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Profiling molecular changes induced by hydrogen treatment of lung allografts prior to procurement

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Abstract

We previously demonstrated that donor treatment with inhaled hydrogen protects lung grafts from cold ischemia/reperfusion (I/R) injury during lung transplantation. To elucidate the mechanisms underlying hydrogen's protective effects, we conducted a gene array analysis to identify changes in gene expression associated with hydrogen treatment. Donor rats were exposed to mechanical ventilation with 98% oxygen and 2% nitrogen or 2% hydrogen for 3h before harvest; lung grafts were stored for 4h in cold Perfadex. Affymetrix gene array analysis of mRNA transcripts was performed on the lung tissue prior to implantation. Pretreatment of donor lungs with hydrogen altered the expression of 229 genes represented on the array (182 upregulated; 47 downregulated). Hydrogen treatment induced several lung surfactant-related genes, ATP synthase genes and stress-response genes. The intracellular surfactant pool, tissue adenosine triphosphate (ATP) levels and heat shock protein 70 (HSP70) expression increased in the hydrogen-treated grafts. Hydrogen treatment also induced the transcription factors C/EBPα and C/EBPβ, which are known regulators of surfactant-related genes. Donor ventilation with hydrogen significantly increases expression of surfactant-related molecules, ATP synthases and stress-response molecules in lung grafts. The induction of these molecules may underlie hydrogen's protective effects against I/R injury during transplantation.

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... One of these is molecular hydrogen, which has both anti-inflammatory and -oxidative effects and is known to modulate signaling. Inhaled hydrogen gas has shown beneficial effects in lung transplantation, e.g., in a rat model of IRI, lung graft injury in a brain-dead donor, pulmonary microvascular endothelial cells during cold storage, and a pig model of ex vivo donor lung perfusion [5][6][7][8][9][10][11][12]. Molecular hydrogen can be easily administered through the oral route using a special apparatus in both clinical and experimental settings [13][14][15][16][17][18][19][20][21]. ...
... Molecular hydrogen has been reported to exert protective effects on experimental lung grafts [5][6][7][8][9][10][11][12]. Donor ventilation with hydrogen increased the expression of surfactantrelated molecules, adenosine triphosphate synthases, and stress response molecules in a lung IRI model [5]. ...
... Molecular hydrogen has been reported to exert protective effects on experimental lung grafts [5][6][7][8][9][10][11][12]. Donor ventilation with hydrogen increased the expression of surfactantrelated molecules, adenosine triphosphate synthases, and stress response molecules in a lung IRI model [5]. Similarly, hydrogen inhibited the secretion of pro-inflammatory mediators and stimulated the release of anti-inflammatory and -apoptotic molecules by suppressing p38 mitogen-activated protein kinase or nuclear factor-κB signaling in an in vitro model [6]. ...
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Objective Bronchiolitis obliterans syndrome arising from chronic airway inflammation is a leading cause of death following lung transplantation. Several studies have suggested that inhaled hydrogen can protect lung grafts from ischemia–reperfusion injury via anti-inflammatory and -oxidative mechanisms. We investigated whether molecular hydrogen-saturated water can preserve lung allograft function in a heterotopic tracheal allograft mouse model of obliterative airway disease Methods Obliterative airway disease was induced by heterotopically transplanting tracheal allografts from BALB/c donor mice into C57BL/6 recipient mice, which were subsequently administered hydrogen water (10 ppm) or tap water (control group) (n = 6 each) daily without any immunosuppressive treatment. Histological and immunohistochemical analyses were performed on days 7, 14, and 21. Results Hydrogen water decreased airway occlusion on day 14. No significant histological differences were observed on days 7 or 21. The cluster of differentiation 4/cluster of differentiation 3 ratio in tracheal allografts on day 14 was higher in the hydrogen water group than in control mice. Enzyme-linked immunosorbent assay performed on day 7 revealed that hydrogen water reduced the level of the pro-inflammatory cytokine interleukin-6 and increased that of forkhead box P3 transcription factor, suggesting an enhancement of regulatory T cell activity. Conclusions Hydrogen water suppressed the development of mid-term obliterative airway disease in a mouse tracheal allograft model via anti-oxidant and -inflammatory mechanisms and through the activation of Tregs. Thus, hydrogen water is a potential treatment strategy for BOS that can improve the outcome of lung transplant patients.
... In both the cardiac death model and brain death model, hydrogen inhalation could effectively reduce the expression level of proinflammatory factors such as IL-8, IL-6, and TNF-α, thus alleviating the lung injury of the donor before extracting the lung [35][36][37]. In addition, Tanaka et al. [38] sequenced hydrogen-pretreated transplant donors and found that hydrogen treatment induced the expression of proteins (including Clara cells) with anti-inflammatory and antioxidant effects and increased intracellular tissue adenosine triphosphate (ATP) and heat shock protein 70 (HSP70) expression levels. Hydrogen treatment also induced surfactants to regulate the expression of C/ EBPA and C/EBPB transcription factors. ...
... Masao et al. preserved donor lungs provided by canine or rat lung transplantation models in hydrogen-rich perfusion fluid. Compared with the control group, donor lungs maintained a higher oxygen partial pressure and had less perivascular edema in the transplanted lung [38]. Hydrogen-rich preservation solutions on the one hand can reduce the expression of proinflammatory cytokine (TNF-α and IL-1β) mRNA and on the other hand can inhibit the expression of 8-OHdG, which is an indicator Hydrogen-rich solution Cold ischemia phase [60] 2 Oxidative Medicine and Cellular Longevity of oxidative stress [39,40]. ...
Article
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Ischemia reperfusion injury (IRI) in organ transplantation has always been an important hotspot in organ protection. Hydrogen, as an antioxidant, has been shown to have anti-inflammatory, antioxidant, and antiapoptotic effects. In this paper, the protective effect of hydrogen against IRI in organ transplantation has been reviewed to provide clues for future clinical studies. 1. Introduction Ischemia reperfusion injury (IRI) is one of the most common clinical complications of organ transplantation [1]. The damage mechanism involves cell ion changes [2], mitochondrial metabolism [3], reactive oxygen species (ROS) system activation [4, 5], various inflammatory reactions [6, 7], and other pathophysiological changes. In severe cases, it may even cause primary graft dysfunction, prolong total hospital stay, and greatly increase mortality risk in solid organ transplant recipients [8–13]. Although various isolated organ protection platforms, such as ex vivo lung perfusion (EVLP) and LifePort Liver Transporter (LIFESPORT), have been developed clinically and extensive research and improvement have been achieved for organ preservation fluid [14–20], IRI cannot be completely prevented. Graft ischemia leads to the harmful production of ROS; however, the reoxygenation process during reperfusion is the reason for the production of most ROS, activation of the complement system, and initiation of inflammatory responses [21]. Occlusion of vascular supply during transplantation leads to severe hypoxia of endothelial cells, which become an important source and target of ROS. Mitochondrial dysfunction, neutrophil initiation, xanthine oxidase, and NADPH oxidase play key roles in this process [22] In turn, excessive oxidizing agents lead to tissue damage and cell death by inducing the peroxidation of DNA, proteins, and lipids. Therefore, use of anti-ROS agents has been an important strategy for reducing IRI during organ transplantation. Hydrogen is widely distributed in nature, with a concentration of 0.00006% in the air [23]. Under physiological states, human intestinal flora can produce a large amount of hydrogen, which participates in human physiological processes and is eventually discharged or metabolized from the lungs to produce nontoxic water [24]. Selective antioxidant function of hydrogen has been demonstrated in previous studies [25]; with the intensification of studies, hydrogen has been proved to exert several effects, such as anti-inflammatory [26–28], antioxidant [29, 30], and antiapoptosis effects [31, 32]. In recent years, the use of hydrogen has become an important part of the use of gases in medical treatments. Hydrogen has been used in various disease models and treatment studies, including IRI in solid organ transplantation. However, the specific mechanism of hydrogen in treating IRI in solid organ transplantation is not completely clear at present. Currently available experiments and studies have found that the mechanism may be related to its selective antioxidant effect and its ability to reduce inflammatory responses and inhibit cell apoptosis. The research progress of its application in solid organ transplantation is summarized below (Table 1). Organ Use-pattern Time Reference Lung 2–3% hydrogen Donor [35–38, 41] Lung Hydrogen-rich solution Cold ischemia phase [39, 40, 48] Lung 2% hydrogen EVLP [43, 45, 46] Lung 3% hydrogen PMVECs [42] Lung 3% hydrogen & CO Cold ischemia phase [46] Lung 2% hydrogen During lung transplantation [47] Liver Hydrogen-rich solution Cold ischemia phase [48, 50] Liver Hydrogen flush after cold storage Cold ischemia phase [49] Liver Hydrogen-rich perfusion fluid Cold ischemia phase [51] Kidney Hydrogen-rich solution Cold ischemia phase [52, 53] Small intestine Hydrogen-rich solution Cold ischemia phase [54, 55] Small intestine Hydrogen-bubbled preservation solution Cold ischemia phase [56] Small intestine 2% hydrogen Perioperative period [57] Heart 1–3% hydrogen 1 h before and after reperfusion [58] Heart Hydrogen-rich water bath Cold ischemia phase [59] Heart Hydrogen-rich solution Cold ischemia phase [60]
... The changes in these molecules may underlie the protective effects of hydrogen against I/R injury during LTx. 15 Preloading hydrogen during organ preservation ameliorates I/R injury of lung grafts In a rat model of LTx, we found that donor lung inflation with 3% hydrogen during the cold ischaemic period could reduce graft MPO activity and serum IL-8 and TNF-a levels, increase PaO 2 /FiO 2 and pulmonary venous oxygen tension (PvO 2 )/FiO 2 , resulting in alleviated lung graft injury and improved function. 16 In 2016, we demonstrated that inflation with CO or H 2 protected against I/R injury in a rat lung transplantation model, and this effect was enhanced by combined CO and H 2 treatment. ...
... The mRNA of dual specificity phos-phatase1, a dual specificity phosphatase that decreases MAPK phosphorylation/activation in the lung grafts, were upregulated by hydrogen, which resulted in reduced protein levels of p38 MAPK, ERK1/2, and JNK in the allografts. 15 We found that the anti-inflammatory effect of hydrogen may be due to a decrease in secretion of proinflammatory cytokines by inhibiting the activation of the p38 MAPK pathway in pulmonary microvascular endothelial cells. 18 Zhai et al. showed that HRS peritoneal injection inhibited the activation and phosphorylation of p38MAPK and NF-jB. ...
Article
Lung grafts may experience multiple injuries during lung transplantation, such as warm ischaemia, cold ischaemia, and reperfusion injury. These injuries all contribute to primary graft dysfunction, which is a major cause of morbidity and mortality after lung transplantation. As a potential selective antioxidant, hydrogen molecule (H2) protects against post-transplant complications in animal models of multiple organ transplantation. Herein, the authors review the current literature regarding the effects of H2 on lung injury from lung transplantation. The reviewed studies showed that H2 improved the outcomes of lung transplantation by decreasing oxidative stress and inflammation at the donor and recipient phases. H2 is primarily administered via inhalation, drinking hydrogen-rich water, hydrogen-rich saline injection, or a hydrogen-rich water bath. H2 favorably modulates signal transduction and gene expression, resulting in the suppression of pro-inflammatory cytokines and excess reactive oxygen species production. Although H2 appears to be a physiological regulatory molecule with antioxidant, anti-inflammatory and anti-apoptotic properties, its exact mechanisms of action remain elusive. Taken together, accumulating experimental evidence indicates that H2 can significantly alleviate transplantation-related lung injury, mainly via inhibition of inflammatory cytokine secretion and reduction in oxidative stress through several underlying mechanisms. Further animal experiments and preliminary human clinical trials will lay the foundation for the use of H2 as a treatment in the clinic.
... We have shown that hydrogen suppresses signaling pathways in allergies[26]and inflammation[27]without directly scavenging reactive oxygen/nitrogen species. Signaling molecules that are modulated by hydrogen include Lyn[26,28], Ras[29], MEK[29,30], ERK[12,24,[29][30][31][32][33][34][35][36][37], p38[12,16,24,27,30,32,33,[35][36][37][38][39][40][41], JNK[13, 24, 27, 30, 32, 33, 35–38, 40, 42–47], ASK1[27,46], Akt[12,29,36,37,48,49], GTPRac1[36], iNOS[27,34,36,[50][51][52], Nox1[36], NF-κB p65 or NF-κB[12, 14, 27, 35–38, 40, 41, 43, 49, 53–75], IκBα[27,40,41,54,60,62,69,73,76], STAT3[65,77,78], NFATc1[12,36,78], c-Fos[36], GSK-3β[48,79], ROCK[80]. Activities and expressions of these molecules are modified by hydrogen. ...
... We have shown that hydrogen suppresses signaling pathways in allergies[26]and inflammation[27]without directly scavenging reactive oxygen/nitrogen species. Signaling molecules that are modulated by hydrogen include Lyn[26,28], Ras[29], MEK[29,30], ERK[12,24,[29][30][31][32][33][34][35][36][37], p38[12,16,24,27,30,32,33,[35][36][37][38][39][40][41], JNK[13, 24, 27, 30, 32, 33, 35–38, 40, 42–47], ASK1[27,46], Akt[12,29,36,37,48,49], GTPRac1[36], iNOS[27,34,36,[50][51][52], Nox1[36], NF-κB p65 or NF-κB[12, 14, 27, 35–38, 40, 41, 43, 49, 53–75], IκBα[27,40,41,54,60,62,69,73,76], STAT3[65,77,78], NFATc1[12,36,78], c-Fos[36], GSK-3β[48,79], ROCK[80]. Activities and expressions of these molecules are modified by hydrogen. ...
Article
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Therapeutic effects of molecular hydrogen for a wide range of disease models and human diseases have been investigated since 2007. A total of 321 original articles have been published from 2007 to June 2015. Most studies have been conducted in Japan, China, and the USA. About three-quarters of the articles show the effects in mice and rats. The number of clinical trials is increasing every year. In most diseases, the effect of hydrogen has been reported with hydrogen water or hydrogen gas, which was followed by confirmation of the effect with hydrogen-rich saline. Hydrogen water is mostly given ad libitum. Hydrogen gas of less than 4 % is given by inhalation. The effects have been reported in essentially all organs covering 31 disease categories that can be subdivided into 166 disease models, human diseases, treatment-associated pathologies, and pathophysiological conditions of plants with a predominance of oxidative stress-mediated diseases and inflammatory diseases. Specific extinctions of hydroxyl radical and peroxynitrite were initially presented, but the radical-scavenging effect of hydrogen cannot be held solely accountable for its drastic effects. We and others have shown that the effects can be mediated by modulating activities and expressions of various molecules such as Lyn, ERK, p38, JNK, ASK1, Akt, GTP-Rac1, iNOS, Nox1, NF-κB p65, IκBα, STAT3, NFATc1, c-Fos, and ghrelin. Master regulator(s) that drive these modifications, however, remain to be elucidated and are currently being extensively investigated.
... Molecular hydrogen could also considerably inhibit apoptosis in rat lung transplantation and inhibit the expression of proapoptotic proteins caspase-3 and caspase-8 in lung grafts, but activate the antiapoptotic proteins Bcl-2 and Bcl-xL, thus stabilizing the mitochondrial outer membrane and terminating the release of cytochrome c into the cytosol via the intrinsic apoptotic pathway Liu et al. 2015b). Additionally, pretreatment of rat donor lungs with molecular hydrogen can induce several lung surfactant-related, ATP synthase, and stress-response genes (Tanaka et al. 2012). ...
Article
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Acute lung injury (ALI) and acute respiratory distress syndrome, which is a more severe form of ALI, are life-threatening clinical syndromes observed in critically ill patients. Treatment methods to alleviate the pathogenesis of ALI have improved to a great extent at present. Although the efficacy of these therapies is limited, their relevance has increased remarkably with the ongoing pandemic caused by the novel coronavirus disease 2019 (COVID-19), which causes severe respiratory distress syndrome. Several studies have demonstrated the preventive and therapeutic effects of molecular hydrogen in the various diseases. The biological effects of molecular hydrogen mainly involve anti-inflammation, antioxidation, and autophagy and cell death modulation. This review focuses on the potential therapeutic effects of molecular hydrogen on ALI and its underlying mechanisms and aims to provide a theoretical basis for the clinical treatment of ALI and COVID-19.
... H 2 can also inhibit the expression of proinflammatory cytokines during the progress of inflammation and has been revealed in many animal models to decrease the overexpression of early proinflammatory cytokines, such as interleukin-(IL-) 1β, IL-6, IL-8, IL-10, tumor necrosis factor-alpha (TNFα) [8], interferon-gamma (INF-γ), and late proinflammatory cytokines, such as high-mobility group box-1 protein (HMGB1) [9]. Tanaka and colleagues [10] conducted a gene array analysis of lung grafts from donor rats pretreated with hydrogen ventilation. The authors described that pretreatment with H 2 obviously elevated the expression of two stress-response proteins: heat shock protein A5 (HSPA5) and dual-specificity phosphatase 1 (DUSP1). ...
Article
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H2 has shown anti-inflammatory and antioxidant ability in many clinical trials, and its application is recommended in the latest Chinese novel coronavirus pneumonia (NCP) treatment guidelines. Clinical experiments have revealed the surprising finding that H2 gas may protect the lungs and extrapulmonary organs from pathological stimuli in NCP patients. The potential mechanisms underlying the action of H2 gas are not clear. H2 gas may regulate the anti-inflammatory and antioxidant activity, mitochondrial energy metabolism, endoplasmic reticulum stress, the immune system, and cell death (apoptosis, autophagy, pyroptosis, ferroptosis, and circadian clock, among others) and has therapeutic potential for many systemic diseases. This paper reviews the basic research and the latest clinical applications of H2 gas in multiorgan system diseases to establish strategies for the clinical treatment for various diseases.
... Nevertheless, these changes in gene expression represent an important effect of molecular hydrogen. For example, gene array analysis of lung allografts revealed that a three-hour pretreatment of 2% hydrogen inhalation in rats altered the expression of 229 genes (182 upregulated and 47 downregulated) (121). ...
Article
There are many situations of excessive production of reactive oxygen species (ROS) such as radiation, ischemia/reperfusion (I/R), and inflammation. ROS contribute to and arises from numerous cellular pathologies, diseases, and aging. ROS can cause direct deleterious effects by damaging proteins, lipids, and nucleic acids as well as exert detrimental effects on several cell signaling pathways. However, ROS are important in many cellular functions. The injurious effect of excessive ROS can hypothetically be mitigated by exogenous antioxidants, but clinically this intervention is often not favorable. In contrast, molecular hydrogen provides a variety of advantages for mitigating oxidative stress due to its unique physical and chemical properties. H2 may be superior to conventional antioxidants, since it can selectively reduce ●OH radicals while preserving important ROS that are otherwise used for normal cellular signaling. Additionally, H2 exerts many biological effects, including anti-oxidation, anti-inflammation, anti-apoptosis, and anti-shock. H2 accomplishes these effects by indirectly regulating signal transduction and gene expression, each of which involve multiple signaling pathways and crosstalk. The Keap1-Nrf2-ARE signaling pathway, which can be activated by H2 , plays a critical role in regulating cellular redox balance, metabolism, and inducing adaptive responses against cellular stress. H2 also influences the crosstalk among the regulatory mechanisms of autophagy and apoptosis, which involve MAPKs, p53, Nrf2, NF-κB, p38 MAPK, mTOR, etc. The pleiotropic effects of molecular hydrogen on various proteins, molecules and signaling pathways can at least partly explain its almost universal pluripotent therapeutic potential.
... 18,19,22 Kawamura et al. 42 found that hydrogen reduced hyperoxic lung injury via the Nrf2 pathway in vivo. Additionally, Tanaka et al. 43 suggested that the protective effect of hydrogen was associated with the upregulation of pulmonary surfactant-related molecules, ATP synthases, and stress-response molecules in lung allograft. Although the signal transduction pathway has not been detected in this study, hydrogen exerted anti-inflammatory, anti-oxidative, and anti-apoptotic effects on graft when applied during the cold ischemia phase. ...
Article
Hydrogen has antioxidant and anti-inflammatory effects on lung ischemia-reperfusion injury when it is inhaled by donor or/and recipient. This study examined the effects of lung inflation with 3% hydrogen during the cold ischemia phase on lung graft function in rats. The donor lung was inflated with 3% hydrogen, 40% oxygen, and 57% nitrogen at 5 mL/kg, and the gas was replaced every 20 min during the cold ischemia phase for 2 h. In the control group, the donor lung was inflated with 40% oxygen and 60% nitrogen at 5 mL/kg. The recipient was euthanized 2 h after orthotropic lung transplantation. The hydrogen concentration in the donor lung during the cold ischemia phase was 1.99-3%. The oxygenation indices in the arterial blood and pulmonary vein blood were improved in the hydrogen group. The inflammation response indices, including lung W/D ratio, the myeloperoxidase activity in the grafts, and the levels of IL-8 and TNF-α in serum, were significantly lower in the hydrogen group (5.2 ± 0.8, 0.76 ± 0.32 U/g, 340 ± 84 pg/mL, and 405 ± 115 pg/mL, respectively) than those in the control group (6.5 ± 0.7, 1.1 ± 0.5 U/g, 443 ± 94 pg/mL, and 657 ± 96 pg/mL, respectively (P < 0.05), and the oxidative stress indices, including the superoxide dismutase activity and the level of malonaldehyde in lung grafts were improved after hydrogen application. Furthermore, the lung injury score determined by histopathology, the cell apoptotic index, and the caspase-3 protein expression in lung grafts were decreased after hydrogen treatment, and the static pressure-volume curve of lung graft was improved by hydrogen inflation. In conclusion, lung inflation with 3% hydrogen during the cold ischemia phase alleviated lung graft injury and improved graft function. © 2015 by the Society for Experimental Biology and Medicine.
... Additionally, recent reports indicated that ventilation with H 2 significantly increased expression of surfactant-related molecules, ATP synthases and stress-response molecules in lung grafts (Tanaka et al., 2012), and that H 2 reduced mRNA levels of osteoclast-specific markers, including tartrate resistant acid phosphatase, calcitonin receptor, cathepsin K, metalloproteinase-9, carbonic anhydrase type-II, and vacuolar-type H + -ATPase (D.Z. . ...
Article
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Molecular hydrogen (H2) has been accepted to be an inert and nonfunctional molecule in our body. We have turned this concept by demonstrating that H2 reacts with strong oxidants such as hydroxyl radical in cells, and proposed its potential for preventive and therapeutic applications. H2 has a number of advantages exhibiting extensive effects: H2 rapidly diffuses into tissues and cells, and it is mild enough neither to disturb metabolic redox reactions nor to affect signaling reactive oxygen species; therefore, there should be no or little adverse effects of H2. There are several methods to ingest or consume H2; inhaling H2 gas, drinking H2-dissolved water (H2-water), injecting H2-dissolved saline (H2-saline), taking an H2 bath, or dropping H2-saline into the eyes. The numerous publications on its biological and medical benefits revealed that H2 reduces oxidative stress not only by direct reactions with strong oxidants, but also indirectly by regulating various gene expressions. Moreover, by regulating the gene expressions, H2 functions as an anti-inflammatory and anti-apoptotic, and stimulates energy metabolism. In addition to growing evidence obtained by model animal experiments, extensive clinical examinations were performed or are under investigation. Since most drugs specifically act to their targets, H2 seems to differ from conventional pharmaceutical drugs. Owing to its great efficacy and lack of adverse effects, H2 has promising potential for clinical use against many diseases.
... Encouragingly, either in the form of gas or saline solution, molecular hydrogen was confirmed effective in treating some very tricky diseases, e.g. ischemia reperfusion injury [4,5], pressure ulcer [6], early neurovascular dysfunction [7]. Since hydrogen is easily available and produces no side effects, the treatment method seems to have a bright future. ...
Article
In recent years, hydrogen molecule as therapeutic antioxidant was found to be useful for the treatment of a number of diseases. To supply hydrogen safely and reliably in the hospital, a patent-pending system was proposed by the authors, including a canister filled with metal hydride, a gas mixing chamber and some other components. The outlet flow of the canister must be controlled within certain accuracy to assure the medical effect of the hydrogen intake, thus was investigated in this work. The mathematical model of hydrogen release process, which couples porous flow, heat and mass transfer was solved using a commercial software package COMSOL Multiphysics 3.5a. The outlet flow dynamics are tested in the cases of convective heating and electrical heating, and great differences are found. For the case of electrical heating that provides constant heat flux, the mass flow rate of H2 showed little temporal variation after the initial transient. Moreover, under certain conditions a PI control strategy was successfully applied to regulate the valve openness for keeping a constant flow rate of H2.
... During I/R, they will eliminate peroxynitrite (and possibly hydroxyl radicals), thereby suppressing inflammation and cellular apoptosis. Concerning the mechanism of the effects of H 2 gas, other possible sites of action of H 2 have been suggested, such as induction of hemoxygenase 1 (4) and heat shock protein (28). These possibilities need to be further investigated. ...
Article
Inhaled nitric oxide (NO) has been reported to decrease the infarct size in cardiac ischemia reperfusion (I-R) injury. However, reactive nitrogen species (RNS) produced by NO causes myocardial dysfunction and injury. Since H2 is reported to eliminate peroxynitrite, it was expected to reduce the adverse effects of NO. In mice, left anterior descending coronary artery ligation for 60 min followed by reperfusion was performed with inhaled NO (80 ppm), H2 (2%), or NO + H2, starting 5 min before reperfusion for 35 min. After 24 hrs, left ventricular function, the infarct size and area at risk (AAR) were assessed. Oxidative stress associated with reactive oxygen species (ROS) was evaluated by staining for 8-hydroxy-2'-deoxyguanosine and 4-hydroxy-2-nonenal, that associated with RNS by staining for nitrotyrosine, and neutrophil infiltration by staining for granulocyte receptor-1. The infarct size/AAR decreased with breathing NO or H2 alone. NO inhalation plus H2 reduced the infarct size/AAR, with significant interaction between the two, reducing ROS and neutrophil infiltration, and improved the cardiac function to normal levels. While nitrotyrosine staining was prominent after NO inhalation alone, it was eliminated after breathing a mixture of H2 with NO. Preconditioning with NO significantly reduced the infarct size/AAR, but not preconditioning with H2. In conclusion, breathing NO + H2 during I-R reduced the infarct size and maintained cardiac function, and reduced the generation of myocardial nitrotyrosine associated with NO inhalation. Administration of NO + H2 gases for inhalation may be useful for planned coronary interventions or for the treatment of I-R injury.
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Ever since molecular hydrogen was first reported as a hydroxyl radical scavenger in 2007, the beneficial effect of hydrogen was documented in more than 170 disease models and human diseases including ischemia/reperfusion injury, metabolic syndrome, inflammation, and cancer. All these pathological damages are concomitant with overproduction of reactive oxygen species (ROS) where molecular hydrogen has been widely demonstrated as a selective antioxidant. Although it is difficult to construe the molecular mechanism of hydrogen's biomedical effect, an increasing number of studies have been helping us draw the picture clearer with days passing by. In this review, we summarized the current knowledge on systemic and cellular modulation by hydrogen treatment. We discussed the antioxidative, anti-inflammatory, and anti-apoptosis effects of hydrogen, as well as its protection on mitochondria and the endoplasmic reticulum, regulation of intracellular signaling pathways, and balancing of the immune cell subtypes. We hope that this review will provide organized information that prompts further investigation for in-depth studies of hydrogen effect.
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Background: Ischemia-reperfusion injury related to lung transplantation is a major contributor to early postoperative morbidity and mortality. We hypothesized that donation after cardiac death donor lungs experience warm ischemic conditions that activate different injurious mechanisms compared with donor lungs that undergo prolonged cold ischemic conditions. Methods: Rat donor lungs were preserved under different cold ischemic times (CIT: 12 hours or 18 hours), or under warm ischemia time (WIT: 3 hours) after cardiac death, followed by single left lung transplantation. Lung function was analyzed during the 2-hours reperfusion period. Microscopic injury, cell death, energy status and inflammatory responses were assessed. Results: Pulmonary oxygenation function was significantly worse in both 18hCIT and WIT groups, accompanied by higher peak airway pressure, acute lung injury scores and expression of cell death markers compared to the 12hCIT control group. In lung tissue, reperfusion induced increased expression levels of interleukin (IL)-1α, IL-1β, IL-6, and chemokines CCL2, CCL3, CXCL1, and CXCL2 in CIT lungs. Notably, these changes were much lower in the WIT group. Additionally, plasma levels of IL-6, IL-18, CCL2 and VEGF were significantly higher, and ATP levels were significantly reduced in warm versus cold ischemic lungs. Conclusions: Compared to 12hCIT, posttransplant pathophysiology deteriorated similarly in both 18hCIT and WIT groups. However, tissue ATP levels and inflammatory profiling differed between warm versus cold ischemic donor lungs. These differences should be carefully considered when developing specific therapeutic strategies to reduce ischemia reperfusion injury in lung transplantation.
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Here we review the literature on the effects of molecular hydrogen (H2) on normal human subjects and patients with a variety of diagnoses, such as metabolic, rheumatic, cardiovascular and neurodegenerative and other diseases, infections and physical and radiation damage as well as effects on aging and exercise. Although the effects of H2 have been studied in multiple animal models of human disease, such studies will not be reviewed in depth here. H2 can be administered as a gas, in saline implants or infusions, as topical solutions or baths or by drinking H2-enriched water. This latter method is the easiest and least costly method of administration. There are no safety issues with hydrogen; it has been used for years in gas mixtures for deep diving and in numerous clinical trials without adverse events, and there are no warnings in the literature of its toxicity or longterm exposure effects. Molecular hydrogen has proven useful and convenient as a novel antioxidant and modifier of gene expression in many conditions where oxidative stress and changes in gene expression result in cellular damage.
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Background: Although the benefits of ex vivo lung perfusion (EVLP) have been globally advocated, the potentially deleterious effects of applying EVLP, in particular activation of proinflammatory cascades and alteration of metabolic profiles, are rarely discussed. This study examined proinflammatory events and metabolic profiles in lung grafts on EVLP and tested whether preconditioning lung grafts with inhaled hydrogen, a potent, cytoprotective gaseous signaling molecule, would alter the lungs' response to EVLP. Methods: Rat heart-lung blocks were mounted on an acellular normothermic EVLP system for 4 hr and ventilated with air or air supplemented with 2% hydrogen. Arterial and airway pressures were monitored continuously; perfusate was sampled hourly to examine oxygenation. After EVLP, the lung grafts were transplanted orthotopically into syngeneic rats, and lung function was examined. Results: Placing lung grafts on EVLP resulted in significant upregulation of the messenger RNAs for several proinflammatory cytokines, higher glucose consumption, and increased lactate production. Hydrogen administration attenuated proinflammatory changes during EVLP through upregulation of the heme oxygenase-1. Hydrogen administration also promoted mitochondrial biogenesis and significantly decreased lactate production. Additionally, in the hydrogen-treated lungs, the expression of hypoxia-inducible factor-1 was significantly attenuated during EVLP. These effects were maintained throughout EVLP and led to better posttransplant lung graft function in the recipients of hydrogen-treated lungs. Conclusions: Lung grafts on EVLP exhibited prominent proinflammatory changes and compromised metabolic profiles. Preconditioning lung grafts using inhaled hydrogen attenuated these proinflammatory changes, promoted mitochondrial biogenesis in the lungs throughout the procedure, and resulted in better posttransplant graft function.
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Mechanical ventilation (MV) can provoke oxidative stress and an inflammatory response, and subsequently cause ventilator-induced lung injury (VILI), a major cause of mortality and morbidity of patients in the intensive care unit. Inhaled hydrogen can act as an antioxidant and may be useful as a novel therapeutic gas. We hypothesized that, owing to its antioxidant and anti-inflammatory properties, inhaled hydrogen therapy could ameliorate VILI. VILI was generated in male C57BL6 mice by performing a tracheostomy and placing the mice on a mechanical ventilator (tidal volume of 30 ml/kg without positive end-expiratory pressure, FiO(2) 0.21). The mice were randomly assigned to treatment groups and subjected to VILI with delivery of either 2% nitrogen or 2% hydrogen in air. Sham animals were given same gas treatments for two hours (n = 8 for each group). The effects of VILI induced by less invasive and longer exposure to MV (tidal volume of 10 ml/kg, 5 hours, FiO(2) 0.21) were also investigated (n = 6 for each group). Lung injury score, wet/dry ratio, arterial oxygen tension, oxidative injury, and expression of pro-inflammatory mediators and apoptotic genes were assessed at the endpoint of two hours using the high-tidal volume protocol. Gas exchange and apoptosis were assessed at the endpoint of five hours using the low-tidal volume protocol. Ventilation (30 ml/kg) with 2% nitrogen in air for 2 hours resulted in deterioration of lung function, increased lung edema, and infiltration of inflammatory cells. In contrast, ventilation with 2% hydrogen in air significantly ameliorated these acute lung injuries. Hydrogen treatment significantly inhibited upregulation of the mRNAs for pro-inflammatory mediators and induced antiapoptotic genes. In the lungs treated with hydrogen, there was less malondialdehyde compared with lungs treated with nitrogen. Similarly, longer exposure to mechanical ventilation within lower tidal volume (10 mg/kg, five hours) caused lung injury including bronchial epithelial apoptosis. Hydrogen improved gas exchange and reduced VILI-induced apoptosis. Inhaled hydrogen gas effectively reduced VILI-associated inflammatory responses, at both a local and systemic level, via its antioxidant, anti-inflammatory and antiapoptotic effects.
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We investigated whether pulmonary surfactant in rat lung transplants recovered during the first week post-transplantation, along with symptoms of the reimplantation response, and whether this recovery was affected by early surfactant treatment. The severity of pulmonary injury was varied by transplanting left lungs with 6-h and 20-h ischemia (n = 12 and 19, respectively). Half of the transplants were treated by instillation of surfactant before reperfusion. Lungs from sham operated, and normal rats (n = 4 and 5, respectively) served as controls. The pulmonary injury severely impaired lung transplant function; 10 of the worst affected animals died. After 1 wk, symptoms of reimplantation response and properties of pulmonary surfactant were assessed. If untreated, the reimplantation response had almost resolved in the 6-h but not in the 20-h ischemia group; pulmonary surfactant, however, continued to be deficient in both ischemia groups (low amounts of surfactant phospholipids and surfactant protein A [SP-A]). Surfactant treatment improved the recovery from injury in the 20-h ischemia group resulting in normal lung function and amounts of surfactant phospholipids. Amounts of SP-A were not improved by surfactant treatment. In conclusion, early surfactant treatment enhances recovery from transplantation injury and is persistently beneficial for pulmonary surfactant in lung transplants.
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Impaired graft function in the postoperative course after lung transplantation (LTx) may in part be due to alterations in pulmonary surfactant. Animal data provide increasing evidence for surfactant abnormalities in the early course after graft reperfusion. However, little is known about the integrity of the surfactant system in human lung transplant recipients. We therefore investigated surfactant properties in bronchoalveolar lavage fluid (BALF) of patients with lung transplants (n = 60) in comparison to that of healthy subjects (n = 10). The phospholipid concentrations of BALF and of surfactant subfractions were measured, and total protein was analyzed. Surface activity was measured with a pulsating bubble surfactometer (PBS). Minimum surface tension was 15.8 +/- 1.1 mN/m in lung transplant recipients, whereas healthy subjects had minimum surface tensions of 3.4 +/- 1.9 mN/m (p = 0.0004). As a marker for potential surfactant inhibition, protein-to-phospholipid (PL) ratios showed no significant differences in the two major study groups. The ratio of small surfactant aggregates to large surfactant aggregates was increased in patients with lung transplants (p = 0.043). Episodes of infection or rejection did not change surface activities, nor did they induce altered ratios of protein to PL or of small to large surfactant aggregates. Surfactant activity did not correlate with pulmonary-function data. Moreover, surface tension showed no correlation with the time after transplantation. Our results suggest a persistent impairment of biophysical surfactant properties after LTx, possibly due to type-II-cell malfunction.
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This review briefly notes recent findings important for understanding the surface mechanical functions of pulmonary surfactant. Currently known surfactant-specific proteins and lipids are discussed, with an eye to their possible functions. Competing models of the alveolar subphase life cycle of surfactant are also presented. It is concluded that, in spite of much effort, we still do not understand the basic molecular mechanisms underlying surfactant's rapid adsorption to the air-water interface.
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Clara cell protein (CC16) is a 15.8-kDa homodimeric protein secreted in large amounts in airways by the non-ciliated bronchiolar Clara cells. This protein increasingly appears to protect the respiratory tract against oxidative stress and inflammation. In vitro, CC16 has been shown to modulate the production and/or the activity of various mediators of the inflammatory response including PLA2, interferon-gamma and tumour necrosis factor-alpha. CC16 has also been found to inhibit fibroblast migration or to bind various endogenous or exogenous substances such as polychlorobiphenyls (PCBs). This protective role is confirmed by studies on transgenic mice, showing that CC16 deficiency is associated with an increased susceptibility of the lung to viral infections and oxidative stress. In humans, a polymorphism of the CC16 gene, localized to a region linked to airway diseases, has recently been discovered in association with an increased risk of developing childhood asthma. Finally, CC16 also presents a major interest as a peripheral marker for assessing the integrity of the lung epithelium. The determination of CC16 in serum is a new non-invasive test to detect Clara cell damage or an increased epithelial permeability in various acute and chronic lung disorders.
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In lung transplantation, reperfusion injury following cold ischemia is one of the crucial problems for recipients. We evaluated the protective effect of adding a combination of ATP and MgCl2 to the preservation solution against lung reperfusion injury following cold ischemia. Using an isolated rat lung perfusion model with fresh rat blood as the perfusate, the rats were divided into five groups (n = 6). In the fresh group, the study lungs were flushed with phosphate-buffered saline (PBS), then immediately reperfused for 120 min. In the control group, the study lungs were flushed with PBS, then cold ischemia was induced for 9 h (4 degrees C), after which reperfusion was performed. In the other three groups, the protocols were the same as for the control group except that ATP and/or MgCl2 were added to the PBS: ATP group (100 microM ATP), MgCl2 group (100 microM MgCl2) and ATP + MgCl2 group (100 microM ATP + 100 microM MgCl2). In the ATP + MgCl2 group, the intrapulmonary shunt fraction, peak airway pressure and wet to dry lung weight ratio were significantly lower than those in the control group. No improvement was observed in the ATP or MgCl2 groups. Histological examination supported these physiological results. In all groups, flush time and lipid peroxide levels in the lungs after cold ischemia did not show any significant differences. The addition of ATP and MgCl2 to the preservation solution attenuated reperfusion injury following cold ischemia in rat lungs.
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The lung is a uniquely vulnerable organ. Residing at the interface of the body and the environment, the lung is optimized for gas exchange, having a very thin, delicate epithelium, abundant blood flow, and a vast surface area. Inherent in this structure is an enormous immunological burden from pathogens, allergens, and pollutants resident in the 11,000 liters of air inhaled daily. Fortunately, protective immune mechanisms act locally in the lung to facilitate clearance of inhaled pathogens and to modulate inflammatory responses. These defensive mechanisms include both innate (nonantibody-mediated) and adaptive (antibody-mediated) systems. The purpose of this commentary is to review briefly the functions of one unique lung innate immune system, pulmonary surfactant, and to highlight the recent findings of Wu et al. (1) described in this issue of the JCI. Wu and colleagues report a new and intriguing innate immune function of surfactant: direct antimicrobial activity.
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During recent years, the biological roles of CCAAT/enhancer binding proteins (C/EBPs) in the lung have started to be uncovered. C/EBPs form a family within the basic region-leucine zipper class of transcription factors. In the lung epithelium C/EBPalpha, -beta, and -delta are expressed. Lung-specific target genes for these transcription factors include the surfactant proteins A and D, the Clara cell secretory protein, and the P450 enzyme CYP2B1. As more information is gathered, a picture is emerging in which C/EBPalpha has a role in regulating proliferation as well as differentiation-dependent gene expression, whereas C/EBPbeta and -delta, in addition to a partly overlapping role in regulating expression of differentiation markers, also seem to be involved in responses to injury and hormones.
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11Beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) increases intracellular glucocorticoid action by converting inactive to active glucocorticoids (cortisol, corticosterone) within cells. It is highly expressed in glucocorticoid target tissues including liver and lung, and at modest levels in adipose tissue and brain. A selective increase in adipose 11beta-HSD1 expression occurs in obese humans and rodents and is likely to be of pathogenic importance in the metabolic syndrome. Here we have used 5' rapid amplificaiton of cDNA ends (RACE) to identify a novel promoter, P1, of the gene encoding 11beta-HSD1. P1 is located 23 kb 5' to the previously described promoter, P2. Both promoters are active in liver, lung, adipose tissue, and brain. However, P1 (encoding exon 1A) predominates in lung and P2 (encoding exon 1B) predominates in liver, adipose tissue, and brain. Adipose tissue of obese leptin-deficient C57BL/6J-Lepob mice showed higher expression only of the P2-associated exon 1B-containing 11beta-HSD1 mRNA variant. In contrast to P2, which is CAAAT/enhancer binding protein (C/EBP)-alpha inducible in transiently transfected cells, the P1 promoter was unaffected by C/EBPalpha in transfected cells. Consistent with these findings, mice lacking C/EBPalpha had normal 11beta-HSD1 mRNA levels in lung but showed a dramatic reduction in levels of 11beta-HSD1 mRNA in liver and brown adipose tissue. These results therefore demonstrate tissue-specific differential regulation of 11beta-HSD1 mRNA through alternate promoter usage and suggest that increased adipose 11beta-HSD1 expression in obesity is due to a selective increase in activity of the C/EBPalpha-regulated P2 promoter.
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The lung is repeatedly exposed to inhaled particles and pathogens that are cleared by the actions of a multi-component innate host defense system. The pulmonary collectins--surfactant proteins A (SP-A) and D (SP-D)--play important roles in innate host defense by binding and clearing invading microbes from the lung. SP-A and SP-D also influence surfactant homeostasis, contributing to the physical structures of lipids in the alveoli and to the regulation of surfactant function and metabolism. In addition to binding and opsonizing infectious pathogens, SP-A and SP-D bind to the surfaces of host defense cells, promoting or inhibiting immune cell activity through multiple cellular pathways. As a consequence of their physiologic functions, SP-A- and SP-D-dependent pathways are targets for clinical therapies designed to limit the proliferation of microoorgansims and to ameliorate inflammation following pulmonary infection.
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Carbon monoxide (CO), a byproduct of heme catalysis by heme oxygenases, has been shown to provide protection against ischemia/reperfusion (I/R) injury. We examined the cytoprotective effect of CO at a low concentration on cold I/R injury of transplanted lung grafts. Orthotopic left lung transplantation was performed in syngenic Lewis to Lewis rat combination. Grafts were preserved in University of Wisconsin solution at 4 degrees C for 6 hours. Donors and/or recipients were exposed to CO (250 ppm) in air for 1 hour before surgery and then continuously post-transplantation. Blood oxygen partial pressure of graft pulmonary veins in the CO-treated group versus the air-treated group was significantly higher. The increase of messenger RNA of inflammatory mediators such as interleukin-6, tumor necrosis factor-alpha, inducible nitric oxide synthase, and cycloooxygenase-2 was markedly inhibited in the CO-treated group. The expression of phosphorylated-extracellular signal-regulated protein kinase 1/2 was significantly reduced in the CO-treated group. CO treatment reduced the number of infiltrating macrophages into the lung grafts. Vascular endothelial cells detected by CD31 stain were well preserved in CO-treated grafts, while those in air-treated grafts were faint and interrupted. These results demonstrate that exogenous low-dose CO treatment of donors and recipients can prevent lung I/R injury and significantly improve function of lung grafts after extended cold preservation and transplantation.
Article
Despite the introduction of low potassium-based preservation strategies for clinical lung transplantation, relevant early graft dysfunction occurs in up to 20% of cases after lung transplantation. This was found to be frequently associated with postreperfusion surfactant dysfunction. We performed a randomized, prospective study investigating the effect of exogenous surfactant instillation into human donor lungs on posttransplant surfactant function and on clinical outcome. Exogenous surfactant was instilled into 15 donor lungs before retrieval via bronchoscopy. Bronchoalveolar lavage fluids were taken before instillation as well as 24 hours after transplantation. Surfactant function, phospholipids, and protein content in bronchoalveolar lavage fluids were assessed and clinical data prospectively recorded. Pulmonary function testing was performed 4 weeks after lung transplantation. Additionally, the best forced expiratory volume in 1 second was determined within the first year after lung transplantation. The control group consisted of 14 patients receiving donor lungs without surfactant instillation in randomized order. Pulmonary function test results were further compared with those of 154 consecutive recipients of bilateral lung transplants, which were not involved in the study (historical control). No deaths occurred during the first year after lung transplantation. Surfactant function in donor lungs was within normal ranges before harvest. In the control group, surfactant function was markedly impaired after reperfusion. This was significantly improved by surfactant substitution. Protein content of the bronchoalveolar lavage fluid in the surfactant group was significantly lower, indicating less leakage through the alveolocapillary membrane. Forced expiratory volume in 1 second after 4 weeks was significantly higher in the surfactant group than in either control group (P = .034 and .01, respectively). Interestingly, the best forced expiratory volume in 1 second during the first year after lung transplantation was significantly higher in both control groups compared with forced expiratory volume measured in examinations 4 weeks after lung transplantation (P = .01). The best forced expiratory volumes in 1 second of control patients were comparable with those in surfactant lungs 4 weeks after transplant. This study indicates a protective effect of exogenous surfactant instillation to donor lungs before retrieval on post-lung transplantation surfactant function and on early clinical outcome. This approach may help to improve the outcome after lung transplantation in the future.
Article
Ischemia-reperfusion injury (IRI) is a prominent cause of primary graft failure after lung transplantation and is associated with an altered surfactant profile. Experimental animal studies have found that replacement with exogenous surfactant administered via fiber-optic bronchoscopy (FOB) enhanced recovery from IRI with improved pulmonary compliance and gas exchange after lung transplantation. We report our clinical experience with FOB instillation of surfactant in severe IRI after human lung transplantation. This study is a retrospective review of 106 consecutive lung or heart-lung transplants performed at a single institution. Severe IRI was defined as diffuse roentgenographic alveolar infiltrates, worsening hypoxemia and decreased lung compliance within 72 hours of lung transplantation. One vial of surfactant (20 mg/ml phospholipid) was instilled into each segmental bronchus upon diagnosis of IRI. Six patients (5 bilateral sequential and 1 re-do heart-lung transplant), mean age 46 years, were diagnosed with IRI and surfactant was administered at a mean of 37 hours (range 2.3 to 98) post-transplant. Mean graft ischemia time was 376 minutes (range 187 to 625) and cardiopulmonary bypass time 174 minutes (range 0 to 210). Mean Pao(2) [mm Hg]/Fio(2) ratio before and 48 hours after surfactant instillation was 70 and 223, respectively. Significant resolution of radiologic infiltrates was evident in all cases within 24 hours. Successful extubation occurred at a mean of 13.5 days and survival is presently 100% at 19 months (range 3 to 54). Bronchoscopic instillation of surfactant improves oxygenation and prognosis after severe IRI in lung transplant recipients. It represents a cost-effective, relatively non-invasive therapeutic alternative to extracorporeal membrane oxygenation.