- Email: [email protected]
Activated Protein C in Ischemia-Reperfusion Injury After Experimental Lung Transplantation
Activated Protein C in Ischemia-Reperfusion Injury After Experimental Lung Transplantation Shin Hirayama, MD,a Marcelo Cypel, MD,a Masaaki Sato, MD,a Masaki Anraku, MD,a Patricia C. Liaw, MD,b Mingyao Liu, MD,a Thomas K. Waddell, MD,a and Shaf Keshavjee, MDa Background: Ischemia-reperfusion injury remains the major cause of early morbidity and mortality after lung transplantation. Activated protein C (APC) has been demonstrated to attenuate various acute inflammation-related injuries in the lung and other organs. Methods: The effect of exogenous APC in lung transplantation was examined using a rat orthotopic lung transplantation model of ischemia-reperfusion injury with 24 hours of cold ischemia. APC was administered to the donor airway before cold pulmonary artery flush, or intravenously to the recipient before reperfusion. Results: The levels of APC in the lung tissue were significantly higher in the intra-airway group compared with the intravenous group and the saline control group (p ⬍ 0.01). Transplanted lung oxygenation was significantly better in the intra-airway APC group at 2 hours after reperfusion compared with controls (PaO2, mean ⫾ SD mm Hg: intra-airway APC, 350.9 ⫾ 85.5; intravenous APC, 241.1 ⫾ 59.3; control, 200.2 ⫾ 37.3; p ⬍ 0.05). No difference was detected in proinflammatory cytokines or thrombin-anti-thrombin complexes in the lung tissue. Histologic examination of the lung injury score or alveolar fibrin deposition did not demonstrate significant differences among groups. Conclusion: Exogenous APC administered to the donor airway attenuates ischemia-reperfusion injury after lung transplantation. This novel administration route sustains high levels of APC in the lung tissue, which should avoid frequent administration and potential systemic side effects of bleeding. Further investigation is necessary to determine the mechanism of the beneficial effect of APC in this setting. J Heart Lung Transplant 2009;28:1180 – 4. Copyright © 2009 by the International Society for Heart and Lung Transplantation.
Lung transplantation has enjoyed increasing success during the last 2 decades. The International Society for Heart and Lung Transplantation Registry reported in 2008 that more than 2,100 lung transplants are performed annually.1 Despite this success, rejection, infection, and primary graft dysfunction are persistent problems and contribute to a mortality rate of 20% to 30% at 1 year.1 Some degree of reperfusion injury develops in most recipients, and severe lung injury or primary graft dysfunction subsequently develops in 10%. These patients require extended support with mechanical ventilation, pharmacologic therapy, and occasionally extra-
From aLatner Thoracic Surgery Laboratories, Toronto General Research Institute, University Health Network, University of Toronto; and bHenderson Research Centre, McMaster University, Hamilton, Ontario, Canada. Submitted March 29, 2009; revised June 19, 2009; accepted June 26, 2009. Reprint requests: Dr. Shaf Keshavjee, Toronto General Hospital, 200 Elizabeth St. 9N947, Toronto, ON, M5G 2C4 Canada. Telephone: 416-340-4010. Fax: 416-340-4556. E-mail: [email protected] Copyright © 2009 by the International Society for Heart and Lung Transplantation. 1053-2498/09/$–see front matter. doi:10.1016/ j.healun.2009.06.026
1180
corporeal membrane oxygenation.2,3 In addition to a high mortality rate, severe reperfusion injury has also been associated with an increased risk of acute rejection4,5 and chronic graft dysfunction represented by bronchiolitis obliterans syndrome.6 Activated protein C (APC) is an active form of the anti-coagulant protein C, the propeptide of which is cleaved by thrombin-thrombomodulin complex on the plasma membrane of endothelial cells, platelets, and monocytes.7 The anti-coagulant protein C pathway is important in inactivation of coagulation factors Va and VIIIa. APC also indirectly promotes fibrinolysis by inactivating plasminogen activation inhibitor-1 (PAI-1).8 APC has potent anti-inflammatory effects by suppressing the release of proinflammatory mediators (eg, tumor necrosis factor [TNF]-␣ and interleukin [IL]-1) from leukocytes and by down-regulating adhesion molecules expressed by endothelial cells.7 Recent studies have demonstrated that APC has an important protective effect in various acute lung injuries. Systemic administration of APC has been reported to prevent the lethal effect of Escherichia coli-associated sepsis in experimental animal models9 and also reduces endotoxin-induced pulmonary vascular injury
The Journal of Heart and Lung Transplantation Volume 28, Number 11
in rats.10 A double-blinded randomized clinical trial has demonstrated that treatment with APC improves the clinical outcome of patients with sepsis.11 Furthermore, a recent report suggests that lower levels of endogenous APC may be associated with a poor outcome of ischemia-reperfusion injury after lung transplantation.12 Given the significance of endogenous activation of protein C in ischemia-reperfusion injury after lung transplantation, supplementation with exogenous APC might be expected to preserve pulmonary function after lung transplantation. This study examined the effect of APC in ischemiareperfusion injury after lung transplantation using a rat orthotopic lung transplantation model and explored the optimal administration route. Because APC has a potent anti-coagulation effect, the potential adverse effect of bleeding after a surgical procedure is a major concern.13 The lung has the unique potential for localized drug delivery through the airway. We thus compared intra-airway administration with conventional intravenous drug administration. We report a significant beneficial effect of intra-airway APC administration on pulmonary function after 24 hours of cold ischemia and reperfusion, without apparent significant adverse side effects. METHODS All animals used in this study 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 prepared by the Institute of Laboratory Animal Resources and published by the National Institutes of Health (NIH Publication No. 86-23, revised 1996). Lung Transplantation Procedure Experiments were performed using male inbred Lewis rats (weight, 250 –300 grams; Charles River Inc, Montreal, QC, Canada). Orthotopic left lung transplantation was performed as described previously.14 The donor lung was preserved for 24 hours at 4°C after being flushed with low potassium dextran solution (Perfadex, Vitrolife, Uppsala, Sweden). A recipient rat was ventilated using a technique of separate ventilation,15 in which the native right lung and the transplanted left lung were ventilated with tidal volumes of 6 ml/kg and 4 ml/kg, respectively (positive end-expiratory pressure, 2 cm H2O; fraction of inspired oxygen, 1.0). Anticoagulation was used only for the donor rat, with systemic intravenous administration of 300 U of heparin (Organon Teknika, Toronto, ON, Canada). Two hours after reperfusion, a blood gas sample was taken from the left pulmonary vein before the transplanted lung was excised for subsequent analysis.
Hirayama et al.
1181
Experimental Groups Recombinant human APC (rh-APC; Drotrecogin Alfa [Activated], Eli Lilly, Indianapolis, IN) was administered according to the study design illustrated in Figure 1. In the intra-airway APC group, the donor rat received rhAPC (0.3 mg/kg) intratracheally 5 minutes before pulmonary artery flush preservation, and the recipient animal received saline through the jugular vein before reperfusion. In the intravenous APC group, the donor rat received saline transtracheally, and the recipient rat received rhAPC (0.3 mg/kg) through the jugular vein before reperfusion. The control group received saline in the donor trachea and recipient jugular vein. Each group comprised 5 transplant recipients. Measurement of APC, Thrombin-Anti-thrombin Complex, and Cytokines The APC assay was performed as previously described in Liaw et al.16 Levels of thrombin–anti-thrombin (TAT) complex in citrated lung homogenate samples were quantified using the Enzygnost TAT micro kit (Dade Behring, Schwalbach, Germany) according to the manufacturer’s instructions. The levels of rat TNF-␣, macrophage-inflammatory protein-2 (MIP-2), rat IL-1, and rat IL-6 were measured using specific kits by enzymelinked immunosorbent assay (Biosource International, Camarillo, CA), as described previously.17 Histological Examination, Lung Injury Scoring, and Quantification of Alveolar Fibrin Deposition Formalin-fixed paraffin-embedded lung tissue was sectioned (5-m thickness) and stained with standard hematoxylin and eosin (H&E) or Martius scarlet blue (MSB) for fibrin. H&E-stained sections were scored for septal thickening, congestion, edema severity, interstitial neutrophil infiltration, intra-alveolar neutrophil in-
Figure 1. Study design. Recombinant human activated protein C (rhAPC; 1 mg/kg) was administered to the donor trachea 5 minutes before flushing, or to the recipient intravenously before reperfusion. FIO2, fraction of inspired oxygen.
1182
Hirayama et al.
The Journal of Heart and Lung Transplantation November 2009
filtration, and intra-alveolar hemorrhage, using a previously described lung-injury scoring system.18 Fibrin deposition in alveolar space was morphometrically quantified using Image J 1.30 software (Wayne Rasband, National Institutes of Health, Bethesda, MD). Statistical Analysis All data are expressed as mean ⫾ standard deviation. One-way analysis of variance (ANOVA) was followed by post hoc Tukey tests to determine statistical significance among groups, except for lung injury scoring, in which a non-parametric Kruskal-Wallis test was used. Values of p ⬍ 0.05 were considered to be significant.
RESULTS Intra-airway Administration of APC Improves Oxygenation after Lung Reperfusion We administered APC to the systemic circulation of the recipient animal before reperfusion or to the airway of the donor lung before flushing. Intra-airway administration resulted in significantly higher levels of APC in the lung tissue 2 hours after reperfusion compared with the other 2 groups (p ⬍ 0.01, Figure 2). Transplanted lung function was also superior in the intra-airway APC group. The intra-airway APC group demonstrated significantly higher PaO2 levels than that of the control group (p ⬍ 0.05) and a trend towards a higher PaO2 than the intravenous group (p ⬎ 0.13, Figure 3). We did not observe any bleeding complications in any of the study groups.
Figure 2. Activated protein C (APC) levels in transplanted lung tissue were measured at 2 hour after reperfusion as indicated in Materials and Methods. Data are expressed as mean ⫾ standard deviation (n ⫽ 5). *p ⬍ 0.01 in one-way analysis of variance, followed by a post hoc Tukey test.
Figure 3. Blood oxygenation levels in the pulmonary graft were measured in the blood drawn from the pulmonary vein of the transplanted left lung at 2 hours after reperfusion with activated protein C (APC). Data are expressed as mean ⫾ standard deviation (n ⫽ 5). *p ⬍ 0.05 in one-way analysis of variance, followed by a post hoc Tukey test.
APC Does Not Change Proinflammatory Cytokines, TAT Complexes, Pulmonary Histology, or Alveolar Fibrin Deposition Because ischemia-reperfusion injury after lung transplantation is considered to be the end result of multifactorial injurious processes that include inflammatory responses and thrombosis formation,19 we explored the potential mechanisms that might mediate the APC attenuation of ischemia-reperfusion injury. Interestingly, APC administration did not significantly change the levels of proinflammatory cytokines (TNF-␣, MIP-2, IL-1, and IL-6) in the lung tissue (control, intravenous APC, intra-airway APC; TNF-␣ (pg/mg): 33.8 ⫾ 11.4, 19.7 ⫾ 8.4, 22.5 ⫾ 14.3; MIP-2: 22.9 ⫾ 12.2, 15.5 ⫾ 5.2, 22.9 ⫾ 16.6; IL-1: 17.9 ⫾ 6.8, 19.5 ⫾ 8.8, 18.9 ⫾ 2.8; and IL-6: 78.8 ⫾ 10.2, 105.5 ⫾ 31.2, 102.9 ⫾ 56.6). APC did not significantly alter the histological score of lung injury in H&E staining (Table 1). We examined the lung wet/dry ratio in each sample but did not detect any significant differences between the groups. Thus, the lung function differences could not be attributed to lung edema formation. Another possible explanation for the protective effect of APC in this setting might be related to reducing fibrin deposition in the alveoli, which could lead to improved gas exchange ability in the lung. However, morphometric quantification of fibrin deposition in alveoli using MSB staining did not demonstrate a significant difference among groups (MSB positive area (%); control, intravenous APC, intra-airway APC; 9.6 ⫾ 1.9. 10.7 ⫾ 2.1, 10.9 ⫾ 0.5; Table 1).
The Journal of Heart and Lung Transplantation Volume 28, Number 11
Hirayama et al.
1183
Table 1. Lung Injury Scores in Rats Variablea Septal thickening Congestion Edema Interstitial PMN Intra-alveolar PMN Intra-alveolar hemorrhage Total score Fibrin deposition % MSB staining
Control group (n ⫽ 4) 1.5 ⫾ 0.2 2.15 ⫾ 0.6 1.75 ⫾ 0.47 2.4 ⫾ 0.48 1.15 ⫾ 0.3 2.1 ⫾ 0.38 11.05 ⫾ 0.72
IV group (n ⫽ 4) 1.7 ⫾ 0.35 1.9 ⫾ 0.48 1.7 ⫾ 0.62 1.9 ⫾ 0.26 1.25 ⫾ 0.44 2.0 ⫾ 0.37 10.45 ⫾ 1.48
IA group (n ⫽ 4) 1.35 ⫾ 0.38 1.65 ⫾ 0.91 1.45 ⫾ 0.44 2.25 ⫾ 0.38 1.15 ⫾ 0.38 1.8 ⫾ 0.99 9.15 ⫾ 2.46
9.6 ⫾ 1.89
10.7 ⫾ 2.11
11 ⫾ 0.54
p-value 0.34 0.61 0.69 0.23 0.91 0.81 0.32 0.50
IA, intra-airway; IV, intravenous; PMN, polymorphonuclear leukocytes; MSB, Martius scarlet blue. a Data are mean ⫾ standard deviation.
APC administration did not change the level of TAT complexes in the lung (TAT complex level (g/mg); control, intravenous APC, intra-airway APC; 13.8 ⫾ 3.0, 13.2 ⫾ 6.8, 12.1 ⫾ 5.4), indicating that APC did not significantly affect intra-alveolar coagulation or fibrinolysis in this model. These results suggest that APC exerted its protective effect in lung ischemia-reperfusion injury through a mechanism other than the well-described anti-inflammatory and anti-coagulation effects of APC. DISCUSSION The lung is unique because of availability of the transairway route to administer therapeutic drugs to the organ. APC is known to have a potent anti-coagulant effect, thus although its therapeutic intent is attractive, systemic administration raises the concern of postsurgical bleeding in clinical application.13 Thus, we explored the potential of the intra-airway delivery route in the present study. Indeed, the intra-airway administration of APC attenuated ischemia-reperfusion injury, demonstrating significantly improved oxygenation compared with the control group and a trend towards improvement compared with the intravenous group. Consistent with these results, we note that local APC administration has previously been demonstrated to attenuate experimental lung fibrosis,20,21 asthmatic inflammation,22 and endotoxin-induced pulmonary inflammation.23,24 These studies underline the beneficial effect of local APC administration in models of lung injury, supporting our findings of potential benefit of this treatment in ischemia-reperfusion–related injury after lung transplantation. The significant beneficial effect of APC on oxygenation appears to be associated with the high level of APC retained in the lung as long as 2 hours after reperfusion (ie, 26 hours after the initial administration to the donor airway). Importantly, because APC is primarily inactivated in the circulation through ␣1antitrypsin, protein C inhibitor, and ␣2-macroglobulin,25 it
is possible that APC is degraded more slowly in the lung, where these degradation pathways are less active and that the lung behaves like a reservoir of APC that is delivered through an endotracheal route. Bleeding complications related to the systemic administration in the intravenous APC group were not evident in these short-term experiments in which APC was administered only once. However, repetitive or continuous injection of APC through this route could well result in an increased risk of bleeding after clinical lung transplantation, as was demonstrated in clinical trials of APC in sepsis.11,26 APC has been described as having potent anti-inflammatory and anti-coagulant effects.25,27 The effect of APC on inflammation has been somewhat controversial. Kotanidou et al24 demonstrated that rhAPC pre-treatment did not attenuate the lipopolysaccharide-induced increases of the proinflammatory cytokines in mice.24 In humans, moreover, administration of rhAPC did not reduce endotoxin-induced cytokine release in the airways.28 Consistent with these findings, we could not detect significant differences among groups, suggesting that the protective effect of APC may not be related to its anti-inflammatory effects in this model. The results of the present study also suggest that the protective effect of APC administered in the airway in the setting of lung transplantation is mediated by a mechanism other than these conventional recognized effects of APC. Surprisingly, we did not observe any significant differences in the histologic manifestation of lung injury such as alveolar hemorrhage, inflammatory cell infiltration, and alveolar wall thickness among groups. A possible mechanism of action of APC that might explain improved blood oxygenation without changing the local lung histology might be related to modifications of vascular tone or microvascular coagulation in the lung microcirculation, which may lead to attenuated regional intrapulmonary shunt and improved oxygenation. Because the present study was limited in the evaluation of intrapulmonary shunt, further investigation
1184
Hirayama et al.
will be necessary to determine the mechanism of APCmediated lung protection after lung transplantation. APC as an agent has limitations in this current study, including cost, bleeding, and a short half-life. Owing to its short half-life in the blood, continuous administration of APC to the recipient will make the treatment more expensive and raise a risk of bleeding. However, we believe airway delivery of APC to the donor lung could minimize those limitations. In conclusion, the present study demonstrates the protective role of APC in ischemia-reperfusion injury after lung transplantation and the novel donor lung intra-airway administration route of APC, which enables sustained high local levels of APC in the lung tissue without systemic administration.
The Journal of Heart and Lung Transplantation November 2009
10.
11.
12.
13.
14.
15.
DISCLOSURE STATEMENT The authors would like to thank Suzanne Beaudin (Department of Medicine, Division of Hematology and Thromboembolism, McMaster University, Canada) for her technical assistance in the APC assay. The authors acknowledge Eli Lilly for providing the activated protein C drug and an unrestricted research grant to support this study.
16.
17.
18.
19.
None of the authors has a financial relationship with a commercial entity that has an interest in the subject of the presented manuscript or other conflicts of interest to disclose.
20.
REFERENCES
21.
1. Christie JD, Edwards LB, Aurora P, et al. Registry of the International Society for Heart and Lung Transplantation: twenty-fifth official adult lung and heart/lung transplantation report—2008. J Heart Lung Transplant 2008;27:957– 69. 2. Fischer S, Bohn D, Rycus P, et al. Extracorporeal membrane oxygenation for primary graft dysfunction after lung transplantation: analysis of the Extracorporeal Life Support Organization (ELSO) registry. J Heart Lung Transplant 2007;26:472–7. 3. Shargall Y, Guenther G, Ahya VN, Ardehali A, Singhal A, Keshavjee S. Report of the ISHLT Working Group on Primary Lung Graft Dysfunction part VI: treatment. J Heart Lung Transplant 2005;24:1489 –500. 4. Howard TK, Klintmalm GB, Cofer JB, Husberg BS, Goldstein RM, Gonwa TA. The influence of preservation injury on rejection in the hepatic transplant recipient. Transplantation 1990;49:103–7. 5. Shackleton CR, Ettinger SL, McLoughlin MG, Scudamore CH, Miller RR, Keown PA. Effect of recovery from ischemic injury on class I and class II MHC antigen expression. Transplantation 1990;49:641–4. 6. Daud SA, Yusen RD, Meyers BF, et al. Impact of immediate primary lung allograft dysfunction on bronchiolitis obliterans syndrome. Am J Respir Crit Care Med 2007;175:507–13. 7. Mosnier LO, Zlokovic BV, Griffin JH. The cytoprotective protein C pathway. Blood 2007;109:3161–72. 8. Idell S. Coagulation, fibrinolysis, and fibrin deposition in acute lung injury. Crit Care Med 2003;31:S213–20. 9. Taylor FB Jr, Chang A, Esmon CT, D’Angelo A, Vigano-D’Angelo S, Blick KE. Protein C prevents the coagulopathic and lethal effects
22.
23.
24.
25. 26.
27.
28.
of Escherichia coli infusion in the baboon. J Clin Invest 1987; 79:918 –25. Murakami K, Okajima K, Uchiba M, et al. Activated protein C prevents LPS-induced pulmonary vascular injury by inhibiting cytokine production. Am J Physiol 1997;272:L197–202. Bernard GR, Vincent JL, Laterre PF, et al. Efficacy and safety of recombinant human activated protein C for severe sepsis. N Engl J Med 2001;344:699 –709. Christie JD, Robinson N, Ware LB, et al. Association of protein C and type 1 plasminogen activator inhibitor with primary graft dysfunction. Am J Respir Crit Care Med 2007;175:69 –74. Abraham E, Laterre PF, Garg R, et al. Drotrecogin alfa (activated) for adults with severe sepsis and a low risk of death. N Engl J Med 2005;353:1332– 41. Hirayama S, Shiraishi T, Shirakusa T, Higuchi T, Miller EJ. Prevention of neutrophil migration ameliorates rat lung allograft rejection. Mol Med 2006;12:208 –13. de Perrot M, Quadri SM, Imai Y, Keshavjee S. Independent ventilation of the graft and native lungs in vivo after rat lung transplantation. Ann Thorac Surg 2005;79:2169 –71. Liaw PC, Esmon CT, Kahnamoui K, et al. Patients with severe sepsis vary markedly in their ability to generate activated protein C. Blood 2004;104:3958 – 64. de Perrot M, Young K, Imai Y, et al. Recipient T cells mediate reperfusion injury after lung transplantation in the rat. J Immunol 2003;171:4995–5002. Murao Y, Loomis W, Wolf P, Hoyt DB, Junger WG. Effect of dose of hypertonic saline on its potential to prevent lung tissue damage in a mouse model of hemorrhagic shock. Shock 2003;20:29 –34. de Perrot M, Liu M, Waddell TK, Keshavjee S. Ischemia-reperfusion-induced lung injury. Am J Respir Crit Care Med 2003;167: 490 –511. Shimizu S, Gabazza EC, Taguchi O, et al. Activated protein C inhibits the expression of platelet-derived growth factor in the lung. Am J Respir Crit Care Med 2003;167:1416 –26. Yasui H, Gabazza EC, Tamaki S, et al. Intratracheal administration of activated protein C inhibits bleomycin-induced lung fibrosis in the mouse. Am J Respir Crit Care Med 2001;163:1660 – 8. Yuda H, Adachi Y, Taguchi O, et al. Activated protein C inhibits bronchial hyperresponsiveness and Th2 cytokine expression in mice. Blood 2004;103:2196 –204. Slofstra SH, Groot AP, Maris NA, Reitsma PH, Cate HT, Spek CA. Inhalation of activated protein C inhibits endotoxin-induced pulmonary inflammation in mice independent of neutrophil recruitment. Br J Pharmacol 2006;149:740 – 6. Kotanidou A, Loutrari H, Papadomichelakis E, et al. Inhaled activated protein C attenuates lung injury induced by aerosolized endotoxin in mice. Vascul Pharmacol 2006;45:134 – 40. Esmon CT. Crosstalk between inflammation and thrombosis. Maturitas 2004;47:305–14. Vincent JL, Bernard GR, Beale R, et al. Drotrecogin alfa (activated) treatment in severe sepsis from the global openlabel trial ENHANCE: further evidence for survival and safety and implications for early treatment. Crit Care Med 2005;33: 2266 –77. Dahlback B, Villoutreix BO. Regulation of blood coagulation by the protein C anticoagulant pathway: novel insights into structure-function relationships and molecular recognition. Arterioscler Thromb Vasc Biol 2005;25:1311–20. Nick JA, Coldren CD, Geraci MW, et al. Recombinant human activated protein C reduces human endotoxin-induced pulmonary inflammation via inhibition of neutrophil chemotaxis. Blood 2004;104:3878 – 85.
Lung transplantation has enjoyed increasing success during the last 2 decades. The International Society for Heart and Lung Transplantation Registry reported in 2008 that more than 2,100 lung transplants are performed annually.1 Despite this success, rejection, infection, and primary graft dysfunction are persistent problems and contribute to a mortality rate of 20% to 30% at 1 year.1 Some degree of reperfusion injury develops in most recipients, and severe lung injury or primary graft dysfunction subsequently develops in 10%. These patients require extended support with mechanical ventilation, pharmacologic therapy, and occasionally extra-
From aLatner Thoracic Surgery Laboratories, Toronto General Research Institute, University Health Network, University of Toronto; and bHenderson Research Centre, McMaster University, Hamilton, Ontario, Canada. Submitted March 29, 2009; revised June 19, 2009; accepted June 26, 2009. Reprint requests: Dr. Shaf Keshavjee, Toronto General Hospital, 200 Elizabeth St. 9N947, Toronto, ON, M5G 2C4 Canada. Telephone: 416-340-4010. Fax: 416-340-4556. E-mail: [email protected] Copyright © 2009 by the International Society for Heart and Lung Transplantation. 1053-2498/09/$–see front matter. doi:10.1016/ j.healun.2009.06.026
1180
corporeal membrane oxygenation.2,3 In addition to a high mortality rate, severe reperfusion injury has also been associated with an increased risk of acute rejection4,5 and chronic graft dysfunction represented by bronchiolitis obliterans syndrome.6 Activated protein C (APC) is an active form of the anti-coagulant protein C, the propeptide of which is cleaved by thrombin-thrombomodulin complex on the plasma membrane of endothelial cells, platelets, and monocytes.7 The anti-coagulant protein C pathway is important in inactivation of coagulation factors Va and VIIIa. APC also indirectly promotes fibrinolysis by inactivating plasminogen activation inhibitor-1 (PAI-1).8 APC has potent anti-inflammatory effects by suppressing the release of proinflammatory mediators (eg, tumor necrosis factor [TNF]-␣ and interleukin [IL]-1) from leukocytes and by down-regulating adhesion molecules expressed by endothelial cells.7 Recent studies have demonstrated that APC has an important protective effect in various acute lung injuries. Systemic administration of APC has been reported to prevent the lethal effect of Escherichia coli-associated sepsis in experimental animal models9 and also reduces endotoxin-induced pulmonary vascular injury
The Journal of Heart and Lung Transplantation Volume 28, Number 11
in rats.10 A double-blinded randomized clinical trial has demonstrated that treatment with APC improves the clinical outcome of patients with sepsis.11 Furthermore, a recent report suggests that lower levels of endogenous APC may be associated with a poor outcome of ischemia-reperfusion injury after lung transplantation.12 Given the significance of endogenous activation of protein C in ischemia-reperfusion injury after lung transplantation, supplementation with exogenous APC might be expected to preserve pulmonary function after lung transplantation. This study examined the effect of APC in ischemiareperfusion injury after lung transplantation using a rat orthotopic lung transplantation model and explored the optimal administration route. Because APC has a potent anti-coagulation effect, the potential adverse effect of bleeding after a surgical procedure is a major concern.13 The lung has the unique potential for localized drug delivery through the airway. We thus compared intra-airway administration with conventional intravenous drug administration. We report a significant beneficial effect of intra-airway APC administration on pulmonary function after 24 hours of cold ischemia and reperfusion, without apparent significant adverse side effects. METHODS All animals used in this study 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 prepared by the Institute of Laboratory Animal Resources and published by the National Institutes of Health (NIH Publication No. 86-23, revised 1996). Lung Transplantation Procedure Experiments were performed using male inbred Lewis rats (weight, 250 –300 grams; Charles River Inc, Montreal, QC, Canada). Orthotopic left lung transplantation was performed as described previously.14 The donor lung was preserved for 24 hours at 4°C after being flushed with low potassium dextran solution (Perfadex, Vitrolife, Uppsala, Sweden). A recipient rat was ventilated using a technique of separate ventilation,15 in which the native right lung and the transplanted left lung were ventilated with tidal volumes of 6 ml/kg and 4 ml/kg, respectively (positive end-expiratory pressure, 2 cm H2O; fraction of inspired oxygen, 1.0). Anticoagulation was used only for the donor rat, with systemic intravenous administration of 300 U of heparin (Organon Teknika, Toronto, ON, Canada). Two hours after reperfusion, a blood gas sample was taken from the left pulmonary vein before the transplanted lung was excised for subsequent analysis.
Hirayama et al.
1181
Experimental Groups Recombinant human APC (rh-APC; Drotrecogin Alfa [Activated], Eli Lilly, Indianapolis, IN) was administered according to the study design illustrated in Figure 1. In the intra-airway APC group, the donor rat received rhAPC (0.3 mg/kg) intratracheally 5 minutes before pulmonary artery flush preservation, and the recipient animal received saline through the jugular vein before reperfusion. In the intravenous APC group, the donor rat received saline transtracheally, and the recipient rat received rhAPC (0.3 mg/kg) through the jugular vein before reperfusion. The control group received saline in the donor trachea and recipient jugular vein. Each group comprised 5 transplant recipients. Measurement of APC, Thrombin-Anti-thrombin Complex, and Cytokines The APC assay was performed as previously described in Liaw et al.16 Levels of thrombin–anti-thrombin (TAT) complex in citrated lung homogenate samples were quantified using the Enzygnost TAT micro kit (Dade Behring, Schwalbach, Germany) according to the manufacturer’s instructions. The levels of rat TNF-␣, macrophage-inflammatory protein-2 (MIP-2), rat IL-1, and rat IL-6 were measured using specific kits by enzymelinked immunosorbent assay (Biosource International, Camarillo, CA), as described previously.17 Histological Examination, Lung Injury Scoring, and Quantification of Alveolar Fibrin Deposition Formalin-fixed paraffin-embedded lung tissue was sectioned (5-m thickness) and stained with standard hematoxylin and eosin (H&E) or Martius scarlet blue (MSB) for fibrin. H&E-stained sections were scored for septal thickening, congestion, edema severity, interstitial neutrophil infiltration, intra-alveolar neutrophil in-
Figure 1. Study design. Recombinant human activated protein C (rhAPC; 1 mg/kg) was administered to the donor trachea 5 minutes before flushing, or to the recipient intravenously before reperfusion. FIO2, fraction of inspired oxygen.
1182
Hirayama et al.
The Journal of Heart and Lung Transplantation November 2009
filtration, and intra-alveolar hemorrhage, using a previously described lung-injury scoring system.18 Fibrin deposition in alveolar space was morphometrically quantified using Image J 1.30 software (Wayne Rasband, National Institutes of Health, Bethesda, MD). Statistical Analysis All data are expressed as mean ⫾ standard deviation. One-way analysis of variance (ANOVA) was followed by post hoc Tukey tests to determine statistical significance among groups, except for lung injury scoring, in which a non-parametric Kruskal-Wallis test was used. Values of p ⬍ 0.05 were considered to be significant.
RESULTS Intra-airway Administration of APC Improves Oxygenation after Lung Reperfusion We administered APC to the systemic circulation of the recipient animal before reperfusion or to the airway of the donor lung before flushing. Intra-airway administration resulted in significantly higher levels of APC in the lung tissue 2 hours after reperfusion compared with the other 2 groups (p ⬍ 0.01, Figure 2). Transplanted lung function was also superior in the intra-airway APC group. The intra-airway APC group demonstrated significantly higher PaO2 levels than that of the control group (p ⬍ 0.05) and a trend towards a higher PaO2 than the intravenous group (p ⬎ 0.13, Figure 3). We did not observe any bleeding complications in any of the study groups.
Figure 2. Activated protein C (APC) levels in transplanted lung tissue were measured at 2 hour after reperfusion as indicated in Materials and Methods. Data are expressed as mean ⫾ standard deviation (n ⫽ 5). *p ⬍ 0.01 in one-way analysis of variance, followed by a post hoc Tukey test.
Figure 3. Blood oxygenation levels in the pulmonary graft were measured in the blood drawn from the pulmonary vein of the transplanted left lung at 2 hours after reperfusion with activated protein C (APC). Data are expressed as mean ⫾ standard deviation (n ⫽ 5). *p ⬍ 0.05 in one-way analysis of variance, followed by a post hoc Tukey test.
APC Does Not Change Proinflammatory Cytokines, TAT Complexes, Pulmonary Histology, or Alveolar Fibrin Deposition Because ischemia-reperfusion injury after lung transplantation is considered to be the end result of multifactorial injurious processes that include inflammatory responses and thrombosis formation,19 we explored the potential mechanisms that might mediate the APC attenuation of ischemia-reperfusion injury. Interestingly, APC administration did not significantly change the levels of proinflammatory cytokines (TNF-␣, MIP-2, IL-1, and IL-6) in the lung tissue (control, intravenous APC, intra-airway APC; TNF-␣ (pg/mg): 33.8 ⫾ 11.4, 19.7 ⫾ 8.4, 22.5 ⫾ 14.3; MIP-2: 22.9 ⫾ 12.2, 15.5 ⫾ 5.2, 22.9 ⫾ 16.6; IL-1: 17.9 ⫾ 6.8, 19.5 ⫾ 8.8, 18.9 ⫾ 2.8; and IL-6: 78.8 ⫾ 10.2, 105.5 ⫾ 31.2, 102.9 ⫾ 56.6). APC did not significantly alter the histological score of lung injury in H&E staining (Table 1). We examined the lung wet/dry ratio in each sample but did not detect any significant differences between the groups. Thus, the lung function differences could not be attributed to lung edema formation. Another possible explanation for the protective effect of APC in this setting might be related to reducing fibrin deposition in the alveoli, which could lead to improved gas exchange ability in the lung. However, morphometric quantification of fibrin deposition in alveoli using MSB staining did not demonstrate a significant difference among groups (MSB positive area (%); control, intravenous APC, intra-airway APC; 9.6 ⫾ 1.9. 10.7 ⫾ 2.1, 10.9 ⫾ 0.5; Table 1).
The Journal of Heart and Lung Transplantation Volume 28, Number 11
Hirayama et al.
1183
Table 1. Lung Injury Scores in Rats Variablea Septal thickening Congestion Edema Interstitial PMN Intra-alveolar PMN Intra-alveolar hemorrhage Total score Fibrin deposition % MSB staining
Control group (n ⫽ 4) 1.5 ⫾ 0.2 2.15 ⫾ 0.6 1.75 ⫾ 0.47 2.4 ⫾ 0.48 1.15 ⫾ 0.3 2.1 ⫾ 0.38 11.05 ⫾ 0.72
IV group (n ⫽ 4) 1.7 ⫾ 0.35 1.9 ⫾ 0.48 1.7 ⫾ 0.62 1.9 ⫾ 0.26 1.25 ⫾ 0.44 2.0 ⫾ 0.37 10.45 ⫾ 1.48
IA group (n ⫽ 4) 1.35 ⫾ 0.38 1.65 ⫾ 0.91 1.45 ⫾ 0.44 2.25 ⫾ 0.38 1.15 ⫾ 0.38 1.8 ⫾ 0.99 9.15 ⫾ 2.46
9.6 ⫾ 1.89
10.7 ⫾ 2.11
11 ⫾ 0.54
p-value 0.34 0.61 0.69 0.23 0.91 0.81 0.32 0.50
IA, intra-airway; IV, intravenous; PMN, polymorphonuclear leukocytes; MSB, Martius scarlet blue. a Data are mean ⫾ standard deviation.
APC administration did not change the level of TAT complexes in the lung (TAT complex level (g/mg); control, intravenous APC, intra-airway APC; 13.8 ⫾ 3.0, 13.2 ⫾ 6.8, 12.1 ⫾ 5.4), indicating that APC did not significantly affect intra-alveolar coagulation or fibrinolysis in this model. These results suggest that APC exerted its protective effect in lung ischemia-reperfusion injury through a mechanism other than the well-described anti-inflammatory and anti-coagulation effects of APC. DISCUSSION The lung is unique because of availability of the transairway route to administer therapeutic drugs to the organ. APC is known to have a potent anti-coagulant effect, thus although its therapeutic intent is attractive, systemic administration raises the concern of postsurgical bleeding in clinical application.13 Thus, we explored the potential of the intra-airway delivery route in the present study. Indeed, the intra-airway administration of APC attenuated ischemia-reperfusion injury, demonstrating significantly improved oxygenation compared with the control group and a trend towards improvement compared with the intravenous group. Consistent with these results, we note that local APC administration has previously been demonstrated to attenuate experimental lung fibrosis,20,21 asthmatic inflammation,22 and endotoxin-induced pulmonary inflammation.23,24 These studies underline the beneficial effect of local APC administration in models of lung injury, supporting our findings of potential benefit of this treatment in ischemia-reperfusion–related injury after lung transplantation. The significant beneficial effect of APC on oxygenation appears to be associated with the high level of APC retained in the lung as long as 2 hours after reperfusion (ie, 26 hours after the initial administration to the donor airway). Importantly, because APC is primarily inactivated in the circulation through ␣1antitrypsin, protein C inhibitor, and ␣2-macroglobulin,25 it
is possible that APC is degraded more slowly in the lung, where these degradation pathways are less active and that the lung behaves like a reservoir of APC that is delivered through an endotracheal route. Bleeding complications related to the systemic administration in the intravenous APC group were not evident in these short-term experiments in which APC was administered only once. However, repetitive or continuous injection of APC through this route could well result in an increased risk of bleeding after clinical lung transplantation, as was demonstrated in clinical trials of APC in sepsis.11,26 APC has been described as having potent anti-inflammatory and anti-coagulant effects.25,27 The effect of APC on inflammation has been somewhat controversial. Kotanidou et al24 demonstrated that rhAPC pre-treatment did not attenuate the lipopolysaccharide-induced increases of the proinflammatory cytokines in mice.24 In humans, moreover, administration of rhAPC did not reduce endotoxin-induced cytokine release in the airways.28 Consistent with these findings, we could not detect significant differences among groups, suggesting that the protective effect of APC may not be related to its anti-inflammatory effects in this model. The results of the present study also suggest that the protective effect of APC administered in the airway in the setting of lung transplantation is mediated by a mechanism other than these conventional recognized effects of APC. Surprisingly, we did not observe any significant differences in the histologic manifestation of lung injury such as alveolar hemorrhage, inflammatory cell infiltration, and alveolar wall thickness among groups. A possible mechanism of action of APC that might explain improved blood oxygenation without changing the local lung histology might be related to modifications of vascular tone or microvascular coagulation in the lung microcirculation, which may lead to attenuated regional intrapulmonary shunt and improved oxygenation. Because the present study was limited in the evaluation of intrapulmonary shunt, further investigation
1184
Hirayama et al.
will be necessary to determine the mechanism of APCmediated lung protection after lung transplantation. APC as an agent has limitations in this current study, including cost, bleeding, and a short half-life. Owing to its short half-life in the blood, continuous administration of APC to the recipient will make the treatment more expensive and raise a risk of bleeding. However, we believe airway delivery of APC to the donor lung could minimize those limitations. In conclusion, the present study demonstrates the protective role of APC in ischemia-reperfusion injury after lung transplantation and the novel donor lung intra-airway administration route of APC, which enables sustained high local levels of APC in the lung tissue without systemic administration.
The Journal of Heart and Lung Transplantation November 2009
10.
11.
12.
13.
14.
15.
DISCLOSURE STATEMENT The authors would like to thank Suzanne Beaudin (Department of Medicine, Division of Hematology and Thromboembolism, McMaster University, Canada) for her technical assistance in the APC assay. The authors acknowledge Eli Lilly for providing the activated protein C drug and an unrestricted research grant to support this study.
16.
17.
18.
19.
None of the authors has a financial relationship with a commercial entity that has an interest in the subject of the presented manuscript or other conflicts of interest to disclose.
20.
REFERENCES
21.
1. Christie JD, Edwards LB, Aurora P, et al. Registry of the International Society for Heart and Lung Transplantation: twenty-fifth official adult lung and heart/lung transplantation report—2008. J Heart Lung Transplant 2008;27:957– 69. 2. Fischer S, Bohn D, Rycus P, et al. Extracorporeal membrane oxygenation for primary graft dysfunction after lung transplantation: analysis of the Extracorporeal Life Support Organization (ELSO) registry. J Heart Lung Transplant 2007;26:472–7. 3. Shargall Y, Guenther G, Ahya VN, Ardehali A, Singhal A, Keshavjee S. Report of the ISHLT Working Group on Primary Lung Graft Dysfunction part VI: treatment. J Heart Lung Transplant 2005;24:1489 –500. 4. Howard TK, Klintmalm GB, Cofer JB, Husberg BS, Goldstein RM, Gonwa TA. The influence of preservation injury on rejection in the hepatic transplant recipient. Transplantation 1990;49:103–7. 5. Shackleton CR, Ettinger SL, McLoughlin MG, Scudamore CH, Miller RR, Keown PA. Effect of recovery from ischemic injury on class I and class II MHC antigen expression. Transplantation 1990;49:641–4. 6. Daud SA, Yusen RD, Meyers BF, et al. Impact of immediate primary lung allograft dysfunction on bronchiolitis obliterans syndrome. Am J Respir Crit Care Med 2007;175:507–13. 7. Mosnier LO, Zlokovic BV, Griffin JH. The cytoprotective protein C pathway. Blood 2007;109:3161–72. 8. Idell S. Coagulation, fibrinolysis, and fibrin deposition in acute lung injury. Crit Care Med 2003;31:S213–20. 9. Taylor FB Jr, Chang A, Esmon CT, D’Angelo A, Vigano-D’Angelo S, Blick KE. Protein C prevents the coagulopathic and lethal effects
22.
23.
24.
25. 26.
27.
28.
of Escherichia coli infusion in the baboon. J Clin Invest 1987; 79:918 –25. Murakami K, Okajima K, Uchiba M, et al. Activated protein C prevents LPS-induced pulmonary vascular injury by inhibiting cytokine production. Am J Physiol 1997;272:L197–202. Bernard GR, Vincent JL, Laterre PF, et al. Efficacy and safety of recombinant human activated protein C for severe sepsis. N Engl J Med 2001;344:699 –709. Christie JD, Robinson N, Ware LB, et al. Association of protein C and type 1 plasminogen activator inhibitor with primary graft dysfunction. Am J Respir Crit Care Med 2007;175:69 –74. Abraham E, Laterre PF, Garg R, et al. Drotrecogin alfa (activated) for adults with severe sepsis and a low risk of death. N Engl J Med 2005;353:1332– 41. Hirayama S, Shiraishi T, Shirakusa T, Higuchi T, Miller EJ. Prevention of neutrophil migration ameliorates rat lung allograft rejection. Mol Med 2006;12:208 –13. de Perrot M, Quadri SM, Imai Y, Keshavjee S. Independent ventilation of the graft and native lungs in vivo after rat lung transplantation. Ann Thorac Surg 2005;79:2169 –71. Liaw PC, Esmon CT, Kahnamoui K, et al. Patients with severe sepsis vary markedly in their ability to generate activated protein C. Blood 2004;104:3958 – 64. de Perrot M, Young K, Imai Y, et al. Recipient T cells mediate reperfusion injury after lung transplantation in the rat. J Immunol 2003;171:4995–5002. Murao Y, Loomis W, Wolf P, Hoyt DB, Junger WG. Effect of dose of hypertonic saline on its potential to prevent lung tissue damage in a mouse model of hemorrhagic shock. Shock 2003;20:29 –34. de Perrot M, Liu M, Waddell TK, Keshavjee S. Ischemia-reperfusion-induced lung injury. Am J Respir Crit Care Med 2003;167: 490 –511. Shimizu S, Gabazza EC, Taguchi O, et al. Activated protein C inhibits the expression of platelet-derived growth factor in the lung. Am J Respir Crit Care Med 2003;167:1416 –26. Yasui H, Gabazza EC, Tamaki S, et al. Intratracheal administration of activated protein C inhibits bleomycin-induced lung fibrosis in the mouse. Am J Respir Crit Care Med 2001;163:1660 – 8. Yuda H, Adachi Y, Taguchi O, et al. Activated protein C inhibits bronchial hyperresponsiveness and Th2 cytokine expression in mice. Blood 2004;103:2196 –204. Slofstra SH, Groot AP, Maris NA, Reitsma PH, Cate HT, Spek CA. Inhalation of activated protein C inhibits endotoxin-induced pulmonary inflammation in mice independent of neutrophil recruitment. Br J Pharmacol 2006;149:740 – 6. Kotanidou A, Loutrari H, Papadomichelakis E, et al. Inhaled activated protein C attenuates lung injury induced by aerosolized endotoxin in mice. Vascul Pharmacol 2006;45:134 – 40. Esmon CT. Crosstalk between inflammation and thrombosis. Maturitas 2004;47:305–14. Vincent JL, Bernard GR, Beale R, et al. Drotrecogin alfa (activated) treatment in severe sepsis from the global openlabel trial ENHANCE: further evidence for survival and safety and implications for early treatment. Crit Care Med 2005;33: 2266 –77. Dahlback B, Villoutreix BO. Regulation of blood coagulation by the protein C anticoagulant pathway: novel insights into structure-function relationships and molecular recognition. Arterioscler Thromb Vasc Biol 2005;25:1311–20. Nick JA, Coldren CD, Geraci MW, et al. Recombinant human activated protein C reduces human endotoxin-induced pulmonary inflammation via inhibition of neutrophil chemotaxis. Blood 2004;104:3878 – 85.