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* Children’s Mercy Hospitals and Clinics, University of Missouri-Kansas City, School of Medicine, Department of Pediatrics, Section of Neonatal-Perinatal Medicine, Kansas City, Missouri
Office of Medical Research, Section of Biostatistics, Kansas City, Missouri
Children’s Hospital of Philadelphia, University of Pennsylvania School of Medicine, Department of Pediatrics, Section of Neonatology, Philadelphia, Pennsylvania
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONREFERENCES |
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Methods. Serial tracheal aspirates collected from birth until extubation in 49 ventilated preterm infants (24-32 weeks’ gestations) were analyzed for MMP-2, MMP-9, TIMP-1, and TIMP-2. Data normalized by TA values of free secretory component of immunoglobulin A were compared for CLD (n = 22) versus no CLD (n = 27). Also, known clinical predictors of CLD (gestational age, birth weight, and sex) were assessed for both groups. Association of predictors with the outcome CLD was assessed by logistic regression.
Results. Mean gestational age was lower in CLD infants, but birth weight and gender were comparable for both groups. CLD infants had significantly lower TIMP-1 level with higher MMP-9/TIMP-1 ratio during the first 2 weeks of life and low TIMP-2 and MMP-2 levels during the first 3 days of life compared with no-CLD infants. Logistic regression analysis indicated that the findings are predictive of CLD.
Conclusions. We conclude that low tracheal aspirate levels of TIMPs, with a high MMP-9/TIMP-1 ratio early in life, are associated with subsequent development of CLD.
Key Words: MMPs • TIMPs • chronic lung disease
Abbreviations: CLD, chronic lung disease • MMP, matrix metalloproteinase • TIMP, tissue inhibitor of metalloproteinase • RDS, respiratory distress syndrome • TAF, tracheal aspirate fluid • EIA, enzyme immunoassay
Chronic lung disease (CLD) remains the most prevalent chronic morbidity afflicting the prematurely born infant. Although advances in therapy have improved survival rates for extremely low birth weight infants, an unfortunate consequence has been the rising incidence of CLD with its serious morbidity implications.1
The mechanisms underlying pathogenesis of CLD are not fully understood, but proteolytic injury to the lungs, from the actions of enzymes released by an influx of activated inflammatory cells, is a major contributing early event.2–4 There is evidence that the proteolytic injury is facilitated through triggering of an imbalance in lung proteinase versus antiproteinase defense.3,4 Studies of this concept to date have focused almost exclusively on the levels of serine proteases, such as elastase, relative to those of their inhibitors 
-1-antitrypsin and secretory leukocyte inhibitor.4,5 Therapeutic and prophylactic intervention strategies for CLD derived from these studies have yielded disappointing results in trials.6
Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases with crucial roles in extracellular matrix remodeling as occurs during lung morphogenesis, growth, and repair after injury.7–9 They are involved in angiogenesis, cell migration, tissue inflammation, degradation, and wound healing.7–9 MMPs proteolytically cleave extracellular matrix components, with some members demonstrating near-total substrate specificity in their actions. MMPs are secreted as zymogens (pro-MMPs) that require proteolysis for activation, and their activities are tightly regulated by specific inhibitors, tissue inhibitors of metalloproteinases (TIMPs) that bind to active MMPs in a 1:1 stoichiometric ratio.7–9
MMP-2 (72-kDa gelatinase or gelatinase A) is secreted mainly by noninflammatory cells (fibroblasts and endothelial and epithelial cells), whereas MMP-9 (92-kDa gelatinase or gelatinase B) is secreted by inflammatory cells (neutrophils, monocyte-macrophages).7–9 TIMP-1 is the specific inhibitor of MMP-9 but has overlapping inhibitory effects on other MMPs, including MMP-2. TIMP-2, however, is the specific inhibitor for MMP-2. The production of MMPs is under transcriptional modulation by several factors, including cytokines, growth factors, and extracellular matrix components.7–9
Imbalance between MMPs and their TIMPs have been implicated in the pathologic tissue degradation and remodeling that occurs in diseases such as asthma, acute respiratory distress syndrome (RDS), pulmonary fibrosis, and emphysema.10–13 However, characterization of MMPs and their TIMPs beyond the first few postnatal days of life in the tracheal aspirate of ventilated preterm infants with evolving CLD has not been reported. The current study was undertaken to investigate the concentrations of MMPs and TIMPs in tracheal aspirates of ventilated preterm infants and the possibility that imbalances in their relative levels may be involved in the development of CLD.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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Measurement of TIMP-1 in Tracheal Aspirate
TIMP-1 levels in the tracheal aspirates was measured using a commercially available enzyme immunoassay (EIA) kit (Amersham, Piscataway, NJ) according to manufacturer’s protocol. A total of 100 mL of serial dilutions of standards and samples was incubated in a 96-well microtiter plate, precoated with monoclonal anti-TIMP-1 antibody for 2 hours at 20°C to 25°C. After washing 4 times, 100 µL of peroxidase-labeled anti-TIMP-1 antibody was added to the wells, followed by additional incubation for 2 hours at 20°C to 25°C. The plate was then aspirated and washed 4 times, followed by detection of bound antibody by incubation with tetra-methyl benzidine substrate. The reaction was quenched after 30 minutes at 20°C to 25°C by addition of 100 µL of 1 M sulfuric acid. The optical density was read at 450 nm using a microplate reader. TIMP-1 concentration in the samples was determined by interpolation from the standard curve. Assays were run in duplicate, and the results were averaged. The assay sensitivity level was 1.25 ng/mL, and the interassay coefficient of variation was 13.6%.
Measurement of TIMP-2 in Tracheal Aspirate
TIMP-2 was measured using a commercially available "double sandwich" EIA kit, Biotrak (Amersham), per the manufacturer’s protocol. A total of 100 mL of peroxidase-conjugated secondary antibody to TIMP- 2, assay buffer, and standard or sample were mixed in a tube, then 100 µL of the mixture was added to a 96-well microtiter plate precoated with monoclonal anti-TIMP-2 antibody and incubated for 2 hours at 20°C to 27°C. Next, the plate was aspirated and washed 4 times, followed by additional incubation for 30 minutes after addition of tetra-methyl benzidine substrate. The reaction was quenched with 100 µL of 1 M sulfuric acid, and the color was read at 450 nm in a plate reader. The concentration of TIMP-2 in the samples was determined by interpolation from the standard curve. Assays were performed in duplicate, and the results were averaged. The assay sensitivity was 3.0 ng/mL, and the interassay coefficient of variations was 4.8%.
Assay of MMP-2 and MMP-9
MMP-2 and MMP-9 were measured with a commercially available EIA kit, Biotrak (Amersham). The kits are designed to measure total, active, or free MMP-2 and MMP-9, respectively, in biological fluids. A total of 100 mL of samples and standards per well of a microtiter plate was assayed according to the manufacturer’s protocol in duplicate, and the results were averaged. The assay sensitivity for MMP-2 and MMP-9 are 0.19 and 0.125 ng/mL, respectively. The interassay coefficient of variations were 17.9% and 20.9% for MMP-2 and MMP-9, respectively.
EIA Soluble Secretory Component of Immunoglobulin A
The concentration of free secretory component of immunoglobulin A (IgA) in the tracheal aspirate samples was assayed using a method established in our laboratory.15 The values obtained were used to normalize that of the MMPs and TIMPs in the samples as previously reported,16 because, unlike albumin, free secretory component of IgA levels in tracheal aspirate are unaffected by increases in lung capillary leak.
Albumin Measurement
Albumin was assayed in the samples using a commercially available kit according to the manufacturer’s protocol (BCA kit; Sigma, St. Louis, MO).
Statistics
Data from the TAF measurements were grouped according to postnatal age at time of collection into days 0 to 3, 4 to 10, 11 to 17, 18 to 24, and 25 to 31 for analysis. Patient characteristic data include gender, birth weight, gestation, surfactant, steroids, indomethacin use, and number of deaths. Categorical data were expressed as percentages and continuous data as mean ± standard deviation unless noted otherwise.
Characteristics of the CLD and the no-CLD groups were compared using 
2, Fisher exact, t, or Mann Whitney U tests as appropriate. Bivariate and multivariate logistic regression analysis was used to assess the effect of the predictors TIMP-1 and TIMP-2 and their ratios with MMPs as well as gestational age, birth weight, and gender at the selected time points, on the risk for developing CLD, the outcome variable. Data on TIMPs and MMPs did not seem to be normally distributed but followed a more normal distribution when transformed to log-2 values. Each level was expressed in a box plot as the median, 25th to 75th percentiles, and 10th to 90th percentiles. We used Pearson linear correlation on normalized data to assess the relationship between variables at the time points. P < .05 was considered significant for all statistical tests.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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Characterization of MMPs and TIMPs in TAF
MMP-2 and MMP-9, as well as their specific inhibitors TIMP-2 and TIMP-1, respectively, were detected in all of the tracheal aspirate samples. Serial measurements from the same patients showed great variability for levels of MMP-2 and MMP-9, as well as TIMP-1 and TIMP-2. TIMP-1 protein had the highest concentration with levels severalfold that of TIMP-2 in the same sample (Fig 1). Also, TIMP-1 was present at concentrations significantly higher than that of MMP-9, but MMP-2 versus TIMP-2 levels were not different. The ratio of MMP-9 to its specific inhibitor TIMP-1 was 10- to 50-fold less than that of MMP-2 to TIMP-2 in individual TAF samples.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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The findings are of clinical importance because they suggest that imbalance in the protease antiprotease system constituted by MMP-9 and its specific tissue inhibitor TIMP-1 may have a role in the pathogenesis of CLD. This has important clinical implications as it raises the prospect of therapeutic and/or prophylactic intervention with anti-MMP agents or TIMP supplementation for CLD.
Previous investigators, including us, have reported data describing the presence of MMPs and TIMPs in TAFs from ventilated preterm infants during the first few days of life.16–20 However, this is the first report characterizing MMP-2, MMP-9, TIMP-1, and TIMP-2, as well as the relationships among these variables, from birth until extubated or development of CLD. Our finding of no difference in TAF MMP-9 level in CLD versus no-CLD infants is consistent with that of Schock and Sweet et al in their recent study but differs from that of Sweet et al in a previous work.17,20
We found lower levels of TIMP-2 and MMP-2 but similar MMP-2/TIMP-2 ratios in TAF samples obtained during the first 3 days of life from infants who later developed CLD compared with those who did not. The observation of low TIMP-2 levels is consistent with that of Cederqvist et al.16 In their study of TAF samples obtained within the first 5 postnatal days in infants with RDS, they reported low TIMP-2 levels in those who either had a poor respiratory outcome or later received a diagnosis of bronchopulmonary dysplasia. Danan et al19 in their study of preterm infants reported a link between low MMP-2 levels at birth and development of CLD. They did not assess TIMP-2 levels and did not detect any association between MMP-9 and TIMP-1 level with development of CLD. Although methodologic differences preclude direct comparison, our findings for MMP-2 and MMP-9 are consistent with their observation but differ with respect to TIMP-1. Our finding of similarity in MMP-2/TIMP-2 ratio of the CLD and no-CLD groups despite lower MMP-2 and TIMP-2 levels in the CLD infants indicates that there was a proportionate decline in the protease and its inhibitor. This finding contrasts with that observed for MMP-9 and TIMP-1, whereby the difference in MMP-9/TIMP-1 ratio between the groups seems to be a consequence of the decreased levels of TIMP-1 in CLD infants. One possible explanation is the absolute dependence of MMP-2 and TIMP-2 production on transcriptional regulation, thus allowing for changes in the levels of both proteins to be tightly controlled. The production of MMP-9 and TIMP-1 is also under transcriptional modulation. However, MMP-9 also exists preformed in neutrophil storage granules that can be released in response to inflammatory stimuli, which would make maintenance of the MMP-9/TIMP-1 ratio a more challenging task.
Extracellular matrix turnover is dependent on the net balance between synthesis and degradation of its constituent proteins such that increased degradation would be expected if the level of proteases rises relative to that of antiproteases, and vice versa. Indeed a high MMP-9/TIMP-1 ratio and low TIMP-1 level have been implicated in the pathogenesis of several pulmonary disorders characterized by extracellular matrix degradation and extensive tissue remodeling, whereas the reverse situation is associated with lung repair and fibrosis.10–13 Increased elastolysis and elevated levels of extracellular matrix constituent proteins including laminin, fibronectin, and elastin breakdown fragments have also been reported in various biological fluids obtained from infants who developed bronchopulmonary dysplasia.21–23 These findings have been attributed to imbalance between elastase and its inhibitors 1 antitrypsin and secretory leukocyte inhibitor.24 However, the current study has shown, for the first time, that infants who develop CLD have a higher MMP-9/TIMP-1 ratio and lower TIMP-1 levels in their TAFs compared with those who do not.
MMPs and their TIMPs have a recognized physiologic role in orchestration of tissue morphogenesis but have also been implicated in pathologic roles in a growing list of disorders that now seems to include CLD. This has important clinical implications because it presents the possibility for therapeutic/prophylactic intervention for CLD using MMP inhibitors or by supplementation of the tissue inhibitor. This approach has demonstrated encouraging results in experimental models of bronchial asthma and lung injury.25
There are several limitations of this study that need to be considered. First, although we have data for total MMP-2 and MMP-9 immunoreactivity in all of the samples, we obtained data for MMP-2 and MMP-9 activity in only a limited number of samples because of insufficient sample volumes. The total level as detected by immunoreactivity includes MMP that is fragmented, bound to TIMPs, or still in the proenzyme form and thus does not reflect activity level. However, other studies implicating imbalance in MMP/TIMP ratio as the basis for disease conditions have relied on the total values.
Second, we restricted the study to preterm infants who were treated with endotracheal intubation because of the need to obtain tracheal aspirate samples for analysis. However, infants who were treated with only continuous positive airway pressure for respiratory support are also at risk for developing CLD. It is not known whether conclusions drawn from our data extend to this population of infants.
Next, is the difficulty presented by the lack of a totally reliable reference protein for normalization of TAF measurements to correct for sample variations that stem from the increased capillary leak that is present in inflamed lungs. The free secretory component of IgA has been used to normalize TIMP and MMPs in tracheal aspirate.16 Although it has its limitations as a reference protein,26 it has been shown to be superior to albumin in this regard and, therefore, was used in this study.27 Furthermore, we also compared values obtained when our data were normalized by TAF albumin, and the results and conclusion were the same.
In summary, we have provided data describing the presence of MMP-2, MMP-9, TIMP-1, and TIMP-2 from birth to the development of CLD in TAF of ventilated preterm infants. Our findings indicate that there is a low level of TIMP-1 with a high MMP-9/TIMP-1 ratio in the first 2 weeks of life, in addition to low levels of TIMP-2 and MMP-2 in the first few days of life in TAF of infants who develop CLD. This suggests that insufficient inhibition of MMP-9 as a result of a low level of TIMPs may be a contributory mechanism to development of CLD in preterm infants; of note, elevated MMP occurs within a few days of birth, a finding consistent with the prevailing view that early lung inflammation is involved in the pathogenesis of CLD. The implications are that early postnatal intervention with MMP antagonists or supplementation of TIMPs could be an effective strategy in the treatment and prophylaxis of CLD. Additional studies are warranted to validate this work and to explore the therapeutic and prophylactic prospects for CLD raised by these findings.
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ACKNOWLEDGMENTS |
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We thank Avital Cnaan, PhD, for statistical assistance; Theresa McDevitt for technical assistance; and Mary S. Bailey for administrative assistance and patience in preparation of this manuscript.
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FOOTNOTES |
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Address correspondence to Ikechukwu I. Ekekezie, MD, Department of Pediatrics, Division of Neonatology, Georgetown University Hospital, 3600 Reservoir Rd, NW, Ste M3400, Washington, DC 20007. E-mail: ie23{at}georgetown.edu
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REFERENCES |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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1-proteinase inhibitor activity in tracheal aspirates during respiratory distress syndrome. Role of inflammation in the pathogenesis of bronchopulmonary dysplasia.
J Clin Invest.1983; 72
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