PERSPECTIVE
NO: More Than Just a Vasodilator in Lung Transplantation

Mallik R. Karamsetty and James R. Klinger

Division of Pulmonary and Critical Care Medicine, Brown University School of Medicine, Rhode Island Hospital, Providence; and Veterans Administration Medical Center, Providence, Rhode Island

Since the discovery of nitric oxide (NO) in biologic systems a scant 15 years ago, an unprecedented number of studies have explored the role of this molecule in the regulation of the pulmonary circulation. Although numerous models have been employed to study the role of NO in the lung, studies examining the effect of NO on the donor lung provide a unique opportunity to better understand the importance of this molecule in maintaining pulmonary vascular homeostasis. It is hard to imagine a worse injury for the lung to endure than removal from the donor, storage on ice for many hours, and eventual transplantation in the recipient. The donor lung usually receives the majority of the pulmonary blood flow and ventilation immediately after transplantation. This is true even with single lung transplantation, because the native lung that remains is often diseased. The result can be severe ischemia-reperfusion (I-R) injury that is often made worse by the high fraction of inspired oxygen (FiO₂) that is needed to oxygenate the patient. Under these circumstances, depletion of any factors important in normal homeostasis of the pulmonary circulation can greatly affect the overall survival and function of the donor lung. Previous studies have demonstrated that NO and cGMP levels fall rapidly in the donor lung immediately after harvest (1). Administration of NO or NO donors at the time of lung harvest can greatly improve function and survival of the lung graft (1). Recent studies exploring the mechanism of this prophylactic effect, such as the one by Minamoto and coworkers in this issue of AJRCMB, are beginning to illuminate the important role that NO has in maintaining normal function in the injured lung.

Cellular Mechanisms Underlying Rejection of Lung Graft

Although the mechanisms responsible for early dysfunction and rejection of lung allografts are not yet fully understood, there are several events that occur during lung transplantation that are likely to be responsible. These include an increase in pulmonary vascular resistance, increased sequestration of polymorphonucelar leukocytes (PMNs), platelet activation, enhanced production of reactive oxygen species (ROS), and increased release of inflammatory cytokines. This is accompanied by increased expression of endothelin-1 (ET-1) (5), a marked decrease in endothelial derived NO production, and lower intracellular cGMP levels (1). At the same time, there is increased expression of inducible nitric oxide synthase (iNOS) and apoptosis of pulmonary vascular endothelial and epithelial cells (6). The importance of each of these abnormalities and their relationship with each other is not completely known.

Reactive Oxygen Species

Reperfusion and ventilation of the previously ischemic donor graft with oxygenated blood and air leads to production of ROS such as superoxide anion, hydrogen peroxide, and the hydroxyl radical. Activated PMNs, alveolar macrophages, and increased iron-generated oxidative stress contribute to increased ROS production (7). Production of ROS is further increased by the high FiO₂ that is often needed to maintain oxygenation in the immediate posttransplant period. Donor lung grafts also have a significant depletion of glutathione, an endogenous scavenger of ROS. Thus, their ability to withstand repetitive ROS-mediated injury during episodes of lung rejection and infection is decreased (11). In addition to direct tissue injury, ROS cause pulmonary vasoconstriction and inactivate NO, thus contributing to endothelial dysfunction in pulmonary arteries (12). Administration of ROS scavengers, such as superoxide dismutase, or inhibitors of xanthine oxidase, a ROS producing enzyme, restore endothelial-NO mediated pulmonary vasodilation in pulmonary arteries isolated from left lung autotransplants (13). Similarly, pretreatment with ROS scavengers or antioxidants has been shown to enhance lung preservation and graft function (8, 15).

Inflammatory Mediators

Sequestration and activation of macrophages, lymphocytes, and neutrophils is increased in allografts during acute rejection (12, 16). These inflammatory cells produce inflammatory cytokines such as interleukin-6 and tumor necrosis factor  (TNF-) (19, 20), which in turn increase the expression of iNOS in the ischemic setting (21). In contrast to the beneficial effects of constitutive eNOS expression in the pulmonary vascular endothelium, activation of iNOS expression in PMNs and alveolar macrophages is associated with increased cell killing and contributes to inflammation during acute graft rejection (22). Increased expression of iNOS has been shown to correlate directly with serum nitrite/nitrate levels in transplanted lung and the concentration of NO in exhaled air has been shown to correlate with the severity of acute lung rejection (23). Treatment with the iNOS inhibitor, aminoguanidine, reduces serum nitrite/nitrate levels, improves gas exchange, and ameliorates acute rejection of the donor graft (22, 24, 25). In addition to increasing expression of iNOS, sequestration of PMNs in the lung leads to generation of thromboxane and contributes to pulmonary vasoconstriction (26).

Apoptosis

Apoptosis of pneumocytes is an early event after lung reperfusion that increases in a time-dependent manner and likely contributes to graft dysfunction (27). The percentage of necrotic cells has been shown to correlate inversely with post-transplant graft function (27). Local ischemia may trigger apoptosis in the lung allograft. In one study, the number of apoptotic cells increased with the length of cold preservation time (28).

Endothelin-1

ET-1 is a potent vasoconstrictor peptide with mitogenic properties that is produced by vascular endothelial cells. Its expression is often increased in response to hypoxia and acute inflammation. Expression of ET-1 and endothelin converting enzyme are increased in donor lungs during acute lung rejection and ET-1 expression returns to control levels at later stages of rejection (29). Protein levels of ET-1 in the plasma and bronchoalveolar lavage fluid (BALF) of the recipient increase soon after transplantation of the lung allograft and decline to control levels one week after transplantation (30). The histologic grade of acute graft rejection has been shown to correlate closely with ET-1 levels on allograft biopsy specimens (31). In addition, the pulmonary vascular proliferative responses to ET-1 and vasoconstrictor responses to ET_B-receptor antagonists are enhanced in donor lungs that develop acute rejection (32). Thus, it appears that ET-1 contributes to acute and chronic pathologic changes seen in graft rejection. Inhibiting the synthesis and antagonizing the effects of ET-1 may lead to improved graft function and survival. ET-1 receptor antagonists given to donors before lung harvest and during reperfusion have been found to improve pulmonary vascular resistance, arterial oxygenation and graft function (33). In addition, ET-1 inhibitors also appear to improve graft function by suppressing iNOS expression and apoptosis (33). Stammberger and colleagues (36) found that combined donor and recipient treatment with an ET-1 antagonist and a platelet activating factor (PAF) antagonist results in superior posttransplant graft function and survival compared with either agent alone, suggesting a synergistic role of ET-1 and PAF in the mediation of reperfusion injury.

Nitric Oxide

It is now well-appreciated that the constitutive expression of endogenous NO by the pulmonary vascular endothelium plays an important role in maintaining homeostasis of many aspects of the pulmonary circulation, including modulation of pulmonary vascular tone, maintenance of an intact pulmonary capillary membrane, and inhibition of platelet aggregation and leukocyte adhesion. Most of these effects are mediated by intracellular cGMP and cGMP-dependent protein kinase (PKG) (1). Previous studies have shown that endogenous NO and cGMP levels fall precipitously in the donor lung immediately after reperfusion (1). Furthermore, expression of endothelial nitric oxide synthase (eNOS) is decreased in lung allografts during acute rejection (23). Consequently, endothelium-dependent NO mediated pulmonary vasodilation in response to various agonists, including acetylcholine and calcium ionophore, is impaired in pulmonary arteries isolated from dysfunctional lung grafts (13). The lack of normal NO production by the pulmonary vascular endothelium is at least partly responsible for the increase in pulmonary vascular resistance in the lung graft. Many studies have demonstrated that administration of NO to the donor lung by various strategies such as adenovirus-mediated eNOS gene transfer (37), early perfusion of the lung graft with NO donors, inhaled NO or essential cofactors for eNOS activity, such as tetrahydrobiopterin, ameliorate I-R injury and improve graft function (1, 4, 16, 38).

Originally, much of the beneficial effect of NO administration was attributed to its acute vasodilator properties and its ability to improve gas exchange by reducing intrapulmonary shunt. However, subsequent studies found that administration of NO to the donor lung is effective even when given well in advance of transplantation. In 1997, Fujino and coworkers (37) reported the beneficial effects of iNO given at the time of graft harvest. Supplementing the preservation solution with cGMP analog has also been shown to improve graft function and improve recipient survival (1). These observations suggest that acute improvements in pulmonary hemodynamics and gas exchange at the time of transplantation are not the only mechanisms by which NO improves graft function. Indeed, the uses of other vasodilators such as hydralazine have not been found to be effective in improving lung graft function despite achieving similar degrees of pulmonary vasodilation (45). Therefore, it is likely that other biologic properties of NO are involved.

Recent studies have shown that NO inhibits adhesion of PMNs to pulmonary artery endothelial cells before and after reperfusion, reduces the sequestration of PMNs into the transplanted lung, and attenuates subsequent tissue injury (46). Early administration of NO or NO donors attenuates the oxidative stress of I-R injury by inhibiting both graft neutrophil and platelet sequestration and activation (3, 45, 46). Again, timing of treatment with NO appears to be critical, because some studies have found that treatment with nitroglycerin (NTG) during flush/preservation, but not during reperfusion, inhibits neutrophil accumulation in the transplanted lung (47).

The other mechanism by which NO may improve graft function is by inhibiting the synthesis of ET-1. NO and cGMP are inhibitors of ET-1 production and counteract its vascular effects (48, 49). Administration of NO donors such as molsidomine (50) reduce the pulmonary expression of ET-1 induced by hypoxia. Overexpression of eNOS reduces pulmonary vascular resistance and pulmonary pressor responses to ET-1 (51). However, the ability of NO to reduce ET-1 expression in lung grafts has not previously been demonstrated. In this issue, Minamoto and coworkers (52), found marked improvement in graft function when the donor lung was flushed with preservation fluid containing the NO donor NTG at the time of harvest in an isogenic rat model of lung transplantation. No improvement was found when the same dose of NTG was given immediately after transplantation into the recipient. Early administration of NTG was associated with a significant reduction in ET-1 expression. Administration of an ET-1 receptor blocker also improved graft function, but not to the same degree as NTG. Although these findings suggest that other mechanisms are involved, they offer strong support for the hypothesis that NO improves graft function by inhibiting ET-1 expression and help explain why early administration of NO to lung grafts may be essential for improved function.

The mechanisms responsible for inducing injury in lung allografts and the role of NO in mitigating these events are summarized in Figure 1. Harvest and cold storage results in ischemia of the donor lung. Reperfusion at the time of transplant results in decreased eNOS expression, depletion of NO and cGMP, and a myriad of injuries that lead to increased pulmonary vascular resistance, increased levels of ROS, cytokines, and ET-1 expression. These, in turn, contribute to pulmonary hypertension, platelet aggregation, and pulmonary recruitment and sequestration of PMNs, which contribute to lung injury and apoptosis. Administration of exogenous NO replaces depleted levels of endogenous NO in the donor lung, which not only improves vascular resistance and gas exchange, but also acts to break the cycle of increased production of the other mediators of lung injury.

View larger version (15K): [in this window] [in a new window]   Figure 1.   Mechanisms of injury to the donor graft in lung transplantation and the inhibitory effect of exogenous NO on these events. See text for explanation.

	Future Implications

Lung transplantation is the ultimate treatment for many patients with progressive lung disease. Unfortunately, much of the success that has been obtained with transplantation of other solid organs has yet to be realized with lung transplantation. One-year survival rates for lung grafts and their recipients remain lower than those for transplantation of any other solid organ. Reperfusion injury in the immediate post-transplantation period occurs in approximately 1 in 5 patients (53, 54). At the same time, the high prevalence of acute lung injury from aspiration, neurogenic pulmonary edema, and ARDS in donors of vital organs greatly limit the number of lungs that are capable of being used. It has been estimated that less than 20% of donors have lungs that are suitable for transplantation. In 1998, 485 patients died on the lung transplant waiting list because of inadequate supply of donor lungs (55). One approach that would greatly increase the number of suitable donor lungs is the use of lungs obtained from non-heart-beating donors (NHBD). The use of NO donors in the preservation fluid of lungs harvested from NHBD has greatly improved the function of these grafts in animal studies and may allow the use of NHBD for human transplantation as well.

Future studies exploring the mechanisms by which NO ameliorates injury to donor grafts may facilitate the development of better methods of preserving lung allograft and improving graft function after transplantation. At the same time, these studies are likely to shed new light on the complex interactions between NO and other vasoactive, mitogenic, and inflammatory substances such as ROS, cytokines, and ET-1 that mediate I-R injury in the lung. As important as NO appears to be in maintaining normal function in native lung, its role may prove to be even greater in preventing injury and dysfunction in the transplanted lung.

	Footnotes

Address correspondence to: James R. Klinger, M.D., Division of Pulmonary, Sleep, and Critical Care Medicine, Rhode Island Hospital, 593 Eddy Street, Providence, RI 02903. E-mail: James_Klinger{at}brown.edu

(Received in original form November 7, 2001).

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