Introduction
Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are common causes of acute respiratory failure with a high mortality rate despite decades of research into these conditions [
1]. Many studies have implicated activation of inflammation and derangement of the coagulation and fibrinolytic pathways in patients with ALI/ARDS. A number of biomarkers of inflammation are associated with poor clinical outcomes in patients with ALI/ARDS, including intercellular adhesion molecule 1 (ICAM-1), IL-6 and IL-8 [
2‐
4]. In patients with ALI/ARDS from a variety of predisposing conditions, higher levels of the proinflammatory cytokines IL-6 and IL-8 predict worse outcomes [
5]. Moreover, levels of ICAM-1 (unpublished data), IL-6 and IL-8 [
5] and levels of other proinflammatory cytokines [
6] are reduced by a low-tidal-volume ventilatory strategy.
In addition to inflammatory markers, markers of dysregulated coagulation and fibrinolysis are predictive of clinical outcomes in patients with ALI/ARDS. Protein C is an endogenous anticoagulant and antiinflammatory protein that is activated by binding to the thrombin–thrombomodulin complex on the endothelium. Lower levels of protein C and higher levels of circulating thrombomodulin are consistent with a procoagulant state [
7]. Fibrinolysis, the process of resolving clot formation, is also impaired in patients with ALI/ARDS [
8,
9]. In a larger multicenter study, higher levels of plasminogen activator inhibitor 1 (PAI-1) and lower levels of protein C in the plasma had a synergistic association with higher mortality in patients with ALI/ARDS [
10]. Protein C levels increased in patients treated with a low-tidal-volume ventilatory strategy in the study.
Given the importance of inflammation and coagulation to the pathogenesis of ALI/ARDS and the demonstrated improvement in biomarkers in these pathways in patients treated with lower tidal volumes, we investigated whether biomarkers of a proinflammatory, procoagulant and antifibrinolytic state remain predictive in the era of routine use of low-tidal-volume ventilation for ALI/ARDS. We chose to test multiple markers within the inflammatory and coagulation cascades that have a carefully considered pathogenetic basis in ALI/ARDS. Our hypothesis was that, despite effective institution of a lung-protective ventilatory strategy, derangement in the plasma levels of biomarkers reflecting inflammation and disordered coagulation and fibrinolysis would be associated with increased mortality in a cohort of prospectively collected patients with ALI/ARDS.
Materials and methods
Subjects and patient samples
Fifty patients who met the American–European Consensus Conference definition for ALI or ARDS [
11] were recruited from both Moffitt-Long University Hospital (33 patients) and San Francisco General Hospital (17 patients) from 2003 to 2006. Patients were recruited for participation within 48 hours of meeting the diagnostic criteria for ALI or ARDS. Informed consent for study participation was obtained from each subject or their designated surrogate. In the case of surrogate consent, follow-up consent was sought from the subject whenever possible. The study was approved by the Committee on Human Research at the University of California San Francisco and was performed in compliance with the mandates of the Helsinki Declaration.
Clinical data, including severity of illness scores and risk factors for the development of ALI/ARDS, were abstracted from the medical record. Ventilator data were also recorded for each subject at the time of collection of the plasma samples. The tidal volume was expressed as the tidal volume per kilogram of predicted body weight [
12]. The primary outcome for the present study was in hospital mortality. The University of California San Francisco Committee on Human Research approved the study protocol.
Plasma biomarker measurements
Plasma samples were collected from each patient at the time of enrollment with the pre hoc intent to study biomarkers of inflammation and coagulation. Blood was collected in heparin tubes and centrifuged for 10 minutes at 3,000 × g. Plasma supernatant was removed from the spun samples and was frozen at -70°C until the time of analysis. The analyses included markers of inflammation, coagulation and fibrinolysis. Specifically, we measured the inflammatory biomarkers ICAM-1, IL-6 and IL-8 and markers of disordered coagulation and fibrinolysis, including protein C, thrombomodulin and PAI-1. An ELISA was used to measure each biomarker in duplicate: ICAM-1, IL-6 and IL-8 (R&D Systems, Minneapolis, MN, USA); thrombomodulin and PAI-1 (American Diagnostica, Stamford, CT, USA); and Protein C (Helena Laboratories, Beaumont, TX, USA).
Statistical analysis
All statistical analyses were performed using STATA software (StataCorp, College Station, TX, USA). All analyses compared survivors with nonsurvivors in this group of patients. For baseline demographics and clinical data, we used chi-square analysis for dichotomous predictor variables and used an unpaired t test to compare survivors and nonsurvivors.
Biomarker values were not normally distributed. Logarithmic transformation of the biomarker data did not normalize the data as assessed by the Shapiro–Wilk test of normalcy. Hence, bivariate analysis of the association between biomarker values and the outcome of mortality was assessed using nonparametric analysis, specifically Mann–Whitney analysis.
We subsequently performed a logistic regression analysis to assess the contribution of demographic, clinical and biomarker data to the outcome of mortality. We confirmed these findings with a stepwise logistic regression model that included sepsis as a condition predisposing to ALI/ARDS to determine significant independent contributors to mortality in ALI/ARDS patients. Sepsis was included in the model since sepsis alone is recognized to contribute to increased mortality [
1] as well as to abnormalities in biomarker levels. Statistical significance for each of these analyses was defined as
P < 0.05.
Discussion
ALI is a complex illness with derangement in multiple metabolic pathways, including inflammation, coagulation and fibrinolysis. Abnormalities of these pathways have been shown in prior evaluations of patients with ALI/ARDS, with the greatest abnormalities presenting in nonsurvivors. These results were obtained, however, before the use of lower tidal volumes and limitations in plateau pressures had been convincingly demonstrated to decrease mortality in clinical ALI/ARDS [
12]. Injurious high tidal volumes alone can cause derangements in coagulation and fibrinolysis, and can trigger an inflammatory response [
17,
18]. To assess abnormalities in inflammation, coagulation and fibrinolysis independent of injurious ventilation, we studied patients at two hospitals that routinely use a low-tidal-volume plateau-pressure-limited ventilatory strategy in patients with ALI/ARDS.
To our knowledge, this is the first study to demonstrate abnormalities in markers of inflammation and impaired coagulation and fibrinolysis remain predictive of increased mortality despite implementation of lung-protective ventilation. Moreover, elevations in IL-8 and ICAM-1 were predictive of increased mortality independent of important clinical predictors and other biomarker abnormalities.
Our findings are consistent with earlier studies of biomarkers in the era prior to routine use of lung-protective mechanical ventilation. ICAM-1 is an adhesion molecule that facilitates trafficking of neutrophils to the lung and is upregulated on the lung endothelial surface during ALI/ARDS [
2]. In patients with ALI/ARDS, higher levels of soluble ICAM-1 in the pulmonary edema fluid were associated with an increased length of mechanical ventilation [
3]. Higher plasma ICAM-1 levels were also associated with mortality in a prospective study of children with ALI/ARDS [
4]. In patients with ALI/ARDS enrolled in a multicenter study of a protective ventilatory strategy, higher baseline levels of IL-6 and IL-8 were associated with increased mortality [
5].
Low levels of protein C showed a strong trend for being independently predictive of worse outcome in ALI/ARDS. These findings were confirmed in a rigorous stepwise backward logistic regression model that included sepsis as a covariate. This result is also consistent with prior work in ALI/ARDS. In a small prospective cohort of patients with ALI/ARDS, lower levels of protein C in pulmonary edema fluid were associated with increased mortality [
7], as were lower plasma levels in a larger multicenter cohort [
10] regardless of the presence or absence of sepsis. We therefore believe that protein C is associated with outcomes in ALI/ARDS and is not simply reflective of higher numbers of patients with sepsis in the nonsurvivor group. This finding suggests that protein C administration in patients with ALI/ARDS may have some benefit; however, a recent phase II, randomized controlled trial of activated protein C administration in patients with ALI/ARDS was stopped early because of lack of efficacy in the treatment group over placebo (Michael Matthay, unpublished data). Further work to understand the role of protein C in ALI/ARDS is therefore indicated.
PAI-1 levels were significantly higher in nonsurvivors than survivors on bivariate analysis. This confirms previous work in ALI examining PAI-1 levels in the era prior to routine use of low-tidal-volume ventilation. Prior work has demonstrated decreased urokinase activity in the air spaces of patients with ALI/ARDS, and this decrease is explained by elevations in levels of PAI-1 [
8]. In a small single-center study, PAI-1 levels in plasma and pulmonary edema samples from patients with ALI/ARDS were associated with higher mortality rates [
9]; this finding was confirmed in a larger multicenter study [
10]. In the current study, the PAI-1 levels did not remain independently predictive on multivariate analyses. This finding may reflect the relatively small sample size.
Lung-protective ventilation, although clearly demonstrated to improve survival in ALI/ARDS [
12], has not been routinely adopted as standard of care [
19,
20]. To confirm that patients in our cohort were ventilated with a low-tidal-volume protocol, we compared the ventilator settings for patients in our study with data from the original ARDS Network trial of lower-tidal-volume ventilation. The ventilator parameters were nearly identical for patients in our study compared with patients in the original trial, with a mean tidal volume of 6.6 ml/kg predicted body weight and a mean inspiratory plateau pressure of 25 cmH
2O. One additional similarity between the current study population and the ARDS Network trial population was that the PaO
2/FiO
2 ratio was lower (worse) in both cases in the groups that survived, although the result was not statistically significant. These data confirm that the PaO
2/FiO
2 ratio is not a good surrogate for outcomes in ALI.
Bivariate analysis of each of biomarker demonstrated that higher levels of IL-8, ICAM-1, thrombomodulin and PAI-1 and lower levels of protein C were significantly associated with increased mortality. The higher levels of IL-8 and ICAM-1 suggest there is greater upregulation of the acute inflammatory process in patients with ALI/ARDS who did not survive their illness. Similarly, lower protein C and higher thrombomodulin levels indicate greater activation of coagulation pathways in patients who died. Finally, the significantly higher level of PAI-1 in patients who died indicates greater impairment of fibrinolysis in these patients. Given that these patients were maintained on lung-protective ventilation, ventilator-induced lung injury is not a probable explanation for the abnormalities of inflammation and coagulation.
Parsons and colleagues [
5] demonstrated in ARDS Network patients that low-tidal-volume ventilation was associated with lower levels of plasma IL-6 and IL-8 levels by day 3 of the study compared with patients maintained on 12 ml/kg. Protein C levels were also normalized to a greater extent in the low-tidal-volume group [
10]. A remaining possibility, however, is that even a lung-protective ventilator strategy is injurious in the acutely injured lung. In a rat model of acid-induced lung injury, Frank and colleagues showed that lung endothelial and epithelial injury were minimized by a reduction in tidal volume to 3 ml/kg compared with 6 or 12 ml/kg [
17], suggesting that even a 6 ml/kg tidal volume might be injurious in some patients.
There are some limitations to our study. First, we studied a relatively small cohort of patients from two hospitals. For this reason, the study may have been underpowered to show a significant association between lower levels of protein C and adverse clinical outcomes in the multivariable analyses. Second, data were not collected on the ventilatory strategy employed prior to enrolment in the study. Patients were enrolled within 48 hours of meeting diagnostic criteria for ALI/ARDS so there was a maximum of 2 days in which patients may have received injurious ventilation. We therefore cannot rule out injurious ventilation prior to enrolment in the study possibly contributing to the findings of the study. Third, the biomarkers we studied were logarithmically transformed to enable statistical analysis. In practice, this means that a large increase in a biomarker such as IL-8 level is associated with a somewhat smaller increased risk of death. The results from the present study are therefore more likely to be useful in understanding the pathogenesis and ongoing injury during ALI/ARDS than as a diagnostic test for individual patients with ALI/ARDS.
In summary, the association of the biologic markers with adverse clinical outcomes does not confirm causality, but rather suggests important in vivo pathways for further study. In addition to clinical utility for prognostication and stratification of patients for enrollment in clinical trials, the clinical measurement of biomarkers may help to elucidate mechanisms of human disease that may have value in designing new therapies for ALI/ARDS.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
DM conceived the study, enrolled the patients, collected the samples, interpreted the data and drafted the manuscript. HJZ assisted with the biostatistical analysis. NW carried out the immunoassays. MAM and LBW conceived the study, participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.