Introduction

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As the lungs are especially susceptible to ischemic injury (1), maintaining vascular homeostasis in the pulmonary graft is a critical determinant of the ultimate success of lung transplantation (2). Nitric oxide (NO) released from endothelial cells is one of the cardinal factors needed to maintain vascular homeostasis, eliciting vascular smooth muscle relaxation (6), preventing neutrophil adherence to the endothelium (7), maintaining endothelial barrier properties (8), and inhibiting platelet aggregation (4) based on guanylate cyclase activation and accumulation of intracellular cGMP (9). We previously reported that endogenous pulmonary NO levels plummet upon the onset of reperfusion, and supplementing preservation solutions with cGMP analogs or nitroglycerin (NTG) at the time of graft harvest enhances NO-related mechanisms of vascular protection immediately following lung transplantation (1). Although other stimulators of the NO pathway, such as sodium nitroprusside (10), added to preservation solutions similarly improve post-transplant lung function, it is not known whether the NO-related beneficial effects accrue if NTG delivery into the graft is restricted to the time immediately before reperfusion.

One potential deleterious effector mechanism which may be activated by the ischemic period is endothelin-1 (ET-1), one of the most potent vasoconstrictor substances known (11). ET-1, synthesized and released by multiple cell types, such as macrophages (12), bronchial epithelial cells (13), bronchial (14) and vascular (15) smooth muscle cells, and endothelial cells (16) in response to several stimuli including hypoxia (17), increased pressure (18), and shear stress (19), may profoundly affect the vasomotor response to exogenous NO. Pulmonary vascular smooth muscle is one of the potential sources of ET-1 production (20). In addition to promoting vasoconstriction, ET-1 also increases vascular permeability (21), promotes coagulation (22), and stimulates neutrophil accumulation (23). A variety of pulmonary pathologic conditions, including pulmonary hypertension (24), pulmonary fibrosis (25), asthma (26), and acute (27) or chronic (28) lung rejection, are partially attributed to ET-1.

NO and ET-1 levels are reciprocally regulated in in vitro studies. Accumulating evidence demonstrates that NO inhibits ET-1 production at the transcriptional level (29), and their balance interacts to modulate vascular tone (30). Nitric oxides' vasodilatory effects may be magnified by suppressing the synthesis of ET-1 (31), and acting as a regulator of ET-1-related vasoconstriction in the setting of lung transplantation. Although blockage of lung graft ET-receptor with antagonist (32, 33) elicits beneficial effects on the graft function associated with ET-mediated ischemia/reperfusion injury, it may be expected to offset the relative balance. If preproendothelin-1 (ppET-1) mRNA in the lung graft is upregulated even during the 4°C cold ischemic period, exogenous NO supplementation would be expected to lead to decreased ET-1 synthesis and possibly maintain vasodilation after lung transplantation. This study was designed (i) to determine whether NTG given immediately before reperfusion would improve post-transplant pulmonary function, as NTG given during preservation does; (ii) to investigate whether NTG supplementation given during preservation confers beneficial effects on lung graft function by suppressing ET-1 expression in the graft; and (iii) to identify the ET-1 dependence of the vasomotor response during preservation and the role of ET-1 in graft function.

	Materials and Methods

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Lung Preservation and Transplantation

Inbred male Lewis rats (250-300 g) were used for all experiments according to a protocol approved by the Institutional Animal Care and Use Committee at Columbia University, in accordance with AAALAC guidelines. Donor rats were given 500 U heparin intravenously, and the lungs were flushed via the IVC with a 30-ml volume of 4°C preservation solution (modified[4] Euro-Collins solution; Baxter Healthcare, Deerfield, IL) at a constant pressure of 20 mm Hg. The time required to flush the lung was recorded as the graft pulmonary vascular resistance at harvest (Harvest PVR). Both of the lungs were harvested and preserved with 10 mm Hg insufflation pressure. The three lobes of the right lung were used for experiments as preserved lung samples (0, 3, and 6 h). After 6 h at 4°C preservation and immediately before transplantation, the left lung was flushed (ex vivo) from the left pulmonary artery (PA) with a 1-ml volume of 25°C normal saline in 3 min by using an infusion pump (KD Scientific Inc., New Hope, PA). As 0.12 ± 0.06 ml volume of infused flush solution remains within the lung graft after flushing (5), the volume of ex vivo flush solution that was used was 1 ml of saline.

Gender/strain/size-matched rats were anesthetized, intubated, and ventilated with 100% O₂. Orthotopic left lung transplantation (34) was performed using a rapid cuff technique. A snare was then passed around the right PA, and 2F sized Millar catheters (Millar Instruments, Houston, TX) were introduced into the main PA and the left atrium (LA). A flow probe (Transonic, Ithaca, NY) was placed around the main PA.

Experimental Groups

Two groups were designated according to the time at which NTG was given, another group was not given NTG, and a fourth group was given an ET-receptor antagonist (n = 12/group). For all solutions containing NTG, the concentration of NTG was 0.1 (mg/ ml), based on its apparent beneficial effects at this level in the same model of lung preservation (4). The Early NTG group was given NTG in the flushing and preservation solution, and the graft was flushed ex vivo with normal saline (25°C, 1 ml/3 min). The Late NTG group was flushed and preserved with EC solution, and then flushed ex vivo with saline containing NTG. The No NTG group was not given NTG in either the preservation or ex vivo flush solution. PD156252 is a nonselective ET-1 receptor antagonist that has a highly affinity for both ET-receptor subtypes (35). To characterize the role of ET-1 in the setting of lung transplantation, an additional group (n = 12) was given PD156252 (5 × 10⁶ M) in the flush/preservation solution, and then flushed ex vivo with saline.

Measurement of Lung Graft Function

The graft PA pressure during the ex vivo flush (ex vivo flush PAP) was recorded (MacLab-Mk III, AD Instruments, Grand Junction, CO). After lung transplantation, recipient hemodynamic parameters (including LA and PA pressures [mm Hg] and PA flow [ml/min]) were measured, and pulmonary vascular resistance (Recipient PVR) was calculated as follows: (mean PA pressure - mean LA pressure)/ PA flow. Baseline measurements were taken 15 min after reperfusion, and then the native right PA was ligated, and serial measurements were taken every 5 min until the time of euthanasia at 30 min or until recipient death, if it preceded the 30-min time point. Comparison of recipient PVRs were performed using PVRs calculated from the last hemodynamic measurements obtained in each group. Arterial oxygen tension (pO₂, mm Hg) was measured at the last time at which the recipient was alive. Recipient death was identified as the point at which the PA flow was under 1 ml/min.

Measurement of Graft Neutrophil Infiltration

To quantify neutrophil accumulation at identical time points between groups, myeloperoxidase (MPO) assays were performed on the transplanted grafts. Because animals in the control group exhibited such poor survival, we elected to obtain lung tissue samples for MPO assays using a shorter preservation period (4 h), which enabled us to reduce the number of experimental animals needed to obtain reperfusion time-matched samples. All recipient animals in each group (n = 5) survived throughout the observation period with this preservation duration. One hour following ligation of the native right PA, the transplanted lungs were removed and snap-frozen until the time of myeloperoxidase (MPO) assay, as previously described (2). Protein content was determined for each sample, and the results were expressed as Abs 460 nm/min/mg protein.

Northern Blotting

To make RNA probes for Northern blotting and in situ hybridization, reverse transcription-polymerase chain reaction (RT-PCR) was performed. Primers for rat preproET-1 were directed at 63-90 and 985-1011 of the cDNA (GenBank M64711), producing a 949-bp product. The forward primer sequence was 5'-CAGAGGCGAT CAGAGCAACCAGACACCA-3'. The reverse primer sequence was 5'-CTACCAGCGGATGCAAACGAAGACAGG-3'. After reverse transcription, PCR was performed (35 cycles at 94°C annealing temperature). The PCR product mixture was extracted and inserted to pGEM-T Easy Vector using the T4 ligation method (Promega, Madison, WI). The RNA expression plasmid was confirmed by enzyme cut and was also sequenced.

In dedicated experiments, fresh/flushed, 3 or 6 h preserved, and transplanted lung grafts were snap-frozen or embedded and stored at 80°C until the time of mRNA extraction or histologic investigation. To detect ET-1 transcripts, 2.0 µg/lane amounts of purified poly(A) mRNA were hybridized with the rat ET-1 (949 bp) cDNA probe labeled with [-³²P]dCTP. Normalized absorption values were obtained by densitometry scanning (Molecular Imager System; Bio-Rad, Hercules, CA) of cDNAs, including -actin bands. Expression was normalized with the baseline expression (fresh/flushed lung) for each experiment.

In Situ Hybridization

The RNA expression plasmid was linearized with SpeI and NcoI enzymes to allow in vitro runoff synthesis of both sense and antisense-oriented RNA probes. Both sense and antisense probes were labeled by transcription with a digoxigenin RNA labeling Kit (Roche, Indianapolis, IN). In situ hybridization was performed as previously described (36).

Western Blotting

Loaded and transferred samples of tissue protein (100 µg/lane) were reacted with a mouse monoclonal anti-human ET-1 (QED Bioscience, San Diego, CA), and a horseradish peroxidase-conjugated goat anti-mouse whole IgG (Sigma, St. Louis, MO). Detection of bands was performed using the enhanced chemiluminescent Western blotting system (Amersham International, Buckinghamshire, England).

Immunohistochemistry

For detecting ET-1, lung sections were incubated with the same primary antibody as in Western blot analysis. Immunodetection was performed using a biotinylated goat anti-mouse IgG antibody and the Vectastain ABC Kit (Vector Laboratories, Inc., Burlingame, CA), and developed in AEC buffer (0.4% AEC in dimethylformamide, 0.1 M sodium acetate buffer, 0.03% H₂O₂) with counter nuclei staining.

Statistics

Data were expressed as mean ± SEM. One way ANOVA and Bonferroni post-hoc test were used to compare different conditions among the four groups. The product limit (Kaplan-Meier) estimate of the cumulative survival was assessed with a Log-rank test to evaluate significance differences. Differences were considered significant at the level of P < 0.05.

	Results

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Harvest PVR

The times required to flush in preservation solution into donor both lungs were measured by delivering identical volumes of the preservation solution at identical flushing pressure as an index of Harvest PVR. The PVR at harvest in the Early NTG group (0.38 ± 0.01 mm Hg/ml/min) was significantly lower than that in the other groups (0.49 ± 0.01 for the Late NTG group; 0.48 ± 0.02 for the No NTG group; 0.44 ± 0.01 for the PD156252 group, P < 0.05 versus the Early NTG group; Figure 1A), which demonstrates NTG given in the flush solutions functions as a vasodilator during harvest.

View larger version (27K): [in this window] [in a new window]   Figure 1.   All lung transplants were performed after ex vivo flush following 6-h 4°C preservation. After measurement of recipient hemodynamics baseline, the native right PA was ligated at time 0, and serial measurements were performed at the indicated points. Graph showing effect of NTG and ET-receptor antagonist on donor PVR at harvest (A), on graft PAP (B), and mean PAP (inset) at the ex vivo flush with 1 ml/3 min of normal saline (No NTG, Early NTG, and PD156252 groups), or saline with supplemental NTG (Late NTG group) immediately before transplantation, and on recipient PVR during the post-transplanted period (C), and at the time of euthanasia at 30 min or recipient death following native right PA ligation (inset). n = 12 for each group; *P < 0.05 versus Early NTG, #P < 0.05 versus PD156252.

Ex Vivo Graft PAP

To determine whether NTG would enhance pulmonary vascular preservation, an ex vivo graft flush with normal saline (25°C, 1 ml/3 min) was performed from the graft PA at the end of preservation period. Representative ex vivo graft PAP recordings during the ex vivo flush (Figure 1B) indicate that both of the No NTG lung and the Late NTG lung have an initial rapid elevation reached around 23 mm Hg, followed by gradual decrease. The Early NTG lung, however, resulted in a lower pressure. PD156252 treatment reduced the ex vivo graft PAP to an intermediary level. The ex vivo graft mean PAP (Figure 1B, inset) of the Early NTG group was less than that of the other groups (5.5 ± 0.6 mm Hg, P < 0.05 versus other groups). Mean PAP for the Late NTG group (11.5 ± 0.9) was similar to the No NTG group (12.0 ± 0.7). Compared with the Late NTG and No NTG groups, the PD156252 group resulted in reduction in the ex vivo graft mean PAP (8.7 ± 0.3, P < 0.05 for both comparison). These data indicate that NTG given to the graft after preservation via an ex vivo flush does not lead to vasorelaxation, and that endogenous ET-1 induced during preservation may contribute to the development of vasoconstriction.

Recipient PVR

Recipient PVRs (Figure 1C) in the Late NTG and No NTG groups were markedly elevated immediately after right PA ligation. The PVRs in the PD156252 group were also elevated 10 min after right PA ligation, whereas those of the Early NTG group were lower and more stable throughout the observation period. The recipient mean PVR (Figure 1C, inset) in the Early NTG group (1.4 ± 0.1 mm Hg/ml/min) after transplantation was significantly less than that of either the Late NTG group (4.2 ± 0.8; P < 0.05) or the No NTG group (5.4 ± 1.2; P < 0.05).

Time Course of ppET-1 mRNA Expression in Preserved and Transplanted Lungs

PreproET-1 (ppET-1) is a primary translation product of ET-1 (37). ET-1 is generated by a biosynthetic pathway from the conversion of two precursors, preproET-1 and pro or bigET-1. ppET-1 mRNA expression was therefore examined, using densitometric values normalized to -actin mRNA levels. These values were then compared with ppET-1 mRNA levels in the fresh/flushed lung (baseline, Figure 2A). Significant ppET-1 mRNA upregulation was observed at the end of the cold ischemic preservation period, and increased further shortly after reperfusion in the grafts preserved with preservation solution alone. Compared with the expression of ppET-1 mRNA in the fresh/ flushed lungs, No NTG lungs showed about a 2-fold increase at the end of preservation, and about a 3.5-fold increase following transplantation. In contrast, Early NTG suppressed the induction of ppET-1 mRNA during preservation and significantly attenuated the induction of ppET-1 mRNA following transplantation. Late NTG, however, did not suppress induction of ppET-1 mRNA during preservation and following transplantation; neither did No NTG. These results indicate that cold ischemia can evoke a significant increase in the levels of ppET-1 mRNA. Although NTG given to the graft at the onset of preservation suppressed ppET-1 mRNA induction, NTG given to the graft immediately before transplantation had no effect on ppET-1 mRNA expression.

View larger version (34K): [in this window] [in a new window]   Figure 2.   Densitometric analysis of lung graft ppET-1 mRNA expression (A) and ET-1 peptide (B) following preservation and reperfusion. ppET-1 mRNA was normalized relative to -actin mRNA, and ET-1 peptide levels were compared with values in the fresh/flushed lung (0; baseline). These results implicated preservation as the primary stimulus and reperfusion as the enhanced stimulus for induction of the ppET-1 gene. n = 10 for each group; *P < 0.05 versus baseline, #P < 0.05 versus reperfused lung in Early NTG (0, baseline; 3, 3 h preservation; 6, 6 h preservation; R, 15 min after reperfusion).

ET-1 Peptide Expression in Lung Grafts during Preservation and after Transplantation

To ascertain the effects of NTG given either early or late during preservation on ET-1 peptide expression in the lung grafts, Western blotting was performed. ET-1 peptide expression in the lung grafts was compared with ET-1 peptide levels in fresh/flushed lungs (baseline), as determined by densitometry (Figure 2B). Compared with ET-1 peptide in preserved lungs at baseline, there were 1.7- and 1.9-fold increases in the Late NTG and the No NTG groups, respectively. The transplanted lungs in the Late NTG and the No NTG groups also contained 7.1 times and 6.6 times more peptide than lungs from the Early NTG group. Only early NTG supplementation significantly decreased the ET-1 peptide expression in the lung after transplantation, compared with the other groups.

Localization of ppET-1 mRNA Expression in the Transplanted Lung

To determine the localization of ppET-1 mRNA in lung grafts after lung transplantation, in situ hybridization was performed using rat antisense (Figures 3A-3C) and sense (Figure 3D) ET-1 probes. Compared with sections taken from Early NTG lungs, transplanted lung tissue from the Late NTG and the No NTG groups demonstrated increased ppET-1 mRNA, which prominently localized to vascular smooth muscle cells, identified by their characteristic histologic appearance as well as -actin immunoreactivity (Figure 3H). ppET-1 mRNA staining was not observed in serial sections probed with the sense probe.

View larger version (109K): [in this window] [in a new window]   Figure 3.   In situ hybridization for ET-1 mRNA, using rat antisense (A-C) and sense (D) ET-1 probe, and immunohistochemistry for ET-1 peptide (E-G) in the transplanted lungs shortly after reperfusion. Sections are shown from lungs in the Early NTG (A and E), Late NTG (B and F), and No NTG (C and G) groups. Corresponding to the localization of the upregulated ET-1 mRNA, transplanted lung tissues from the Late NTG and No NTG groups stained intensely in the endothelial-lining and the smooth muscle layer of pulmonary arteries, defined by immunohistochemistry for smooth muscle -actin (arrow, endothelial layer; arrowhead, smooth muscle layer; H). ET-1 mRNA staining was not observed in serial sections probed with the sense probe (D). Bar = 50 µm.

Localization of ET-1 Peptide Expression in the Transplanted Lung

ET-1 peptide localization in the lung graft was ascertained by immunohistochemistry. Corresponding to the localization of the up-regulated ET-1 mRNA, transplanted lung tissues from the Late NTG and the No NTG groups were intensely stained in the endothelial lining and the smooth muscle areas of large vessels, compared with low levels of ET-1 detection in the Early NTG group (Figures 3E-3G). To ascertain whether ET-1 expression varies with vascular location, we also histologically examined the presence of ET-1 in smaller caliber vessels. Immunohistochemical detection of ET-1 in smaller pulmonary arteries was similar to that of the large vessels, and was suppressed by provision of NTG early on during preservation (Figures 4A-4C). The induction of ET-1 following lung transplantation was increased in the endothelial lining and the smooth muscle regions of large as well as small vessels; early NTG supplementation to the lung graft markedly reduced ET-1 expression after reperfusion.

View larger version (81K): [in this window] [in a new window]   Figure 4.   Immunoreactivity for ET-1 in smaller vessels demonstrates that localization of ET-1 is similar to that of large vessels. The smaller caliber pulmonary arteries in the Late NTG (B) and No NTG (C) grafts show the presence of strong ET-1 expression in both the endothelial and smooth muscle layers; provision of NTG early on in the preservation period suppressed expression (A). Bar = 50 µm.

Quantification of Neutrophil Infiltration

To evaluate the effect of timing NTG supplementation or ET-receptor blockade on neutrophil accumulation in the transplanted lung, myeloperoxidase assays were performed (Figure 5A). The lung grafts of the Late NTG and the No NTG groups demonstrated increased recruitment of neutrophils following transplantation, whereas the grafts in the Early NTG group demonstrated reduced neutrophil accumulation. PD156252 treatment also resulted in diminished neutrophil recruitment in the transplanted lung. These findings suggest that endogenous ET in lung graft may participate in modulation of neutrophil recruitment after transplantation.

View larger version (20K): [in this window] [in a new window]   Figure 5.   Effects of NTG or PD156252 on graft neutrophil accumulation (A; n = 5 for each group), arterial oxygenation (B; n = 12), and recipient survival (C; n = 12). Sample lung grafts for the MPO assay were taken after 4 h preservation and 1 h reperfusion following ligation of the native right PA, so that all recipient animals in each group could survive throughout the observation period. Blood samples were taken at the time of euthanasia at 30 min or recipient death following native right PA ligation. *P < 0.05 versus Early NTG, #P < 0.05 versus PD156252.

Arterial Blood Gas Analysis

Arterial blood samples were taken 30 min after ligation of the native right PA or at the time of recipient death. Although pO₂ remained excellent in the Early NTG group, arterial oxygenation deteriorated in the other groups (Figure 5B).

Survival

Because NTG reduces ischemia/reperfusion injury when given to donor lungs at harvest (4), experiments were performed to establish the effect of timing of NTG supplementation on lung graft recipient survival (Figure 5C). The effect of ET-receptor blockade on survival was also assessed. Recipients in the Early NTG group exhibited 100% survival during the 30-min observation period. However, survival was significantly diminished in the No NTG group (25%). Late NTG treatment did not improve recipient survival (17%). These results suggest that the timing of NTG exposure to the lung graft is a critical determinant of recipient survival. The PD156252 group resulted in a partial improvement of survival (50%), that implies that the ET-1 induced in the lung graft exerts a significant detrimental influence on recipient survival.

	Discussion

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Among the numerous factors maintaining pulmonary vascular homeostatic properties, NO released from endothelial cells serves as an endothelium-derived relaxing factor which elicits vasorelaxation, caused by guanylate cyclase activation and synthesis of cGMP (9). Pulmonary transplantation following preservation can be thought of as a NO/cGMP deficiency state, and we have previously reported that supplementing preservation solutions with cGMP analogs (2) or NTG (3) improved lung graft function following transplantation by stimulating the NO/cGMP pathway during the preservation period. It is not known, however, when the NO/cGMP pathway must be replenished in the setting of lung transplantation to be effective. Furthermore, the mechanism(s) by which NTG added to preservation solution confers benefit, despite being promptly washed out from the pulmonary vasculature at the time of reperfusion, remains incompletely understood.

In this study, we focused on vascular tone as one of the NO/cGMP-related vascular properties associated with lung graft preservation. When NTG was given during the initial pulmonary flush at the time of lung harvest, PVR after lung transplantation was significantly decreased. On the other hand, NTG given at the end of preservation did not reduce recipient PVR. NTG given during the ex vivo flush should theoretically exert similar vasodilatory effects on the pulmonary vasculature, but there was no difference between the PVR of the control animals and those which received NTG during an ex vivo immediate graft flush. This suggests that, at least in terms of vascular resistance, the vasoconstrictor phenotype of the pulmonary vasculature is not altered after it has already become established during the preservation period by giving NTG at a delayed time point.

To understand the vascular response of the graft to an exogenous NO donor, we investigated the role of endothelin, one of the most potent vasoconstrictors known. Both NO and ET-1 play a pivotal role to control vascular tone, and are in reciprocal balance (30). The mechanistic balance of vasodilator NO and vasoconstrictor ET-1 largely determines the regulation of pulmonary vasomotor tone. Although NO and ET-1 are involved in the adaptation of vascular tone to control pulmonary blood flow (38), the relative balance during the cold ischemic period or early post-transplant period on graft PVR is not clearly defined. Our results revealed a striking increase in the expression of ppET-1 mRNA and ET-1 peptide following transplantation; even during cold preservation, ppET-1 mRNA was significantly suppressed by the presence of NTG at the onset of preservation. However, similar suppressive effects on ET-1 induction were not seen by giving NTG to the graft immediately before transplantation. Early NTG, but not Late NTG, exerts an important modulatory effect on vascular tone by suppressing induction of ET-1. During 6 h preservation, sufficient priming for injury has occurred so as to make delayed NTG administration futile. These data indicate that cold ischemia, without reperfusion, can evoke a significant increase in levels of ppET-1 mRNA and accelerate ET-1 peptide production after reperfusion, and exogenous NO given early during the preservation period enhances post-transplant pulmonary function at least in part by the prevention of ET-1 synthesis.

PD156252 is a potent nonselective inhibitor of ET-mediated responses (35), acting on both ET_A and ET_B receptor subtypes. Both of these receptors are localized on smooth muscle cells, and are linked to vasoconstriction (20). The improved graft function and survival observed when PD156252 was given at the onset of preservation indicates that ET-1 is at least partly responsible for vasoconstriction induced during preservation. Among the mechanisms responsible for graft damage elicited by preservation, ET-1 induced during preservation may aggravate graft injury. The enhanced induction of ET-1 following reperfusion undoubtedly contributed to elevation of recipient PVR, which may lead to increase in vascular permeability and hemodynamic abnormalities in post-transplant lung graft function and result in primary graft failure. The localization of ppET-1 mRNA or ET-1 peptide in the graft following reperfusion supports our hypothesis that the optimal timing of NTG administration to the lung graft should be early during the ischemic period. It also suggests that upregulated ET-1, predominantly in the smooth muscle layer of large as well as small pulmonary arteries, may contribute directly to vasoconstriction and subsequent elevation of graft PVR. ET mediated vasoconstriction may contribute to the initial vascular disturbance, which leads to irreversible vasoconstriction and eventual damage to the lung graft. Attenuation of this process by giving NTG to the graft at an early stage, such as early during preservation, can improve vasomotor function of the graft by downregulating the subsequent induction of ET-1.

The beneficial effects of NTG supplementation are not exclusively due to its actions as a vasodilator (4). Early NTG inhibited neutrophil adherence to vessels, improved oxygenation of the transplanted graft, and thereby ameliorated recipient survival. Based on the beneficial effects of NO, administration of NO or an NO donor decreases neutrophil adherence to endothelium by reducing expression of adhesion molecules on vascular endothelium (39), decreases vascular permeability by preventing endothelial capillary leakage (8), and improves oxygenation and recipient survival (4). On the other hand, Late NTG did not mitigate damage to the lung graft. Besides a loss of available endogenous NO during the cold ischemic period, induction of ET-1 may contribute in other ways to primary failure of the lung graft. PD156252 treatment attenuated graft vasoconstriction during preservation, and neutrophil accumulation in the transplanted lungs. ET-1 is not only a potent vasoconstrictor, but also stimulates neutrophil adhesion to endothelial cells (23) and increases vascular permeability (21), promoting lung edema. Increased or enhanced expression of ET-1 following preservation and reperfusion can be considered an early step in the development of delayed graft function.

In summary, these results suggest that loss of endogenous NO in the graft during preservation leads to upregulation of ET-1 expression in the graft vasculature and subsequent irreversible vasoconstriction, which could not be altered even with the administration of NTG to the lung immediately before reperfusion. In contrast, supplementing NTG at the onset of preservation maintained vasomotor function through the preservation to early post-transplant periods, suppressed induction of ET-1, and provided functional benefits to the transplanted lung, such as vasodilation, reduced neutrophil infiltration, and improved arterial oxygenation. Taken together, these improved vascular properties ultimately accrued to benefit recipient survival.