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.

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.

Ideation and assessment of a nitric oxide delivery system for spontaneously breathing subjects.

BACKGROUND: There is an increasing interest in safely delivering high dose of inhaled nitric oxide (NO) as an antimicrobial and antiviral therapeutics for spontaneously breathing patients. A novel NO delivery system is described. METHODS: We developed a gas delivery system that utilizes standard respiratory circuit connectors, a reservoir bag, and a scavenging chamber containing calcium hydroxide. The performance of the system was tested using a mechanical lung, assessing the NO concentration delivered at varying inspiratory flows. Safety was assessed in vitro and in vivo by measuring nitrogen dioxide (NO2) levels in the delivered NO gas. Lastly, we measured the inspired and expired NO and NO2 of this system in 5 healthy subjects during a 15-min administration of high dose NO (160 parts-per-million, ppm) using our delivery system. RESULTS: The system demonstrated stable delivery of prescribed NO levels at various inspiratory flow rates (0-50 L/min). The reservoir bag and a high flow of entering air minimized the oscillation of NO concentrations during inspiration on average 4.6 ppm for each 10 L/min increment in lung inspiratory flow. The calcium hydroxide scavenger reduced the inhaled NO2 concentration on average 0.9 ppm (95% CI -1.58, -0.22; p = .01). We performed 49 NO administrations of 160 ppm in 5 subjects. The average concentration of inspired NO was 164.8±10.74 ppm, with inspired NO2 levels of 0.7±0.13 ppm. The subjects did not experience any adverse events; transcutaneous methemoglobin concentrations increased from 1.05±0.58 to 2.26±0.47%. CONCLUSIONS: The system we developed to administer high-dose NO for inhalation is easy to build, reliable, was well tolerated in healthy subjects.

Phototherapy and extracorporeal membrane oxygenation facilitate removal of carbon monoxide in rats.

Inhaled carbon monoxide (CO) displaces oxygen from hemoglobin, reducing the capacity of blood to carry oxygen. Current treatments for CO-poisoned patients involve administration of 100% oxygen; however, when CO poisoning is associated with acute lung injury secondary to smoke inhalation, burns, or trauma, breathing 100% oxygen may be ineffective. Visible light dissociates CO from hemoglobin. We hypothesized that the exposure of blood to visible light while passing through a membrane oxygenator would increase the rate of CO elimination in vivo. We developed a membrane oxygenator with optimal characteristics to facilitate exposure of blood to visible light and tested the device in a rat model of CO poisoning, with or without concomitant lung injury. Compared to ventilation with 100% oxygen, the addition of extracorporeal removal of CO with phototherapy (ECCOR-P) doubled the rate of CO elimination in CO-poisoned rats with normal lungs. In CO-poisoned rats with acute lung injury, treatment with ECCOR-P increased the rate of CO removal by threefold compared to ventilation with 100% oxygen alone and was associated with improved survival. Further development and adaptation of this extracorporeal CO photo-removal device for clinical use may provide additional benefits for CO-poisoned patients, especially for those with concurrent acute lung injury.

Pulmonary Phototherapy to Treat Carbon Monoxide Poisoning in Rats.

BACKGROUND: Carbon monoxide (CO) poisoning is a common cause of poison-related mortality. CO binds to hemoglobin in the blood to form carboxyhemoglobin (COHb), impairing oxygen delivery to peripheral tissues. Current treatment of CO-poisoned patients involves oxygen administration to rapidly remove CO and restore oxygen delivery. Light dissociates CO from COHb with high efficiency. Exposure of murine lungs to visible laser-generated light improved the CO elimination rate in vivo. The aims of this study were to apply pulmonary phototherapy to a larger animal model of CO poisoning, to test novel approaches to light delivery, and to examine the effect of chemiluminescence-generated light on the CO elimination rate. METHODS: Anesthetized and mechanically ventilated rats were poisoned with CO and subsequently treated with air or oxygen combined with or without pulmonary phototherapy delivered directly to the lungs of animals at thoracotomy, via intrapleural optical fibers or generated by a chemiluminescent reaction. RESULTS: Direct pulmonary phototherapy dissociated CO from COHb reducing COHb half-life by 38%. Early treatment with phototherapy in critically CO poisoned rats improved lactate clearance. Light delivered to the lungs of rats via intrapleural optical fibers increased the rate of CO elimination without requiring a thoracotomy, as demonstrated by a 16% reduction in COHb half-life. Light generated in the pleural spaces by a chemiluminescent reaction increased the rate of CO elimination in rats breathing oxygen, reducing the COHb half-life by 12%. CONCLUSIONS: Successful application of pulmonary phototherapy in larger animals and humans may represent a significant advance in the treatment of CO-poisoned patients.

Pulmonary Phototherapy for Treating Carbon Monoxide Poisoning.

RATIONALE: Carbon monoxide (CO) exposure is a leading cause of poison-related mortality. CO binds to Hb, forming carboxyhemoglobin (COHb), and produces tissue damage. Treatment of CO poisoning requires rapid removal of CO and restoration of oxygen delivery. Visible light is known to effectively dissociate CO from Hb, with a single photon dissociating one CO molecule. OBJECTIVES: To determine whether illumination of the lungs of CO-poisoned mice causes dissociation of COHb from blood transiting the lungs, releasing CO into alveoli and thereby enhancing the rate of CO elimination. METHODS: We developed a model of CO poisoning in anesthetized and mechanically ventilated mice to assess the effects of direct lung illumination (phototherapy) on the CO elimination rate. Light at wavelengths between 532 and 690 nm was tested. The effect of lung phototherapy administered during CO poisoning was also studied. To avoid a thoracotomy, we assessed the effect of lung phototherapy delivered to murine lungs via an optical fiber placed in the esophagus. MEASUREMENTS AND MAIN RESULTS: In CO-poisoned mice, phototherapy of exposed lungs at 532, 570, 592, and 628 nm dissociated CO from Hb and doubled the CO elimination rate. Phototherapy administered during severe CO poisoning limited the blood COHb increase and improved the survival rate. Noninvasive transesophageal phototherapy delivered to murine lungs via an optical fiber increased the rate of CO elimination while avoiding a thoracotomy. CONCLUSIONS: Future development and scaling up of lung phototherapy for patients with CO exposure may provide a significant advance for treating and preventing CO poisoning.

Autologous transfusion of stored red blood cells increases pulmonary artery pressure.

RATIONALE: Transfusion of erythrocytes stored for prolonged periods is associated with increased mortality. Erythrocytes undergo hemolysis during storage and after transfusion. Plasma hemoglobin scavenges endogenous nitric oxide leading to systemic and pulmonary vasoconstriction. OBJECTIVES: We hypothesized that transfusion of autologous blood stored for 40 days would increase the pulmonary artery pressure in volunteers with endothelial dysfunction (impaired endothelial production of nitric oxide). We also tested whether breathing nitric oxide before and during transfusion could prevent the increase of pulmonary artery pressure. METHODS: Fourteen obese adults with endothelial dysfunction were enrolled in a randomized crossover study of transfusing autologous, leukoreduced blood stored for either 3 or 40 days. Volunteers were transfused with 3-day blood, 40-day blood, and 40-day blood while breathing 80 ppm nitric oxide. MEASUREMENTS AND MAIN RESULTS: The age of volunteers was 41 ± 4 years (mean ± SEM), and their body mass index was 33.4 ± 1.3 kg/m(2). Plasma hemoglobin concentrations increased after transfusion with 40-day and 40-day plus nitric oxide blood but not after transfusing 3-day blood. Mean pulmonary artery pressure, estimated by transthoracic echocardiography, increased after transfusing 40-day blood (18 ± 2 to 23 ± 2 mm Hg; P < 0.05) but did not change after transfusing 3-day blood (17 ± 2 to 18 ± 2 mm Hg; P = 0.5). Breathing nitric oxide decreased pulmonary artery pressure in volunteers transfused with 40-day blood (17 ± 2 to 12 ± 1 mm Hg; P < 0.05). CONCLUSIONS: Transfusion of autologous leukoreduced blood stored for 40 days was associated with increased plasma hemoglobin levels and increased pulmonary artery pressure. Breathing nitric oxide prevents the increase of pulmonary artery pressure produced by transfusing stored blood. Clinical trial registered with www.clinicaltrials.gov (NCT 01529502).

Transfusion of stored autologous blood does not alter reactive hyperemia index in healthy volunteers.

BACKGROUND: Transfusion of human blood stored for more than 2 weeks is associated with increased mortality and morbidity. During storage, packed erythrocytes progressively release hemoglobin, which avidly binds nitric oxide. We hypothesized that the nitric oxide mediated hyperemic response after ischemia would be reduced after transfusion of packed erythrocytes stored for 40 days. METHODS AND RESULTS: We conducted a crossover randomized interventional study, enrolling 10 healthy adults. Nine volunteers completed the study. Each volunteer received one unit of 40-day and one of 3-day stored autologous leukoreduced packed erythrocytes, on different study days according to a randomization scheme. Blood withdrawal and reactive hyperemia index measurements were performed before and 10 min, 1 h, 2 h, and 4 h after transfusion. The reactive hyperemia index during the first 4 h after transfusion of 40-day compared with 3-day stored packed erythrocytes was unchanged. Plasma hemoglobin and bilirubin concentrations were higher after transfusion of 40-day than after 3-day stored packed erythrocytes (P = 0.02, [95% CI difference 10-114 mg/l] and 0.001, [95% CI difference 0.6-1.5 mg/dl], respectively). Plasma concentrations of potassium, lactate dehydrogenase, haptoglobin, and cytokines, as well as blood pressure, did not differ between the two transfusions and remained within the normal range. Plasma nitrite concentrations increased after transfusion of 40-day stored packed erythrocytes, but not after transfusion of 3-day stored packed erythrocytes (P = 0.01, [95% CI difference 0.446-0.66 µM]). CONCLUSIONS: Transfusion of autologous packed erythrocytes stored for 40 days is associated with increased hemolysis, an unchanged reactive hyperemia index, and increased concentrations of plasma nitrite.

Pulmonary hypertension in lambs transfused with stored blood is prevented by breathing nitric oxide.

BACKGROUND: During extended storage, erythrocytes undergo functional changes. These changes reduce the viability of erythrocytes leading to release of oxyhemoglobin, a potent scavenger of nitric oxide. We hypothesized that transfusion of ovine packed erythrocytes (PRBC) stored for prolonged periods would induce pulmonary vasoconstriction in lambs, and that reduced vascular nitric oxide concentrations would increase this vasoconstrictor effect. METHODS: We developed a model of autologous stored blood transfusion in lambs (n = 36). Leukoreduced blood was stored for either 2 days (fresh PRBC) or 40 days (stored PRBC). Fresh or stored PRBC were transfused into donors instrumented for awake hemodynamic measurements. Hemodynamic effects of PRBC transfusion were also studied after infusion of N-nitro-L-arginine methyl-ester (25 mg/kg) or during inhalation of nitric oxide (80 ppm). RESULTS: Cell-free hemoglobin levels were higher in the supernatant of stored PRBC than in supernatant of fresh PRBC (Mean ± SD, 148 ± 20 vs. 41 ± 13 mg/dl, respectively, P < 0.001). Pulmonary artery pressure during transfusion of stored PRBC transiently increased from 13 ± 1 to 18 ± 1 mmHg (P < 0.001) and was associated with increased plasma hemoglobin concentrations. N-nitro-L-arginine methyl-ester potentiated the increase in pulmonary arterial pressure induced by transfusing stored PRBC, whereas inhalation of nitric oxide prevented the vasoconstrictor response. CONCLUSIONS: Our results suggest that patients with reduced vascular nitric oxide levels because of endothelial dysfunction may be more susceptible to adverse effects of transfusing blood stored for prolonged periods. These patients might benefit from transfusion of fresh PRBC, when available, or inhaled nitric oxide supplementation to prevent the pulmonary hypertension associated with transfusion of stored PRBC.

Inhaled nitric oxide enables artificial blood transfusion without hypertension.

BACKGROUND: One of the major obstacles hindering the clinical development of a cell-free, hemoglobin-based oxygen carrier (HBOC) is systemic vasoconstriction. METHODS AND RESULTS: Experiments were performed in healthy mice and lambs by infusion of either murine tetrameric hemoglobin (0.48 g/kg) or glutaraldehyde-polymerized bovine hemoglobin (HBOC-201, 1.44 g/kg). We observed that intravenous infusion of either murine tetrameric hemoglobin or HBOC-201 induced prolonged systemic vasoconstriction in wild-type mice but not in mice congenitally deficient in endothelial nitric oxide (NO) synthase (NOS3). Treatment of wild-type mice by breathing NO at 80 ppm in air for 15 or 60 minutes or with 200 ppm NO for 7 minutes prevented the systemic hypertension induced by subsequent intravenous administration of murine tetrameric hemoglobin or HBOC-201 and did not result in conversion of plasma hemoglobin to methemoglobin. Intravenous administration of sodium nitrite (48 nmol) 5 minutes before infusion of murine tetrameric hemoglobin also prevented the development of systemic hypertension. In awake lambs, breathing NO at 80 ppm for 1 hour prevented the systemic hypertension caused by subsequent infusion of HBOC-201. CONCLUSIONS: These findings demonstrate that HBOC can cause systemic vasoconstriction by scavenging NO produced by NOS3. Moreover, in 2 species, inhaled NO administered before the intravenous infusion of HBOC can prevent systemic vasoconstriction without causing methemoglobinemia.

Soluble guanylate cyclase activator reverses acute pulmonary hypertension and augments the pulmonary vasodilator response to inhaled nitric oxide in awake lambs.

BACKGROUND: Inhaled nitric oxide (NO) is a potent and selective pulmonary vasodilator, which induces cGMP synthesis by activating soluble guanylate cyclase (sGC) in ventilated lung regions. Carbon monoxide (CO) has also been proposed to influence smooth muscle tone via activation of sGC. We examined whether direct stimulation of sGC by BAY 41-2272 would produce pulmonary vasodilation and augment the pulmonary responses to inhaled NO or CO. METHODS AND RESULTS: In awake, instrumented lambs, the thromboxane analogue U-46619 was intravenously administered to increase mean pulmonary arterial pressure to 35 mm Hg. Intravenous infusion of BAY 41-2272 (0.03, 0.1, and 0.3 mg x kg(-1) x h(-1)) reduced mean pulmonary arterial pressure and pulmonary vascular resistance and increased transpulmonary cGMP release in a dose-dependent manner. Larger doses of BAY 41-2272 also produced systemic vasodilation and elevated the cardiac index. N(omega)-nitro-l-arginine methyl ester abolished the systemic but not the pulmonary vasodilator effects of BAY 41-2272. Furthermore, infusing BAY 41-2272 at 0.1 mg x kg(-1) x h(-1) potentiated and prolonged the pulmonary vasodilation induced by inhaled NO (2, 10, and 20 ppm). In contrast, inhaled CO (50, 250, and 500 ppm) had no effect on U-46619-induced pulmonary vasoconstriction before or during administration of BAY 41-2272. CONCLUSIONS: In lambs with acute pulmonary hypertension, BAY 41-2272 is a potent pulmonary vasodilator that augments and prolongs the pulmonary vasodilator response to inhaled NO. Direct pharmacological stimulation of sGC, either alone or in combination with inhaled NO, may provide a novel approach for the treatment of pulmonary hypertension.