Zahra Gharraee

Effects of Antenatal Betamethasone on Maternal and Fetoplacental Matrix Metalloproteinases 2 and 9 Activities in Human Singleton Pregnancies

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.

467 EFFECT OF INDOMETHACIN AND IBUPROFEN ON RAT LUNG VASCULAR ENDOTHELIAL GROWTH FACTOR AND SOLUBLE VASCULAR ENDOTHELIAL GROWTH FACTOR RECEPTORS DURING EARLY POSTNATAL DEVELOPMENT.

Vascular endothelial growth factor (VEGF) is a potent endothelial cell mitogen that is highly involved in lung microvasculature proliferation, permeability, and maturation. Its overexpression in the neonatal lung results in pulmonary hemorrhage and inflammation. Soluble VEGF receptors (sVEGFR-1, sVEGFR-2) are the negative regulators in the VEGF signaling pathway. Indomethacin (Indo) is administered to premature newborn infants for ductus arteriosus closure. However, its use is associated with increased pulmonary vascular resistance. The purpose of this study was to compare the effects of early administration of Indo and Ibu on VEGF and its soluble receptors in the developing rat lungs. Sprague-Dawley rats received IP injections at birth (P0), postnatal day 1 (P1), and P2 of either saline (Sal); 0.2 mg/kg IN on P0 followed by 0.1 mg/kg on P1 and P2; 1.0 mg/kg IN on P0 followed by 0.5 mg/kg on P1 and P2; 10 mg/kg IB on P0 followed by 5 mg/kg on P1 and P2; and 50 mg/kg IB on P0 followed by 25 mg/kg on P1 and P2. At P14 and P21 (time of microvascular maturation), lung homogenates were assessed for VEGF, sVEGFR-1 and sVEGFR-2. Untreated term rat lungs were also examined. VEGF levels increased at P14 and P21 (p < .001 vs term) whereas sVEGFR-1 levels remained unchanged and were 30- to 50-fold higher than sVEGFR-2. sVEGFR-2 levels peaked at P14 (p < .001 vs term) and decreased at P21 (p < .01 vs. term and P14). Indo and Ibu had no effect on VEGF levels at P14; however, at P21, VEGF levels increased with low-dose Indo (p < .05 vs Sal). Similarly, sVEGFR-1 levels remained unchanged at P14. In contrast, sVEGFR-1 levels were elevated at P21 in the high-dose Indo group (p < .001 vs low-dose Ibu and Indo). Low and high doses of Indo had significant suppressive effects on VEGFR-2 at P14 (p < .01 vs Sal), although the effect of Indo was more potent and was sustained until P21 (p < .01 vs Sal). Soluble VEGFR-2 may play an important role during the time of lung microvascular maturation. Its suppression at P21 with Indo may result in VEGF overexpression. Ibu does not exhibit similar suppressive effects at this crucial time of lung development and therefore should be considered as an alternate therapy for ductus arteriosus closure.

158 ANTENATAL INDOMETHACIN SUPPRESSES LUNG MATRIX METALLOPROTEINASE ACTIVITY AND VASCULAR ENDOTHELIAL GROWTH FACTOR LEVELS IN FETAL RABBITS.

Oral indomethacin (OI) has been shown to increase saccular wall mass and decrease airspace in fetal rat lungs, implicating an etiologic relationship with persistent pulmonary hypertension (PPHN). Normal fetal lung maturation is associated with increased breakdown and remodeling of the lung basement membrane and extracellular matrix, as well as microvascular maturation. These processes are regulated, in part, by matrix metalloproteinases (MMPs) and vascular endothelial growth factor (VEGF). We tested the hypothesis that maternal administration of OI suppresses MMP activity and VEGF levels in fetal rabbit lungs, in a rabbit model for RU486-induced preterm birth. We also examined and compared the effects of vaginally administered indomethacin. Pregnant rabbits (n = 6/group) were injected with a single 50 mg IM dose of RU486 at 24 days of a 30- to 32-day gestation. Immediately following, the animals received either (1) OI (20 mg in a vehicle suspension); (2) a mixture of methylcellulose and polysorbate 80 (OV, vehicle); a vaginal suppository of indomethacin (VI, 20 mg in cocoa butter); or a suppository of cocoa butter only (placebo, VP), once daily for 2 days. At delivery, lung homogenates were examined for MMP-2 and MMP-9 activity, tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 levels, and VEGF levels. Lung MMP-9 activity (ng/mg protein) was suppressed with OI (0.11 ± 0.003 vs 0.17 ± 0.005, p < .001) and VI (0.093 ± 0.008 vs 0.25 ± 0.008, p < .01) compared to OV and VP, respectively. In contrast, TIMP-1 (3.7 ± 0.87 vs 0.89 ± 0.09, p < .01) and TIMP-2 (43.3 ± 2.7 vs 22.4 ± 2.9, p < .001) levels (ng/mg protein) were elevated with OI only compared to OV resulting in suppression of MMP-2/TIMP-2 (0.23 ± 0.01 vs 0.55 ± 0.01, p < .001) and MMP-9/TIMP-1 (0.07 ± 0.002 vs 0.21 ± 0.007, p < .001) ratios. Lung VEGF levels (pg/mg protein) were suppressed with both OI (1.79 ± 0.11 vs 2.5 ± 0.07, p < .05) and VI (2.2 ± 0.2 vs 2.9 ± 0.16, p < .05). We conclude that the combined suppression of lung MMP activity and VEGF synthesis with OI may be responsible for immature, thickened saccular walls and the development of PPHN.

271 ONTOGENY OF VASCULAR ENDOTHELIAL GROWTH FACTOR, INSULIN-LIKE GROWTH FACTOR-I, AND GROWTH HORMONE IN THE RAT VITREOUS FLUID, RETINAL HOMOGENATES, AND SYSTEMIC CIRCULATION: RELATIONSHIP TO PHYSICAL GROWTH AND RETINAL DEVELOPMENT

Vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF)-I, and growth hormone (GH) are important regulators of physical growth and have been shown to play key roles in retinal angiogenesis and development. In premature infants, low serum IGF-I and high GH levels in the early postnatal period were reported to be associated with severe retinopathy of prematurity (ROP). We examined the ontogenic profile of VEGF, IGF-I and GH in rat vitreous fluid, retinal homogenates and systemic circulation from birth to 21 days postnatal age (weaning, P21). Sprague Dawley rats (litter size =15 pups) were sacrificed at P0, P7, P14 and P21 (3 litters/group). At sacrifice, the pups were weighed and measured for linear growth (nose to tail length). Vitreous fluid, retinal homogenates, and serum were analyzed for VEGF, IGF-I and GH by enzyme immunoassay. VEGF levels were 10-fold higher in the vitreous (pg/mL) than serum (pg/mL) at all stages of development. Vitreous and serum VEGF levels progressively declined at P7, P14 and P21 (p≤0.05 to p≤0.001) compared to term, however in the retinal homogenates, VEGF levels (pg/mg protein) increased with the highest concentration at P21 (p≤0.05 vs term). Vitreous IGF-I levels were decreased at P7 through P14 (p≤0.05) compared to term. Vitreous GH levels were 10-fold lower than serum levels and were decreased at P14 and P21 (p≤0.001) compared to P7. Despite a trend for increasing IGF-I and decreasing GH in retinal homogenates, no appreciable changes were detected with advancing postnatal age. Similarly, serum IGF-I levels increased with postnatal age (P14 and P21: p≤0.05 vs term), whereas serum GH levels were decreased at P7 (p≤0.05), P14 (p≤0.01), and P21 (p≤0.01) compared to term. In rats, retinal development occurs postnatally by P14. Our data demonstrate that during normal retinal development, VEGF, IGF-I and GH decrease in the vitreous; and VEGF and IGF-I increase, while GH decreases in the retinal homogenates. A quite different ontogenic pattern is noted in the systemic circulation. VEGF and GH decrease, while IGF-I increases with advancing physical growth. We therefore conclude that any future therapy for ROP should consider changes occurring in the eye rather than the systemic compartment.

466 A SINGLE COURSE OF ANTENATAL BETAMETHASONE INCREASES MATRIX METALLOPRPTEINASE-2 IN MATERNAL BLOOD AND PLACENTA

A single course of antenatal betamethasone (B) is administered to women with threatened preterm delivery to advance fetal lung maturation. Lung maturation is associated with increased breakdown and remodeling of the lung basement membrane and extracellular matrix. These processes are regulated by matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) which are present in the fetomaternal membranes of the placenta. We examined the effects of a single course of antenatal B on maternal and placental MMPs and TIMPs. A prospective pilot study of three groups of pregnant patients was conducted. Group I (n=21): women who were antenatally treated with a single course of B, and who delivered ≤36 weeks gestation; Group II (n=7): untreated women who delivered ≤36 weeks (preterm controls); and Group III (n=15): untreated women who delivered ≥38 weeks (term controls). Group I was subdivided into women delivering: a) ≤2 weeks (IA, n=13); and b) ≥2 weeks (IB, n=8) post B treatment. Maternal blood and placental samples were collected at the time of delivery for MMP-2, MMP-9, TIMP-1 and TIMP-2 levels. Placental levels were standardized using total cellular protein levels. Maternal MMP-2 levels (ng/mL) were significantly lower in Group II (14.5±1.2, p≤0.05) than Groups IA (17.9±0.67) IB (18.4±0.6), and III (18.2±0.83). Maternal MMP-9 and TIMP-1 remained unchanged. Maternal TIMP-2 was suppressed in Group IA (38.6±10.0, p≤0.05) and Group II (38.5±4.5, p≤0.05) compared to Group III (83.7±12.3), however, in Group IB, the levels were increased (65.0±7.9, p≤0.01) compared to Groups IA and II. As a result, a higher MMP-2 to TIMP-2 ratio was noted in Group IA (0.54±0.06, p≤0.01) than Groups IB (0.32±0.04) and III (0.33±0.04); and a lower ratio was noted in Group IB (0.32±0.04, p≤0.05) than Group II (0.40±0.04). In the placenta, MMP-2 levels were increased in Group IA (117.3±3.9, p≤0.05) compared to Groups II (100.8±6.0) and III (104.5±3.7). Interestingly, TIMP-1 levels were 10-fold higher in maternal blood than placenta, whereas TIMP-2 levels were 2-fold greater in placenta than maternal blood. Data are mean±SEM. A single course of antenatal B acutely enhances maternal and placental MMP-2 synthesis and maternal MMP-2/TIMP-2 ratio. Increased MMP-2 activity may result in increased collagen breakdown, and suggests a mechanism for Bs effect on fetal lung maturation. Decreased MMP-2 by 2 weeks post B treatment reflects abatement of the effects of antenatal B known to occur by 7-10 days.