J Korean Med Sci.  2015 Sep;30(9):1295-1301. 10.3346/jkms.2015.30.9.1295.

Deferoxamine Improves Alveolar and Pulmonary Vascular Development by Upregulating Hypoxia-inducible Factor-1alpha in a Rat Model of Bronchopulmonary Dysplasia

• Choi CW
• Lee J
• Lee HJ
• Park HS
• Chun YS
• Kim BI

• KMID: 2344155
• DOI: http://doi.org/10.3346/jkms.2015.30.9.1295

Abstract

Fetal lung development normally occurs in a hypoxic environment. Hypoxia-inducible factor (HIF)-1alpha is robustly induced under hypoxia and transactivates many genes that are essential for fetal development. Most preterm infants are prematurely exposed to hyperoxia, which can halt hypoxia-driven lung maturation. We were to investigate whether the HIF-1alpha inducer, deferoxamine (DFX) can improve alveolarization in a rat model of bronchopulmonary dysplasia (BPD). A rat model of BPD was produced by intra-amniotic lipopolysaccharide (LPS) administration and postnatal hyperoxia (85% for 7 days), and DFX (150 mg/kg/d) or vehicle was administered to rat pups intraperitoneally for 14 days. On day 14, the rat pups were sacrificed and their lungs were removed and examined. A parallel in vitro study was performed with a human small airway epithelial cell line to test whether DFX induces the expression of HIF-1alpha and its target genes. Alveolarization and pulmonary vascular development were impaired in rats with BPD. However, DFX significantly ameliorated these effects. Immunohistochemical analysis showed that HIF-1alpha was significantly upregulated in the lungs of BPD rats treated with DFX. DFX was also found to induce HIF-1alpha in human small airway epithelial cells and to promote the expression of HIF-1alpha target genes. Our data suggest that DFX induces and activates HIF-1alpha, thereby improving alveolarization and vascular distribution in the lungs of rats with BPD.

Keyword

Alveolarization; Bronchopulmonary Dysplasia; Deferoxamine; Hypoxia-Inducible Factor

MeSH Terms

Animals
Bronchopulmonary Dysplasia/*drug therapy/*metabolism/pathology
Deferoxamine/*administration & dosage
Female
Hypoxia-Inducible Factor 1, alpha Subunit/*metabolism
Male
Pulmonary Alveoli/drug effects/*growth & development/metabolism/pathology
Pulmonary Veins/drug effects/*growth & development/pathology
Rats
Rats, Sprague-Dawley
Treatment Outcome
Up-Regulation/drug effects
Deferoxamine
Hypoxia-Inducible Factor 1, alpha Subunit

Figure

• 

  Fig. 1 Representative photomicrographs of rat lungs on day 14 (A). H&E staining; magnification, 100 ×. Scale bar indicates 200 µm. Morphometric data of rat lungs on day 14 are shown (B, C). n = 5-8 per group. *P <0.05. BPD, bronchopulmonary dysplasia; V, vehicle; DFX, deferoxamine.

• 

  Fig. 2 Representative photomicrographs illustrating the results of immunohistochemical analysis of PECAM-1 in rat lungs on day 14 (A). Arrows indicate pulmonary vessels stained with PECAM-1. Magnification, 400 ×. Scale bar indicates 50 µm. The number of pulmonary vessels per high-power field (HPF) (B) and pulmonary vascular density (C) are displayed in a bar graph. n = 5-8 per group. *P < 0.05. PECAM, platelet endothelial cell adhesion molecule; BPD, bronchopulmonary dysplasia; V, vehicle; DFX, deferoxamine.

• 

  Fig. 3 Representative photomicrographs illustrating the results of immunofluorescence analysis of HIF-1α in rat lung on day 14 (A). Treatment with DFX recovered the expression of HIF-1α in the lungs of BPD rats. Tissue sections were serially cut at 4 µm and subjected to immunofluorescence staining of HIF-1α. Lung tissues in the four experimental groups were homogenized, and 20 µg of protein was subjected to Western blotting using an anti-HIF-1α antibody (B). Tubulin was used as a loading control. DFX, deferoxamine; HIF, hypoxia-inducible factor; BPD, bronchopulmonary dysplasia; V, vehicle; DAPI, 4',6-diamidino-2-phenylindole.

• 

  Fig. 4 Deferoxamine induced HIF-1α in a dose-dependent manner in human small airway epithelial cells (HSAEpCs) (A). HSAEpCs were incubated with 10, 20, 30 or 65 µM DFX for 8 hr, and HIF-1α expression was evaluated by Western blotting. HSAEpCs were incubated with 65 µM DFX for the indicated times (B). The expression of HIF-1α was analyzed by Western blotting. DFX, deferoxamine; HIF, hypoxia-inducible factor.

• 

  Fig. 5 Deferoxamine-induced HIF-1α was active. Deferoxamine activated VEGF promoter (A). Human small airway epithelial cells (HSAEpCs) were co-transfected with 1 µg of VEGF promoter-luciferase plasmid and β-galactosidase plasmid. After 48 hr, the cells were treated with 10 or 65 µM DFX for 16 hr and lysed for the luciferase assay. Luciferase activity was normalized to β-galactosidase activity. Bars represent the mean±SD (n = 4) of luciferase activity. Deferoxamine induces the mRNA expression of HIF-1α target genes (B, C). Cells were treated with 65 µM DFX for the indicated times and subsequently harvested. Total RNAs were extracted from the cells, and the mRNA levels of CA9 (B), LOX (C), and 18S were analyzed by RT-qPCR. Each bar represents the mean±SD of four separate experiments. *P < 0.05. DFX, deferoxamine; HIF, hypoxia-inducible factor.

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