Abstract
Background A single course of antenatal betamethasone is administered to women at risk of preterm labor to advance fetal lung maturation. Matrix metalloproteinases (MMPs) are collagen-degrading enzymes that remodel extracellular matrix components during lung development. We tested the hypothesis that the effects of betamethasone on fetal lung maturation involve changes in MMP activity.
Methods We conducted a prospective, observational pilot study of three groups of singleton pregnancies. Group 1 (n = 21) was composed of women who were antenatally treated with a single course of betamethasone and who delivered < 37 weeks of gestation, group 2 (n = 7) was composed of matched untreated women who delivered < 37 weeks of gestation, and group 3 (n = 15) was composed of untreated women who delivered > 37 weeks of gestation. Maternal blood, mixed cord blood, and placental samples were collected at the time of delivery for MMP-2 and MMP-9 activity and tissue inhibitor of metalloproteinases (TIMP)-1 and -2 levels.
Results MMP-2 activity was significantly higher in the maternal, placental, and fetal compartments in group 1 compared with group 2 (p < .05). TIMP-2 levels were lower in groups 1 and 2 compared with group 3. Maternal TIMP-2 levels were higher (p < 0.003), whereas fetal TIMP-1 (p < .01) and MMP-9 to TIMP-1 ratios (p < .05) were lower when delivery was delayed more than 2 weeks following betamethasone treatment.
Conclusion We conclude that elevated MMP-2 activity in the maternal and fetoplacental compartments may suggest a mechanism, in part, for betamethasone-induced fetal lung maturation.
A single two-dose course of antenatal betamethasone given 24 hours apart is administered to women at risk of preterm birth before 34 weeks of gestation to advance fetal lung maturation and reduce the incidence of respiratory distress syndrome, intraventricular hemorrhage, the need for surfactant therapy, and mortality in the neonate.1-3Normal fetal lung development requires the generation of a large respiratory surface, which is associated with remodeling of the extracellular matrix (ECM). Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) are a family of proteolytic enzymes that digest and degrade the ECM components and therefore are important in the morphogenesis of all fetal organs.4,5
The gelatinases MMP-2 (gelatinase A) and MMP-9 (gelatinase B) degrade type IV collagen, the major constituent of lung basement membranes.6,7MMPs form a 1:1 covalent complex with TIMPs, which regulate their activities.8A balance between the MMPs and TIMPs is necessary for normal matrix turnover. TIMP-1 inhibits the active form of all MMPs and the latent form of MMP-9 (pro-MMP-9) and is the most widely distributed TIMP.9TIMP-2 binds to both forms of MMP-2, although its inhibitory effect over the other MMPs is significantly lower.10MMP-2 and -9 are actively secreted from placental villous tissue in the first trimester, whereas there is a constant production of TIMPs (especially TIMP-2) throughout the gestational period.11
One of the known mechanisms for glucocorticoid-induced maturation of fetal lungs involves stimulation of the pulmonary surfactant system. However, studies have shown that antenatal glucocorticoids interfere with the process of alveolarization.12-14The beneficial effects of a single course of antenatal glucocorticoids on the fetus begin within 24 hours after administration but may last up to 7 days after treatment.15-17Although this treatment appears to be safe, there are no studies examining the effects of a single course of antenatal glucocorticoids on factors that regulate ECM remodeling during lung development. In the present study, we conducted a prospective, observational, pilot study to test the hypothesis that betamethasone-induced fetal lung maturation is mediated by changes in MMP activity. Furthermore, to determine whether the effects of antenatal betamethasone on MMPs and TIMPs are sustained when delivery is delayed for over 1 week, we compared the responses between patients who delivered < 2 weeks and > 2 weeks following betamethasone treatment.
MATERIAL AND METHODS
Patients
The study was approved by the Institutional Review Board of Long Beach Memorial Medical Center. Informed consent was obtained from all women enrolled in the study. Authorization for use and disclosure of protected health information according to the Health Information Portability and Accountability Act guidelines was also obtained. We conducted a prospective, observational pilot study on 43 singleton pregnancies over a 1-year period. Patients with preeclampsia, eclampsia, diabetes, or clinical chorioamnionitis were excluded. Placentas are not routinely examined for histologic chorioamnionitis but are examined at the discretion of each patient's physician based on evidence of clinical chorioamnionitis. The study consisted of three groups: group 1 (n = 21) was composed of women who were antentally treated with betamethasone for threatened premature delivery before 34 weeks of gestation and who delivered before 37 weeks of gestation. Betamethasone treatment consisted of two doses (12 mg/dose) given by intramuscular injection 24 hours apart. Preterm labor was defined as the presence of regular uterine contractions associated with cervical dilation at a gestational age of less than 37 weeks. To determine whether the effects of betamethasone on MMP activity and TIMP levels were sustained if pregnancy was delayed, group 1 was subdivided into patients delivering < 2 weeks (Group 1A, n = 13) and > 2 weeks (group 1B, n = 8) following treatment. Group 2 (n = 7) was composed of untreated women who delivered before 37 weeks of gestation and who were matched with group 1 for maternal race (as determined by maternal medical records) and gestational age. Group 3 (n = 15) was composed of untreated women who delivered > 37 weeks of gestation. Maternal data included age, mode of delivery, race, and preterm premature rupture of membranes (PPROM). Infant outcome data included gestational age at delivery, gender, birth weight, head circumference, body length (crown to heel), Apgar scores at 1 and 5 minutes, morbidities, mortality, and length of hospital stay.
Sample Collection
Maternal venous blood (2 mL) was obtained at the time of delivery using a 25-gauge phlebotomy needle and syringe (Vacutainer, Becton-Dickinson, Franklin Lakes, NJ). Mixed umbilical cord blood (approximately 1.0 mL) was also obtained at the time of delivery. Blood samples were collected in sterile tubes on ice and allowed to coagulate for 30 minutes. Samples were centrifuged at 3,000 rpm at 4 °C for 20 minutes. The resulting serum was frozen at −20°C until analysis. Fresh placental samples (100-200 mg) excised from the maternal-fetal interface were obtained in the delivery room and placed in sterile polypropylene tubes containing ice-cold 50 mM Tris-HCl buffer, pH 7.4 on ice, and taken to the laboratory for processing. For tissue homogenates, samples were rinsed several times in ice-cold buffer to remove blood elements, after which they were homogenized in ice-cold 50 mM Tris-HCl buffer, pH 7.4. The samples were centrifuged at 5,000 rpm at 4°C for 45 minutes, and a portion of the supernatant was removed for determination of total cellular protein levels. After filtration, the supernatant was frozen at −20°C until assay.
Assay of MMP Activity and TIMP Levels
Activity of MMP-2 and MMP-9 was determined in the serum and placental tissue homogenates using commercially available human Biotrak enzyme-linked immunosorbent assay (ELISA) activity kits (Amersham Pharmacia Biotech, Piscataway, NJ), as previously described.18,19This method for determining MMP-2 and -9 activity was previously validated and found to have several advantages over zymography, ELISA, and Western blotting.20-22Briefly, serum samples were diluted 1:1 in an assay buffer (provided in the kit). The activity assay is a specific and precise quantitative method for determination of active or pro-MMP-2 or -MMP-9 in serum and tissue homogenates. The assay recognizes the pro and active forms of MMP-2 and MMP-9 and does not cross-react with other MMPs or TIMPs. Standards and samples were incubated in microtiter wells precoated with anti-MMP-2 or anti-MMP-9 antibody at 2 to 8°C overnight. Any MMP-2 or -9 present was bound to the wells, and other components in the sample were removed by washing and aspiration. Endogenous levels of free active MMP-2 or -9 or total levels of free MMPs in the sample were detected. To measure total MMPs, bound MMPs in their pro form were activated using p-aminophenylmercuric acetate. Active MMPs were detected through activation of the modified pro detection enzyme. The concentrations of active MMPs in the sample were measured by extrapolation from a standard curve, which ranges from 0 to 12 ng/mL (MMP-2) and 0 to 4 ng/mL (MMP-9). MMP-2 or -9 activity was directly proportional to the generation of color and was represented by the rate of change of absorbance at 405 nm calculated as Abst = 2-Abst = 0/incubation time2× 1,000.
TIMP-1 and TIMP-2 levels were measured using human Biotrak ELISA kits from Amersham Biosciences. The assays use a two-site sandwich format. Standards and samples were incubated in microtiter wells precoated with anti-TIMP-1 or anti-TIMP-2 antibody at room temperature for 2 hours. Any TIMPs present in the samples were bound to the wells, and other components were removed by washing and aspiration. TIMPs were detected by peroxidase antibody to TIMP-1 or -2. The amount of peroxidase bound to each well was determined by the addition of tetramethylenbenzidine (TMB) substrate. The concentration of TIMPs in the sample was determined by extrapolation from a standard curve, which ranged from 0 to 50 ng/mL for TIMP-1 and 0 to 128 ng/mL for TIMP-2. The inter- and intra-assay variability for MMPs and TIMPs ranged between 5 and 12% and 2 and 16%, respectively. The sensitivities were 0.5, 0.5, 1.25, and 3.0 ng/mL for MMP-2, MMP-9, TIMP-1, and TIMP-2, respectively.
Protein Assay
On the day of the assay, the placental samples were homogenized and centrifuged at 5,000 rpm at 4°C for 20 minutes. The supernatant (10 μL) was used to assay total cellular protein by the dye-binding Bio-Rad protein assay (Bio-Rad, Hercules, CA) with bovine plasma albumin as a standard. The standard curve was linear from 0.05 to 1.45 mg/mL of protein.
Statistical Analysis
Statistical analyses were performed using GraphPad Prizm (GraphPad Software Inc., San Diego, CA). Categorical data were analyzed by uncorrected chi-square or Fisher exact test for cells that contained five or more observations. For normally distributed variables, Student's t-test was used to analyze data between the groups delivering < 2 weeks (group 1A) and > 2 weeks (group 1B) following betamethasone treatment. Wilcoxon rank sum test was used for non-normal variables, following Levene's test. Analysis of variance was used to determine differences among groups 1, 2, and 3 for normally distributed data, and Kruskal-Wallis test was used for non-normally distributed data, following Bartlett's test. Post hoc analysis was performed using the Student-Newman-Keuls test for significance. Significance was set at p < .05, and data are reported as mean ± SD, where applicable. All analyses were two-tailed.
RESULTS
Patient Characteristics
Fifty-three women with singleton pregnancies were enrolled in the study. Of those, 10 were excluded for various reasons, including samples not collected (n = 4), delivery at another hospital (n = 2), or delivery at > 37 weeks with betamethasone treatment (n = 4). The rate of cesarean delivery was comparable among the groups, as were race and infant gender. As expected, the incidence of PPROM was higher in groups 1 (62%, p < .05) and 2 (86%, p < .05) than in group 3. The mean gestational age was 30.5 ± 8.2, 33.9 ± 1.6, and 39.9 ± 1.3 weeks, and the mean birth weight was 2,011.7 ± 595.3, 2,364.7 ± 395.0, and 3,655.2 ± 628.2 g for groups 1, 2, and 3, respectively. Maternal age was significantly lower in group 1 (24.5 ± 4.6 years, p < .01) compared with group 3 (31.0 ± 4.0 years) but not group 2 (28.1 ± 6.8 years). Maternal characteristics comparing the preterm groups (group 1A treated patients delivering < 2 weeks following betamethasone treatment, group 1B treated patients delivering > 2 weeks following betamethasone treatment, and group 2 untreated preterm patients) are listed in Table 1. Maternal characteristics were similar among the groups except for the number of hours of rupture of membranes. Infant characteristics and clinical outcomes were similar among the preterm groups (Table 2).
MMPs, TIMPs and Their Ratios in Maternal Serum
MMP-2 and MMP-9, as well as their specific inhibitors, TIMP-2 and TIMP-1, respectively, were detected in all of the maternal serum samples. Figure 1 shows that maternal MMP-2 activity levels (ng/mL) were significantly lower in group 2 (14.5 ± 3.2, p < .05) than in groups 1 (18.1 ± 2.1) and 3 (18.2 ± 3.2). There were no differences in maternal MMP-2 levels between groups 1A and 1B (Table 3). Maternal TIMP-2 levels (ng/mL) were decreased in groups 1 (48.6 ± 21.5, p < .01) and 2 (38.5 ± 11.9, p < .01) compared with group 3 (83.7 ± 47.3) (Figure 2). Patients who delivered < 2 weeks following betamethasone treatment had lower maternal TIMP-2 levels (38.6 ± 14.5, p < .01) compared with those who delivered > 2 weeks (65.0 ± 22.3) (see Table 3). Maternal serum MMP-2 to TIMP-2 ratios remained comparable among the groups (Figure 3) and between groups 1A and 1B (see Table 3). Whereas maternal serum MMP-9 activity levels (Figure 4) and TIMP-1 levels (Figure 5) did not differ significantly among the groups, MMP-9 to TIMP-1 ratios were suppressed in group 1 in response to betamethasone treatment (0.11 ± 0.09, p < .05) compared with group 3 (0.18 ± 0.08) (Figure 6). Maternal MMP-9 to TIMP-1 ratios were not different between groups 1A and 1B (see Table 3).
MMPs, TIMPs, and Their Ratios in Placental Tissue
MMP-2 activity levels (ng/mg protein) in placental tissue were higher (115.2 ± 13.7, p < .05) compared with group 3 (104.5 ± 14.3) but not group 2, despite lower levels (100.8 ± 15.88) (see Figure 1). Whereas no differences in placental TIMP-2 levels were detected among the groups (see Figure 2), placental MMP-2 to TIMP-2 ratios were higher in group 1 (2.64 ± 1.56, p < .05) compared with group 3 (1.36 ± 0.39) (see Figure 3). No significant differences were detected for placental MMP-9, TIMP-1, and MMP-9 to TIMP-1 ratios among groups 1, 2, and 3 (see Figures 4 to 6). There were no differences in placental MMPs, TIMPs, or their ratios between groups 1A and 1B (data not shown).
MMPs, TIMPs, and Their Ratios in Mixed Umbilical Cord Serum
MMP-2 activity levels (ng/mL) in mixed umbilical cord serum was significantly lower in group 2 (23.3 ± 8.5, p < .05) compared with groups 1 (29.8 ± 6.1) and 3 (31.1 ± 6.3) (see Figure 1). Umbilical cord TIMP-2 levels (ng/mL) were lower in groups 1 (22.0 ± 29.8, p < .001) and 2 (32.0 ± 27.8, p < .01) compared with group 3 (66.2 ± 25.2) (see Figure 2). Mixed cord blood MMP-2 and TIMP-2 ratios were non significantly higher in group 1 (1.6 ± 4.3, compared with group 2 (0.35 ± 0.4) and group 3 (0.7 ± 0.8) (see Figure 3). There were no differences in MMP-2, TIMP-2, or their ratio between groups 1A and 1B (see Table 3). Similarly, MMP-9 activity levels (ng/mL) were significantly lower in group 2 (3.1 ± 1.9, p < .05) compared with groups 1 (4.5 ± 2.6) and 3 (6.9 ± 3.4) (see Figure 4). Although no differences in mixed umbilical cord serum TIMP-1 levels were noted among the groups (see Figure 5), there was a substantial reduction in MMP-9 to TIMP-1 ratios in groups 1 (0.056 ± 0.03, p < .05) and 2 (0.037 ± 0.02) compared with group 3 (0.08 ± 0.037) (see Figure 6). Comparison between the treated groups revealed that mixed umbilical cord serum TIMP-1 levels and MMP-9 to TIMP-1 ratios were higher in group 1A (82.8 ± 6.1, p < .01 and 0.07 ± 0.023, p < .05, respectively) compared with group 1B (72.8 ± 9.9 and 0.05 ± 0.016, respectively) (see Table 3).
DISCUSSION
Our findings suggest that betamethasone treatment results in increased MMP-2 activity in the maternal and fetoplacental compartments, as well as higher MMP-2 to TIMP-2 ratios in the fetoplacental compartment. Moreover, the findings of decreased fetal MMP-2 activity in conjunction with low TIMP-2 levels in the non-betamethasone-treated preterm group may suggest that these infants may be at risk of chronic lung disease, as demonstrated by Ekekezie and colleagues,23and bronchopulmonary dysplasia, as reported by Danan and colleagues.24Considering the important role of MMP-2 in lung morphogenesis, these data may suggest a mechanism, in part, for the effect of betamethasone on accelerated fetal lung maturation. Lower maternal TIMP-2 levels when delivery is delayed more than 2 weeks following betamethasone treatment and higher fetal MMP-9 to TIMP-1 ratios when delivery occurs at the time of maximum beneficial effects of betamethasone further underscore the importance of the treatment to delivery interval.15
The most significant effect of antenatal betamethasone was increased MMP-2 activity, which was comparable with term levels in all compartments. This finding proves our hypothesis that betamethasone-induced accelerated fetal lung maturation may involve its effects on MMPs. The role of MMPs in fetal lung maturation is well known. A comprehensive study of MMPs and TIMPs during human lung development showed that MMP-2 is the dominant proteolytic enzyme that was coexpressed with TIMP-2, suggesting that MMP-2 and TIMP-2 are key regulators of lung development.25Normal fetal lung development involves increased alveolar surfaces consisting of type I alveolar epithelial cells and alveolar capillary endothelial cells.26These processes are regulated by a balance between MMP-2 and its inhibitor, TIMP-2, which are highly expressed and activated during fetal lung development.27-29It has been suggested that activated MMP-2 in alveolar epithelial cells contributes to the formation of a large surface area.5Antenatal betamethasone is a potent lung-maturing agent. However, improved lung function with its use was shown to be associated with increased alveolar size, decreased alveolar number, and decreased septation in fetal lambs.13Similar characteristics develop with postnatal glucocorticoids.14We suggest that these unique characteristics associated with glucocorticoid-induced precocious lung development may be due to increased collagen breakdown through activated MMP-2. Our present findings corroborate recent evidence emerging from our laboratory that suggests that glucocorticoids interfere with the balance between MMPs and their tissue inhibitors in developing rat lungs.18To our knowledge, this is the first report of an association between betamethasone and MMP-2 activity in the setting of preterm pregnancy.
Although MMP-9 appears to play a less significant role during early fetal lung development, it has been detected in bronchial epithelial cells and type II alveolar cells in the late stages of lung development.5MMP-9 is secreted by inflammatory cells, including neutrophils and macrophages,30and its beneficial role involves cell spreading and migration.31In the present study, we found decreased fetal MMP-9 activity in the non-betamethasone-treated preterm group. This finding may be a consequence of prematurity because the number of alveolar macrophages is low in premature neonates.32Coexpression of MMP-2, MMP-9, and TIMP-1 is associated with intense remodeling, rapid cell proliferation, and collagen breakdown.33In the third trimester, the placenta secretes large amounts of MMP-9 and TIMP-1 to coincide with tissue remodeling and growth.34In our study, we found that MMP-9 activity in the maternal serum and placenta was comparable among the groups, although a nonsignificant trend for higher levels was noted in the term group. Given that all of our patients were in active labor, this finding, as well as that of others,35suggests that MMP-9 is similarly up-regulated in preterm and term placenta at the time of labor and is not affected by betamethasone.
TIMP-2 is a specific inhibitor of MMP-2 and is produced in the placenta throughout gestation.36Low TIMP levels at the onset of labor are responsible for membrane breakdown and detachment of fetal tissues and may well be responsible for the incidence of PPROM in our preterm groups. The maximum beneficial effects of betamethasone begin at 24 hours after treatment and may last up to 7 days post-treatment. However, some pregnancies are delayed a week or more after the effects have abated. Our results demonstrate that patients who deliver after the effects of betamethasone have subsided have higher maternal TIMP-2 levels and lower fetal MMP-9 to TIMP-1 ratios. These findings may demonstrate the importance of the treatment to delivery interval. A meta-analysis of antenatal steroid therapy demonstrated that babies delivered between 24 hours and 7 days after antenatal steroid therapy show more marked benefit.15A more recent study showed that infants exposed to a single course of antenatal steroids and delivered more than 7 days after initiation of treatment were associated with an increased need for short-term respiratory support.37
Although our preliminary pilot study has clinical implications, it suffers from several limitations, including a small sample size. A lack of statistical power resulted in nonsignificant trends, particularly with respect to group 2. Also, we did not examine MMP activity and TIMP levels in venous and arterial umbilical cord serum to more accurately reflect fetal responses. The use of zymography was not employed for qualitative MMP activity. However, the quantitative BiotraK activity assay employed in this report has been proven to be more sensitive and specific.20,21In conclusion, antenatal betamethasone appears to activate MMP-2 in the maternal and fetoplacental compartments, suggesting a mechanism, in part, for accelerated lung maturation and abnormal alveolar growth associated with glucocorticoids. Preterm pregnancies appear to be characterized by low maternal and fetal TIMP-2 levels, which may contribute to increased ECM breakdown and PPROM. Understanding the mechanisms of glucocorticoid effects on lung structure and function will help identify possible therapeutic agents for respiratory diseases of the newborn.
ACKNOWLEDGMENT
We thank Mrs. Pamela J. Rumney, RNC, for her participation in patient consent and sample collection.