Clinical criteria used to evaluate donor lungs prior to transplantation may not be adequate as many lungs rejected for transplant appear to have normal alveolar epithelial function.1
Biological markers of lung endothelial and alveolar epithelial injury may have clinical value in determining the severity of alveolar barrier injury in potential transplant donor lungs or in patients with ALI. Prior studies have shown a relationship between elevated levels of vWF:Ag and poor clinical outcomes in ALI,12,13
and recent work has suggested that elevated plasma levels of RAGE may reflect the severity of alveolar epithelial injury in patients following lung transplantation.14
More importantly, intact alveolar barrier function has been associated with lower mortality in patients with ALI5
and in patients with posttransplant pulmonary edema.6
We recently evaluated the effect of perfusion and β-adrenergic agonist therapy on AFC in a novel preparation of ex vivo
perfused human lungs.9
In that study, RAGE levels were measured in only nine lungs, but a marked elevation in RAGE levels was observed in three lungs without measurable AFC. Therefore, the main objective of this study was to prospectively determine if levels of RAGE correlated with AFC in a larger number of donor lungs (N = 30) declined for transplantation. Our primary finding was that elevated airspace levels of RAGE were significantly correlated with impaired AFC in lungs declined for transplantation.
RAGE is a member of the immunoglobulin super-family of cell surface molecules that has been implicated as a biological marker of cellular injury in multiple pathologic conditions such as diabetes, amyloidosis, and cancer, although the role of RAGE in the pathogenesis of these conditions is uncertain.15,16
Proteolysis of full-length RAGE during tissue injury accounts for the release of the soluble 48-kd isoform into the alveolar space. In the lung, the majority of soluble RAGE is released from the epithelium. Recent studies have demonstrated that RAGE expression in the lung is primarily limited to alveolar type I epithelial cells7,17
and RAGE is not expressed at high levels in human pulmonary vascular endothelial cell lines.7
Previous clinical studies have shown that RAGE levels are higher in pulmonary edema fluid from patients with ALI than in edema fluid from patients with hydrostatic pulmonary edema.7
In addition, plasma RAGE levels in ALI/ARDS patients are significantly higher than in healthy volunteers and patients with hydrostatic pulmonary edema. Therefore, an increase in RAGE levels may reflect injury to alveolar type I epithelial cells.
In the lungs we studied, the RAGE concentrations globally differed based on the reasons for rejecting the lungs for transplantation in patients. Because of the small number of lungs in each group we failed to show which reason for rejection is statistically different from the others. We hypothesize that there is a trend for higher RAGE levels in possibly injured lungs (suspicion of pulmonary edema, or suspicion of active infection or inflammation). The RAGE levels tend to be lower in lungs with no apparent gross injury but only a potential risk due to a previous history of infection (eg, hepatitis virus or HIV) or tobacco abuse.
While elevated levels of RAGE in the airspaces were associated with impaired AFC in our study, perfusate RAGE levels were not associated with alveolar epithelial function. The lack of significant correlation between perfusate RAGE and AFC in our data may be explained by the dilution, in the perfusion circuit, of RAGE proteins principally released in the alveolar compartment, which must then cross the injured capillary-alveolar barrier to reach the vascular compartment. In a previous study with this model,9
we found elevated levels of RAGE in the perfusion solution of lungs with essentially no intact AFC (20% of the studied lungs). The lungs we used in the present series were in better condition, as only 3% of the studied lungs had no evidence of net AFC. This difference between the two studies may be explained by the fact that we were more reluctant to use lungs that had more macroscopic evidence of injury in the current study, thus reducing the magnitude of RAGE measurements in the perfusate.
We also found that longer cold ischemic time was associated with higher levels of RAGE and submaximal AFC (). Although some previous studies concluded that graft ischemic time was not an independent predictor of increased adverse outcomes18,19
except with older donors,20
other studies have shown that lung dysfunction and clinical outcomes may be influenced by cold ischemic time. For example, Fischer et al21
reported an association between cold preservation times and post-transplant lung function in rat lung transplants. In addition, Ware et al6
reported a correlation between graft ischemic time and capillary-alveolar protein permeability in patients with reperfusion edema after lung transplantation. Snell et al22
studied 106 transplant patients and reported poorer outcomes with graft ischemic times beyond 5 h. Finally, Thabut et al23
found, in a cohort of 505 patients with lung transplantation, a close relationship between graft ischemic time and both early gas exchange and long-term survival.
The lungs that we received were not used for lung transplantation for a variety of clinical reasons, thus creating a natural experiment in which ischemia time varied substantially. Based on prior studies,1,9
we knew that these lungs may be normal, mildly injured, or more severely injured. Thus we designed this study of AFC in the human lung anticipating that there would be a spectrum of normal to injured lungs and taking advantage of the various degrees of injury to test for a correlation between biomarkers and alveolar epithelial function, as reflected by the rate of AFC. The correlations demonstrated in this study between RAGE levels and both AFC and ischemia time suggest that increased ischemia time negatively impacts alveolar epithelial function.
The lack of correlation between airspace and perfusate vWF:Ag levels and AFC serves as a useful negative control for our studies of RAGE. Plasma vWF:Ag levels are an early biochemical marker of endothelial injury and predict the development of ALI in patients with nonpulmonary sepsis syndrome.13,24
The degree of endothelial injury is associated with poorer outcomes in patients with ALI. In a study of 559 adult patients with ALI/ARDS, plasma levels of vWF:Ag were significantly higher in non-survivors.13
Flori et al25
found that plasma vWF:Ag levels in pediatric patients with ALI were associated with an increased risk of death and prolonged mechanical ventilation. In the present study, AFC was not associated with circulating vWF:Ag levels. Consistent with our previous study,9
these data suggest that lung endothelial injury is not a major determinant of AFC in this preparation. Separate measures of markers of endothelial and epithelial injury may provide additional information on the severity of lung injury and improve pretransplant assessment of donor lungs.
This study has some limitations. First, the number of human lungs available for study is by necessity rather small; thus, outlying data points may significantly impact the results. To address this issue, we used nonparametric statistical methods, which analyze the distribution of data based on rank rather than absolute value. In addition, the small sample size limits our ability to draw conclusions about nonsignificant associations, due to the risk of a type II error. Second, the method of lung preservation used in this study differs from methods used in studies by other groups,2-4,26
most of which were designed to keep lungs in perfect condition for possible re-transplantation. In these studies, the lungs were flushed with preservation solutions during the procurement surgery and were reperfused with a physiologic solution with RBCs. These differing methods of preservation may affect biomarker levels in either the airspaces, the perfusate, or both.
Clinical criteria inadequately predict preserved AFC and graft function in donor lungs. We found that elevated airspace RAGE levels were a marker for impaired AFC in donor lungs rejected for transplant. These data support the hypothesis that RAGE is a valuable biological marker of alveolar epithelial injury and function.
Several articles have been recently published about successful transplantation of lungs from marginal donors (ie
, non-heart-beating donors or lungs previously rejected for transplantation).2-4
In these publications, evaluation criteria are based on hemodynamic values and gas exchange capacities of the lungs. In this context, clinically relevant biomarkers of lung injury may be useful in the assessment of lungs prior to transplantation, as well as in selecting patients for clinical trials with ALI.