| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Eur J Cardiothorac Surg 2005;28:581-587
© 2005 Elsevier Science NL
Original articles |
Department of Cardio-thoracic Surgery, Graduate School of Medicine, Tokyo Medical and Dental University, Yushima 1-5-45, Bunkyo-ku, Tokyo 113-8519, Japan
Received 8 March 2005; received in revised form 27 May 2005; accepted 1 June 2005.
* Corresponding author. Tel.: +81 3 5803 5270; fax: +81 3 5803 0141. (Email: t-shim.tsrg{at}tmd.ac.jp).
Abstract
Objective: Ischemia–reperfusion injury is a major factor in the early phase of lung transplantation. We hypothesized that aprotinin, a nonspecific serine protease inhibitor, attenuates ischemia–reperfusion lung injury by inhibiting the inflammatory response and suppressing NADPH oxidase. Methods: We used an isolated rat lung model to test the above. A Control group was immediately perfused with fresh heparinized allogeneic blood after lung harvest without an ischemic period. Study lungs were flushed with low-potassium dextran (LPD) solution and stored for 18h at 4°C then divided into two groups: the LPD group was flushed with LPD solution only, and the LPD+A group was flushed with LPD solution +200KIU/ml aprotinin. Lungs in all three groups were then reperfused with fresh heparinized allogeneic blood for 120min at 37°C. Results: Throughout reperfusion, PO2 levels in the LPD+A group were similar to those in the Control group; although in the LPD group, PO2 levels were significantly lower (P<0.05). Tissue MDA levels were significantly higher in the LPD group than the Control and LPD+A groups (P<0.05). IL-8 levels were significantly higher in the LPD group than the Control group (P<0.05), while in the LPD+A group they were similar to those in the Control group. Histological evaluation showed interstitial edema accompanied by neutrophil extravasation in the LPD group, whereas this effect was modest in the LPD+A group. An additional study of ischemia–reperfusion in an alveolar macrophage culture showed that the activitvation of NADPH oxidase, and translocation of p47phox from the cytosol to the membrane were suppressed in aprotinin group. Conclusions: Aprotinin attenuates ischemia–reperfusion lung injury by inhibiting the early inflammatory response, neutrophil extravasation and the production of oxygen free radicals through inhibition of the activation of the NADPH oxidase. The inhibition of p47phox translocation in alveolar macrophage seemed involved in this mechanism of aprotinin.
Key Words: Aprotinin • Lung • Ischemia–reperfusion
1. Introduction
Lung transplantation has become an established therapeutic option in a selected number of patients with progressive end-stage lung disease. The 1-year actuarial survival is approximately 70%, and most of the deaths occur within 30 days of transplantation. The main cause of death during the first postoperative month is nonspecific graft failure secondary to ischemia–reperfusion lung injury [1,2], despite many attempts have been made to reduce ischemia–reperfusion lung injury: experimentally, improvement of preservation solution, adequate setting of flushing solution, reperfusion pressure and ventilation, and in clinical trials, inhaled Nitric oxide, administration of prostaglandins, complement inhibition, the antagonist of platelet-activating factor and a surfactant therapy [3]. Lung injury induced by ischemia–reperfusion is characterized by increased pulmonary vascular resistance, decreased oxygenation capacity, worsened compliance, and edema formation, and is thought to be neutrophil-mediated organ injury [4]. However, recent studies have revealed the importance of alveolar macrophages. In the early phase of reperfusion, alveolar macrophages are activated as resident cells secreting proinflammatory cytokines. When triggered by ischemia–reperfusion, NADPH oxidase, which mainly exists in macrophages and neutrophils, causes heavy phosphorylation of the cytosolic component p47phox, which subsequently forms a cytosolic complex with another component, p67phox, then translocates to the membrane. As a result, NADPH oxidase produces a burst of oxygen free radicals [5,6]. This event subsequently results in overproduction of reactive oxygen species (ROS), which causes alveolar tissue damage. Ischemia–reperfusion also stimulates matrix metalloproteinases (MMPs) and affects basement membranes, further promoting neutrophil extravasation into the alveolar interstitial space [7].
Aprotinin, a nonspecific serine protease inhibitor, has been extensively used in cardiac operations to reduce postoperative blood loss [4,8–12]. In a recent report, neutrophil extravasation in association with ROS production, a necessary step in the inflammatory cascade, was inhibited by aprotinin in the mesenteric microcirculation [8], or during cardiopulmonary bypass [9]. These results suggest that aprotinin attenuates lung reperfusion injury, although its mechanism remains unknown [4,13].
The present study was designed to test the following hypothesis using an isolated rat lung model: aprotinin used as an additive to flush-preservation solution, ameliorates ischemia–reperfusion injury by suppressing alveolar macrophages to induce proinflammatory cytokines, and to promote neutrophil migration. These cytokines, and other signals, could also stimulate phosphorylation of NADPH oxidase and subsequent ROS generation.
2. Material and methods
2.1 Experimental protocol
Male Sprague–Dawley rats weighing 350–450g were obtained from Sankyo Labo Service Co., Inc. (Tokyo, Japan). All animals received humane care in compliance with the ‘Guide for the Care and Use of Laboratory Animals (National Institute of Health Publication Nos. 86-23, revised 1985)’, and this study was approved by the Institutional Committee for Experimental Research in our University.
To assess ischemia–reperfusion injury, isolated lungs were assigned to three groups. Lungs in the Control group (n=7) were immediately reperfused with fresh heparinized allogeneic blood for 120min after harvest without an ischemic period. Lungs in the other two groups were flushed with low-potassium dextran (LPD) solution through the main pulmonary artery and stored for 18h at 4°C. They were then divided into two groups: lungs in the LPD group (n=7) were flushed with LPD solution alone, and lungs in the LPD+A group (n=7) were flushed with LPD solution mixed with 200KIU/ml of aprotinin. After 18h of ischemia, each lung in the LPD and LPD+A groups was reperfused with fresh heparinized allogeneic blood for 120min in a warm chamber kept at 37°C.
2.2 Procedure for lung harvesting
Animals were anesthetized with 50mg/kg injections of intraperitoneal ketamine hydrochloride. A 14-gauge angiocatheter was then inserted into the trachea by cervical tracheotomy and secured with 3-0 braided silk ligatures. Animals were ventilated with 95% O2+5% CO2 at a tidal volume of 3ml and a rate of 50breath/min with a positive end-expiratory pressure (PEEP) of 3cm water using a volume-limited ventilator (model SN-480-7; Shinano Ika, Tokyo, Japan). After an abdominal midline incision, heparin (1000U/kg) was injected into the inferior vena cava. Blood samples were collected from the right common iliac artery for measurements of PO2 to assess oxygenation capacity in vivo before harvest. Then, a median sternotomy and thymectomy was performed to expose the heart–lung block. A cannula was placed into the main pulmonary artery through the right ventricular outflow tract and secured with 3-0 braided silk sutures through the transverse sinus. The left atrium and left ventricle were amputated to vent blood. The Control group was immediately reperfused after harvesting without flushing with LPD solution, but the study lungs were flushed with 20ml of LPD solution at 4°C through the main pulmonary artery from a height of 25cm. After flushing, the heart–lung block, with the lung inflated, was harvested and stored at 4°C for 18h.
2.3 Reperfusion
Additional male Sprague–Dawley rats served as fresh blood donors from which heparinized blood (1000U/kg) was collected. Heart–lung blocks were mounted in a warm chamber kept at 37°C (Fig. 1
). The perfusion circuit was primed with 16ml of heparinized blood adjusted to a hematocrit of 20% with modified Krebs–Henseleit buffer solution (NaCl: 118mM, KCl: 4.7mM, KH2PO4: 1.2mM, NaHCO3: 24mM, MgSO4·7H2O: 1.2mM, glucose: 11.0mM, CaCl2·H2O: 1.7mM); sodium bicarbonate was added to maintain the pH at 7.4–7.5. Blood from the left atrium and left ventricle was drained into the chamber and circulated to the heart–lung block through a membrane oxygenator (Senko Ika, Tokyo, Japan) using a roller pump (model 7518-10 Masterflex pump system, Cole-Palmer Instrument Company, Chicago, IL). Lungs were ventilated with 95% O2/5% CO2 at a tidal volume of 3ml and a rate of 50breathes/min with a PEEP of 3cm water. The rate of perfusion blood flow was gradually increased to 8ml/min in the first 15min then continued at a constant rate during reperfusion. Deoxygenation of perfusion blood into the pulmonary artery was done with 95% N2/5% CO2 delivered through a membrane oxygenator to adjust the PO2 to 40–50mmHg.
|
After reperfusion, the lung blocks were immediately flushed with 20ml of normal saline solution through the main pulmonary artery to washout the blood, and then kept in liquid-nitrogen and frozen at –80°C until being measured for tissue MDA levels and gelatin zymography. Lungs for histological examination were fixed with 10% formalin.
2.5 Measurement of malondialdehyde (MDA) in the lung tissue
Right lung tissue samples from each group were used for measurements of lipid peroxidation. The thiobarbituric acid (TBA) test was used for measuring levels of tissue malondialdehyde (MDA), a last product of lipid peroxidation that reflects free radical oxygen. Lung tissue was homogenized in 1.15% KCl solution then 100µl of 10% homogenized sample was prepared, and 0.2ml of 8.1% sodium dodecyl sulfate (SDS), 1.5ml of 20% acetate acid, 1.5ml of TBA solution and 0.7ml of distilled water were added to the sample. The mixture was kept in a boiling water bath for 1h. After these procedures, the mixture was cooled in tap water for 10min, then 1.0ml of distilled water and 5.0ml of N-butanol/pyridine mixture were added. The mixture was centrifuged at 3000rpm for 15min, and the N-butanol/pyridine phase (supernatant fluid) was measured for absorbance of the colored complex; results were expressed as per gram of wet tissue.
2.6 Cytokine levels in the lung tissue
The extracted protein from the lung tissue was also used for measuring tissue cytokine levels. Concentrations of TNF-
, IL-1ß and IL-8 were determined by a solid-phase sandwich Enzyme-Linked Immunosorbent Assay (ELISA).
2.7 Gelatin zymography
Left lung tissues from each group were used for gelatin zymography to assay MMP expression as previously described by Kunugi et al. [14]. Briefly, frozen lung tissue was homogenized then 10µg of total protein were subjected to electrophoresis through 10% polyacrylamide gels containing 0.1% gelatin. After electrophoresis, the gels were washed in 2.5% Triton X-100 to remove sodium dodecyl sulfate, incubated for 18h at 37°C then stained with 0.1% Coomassie Brilliant Blue R250. Enzyme activity appeared as clear bands against a blue background, and densitometric analysis of the bands was performed on a Macintosh computer using NIH image software.
2.8 Histological evaluation
Reperfused lungs from each group were also histologically examined. After completion of reperfusion, lungs were immediately fixed in 10% neutral buffered formalin then embedded in a paraffin block. Four-micrometer thin sections were then cut from the paraffin blocks and stained with hematoxylin and eosin (HE). Neutrophils were counted in 20 different magnification areas (x400 magnification, OLYMPUS AX-80 0.1mm2) for each group then the total numbers of extravasated neutrophils were divided by 20 to find the average number per area.
2.9 Study of NADPH oxidase
Changes in NADPH oxidase were studied in vitro using alveolar macrophages obtained by bronchoalveolar lavage (BAL). Animals were anesthetized and a 14-gauge angiocatheter was inserted into the trachea. A median sternotomy was then performed followed by intratracheal lavage of the lungs with 10 injections of 5ml of cold phosphate-buffered saline (PBS). At least 90% of fluid was recovered from each sample. The lavage samples were immediately centrifuged at 3000rpm for 15min, and the pellets were collected.
2.10 Alveolar macrophages culture
With the ischemia groups, pellets were incubated in LPD solution at 4°C for 2h then reoxygenated in the incubator at 37°C for 1h in RPMI 1640 medium (0.1% BSA, 1% wt/vol penicillin and streptomycin) while exposed to 95% O2/5% CO2. They were then divided into two groups: the IR group was incubated in LPD solution alone at 4°C for 2h and the IR+A group was incubated in LPD solution mixed with 200KIU/ml of aprotinin at 4°C for 2h, then they were both incubated at 37°C for 1h in RPMI 1640 medium. The non-IR group was immediately incubated at 37°C for 1h in the RPMI 1640 medium without ischemia and served as a control.
2.11 Immunofluorescence for the components of NADPH oxidase
After incubation, cells from each group were fixed in 95% methanol, and immunohistochemistry was performed. Polyclonal goat antibodies to p22phox (sc-11712), p47phox (sc-7660), p67phox (sc-7663) and gp91phox (sc-11712) were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). First, cells were incubated with antibodies (1:400) for 2h at 4°C then with FITC conjugated polyclonal rabbit anti-goat secondary antibody (Code-Nr. F0250; 1:250 dilution, DAKO, Copenhagen, Denmark) at room temperature for 1h.
2.12 Statistical analysis
Results are expressed as the mean±standard error of the mean (SEM). Data were analyzed by analysis of variance (ANOVA). If ANOVA showed an overall difference, posthoc comparisons were performed with the Bonferroni–Dunn test for paired or unpaired data as appropriate. P<0.05 was considered statistically significant.
3. Results
3.1 Lung function during reperfusion
In vivo PO2 levels before the lung harvest were the same in all groups. Throughout the 120min of reperfusion, PO2 levels in the LPD+A group was preserved similarly as the control group, whereas PO2 levels in the LPD group were lower than in the other two groups (P<0.05) (Fig. 2
). Peak airway pressure (pAwP) in the LPD+A group was significantly lower than in the LPD group (P<0.05) after 30min of reperfusion (Table 1
). Mean pulmonary artery pressure (mPAP) showed the similar values between the groups during reperfusion, except the abnormally high starting values in the both ischemic groups (Table 1).
|
|
levels were 47.4±12.1, 25.9±4.1 and 38.8±3.1µg/g in the LPD, Control and LPD+A groups, respectively; these differences were not significant. IL-1ß levels were 22.2±3.1, 14.5±1.2 and 18.5±1.5µg/g in the LPD, Control and LPD+A groups; these differences were not significant. IL-8 levels were 529.60±116.1µg/g in the LPD group compared with 260.4±36.0µg/g in the Control group (P<0.05), while in the LPD+A group the IL-8 level was 320.0±15.2µg/g, which was similar to that in the Control group, but lower than that in the LPD group (P=0.07) (Fig. 3
).
|
|
|
|
The remarkable effect of aprotinin as an additive to flush-preservation solution for lung was revealed in the present study, which involved 18-h lung preservation at 4°C followed by normothemic reperfusion with blood. The MDA and IL-8 levels in the lung tissue after reperfusion were well suppressed in aprotinin group, which accords with the observation in the collected alveolar macrophages that the activation of NADPH oxidase and the translocation of p47phox were suppressed. Levels of malondialdehyde (MDA), a last product of lipid peroxidation, are a good indicator of tissue oxidation. The aprotinin-treated lungs showed significantly better oxygenation capacities, similar to the non-ischemic lungs, than the LPD-treated lungs throughout the reperfusion period. This preservative effect of aprotinin was also confirmed by the prevention of increase in peak airway pressure in aprotinin group. However, we could not detect the aprotinin effect on mean pulmonary artery pressure, probably because it was largely influenced by abnormally high starting value after cold preservation. Furthermore, we observed that lungs rendered cold ischemia took nearly 15min to show homogeneously red to pink color in the lug surface. These results suggest that firstly, cold ischemic preservation followed by reprfusion is responsible to interstitial edema and neutrophil extravasation into alveoli, secondly, aprotinin prevented these pathological changes (Fig. 5).
The importance of alveolar macrophages in ischemia–reperfusion lung injury has recently been revealed. In lung transplantation, alveolar macrophages are activated in the early phase of reperfusion as resident cells [15,16], which secrete proinflammatory cytokines that prompt neutrophil attraction. In this state, neutrophils attach to vascular endothelial cells, which are subsequently activated and invade the alveolar interstitial space (extravasation) where they induce production of ROS, resulting in lung parenchyma damage. Fisher et al. demonstrated that early phase lung transplant reperfusion injury is mediated by donor pulmonary macrophages, while late injury is induced by recipient circulating neutrophils [15]. Hypoxia induces macrophages to release proinflammatory cytokines. The proinflammatory cytokines TNF-, IL-1ß and IL-8 are thought to initiate a cascade of events that lead to the expression of adhesion molecules on endothelial cells, neutrophil activation and sequestration [17]. TNF- and IL-1ß appear to promote injury by altering expression of proinflammatory and anti-inflammatory cytokines and influencing tissue neutrophil recruitment [18]. Especially, IL-8 has such a potent chemoattractant activity for neutrophils that it can induce neutrophil migration through adhesion at the vascular endothelium. NADPH oxidase, which produces free radicals, exists in alveolar macrophages and neutrophils. When activation NADPH oxidase takes place, p47phox, p67phox and p40phox translocate from cytosol to the membranes, where they become associated with b558, which is composed of gp91phox and p22phox, and are involved in the assembly of the catalytically active oxidase [5]. NADPH is activated by the phosphorylation of multiple serine residues in p47 phox [6]. In our study, we tested the effect of aprotinin in vitro using macrophages obtained by broncho-alveolar lavage (BAL). Immunohistological staining suggested that aprotinin inhibited localizing of p47phox from the cytosol to the membranes, although the present study was not designed to determine whether aprotinin inhibits the activation of NADPH oxidase directly or via another signal transduction pathway. In this regard, further studies including molecular details are waited.
Aprotinin, a serine protease inhibitor, reduces perioperative blood loss in cardiac surgery [4,8,9–11], by blocking enzymes such as trypsin, plasmin and kallikrein, leading to preserve platelet function and reduce anticoagulant activity of endothelial cells [12]. Aprotinin is also thought to modify the inflammatory response to major operations by inhibiting neutrophil activation and/or extravasation [9]. As for the organ preservation, it has been demonstrated that aprotinin is effective in preserving adenine nucleotide and adenosine triphosphate levels during prolonged hypothermic cardioplegic preservation followed by reperfusion [19]. For the experimental lung preservation, it has been shown that aprotinin added into preservation solution attenuates ischemia–reperfusion lung injury assessed by oxygen capacity [4] and compliance and capillary permeability as well [13]. For the working mechanism of aprotinin, the inhibition of neutrophil migration through pulmonary endothelial layers has been shown in the mesenteric microcirculation of rats [8] and in lung tissues after cardiopulmonary bypass [10], associated with the decrease of neutrophil extravasation into the alveolar space by BAL and alveolar histoligical injury [10]. Rahman et al. reported that aprotinin added to the prime solution during CPB reduced lung tissue MDA levels [9]. Furthermore, Eren et al. reported that the addition of aprotinin to lung protection solution significantly reduced tissue MDA levels in an in situ normothermic 2-h ischemic lung model [10], which is consistent with the present results. However, the mechanism of neutrophil extravasation and prevention of ROS has not been clearly elucidated. In cardiac operations using cardiopulmonary bypass (CPB), controversy exists with regards to the effect of aprotinin, namely, whether aprotinin reduces post-CPB IL-8 levels significantly [11,20], or insignificantly [12]. In our ex vivo study, tissue TNF- and IL-1ß were detected at very low levels and were not significantly different among the Control, LPD and LPD+A groups. However, tissue IL-8 levels were significantly higher in the LPD group than the Control group. Tissue IL-8 levels in the LPD+A group, which was treated with an aprotinin dose of 200KIU/l, were similar to those in the Control group and tended to be lower than those in the LPD group (P=0.07). Asimakopoulos et al. demonstrated that aprotinin, especially in high doses, attenuated the effect of regular concentrations of IL-8 in vitro during observations of neutrophil transmigration through HUVEC monolayers [8]. Our results, namely, that the addition of aprotinin to lung preservation solution reduced tissue IL-8 levels after ischemia–reperfusion, suggest that aprotinin directly inhibits the secretion of IL-8 from alveolar macrophages, endothelial and epithelial cells.
Matrix metalloproteinases (MMPs) is also stimulated by ischemic-reperfusion in response to various pathways, and MMPs affect basement membranes [7]. Lung basement membranes are composed of an extracellular matrix that includes type IV collagen [21]. The repair and reconstruction of lung tissues requires destruction of basement membranes, and MMP-2 and MMP-9, which specifically decompose type-IV collagen, are thought to play an important role in this. The most potent stimulus of MMPs is oxidative stress, which affects MMP-9 gene expression via oxidant-induced NF-
ß activity [22]. The degradation of basement membranes promotes neutrophil extravasation. Yano et al. reported that MMP-9, but not MMP-2, might play an important role in ischemia–reperfusion lung injury [7]. Our gelatin zymography data also showed that MMP-9 levels both in the LPD and LPD+A groups were greater than those in the Control group, but MMP-2 levels were the same in all three groups. Keck et al. also demonstrated that aprotinin did not inhibit gelatinolytic activity in a pancreatitis-associated lung injury model in rat [23]. This result suggests that aprotinin did not affect MMP-2 and MMP-9 activity directly, and that another mechanism might have inhibited neutrophil extravasation.
In conclusion, aprotinin attenuates ischemia–reperfusion lung injury by inhibiting neutrophil extravasation and the production of IL-8 and ROS, but not MMP activity. ROS production appears to be suppressed by a mechanism whereby aprotinin inhibited the activation of NADPH oxidase. Before clinical application, however, the effect of aprotinin observed in the present study should be re-studied by the vivo study in the presence of host inflammatory and immune system. Also, clinical use of aprotinin should be cautiously examined in the regard of its possible prothrombotic action in the endothelial layers, and anaphylactic reaction [24,25].
References
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| ANN THORAC SURG | ASIAN CARDIOVASC THORAC ANN | EUR J CARDIOTHORAC SURG |
| J THORAC CARDIOVASC SURG | ICVTS | ALL CTSNet JOURNALS |
