Inhibition of nuclear factor kappaB by IkappaB superrepressor gene transfer ameliorates ischemia-reperfusion injury after experimental lung transplantation.

OBJECTIVES: Ischemia-reperfusion injury after lung transplantation is associated with significant morbidity and mortality. The activation of the transcription factor nuclear factor kappaB 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 kappaB inhibition on experimental lung transplant ischemia-reperfusion injury with gene transfer of the nuclear factor kappaB inhibitor IkappaB in a superrepressor form (IkappaBSR). 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 beta-galactosidase control or IkappaBSR 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 kappaB 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 IkappaBSR resulted in significantly improved oxygenation compared with that seen in control tissue 24 hours after transplantation. IkappaBSR-treated lungs also demonstrated less pulmonary edema and reduced neutrophil infiltration 24 hours after reperfusion. Nuclear factor kappaB activation and apoptotic cell death induction 2 hours after transplantation was significantly reduced in IkappaBSR-treated lungs compared with in control lungs. CONCLUSIONS: Inhibition of nuclear factor kappaB activation by IkappaBSR gene transfer improves transplanted lung graft oxygenation, decreases pulmonary edema and neutrophil sequestration, and reduces apoptotic cell death after experimental lung transplantation.

Recipient intramuscular administration of naked plasmid TGF-beta1 attenuates lung graft reperfusion injury.

BACKGROUND: Gene therapy may be an effective strategy for modulating lung graft ischemia-reperfusion injury. We investigated whether recipient intramuscular (IM) naked plasmid gene transfer of transforming growth factor beta1-active (TGF-beta1-active) ameliorates lung graft ischemia-reperfusion injury. METHODS: Preliminary studies in F344 rats demonstrated that gastrocnemius muscle transfection of TGF-beta1-active produced muscle and plasma protein expression at 24 and 48 hours after transfection. Recipients (n = 8) received IM injection of naked plasmid-encoding chloramphenicol acetyl transferase (CAT), TGF-beta1-latent or TGF-beta1-active, respectively, at 24 or at 48 hours before left lung transplantation. We did not treat the control group before transplantation (18-hour cold ischemia). Donor lungs were flushed with low-potassium dextran-1% glucose and stored for 18 hours at 4 degrees C. All groups were killed at 24 hours after transplantation. Immediately before killing the animals, we clamped the contralateral right hilum and assessed graft function. We measured wet-to-dry ratio (W/D), myeloperoxidase, pro-inflammatory cytokines (interleukin 1 [IL-1], tumor necrosis factor alpha [TNF-alpha], interferon-gamma [INF-gamma], and IL-2) and performed immunohistochemistry. RESULTS: Arterial oxygenation was greatest in the recipient group transfected with TGF-beta1-active at 24 hours before transplantation compared with CAT, TGF-beta1-latent, and 18-hour cold ischemia groups (p < 0.01). The W/D ratio and myeloperoxidase decreased in both 24- and 48-hour groups, with TGF-beta1-active compared with CAT, and 18-hour cold ischemia groups (W/D, p < 0.02 and p < 0.004, respectively; myeloperoxidase, p < 0.05 and p < 0.01, respectively). All pro-inflammatory cytokines decreased in the 24-hour TGF-beta1-active group compared with CAT, TGF-beta1-latent, 18-hour and 1-hour cold ischemia, and non-treated lung groups (IL-1beta, p < 0.03; TNF-alpha, p < 0.02; IFN-gamma, p < 0.001; IL-2, p < 0.0001). In 24- and 48-hour groups with TGF-beta1-active, immunohistochemistry showed marked staining of Type I and Type II alveolar cells and of macrophages from the apical to the caudal sections of the lung grafts. CONCLUSIONS: Recipient IM administration of naked plasmid encoding TGF-beta1-active before transplantation ameliorates lung isograft reperfusion injury after prolonged ischemia.

Intramuscular gene transfer of interleukin-10 reduces neutrophil recruitment and ameliorates lung graft ischemia-reperfusion injury.

Interleukin-10 (IL-10) has potent anti-inflammatory properties but its direct effects on neutrophil trafficking in lung transplant ischemia-reperfusion (I/R) injury are unknown. This study was performed to determine if recipient intramuscular IL-10 gene transfer reduces neutrophil infiltration in lung isografts and ameliorates I/R injury. Twenty-four hours before transplantation, recipient rodents received intramuscular injection with 1 x 10(10) plaque-forming units (pfu) adenovirus encoding human IL-10 (hIL-10), 1 x 10(10) pfu adenovirus control encoding p-galactosidase, or saline. Gene expression in muscle and plasma was confirmed. Lung grafts were harvested, stored at 4 degrees C for 18h, and assessed 24 h after transplantation. Peak muscle and plasma expression of hIL-10 was achieved 24h after gene transfer and returned to baseline by 7 days (p < 0.05 vs. controls). Gene transfer of hIL-10 reduced neutrophil sequestration and emigration in lung grafts as measured by morphometry and myeloperoxidase activity (p < 0.03 vs. controls). Furthermore, hIL-10 improved graft oxygenation and reduced lung edema (p <0.01 vs. controls). Intramuscular gene transfer of hIL-10 releases hIL-10 protein into plasma and reduces neutrophil sequestration and emigration in lung isografts. This is associated with a reduction in I/R injury with improved isograft oxygenation and reduced tissue edema. Intramuscular gene transfer may be a useful strategy to reduce clinical l/R injury.

Recipient intramuscular cotransfection of naked plasmid transforming growth factor beta1 and interleukin 10 ameliorates lung graft ischemia-reperfusion injury.

OBJECTIVE: Multiple gene transfer might permit modulation of concurrent biochemical pathways involved in lung graft ischemia-reperfusion injury. In this study we analyzed whether recipient intramuscular naked plasmid cotransfection of transforming growth factor beta(1) and interleukin 10 would result in amelioration of lung graft ischemia-reperfusion injury. METHODS: Forty-eight hours before transplantation, 6 groups (n = 6) of F344 rats received intramuscular injection of naked plasmid encoding chloramphenicol acetyltransferase, chloramphenicol acetyltransferase plus beta-galactosidase, transforming growth factor beta(1), interleukin 10, or transforming growth factor beta(1) plus interleukin 10 or were not treated. Donor lungs were flushed and stored for 18 hours at 4 degrees C before transplantation. Twenty-four hours later, grafts were assessed immediately before the animals were killed. Arterial oxygenation, wet/dry ratio, myeloperoxidase, and proinflammatory cytokines (interleukin 1, tumor necrosis factor alpha, interferon gamma, and interleukin 2) were measured, and immunohistochemistry was performed. RESULTS: For lung graft function, the arterial oxygenation was considerably higher in the cotransfected group receiving transforming growth factor beta(1) plus interleukin 10 compared with that in all other groups (P < or =.03). The wet/dry ratio, reflecting lung edema, was reduced in the cotransfected group compared with that in control animals (nontreated, P <.02; chloramphenicol acetyltransferase, P <.03; chloramphenicol acetyltransferase plus beta-galactosidase, P <.01). Myeloperoxidase, which measures neutrophil sequestration, was also reduced with cotransfection compared with that seen in control animals (P < or =.03). All proinflammatory cytokines were decreased in the cotransfected group compared with those in all other groups (interleukin 1beta, P <.04; tumor necrosis factor alpha, P <.002; interferon gamma, P <.0001; interleukin 2, P <.03). These results indicate that cotransfection provides a synergistic benefit in graft function versus either cytokine alone, neutrophil sequestration, or inflammatory cytokine expression. Immunohistochemistry showed positive staining of transforming growth factor beta(1) plus interleukin 10 in type I and II pneumocytes and localized edema fluid. CONCLUSIONS: Recipient intramuscular naked plasmid cotransfection of transforming growth factor beta(1) and interleukin 10 provides a synergistic effect in ameliorating lung reperfusion injury after prolonged ischemia.