Beneficial effects of novel nitric oxide donor (FK409) on pulmonary ischemia-reperfusion injury in rats

EXPERIMENTAL TRANSPLANTATION

Beneficial Effects of Novel Nitric Oxide Donor (FK409) on Pulmonary Ischemia-Reperfusion Injury in Rats Izumi Takeyoshi, MD,a Yoshimi Otani, MD,a Daisuke Yoshinari, MD,a Yoshiyuki Kawashima, MD,a Susumu Ohwada, MD,a Koshi Matsumoto, MD,b and Yasuo Morishita, MDa Background: Nitric oxide (NO) seems to play an important role in tissue injury during reperfusion of the lung. FK409 is the first spontaneous NO donor that increases plasma guanosine 3⬘:5⬘-cyclic monophosphate. It is reported that FK409 prevented myocardial infarction following occlusion and reperfusion in rat coronary arteries. In this study, we evaluated the effects of FK409 on pulmonary ischemia-reperfusion injury in an in situ warm ischemia model of rats. Methods: Animals were divided into 2 groups: the FK409 study group that was administered FK409 (0.4 mg/kg) before reperfusion and the control group, administered a saline vehicle only. Following a thoracotomy, the bronchus, pulmonary artery and vein were separately clamped for 1 hour. Arterial oxygen tension (PaO2), arterial oxygen saturation (SaO2), and endothelin-I (ET-I) were measured after 2 hours of reperfusion. Histologic and immunohistochemical studies were performed; polymorphonuclear neutrophils (PMNs) were counted after 2 hours of reperfusion. Results: PaO2, SaO2, ET-I after 2 hours of reperfusion and the 7-day survival rate were significantly (p ⬍ 0.05) better in the FK409 group than the control group. Histologic damage was reduced in the FK409 group compared with the control group. PMN infiltration was also significantly (p ⬍ 0.05) lower in the FK409 group than in the control group. Conclusion: FK409 seems to protect against ischemia-reperfusion injury of the lung. This effect may be related to a homeostatic effect on pulmonary vascular beds and prevention of PMN sequestration. J Heart Lung Transplant 2000;19:185–192.

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oncerns have arisen about reperfusion injury, the biochemical changes that occur during ischemia and increase tissue damage when the blood supply is reestablished. Reperfusion injury is thought to result from disturbances to pulmonary microcircula-

tion.1 Lung transplantation has been an effective treatment for patients with end-stage lung disease, and recently short-term survival rates have improved. Ischemia-reperfusion injury results in the deterioration of pulmonary function after lung

From the aSecond Department of Surgery, Gunma University School of Medicine, 3-39-15 Showa-Machi, Maebashi, Gunma 371-8511, Japan, and bDepartment of Pathology, Nippon Medical School, 1-396 Kosugi-Chou, Nakahara-ku, Kawasaki, Kanagawa 211-8533, Japan. Submitted June 10, 1999; accepted October 5, 1999.

Reprint requests: Izumi Takeyoshi, M.D., 3-39-15 Showa-Machi, Maebashi, Gunma 371-8511, JAPAN Tel: ⫹81-27-220-8242, Fax: ⫹81-27-220-8250. Copyright © 2000 by the International Society for Heart and Lung Transplantation. 1053-2498/00/$–see front matter PII S1053-2498(99)00113-8

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transplantation. The ability to preserve pulmonary grafts in clinical lung transplantation lags behind that of other solid organs such as the kidney and liver. The acceptable ischemic time for lung grafts remains at 4 to 7 hours.2,3 The inhibition of ischemia-reperfusion injury may extend that preservation time. The FK409 ((⫾)-(E)-ethyl-2-((E)-hydroxyimino)5-nitro-3-hexeneamide; Fujisawa Pharmaceutical Co. Ltd., Osaka) is a semi-synthetic fermentation product of Streptomyces griseosporeus with vasodilating activities resulting from its nitric oxide-donating ability.4 The biologic actions of FK409 are thought to be due to endogenous nitric oxide (NO) released from the compound.5 FK409 is the first NO donor that increases the plasma guanosine 3⬘:5⬘-cyclic monophosphate (cyclic GMP) levels.6 It has been reported that FK409 prevented myocardial infarction following occlusion and reperfusion in a rat and dog coronary artery.7,8 This NO donor may improve early lung function and may also increase the supply of available lung grafts by extending the preservation time. In this study, the effect of FK409 on pulmonary ischemia-reperfusion injury was investigated in an in situ warm ischemia model of the rat lung.

MATERIALS AND METHODS Experimental model Male Wistar rats weighing 220 to 280 g were prepared for this study. All rats received humane care according to 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. 85-23, revised 1985). Rats were anesthetized with pentobarbital sodium (50 mg/kg) intraperitoneal administration. Following intubation of a 16-gauge intravenous catheter into the trachea, ventilation was maintained using a Harvard Model 683 ventilator (Harvard Apparatus, Inc., South Natick, MA) with a tidal volume of 2.5 ml in room air at a respiratory rate of 80 cycles/min. Positive end-expiratory pressure of 2 cm H2O was applied to prevent lung collapse. FK409 (0.4 mg/kg) or a saline vehicle was intravenously administered through the penile vein in a bolus fashion 5 minutes before reperfusion. A thoracotomy was done at the left fourth intercostal space. After intravenous injection of 500 IU/kg of heparin, the bronchus, pulmonary artery and vein were separately clamped with microclips for 1 hour. Following the release of the clips, the thorax was closed. Rats were divided

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into two groups: the FK409 group (n ⫽ 8) and the control group (n ⫽ 10). All rats were observed for a week or until death. As an additional experiment, the FK409 group (n ⫽ 6) was compared with the control group (n ⫽ 6) 2 hours after reperfusion by using such parameters as arterial oxygen saturation (SaO2), arterial oxygen tension (PaO2) and endothelin-I (ET-I), including histologic examination.

Blood gas analysis Blood was sampled by puncture via the ascending aorta following a 5-minute clamping of the right hilum. SaO2 and PaO2 were analyzed with the ABL 520 Blood Gas System (Radiometer Co., Copenhagen).

Endothelin-I measurement ET-I levels of arterial blood were measured 2 hours after reperfusion. Blood samples for ET-I measurement were drawn into ice-chilled tubes containing K2EDTA 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 with octylsilane-silica cartridges, containing 2 ml of 60% acetonitrile and 0.09% trifluoroacetic acid and evaporated by a centrifugal concentrator. The dried residue was reconstituted in the assay buffer and subjected to radioimmunoassay; 0.1 ml assay buffer and 0.1 ml anti-ET-I serum at a final dilution of 1:300,000 were incubated at 4° C for 20 hours, followed by the late addition of 0.05 ml 125 I-endothelin-I 83 pmol/ml with a specific activity of 74 TBq/mmol (Amersham International, Buckinghamshire, UK), then further incubation at 4° C for 48 hours. Separation of bound ligands from the free ligands was performed by the double-antibody/ polyethylene glycol method.9

Histological study After 2 hours of reperfusion, lung specimens were harvested for histologic examination. Specimens were fixed in 10% formalin. Tissues were dehydrated, embedded in paraffin, cut into 3 to 5 ␮m sections and mounted. After the tissues were removed from paraffin, they were stained with hematoxylin and eosin for histopathologic examination and with neutrophil estelase to count polymorphonuclear neutrophils (PMNs). PMNs were counted with a light microscope at ⫻400 magnification. PMNs were counted in 10 fields for each specimen. Alveoli were also counted, and the data were expressed as PMNs per alveolus. The PMN counts were done by a single pathologist who was blind to the details about each specimen. Immunohisto-

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FIGURE 1 Blood gas assessment. SaO2 (a) and PaO2 levels (b) after 2 hours of reperfusion were significantly better in the FK409 group than in the control group. *p ⬍ 0.05.

chemical staining for inducible NO synthase (iNOS) and endothelial NOS (eNOS) were also performed, using the streptoavidin-biotin method.

Statistical analyses The 1-week survival rate was calculated by KaplanMeier method and analyzed using the log-rank method. All other results are expressed as the mean ⫾ SD. Statistical significance was determined by the Student’s t-test. A p value of less than 0.05 was considered statistically significant.

control group. There was a significant (p ⬍ 0.05) difference between the groups (Figure 1).

Endothelin-I measurement After 2 hours of reperfusion, ET-I levels were significantly (p ⬍ 0.05) higher in the control group (10.3 ⫾ 0.4 pg/ml) than in the FK409 group (8.6 ⫾ 0.3 pg/ml) (Figure 2).

RESULTS Survival rate Seven of 8 rats in the FK409 group survived over 1 week and the survival rate was 88%. In the control group, only 2 of 10 rats survived for more than 1 week and the remaining 8 died within 3 days. The 1-week survival rate was 20%. The difference in the survival rate between the FK409 group and the control group was significant (p ⬍ 0.01).

Arterial oxygen saturation and arterial oxygen tension SaO2 levels after 2 hours of reperfusion were 100.0 ⫾ 0% in the FK409 group and 89.3 ⫾ 3.4% in the control group. There was a significant (p ⬍ 0.05) difference between the groups. PaO2 levels after 2 hours of reperfusion were 147.7 ⫾ 24.7 mm Hg in the FK409 group and 96.7 ⫾ 11.0 mm Hg in the

FIGURE 2 Endothelin-I measurement. After 2 hours

of reperfusion, ET-I levels were significantly higher in the control group than in the FK409 group. *p ⬍ 0.05.

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FIGURE 4 PMNs. PMN infiltration was also

significantly higher in the control group than in the FK409 group. *p ⬍ 0.05.

the control group (1.52 ⫾ 0.13/alveolus) than in the FK409 group (1.00 ⫾ 0.08/alveolus) (Figure 4). In immunohistochemical observation, both FK409 and control groups showed a positive staining for eNOS, and were negative for iNOS (Figure 5).

DISCUSSION

FIGURE 3 Histologic examination. In histologic

examination after 2 hours of reperfusion, both alveolar damage with edema and interstitial thickening localized along the alveolar duct were observed in the control group (a), whereas only slight interstitial edema was observed in the FK409 group rats (b) (Hematoxylin and eosin stain, original magnification ⫻ 50).

Histologic examination Histologic examination after 2 hours of reperfusion showed both alveolar damage with edema and interstitial thickening localized along the alveolar duct in the control group, whereas only slight interstitial edema was observed in the FK409 group rats (Figure 3). Lung specimens from the control group rats at the time of death showed alveolar damage with interstitial edema, neutrophil and leukocyte infiltration, hyaline membranes localized along the predominant alveolar ducts and collapsed alveoli. PMN infiltration was also significantly (p ⬍ 0.05) higher in

The effects of NO on ischemia-reperfusion injury have been widely interpreted. NO may attenuate tissue injury by maintaining circulation as an endothelium-derived relaxing factor,10,11 directly scavenging superoxide,12 attenuating leukocyte adhesion13 and inhibiting platelet aggregation.14 NO promotes organ protection by improving organ blood flow in ischemia-reperfusion injury.15 Some reports state that inhaling exogenous NO can mediate vasodilation and improve pulmonary function after ischemia reperfusion and transplantation of the lung.16 –20 This protective effect is due to the prevention of ischemia-reperfusion injury inducing pulmonary vasoconstriction, inhibition of neutrophil sequestration in the reperfused lung16,17,19 and suppression of oxygen free radicals.18 NO promptly reacts with the superoxide anion to form oxidant peroxynitrite, a potent oxidant that would likely increase ischemia-reperfusion injury.21 Also, there are some reports of harmful effects of enhanced NO production generated by iNOS.22 An extremely high amount of NO has been reported to have potentially toxic effects, depress mitochondrial respiration23

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FIGURE 5 Immunohistochemistry. Both FK409 (a) and control groups (b) showed a

positive staining for eNOS, and were negative for iNOS. (a) FK409; eNOS, (b) control; eNOS, (c) FK409; iNOS, (d) control; iNOS.

and inhibit total protein synthesis.24 iNOS increases steadily, with peak activity at 8 hours after TNF-␣ stimulation.25 Similarly, it has been shown that the peak activity of iNOS ranges from 12 to 24 hours after reperfusion injury.26,27 It was also reported that during rejection expression of iNOS in immunohistochemistry increased. The return to low levels during periods of non-rejection in human lung transplantation, and the non or low level expression of iNOS after lung transplantation indicated that ischemia and reperfusion injury were not involved in the up-regulation of the enzyme.28 Thus, in our experiment neither the FK409 nor the control groups stained positive for iNOS after 2 hours of reperfusion. Several studies have shown the protective action of L-arginine in reperfusion injury. Elevated arginase in the tissue may be responsible for the inacti-

vation of the L-arginine/NO pathway; however, there are some limitations to using L-arginine. Specifically, increased arginase might destroy much of the L-arginine given.29 In addition, only the constitutive form of NOS might be available for the conversion of L-arginine to NO during the immediate reperfusion period.29 Moreover, even in the presence of large amounts of L-arginine, sufficient amounts of NO were not generated in the immediate reperfusion period of the liver.30 FK409 releases NO spontaneously, demonstrating potent vasorelaxant and antiplatelet effects.18 FK409 produces vasorelaxation by increasing intracellular cyclic GMP,31 and inhibiting platelet aggregation in vitro and thrombus formation in the rat extracorporeal shunt model.5 The mechanism for releasing NO from FK409 is not clear, but it was reported that NO of 1 to 1.5 mol/liter was released from 1 mol/liter of

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FK409 in vitro.32,33 Thus, it seems that FK409 might be the ideal NO donor since it releases sufficient amounts of NO immediately when needed without sustained release over long periods.29 In our immunohistochemical observation, after 2 hours of reperfusion both FK409 and control groups showed a positive staining for eNOS. However, we could not detect any difference between the FK409 and control groups. To gain a protective effect from L-arginine, an intact NOS system is essential in the reperfusion organ29; however, FK409 releases sufficient amounts of NO immediately when needed without sustained release over long periods. Moreover, eNOS expression has been detected in a normal human lung.34 Thus, a difference of eNOS expression between the FK409 and control groups may not have been detected. FK409 had a more potent anti-platelet effect than doses of isosorbide dinitrate, the most popular orally active NO donor used to treat ischemic cardiovascular disease.6 Platelet aggregation and neutrophil adherence may be factors in microcirculatory failure.35,36 Moreover, oxygen free radicals released from infiltrated neutrophils damage the endothelium.37 The protective effects of NO are consistently related to the prevention of pulmonary sequestration of PMNs.38 Indeed, PMNs play a pivotal role in ischemia-reperfusion injury by becoming activated, adhering to the endothelium and releasing reactive oxygen species into the surrounding tissue.39,40 Since the PMN-endothelium interaction is the central mechanism in ischemia-reperfusion inducing lung endothelial injury,41,42 we assessed lung sequestration of PMNs in the present study. PMN infiltration was significantly lower in the FK409 group than in the control group. Vasoconstriction is one explanation for the microcirculation disturbance found in ischemia-reperfusion injury, and ET-I is one of the most potent endothelium-derived contracting factors.43 ET-I is released during hypoxia44 and contributes to ischemia-reperfusion injury.1 ET-I is also generated in physiologic disorders of the vascular system.45 NO influences vascular tone via the regulated reciprocal production of ET-I in vasculatures.46 On the other hand, the elimination of ET-I from the blood mostly results from the removal of peptides by lung, kidney and liver parenchyma.47 ET-I levels were significantly lower in the FK409 group than in the control group. We did not conclude that FK409 directly inhibited ET-I production as antagonistic action of NO; however, this study suggests that ET-I is released secondarily by endothelial cell damage that

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induces ischemia-reperfusion injury, and that FK409 ameliorates endothelial injury and microcirculation. Some researchers reported that the increased vascular permeability, alveolar damage, alveolar collapse and interstitial edema observed in ischemiareperfusion injury in the lung resulted in collapsed circulation, reduced oxygen-exchange capability and increased pulmonary vascular resistance.48,49 Kamoshita and co-workers50 reported that histologic findings after reperfusion exhibited marked collapse of capillaries, intra-alveolar hemorrhage and interstitial edema in the subject animals. In the present study, histologic findings after 2 hours of reperfusion in the control group revealed alveolar damage, interstitial edema and hyaline membranes localized along the alveolar duct; the damage was less severe in the FK409 group. FK409 was intravenously administered at 0.4 mg/kg before reperfusion. In a rat methacholineinduced coronary vasospasm model, FK409 administration suppressed the elevation of the ST segment on epicardial electrocardiograms dose-dependently and significantly at 0.1 mg/kg. On the other hand, FK409 significantly decreased mean blood pressure at a dose of 1 mg/kg.6 FK409 appears to decrease the mean blood pressure via an increase in cGMP due to spontaneously released NO.51 Thus, we selected 0.4 mg/kg of FK409 as the effective dosage, because it did not depress the mean blood pressure. In a recent study, Dhar and co-workers52 also reported that a low dose (0.4 mg/kg) of FK409 had a significant protective effect on hepatic ischemia-reperfusion injury in rats. Murakami and colleagues19 reported that inhaled NO, which was used during ischemia, during reperfusion, or both, had a beneficial effect on ischemia-reperfusion injury occurring after a warm lung ischemia-reperfusion, and the best results were obtained when NO was applied successively during periods of both ischemia and reperfusion. In this study, FK409 was intravenously administered 5 minutes before reperfusion to improve microcirculation after reperfusion. The bolus infusion of FK409 before reperfusion was effective. An experimental study was conducted to determine the effects of FK409 on ischemia-reperfusion injury of the lung by using of an in situ warm lung ischemia model in rats. SaO2, PaO2, and ET-I levels 2 hours after reperfusion were significantly better in the FK409 group than in the control group. In histologic study, there was less pulmonary injury in the FK409 group than in the control group after 2 hours of reperfusion. Furthermore, PMN infiltration was significantly lower in the FK409 group than

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in the control group. The 7-day survival rate was 88% in the FK409 group and 20% in the control group, with a statistically significant difference between the groups. In conclusion, this drug provides organic protection against pulmonary ischemia-reperfusion injury. This effect may be related to creation of a homeostatic effect in pulmonary vascular beds, and prevention of PMN sequestration. The authors wish to express sincere thanks to Fujisawa Pharmaceutical Co., Ltd. for supplying the FK409 used for this study.

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