Conceived and designed the experiments: MK JPC. Performed the experiments: MK YL. Analyzed the data: MK RO AG JPC. Contributed reagents/materials/analysis tools: MK JPC. Wrote the paper: MK RO AG JPC.
The authors have declared that no competing interests exist.
Matrix metalloproteinases (MMP) -8 and -9 may play key roles in the modulation of neutrophilic lung inflammation seen in pediatric Acute Respiratory Distress Syndrome (ARDS). We aimed to perform a comprehensive analysis of MMP-8 and MMP-9 activity in tracheal aspirates of pediatric ARDS patients compared with non-ARDS controls, testing whether increased MMP-8 and -9 activities were associated with clinical outcomes.
Tracheal aspirates were collected from 33 pediatric ARDS patients and 21 non-ARDS controls at 48 hours of intubation, and serially for those who remained intubated greater than five days. MMPs, tissue inhibitor of metalloproteinases (TIMPs), human neutrophil elastase (HNE) and myeloperoxidase (MPO) activity were measured by ELISA, and correlated with clinical indicators of disease severity such as PRISM (Pediatric Risk of Mortality) scores, oxygen index (OI), multi-organ system failure (MOSF) and clinical outcome measures including length of intubation, ventilator-free days (VFDs) and mortality in the Pediatric Intensive Care Unit (PICU).
Active MMP-9 was elevated early in pediatric ARDS subjects compared to non-ARDS controls. Higher MMP-8 and active MMP-9 levels at 48 hours correlated with a longer course of mechanical ventilation (r = 0.41, p = 0.018 and r = 0.75, p<0.001; respectively) and fewer number of VFDs (r = −0.43, p = 0.013 and r = −0.76, p<0.001; respectively), independent of age, gender and severity of illness. Patients with the highest number of ventilator days had the highest levels of active MMP-9. MMP-9 and to a lesser extent MMP-8 activities in tracheal aspirates from ARDS subjects were sensitive to blockade by small molecule inhibitors.
Higher MMP-8 and active MMP-9 levels at 48 hours of disease onset are associated with a longer duration of mechanical ventilation and fewer ventilator-free days among pediatric patients with ARDS. Together, these results identify early biomarkers predictive of disease course and potential therapeutic targets for this life threatening disease.
Acute Lung Injury (ALI), and in its most severe form, Acute Respiratory Distress Syndrome (ARDS) is a common, life-threatening cause of respiratory failure in children. Recent studies support a role for neutrophil-derived MMP-8 and -9, and an imbalance with their natural inhibitors, TIMPs in the pathogenesis of adult ALI and ARDS
This study was performed in the PICU at The Children's Hospital of Alabama with approval from the Institutional Review Board at the University of Alabama at Birmingham and written informed consent provided by legal guardians of the subject.
ARDS subjects were children (≤17 years of age) who required mechanical ventilation and met clinical criteria for ARDS, including hypoxemia as defined by partial pressure of oxygen in arterial blood (PaO2)/fraction of oxygen in inspired air (FiO2) ≤200 mm Hg, bilateral infiltrates on chest radiograph and no clinical evidence of left atrial hypertension
Clinical data collected included patient demographics, diagnosis associated with the onset of ARDS, radiographic findings, arterial blood gases, OI, PaO2/FiO2 ratios, blood and tracheal aspirate culture data, PRISM scores, MOSF
Endotracheal tube aspirates were collected on ice and centrifuged at 1000 RPM for 10 minutes, with separation of pellet from supernatant. Once the supernatant was collected, protein concentration was measured (Catalog # 5000112; Bio-Rad), and then separate aliquots were saved at 4°C for quantitative analysis of MMP-8, MMP-9, TIMP-1, MPO and HNE.
Zymography was performed on tracheal aspirates using a modified technique
MMP-9 activity was quantified using a fluorometric assay (R&D Systems # F9M00). 1 mM of Aminophenylmercuric acetate (APMA), a chemical activator of MMP-9 was added to selected samples to determine the total concentration of MMP-9 present, including pro-MMP-9. Complementary samples without the addition of APMA measured the amount of endogenously active MMP-9 present in the samples.
MMP-8, TIMP-1(R&D Systems # DMP800 and DTM100; respectively), MPO and HNE concentrations (Calbiochem # 475919 and CBA016; respectively) were measured using sandwich enzyme immunoassay techniques. MMP-8 activity was not routinely measured due to variable availability of standardized MMP-8 activity ELISA kits.
Tracheal aspirates from ARDS subjects with high MMP-8 and -9 activity were incubated with EDTA (5 mM), MMP-8 specific inhibitor (Calbiochem #44237 at 30 ng/ml), MMP-9 specific inhibitor (Calbiochem #444278 at 50 ng/ml) and doxycyline (100 mcg/ml) or vehicle (DMSO, 1∶1000) for 2 hours followed by MMP-8 and -9 activity measurement (R&D Systems).
Descriptive statistics were computed for each study variable of interest, including means, medians, standard error of the means (SEM), and ranges. Since the distributions of the data from the tracheal aspirate inflammatory markers deviated from a normal distribution, these data were logarithmically transformed using a log10 scale prior to statistical analysis. The log-transformed variables were determined to follow a normal distribution through the use of the Kolmogorov-Smirnov test and normal probability plots. Demographic and diagnostic comparisons between ARDS and control subjects were performed using the unpaired t-test for quantitative variables and the two-group chi-square test (or Fisher's exact test as needed) for categorical variables. Overall comparisons between ARDS and control subjects were performed using the unpaired t-test, while comparisons including covariates of interest (two disease etiologies, three disease severities, age, and gender) were performed using analysis of covariance. Comparisons between 48 hour and Day 6 measurements of total MMP-8, MMP-9 and active MMP-9 were performed using the paired t-test. Pearson correlation analysis was used to determine correlations between pairs of quantitative variables and Spearman correlation analysis was used to determine correlations between categorical variables (MOSF and gender) and quantitative variables. Simple and multiple linear regression analyses were used to assess the ability of the markers (active MMP-9, total MMP-9, percent active MMP-9, MMP-8, TIMP-1, MMP-9: TIMP-1, HNE, and MPO) to predict the outcome measures (days of intubation and ventilator-free days). Covariates included in the multiple regression analyses were age, gender, disease etiologies, P/F ratio, OI index, PRISM scores and MOSF. One-way analysis of variance was used to perform comparisons between the four quartiles for ARDS subjects. The Tukey-Kramer multiple comparisons test was then used to determine which specific pairs of means were significantly different. Where possible, nonparametric statistical analyses (including the Wilcoxon rank-sum test, the Wilcoxon signed rank test, the Kruskal-Wallis test, and Spearman correlation analyses) corresponding to the above analyses were performed, and these analyses yielded results that are similar to those obtained by the parametric analyses described above. Statistical tests were two-sided and were performed using a 5% significance level (i.e. alpha = 0.05). Statistical analyses were performed with the use of SAS software (version 9.2; SAS Institute, Inc., Cary, NC).
A summary of demographic and diagnostic information regarding the pediatric ARDS and control subjects is shown in
Control | ARDS | P | |
|
21 | 33 | |
|
6.5±1.4(4 weeks - 17 years) | 6.6±1.1(6 weeks - 17 years) | 0.95 |
|
6∶15 | 14∶19 | 0.30 |
|
4.3±0.6 (1 day - 10 days) | 11±1.3 (4 days – 33 days) | <0.001 |
|
23.7±0.6(18 days – 27 days) | 17.2±1.2(0 days – 24 days) | <0.001 |
|
7.7±1.6 | 12.5±1.2 | 0.018 |
|
5.7±1.2 | 10.2±0.8 | 0.003 |
|
0 | 13 (39%) | <0.001 |
|
0 | 1 (3%) | 1.0 |
|
0 | 20 (61%) | <0.001 |
|
0 | 27 (82%) | <0.001 |
|
Closed head injury (9)Seizure (3)Post laryngotrachealreconstruction (3)‡Others (6) | Bacterial pneumonia (8)Viral pneumonia (11)Aspiration (2)Pulmonary hemorrhage (2)Sepsis (6)Candidal pneumonia (1)Eosinophilic pneumonia (1)Smoke inhalation (1)Pulmonary contusion (1) |
*Pediatric Risk of Mortality; † Multi-organ System Failure; ‡ventricular shunt failure, post spinal fusion, choking, arrhythmia, spinal muscular atrophy, esophagitis.
Tracheal aspirates from ARDS subjects were initially probed for MMP-9 activity by zymography.
A. Zymogram (7.5% SDS non-reduced gel) demonstrates robust gelatinase activity in tracheal aspirates from ARDS subjects. Each lane represents a sample obtained from separate ARDS subjects at 48 hours of disease onset and intubation. The higher molecular weight band at 135 kDa (black arrow) likely represents lipocalin:MMP-9 complexes, while the 82 KDa band (white arrow) corresponds with active MMP-9 isoforms. B. Endogenously active MMP-9 levels measured in tracheal aspirates of ARDS subjects at 48 hours of disease onset and intubation were elevated (16-fold) compared with control subjects (
As surrogates of pulmonary neutrophil influx, MPO and HNE concentrations in tracheal aspirates were measured in all subjects. MPO levels from ARDS patients were found to be approximately 7-fold higher than in controls (MPO in ARDS mean±SEM = 9262±1696 ng/mg vs. control = 1396±439 ng/mg;
r | P | |
|
0.63 | <0.001 |
|
0.47 | 0.006 |
|
0.46 | 0.007 |
|
0.56 | <0.001 |
|
0.67 | <0.001 |
|
0.38 | 0.031 |
Note: All correlations are indicative of positive relationships between these pairs of variables.
TIMP-1 concentrations were slightly lower in the ARDS subjects compared to controls (mean±SEM = 113±15 ng/mg vs.144±27 ng/mg, respectively; p = 0.43), contributing to higher MMP-9:TIMP-1 activity ratios in ARDS subjects compared to controls (mean±SEM = 2.7±0.6 vs. 1.4±0.5, respectively;
At 48 hours of intubation and ARDS onset, increasing active MMP-9 levels and fraction of active MMP-9 (active MMP-9/total MMP-9) had strong positive correlation (r = 0.75;
No significant correlations were identified between total MMP-9, TIMP-1, MMP-9:TIMP-1 ratios, HNE or MPO with duration of mechanical ventilation and VFDs in the ARDS subjects. Similarly, no clear relationship was observed between the measured biomarkers and clinical indicators of disease severity (P/F ratios, OI, PRISM 12, PRISM 24 and MOSF). In the control non-ARDS group, no correlation was observed between the tracheal aspirate biomarkers (active MMP-9, total MMP-9, percent active MMP-9, MMP-8, TIMP-1, MMP-9:TIMP-1, HNE and MPO) and clinical markers of disease severity and outcome measures (P/F ratios, OI, PRISM 12, PRISM 24, MOSF, duration of intubation and VFDs).
To further examine relationships between duration of intubation and measured MMP-8 and MMP-9 levels at 48 hours of ARDS, we stratified ARDS subjects into quartiles based on number of ventilator days (
1st Quartile(n = 8) | 2nd Quartile(n = 8) | 3rd Quartile(n = 8) | 4th Quartile(n = 9) | Overall p-value | |
|
21.9±2.9a | 10.5±0.6b | 7.7±0.4b,c | 4.8±0.2c,d | <0.001 |
|
67.5±12.1a | 9.7±2.5b | 7.1±1.7b | 7.4±1.2b | <0.001 |
|
176±68 | 190±55 | 278±82 | 80±17 | 0.31 |
|
52.8±8.7a | 14.5±7.3b | 5.5±1.6b | 11.5±2.0b | <0.001 |
|
116±33 | 106±32 | 136±32 | 89±27 | 0.64 |
|
3.3±1.4 | 3.9±2.0 | 2.3±0.6 | 1.4±0.3 | 0.79 |
|
1595±505 | 1227±476 | 729±261 | 281±82 | 0.087 |
|
15.7±9.2 | 22.5±12.3 | 7.1±2.9 | 5.3±2.0 | 0.72 |
|
11562±3646 | 11638±5581 | 7576±1672 | 6481±1215 | 0.62 |
*Active MMP-9/Total MMP-9 in %.
Data are presented as means ± SEM. Means in a row with different superscript letters are significantly different, p<0.05. Only active MMP-9 and fraction of active MMP-9/total MMP-9 demonstrated positive correlation with length of intubation (p<0.001), while total MMP-8 demonstrated a trend towards correlation with length of intubation.
Due to consistent covariate analysis demonstrating a significant relationship between MMP-9 activity and the length of mechanical ventilation, we analysed MMP-9 activity profile with ARDS progression. Of the 33 ARDS subjects, 25 went on to require more than five days of mechanical ventilation and continued to meet the diagnostic criteria for ARDS. In this group of patients, a second sample was obtained between 5–10 days of intubation (mean±SEM = 7.1±0.3 days, median = 6 days). The concentrations of active MMP-9 in these second samples were nearly 6-fold higher than at 48 hours (
A: Increase in active MMP-9 with ARDS progression. Total MMP-9 remained elevated in ARDS patients intubated for >5 days (n = 25). With ARDS progression, approximately 80% of MMP-9 was found in the active form (prior to APMA stimulation). Active MMP-9 at Day 6 of ARDS was 6-fold higher than at 48 hours of disease (mean±SEM 176±61 ng/mg vs. 30±16 ng/mg at 48 hrs, respectively; *
To determine if small molecule inhibitors of MMPs could normalize MMP-8 and MMP-9 activity in pediatric ARDS tracheal aspirates, samples from subjects with high MMP-8 and MMP-9 activity (n = 4) were treated with specific and nonspecific MMP inhibitors, and MMP activity was measured. MMP-8 activity was reduced by approximately 40% with EDTA and a specific MMP-8 inhibitor (
A. Tracheal aspirates from ARDS subjects (n = 4) with high MMP-8 activity were examined in the presence of different MMP inhibitors. MMP-8 was reduced by 40–50% relative to basal activity when incubated with 5 mM of EDTA (†
ARDS is a syndrome of severe pulmonary dysfunction and refractory hypoxemia that results from a massive neutrophilic inflammatory response
In this study, we examined MMP-8 and MMP-9 levels in pediatric ARDS, exploring relationships between their concentrations, activity and modulation with disease progression. These two MMPs were chosen based on our previous work in which comprehensive MMP screening demonstrated that MMP-8 and 9 were among a limited number of MMPs found to be elevated in pediatric ALI
In the current report, both MMP-8 and -9 had positive correlations with MPO and HNE, enzymes present in abundance in neutrophil granules, suggesting that neutrophils were an important source of the MMPs. In comparison to MMP-8, MMP-9 had a weaker correlation with MPO and HNE. Since more than one-third of our ARDS subjects had viral induced ARDS, epithelia derived MMP-9 {described in respiratory viral infections
To date, there have been contradictory reports regarding whether MMP-9 is protective or harmful in lung injury models
Elevated MMP-8 and MMP -9 activity in ARDS lung secretions could be reduced by treatment with MMP inhibitory molecules ex vivo (
Patients who were on steroids or immunosuppressive medications prior to intubation (both for control and ARDS subjects) were excluded from the study secondary to the potential effect of these medications on MMP expression and activity. However, out of the 33 enrolled ARDS subjects, three received hydrocortisone (100 mg/m2/day) for shock and five received solumedrol (2 mg/kg/day) for wheezing within the first two days of intubation. No correlation was found between the use of steroids and MMP expression or with markers of disease severity and outcome measures. None of the control subjects received steroids during the course of their intubation. However, due to the small number of patients who received steroids in our cohort, firm conclusions cannot be drawn.
One limitation of this study is that it was performed in one tertiary pediatric medical institution only. Future examination of MMP-8 and -9 as biomarkers of pediatric ARDS would benefit from a larger multicenter approach. Although this study demonstrated up-regulation of active MMP-9 in pediatric ARDS subjects and association between increased MMP-8 and MMP-9 early in disease course with duration of mechanical ventilation, it is crucial to recognize that examination of MMP-8/9 levels with other previously recognized ARDS biomarkers such as plasma von Willebrand factor antigen, plasminogen activator inhibitor-1 and B-type natriuretic peptide
The results of this study confirm our previous reports that MMP-8 and -9 are increased in pediatric ARDS, and provide evidence that early elevation of MMP-8 and MMP-9 levels, particularly active MMP-9 predicts the duration of mechanical ventilation. This relationship is independent of age, gender, disease etiology and disease severity. These findings support a role of protease dysregulation in the pathogenesis of pediatric ARDS, and suggest that elevated MMP-8 and/or active MMP-9 may have a prognostic value in pediatric ARDS patients. Larger prospective studies are warranted to confirm these findings with a goal of determining whether MMP-8/9 are appropriate targets to modify patient outcomes.
The authors would like to thank Tajuanna Lucious for assisting with clinical data collection.