Brown Adipose Tissue and BMP3b Decrease Injury in Cardiac Ischemia-Reperfusion.

BACKGROUND: Despite advances in treatment, myocardial infarction (MI) is a leading cause of heart failure and death worldwide, with both ischemia and reperfusion (I/R) causing cardiac injury. A previous study using a mouse model of nonreperfused MI showed activation of brown adipose tissue (BAT). Recent studies showed that molecules secreted by BAT target the heart. We investigated whether BAT attenuates cardiac injury in I/R and sought to identify potential cardioprotective proteins secreted by BAT. METHODS: Myocardial I/R surgery with or without BAT transplantation was performed in wild-type (WT) mice and in mice with impaired BAT function (uncoupling protein 1 [Ucp1]-deficient mice). To identify potential cardioprotective factors produced by BAT, RNA-seq (RNA sequencing) was performed in BAT from WT and Ucp1-/- mice. Subsequently, myocardial I/R surgery with or without BAT transplantation was performed in Bmp3b (bone morphogenetic protein 3b)-deficient mice, and WT mice subjected to myocardial I/R were treated using BMP3b. RESULTS: Dysfunction of BAT in mice was associated with larger MI size after I/R; conversely, augmenting BAT by transplantation decreased MI size. We identified Bmp3b as a protein secreted by BAT after I/R. Compared with WT mice, Bmp3b-deficient mice developed larger MIs. Increasing functional BAT by transplanting BAT from WT mice to Bmp3b-deficient mice reduced I/R injury whereas transplanting BAT from Bmp3b-deficient mice did not. Treatment of WT mice with BMP3b before reperfusion decreased MI size. The cardioprotective effect of BMP3b was mediated through SMAD1/5/8. In humans, the plasma level of BMP3b increased after MI and was positively correlated with the extent of cardiac injury. CONCLUSIONS: The results of this study suggest a cardioprotective role of BAT and BMP3b, a protein secreted by BAT, in a model of I/R injury. Interventions increasing BMP3b levels or targeting Smad 1/5 may represent novel therapeutic approaches to decrease myocardial damage in I/R injury.

Matrix Gla Protein Levels Are Associated With Arterial Stiffness and Incident Heart Failure With Preserved Ejection Fraction.

OBJECTIVE: Arterial stiffness is a risk factor for cardiovascular disease, including heart failure with preserved ejection fraction (HFpEF). MGP (matrix Gla protein) is implicated in vascular calcification in animal models, and circulating levels of the uncarboxylated, inactive form of MGP (ucMGP) are associated with cardiovascular disease-related and all-cause mortality in human studies. However, the role of MGP in arterial stiffness is uncertain. Approach and Results: We examined the association of ucMGP levels with vascular calcification, arterial stiffness including carotid-femoral pulse wave velocity (PWV), and incident heart failure in community-dwelling adults from the Framingham Heart Study. To further investigate the link between MGP and arterial stiffness, we compared aortic PWV in age- and sex-matched young (4-month-old) and aged (10-month-old) wild-type and Mgp+/- mice. Among 7066 adults, we observed significant associations between higher levels of ucMGP and measures of arterial stiffness, including higher PWV and pulse pressure. Longitudinal analyses demonstrated an association between higher ucMGP levels and future increases in systolic blood pressure and incident HFpEF. Aortic PWV was increased in older, but not young, female Mgp+/- mice compared with wild-type mice, and this augmentation in PWV was associated with increased aortic elastin fiber fragmentation and collagen accumulation. CONCLUSIONS: This translational study demonstrates an association between ucMGP levels and arterial stiffness and future HFpEF in a large observational study, findings that are substantiated by experimental studies showing that mice with Mgp heterozygosity develop arterial stiffness. Taken together, these complementary study designs suggest a potential role of therapeutically targeting MGP in HFpEF.

UBA6 and NDFIP1 regulate the degradation of ferroportin.

Hepcidin regulates iron homeostasis by controlling the level of ferroportin, the only membrane channel that facilitates export of iron from within cells. Binding of hepcidin to ferroportin induces the ubiquitination of ferroportin at multiple lysine residues and subsequently causes the internalization and degradation of the ligand-channel complex within lysosomes. The objective of this study was to identify components of the ubiquitin system that are involved in ferroportin degradation. A HepG2 cell line, which inducibly expresses ferroportingreen fluorescent protein (FPN-GFP), was established to test the ability of small interfering (siRNA) directed against components of the ubiquitin system to prevent BMP6- and exogenous hepcidin-induced ferroportin degradation. Of the 88 siRNA directed against components of the ubiquitin pathway that were tested, siRNA-mediated depletion of the alternative E1 enzyme UBA6 as well as the adaptor protein NDFIP1 prevented BMP6- and hepcidin-induced degradation of ferroportin in vitro. A third component of the ubiquitin pathway, ARIH1, indirectly inhibited ferroportin degradation by impairing BMP6-mediated induction of hepcidin. In mice, the AAV-mediated silencing of Ndfip1 in the murine liver increased the level of hepatic ferroportin and increased circulating iron. The results suggest that the E1 enzyme UBA6 and the adaptor protein NDFIP1 are involved in iron homeostasis by regulating the degradation of ferroportin. These specific components of the ubiquitin system may be promising targets for the treatment of iron-related diseases, including iron overload and anemia of inflammation.

Hyperbaric phototherapy augments blood carbon monoxide removal.

BACKGROUND AND OBJECTIVES: Carbon monoxide (CO) poisoning is responsible for nearly 50,000 emergency department visits and 1200 deaths per year. Compared to oxygen, CO has a 250-fold higher affinity for hemoglobin (Hb), resulting in the displacement of oxygen from Hb and impaired oxygen delivery to tissues. Optimal treatment of CO-poisoned patients involves the administration of hyperbaric 100% oxygen to remove CO from Hb and to restore oxygen delivery. However, hyperbaric chambers are not widely available and this treatment requires transporting a CO-poisoned patient to a specialized center, which can result in delayed treatment. Visible light is known to dissociate CO from carboxyhemoglobin (COHb). In a previous study, we showed that a system composed of six photo-extracorporeal membrane oxygenation (ECMO) devices efficiently removes CO from a large animal with CO poisoning. In this study, we tested the hypothesis that the application of hyperbaric oxygen to the photo-ECMO device would further increase the rate of CO elimination. STUDY DESIGN/MATERIAL AND METHODS: We developed a hyperbaric photo-ECMO device and assessed the ability of the device to remove CO from CO-poisoned human blood. We combined four devices into a "hyperbaric photo-ECMO system" and compared its ability to remove CO to our previously described photo-ECMO system, which was composed of six devices ventilated with normobaric oxygen. RESULTS: Under normobaric conditions, an increase in oxygen concentration from 21% to 100% significantly increased CO elimination from CO-poisoned blood after a single pass through the device. Increased oxygen pressure within the photo-ECMO device was associated with higher exiting blood PO2 levels and increased CO elimination. The system of four hyperbaric photo-ECMO devices removed CO from 1 L of CO-poisoned blood as quickly as the original, normobaric photo-ECMO system composed of six devices. CONCLUSION: This study demonstrates the feasibility and efficacy of using a hyperbaric photo-ECMO system to increase the rate of CO elimination from CO-poisoned blood. This technology could provide a simple portable emergency device and facilitate immediate treatment of CO-poisoned patients at or near the site of injury.

Veno-venous extracorporeal blood phototherapy increases the rate of carbon monoxide (CO) elimination in CO-poisoned pigs.

BACKGROUND AND OBJECTIVES: Carbon monoxide (CO) inhalation is the leading cause of poison-related deaths in the United States. CO binds to hemoglobin (Hb), displaces oxygen, and reduces oxygen delivery to tissues. The optimal treatment for CO poisoning in patients with normal lung function is the administration of hyperbaric oxygen (HBO). However, hyperbaric chambers are only available in medical centers with specialized equipment, resulting in delayed therapy. Visible light dissociates CO from Hb with minimal effect on oxygen binding. In a previous study, we combined a membrane oxygenator with phototherapy at 623 nm to produce a "mini" photo-ECMO (extracorporeal membrane oxygenation) device, which improved CO elimination and survival in CO-poisoned rats. The objective of this study was to develop a larger photo-ECMO device ("maxi" photo-ECMO) and to test its ability to remove CO from a porcine model of CO poisoning. STUDY DESIGN/MATERIALS AND METHODS: The "maxi" photo-ECMO device and the photo-ECMO system (six maxi photo-ECMO devices assembled in parallel), were tested in an in vitro circuit of CO poisoning. To assess the ability of the photo-ECMO device and the photo-ECMO system to remove CO from CO-poisoned blood in vitro, the half-life of COHb (COHb-t1/2 ), as well as the percent COHb reduction in a single blood pass through the device, were assessed. In the in vivo studies, we assessed the COHb-t1/2 in a CO-poisoned pig under three conditions: (1) While the pig breathed 100% oxygen through the endotracheal tube; (2) while the pig was connected to the photo-ECMO system with no light exposure; and (3) while the pig was connected to the photo-ECMO system, which was exposed to red light. RESULTS: The photo-ECMO device was able to fully oxygenate the blood after a single pass through the device. Compared to ventilation with 100% oxygen alone, illumination with red light together with 100% oxygen was twice as efficient in removing CO from blood. Changes in gas flow rates did not alter CO elimination in one pass through the device. Increases in irradiance up to 214 mW/cm2 were associated with an increased rate of CO elimination. The photo-ECMO device was effective over a range of blood flow rates and with higher blood flow rates, more CO was eliminated. A photo-ECMO system composed of six photo-ECMO devices removed CO faster from CO-poisoned blood than a single photo-ECMO device. In a CO-poisoned pig, the photo-ECMO system increased the rate of CO elimination without significantly increasing the animal's body temperature or causing hemodynamic instability. CONCLUSION: In this study, we developed a photo-ECMO system and demonstrated its ability to remove CO from CO-poisoned 45-kg pigs. Technical modifications of the photo-ECMO system, including the development of a compact, portable device, will permit treatment of patients with CO poisoning at the scene of their poisoning, during transit to a local emergency room, and in hospitals that lack HBO facilities.

Hypoxia ameliorates brain hyperoxia and NAD+ deficiency in a murine model of Leigh syndrome.

Leigh syndrome is a severe mitochondrial neurodegenerative disease with no effective treatment. In the Ndufs4-/- mouse model of Leigh syndrome, continuously breathing 11% O2 (hypoxia) prevents neurodegeneration and leads to a dramatic extension (~5-fold) in lifespan. We investigated the effect of hypoxia on the brain metabolism of Ndufs4-/- mice by studying blood gas tensions and metabolite levels in simultaneously sampled arterial and cerebral internal jugular venous (IJV) blood. Relatively healthy Ndufs4-/- and wildtype (WT) mice breathing air until postnatal age ~38 d were compared to Ndufs4-/- and WT mice breathing air until ~38 days old followed by 4-weeks of breathing 11% O2. Compared to WT control mice, Ndufs4-/- mice breathing air have reduced brain O2 consumption as evidenced by an elevated partial pressure of O2 in IJV blood (PijvO2) despite a normal PO2 in arterial blood, and higher lactate/pyruvate (L/P) ratios in IJV plasma revealed by metabolic profiling. In Ndufs4-/- mice, hypoxia treatment normalized the cerebral venous PijvO2 and L/P ratios, and decreased levels of nicotinate in IJV plasma. Brain concentrations of nicotinamide adenine dinucleotide (NAD+) were lower in Ndufs4-/- mice breathing air than in WT mice, but preserved at WT levels with hypoxia treatment. Although mild hypoxia (17% O2) has been shown to be an ineffective therapy for Ndufs4-/- mice, we find that when combined with nicotinic acid supplementation it provides a modest improvement in neurodegeneration and lifespan. Therapies targeting both brain hyperoxia and NAD+ deficiency may hold promise for treating Leigh syndrome.

Inhaled high dose nitric oxide is a safe and effective respiratory treatment in spontaneous breathing hospitalized patients with COVID-19 pneumonia.

BACKGROUND: Inhaled nitric oxide (NO) is a selective pulmonary vasodilator. In-vitro studies report that NO donors can inhibit replication of SARS-CoV-2. This multicenter study evaluated the feasibility and effects of high-dose inhaled NO in non-intubated spontaneously breathing patients with Coronavirus disease-2019 (COVID-19). METHODS: This is an interventional study to determine whether NO at 160 parts-per-million (ppm) inhaled for 30 min twice daily might be beneficial and safe in non-intubated COVID-19 patients. RESULTS: Twenty-nine COVID-19 patients received a total of 217 intermittent inhaled NO treatments for 30 min at 160 ppm between March and June 2020. Breathing NO acutely decreased the respiratory rate of tachypneic patients and improved oxygenation in hypoxemic patients. The maximum level of nitrogen dioxide delivered was 1.5 ppm. The maximum level of methemoglobin (MetHb) during the treatments was 4.7%. MetHb decreased in all patients 5 min after discontinuing NO administration. No adverse events during treatment, such as hypoxemia, hypotension, or acute kidney injury during hospitalization occurred. In our NO treated patients, one patient of 29 underwent intubation and mechanical ventilation, and none died. The median hospital length of stay was 6 days [interquartile range 4-8]. No discharged patients required hospital readmission nor developed COVID-19 related long-term sequelae within 28 days of follow-up. CONCLUSIONS: In spontaneous breathing patients with COVID-19, the administration of inhaled NO at 160 ppm for 30 min twice daily promptly improved the respiratory rate of tachypneic patients and systemic oxygenation of hypoxemic patients. No adverse events were observed. None of the subjects was readmitted or had long-term COVID-19 sequelae.

Deficiency of bone morphogenetic protein-3b induces metabolic syndrome and increases adipogenesis.

Bone morphogenetic protein (BMP) receptor signaling is critical for the regulation of the endocrine system and cardiovascular structure and function. The objective of this study was to investigate whether Bmp3b, a glycoprotein synthetized and secreted by adipose tissue, is necessary to regulate glucose and lipid metabolism, adipogenesis, and cardiovascular remodeling. Over the course of 4 mo, Bmp3b-knockout (Bmp3b-/-) mice gained more weight than wild-type (WT) mice. The plasma levels of cholesterol and triglycerides were higher in Bmp3b-/- mice than in WT mice. Bmp3b-/- mice developed insulin resistance and glucose intolerance. The basal heart rate was higher in Bmp3b-/- mice than in WT mice, and echocardiography revealed eccentric remodeling in Bmp3b-/- mice. The expression of adipogenesis-related genes in white adipose tissue was higher in Bmp3b-/- mice than in WT control mice. In vitro studies showed that Bmp3b modulates the activity of the C/ebpα promoter, an effect mediated by Smad2/3. The results of this study suggest that Bmp3b is necessary for the maintenance of homeostasis in terms of age-related weight gain, glucose metabolism, and left ventricular (LV) remodeling and function. Interventions that increase the level or function of BMP3b may decrease cardiovascular risk and pathological cardiac remodeling.

Intratracheal injection of nitric oxide, generated from air by pulsed electrical discharge, for the treatment of pulmonary hypertension in awake ambulatory lambs.

OBJECTIVES: To test the feasibility, safety, and efficacy of intratracheal delivery of nitric oxide (NO) generated from air by pulsed electrical discharge via a Scoop catheter. STUDY DESIGN: We studied healthy 3- to 4-month-old lambs weighing 34 ± 4 kg (mean ± SD, n = 6). A transtracheal Scoop catheter was inserted through a cuffed tracheostomy tube. U46619 was infused to increase mean pulmonary arterial pressure (mPAP) from 16 ± 1 to 32 ± 3 mmHg (mean ± SD). Electrically generated NO was delivered via the Scoop catheter to awake lambs. A sampling line, to monitor NO and nitrogen dioxide (NO2) levels, was placed in the distal trachea of the lambs. The effect of varying doses of electrically generated NO, produced continuously, on pulmonary hypertension was assessed. RESULTS: In awake lambs with acute pulmonary hypertension, NO was continuously delivered via the Scoop catheter at 400 ml/min. NO induced pulmonary vasodilation. NO2 levels, measured in the trachea, were below 0.5 ppm at intratracheal NO doses of 10-80 ppm. No changes were detected in the levels of methemoglobin in blood samples before and after 5 min of NO breathing. CONCLUSIONS: Continuously delivering electrically generated NO through a Scoop catheter produces vasodilation of the pulmonary vasculature of awake lambs with pulmonary hypertension. Transtracheal NO delivery may provide a long-term treatment for patients with chronic pulmonary hypertension as an outpatient without requiring a mask or tracheal intubation.

The role of hepcidin and iron homeostasis in atherosclerosis.

Atherosclerotic cardiovascular disease is a major burden on global health and a leading cause of death worldwide. The pathophysiology of this chronic disease is complex, involving inflammation, lipoprotein oxidation and accumulation, plaque formation, and calcification. In 1981, Dr. Jerome Sullivan formulated the 'Iron Hypothesis', suggesting that higher levels of stored iron promote cardiovascular diseases, whereas iron deficiency may have an atheroprotective effect. This hypothesis has stimulated research focused on clarifying the role of iron in the development of atherosclerosis. However, preclinical and clinical studies have produced contradictory results and the observation that patients with hemochromatosis do not appear to have an increased risk of atherosclerosis seemed incongruous with Sullivan's initial hypothesis. The 'paradox' of systemic iron overload not being accompanied by an increased risk for atherosclerosis led to a refinement of the iron hypothesis focusing on intracellular macrophage iron. More recent in vitro and animal studies have elucidated the complex signaling pathways regulating iron, with a particular focus on hepcidin, the master regulator of body iron homeostasis. Bone morphogenetic protein (BMP) signaling is the major pathway that is required for induction of hepcidin expression in response to increasing levels of iron. Strong links between iron homeostasis, BMP signaling, inflammation and atherosclerosis have been established in both mechanistic and human studies. This review summarizes the current understanding of the role of iron homeostasis and hepcidin in the development of atherosclerosis and discusses the BMP-hepcidin-ferroportin axis as a novel therapeutic target for the treatment of cardiovascular disease.

Hepcidin Deficiency Protects Against Atherosclerosis.

Objective- Inflammatory stimuli enhance the progression of atherosclerotic disease. Inflammation also increases the expression of hepcidin, a hormonal regulator of iron homeostasis, which decreases intestinal iron absorption, reduces serum iron levels and traps iron within macrophages. The role of macrophage iron in the development of atherosclerosis remains incompletely understood. The objective of this study was to investigate the effects of hepcidin deficiency and decreased macrophage iron on the development of atherosclerosis. Approach and Results- Hepcidin- and LDL (low-density lipoprotein) receptor-deficient ( Hamp-/-/ Ldlr-/-) mice and Hamp+/+/ Ldlr-/- control mice were fed a high-fat diet for 21 weeks. Compared with control mice, Hamp-/-/ Ldlr-/- mice had decreased aortic macrophage activity and atherosclerosis. Because hepcidin deficiency is associated with both increased serum iron and decreased macrophage iron, the possibility that increased serum iron was responsible for decreased atherosclerosis in Hamp-/-/ Ldlr-/- mice was considered. Hamp+/+/ Ldlr-/- mice were treated with iron dextran so as to produce a 2-fold increase in serum iron. Increased serum iron did not decrease atherosclerosis in Hamp+/+/ Ldlr-/- mice. Aortic macrophages from Hamp-/-/ Ldlr-/- mice had less labile free iron and exhibited a reduced proinflammatory (M1) phenotype compared with macrophages from Hamp+/+/ Ldlr-/- mice. THP1 human macrophages treated with an iron chelator were used to model hepcidin deficiency in vitro. Treatment with an iron chelator reduced LPS (lipopolysaccharide)-induced M1 phenotypic expression and decreased uptake of oxidized LDL. Conclusions- In summary, in a hyperlipidemic mouse model, hepcidin deficiency was associated with decreased macrophage iron, a reduced aortic macrophage inflammatory phenotype and protection from atherosclerosis. The results indicate that decreasing hepcidin activity, with the resulting decrease in macrophage iron, may prove to be a novel strategy for the treatment of atherosclerosis.

Establishment of international autoantibody reference standards for the detection of autoantibodies directed against PML bodies, GW bodies, and NuMA protein.

Objectives: Reference materials are important in the standardization of autoantibody testing and only a few are freely available for many known autoantibodies. Our goal was to develop three reference materials for antibodies to PML bodies/multiple nuclear dots (MND), antibodies to GW bodies (GWB), and antibodies to the nuclear mitotic apparatus (NuMA). Methods: Reference materials for identifying autoantibodies to MND (MND-REF), GWB (GWB-REF), and NuMA (NuMA-REF) were obtained from three donors and validated independently by seven laboratories. The sera were characterized using indirect immunofluorescence assay (IFA) on HEp-2 cell substrates including two-color immunofluorescence using antigen-specific markers, western blot (WB), immunoprecipitation (IP), line immunoassay (LIA), addressable laser bead immunoassay (ALBIA), enzyme-linked immunosorbent assay (ELISA), and immunoprecipitation-mass spectrometry (IP-MS). Results: MND-REF stained 6-20 discrete nuclear dots that colocalized with PML bodies. Antibodies to Sp100 and PML were detected by LIA and antibodies to Sp100 were also detected by ELISA. GWB-REF stained discrete cytoplasmic dots in interphase cells, which were confirmed to be GWB using two-color immunofluorescence. Anti-Ge-1 antibodies were identified in GWB-REF by ALBIA, IP, and IP-MS. All reference materials produced patterns at dilutions of 1:160 or greater. NuMA-REF produced fine speckled nuclear staining in interphase cells and staining of spindle fibers and spindle poles. The presence of antibodies to NuMA was verified by IP, WB, ALBIA, and IP-MS. Conclusions: MND-REF, GWB-REF, and NuMA-REF are suitable reference materials for the corresponding antinuclear antibodies staining patterns and will be accessible to qualified laboratories.

Impaired hypoxic pulmonary vasoconstriction in a mouse model of Leigh syndrome.

Hypoxic pulmonary vasoconstriction (HPV) is a physiological vasomotor response that maintains systemic oxygenation by matching perfusion to ventilation during alveolar hypoxia. Although mitochondria appear to play an essential role in HPV, the impact of mitochondrial dysfunction on HPV remains incompletely defined. Mice lacking the mitochondrial complex I (CI) subunit Ndufs4 ( Ndufs4-/-) develop a fatal progressive encephalopathy and serve as a model for Leigh syndrome, the most common mitochondrial disease in children. Breathing normobaric 11% O2 prevents neurological disease and improves survival in Ndufs4-/- mice. In this study, we found that either genetic Ndufs4 deficiency or pharmacological inhibition of CI using piericidin A impaired the ability of left mainstem bronchus occlusion (LMBO) to induce HPV. In mice breathing air, the partial pressure of arterial oxygen during LMBO was lower in Ndufs4-/- and in piericidin A-treated Ndufs4+/+ mice than in respective controls. Impairment of HPV in Ndufs4-/- mice was not a result of nonspecific dysfunction of the pulmonary vascular contractile apparatus or pulmonary inflammation. In Ndufs4-deficient mice, 3 wk of breathing 11% O2 restored HPV in response to LMBO. When compared with Ndufs4-/- mice breathing air, chronic hypoxia improved systemic oxygenation during LMBO. The results of this study show that, when breathing air, mice with a congenital Ndufs4 deficiency or chemically inhibited CI function have impaired HPV. Our study raises the possibility that patients with inborn errors of mitochondrial function may also have defects in HPV.

Low guanylyl cyclase activity in Weddell seals: implications for peripheral vasoconstriction and perfusion of the brain during diving.

Nitric oxide (NO) is a potent vasodilator, which improves perfusion and oxygen delivery during tissue hypoxia in terrestrial animals. The vertebrate dive response involves vasoconstriction in select tissues, which persists despite profound hypoxia. Using tissues collected from Weddell seals at necropsy, we investigated whether vasoconstriction is aided by downregulation of local hypoxia signaling mechanisms. We focused on NO-soluble guanylyl cyclase (GC)-cGMP signaling, a well-known vasodilatory transduction pathway. Seals have a lower GC protein abundance, activity, and capacity to respond to NO stimulation than do terrestrial mammals. In seal lung homogenates, GC produced less cGMP (20.1 ± 3.7 pmol·mg protein-1·min-1) than the lungs of dogs (-80 ± 144 pmol·mg protein-1·min-1 less than seals), sheep (-472 ± 96), rats (-664 ± 104) or mice (-1,160 ± 104, P < 0.0001). Amino acid sequences of the GC enzyme α-subunits differed between seals and terrestrial mammals, potentially affecting their structure and function. Vasoconstriction in diving Weddell seals is not consistent across tissues; perfusion is maintained in the brain and heart but decreased in other organs such as the kidney. A NO donor increased median GC activity 49.5-fold in the seal brain but only 27.4-fold in the kidney, consistent with the priority of cerebral perfusion during diving. Nos3 expression was high in the seal brain, which could improve NO production and vasodilatory potential. Conversely, Pde5a expression was high in the seal renal artery, which may increase cGMP breakdown and vasoconstriction in the kidney. Taken together, the results of this study suggest that alterations in the NO-cGMP pathway facilitate the diving response.

Arginase impairs hypoxic pulmonary vasoconstriction in murine endotoxemia.

BACKGROUND: Hypoxic pulmonary vasoconstriction (HPV) optimizes the match between ventilation and perfusion in the lung by reducing blood flow to poorly ventilated regions. Sepsis and endotoxemia impair HPV. We previously showed that nitric oxide synthase 2 (NOS2) is required, but not sufficient, for the effect of endotoxin on HPV. The aim of the current study was to identify additional factors that might contribute to the impairment of HPV during endotoxemia. METHODS: Gene expression profiling was determined using pulmonary tissues from NOS2-deficient (NOS2-/-) and wild-type mice subjected to endotoxin or saline challenge (control). HPV was accessed as the percentage increase in left pulmonary vascular resistance (LPVR) in response to left main bronchus occlusion (LMBO) in wild-type mice. RESULTS: Among the 22,690 genes analyzed, endotoxin induced a greater than three-fold increase in 59 and 154 genes in the lungs of wild-type and NOS2-/- mice, respectively. Of all the genes induced by endotoxin in wild-type mice, arginase 1 (Arg1) showed the greatest increase (16.3-fold compared to saline treated wild-type mice). In contrast, endotoxin did not increase expression of Arg1 in NOS2-/- mice. There was no difference in the endotoxin-induced expression of Arg2 between wild-type and NOS2-deficient mice. We investigated the role of arginase in HPV by treating the mice with normal saline or the arginase inhibitor Nω-hydroxy-nor-L-arginine (norNOHA). In control mice (in the absence of endotoxin) treated with normal saline, HPV was intact as determined by profound LMBO-induced increase in LPVR (121 ± 22% from baseline). During endotoxemia and treatment with normal saline, HPV was impaired compared to normal saline treated control mice (33 ± 9% vs. 121 ± 22%, P < 0.05). HPV was restored in endotoxin-exposed mice after treatment with the arginase inhibitor norNOHA as shown by the comparison to endotoxemic mice treated with normal saline (113 ± 29% vs, 33 ± 9%, P < 0.05) and to control mice treated with normal saline (113 ± 29% vs, 121 ± 22%, P = 0.97). CONCLUSIONS: The results of this study suggest that endotoxemia induces Arg1 and that arginase contributes to the endotoxin-induced impairment of HPV in mice.

Cross-linked hemoglobin bis-tetramers from bioorthogonal coupling do not induce vasoconstriction in the circulation.

BACKGROUND: Hemoglobin-based oxygen carriers (HBOCs) are potential alternatives to red blood cells in transfusions. Clinical trials using early versions of HBOCs noted adverse effects that appeared to result from removal of the vasodilator nitric oxide (NO). Previous reports suggest that size-enlarged HBOCs may avoid NO-rich regions along the vasculature and therefore not cause vasoconstriction and hypertension. STUDY DESIGN AND METHODS: Hemoglobin (Hb) bis-tetramers (bis-tetramers of hemoglobin that are prepared using CuAAC chemistry [BT-Hb] and bis-tetramers of hemoglobin that are specifically acetylated and prepared using CuAAC chemistry [BT-acHb]) can be reliably produced by a bio-orthogonal cyclo-addition approach. We considered that an HBOC derived from chemical coupling of two Hbs would be sufficiently large to avoid NO scavenging and related side effects. The ability of intravenously infused BT-Hb and BT-acHb to remain in the circulation without causing hypertension were determined in wild-type (WT) and diabetic (db/db) mouse models. RESULTS: In WT mice, the coupled oxygen-carrying proteins retained their function over several hours after administration. No significant changes in systolic blood pressure from baseline were observed after intravenous infusion of BT-Hb or BT-acHb in awake WT and db/db mice. In contrast, infusion of native Hb or cross-linked Hb tetramers in both animal models induced systemic hypertension. CONCLUSION: The results of this study indicate that bis-tetrameric HBOCs derived from the bio-orthogonal cyclo-addition process are likely to overcome clinical issues that arise from NO scavenging by Hb derivatives.

A Triazole Disulfide Compound Increases the Affinity of Hemoglobin for Oxygen and Reduces the Sickling of Human Sickle Cells.

Sickle cell disease is an inherited disorder of hemoglobin (Hb). During a sickle cell crisis, deoxygenated sickle hemoglobin (deoxyHbS) polymerizes to form fibers in red blood cells (RBCs), causing the cells to adopt "sickled" shapes. Using small molecules to increase the affinity of Hb for oxygen is a potential approach to treating sickle cell disease, because oxygenated Hb interferes with the polymerization of deoxyHbS. We have identified a triazole disulfide compound (4,4'-di(1,2,3-triazolyl)disulfide, designated TD-3), which increases the affinity of Hb for oxygen. The crystal structures of carboxy- and deoxy-forms of human adult Hb (HbA), each complexed with TD-3, revealed that one molecule of the monomeric thiol form of TD-3 (5-mercapto-1H-1,2,3-triazole, designated MT-3) forms a disulfide bond with ß-Cys93, which inhibits the salt-bridge formation between ß-Asp94 and ß-His146. This inhibition of salt bridge formation stabilizes the R-state and destabilizes the T-state of Hb, resulting in reduced magnitude of the Bohr effect and increased affinity of Hb for oxygen. Intravenous administration of TD-3 (100 mg/kg) to C57BL/6 mice increased the affinity of murine Hb for oxygen, and the mice did not appear to be adversely affected by the drug. TD-3 reduced in vitro hypoxia-induced sickling of human sickle RBCs. The percentage of sickled RBCs and the P50 of human SS RBCs by TD-3 were inversely correlated with the fraction of Hb modified by TD-3. Our study shows that TD-3, and possibly other triazole disulfide compounds that bind to Hb ß-Cys93, may provide new treatment options for patients with sickle cell disease.

Development of a portable mini-generator to safely produce nitric oxide for the treatment of infants with pulmonary hypertension.

OBJECTIVES: To test the safety of a novel miniaturized device that produces nitric oxide (NO) from air by pulsed electrical discharge, and to demonstrate that the generated NO can be used to vasodilate the pulmonary vasculature in rabbits with chemically-induced pulmonary hypertension. STUDY DESIGN: A miniature NO (mini-NO) generator was tested for its ability to produce therapeutic levels (20-80 parts per million (ppm)) of NO, while removing potentially toxic gases and metal particles. We studied healthy 6-month-old New Zealand rabbits weighing 3.4 ±â€¯0.4 kg (mean ±â€¯SD, n = 8). Pulmonary hypertension was induced by chemically increasing right ventricular systolic pressure to 28-30 mmHg. The mini-NO generator was placed near the endotracheal tube. Production of NO was triggered by a pediatric airway flowmeter during the first 0.5 s of inspiration. RESULTS: In rabbits with acute pulmonary hypertension, the mini-NO generator produced sufficient NO to induce pulmonary vasodilation. Potentially toxic nitrogen dioxide (NO2) and ozone (O3) were removed by the Ca(OH)2 scavenger. Metallic particles, released from the electrodes by the electric plasma, were removed by a 0.22 µm filter. While producing 40 ppm NO, the mini-NO generator was cooled by a flow of air (70 ml/min) and the external temperature of the housing did not exceed 31 °C. CONCLUSIONS: The mini-NO generator safely produced therapeutic levels of NO from air. The mini-NO generator is an effective and economical approach to producing NO for treating neonatal pulmonary hypertension and will increase the accessibility and therapeutic uses of life-saving NO therapy worldwide.

Pharmacological preconditioning with inhaled nitric oxide (NO): Organ-specific differences in the lifetime of blood and tissue NO metabolites.

BACKGROUND: Endogenous nitric oxide (NO) may contribute to ischemic and anesthetic preconditioning while exogenous NO protects against ischemia-reperfusion (I/R) injury in the heart and other organs. Why those beneficial effects observed in animal models do not always translate into clinical effectiveness remains unclear. To mitigate reperfusion damage a source of NO is required. NO inhalation is known to increase tissue NO metabolites, but little information exists about the lifetime of these species. We therefore sought to investigate the fate of major NO metabolite classes following NO inhalation in mice in vivo. METHODS: C57BL/6J mice were exposed to 80 ppm NO for 1 h. NO metabolites were measured in blood (plasma and erythrocytes) and tissues (heart, liver, lung, kidney and brain) immediately after NO exposure and up to 48 h thereafter. Concentrations of S-nitrosothiols, N-nitrosamines and NO-heme products as well as nitrite and nitrate were quantified by gas-phase chemiluminescence and ion chromatography. In separate experiments, mice breathed 80 ppm NO for 1 h prior to cardiac I/R injury (induced by coronary arterial ligation for 1 h, followed by recovery). After sacrifice, the size of the myocardial infarction (MI) and the area at risk (AAR) were measured. RESULTS: After NO inhalation, elevated nitroso/nitrosyl levels returned to baseline over the next 24 h, with distinct multi-phasic decay profiles in each compartment. S/N-nitroso compounds and NO-hemoglobin in blood decreased exponentially, but remained above baseline for up to 30min, whereas nitrate was elevated for up to 3hrs after discontinuing NO breathing. Hepatic S/N-nitroso species concentrations remained steady for 30min before dropping exponentially. Nitrate only rose in blood, liver and kidney; nitrite tended to be lower in all organs immediately after NO inhalation but fluctuated considerably in concentration thereafter. NO inhalation before myocardial ischemia decreased the ratio of MI/AAR by 30% vs controls (p = 0.002); only cardiac S-nitrosothiols and NO-hemes were elevated at time of reperfusion onset. CONCLUSIONS: Metabolites in blood do not reflect NO metabolite status of any organ. Although NO is rapidly inactivated by hemoglobin-mediated oxidation in the circulation, long-lived tissue metabolites may account for the myocardial preconditioning effects of inhaled NO. NO inhalation may afford similar protection in other organs.