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During ischemia reperfusion (IR) injury, high mobility group box 1 (HMGB1), a chromatin binding protein, is released from necrotic cells and triggers inflammatory responses. We assessed the therapeutic effect of a neutralizing anti-HMGB1 monoclonal antibody (mAb) on lung IR injury. A murine hilar clamp model of IR was used, where mice were divided into sham and IR groups with intravenous administration of anti-HMGB 1 mAb or control mAb. We analyzed the effect of anti-HMGB1 mAb against IR injury by assessing lung oxygenation, lung injury score, neutrophil infiltration, expression of proinflammatory cytokines and chemokines, levels of mitogen-activated protein kinase (MAPK) signaling, and measurement of apoptotic cells. Anti-HMGB1 mAb significantly decreased the plasma level of HMGB1 elevated by IR. The severity of IR injury represented by oxygenation capacity, lung injury score, and neutrophil infiltration was significantly improved by anti-HMGB1 mAb treatment. The expression of proinflammatory factors, including IL-1β, IL-6, IL-12, TNF-α, CXCL-1, and CXCL-2, and phosphorylation of p38 MAPK were both significantly reduced by anti-HMGB1 mAb treatment. Furthermore, anti-HMGB1 mAb treatment suppressed apoptosis, as determined through TUNEL assays. Overall, anti-HMGB1 mAb ameliorated lung IR injury by reducing inflammatory responses and apoptosis. Our findings indicate that anti-HMGB1 mAb has potential for use as a therapeutic to improve IR injury symptoms during lung transplantation., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Ischemia-reperfusion injury (IRI) after lung transplantation mainly contributes to the development of primary graft dysfunction. The Sprouty-related EVH1-domain-containing (SPRED) protein family inhibits the mitogen activated protein kinase/extracellular-signal-regulated kinase (MAPK/ERK) pathway. Our study was aimed at examining the role of SPRED2 in IRI in mice that received orthotopic lung transplantation. Syngeneic mouse lung transplantation was performed in wild-type C57BL/6 J (WT) mice and Spred2 knockout (Spred2-/-) mice on the C57BL/6 J background from the WT donor. Four hours after reperfusion, blood gas analysis was performed, and lung grafts were sacrificed and analyzed. By using arterial oxygen tension measurements and histological evaluation using Lung Injury Score, we revealed more severe IRI in the grafts transplanted to Spred2 -/- recipients, which manifested as exacerbated airway epithelial cell damage, interstitial edema with hemorrhage and neutrophil infiltration. Intragraft ERK1/2 activation and expression levels of proinflammatory cytokines and chemokines in Spred2 -/- recipients were higher than those in WT recipients. SPRED2 plays an important role in protecting the lungs from IRI in lung transplantation recipients. We suggest that focused treatments suppressing the activity of the MAPK/ERK pathway in transplantation recipients could be the potential therapeutic option for the prevention of lung IRI., (Copyright © 2019. Published by Elsevier B.V.)

Objectives: The role of intraoperative cardiopulmonary bypass (CPB) in lung transplant (LTx) surgery is controversial. CPB enables slow pulmonary reperfusion and initial ventilation with low oxygen concentrations, both theoretically protective of transplanted lungs. In this study, we explored clinical outcomes following extended criteria donor LTx surgery implementing a thoroughly protective allograft reperfusion strategy using CPB., Methods: Thirty-nine consecutive adult patients who underwent bilateral LTx with elective CPB and protective allograft reperfusion were reviewed. Bilaterally implanted lungs were reperfused simultaneously, via slow CPB flow reduction and initial ventilation with 21% oxygen and nitric oxide, followed by a brief modified ultrafiltration. During weaning from CPB, mean pulmonary arterial pressure was strictly maintained at 10-15 mmHg by controlling CPB and pulmonary flow. The clinical outcomes in 23 patients who received lungs from extended criteria donors (ECD group) were elucidated and compared to 16 patients undergoing LTx from standard criteria donors (SCD group)., Results: No life-threatening deterioration was observed to graft functionality during the first 72 h after LTx in the ECD group; however, only one patient required post-transplant extracorporeal membrane oxygenation. In three of 23 ECD LTx patients (12%), surgical revision for bleeding was required. Survival outcomes for the ECD group were favorable, with 100% survival at 6-months, 87.0% at 1-year, and 80.7% at 5-years. Outcomes in the ECD group were comparable to those in the SCD group., Conclusions: Despite a certain extent of risk associated with full-dose heparinization, use of CPB does not undermine survival outcomes after ECD LTx surgery if protective allograft reperfusion is securely performed.

Purpose: Inflammatory changes during lung ischemia-reperfusion injury (IRI) are related to the activation of the extracellular signal-regulated kinase (ERK)1/2 signaling pathway. Sprouty-related EVH1 (enabled/vasodilator-stimulated phosphoprotein homology 1)-domain-containing proteins (SPREDs) are known inhibitors of ERK1/2 signaling. The role of SPRED2 in lung IRI was examined in a left hilar clamp mouse model., Methods: C57BL/6 wild-type (WT) and Spred2 -/- mice were used in the left hilar clamp model. Experimental groups underwent 30 min of left hilar clamping followed by 1 h of reperfusion. U0126, an ERK1/2 inhibitor, was administered to Spred2 -/- mice with reperfused lungs., Results: The partial pressures of oxygen of the Spred2 -/- mice after reperfusion were significantly worse than those of WT mice (p < 0.01). Spred2 -/- mice displayed more severe injuries than WT mice with increased neutrophil infiltration observed by a histological evaluation and flow cytometry (p < 0.001). This severe inflammation was inhibited by U0126. In addition, the rate of ERK1 activation was significantly higher in the lungs of Spred2 -/- mice after reperfusion than in WT mice according to a Western blot analysis (p < 0.05)., Conclusion: The activation of the ERK1/2 signaling pathway influences the severity of lung IRI, causing inflammation with neutrophil infiltration. SPRED2 may be a promising target for the suppression of lung IRI.

Objective: Ex vivo lung perfusion (EVLP) has been used not only for graft evaluation but also for graft reconditioning prior to lung transplantation. Inflammatory cells such as neutrophils may cause additional graft injury during EVLP. Neutrophil elastase inhibitors protect lungs against neutrophil-induced lung injury, such as acute respiratory distress syndrome. This study aimed to investigate the effect of a neutrophil elastase inhibitor during EVLP., Methods: EVLP was performed for 4 h in bilateral pig lungs that had previously experienced warm ischemia for 2 h with or without a neutrophil elastase inhibitor (treated and control groups, respectively; n = 6). Following EVLP, the left lung was transplanted into a recipient pig, and this was followed by observation for 4 h. Pulmonary functions were observed both during EVLP and during the early post-transplant stage., Results: During EVLP, decreases in neutrophil elastase levels (P < 0.001), the wet-dry weight ratio (P < 0.05), and pulmonary vascular resistance (P < 0.01) and increases in the PaO2/FiO2 ratio (P < 0.01) and pulmonary compliance (P < 0.05) were observed in the treated group. After transplantation, decreased pulmonary vascular resistance (P < 0.05) was observed in the treated group., Conclusions: A neutrophil elastase inhibitor attenuated the inflammatory response during EVLP and may decrease the incidence of lung reperfusion injury after transplantation.

Background: Early growth response-1 (Egr-1) has been shown to be a trigger-switch transcription factor that is involved in lung ischemia-reperfusion injury (IRI)., Methods: Mouse lung transplants were performed in wild-type (WT) C57BL/6 and Egr1-knockout (KO) mice in the following donor → recipient combinations: WT → WT, KO → WT, WT → KO, and KO → KO to determine whether the presence of Egr-1 in the donor or recipient is the most critical factor for IRI. Pulmonary grafts were retrieved after 18 hours of ischemia after 4 hours of reperfusion. We analyzed graft function by analyzing arterial blood gas and histology in each combination and assessed the effects of Egr1 depletion on inflammatory cytokines that are regulated by Egr-1 as well on polymorphonuclear neutrophil (PMN) infiltration., Results: Deletion of Egr1 improved pulmonary graft function in the following order of donor → recipient combinations: WT → WT < WT → KO < KO → WT < KO → KO. Polymerase chain reaction assays for Il1B, Il6, Mcp1, Mip2, Icam1, and Cox2 showed significantly lower expression levels in the KO → KO group than in the other groups. Immunohistochemistry demonstrated clear Egr-1 expression in the nuclei of pulmonary artery endothelial cells and PMN cytoplasm in the WT grafts. Flow cytometry analysis showed that Egr1 deletion reduced PMN infiltration and that the extent of reduction correlated with graft function., Conclusions: Both graft and recipient Egr-1 played a role in lung IRI, but the graft side contributed more to this phenomenon through regulation of PMN infiltration. Donor Egr-1 expression in pulmonary artery endothelial cells may play an important role in PMN infiltration, which results in IRI after lung transplantation.

Purpose: Ischemia reperfusion injury (IRI) remains a significant cause of morbidity and mortality after lung transplantation. Early growth response-1 (EGR1) drives the expression of inflammatory mediators and has an important role in IRI. We hypothesized that the severe IRI caused by a long preservation induces a specific expression pattern of EGR1 and its target genes which would correlate with the lung graft function., Methods: SD rat lungs were preserved at 4 °C for 3 or 18 h, then transplanted and reperfused. Pulmonary grafts were evaluated for the blood gas oxygenation and pathological findings. The intra-graft mRNA levels of EGR1 and its downstream target genes were measured by real-time PCR. A Western blotting analysis of the EGR1 expression was used to validate the changes in the protein level., Results: There was upregulation of EGR1, MIP-2 and PAI-1 when there was prolonged hypothermic preservation. The expression levels of MIP-2 and PAI-1 were observed to increase for up to 4 h in the 18 h preserved lungs. There were no differences in the expression levels of IL-1β and ICAM-1 between the lungs subjected to short and long periods of ischemia., Conclusions: Our data showed that prolonged hypothermic graft preservation deteriorates the pulmonary graft function, which was associated with the induction of EGR1 and its downstream target genes, which may aggravate IRI following lung transplantation.

Background: Although lung transplantation from donation after cardiac death (DCD), especially uncontrolled DCD, is limited by warm ischemic periods, the molecular mechanism of warm ischemia-reperfusion-injury (IRI) has not been well elucidated. The purpose of this study was to clarify the particular longitudinal mechanisms of molecular factors involved in warm IRI., Methods: Cold ischemic-time (CIT)-group lungs were retrieved and subjected to 3-h of cold preservation, whereas warm ischemic-time (WIT)-group lungs were retrieved after 3-h of warm ischemia. Orthotopic rat lung transplantation was performed and the grafts were reperfused for 1 or 4-h. The graft functions, gene expression, and activation of inflammatory molecules in the grafts were analyzed. Exhaled-carbon-monoxide-concentration (ExCO-C) was measured during reperfusion., Results: Only the WIT-group showed obvious primary graft dysfunction at 1-h reperfusion, but the graft function was recovered during 4-h reperfusion. Most of pro-inflammatory cytokines and stress-induced molecules showed different expression and activation patterns between CIT and WIT groups. In the WIT-group, the expressions of anti-inflammatory molecules, IL-10 and HO-1, were significantly increased at 1-h reperfusion compared to the CIT-group, and these high levels were maintained through 4-h reperfusion. Furthermore, ExCO-C levels in the WIT-group increased immediately after reperfusion compared to the CIT-group., Conclusions: This study indicates that warm IRI may involve a different mechanism than cold IRI and anti-inflammatory pathways may play important roles in the graft recovery after lung transplantation from uncontrolled DCD., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Objectives: Accepting organs donated after cardiac death (DCD) is an effective approach to the donor shortage. However, lung transplantations from DCD donors show severe rapid pulmonary graft dysfunction (PGD) followed by warm ischemia-reperfusion injury (IRI). This study sought to clarify the molecular mediators in warm IRI, including activation of mitogen-activated protein kinase (MAPK) and the downstream cascades., Methods: We performed single left lung transplantation using organs from male Sprague-Dawley rats after 0 (CIT group), 30 (30WIT group), or 180 (180WIT group) minutes of warm ischemia time. Pulmonary graft functions were estimated by blood gas analysis. At 1 hour after reperfusion, the phosphorylation status of MAPKs (ERK, p38, and JNK) and the gene expression levels of transcription factors (Egr-1 and ATF-3) and immune mediators (MCP-1, MIP-2, PAI-1, ICAM-1, TNF-α, IL-1β, IL-6, and COX-2) in the grafts were examined using Western blotting and real-time polymerase chain reaction assays., Results: Severe PGD was observed in the 180WIT group compared with transplanted lungs in the other groups, which exhibited good pulmonary graft function. ERK and JNK activations, as well as mRNA levels of transcription factors (Egr-1 and ATF3) significantly increased with greater warm ischemic times. The pattern of JNK activation correlated with the severity of PGD. MCP-1, ICAM-1, IL-1β, IL-6, and COX-2 were also up-regulated among the 180WIT group, although MIP-2 and PAI-1 showed no significant differences among the groups., Conclusions: We suggest that the ERK and JNK pathways may play important roles to induce the injury caused by prolonged warm ischemia followed by reperfusion in the setting of lung transplantation from DCD donors., (Copyright © 2011 Elsevier Inc. All rights reserved.)

This report describes a new experimental procedure, a rat unilateral, orthotopic lung transplantation with cold storage, and evaluates its relevancy and reliability to study the early events during cold ischemia/reperfusion (I/R) injury. This model, using the cuff technique, does not require extensive training and is relatively easy to be established. The model can induce reproducible degrees of pulmonary graft injury including impaired gas exchange, proinflammatory cytokine upregulation, or inflammatory infiltrates, depending on the preservation time. The results are consistent with the previous clinical evidence, thus suggesting that this model is a valid and reliable animal model of cold I/R injury.

Ischemia-Reperfusion (I/R) injury after lung transplantation (LTx) can lead to significant morbidity and mortality in recipients. In an attempt to improve our understanding of molecular mechanisms of I/R injury, we examined the changes in gene expression levels in a rat lung transplant model using oligonucleotide microarrays. Lewis rat lung grafts were stored for 6 or 24 h followed by transplantation and reperfusion for 2 h. Lung tissues were taken before and after flushing the grafts, before implantation, and after 2 h of reperfusion. RNA samples were examined with Affymetrix rat microarray chips and RT-PCR was performed to validate significant changes in gene expression. Microarray analysis showed 404 genes that were up-regulated more than 2-fold after reperfusion compared to cold ischemic lungs, and 187 genes that were down-regulated. Using RT-PCR, we confirmed the response pattern of several specific gene transcripts from the microarray analysis. Among these, up-regulation in transcripts of transcription factors, adhesion molecules, pro-coagulant factors and pro-inflammatory cytokines were identified. The differential gene regulation during the I/R process can be considered as molecular signatures for the changes of cellular metabolism, functions and injury. Reperfusion-induced genes related to inflammatory response may contribute to graft dysfunction in LTx.

Interleukin-10 (IL-10) gene transfection of donor lungs prior to transplantation is an attractive strategy to reduce ischemia-reperfusion induced lung injury. However, experimental data with gene therapy in large animal models of lung transplantation are generally lacking. We have developed a simple clinically applicable technique for adenoviral-mediated gene delivery of human IL-10 to the lung of large animals that provides homogenous gene expression after 12-24 h of transfection. Using this technique of gene delivery, we have studied the dynamics of adenoviral gene delivery to the lung in the setting of lung transplantation. Although there is a persistent inflammatory response to the adenoviral vector, we achieved significant expression of human IL-10 in lung tissue before lung retrieval to obviate the deleterious impact of the adenoviral vector on the donor lung. The administration of adenoviral-mediated human IL-10 to the donor lung reduced ischemia-reperfusion injury and improved graft function after lung transplantation in this pig lung transplantation model. Transfection of adenoviral-mediated human IL-10 to the donor lung prevented the release of inflammatory cytokines such as IL-6 in lung tissue and plasma. We have demonstrated that IL-10 gene therapy has significant potential to prevent or treat the inflammatory response associated with ischemia-reperfusion injury in lung transplantation. In the future, IL-10 gene therapy could also be used for immunomodulation or tolerance induction.

Lung ischemia reperfusion (IR) injury increases the mortality and morbidity in patients undergoing lung transplantation. Inflammatory mediators called Damage Associated Molecular Patterns (DAMPs) concern with inflammatory response to develop the organ damages during IR injury and High Mobility Group Box 1 (HMGB1) is one of the most important DAMPs. We had previously developed anti-HMGB1 monoclonal antibody (mAb) and assessed the therapeutic effect of the antibody in lung IR injury. A mice hilar clamp model of IR was used, and animals were divided into sham groups and IR groups with or without administration of anti-HMGB 1 mAb. We analyzed the effect of anti-HMGB 1 mAb against IR injury by assessing lung oxygenation, injury, neutrophil infiltrations, expression of proinflammatory cytokines and chemokines, MAPK signaling and apoptosis. The severity of IR injury represented by oxygenation capacity, lung injury score and neutrophil infiltrations was significantly improved by anti-HMGB1 mAb treatment. The expressions of proinflammatory factors including IL-1b, IL-6, IL-12, TNFα, CXCL1 and CXCL2 and phosphorylation of p38 MAPK were significantly reduced by the anti-HMGB1 mAb treatment. Furthermore, the antibody decreased TUNEL positive cells to detect apoptosis. Anti-HMGB1 mAb obviously ameliorated lung IR injury by reduction of inflammatory responses and apoptosis. Our findings indicate that anti-HMGB1 mAb have a potential as therapeutic drug to improve IR injury in lung transplantation. [ABSTRACT FROM AUTHOR]