INTRODUCTION

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Lung transplantation has evolved into an accepted therapeutic option for selected patients with end-stage pulmonary and cardiopulmonary disease (1). A 1-yr actuarial survival of 62% has been reported (2). Despite this success, the deleterious effects of ischemia-reperfusion injury remain a major contributing factor to early and late postoperative complications and limited survival following lung transplantation. Early graft failure, with a mortality rate as high as 25% within the first 3 mo, can be attributed either directly or indirectly to ischemia- reperfusion injury (2, 3).

Severe forms of ischemia-reperfusion injury present as pulmonary edema, hypoxia, and increased pulmonary vascular resistance (PVR), and result in pulmonary hypertension and right-sided heart failure (4, 5). It is assumed that recruited inflammatory cells, accompanied by dysfunctional vasculoendothelial cells, may produce a variety of chemical mediators leading to acute pulmonary graft dysfunction. However, the exact mechanism underlining altered vasoreactivity and increased vessel-wall permeability in lung grafts remains unclear.

We have previously demonstrated increased expression of endothelin-1 (ET-1), a potent vasoconstrictor, bronchoconstrictor, and mitogenic peptide (6), in pulmonary endothelial and epithelial cells in conditions associated with increased PVR and cellular proliferation, such as pulmonary hypertension (7) and pulmonary fibrosis (8). We subsequently demonstrated increased expression of ET-1 in bronchoalveolar lavage fluid (BALF) and plasma, which was associated with increased PVR and early allograft dysfunction (9, 10). Several studies have suggested a role for ET-1 in mediating other posttransplant complications, such as acute rejection (11) and bronchiolitis obliterans (12). Recently developed endothelin-receptor antagonists have been shown to be effective in preventing ischemia-reperfusion injury in different organ systems (13). Among these antagonists is SB209670, a potent mixed endothelin A/endothelin B (ETA/ETB) receptor antagonist demonstrating numerous vasculoprotective properties (17).

The purpose of the present study was to evaluate the possible involvement of ET-1 in mediating ischemia-reperfusion injury in donor lungs following prolonged cold ischemia of these lungs. We investigated whether treatment of lung allografts with SB209670 could ameliorate early allograft dysfunction and improve survival in a canine model of lung allotransplantation.

	METHODS

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Experimental Design

Successfully allotransplanted animals (n = 11) received intravenous infusion of either ET-1 receptor antagonist (15 µg/kg/min; treatment group, n = 6) or saline (control group, n = 5) in a blinded fashion. Solutions were administered 30 min before reperfusion of the transplanted lung, and were continued for up to 6 h after transplantation. Hemodynamic measurements, blood gas measurements, and plasma samples were obtained at T = 0 h, 1 h, 3 h, and 6 h after transplantation, with the animal functioning solely on the newly transplanted lung. This was achieved by applying a right pulmonary artery snare 15 to 20 min before each of the designated time intervals.

SB209670 solution (SmithKline-Beecham Pharmaceutical Co. Ltd., Philadelphia, PA), a nonpeptide, nonselective ET(A)/ET(B)- receptor antagonist, was freshly prepared on the day of transplantation by dissolving the antagonist in normal saline.

Donor Lung Harvest

Heart-lung blocks were harvested from 11 donor dogs (25 to 30 kg) as previously described (22). In brief, donor animals underwent pulmonary artery (PA) flushing with 3 L of cold, modified Eurocollins solution (3 mEq/L sodium bicarbonate + 12.8 mEq/L magnesium sulfate + 3 ml/L heparin) at a perfusion pressure of approximately 30 mm Hg, with the lungs ventilated. The heart-lung blocks were excised and stored in cold, modified Eurocollins solution at 4° C for 18 h in a semiinflated state.

Recipient Operation

On the day of surgery, size- and weight-matched animals received 30 mg/kg of oral cyclosporine A, 3 mg/kg of azathioprine, and 10 mg/kg of methylprednisolone intravenously. Animals were mechanically ventilated with 100% oxygen at a flow rate of 0.5 L/s. Neuromuscular blocking agents were not used in this study. Animals were then prepared for hemodynamic monitoring. The heart rate (HR) was measured with a continuous electrocardiographic monitor (Model 90903A; Space Labs Inc., Redmond, WA). Invasive arterial blood pressure (BP) measurement was done with femoral artery cannulation, and the pulmonary arterial pressure (Ppa), pulmonary capillary wedge pressure (PCWP), and central venous pressure (CVP) were measured with a Swan-Ganz catheter positioned in the pulmonary artery (Space Labs Inc.). Cardiac output (CO) was also measured with a Swan-Ganz catheter, using a thermal dilution technique (Space Labs Inc.) From these data, pulmonary vascular resistance (PVR) and systemic vascular resistance (SVR) were calculated as PVR = (mPpa  mPCWP)/CO × 80 dyn · s/cm⁵, and SVR = (mBP  mCVP)/CO × 80 dyn · s/cm⁵. Arterial oxygen saturation (%O₂) was measured with a sensor probe (Pulse-Oxymeter; Criticare Systems Inc., Redmond, WA) fixed on the tongue. Arterial and mixed venous blood samples were obtained from the femoral artery and vein, respectively, for blood-gas and plasma ET-1 analyses. Complete blood-gas analysis was performed with an automated blood-gas analyzer (Nova Biomedical, Waltham, MA). Body temperature was monitored and maintained with warming blankets and heating lamps.

Animals underwent a left posterolateral thoracotomy through the fifth intercostal space, followed by a left pneumonectomy. The right PA was encircled with umbilical tape and snared, and a semirigid rubber catheter was used to obtain isolated perfusion of the left lung allograft. Left lung transplantation was performed as usual, by anastomosing the left atrial cuff, PA, and bronchus of the donor and recipient. Throughout the experiment, animals received mechanical ventilation with an FI_O2 of 1.0, positive end-expiratory pressure (PEEP) of 5 cm H₂O, and flow rate of 0.5 L/s. After hemodynamic stability was ascertained, the chest cavity was closed by approximating the ribs, and the animals were repositioned in a supine position to obtain baseline posttransplant measurements (T = 0). Animals were then monitored continuously for the duration of the experiment. At the end of the study (6 h after transplantation), animals were killed with an intravenous bolus injection of concentrated potassium chloride solution.

All animals were given humane care in compliance with the Animal Care Committee regulations of the Montreal General Hospital and McGill University, as well as 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 National Academy of Sciences and published by the National Institutes of Health (NIH Publication No. 85-23, revised 1985).

Histology and Immunohistochemistry

Open-lung biopsies of the transplanted left and native right lung were obtained at the end of each experiment for histologic and wet-to-dry-weight ratio 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 of 4-µm thickness were cut from several levels of each block, mounted on glass slides, and stained with hematoxylin and eosin (H&E) for histologic examination. Paraffin-embedded step sections of biopsy specimens not stained for histologic diagnosis were immunostained with antiserum to ET-1, using a modification of the avidin-biotin-peroxidase method (7). Briefly, sections were dewaxed in toluene and dehydrated in ethanol. To block endogenous peroxidase activity, the sections were immersed in 2% hydrogen peroxide. The sections were permeabilized in 0.2% Triton X-100 and incubated with 10% normal goat serum to reduce nonspecific binding of the antiserum. The serum was drained and sections were incubated with the ET-1 antiserum overnight at 4° C. After three washes in phosphate-buffered saline (PBS), sections were incubated with biotin-conjugated goat antirabbit IgG for 60 min, rewashed in PBS, and then incubated with the avidin-biotin-peroxidase complex (Vectastain Elite kit; Vector Laboratories, Burlingame, CA) for 60 min at room temperature. Sites of immunoreactivity were visualized with diaminobenzidine (DAB) and hydrogen peroxide. The preparations were then counterstained with hematoxylin, dehydrated, cleared, and covered with glass coverslips. Negative-control experiments involved incubation of sections with antiserum neutralized with the respective antigen, or with normal serum instead of the first-layer antiserum.

Measurement of Plasma ET-1

Arterial and venous plasma ET-1 levels were measured with reverse-phase high-performance liquid chromatography (HPLC). In brief, samples were injected into a C18 reverse-phase HPLC column, which was equilibrated with 18% (vol/vol) acetonitrile and 0.1% (vol/vol) TFA and maintained at 40° C. The column was eluted at a flow rate of 1 ml/min with an 18% to 30% linear gradient of acetonitrile in 0.1% trifluoroacetate (TFA) over 12 min, followed by a 30 to 36% linear gradient over an additional 12 min. The peptide was detected by UV absorbance at 210 nm. Synthetic ET-1 (amino acids 1 to 21) eluted at 23 min; HPLC fractions (0.5 ml) were collected and evaporated to dryness under vacuum. The dried residue from each fraction was reconstituted in enzyme-linked immunosorbent assay (ELISA) buffer and subjected to assay for mature ET-1. We consistently recovered > 90% of the immunoreactive mature ET-1 contained in the plasma as a single peak that eluted at 23 min. No other immunoreactive peaks were detected.

Statistical Analysis

All results are expressed as means ± SE. Comparison between groups and between different time intervals was done through analysis of variance (ANOVA). A value of p < 0.05 was considered statistically significant.

	RESULTS

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Hemodynamic Measurements and Survival

No significant difference was observed between the two study groups with regard to weight, total ischemia time, pretransplant hemodynamic parameters, or blood gas tensions. In total, 12 lung transplants were performed in the study. One animal was excluded because of fatal surgical complications (femoroiliac artery dissection and retroperitoneal hematoma).

All animals survived the first 3 h after transplantation, but at 6 h after transplantation, survival in the control group was two of five (40%) animals, compared with six of six (100%) in the treatment group. In the latter group, animals maintained stable hemodynamic parameters and lower peak inspiratory pressure at the end of the study (Table 1). In the treatment group, posttransplant measurements of mPpa, systemic blood pressure, and PVR at T = 0 were slightly lower than those of the control group (Table 1). In contrast, the arterial oxygen tension was higher in the treated allografts (Table 1). After 1 h of reperfusion, the PVR in control animals (715 ± 187 dyn · s/cm⁵) was significantly greater than in treated allografts (297 ± 40 dyn · s/cm⁵) (p = 0.036; Figure 1). Concurrently, no statistical difference between the two groups was detected in SVR (control: 2,938 ± 154 dyn · s/cm⁵, treatment: 2,733 ± 486 dyn · s/cm⁵) (Table 1). These findings were coupled with a continued trend of stable arterial oxygen tension in the treatment group. The PVR remained significantly higher at 3 h after transplantation in control animals than in those in the treatment group (Table 1; Figure 1).

View larger version (16K): [in this window] [in a new window]   Figure 1.   PVR in control group was significantly higher than that in treatment group at 1 and 3 h after transplantation. Only two animals in the control group survived for 6 h after transplantation, and were not included in this figure. Their mean PVR was higher than 1,200 dynes · s/cm5; *p < 0.05.

Lung tissue wet-to-dry-weight ratio analysis demonstrated significantly higher water content in the control allografted lungs than in the contralateral native and treated allografted lungs (Figure 2). No significant difference in wet-to-dry-weight ratio was observed between contralateral native lung tissue (4.6 ± 0.21) and treated transplanted lungs (5.4 ± 0.48).

View larger version (36K): [in this window] [in a new window]   Figure 2.   Wet-to-dry lung weight ratio was significantly higher in control group than in treatment group or contralateral native lung (normal explanted lung); *p < 0.001, p < 0.004.

Histology and Immunohistochemistry

Histologic sections from the control group showed signs of severe pulmonary edema, hemorrhage, alveolar septal thickening, and increased number of inflammatory cells. In comparison, lung sections from the treatment group showed normal alveolar architecture with minimal inflammatory infiltration and only foci of pulmonary edema (Figure 3). Immunostaining for ET-1 was seen in the airway epithelium and in smooth-muscle cells, and in the vascular endothelium throughout the lung. The intensity of ET-1 immunoreactivity appeared to be more prominent over lung sections from the treatment than from the control group (Figure 3).

View larger version (84K): [in this window] [in a new window]   Figure 3.   Histologic and immunohistochemical sections through lungs of animals in the control and treatment groups. (A to D) H&E-stained sections from control (A and B) and treatment (C and D) groups. Note the presence of pulmonary edema, hemorrhage, and increased inflammatory-cell infiltration in the control group as compared with the normal lung architecture in the treatment group. (E to J ) Immunohistochemical localization of ET-1 in control (E and F ) and treatment (G to J ) groups. Note the presence of ET-1 immunostaining in the airway epithelium (E and G), smooth-muscle cells (G), and vascular endothelium (curved arrow) of a pulmonary artery (H). (I ) Presence of little ET-1 immunostaining in a parenchymal vein. ( J ) High-power magnification of ET-1 immunostaining in endothelium of pulmonary artery (arrow). (F ) Negative control section, showing absence of ET-1 immunostaining. Magnifications: A to G: ×130; H and I : ×250; J: ×400.

Arterial and Venous Plasma ET-1 Levels

ET-1 levels following administration of SB209670 were elevated in peripheral arterial and venous plasma samples (Table 2). Pretreatment of recipients with the antagonist resulted in a significant increase in ET-1 in baseline arterial (15.1 ± 2.1 versus 1.0 ± 0.2, p = 0.0002) and venous (19.2 ± 2.2 versus 1.6 ± 0.3, p < 0.0001) plasma values as compared with those of control animals. Arterial plasma ET-1 levels remained significantly higher in antagonist-treated animals at 1 h (22.6 ± 2.5 versus 1.9 ± 0.6, p < 0.0001) and 3 h (29.7 ± 2.6 versus 1.2 ± 0.3, p < 0.0001) after transplantation. A similar trend was also observed in venous samples (Table 2). Group comparison of arterial versus venous plasma ET-1 concentrations did not reveal any significant differences in either control or antagonist-treated animals.

	DISCUSSION

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Ischemia-reperfusion injury is often associated with increased PVR and pulmonary edema (4). Although the mechanisms involved are multifactorial, one important mediator is likely to be the potent vasoconstrictor peptide ET-1. The latter is produced by numerous cells in the lung, and is known to cause increases in PVR and vascular permeability (6). Furthermore, we have previously demonstrated increased levels of ET-1 shortly after allotransplantation, which were associated with increased PVR in both humans and animal lung-transplant recipients (9, 10). In the present study we showed that administration of the ET-receptor blocker SB209670 improves lung allograft function and survival following prolonged ischemia. We observed improvement in PVR, lung edema, and oxygen tension following SB209670 administration. These findings suggest an important role for ET-1 in ameliorating lung injury following a long period of ischemia-reperfusion, and further suggest that ET-receptor antagonists may constitute important therapeutic tools for the prevention of early graft dysfunction.

The lung is a major site for ET-1 synthesis, production, processing, and clearance (6). We were the first to demonstrate localization of ET-1 in the developing human lung (23). Subsequently, several laboratories reported the expression of this peptide in normal and diseased lungs of human and experimental animals (6). ET-1 is produced in pulmonary epithelial cells, smooth-muscle cells, endothelial cells, macrophages, fibroblasts, and neutrophils. We demonstrated increased expression of ET-1 in lungs of patients with pulmonary hypertension, and showed a significant correlation between this increased ET-1 expression an of increased PVR in patients with plexogenic pulmonary arteriopathy (7). We also demonstrated increased production and release of ET-1 in BALF and blood plasma in lung-transplant recipients immediately (within 4 to 6 h) after lung allotransplantation (9, 10). In accord with our previous findings, we observed an increase in plasma levels of ET-1 in the control group in the present study. Both venous and arterial ET-1 levels were increased. In the treatment group, greater levels of ET-1 were detected in the venous and arterial blood plasma following administration of SB209670. This is consistent with previous reports of effects of the same and other ET receptor antagonists, which may reflect both increased production and impaired clearance of ET-1 due to blockage of the ET receptors (24, 25). Indeed, lungs and kidneys are known to be the major sites of ET-1 clearance (6).

Both ET receptors, ETA and ETB, are expressed in the lung. The site and function of each receptor varies among species (6). In general, ETA is expressed mainly on airway and vascular smooth-muscle cells, and is believed to mediate vasoconstriction. ETB, on the other hand, is expressed on the vascular endothelium and is believed to mediate vasorelaxation through the release of nitric oxide and prostaglandin. However recent reports indicated that ETB may also have some vasoconstrictive properties (26). We therefore used a well-characterized ETA/ETB-receptor antagonist that has previously been shown to effectively block ET receptors in several systems. Indeed, administration of SB209670 has been shown to be beneficial in attenuating acute-hypoxia-induced pulmonary hypertension (21) and angioplasty-induced neointima formation (20), and in preventing cyclosporine-induced renal vasoconstriction (19) and radiocontrast-induced nephrotoxicity (18). Furthermore, ET-receptor antagonists have been shown to reduce ischemia-reperfusion injury in several organs (14). Together, these findings suggest an imminent use of ET-receptor antagonist in clinical transplantation.

Injection of ET-1 into rat lungs resulted in a significant increase in PVR without affecting pulmonary arterial pressure or CO (27). In the present study, administering SB209670 reduced PVR without affecting Ppa, SVR, or blood pressure. Blockage of ET receptors may directly cause vasorelaxation of pulmonary vessels by blocking sites of ET-1 binding on vascular smooth-muscle cells, or may indirectly cause vasorelaxation by enhancing the release of other vasomediators such as prostaglandins and/or nitric oxide (NO). Previous studies have shown that inhibition of ET-1 enhances the release of these vasomediators. Similarly, inhibition of NO or prostaglandin release enhances ET-1 expression (6). It is unlikely that the level of ET-1 increase seen in the present study can by itself induce such a dramatic increase in PVR. It is noteworthy that reperfusion induces a number of other vasoconstrictor substances, including thromboxane. Given this, ET-1 may act synergistically with other vasoconstrictors to increase PVR in transplanted lungs. This hypothesis is supported by the fact that blockage of ET receptors in the present study did not completely normalize PVR in lung-allotransplant recipients. Additionally, the mechanism of increased production of ET-1 in this model may be related to alteration in blood flow and to hypoxia, both of which are known to induce release and expression of this peptide by the vascular endothelium (6).

Besides reducing PVR, treatment with SB209670 in a blinded manner was associated with 100% survival, as compared with 40% survival in the untreated group at 6 h after transplantation. Interestingly, the wet-to-dry-weight ratio of the treatment group was similar to that of contralateral native lungs, suggesting an important role for ET-1 in mediating pulmonary edema associated with ischemia-reperfusion injury. This is further supported by the findings that activation of the ETA receptor increases microvascular permeability, and that infusion of ET-1 augments albumin escape into the lungs and raises the pulmonary filtration rate, and by the recent report that blockage of ETA receptors improves gas exchange and pulmonary edema after 60 min of warm ischemia and 90 min of reperfusion (6, 14, 28, 29). Interestingly, our data are consistent with the recent findings of Khimenko and colleagues (14), and with the latter suggest that ETA is the ET-1 receptor responsible for the increased vascular permeability associated with ischemia-reperfusion, without causing endothelial-cell damage. In a recent report, Okada and colleagues (13) observed no increase in ET-1 release following warm ischemia; however, there was a significant increase following reperfusion. This may explain the limited increase in ET-1 release in our baseline measurement (T = 0). The mechanism of improved survival in the treatment group may be directly related to improvement in gas exchange and pulmonary edema. Indeed, the single most important cause of death in early graft dysfunction is the development of severe pulmonary edema.

Besides causing endothelial damage, increased vascular permeability, and increased PVR, reperfusion injury is associated with sequestration of inflammatory cells (4). We observed increase numbers of parenchymal and intravascular inflammatory cells in our control group. In comparison, the presence of inflammatory cells was far less evident in the treatment group. Inflammatory cells contribute to ischemia- reperfusion-associated lung injury through the production of oxygen free radicals, endothelial injury, and increased vascular permeability. Indeed, ET-1 has been shown to induce thromboxane formation, superoxide generation, and leukocyte aggregation (30, 31). Therefore, blockage of ET receptors and the subsequent reduction in inflammatory-cell sequestration may protect the transplanted lung from oxidant injury. Our findings are supported by the recent report that blockage of ET receptors inhibits the inflammatory response in rat lungs following instillation of Sephadex (32).

In summary, we provide strong evidence for a pivotal role for the potent vasoconstrictor peptide ET-1 in early lung graft dysfunction following prolonged ischemia. Our data show that blockage of ET receptors with the mixed ETA/ETB-receptor blocker SB209670 improves survival, pulmonary graft function, and hemodynamics. These findings are of significant importance in the management of early graft dysfunction following prolonged lung-allograft ischemia.