Anne Chetty

Insulin-like Growth Factor-I signaling mechanisms, type I collagen and alpha smooth muscle actin in human fetal lung fibroblasts.

Bronchial wall remodeling is a major morbidity component in oxidant injury in bronchopulmonary dysplasia (BPD) and asthma. Hypothesis: IGF-1 enhances alpha smooth muscle expression and collagen synthesis in developing lung fibroblasts leading to fibrosis through nuclear NF-kB -dependent transcription. We studied NF-kB dependent transcription by transfecting HFLF with a NF-kB responsive promoter driving the luciferase gene and treating with IGF-1 (100 ng/mL) and measuring luciferase activity. We exposed cells to the PI-3 kinase inhibitor or the Erk1/2 inhibitor one hr before stimulating with IGF-1. We also used IGF-1 receptor antibody to inhibit the action of IGF-1 and studied its effect on alpha-sma and type I collagen. IGF-1 treatment significantly increased luciferase activity. This was attenuated by PI-3 kinase and MAP-Kinase inhibitors. Western blot analysis showed PI-3 kinase mediates IGF-1 activation of NF-kB independent of IKB phosphorylation. We found an up-regulation of phospho NF-kB in the nuclear extract compared with total NFKB showing that IGF-1 regulates NF-kB transcriptional activity downstream of NF-kB nuclear translocation. IGF-1-induced increase in alpha-sma expression and type-I collagen was significantly inhibited by pretreatment with LY294002 and IGF-1 receptor antibody. IGF-1 cell signaling leading to collagen synthesis in fetal lung fibroblasts is mediated by PI3 Kinase acting through NF-kB in HFLF.

Role of matrix metalloprotease-9 in hyperoxic injury in developing lung.

Matrix metalloprotease-9 (MMP-9) is increased in lung injury following hyperoxia exposure in neonatal mice, in association with impaired alveolar development. We studied the role of MMP-9 in the mechanism of hyperoxia-induced functional and histological changes in neonatal mouse lung. Reduced alveolarization with remodeling of ECM is a major morbidity component of oxidant injury in developing lung. MMP-9 mediates oxidant injury in developing lung causing altered lung remodeling. Five-day-old neonatal wild-type (WT) and MMP-9 (-/-) mice were exposed to hyperoxia for 8 days. The lungs were inflation fixed, and sections were examined for morphometry. The mean linear intercept and alveolar counts were evaluated. Immunohistochemistry for MMP-9 and elastin was performed. MMP-2, MMP-9, type I collagen, and tropoelastin were measured by Western blot analysis. Lung quasistatic compliance was studied in anaesthetized mice. MMP-2 and MMP-9 were significantly increased in lungs of WT mice exposed to hyperoxia compared with controls. Immunohistochemistry showed an increase in MMP-9 in mesenchyme and alveolar epithelium of hyperoxic lungs. The lungs of hyperoxia-exposed WT mice had less gas exchange surface area and were less compliant compared with room air-exposed WT and hyperoxia-exposed MMP-9 (-/-) mice. Type I collagen and tropoelastin were increased in hyperoxia-exposed WT with aberrant elastin staining. These changes were ameliorated in hyperoxia-exposed MMP-9 (-/-) mice. MMP-9 plays an important role in the structural changes consequent to oxygen-induced lung injury. Blocking MMP-9 activity may lead to novel therapeutic approaches in preventing bronchopulmonary dysplasia.

EPITHELIAL-MESENCHYMAL INTERACTION AND INSULIN-LIKE GROWTH FACTORS IN HYPEROXIC LUNG INJURY

To investigate the role of epithelial-mesenchymal interaction on oxygen-induced lung injury, we used a coculture model with lung fibroblasts (FB) embedded between 2 layers of collagen gel with and without human tracheobronchial epithelial cells (HTBE), and studied the effect of hyperoxia on the directed migration of FB towards epithelial cells and proliferation of fetal lung FB. The expression of insulin-like growth factor (IGF)-I, -II, and -IIR mRNAs and proteins was studied in FB and HTBE cells cultured separately in 95% oxygen and 5% CO2 for 48 hours. There was a significant increase in directional migration of FB in coculture with epithelial cells when exposed to 95% oxygen and 5% CO2 (P = .04 compared to cocultures without oxygen exposure). Hyperoxia stimulated the proliferation of fibroblasts cocultured with HTBE cells (0.75 +/- 0.05 x 10(6) cells per well) as compared to control (0.47 +/- 0.03 x 10(6) cells per well; P = .01). This was inhibited by anti-IGF-I antibody (69 +/- 2% of hyperoxia alone; P = .002). Western blot showed a significant increase in IGF-I protein in epithelial cells (P = .02). IGF-I mRNA was increased in HTBE cells after hyperoxia (P = .003). In conclusion, HTBE cells modulate lung FB migration and proliferation in response to hyperoxia exposure. This is mediated in part by IGF-I produced by epithelial cells.

The role of IL‐6 and IL‐11 in hyperoxic injury in developing lung

We examined the cytoprotective effect of interleukin‐6 (IL‐6) and interleukin‐11 (IL‐11) during oxidant injury in neonatal lung and the regulators of cell death in vitro and in vivo after oxidant exposure. Type II cells from day 21 fetal neonatal rat lungs were treated with varying concentrations of either IL‐6 or IL‐11 for 24 hr prior to exposure to H2O2. Three‐day‐old transgenic lung‐specific IL‐11 and IL‐6 overexpressing and wild type (WT) mouse pups were exposed to hyperoxia or room air for 3 days. Type II cells exposed to either IL‐6 or IL‐11 prior to oxidant injury exhibited improved survival compared to controls, 67% ± 2.6 survivals in IL‐6 pretreated cells compared to 48% ± 1.6 in control; 63% ± 3 survivals in IL‐11 pretreated cells compared to 49% ± 2.6 in control. The number of TUNEL positive cells in hyperoxia‐exposed lungs was increased compared to room air animals (27 ± 0.9 vs. 4 ± 0.4; mean ± SEM; P < 0.05). In contrast, the number of TUNEL positive cells was reduced in hyperoxia‐exposed lungs from IL‐11 (+) mice (15.2 ± 2.2; mean ± SEM; P < 0.05). There was an enhanced accumulation of Bcl‐2 and reduction of Bax protein in hyperoxia‐exposed IL‐11 (+) compared to room air‐exposed mice. This was not seen in hyperoxia‐exposed IL‐6 (+) pups. An increase in caspase‐3 was seen in hyperoxia‐exposed lungs of WT pups compared to IL‐11 (+) pups. IL‐11 and IL‐6 provide protective effects against oxidant‐mediated injury in fetal type II cells and IL‐11 provides protection in vivo by down‐regulation of caspase‐mediated cell death. Pediatr Pulmonol. 2008; 43:297–304.

Pigment Epithelium–Derived Factor Mediates Impaired Lung Vascular Development in Neonatal Hyperoxia

Bronchopulmonary dysplasia is a chronic lung disease of preterm infants characterized by arrested microvascularization and alveolarization. Studies show the importance of proangiogenic factors for alveolarization, but the importance of antiangiogenic factors is unknown. We proposed that hyperoxia increases the potent angiostatin, pigment epithelium-derived factor (PEDF), in neonatal lungs, inhibiting alveolarization and microvascularization. Wild-type (WT) and PEDF(-/-) mice were exposed to room air (RA) or 0.9 fraction of inspired oxygen from Postnatal Day 5 to 13. PEDF protein was increased in hyperoxic lungs compared with RA-exposed lungs (P < 0.05). In situ hybridization and immunofluorescence identified PEDF production primarily in alveolar epithelium. Hyperoxia reduced alveolarization in WT mice (P < 0.05) but not in PEDF(-/-) mice. WT hyperoxic mice had fewer platelet endothelial cell adhesion molecule (PECAM)-positive cells per alveolus (1.4 ± 0.4) than RA-exposed mice (4.3 ± 0.3; P < 0.05); this reduction was absent in hyperoxic PEDF(-/-) mice. The interactive regulation of lung microvascularization by vascular endothelial growth factor and PEDF was studied in vitro using MFLM-91U cells, a fetal mouse lung endothelial cell line. Vascular endothelial growth factor stimulation of proliferation, migration, and capillary tube formation was inhibited by PEDF. MFLM-91U cells exposed to conditioned medium (CM) from E17 fetal mouse lung type II (T2) cells cultured in 0.9 fraction of inspired oxygen formed fewer capillary tubes than CM from T2 cells cultured in RA (hyperoxia CM, 51 ± 10% of RA CM, P < 0.05), an effect abolished by PEDF antibody. We conclude that PEDF mediates reduced vasculogenesis and alveolarization in neonatal hyperoxia. Bronchopulmonary dysplasia likely results from an altered balance between pro- and antiangiogenic factors.

Modulation of IGF-binding protein-2 and -3 in hyperoxic injury in developing rat lung.

Retinoids play an important role in lung development and repair. We showed that retinoic acid (RA) inhibits O2-induced fibroblast proliferation in rat lung explants. IGF-1, which enhances the proliferation of human fetal lung fibroblasts and stimulates collagen production during lung injury, has an important role in the lung injury/repair process. Interactions of IGF-1 with its receptor are modulated by IGF-binding proteins IGFBPs. We hypothesized that RA alters IGFBP-2 and -3 in hyperoxia-exposed neonatal lung and alters collagen production. Neonatal rat lungs were cultured in room air or 95% O2 and 5% CO2 for 3 d with or without RA. IGFBP-2 and -3 were measured both in culture medium and in lung tissue. Type I collagen and procollagen propeptide were analyzed in the lung tissue. Hyperoxia induced an increase in type I collagen that was significantly inhibited in the presence of RA. IGFBP-2 and IGFBP-3 in the lungs were decreased in hyperoxia but significantly increased in hyperoxia plus RA. In the culture medium, IGFBP-2 and -3 were not increased with hyperoxia but significantly increased in the presence of RA plus hyperoxia. There was no increase in IGFBP-3 RNA transcript after RA treatment in either room air or O2 exposure. In conclusion, RA modulates the secreted IGFBP-2 and -3 during O2 exposure and inhibits the increase in collagen that occurs during lung injury. We speculate that RA protects against O2-induced neonatal lung injury through modulation of the IGFBPs.

Oxygen-Induced Fibroblast Proliferation in Human Fetal Lung Cells Is Mediated by Insulin-Like Growth Factor-1

Oxygen-Induced Fibroblast Proliferation in Human Fetal Lung Cells Is Mediated by Insulin-Like Growth Factor-1