Metabolic and Proteomic Divergence Is Present in Circulating Monocytes and Tissue-Resident Macrophages from Berkeley Sickle Cell Anemia and ß-Thalassemia Mice.

Sickle cell disease and ß-thalassemia represent hemoglobinopathies arising from dysfunctional or underproduced ß-globin chains, respectively. In both diseases, red blood cell injury and anemia are the impetus for end organ injury. Because persistent erythrophagocytosis is a hallmark of these genetic maladies, it is critical to understand how macrophage phenotype polarizations in tissue compartments can inform on disease progression. Murine models of sickle cell disease and ß-thalassemia allow for a basic understanding of the mechanisms and provide for translation to human disease. A multi-omics approach to understanding the macrophage metabolism and protein changes in two murine models of ß-globinopathy was performed on peripheral blood mononuclear cells as well as spleen and liver macrophages isolated from Berkley sickle cell disease (Berk-ss) and heterozygous B1/B2 globin gene deletion (Hbbth3/+) mice. The results from these experiments revealed that the metabolome and proteome of macrophages are polarized to a distinct phenotype in Berk-ss and Hbbth3/+ compared with each other and their common-background mice (C57BL6/J). Further, spleen and liver macrophages revealed distinct disease-specific phenotypes, suggesting that macrophages become differentially polarized and reprogrammed within tissue compartments. We conclude that tissue recruitment, polarization, and metabolic and proteomic reprogramming of macrophages in Berk-ss and Hbbth3/+ mice may be relevant to disease progression in other tissue.

Effects of living at moderate altitude on pulmonary vascular function and exercise capacity in mice with sickle cell anaemia.

KEY POINTS: Sickle cell disease (SCD) results in cardiopulmonary dysfunction, which may be exacerbated by prolonged exposure to environmental hypoxia. It is currently unknown whether exposure to mild and moderate altitude exacerbates SCD associated cardiopulmonary and systemic complications. Three months of exposure to mild (1609 m) and moderate (2438 m) altitude increased rates of haemolysis and right ventricular systolic pressures in mice with SCD compared to healthy wild-type cohorts and SCD mice at sea level. The haemodynamic changes in SCD mice that had lived at mild and moderate altitude were accompanied by changes in the balance between pulmonary vascular endothelial nitric oxide synthase and endothelin receptor expression and impaired exercise tolerance. These data demonstrate that chronic altitude exposure exacerbates the complications associated with SCD and provides pertinent information for the clinical counselling of SCD patients. ABSTRACT: Exposure to high altitude worsens symptoms and crises in patients with sickle cell disease (SCD). However, it remains unclear whether prolonged exposure to low barometric pressures exacerbates SCD aetiologies or impairs quality of life. We tested the hypothesis that, relative to wild-type (WT) mice, Berkley sickle cell mice (BERK-SS) residing at sea level, mild (1609 m) and moderate (2438 m) altitude would have a higher rate of haemolysis, impaired cardiac function and reduced exercise tolerance, and that the level of altitude would worsen these decrements. Following 3 months of altitude exposure, right ventricular systolic pressure was measured (solid-state transducer). In addition, the adaptive balance between pulmonary vascular endothelial nitric oxide synthase and endothelin was assessed in lung tissue to determine differences in pulmonary vascular adaptation and the speed/duration relationship (critical speed) was used to evaluate treadmill exercise tolerance. At all altitudes, BERK-SS mice had a significantly lower percentage haemocrit and higher total bilirubin and free haemoglobin concentration (P < 0.05 for all). right ventricular systolic pressures in BERK-SS were higher than WT at moderate altitude and also compared to BERK-SS at sea level (P < 0.05, for both). Critical speed was significantly lower in BERK-SS at mild and moderate altitude (P < 0.05). BERK-SS demonstrated exacerbated SCD complications and reduced exercise capacity associated with an increase in altitude. These results suggest that exposure to mild and moderate altitude enhances the progression of SCD in BERK-SS mice compared to healthy WT cohorts and BERK-SS mice at sea level and provides crucial information for the clinical counselling of SCD patients.

Impact of cell-free hemoglobin on contracting skeletal muscle microvascular oxygen pressure dynamics.

Free hemoglobin (Hb) associated with hemolysis extravasates into vascular tissue and depletes nitric oxide (NO), which leads to impaired vascular function and could impair skeletal muscle metabolic control during exercise. We tested the hypothesis that: 1) free Hb would extravasate into skeletal muscle tissue, reducing the contracting skeletal muscle O2 delivery/O2 utilization ratio (microvascular PO2, PO2mv) to a similar extent as that observed following NO synthase (NOS) blockade, and 2) that the Hb scavenging protein haptoglobin (Hp) would prevent Hb extravasation and inhibit these skeletal muscle tissue effects. PO2mv was measured in eight rats (phosphorescence quenching) at rest and during 180 s of electrically induced (1-Hz) twitch spinotrapezius muscle contractions (experiment 1). A second group of seven rats was also used to investigate the effects of Hb + Hp (experiment 2). For both experiments, measurements were made: 1) during control conditions, 2) following a bolus infusion of either Hb (50 mg/kg) or Hb + Hp (50 mg/kg), and 3) following local superfusion of NG-nitro-l-arginine methyl ester (L-NAME; 10 mg/kg). Additional experiments were completed to visualize Hb extravasation into the muscular tissue using Click chemistry techniques. There were no significant differences in the PO2mv observed at rest for any condition in either experiment (p > 0.05 for all). In experiment 1, both Hb and L-NAME reduced the PO2mv significantly during the steady-state of muscle contractions when compared to control conditions with no differences between Hb and L-NAME (control: 24 ±â€¯1, Hb: 21 ±â€¯1, L-NAME: 20 ±â€¯1 mmHg, p < 0.05). In experiment 2, only L-NAME resulted in a significantly lower PO2mv during the steady-state of muscle contractions (control: 25 ± 1, Hb + Hp: 22 ± 2, L-NAME: 18 ± 1 mmHg, p < 0.05). Free Hb lowered the blood-myocyte O2 driving force to a level not significantly different from L-NAME. However, infusing Hb bound to Hp resulted in no significant differences in steady-state PO2mv during muscle contractions when compared to control. Surprisingly, we did not observe Hb accumulation in skeletal muscle tissue. Taken together these data suggests that free Hb impairs O2 delivery/utilization via a NO scavenging effect. Furthermore, the unchanged PO2mv steady-state observed following Hb + Hp further indicates that vascular compartmentalization of Hb by the scavenger protein haptoglobin may improve skeletal muscle metabolic control and potentially exercise tolerance in those afflicted with hemolytic diseases.

Probing cytoplasmic peroxide metabolism in Shewanella oneidensis.

The facultative anaerobe Shewanella oneidensis respires an extensive set of electron acceptors and, as a consequence, can leak electrons to produce reactive oxygen species such as hydrogen peroxide (H2O2). However, the effects of respiration on cytoplasmic redox homeostasis are poorly characterized in comparison. In the present study, the H2O2 sensor HyPer-3 was deployed to interrogate cytoplasmic peroxide levels of both wild-type and gene deletion mutants lacking peroxide scavenging enzymes following exposure to H2O2. HyPer-3 signals were validated in the S. oneidensis wild-type strain and exhibited a dynamic range of 0-250 µM H2O2. As reported by the HyPer-3 sensor, the cytoplasm of H2O2-perturbed mutant strains lacking periplasmic glutathione peroxidase (PgpD) and double deletion mutants lacking catalase (KatB) and bifunctional catalase-peroxidases (KatG1 or KatG2) contained high H2O2 concentrations. The high cytoplasmic H2O2 concentrations correlated with impaired H2O2 removal rates displayed by the mutant strains. Results of the present study provide the first in vivo interrogation of the redox environment of the S. oneidensis cytoplasm with HyPer-3 sensors and indicate that proper redox conditions in minimal growth medium are maintained by the concerted action of both well-known (periplasmic PgpD, cytoplasmic KatB and KatG1) and previously overlooked (cytoplasmic KatG2) peroxidases and catalases.

Moderate hypoxia induces metabolic divergence in circulating monocytes and tissue resident macrophages from Berkeley sickle cell anemia mice.

Introduction: Human and murine sickle cell disease (SCD) associated pulmonary hypertension (PH) is defined by hemolysis, nitric oxide depletion, inflammation, and thrombosis. Further, hemoglobin (Hb), heme, and iron accumulation are consistently observed in pulmonary adventitial macrophages at autopsy and in hypoxia driven rodent models of SCD, which show distribution of ferric and ferrous Hb as well as HO-1 and ferritin heavy chain. The anatomic localization of these macrophages is consistent with areas of significant vascular remodeling. However, their contributions toward progressive disease may include unique, but also common mechanisms, that overlap with idiopathic and other forms of pulmonary hypertension. These processes likely extend to the vasculature of other organs that are consistently impaired in advanced SCD. Methods: To date, limited information is available on the metabolism of macrophages or monocytes isolated from lung, spleen, and peripheral blood in humans or murine models of SCD. Results: Here we hypothesize that metabolism of macrophages and monocytes isolated from this triad of tissue differs between Berkley SCD mice exposed for ten weeks to moderate hypobaric hypoxia (simulated 8,000 ft, 15.4% O2) or normoxia (Denver altitude, 5000 ft) with normoxia exposed wild type mice evaluated as controls. Discussion: This study represents an initial set of data that describes the metabolism in monocytes and macrophages isolated from moderately hypoxic SCD mice peripheral lung, spleen, and blood mononuclear cells.

Extracellular Vesicle Size Reveals Cargo Specific to Coagulation and Inflammation in Pediatric and Adult Sickle Cell Disease.

Aberrant coagulation in sickle cell disease (SCD) is linked to extracellular vesicle (EV) exposure. However, there is no consensus on the contributions of small EVs (SEVs) and large EVs (LEVs) toward underlying coagulopathy or on their molecular cargo. The present observational study compared the thrombin potential of SEVs and LEVs isolated from the plasma of stable pediatric and adult SCD patients. Further, EV lipid and protein contents were analyzed to define markers consistent with activation of thrombin and markers of underlying coagulopathy. Results suggested that LEVs-but not SEVs-from pediatrics and adults similarly enhanced phosphatidylserine (PS)-dependent thrombin generation, and cell membrane procoagulant PS (18:0;20:4 and 18:0;18:1) were the most abundant lipids found in LEVs. Further, LEVs showed activated coagulation in protein pathway analyses, while SEVs demonstrated high levels of cholesterol esters and a protein pathway analysis that identified complement factors and inflammation. We suggest that thrombin potential of EVs from both stable pediatric and adult SCD patients is similarly dependent on size and show lipid and protein contents that identify underlying markers of coagulation and inflammation.

Murine models of sickle cell disease and beta-thalassemia demonstrate pulmonary hypertension with distinctive features.

Sickle cell anemia and ß-thalassemia intermedia are very different genetically determined hemoglobinopathies predisposing to pulmonary hypertension. The etiologies responsible for the associated development of pulmonary hypertension in both diseases are multi-factorial with extensive mechanistic contributors described. Both sickle cell anemia and ß-thalassemia intermedia present with intra and extravascular hemolysis. And because sickle cell anemia and ß-thalassemia intermedia share features of extravascular hemolysis, macrophage iron excess and anemia we sought to characterize the common features of the pulmonary hypertension phenotype, cardiac mechanics, and function as well as lung and right ventricular metabolism. Within the concept of iron, we have defined a unique pulmonary vascular iron accumulation in lungs of sickle cell anemia pulmonary hypertension patients at autopsy. This observation is unlike findings in idiopathic or other forms of pulmonary arterial hypertension. In this study, we hypothesized that a common pathophysiology would characterize the pulmonary hypertension phenotype in sickle cell anemia and ß-thalassemia intermedia murine models. However, unlike sickle cell anemia, ß-thalassemia is also a disease of dyserythropoiesis, with increased iron absorption and cellular iron extrusion. This process is mediated by high erythroferrone and low hepcidin levels as well as dysregulated iron transport due transferrin saturation, so there may be differences as well. Herein we describe common and divergent features of pulmonary hypertension in aged Berk-ss (sickle cell anemia) and Hbbth/3+ (intermediate ß-thalassemia) mice and suggest translational utility as proof-of-concept models to study pulmonary hypertension therapeutics specific to genetic anemias.

Evidence supporting a role for circulating macrophages in the regression of vascular remodeling following sub-chronic exposure to hemoglobin plus hypoxia.

Macrophages are a heterogeneous population with both pro- and anti-inflammatory functions play an essential role in maintaining tissue homeostasis, promoting inflammation under pathological conditions, and tissue repair after injury. In pulmonary hypertension, the M1 phenotype is more pro-inflammatory compared to the M2 phenotype, which is involved in tissue repair. The role of macrophages in the initiation and progression of pulmonary hypertension is well studied. However, their role in the regression of established pulmonary hypertension is not well known. Rats chronically exposed to hemoglobin (Hb) plus hypoxia (HX) share similarities to humans with pulmonary hypertension associated with hemolytic disease, including the presence of a unique macrophage phenotype surrounding distal vessels that are associated with vascular remodeling. These lung macrophages are characterized by high iron content, HO-1, ET-1, and IL-6, and are recruited from the circulation. Depletion of macrophages in this model prevents the development of pulmonary hypertension and vascular remodeling. In this study, we specifically investigate the regression of pulmonary hypertension over a four-week duration after rats were removed from Hb + HX exposure with and without gadolinium chloride administration. Withdrawal of Hb + HX reversed systolic pressures and right ventricular function after Hb + Hx exposure in four weeks. Our data show that depleting circulating monocytes/macrophages during reversal prevents complete recovery of right ventricular systolic pressure and vascular remodeling in this rat model of pulmonary hypertension at four weeks post exposure. The data presented offer a novel insight into the role of macrophages in the processes of pulmonary hypertension regression in a rodent model of Hb + Hx-driven disease.

Hemopexin dosing improves cardiopulmonary dysfunction in murine sickle cell disease.

Hemopexin (Hpx) is a crucial defense protein against heme liberated from degraded hemoglobin during hemolysis. High heme stress creates an imbalance in Hpx bioavailability, favoring heme accumulation and downstream pathophysiological responses leading to cardiopulmonary disease progression in sickle cell disease (SCD) patients. Here, we evaluated a model of murine SCD, which was designed to accelerate red blood cell sickling, pulmonary hypertension, right ventricular dysfunction, and exercise intolerance by exposure of the mice to moderate hypobaric hypoxia. The sequence of pathophysiology in this model tracks with circulatory heme accumulation, lipid oxidation, extensive remodeling of the pulmonary vasculature, and fibrosis. We hypothesized that Hpx replacement for an extended period would improve exercise tolerance measured by critical speed as a clinically meaningful therapeutic endpoint. Further, we sought to define the effects of Hpx on upstream cardiopulmonary function, histopathology, and tissue oxidation. Our data shows that tri-weekly administrations of Hpx for three months dose-dependently reduced heme exposure and pulmonary hypertension while improving cardiac pressure-volume relationships and exercise tolerance. Furthermore, Hpx administration dose-dependently attenuated pulmonary fibrosis and oxidative modifications in the lung and myocardium of the right ventricle. Observations in our SCD murine model are consistent with pulmonary vascular and right ventricular pathology at autopsy in SCD patients having suffered from severe pulmonary hypertension, right ventricular dysfunction, and sudden cardiac death. This study provides a translational evaluation supported by a rigorous outcome analysis demonstrating therapeutic proof-of-concept for Hpx replacement in SCD.

The effect of dietary nitrate supplementation on the speed-duration relationship in mice with sickle cell disease.

Sickle cell disease (SCD) causes exercise intolerance likely due to impaired skeletal muscle function and low nitric oxide (NO) bioavailability. Dietary nitrate improves hemodynamic and metabolic control during exercise in humans and animals. The purpose of this investigation was to assess the impact of nitrate supplementation on exercise capacity as measured by the running speed to exercise duration relationship [critical speed (CS)]in mice with SCD. We tested the hypothesis that nitrate supplementation via beetroot juice (BR) would attenuate the exercise intolerance observed in mice with SCD. Ten wild-type (WT) and 18 Berkley sickle-cell mice (BERK) received water (WT: n = 10, BERK: n = 10) or nitrate-rich BR (BERK+BR: n = 8, nitrate dose 1 mmol/kg/day) for 5 days. Following the supplementation period, all mice performed 3-5 constant-speed treadmill tests that resulted in exhaustion within 1.5 to 20 min. Time to exhaustion vs. treadmill speed was fit to a hyperbolic model to determine CS. CS was significantly lower in BERK vs. WT and BERK+BR with no significant difference between WT and BERK+BR (WT: 36.6 ± 1.6, BERK: 23.8 ± 1.5, BERK+BR: 31.1 ± 2.1 m/min, P < 0.05). Exercise tolerance, measured via CS, was significantly lower in BERK mice relative to WT. However, BERK mice receiving 5 days of nitrate supplementation exhibited no difference in exercise tolerance when compared with WT. These results support the potential utility of a dietary nitrate intervention to improve functionality in SCD patients.NEW & NOTEWORTHY Sickle cell disease compromises muscle O2 delivery resulting in exercise intolerance. Dietary nitrate supplementation increases skeletal muscle blood flow during exercise and may improve exercise capacity in a mouse model of sickle cell disease. We investigated the effects of dietary nitrate supplementation on exercise tolerance in a mouse model of sickle cell disease using the treadmill speed-duration relationship (critical speed). Mice with sickle cell disease provided with a dietary nitrate supplement had a critical speed not significantly different from healthy wild-type mice.

Pre-clinical assessment of a water-in-fluorocarbon emulsion for the treatment of pulmonary vascular diseases.

Hypoxic pulmonary vasoconstriction (HPV) is a well-characterized vascular response to low oxygen pressures and is involved in life-threatening conditions such as high-altitude pulmonary edema (HAPE) and pulmonary arterial hypertension (PAH). While the efficacy of oral therapies can be affected by drug metabolism, or dose-limiting systemic toxicity, inhaled treatment via pressured metered dose inhalers (pMDI) may be an effective, nontoxic, practical alternative. We hypothesized that a stable water-in-perfluorooctyl bromide (PFOB) emulsion that provides solubility in common pMDI propellants, engineered for intrapulmonary delivery of pulmonary vasodilators, reverses HPV during acute hypoxia (HX). Male Sprague Dawley rats received two 10-min bouts of HX (13% O2) with 20 min of room air and drug application between exposures. Treatment groups: intrapulmonary delivery (PUL) of (1) saline; (2) ambrisentan in saline (0.1 mg/kg); (3) empty emulsion; (4) emulsion encapsulating ambrisentan or sodium nitrite (NaNO2) (0.1 and 0.5 mg/kg each); and intravenous (5) ambrisentan (0.1 mg/kg) or (6) NaNO2 (0.5 mg/kg). Neither PUL of saline or empty emulsion, nor infusions of drugs prevented pulmonary artery pressure (PAP) elevation (32.6 ± 3.2, 31.5 ± 1.2, 29.3 ± 1.8, and 30.2 ± 2.5 mmHg, respectively). In contrast, PUL of aqueous ambrisentan and both drug emulsions reduced PAP by 20-30% during HX, compared to controls. IL6 expression in bronchoalveolar lavage fluid and whole lung 24 h post-PUL did not differ among cohorts. We demonstrate proof-of-concept for delivering pulmonary vasodilators via aerosolized water-in-PFOB emulsion. This concept opens a potentially feasible and effective route of treating pulmonary vascular pathologies via pMDI.

An Hb-mediated circulating macrophage contributing to pulmonary vascular remodeling in sickle cell disease.

Circulating macrophages recruited to the lung contribute to pulmonary vascular remodeling in various forms of pulmonary hypertension (PH). In this study we investigated a macrophage phenotype characterized by intracellular iron accumulation and expression of antioxidant (HO-1), vasoactive (ET-1), and proinflammatory (IL-6) mediators observed in the lung tissue of deceased sickle cell disease (SCD) patients with diagnosed PH. To this end, we evaluated an established rat model of group 5 PH that is simultaneously exposed to free hemoglobin (Hb) and hypobaric hypoxia (HX). Here, we tested the hypothesis that pulmonary vascular remodeling observed in human SCD with concomitant PH could be replicated and mechanistically driven in our rat model by a similar macrophage phenotype with iron accumulation and expression of a similar mixture of antioxidant (HO-1), vasoactive (ET-1), and inflammatory (IL-6) proteins. Our data suggest phenotypic similarities between pulmonary perivascular macrophages in our rat model and human SCD with PH, indicating a potentially novel maladaptive immune response to concomitant bouts of Hb and HX exposure. Moreover, by knocking out circulating macrophages with gadolinium trichloride (GdCl3), the response to combined Hb and hypobaric HX was significantly attenuated in rats, suggesting a critical role for macrophages in the exacerbation of SCD PH.