Low levels of tissue inhibitor of metalloproteinase-2 at birth may be associated with subsequent development of bronchopulmonary dysplasia in preterm infants

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Abstract

Introduction

In recent years, the quality of neonatal care in Korea has improved, resulting in a significant increase in the survival rate of neonates at high risk of mortality1). The improved survival of these infants has likely led to the increased incidence of a common corollary, bronchopulmonary dysplasia (BPD)2). BPD has long-lasting effects including poor neurodevelopmental outcomes and long-term pulmonary sequela. To date, there are few interventional therapies available to prevent or treat BPD3). Inflammation has been found to be central to the pathogenesis of BPD, but, unfortunately, this understanding has not yet translated into useful therapies.

The mechanisms underlying the pathogenesis of BPD have not been fully established, but proteolytic damage to the lungs from enzymes released by an influx of activated inflammatory cells is known to play a major role in the early stages. Furthermore, evidence suggests that proteolytic injury is expedited by an imbalance in lung proteinase versus antiproteinase defense456).

Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases that play an important role in the extracellular matrix remodeling that occurs during embryogenesis7), lung morphogenesis8), inflammation9), cardiovascular diseases10), metabolic disorders11), repair after injury12), and cancer13). A total of 25 MMP family members have been identified thus far, although not all are found in humans14). MMP-9 degrades collagen IV, a major component of the basement membrane of airways615). MMP-8 is another member of the MMP family that degrades type I collagen1617); it is associated with acute lung injury18192021) as well as chronic lung diseases such as asthma22) and BPD5). The activity of MMPs is controlled by specific tissue inhibitors of metalloproteinases (TIMPs)23). Previous studies have suggested that MMP-8, MMP-9, TIMP-2, TIMP-1, and their imbalanced ratios in bronchoalveolar lavage fluid (BALF) and blood may play a role in the pathogenesis of BPD, but the results of these studies have been inconsistent4562425262728).

The aims of the present study were to assess levels of MMP-8, MMP-9, TIMP-2, and TIMP-1 at birth in the serum of preterm infants and to determine whether these levels are associated with subsequent development of BPD.

Materials and methods

Results

1. Population characteristics and comparison of clinical parameters between preterm infants with and without BPD

The demographic and clinical characteristics and laboratory findings of the study population are presented in Table 1. Twenty-four preterm infants developed BPD and 38 did not. Of the 24 BPD infants, 16, six, and two infants developed mild, moderate, and severe BPD, respectively. The mean birth weights of the BPD and no BPD groups were 1,125.0±117.8 g and 1,181.8±121.4 g, respectively (P=0.923). The median gestational ages of BPD and no BPD subjects were 28⁺² (26⁺³-31⁺⁶) weeks and 29⁺¹ (26⁺²-31⁺⁶) weeks, respectively (P=0.875). BPD subjects were more likely to have mothers with proteinuric preeclampsia (16 [66.7%] vs. 10 [27.3%], P=0.036) and to develop IVH≥grade 3 (8 [33.3%] vs. 0 [0%], P=0.011). The BPD group had a longer mean duration of mechanical ventilation (21±13 days vs. 10±11 days, P=0.006) and oxygen supplementation (33±3 days vs. 17±18 days, P=0.002) compared with the no BPD group. There were no significant differences in sex, premature rupture of membranes, chorioamnionitis, Apgar scores at 1 and 5 minutes, multiplicity, antenatal steroid use, RDS, number of surfactant replacement treatments, or the incidence of PDA, pulmonary hemorrhage, NEC, or culture-proven sepsis between the two groups.

Table 1

Demographic and clinical characteristics of the infants

2. Comparison of MMPs, TIMPs, and their ratios between preterm infants with and without BPD

Preterm infants with BPD had significantly lower TIMP-2 levels at birth compared with those without BPD (138.1±23.0 ng/mL vs. 171.8±44.1 ng/mL, P=0.027) (Fig. 1C). No significant differences were observed in MMP-8 (2.0±2.2 ng/mL vs. 22.5±58.4 ng/mL, P=0.405) (Fig. 1A), MMP-9 (180.9±87.4 ng/mL vs. 285.4±533.7 ng/mL, P=0.165) (Fig. 1B), TIMP-1 (179.6±25.5 ng/mL vs. 176.8±44.2 ng/mL, P=0.692) (Fig. 1D) or their ratios between the two groups.

Fig. 1

Serum concentrations of MMP-8 (A), MMP-9 (B), TIMP-2 (C), and TIMP-1 (D) at birth in bronchopulmonary dysplasia (BPD) versus no BPD preterm infants. Preterm infants who developed BPD had significantly lower levels of TIMP-2 at birth compared with those who did not develop BPD. MMP, matrix metalloproteinase; TIMP, tissue inhibitor of metalloproteinase. Values are presented as mean±standard deviation.

3. Risk factors for the subsequent development of BPD in preterm infants

Multivariate logistic regression analysis was performed to evaluate independent relationships between the development of BPD and clinical and laboratory parameters. The results suggest that after adjusting for sex, birth weight, gestational age, proteinuric preeclampsia, and IVH, TIMP-2 level was significantly and inversely associated with subsequent development of BPD in preterm infants (β=-0.063, P=0.041) (Table 2).

Table 2

Assessment of the relationships between the development of bronchopulmonary dysplasia and clinical and laboratory variables by using multivariate logistic regression analysis

Discussion

In this prospective study, we characterized the presence of MMP-8, MMP-9, TIMP-2, and TIMP-1 in the serum of preterm infants and found that a low level of TIMP-2 at birth in preterm infants may be associated with the subsequent development of BPD. To the best of our knowledge, the present study is the first to identify a significant association between low serum TIMP-2 concentration at birth and subsequent development of BPD in preterm infants.

The results of the present study correspond in part with those of previous studies. A study by Ekekezie et al.4) evaluated MMP-2, MMP-9, TIMP-2, and TIMP-1 levels in tracheal aspirate fluid samples obtained serially from birth until extubation in 49 ventilated preterm infants; they found low TIMP-1 levels and a 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 in infants with BPD when compared to those without BPD. Another study by Cederqvist et al.5) analyzed tracheal aspirate fluids obtained within the first 5 postnatal days in newborns with RDS and observed lower TIMP-2 levels in those who experienced poor respiratory outcome (i.e., infants who developed BPD and those who died of severe respiratory distress) when compared to those who did not, but there were no observed associations between the development of BPD and MMP-2, MMP-9, or TIMP-2 levels. BALF is likely influenced by dilutional effects, however, and therefore MMP and TIMP measurements from BALF may not reflect the true physiologic status of newborns. The present study is unique in that MMPs and TIMPs were collected from the serum of preterm infants, which will not be affected by dilutional effects that are a major concern in studies assessing MMPs and TIMPs on BALF.

The mechanism underlying the relationship between low TIMP-2 level at birth in the serum of preterm infants and the development of BPD remains unclear. Possible underlying mechanisms for this association are as follows: First, TIMP-2 production depends on transcriptional regulation, thereby allowing for changes in the protein level to be tightly controlled. Hence, if TIMP-2 is not produced normally in response to regulatory signals, MMP-8 may degrade the extracellular matrix to a greater extent than it should. Second, TIMP-2 plays a unique role among TIMP family members in its capacity to suppress the proliferation of endothelial cells via metalloproteinase-independent mechanisms333435). Consequently, TIMP-2 may play an important role in maintaining tissue homeostasis by suppressing the proliferation of lung tissues in response to angiogenic factors, and by mediating protease activity in the lungs through extracellular matrix remodeling.

There are limitations to this study that need to be addressed. First, although we have data on total MMP-8 and MMP-9 immunoreactivity for all of the samples, we did not obtain data for MMP-8 and MMP-9 activity due to insufficient sample volume. The total level as determined from immunoreactivity consists of MMP that is fragmented, bound to TIMPs, or still in proenzyme form and thus does not reflect activity levels; however, other studies investigating MMPs, TIMPs, and their ratios as the basis for disease status have also based their analysis on these total values. Second, we did not obtain samples serially and therefore were not able to monitor changes in MMPs, TIMPs, or their ratios over time.

In conclusion, we described the presence of MMP-8, MMP-9, TIMP-2, and TIMP-1 at birth in the serum of preterm infants. Our findings indicate that there was a lower level of TIMP-2 at birth in the serum of preterm infants who subsequently developed BPD compared with those who did not. This finding suggests that a low serum TIMP-2 level at birth may play a role in the development of BPD in preterm infants by contributing to early lung inflammation. Thus, early postnatal supplementation of TIMPs could be an effective strategy in the treatment and prophylaxis of BPD. Further research is warranted to confirm this finding and to explore the possibility of TIMPs as a therapeutic and prophylactic option for BPD.

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