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The effect of FK409—a nitric oxide donor—on canine lung transplantation
The Effect of FK409 —a Nitric Oxide Donor— on Canine Lung Transplantation Yutaka Sunose, MD,a Izumi Takeyoshi, MD,a Susumu Ohwada, MD,a Shigeru Iwazaki, MD,a Masaaki Aiba, MD,a Naoki Tomizawa, MD,a Hirofumi Tsutsumi, MD,a Noboru Oriuchi, MD,b Koshi Matsumoto, MD,c and Yasuo Morishita, MDa Background: Nitric oxide (NO) is known to have beneficial effects in ischemiareperfusion (I/R) injury through maintaining endothelial integrity, inhibiting leukocyte adhesion and platelet aggregation, and inducing vasodilation. The effect of FK409 (FK), a spontaneous NO donor, was investigated in a canine lung transplantation model. Methods: Ten pairs of weight-matched dogs were used. Five pairs were assigned to the FK group, to which FK (5 g/kg/min) was administered intravenously from 30 minutes prior to ischemia until the induction of ischemia in the donor, and from 15 minutes prior to reperfusion until 45 minutes after reperfusion in the recipient. The others were assigned to the control group. After 8-hour preservation in 4° C Euro-Collins solution, orthotopic single-lung transplantation was performed. During a 5-minute clamping test of the right pulmonary artery, left pulmonary arterial pressure (L-PAP), left pulmonary vascular resistance (L-PVR), arterial oxygen pressure (PaO2), and alveolar-arterial oxygen pressure difference (A-aDO2) were measured. The lung specimens were harvested for histologic study, and polymorphonuclear neutrophils (PMNs) were counted. Pulmonary perfusion and ventilation scintigraphy (Tc-99m-MAA and Xe-133) were performed. Results: PAP, L-PVR, PaO2, and A-aDO2 revealed significantly (p ⬍ 0.05) better function in the FK group than in the control group. Histologically, edema was more mild, and PMN infiltration was significantly (p ⬍ 0.05) lower in the FK group than in the control group. Xe-133 and Tc-99m–MAA were widely distributed throughout the graft lung in the FK group. The 2-day survival rate was 100% in the FK group, which was significantly (p ⬍ 0.05) better than the rate (40%) in the control group. Conclusions: FK appears to generate a protective effect on I/R injury in lung transplantation. J Heart Lung Transplant 2000;19:298–309.
L
ung transplantation is an acceptable therapeutic approach for patients with end-stage lung disease, and the short-term survival rates have recently im-
proved.1,2 However, donor lungs are particularly vulnerable to ischemia, and tolerable ischemic time is reported to be shorter than that of other trans-
From Second Department of Surgery,a Gunma University School of Medicine, Gunma, Japan; Department of Nuclear Medicine,b Gunma University School of Medicine, Gunma, Japan; and Department of Pathology,c Nippon Medical School, Kanagawa, Japan. Submitted July 26, 1999; accepted December 10, 1999. Reprint requests: Izumi Takeyoshi, MD, Second Department of Surgery, Gunma University School of Medicine, 3-39-15
Showa-Machi, Maebashi, Gunma 371-8511, Japan. Telephone: ⫹81-27-220-8242. Fax: ⫹81-27-220-8250. E-mail: takeyosi@ akagi.sb.gunma-u.ac.jp. Copyright © 2000 by the International Society for Heart and Lung Transplantation. 1053-2498/00/$–see front matter PII S1053-2498(99)00140-0
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plantable solid organs.3,4 Ischemia-reperfusion (I/R) injury is a significant factor for morbidity, and it may result in deterioration of the graft function or in primary graft failure after lung transplantation. Its mortality rate is as high as 20% to 25%, and it is the second most frequent cause of death within 90 days after transplantation.3,5,6 The acceptable ischemic time for lung grafts is now thought to be between 4 and 7 hours,7,8 although inhibiting I/R injury at the point of donor organ harvesting,3 graft preservation,9,10 and recipient reperfusion11 may prolong this time and can be expected to result in safer transplantation. Nitric oxide (NO) is endogenously and constitutively produced in vascular endothelial cells, and protects against I/R injury by maintaining vascular integrity.12 FK409 (FK) ([⫾]-[E]4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide; Fujisawa Pharmaceutical Co. Ltd.; Osaka) is an organic NO donor, which spontaneously releases NO.13 FK409 has potent vasorelaxant and antiplatelet effects,13 and FK produces these effects through elevating the plasma cyclic guanosine monophosphate (cGMP) level.14 We have previously reported the protective effect of intravenous administration of FK in canine hepatic warm-ischemia and reperfusion models.15 In this study, we evaluated the effect of FK on pulmonary I/R injury in a canine lung transplantation model.
MATERIALS AND METHODS Animals Twenty adult mongrel dogs weighing 8 to 14 kg were used in this study. The animals received a standard commercial diet and were allowed free access to food and water until 12 hours prior to surgery. All the animals received humane care in compliance with the Principles of Laboratory Animal Care, formulated by the National Society for Medical Research, and the Guide for the Care and Use of Laboratory Animals, published by the National Institutes of Health (NIH publication No. 86-23, revised 1985). This experimental study was performed with the approval of the Animal Care and Experimental Committee, Gunma University, Showa Campus.
Donor Procedure After administering ketamine hydrochloride (10 mg/ kg, intramuscular injection), the animals were anesthetized with pentobarbital sodium (10 mg/kg) and pancuronium bromide (0.2 mg/kg), then intubated
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and mechanically ventilated at a tidal volume of 20 ml/kg and a rate of 15 breaths/min. Positive endexpiratory pressure was controlled at 5.0 cm H2O, and the inspired O2 fraction was 0.5. Anesthesia was maintained with inhalation of 1% to 2% halothane, and muscular relaxation was obtained with additional pancuronium bromide of 0.1 mg/kg. An arterial line inserted into the right carotid artery monitored blood pressure and blood gases. The right external jugular vein was used as a venous infusion line. A left thoracotomy was performed at the fifth intercostal space. The aorta, pulmonary artery, and trachea were isolated. Sodium heparin (300 U/kg) was administered systemically. After clamping the main pulmonary artery, a catheter was inserted into the main pulmonary artery while ligating the right pulmonary artery, and the left lung was flushed with 4° C Euro-Collins solution (50 ml/kg). Simultaneously, the lung was cooled topically by immersing it in saline ice slush. During flushing, the lungs were ventilated continuously. After the flush, the heartlung block was excised and preserved in 4° C EuroCollins solution for 8 hours. During preservation, the lungs were kept inflated at 25 cm H2O of airway pressure. After 8-hour preservation, the left lung was dissected from the heart-lung block in a basin containing cold Euro-Collins solution.
Recipient Procedure Recipient dogs were anesthetized and ventilated, and catheters were inserted in the same manner as in the donors. After thoracotomy, a left pneumonectomy was performed. Then the donor lung was orthotopically transplanted in the following way: The left atrial anastomosis was performed with running 5-0 Prolene everting sutures. The arterial and bronchial anastomoses were performed with running 6-0 and 4-0 Prolene sutures, respectively. After completion of these anastomoses, the lung was ventilated and reperfused. Thirty minutes after reperfusion, a 5-minute clamping test of the right pulmonary artery was performed, and specimens from the graft lung were harvested. The chest was then closed and a drainage tube left in place. The animals were observed until 2 days after the operation and then sacrificed. Lung specimens were harvested 2 days after reperfusion or at the time of death for histopathologic study.
Experimental Design Ten weight-matched pairs of adult mongrel dogs were randomly divided into 2 groups. Five pairs were assigned to the FK group, in which FK (5
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g/kg/min) was administered to the donor from 30 minutes prior to ischemia until the induction of ischemia, and to the recipient from 15 minutes prior to reperfusion until 45 minutes after reperfusion. Five pairs were assigned to the control group (n ⫽ 5), in which physiologic saline solution was administered in the same manner.
Hemodynamics and Blood Gas Measurements During the experiment, arterial blood pressure (ABP) was monitored continuously. The ABP of the recipient dogs with FK administration immediately, 30 minutes and 2 hours after reperfusion was compared with their status before FK administration and with the control group. A 5-minute clamping test of the right pulmonary artery was performed prior to ischemia in the donor, and 30 minutes after reperfusion in the recipient. During the test, the left pulmonary arterial pressure (L-PAP) was measured by inserting a 24-gauge needle in the main pulmonary artery and connecting it to a transducer (Spectramed TA 1017, San-ei Co.; Tokyo). The left atrial pressure (LAP) was measured via a catheter inserted into the left atrium through the appendage and connected to the transducer. The cardiac output (CO) was measured by placing an electromagnetic blood flowmeter (MF V-3100, Nihonkohden Co.; Tokyo) on the ascending aorta. The left pulmonary vascular resistance (LPVR) during the 5-minute clamping test was calculated using the following formula: L-PVR (dynes 䡠 sec 䡠 cm ⫺ 5) ⫽ (mean L-PAP (mm Hg) ⫺ mean LAP (mmHg)) ⫻ 79.92/CO (L/min).
At the same time, arterial blood samples were taken to measure arterial oxygen pressure (PaO2) and alveolar-arterial oxygen pressure difference (AaDO2). The PaO2 was determined by a blood gas analyzer (Stat Profile M, Nova Biomedical Co.; Waltham, Massachusetts), and the A-aDO2 was calculated using the following formula: A-aDO2 (mm Hg) ⫽ PiO2 ⫺ PacO2/0.8 ⫺ PaO2, Pio2 ⫽ (760 ⫺ 47) ⫻ Fio2.
Nitrite and Nitrate Measurements The serum NO levels (nitrite and nitrate) were measured before ischemia; before reperfusion (endischemia); and immediately, 30 minutes, 2 hours, and 6 hours, after reperfusion. Using an enzymatic assay kit (Cayman Chemical Company, Ann Arbor; Michigan), nitrite and nitrate were measured in
plasma samples, making use of the 2-step Griess reaction. The first step involves converting nitrate to nitrite using nitrate reductase. The second step is the addition of the Griess reagents, which convert nitrite to an azo compound. For nitrate measurements, 40-ml aliquots of plasma were placed into wells, adjusting the volume to 80 ml with the assay buffer solution. Ten ml of enzyme cofactor and 10 ml of nitrate-reductase cofactor mixture were also added and left to incubate for 3 hours at room temperature. After the process, 100 ml of Griess reagents were added to each of the wells, and the wells were left at room temperature for 10 minutes to develop the color. Then the absorbance at 540 nm was read using a plate reader. To measure nitrite, the same procedure was performed, excluding the first step. The results were expressed as the total amounts of nitrite and nitrate.
Endothelin-1 Measurements The endothelin-1 (ET-1) levels in arterial blood were measured 2 hours after reperfusion. Blood samples for the ET-1 measurement were drawn into ice-chilled tubes containing K2 ethylenediaminetetraacetic acid and Trasylol, and the plasma was immediately separated by centrifugation at 4° C and stored at ⫺80° C until the assay. Plasma samples were extracted in octylsilane-silica cartridges with 2 ml 60% acetonitrile and 0.09% trifluoroacetic acid, and evaporated in a centrifugal concentrator. The dried residue was reconstituted in the assay buffer and subjected to radioimmunoassay. A total of 0.1 ml assay buffer and 0.1 ml antiendothelin-1 serum at a final dilution of 1:300,000 was incubated at 4° C for 20 hours. Then 0.05 ml of 83 pmol/ml 125I-endothelin-1 with a specific activity of 74 TBq/mmol (Amersham International, Buckinghamshire; United Kingdom) was added and incubated at 4° C for 48 hours. The bound and the free ligands were separated by the double-antibody/polyethylene glycol method.
Histopathologic Studies and Polymorphonuclear Neutrophil Measurements Lung specimens were harvested for histopathologic examination 30 minutes after reperfusion, and 2 days after reperfusion (at the time of sacrifice) or at the time of death, and were fixed in 10% formalin. Tissues were dehydrated, embedded in paraffin, cut into 3 to 5 m sections, and mounted. After deparafinizing, the tissues were stained with hematoxylin and eosin, and also stained with naphthol AS-D chloroacetate esterase. The polymorphonu-
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clear neutrophil (PMNs) were identified by positive staining and morphology, and were counted under ⫻400 magnification. At the same time, the alveoli were also counted, and the data were expressed as PMNs/alveolus. A single pathologist, blind to the details of each specimen, performed the measurements.
Radiologic Studies To investigate the in situ function of the lung graft, pulmonary perfusion and ventilation scintigraphy were performed 6 hours after reperfusion. In the perfusion scan, Tc-99m–macro agglutinated albumin (MAA) (1 mCi) was administered intravenously. Then a Tc-99m-MAA scintillation image was taken to investigate the blood perfusion of the lung graft. In the ventilation scan, Xe-133 (3 mCi) was administered through inhalation under spontaneous respiration. A Xe-133 scintillation image was also made to investigate the ventilatory capacity of the lung graft.
Statistical Analysis All the values are expressed as the mean ⫾ the standard error of the mean. Statistical analysis was performed using the Mann-Whitney U test and analysis of variance (ANOVA). The animal survival rate was determined using the Kaplan-Meier method, and the log-rank test was used to determine significance. A p value ⬍ 0.05 was considered to be statistically significant.
RESULTS No significant differences were measured in preservation time (cold ischemic time) or operating time (warm ischemic time) between the FK group and the control group. There were also no significant differences in the mean ABP, CO, L-PAP, L-PVR, PaO2, or A-aDO2 of the FK and control groups before ischemia in the donor. The systemic hemodynamics (ABP) were measured in the recipient before and after the administration of FK. The mean ABP 30 minutes before ischemia (before the administration of FK), and immediately after and 30 minutes after reperfusion (during the administration of FK), was 162 ⫾ 10, 107 ⫾ 10, and 114 ⫾ 12 mm Hg, respectively, in the FK group; and 158 ⫾ 12, 140 ⫾ 8, and 116 ⫾ 14 mm Hg, respectively, in the control group. In the FK group, the mean ABP levels immediately after and 30 minutes after reperfusion were significantly (p ⫽ 0.018 and 0.005) lower than the pre-ischemic level, but the mean ABP remained over 100 mm Hg. The
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mean ABP immediately after reperfusion was significantly (p ⫽ 0.046) lower in the FK group than in the control group, but 30 minutes after reperfusion there was no significant (p ⫽ 0.899) difference between the groups. The mean ABP 2 hours after reperfusion improved to 140 ⫾ 13 mm Hg in the FK group (Figure 1). A 5-minute clamping test of the right pulmonary artery was performed prior to inducing ischemia and 30 minutes after reperfusion. During the test, LPAP, L-PVR, CO, PaO2, and A-aDO2 were measured simultaneously. The L-PAP in the control group was significantly (p ⫽ 0.043) higher after 30 minutes of reperfusion than in the pre-ischemic condition. Whereas, in the FK group, the L-PAP after 30 minutes of reperfusion was not significantly (p ⫽ 0.56) higher than in the pre-ischemic condition of the donor lung. After 30 minutes of reperfusion, the L-PAP was significantly (p ⫽ 0.035) lower in the FK group than in the control group. The L-PVR in the control group was significantly (p ⫽ 0.043) higher after 30 minutes of reperfusion than in the pre-ischemic condition of the donor lung. On the other hand, in the FK group, the L-PVR after 30 minutes of reperfusion was not significantly (p ⫽ 0.685) higher than in the pre-ischemic condition. After 30 minutes of reperfusion, the L-PVR was significantly (p ⫽ 0.009) lower in the FK group than in the control group. The CO in the control group was significantly (p ⫽ 0.028) lower after 30 minutes of reperfusion than in the pre-ischemic condition. There was no significant (p ⫽ 0.272) difference in the CO after 30 minutes of reperfusion between the FK group and the control group. The PaO2 in the control group was significantly (p ⫽ 0.043) lower 30 minutes after reperfusion than the pre-ischemic level. The PaO2 30 minutes after reperfusion was significantly (p ⫽ 0.047) higher in the FK group than in the control group. The A-aDO2 in the control group was significantly (p ⫽ 0.043) higher 30 minutes after reperfusion than the pre-ischemic level. The A-aDO2 30 minutes after reperfusion was significantly (p ⫽ 0.047) lower in the FK group than in the control group (Table I). The serum NO levels (nitrite and nitrate) were measured before, during, and after administration of FK. The serum NO levels during FK administration (at end-ischemia, at reperfusion, and at 30 minutes after reperfusion) in the FK group increased beyond the levels before ischemia, and remained higher than in the control group. The serum NO levels in the control group tended to decrease after ischemia and reperfusion (Figure 2).
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FIGURE 1 Changes in mean arterial blood pressure before, during, and after
administration of FK or vehicle in the recipient dogs. Data are expressed as mean ⫾ SEM. # p ⬍ 0.05 vs pre-ischemic level *p ⬍ 0.05 vs Control group Pre, pre-ischemia; Reperf, reperfusion; 30 min, 30 min after reperfusion; 2 hr, 2 hr after reperfusion.
The serum ET-1 level 2 hours after reperfusion was significantly (p ⫽ 0.043) lower in the FK group (41.9 ⫾ 8.0 pg/ml) than in the control group (80.3 ⫾ 15.1 pg/ml). Histologically, only slight interstitial edema was observed in the FK group, whereas interstitial, alveolar, and alveolar-septal edema along with inflammatory cell infiltration was observed in the control group after 30 minutes of reperfusion (Figure 3). At the time of death, severe alveolar damage with
edema was observed in the control group. These findings were obviously slight in the FK group 2 days after reperfusion (at the time of sacrifice) (Figure 4). The PMN infiltration was significantly (p ⫽ 0.028) lower in the FK group (0.66 ⫾ 0.16 PMNs/ alveolus) than in the control group (1.42 ⫾ 0.12 PMNs/alveolus). The perfusion scintigram, which was performed 6 hours after reperfusion, revealed sufficient perfusion of the lung graft in the FK group (Figure 5A).
TABLE I Levels of hemodynamics and gas exchange Before Ischemia
L-PAP (mm Hg) L-PVR (dynes 䡠 sec 䡠 cm⫺5) CO (L/min) PaO2 (mm Hg) A-aDO2 (mm Hg)
30 Minutes after Reperfusion
Control
FK
Control
FK
29.0 ⫾ 4.2 2,641 ⫾ 397 1.30 ⫾ 0.12 175 ⫾ 12 144 ⫾ 13
32.0 ⫾ 2.9 2,575 ⫾ 359 1.12 ⫾ 0.09 169 ⫾ 14 151 ⫾ 15
53.8 ⫾ 2.8# 7,681 ⫾ 1,352# 0.72 ⫾ 0.19# 76 ⫾ 13# 243 ⫾ 12#
34.0 ⫾ 5.5* 3,341 ⫾ 339* 0.82 ⫾ 0.09 119 ⫾ 14* 202 ⫾ 10*
MEAN ⫾ SEM # P ⬍ 0.05 vs preischemic level *P ⬍ 0.05 vs Control A-aDO2, alveolar-arterial oxygen pressure difference; CO, cardiac output; L-PAP, left pulmonary artery pressure; L-PVR, left pulmonary vascular resistance; PaO2, arterial oxygen pressure.
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FIGURE 2 Changes in serum nitric oxide (nitrite and nitrate) levels before, during, and
after administration of FK or vehicle in the recipient dogs. Data are expressed as mean ⫾ SEM. #p ⬍ 0.05 vs pre-ischemic level *p ⬍ 0.05 vs control group Pre, pre-ischemia; Endisch, end-ischemia; Reperf, reperfusion; 30 min, 30 min after reperfusion; 2 hr, 2 hr after reperfusion; 6 hr, 6 hr after reperfusion.
In the ventilation scintigram, which was performed at the same time under spontaneous respiration, Xe-133 was widely distributed throughout the lung field of the graft in the FK group (Figure 5B). On the other hand, only slight accumulation was observed in either scintigram in the control group. All 5 animals in the FK group survived for 48 hours, whereas in the control group 3 animals died within 12 hours. The 2-day survival rate was 100% in the FK group and 40% in the control group. The survival rate in the FK group was significantly (p ⫽ 0.049) higher than in the control group (Figure 6). In the control group, because of pulmonary edema, the dying animals discharged significant amounts of foamy bloody sputum from the trachea. They seemed to die from severe pulmonary edema, which caused respiratory failure.
DISCUSSION The various effects of NO on I/R injury or in lung transplantation have been widely interpreted.9,11 Nitric oxide may attenuate tissue injury by mediating vasodilation,12 directly scavenging superoxides,16 attenuating leukocyte adhesion and activation,17 in-
hibiting platelet aggregation,18 and maintaining vascular endothelial integrity.11,19 The administration of NO in pulmonary I/R injury may contribute to inhibiting the elevation of PVR,3,20 the infiltration of neutrophils, the formation of neutrophil and platelet plugs, and pulmonary edema formation.21,22,23 Several studies have demonstrated that the infusion of L-arginine, which is a precursor of NO, at the time of reperfusion can drastically reduce I/R injury,24 whereas the infusion of NG-nitro-L-arginine methyl ester (L-NAME), which is a specific inhibitor of NO synthesis, promotes injury.2 On the contrary, NO is known to react with superoxide anion to form highly toxic and harmful peroxynitrite, which aggravates I/R injury.25,26 Because NO has both beneficial and harmful effects, these conflicting effects must be considered in the use of NO. FK409 is a semisynthetic fermentation product of Streptomyces griseosporeus, with vasodilating activities resulting from its nitric oxide– donating ability.27 The biologic actions of FK are due to released NO, such as endogenous NO.28 FK409 releases NO spontaneously, causing potent vasorelaxant and antiplatelet effects, and inhibition of neutrophil se-
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FIGURE 3 Histopathologic findings of the graft lung 30 minutes after reperfusion in the control group (A) and in the FK group (B). (Hematoxylin and eosin, original magnification ⫻100.)
questration.29 FK409 produces vasorelaxation via an increase in intracellular cGMP,30 and inhibits platelet aggregation in vitro, and thrombus formation in the rat extracorporeal shunt model.27 It has been reported that 1 to 1.5 mols NO are released from 1 mol of FK in vitro,31,32 and that FK is rapidly inactivated in the blood after being bound to hemoglobin without sustained release.24 FK409 might be an ideal NO donor because it instantly releases NO in a dose-dependent manner and immediately loses its biochemical activity,31 so that it cannot release excessive NO and harmful products of NO, such as peroxynitrite, are not produced in significant quantity. In the process of I/R injury, neutrophil sequestration plays a pivotal role. After the onset of reperfusion, oxygen free radicals are induced and the endothelium is damaged.33 Subsequently, neutrophil adhere to the damaged endothelium and then further adhesion, activation, and migration are increasingly induced.34 In this process NO has inhibitory
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FIGURE 4 Histopathologic findings of the graft lung 2 days after reperfusion in the control group (at the time of death) (A) and in the FK group (at the time of sacrifice) (B). (Hematoxylin and eosin, original magnification ⫻100.)
effects on the endothelium-neutrophil interaction, through down-regulation of the expression of adhesion molecules both on endothelium and neutrophils, such as inhibition of integrins on endothelium or P-selectin on neutrophils.35 Moreover, NO modulates release of inflammatory cytokines (tumor necrosis factor-␣ and interleukin-1) or arachidonic acid degradation products (thromboxane A2 and leukotriene B4 [LTB4]) from macrophages as a cause of PMN activation.36,37 In many reports, activated neutrophil infiltration is emphasized as an important factor causing lung injury.38,39 Considering these facts, we measured PMN infiltration and found that PMN infiltration was significantly reduced in the FK group. In lung injury, the extreme vulnerability of the lung is represented by the state of gas exchange, hemodynamics, and pulmonary edema.24 In our experiment, the gas exchange capacity was evaluated with the PaO2 and A-aDO2. Hemodynamics was
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FIGURE 5 Perfusion and ventilation scintigrams 6
hours after reperfusion in the FK group. In the perfusion scan Tc-99m-macro agglutinated albumin was administered intravenously (A), whereas in the ventilation scan Xe-133 was administered through inhalation (B).
estimated with the L-PAP and L-PVR. Histologic findings quantified pulmonary edema. Based on the PaO2 and A-aDO2, the FK group showed significantly (p ⬍ 0.05) better function than the control group after 30 minutes of reperfusion. The FK group had significantly (p ⬍ 0.05) lower L-PAP and L-PVR after 30 minutes of reperfusion. Furthermore, in the histologic study, the FK group showed a greater reduction of pulmonary edema and PMN infiltration than did the control group after 30 minutes of reperfusion. Some researchers have re-
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ported increased vascular permeability, alveolar damage, and interstitial edema morphologic in I/R injury in the lung, which has resulted in collapsed circulation, reduced oxygen-exchange capability, and increased pulmonary vascular resistance.40,41 Others have reported histologic damage such as perivascular, interstitial, or alveolar edema, and collapse of capillaries or intra-alveolar hemorrhage, according to the severity of the injury.42 Our study showed less edema in the FK group after 30 minutes of reperfusion. Alveolar damage with severe interstitial edema was observed in the control group at the time of death, whereas the damage was not very severe in the FK group 2 days post-operatively. To investigate in situ lung function, we performed perfusion and ventilation scintigraphy under spontaneous respiration. In both scintigrams, the graft lung functioned adequately 6 hours after reperfusion in the FK group. From this result, we can assume that the good hemodynamics, blood-gas exchange, and survival in the FK group are attributed to the graft lung function. In our study, FK provided organic protection in lung transplantation. To investigate the contribution of FK in this model, we measured the plasma NO levels. Nitric oxide is constitutively produced and exists on the surface of endothelial cells, where it contributes to endothelial homeostasis.43 On the induction of I/R, superoxide radicals are abruptly produced and endothelial dysfunction occurs; simultaneously the endogenous NO levels plummet, then further injury is caused by PMNs.44 Administering exogenous NO just before or after reperfusion ameliorates reperfusion injury by supplementing the decreased endogenous NO. In our experiment, the NO levels gradually decreased after ischemia in the control group. Quenching superoxide radicals may reduce endogenous NO. However, in the FK group, the NO levels maintained a higher level during reperfusion, when FK was continuously administered, than before ischemia. According to our data, maintaining a high NO level with exogenous NO supplementation contributes to the amelioration of I/R injury by maintaining endothelial homeostasis. Endothelin-1, a 21-amino-acid peptide derived from vascular endothelial cells, is one of the most potent vasoconstrictive factors.45 There are 3 ET isoforms (ET-1, ET-2, and ET-3) and 2 ET receptors (ETA receptor and ETB receptor). ETA receptor, which has high affinity to ET-1, exists on vascular smooth-muscle cells and cardiovascular tissues and mediates vasoconstriction, whereas ETB receptor, which has no selective affinity, exists on
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FIGURE 6 Survival curve expressed using the Kaplan-Meier method. Significance of 2-day survival rate was determined by the log-rank test.
vascular endothelial cells and mediates vasoconstriction (ETB2 subtype) or vasodilation (ETB1 subtype).46,47 Vasoconstriction is one explanation for the microcirculatory disturbance in I/R injury,48 and ET-1 is responsible for the increase of PVR and microvascular permeability in pulmonary I/R injury.49 Endothelin-1 has the potential to activate PMNs through platelet-activating factor and LTB4 release from macrophages or endothelial cells.50 Endothelin-1 also contributes to the accumulation of PMNs by up-regulating the surface expression of adhesion molecules both on neutrophils and endothelial cells.51 Moreover, the lung has the highest tissue ET-1 levels.52 Therefore we assessed the contribution of ET-1 to the pulmonary I/R model. Our study revealed that the ET-1 level was significantly lower in the FK group than in the control group 2 hours after reperfusion. The hemodynamics data also implied microcirculatory disturbance in the control group. With respect to these results, some investigators have stated that NO could modulate organ injury by its inhibitory action on endothelin synthesis51 or that NO influences vascular tone via the reciprocal production of ET-1.52 Moreover, ET-1 level is regarded as a useful parameter of endothelial cell damage.53 Though we could not demonstrate whether FK directly inhibited ET-1 production, imbalance between NO and ET-1,
*p ⬍ 0.05 vs control group.
which is thought to aggravate I/R injury, was improved in the FK group.54 This study suggests that ET-1 was released as a result of endothelial cell damage and induced I/R injury, whereas FK contributed to the reduction of endothelial injury and microcirculation, based on the significantly lower levels of ET-1 in the FK group than in the control group. Systemic hypotension often becomes a problem because of the potent vasodilative effect of NO.11,45 In our preliminary hepatic I/R model, when FK was administered at a dose of 5 g/kg/min, the mean ABP did not fall below 70 mm Hg, a level that might lead to severe reperfusion injury due to unstable systemic hemodynamics.15 Moreover, the same dose of FK was sufficiently effective on the canine lung warm-ischemia model,55 a dosage of 5 g/kg/min was therefore applied in our experiment. Five g/kg/min of FK did not decrease the mean ABP below 100 mm Hg (either immediately after or 30 minutes after reperfusion), despite the influence of I/R injury. The ABP then gradually increased to the same level as in the control group, from 30 minutes after reperfusion to 2 hours after reperfusion. The ABP change is reversible with the administration of FK at 5 g/kg/min, and systemic perfusion failure did not occur in our pulmonary transplantation model. In this experimental model, FK was effectively and safely administered intravenously.
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Although NO has also been administered by inhalation in some experimental transplantation models, intravenous administration is thought preferable. During inhalation of NO (a dosage of 20 to 80 ppm is often used), it has been reported that gaseous NO was delivered only to the ventilated alveoli and improved blood flow to those alveoli, so it effectively improved ventilation and perfusion matching.56 Because it is locally absorbed and inactivated immediately after binding to hemoglobin in the blood, systemic blood pressure does not decrease.57 Nevertheless, Hausen and coworkers reported that inhaled NO was not as effective because gaseous NO cannot reach alveoli with microatelectasis, therefore in these alveoli I/R injury is not reduced.58 In a clinical situation, donor lungs might have large areas of microatelectasis, because they are often mechanically ventilated for a long time and are often left lying in a flat position. Moreover, King and colleagues revealed that gaseous NO is itself lipophilic in nature, and insufficient amounts are delivered through the edematous interstitium. On the other hand, NO supplied intravascularly overcomes this interstitial barrier, because it is linked to a hydrophilic carrier compound.59 Therefore, FK reaches the entire pulmonary vascular bed, even though there may be insufficient ventilation. To ensure the exogenously administered NO has a sufficient effect, FK is intravenously administered both before ischemia in the donor and before reperfusion in the recipient. The administration of NO before clamping in the donor can have several perceived beneficial effects on the graft. First, because of the vasodilative effect of NO, the donor lung vasculature is rapidly and completely flushed and cooled, which contributes to reduction of warm ischemia and to uniform distribution of the preservation solution.60 Second, because of the radical scavenging effect of NO, oxidative stress, which may occur during the preservation and subsequent reperfusion periods diminishes.3,20 On the other hand, when NO is induced just before or after reperfusion, the supplemental NO can scavenge reactive oxygen species, inhibit the adhesion and activation of PMNs, prohibit platelet aggregation, and induce vasodilation. These effects lead to a reduction in endothelial injury and microcirculatory disturbance, and lessen the pulmonary I/R injury.57 Therefore, we administered FK before ischemia and during the reperfusion period. Prostaglandin E1 (PGE1) or prostacyclin (PGI2) is now routinely used in clinical lung transplantation to improve initial flush and distribution of preservation
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solution,61 and our study does not represent the standard method. Though PGE1 and PGI2 have pharmacologic properties of pulmonary vasodilation, antineutrophil and antiplatelet functions, and endothelial-barrier stabilization, their vasodilative effects are markedly reduced in such high-potassium conditions as in Euro-Collins solution.61,62 It has been reported that NO could accomplish more appropriate perfusion than PGE1 and PGI2, because its vasodilation is mediated by the cGMP pathway, which is independent of high potassium conditions.63 Though the initial graft flush might be remarkably inadequate in the low-temperature and high-potassium vasoconstrictive conditions of our control lung without prostaglandins, NO donor was used instead of prostaglandins to investigate the effect of NO compared with the non-treated control group. Though many studies have been reported concerning the beneficial effects of FK on I/R injury in the lung,55 liver,15,64 kidney,65 and heart,66 this is the first experimental model of FK in lung transplantation. In conclusion, FK ameliorates I/R injury by maintaining pulmonary vascular homeostasis and inhibiting PMN sequestration. The NO donor, FK409, may have clinical applications in lung transplantation because of these effects. REFERENCES 1. McFadden PM, Emory WB. Lung transplantation. Surg Clin North Am 1998;78:749 – 62. 2. Kirk AJ, Colquhoun IW, Dark JH. Lung preservation: a review of current practice and future directions. Ann Thorac Surg 1993;56:990 –1000. 3. Cooper JD. Current status of lung transplantation. Transplant Proc 1991;23:2107–14. 4. Theodore J, Lewiston N. Lung transplantation comes of age. N Engl J Med 1990;332:772– 4. 5. Keck BM, Bennett LE, Fiol BS, Daily OP, Novick RJ, Hosenpud JD. Worldwide thoracic organ transplantation: a report from the UNOS/ISHLT International Registry for thoracic organ transplantation. Clin Transplant 1996:31– 45. 6. Hosenpud JD, Novick RJ, Bennett LE, Keck BM, Fiol B, Daily OP. The Registry of the International Society for Heart and Lung Transplantation: thirteenth official report-1996. J Heart Lung Transplant 1996;15:655–74. 7. Cooper JD. Lung transplantation: a new era. Ann Thorac Surg 1987;44:447– 8. 8. Keenan RJ, Griffith BP, Kormos RL, Armitage JM, Hardesty RL. Increased perioperative lung preservation injury with lung procurement by Euro-Collins flush. J Heart Lung Transplant 1991;10:650 –5. 9. Naka Y, Roy DK, Smerling AJ, et al. Inhaled nitric oxide fails to confer the pulmonary protection provided by distal stimulation of the nitric oxide pathway at the level of cyclic guanosine monophosphate. J Thorac Cardiovasc Surg 1995; 10:1434 – 41.
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10. Struber M, Ehlers KA, Nilsson FN, Miller VM, McGregor CGA, Haverich A. Effect of lung preservation with EuroCollins and University of Wisconsin solutions on endothelium dependent relaxation. Ann Thorac Surg 1997;63:1428 – 35. 11. Takashima S, Date H, Aoe M, Yamashita M, Andou A, Shimizu N. Effects of inhaled nitric oxide in canine lung transplantation from non-heart-beating donor. Jpn J Thorac Cardiovasc Surg 1998;46:657– 63. 12. Palmer RMJ, Ferrige AG, Moncada S. Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature 1987;327:524 – 6. 13. Kita Y, Sugimoto T, Hirasawa Y, Yoshida K, Maeda K. Close correlation of the cardioprotective effect of FK409, a spontaneous NO releaser, with an increase in plasma cyclic GMP level. Br J Pharmacol 1994;113:5– 6. 14. Isono T, Sato N, Koibuchi Y, et al. Effect of FK409, a novel nitric oxide donor, on acute experimental myocardial ischemia. Jpn J Pharmacol 1993;62:315–24. 15. Aiba M, Takeyoshi I, Ohwada S, et al. The effects of FK409, a novel nitric oxide donor, on extended liver resection with ischemia in dogs. Gastroenterology 1998;114:A1200 –1. 16. Huie RE, Padmaja S. The reaction of NO with superoxide. Free Radic Res Commun 1993;18:195–9. 17. Kubes P, Suzuki M, Granger DN. Nitric oxide: an endogenous modulator of leukocyte adhesion. Proc Natl Acad Sci USA 1991;88:4651–5. 18. Pronai L, Ichimori K, Nozaki H, et al. Investigation of the existence and biological role of L-arginine/nitric oxide pathway in human platelets by spin-trapping/EPR studies. Eur J Biochem 1991;202:923–30. 19. Yamashita M, Schmid RA, Ando K, Cooper JD, Patterson GA. Nitroprusside ameliorates lung allograft reperfusion injury. Ann Thorac Surg 1996;62:791–7. 20. Murakami S, Bacha EA, Herve P, et al. Prevention of reperfusion injury by inhaled nitric oxide in lung harvested from non-heart-beating donors. Ann Thorac Surg 1996;62: 1632– 8. 21. Bacha EA, Herve P, Murakami S, et al. Lasting beneficial effect of short-term inhaled nitric oxide on graft function after lung transplantation. Paris-Sud University Lung Transplantation Group. J Thorac Cardiovasc Surg 1996;112:590 – 8. 22. Eppinger MJ, Ward PA, Jones ML, Bolling SF, Deeb GM. Disparate effects of nitric oxide on lung ischemia-reperfusion injury. Ann Thorac Surg 1995;60:1169 –75. 23. Lefer DJ, Nakanishi K, Vinten-Johansen J. Endothelial and myocardial cell protection by a cysteine-containing nitric oxide donor after myocardial ischemia and reperfusion. J Cardiovasc Pharmacol 1993;22:S34 – 43. 24. Ohmori H, Dhar DK, Nakashima Y, Hashimoto M, Masumura S, Nagasue N. Beneficial effects of FK409, a novel nitric oxide donor, on reperfusion injury of rat liver. Transplantation 1998;66:579 – 85. 25. Freeman B. Free radical chemistry of nitric oxide, looking at the dark side. Chest 1994;105:79S– 84S. 26. Cross CE, Van der Vliet A, O’Neill C, Eiserich JP. Reactive oxygen species and the lung. Lancet 1994;344:930 –3. 27. Hino M, Iwami M, Okamoto M, et al. FK409, a novel vasodilator isolated from the acid-treated fermentation broth of Streptomyces griseosporeus. I. Taxonomy, fermentation, isolation, and physico-chemical and biological characteristics. J Antibiot 1989;42:1578 – 83. 28. Hirasawa Y, Nishio M, Maeda K, Yoshida K, Kita Y.
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Comparison of antiplatelet effects of FK409, a spontaneous nitric oxide releaser, with those of TRK-100, a prostacyclin analogue. Eur J Pharmacol 1995;272:39 – 43. Fukahara K, Murakami A, Watanabe G, Kotoh K, Misaki T. Inhaled nitric oxide after lung ischemia reperfusion: effect on hemodynamics and oxygen free radical scavenger system. Eur J Cardiothorac Thorac Surg 1997;11:343–9. Yamada H, Yoneyama F, Satoh K, Taira N. Comparison of the effects of the novel vasodilator FK409 with those of nitroglycerin in isolated coronary artery of the dog. Br J Pharmacol 1991;103:1713– 8. Kato M, Nishino S, Ohno M, et al. New reagents for controlled release of nitric oxide. Structure-stability relationships. Bioorg Med Chem Lett 1996;6:33– 8. Decout JL, Roy B, Fontecave M, et al. Decomposition of FK409, a new vasodilator: identification of nitric oxide as a metabolite. Bioorg Med Chem Lett 1995;5:973– 8. Jaeschke H, Farhood A. Neutrophil and Kupffer cell-induced oxidant stress and ischemia-reperfusion injury in rat liver. Am J Physiol 1991;260:G355– 62. Conke JP, Tsao PS. Cytoprotective effects of nitric oxide. Circulation 1993;88:2451– 4. Shimamura T, Zhu Y, Zhang S, et al. Protective role of nitric oxide in ischemia and reperfusion injury of the liver. J Am Coll Surg 1999;188:43–52. Meldrum D, Shames BD, Meng X, et al. Nitric oxide downregulates lung macrophage inflammatory cytokine production. Ann Thorac Surg 1998;66:313–7. Hotter G, Closa D, Pi F, et al. Nitric oxide and arachidonate metabolism in ischemia-reperfusion associated with pancreas transplantation. Transplantation 1995;59:417–21. Barbotin-Larrieu F, Mazmanian M, Baudet B, et al. Prevention of ischemia reperfusion lung injury by inhaled nitric oxide in neonatal piglets. J Appl Physiol 1996;80:782– 8. Mazmanian M, Baudet B, Brink C, Cerrina J, Kirkiacharian S, Weiss M. Methylene blue potentiates vascular reactivity in isolated rat lungs. J Appl Physiol 1989;66:1040 –5. Johnson J, Brigham KL, Jesmok G, Meyrick B. Morphologic changes in lungs of anesthetized sheep following intravenous infusion of recombinant tumor necrosis factor alpha. Am Rev Respir Dis 1991;144:179 – 86. Stephens KE, Ishizaka A, Larrick JW, Raffin TA. Tumor necrosis factor causes increased pulmonary permeability and edema. Am Rev Respir Dis 1988;137:1364 –70. Ohwada S, Tomizawa N, Takahashi T, et al. Effects of a specific neutrophil elastase inhibitor (ONO-5046 Na) and neutrophil depletion using G-1 column on lung reperfusion injury in dogs. Transplant Proc 1996;28:1826 –7. Pinsky DJ, Mehmet CO, Koga S, et al. Cardiac preservation is enhanced in a heterotopic rat transplant model by supplementing the nitric oxide pathway. J Clin Invest 1994;93: 2291–7. Lefer AM, Siegfried MR, Xin-liang A. Protection of ischemia-reperfusion injury by sydnonimine NO donors via inhibition of neutrophil-endothelium interaction. J Cardiovasc Pharmacol 1993;22:S27–33. Yanagisawa M, Kurihara H, Kimura S, et al. A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature 1988;332:411–5. Rubanyi GM, Polokoff MA. Endothelins: molecular biology, biochemistry, pharmacology, physiology, and pathophysiology. Pharmacol Rev 1994;46:325– 415.
The Journal of Heart and Lung Transplantation Volume 19, Number 3 47. Pollock DM, Keith TL, Highsmith RF. Endothelin receptors and calcium signaling. FASEB J 1995;9:1196 –204. 48. Murakami S, Bacha EA. Tumor necrosis factor causes increased pulmonary permeability and edema. Am Rev Respir Dis 1988;137:1364 –70. 49. Khimenko PL, Moore TM, Taylor AE. Blocked ETA receptors prevent ischemia and reperfusion injury in rat lungs. J Appl Physiol 1996;80:203–7. 50. Takayasu-Okishio M, Terashita Z, Kondo K. Endothelin-1 and platelet-activating factor stimulate thromboxane A2 biosynthesis in rat vascular smooth muscle cells. Biochem Pharmacol 1990;40:2713–7. 51. Peralta C, Closa D, Hotter G, Gelpi E, Prats N, RoselloCatafau S. Liver ischemic preconditioning is mediated by the inhibitory action of nitric oxide on endothelin. Biochem Biophys Res Commun 1996;229:264 –70. 52. Mizutani H, Minamoto K, Aoe M, et al. Expression of endothelin-1 and effects of an endothelin receptor antagonist, TAK-044, at reperfusion after cold preservation in canine lung transplantation model. J Heart Lung Transplant 1998;17:835– 45. 53. Kourembanas S, McQuillan LP, Leung GK, Faller DV. Nitric oxide regulates the expression of vasoconstrictors and growth factors by vascular endothelium under both normoxia and hypoxia. J Clin Invest 1993;92:99 –104. 54. Hiramatsu T, Forbess J, Miura T, Roth SJ, Cioffi MA, Mayer JE. Effects of endothelin-1 and endothelin-A receptor antagonist on recovery after hypothermic cardioplegic ischemia in neonatal lamb hearts. Circulation 1995;92:II400 – 4. 55. Tanahashi Y, Takeyoshi I, Aiba M, Ohwada S, Matsumoto K, Morishita Y. The effects of FK409 on pulmonary ischemiareperfusion injury in dogs. Transplant Int 1999;12:401–7. 56. Naka Y, Chowdhury NC, Liao H, Mehmet CO, Michler RE, Pinsky DJ. Enhanced preservation of orthotopically transplanted rat lungs by nitroglycerin but not hydralazine. Circ Res 1995;76:900 – 6.
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57. Bhabra MS, Hopkinson DN, Shaw TE, Hooper TL. Lowdose nitric oxide inhalation during initial reperfusion enhances rat lung graft function. Ann Thorac Surg 1997;63: 339 – 44. 58. Hausen B, Muller P, Bahra M, Ramsamoij R, Hewitt CW. Donor pretreatment with intravenous prostacyclin versus inhaled nitric oxide in experimental lung transplantation. Transplantation 1996;62:1714 –9. 59. King RC, Binns OAR, Kanithanon RC, et al. Low-dose sodium nitroprusside reduces pulmonary reperfusion injury. Ann Thorac Surg 1997;63:1398 – 404. 60. Bhabra MS, Hopkinson DN, Shaw TE, Hooper TL. Relative importance of prostaglandin/cyclic adenosine monophosphate and nitric oxide/cyclic guanosine monophosphate pathways in lung preservation. Ann Thorac Surg 1996;62:1494 –9. 61. Sasaki S, Yasuda K, McCully JD, Locicero J. III. Does PGE1 attenuate potassium-induced vasoconstriction in initial pulmonary artery flush on lung preservation? J Heart Lung Transplant 1999;18:139 – 42. 62. DeCampos KN, Keshavjee S, Liu M, Slutsky AS. Preservation of rapid perfusion-induced lung injury with prostaglandin Ej during the initial period of reperfusion. J Heart Lung Transpl 1998;17:1121– 8. 63. Naka Y, Roy DK, Liao H, et al. cAMP-mediated vascular protection in an orthotopic rat lung transplant model. Circ Res 1996;79:773– 83. 64. Dhar DK, Yamanoi A, Ohmori H, et al. Modulation of endothelin and nitric oxide: a rational approach to improve canine hepatic microcirculation. Hepatology 1998;28:782– 8. 65. Matsumura Y, Nishiura M, Deguchi S, Hashimoto N, Ogawa T, Seo R. Protective effect of FK409, a spontaneous nitricoxide releaser, on ischemic acute renal failure in rats. J Pharmacol Exp Ther 1998;287:1084 –91. 66. Yamada H, Yoneyama F, Satoh K, Taira N. Cardiohemodynamic effect of FK409, a novel highly potent nitrovasodilator, in anesthetized dog. Eur J Pharmacol 1991;205:81–3.
L
ung transplantation is an acceptable therapeutic approach for patients with end-stage lung disease, and the short-term survival rates have recently im-
proved.1,2 However, donor lungs are particularly vulnerable to ischemia, and tolerable ischemic time is reported to be shorter than that of other trans-
From Second Department of Surgery,a Gunma University School of Medicine, Gunma, Japan; Department of Nuclear Medicine,b Gunma University School of Medicine, Gunma, Japan; and Department of Pathology,c Nippon Medical School, Kanagawa, Japan. Submitted July 26, 1999; accepted December 10, 1999. Reprint requests: Izumi Takeyoshi, MD, Second Department of Surgery, Gunma University School of Medicine, 3-39-15
Showa-Machi, Maebashi, Gunma 371-8511, Japan. Telephone: ⫹81-27-220-8242. Fax: ⫹81-27-220-8250. E-mail: takeyosi@ akagi.sb.gunma-u.ac.jp. Copyright © 2000 by the International Society for Heart and Lung Transplantation. 1053-2498/00/$–see front matter PII S1053-2498(99)00140-0
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plantable solid organs.3,4 Ischemia-reperfusion (I/R) injury is a significant factor for morbidity, and it may result in deterioration of the graft function or in primary graft failure after lung transplantation. Its mortality rate is as high as 20% to 25%, and it is the second most frequent cause of death within 90 days after transplantation.3,5,6 The acceptable ischemic time for lung grafts is now thought to be between 4 and 7 hours,7,8 although inhibiting I/R injury at the point of donor organ harvesting,3 graft preservation,9,10 and recipient reperfusion11 may prolong this time and can be expected to result in safer transplantation. Nitric oxide (NO) is endogenously and constitutively produced in vascular endothelial cells, and protects against I/R injury by maintaining vascular integrity.12 FK409 (FK) ([⫾]-[E]4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide; Fujisawa Pharmaceutical Co. Ltd.; Osaka) is an organic NO donor, which spontaneously releases NO.13 FK409 has potent vasorelaxant and antiplatelet effects,13 and FK produces these effects through elevating the plasma cyclic guanosine monophosphate (cGMP) level.14 We have previously reported the protective effect of intravenous administration of FK in canine hepatic warm-ischemia and reperfusion models.15 In this study, we evaluated the effect of FK on pulmonary I/R injury in a canine lung transplantation model.
MATERIALS AND METHODS Animals Twenty adult mongrel dogs weighing 8 to 14 kg were used in this study. The animals received a standard commercial diet and were allowed free access to food and water until 12 hours prior to surgery. All the animals received humane care in compliance with the Principles of Laboratory Animal Care, formulated by the National Society for Medical Research, and the Guide for the Care and Use of Laboratory Animals, published by the National Institutes of Health (NIH publication No. 86-23, revised 1985). This experimental study was performed with the approval of the Animal Care and Experimental Committee, Gunma University, Showa Campus.
Donor Procedure After administering ketamine hydrochloride (10 mg/ kg, intramuscular injection), the animals were anesthetized with pentobarbital sodium (10 mg/kg) and pancuronium bromide (0.2 mg/kg), then intubated
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and mechanically ventilated at a tidal volume of 20 ml/kg and a rate of 15 breaths/min. Positive endexpiratory pressure was controlled at 5.0 cm H2O, and the inspired O2 fraction was 0.5. Anesthesia was maintained with inhalation of 1% to 2% halothane, and muscular relaxation was obtained with additional pancuronium bromide of 0.1 mg/kg. An arterial line inserted into the right carotid artery monitored blood pressure and blood gases. The right external jugular vein was used as a venous infusion line. A left thoracotomy was performed at the fifth intercostal space. The aorta, pulmonary artery, and trachea were isolated. Sodium heparin (300 U/kg) was administered systemically. After clamping the main pulmonary artery, a catheter was inserted into the main pulmonary artery while ligating the right pulmonary artery, and the left lung was flushed with 4° C Euro-Collins solution (50 ml/kg). Simultaneously, the lung was cooled topically by immersing it in saline ice slush. During flushing, the lungs were ventilated continuously. After the flush, the heartlung block was excised and preserved in 4° C EuroCollins solution for 8 hours. During preservation, the lungs were kept inflated at 25 cm H2O of airway pressure. After 8-hour preservation, the left lung was dissected from the heart-lung block in a basin containing cold Euro-Collins solution.
Recipient Procedure Recipient dogs were anesthetized and ventilated, and catheters were inserted in the same manner as in the donors. After thoracotomy, a left pneumonectomy was performed. Then the donor lung was orthotopically transplanted in the following way: The left atrial anastomosis was performed with running 5-0 Prolene everting sutures. The arterial and bronchial anastomoses were performed with running 6-0 and 4-0 Prolene sutures, respectively. After completion of these anastomoses, the lung was ventilated and reperfused. Thirty minutes after reperfusion, a 5-minute clamping test of the right pulmonary artery was performed, and specimens from the graft lung were harvested. The chest was then closed and a drainage tube left in place. The animals were observed until 2 days after the operation and then sacrificed. Lung specimens were harvested 2 days after reperfusion or at the time of death for histopathologic study.
Experimental Design Ten weight-matched pairs of adult mongrel dogs were randomly divided into 2 groups. Five pairs were assigned to the FK group, in which FK (5
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g/kg/min) was administered to the donor from 30 minutes prior to ischemia until the induction of ischemia, and to the recipient from 15 minutes prior to reperfusion until 45 minutes after reperfusion. Five pairs were assigned to the control group (n ⫽ 5), in which physiologic saline solution was administered in the same manner.
Hemodynamics and Blood Gas Measurements During the experiment, arterial blood pressure (ABP) was monitored continuously. The ABP of the recipient dogs with FK administration immediately, 30 minutes and 2 hours after reperfusion was compared with their status before FK administration and with the control group. A 5-minute clamping test of the right pulmonary artery was performed prior to ischemia in the donor, and 30 minutes after reperfusion in the recipient. During the test, the left pulmonary arterial pressure (L-PAP) was measured by inserting a 24-gauge needle in the main pulmonary artery and connecting it to a transducer (Spectramed TA 1017, San-ei Co.; Tokyo). The left atrial pressure (LAP) was measured via a catheter inserted into the left atrium through the appendage and connected to the transducer. The cardiac output (CO) was measured by placing an electromagnetic blood flowmeter (MF V-3100, Nihonkohden Co.; Tokyo) on the ascending aorta. The left pulmonary vascular resistance (LPVR) during the 5-minute clamping test was calculated using the following formula: L-PVR (dynes 䡠 sec 䡠 cm ⫺ 5) ⫽ (mean L-PAP (mm Hg) ⫺ mean LAP (mmHg)) ⫻ 79.92/CO (L/min).
At the same time, arterial blood samples were taken to measure arterial oxygen pressure (PaO2) and alveolar-arterial oxygen pressure difference (AaDO2). The PaO2 was determined by a blood gas analyzer (Stat Profile M, Nova Biomedical Co.; Waltham, Massachusetts), and the A-aDO2 was calculated using the following formula: A-aDO2 (mm Hg) ⫽ PiO2 ⫺ PacO2/0.8 ⫺ PaO2, Pio2 ⫽ (760 ⫺ 47) ⫻ Fio2.
Nitrite and Nitrate Measurements The serum NO levels (nitrite and nitrate) were measured before ischemia; before reperfusion (endischemia); and immediately, 30 minutes, 2 hours, and 6 hours, after reperfusion. Using an enzymatic assay kit (Cayman Chemical Company, Ann Arbor; Michigan), nitrite and nitrate were measured in
plasma samples, making use of the 2-step Griess reaction. The first step involves converting nitrate to nitrite using nitrate reductase. The second step is the addition of the Griess reagents, which convert nitrite to an azo compound. For nitrate measurements, 40-ml aliquots of plasma were placed into wells, adjusting the volume to 80 ml with the assay buffer solution. Ten ml of enzyme cofactor and 10 ml of nitrate-reductase cofactor mixture were also added and left to incubate for 3 hours at room temperature. After the process, 100 ml of Griess reagents were added to each of the wells, and the wells were left at room temperature for 10 minutes to develop the color. Then the absorbance at 540 nm was read using a plate reader. To measure nitrite, the same procedure was performed, excluding the first step. The results were expressed as the total amounts of nitrite and nitrate.
Endothelin-1 Measurements The endothelin-1 (ET-1) levels in arterial blood were measured 2 hours after reperfusion. Blood samples for the ET-1 measurement were drawn into ice-chilled tubes containing K2 ethylenediaminetetraacetic acid and Trasylol, and the plasma was immediately separated by centrifugation at 4° C and stored at ⫺80° C until the assay. Plasma samples were extracted in octylsilane-silica cartridges with 2 ml 60% acetonitrile and 0.09% trifluoroacetic acid, and evaporated in a centrifugal concentrator. The dried residue was reconstituted in the assay buffer and subjected to radioimmunoassay. A total of 0.1 ml assay buffer and 0.1 ml antiendothelin-1 serum at a final dilution of 1:300,000 was incubated at 4° C for 20 hours. Then 0.05 ml of 83 pmol/ml 125I-endothelin-1 with a specific activity of 74 TBq/mmol (Amersham International, Buckinghamshire; United Kingdom) was added and incubated at 4° C for 48 hours. The bound and the free ligands were separated by the double-antibody/polyethylene glycol method.
Histopathologic Studies and Polymorphonuclear Neutrophil Measurements Lung specimens were harvested for histopathologic examination 30 minutes after reperfusion, and 2 days after reperfusion (at the time of sacrifice) or at the time of death, and were fixed in 10% formalin. Tissues were dehydrated, embedded in paraffin, cut into 3 to 5 m sections, and mounted. After deparafinizing, the tissues were stained with hematoxylin and eosin, and also stained with naphthol AS-D chloroacetate esterase. The polymorphonu-
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clear neutrophil (PMNs) were identified by positive staining and morphology, and were counted under ⫻400 magnification. At the same time, the alveoli were also counted, and the data were expressed as PMNs/alveolus. A single pathologist, blind to the details of each specimen, performed the measurements.
Radiologic Studies To investigate the in situ function of the lung graft, pulmonary perfusion and ventilation scintigraphy were performed 6 hours after reperfusion. In the perfusion scan, Tc-99m–macro agglutinated albumin (MAA) (1 mCi) was administered intravenously. Then a Tc-99m-MAA scintillation image was taken to investigate the blood perfusion of the lung graft. In the ventilation scan, Xe-133 (3 mCi) was administered through inhalation under spontaneous respiration. A Xe-133 scintillation image was also made to investigate the ventilatory capacity of the lung graft.
Statistical Analysis All the values are expressed as the mean ⫾ the standard error of the mean. Statistical analysis was performed using the Mann-Whitney U test and analysis of variance (ANOVA). The animal survival rate was determined using the Kaplan-Meier method, and the log-rank test was used to determine significance. A p value ⬍ 0.05 was considered to be statistically significant.
RESULTS No significant differences were measured in preservation time (cold ischemic time) or operating time (warm ischemic time) between the FK group and the control group. There were also no significant differences in the mean ABP, CO, L-PAP, L-PVR, PaO2, or A-aDO2 of the FK and control groups before ischemia in the donor. The systemic hemodynamics (ABP) were measured in the recipient before and after the administration of FK. The mean ABP 30 minutes before ischemia (before the administration of FK), and immediately after and 30 minutes after reperfusion (during the administration of FK), was 162 ⫾ 10, 107 ⫾ 10, and 114 ⫾ 12 mm Hg, respectively, in the FK group; and 158 ⫾ 12, 140 ⫾ 8, and 116 ⫾ 14 mm Hg, respectively, in the control group. In the FK group, the mean ABP levels immediately after and 30 minutes after reperfusion were significantly (p ⫽ 0.018 and 0.005) lower than the pre-ischemic level, but the mean ABP remained over 100 mm Hg. The
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mean ABP immediately after reperfusion was significantly (p ⫽ 0.046) lower in the FK group than in the control group, but 30 minutes after reperfusion there was no significant (p ⫽ 0.899) difference between the groups. The mean ABP 2 hours after reperfusion improved to 140 ⫾ 13 mm Hg in the FK group (Figure 1). A 5-minute clamping test of the right pulmonary artery was performed prior to inducing ischemia and 30 minutes after reperfusion. During the test, LPAP, L-PVR, CO, PaO2, and A-aDO2 were measured simultaneously. The L-PAP in the control group was significantly (p ⫽ 0.043) higher after 30 minutes of reperfusion than in the pre-ischemic condition. Whereas, in the FK group, the L-PAP after 30 minutes of reperfusion was not significantly (p ⫽ 0.56) higher than in the pre-ischemic condition of the donor lung. After 30 minutes of reperfusion, the L-PAP was significantly (p ⫽ 0.035) lower in the FK group than in the control group. The L-PVR in the control group was significantly (p ⫽ 0.043) higher after 30 minutes of reperfusion than in the pre-ischemic condition of the donor lung. On the other hand, in the FK group, the L-PVR after 30 minutes of reperfusion was not significantly (p ⫽ 0.685) higher than in the pre-ischemic condition. After 30 minutes of reperfusion, the L-PVR was significantly (p ⫽ 0.009) lower in the FK group than in the control group. The CO in the control group was significantly (p ⫽ 0.028) lower after 30 minutes of reperfusion than in the pre-ischemic condition. There was no significant (p ⫽ 0.272) difference in the CO after 30 minutes of reperfusion between the FK group and the control group. The PaO2 in the control group was significantly (p ⫽ 0.043) lower 30 minutes after reperfusion than the pre-ischemic level. The PaO2 30 minutes after reperfusion was significantly (p ⫽ 0.047) higher in the FK group than in the control group. The A-aDO2 in the control group was significantly (p ⫽ 0.043) higher 30 minutes after reperfusion than the pre-ischemic level. The A-aDO2 30 minutes after reperfusion was significantly (p ⫽ 0.047) lower in the FK group than in the control group (Table I). The serum NO levels (nitrite and nitrate) were measured before, during, and after administration of FK. The serum NO levels during FK administration (at end-ischemia, at reperfusion, and at 30 minutes after reperfusion) in the FK group increased beyond the levels before ischemia, and remained higher than in the control group. The serum NO levels in the control group tended to decrease after ischemia and reperfusion (Figure 2).
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FIGURE 1 Changes in mean arterial blood pressure before, during, and after
administration of FK or vehicle in the recipient dogs. Data are expressed as mean ⫾ SEM. # p ⬍ 0.05 vs pre-ischemic level *p ⬍ 0.05 vs Control group Pre, pre-ischemia; Reperf, reperfusion; 30 min, 30 min after reperfusion; 2 hr, 2 hr after reperfusion.
The serum ET-1 level 2 hours after reperfusion was significantly (p ⫽ 0.043) lower in the FK group (41.9 ⫾ 8.0 pg/ml) than in the control group (80.3 ⫾ 15.1 pg/ml). Histologically, only slight interstitial edema was observed in the FK group, whereas interstitial, alveolar, and alveolar-septal edema along with inflammatory cell infiltration was observed in the control group after 30 minutes of reperfusion (Figure 3). At the time of death, severe alveolar damage with
edema was observed in the control group. These findings were obviously slight in the FK group 2 days after reperfusion (at the time of sacrifice) (Figure 4). The PMN infiltration was significantly (p ⫽ 0.028) lower in the FK group (0.66 ⫾ 0.16 PMNs/ alveolus) than in the control group (1.42 ⫾ 0.12 PMNs/alveolus). The perfusion scintigram, which was performed 6 hours after reperfusion, revealed sufficient perfusion of the lung graft in the FK group (Figure 5A).
TABLE I Levels of hemodynamics and gas exchange Before Ischemia
L-PAP (mm Hg) L-PVR (dynes 䡠 sec 䡠 cm⫺5) CO (L/min) PaO2 (mm Hg) A-aDO2 (mm Hg)
30 Minutes after Reperfusion
Control
FK
Control
FK
29.0 ⫾ 4.2 2,641 ⫾ 397 1.30 ⫾ 0.12 175 ⫾ 12 144 ⫾ 13
32.0 ⫾ 2.9 2,575 ⫾ 359 1.12 ⫾ 0.09 169 ⫾ 14 151 ⫾ 15
53.8 ⫾ 2.8# 7,681 ⫾ 1,352# 0.72 ⫾ 0.19# 76 ⫾ 13# 243 ⫾ 12#
34.0 ⫾ 5.5* 3,341 ⫾ 339* 0.82 ⫾ 0.09 119 ⫾ 14* 202 ⫾ 10*
MEAN ⫾ SEM # P ⬍ 0.05 vs preischemic level *P ⬍ 0.05 vs Control A-aDO2, alveolar-arterial oxygen pressure difference; CO, cardiac output; L-PAP, left pulmonary artery pressure; L-PVR, left pulmonary vascular resistance; PaO2, arterial oxygen pressure.
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FIGURE 2 Changes in serum nitric oxide (nitrite and nitrate) levels before, during, and
after administration of FK or vehicle in the recipient dogs. Data are expressed as mean ⫾ SEM. #p ⬍ 0.05 vs pre-ischemic level *p ⬍ 0.05 vs control group Pre, pre-ischemia; Endisch, end-ischemia; Reperf, reperfusion; 30 min, 30 min after reperfusion; 2 hr, 2 hr after reperfusion; 6 hr, 6 hr after reperfusion.
In the ventilation scintigram, which was performed at the same time under spontaneous respiration, Xe-133 was widely distributed throughout the lung field of the graft in the FK group (Figure 5B). On the other hand, only slight accumulation was observed in either scintigram in the control group. All 5 animals in the FK group survived for 48 hours, whereas in the control group 3 animals died within 12 hours. The 2-day survival rate was 100% in the FK group and 40% in the control group. The survival rate in the FK group was significantly (p ⫽ 0.049) higher than in the control group (Figure 6). In the control group, because of pulmonary edema, the dying animals discharged significant amounts of foamy bloody sputum from the trachea. They seemed to die from severe pulmonary edema, which caused respiratory failure.
DISCUSSION The various effects of NO on I/R injury or in lung transplantation have been widely interpreted.9,11 Nitric oxide may attenuate tissue injury by mediating vasodilation,12 directly scavenging superoxides,16 attenuating leukocyte adhesion and activation,17 in-
hibiting platelet aggregation,18 and maintaining vascular endothelial integrity.11,19 The administration of NO in pulmonary I/R injury may contribute to inhibiting the elevation of PVR,3,20 the infiltration of neutrophils, the formation of neutrophil and platelet plugs, and pulmonary edema formation.21,22,23 Several studies have demonstrated that the infusion of L-arginine, which is a precursor of NO, at the time of reperfusion can drastically reduce I/R injury,24 whereas the infusion of NG-nitro-L-arginine methyl ester (L-NAME), which is a specific inhibitor of NO synthesis, promotes injury.2 On the contrary, NO is known to react with superoxide anion to form highly toxic and harmful peroxynitrite, which aggravates I/R injury.25,26 Because NO has both beneficial and harmful effects, these conflicting effects must be considered in the use of NO. FK409 is a semisynthetic fermentation product of Streptomyces griseosporeus, with vasodilating activities resulting from its nitric oxide– donating ability.27 The biologic actions of FK are due to released NO, such as endogenous NO.28 FK409 releases NO spontaneously, causing potent vasorelaxant and antiplatelet effects, and inhibition of neutrophil se-
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FIGURE 3 Histopathologic findings of the graft lung 30 minutes after reperfusion in the control group (A) and in the FK group (B). (Hematoxylin and eosin, original magnification ⫻100.)
questration.29 FK409 produces vasorelaxation via an increase in intracellular cGMP,30 and inhibits platelet aggregation in vitro, and thrombus formation in the rat extracorporeal shunt model.27 It has been reported that 1 to 1.5 mols NO are released from 1 mol of FK in vitro,31,32 and that FK is rapidly inactivated in the blood after being bound to hemoglobin without sustained release.24 FK409 might be an ideal NO donor because it instantly releases NO in a dose-dependent manner and immediately loses its biochemical activity,31 so that it cannot release excessive NO and harmful products of NO, such as peroxynitrite, are not produced in significant quantity. In the process of I/R injury, neutrophil sequestration plays a pivotal role. After the onset of reperfusion, oxygen free radicals are induced and the endothelium is damaged.33 Subsequently, neutrophil adhere to the damaged endothelium and then further adhesion, activation, and migration are increasingly induced.34 In this process NO has inhibitory
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FIGURE 4 Histopathologic findings of the graft lung 2 days after reperfusion in the control group (at the time of death) (A) and in the FK group (at the time of sacrifice) (B). (Hematoxylin and eosin, original magnification ⫻100.)
effects on the endothelium-neutrophil interaction, through down-regulation of the expression of adhesion molecules both on endothelium and neutrophils, such as inhibition of integrins on endothelium or P-selectin on neutrophils.35 Moreover, NO modulates release of inflammatory cytokines (tumor necrosis factor-␣ and interleukin-1) or arachidonic acid degradation products (thromboxane A2 and leukotriene B4 [LTB4]) from macrophages as a cause of PMN activation.36,37 In many reports, activated neutrophil infiltration is emphasized as an important factor causing lung injury.38,39 Considering these facts, we measured PMN infiltration and found that PMN infiltration was significantly reduced in the FK group. In lung injury, the extreme vulnerability of the lung is represented by the state of gas exchange, hemodynamics, and pulmonary edema.24 In our experiment, the gas exchange capacity was evaluated with the PaO2 and A-aDO2. Hemodynamics was
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FIGURE 5 Perfusion and ventilation scintigrams 6
hours after reperfusion in the FK group. In the perfusion scan Tc-99m-macro agglutinated albumin was administered intravenously (A), whereas in the ventilation scan Xe-133 was administered through inhalation (B).
estimated with the L-PAP and L-PVR. Histologic findings quantified pulmonary edema. Based on the PaO2 and A-aDO2, the FK group showed significantly (p ⬍ 0.05) better function than the control group after 30 minutes of reperfusion. The FK group had significantly (p ⬍ 0.05) lower L-PAP and L-PVR after 30 minutes of reperfusion. Furthermore, in the histologic study, the FK group showed a greater reduction of pulmonary edema and PMN infiltration than did the control group after 30 minutes of reperfusion. Some researchers have re-
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ported increased vascular permeability, alveolar damage, and interstitial edema morphologic in I/R injury in the lung, which has resulted in collapsed circulation, reduced oxygen-exchange capability, and increased pulmonary vascular resistance.40,41 Others have reported histologic damage such as perivascular, interstitial, or alveolar edema, and collapse of capillaries or intra-alveolar hemorrhage, according to the severity of the injury.42 Our study showed less edema in the FK group after 30 minutes of reperfusion. Alveolar damage with severe interstitial edema was observed in the control group at the time of death, whereas the damage was not very severe in the FK group 2 days post-operatively. To investigate in situ lung function, we performed perfusion and ventilation scintigraphy under spontaneous respiration. In both scintigrams, the graft lung functioned adequately 6 hours after reperfusion in the FK group. From this result, we can assume that the good hemodynamics, blood-gas exchange, and survival in the FK group are attributed to the graft lung function. In our study, FK provided organic protection in lung transplantation. To investigate the contribution of FK in this model, we measured the plasma NO levels. Nitric oxide is constitutively produced and exists on the surface of endothelial cells, where it contributes to endothelial homeostasis.43 On the induction of I/R, superoxide radicals are abruptly produced and endothelial dysfunction occurs; simultaneously the endogenous NO levels plummet, then further injury is caused by PMNs.44 Administering exogenous NO just before or after reperfusion ameliorates reperfusion injury by supplementing the decreased endogenous NO. In our experiment, the NO levels gradually decreased after ischemia in the control group. Quenching superoxide radicals may reduce endogenous NO. However, in the FK group, the NO levels maintained a higher level during reperfusion, when FK was continuously administered, than before ischemia. According to our data, maintaining a high NO level with exogenous NO supplementation contributes to the amelioration of I/R injury by maintaining endothelial homeostasis. Endothelin-1, a 21-amino-acid peptide derived from vascular endothelial cells, is one of the most potent vasoconstrictive factors.45 There are 3 ET isoforms (ET-1, ET-2, and ET-3) and 2 ET receptors (ETA receptor and ETB receptor). ETA receptor, which has high affinity to ET-1, exists on vascular smooth-muscle cells and cardiovascular tissues and mediates vasoconstriction, whereas ETB receptor, which has no selective affinity, exists on
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FIGURE 6 Survival curve expressed using the Kaplan-Meier method. Significance of 2-day survival rate was determined by the log-rank test.
vascular endothelial cells and mediates vasoconstriction (ETB2 subtype) or vasodilation (ETB1 subtype).46,47 Vasoconstriction is one explanation for the microcirculatory disturbance in I/R injury,48 and ET-1 is responsible for the increase of PVR and microvascular permeability in pulmonary I/R injury.49 Endothelin-1 has the potential to activate PMNs through platelet-activating factor and LTB4 release from macrophages or endothelial cells.50 Endothelin-1 also contributes to the accumulation of PMNs by up-regulating the surface expression of adhesion molecules both on neutrophils and endothelial cells.51 Moreover, the lung has the highest tissue ET-1 levels.52 Therefore we assessed the contribution of ET-1 to the pulmonary I/R model. Our study revealed that the ET-1 level was significantly lower in the FK group than in the control group 2 hours after reperfusion. The hemodynamics data also implied microcirculatory disturbance in the control group. With respect to these results, some investigators have stated that NO could modulate organ injury by its inhibitory action on endothelin synthesis51 or that NO influences vascular tone via the reciprocal production of ET-1.52 Moreover, ET-1 level is regarded as a useful parameter of endothelial cell damage.53 Though we could not demonstrate whether FK directly inhibited ET-1 production, imbalance between NO and ET-1,
*p ⬍ 0.05 vs control group.
which is thought to aggravate I/R injury, was improved in the FK group.54 This study suggests that ET-1 was released as a result of endothelial cell damage and induced I/R injury, whereas FK contributed to the reduction of endothelial injury and microcirculation, based on the significantly lower levels of ET-1 in the FK group than in the control group. Systemic hypotension often becomes a problem because of the potent vasodilative effect of NO.11,45 In our preliminary hepatic I/R model, when FK was administered at a dose of 5 g/kg/min, the mean ABP did not fall below 70 mm Hg, a level that might lead to severe reperfusion injury due to unstable systemic hemodynamics.15 Moreover, the same dose of FK was sufficiently effective on the canine lung warm-ischemia model,55 a dosage of 5 g/kg/min was therefore applied in our experiment. Five g/kg/min of FK did not decrease the mean ABP below 100 mm Hg (either immediately after or 30 minutes after reperfusion), despite the influence of I/R injury. The ABP then gradually increased to the same level as in the control group, from 30 minutes after reperfusion to 2 hours after reperfusion. The ABP change is reversible with the administration of FK at 5 g/kg/min, and systemic perfusion failure did not occur in our pulmonary transplantation model. In this experimental model, FK was effectively and safely administered intravenously.
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Although NO has also been administered by inhalation in some experimental transplantation models, intravenous administration is thought preferable. During inhalation of NO (a dosage of 20 to 80 ppm is often used), it has been reported that gaseous NO was delivered only to the ventilated alveoli and improved blood flow to those alveoli, so it effectively improved ventilation and perfusion matching.56 Because it is locally absorbed and inactivated immediately after binding to hemoglobin in the blood, systemic blood pressure does not decrease.57 Nevertheless, Hausen and coworkers reported that inhaled NO was not as effective because gaseous NO cannot reach alveoli with microatelectasis, therefore in these alveoli I/R injury is not reduced.58 In a clinical situation, donor lungs might have large areas of microatelectasis, because they are often mechanically ventilated for a long time and are often left lying in a flat position. Moreover, King and colleagues revealed that gaseous NO is itself lipophilic in nature, and insufficient amounts are delivered through the edematous interstitium. On the other hand, NO supplied intravascularly overcomes this interstitial barrier, because it is linked to a hydrophilic carrier compound.59 Therefore, FK reaches the entire pulmonary vascular bed, even though there may be insufficient ventilation. To ensure the exogenously administered NO has a sufficient effect, FK is intravenously administered both before ischemia in the donor and before reperfusion in the recipient. The administration of NO before clamping in the donor can have several perceived beneficial effects on the graft. First, because of the vasodilative effect of NO, the donor lung vasculature is rapidly and completely flushed and cooled, which contributes to reduction of warm ischemia and to uniform distribution of the preservation solution.60 Second, because of the radical scavenging effect of NO, oxidative stress, which may occur during the preservation and subsequent reperfusion periods diminishes.3,20 On the other hand, when NO is induced just before or after reperfusion, the supplemental NO can scavenge reactive oxygen species, inhibit the adhesion and activation of PMNs, prohibit platelet aggregation, and induce vasodilation. These effects lead to a reduction in endothelial injury and microcirculatory disturbance, and lessen the pulmonary I/R injury.57 Therefore, we administered FK before ischemia and during the reperfusion period. Prostaglandin E1 (PGE1) or prostacyclin (PGI2) is now routinely used in clinical lung transplantation to improve initial flush and distribution of preservation
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solution,61 and our study does not represent the standard method. Though PGE1 and PGI2 have pharmacologic properties of pulmonary vasodilation, antineutrophil and antiplatelet functions, and endothelial-barrier stabilization, their vasodilative effects are markedly reduced in such high-potassium conditions as in Euro-Collins solution.61,62 It has been reported that NO could accomplish more appropriate perfusion than PGE1 and PGI2, because its vasodilation is mediated by the cGMP pathway, which is independent of high potassium conditions.63 Though the initial graft flush might be remarkably inadequate in the low-temperature and high-potassium vasoconstrictive conditions of our control lung without prostaglandins, NO donor was used instead of prostaglandins to investigate the effect of NO compared with the non-treated control group. Though many studies have been reported concerning the beneficial effects of FK on I/R injury in the lung,55 liver,15,64 kidney,65 and heart,66 this is the first experimental model of FK in lung transplantation. In conclusion, FK ameliorates I/R injury by maintaining pulmonary vascular homeostasis and inhibiting PMN sequestration. The NO donor, FK409, may have clinical applications in lung transplantation because of these effects. REFERENCES 1. McFadden PM, Emory WB. Lung transplantation. Surg Clin North Am 1998;78:749 – 62. 2. Kirk AJ, Colquhoun IW, Dark JH. Lung preservation: a review of current practice and future directions. Ann Thorac Surg 1993;56:990 –1000. 3. Cooper JD. Current status of lung transplantation. Transplant Proc 1991;23:2107–14. 4. Theodore J, Lewiston N. Lung transplantation comes of age. N Engl J Med 1990;332:772– 4. 5. Keck BM, Bennett LE, Fiol BS, Daily OP, Novick RJ, Hosenpud JD. Worldwide thoracic organ transplantation: a report from the UNOS/ISHLT International Registry for thoracic organ transplantation. Clin Transplant 1996:31– 45. 6. Hosenpud JD, Novick RJ, Bennett LE, Keck BM, Fiol B, Daily OP. The Registry of the International Society for Heart and Lung Transplantation: thirteenth official report-1996. J Heart Lung Transplant 1996;15:655–74. 7. Cooper JD. Lung transplantation: a new era. Ann Thorac Surg 1987;44:447– 8. 8. Keenan RJ, Griffith BP, Kormos RL, Armitage JM, Hardesty RL. Increased perioperative lung preservation injury with lung procurement by Euro-Collins flush. J Heart Lung Transplant 1991;10:650 –5. 9. Naka Y, Roy DK, Smerling AJ, et al. Inhaled nitric oxide fails to confer the pulmonary protection provided by distal stimulation of the nitric oxide pathway at the level of cyclic guanosine monophosphate. J Thorac Cardiovasc Surg 1995; 10:1434 – 41.
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