Arkadi Beloiartsev

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 NG-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. NG-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 Attenuates the Adverse Effects of Transfusing Stored Syngeneic Erythrocytes in Mice with Endothelial Dysfunction after Hemorrhagic Shock

Background:The authors investigated whether transfusion with stored erythrocytes would increase tissue injury, inflammation, oxidative stress, and mortality (adverse effects of transfusing stored erythrocytes) in a murine model of hemorrhagic shock. They tested whether the adverse effects associated with transfusing stored erythrocytes were exacerbated by endothelial dysfunction and ameliorated by inhaling nitric oxide. Methods:The authors studied mice fed a high-fat diet (HFD-fed; to induce endothelial dysfunction) or a standard diet for 4–6 weeks. Mice were subjected to 90 min of hemorrhagic shock, followed by resuscitation with leukoreduced syngeneic erythrocytes stored less than 24 h (fresh erythrocytes) or stored for 2 weeks (stored erythrocytes). Results:In standard-diet–fed mice at 2 h after resuscitation, transfusion with stored erythrocytes increased tissue injury more than transfusion with fresh erythrocytes. The adverse effects of transfusing stored erythrocytes were more marked in HFD-fed mice and associated with increased lactate levels and short-term mortality. Compared with fresh erythrocytes, resuscitation with stored erythrocytes was associated with a reduction in P50, increased plasma hemoglobin levels, and increased indices of inflammation and oxidative stress, effects that were exacerbated in HFD-fed mice. Inhaled nitric oxide reduced tissue injury, lactate levels, and indices of inflammation and oxidative stress and improved short-term survival in HFD-fed mice resuscitated with stored erythrocytes. Conclusions:Resuscitation with stored erythrocytes adversely impacts outcome in mice with hemorrhagic shock, an effect that is exacerbated in mice with endothelial dysfunction. Inhaled nitric oxide reduces tissue injury and improves short-term survival in HFD-fed mice resuscitated with stored erythrocytes.

Adverse effects of hemorrhagic shock resuscitation with stored blood are ameliorated by inhaled nitric oxide in lambs

Objectives:Transfusion of stored RBCs is associated with increased morbidity and mortality in trauma patients. Plasma hemoglobin scavenges nitric oxide, which can cause vasoconstriction, induce inflammation, and activate platelets. We hypothesized that transfusion of RBCs stored for prolonged periods would induce adverse effects (pulmonary vasoconstriction, tissue injury, inflammation, and platelet activation) in lambs subjected to severe hemorrhagic shock and that concurrent inhalation of nitric oxide would prevent these adverse effects. Design:Animal study. Setting:Research laboratory at the Massachusetts General Hospital, Boston, MA. Subjects:Seventeen awake Polypay-breed lambs. Interventions:Lambs were subjected to 2 hours of hemorrhagic shock by acutely withdrawing 50% of their blood volume. Lambs were resuscitated with autologous RBCs stored for 2 hours or less (fresh) or 39 ± 2 (mean ± SD) days (stored). Stored RBCs were administered with or without breathing nitric oxide (80 ppm) during resuscitation and for 21 hours thereafter. Measurements and Main Results:We measured hemodynamic and oxygenation variables, markers of tissue injury and inflammation, plasma hemoglobin concentrations, and platelet activation. Peak pulmonary arterial pressure was higher after resuscitation with stored than with fresh RBCs (24 ± 4 vs 14 ± 2 mm Hg, p < 0.001) and correlated with peak plasma hemoglobin concentrations (R2 = 0.56, p = 0.003). At 21 hours after resuscitation, pulmonary myeloperoxidase activity was higher in lambs resuscitated with stored than with fresh RBCs (11 ± 2 vs 4 ± 1 U/g, p = 0.007). Furthermore, transfusion of stored RBCs increased plasma markers of tissue injury and sensitized platelets to adenosine diphosphate activation. Breathing nitric oxide prevented the pulmonary hypertension and attenuated the pulmonary myeloperoxidase activity, as well as tissue injury and sensitization of platelets to adenosine diphosphate. Conclusions:Our data suggest that resuscitation of lambs from hemorrhagic shock with autologous stored RBCs induces pulmonary hypertension and inflammation, which can be ameliorated by breathing nitric oxide.

Deletion of the Murine Cytochrome P450 Cyp2j Locus by Fused BAC-Mediated Recombination Identifies a Role for Cyp2j in the Pulmonary Vascular Response to Hypoxia

Epoxyeicosatrienoic acids (EETs) confer vasoactive and cardioprotective functions. Genetic analysis of the contributions of these short-lived mediators to pathophysiology has been confounded to date by the allelic expansion in rodents of the portion of the genome syntenic to human CYP2J2, a gene encoding one of the principle cytochrome P450 epoxygenases responsible for the formation of EETs in humans. Mice have eight potentially functional genes that could direct the synthesis of epoxygenases with properties similar to those of CYP2J2. As an initial step towards understanding the role of the murine Cyp2j locus, we have created mice bearing a 626-kb deletion spanning the entire region syntenic to CYP2J2, using a combination of homologous and site-directed recombination strategies. A mouse strain in which the locus deletion was complemented by transgenic delivery of BAC sequences encoding human CYP2J2 was also created. Systemic and pulmonary hemodynamic measurements did not differ in wild-type, null, and complemented mice at baseline. However, hypoxic pulmonary vasoconstriction (HPV) during left mainstem bronchus occlusion was impaired and associated with reduced systemic oxygenation in null mice, but not in null mice bearing the human transgene. Administration of an epoxygenase inhibitor to wild-type mice also impaired HPV. These findings demonstrate that Cyp2j gene products regulate the pulmonary vascular response to hypoxia.

Exposure of stored packed erythrocytes to NO prevents transfusion-associated pulmonary hypertension

Background:Transfusion of packed erythrocytes stored for a long duration is associated with increased pulmonary arterial pressure and vascular resistance. Prolonged storage decreases erythrocyte deformability, and older erythrocytes are rapidly removed from the circulation after transfusion. The authors studied whether treating stored packed ovine erythrocytes with NO before transfusion could prevent pulmonary vasoconstriction, enhance erythrocyte deformability, and prolong erythrocyte survival after transfusion. Methods:Ovine leukoreduced packed erythrocytes were treated before transfusion with either NO gas or a short-lived NO donor. Sheep were transfused with autologous packed erythrocytes, which were stored at 4°C for either 2 (“fresh blood”) or 40 days (“stored blood”). Pulmonary and systemic hemodynamic parameters were monitored before, during, and after transfusion. Transfused erythrocytes were labeled with biotin to measure their circulating lifespan. Erythrocyte deformability was assessed before and after NO treatment using a microfluidic device. Results:NO treatment improved the deformability of stored erythrocytes and increased the number of stored erythrocytes circulating at 1 and 24 h after transfusion. NO treatment prevented transfusion-associated pulmonary hypertension (mean pulmonary arterial pressure at 30 min of 21 ± 1 vs. 15 ± 1 mmHg in control and NO–treated packed erythrocytes, P < 0.0001). Washing stored packed erythrocytes before transfusion did not prevent pulmonary hypertension. Conclusions:NO treatment of stored packed erythrocytes before transfusion oxidizes cell-free oxyhemoglobin to methemoglobin, prevents subsequent NO scavenging in the pulmonary vasculature, and limits pulmonary hypertension. NO treatment increases erythrocyte deformability and erythrocyte survival after transfusion. NO treatment might provide a promising therapeutic approach to prevent pulmonary hypertension and extend erythrocyte survival.

Soluble epoxide hydrolase deficiency or inhibition enhances murine hypoxic pulmonary vasoconstriction after lipopolysaccharide challenge.

Hypoxic pulmonary vasoconstriction (HPV) is the response of the pulmonary vasculature to low levels of alveolar oxygen. HPV improves systemic arterial oxygenation by matching pulmonary perfusion to ventilation during alveolar hypoxia and is impaired in lung diseases such as the acute respiratory distress syndrome (ARDS) and in experimental models of endotoxemia. Epoxyeicosatrienoic acids (EETs) are pulmonary vasoconstrictors, which are metabolized to less vasoactive dihydroxyeicosatrienoic acids (DHETs) by soluble epoxide hydrolase (sEH). We hypothesized that pharmacological inhibition or a congenital deficiency of sEH in mice would reduce the metabolism of EETs and enhance HPV in mice after challenge with lipopolysaccharide (LPS). HPV was assessed 22 h after intravenous injection of LPS by measuring the percentage increase in the pulmonary vascular resistance of the left lung induced by left mainstem bronchial occlusion (LMBO). After LPS challenge, HPV was impaired in sEH+/+, but not in sEH-/- mice or in sEH+/+ mice treated acutely with a sEH inhibitor. Deficiency or pharmacological inhibition of sEH protected mice from the LPS-induced decrease in systemic arterial oxygen concentration (PaO2 ) during LMBO. In the lungs of sEH-/- mice, the LPS-induced increase in DHETs and cytokines was attenuated. Deficiency or pharmacological inhibition of sEH protects mice from LPS-induced impairment of HPV and improves the PaO2 after LMBO. After LPS challenge, lung EET degradation and cytokine expression were reduced in sEH-/- mice. Inhibition of sEH might prove to be an effective treatment for ventilation-perfusion mismatch in lung diseases such as ARDS.

Pulmonary hypertension after prolonged hypoxic exposure in mice with a congenital deficiency of Cyp2j.

Cytochrome P450 epoxygenase-derived epoxyeicosatrienoic acids contribute to the regulation of pulmonary vascular tone and hypoxic pulmonary vasoconstriction. We investigated whether the attenuated acute vasoconstrictor response to hypoxic exposure of Cyp2j(-/-) mice would protect these mice against the pulmonary vascular remodeling and hypertension associated with prolonged exposure to hypoxia. Cyp2j(-/-) and Cyp2j(+/+) male and female mice continuously breathed an inspired oxygen fraction of 0.21 (normoxia) or 0.10 (hypoxia) in a normobaric chamber for 6 weeks. We assessed hemoglobin (Hb) concentrations, right ventricular (RV) systolic pressure (RVSP), and transthoracic echocardiographic parameters (pulmonary acceleration time [PAT] and RV wall thickness). Pulmonary Cyp2c29, Cyp2c38, and sEH mRNA levels were measured in Cyp2j(-/-) and Cyp2j(+/+) male mice. At baseline, Cyp2j(-/-) and Cyp2j(+/+) mice had similar Hb levels and RVSP while breathing air. After 6 weeks of hypoxia, circulating Hb concentrations increased but did not differ between Cyp2j(-/-) and Cyp2j(+/+) mice. Chronic hypoxia increased RVSP in Cyp2j(-/-) and Cyp2j(+/+) mice of either gender. Exposure to chronic hypoxia decreased PAT and increased RV wall thickness in both genotypes and genders to a similar extent. Prolonged exposure to hypoxia produced similar levels of RV hypertrophy in both genotypes of either gender. Pulmonary Cyp2c29, Cyp2c38, and sEH mRNA levels did not differ between Cyp2j(-/-) and Cyp2j(+/+) male mice after breathing at normoxia or hypoxia for 6 weeks. These results suggest that murine Cyp2j deficiency does not attenuate the development of murine pulmonary vascular remodeling and hypertension associated with prolonged exposure to hypoxia in mice of both genders.

Hemoglobin infusion does not alter murine pulmonary vascular tone

Plasma hemoglobin (Hb) scavenges endothelium-derived nitric oxide (NO), producing systemic and pulmonary vasoconstriction in many species. We hypothesized that i.v. administration of murine cell-free Hb would produce pulmonary vasoconstriction and enhance hypoxic pulmonary vasoconstriction (HPV) in mice. To assess the impact of plasma Hb on basal pulmonary vascular tone in anesthetized mice we measured left lung pulmonary vascular resistance (LPVRI) before and after infusion of Hb at thoracotomy. To confirm the findings obtained at thoracotomy, measurements of right ventricular systolic pressure (RVSP) and systemic arterial pressure (SAP) were obtained in closed-chest wild-type mice. To elucidate whether pretreatment with Hb augments HPV we assessed the increase in LPVRI before and during regional lung hypoxia produced by left mainstem bronchial occlusion (LMBO) in wild-type mice pretreated with Hb. Infusion of Hb increased SAP but did not change pulmonary arterial pressure (PAP), left lung pulmonary arterial flow (QLPA) or LPVRI in either wild-type or diabetic mice with endothelial dysfunction. Scavenging of NO by plasma Hb did not alter HPV in wild-type mice. Inhibition of NO synthase with l-NAME did not change the basal LPVRI, but augmented HPV during LMBO. Our data suggest that scavenging of NO by plasma Hb does not alter pulmonary vascular tone in mice. Therefore, generation of NO in the pulmonary circulation is unlikely to be responsible for the low basal pulmonary vascular tone of mice.