N. Waki

Suppression of Inflammatory Cytokines During Ex Vivo Lung Perfusion With an Adsorbent Membrane

BACKGROUND Lung grafts can be perfused ex vivo for 2 hours without edema formation; however, prolonged ex vivo lung perfusion (EVLP) eventually induces lung injury. This study evaluated the change in proinflammatory cytokines of the perfusate during EVLP and investigated the effect of cytokine removal using an adsorbent membrane. METHODS Porcine heart-lung blocks were harvested after electrically induced cardiac arrest and underwent 12-hour EVLP with an adsorbent membrane (membrane group: n = 5) and without an adsorbent membrane (control group: n = 6). RESULTS In the control group, both tumor necrosis factor-alpha and interleukin 8 levels were elevated in the perfusate 2 hours after perfusion. Although tumor necrosis factor-alpha and interleukin 8 levels were significantly lower in the membrane group than in the control group during the EVLP period, there was no significant difference in oxygenation, pulmonary vascular resistance, edema formation, or myeloperoxidase activity between the two groups. CONCLUSIONS Tumor necrosis factor-alpha and interleukin 8 levels of the perfusate were elevated during EVLP. Although adverse effects of these inflammatory cytokines were anticipated, removal of inflammatory cytokines by the adsorbent membrane did not improve lung function during prolonged EVLP. Factors other than the cytokines may play a major role in causing lung injury during EVLP. Further research is needed to investigate the real mechanism of lung graft injury during prolonged EVLP and to establish longer EVLP duration for graft treatment. This strategy could contribute to the salvage of potentially damaged lungs, especially from cardiac death donors, and to expansion of the donor pool.

Early Growth Response-1 Plays an Important Role in Ischemia-Reperfusion Injury in Lung Transplants by Regulating Polymorphonuclear Neutrophil Infiltration.

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.

Activations of mitogen-activated protein kinases and regulation of their downstream molecules after rat lung transplantation from donors after cardiac death.

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.

Egr1: a novel target for ameliorating acute allograft rejection in an experimental lung transplant model †

OBJECTIVES Acute allograft rejection is one of the significant complications occurring in lung transplant recipients. Early growth response-1 (Egr-1), zinc-finger-type transcription factor, is known as a master switch regulator of diverse chemical mediators. We used an orthotopic mouse model of left lung transplant to elucidate the function of Egr-1 in acute pulmonary rejection. METHODS Left lung grafts retrieved from C57BL/6 wild mice or C57BL/6 Egr-1-null mice were orthotopically transplanted into BALB/c mice; the lungs were harvested at day 1, 3, 5 or 7 after lung transplantation. The grade of acute rejection was histopathologically evaluated. The intragraft gene expression levels of Egr-1 and downstream target mediators were quantitatively measured by real-time polymerase chain reaction. Immunohistochemical analysis was used to determine the location and distribution of the Egr-1 protein in the pulmonary graft. RESULTS Severe acute rejection was observed in allografts from wild-type mice at 5 days after transplantation. Only minimal rejection was seen in the lung graft from Egr-1-null donor mice at 5 days after transplantation. Strong upregulation of Egr-1 mRNA transcripts was observed at day 1, which then decreased during the next 5 days. The mRNA of Egr-1 target mediators [interleukin-1-beta (IL-1β), monocyte chemotactic protein-1 (MCP-1) and plasminogen activator inhibitor-1] reached maximal levels at day 5. Egr-1-null allografts exhibited significantly lower expressions of IL-1β and MCP-1 mRNA (P < 0.05). CONCLUSIONS Our study showed that deletion of Egr-1 in lung allografts ameliorates severe acute rejection with the reduction of expression levels of chemical mediators, implying a new possible strategy for treating acute pulmonary allograft rejection.

Peculiar mechanisms of graft recovery through anti-inflammatory responses after rat lung transplantation from donation after cardiac death

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.

THE VARIETY ACTIVATIONS OF MITOGEN-ACTIVATED PROTEIN KINASES (MAPKS) AND REGULATIONS OF THEIR DOWN-STREAM MOLECULES AFTER RAT LUNG TRANSPLANTATION FROM NON-HEART-BEATING DONORS (NHBDS): 2597

Introduction: To extend the adaptation to non-heart-beating donors (NHBDs) is an effective solution for donor shortage. Although using the NHBDs have a problem of severe and rapid pulmonary graft dysfunction (PGD) followed by warm ischemia-reperfusion injury (IRI). The aim of this study is to clarify the peculiar mechanisms in molecular factors through warm IRI, and we hypothesize that activation of mitogen-activated protein kinases (MAPKs) and their down-stream molecular factors make important roles in warm IRI. Methods: The NHBDs lungs from male Sprague-Dawley rats were retrieved after 0, 30 and 180 minutes warm ischemic time (WIT) in the closed thoracic cavity of cadaveric rats, and single left lung transplantation was performed at the same total ischemic time point in each group (0WIT, 30WIT and 180WIT; n=5). One hour after reperfusion, the right hilum was clamped, the graft pulmonary function was assessed with blood gas analysis. The phosphorylation status of MAPKs (ERK, p38 and JNK) were estimated by western blot analysis. The gene expression levels of their down-stream transcription factors (Egr-1 and ATF3) and pro-inflammatory molecular factors (MCP-1, MIP-2, PAI-1, ICAM-1, TNFα, IL-1β, IL-6, COX2) in the grafts were examined using reverse transcriptase-polymerase chain reaction (RT-PCR). Results: 180WIT group showed significant PGD compared to the other groups which displayed good puimonary graft function (0WIT, 30WIT, 180WIT, mean±SE mmHg; 608.9 ± 104.6, 511.6 ± 62.5, and 177.8 ± 93.5, respectively; 0WIT, 30WIT vs 180WIT: p<0.01). Phosphorylations of ERK and JNK were significantly increased in 180WIT group, however only the JNK activation had the same pattern of elevation as PGD (0WIT, 30WIT, 180WIT, mean±SE fold; ERK: 3.5±2.0, 5.7±1.7, 11.4±5.0, JNK: 1.9±0.8, 2.6±2.5, 14.0±6.2; 0WIT, 30WIT vs 180WIT: p<0.01). The expression levels of transcription factors known to be regulated by both MAPKs showed the same increased pattern (0WIT, 30WIT, 180WIT mean±SE fold; Egr-1: 14.6±3.8, 15.0±3.4, 38.2±6.8, ATF3: 11.6±2.0, 13.0±4.1, 25.0±4.6; 0WIT, 30WIT vs 180WIT: p<0.01). MCP-1, ICAM-1, IL-1β, IL-6 and COX2 were also up-regulated in 180WIT group, though MIP-2 and PAI-1 showed no significant differences between three groups. Conclusions: The activations of ERK and JNK pathways were seemed in warm IRI. We speculate that ERK and JNK pathways cause the severe primary graft dysfunction by up-regulating the Egr-1 and its down-stream molecules after lung transplantation from NHBDs, and JNK pathway may induce the peculiar injury caused by warm ischemia.