Endotoxin Alters Early Fetal Lung Morphogenesis

Christopher S. Muratore, Francois I. Luks, Yonghong Zhou, Mark Harty, Jonathan Reichner, Thomas Tracy

Research output: Contribution to journalArticle

8 Citations (Scopus)

Abstract

Background: The effects of immaturity and hypoplasia of the premature lung can be affected by proinflammatory stimuli in late gestation or the postnatal period from acute lung injury secondary to intensive ventilatory management or the metabolic consequences of surgery. These stimuli alter alveolarization and contribute to bronchopulmonary dysplasia. While prior research has focused primarily on late gestational effects of inflammation on alveolar development, we sought to study whether early gestational exposure to endotoxin affects branching morphogenesis, during the critical pseudoglandular stage of lung development. Method: Gestational day 15 (E15) fetal rat lung explants (term = 22 d) were treated with either 200 ng/mL or 2 μg/mL lipopolysaccharides (LPS) with controls and examined daily by phase microscopy. After 5 d, explants were fixed in 4% formaldehyde, paraffin embedded, and sectioned at 5 μm in the coronal plane. Immunohistochemical analysis was performed with platelet endothelial cell adhesion molecule (PECAM) to define endothelial cells, vascular endothelial growth factor (VEGF) to examine endothelial mitogenesis, and COX-2 antibodies as a marker for prostaglandin synthesis. Real-time PCR examined inducible nitric oxide synthase (iNOS), FGF9, FGF10, and FGFr2 gene expression. Air space fraction and airway epithelium were analyzed with Image J software. Results: Phase contrast microscopy and hematoxylin-eosin histology revealed progressive, dose-related changes in air sac contraction and interstitial thickening. Compared with control E15 explants, day 5 explants incubated with high dose LPS demonstrated thickened and shrunken airway sacs with stunted branching and increased matrix deposition in interstitial areas. By immunohistochemical staining, COX-2 was quantitatively increased after high dose LPS exposure, while PECAM was reduced. VEGF expression was unaltered. LPS increased iNOS, but decreased FGF9, FGF10, and FGFr2 gene expression. Conclusions: These data support evidence for an inflammatory effect of LPS on the early phase of lung development in the fetal rat, affecting branching morphogenesis during the pseudoglandular phase. Fetal endothelial cells are clearly affected, while COX-2 elevation suggests activation of an as yet undefined fetal pulmonary inflammatory cascade. We speculate that proinflammatory stimuli may ultimately lead to abnormal pulmonary development via fibroblastic growth factor (FGF)-directed mechanisms that affect epithelial-mesenchymal interaction and differentiation at a much earlier gestational age than was previously recognized.

Original languageEnglish (US)
Pages (from-to)225-230
Number of pages6
JournalJournal of Surgical Research
Volume155
Issue number2
DOIs
StatePublished - Aug 1 2009

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Morphogenesis
Endotoxins
Lipopolysaccharides
Lung
Endothelial Cells
Cell Adhesion Molecules
Nitric Oxide Synthase Type II
Vascular Endothelial Growth Factor A
Blood Platelets
Air Sacs
Gene Expression
Phase-Contrast Microscopy
Bronchopulmonary Dysplasia
Bariatric Surgery
Acute Lung Injury
Hematoxylin
Eosine Yellowish-(YS)
Fetal Development
Paraffin
Formaldehyde

All Science Journal Classification (ASJC) codes

  • Surgery

Cite this

Muratore, C. S., Luks, F. I., Zhou, Y., Harty, M., Reichner, J., & Tracy, T. (2009). Endotoxin Alters Early Fetal Lung Morphogenesis. Journal of Surgical Research, 155(2), 225-230. https://doi.org/10.1016/j.jss.2008.06.043
Muratore, Christopher S. ; Luks, Francois I. ; Zhou, Yonghong ; Harty, Mark ; Reichner, Jonathan ; Tracy, Thomas. / Endotoxin Alters Early Fetal Lung Morphogenesis. In: Journal of Surgical Research. 2009 ; Vol. 155, No. 2. pp. 225-230.
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abstract = "Background: The effects of immaturity and hypoplasia of the premature lung can be affected by proinflammatory stimuli in late gestation or the postnatal period from acute lung injury secondary to intensive ventilatory management or the metabolic consequences of surgery. These stimuli alter alveolarization and contribute to bronchopulmonary dysplasia. While prior research has focused primarily on late gestational effects of inflammation on alveolar development, we sought to study whether early gestational exposure to endotoxin affects branching morphogenesis, during the critical pseudoglandular stage of lung development. Method: Gestational day 15 (E15) fetal rat lung explants (term = 22 d) were treated with either 200 ng/mL or 2 μg/mL lipopolysaccharides (LPS) with controls and examined daily by phase microscopy. After 5 d, explants were fixed in 4{\%} formaldehyde, paraffin embedded, and sectioned at 5 μm in the coronal plane. Immunohistochemical analysis was performed with platelet endothelial cell adhesion molecule (PECAM) to define endothelial cells, vascular endothelial growth factor (VEGF) to examine endothelial mitogenesis, and COX-2 antibodies as a marker for prostaglandin synthesis. Real-time PCR examined inducible nitric oxide synthase (iNOS), FGF9, FGF10, and FGFr2 gene expression. Air space fraction and airway epithelium were analyzed with Image J software. Results: Phase contrast microscopy and hematoxylin-eosin histology revealed progressive, dose-related changes in air sac contraction and interstitial thickening. Compared with control E15 explants, day 5 explants incubated with high dose LPS demonstrated thickened and shrunken airway sacs with stunted branching and increased matrix deposition in interstitial areas. By immunohistochemical staining, COX-2 was quantitatively increased after high dose LPS exposure, while PECAM was reduced. VEGF expression was unaltered. LPS increased iNOS, but decreased FGF9, FGF10, and FGFr2 gene expression. Conclusions: These data support evidence for an inflammatory effect of LPS on the early phase of lung development in the fetal rat, affecting branching morphogenesis during the pseudoglandular phase. Fetal endothelial cells are clearly affected, while COX-2 elevation suggests activation of an as yet undefined fetal pulmonary inflammatory cascade. We speculate that proinflammatory stimuli may ultimately lead to abnormal pulmonary development via fibroblastic growth factor (FGF)-directed mechanisms that affect epithelial-mesenchymal interaction and differentiation at a much earlier gestational age than was previously recognized.",
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Muratore, CS, Luks, FI, Zhou, Y, Harty, M, Reichner, J & Tracy, T 2009, 'Endotoxin Alters Early Fetal Lung Morphogenesis', Journal of Surgical Research, vol. 155, no. 2, pp. 225-230. https://doi.org/10.1016/j.jss.2008.06.043

Endotoxin Alters Early Fetal Lung Morphogenesis. / Muratore, Christopher S.; Luks, Francois I.; Zhou, Yonghong; Harty, Mark; Reichner, Jonathan; Tracy, Thomas.

In: Journal of Surgical Research, Vol. 155, No. 2, 01.08.2009, p. 225-230.

Research output: Contribution to journalArticle

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T1 - Endotoxin Alters Early Fetal Lung Morphogenesis

AU - Muratore, Christopher S.

AU - Luks, Francois I.

AU - Zhou, Yonghong

AU - Harty, Mark

AU - Reichner, Jonathan

AU - Tracy, Thomas

PY - 2009/8/1

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N2 - Background: The effects of immaturity and hypoplasia of the premature lung can be affected by proinflammatory stimuli in late gestation or the postnatal period from acute lung injury secondary to intensive ventilatory management or the metabolic consequences of surgery. These stimuli alter alveolarization and contribute to bronchopulmonary dysplasia. While prior research has focused primarily on late gestational effects of inflammation on alveolar development, we sought to study whether early gestational exposure to endotoxin affects branching morphogenesis, during the critical pseudoglandular stage of lung development. Method: Gestational day 15 (E15) fetal rat lung explants (term = 22 d) were treated with either 200 ng/mL or 2 μg/mL lipopolysaccharides (LPS) with controls and examined daily by phase microscopy. After 5 d, explants were fixed in 4% formaldehyde, paraffin embedded, and sectioned at 5 μm in the coronal plane. Immunohistochemical analysis was performed with platelet endothelial cell adhesion molecule (PECAM) to define endothelial cells, vascular endothelial growth factor (VEGF) to examine endothelial mitogenesis, and COX-2 antibodies as a marker for prostaglandin synthesis. Real-time PCR examined inducible nitric oxide synthase (iNOS), FGF9, FGF10, and FGFr2 gene expression. Air space fraction and airway epithelium were analyzed with Image J software. Results: Phase contrast microscopy and hematoxylin-eosin histology revealed progressive, dose-related changes in air sac contraction and interstitial thickening. Compared with control E15 explants, day 5 explants incubated with high dose LPS demonstrated thickened and shrunken airway sacs with stunted branching and increased matrix deposition in interstitial areas. By immunohistochemical staining, COX-2 was quantitatively increased after high dose LPS exposure, while PECAM was reduced. VEGF expression was unaltered. LPS increased iNOS, but decreased FGF9, FGF10, and FGFr2 gene expression. Conclusions: These data support evidence for an inflammatory effect of LPS on the early phase of lung development in the fetal rat, affecting branching morphogenesis during the pseudoglandular phase. Fetal endothelial cells are clearly affected, while COX-2 elevation suggests activation of an as yet undefined fetal pulmonary inflammatory cascade. We speculate that proinflammatory stimuli may ultimately lead to abnormal pulmonary development via fibroblastic growth factor (FGF)-directed mechanisms that affect epithelial-mesenchymal interaction and differentiation at a much earlier gestational age than was previously recognized.

AB - Background: The effects of immaturity and hypoplasia of the premature lung can be affected by proinflammatory stimuli in late gestation or the postnatal period from acute lung injury secondary to intensive ventilatory management or the metabolic consequences of surgery. These stimuli alter alveolarization and contribute to bronchopulmonary dysplasia. While prior research has focused primarily on late gestational effects of inflammation on alveolar development, we sought to study whether early gestational exposure to endotoxin affects branching morphogenesis, during the critical pseudoglandular stage of lung development. Method: Gestational day 15 (E15) fetal rat lung explants (term = 22 d) were treated with either 200 ng/mL or 2 μg/mL lipopolysaccharides (LPS) with controls and examined daily by phase microscopy. After 5 d, explants were fixed in 4% formaldehyde, paraffin embedded, and sectioned at 5 μm in the coronal plane. Immunohistochemical analysis was performed with platelet endothelial cell adhesion molecule (PECAM) to define endothelial cells, vascular endothelial growth factor (VEGF) to examine endothelial mitogenesis, and COX-2 antibodies as a marker for prostaglandin synthesis. Real-time PCR examined inducible nitric oxide synthase (iNOS), FGF9, FGF10, and FGFr2 gene expression. Air space fraction and airway epithelium were analyzed with Image J software. Results: Phase contrast microscopy and hematoxylin-eosin histology revealed progressive, dose-related changes in air sac contraction and interstitial thickening. Compared with control E15 explants, day 5 explants incubated with high dose LPS demonstrated thickened and shrunken airway sacs with stunted branching and increased matrix deposition in interstitial areas. By immunohistochemical staining, COX-2 was quantitatively increased after high dose LPS exposure, while PECAM was reduced. VEGF expression was unaltered. LPS increased iNOS, but decreased FGF9, FGF10, and FGFr2 gene expression. Conclusions: These data support evidence for an inflammatory effect of LPS on the early phase of lung development in the fetal rat, affecting branching morphogenesis during the pseudoglandular phase. Fetal endothelial cells are clearly affected, while COX-2 elevation suggests activation of an as yet undefined fetal pulmonary inflammatory cascade. We speculate that proinflammatory stimuli may ultimately lead to abnormal pulmonary development via fibroblastic growth factor (FGF)-directed mechanisms that affect epithelial-mesenchymal interaction and differentiation at a much earlier gestational age than was previously recognized.

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