Synergistic protection in lung ischemia-reperfusion injury with calcineurin and thrombin inhibition.

BACKGROUND: Ischemia-reperfusion injury impairs lung transplant outcomes. The transcription factors, activator protein-1, and nuclear factor kappa B, are activated early in reperfusion and drive the development of injury. Thrombin inhibition with hirudin, and calcineurin inhibition with tacrolimus have independently been shown to ameliorate lung ischemia-reperfusion injury by reducing activator protein-1 and nuclear factor kappa B activation, respectively. However, high doses were required to achieve protection using individual agents, raising concerns about potential toxicities. We sought to determine if low-dose combination therapy reduced injury through synergistic inhibition of pretranscriptional signaling events. METHODS: Rats were pretreated with either intravenous hirudin or tacrolimus at low doses or high doses, or both at low doses, prior to undergoing left lung ischemia and reperfusion. Lungs were assessed for markers of lung injury, including bronchoalveolar lavage cytokine-chemokine content and transcription factor transactivation of activator protein-1 and nuclear factor kappa B. RESULTS: High-dose monotherapy with hirudin or tacrolimus reduced lung injury and transactivation of activator protein-1 and nuclear factor kappa B activation, respectively, whereas low-dose monotherapy with either agent did not alter transcription factor activation or lung injury compared with positive controls. Low-dose combination therapy was more protective than high-dose monotherapy with either drug, and correlated with a reduction in activation of both transcription factors and their associated cytokines. CONCLUSIONS: The significant decrease in lung injury severity and transcription factor activation with combined pathway inhibition suggests pretranscriptional signaling redundancy between the calcineurin and thrombin dependent pathways in lung reperfusion injury.

Alveolar macrophage secretory products effect type 2 pneumocytes undergoing hypoxia-reoxygenation.

BACKGROUND: Activation of the alveolar macrophage is centrally important to the development of lung ischemia reperfusion injury. Alveolar macrophages and type 2 pneumocytes secrete a variety of proinflammatory mediators in response to oxidative stress. The manner in which they interact and how the macrophage may influence pneumocyte responses in lung ischemia reperfusion injury is unknown. Utilizing an in vitro model of hypoxia and reoxygenation, we sought to determine if the proinflammatory response of type 2 pneumocytes to oxidative stress would be amplified by alveolar macrophage secretory products. METHODS: Cultured pneumocytes were exposed to control media or media from cultured macrophages exposed to hypoxia and reoxygenation. Pneumocytes were subsequently subjected to hypoxia and reoxygenation and assessed for both nuclear translocation of nuclear factor kappa B and inflammatory cytokine and chemokine secretion. To examine for any reciprocal interactions, we reversed the experiment, exposing macrophages to conditioned pneumocyte media. RESULTS: In the presence of media from stimulated macrophages, production of proinflammatory mediators by type 2 pneumocytes was dramatically enhanced. In contrast, exposure of the macrophage to conditioned pneumocyte media had an inhibitory effect on macrophage responses subsequently exposed to hypoxia and reoxygenation. CONCLUSIONS: The alveolar macrophage drives the development of lung reperfusion injury in part through amplification of the inflammatory response of type 2 pneumocytes subjected to hypoxia and reoxygenation.

Stress-activated protein kinase inhibition to ameliorate lung ischemia reperfusion injury.

OBJECTIVE: Inhibition of cytokines offers modest protection from injury in animal models of lung ischemia-reperfusion. Improved strategies would selectively inhibit the transcriptional activation response to oxidative stress. Mitogen-activated protein kinases (p38, c-jun N-terminal kinase, extracellular signal-regulated kinase) have been shown to be activated after oxidative stress and in animal models of acute inflammatory lung injury. We hypothesized that mitogen-activated protein kinase inhibition would block downstream transcriptional activation, providing robust protection from lung ischemia-reperfusion injury. METHODS: Experimental rats received inhibitors of p38, c-jun kinase, or extracellular signal-regulated kinase before in situ left lung ischemia-reperfusion. Immunohistochemistry localized cellular sites of mitogen-activated protein kinase activation. Several markers of lung injury were assessed. Enzyme-linked immunosorbent assay measured soluble cytokine and chemokine contents. Western blotting assessed mitogen-activated protein kinase phosphorylation. Electromobility shift assays measured transcription factor nuclear translocation. RESULTS: Immunohistochemistry localized p38 and c-jun kinase activations in positive controls to alveolar macrophages. Extracellular signal-regulated kinase was activated in endothelial and epithelial cells. Animals treated with p38 or c-jun kinase inhibitor demonstrated significant reductions in transcription factor activation and markers of lung injury. Extracellular signal-regulated kinase inhibition was not protective. Western blotting confirmed inhibitor specificity. CONCLUSION: Inhibition of p38 and c-jun kinase provided significant protection from injury. The alveolar macrophage appears to be the key coordinator of injury in response to oxidative stress. Therapeutically targeting specific cell population (macrophage) responses to oxidative stress has the potential benefit of reducing lung reperfusion injury severity while leaving host immune responses intact.

Alveolar macrophage secretory products augment the response of rat pulmonary artery endothelial cells to hypoxia and reoxygenation.

BACKGROUND: Endothelial cell activation is an important response to ischemia and reperfusion in a variety of vascular beds. Endothelial cells secrete a multitude of proinflammatory mediators and express adhesion molecules that promote leukocyte recruitment into injured tissues. Pulmonary artery endothelial cell response to lung ischemia-reperfusion injury does not appear robust enough to drive the development of lung injury independently. Rather, the alveolar macrophage is the key cell in the development of ischemia-reperfusion injury of the lung. Macrophages are known to be a rich source of inflammatory mediators, but the precise mechanism whereby they amplify injury is unknown. The aim of this study was to determine whether alveolar macrophage secretory products amplify the response of the endothelial cell using an in vitro model of lung reperfusion injury. METHODS: Macrophages were exposed to hypoxia and reoxygenation and the media collected. Cultured endothelial cells were then exposed to macrophage media and maintained at normoxia or subjected to hypoxia and reoxygenation. To assess any reciprocal effects of endothelial cell products on macrophage activation, macrophages were likewise exposed to activated endothelial cell media. RESULTS: Exposure of endothelial cells to activated alveolar macrophage media enhanced chemokine secretion in response to hypoxia and reoxygenation. In the reciprocal experiment, activated endothelial cell media increased the production of macrophage inflammatory protein 1alpha from macrophages. CONCLUSIONS: Alveolar macrophages drive the development of lung reperfusion injury, by enhancing the production of proinflammatory chemokines from endothelial cells, which impart a degree of positive feedback on alveolar macrophages.

Poly (ADP) ribose synthetase inhibition in alveolar macrophages undergoing hypoxia and reoxygenation.

BACKGROUND: Inhibition of the nuclear enzyme poly ribose synthetase (PARS) protects against in vivo lung ischemia reperfusion injury (LIRI). The effectiveness of intratracheal treatment suggests that PARS inhibition may primarily modulate alveolar macrophage (AM) activation. These studies attempted to characterize the effects of PARS on AM activation in response to oxidative stress. METHODS: Primary cultures of AM were rendered hypoxic for 2 h and reoxygenated for up to 4 h. Cells were preincubated with INO-1001, a specific PARS inhibitor 1 h prior to hypoxia. Gel shift assays characterized nuclear factor kappa B (NFkappaB), and enzyme linked immunosorbent assay quantitated chemokine/cytokine protein secretion. RESULTS: Hypoxia and reoxygenation resulted in an increase in the early nuclear translocation of NFkappaB, and an increase in the secretion of the cytokine tumor necrosis factor-alpha (TNF-alpha), chemokines macrophage inflammatory protein (MIP-1alpha), monocyte chemoattractant protein one (MCP-1) and cytokine induced neutrophil chemoattractant (CINC). Pretreatment of AM with INO-1001 decreased both the early translocation of NFkappaB and the production of TNF-alpha (p<0.05) and MIP-1alpha p=0.02, but did not affect CINC or MCP-1 production. CONCLUSIONS: These findings indicate that PARS inhibition in the AM blunts their response to oxidative stress and may help explain the protective effects of intratracheal PARS inhibition in LIRI.

Interleukin-6 regulation of direct lung ischemia reperfusion injury.

BACKGROUND: Lung ischemia reperfusion injury continues to adversely affect patient and graft survival after transplantation. While the role of interleukin-6 has been studied in ischemia-reperfusion models of intestine, liver, and heart, its participation in lung reperfusion injury has not been characterized. METHODS: We administered recombinant interleukin-6 to rat lungs through the intratracheal route before inducing left lung ischemia and reperfusion. Multiple in-vivo indicators of left lung injury were studied, as were transactivation patterns for nuclear factor kappa B and signal transduction and activators of transcription-3. Downstream effects on the elaboration of proinflammatory chemokines and cytokines were also studied. RESULTS: Recombinant interleukin-6 reduced endothelial disruption and neutrophil sequestration in left lung and alveolar spaces, resulting in improved oxygenation after ischemia and 4 hours of reperfusion. This protection was associated with decreased nuclear factor kappa B and signal transduction and activators of transcription-3 nuclear translocation early in reperfusion, and diminished proinflammatory mediator secretion late in reperfusion. CONCLUSIONS: Further studies focusing on the effects of recombinant interleukin-6 in large animal models are warranted, as this may be a novel strategy to improve outcomes after lung transplantation. Intratracheal administration may focus its efficacy on the lung while reducing effects on other organ systems during organ procurement.

Crosstalk between thrombosis and inflammation in lung reperfusion injury.

BACKGROUND: Activation of extravascular coagulation has been reported in acute lung injury models of sepsis and acute respiratory distress syndrome. Thrombin, the main effector protease of extravascular coagulation, activates proinflammatory cell types, including macrophages, endothelial cells, and neutrophils, each of which participates in lung ischemia-reperfusion injury. We used hirudin, a potent, specific direct thrombin inhibitor, to define the role of thrombin in lung ischemia-reperfusion injury. METHODS: Rats were pretreated with hirudin 30 minutes before warm, in situ left lung ischemia and reperfusion. Multiple in vivo assessments of lung injury were determined, and mechanistic studies assessed transcriptional regulation early in reperfusion and proinflammatory protein secretion late in reperfusion. Immunohistochemistry localized thrombin activation. RESULTS: Thrombin localized to macrophages and endothelial and epithelial cells early in reperfusion. Hirudin significantly limited lung ischemia-reperfusion injury-induced derangements in vascular permeability and intraalveolar inflammatory cell sequestration, resulting in improved arterial oxygenation after ischemia and 4 hours of reperfusion. The protection was transcriptionally mediated by attenuated activator protein-1 and early growth response-1 transactivation, but not nuclear factor kappa B transactivation. This was associated with reduced chemokine, but not tumor necrosis factor alpha, secretion late in reperfusion. CONCLUSIONS: Thrombin promotes lung ischemia-reperfusion injury, as hirudin protected against experimental acute lung injury. Hirudin conferred protection through a mechanism independent of nuclear factor kappa B and tumor necrosis factor alpha, suggesting that its effects may be mediated by a parallel, synergistic inflammatory pathway through activator protein-1 and early growth response-1.

Poly (ADP) ribose polymerase inhibition improves rat cardiac allograft survival.

BACKGROUND: Heart transplantation is an accepted treatment modality for end-stage heart failure. However, acute cellular rejection (ACR) continues to be a morbid complication. Recently a novel mechanism of inflammatory allograft injury has been characterized which involves overactivation of the nuclear enzyme poly (ADP-ribose) polymerase (PARP). In the present studies, we compared the efficacy of INO-1001, a novel, potent PARP inhibitor, in limiting ACR with and without adjuvant low-dose cyclosporine (CSA). METHODS: Heterotopic heart transplantation was performed utilizing Brown-Norway strains as donors and Lewis rats as recipients. Groups received daily intraperitoneal injections of: vehicle, low-dose CSA, low-dose INO-1001, high-dose INO-1001, and low-dose CSA combined with high-dose INO-1001. Additional animals were sacrificed on postoperative Day 5 for histologic assessments of allograft inflammation, including immunohistochemistry for nitrotyrosine and poly (ADP-ribose) (the product of PARP) staining. RESULTS: PARP inhibition significantly prolonged allograft survival relative to vehicle controls. The combination of low-dose CSA and INO-1001 resulted in a marked increase in allograft survival and significant reductions in allograft rejection scores. This was associated with decreased nitrotyrosine and PAR staining in transplanted cardiac allografts. CONCLUSIONS: Pharmacologic inhibition of INO-1001 prolongs allograft survival in a dose-dependent fashion in a rodent model of heart transplantation. PARP inhibitors may permit reductions in the dose of CSA needed for adequate immunosuppression after heart transplantation.

The role of CC and CXC chemokines in cardiac allograft rejection in rats.

Acute cellular rejection is due in part to an upregulation of chemokine genes, resulting in eventual cell-mediated cytotoxicity. The role of chemokines in acute cardiac allograft rejection is not fully characterized presently. These studies compared the patterns of expression for multiple chemokines in rodent cardiac allograft rejection. Allogeneic transplants were performed from Brown-Norway donors to Lewis recipients. Survival studies utilized daily administration of neutralizing antisera to MCP-1, CINC, and MIP-1alpha. Patterns of mRNA and protein expression were determined by Northern blots and immunohistochemistry. Allogeneic controls rejected at mean of 6.5 days. Neutralization of MCP-1 (10.8 days, P<0.001) and MIP-1alpha (7.5 days, P=0.004) function, but not CINC (6.2 days, P>0.05), significantly prolonged allograft survival. Message expression for the beta chemokines studied were increased by day 2 and continued to increase until day 6 just before rejection, while CINC levels did not change as dramatically after day 2. Chemokine protein levels mirrored mRNA patterns by IHC analysis. MCP-1 and MIP-1alpha appear to play regulatory roles in cardiac allograft rejection, while CINC is expressed, but not functional, in injury development. Beta chemokine activity should be studied further in hope of developing more targeted immunosuppression, or identifying specific chemokines that may be useful for immunosurveillance purposes.

Obliterative airway disease in rat tracheal allografts requires tumor necrosis factor alpha.

Obliterative bronchiolitis is the major complication affecting long-term lung transplant survivors. Tumor necrosis factor-alpha (TNF-alpha) promotes inflammation and fibrosis in chronic lung injury models. These experiments defined the role of TNF-alpha in an established model of obliterative airway disease (OAD). Rat tracheas were transplanted from Brown-Norway donors into Lewis recipients, and explanted on days 7 and 14. Treated groups received either anti-TNF-alpha antibodies or a novel TNF-alpha translational inhibitor, RDP-58, beginning either immediately or on post-transplant day 7. Morphometry assessed epithelial loss and luminal obliteration, while separate tracheas were processed for TNF-alpha mRNA expression by RQRT-PCR or protein localization/expression by immunohistochemistry. EMSAs evaluated NFkappaB activation. 14-day control allografts averaged 58% occlusion and 98% epithelial loss. These parameters were significantly improved with TNF-alpha inhibition, averaging 32% luminal obliteration and 37% epithelial preservation. TNF-alpha mRNA expression increased at 14-days relative to native tracheas, and was unchanged by RDP-58 treatment. However, TNF-alpha protein expression, localized to the mucosa/submucosa, was markedly reduced with RDP-58, and resulted in diminished global NFkappaB activation in allografts. Delayed RDP treatment reduced disease progression during the second week, as luminal occlusion increased from 26% to only 35%, while respiratory epithelium persisted at 21%. TNF-alpha promotes the development of OAD in tracheal allografts via an NFkappaB-dependent mechanism, and its inhibition may prove beneficial clinically.

Cyclosporine modulates the response to hypoxia-reoxygenation in pulmonary artery endothelial cells.

BACKGROUND: Depletion of macrophages, neutrophils, or lymphocytes confers only partial protection against experimental lung reperfusion injury, suggesting that inflammatory responses in other cell types contribute to tissue injury. Endothelial cell activation has previously been shown to be critical to the development of ischemia-reperfusion injury in other vascular beds. Furthermore, cyclosporine (CSA) reduces in vivo lung reperfusion injury through attenuated secretion of proinflammatory mediators. These studies determined whether pulmonary artery endothelial cells (PAEC), subjected to hypoxia and reoxygenation, promote inflammation and whether CSA afforded any modulation of that response. METHODS: Isolated rat PAEC were subjected in vitro to 2 hours hypoxia followed by up to 4 hours reoxygenation. Cells were pretreated with CSA or a cremaphor vehicle. Differences in activation of signaling kinases and transcription factors were assessed, as was cytokine-chemokine protein secretion. RESULTS: There was significant signaling kinase (extracellular signal regulated kinase [ERK 1/2]) activation by 15 minutes reoxygenation, which was temporally associated with marked activation of the transcription factors nuclear factor kappa B (NFkappaB) and early growth response one (EGR-1). At 4 hours reoxygenation there were significant increases in chemokine protein secretion. The CSA decreased ERK 1/2 phosphorylation and significantly attenuated transcription factor transactivation at 15 minutes reoxygenation. The CSA was found to be selective in reducing cytokine-chemokine elaboration at 4 hours reoxygenation. CONCLUSIONS: Hypoxia-reoxygenation induces ERK 1/2 phosphorylation, as well as transactivation of the transcription factors NFkappaB and EGR-1 in PAEC. Cyclosporine selectively reduces proinflammatory mediator secretion, likely by transcriptional regulation through NFkappaB and EGR-1. This is the first demonstration of ERK 1/2 inhibition afforded by CSA.