Pediatric Pulmonology 35:456–462 (2003)
Early Postnatal Dexamethasone Influences Matrix Metalloproteinase-2 and -9, and Their Tissue Inhibitors in the Developing Rat Lung Arwin M. Valencia, MD,1,2 Kay D. Beharry, BSc,3 Jorge G. Ang, MD,1,2 Kamakshi Devarajan, MD,1,2 Richard Van Woerkom, MD,1,2 Maria Abrantes, MD,1,2 Kenji Nishihara, BA,4 Eileen Chang, BS,4 Joshua Waltzman,4 and Houchang D. Modanlou, MD1* Summary. In order to test the hypothesis that early postnatal exposure to dexamethasone (Dex) influences matrix metalloproteinases (MMP)-2 and -9, as well as their tissue inhibitors (TIMP-1 and -2) in the developing rat lung, newborn rats (3 litters/group) were treated with low Dex (0.1 mg/kg/ day, IM), high Dex (0.5 mg/kg/day), or equivalent volumes of saline at 5 days postnatal age (P5), P6, and P7. Lung weight and lung MMP and TIMP levels were determined at sacrifice (7 days postinjection, P14; at weaning, P21; and at adolescence, P45, n ¼ 10/group and time). Dex did not adversely affect lung weight or lung MMP-2 levels, which peaked in all groups at P21 and then fell by P45. In contrast, Dex decreased TIMP-2 at all time intervals, but achieved statistical significance only at P45. An imbalance in MMP-2/TIMP-2 ratio was noted at P21, with elevations occurring in the low and high Dex-treated groups. Lung MMP-9 levels remained comparable with controls during low Dex treatment. However, high Dex exposure resulted in elevated lung MMP-9 levels at P21 and P45. Lung TIMP-1 levels increased only with high Dex exposure at P14 and P21, whereas the lung MMP-9/TIMP-1 ratio was elevated at P21 in the high Dex group, and at P45 in both Dex-treated groups. These data provide evidence that early postnatal dexamethasone results in an imbalance between gelatinase-A and -B, and their tissue inhibitors in the developing rat lung. These changes may be responsible, in part, for some of the known maturational effects of steroids on lung structure in the newborn. Pediatr Pulmonol. 2003; 35:456–462. ß 2003 Wiley-Liss, Inc. Key words: dexamethasone; lung; matrix metalloproteinases; tissue inhibitors of metalloproteinases.
INTRODUCTION
Postnatal glucocorticoids such as dexamethasone (Dex) have been used to improve pulmonary function in premature newborn infants at risk for chronic lung disease (CLD). The pulmonary benefits of Dex are demonstrated by several randomized controlled trials, which showed improved pulmonary function and decreased duration of ventilator dependency in preterm infants.1–3 However, studies in rats demonstrated that the normal process of alveolarization which occurs prior to microvascular maturation was suppressed in order to induce precocious microvascular maturation.4 At 1 week post-Dex withdrawal, microvascular maturation ceases, and the delayed process of alveolarization is reestablished. This catch-up alveolarization resulted in larger and fewer alveoli, i.e., characteristics similar to CLD. These morphologic changes may be linked with alterations in lung matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs). MMPs are mediators of tissue matrix degradation. They belong to a family of zinc endopeptidases that are collectively capable of degrading essentially all extracellular matrix (ECM) components,5 and as such, play a ß 2003 Wiley-Liss, Inc.
1 Division of Neonatal-Perinatal Medicine, Department of Pediatrics, University of California, Irvine Medical Center, Orange, California. 2
Department of Pediatrics, Miller Children’s Hospital, Long Beach Memorial Medical Center, Long Beach, California. 3 Division of Maternal-Fetal Medicine, Women’s Hospital, Long Beach Memorial Medical Center, Long Beach, California. 4 Research Administration, Long Beach Memorial Medical Center, Long Beach, California.
This paper was presented in part, at the 2002 Pediatric Academic Societies Meeting, Baltimore, MD. Grant sponsor: Long Beach Memorial Medical Center Foundation. *Correspondence to: Houchang D. Modanlou, M.D., Division of NeonatalPerinatal Medicine, Fellowship Program, University of California, Irvine Medical Center, 101 The City Drive, Building 2, Route 81, Orange, CA 92868. E-mail:
[email protected] Received 20 August 2002; Accepted 23 January 2003. DOI 10.1002/ppul.10293 Published online in Wiley InterScience (www.interscience.wiley.com).
Early Postnatal Steroids Alter Rat Lung MMPs 6,7
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major role in inflammation, tissue remodeling, and angiogenesis.9 Of particular interest for both neonatologists and pulmonologists is the active role of MMPs in lung development, particularly in the process of alveolarization and microvascular maturation.10–12 MMP-2 (gelatinase-A) appears to play a key role in the remodeling of the neonatal lung structure during alveolarization, whereas MMP-9 (gelatinase-B) has been shown to be involved in the latter part of microvascular maturation and decreased fibrosis.13–16 The regulation of MMPs occur through intracellular-, extracellular-, or cell surfacemediated proteolytic mechanisms involving a specific family of tissue inhibitors of metalloproteinases (TIMPs), which maintains the balance between the synthesis and degradation of ECM components required for maintenance of lung structure and function.17–22 Loss of regulated turnover may result in a number of different lung pathologies. Studies have shown that decreased MMP activity is associated with the development of pulmonary fibrosis, whereas excessive activity can result in the destruction of the lung architecture, as seen in emphysema.14 With the knowledge that Dex influences alveolarization and microvascular maturation, and considering the specific roles of MMPs in lung development, we examined the hypothesis that alterations in the processes of alveolarization and microvascular maturation following early Dex treatment are mediated, in part, by changes in lung MMP-2 and -9, as well as their tissue inhibitors. To test our hypothesis, we examined and compared the effects of low and high doses of Dex on MMP-2 and -9 and TIMP1 and -2 in the rat lung at 1 week post-Dex exposure (postnatal age 14 days (P14), which coincides with the timing of catch-up in alveolarization), P21 (weaning from the dam), and P45 (adolescence). MATERIALS AND METHODS
All experiments were conducted according to the guidelines of the Institutional Review Board and Institutional Animal Care and Use Committee of Long Beach Memorial Medical Center (Long Beach, CA). Animals were housed and treated according to the guidelines outlined by the National Research Council Guide for the Care and Use of Laboratory Animals.23 Euthanasia of animals was conducted according to the guidelines of the American Veterinary Medical Association.24 Experimental Design
Timed pregnant Sprague-Dawley rats (200–300 g body weight) were purchased from Charles River (Hollister, CA) at 19 days of gestation. The pregnant rats were housed under controlled environmental conditions,23 with free access to food and water until delivery at 22 days of gestation. The newborn pups were undisturbed for 5 days
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to allow for stabilization with the dam. At P5, pups from 27 litters (6–17 pups/litter) were pooled (to eliminate litter differences), weighed, and randomly assigned to receive Dex sodium phosphate (0.5 mg/kg IM), 0.1 mg/kg Dex, or equivalent volume saline, i.m., at P5, P6, and P7. At time of randomization, litter sizes were normalized to 10 pups/ litter. The total number of litters in each treatment group was 9 (low Dex, high Dex, and saline), and the total number of litters at each time was 3. Ten pups from the 3 litters in each group were randomly chosen and sacrificed on P14 (1 week post-Dex administration), P21 (weaning from the dam), and P45 (adolescence). At time of sacrifice, total body weight was determined. The lungs were removed and weighed, and 0.5-g biopsies were placed in sterile tubes and frozen at 808C until analysis. Assay of MMPs and TIMPs
Lung tissue samples were examined for MMP-2 and -9, and TIMP-1 and -2 levels, using a commercially available enzyme-linked immunosorbent assay kit (Amersham Pharmacia Biotech, Piscataway, NJ). The samples (0.5 g/ml) were homogenized in 0.1 M Tris-HCl (pH 7.5) containing 10 mM CaCl2 on ice. Samples were centrifuged at 48C for 45 min, and the supernatant was assayed for MMP-2, MMP-9, TIMP-1, and TIMP-2. The inter- and intraassay variability for MMP-2, MMP-9, TIMP-1, and TIMP-2 ranged between 5–12%, 5–10%, 8–16%, and 2–6%, respectively, with the lowest limit of detectability set at 0 ng/ml for each assay. Protein Assay
Lung tissue MMP and TIMP levels were standardized as a function of total cellular protein levels. Therefore, a portion (10 ml) of supernatant was utilized for total cellular protein determination. Total cellular protein levels were determined in the supernatant by the Bradford method (Bio-Rad protein assay kit), using bovine serum albumin as a standard. Statistical Analysis
Statistical analyses were accomplished with the use of GraphPad Instat (GraphPad, San Diego, CA). One-way analysis of variance was employed to examine differences among groups at each experimental time for each measured variable (MMPs and TIMPs). Post hoc analysis was accomplished using the Student-Newman-Keuls test for significance. All data were expressed as means SEM. A P-value of less than 0.05 was considered significant. RESULTS
Pooling and randomization resulted in equal distribution of runts among groups. There was no evidence of fetal mortality or postnatal death. No more than 10 pups were
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assigned to each dam, which accepted all pups as her own despite pooling. There were no differences in total body weight (grams) among groups prior to treatment (P5) or at 1 week posttreatment (P14). However, at P21, both low (39.3 0.6, P < 0.001, n ¼ 10) and high (37.8 0.4, P < 0.001, n ¼ 10) doses of Dex resulted in decreased body weight compared to controls (45.5 0.4, n ¼ 10). At P45, the animals were growth-adjusted. Effect of Dexamethasone on Lung Weight
Lung weights (grams) in the Dex-treated groups were determined at sacrifice and compared with those of agematched controls (Fig. 1). There was no significant effect on lung weight in any of the treated groups at P14 (control, 0.45 0.008; low dose, 0.5 0.1; high dose, 0.43 0.02; n ¼ 10/group), P21 (control, 0.44 0.01; low dose, 0.44 0.02; high dose, 0.45 0.02; n ¼ 10/group), or P45 (control, 1.15 0.04; low dose, 1.12 0.06; high dose, 0.99 0.04; n ¼ 10/group). Lung to body weight ratios (Fig. 2) increased in the animals exposed to Dex at P21 (low dose, 0.011 0.002; high dose, 0.013 0.0004; P < 0.01) vs. control (0.0096 0.003). Effect of Dexamethasone on Lung MMP-2 Levels
MMP-2 levels (ng/mg protein) in the lung homogenates were elevated, though not statistically, in the treated groups (low dose, 9.1 2.1; high dose, 10.4 1.7) compared to the control group (6.5 0.8). MMP-2 levels were comparable among the groups at P14 and P45 (Fig. 3).
Fig. 2. Effects of early postnatal dexamethasone (Dex) administration on ratio of lung weight to body weight. Rats were administered low (0.1 mg/kg, i.m.) or high (0.5 mg/kg, i.m.) doses of Dex or saline (i.m.), at 5, 6, and 7 days postnatal age. Total body and lung weights were determined at sacrifice (1 week post-Dex, P14; at weaning, P21; and at adolescence, P45). Data are expressed as means SEM, n ¼ 10 in each group.
control (9.11 0.54). Dex treatment had no notable effects on lung TIMP-2 levels at P14 or P21. Effect of Dexamethasone on Lung MMP-2/TIMP-2 Ratio
Dex treatment increased the MMP-2/TIMP-2 ratio at P21 by 85% (low dose, 1.74 0.24, P < 0.05) and 168% (high dose, 2.52 0.23, P < 0.01) compared to controls (0.94 0.07) (Fig. 5). No significant effects were detected at P14 or P45. Effect of Dexamethasone on Lung MMP-9 Levels
Effect of Dexamethasone on Lung TIMP-2 Levels
Figure 4 illustrates that mean lung TIMP-2 levels (ng/mg protein) were decreased in treated groups at all time intervals. However, statistical significance in TIMP-2 deficits was achieved only at P45 (low dose, 5.1 0.81, P < 0.01; high dose, 3.62 0.32, P < 0.001) compared to
A different response pattern from lung MMP-2 (ng/mg protein) emerged with respect to lung MMP-9 levels (Fig. 6). High Dex treatment was associated with increased lung MMP-9 levels at P21 (2.76 0.2 ng/mg protein, P < 0.01) and P45 (4.48 1.0 ng/mg protein, P < 0.05) compared to control (P21, 1.87 0.11 ng/mg protein; P45,
Fig. 1. Effect of early postnatal dexamethasone (Dex) administration on lung weight (grams). Rats were administered low (0.1 mg/kg, i.m.) or high (0.5 mg/kg, i.m.) doses of Dex or saline, at 5, 6, and 7 days postnatal age. Lung weights were determined at sacrifice (1 week post-Dex, P14; at weaning, P21; and at adolescence, P45). Data are expressed as means SEM, n ¼ 10 in each group.
Fig. 3. Effects of early postnatal dexamethasone (Dex) administration on lung MMP-2 levels. Rats were administered low (0.1 mg/ kg, i.m.) or high (0.5 mg/kg, i.m.) doses of Dex or saline (i.m.), at 5, 6, and 7 days postnatal age. Lung MMP-2 levels were determined at 1 week post-Dex (P14); at weaning (P21); and at adolescence (P45). Data are expressed as means SEM, n ¼ 10 in each group.
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Fig. 4. Effects of early postnatal dexamethasone (Dex) administration on lung TIMP-2 levels. Rats were administered low (0.1 mg/kg, i.m.) or high (0.5 mg/kg, i.m.) doses of Dex or saline (i.m.), at 5, 6, and 7 days postnatal age. Lung TIMP-2 levels were determined at 1 week post-Dex (P14); at weaning (P21); and at adolescence (P45). Data are expressed as means SEM, n ¼ 10 in each group.
Fig. 6. Effects of early postnatal dexamethasone (Dex) administration on lung MMP-9 levels. Rats were administered low (0.1 mg/ kg, i.m.) or high (0.5 mg/kg, i.m.) doses of Dex or saline (i.m.), at 5, 6, and 7 days postnatal age. Lung MMP-9 levels were determined at 1 week post-Dex (P14); at weaning (P21); and at adolescence (P45). Data are expressed as means SEM, n ¼ 10 in each group.
1.76 0.13 ng/mg protein) and low-dose (P21, 1.96 0.05 ng/mg protein; P45, 2.03 0.07 ng/mg protein) groups. MMP-9 levels were comparable among the groups at P14. Low Dex exposure did not influence MMP-9 levels at any time interval.
Dex exposure resulted in an increased lung MMP-9/ TIMP-1 ratio at P21 (1.34 0.05, P < 0.05) compared to the control (1.17 0.05) and low dose (1.02 0.1) groups. At P45, elevations in lung MMP-9/TIMP-1 ratio were noted in both treated groups (low dose, 1.2 0.02, P 0.01; high dose, 1.1 0.09, P < 0.05) compared to controls (0.9 0.02). Lung MMP-9/TIMP-1 ratios were comparable among the groups at P14.
Effect of Dexamethasone on Lung TIMP-1 Levels
Lung TIMP-1 levels (ng/mg protein) were affected only by high Dex exposure (Fig. 7) at P14 (2.0 0.09, P < 0.05) and P21 (2.1 0.07, P < 0.01) compared to the control (P14, 7.7 0.04; P21, 1.65 0.07) and lowdose (P14, 1.9 0.1; P21, 1.9 0.08) groups. Although lung TIMP-1 levels were visibly lower in the treated groups at P45 compared to controls, no differences were detected.
DISCUSSION
A response pattern similar to lung MMP-9 levels was noted for the lung MMP-9/TIMP-1 ratio (Fig. 8). High
The present study is the first to examine the relationship between the effects of postnatal Dex withdrawal on the enzymatic activity of MMPs during the timing of alveolarization and microvascular maturation in the rat lung. We focused specifically on gelatinases A and B or type IV collagenases and their inhibitors, because type IV collagen is the major constituent of basement membranes. We demonstrated that approximately 1 week following postnatal Dex withdrawal (a time coinciding with catch-up of
Fig. 5. Effects of early postnatal dexamethasone (Dex) administration on ratio of lung MMP-2 to TIMP-2 levels. Rats were administered low (0.1 mg/kg, i.m.) or high (0.5 mg/kg, i.m.) doses of Dex or saline (i.m.), at 5, 6, and 7 days postnatal age. Lung MMP-2 to TIMP-2 ratios were determined at 1 week post-Dex (P14); at weaning (P21); and at adolescence (P45). Data are expressed as means SEM, n ¼ 10 in each group.
Fig. 7. Effects of early postnatal dexamethasone (Dex) administration on lung TIMP-1 levels. Rats were administered low (0.1 mg/kg, i.m.) or high (0.5 mg/kg, i.m.) doses of Dex or saline (i.m.), at 5, 6, and 7 days postnatal age. Lung TIMP-1 levels were determined at 1 week post-Dex (P14); at weaning (P21); and at adolescence (P45). Data are expressed as means SEM, n ¼ 10 in each group.
Effect of Dexamethasone on Lung MMP-9/TIMP-1 Ratio
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Fig. 8. Effects of early postnatal dexamethasone (Dex) administration on ratio of lung MMP-9 to TIMP-1 levels. Rats were administered low (0.1 mg/kg, i.m.) or high (0.5 mg/kg, i.m.) doses of Dex or saline (i.m.), at 5, 6, and 7 days postnatal age. Lung MMP-1 to TIMP-1 ratios were determined at 1 week post-Dex (P14); at weaning (P21); and at adolescence (P45). Data are expressed as means SEM, n ¼ 10 in each group.
delayed alveolarization), there was an imbalance between MMP-2 and -9 and their inhibitors toward elevated MMP activity. These changes will most likely determine the later mechanical and functional properties of the lung tissue.25,26 Previously, Tschanz et al.4 demonstrated accelerated microvascular maturation concurrent with suppression of alveolarization during the first 2 weeks of postnatal Dex administration in rats. By 1 week of postnatal Dex withdrawal, there was a reversal in microvascular maturation concurrent with catch-up in delayed alveolarization. By P60, the lungs developed characteristics similar to CLD (emphysematous, with larger and fewer alveoli). The present study clearly demonstrates that an imbalance between type IV collagenases and their inhibitors occurs simultaneously with these reported adverse events. MMPs and TIMPs are produced ubiquitously in the body. However, in the lungs, MMPs are produced by structural cells such as fibroblasts, endothelial and epithelial cells,27 and alveolar macrophages.28–30 Dex has been shown to influence these cell types: specifically, suppression of bronchoalveolar macrophages31,32 and precocious induction of functional type II alveolar cells33 and microvascular maturation.34 The doses of Dex used in the present study (0.1 mg/kg/day and 0.5 mg/kg/day) were determined based on the doses used in the neonatal intensive care unit.35,36 The first test of the hypothesis that MMPs are involved in the events associated with Dex withdrawal is demonstrated by the response pattern of lung MMP-2 levels and MMP-2 to TIMP-2 ratio, which simulated that of lung to body weight ratio. It was interesting to note that neither the low nor high doses had any significant influence on absolute lung MMP-2 levels at any age, despite the trend toward higher levels at P21. Conversely, Dex caused lower TIMP-2 levels at all time intervals, though significance was achieved only at P45, with no differences between the effects of low or high
doses, suggesting selective targeting of the inhibitor, TIMP-2, by Dex, and thus increasing the remodeling activity of MMP-2. The increased MMP-2 activity may be partly responsible for the changes in microvascular maturation and alveolarization previously noted by Tschanz et al.4 The effects of dexamethasone on MMP-2 activity were similar at P21 regardless of dose, suggesting that the use of lower doses should be considered in those cases where Dex is still being used as a therapeutic intervention for CLD. The second test of our hypothesis is demonstrated by the response patterns of MMP-9 and TIMP-1. The most interesting finding was that increments in lung MMP-9 and TIMP-1 levels occurred only in response to high-dose Dex. Elevated levels of MMP-9 were found in brochoalveolar lavage fluid from patients with acute respiratory distress syndrome.37 Increases in MMP-9 secondary to high doses of Dex may contribute to the development of lung characteristics similar to CLD, as demonstrated by Tschanz et al.4 and Okajima et al.34 Indeed, an imbalance between MMP-9 and TIMP-1, favoring MMP-9, was detected as early as P21, 2 weeks after the high Dex dose, and continued until P45 in both treatment groups. Given the important role of MMP-9 in inflammation, the possibility of its measurement as a pulmonary teratologic endpoint warrants investigation. During the perinatal period, human lungs undergo several phases of development. Establishment of a surfactant system, an increase in the number of alveoli (through division of existing alveoli), alveolar wall thinning, and capillary fusion results in microvascular maturation.38 In humans, alveolarization occurs mainly between week 35 of gestation and approximately the third postnatal year. Microvascularization, on the other hand, begins at time of birth and is completed by 5 years of age.38 At birth, the newborn rat lungs are equivalent in development to those of a human fetus at 28 weeks of gestation. Therefore, the rat’s lung develops postnatally, since birth precedes the process of alveolarization. Alveolarization in the rat’s lungs takes place predominantly within the first 2 weeks of life, with microvascular maturation occurring during week 3 of life.39 It is important to note that the high dose of Dex impacted MMP-9 activity at the time when microvascularization in rats is known to occur, while the effects of the low dose of Dex on MMP-9 activity occurred only at P45, when alveolarization is presumed to be complete. One limitation of this study is that we did not examine morphologic changes in the lungs. However, our observations, combined with those of Tschanz et al.,4 provide further evidence to support the growing disfavor towards the use of high Dex doses in CLD, but on the other hand, may suggest a role for low doses. In conclusion, this study establishes that early postnatal Dex has significant effects on the enzymatic activity of
Early Postnatal Steroids Alter Rat Lung MMPs
lung MMPs at a critical time of lung development. Because type IV collagen is the major constituent of basement membranes, precocious remodeling and matrix destruction by type IV collagenases may contribute to morphologic changes associated with lung damage and subsequent CLD. Further studies are required to evaluate the predictive value of MMPs and their inhibitors, to identify infants at risk for CLD. REFERENCES 1. Halliday HL, Ehrenkranz RA. Moderately early (7–14 days) postnatal corticosteroids for preventing chronic lung disease in preterm infants (Cochrane review). Cochrane Database Syst Rev 2001;1:CD001144. 2. Arias-Camison JM, Lau J, Cole CH, Frantz ID III. Meta-analysis of dexamethasone therapy started in the first 15 days of life for prevention of chronic lung disease in premature infants. Pediatr Pulmonol 1999;28:167–174. 3. Bhuta T, Ohlsson A. Systematic review and meta-analysis of early postnatal dexamethasone for prevention of chronic lung disease. Arch Dis Child Fetal Neonatal Ed 1998;79:26–33. 4. Tschanz SA, Damke BM, Burri PH. Influence of postnatally administered glucocorticoids on rat lung growth. Biol Neonate 1995;68:229–245. 5. Massova I, Kotra LP, Fridman R, Mobasher S. Matrix metalloproteinases: structure evolution and diversification. FASEB J 1998;12:1075–1095. 6. Woessner J Jr. Matrix metalloproteinases and their inhibitors in connective tissue remodeling. FASEB J 1991;5:2145–2154. 7. Matrisan LM. The matrix-degrading metalloproteinases. Bioessays 1992;14:455–463. 8. Roche WR, Beasley R, Williams JH, Holgate ST. Subepithelial fibrosis in the bronchi of asthmatics. Lancet 1989;1:520–524. 9. Mignatti P, Tsuboi R, Robbins E, Rifkin DB. In vitro angiogenesis on the human amniotic membrane: requirement for basic fibroblast growth factor-induced proteinases. Bioassays 1989;14:455– 463. 10. Fukuda Y, Ferran VJ, Crystal RG. The development of alveolar septa in fetal sheep lung: ultrastructural and immunohistochemical study. Am J Anat 1983;167:405–439. 11. Heine UI, Munoz EF, Flanders KC, Roberts AB, Spora MB. Colocalization of TGF-beta 1 and collagen I and III, fibronectin and glycosaminoglycans during lung branching morphogenesis. Development 1990;109:29–36. 12. Roman J, McDonald JA. Expression of fibronectin, the intedrin alpha 5, and alpha-smooth muscle action in heart and lung development. Am J Respir Cell Mol Biol 1992;6:472–480. 13. Fukuda Y, Ishizaki M, Kudoh S, Kitaichi M, Yamanaka N. Localization of matrix metalloproteinases-1, -2, and -9, and tissue inhibitor of metalloproteinases-2 in interstitial lung diseases. Lab Invest 1998;78:687–698. 14. Katzentein AL, Myers JL. Idiopathic pulmonary fibrosis. Clinical relevance of pathologic classification. Am J Respir Crit Care Med 1998;157:1301–1315. 15. Crouch E. Pathobiology and pulmonary fibrosis. Am J Physiol Lung Cell Mol Physiol 1990;259:159–184. 16. Raghu G, Striker L, Hudson LD, Striker GE. Extracellular matrix in normal and fibrotic lungs. Am Rev Respir Dis 1985;131:281– 289. 17. Birkedal-Hansen H, Moore WG, Bodden MK, Windsor LJ, Birkedal-Hansen B, De Carlo A, Engler JA. Matrix metalloproteinases: a review. Crit Rev Oral Biol Med 1993;4:197–250.
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