|Year : 2013 | Volume
| Issue : 4 | Page : 209-213
|The relationship between the first episode of wheezing and matrix metalloproteinases-9 and MMP-2 and tissue inhibitors of MMP-1 levels in preterm infants
Rabia Gonul Sezer1, Gokhan Aydemir2, Abdulkadir Bozaykut1, Serdar Hira3, Ilhan Asya Tanju2, ÷mer ÷zcan3
1 Department of Pediatrics, Zeynep Kamil Maternity and Children's Disease Training and Research Hospital, Uskudar 34668, Istanbul, Turkey
2 Department of Pediatrics, GATA Teaching Hospital, Uskudar 34668, Istanbul, Turkey
3 Department of Biochemistry, GATA Teaching Hospital, Uskudar 34668, Istanbul, Turkey
|Date of Submission||11-May-2013|
|Date of Acceptance||04-Jul-2013|
|Date of Web Publication||20-Sep-2013|
Rabia Gonul Sezer
Zeynep Kamil Hastanesi, Arakiyeci Haci Mehmet Mah., Op. Dr. Burhanettin ‹stŁnel Caddesi, Uskudar 34668, Istanbul
| Abstract|| |
Aims: Matrix metalloproteinases (MMP) have been associated with neonatal lung morbidity and MMP dysregulation contributes to the pathology of chronic and acute lung disorders. Most of the previous studies were performed in the 1 st weeks of life of the preterm newborns. There are no data on the serum levels of MMP-2, MMP-9 or tissue inhibitors of matrix metalloproteinases (TIMP-1) from preterm infants recovering from lung morbidities. We aimed to compare MMP-2, MMP-9 and TIMP-1 levels in preterm and term infants hospitalized with their first episode of wheezing.
Methods: We prospectively evaluated 18 preterm infants with a history of chronic lung disease, respiratory distress syndrome or oxygen therapy and 14 age- and sex-matched term infants who were admitted for a first episode of wheezing. We quantified total serum concentrations of MMP-2, MMP-9 and TIMP-1 to assess whether these serum markers levels were associated with the first episode of wheezing in infants with a history of oxygen therapy during the neonatal period.
Results: Upon hospitalization, MMP-2 and TIMP-1 levels were higher in preterm infants than in term infants. In contrast, there was no significant relationship between MMP-9 levels or the MMP-9/TIMP-1 ratio between preterm and term infants. The area under the receiver operating characteristic curve for MMP-2 was 0.70 (95% confidence interval [CI] 0.51-0.89). The area under the curve for TIMP-1 was 0.78 (95% CI 0.61-0.94). MMP-9, MMP-2 and TIMP-1 levels did not correlate with gestational age, gender or severity of wheezing.
Conclusion: The negative proportion of MMP-9 to TIMP-1 that we detected in term infants was not present in preterm infants. The balance of MMP-9 to TIMP-1 may have been disrupted by lung damage in the premature infants. Overproduction of MMP-2 and TIMP-1 in the serum may be associated with the pathogenesis of wheezing in preterm infants.
Keywords: Matrix metalloproteinases, matrix metalloproteinases-2, matrix metalloproteinases-9, preterm, tissue inhibitors of matrix metalloproteinases-1, wheezing
|How to cite this article:|
Sezer RG, Aydemir G, Bozaykut A, Hira S, Tanju IA, ÷zcan ÷. The relationship between the first episode of wheezing and matrix metalloproteinases-9 and MMP-2 and tissue inhibitors of MMP-1 levels in preterm infants. Ann Thorac Med 2013;8:209-13
|How to cite this URL:|
Sezer RG, Aydemir G, Bozaykut A, Hira S, Tanju IA, ÷zcan ÷. The relationship between the first episode of wheezing and matrix metalloproteinases-9 and MMP-2 and tissue inhibitors of MMP-1 levels in preterm infants. Ann Thorac Med [serial online] 2013 [cited 2020 Oct 24];8:209-13. Available from: https://www.thoracicmedicine.org/text.asp?2013/8/4/209/118506
Advances in neonatal medicine have resulted in the survival of extremely preterm, very low-birth weight newborns. An increasing number of preterm survivors are developing chronic lung disease (CLD).  The features of CLD of early infancy include alveolar hypoplasia, peribronchial fibrosis, alveolar septal fibrosis, hypertensive vascular changes, airway muscle thickening and abnormalities of elastic fiber architecture. , The early abnormal lung development in patients with CLD may prevent subsequent lung growth and development.  The long-term respiratory consequences of CLD can extend beyond childhood.  Preterm infants have a tendency to wheeze throughout early childhood and these children experience viral bronchiolitis, which necessitates frequent hospital readmissions. 
Matrix metalloproteinases (MMP) are enzymes that are able to degrade collagen, fibronectin, elastin and laminin, which are all components of the extracellular matrix. The proteolytic activity of MMPs is counteracted by tissue inhibitors of matrix metalloproteinases (TIMPs). MMPs function in growth and vascular remodeling during wound healing and inflammation. , MMPs disrupt the integrity of the basement membranes of alveolar epithelial cells. , MMPs can function in both destruction and the reorganization of alveolar epithelia. In animal models, MMP-2 and MMP-9 appear to be involved in early stages of rabbit lung development and a dramatic increase in MMP-2 occurs during the late prenatal lung development and in postnatal lungs. 
MMP dysregulation contributes to the pathology of chronic and acute lung disorders, including bronchopulmonary dysplasia,  respiratory distress syndrome (RDS) of newborn,  asthma  and emphysema. ,, In addition, the impact of oxygen toxicity, barotrauma and volutrauma caused by mechanical ventilation disrupts the structure of the lungs and initiates an inflammatory cascade.  Inflammation is the physiologic hallmark of respiratory morbidity and mortality in preterm infants, with neutrophils and macrophages releasing proteolytic enzymes, such as MMPs.  The increased number of neutrophils, proteinases (like MMP-9) and anti-proteinases increases the likelihood of developing CLD and postnatal infections and represents the primary trigger for initiating the inflammatory process.  It is known that MMP-9 is increased in the lungs of preterm babies who develop CLD and that in these children, there is an imbalance between MMP-9 and its inhibitor, TIMP-1. 
This study examines the relationship between metalloproteinases and their inhibitors in preterm infants who are experiencing their first attack of wheezing after discharge from the neonatal intensive care unit. We hypothesized that a unique MMP and TIMP profile exists in the serum of preterm infants who have recovered from RDS and/or CLD. Most of the previous studies were performed in the 1 st weeks of life of the preterm newborns. There are no data on the serum levels of MMP-2, MMP-9, or TIMP-1 from preterm infants recovering from lung morbidities. The aims of this study were (1) to serially measure plasma levels of MMP-2, MMP-9 and TIMP-1 in premature infants who have recovered from RDS and/or CLD and have been re-hospitalized for a first episode of wheezing; (2) to examine MMP-2, MMP-9 and TIMP-1 levels and their relationships to C-reactive protein (CRP), complete blood count, blood gases and postnatal history; and (3) to compare MMP-2, MMP-9 and TIMP-1 levels in preterm infants with those of term infants.
| Methods|| |
This hospital-based, prospective observational study was conducted from September 2010 to October 2011 in Istanbul, Turkey. Informed consent was obtained from the parents of all subjects and the study was approved by the local Ethics Committee.
Patients who were admitted with a first episode of wheezing were included if they met the inclusion criteria: A preceding viral upper respiratory infection that was followed by wheezing and crackles on auscultation; a viral respiratory infection was diagnosed on clinical grounds; the patient was a preterm infant (<37 weeks gestation at birth) who had developed and recovered from CLD of prematurity or neonatal RDS or had received oxygen treatment. The patients were symptom free and breathing room air upon discharge from the neonatal intensive care unit. All patients were treated and discharged from the same neonatal intensive care unit. We collected data on the use of mechanical ventilation, gestation, birth weight, gender and the number of doses of surfactant administered.
Patients were excluded if they experienced lung morbidities other than wheezing, were born at ≥37 weeks, were younger than 1 month or older than 6 months, had a history of recurrent wheezing episodes, had consolidation or atelectasis on a chest roentgenogram or had proven immune deficiency or severe neurological disease. In addition, infants were excluded if their parents did not provide informed consent. None of the study participants received steroids or other immunosuppressive medications during the study.
On admission, blood was drawn to check the complete blood count, CRP and blood gases. In addition, blood was obtained within 24 h of admission and separated and frozen at −70°C for analysis of MMP-2, MMP-9 and TIMP-1. On admission and discharge, serum samples were obtained and aliquoted.
Supportive care, including oxygen supplementation, aspiration and hydration, was provided to all patients when necessary. Pulses, respiratory rates and oxygen saturation were measured using a bedside monitor (SC 6002XL Multiparameter Monitor, Siemens, Germany). The severity of the wheezing was assessed using respiratory rate, pulse, oxygen saturation and length of hospitalization.
The second group was comprised of term infants with no known lung disease who were hospitalized for their first episode of wheezing. Patients were matched for age, gender and severity of wheezing. As with the preterm infants, blood was drawn for complete blood count, CRP and blood gases on admission. In addition, blood was obtained within 24 h of admission and separated and frozen at −70°C for analysis of MMP-2, MMP-9 and TIMP-1 concentrations.
Measurement of MMP-2, MMP-9 and TIMP-1 concentrations
Plasma levels of MMPs were assessed using the enzyme-linked immunosorbent assay (ELISA) kits (RayBio® Human MMP-2, MMP-9, TIMP-1 ELISA Kit; Ray-Bio Catalog#: ELH-MMP2-001, ELH-MMP9-001, ELH-TIMP1-001). Protein concentrations were measured by ELISA (CA-2000, CIOM Medical Co., Ltd., Jilin, China) according to the manufacturer's instructions. The manufacturer's minimum detectable concentration for individual kits was <3.5 ng/ml for MMP-2, 10 pg/ml for MMP-9 and 40 pg/ml for TIMP-1.
Serum CRP concentrations were measured using a nephelometric method (IMMAGE 800, Beckman Coulter, Inc., Brea, USA) and values >1 mg/dl were considered abnormal.
Statistical analysis was performed using the Statistical Package for Social Sciences (version 17.0; SPSS Inc., Chicago, IL, USA). MMP-9, MMP-2 and TIMP-1 levels were not normally distributed so non-parametric tests were used to assess for differences between the two groups. Differences between groups were analyzed with the Mann-Whitney U test. The two consecutive measurements in the preterm group were analyzed with paired t-test. Correlations between groups were performed using Spearman correlation coefficients. Receiver operating characteristic curves (ROC) were used for MMP and TIMP levels. A P < 0.05 was considered to be significant.
| Results|| |
Characteristics of the preterm and term groups
A total of 18 infants who were born at ≤33 weeks (24-33 weeks) were recruited to the preterm group. Of those, 13 had recovered from RDS and 4 of those infants had also recovered from CLD. The five remaining infants had received oxygen treatment during the neonatal period. Fifty blood samples were collected: 36 from the preterm group and 14 from the term group. Both groups had similar severity of wheezing as assessed by respiratory rate, oxygen saturation and length of stay. None of the patients required mechanical ventilation.
Arterial blood gases were tested in every patient and there was no difference between the term and preterm infants (P > 0.05). The laboratory results of the preterm and term groups are given in [Table 1].
|Table 1: Comparison of the laboratory results and MMP-2, MMP-9, TIMP-1 levels in preterm and term infants|
Click here to view
MMP-2, MMP-9 and TIMP-1 concentrations
Serum MMP-2 and TIMP-1 levels were higher in preterm patients than in term patients (P = 0.04 and P = 0.007, respectively) [Figure 1].
|Figure 1: Comparison of matrix metalloproteinases (MMP)-2, MMP-9 and tissue inhibitors of MMP-1 levels (ng/ml) in preterm and term infants|
Click here to view
In the preterm group, the two consecutive measurements of MMP-9 levels were significantly different (P = 0.03). The median MMP-9, MMP-2 and TIMP-1 concentrations of two consecutive measurements in the preterm group and the results of term group are given in [Table 1].
There was a negative correlation between MMP-9 and TIMP-1 levels in term infants (r = −0.81, P < 0.001), whereas there was no correlation in these levels in preterm infants. CRP levels were correlated with MMP-9 and TIMP-1 levels in the term group (r = −0.80, P < 0.001; r = 0.76, P = 0.001, respectively).
Upon admission to the hospital, MMP-2 levels were inversely correlated with respiratory rate (r = −0.68, P = 0.002) and percentage of neutrophils (r = −0.47, P = 0.04) in preterm infants. At the time of discharge, MMP-2 levels correlated with CRP levels (r = −0.61, P = 0.007).
The area under the ROC curve for MMP-2 was 0.70 (95% confidence interval [CI] 0.51-0.89, P = 0.04). The area under the curve for TIMP-1 was 0.78 (95% CI 0.61-0.94, P = 0.007) [Figure 2].
|Figure 2: Receiver operating curve for the specificity and sensitivity of matrix metalloproteinases-2 and tissue inhibitors of matrix metalloproteinases-1 measurements|
Click here to view
MMP-9, MMP-2 and TIMP-1 levels were not correlated with gestational age. There was no difference in the concentration levels of boys versus girls. There was no relationship between proteinases and gestational age; birth weight; postnatal history of intubation; continuous positive airway pressure, hood or oxygen therapy; number of surfactant replacement therapies or history of CLD. Likewise, there was no association between the proteinases and age and weight at the time of study, heart rate, oxygen saturation at admission, duration of hospitalization, white blood cell count, hemoglobin, pH, PCO 2 or HCO 3 levels.
Potential confounders that may affect proteinase levels include age, gender, birth weight, CRP level, complete blood count and blood gases analysis. These clinical parameters were not significantly different between the preterm and term groups.
| Discussion|| |
The principle finding of this study was that MMP-2 and TIMP-1 serum concentrations were elevated in preterm infants who presented for a first episode of wheezing. However, MMP-9 and the MMP-9/TIMP-1 ratio were not elevated in these infants.
MMPs includes collagenases (MMP-1, MMP-8 and MMP-13), stromelysins (MMP-3, MMP-7 and MMP-10) and gelatinases (MMP-2 and MMP-9). , Gelatinases degrade the extracellular matrix, regulate proinflammatory cytokines and contribute to tissue remodeling in the lungs. MMP-2 is secreted by many cells of mesenchymal origin and is unique among MMPs in that it is not activated with elastase, cathepsin G, plasmins or urokinase. It is activated only on the cell surface.  MMP-9, also known as gelatinase-B, is a type 4 collagenase secreted by leukocytes, , and excess MMP-9 activity and hyperoxic lung damage are closely linked. ,,,, TIMP-1 is produced by connective tissue cells and macrophages and irreversibly binds and inactivates MMP-9. , It has been suggested that TIMP-1 may have a role in postnatal lung adaptation. Furthermore, a high ratio of MMP-9 to TIMP-1 is thought to be a risk factor for lung damage. , One study that examined the longitudinal profile of MMP-9 and the MMP-9/TIMP-1 ratio showed episodic peaks in the level of this proteinase and its inhibitor.  Infections triggering the proteinase cascade lead to lung injury and CLD development.  The negative proportion of MMP-9 to TIMP-1 that we detected in term infants was not present in preterm infants. The balance of MMP-9 to TIMP-1 may have been disrupted by lung damage in the premature infants.
Previous studies examined MMP-9 and the MMP-9/TIMP-1 complex in bronchoalveolar lavage specimens of ventilated preterm infants and identified relationships between CLD development and proteinase release.  Studies of MMP levels in RDS and CLD patients have produced conflicting results. ,,, Dik et al.  reported increased MMP-9 levels in patients with RDS, but Sweet et al.  and Ekekezie et al.  did not find a difference. In another study, median MMP-9 levels were significantly higher in infants with RDS up to 4 days after birth, but in infants with CLD, levels increased after a week.  Increased MMP-9 activity is associated with better pulmonary outcomes in infants with RDS, suggesting that MMP-9 plays a protective role.  A higher ratio of MMP-9 to TIMP-1 in bronchoalveolar lavage specimens from infants with CLD was reported. , An imbalance in MMP-9 and TIMP-1 can lead to neonatal lung disease and the development of CLD. , These controversial results on the harms and benefits associated with MMP-9 should be highlighted. In this study, we compared two groups of infants that were matched for age and gender and differed only with respect to their gestational age at birth and postnatal history. We studied proteinase levels in the peripheral blood so that the results could be related to the timing of the study and to the type of specimen tested. Moreover, most of the previous studies were performed in the 1 st weeks of life of the preterm newborns. Our study included the infancy period, which has not been studied before.
A study of 1059 preterm infants with birth weights <1000 g detected a significant negative correlation between MMP-9 levels on day one of life and administered FiO 2 on day three.  This was contrary to studies that had shown a relationship between high MMP-9 levels and hyperoxia.  It is not appropriate to compare studies that have used different technologies such as zymography, ELISA and luminex. In addition, there are limited normative data on the plasma activity of the MMPs. 
Endotracheal aspirates of children with respiratory failure have revealed increased MMP-9 concentrations compared with the controls, yet MMP-2 levels did not differ.  MMP-2 levels were significantly increased from day zero to day one in the tracheal secretions of infants who subsequently developed CLD, suggesting that MMP-2 plays a role in the pathogenesis of CLD.  Compared with term patients, we detected increased MMP-2 levels, but no difference in MMP-9 levels, in preterm infants in the acute phase of wheezing. The inverse correlation may be due to the active secretion of MMPs from cells of the lungs that was not reflected by serum levels.
One limitation of our study is that we studied systemic rather than tissue MMP and TIMP levels. Nonetheless, we demonstrated significant differences in serum MMP-2 and TIMP-1 levels. Our patient group was not on mechanical ventilation, so obtaining tracheal samples would not have been ethical. In addition, the viral etiology for the episode of wheezing was not detected, which may have altered serum proteinase levels. Furthermore, we do not know the baseline MMP and TIMP levels before the wheezing attack. Despite our limited sample size and lack of a control group of premature born infants without a wheezing episode, we observed significantly higher levels of MMP-2 and TIMP-1 in preterm infants than in term infants, which may be useful for predicting lung morbidity during early infancy.
Disruption of the balance between MMP-9 and TIMP-1 and overproduction of MMP-2 and TIMP-1 in the serum may be associated with the pathogenesis of wheezing in preterm infants recovering from RDS and/or CLD.
| References|| |
|1.||Bland RD. Neonatal chronic lung disease in the post-surfactant era. Biol Neonate 2005;88:181-91. |
|2.||Husain AN, Siddiqui NH, Stocker JT. Pathology of arrested acinar development in postsurfactant bronchopulmonary dysplasia. Hum Pathol 1998;29:710-7. |
|3.||Thébaud B, Ladha F, Michelakis ED, Sawicka M, Thurston G, Eaton F, et al. Vascular endothelial growth factor gene therapy increases survival, promotes lung angiogenesis, and prevents alveolar damage in hyperoxia-induced lung injury: Evidence that angiogenesis participates in alveolarization. Circulation 2005;112:2477-86. |
|4.||Elder DE, Hagan R, Evans SF, Benninger HR, French NP. Recurrent wheezing in very preterm infants. Arch Dis Child Fetal Neonatal Ed 1996;74:F165-71. |
|5.||Sweet DG, Halliday HL, Warner JA. Airway remodelling in chronic lung disease of prematurity. Paediatr Respir Rev 2002;3:140-6. |
|6.||Chakrabarti S, Patel KD. Matrix metalloproteinase-2 (MMP-2) and MMP-9 in pulmonary pathology. Exp Lung Res 2005;31:599-621. |
|7.||Fukuda Y, Ishizaki M, Okada Y, Seiki M, Yamanaka N. Matrix metalloproteinases and tissue inhibitor of metalloproteinase-2 in fetal rabbit lung. Am J Physiol Lung Cell Mol Physiol 2000;279:L555-61. |
|8.||Sweet DG, Curley AE, Chesshyre E, Pizzotti J, Wilbourn MS, Halliday HL, et al. The role of matrix metalloproteinases-9 and -2 in development of neonatal chronic lung disease. Acta Paediatr 2004;93:791-6. |
|9.||Cederqvist K, Sorsa T, Tervahartiala T, Maisi P, Reunanen K, Lassus P, et al. Matrix metalloproteinases-2, -8, and -9 and TIMP-2 in tracheal aspirates from preterm infants with respiratory distress. Pediatrics 2001;108:686-92. |
|10.||Tanaka H, Miyazaki N, Oashi K, Tanaka S, Ohmichi M, Abe S. Sputum matrix metalloproteinase-9: Tissue inhibitor of metalloproteinase-1 ratio in acute asthma. J Allergy Clin Immunol 2000;105:900-5. |
|11.||Davies PL, Spiller OB, Beeton ML, Maxwell NC, Remold-O'Donnell E, Kotecha S. Relationship of proteinases and proteinase inhibitors with microbial presence in chronic lung disease of prematurity. Thorax 2010;65:246-51. |
|12.||Sweet DG, Pizzotti J, Wilbourn M, Halliday HL, Warner JA. Matrix metalloproteinase-9 (MMP-9) in the airways of infants at risk of developing chronic lung disease (CLD). Eur Respir J 1999;14 Suppl 30:248s. |
|13.||O'Connor CM, FitzGerald MX. Matrix metalloproteases and lung disease. Thorax 1994;49:602-9. |
|14.||Dik WA, van Kaam AH, Dekker T, Naber BA, Janssen DJ, Kroon AA, et al. Early increased levels of matrix metalloproteinase-9 in neonates recovering from respiratory distress syndrome. Biol Neonate 2006;89:6-14. |
|15.||Ekekezie II, Thibeault DW, Simon SD, Norberg M, Merrill JD, Ballard RA, et al. Low levels of tissue inhibitors of metalloproteinases with a high matrix metalloproteinase-9/tissue inhibitor of metalloproteinase-1 ratio are present in tracheal aspirate fluids of infants who develop chronic lung disease. Pediatrics 2004;113:1709-14. |
|16.||Minoo P, Penn R, deLemos DM, Coalson JJ, deLemos RA. Tissue inhibitor of metalloproteinase-1 mRNA is specifically induced in lung tissue after birth. Pediatr Res 1993;34:729-34. |
|17.||Natarajan G, Shankaran S, McDonald SA, Das A, Stoll BJ, Higgins RD, et al. Circulating beta chemokine and MMP 9 as markers of oxidative injury in extremely low birth weight infants. Pediatr Res 2010;67:77-82. |
|18.||Schock BC, Sweet DG, Ennis M, Warner JA, Young IS, Halliday HL. Oxidative stress and increased type-IV collagenase levels in bronchoalveolar lavage fluid from newborn babies. Pediatr Res 2001;50:29-33. |
|19.||Schulz CG, Sawicki G, Lemke RP, Roeten BM, Schulz R, Cheung PY. MMP-2 and MMP-9 and their tissue inhibitors in the plasma of preterm and term neonates. Pediatr Res 2004;55:794-801. |
|20.||Winkler MK, Foldes JK, Bunn RC, Fowlkes JL. Implications for matrix metalloproteinases as modulators of pediatric lung disease. Am J Physiol Lung Cell Mol Physiol 2003;284:L557-65. |
|21.||Hadchouel A, Decobert F, Franco-Montoya ML, Halphen I, Jarreau PH, Boucherat O, et al. Matrix metalloproteinase gene polymorphisms and bronchopulmonary dysplasia: Identification of MMP16 as a new player in lung development. PLoS One 2008;3:e3188. |
[Figure 1], [Figure 2]
|This article has been cited by|
||The Association of Nailfold Capillaroscopy with Systemic Matrix Metalloproteinase-9 Concentration in Normal-Tension Glaucoma
| ||Na Young Lee,Hae-Young Lopilly Park,Sung-Hwan Park,Chan Kee Park |
| ||Current Eye Research. 2015; : 1 |
|[Pubmed] | [DOI]|