Effect of Endothelin Receptor Antagonist on Lung Allograft Apoptosis and NOSII Expression

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1 Effect of Endothelin Receptor Antagonist on Lung Allograft Apoptosis and NOSII Expression Matthew J. Shaw, MSc, Hani Shennib, MD, Nicolas Bousette, MSc, Eliot H. Ohlstein, PhD, and Adel Giaid, PhD Departments of Surgery and Pathology, The Montreal General Hospital, McGill University, Montreal, Quebec, Canada, and SmithKline Beecham, King of Prussia, Pennsylvania Background. It is postulated that apoptosis contributes to ischemia-reperfusion graft dysfunction after lung transplantation. The purpose of this study was to determine whether the improvement in lung function that we previously observed with the use of an endothelin-1 (ET-1) receptor antagonist after ischemia-reperfusion injury is associated with a reduction in inducible nitric oxide synthase (NOSII) expression and programmed cell death. Methods. Left lung canine allotransplantation was performed. Harvested lung blocks were preserved with modified Eurocollins solution and stored at 4 C for 18 to 20 hours. Lung allografts were tested for the expression of NOSII by immunohistochemistry, and extent of apoptosis by terminal dutp nick end-labeling (TUNEL). Animals blindly received either an intravenous infusion of saline (control) or the ET-1 receptor antagonist (SB209670) (15 g/kg/min). Infusion began 30 minutes pretransplantation and continued to 6 hours posttransplantation. Results. Immunohistochemical analysis demonstrated significantly stronger NOSII immunostaining in the allografts of the saline control group (36.5% 3.6%) compared with native right lungs (6.9% 1.3%, p < 0.001) or the ET-receptor antagonist treatment group (9.6% 1.4%, p < 0.001). The TUNEL staining revealed a significantly stronger labeling in the allografts of the saline treatment control group (40.7% 6.2%) compared with native right lungs (5.0% 0.6%, p < 0.005) or the ET receptor antagonist treatment group (14.1% 2.8%, p < 0.01). Conclusions. We conclude that treatment of lung allografts with the ET-1 receptor antagonist SB reduces the area of NOSII expression and the extent of apoptosis, factors known to contribute to the process of prolonged ischemia-reperfusion injury. (Ann Thorac Surg 2001;72:386 90) 2001 by The Society of Thoracic Surgeons Lung transplantation is an important treatment modality for end-stage lung disease. However, posttransplant graft dysfunction represents serious challenges to recipient management. Graft dysfunction has many causes including ischemic/reperfusion injury, which is associated with a high early mortality rate [1]. Ischemic/ reperfusion injury presents as increased pulmonary vascular resistance (PVR), hypoxia, and pulmonary edema. These characteristics are generally associated with vasoconstriction, bronchoconstriction, vascular leakage, and increased inflammatory cell infiltrate. Interestingly, endothelin-1 (ET-1), a 210 amino acid peptide, has been shown to mediate most of these pathologic processes [2]. Biological actions of ET-1 are mediated mainly by two related receptors, ET-A and ET-B. ET-1 is expressed in vascular endothelial cells, alveolar macrophages, pulmonary epithelial cells, and vascular smooth muscle cells [3]. Clearance of this factor is known to occur mainly in the lungs and kidneys [3]. The fact that ET-1 antagonism prevented the infiltration of inflammatory cells in an Presented at the Poster Session of the Thirty-sixth Annual Meeting of The Society of Thoracic Surgeons, Fort Lauderdale, FL, Jan 31 Feb 2, Address reprint requests to Dr Giaid, The Montreal General Hospital, 1650 Cedar Ave, Suite L3-314, Montreal, PQ H3G 1A4, Canada; adel.giaid@mcgill.ca. airway inflammation model [4] suggests that it also has chemoattractant properties. This is also supported by the observation that a drastic increase in ET-1 mrna can be detected before any sign of the inflammatory response [5]. ET-1 has also been shown to induce the release of many proinflammatory cytokines including TNF-, IL- 1, IL-6, and IL-8 [3]. Activation of ETB receptor has been shown to induce the release of the free radical nitric oxide (NO) [3]. The latter is an important antimicrobial factor; however, when present in high concentrations, it can be cytotoxic to host cells [6]. Indeed, activation of the inducible form of nitric oxide synthase (NOSII) has been shown to induce apoptosis [7]. We have recently shown that the use of the ETA/ETB receptor antagonist SB improves lung function in the canine lung allograft model [8]. Specifically, SB showed improvements in lung edema, oxygen tension, PVR, and early mortality rates [8]. The aim of the present study was to determine the level of NOSII expression and the degree of apoptosis in the SB treated lung allografts compared to those of saline-treated animals. Material and Methods The experimental design comprised two groups of dogs. Both groups underwent the same surgical operation; one 2001 by The Society of Thoracic Surgeons /01/$20.00 Published by Elsevier Science Inc PII S (01)

2 Ann Thorac Surg SHAW ET AL 2001;72: ENDOTHELIN IN CELL APOPTOSIS 387 group received an infusion of saline solution (control, n 5), whereas the other group received saline solution containing the ET-1 receptor antagonist SB (n 6). The solutions were administered 30 minutes before reperfusion of the transplanted lung, and continued to 6 hours posttransplantation. Tissue samples as well as the native right lungs were collected from both groups. Details of the surgical procedure used for the donor lung harvest and recipient operation are as described elsewhere [8]. Immunohistochemistry Lung biopsies (1 cm 2 ) were taken 6 hours after initiation of reperfusion. Samples for immunohistochemistry were fixed in 4% paraformaldehyde, stored in 30% ethanol, embedded in paraffin, and cut on a microtome (5 m thick). Immunohistochemical staining for NOS II was performed using the avidin-biotin peroxidase method [9]. Paraffin sections were dewaxed in toluene for 20 minutes and rehydrated with 100%, 90%, 70%, and 50% alcohol for 2 minutes each. All sections were then immersed in PBS solution for 5 minutes and then in 2% hydrogen peroxide solution to block endogenous peroxidase activity for 30 minutes. The sections were then washed three times in PBS solution for 5 minutes. The sections were permeabilized in 0.2% Triton in 0.1 mol/l PBS (ph 7.4) for 30 minutes and washed three times in PBS for 5 minutes. Sections were then incubated in 10% normal goat serum (NGS) for 30 minutes at room temperature, after which they were incubated overnight at 4 C with the mouse anti-nos II antiserum (Bio/Can Scientific, Mississauga, ON, Canada). The sections were washed three times in PBS for 5 minutes after the cold storage, and incubated for 45 minutes with biotinylated goat-antimouse-igg (1:200) at room temperature. They were then washed three times again in PBS for 5 minutes, and incubated with the avidin-biotin-peroxidase complex (Vectastain Elite Kit, Vector Laboratories, Burlingame, CA) for 45 minutes at room temperature. The sections were then washed in PBS for 5 minutes. Sites of immunostaining were visualized by developing sections in 0.025% diaminobenzidine and 0.03% peroxide for 2 to 3 minutes. The sections were then returned to the PBS solution and subsequently immersed in cold running tap water for 5 minutes. The sections were then dehydrated through 50%, 70%, 90%, and 100% ethanol for 2 minutes each, and then cleared in toluene. Finally, the sections were mounted with Permount and glass cover slips and allowed to dry. The specificity of the immunostaining was confirmed using negative control experiments in which anti-nos II antibody was substituted with NGS or by immunoabsorption of the antisera to its antigen. No counterstain was used to facilitate computer quantitative analysis. Quantitation of NOS II expression was accomplished in a blinded fashion using NIH Image 1.44 (US National Institutes of Health, Bethesda, MD). For each slide (n 3 sections/specimen), five high-power fields were photographed and imported into Image The integrated density for the five high-power field views was calculated and the integrated density of the negative control slide was subtracted. The integrated density was calculated as N (mean background) where N is number of pixels in the selection, mean is the average gray value of the pixels within the selection, and background is the modal gray value (most common pixel value) after smoothing the histogram. This formula assumed that the background is lighter (has lower pixel values) than the object being measured. The mean of the five slides for each particular tissue (eg, 6 hours of ischemia with 2 hours of perfusion) was taken the standard error, and the values were defined as arbitrary units (AU). Terminal dutp Nick-End Labeling Open-lung biopsies of the transplanted left and native right lungs were obtained at the end of each experiment for terminal dutp nick-end labeling (TUNEL) analysis. Normal lung biopsies were obtained from the extracted native left lung. Tissue specimens were inflated and fixed in 4% paraformaldehyde, which was switched to 30% ethanol, and were embedded in paraffin. Sections 5 m thick were cut from several levels of each block and mounted on glass slides. Apoptotic cells were labeled by enzymatic in situ end labeling of apoptosis-induced DNA strand breaks using a commercial kit (In Situ Cell Death Detection Kit, Boehringer Mannheim, Laval, Quebec, Canada). This process is also referred to as TUNEL. Briefly, cryostat sections of 4% paraformaldehyde-fixed specimens were rinsed in PBS for 30 minutes, then immersed in 2% hydrogen peroxide for 30 minutes at room temperature. The slides were permeabilized in proteinase K for 30 minutes at 37 C after PBS washing. The slides were then washed with PBS and incubated with terminal deoxynucleotidyl transferase (TDT) and nucleotides in a humid atmosphere for 60 minutes at 37 C. The slides were washed with PBS and incubated with Converter-POD (antifluorescein antibody, Fab fragment from sheep, conjugated with horseradish peroxidase) for 30 minutes at 37 C. After PBS wash, sites of immunostaining were visualized by developing sections in 0.025% diaminobenzidine and 0.03% peroxide. The specificity of labeling was confirmed using negative control experiments in which terminal transferase was substituted with nucleotide labeling mixture. No counterstain was used to facilitate computer quantitative analysis. Quantitation of TUNEL staining was accomplished in a blinded fashion using Image-Pro Plus (MediaCybernetics, Silver Spring, MD). For each slide (n 3 sections/ specimen), five high-power fields were imported into Image-Pro Plus. This was facilitated by means of a video camera attached to the Olympus BX60 microscope, (Olympus, Tokyo, Japan) which imported the image. The image was then digitized, dividing the image into pixels. A ratio percentage of positively stained area to negatively stained area was calculated by the program. The mean of the slides for each particular group was taken SE.

3 388 SHAW ET AL Ann Thorac Surg ENDOTHELIN IN CELL APOPTOSIS 2001;72: Fig 1. Immunohistochemical localization of inducible nitric oxide synthase (NOSII) in the two experimental groups. Panel A represents a lung section from the ET receptor antagonist (SB209670) treatment group showing minimal staining for NOSII. Panel B shows a lung section from the saline-treated (control) group with strong NOSII immunostaining in epithelial cells. (Magnification 40.) Statistical Analysis All results are expressed as means SE. Differences between groups were assessed by analysis of variance (ANOVA) using a commercial program. A p value of 0.05 or less was considered statistically significant. Results Using antiserum against NOSII on tissue samples from the canine allografts, we observed NOSII immunoreactivity in the airway epithelium, lung parenchyma, and macrophages, as well as endothelial and vascular smooth muscle cells. The lung allografts that received the saline (control) treatment had significantly higher levels of NOSII immunostaining compared with the native right lungs. Specifically, 36.5% 3.6% of the tissue area was stained for NOSII in the saline control group, compared with 6.9% 1.3% in the native right lung (p 0.001) (Fig 1). Intensity of NOSII immunostaining in the ETA/ETB receptor antagonist group was significantly reduced compared with the saline control group, and was comparable to that of the native right lung (9.6% 0.8%; p 0.001) (Fig 2). The negative control sections showed no staining. The use of the TUNEL technique showed that the saline control group had a higher level of apoptosis as compared with the native right lung, in which staining was found mostly in the airway epithelium and lung parenchyma (Fig 3). Specifically, the saline group had 40.67% 6.16% of the tissue area labeled, whereas only a low 5.00% 0.56% of the native right lung was stained (p 0.005). The treatment group receiving SB showed reduced staining and had only 14.13% 1.8% staining, which was significantly reduced compared with the control saline group (p 0.05). The negative control sections showed no labeling. Comment Ischemia-reperfusion injury in the lung is usually associated with an increase in PVR and pulmonary edema. The basic mechanisms of IR injury during lung preservation have yet to be elucidated. One important mediator of IR injury is likely to be the potent vasconstrictor peptide ET-1, which is produced by numerous cell types in the lung such as vascular endothelial cells, airway epithelial cells, polymorphonuclear inflammatory cells, and smooth muscle cells [3, 9]. ET-1 is known to induce an increase in PVR and vascular permeability in the lung [3]. Recently, the importance of apoptosis and its relation to IR injury has become recognized. Apoptosis associated specifically with IR injury has been reported in several organs including the heart [10], kidney [11], and liver [12]. There is still controversy, however, over the relative contributions of ischemia and reperfusion in the induction of apoptosis. The principal findings of this study were that there was a significant decrease in NOSII immunostaining in the inflammatory and epithelial cells of lung allografts treated with the ET-1 receptor antagonist SB209670, compared with the saline control group. In addition, the TUNEL immunostaining demonstrated a significant reduction of apoptosis in the airway epithelium and parenchyma of the ET-1 receptor antagonist treated group compared with the saline control group. We have previously demonstrated increased levels of ET-1 in bronchoalveolar lavage fluid and plasma after allotransplantation in both humans and animal lung transplant recipients [13]. This was associated with increased PVR and early graft dysfunction. Recently developed endothelin receptor antagonists have been shown to be effective in ameliorating IR injury in various organ systems [14]. Subsequently we demonstrated that the administration of the endothelin receptor antagonist SB209670, a potent mixed endothelin A/endothelin B

4 Ann Thorac Surg SHAW ET AL 2001;72: ENDOTHELIN IN CELL APOPTOSIS 389 Fig 2. Quantitative analysis of the terminal dutp nick-end labeling (TUNEL) and inducible nitric oxide synthase (NOSII) immunostaining. Upper panel represents the mean SE (in %) of the area of TUNEL staining in the lung allograft saline-treated group, endothelin (ET) receptor antagonist (SB209670) treated lung allograft group, native right lung. *p Lower panel represents the mean SE (in %) of the area of NOSII immunostaining in the lung allograft saline-treated group, ET receptor antagonist (SB209670) treated lung allograft group, and native right lung. *p 0.01 saline vs SB209670; **p saline vs native right lung. (ETA/ETB) receptor antagonist that demonstrates numerous vasculoprotective properties, improves lung allograft function and survival after prolonged ischemia [8]. Taken together, these findings point to an important role for ET-receptor antagonists in the prevention of early lung graft dysfunction after prolonged ischemia, the mechanism of which may involve a reduction in NOSII expression and cell apoptosis. There are various theories on how pulmonary IR injury could trigger apoptosis. One theory involves the generation of nitric oxide (NO) produced by inducible nitric oxide synthase (NOS II). NO is an important free radical that is known to modulate lung function [15]. Numerous research articles have described the physiologic and pathophysiologic functions of NO, including its role in neurotransmission, cardiovascular smooth muscle relaxation, and microbicide [15]. NO is produced from one of three isoforms of nitric oxide synthase (NOS). Each NOS isoform has a specialized regulatory mechanism and is a product of a unique gene. Two of these isoforms, endothelial NOS (NOS III) and neuronal NOS (NOS I), are constitutive; the other type of NOS isoform (NOS II), is inducible. NO generated from macrophages demonstrates cytotoxic activity such as tumoricidal and antimicrobial effects [16]. Nitric oxide generated by the enzyme NOS II in macrophages has been shown to cause induction of apoptosis in cardiac allografts in vivo [17]. Szabolcs and colleagues [18] reported a matching increase in NOS II expression and apoptosis over several days. In this study we have demonstrated a significant reduction in the immunostaining for NOSII in the allografts of the treatment group compared with that of the saline control group. ET receptor antagonists have been shown to inhibit the increase in eosinophils in bronchoalveolar lavage fluid and reduce the inflammatory reaction in lung tissue [4]. These findings offer a poignant explanation for the observed decrease in the level of immunostaining that we observed in the treatment group. As the receptor antagonist reduces the level of inflammation, it will consequently effect a decrease in the level of cytokines. This reduced level of cytokines allows a decrease in cytokine mediated NOSII activation. In addition there has recently been evidence suggesting that ET-1 directly mediates nitric oxide synthase activation, via an ETB receptor G protein pathway [6]. Therefore blockage of the ETB receptor may also directly reduce NOSII activation. In situ nick-end labeling, or TUNEL, has been used to detect apoptosis in tissue sections [19]. The information it provides is valuable because of the selectivity of in situ nick-end labeling technique for apoptotic nuclei, as these contain a far greater degree of DNA fragmentation than necrotic cells [20]. It has been demonstrated that DNA fragmentation can occur without significant changes in morphology, even though the accompanying morphologic changes of apoptosis or necrosis aid the determination of the type of cell death [21]. The nucleosomal cleavage of DNA is due to the action of unique endonucleases [22]. In this study, the TUNEL staining showed a significant reduction in labeling in the allografts of the ET-receptor antagonist treatment group compared to the saline receiving control group. This suggests that some of the beneficial effects of the ETA/ETB receptor antagonists on organ function may be related to the reduction of cell death associated with prolonged ischemia/reperfusion injury. In summary, the results of this study strongly support an important role for ET-1 in lung injury after ischemiareperfusion injury, and further suggest that ET-receptor antagonists may constitute important therapeutic tools for the prevention of early graft dysfunction. Our findings indicate that the treatment of lung allografts with the mixed ETA/ETB receptor antagonist SB can ame-

5 390 SHAW ET AL Ann Thorac Surg ENDOTHELIN IN CELL APOPTOSIS 2001;72: Fig 3. Terminal dupt nick-end labeling (TUNEL) immunostaining of lung sections from the two experimental groups. Panel A represents a section from a lung allograft in the saline (control) group showing abundant degree of apoptotic nuclei. Panel B represents a section from a lung allograft in the endothelin (ET) antagonist (SB209670) treatment group showing fewer apoptotic nuclei. (Magnifications 40.) liorate lung injury possibly through a reduction in the level of NOSII expression and apoptosis after prolonged ischemia and reperfusion. This work was supported in part by the Medical Research Council of Canada. Doctor Adel Giaid is a recipient of a scholarship from the Fond du Recherche en Santé du Québec. References 1. Hosenpud JD, Novick RJ, Breen TJ, Keck B, Daily P. The Registry of the International Society for Heart and Lung Transplantation: twelfth official report J Heart Lung Transplant 1995;14: Okada M, Yamashita C, Okada M, et al. Contribution of endothelin-1 to warm ischemia/reperfusion injury of the rat lung. Am J Respir Crit Care Med 1995;152: Michael JR, Markewitz BA. Endothelins and the lung. Am J Respir Crit Care Med 1996;154: Finsnes F, Skjonsberg OH, Tonnessen T, et al. Endothelin production and effects of endothelin antagonism during experimental airway inflammation. Am J Respir Crit Care Med 1992;155: Finsnes F, Christensen G, Lyberg T, Sejersted OM, Skjonsberg OH. Increased synthesis and release of endothelin-1 during the initial phase of airway inflammation. Am J Respir Crit Care Med 1998;158: Szabolcs MJ, Ravalli S, Minanov O, Sciacca RR, Michler RE, Cannon PJ. Apoptosis and increased expression of inducible nitric oxide synthase in human allograft rejection. Transplantation 1998;65: Arstall MA, Sawyer DB, Fukazawa R, Kelly RA. Cytokinemediated apoptosis in cardiac myocytes: the role of inducible nitric oxide synthase induction and peroxynitrite generation. Circ Res 1999;85: Shennib H, Lee AG, Kuang JO, et al. Efficacy of administering an endothelin-receptor antagonist SB in ameliorating ischemia-reperfusion injury in lung allografts. Am J Respir Crit Care Med 1998;157: Giaid A, Yanagisawa M, Langleban D, et al. Expression of endothelin-1 in lungs of patients with pulmonary hypertension. N Engl J Med 1993;328: Gottlieb RA, Burleson KO, Kloner RA, et al. Reperfusion injury induces apoptosis in rabbit cardiomyocytes. J Clin Invest 1994;94: Burns AT, Davies DR, McLaren AJ, et al. Apoptosis in ischemia reperfusion injury of human renal allografts. Transplantation 1998;66: Sasaki H, Matsuno T, Ishikawa T, et al. Activation of apoptosis during early phase of reperfusion after liver transplantation. Transplant Proc 1997;29: Shennib H, Serrick C, Saleh D, Reis A, Stewart DJ, Giaid A. Plasma endothelin-1 levels in human lung transplant recipients. J Cardiovasc Pharmacol 1995;26(Suppl 3):S Shennib H, Kuang JQ, Ohlstein EH, Giaid A. Endothelin receptor antagonist improves pulmonary hemodynamics during lung ischemia/reperfusion injury. Transplantation 1998;66: Dugas B, Debre P, Moncada S. Nitric oxide, a vital poison inside the immune and inflammatory network. Res Immunol 1995;146: Nathan C, Hibbs JB. Role of nitric oxide synthesis in macrophage antimicrobial activity. Curr Opin Immunol 1991;3: Suzuki H, Wildhirt SM, Dudek RR, et al. Induction of apoptosis in myocardial infarction and its possible relationship to nitric oxide synthase in macrophages. Tissue Cell 1996;28: Szabolcs M, Michler RE, Yang X, et al. Apoptosis of cardiac myocytes during cardiac allograft rejection. Circulation 1996; 94: Wijsman JH, Jonker RR, Keijzer R, et al. A new method to detect apoptosis in paraffin sections: ISEL of fragmented DNA. J Histochem Cytochem 1993;41: Ansari B, Coates BD, Greenstein, et al. In situ end labeling detects DNA strand breaks in apoptosis and other physiological and pathological states. J Pathol 1993;170: Zakeri ZF, Quaglino D, Latham T, et al, Delayed internucleosomal DNA fragmentation in programmed cell death. FASEB J 1993;7: Wyllie AH. Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation. Nature 1980;284:555 6.