retention and recovery from ischemia-reperfusion injury. To test the hypothesis that polymerization of HSA can improve recovery from ischemia-reperfusion injury, we studied how exchange transfusion of 20% of the blood volume with HSA or PolyHSA immediately before reperfusion can affect local ischemic tissue microhemodynamics, vascular integrity, and tissue viability in a hamster dorsal window chamber model. Microvascular flow and functional capillary density were maintained in animals exchanged with PolyHSA compared with HSA. Likewise, exchange transfusion with PolyHSA preserved vascular permeability measured with extravasation of fluorescently labeled dextran. The intravascular retention time of the exchanged PolyHSA was significantly longer compared with the intravascular retention time of HSA. Lastly, the viability of tissue subjected to ischemia-reperfusion injury increased in animals exchanged with PolyHSA compared with HSA. Therefore maintenance of microvascular perfusion, improvement in vascular integrity, and reduction in tissue damage resulting from reperfusion with PolyHSA suggest that PolyHSA is a promising fluid therapy to improve outcomes of ischemia-reperfusion injury.NEW & NOTEWORTHY Polymerized human serum albumin reduced reperfusion injury and preservers microvascular hemodynamics. Polymerized human serum albumin reduces fluid extravasation and prevents fluid extravasation. Consequently, the tissue viability of ischemic tissue is preserved by polymerized human serum.

Aerobic Physical Exercise Improves Exercise Tolerance and Fasting Glycemia Independent of Body Weight Change in Obese Females.

Obesity is associated with increased risk of several chronic diseases and the loss of disease-free years, which has increased the focus of much research for the discovery of therapy to combat it. Under healthy conditions, women tend to store more fat in subcutaneous deposits. However, this sexual dimorphism tends to be lost in the presence of comorbidities, such as type 2 diabetes mellitus (T2DM). Aerobic physical exercise (APE) has been applied in the management of obesity, however, is still necessary to better understand the effects of APE in obese female. Thus, we investigated the effect of APE on body weight, adiposity, exercise tolerance and glucose metabolism in female ob/ob mice. Eight-weeks-old female wild-type C57BL/6J and leptin-deficient ob/ob mice (Lepob) were distributed into three groups: wild-type sedentary group (Wt; n = 6), leptin-deficient sedentary group (LepobS; n = 5) and leptin-deficient trained group (LepobT; n = 8). The LepobT mice were subjected to 8 weeks of aerobic physical exercise (APE) at 60% of the maximum velocity achieved in the running capacity test. The APE had no effect in attenuating body weight gain, and did not reduce subcutaneous and retroperitoneal white adipose tissue (SC-WAT and RP-WAT, respectively) and interscapular brown adipose tissue (iBAT) weights. The APE neither improved glucose intolerance nor insulin resistance in the LepobT group. Also, the APE did not reduce the diameter or the area of RP-WAT adipocytes, but the APE reduced the diameter and the area of SC-WAT adipocytes, which was associated with lower fasting glycemia and islet/pancreas area ratio in the LepobT group. In addition, the APE increased exercise tolerance and this response was also associated with lower fasting glycemia in the LepobT group. In conclusion, starting APE at a later age with a more severe degree of obesity did not attenuate the excessive body weight gain, however the APE promoted benefits that can improve the female health, and for this reason it should be recommended as a non-pharmacological therapy for obesity.

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.

Oxidative phenotype induced by aerobic physical training prevents the obesity-linked insulin resistance without changes in gastrocnemius muscle ACE2-Angiotensin(1-7)-Mas axis.

BACKGROUND: We investigate the effect of aerobic physical training (APT) on muscle morphofunctional markers and Angiotensin Converting Enzyme 2/Angiotensin 1-7/Mas receptor (ACE2/Ang 1-7/Mas) axis in an obesity-linked insulin resistance (IR) animal model induced by cafeteria diet (CAF). METHODS: Male C57BL/6J mice were assigned into groups CHOW-SED (chow diet, sedentary; n = 10), CHOW-TR (chow diet, trained; n = 10), CAF-SED (n = 10) and CAF-TR (n = 10). APT consisted in running sessions of 60 min at 60% of maximal speed, 5 days per week for 8 weeks. RESULTS: Trained groups had lower body weight and adiposity compared with sedentary groups. CAF-TR improved the glucose and insulin tolerance tests compared with CAF-SED group (AUC = 28.896 ± 1589 vs. 35.200 ± 1076 mg dL-1 120 min-1; kITT = 4.1 ± 0.27 vs. 2.5 ± 0.28% min-1, respectively). CHOW-TR and CAF-TR groups increased exercise tolerance, running intensity at which VO2 max was reached, the expression of p-AMPK, p-ACC and PGC1-α proteins compared with CHOW-SED and CAF-SED. Mithocondrial protein expression of Mfn1, Mfn2 and Drp1 did not change. Lipid deposition reduced in CAF-TR compared with CAF-SED group (3.71 vs. 5.53%/area), but fiber typing, glycogen content, ACE2 activity, Ang 1-7 concentration and Mas receptor expression did not change. CONCLUSIONS: The APT prevents obesity-linked IR by modifying the skeletal muscle phenotype to one more oxidative independent of changes in the muscle ACE2/Ang 1-7/Mas axis.

High fat high sucrose diet-induced dyslipidemia in guinea pigs.

Easy access to high-calorie and fat-dense fast food has resulted in unhealthy dietary and lifestyle changes worldwide, which affects both developed and developing economies. This predisposes populations to a considerable number of metabolic and inflammatory conditions, such as diabetes, nonalcoholic fatty liver disease (NAFLD), and cardiovascular disease (CVD). Guinea pigs have been proposed as a model to study high-fat diet-induced metabolic disease due to their similar antioxidant metabolism and lipid profile to humans, and their susceptibility to atherosclerosis and endothelial disease. This study aims to evaluate cardiovascular and metabolic disorders induced by high-fat high-sucrose diet (HFHSD) in guinea pigs. Two to three-week-old male guinea pigs were fed a normal diet (ND) or HFHSD for 12 wk. Guinea pigs fed a HFHSD developed glucose intolerance, dyslipidemia, and liver, cardiac, and kidney damage. However, hypertension, dysautonomia, endothelial disease, and obesity were absent in these HFHSD guinea pigs. Taken together, these results show that guinea pigs fed a HFHSD are a nonobese model of metabolic disorders, resulting in important cardiac damage. Moreover, our findings suggest that NAFLD may be an important risk factor for diet-induced CVD.NEW & NOTEWORTHY In this study, we show a new animal model for diet-induced disease metabolic disorders without obesity in guinea pigs. Moreover, results suggest a strong relation between liver disease and increased cardiovascular risks.

Resuscitation from hemorrhagic shock after traumatic brain injury with polymerized hemoglobin.

Traumatic brain injury (TBI) is often accompanied by hemorrhage, and treatment of hemorrhagic shock (HS) after TBI is particularly challenging because the two therapeutic treatment strategies for TBI and HS often conflict. Ischemia/reperfusion injury from HS resuscitation can be exaggerated by TBI-induced loss of autoregulation. In HS resuscitation, the goal is to restore lost blood volume, while in the treatment of TBI the priority is focused on maintenance of adequate cerebral perfusion pressure and avoidance of secondary bleeding. In this study, we investigate the responses to resuscitation from severe HS after TBI in rats, using fresh blood, polymerized human hemoglobin (PolyhHb), and lactated Ringer's (LR). Rats were subjected to TBI by pneumatic controlled cortical impact. Shortly after TBI, HS was induced by blood withdrawal to reduce mean arterial pressure (MAP) to 35-40 mmHg for 90 min before resuscitation. Resuscitation fluids were delivered to restore MAP to ~ 65 mmHg and animals were monitored for 120 min. Increased systolic blood pressure variability (SBPV) confirmed TBI-induced loss of autoregulation. MAP after resuscitation was significantly higher in the blood and PolyhHb groups compared to the LR group. Furthermore, blood and PolyhHb restored diastolic pressure, while this remained depressed for the LR group, indicating a loss of vascular tone. Lactate increased in all groups during HS, and only returned to baseline level in the blood reperfused group. The PolyhHb group possessed lower SBPV compared to LR and blood groups. Finally, sympathetic nervous system (SNS) modulation was higher for the LR group and lower for the PolyhHb group compared to the blood group after reperfusion. In conclusion, our results suggest that PolyhHb could be an alternative to blood for resuscitation from HS after TBI when blood is not available, assuming additional testing demonstrate similar favorable results. PolyhHb restored hemodynamics and oxygen delivery, without the logistical constraints of refrigerated blood.

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.

Control of systemic inflammation through early nitric oxide supplementation with nitric oxide releasing nanoparticles.

Amelioration of immune overactivity during sepsis is key to restoring hemodynamics, microvascular blood flow, and tissue oxygenation, and in preventing multi-organ dysfunction syndrome. The systemic inflammatory response syndrome that results from sepsis ultimately leads to degradation of the endothelial glycocalyx and subsequently increased vascular leakage. Current fluid resuscitation techniques only transiently improve outcomes in sepsis, and can cause edema. Nitric oxide (NO) treatment for sepsis has shown promise in the past, but implementation is difficult due to the challenges associated with delivery and the transient nature of NO. To address this, we tested the anti-inflammatory efficacy of sustained delivery of exogenous NO using i.v. infused NO releasing nanoparticles (NO-np). The impact of NO-np on microhemodynamics and immune response in a lipopolysaccharide (LPS) induced endotoxemia mouse model was evaluated. NO-np treatment significantly attenuated the pro-inflammatory response by promoting M2 macrophage repolarization, which reduced the presence of pro-inflammatory cytokines in the serum and slowed vascular extravasation. Combined, this resulted in significantly improved microvascular blood flow and 72-h survival of animals treated with NO-np. The results from this study suggest that sustained supplementation of endogenous NO ameliorates and may prevent the morbidities of acute systemic inflammatory conditions. Given that endothelial dysfunction is a common denominator in many acute inflammatory conditions, it is likely that NO enhancement strategies may be useful for the treatment of sepsis and other acute inflammatory insults that trigger severe systemic pro-inflammatory responses and often result in a cytokine storm, as seen in COVID-19.

Balance between oxygen transport and blood rheology during resuscitation from hemorrhagic shock with polymerized bovine hemoglobin.

Alternatives to blood for use in transfusion medicine have been investigated for decades. An ideal alternative should improve oxygen (O2)-carrying capacity and O2 delivery and support microvascular blood flow. Previous studies have shown that large-molecular diameter hemoglobin (Hb)-based oxygen carriers (HBOCs) based on polymerized bovine Hb (PolybHb) reduce the toxicity and vasoconstriction of first-generation HBOCs by increasing blood and plasma viscosity and preserving microvascular perfusion. The objective of this study was to examine the impact of PolybHb concentration and therefore O2-carrying capacity and solution viscosity on resuscitation from hemorrhagic shock in rats. PolybHb was diafiltered on a 500-kDa tangential flow filtration (TFF) module to remove low-molecular weight (MW) PolybHb molecules from the final product. Rats were hemorrhaged and maintained in hypovolemic shock for 30 min before transfusion of PolybHb at 10 g/dL (PHB10), 5 g/dL (PHB5), or 2.5 g/dL (PHB2.5) concentration, to restore blood pressure to 90% of the animal's baseline blood pressure. Resuscitation restored blood pressure and cardiac function in a PolybHb concentration-dependent manner. Parameters indicative of the heart's metabolic activity indicated that the two higher PolybHb concentrations better restored coronary O2 delivery compared with the low concentration evaluated. Markers of organ damage and inflammation were highest for PHB10, whereas PHB5 and PHB2.5 showed similar expression of these markers. These studies indicate that a concentration of ~5 g/dL of PolybHb may be near the optimal concentration to restore cardiac function, preserve organ function, and mitigate the toxicity of PolybHb during resuscitation from hemorrhagic shock.NEW & NOTEWORTHY Large-molecular diameter polymerized bovine hemoglobin avoided vasoconstriction and impairment of cardiac function during resuscitation from hemorrhagic shock that was seen with previous hemoglobin-based O2 carriers by increasing blood viscosity in a concentration-dependent manner. Supplementation of O2-carrying capacity played a smaller role in maintaining cardiac function than increased blood and plasma viscosity.

Resuscitation From Hemorrhagic Shock With Fresh and Stored Blood and Polymerized Hemoglobin.

BACKGROUND: Hemoglobin (Hb)-based oxygen carriers (HBOCs) have been proposed as alternatives to blood for decades. Previous studies demonstrated that large molecular diameter HBOCs based on polymerized bovine Hb (PolybHb) attenuate Hb side-effects and toxicity. The objective of this study was to test the safety and efficacy of tense state PolybHb after long-term storage. METHODS AND RESULTS: PolybHb was subjected to diafiltration to remove low molecular weight (< 500 kDa) species and stored for 2 years. PolybHb was studied in parallel with blood, collected from rats and stored leukodepleted under blood bank conditions for 3 weeks. Rats were hemorrhaged and resuscitated to 90% of the blood pressure before the hemorrhage with fresh blood, stored blood, fresh PolybHb, or 2-year-stored PolybHb. Hemorrhagic shock impaired oxygen delivery and cardiac function. Resuscitation restored blood pressure and cardiac function, but stored blood required a significantly larger transfusion volume to recover from shock compared with fresh blood and PolybHb (fresh and stored). Stored blood transfusion elevated markers of organ damage compared with all other groups. CONCLUSIONS: These studies indicate that large molecular diameter PolybHb is as efficacious as fresh blood in restoring cardiac function and confirm the lack of degradation of PolybHb's safety or efficacy during long-term storage.

Polymerized Hemoglobin With Increased Molecular Size Reduces Toxicity in Healthy Guinea Pigs.

Hemoglobin (Hb)-based oxygen (O2) carriers (HBOCs) have been developed as an alternative to red blood cells (RBCs) for use in transfusion medicine. HBOCs have many benefits over RBCs; however, previous generations of HBOCs failed in clinical trials due to unanticipated cardiotoxicity. These problems likely originated from vasoconstriction, hypertension, oxidative stress, and the presence of low-molecular-weight (MW) Hb species in the HBOC formulation. Therefore, the objective of this study is to compare the toxicity of small-MW Polymerized bovine Hb (SPolyHb) to large-MW Polymerized bovine Hb (LPolyHb) in guinea pigs, since they lack the ability to synthesize vitamin C and are more sensitive to oxidative stress than other preclinical animal models. The two PolyHbs used in this study have similar molecular diameters (72 and 69 nm, respectively), but the SPolyHb included approximately 15% Hb polymers with MW below 256 kDa, which were significantly removed from LPolyHb. Solutions were injected as a hypervolemic (topload) infusion of 10% of the blood volume into animals. SPolyHb caused a 50% elevation in mean arterial pressure (MAP) from the baseline, while LPolyHb caused only a small increase in MAP. Both PolyHbs also increased markers of organ damage and tissue and systemic inflammation compared to controls. SPolyHb caused significant changes in tissue function and vital organ toxicity markers compared to LPolyHb, specifically markers related to kidney, liver, and lung injury and systemic inflammation and iron transport by the reticuloendothelial system. LPolyHb had a longer half-life than SPolyHb, which correlates with observations made in the reticuloendothelial and iron transport systems. These studies indicate that the molecular size of PolyHb determines vasoactivity, circulation time, mechanism of elimination, toxicity, and inflammation induced by its infusion.

Increased Hemoglobin Oxygen Affinity With 5-Hydroxymethylfurfural Supports Cardiac Function During Severe Hypoxia.

Acclimatization to hypoxia or high altitude involves physiological adaptation processes, to influence oxygen (O2) transport and utilization. Several natural products, including aromatic aldehydes and isothiocyanates stabilize the R-state of hemoglobin (Hb), increasing Hb-O2 affinity and Hb-O2 saturation. These products are a counter intuitive therapeutic strategy to increase O2 delivery during hypoxia. 5-Hydroxymethylfurfural (5-HMF) is well known Amadori compound formed during the Maillard reaction (the non-enzymatic browning and caramelization of carbohydrate-containing foods after thermal treatment), with well documented effects in Hb-O2 affinity. This study explores the therapeutic potential of 5-HMF on left ventricular (LV) cardiac function (LVCF) during hypoxia. Anesthetized Golden Syrian hamsters received 5-HMF i.v., at 100 mg/kg and were subjected to stepwise increased hypoxia (15, 10, and 5%) every 30 min. LVCF was assessed using a closed chest method with a miniaturized conductance catheter via continuous LV pressure-volume (PV) measurements. Heart hypoxic areas were studied using pimonidazole staining. 5-HMF improved cardiac indices, including stroke volume (SV), cardiac output (CO), ejection fraction (EF), and stroke work (SW) compared to the vehicle group. At 5% O2, SV, CO, EF, and SW were increased by 53, 42, 33, and 51% with 5-HMF relative to vehicle. Heart chronotropic activity was not statistically changed, suggesting that differences in LV-CF during hypoxia by 5-HMF were driven by volume dependent effects. Analysis of coronary blood flow and cardiac muscle metabolism suggest no direct pharmacological effects from 5-HMF, therefore these results can be attributed to 5-HMF-dependent increase in Hb-O2 affinity. These studies establish that naturally occurring aromatic aldehydes, such as 5-HMF, produce modification of hemoglobin oxygen affinity with promising therapeutic potential to increase O2 delivery during hypoxic hypoxia.

Post-weaning Exposure to High-Fat Diet Induces Kidney Lipid Accumulation and Function Impairment in Adult Rats.

Aim: We investigated the kidney morphofunctional consequences of high-fat diet intake since post-weaning in adult rats. Main Methods: Male Wistar rats were divided into two groups: ND (normal diet; n = 10) and HD (high-fat diet; n = 10). The high-fat diet was introduced post-weaned and animals were followed for 8 weeks. Key Findings: HD group did not change body weight gain even though food consumption has decreased with no changes in caloric consumption. The HD group showed glucose intolerance and insulin resistance. The glomerular filtration rate (GFR) was decreased in vivo (ND: 2.8 ± 1.01; HD: 1.1 ± 0.14 ml/min) and in the isolated perfusion method (34% of decrease). Renal histological analysis showed a retraction in glomeruli and an increase in kidney lipid deposition (ND: 1.5 ± 0.17 HD: 5.9 ± 0.06%). Furthermore, the high-fat diet consumption increased the pro-inflammatory cytokines IL-6 (ND: 1,276 ± 203; HD: 1,982 ± 47 pg/mL/mg) and IL-1b (ND: 97 ± 12 HD: 133 ± 5 pg/mL/mg) without changing anti-inflammatory cytokine IL-10. Significance: Our study provides evidence that high-fat diet consumption leads to renal lipid accumulation, increases inflammatory cytokines, induces glomeruli retraction, and renal dysfunction. These damages observed in the kidney could be associated with an increased risk to advanced CKD in adulthood suggesting that reduction of high-fat ingestion during an early period of life can prevent metabolic disturbances and renal lipotoxicity.

Aerobic exercise training prevents obesity and insulin resistance independent of the renin angiotensin system modulation in the subcutaneous white adipose tissue.

We investigate the effects of aerobic exercise training (AET) on the thermogenic response, substrate metabolism and renin angiotensin system (RAS) in the subcutaneous white adipose tissue (SC-WAT) of mice fed cafeteria diet (CAF). Male C57BL/6J mice were assigned into groups CHOW-SED (chow diet, sedentary; n = 10), CHOW-TR (chow diet, trained; n = 10), CAF-SED (CAF, sedentary; n = 10) and CAF-TR (CAF, trained; n = 10). AET consisted in running sessions of 60 min at 60% of maximal speed, five days per week for eight weeks. The CAF-SED group showed higher body weight and adiposity, glucose intolerance and insulin resistance (IR), while AET prevented such damages in CAF-TR group. AET reduced the p-AKT/t-AKT ratio and increased ATGL expression in CHOW-TR and CAF-TR groups and increased t-HSL and p-HSL/t-HSL ratio in CAF-TR. AET prevented adipocyte hypertrophy in CAF-TR group and increased UCP-1 protein expression only in CHOW-TR. Serum ACE2 increased in CHOW-TR and CAF-TR groups, and Ang (1-7) increased in the CHOW-TR group. In the SC-WAT, CAF-TR group increased the expression of AT1, AT2 and Mas receptors, whereas CHOW-TR increased Ang (1-7) and Ang (1-7)/Ang II ratio in SC-WAT. No changes were observed in ACE and Ang II. Positive correlations were observed between UCP-1 and kITT (r = 0.6), between UCP-1 and Ang (1-7) concentration (r = 0.6), and between UCP-1 and Ang (1-7)/Ang II ratio (r = 0.7). In conclusion, the AET prevented obesity and IR, reduced insulin signaling proteins and increased lipolysis signaling proteins in the SC-WAT. In addition, the CAF diet precludes the AET-induced thermogenic response and the partial modulation of the RAS suggests that the protective effect of AET against obesity and IR could not be associated with SC-WAT RAS.

Disruption of beta3 adrenergic receptor increases susceptibility to DIO in mouse.

The brown adipose tissue (BAT) mediates adaptive changes in metabolic rate by responding to the sympathetic nervous system through ß-adrenergic receptors (AR). Here, we wished to define the role played by the ARß3 isoform in this process. This study focused on the ARß3 knockout mice (ARß3KO), including responsiveness to cold exposure, diet-induced obesity, intolerance to glucose, dyslipidaemia and lipolysis in white adipose tissue (WAT). ARß3KO mice defend core temperature during cold exposure (4°C for 5 h), with faster BAT thermal response to norepinephrine (NE) infusion when compared with wild-type (WT) mice. Despite normal BAT thermogenesis, ARß3KO mice kept on a high-fat diet (HFD; 40% fat) for 8 weeks exhibited greater susceptibility to diet-induced obesity, markedly increased epididymal adipocyte area with clear signs of inflammation. The HFD-induced glucose intolerance was similar in both groups but serum hypertriglyceridemia and hypercholesterolemia were less intense in ARß3KO animals when compared with WT controls. Isoproterenol-induced lipolysis in isolated white adipocytes as assessed by glycerol release was significantly impaired in ARß3KO animals despite normal expression of key proteins involved in lipid metabolism. In conclusion, ARß3 inactivation does not affect BAT thermogenesis but increases susceptibility to diet-induced obesity by dampening WAT lipolytic response to adrenergic stimulation.