Jamal Hasan

Soluble Vascular Endothelial Growth Factor Receptor 1 in Tracheal Aspirate Fluid of Preterm Neonates at Birth May Be Predictive of Bronchopulmonary Dysplasia/Chronic Lung Disease

OBJECTIVE. We tested the hypothesis that soluble vascular endothelial growth factor receptors are involved in the development of bronchopulmonary dysplasia/chronic lung disease. PATIENTS AND METHODS. Neonates with a birth weight of ≤1500 g and/or ≤30 weeks’ gestation, with respiratory failure, requiring O2 and mechanical ventilation within 24 hours, were eligible. Tracheal aspirate fluid samples were collected from 65 neonates before surfactant and/or assisted mechanical ventilation (baseline), at 3 and 7 days after birth, and weekly thereafter until extubation. Samples were analyzed for total vascular endothelial growth factor, soluble vascular endothelial growth factor receptor 1 and 2 levels and compared in infants with bronchopulmonary dysplasia/chronic lung disease (n = 31) versus those with no bronchopulmonary dysplasia/chronic lung disease (n = 34). RESULTS. Mean gestational age and birth weight were lower in infants with bronchopulmonary dysplasia/chronic lung disease. At baseline, vascular endothelial growth factor levels in the tracheal aspirate fluid were significantly lower, whereas soluble vascular endothelial growth factor receptor 1 levels were higher in the bronchopulmonary dysplasia/chronic lung disease infants compared with infants with no bronchopulmonary dysplasia/chronic lung disease. Vascular endothelial growth factor levels progressively increased from baseline to 4 weeks in all of the infants developing bronchopulmonary dysplasia/chronic lung disease. Conversely, soluble vascular endothelial growth factor receptor 1 declined in both groups from baseline to 5 weeks of age. Similarly, soluble vascular endothelial growth factor receptor 2 declined from baseline to 5 weeks in the control infants, but there were significant increases at 3 and 4 weeks in infants developing bronchopulmonary dysplasia/chronic lung disease. CONCLUSIONS. We speculate that low vascular endothelial growth factor levels in tracheal aspirate fluid, concurrent with elevated soluble vascular endothelial growth factor receptor 1 levels on the first day of life, are biological markers for the development of bronchopulmonary dysplasia/chronic lung disease in very low birth weight infants requiring O2 and assisted mechanical ventilation.

Reactive Oxygen Species, Biomarkers of Microvascular Maturation and Alveolarization, and Antioxidants in Oxidative Lung Injury

The lungs of extremely low gestational age neonates (ELGANs) are deficient in pulmonary surfactant and are incapable of efficient gas exchange necessary for successful transition from a hypoxic intrauterine environment to ambient air. To improve gas exchange and survival, ELGANs often receive supplemental oxygen with mechanical ventilation which disrupts normal lung developmental processes, including microvascular maturation and alveolarization. Factors that regulate these developmental processes include vascular endothelial growth factor and matrix metalloproteinases, both of which are influenced by generation of oxygen byproducts, or reactive oxygen species (ROS). ELGANs are also deficient in antioxidants necessary to scavenge excessive ROS. Thus, the accumulation of ROS in the preterm lungs exposed to prolonged hyperoxia, results in inflammation and development of bronchopulmonary dysplasia (BPD), a form of chronic lung disease (CLD). Despite advances in neonatal care, BPD/CLD remains a major cause of neonatal morbidity and mortality. The underlying mechanisms are not completely understood, and the benefits of current therapeutic interventions are limited. The association between ROS and biomarkers of microvascular maturation and alveolarization, as well as antioxidant therapies in the setting of hyperoxia-induced neonatal lung injury are reviewed in this article.

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.

26 Comparative Effects of Ibuprofen (IB) and Indomethacin (IN) for Suppression of Retinal VEGF Signaling in Rats

Background/Aims: VEGF is an angiogenic factor involved in retinal development through interaction with VEGFR1 and VEGFR2. Neuropilin (Npn) has been shown to enhance VEGF164 affinity for VEGFR2. Pigment epithelium-derived factor (PEDF) is an angiogenic antagonist that is upregulated after retinal vascularization is complete. IB and IN have been shown to improve oxygen-induced retinopathy in mice. We hypothesized that IB is more effective than IN for suppression of retinal VEGF signaling in rats.Methods: Newborn rats (n=3 litters/groups; 15 pups/litter) received IP injections of either saline (Sal), IB (10 mg/Kg), IB (50 mg/Kg), IN (0.2 mg/Kg), or IN (1.0 mg/Kg) on days 1, 2 and 3 postnatal age. At P14, the pups were sacrificed and retinal mRNA expression of VEGF188, VEGF164, VEGF120, VEGFR1, VEGFR2, Npn1, Npn2 and PEDF were examined.Results: Transcripts for VEGF164 were more abundant in the rat retina than VEGF188 and VEGF120. While no differences in VEGF188 mRNA expression were detected among the groups, VEGF164 transcripts were decreased with 50 mg IB (p<0.05 vs Sal), and both doses of IN (p<0.01 vs Sal). VEGF120 was suppressed only with 50 mg IB (p<0.01 vs Sal). Transcripts for Npn1 and Npn2 were co-expressed and abundant in the retina. Npn1 mRNA remained unchanged but Npn2 mRNA was significantly increased with 1.0 mg IN (p<0.01 vs Sal). IB and IN had no significant effects on retinal VEGFR1 or PEDF mRNA expression.Conclusions: Abundant expression of Npn1 and Npn2 in the retina suggests their involvement in retinal development. Although IN suppressed retinal VEGF164 transcripts, it had no effect on VEGFR1 and VEGFR2. In contrast, the high dose of IB suppressed VEGF164, VEGF120, and VEGFR2. IB appears to be more effective than IN for suppression of retinal VEGF signaling in rats, suggesting a possible novel therapy for retinal neovascularization.

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.