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J Thorac Cardiovasc Surg 2005;130:194-201
© 2005 The American Association for Thoracic Surgery
Cardiothoracic Transplantation |
B by IB superrepressor gene transfer ameliorates ischemia-reperfusion injury after experimental lung transplantation
a Division of Cardiothoracic Surgery, Department of Surgery, Washington University School of Medicine, St Louis, Mo.
b Department of Genetics, Washington University School of Medicine, St Louis, Mo.
Received for publication July 16, 2004; revisions received February 2, 2005; accepted for publication February 16, 2005. * Address for reprints: G. Alexander Patterson, MD, 3108 Queeny Tower, One Barnes-Jewish Hospital Plaza, St Louis, MO 63110-1013. (Email: pattersona{at}msnotes.wustl.edu).
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Abstract |
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B is central to the 2 important pathways that characterize ischemia-reperfusion injury, namely the inflammatory response and apoptosis. The purpose of this study was to determine the effects of nuclear factor B inhibition on experimental lung transplant ischemia-reperfusion injury with gene transfer of the nuclear factor B inhibitor IB in a superrepressor form (IBSR).
METHODS: An orthotopic left lung transplant model in isogeneic rats was used, with 18 hours of prolonged cold storage of donor lung grafts used to create severe ischemia-reperfusion injury. Donor rats underwent endobronchial gene transfection with saline alone or adenovirus encoding either ß-galactosidase control or IBSR 48 hours before harvest. The function of transplanted lung grafts was assessed on the basis of isolated graft oxygenation, wet/dry lung weight ratio, and myeloperoxidase activity. Nuclear factor B activation was assessed by means of enzyme-linked immunosorbent assay. Apoptotic cell death was assessed by evaluating the levels of histone-associated DNA fragments and caspase-3 activity.
RESULTS: Treatment of donor lung grafts with IBSR resulted in significantly improved oxygenation compared with that seen in control tissue 24 hours after transplantation. IBSR-treated lungs also demonstrated less pulmonary edema and reduced neutrophil infiltration 24 hours after reperfusion. Nuclear factor B activation and apoptotic cell death induction 2 hours after transplantation was significantly reduced in IBSR-treated lungs compared with in control lungs.
CONCLUSIONS: Inhibition of nuclear factor B activation by IBSR gene transfer improves transplanted lung graft oxygenation, decreases pulmonary edema and neutrophil sequestration, and reduces apoptotic cell death after experimental lung transplantation.
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Introduction |
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Despite of these improvements, however, ischemia-reperfusion (I/R) injury continues to cause significant morbidity and mortality in lung transplantation.
I/R injury after lung transplantation is a form of acute lung injury characterized by a dynamic and well-orchestrated combination of events involving a number of cell types and cytokine mediators. I/R injury after lung transplantation is thought to be a biphasic process. The initial phase of injury occurs soon after reperfusion (within 30 minutes) and is mediated primarily by activation of resident alveolar macrophages in the donor lung, which then produce several proinflammatory cytokines and chemokines, including tumor necrosis factor (TNF) 
, interleukin (IL) 1ß, IL-8, and macrophage inflammatory protein 2. These mediators result in the recruitment and activation of recipient neutrophils to the transplanted lung, eliciting a subsequent late phase of injury that generally occurs 4 to 6 hours after reperfusion. Activated neutrophils produce a number of proteolytic enzymes and reactive oxygen species that produce end-organ damage in the allograft.
2,3
Two important pathways that contribute significantly to the pathogenesis of I/R injury are the proinflammatory response and cell death in the form of apoptosis. In recent years, mechanisms leading to increased gene expression and biosynthesis of proinflammatory and apoptotic mediators have been well characterized. Nuclear factor B (NFB) is one such important rapid-response transcription factor that is critical to both the regulation of apoptosis and the maximal expression of many proinflammatory mediators.
4
The activation of NFB has been shown to be central to the development of pulmonary inflammation and important in the pathogenesis of acute lung injury typical of I/R injury. In lung transplantation Ross and colleagues
5
recently reported that NFB activation is an important event in initiating I/R injury in a porcine lung transplant model.
In unstimulated cells, NFB is bound to an inhibitor, IB, in the cytoplasm. IB prevents NFB from entering the nucleus, where it regulates gene transcription.
6
When cells are stimulated, activation signals, such as reactive oxygen species, IL-1, and TNF-, lead to signaling pathways involving the NFB/IB complex. By a specific kinase named IKK, IB is phosphorylated, then ubiquitinated, and finally rapidly degraded by proteasomes. After degradation of IB, NFB is released and enters the nucleus, where it binds to specific sequences in the promoter regions of target genes. The rapid transcription of proinflammatory cytokines (TNF-, IL-1, and IL-6) and chemokines (IL-8 and macrophage inflammatory protein 1) begins, and these mediators stimulate neutrophil recruitment. Furthermore, NFB induces the production of cellular adhesion molecules (intercellular adhesion molecule 1 and E-selectin), which promote the binding and emigration of sequestered neutrophils. A positive feedback loop is created because IL-1 and TNF- are important activators of IB, but they are also targets for NFB-mediated transcription. In addition, NFB regulates gene expression in apoptosis.
3,6
Programmed cell death, or apoptosis, can be initiated by a wide variety of stimuli, including I/R injury. Apoptosis has been reported to occur in animal models of I/R injury in the heart, kidney, retina, brain, liver, and adrenal gland.
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In both experimental and clinical lung transplantation, it has been shown that up to 30% of alveolar type II pneumocytes undergo apoptotic cell death after reperfusion.
8
The presence of apoptosis and its effect on lung function after transplantation is still unclear, however.
In different cell types, NFB transcription has been reported to regulate the apoptosis pathway by either inducing or inhibiting it.
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Whether NFB promotes or inhibits apoptosis depends on the specific cell type and the inducing stimulus.
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A number of studies report that NFB activation can have a proapoptotic effect by means of downregulation of expression of antiapoptotic members of the bcl-2 gene family.
9,10
Although some recent studies have reported on the separate contributions of both NFB activation and apoptosis to I/R injury in lung transplant models,
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the effect of NFB activation on levels of apoptosis and lung function after lung transplantation has not been definitively studied.
We have recently demonstrated that several gene transfer strategies, such as overexpression of IL-10, heat shock protein 70, or soluble inhibitory receptors for TNF-, protect lung grafts from I/R injury.
12–17
Preliminary data from our laboratory indicate that these strategies act in part through a final common pathway of NFB inhibition. A superrepressor form of IB (IBSR) containing serine-to-alanine mutations at amino acids 32 and 36 has been described, which inhibits signal-induced phosphorylation and subsequent degradation of IB.
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In this study we investigated the effect of NFB inhibition by IBSR gene transfer on I/R injury after experimental lung transplantation. We also attempted to further elucidate the complex relationship between NFB activation, levels of apoptotic cell death, and graft function in lung transplantation.
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Materials and Methods |
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Adenoviral Vectors
Adenovirus encoding IB superrepressor
Adenovirus encoding IB superrepressor (Ad5IBSR) is a recombinant, replication-deficient adenovirus encoding a dominant negative superrepressor form of IB. It is derived from E1-deleted adenovirus type 5 and driven by the constitutive cytomegalovirus promoter. The IB gene contains an extra 27-bp DNA nucleotide coding for a peptide derived from the hemagglutinin gene to discriminate between endogenous and exogenous IB. Ad5IBSR was purchased from the Gene Therapy Center Viral Vector Core Facility, University of North Carolina, with permission from Dr David A. Brenner.
Adenovirus encoding ß-galactosidase
Ad5LacZ is an adenovirus control vector that produces a nonfunctional carbohydrate product. First-generation replication-deficient adenovirus serotype 5 carrying the Escherichia coli LacZ gene encoding for ß-galactosidase (ß-gal) and driven by the constitutive cytomegalovirus promoter was used in this study. Ad5LacZ was purchased from the Gene Therapy Center Viral Vector Core Facility at the University of North Carolina, Chapel Hill, North Carolina.
Purified viral aliquots were stored at –80°C in 10% glycerol buffered with 10 mmol/L tris (hydroxymethyl) aminomethane, 140 mmol/L sodium chloride, and 1 mmol/L magnesium chloride. These stocks were thawed and diluted in 0.1 mL of sterile isotonic sodium chloride solution immediately before use.
Donor Endobronchial Gene Transfection
Endobronchial gene transfection in rats is a technique that was developed in our laboratory and has been reported by us in detail previously.
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In brief, animals were anesthetized, intubated, and mechanically ventilated, and a right thoracotomy was performed. The carina was dissected, and a catheter was introduced through the endotracheal tube selectively into the left main bronchus. Adenoviral vector, 2 x 107 plaque-forming units, encoding either ß-gal or IBSR or 0.1 mL of saline (vehicle alone) was then instilled through the catheter. The dose of 2 x 107 plaque-forming units was chosen on the basis of previous studies in our laboratory.
15
After 10 minutes of bilateral ventilation after instillation, the left main bronchus was clamped for 60 minutes at the end-inspiratory phase of mechanical ventilation. After the left main bronchus was unclamped, the right thoracotomy was closed, and a temporary chest tube was placed. The chest tube was removed after recovery from anesthesia. Forty-eight hours after transfection, donor lungs were harvested for all experiments.
Orthotopic Left Lung Transplantation
We used the modified cuff technique for orthotopic left lung transplantation, which is well established in our laboratory and has been described in detail in previous reports.
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In brief, after general anesthesia with intraperitoneal pentobarbital (65 mg/kg), mechanical ventilation, systemic heparinization (300 units), and median laparosternotomy, donor rat lungs were flushed through the main pulmonary artery with 20 mL of cold (4°C) low-potassium dextran glucose solution at 20 cm H2O pressure. The heart-lung block was then removed with the lungs inflated at end-tidal volume. The left lung graft was isolated, prepared, and stored in low-potassium dextran glucose at 4°C until transplantation.
For transplantation, recipient animals were anesthetized, intubated with a 14-gauge catheter, and underwent a left thoracotomy. The pulmonary vessels and bronchus were anastomosed with a modification of the previously described cuff technique.
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Functional Assessment of Transplanted Lung Grafts
Recipient rats were killed, and the function of left lung grafts was assessed 24 hours after transplantation. Recipient animals in each group were reanesthetized with pentobarbital. After tracheostomy, the animals were mechanically ventilated with 100% oxygen, and laparosternotomy was performed. The right main bronchus and pulmonary artery were clamped to isolate the left lung graft. The animals were ventilated for 5 minutes at a tidal volume of 1.5 mL, a respiratory rate of 100 breaths/min, and 1.0 cm H2O of positive end-expiratory pressure. Arterial blood gas analysis was performed with blood samples obtained from the ascending aorta. The lungs were flushed with 20 mL of cold (4°C) saline solution, and the lung graft was excised and divided into sections.
Myeloperoxidase Activity
Quantitative myeloperoxidase activity was determined as previously described.
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Optical density was measured at 460 nm with a spectrophotometer (model PMQ II; Carl Zeiss, Oberkochen, Germany). Color development was linear from 5 minutes to 20 minutes. One unit of enzyme activity was defined as 1.0 optical density unit per minute per milligram of tissue protein at room temperature.
NFB p65 Transcription Factor Assay
NFB activation was measured in nuclear extracts of lung homogenates by a nuclear extraction kit (Active Motif North America, Carlsbad, Calif) per the manufacturer’s protocol.
NFB binding to B sites was assessed by using the Trans-AM NFB p65 transcription factor assay kit (Active Motif North America), according to the manufacturer’s instructions. In this assay an oligonucleotide containing the NFB consensus site is attached to a 96-well plate. The active form of NFB contained in cell extracts specifically binds to this oligonucleotide and can be revealed by incubation with antibodies by using enzyme-linked immunosorbent assay technology with absorbance reading.
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Determination of Histone-Associated DNA Fragments and Caspase-3 Activity
To evaluate induction of apoptosis, levels of histone-associated DNA fragments were determined in lung homogenates.
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DNA fragmentation was quantified by measuring histone-associated DNA fragments with an ELISA kit (Cell Death Detection ELISAplus; Roche Diagnostics, Mannheim, Germany), according to the manufacturer’s instructions. Lung samples were homogenized with T-PER Extraction Reagent (Pierce Biotechnology, Rockford, Ill). To confirm results obtained with ELISA, active caspase-3 activity was determined in situ by means of immunohistochemistry with the Biotin Tyramide Signal Amplification kit (Perkin-Elmer Life Sciences, Boston, Mass).
Statistical Analysis
All values are described as means ± standard deviation. Statistical analysis was performed with 1-way analysis of variance for multiple group comparisons, with pairwise comparisons performed with the Fisher protected least significant difference (PLSD) test. Student t tests were used for 2-group comparisons. All analyses were performed on StatView software (SAS Institute, Inc, Cary NC).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B Activation and Apoptotic Cell Death in Lungs After Cold Preservation and TransplantationB was measured at various time points after reperfusion after 18 hours of cold preservation to evaluate the time point of maximal NFB activation in our model. NFB was activated 30 minutes after reperfusion, and there was no significant difference in NFB activation between lung grafts from the time points between 30 minutes after reperfusion and 4 hours after reperfusion (Figure 1). Maximal NFB activation was detected from 30 minutes to 4 hours after reperfusion.
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B activation and apoptosis induction in I/R injury after experimental lung transplantation, we used quantitative analyses for both NFB activation and apoptosis in lungs after cold preservation for various time periods and subsequent transplantation. All lungs were sampled after 2 hours of reperfusion because this has been shown to be the time point of maximal NFB activation (Figure 1) and apoptotic cell death.
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Figure 2
shows NFB activation and apoptotic cell death induction in transplantation after varying amounts of cold preservation, ranging from 1 hour (immediate transplantation) to 18 hours. Additional groups titled Normal and 18hCP were used to study normal lungs and lungs after 18 hours of cold preservation alone without transplantation.
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B and apoptotic cell death in transplanted lungs. NFB activation and apoptotic cell death are not observed during cold preservation for 18 hours without transplantation (compare the 18hCP group between Figure 2, A, and Figure 2, B). However, NFB activation begins after transplantation alone with minimal cold preservation (Figure 2, A, 1-hour group), whereas activation of apoptotic cell death is not observed at this time (Figure 2, B, 1-hour group). Significant NFB activation and apoptotic cell death are observed in transplanted lungs with more than 6 hours of cold preservation (Figure 2, A, 6 hours to 18 hours, and Figure 2, B, 6 hours to 18 hours). These data indicate that events occurring because of transplantation itself (minimum cold preservation) can trigger the activation of NFB, whereas additional events occurring during cold preservation are required to trigger apoptotic cell death. In this study, the induction of apoptotic cell death occurred in lungs after 6 hours or more of cold preservation and not in lungs preserved only for 1 hour, although it is unknown whether any time points between 1 and 6 hours of cold preservation would be sufficient to induce apoptotic cell death.
NFB Inhibition Ameliorates Lung Transplant I/R Injury
Functional assessment of recipient lung grafts was performed 24 hours after reperfusion to examine whether direct inhibition of NFB activity by IBSR gene transfer ameliorates lung transplant I/R injury. To evaluate the influence of IBSR on NFB activation in I/R injury after experimental lung transplantation, we analyzed the activation of NFB. Reduced activation of NFB in lungs transplanted after IBSR gene transfection was confirmed (Figure 3). Next we confirmed the improvement of transplanted lung function after Ad5IBSR gene transfection by isolated graft oxygenation (Figure 4, A), myeloperoxidase activity (Figure 4, B), and wet/dry lung weight ratio (Figure 4, C).
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BSR-treated group was significantly higher than that for the saline-treated and ß-gal-treated groups (Figure 4, A). Myeloperoxidase activity is a reflection of tissue neutrophil sequestration. Myeloperoxidase activity levels were lower in the IBSR-treated group than in the saline-treated and ß-gal-treated groups, although this did not reach statistical significance (Figure 4, B). The wet/dry lung weight ratio is an indicator of tissue edema, Lungs in the IBSR-treated group had lower wet/dry lung weight ratios than those of either the saline-treated or ß-gal-treated groups, although this difference also did not reach statistical significance (Figure 4, C).
Apoptosis Induction Was Significantly Reduced After IBSR Gene Transfection
To evaluate the influence of NFB activation on apoptosis in I/R injury after experimental lung transplantation, we analyzed the induction of apoptotic cell death in the IBSR-treated and ß-gal-treated groups. Apoptotic cell death, as measured by means of histone-associated DNA fragments, was significantly reduced in the group treated with IBSR (Figure 5, A). Immunohistochemical analysis for active caspase-3 activity, another indicator of apoptotic cell death, also showed a marked decrease in apoptotic cell death in the IBSR-treated group (Figure 5, B). These data show that NFB inhibition reduces apoptotic cell death in I/R injury after experimental lung transplantation.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B activation ameliorates lung I/R injury. We have demonstrated that several gene transfer strategies protect lung grafts from I/R injury.
12–17
Preliminary data from our laboratory suggest that these strategies act in part through the final common mechanism of inhibition of NFB activation (unpublished results). In this study, we were able to demonstrate significant improvements in isolated graft oxygenation in transplanted lungs after the inhibition of NFB activation by IBSR gene transfer in experimental lung transplant I/R injury (Figure 3). Ross and coleagues
5
showed that inhibiting NFB with a nonspecific NFB inhibitor, pyrrolidine dithiocarbamate, attenuates I/R injury in a porcine lung transplantation model. Together with our findings, these results demonstrate that NFB is a major factor in the pathogenesis of lung transplant I/R injury and might provide a useful target for therapy to prevent I/R injury.
We were unable to demonstrate a significant reduction in myeloperoxidase activity and wet/dry weight ratio in treated lungs in this study, despite a significant reduction in NFB activation and apoptotic cell death in these grafts. It is unclear whether these results imply differential effects on apoptosis and inflammation pathways mediated by IBSR gene therapy. The most likely explanation for these differences is that these factors were measured in transplanted grafts after 24 hours of reperfusion. We and others have been able to show that many of the inflammatory changes in transplanted grafts begin to resolve by 24 hours after reperfusion. Therefore, it is likely that neutrophil activity and pulmonary edema in untreated control subjects is beginning to improve by this point and therefore is not significantly different from that of treated lung grafts. Another factor that might have contributed to these differences is adenoviral distribution after transfection. We have shown in prior publications that adenoviral transfection after endobronchial transfection is patchy rather than homogenous, and therefore depending on which portion of transfected grafts was used for different assays, effects of treatment on measured parameters might vary.
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NFB Functions as a Factor Promoting Apoptosis in Lung Transplantation
We demonstrated a significant reduction in apoptotic cell death after inhibiting NFB (Figure 5), suggesting that NFB activation contributes to I/R injury by promoting apoptotic cell death. Other researchers have shown that NFB promotes apoptosis in a variety of neuronal cell injury models.
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Meldrum and associates
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showed that NFB inhibition by pyrrolidine dithiocarbamate prevents simulated ischemia-induced renal tubular cell apoptosis and that NFB activation is a proapoptotic signal in their model of simulated ischemia. Our results are similar to these conclusions in our in vivo lung transplant I/R injury model.
Two Independent Stimuli Activate Apoptosis in Lung Transplantation
Our results indicate that significant NFB activation occurs after transplantation alone (with minimal cold ischemia), whereas apoptotic cell death is activated only after transplantation after cold preservation of at least 6 hours in our experiment (Figure 2). We were also able to demonstrate that apoptotic cell death is significantly reduced by NFB inhibition (Figure 5). Taken together, these data indicate that there are at least 2 events necessary to activate apoptotic cell death in transplanted lungs. One event is triggered by transplantation by itself, which is most likely through an NFB-dependent pathway because NFB is activated by transplantation by itself. The other event is triggered during cold preservation. The time of cold preservation required to induce apoptotic cell death was found to be 6 hours or more, although time points between 1 and 6 hours of cold preservation were not evaluated in this study. In an experimental model of lung transplantation, Fischer and coworkers
25
demonstrated that with shorter periods of ischemia (6 or 12 hours), the mode of cell death was primarily apoptosis. Our results also demonstrate that apoptotic cell death is induced by 6 hours or more of cold preservation and reperfusion. There are two known pathways for apoptosis: the mitochondrial pathway and the death receptor pathway. The suggestion of these two events being necessary for the induction of apoptotic cell death might somehow be related to each of these two different pathways.
In conclusion, this study demonstrates that NFB inhibition with gene therapy prevents the induction of apoptosis and ameliorates I/R injury in an experimental rodent lung transplantation model. The present results suggest that NFB activation is a major factor in inciting I/R injury in lung transplantation by acting as a factor promoting apoptotic cell death and that at least two signals, one of which involves NFB activation, might be necessary for the induction of apoptosis in lung transplantation. The application of gene therapy as a therapeutic strategy for lung transplantation will become more realistic as we solve the complicated mechanisms of the inflammatory cascade and apoptosis.
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Acknowledgments |
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BSR. We also thank Richard B. Schuessler, PhD, for his guidance regarding statistical analysis; Camila Pflederer for her invaluable assistance with the assays used in this study; and Takashi Suda, MD, and Tsutomu Tagawa, MD, for their assistance with the technique of rat lung transplantation. |
Footnotes |
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References |
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